KR970010460B1 - Separation method for 5-(3-(2-4-dihydroxy phenyl)propyl)-3,4- bis (3-methyl-2-butenyl)-1,2-benzenediol - Google Patents

Abstract

Process for separating 5- (3- (2- 4- dehydroxy phenyl) prophyl) - 3,4- bis (3- methyl - 2- butenyl)- 1,2- benzendiol which is used to make compositions for whitening skin, antioxidizing or antibrowning comprises the steps of: extracting from bark or velamen of paper mulberry by using a polar solvent; condensing the extract; partitioning it with a oil-soluble solvent and water; chromatographing the fraction of the oil-soluble solvent layer on Sephadex LH-20, a column packed silica and a reversed-phase column, subsequently to separate and refine the final compound.

Description

Separation method of 5- (3- (2,4-dihydroxyphenyl) propyl) -3,4-bis (3-methyl-2-butenyl) -1,2-benzenediol and composition containing the same

1 is a ¹ H-NMR spectrum of 5- (3- (2,4-dihydroxyphenyl) propyl) -3,4-bis (3-methyl-2-butenyl) -1,2-benzenediol .

2 is a ¹³ C-NMR spectrum of 5- (3- (2,4-dihydroxyphenyl) propyl) -3,4-bis (3-methyl-2-butenyl) -1,2-benzenediol .

3 is an IR spectrum of 5- (3- (2,4-dihydroxyphenyl) propyl) -3,4-bis (3-methyl-2-butenyl) -1,2-benzenediol.

4 is an MS spectrum of 5- (3- (2,4-dihydroxyphenyl) propyl) -3,4-bis (3-methyl-2-butenyl) -1,2-benzenediol.

5 is the UV spectrum of 5- (3- (2,4-dihydroxyphenyl) propyl) -3,4-bis (3-methyl-2-butenyl) -1,2-benzenediol.

FIG. 6 shows the tyrosinase activity inhibitory activity of 5- (3- (2,4-dihydroxyphenyl) propyl) -3,4-bis (3-methyl-2-butenyl) -1,2-benzenediol It is a graph.

7 is a graph showing the antioxidant activity of 5- (3- (2,4-dihydroxyphenyl) propyl) -3,4-bis (3-methyl-2-butenyl) -1,2-benzenediol .

8 shows polyphenol oxidase activity inhibition of 5- (3- (2,4-dihydroxyphenyl) propyl) -3,4-bis (3-methyl-2-butenyl) -1,2-benzenediol Graph showing the action.

The present invention provides a method for separating and purifying 5- (3- (2,4-dihydroxyphenyl) propyl) -3,4-bis (3-methyl-2-butenyl) -1,2-benzenediol and so It relates to the use of the separated material.

More specifically, the present invention provides 5- (3- (2,4-dihydroxyphenyl) propyl) -3,4-bis () in bark or root bark of Broussonetia plants. It relates to a method for separating and purifying 3-methyl-2-butenyl) -1,2-benzenediol and a skin lightening composition, an antioxidant composition and a browning prevention composition containing the compound as an active ingredient.

The blackening of the skin is caused by the reaction of skin cells to internal and external factors. Phenomenologically, a series of oxidation processes, followed by the action of tyrosine and tyrosinase on tyrosine in cells called melanocytes in the basal layer of the skin's epidermal tissue. This creates an excess of melanin and accumulates in the skin. Tyrosinase is the most important enzyme in the skin's blackening process that makes tyrosine, an amino acid, into dopa and dopaquinone, the intermediate products of melanin polymer production. As the melanin production process is the action of tyrosinase and oxidation of melanin polymer after dopaquinone, the melanin-preventing substances have a tyrosinase inhibitory effect or an antioxidant effect.

The desire for improvement and treatment of pigmented skin diseases such as blackened skin and freckle freckles is particularly great for women. Actually, ascorbic acid, kojic acid, glutathione, hydroquinone, etc. have tyrosinase inhibitory effect or antioxidant effect, so whitening cosmetics or drugs containing them are commercially available, but ascorbic acid, hydroquinone, glutathione, etc. are very unstable and easily oxidized. There is a problem of discoloration or deterioration easily in the formulation. Glutathione has its own peculiar smell, and kojic acid is not satisfactory in terms of whitening effect, and ascorbic acid and hydroquinone have been severely restricted in use due to severe skin side effects.

In recent years, the tendency to search for substances having a whitening effect in natural products is prominent, and it has been found that sangbaekpi (Japanese Patent Application No. 55-44375, Cow 64-26507, Cow 64-83009, Pyeong 1-256587, Licorice (Japanese Patent Application Laid-Open No. 60-60). 214721, small 63-23809, small 64-63506, flat 1-149706), peony (Japanese Patent Laid-Open No. 61-246109), cinnamon (Japanese Patent Laid-Open No. 63-30403), red ginseng, Ho Jang-geun, oman manza, Sanyu It has already been reported to inhibit melanin production in plant extracts, eg, Hogo vinegar, etc. However, these extracts have color effects, so they have problems in formulation and do not know exactly the active ingredients, so they have the same effect when manufacturing products. It is difficult to expect and the possibility of containing unwanted components in the extract cannot be excluded.

On the other hand, the present inventors have confirmed that the extract of the mulberry has a whitening effect and has applied for a patent (Patent Publication Nos. 92-021129 and 92-1218), and while continuing to study the active ingredient of the The material has been identified by pure separation and its properties and additional effects have been found to complete the present invention.

Accordingly, the present invention aims to isolate 5- (3- (2,4-dihydroxyphenyl) propyl) -3,4-bis (3-methyl-2-butenyl) -1,2-benzenediol and It is to provide a method for purification.

Another object of the present invention is the skin containing 5- (3- (2,4-dihydroxyphenyl) propyl) -3,4-bis (3-methyl-2-butenyl) -1,2-benzenediol It is to provide a composition for whitening.

Another object of the present invention is to contain 5- (3- (2,4-dihydroxyphenyl) propyl) -3,4-bis (3-methyl-2-butenyl) -1,2-benzenediol It is to provide an antioxidant composition.

Another object of the invention is the addition of 5- (3- (2,4-dihydroxyphenyl) propyl) -3,4-bis (3-methyl-2-butenyl) -1,2-benzenediol It is to provide a method for preventing browning phenomenon of food, cosmetic composition or pharmaceutical composition characterized in that.

Another object of the invention is to contain 5- (3- (2,4-dihydroxyphenyl) propyl) -3,4-bis (3-methyl-2-butenyl) -1,2-benzenediol It is to provide a cosmetic composition.

Hereinafter, the present invention will be described in more detail.

5- (3- (2,4-dihydroxyphenyl) propyl) -3,4-bis (3-methyl-2-butenyl) -1,2-benzenediol component used as an active ingredient in the present invention Nomura et al., Japan, is one of several isoprenenylated 1,3-diphenylpropane derivatives isolated from the roots of Broussonetia Kazinoki [Chem. Pharm. Bull., 34 (5) pp 1968-1979 (1986)], it was first identified by the present inventors that the compound has tyrosinase activity inhibition, polyphenooxidase activity inhibition and antioxidant activity.

According to the present invention, 5- (3- (2,4-dihydroxyphenyl) propyl) -3,4-bis (3-, which is a natural whitening substance having an excellent inhibitory effect on tyrosinase activity and a powerful antioxidant from the hawthorn plants A method for separating and purifying methyl-2-butenyl) -1,2-benzenediol is provided. The separation method known by Nomura et al. Has a disadvantage in that a large amount cannot be separated as a separation method using a preparative TLC, but the present inventors are widely distributed in Korea using solvent distribution and a series of column chromatography methods. 5- (3- (2,4-dihydroxyphenyl) propyl) -3,4-bis (3-methyl-2-butenyl) -1,2-benzene in large amounts from the stem or root bark of the camphor A method by which diols can be isolated and purified has been established.

The separation and purification method of 5- (3- (2,4-dihydroxyphenyl) propyl) -3,4-bis (3-methyl-2-butenyl) -1,2-benzenediol will be described in more detail. Extraction is carried out by adding a polar solvent such as ethanol, methanol, propanol, acetone alone, or a mixture thereof to the bark and root bark of the hawthorn plant, and at room temperature or by heating. The extract is concentrated to add an oil-soluble solvent such as ethyl acetate, toluene, normal hexane, and water to the extract obtained. In this case, Licrosorb RP-18, which is a reverse phase distribution column filled with Sephadex LH-20 column, silica filled column, and reverse phase distribution column, for example, octadecylated, with respect to the material remaining in the fat-soluble solvent layer. Chromatography using a column is carried out sequentially with the following conditions as an indicator of homeostasis and inhibitory activity of tyrosinase activity to isolate the active ingredient and recrystallize the material.

* Column Condition:

First column-Sephadex LH-20 packed column

(Solvent) Chloroform: Methanol = 1: 1

Second column-silica packed column

(Solvent) Chloroform: Methanol = 8: 1

Third Column-Lichrosorb RP-18

(Solvent) Acetonitrile: Water (50:50)

5- (3- (2,4-dihydroxyphenyl) propyl) -3,4-bis (3-methyl-2-butenyl) -1,2-benzenediol isolated and purified in the same manner as described above It has the following structural formula.

5- (3- (2,4-dihydroxyphenyl) propyl) -3,4-bis (3-methyl-2-butenyl) -1,2-benzenediol has an excellent inhibitory effect on tyrosinase activity. In other words, IC ₅₀ , a concentration that inhibits mushroom tyrosinase activity by 50%, is about 50 μM. Considering that IC ₅₀ of kojic acid, known as an excellent tyrosinase inhibitor, is about 100 μM, its tyrosinase activity inhibition effect is about ₅₀ μM. You can see how excellent it is.

Therefore, 5- (3- (2,4-dihydroxyphenyl) propyl) -3,4-bis (3-methyl-2-butenyl) -1,2-benzenediol can be used as an effective whitening material.

5- (3- (2,4-dihydroxyphenyl) propyl) -3,4-bis (3-methyl-2-butenyl) -1,2-benzenediol also shows excellent antioxidant activity. Antioxidant effect of this substance has IC ₅₀ was tried so DDPH radical scavenging effect appeared about 10μM, more general vitamin E (IC = 35μM) is used as an antioxidant, vitamin C (IC = 30μM), hydroquinone (IC ₅₀ = 25μM) It can be seen that its antioxidant effect is excellent material. In fact, this material was formulated into cosmetics and meat products to test skin stability, whitening effect, and skin blackening, and it was confirmed that it had a safe and significant whitening effect on skin.

In addition, polyphenol compounds are contained in plant foods, which promote oxidation by polyphenol oxidase and cause discoloration called browning. 5- (3- (2,4-dihydroxyphenyl) propyl) -3,4-bis (3-methyl-2-butenyl) -1,2-benzenediol is a polyphenol, an enzyme involved in browning Since it has a function of inhibiting the activity of oxidase, it can be applied as a food additive for preventing discoloration of food.

5- (3- (2,4-dihydroxyphenyl) propyl) -3,4-bis (3-methyl-2-butenyl) -1,2-benzenediol for this purpose, namely whitening effect, antioxidant action And when used for browning prevention, the amount thereof added varies depending on the purpose of use, the formulation or use of the product, and can be easily determined by those skilled in the art, but may be used, for example, in an amount of 0.01 to 50% based on the total weight of the final composition. have.

Hereinafter, the present invention will be described in more detail with reference to examples.

Example 1

Root bark of Burberrys (Broussonetia Kazinoki) was added to 300 g of air-dried 2000 ml of 95% aqueous ethanol and heated at 70 ° C. for 4 hours. Subsequently, filtrate was obtained, and the solution was evaporated. 500 ml of water and ethyl acetate were added to about 38 g of the extract, and the mixture was partitioned to obtain an ethyl acetate layer. Sepadex LH 20 packed column (solvent; chloroform: methanol = 1: 1) and column-silica packed column (solvent; chloroform: methanol = 8: 1) were applied to about 24 g of the material obtained by evaporating the ethyl acetate layer. And chromatography using a Lichrosorb RP-18 column (solvent; acetonitrile: water = 50: 50). The final fraction containing the desired material was evaporated and recrystallized from benzene to give 5- (3- (2,4-dihydroxyphenyl) propyl) -3,4-bis (3-methyl-2-butenyl) -1, About 1.1 g of 2-benzenediol was prepared. (Yield 0.37%)

Example 2

Root bark of Broussonetia Paphrifera was added to 300 g of acetone, and 2000 ml of acetone was added thereto and then immersed at room temperature for 7 days. The filtrate was filtered, and the filtrate was evaporated. Each 500 ml of normal hexane was added and distributed, and the normal hexane layer was obtained. Chromatography was carried out in the same manner as in Example 1 with respect to about 19 g of the material obtained by evaporating the normal hexane layer. The final fraction containing the desired material was evaporated and recrystallized from benzene to give 5- (3- (2,4-dihydroxyphenyl) propyl) -3,4-bis (3-methyl-2-butenyl) -1, About 1.2 g of 2-benzenediol was prepared. (Yield 0.4%).

Example 3

Root bark of Burberrys (Broussonetia Kazinoki) was added to 300 g of air dried 2000 ml of 95% water-soluble propanol and heated at 70 ° C. for 4 hours. Subsequently, filtrate was obtained to obtain a filtrate, and then 500 ml of water and toluene were added and distributed to about 31 g of the extract obtained by evaporation of the solution. About 13 g of the material obtained by evaporating this toluene layer was chromatographed similarly to Example 1. The final fraction containing the desired material was evaporated and recrystallized from benzene to give 5- (3- (2,4-dihydroxyphenyl) propyl) -3,4-bis (3-methyl-2-butenyl) -1, About 0.6 g of 2-benzenediol was prepared. (Yield 0.2%).

Example 4

Structural analysis

To identify the structure of 5- (3- (2,4-dihydroxyphenyl) propyl) -3,4-bis (3-methyl-2-butenyl) -1,2-benzenediol obtained in Example 1 Instrumental analysis was performed.

Melting point measurements include the Coma Hoover Capillary melting point apparatus, the UV spectrum is the Cecil 5501 UV spectrophotometer, the IR spectrum is the Bio-Red FTS-40 spectrometer, ¹ H and ¹³ C-NMR spectra were measured using a Brookler AM 500 spectrometer and MS spectra using a VG Trio-2 (Unit 2,000 AMU).

The results are as follows, and the materials obtained in Examples 2 to 4 also showed the same results.

Melting Point: 108 ~ 109 ℃

UV λ _max ^ethanol (log ε): 283.7 (2.457)

IR ν _max cm ^-1 (KBr): 3650.6, 3502.0, 1609.1

¹ H-NMR (DMSO-d _s ): δ, ppm from TMS 6.21 (1H, d, J = 8 Hz), 6.35 (1H, s), 6.87 (1H, d, J = 8 Hz)

¹³ C-NMR (DMSO-d _s ): ppm 17.7, 17.7, 25.2, 25.4, 25.4, 27.0, 29.2, 31.7, 32.3, 102.3, 105.7, 113.6, 118.6, 124.0, 124.6, 126.8, 128.2, 129.4, 129.6, 129.8, 130.7, 141, 142.5, 155.7, 156.1

MS m / z: 340, 204, 189, 161, 123, 69

[Test Example 1]

Tyrosinase Activity

Inhibition of tyrosinase activity of 5- (3- (2,4-dihydroxyphenyl) propyl) -3,4-bis (3-methyl-2-butenyl) -1,2-benzenediol was determined as follows. It was compared with the effect of crude root extract, ethyl acetate soluble extract, ascorbic acid and kojic acid.

1) Experiment Method

A 0.3 mg / mL aqueous solution of tyrosine and 0.1 mol of potassium-phosphate buffer solution (pH 6.8) were each added to the test tube, and 0.9 mL of the appropriate concentration of tyrosinase activity inhibitor to be tested was added to the test group, and the control group was tyrosinase activity inhibitor. Put only the dissolved solvent. After mixing well, add 0.1 ml of each solution of mushroom-derived tyrosinase (Sigma Co., Ltd .: 2,000 units / ml), and react for 10 minutes in a 37 ° C thermostat. It was. With respect to the measured absorbance, the tyrosinase activity inhibitory effect was measured using the following equation.

2) Experiment result

IC (50% inhibitory concentration): the amount of sample that inhibits the reaction by 50% in the reaction system

[Test Example 2]

Antioxidative effect

The antioxidant effect of 5- (3- (2,4-dihydroxyphenyl) propyl) -3,4-bis (3-methyl-2-butenyl) -1,2-benzenediol was measured as follows. It was compared with the effects of known antioxidants such as hydroquinone, vitamin E, ascorbic acid, caffeic acid, 3,4-dihydroxy benzoic acid.

1) Experiment Method

Into a 100 μM DPPH (Dipheny1 picryl Hydrazile) ethanol solution, 1.9 ml of a sample solution to be tested at a suitable concentration was added and reacted at 37 ° C. for 30 minutes. After the reaction, the absorbance of the reaction solution was measured at 515 nm with an absorbance photometer.

2) Experiment result

[Test Example 3]

Skin safety

In order to test the skin safety, a human patch test was performed on 20 healthy men and women (10 males, 10 females) as follows.

1) Experiment Method

5- (3- (2,4-dihydroxyphenyl) propyl) separated in Examples 1 to 3 on the same cosmetic base as Formulation Example 7 described later, after wiping the forearm of the test subject with 70% ethanol well. Nourishing cream sample (4-6) and ethanol bath in Example 1, wherein -3,4-bis (3-methyl-2-butenyl) -1,2-benzenediol was combined in an amount of 0.2% (dry weight) A Finn Chamber containing 20 microliters of nourishing cream samples (Sample Nos. 2 and 3) containing 1% of extract and 1% of ethyl acetate extract was closed and attached for 24 hours using an adhesive tape. As a control (Sample No. 1), a cosmetic base containing no active substance described above was used. After 24 hours, the adhesive tape and the pin chamber were removed, and the sample remaining on the skin with a gauze was lightly removed. Then, the skin condition (erythema, edema, papules) was observed, and again after 24 hours.

2) Test result

*) Number of subjects who showed positive response / total number of test subjects

As shown in the table above it contains 5- (3- (2,4-dihydroxyphenyl) propyl) -3,4-bis (3-methyl-2-butenyl) -1,2-benzenediol The cream of the present invention was confirmed to have no irritation to human skin.

[Test Example 4]

Pigmentation inhibitory effect

1) Experiment Method

A region of 2 × 2 cm 2 was set in the lower arms of both arms of 20 healthy men and women. Put the aluminum foil on the arm so that the ultraviolet ray is irradiated only on the test target area, and at the distance of 10cm, at the same time 0.8 × 10 erg / cm 3 / circuit was examined three times in a row once daily. 70% of the test subjects were well washed with an isopropyl alcohol solution before irradiation. Preparation containing 5- (3- (2,4-dihydroxyphenyl) propyl) -3,4-bis (3-methyl-2-butenyl) -1,2-benzenediol at the site of ultraviolet irradiation after irradiation The cosmetic of Example 9 was applied three times a day. After four weeks of application, the degree of pigmentation was measured by naked eye, and the treatment group inhibited pigmentation compared to the control group, and evaluated in three stages of remarkable effect, effectiveness, and invalidity.

2) Experiment result

[Test Example 5]

5- (3- (2,4-dihydroxyphenyl) propyl) -3,4-bis (3-methyl-2-butenyl) -1,2-benzenediol for 20 women with blemishes on the facial area The blemish healing effect of the cream containing was tested as follows.

1) Experiment Method

Twenty female subjects in their 20s and 50s with facial signs and no other skin diseases had 5- (3- (2,4-dihydroxyphenyl) propyl) -3,4-bis (3) on the left side of the face. The cream (control) containing no methyl-2-butenyl) -1,2-benzenediol, and the cream of Formulation Example 9 were applied twice a day for 8 weeks on the right side, and the result was visually observed. Observed by. As a result of the observation, the degree of discoloration of the blemished portion of the cream-coated portion of Formulation Example 9 was evaluated in three stages of remarkable effect, effectiveness, and invalidity, compared with the control-coated portion.

2) Experiment result

[Test Example 6]

Polyphenol oxidase activity inhibitory effect measurement

1) Experiment Method

2.0 ml of 0.05 M MES-NaOH buffer (pH 5.5), 0.5 ml of 0.5 mM syringadazine (Sigma) dissolved in ethanol and 0.5 ml of ethanol or aqueous solution containing polyphenol oxidase activity inhibitors shown in Table 6 and sigma 0.2 ml (500 units / ml) of the Lacase enzyme solution purchased from the company was mixed and reacted at 30 ° C. for 5 minutes, and the absorbance was measured at 530 nm. The reaction was initiated by adding cyringalazine to the reaction solution.

2) Experiment result

5- (3- (2,4-dihydroxyphenyl) propyl) -3,4-bis (3-methyl-2-butenyl) -1,2-benzenediol is added at different concentrations, and this substance Inhibition of enzyme activity was measured using the reaction solution without the addition as a control. As a result, IC = 0.40 µM, which is a concentration that inhibits 50% of enzyme activity, was obtained (Fig. 8).

[Test Example 7]

Browning prevention activity

5- (3- (2,4-dihydroxyphenyl) propyl) -3,4-bis (3-methyl-2-butenyl) -1,2-benzenediol was tested for browning activity as follows. Evaluated.

1) Experiment Method

After grinding 0.5kg of apples, they were allowed to stand for one day, and the filtrated apple juice was divided into 50 parts of 50 g each, and then fractions were divided into 5- (3- (2,4-dihydroxyphenyl) propyl) -3,4-bis (3-). Methyl-2-butenyl) -1,2-benzenediol was added at 0, 1, 2, 4, or 8 μg, respectively, and the apple juice fractions were stored in a 37 ° C. constant temperature room to observe discoloration over time. The degree of discoloration was measured using a chrominance spectrophotometer (Ultrascan spectrophotometer, manufacturer: Hunter Associates Laboratory, Inc.) The values were compared and measured. Where △ E = [(△ L ) + (△ a ) + (△ b ) ] Indicates. The measurement was performed three times and averaged. Table 6 below shows the results. Browning phenomenon in proportion to the amount of 5- (3- (2,4-dihydroxyphenyl) propyl) -3,4-bis (3-methyl-2-butenyl) -1,2-benzenediol added It can be seen that the decrease significantly.

[Example 1-3]

Softener (Skin Lotion)

Preparation Example 4-6

Nourishing Cosmetic (Milkkin Lotion)

[Example 7-9]

Nutrition Cream

Preparation Example 10-12

Massage cream

Preparation Example 13-15

pack

[Example 16-18]

Ointment

Preparation Example 19-21

oil

Claims (5)

Extracting the bark or the bark of the hawthorn plant with a polar solvent, obtaining a concentrated extract by concentrating the extract, distributing it by adding a fat-soluble solvent and water to the concentrated extract and separating the fat-soluble solvent layer from the Sephadex (Sephadex) 5- (3- (2,4-dihydroxyphenyl) propyl) -3,4-bis, comprising the steps of sequentially performing an LH-20 column, a silica packed column and a reverse phase distribution column Separation and Purification Method of (3-methyl-2-butenyl) -1,2-benzenediol. The method of claim 1, wherein the first column is a Sephadex LH 20 packed column, the solvent is a chloroform: methanol (1: 1) mixture, and the second column, the silica packed column is chloroform: methanol. (8: 1) mixture, and the third reverse phase distribution column is a Lichrosorb RP-18 column, wherein the solvent is an acetonitrile: water (50:50) mixture. A composition for skin whitening containing 5- (3- (2,4-dihydroxyphenyl) propyl) -3,4-bis (3-methyl-2-butenyl) -1,2-benzenediol. An antioxidant composition containing 5- (3- (2,4-dihydroxyphenyl) propyl) -3,4-bis (3-methyl-2-butenyl) -1,2-benzenediol. Foods and cosmetics characterized by the addition of 5- (3- (2,4-dihydroxyphenyl) propyl) -3,4-bis (3-methyl-2-butenyl) -1,2-benzenediol A method of preventing browning of a composition or a pharmaceutical composition.