KR20160082675A - Composition for skin conditions improvement comprising fractions of honeybush extracts and compounds derived from the same - Google Patents

Abstract

The present invention relates to a composition for improving skin conditions, comprising a solvent fraction of a honeybush extract as an active ingredient, so as to prevent skin aging or reduce skin wrinkles. The fraction or the compound derived from the fraction has excellent effects in reducing skin wrinkles generated from skin damage increased by ultraviolet radiation, thereby being efficiently used for a health functional food composition, a cosmetic material, a pharmaceutical composition for improving skin conditions.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a composition for improving skin comprising honeycomb extract fractions and a compound derived therefrom.

The present invention relates to a composition for preventing skin aging or improving skin wrinkles containing a fraction of honeybush extract and a compound derived therefrom as an active ingredient.

Skin aging occurs through two processes: intrinsic natural aging that occurs naturally as you get older, and extrinsic photoaging by exposure to ultraviolet light. Although both natural aging and photoperiod have wrinkles, and Langerhans' cells (skin cells), which are skin immunity cells, and dermal cell components are reduced, there are many common features. However, in the case of photoaging, skin thickness increases and elastic fibers increase, In aging, there are other features that make the skin thinner. Recently, there has been a growing interest in prevention / treatment of photoaging related to exposure to ultraviolet rays, which is a field of interest from the younger generation to the elderly, unlike natural aging, which has a direct relationship with age.

The main cause of endogenous natural aging is the accumulation of damage to the constituents of our body due to the reactive reactive oxygen radicals produced in the metabolic process of our body. On the other hand, extrinsic photoaging is a result of skin damage caused by ultraviolet rays. One of the typical symptoms is wrinkle. Excessive ultraviolet exposure is always caused by ozone depletion resulting from various air pollution like modern society. It is a reality that is becoming a concern. If exposure to ultraviolet light is continued, oxidative stress occurs, which increases radicals in the body and destroys collagen, elastin, and hyaluronic acid, which are connective tissues of the dermis. (Wrinkles) in the skin of the skin. In addition, these mechanisms oxidize the lipid part of the cell membrane and cause cell destruction, which can lead to diseases such as dermatitis, acne or skin cancer.

On the other hand, the most excellent cosmetic products currently used for skin wrinkles are retinoid products, which have an advantageous effect. However, there are side effects that discoloration and deterioration occur when prescribed to a product and skin irritation occurs even when used in small amounts, And the reduction of the effect is recognized as a big problem. Therefore, it is urgent to develop a safe product that can be replaced. That is, a chemical synthetic substance such as a retinoid is effective in terms of function but may cause skin irritation due to chemical change (rancidity). Therefore, in order to prevent skin aging by removing active oxygen in the skin without such skin irritation, Functional cosmetics or health functional foods using natural vegetable raw materials having excellent safety with less side effects have been actively developed.

Honeybush ( Cyclopia intermedia , honeybush) is an evergreen shrub belonging to Melianthaceae (Melianthaceae), a plant that grows only in a narrow region of the ridge of the eastern coast of the Cape region of South Africa. It is a herb to be. The name 'Honeybush' is the name of the flower, because it has a scent of honey. It is similar to rooibos, but the flavor is much more sweet. Honeybush has few tannins, no caffeine, and a variety of minerals (eg iron, potash, calcium, copper, zinc, magnesium).

Honeybush's efficacy has long been used to treat colds, insomnia and stomach pains, and other anti-diabetic effects have been reported. Further, a patent (Korean Patent Registration No. 10-1295368) is disclosed for a composition for improving skin wrinkles containing a water extract of Honeybush or a fermentation broth thereof as an active ingredient, but a fraction further fractionated by a specific solvent or a derivative thereof The effect on one compound is not known.

That is, it can not be easily deduced that only the initiation of the water extract of Honeybush or its fermentation broth has any effect on the skin-improving effect of any of these fractions. Specific experimental data must be provided in order to prove such effects.

Accordingly, the present inventors have intensively studied to develop a natural medicament having an excellent skin wrinkle-reducing effect without side effects. As a result, it has been found that the fractions of honeybush extract and the compound derived therefrom have an ultraviolet protecting effect and photoaging factors (MMP-1 and MMP -9) suppressing effect, and confirmed that skin aging can be prevented or skin wrinkles can be improved due to ultraviolet rays or the like, thereby completing the present invention.

It is an object of the present invention to provide a composition for improving skin comprising a fraction of Honeybush extract and a compound derived therefrom as an active ingredient.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

The present invention provides a composition for skin improvement comprising a fraction of Honey bush extract as an active ingredient.

The extract may be extracted with water, a C ₁ to C ₆ lower alcohol, or a mixed solvent thereof.

The fraction may be dichloromethane, ethyl acetate, or water fraction of a honeybee extract.

The fraction may be an eluted product obtained by adsorbing Honeybush extract with Diaion HP-20 resin as an adsorbent.

The eluate may be eluted with water, a C ₁ to C ₆ lower alcohol, or a mixed solvent thereof.

The volume of the fraction may be 10 μg / ml to 200 μg / ml.

In one embodiment of the invention, one or more compounds selected from the group consisting of hesperidin, 5,7-dihydroxychromone, 4-hydroxybenzaldehyde, 4-hydroxyacetophenone, hesperetin and protocatechuic acid A composition for improving skin comprising a compound as an active ingredient.

The compound can be isolated from the fraction of Honeybush extract.

The dose of the compound may be 10 μg / ml to 200 μg / ml.

The composition may be for preventing skin aging or improving skin wrinkles.

The composition may be for preventing skin aging or improving skin wrinkles by inhibiting matrix metalloproteinase-1 (MMP-1) and matrix metalloproteinase-9 (MMP-9).

The composition may be a pharmaceutical composition, a cosmetic composition or a health functional food composition.

The composition may be formulated into powders, powders, granules, beverages, tablets, capsules, suspensions, emulsions, syrups, aerosols, transdermal preparations, suppositories or injection solutions.

The specific fractions of the honeybush extract of the present invention have an effect of reducing skin wrinkles which are increased by irradiation with ultraviolet rays and suppressing the destruction reaction of collagen tissues to improve skin wrinkles, It is possible to provide a new therapeutic agent and a therapeutic method that goes beyond existing natural skin improving agents.

1 is a graph showing the HPLC pattern analysis results of the (a) dichloromethane fraction and (b) the Diaion HP-20 water fraction.
2 is a graph showing the results of the cytotoxicity evaluation for the compounds 1 to 6.

The present invention provides a composition for improving skin comprising a fraction of Honey bush extract as an active ingredient.

In the present invention, honeybees can be used without limitation, such as cultivated or commercially available ones, and it is preferable to use leaves, seeds, fruits, roots or aerial parts, but not always limited thereto.

In the present invention, it is preferable, but not limited, to use water, an alcohol or a mixture thereof as the honeycomb extraction solvent. As the alcohol, C ₁ to C ₆ lower alcohols are preferably used, and as the lower alcohol, ethanol or methanol is preferably used.

In the present invention, as the honeybee bush extraction method, it is preferable to use shaking extraction or reflux extraction, and more preferably, using reflux cooling extraction, but not always limited thereto. Specifically, the extraction solvent is preferably added by 2 to 10 times the amount of the honeycomb bush, and the extraction temperature is preferably from room temperature (25 ° C) to 100 ° C, but not limited thereto, and the extraction time is 1 to 3 Hour, and the number of times of extraction is preferably 1 to 3 times, but is not limited thereto.

In the present invention, the organic solvent fraction of the Honeybush extract can be prepared by adding an organic solvent to the concentrate of the extract to prepare fractions, using an extraction method using a known organic solvent. The organic solvent is preferably water, dichloromethane, ethyl acetate, water-saturated butanol, 70-100% ethanol, hexane or a mixture thereof, more preferably water, dichloromethane or ethyl acetate. I never do that.

In the present invention, the organic solvent fraction of the honeybush extract may be an eluate obtained by adsorbing the honeycomb extract with a porous adsorbent. At this time, there is no limitation on the porous adsorbent, but it is preferable that Diaion HP-20 resin (resin) is used as an ion exchange resin. Diaion HP 20 is a high porosity type synthetic adsorbent of styrene and divinyl benzene (DVB) copolymer. It has a large number of fine pores and high surface area. It is excellent and suitable for adsorption of large molecules (MW 1,500 or more) because of its relatively large pores, and elution of the adsorbed material is easy. Elution of the adsorbed material may be performed using an acid, an alkali, a polar solvent and the like, and particularly preferably eluted with water, a C ₁ to C ₆ lower alcohol, or a mixed solvent thereof.

In the present invention, the fraction of Honeybush extract is selected from the group consisting of hesperidin, 5,7-dihydroxychromone, 4-hydroxybenzaldehyde, 4-hydroxyacetophenone, hesperetin and protocatechuic acid 0.001 to 10.0 parts by weight of hesperidin, 0.01 to 5.0 parts by weight of 5,7-dihydroxychromone, and 0.01 to 5 parts by weight of 4- But are not limited to, 0.005 to 2.0 parts by weight of hydroxybenzaldehyde, 0.01 to 4.0 parts by weight of 4-hydroxyacetophenone, 0.005 to 5.0 parts by weight of hesperitin, and 0.0005 to 5.0 parts by weight of protocatechuic acid.

In the present invention, the volume of the fraction of the honeybee extract is preferably 10 μg / ml to 200 μg / ml, but is not limited thereto.

The present invention also provides a pharmaceutical composition comprising at least one compound selected from the group consisting of hesperidin, 5,7-dihydroxychromone, 4-hydroxybenzaldehyde, 4-hydroxyacetophenone, hesperetin and protocatechuic acid And a composition for improving skin comprising the composition.

In the present invention, the compound may be isolated from the fraction of Honeybush extract.

Hesperidin and Hesperitin are flavanone glycosides in flavonoid pigments which are present in many citrus fruits. They inhibit the formation of lipid peroxides and have antioxidative effects such as delayed aging, anti-inflammatory effects, capillary protection And anti-cancer effects, and it is known to lower cholesterol.

5,7-dihydroxychromone is an abundant compound in the peanut shell and has an antifungal effect and is known to lower blood sugar. The effect of protecting brain cells is also known.

4-Hydroxybenzaldehyde (4-hydroxybenzaldehyde) is a phenolic compound that exists in many horses, and has anti-inflammatory and anti-angiogenic effects and is known to have a pain-suppressing action.

4-hydroxyacetophenone, also known as piceol, is a compound isolated from a plant called Picea ebies, Norway spruces, and has not yet been reported for its efficacy or mechanism.

Protocatechuic acid is a phenolic compound present in many green tea and hibiscus, and has antioxidant and anti-inflammatory activity. It is known that it protects against hepatotoxicity, promotes cell proliferation, and prevents apoptosis.

In the present invention, the dose of the compound is preferably 10 μg / ml to 200 μg / ml, but is not limited thereto.

In the present invention, the fractions of the present invention and the compounds derived therefrom inhibit skin death by ultraviolet rays through the MTS assay. The MTS assay is a well-known method for assessing cell viability (toxicity) as well as MTT assays, in which the yellow MTS tetrazolium is transformed into a brownish-colored purple powdery mica by dehydrogenase of living mitochondria formazan) is a method for measuring the absorbance at the time of conversion.

Thus, the higher the cell survival rate, the better the effect of wrinkle improvement and the like. In Experiment Example 1, it was confirmed that the honeycomb fraction of the present invention had significantly higher cell survival rate than the simple extract.

Further, in the present invention, it was confirmed that the fractions of the present invention improve photoaging (skin wrinkles) by ultraviolet rays through MMP-1 and MMP-9 assay. The matrix metalloproteinase (MMP), one of the zinc-dependent endoprotease systems, shares a wide range of substrates and is the main enzyme for extracellular matrix proteolysis of the dermis. In particular, ultraviolet light induces several types of MMPs, including MMP-1, which degrades Type I and III collagen, and MMP-9, which degrades collagen fragments. Excessive MMP secretion is induced in cells stimulated by ultraviolet rays or the like, that is, keratinocytes, fibroblasts of dermis, etc., causing degradation of extracellular matrix proteins, thereby causing skin aging and clinically wrinkling.

Therefore, the higher inhibitory activity of MMP-1 and MMP-9 means that the effect of improving wrinkles is better. In Examples 2 and 3 of the present invention, the honeycomb fraction of the present invention has the above inhibitory activity Respectively.

The pharmaceutical compositions of the present invention may further comprise components such as conventional therapeutically active ingredients, other adjuvants, pharmaceutically acceptable carriers, and the like. The pharmaceutically acceptable carrier includes saline, sterilized water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and the like.

The cosmetic composition of the present invention may be in the form of a solution, a gel, a solid or a paste anhydrous product, an emulsion obtained by dispersing an oil phase in an aqueous phase, a suspension, a microemulsion, a microcapsule, a microgranule or an ionic (liposome) , Creams, skins, lotions, powders, ointments, essences, sprays or conical sticks. It can also be prepared in the form of a foam or in the form of an aerosol composition further containing a compressed propellant.

The health functional food composition of the present invention can be used as it is or in combination with other food or food ingredients, and can be suitably used according to conventional methods. There is no particular limitation on the kind of the food, but it is not limited to the kind of food such as caramel, meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, , An alcoholic beverage and a vitamin complex, and includes all the health foods in a conventional sense.

The term "individual" as used herein refers to a subject in need of treatment for a disease, and more specifically refers to a human or non-human primate, mouse, rat, dog, cat, It means mammals. The term "pharmaceutically effective amount" as used herein refers to the type and severity of the disease to be treated, the age and sex of the patient, the sensitivity to the drug, the time of administration, the route of administration and the rate of release, Can be readily determined by those skilled in the art in a quantity that is determined by well-known factors in the art and can be maximized without adverse effects taking all of the factors into consideration.

The composition of the present invention is not limited as long as it can reach the target tissues. For example, oral administration, arterial injection, intravenous injection, percutaneous injection, intranasal administration, transbronchial administration, or intramuscular administration.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

[Comparative Example] Preparation of Honeybee extract

Comparative Example 1. Honey bush water extract

One kilogram of honey bush was selected and placed in a reflux cooling extractor, and 5 to 10 times as much purified water was added thereto. The extract was extracted twice with reflux cooling for 1 to 4 hours at 80 to 100 ° C to obtain an extract. The mixture was filtered and the filtrate was concentrated under reduced pressure using a vacuum condenser (EYELA N-1000, EYELA Ltd., JAPAN) at 70 ° C or lower to obtain a crude extract (yield: 21%).

Comparative Example 2. Honeybush 70% ethanol extract

One kilogram of honey bush was selected, put in a reflux cooling extractor, and 5 to 10 times of 70% ethanol was added and extracted twice at 70 to 90 ° C for 1 to 4 hours by reflux cooling to obtain an extract. The mixture was filtered and the filtrate was concentrated under reduced pressure with a vacuum condenser (EYELA N-1000, EYELA Ltd., JAPAN) at 70 ° C or lower to obtain crude extract (yield: 22%).

Comparative Example 3. Honeybush 100% ethanol extract

One kilogram of honey bush was selected and placed in a reflux cooling extractor. Five to ten times of 100% ethanol was added and extracted twice with reflux cooling for 1 to 4 hours at 60 to 90 ° C to obtain an extract. The mixture was filtered and the filtrate was concentrated under reduced pressure using a vacuum condenser (EYELA N-1000, EYELA Ltd., JAPAN) at 60 ° C or lower to obtain a crude extract (yield of 12%).

[Example 1-15] Production of solvent fractions of Honeybush extract

Example 1. Preparation of hexane fractions of water extract

20 g of the water extract of Comparative Example 1 was suspended in 800 mL of distilled water and the solvent fraction was performed three times using the same amount of n-hexane. The fraction obtained was concentrated under reduced pressure at 40 DEG C or less to obtain a hexane fraction (yield: 3.5%). .

Example 2. Dichloromethane fraction

After the hexane fraction of Example 1 was obtained, the remaining water layer was subjected to three solvent fractions using 800 mL of dichloromethane. The fractions thus obtained were concentrated under reduced pressure at 40 DEG C or less to obtain a dichloromethane fraction (yield 1.7%).

Example 3. Ethyl acetate fraction

After the dichloromethane fraction of Example 2 was prepared, the remaining water layer was subjected to three solvent fractions using 800 mL of ethyl acetate. The fractions thus obtained were concentrated under reduced pressure at 50 DEG C or less to obtain ethyl acetate fractions (yield of 2.9% ).

Example 4. Water saturated butanol fraction

After the ethyl acetate fraction of Example 3 was prepared, the remaining water layer was subjected to three solvent fractions using 800 mL of water-saturated butanol. The fraction obtained was concentrated under reduced pressure at 70 ° C or lower to obtain a water saturated butanol fraction 13.5%).

Example 5. Water fraction

After the water saturated butanol fraction of Example 4 was prepared, the remaining water layer was concentrated under reduced pressure at 70 캜 or lower to obtain a water fraction (yield: 68.8%).

Example 6. Preparation of hexane fractions of 70% ethanol extracts

20 g of the 70% ethanol extract of Comparative Example 2 was suspended in 800 mL of distilled water, and the solvent fraction was performed three times using the same amount of n-hexane. The fraction thus obtained was concentrated under reduced pressure at 40 DEG C or less to obtain a hexane fraction %).

Example 7. Dichloromethane fraction

After the hexane fraction of Example 6 was prepared, the remaining water layer was subjected to three solvent fractions using 800 mL of dichloromethane. The fractions thus obtained were concentrated under reduced pressure at 40 ° C or lower to obtain a dichloromethane fraction (yield: 3.5%). .

The HPLC pattern analysis results of the dichloromethane fractions are shown in Fig. 1 (a).

Specifically, 27.14 g of the dichloromethane fraction was subjected to silica gel column chromatography eluting with hexane: ethyl acetate = 3: 1 initial conditions, and 7 fractions were obtained. Fraction 4 (2.35 g) was eluted with methanol: dichloromethane = 1: 1 and then centrifuged (3000 rpm, 3 min) to obtain a supernatant, which was then concentrated under reduced pressure. The supernatant portion was subjected to Sephadex LH-20 column chromatography under a condition of methanol: dichloromethane = 1: 1, and a total of 7 fractions were obtained. The double fraction 5 was filtered with methanol to take up only the eluted fraction and purified by prep-HPLC (Eurospher 100-10 C18, 250 x 20 mm, 10 ml / min) under the conditions of acetonitrile: 0.1% TFA water = 10: To obtain Compound 1 (8.6 mg, 0.032%), Compound 2 (21.21 mg, 0.078%) and Compound 3 (29.13 mg, 0.11%). Compound No. 4 (11.41 mg, 0.04%) was obtained by performing prep-HPLC (Eurospher 100-10 C18, 250 x 20 mm, 10 ml / min) under the conditions of acetonitrile: 0.1% TFA water = 23:77.

Compounds isolated from the dichloromethane fractions were identified using NMR (Bruker model digital AVANCCE III, 400 MHz) and UPLC-UV-MS (Waters, ESI, negative mode).

Spectroscopic data of Compound 1 are shown below: ¹ H-NMR (MeOH, 400 MHz):? 6.95-6.88 (2H, m), 7.81-7.74 (2H, m), 9.77 (1H, s); ¹³ C-NMR (MeOH, 100 MHz):? 192.83, 165.18, 133.44, 130.30, 116.85, m / z 121 [M + H] ^- . Based on the above spectroscopic results, it was confirmed that Compound 1 was 4-Hydroxybenzaldehyde (Scholars Research Library Der Pharmacia Lettre (2012), 4 (6) ): 1817-1820):

[Chemical Formula 1]

.

.

Spectroscopic data of Compound 2 is shown below. ¹ H NMR (MeOD, 400 MHz):? 2.51 (1H, s), 6.87-6.80 (2H, m), 7.93-7.83 (2H, m); ¹³ C NMR (MeOD, 400 MHz): δ 199.49, 163.91, 132.12, 130.14, 116.19, 26.27, m / z 135 [M + H] ^- . Based on the above spectroscopic results, it was confirmed that Compound 2 was 4-Hydroxyacetophenone represented by the following Chemical Formula 2 (Natural Product Sciences (2010) 16 (4): 203-206 ):

(2)

.

.

Spectroscopic data of Compound 3 is shown below. _{^{1 H NMR (DMSO-d 6}} , 400MHz): δ 1.88 (1H, dd, J = 17.1, 3.0Hz), 2.24 (1H, dd, J = 17.1, 12.7Hz), 3.04 (3H, s), 4.48 ( (2H, dd, J = 10.9, 2.5Hz), 6.13 (1H, t, J = 2.9Hz, 1H), 5.08 ); ¹³ C NMR (MeOD, 100 MHz): δ 188.11, 158.84, 155.94, 155.25, 139.82, 138.23, 123.59, 109.51, 105.01, 103.00, 93.86, 87.57, 86.69, 70.77, 46.89, 34.55. In the case of this compound, it was confirmed that the same Rt value and UV spectrum were also the same when compared with HPLC by purchasing the standard product (Hesperetin, Sigma-Aldrich). When comparing the spectroscopic analysis data with the prior art, it can be confirmed that it is hesperetin represented by the following formula (3) (Journal of the Science of Food and Agriculture (2015)):

(3)

.

.

Spectroscopic data of compound 4 is shown below. ¹ H NMR (DMSO-d ₆ , 400 MHz):? 6.20 (1H, s), 6.25 (1H, d, J = 5.7 Hz), 6.37 (1H, d, J = 1.2 Hz); _{^{13 C NMR (DMSO-d 6}} , 100MHz): δ 181.33, 164.44, 161.62, 157.83, 157.54, 110.51, 104.91, 99.04, 94.01 m / z 177 [M + H] -. Based on the above spectroscopic results, it was confirmed that the compound 4 was 5,7-dihydroxychromone represented by the following formula (4) (Phytochemistry (1994) Vol. 4, pp. 1043-1045):

[Chemical Formula 4]

.

.

The cytotoxicity of compounds 1 to 4 was evaluated, and no cytotoxicity was observed up to a concentration of 200 ug / ml (see FIG. 2).

Example 8. Ethyl acetate fraction

After the dichloromethane fraction of Example 7 was prepared, the remaining water layer was subjected to three solvent fractions using 800 mL of ethyl acetate. The fractions obtained were concentrated under reduced pressure at 50 DEG C or less to obtain ethyl acetate fractions (yield 4.9% ).

Example 9. Water saturated butanol fraction

After the ethyl acetate fraction of Example 8 was prepared, the remaining water layer was subjected to solvent fractionation three times using 800 mL of water-saturated butanol. The resulting fraction was concentrated under reduced pressure at 70 ° C or lower to obtain a water saturated butanol fraction 12.4%).

Example 10. Water fraction

After the water saturated butanol fraction of Example 9 was prepared, the remaining aqueous layer was concentrated under reduced pressure at 70 ° C or lower to obtain a water fraction (yield: 58.7%).

Example 11. Preparation of hexane fractions of 100% ethanol extracts

20 g of the 100% ethanol extract of Comparative Example 3 was suspended in 800 mL of distilled water and the solvent fraction was performed three times using the same amount of n-hexane. The fraction obtained was concentrated under reduced pressure at 40 DEG C or less to obtain a hexane fraction (yield: 10.4 %).

Example 12. Dichloromethane fraction

After the hexane fraction of Example 11 was prepared, the remaining water layer was subjected to three solvent fractions using 800 mL of dichloromethane. The fractions thus obtained were concentrated under reduced pressure at 40 DEG C or less to obtain a dichloromethane fraction (yield: 9.9%). .

Example 13. Ethyl acetate fraction

After the dichloromethane fraction of Example 12 was prepared, the remaining water layer was subjected to three solvent fractions using 800 mL of ethyl acetate. The fractions thus obtained were concentrated under reduced pressure at 50 DEG C or lower to obtain an ethyl acetate fraction (yield: 8.6% ).

Example 14. Water saturated butanol fraction

After the ethyl acetate fraction of Example 13 was prepared, the remaining water layer was subjected to three solvent fractions using 800 mL of water-saturated butanol. The obtained fractions were concentrated under reduced pressure at 70 ° C or lower to obtain a water saturated butanol fraction 25.9%).

Example 15. Water fraction

After the water-saturated butanol fraction of Example 14 was prepared, the remaining aqueous layer was concentrated under reduced pressure at 70 ° C or lower to obtain a water fraction (yield: 48.7%).

[Example 16-30] Preparation of Diaion HP-20 fraction of Honeybush extract

Example 16. Preparation of Diaion HP-20 water fraction of water extract

21 g of the water extract of Comparative Example 1 was suspended in 5 to 20 times as much distilled water, and fractions were prepared using Diaion HP-20 resin as a porous adsorbent.

Specifically, 400 to 600 mL of Diaion HP-20 resin was activated with methanol three times its volume, filled in a column having a diameter of 7 cm and a height of 60 cm, and then poured into methanol until the resin was completely precipitated. Thereafter, the amount of the distilled water was sequentially increased to be replaced with the final distilled water, and then the suspension of the water extract of Comparative Example 1 was loaded.

Thereafter, the loaded column was eluted with distilled water 5 to 10 times the resin volume, and the eluate was concentrated under reduced pressure at 70 占 폚 or less to obtain a water fraction (yield: 51.7%).

The HPLC pattern analysis results of Diaion HP-20 water fraction are shown in FIG. 1 (b).

Specifically, the compound was separated and purified from the Diaion HP-20 water fraction.

Approximately 14 g of the Diaion HP-20 water fraction was dissolved in 300 ml of water, and 240 ml of n-butanol was added thereto. The resulting mixture was divided into three portions. Only the butanol layer was separated and concentrated under reduced pressure. 730 mg of this product was eluted from silica gel column chromatography using chloroform: methanol: water at a ratio of 10: 3: 1 to 5: 4: 1 in total to obtain a total of 7 small fractions. Of these, 108 mg of small fraction 3 was carried out using Sephadex LH20 column chromatography under methanol solvent conditions. As a result, Compound 5 (50 mg, 0.36%) was finally separated and spectroscopic data of this compound is shown below. _{^{1 H NMR (CD 3 OD,}} 600MHz): δ 6.79 (1H, d, J = 8.2Hz), 7.43 (1H, dd, J = 8.2Hz, J = 1.4Hz), 7.44 (1H, d, J = 8.2 Hz); _{^{13 C NMR (CD 3 OD,}} 150MHz): δ 170.63, 151.55, 146.08, 124.06, 123.34, 117.84, 115.88; ^{m / z 153 [MH] -} , MS / MS m / z 109 [MH-CO 2] -. Based on the above spectroscopic results, it was confirmed that Compound 5 was protocatechuic acid represented by Formula 5 (Chromatographia (2007) Vol 65 (1/2), pp. 1-7) :

[Chemical Formula 5]

.

.

Hesperidin represented by the formula (6) was used as a standard without a separate purification because there were commercialized standard products:

[Chemical Formula 6]

.

.

Assessment of cytotoxicity against Compound 5 and Compound 6 showed no cytotoxicity up to a concentration of 200 ug / ml (see FIG. 2).

Example 17. 20% methanol fraction

After elution of Example 16 was completed, the mixture was eluted again with 20% methanol and concentrated under reduced pressure at 70 ° C or lower to obtain a 20% methanol fraction (yield: 9.2%).

Example 18. 40% methanol fraction

After elution of Example 17 was completed, the mixture was further eluted with 40% methanol and concentrated under reduced pressure at 70 ° C or lower to obtain a 40% methanol fraction (yield: 23.4%).

Example 19. 70% methanol fraction

After elution of Example 18 was completed, elution was further carried out using 70% methanol, followed by concentration under reduced pressure at 70 ° C or lower to obtain a 70% methanol fraction (yield: 13.2%).

Example 20. 100% methanol fraction

After the elution of Example 19 was completed, the reaction mixture was eluted again with 100% methanol and concentrated under reduced pressure at 70 ° C or lower to obtain a 100% methanol fraction (1.1% yield)

Example 21. Preparation of Diaion HP-20 Water Fraction of 70% Ethanol Extract

19 g of the 70% ethanol extract of Comparative Example 2 was suspended in 5 to 20 times of distilled water, and fractions were prepared using Diaion HP-20 resin as a porous adsorbent.

Specifically, 400 to 600 mL of Diaion HP-20 resin was activated with methanol three times its volume, filled in a column having a diameter of 7 cm and a height of 60 cm, and then poured into methanol until the resin was completely precipitated. Thereafter, the amount of distilled water was sequentially increased to be replaced with the final distilled water, and then the 70% ethanol extract suspension of Comparative Example 2 was loaded.

Thereafter, the loaded column was eluted with 5 to 10 times as much volume of resin as the distilled water, and the eluate was concentrated under reduced pressure at 70 ° C or lower to obtain a water fraction (yield: 41.5%).

Example 22. 20% methanol fraction

After elution of Example 21 was completed, the mixture was eluted again with 20% methanol and concentrated under reduced pressure at 70 ° C or lower to obtain a 20% methanol fraction (yield: 7.8%).

Example 23. 40% methanol fraction

After elution of Example 22 was completed, it was eluted again with 40% methanol and concentrated under reduced pressure at 70 ° C or lower to obtain a 40% methanol fraction (yield: 18.9%).

Example 24. 70% methanol fraction

After elution of Example 23 was completed, elution was further carried out using 70% methanol, followed by concentration under reduced pressure at 70 DEG C or less to obtain a 70% methanol fraction (yield 16.5%).

Example 25. 100% methanol fraction

After the elution of Example 24 was completed, elution was again carried out using 100% methanol, and the filtrate was concentrated under reduced pressure at 70 ° C or lower to obtain a water fraction (yield: 4.2%).

Example 26. Preparation of Diaion HP-20 water fraction of 100% ethanol extract

11 g of the 100% ethanol extract of Comparative Example 3 was suspended in 5 to 20 times of distilled water, and fractions were prepared using Diaion HP-20 resin as a porous adsorbent.

Specifically, 400 to 600 mL of Diaion HP-20 resin was activated with methanol three times its volume, filled in a column having a diameter of 7 cm and a height of 60 cm, and then poured into methanol until the resin was completely precipitated. Thereafter, the amount of distilled water was sequentially increased to be replaced with the final distilled water, and then the 70% ethanol extract suspension of Comparative Example 2 was loaded.

Thereafter, the loaded column was eluted with distilled water 5 to 10 times the resin volume, and the eluate was concentrated under reduced pressure at 70 占 폚 or less to obtain a water fraction (yield: 11.5%).

Example 27. 20% methanol fraction

After elution of Example 26 was completed, the mixture was eluted again with 20% methanol and concentrated under reduced pressure at 70 ° C or lower to obtain a 20% methanol fraction (yield: 7.2%).

Example 28. 40% methanol fraction

After the elution of Example 27 was completed, the reaction mixture was eluted again with 40% methanol and concentrated under reduced pressure at 70 ° C or lower to obtain a 40% methanol fraction (yield: 13.9%).

Example 29. A 70% methanol fraction

After the elution of Example 28 was completed, elution was further carried out using 70% methanol, followed by concentration under reduced pressure at 70 ° C or lower to obtain a 70% methanol fraction (yield: 10.3%).

Example 30. 100% methanol fraction

After the elution of Example 29 was completed, elution was further carried out using 100% methanol, followed by concentration under reduced pressure at 70 ° C or lower to obtain a methanol fraction (yield of 7.4%).

[Experimental Example]

Experimental Example 1: Protective Effect of Honeybush Fraction in UV-B Treated Cells

To confirm whether the solvent fraction of the honeybush extract inhibited the death of cells treated with UV-B, the cell survival rate was evaluated by a known MTS assay method.

First, keratinocyte HaCaT (human keratinocyte) cell line was cultured in DMEM (Dulbecco's Modified Eagle's Medium) medium containing 10% FBS and 1% penicillin-streptomycin at 37 ° C in a 5% CO ₂ incubator, Cells were seeded in 96-well plates at 5 × 10 ⁴ cells / well.

After the cells were divided into 96-well plates and cultured for 24 hours in such a manner that the cells were completely attached to the cells, the extracts of Comparative Examples 1-3 and the fractions of Examples 1-30 were mixed at 10, 25, 50, ml and cultured again for 24 hours.

After replacing the cells with serum-free medium, UV-B was treated with 20mJ / cm ² for 30 minutes, replaced with medium containing MTS (Promega) reagent and absorbance was measured at 490nm (SpectraMax, Molecular Devices) The relative cell viability was then calculated for the control without UV-B treatment, and the results are shown in Tables 1 and 2 below.

In addition, cytotoxicity did not occur when the honeycomb fraction of Example 30 was treated at 10, 50, 100, and 200 ug / ml concentration in Example 1 in a cell line not irradiated with UV-B.

Protective Effect of Honeybush Solvent Fraction in UV-Treated Skin Cells (Unit:% Cell Survival) 10 [mu] g / ml 25 μg / ml 50 μg / ml 100 [mu] g / ml Comparative Example 1 26 30 52 59 Comparative Example 2 40 43 34 45 Comparative Example 3 38 41 39 35 Example 1 32 40 51 53 Example 2 22 32 32 60 Example 3 40 44 57 86 Example 4 37 35 38 57 Example 5 29 33 31 44 Example 6 32 35 41 39 Example 7 60 61 72 88 Example 8 49 54 56 77 Example 9 41 41 56 63 Example 10 32 33 39 39 Example 11 43 46 38 49 Example 12 69 72 78 65 Example 13 77 77 75 80 Example 14 56 56 60 65 Example 15 42 42 52 47

As shown in Table 1, in the case of the solvent fraction by polarity, in Example 7, which is a fraction of dichloromethane, compared with the 70% ethanol extract (Comparative Example 2), the low concentration and high concentration of 10 to 100 ug / Respectively.

In comparison with the 100% ethanol extract (Comparative Example 3), the low concentration and high concentration of 10 to 100 ug / ml showed excellent cell protection effect even in Example 13 which was the ethyl acetate fraction.

Protective Effect of Honeybush Diaion HP-20 Fraction in Ultraviolet-Treated Skin Cells (Unit:% Cell Survival) 10 [mu] g / ml 25 μg / ml 50 μg / ml 100 [mu] g / ml Comparative Example 1 26 30 52 59 Comparative Example 2 40 43 34 45 Comparative Example 3 38 41 39 35 Example 16 73 68 65 64 Example 17 68 63 70 82 Example 18 22 24 26 35 Example 19 34 46 64 72 Example 20 34 40 34 39 Example 21 33 38 32 29 Example 22 31 35 33 28 Example 23 26 24 27 41 Example 24 61 59 68 92 Example 25 30 33 35 52 Example 26 26 35 41 55 Example 27 37 47 51 52 Example 28 39 61 52 59 Example 29 42 48 51 52 Example 30 40 34 41 47

In addition, in the case of the fraction using Diaion HP-20 resin, it was confirmed that the water-eluting fraction of Example 16 and the 20% methanol eluate fraction of Example 17 had an excellent cytoprotective effect in comparison with the water extract of Comparative Example 1 .

In addition, in comparison with the 70% ethanol extract of Comparative Example 2, Example 24, which was a 70% methanol eluate fraction, also showed excellent cytoprotective effect.

Experimental Example 2: MMP-1 Inhibitory Activity of Honeybush Fractions in UV-B Treated Cells

The inhibition of MMP-1 activity was evaluated by a known MMP-1 assay method in order to confirm whether the solvent fraction of the honeybush extract inhibited wrinkle formation of skin cells treated with UV-B.

First, keratinocyte HaCaT (human keratinocyte) cell line was cultured in DMEM (Dulbecco's Modified Eagle's Medium) medium containing 10% FBS and 1% penicillin-streptomycin at 37 ° C in a 5% CO ² incubator, Cells were seeded in 96-well plates at 5 × 10 ⁴ cells / well.

After the cells were divided into 96-well plates and cultured for 24 hours in such a manner that the cells were completely attached to the cells, the extracts of Comparative Examples 1-3 and the fractions of Examples 1-30 were mixed at 10, 25, 50, ml and cultured again for 24 hours.

Subsequently, the cells were replaced with serum-free medium, UV-B was treated at 20 mJ / cm ² for 30 minutes, and the cells were harvested and assayed using an MMP-1 kit. After the completion of the reaction, the absorbance at 450 nm was measured to calculate the inhibition rate of MMP-1 activity. The results are shown in Tables 3 and 4 below.

(%) Inhibition of MMP-1 activity of the solvent fraction of the honeybee in skin treated with ultraviolet rays 10 [mu] g / ml 25 μg / ml 50 μg / ml 100 [mu] g / ml Comparative Example 1 60 44 56 67 Comparative Example 2 54 57 68 42 Comparative Example 3 54 23 43 26 Example 1 35 32 45 49 Example 2 30 39 77 105 Example 3 76 66 73 80 Example 4 58 63 86 103 Example 5 107 119 95 126 Example 6 5 14 -8 26 Example 7 43 50 51 55 Example 8 -76 -31 37 63 Example 9 -68 -126 -105 -82 Example 10 -42 16 39 100 Example 11 -25 8 14 11 Example 12 -96 12 4 35 Example 13 -4 27 15 -42 Example 14 4 -23 -19 19 Example  15 41 78 119 81

As shown in Table 3, it was confirmed that the expression of MMP-1 was strongly inhibited in Example 5, which is a water fraction, of the solvent fraction than that of Comparative Example 1 which is a water extract. In addition, as compared with the ethanol extract of Comparative Example 3, it was confirmed that the water fraction, Example 15, exhibited strong MMP-1 inhibitory activity.

Inhibition rate (%) of MMP-1 activity of Honeybush Diain HP-20 fraction in UV-treated skin cells 10 [mu] g / ml 25 μg / ml 50 μg / ml 100 [mu] g / ml Comparative Example 1 60 44 56 67 Comparative Example 2 54 57 68 42 Comparative Example 3 54 23 43 26 Example 16 46 84 100 89 Example 17 3 51 95 116 Example 18 47 -15 51 76 Example 19 91 89 105 95 Example 20 55 71 11 -18 Example 21 82 107 106 107 Example 22 20 23 21 17 Example 23 One -6 39 13 Example 24 46 86 120 126 Example 25 66 74 104 116 Example 26 16 28 51 52 Example 27 41 43 59 54 Example 28 15 -20 45 36 Example 29 56 45 75 63 Example 30 21 63 43 51

In addition, in the case of the fraction using Diaion HP-20 resin, in comparison with the water extract of Comparative Example 1, the water-eluting fraction of Example 16, the 20% methanol eluate fraction of Example 17, and the 70% 19 showed strong MMP-1 inhibitory activity.

Also, in comparison with the 70% ethanol extract of Comparative Example 2, the water-eluting fraction Example 21, the 70% methanol eluting fraction Example 24, and the 100% methanol eluate fraction Example 25 also showed excellent MMP-1 inhibitory activity Respectively.

Experimental Example 3: MMP-9 Inhibitory Activity of Honeybush Fractions in UV-B Treated Cells

(Examples 5, 7, 13, 15, 16, 17, 19, 21, 24 and 25), which are excellent in the effect thereof, based on the cytoprotective effect of Experimental Example 1 and the MMP- Was selected to measure the final inhibitory activity of MMP-9, thereby clearly demonstrating the photoaging (wrinkle reduction) effect.

First, keratinocyte HaCaT (human keratinocyte) cell line was cultured in DMEM (Dulbecco's Modified Eagle's Medium) medium containing 10% FBS and 1% penicillin-streptomycin at 37 ° C in a 5% CO ² incubator, Cells were seeded in 96-well plates at 5 × 10 ⁴ cells / well.

The fractions of Examples 5, 7, 13, 15, 16, 17, 19, 21, 24 and 25 were cultured for 10 hours in a 96 well plate and then cultured for 24 hours in such a manner that the cells were completely attached to the cells. , 50, 100 ug / ml and cultured for another 24 hours.

Subsequently, the cells were replaced with serum-free medium, UV-B was treated at 20 mJ / cm ² for 30 minutes, and the cells were harvested and assayed using an MMP-9 kit. After all the reactions were completed, the absorbance at 450 nm was measured to calculate the inhibition rate of MMP-9 activity. The results are shown in Table 5 below.

(%) Inhibition of MMP-9 activity of Honeybush fractions in UV-treated skin cells 10 [mu] g / ml 50 μg / ml 100 [mu] g / ml Example 5 14 122 100 Example 7 109 90 107 Example 13 106 106 95 Example 15 103 89 107 Example 16 59 91 125 Example 17 53 100 86 Example 19 70 124 103 Example 21 56 67 90 Example 24 115 84 101 Example 25 79 87 95

Experimental Example 4: Cytoprotective effect of honey-bush fraction-derived compounds in UV-B treated cells

The cytoprotective effects of the compounds derived from the honey-bush fraction (Example 7 and Example 16) in the UV-treated cells are shown in Table 6.

Protective Effect of Honey Bush-derived Compound in UV-Treated Skin Cells (Unit:% Cell Survival) compound 10 [mu] g / ml 50 μg / ml 100 [mu] g / ml 200 [mu] g / ml Hesperidin 77 89 92 99 5,7-dihydroxychromone 25 28 29 28 4-hydroxybenzaldehyde 29 28 32 45 4-hydroxyacetophenone 28 33 31 35 Hesperrettin 38 53 59 87 Protocateque Iksan 45 55 56 73

As shown above, hesperidin, which is a compound derived from Example 16, showed excellent cytoprotective effect from a low concentration (10 ug / ml). In addition, it was confirmed that protocatequaquacans had a cytoprotective effect when treated at a high concentration.

It was also confirmed that the hES ferritin, which is a compound derived from Example 7, has a cytoprotective effect at a high concentration.

Experimental Example 5: MMP-1 Inhibitory Activity of Honeybush Fraction-Derived Compounds in UV-B Treated Cells

Table 7 shows the MMP-1 inhibitory effect of the compounds derived from the honey-bush fraction (Example 7 and Example 16) in UV-treated cells.

(%) Of MMP-1 activity of honeycomb-derived compounds in skin cells treated with ultraviolet rays compound 10 [mu] g / ml 50 μg / ml 100 [mu] g / ml 200 [mu] g / ml Hesperidin 59 83 88 99 5,7-dihydroxychromone -9 -7 6 30 4-hydroxybenzaldehyde 21 27 25 39 4-hydroxyacetophenone 86 94 109 114 Hesperrettin 106 107 114 113 Protocateque Iksan 93 98 98 105

As shown above, hesperidin, which is a compound derived from Example 16, showed strong MMP-1 inhibitory activity at a concentration of 50 ug / ml. In the case of protocatechuic acid, it was also confirmed that it has a strong inhibitory activity from low concentration of 10 ug / ml to high concentration.

In the case of 4-hydroxyacetophenone and hesperitin, which are derived from Example 7, it was confirmed that MMP-1 inhibitory activity was low at a low concentration.

Experimental Example 6: MMP-9 inhibitory activity of the compounds derived from the honeycomb fraction in UV-B treated cells

Table 8 shows the MMP-9 inhibitory effect of the compounds derived from the honeycomb fraction (Example 7 and Example 16) in UV-treated cells.

(%) Of MMP-9 activity of the honeycomb-derived compound in UV-treated skin cells compound 10 [mu] g / ml 50 μg / ml 100 [mu] g / ml 200 [mu] g / ml Hesperidin 61 85 77 95 5,7-dihydroxychromone 93 103 104 105 4-hydroxybenzaldehyde 80 99 103 111 4-hydroxyacetophenone 95 97 101 109 Hesperrettin 79 86 103 115 Protocateque Iksan 99 105 114 104

As shown above, hesperidin, which is a compound derived from Example 16, showed an inhibitory activity exceeding 80% with respect to MMP-9 at a concentration of 50 ug / ml. In addition, it was confirmed that protocatechuic acid has strong inhibitory activity from low concentration of 10 ug / ml to high concentration.

In the case of the compounds derived from Example 7, 5,7-dihydroxychromone, 4-hydroxybenzaldehyde, 4-hydroxyacetophenone and hesperitin, a strong inhibitory activity against MMP-9 from low to high concentrations Respectively.

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

Claims (13)

A composition for improving skin, comprising a fraction of Honeybush extract as an active ingredient. The composition of claim 1, wherein the extract is extracted with water, a C ₁ to C ₆ lower alcohol, or a mixed solvent thereof. The composition of claim 1, wherein said fraction is dichloromethane, ethyl acetate, or water fraction of a honey bee extract. The composition of claim 1, wherein the fraction is an eluate after adsorption of the Honeybush extract with Diaion HP-20 resin as an adsorbent. The composition of claim 4, wherein the eluate is eluted with water, a C ₁ to C ₆ lower alcohol, or a mixed solvent thereof. The composition of claim 1, wherein the fraction has a volume of from 10 μg / ml to 200 μg / ml. A skin containing at least one compound selected from the group consisting of hesperidin, 5,7-dihydroxychromone, 4-hydroxybenzaldehyde, 4-hydroxyacetophenone, hesperetine and protocatechuic acid as an active ingredient / RTI > 8. The composition of claim 7, wherein said compound is isolated from a fraction of Honeybush extract. 8. The method of claim 7,
Wherein the dose of said compound is 10 [mu] g / ml to 200 [mu] g / ml. 8. The composition according to any one of claims 1 to 7, wherein the composition is for preventing skin aging or improving skin wrinkles. 11. The composition of claim 10, wherein the composition is for preventing skin aging or improving skin wrinkles by inhibiting matrix metalloproteinase-1 (MMP-1) and matrix metalloproteinase-9 (MMP-9). 8. The composition according to any one of claims 1 to 7, wherein the composition is a pharmaceutical composition, a cosmetic composition or a health functional food composition. The composition of claim 1 or 7, wherein the composition is formulated as a powder, powder, granule, beverage, tablet, capsule, suspension, emulsion, syrup, aerosol, Composition.

Images