KR100741424B1 - Angoroside c with preventing or improving activity of skin wrinkle - Google Patents

Abstract

A composition for preventing or improving skin wrinkle comprising angoroside C is provided to eliminate side effects in the application of angoroside C, and improve skin wrinkle-preventing or improving effects. The composition for preventing or improving skin wrinkle comprises 0.05-5%(w/v) of angoroside C isolated from the extract of Scrophularia koraiensis Naka, Scrophulariaceae plant selected from Scrophularia ningpoensis, Scrophularia lepidota roots, Scrophularia scorodonia, Scrophularia ningpoensis Hemsl and Scrophularia ilwensis, or Verbascum spinosum wherein the extract is prepared by extracting the plant with C1-C4 lower alcohol, solubilizing the extract in water, fractionating the solution with ethylacetate, and subjecting the ethylacetate fraction to chromatography. The composition is a cosmetic composition having a formulation of skin, suspension or emulsion.

Description

ANGOROSIDE C with PREVENTING OR IMPROVING ACTIVITY OF SKIN WRINKLE}

1 is a diagram schematically showing a process for separating and purifying angoroside C from plants.

2, 3 and 4 are photographs showing the degree of improvement of skin wrinkles of hairless rats when the fractions obtained in various separation and purification processes of Example 1 were treated on the skin wrinkles of the back of hairless mice caused by UV irradiation.

FIG. 5 shows the change in the extent of wrinkle formation after treatment of fractions obtained in various separation and purification processes of Example 1 to skin wrinkles of hairless mice caused by UV irradiation using the Donald methods (photochemistry and photobiology 1987). One result is graphed.

Figure 6 shows the ¹ H NMR spectroscopy results to reveal the chemical structure of the fraction H2 of the fraction obtained in Example 1 was found to have the best effect of improving the wrinkles generated by UV light in the back of the hairless rat.

Figure 7 shows the ¹³ C NMR spectroscopy results of the fraction H-2 of Example 1.

8 shows the results of HMQC spectroscopy analysis of the fraction H-2 of Example 1 and the mass spectrometry of FM MS.

FIG. 9 shows the IR absorbance, UV absorbance, specific light intensity, mass spectrometry, and chemical structure of Fraction H-2 of Example 1. FIG.

[Industrial use]

The present invention relates to a composition for preventing or treating skin wrinkles containing angoroside C or angoroside C-containing plant extract as an active ingredient, and to a method for separating and purifying angoroside C from plants. The composition can be applied to the manufacture of various pharmaceuticals or cosmetics.

[Private Technology]

The skin consists of the upper layer, epidermis (thickness about 0.1 mm), lower layer and dermis (thickness about 1 to 4 mm), basal layer. The causes of the appearance of wrinkles on the skin can be largely divided into the generation of wrinkles due to natural aging and the generation of wrinkles by exposure to ultraviolet light (photoaging). to be. Common phenomena in aging due to the above two causes are the reduction and modification of collagen and elastin, which are the major constituent proteins in the dermis, and the decrease of hyaluronic acid, which is the substrate of the dermis. You lose. Previous studies have shown that the reduction and modification of these major proteins, collagen and elastin, are directly involved in the formation of wrinkles and the deterioration of skin elasticity.

There are various types of skin aging inhibitors known to date according to their mechanisms for inhibiting skin aging. Typically, collagen production promoters, elastin activators, hyaluronic acid production enhancers, free radical scavengers, cell proliferators, photoaging inhibitors, keratinocyte proliferation Inhibitors, humectants, inhibitors of harmful free radical production, and cellular regeneration materials. In addition, currently applied skin wrinkle improvement methods to prevent the formation of wrinkles by antioxidants such as vitamin C, E, carotenoids that inhibit the formation or reaction of free radicals, increase collagen biosynthesis in the dermis such as retinol Or a method of injecting collagen into the skin or injecting botulinum toxin into the skin. However, vitamins C, E, carotenoids, and retinol are all easily broken at room temperature, and retinol is known to have side effects on the skin at higher concentrations. Collagen injections and toxin injections can also provide temporary relief and serious side effects There is a problem.

In order to solve the above problems, the present invention aims to provide a composition having an excellent skin wrinkle prevention or treatment effect without side effects.

More specifically, it is an object of the present invention to provide a composition for preventing or treating skin wrinkles containing Angoroside C as an active ingredient.

Still another object of the present invention is to provide a composition for preventing or treating skin wrinkles, which includes a plant extract containing angoside C.

Another object of the present invention is to provide a method for separating and purifying angoroside C from a plant.

The present invention relates to a composition for preventing or treating skin wrinkles containing angoroside C or angoroside C-containing plant extract as an active ingredient, and to a method for separating and purifying angoroside C from plants. The composition can be applied to the manufacture of various pharmaceuticals or cosmetics.

The present invention selects several plant extracts that are effective in improving skin wrinkles among various plant extracts, and selects the fraction having the most skin wrinkle improvement effect among various fractions obtained in the extraction process of the selected plant extracts. , By analyzing the components of the fraction, and revealed that the component is Angoroside C, to prove the skin wrinkle improvement activity of Angoroside C to complete the present invention.

First, the present invention relates to a composition for preventing or treating skin wrinkles containing angoroside C as an active ingredient.

Angoroside C is known as one of the phenylpropanoid glycosides. Phenylpropanoid glycosides are found in many types of plants and are antibacterial, antiviral, analgesic, antispasmodic, neuroprotective, and cytostatic. It has been used as a traditional medicine, such as anti-inflammatory and radical scavenger activity. However, there is no report on the skin wrinkle prevention and improvement activity of Angoroside C.

Thus, the present invention is to reveal the skin wrinkle prevention and improvement effect of Angoroside C. Therefore, the composition for preventing or treating skin wrinkles of the present invention may contain only angoroside C as a pure substance. In addition, the composition for preventing or treating skin wrinkles of the present invention is an angroside C based on the total composition of 0.01 w / v% or more, preferably 0.05 to 5 w / v%, more preferably 0.1 to 1 w / It may be contained in the amount of v%. If the angoroside C content is less than the above range, the skin wrinkle improvement effect is insignificant. If the angoroside C content is more than the above range, even if the angoroside C content increases, the corresponding skin wrinkle improvement effect may not be obtained, which may be economical.

The present invention Angoroside C may be isolated and purified from plant extracts, for example, Ssam ( Scrophularia koraiensis Naka), Scrophularia ningpoensis , Scrophularia buergeriana, Scrophularia lepidota roots, Scrophularia scorodonia , Scrophularia ningpoensis Hemsl and Scrophularia (Scrophulariaceae) plant, Ballota nigra , Verbascum spinosum , Buddleja species and the like can be separated and purified from the extract.

The present invention also relates to a composition for preventing or treating skin wrinkles containing a plant extract containing angoroside C. The plant extract is tohyeonsam (Scrophularia koraiensis Naka), Scrophularia ningpoensis , Scrophularia buergeriana, Scrophularia lepidota roots, Scrophularia scorodonia, Scrophularia ningpoensis Hemsl and figwort selected from the group consisting of Scrophularia ilwensis科(Scrophulariaceae) plant, Ballota nigra, Verbascum spinosum and Buddleja species It may be an extract of a plant selected from the group consisting of. The plant extract is composed of an ethyl acetate fraction obtained by adding ethyl acetate to a suspension obtained by dissolving the alcohol extracts of the above-listed plants in water, and a fraction obtained from a methanol column by chromatography of a water fraction except for the ethyl acetate fraction. It may include one or more selected from the group. The alcohol may be a lower alcohol having 1 to 4 carbon atoms, ethanol is preferred.

The content of angoroside C contained in the composition for preventing or treating skin wrinkles containing the plant extract containing angoroside C is preferably 0.01 w / v% or more based on the total composition, and 0.05 to 5 w / It is more preferable that it is v% range, and it is still more preferable that it is 0.1-1 w / v%. If the angoroside C content is less than the above range, the skin wrinkle improvement effect is insignificant. If the angoroside C content is more than the above range, even if the angoroside C content increases, the corresponding skin wrinkle improvement effect may not be obtained, which may be uneconomical.

The composition for preventing or treating skin wrinkles containing angoroside C of the present invention may be formulated in pharmaceutical form. When formulated as such, it may further contain one or more pharmaceutically acceptable carriers. The carrier may include, but is not limited to, saline, buffered saline, water, glycerol and ethanol, and any suitable agent known in the art (Remington's Pharmaceutical Science (Recent Edition), Mack Publishing Company, Easton PA) may be used. Do. In addition, depending on the type of medicament desired, all pharmaceutically acceptable fillers, extenders, binders, wetting agents, sweeteners, fragrances, preservatives, disintegrants, surfactants, diluents and other excipients conventionally used in the manufacture of a medicament may be used. It may further contain one or more additives selected from the group. The medicaments according to the invention may be prepared in unit dosage form, for example capsules, suppositories or ampoules.

In addition, the dosage form of the medicament according to the present invention may be oral and parenteral, but parenteral administration such as transdermal administration is preferred in order to obtain a direct effect on skin wrinkle improvement. The pharmaceutical agent according to the present invention may be in the form of a suspension, a solution, an emulsion, or the like as a liquid preparation for transdermal administration.In addition to water, liquid paraffin, which are conventionally used simple diluents, various excipients such as wetting agents, sweeteners, Fragrance, preservative, and the like. The dosage of the other drug in the present invention may be differently applied according to the age, sex, skin condition, absorbency of the active ingredient in the body, inactivation rate, concomitant drug, and the like. It may be administered in an amount of 0.1 to 1 ml per cm ² , for example, 1 ml of the drug may be administered to the skin 4 cm ² wide (based on 1 μg / ml water of angoroside C content), but is not limited thereto.

In addition, the composition for preventing or treating skin wrinkles of the present invention can be used in the manufacture of cosmetics. Cosmetics according to the invention may be in the form of skins, suspensions, emulsions, the preparation of which is in accordance with conventional methods. Cosmetics according to the present invention may further contain various excipients, for example, wetting agents, sweeteners, fragrances, preservatives, etc., in addition to water, liquid paraffin, which is a conventional simple diluent, and may include various cosmetic bases.

The present invention also relates to a method for separating and purifying Angoroside C from plants. More specifically, the method for separating and purifying Angoroside C of the present invention

Grinding the plant dry matter,

Lower alcohol having 1 to 4 carbon atoms was added to the obtained plant ground product to obtain an alcohol extraction fraction,

The obtained alcohol extract fractions were dried, dissolved with lower alcohols having 1 to 4 carbon atoms and the dried product was then suspended in water to prepare a suspension;

Adding ethyl acetate to the suspension to obtain an ethyl acetate extraction fraction layer and a water layer;

Column chromatography of the obtained water layer to pour methanol to obtain the fraction obtained.

The plant is in tohyeonsam (Scrophularia koraiensis Naka), Scrophularia ningpoensis , Scrophularia buergeriana, Scrophularia lepidota roots, Scrophularia scorodonia, Scrophularia ningpoensis Hemsl and Scrophularia Scrophulariaceae selected from the group consisting of ilwensis科(Scrophulariaceae) plant, Ballota nigra, Verbascum spinosum and Buddleja species It may be selected from the group consisting of, the plant dry is not affected by the plant parts, may be the leaves, stems, roots, flowers or the whole plant of the plant, preferably the root portion of the plant can be used. The column chromatography may be performed using a Diaion HP 25 resin (synthetic adsorbent) column.

 EXAMPLE

Hereinafter, a preferred embodiment of the present invention. The following examples are only intended to illustrate the present invention more specifically, but the scope of the present invention is not limited to these examples.

In vivo models using hairless mice have been accepted as a common method for performing skin wrinkle studies. In the following examples, the anti-wrinkle and anti-wrinkle effect of Angoroside C was identified through in vivo experiments using hairless mice. One of the preferred embodiments of the present invention is a method of purifying Angoroside C from a plant and a method of identifying the chemical structure of the purified material to prove that it is Angoroside C. Separation and structure analysis is also possible by the method.

Example 1 Separation and Purification of Angoroside C

In this example, angoroside C was isolated and purified using the root of Scrophularia koraiensis Naka . The separation and purification process of this embodiment is shown in FIG.

After grinding the dried 200 g of Tohsam root finely, reflux extraction with 2 liters of 70% alcoholic alcohol ethanol for 3 hours at 80 ℃, and then remove the ethanol components by using a vacuum rotary evaporator to concentrate the fraction and concentrated by fractionation. A was obtained. The fraction A thus obtained was dissolved in 500 ml of 80% food alcohol ethanol, and the insoluble portion was separated by Shephadex G50 and separated into fractions of 30 kd or less (fraction B) and fractions of 30 kd or more (fraction C). Meanwhile, the part (fraction D) dissolved in 80% ethanol was completely removed by using a vacuum rotary evaporator, concentrated, and then suspended in 500 ml of water to form a suspension. 500 ml of ethyl acetate was added to the suspension, which was then extracted three times using a separatory funnel to obtain an ethyl acetate layer and a water layer. The ethyl acetate layer (fraction E) was partitioned with methylene chloride: ethyl acetate: butanol: water (3: 7: 5: 5) mixed solvent, and the lower layer was evaporated and concentrated to obtain fraction F. On the other hand, the water layer obtained by the ethyl acetate extraction is poured into a Diaion HP 25 resin (synthetic adsorbent) column, water 100% (fraction I), water 75 vol% + methanol 25 vol%, water 50 vol% + methanol 50 vol %, 25 vol% water + 75 vol% methanol and 100% methanol were flowed to each column, and the solution which passed through each column was collected. The solution (fraction H) obtained through the column with 100% methanol poured through was poured into a Diaion HP20 resin column, and fractionated while flowing into methanol to fractionate the first peak (fraction H-1) and the second peak (fraction H-2). Two peaks of were obtained.

Example 2: Determination of wrinkle removal efficacy using hairless mice

Fractions F, H1 and H2 obtained above were used to confirm wrinkle prevention and improvement efficacy in hairless mice. The hairless mice used in this experiment were purchased from Charles River, and the age was 6 weeks and the experimental period was 8 weeks. Considering the extent of wrinkles in hairless rats, young rats (6 weeks old) were not used. During the experiment period, the environment was maintained at 23 ㅁ 2 ℃ and relative humidity 55 ㅁ 10%. The animals were reared in four cages for 12 hours under artificial lighting. Feed and drinking water for experimental animals were supplied freely. In order to verify the anti-wrinkle effect in the breeding mice, the dilution aqueous solution of diluting the fractions F, H, H1 and H2 to 1/50, 1/500 in purified water, etc. Daily (approximately 1 ml of the drug to 4 cm ² wide skin).

In order to develop wrinkles in the hairless mice, the 6-week-old hairless mice were exposed to UV 3 times a week for 8 weeks, and UVA was irradiated at 14 J / cm 2 and UVB at 20 mJ / cm 2. Wrinkle formation was measured using Donald methods (photochemistry and photobiology 1987). As the normal control group, the headless rat group which was not exposed to UV was used, and as the negative control group, the headless rat group which was not treated after UV exposure was used, and the enbutyric acid known to have an anti wrinkle effect after exposure to UV as a positive control group ( n-butyric acid; n-BA, Korean Patent 0519890) was used as a group of headless mice treated with a concentration of 5mM, and a group of headless rats exposed to UV and treated with the fractions as an experimental group.

Figures 2, 3 and 4 are photographs showing the degree of wrinkles of hairless rats that are changed when the fractions of the various purification processes are treated on the skin wrinkles generated by UV irradiation. UV control of Figure 2 is a wrinkle photo of the negative control hairless mice that were wrinkled by UV exposure, wrinkled pictures of the positive control hairless mice treated with 5 mM n-BA and 1/50 and 1 / of the fraction H-2 The wrinkles of the experimental group treated with 500 dilute aqueous solutions were shown. Figure 3 shows a photograph of the wrinkles of experimental group hairless mice treated with 1/50 and 1/500 dilute aqueous solution of Fraction H and 1/50 and 1/500 dilute aqueous solution of Fraction H-1, respectively. Figure 4 of the experimental group treated with 1/50 and 1/500 dilute aqueous solution of the fraction F, the control treated with 0.3 μM TSA (Trichostatic acid) and the normal control group of rats that were not treated with any substance without UV exposure It is showing a picture of wrinkles.

5 is an experimental group treated with 1/50 and 1/500 dilute aqueous solution of fractions F, H, H-1 and H-2, respectively, 0.3 μM TSA treated control, 5 mM n-BA treated control, negative control and This is a graph showing the results of measuring wrinkles over a period of 8 weeks using Donald methods (photochemistry and photobiology 1987) of the normal control group.

As can be seen in Figure 2 to 5, the fractions F, H, H1 and H2 separated and purified in the present invention all showed some degree of wrinkle improvement effect, the effect is H-2> H> H-1 > F. In particular, fraction H-2 was shown to have an excellent wrinkle improvement effect comparable to the existing n-BA known to have excellent wrinkle improvement effect. In addition, the fractions H, H-1, H-2, and F separated and purified in the present invention do not cause any side effects such as erythema and edema when they are all treated on the skin of the back of hairless mice for 8 weeks. It was confirmed that it is a safe fraction without toxicity.

Example 3: Structural Analysis of Purified Fraction H-2 with Excellent Wrinkle Improvement Efficacy

In order to clarify the chemical structure of fraction H-2, which was shown to have the best effect of improving wrinkles in Example 2, ¹ H NMR spectroscopy, ¹³ C NMR spectroscopy, HMQC spectroscopy, and FM MS analysis were performed, respectively. To Fig. 8 (Fig. 6: ¹ H NMR spectroscopy results, Fig. 7: ¹³ C NMR spectroscopy results, Figure 8: HMQC spectroscopy and FM MS analysis results). In addition, IR absorbance, UV absorbance, specific light absorbance and mass of fraction H-2 were analyzed and the results are shown in FIG. 9.

As a result of the structural analysis shown in FIG. 9, H-2 obtained in the present invention was found to be angoroside C. As shown in Example 2, the angoroside C has an excellent effect on improving skin wrinkles, it was shown that there is no skin toxicity. Angoroside C has already been reported in several reports for isolated tablets and other physiological activities, but has not yet been reported for skin wrinkle prevention and amelioration activity.

As described above, the present invention can be usefully applied to functional drugs, cosmetics, and the like for inhibiting wrinkles by providing a composition having angoroside C as an active ingredient and having a wrinkle preventing and improving effect.

All simple modifications or changes of the present invention can be easily carried out by those skilled in the art, and all such modifications or changes can be seen to be included in the scope of the present invention.

Claims (14)

Composition for preventing or improving skin wrinkles containing angoroside C as an active ingredient. The composition of claim 1 wherein the angoroseside C content is from 0.05 to 5 w / v%. According to claim 1, wherein said and-blast side C is tohyeonsam (Scrophularia koraiensis Naka), Scrophularia ningpoensis, Scrophularia lepidota roots, Scrophularia scorodonia, S crophularia ningpoensis Hemsl and figwort selected from the group consisting of Scrophularia ilwensis科(Scrophulariaceae) plant, or Verbascum The composition is isolated and purified from the extract of the spinosum plant. And therefore it tohyeonsam containing side-C (Scrophularia koraiensis Naka), Scrophularia ningpoensis, Scrophularia lepidota roots, Scrophularia scorodonia, S crophularia ningpoensis figwort selected from the group consisting of Hemsl and Scrophularia ilwensis科(Scrophulariaceae) comprises a plant, or Verbascum spinosum plant extracts Composition for preventing or improving skin wrinkles. The composition of claim 4, wherein the angoroside C content is from 0.05 to 5 w / v%. The composition of claim 4, wherein the plant extract is Scrophularia koraiensis Naka . The method of claim 6, wherein the plant extract is chromatographed with an ethyl acetate fraction obtained by adding ethyl acetate to a suspension obtained by dissolving a lower alcohol extract having 1 to 4 carbon atoms of the plant in water, and the water fraction except for the ethyl acetate fraction. And one or more selected from the group consisting of fractions obtained by methanol column. delete delete delete A cosmetic composition comprising the composition according to any one of claims 1 to 7. The cosmetic composition according to claim 11, wherein the cosmetic composition is in the form of a skin, a suspension or an emulsion. Tohyeonsam (Scrophularia koraiensis Naka) containing and therefore the side C, Scrophularia ningpoensis, the dry product of Scrophularia lepidota roots, Scrophularia scorodonia, S crophularia ningpoensis Hemsl and figwort科(Scrophulariaceae) plant, or Verbascum spinosum plant selected from the group consisting of Scrophularia ilwensis Crushed, Lower alcohol having 1 to 4 carbon atoms was added to the obtained plant ground product to obtain an alcohol extraction fraction, The obtained alcoholic extract fractions were dried and then dissolved with a lower alcohol having 1 to 4 carbon atoms, dried again, and then suspended in water to prepare a suspension. Ethyl acetate was added to the suspension to obtain an ethyl acetate extract fractionation layer and a water layer, Column chromatography of the obtained water layer to pour methanol to obtain the obtained fractions. A method for separating and purifying Angoroside C from plants. The method of claim 13, wherein the plant is Scrophularia koraiensis Naka .

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