KR101356797B1 - Extract Containing Acteoside Derived from Lippia citriodora and Cosmetic Composition Containing the Same - Google Patents

Abstract

The present invention relates to a lemon verbena-derived actioside-containing extract and a functional cosmetic composition comprising the extract, and more particularly to a lemon verbena extract containing a high content of actioside through a specific continuous process manufacturing method. The present invention relates to a lemon verbena extract containing a high content of actioside prepared by the method and a functional cosmetic composition comprising the same.
The cosmetic composition of the present invention contains a high content of actioside, which has been separated and purified from lemon verbena through solvent extraction, concentration, solvent fractionation and recrystallization as an active ingredient, and has anti-oxidation, whitening, anti-wrinkle, anti-inflammatory, antibacterial and atopy. It can be manufactured and applied as a cosmetic composition having excellent effects such as improvement and UV protection.

Description

Extract containing lemon verbena derived actioside and functional cosmetic composition comprising same {Extract Containing Acteoside Derived from Lippia citriodora and Cosmetic Composition Containing the Same}

The present invention relates to a lemon verbena-derived actioside-containing extract and a functional cosmetic composition comprising the extract, and more particularly to a lemon verbena extract containing a high content of actioside through a specific continuous process manufacturing method. The present invention relates to a lemon verbena extract containing a high content of actioside prepared by the method and a functional cosmetic composition comprising the same.

In human skin, various physicochemical changes occur in the aging process, and the causes are largely divided into intrinsic aging and photo-aging. Free radicals can be caused by activation of ultraviolet rays, stress, disease state, environmental factors, wounds, and aging. If such state is deepened, the antioxidant defense network existing in the living body is destroyed, And aging. That is, lipids, proteins, polysaccharides and nucleic acids, which are major constituents of the skin, are oxidized to destroy skin cells and tissues, resulting in skin aging. Especially, the oxidation of protein breaks down collagen, hyaluronic acid, elastin, proteoglycan and fibronectin, which are connective tissues of skin, And if this gets worse, DNA mutation leads to mutations, cancer induction, and immune deficiency. Therefore, it is necessary to protect the cell membrane by destroying the free radicals mediated by free radicals, ultraviolet rays, and inflammation that occur during the metabolism of the body, and to regenerate already damaged cells by active metabolism, It can quickly recover and maintain healthy skin.

 In the aging process, not only free radicals but also enzymes such as MMP are involved. Synthesis and degradation of extracellular matrix such as collagen are controlled appropriately in the body, but their synthesis is reduced as aging progresses and collagen degradation enzymes such as matrix metalloproteinase MMP) is promoted and skin elasticity is lowered and wrinkles are formed. These degrading enzymes are also activated by ultraviolet irradiation.

Therefore, researches on substances with whitening, anti-aging, anti-wrinkle and antioxidant effects have been actively conducted. However, existing chemicals are used for various problems such as toxicity, low activity, limit of use and side effects of overdose. As part of the development of materials that are less likely to have side effects, researches on extracting active ingredients from natural plants are active. However, even in the case of natural products, there are many problems in using cosmetics or medicines at an effective concentration or more in terms of safety, stability, and discoloration possibility. .

On the other hand, Lippia citriodora , also known as deodorant tree, is a non-cold resistant deciduous tree with a height of 60 to 150 cm, which is a herb of a perennial plant, a monocotyledonous plant, It has strong lemon fragrance throughout. Leaf length is 7 ~ 12cm, long lanceolate, pointed end. Leaves are 3 ~ 4 in the stem. The flowers are white or light purple in summer, and small ones are densely packed and run on cones.

Actioside is a compound belonging to phenylpropanoid glycoside, which has been reported for various physiological activities such as antioxidant, anti-inflammatory, antibacterial and immunosuppressive effects. In the prior art, there is a liquid leaching tea and a method of manufacturing the same (Korean Patent No. 10-0899890) by squeezing the fragrance of the lemon on the lemon verbena leaf, and the flavor lasts for a long time. There is no disclosure of lemon verbena extract containing actiosides and functional cosmetic compositions comprising the same.

Accordingly, the present inventors have made efforts to develop a natural product extract having the effects of skin aging, whitening, etc. among the various natural product extracts that have not been known so far, isolate the extract containing lemon verbena-derived actioside, the extract is antioxidant, whitening The present invention was completed by confirming that it has effects such as wrinkle improvement, anti-inflammatory, antibacterial, atopy improvement and UV protection.

It is an object of the present invention to provide a lemon verbena extract containing a lemon verbena-derived high content of actioside, which is excellent in antioxidant, whitening, anti-wrinkle, anti-inflammatory, antibacterial, atopic improvement and UV protection, and a cosmetic composition comprising the same. It is.

In order to achieve the above object, the present invention comprises the steps of (a) extracting actioside by adding a solvent to lemon verbena, and then concentrating; (b) adding at least two solvents selected from the group consisting of water, ethanol, methanol, butanol, ethyl acetate, ethyl ether, chloroform and hexane to the concentrated extract of step (a) to obtain an actioside fraction; ; (c) removing the organic solvent of step (b) and secondarily concentrating the organic solvent to prepare a fractionally concentrated powder; (d) dissolving the fractionally concentrated powder of step (c) in a solvent, removing the pigment and chlorophyll using a column; (e) precipitating and recrystallizing the pigment and chlorophyll-extracted extract by adjusting the pressure and temperature; and (f) spray-dried or freeze-dried the pellet precipitated through the recrystallization to obtain a powdery actioside. It provides a method for producing a lemon verbena extract containing a high content of actioside comprising the step of.

The present invention also provides a cosmetic composition prepared by the above production method, comprising a lemon verbena extract containing a high content of actioside as an active ingredient.

The cosmetic composition of the present invention contains a lemon verbena extract containing a high content of actioside as an active ingredient in an optimum production yield and manufacturing process, the lemon verbena extract containing a high content of actioside derived from lemon verbena It can be prepared and applied as a cosmetic composition excellent in antioxidant effect, whitening effect, wrinkle improvement effect, anti-inflammatory effect, antibacterial effect and atopy improvement effect.

1 relates to a method for producing a lemon verbena extract containing lemon verbena derived actioside.
2 relates to HPLC chromatography of lemon verbena extract containing lemon verbena derived actiosides.
Figure 3 relates to the UV spectrum of lemon verbena extract containing lemon verbena derived actiosides.
Figure 4 relates to the in vitro SPF measurement chromatography of lemon verbena extract containing lemon verbena derived actiosides.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

In one aspect, the present invention comprises the steps of (a) extracting actioside by adding a solvent to lemon verbena, and then concentrating; (b) adding at least two solvents selected from the group consisting of water, ethanol, methanol, butanol, ethyl acetate, ethyl ether, chloroform and hexane to the concentrated extract of step (a) to obtain an actioside fraction; ; (c) removing the organic solvent of step (b) and secondarily concentrating the organic solvent to prepare a fractionally concentrated powder; (d) dissolving the fractionally concentrated powder of step (c) in a solvent, removing the pigment and chlorophyll using a column; (e) precipitating and recrystallizing the pigment and chlorophyll-extracted extract by adjusting the pressure and temperature; and (f) spray-dried or freeze-dried the pellet precipitated through the recrystallization to obtain a powdery actioside. It relates to a method for producing lemon verbena extract containing actioside comprising the step of.

In one embodiment of the present invention is to elute the actioside from the lemon verbena through the extraction step, the optimal extraction step according to the present invention is the weight of lemon verbena at the rate of 0.5 ~ 20.0% water, ethanol, methanol, butanol, ethyl It is added to a solvent such as acetate, ethyl ether, chloroform and hexane to extract 0.5-5 hours. The lemon verbena extract eluted through the extraction step is subjected to the first concentration step to remove the solvent, it can be applied by various methods such as heat drying, reduced pressure drying, spray drying and freeze drying.

The lemon verbena extract obtained through the primary concentration step is subjected to a fractionation step of separating the compounds according to the polarity. A solvent such as water, ethanol, methanol, butanol, ethyl acetate, ethyl ether, chloroform and hexane is used. After the fractionation step, the fraction is subjected to a second concentration step for removing the organic solvent. The fraction may be applied by various methods such as heat drying, vacuum drying, spray drying and freeze drying.

After the second concentration step, the fractionally concentrated powder is subjected to a column step of removing the dye and chlorophyll. The resin is suitable for a resin column using an adsorbent resin. The resin is selected from the group consisting of Amberlite XAD resin, Dow X resin, Suferite DAX resin, Diaion HP-10, HP-20, HP-30, HP-40 and HP-50 resin. After the above-mentioned column steps, a recrystallization step is carried out by adjusting the pressure and temperature of the column passing liquid by precipitation. The recrystallization is carried out under ultra low temperature and ultra high pressure conditions including -300 to 25 ° C and 0.5 to 800 MPa. The recrystallized precipitate pellet is subjected to the step of obtaining a lemon verbena extract containing a high content of actioside powder by spray drying or lyophilization, can be used by concentration by centrifugation, filter and vacuum concentration method. The lemon verbena extract containing the powdery high content of actioside was obtained by spray drying and freeze drying.

Lemon verbena extract containing high content of actioside obtained in another embodiment of the present invention is analyzed by high pressure liquid chromatography (HPLC) for qualitative and quantitative analysis of actioside of the sample according to the preparation method. Was carried out. As a result, it was confirmed that the content of actioside was increased as the extraction, concentration, fraction, column, and recrystallization step were increased from lemon verbena, and a high content of actioside of more than 99% was obtained through the ultra low temperature and ultra high pressure recrystallization step. It was confirmed that the lemon verbena extract containing could be prepared.

In the present invention, the extracting step (a) may be characterized in that the extraction of lemon verbena at 0.5 to 20.0% ratio for 0.5 to 5 hours, the solvent of step (a) is water, ethanol, methanol, butanol It may be characterized in that at least one solution selected from the group consisting of ethyl acetate, ethyl ether, chloroform and hexane.

In the present invention, in the concentration step of (a) and (c) it may be characterized by using a method such as heat drying, reduced pressure drying, spray drying and freeze drying, Actio obtained in step (b) The fraction containing the side may be characterized in that the ethyl acetate fraction.

In the present invention, the step (d) may be a resin column using an adsorbent resin. The resin of step (d) may be selected from the group consisting of Amberlite XAD resin, Dow X resin, Suferite DAX resin, HP-10, HP-20, HP-30, HP-40 and HP-50 resin.

In the present invention, the step (e) may be characterized by recrystallization under ultra-low temperature, ultra-high pressure conditions including -300 ~ 25 ℃ and 0.5 ~ 800MPa, the precipitated high content of the liquid (f) step In order to obtain a lemon verbena extract containing a thioside may be characterized by using a spray drying or lyophilization after concentration by centrifugation, a filter and a vacuum concentration method.

In an embodiment of the present invention, after extracting lemon verbena at 99% ethanol aqueous solution at 10% temperature for 2 hours under the condition of 80 ℃, an extract filtrate was obtained using an 80 mesh filter and a 1.2㎛ filter. The obtained filtrate was concentrated at 65 ° C. under 20 hPa for 1 hour, and then, 10 times by weight of distilled water was added to the concentrated powder to disperse the mixture evenly, and then fractionated hexane, ethyl acetate, and n-butanol were used. Fractionation was performed in turn to obtain an ethyl acetate layer. The obtained ethyl acetate fractional filtrate was concentrated for 1 hour under 65 ° C. and 20 hPa conditions, and the obtained concentrated powder was dispersed again in 10-fold weight of distilled water and finally 70% to 99.5% ethanol using a DIION HP-20 column. A column solution was obtained and subjected to cryogenic recrystallization for 2 hours under -90 ° C. This was centrifuged using a centrifuge for 15 minutes under conditions of 8,000rpm and 15 ° C., filtered through a 1.2 μm filter, and finally dried at 60 ° C. for 1 hour to extract lemon verbena containing a high content of actioside. A powder was obtained.

In another aspect, the present invention relates to a lemon verbena extract prepared by the above production method and containing a high content of actioside.

In another embodiment of the present invention, the free radical scavenging test and the active oxygen scavenging test were conducted to confirm the antioxidant effect in the skin aging improvement effect assay test for lemon verbena extract containing a high content of actioside. As a result, the obtained lemon verbena extract containing high content of actioside showed an antioxidant capacity similar to that of L-ascorbic acid, a powerful synthetic antioxidant, and the concentration of 50% free radical scavenging ratio was The concentration of 6 ㎍ / ㎖, 50% active oxygen removal rate was 5 ㎍ / ㎖ was confirmed that the antioxidant effect was excellent at a trace concentration.

In another embodiment of the present invention, a tyrosinase deactivation test was conducted to assay the whitening effect of lemon verbena extract containing a high content of actioside. As a result, the obtained lemon verbena extract containing high content of actioside showed a similar whitening effect to arbutin, which is known to have excellent whitening effect, and the concentration of 50% of tyrosinase activity inhibition rate was 0.031 mg. It was confirmed that the whitening effect was excellent at a trace concentration of / ml. In addition, as a result of measuring the melanin biosynthesis inhibitory effect of B16F1 melanocytes, the concentration of 50% of melanin synthesis inhibitory effect of lemon verbena extract containing a high content of actioside was 0.08%, arbutin and oil-soluble licorice known as a whitening agent. It was confirmed that similar or superior to the extract.

In another embodiment of the present invention, in order to test the effect of inhibiting and improving the skin wrinkles of lemon verbena extract containing a high content of actioside, an elastase deactivation test was conducted. The concentration of 50% of the elastase activity inhibition rate of the contained lemon verbena extract was 0.20 mg / ml, and it was confirmed that the inhibitory effect of elastase was better than 0.38 mg / ml of retinol, which is known to be excellent in wrinkle improvement. In addition, as a result of measuring the collagen synthesis effect in order to test the skin wrinkle improvement effect of lemon verbena extract containing high content of actioside, the obtained lemon verbena extract containing high content of actioside has a collagen synthesis effect. It showed a collagen synthesis effect similar to ascorbic acid known to be excellent, it was confirmed that the excellent skin wrinkle suppression and improvement effect of lemon verbena extract containing a high content of actioside.

In another embodiment of the present invention, enzyme immunoassay (ELISA) was performed to measure the concentration of MMP-1 after UV irradiation and sample addition of lemon verbena extract containing a high content of actioside. Inhibition of MMP-1 induced expression by UV irradiation showed more than 48% inhibition rate compared to the control group without treatment, which was superior to the inhibition rate of retinol used as a control, and lemon verbena containing high content of actioside As a result of evaluating the cytotoxicity of the extract by UV irradiation, lemon verbena extract containing high content of actioside exhibited a cytotoxicity of 41% at 0.1% concentration, effectively protecting against cytotoxicity by UV rays. I could see. As a result of evaluating the inhibitory effect of inflammatory cytokine expression expressed by UV irradiation of lemon verbena extract containing high content of actioside, lemon verbena extract containing high content of actioside was found to be inflammatory cytokine induced by UV light. The production of IL-1α was suppressed to 41% at a concentration of 0.1%, which was found to effectively protect against inflammation caused by ultraviolet rays at low concentrations. In addition, the UV protection effect assay test for lemon verbena extract containing a high content of actioside in As a result of measuring the sunscreen effect by using the in vitro SPF analyzer (Optometrics SPF-290S), lemon verbena extract containing high content of actioside was more effective than the OMC, which is a powerful synthetic organic sunscreen, ) Could be confirmed.

The antibacterial effect assay test for lemon verbena extract containing a high content of actioside of the present invention was carried out by agar serial dilution method and paper disc method. As a result, lemon verbena extract containing a high content of actioside was able to confirm superior antimicrobial activity against four kinds of bacteria, yeast and filamentous fungus than methyl paraben, a powerful synthetic preservative.

In another aspect, the present invention relates to a cosmetic composition containing a lemon verbena extract containing a high content of actioside as an active ingredient.

In another embodiment of the present invention to prepare a nourishing cream containing a lemon verbena extract containing a high content of actioside and a lemon verbena extract containing a high content of actioside, prepared nutrition cream The whitening effect of 30 women over 25 years of age with freckles, freckles and pigmentation were used for 12 weeks. (ΔL) was measured. As a result of the measurement, the nutritional cream containing lemon verbena extract containing high content of actioside was found to have excellent whitening effect compared to the nutrition cream without addition of lemon verbena extract containing high content of actioside. there was.

In another embodiment of the present invention, the anti-wrinkle effect is measured using a nourishing cream containing a lemon verbena extract containing a high content of actioside and a nourishing cream containing a lemon verbena extract containing a high content of actioside To prepare 10 women over 30 years of age, use the nourishing cream on each eye wrinkle for 12 weeks, and then use the image analysis to measure the wrinkles around the eyes at 4, 8, and 12 weeks. Was measured. As a result of the measurement, the nutrition cream with lemon verbena extract containing high content of actioside was confirmed to have an excellent wrinkle improvement effect compared with the nutrition cream without addition of lemon verbena extract containing high content of actioside. Could.

In another embodiment of the present invention using a nutrition cream containing a lemon verbena extract containing a high content of actioside and a lemon cream or a lemon cream containing a high content of actioside does not measure the atopic improvement effect It was. As a result, the nutritional cream containing lemon verbena extract containing high content of actioside was recovered after skin barrier damage compared to the nutrition cream without adding lemon verbena extract containing high content of actioside. Was confirmed to rise.

In the cosmetic composition of the present invention, the content of the lemon verbena extract containing the actioside may be 0.0005 to 10% by weight based on the total weight of the cosmetic composition.

In the present invention, the cosmetic composition may be selected from cosmetics, gels, water-soluble liquids, creams, essences, oil-in-water (O / W) or water-in-oil (W / O) formulations.

In another aspect, the present invention relates to a topical skin composition containing a lemon verbena extract containing a high content of actioside as an active ingredient.

Lemon verbena extract containing the actioside obtained by the above specific continuous process of the present invention and the cosmetic composition comprising the same shows the effect of excellent antioxidant, whitening, wrinkle improvement, anti-inflammatory, antibacterial, UV protection and atopy improvement. In addition, the cosmetic composition containing lemon verbena extract containing actioside may be prepared in various formulations such as cosmetics, essences, creams, packs, patches, skin adhesive gels, foundations, makeup bases, It can be applied to conventional cosmetic preparation methods. Specifically, it can be applied in various form such as liquid, cream, paste, and solid form, and may include various conventional auxiliary agents and carriers suitable for each of the formulations and well known in the art.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Example  One : Lemon verbena origin Actioside  purchase

Lemon verbena was added to a 99% ethanol aqueous solution at 10% by weight and extracted in a reflux extractor equipped with a cooling capacitor for 2 hours under a temperature of 80 ° C., followed by filtration with an 80 mesh filter and a 1.2 μm filter to obtain an extract filtrate. The obtained extraction filtrate was concentrated using a vacuum concentrator (Rotary Evaporator NE-series, Eyela, Japan) under the conditions of 65 캜 and 20 hPa for 1 hour to obtain a concentrated powder. The sample thus obtained was called "sample 1" and used in some test examples.

Example  2 : Lemon verbena origin Actioside  purchase

Lemon verbena was added to a 99% ethanol aqueous solution at 10% by weight and extracted in a reflux extractor equipped with a cooling capacitor for 2 hours under a temperature of 80 ° C., followed by filtration with an 80 mesh filter and a 1.2 μm filter to obtain an extract filtrate. The obtained filtrate was concentrated for 1 hour at 65 ° C. and 20 hPa using a vacuum concentrator. 10 times weight of distilled water was added to the concentrated powder, and then dispersed evenly. , Ethyl acetate and n-butanol were fractionated one by one to obtain an ethyl acetate layer. The obtained ethyl acetate fractional filtrate was concentrated for 1 hour under a vacuum concentrator at 20 ° C. at 65 ° C. for 1 hour to obtain a concentrated powder, which was obtained as “sample 2” and used in some test examples.

Example  3: Lemon verbena origin Actioside  purchase

Lemon verbena was added to a 99% ethanol aqueous solution at 10% by weight and extracted in a reflux extractor with a cooling capacitor for 2 hours under a temperature of 80 ° C., followed by filtration with an 80 mesh filter and a 1.2 μm filter to obtain an extract filtrate. The obtained filtrate was concentrated for 1 hour at 65 ° C. and 20 hPa using a vacuum concentrator. 10 times weight of distilled water was added to the concentrated powder, and then dispersed evenly. , Ethyl acetate and n-butanol were fractionated one by one to obtain an ethyl acetate layer. The obtained ethyl acetate fractional filtrate was concentrated for 1 hour at 65 ° C. and 20 hPa using a vacuum concentrator, and the obtained concentrated powder was dispersed again in 10-fold weight of distilled water and filled in a DIION HP-20 column, and Chromatography was performed by gradient using 99.5% ethanol. Finally, 70% to 99.5% ethanol column solution was received under a flow rate of 20 ml / min and concentrated using a vacuum concentrator at 65 ° C. and 20 hPa for 1 hour to obtain a concentrated powder. 3 "and was used in some test examples.

Example  4 : Lemon verbena origin Actioside  purchase

Lemon verbena was added to a 99% ethanol aqueous solution at 10% by weight and extracted in a reflux extractor equipped with a condenser for 2 hours under a temperature of 80 ° C., followed by filtration with an 80 mesh filter and a 1.2 μm filter to obtain an extract filtrate. The obtained filtrate was concentrated for 1 hour at 65 ° C. and 20 hPa using a vacuum concentrator. 10 times weight of distilled water was added to the concentrated powder to disperse evenly, followed by fractional hexane. , Ethyl acetate and n-butanol were fractionated one by one to obtain an ethyl acetate layer. The obtained ethyl acetate fractional filtrate was concentrated for 1 hour at 65 ° C. and 20 hPa using a vacuum concentrator, and the obtained concentrated powder was dispersed again in 10-fold weight of distilled water and filled in a DIION HP-20 column, and Chromatography was performed by gradient using 99.5% ethanol. Ultra high pressure recrystallization under 100MPa, 25 ℃ for 1 hour using ultra high pressure device (TFS-2L, TOYO KOATSU CO., LTD., Japan) after receiving 70% ~ 99.5% ethanol column solution under flow rate 20ml / min Treated. The mixture was centrifuged at 8,000 rpm and 15 ° C for 15 minutes using a centrifugal separator (Supra 22K, Hanil, Korea), filtered through a 1.2 μm filter, and finally dried in a dry oven at 60 ° C. for 1 hour, Was obtained. The thus obtained sample was called "sample 4 ", and used in some test examples.

Example  5: Lemon verbena origin Actioside  purchase

Lemon verbena was added to a 99% ethanol aqueous solution at 10% by weight and extracted in a reflux extractor with a cooling capacitor for 2 hours under a temperature of 80 ° C., followed by filtration with an 80 mesh filter and a 1.2 μm filter to obtain an extract filtrate. The obtained filtrate was concentrated for 1 hour at 65 ° C. and 20 hPa using a vacuum concentrator. 10 times weight of distilled water was added to the concentrated powder to disperse evenly, followed by fractional hexane. , Ethyl acetate and n-butanol were fractionated one by one to obtain an ethyl acetate layer. The obtained ethyl acetate fractional filtrate was concentrated for 1 hour at 65 ° C. and 20 hPa using a vacuum concentrator, and the obtained concentrated powder was dispersed again in 10-fold weight of distilled water and filled in a DIION HP-20 column, and Chromatography was performed by gradient using 99.5% ethanol. Finally, 70% to 99.5% ethanol column solution was received under a flow rate of 20 ml / min, and cryogenic recrystallization was performed for 2 hours under -90 ° C using an ultra low temperature apparatus (DF-9010, Ilshin, Korea). This was centrifuged at 8,000 rpm and 15 ° C for 15 minutes using a centrifuge, filtration was performed with a 1.2 μm filter, and finally dried in a dry oven at 60 ° C. for 1 hour to obtain a powder. Was called "sample 5 ", and used in some test examples.

Test Example  One: HPLC Using Actioside  Content analysis

In order to confirm the change in the content of actioside according to the preparation method of lemon verbena extract containing actioside of the present invention, the content analysis using HPLC of each sample was performed as follows.

For qualitative and quantitative analysis of the actioside of the sample prepared according to the preparation method according to the above example, the analysis was performed using high pressure liquid chromatography (HPLC). Samples were prepared at appropriate concentrations with each solvent, filtered through a 0.2 μm filter, pretreated and injected into the instrument. The instrument was analyzed in the whole UV range using a UV detector using HPLC (Agilent 1200 series system, Agilent, USA) and analyzed using a C ₁₈ column (Zorbax XDB-C ₁₈ 4.6 x 250 mm, Agilent, USA) using a gradient of water containing 2% acetic acid and acetonitrile as a solvent. All of the solvents used in the analysis were HPLC grade reagents, and standard curves were obtained and quantitative analysis was performed. As a result, it can be seen that the content of actioside increases as the extraction, concentration, fraction, column, and recrystallization steps are increased from lemon verbena. It was confirmed that lemon verbena extract containing could be prepared.

Content analysis using HPLC Manufacturing method steps Name of sample Actioside content (%) Extraction, concentration Sample 1 2.5 Extraction, concentration, fraction Sample 2 11.4 Extraction, concentration, fraction, column Sample 3 32.9 Extraction, concentration, fraction, column, ultrahigh pressure recrystallization Sample 4 99.4 Extraction, concentration, fractionation, column, cryogenic recrystallization Sample 5 99.3

Test Example  2: Antioxidant effect measurement

A free radical scavenging test and an active oxygen scavenging test were carried out to confirm the antioxidant effect by the skin aging improvement effect assay test on lemon verbena extract containing a high content of actioside obtained in Example (Sample 5) of the present invention. It was.

In the free radical scavenging test, the absorbance of the stable DPPH exhibits the maximum absorbance at 540 nm, and the free radical DPPH is erased by the sample to become purple to transparent color, and as the free radical scavenging rate increases, the absorbance at the 540 nm wavelength is increased. It was carried out by the test method by applying the decrease.

In 1 ml of 0.1 mM DPPH (2,2-diphenyl-1-picryl-hydrazyl radical, Sigma) solution, lemon verbena extract containing the high content of actioside obtained in Example 5 was diluted in methanol at an appropriate concentration. After mixing 1ml, and allowed to stand at 37 ℃ for 15 minutes, the absorbance at 540nm wavelength was measured using a microplate reader (UVT-06685, Thermo max, USA).

In the free radical scavenging test, 1 ml of DPPH and 1 ml of methanol were added in the same manner as in the control group (Table 2), and 1 ml of methanol and 1 ml of the sample were added to obtain the respective color correction values for the sample and the control. SC ₅₀ is the concentration of the sample required to remove 50% of the free radicals, and the smaller the value, the higher the antioxidant activity.

In the active oxygen scavenging test, the change of absorbance by the oxidation of nitroblue tetrazolium (NBT) by reactive oxygen species using active oxygen generation by enzyme reaction of xanthine / xanthine oxidase (Sigma) By measuring, it is possible to know the scavenging ability of the active oxygen.

0.1 ml of sodium carbonate (Na ₂ CO ₃ ), 0.1 ml of xanthine (Sigma), 0.1 ml of ethylenediamine tetraacetic acid (EDTA), 0.1 ml of bovine serum albumin (BSA, Sigma) And 0.1 ml of the sample were placed in a vortex mixer (Type 37600 Mixer, Mini Mix, USA) and allowed to stand for 10 minutes at 25 ° C. After vortexing, 0.1 ml of xanthine oxidase was added, The reaction was stopped by adding 6 mM CuCl ₂ , and the absorbance was measured at a wavelength of 540 nm using a microplate reader (Table 2).

In the active oxygen scavenging activity test, the control group was measured in the same manner by adding tertiary distilled water instead of the sample solution. .

As a result, the obtained lemon verbena extract containing a high content of actioside showed an antioxidant capacity similar to that of L-ascorbic acid, a powerful synthetic antioxidant, and the concentration of 50% of free radical scavenging rate was 6 µg. The concentration of / ㎖, active oxygen removal rate of 50% was 5 ㎍ / ㎖ was confirmed that the antioxidant effect is very excellent at a trace concentration.

[Equation 1]

Free radical scavenging rate (%) = 100 - ((absorbance of sample / absorbance of control) x 100)

&Quot; (2) "

(%) = 100 - ((absorbance of sample / absorbance of control) x 100)

Antioxidant Effect of Lemon Verbena Extract Containing High Content of Actioside Name of sample Antioxidative effect Free radical scavenging rate SC ₅₀ (ug / ml) The active oxygen scavenging rate IC ₅₀ (ug / ml) Sample 5 6 5 Ascorbic acid
(L-ascorbic acid) 5 7

Test Example  3: Tairosineiz  Active inhibition test

In order to assay the whitening effect of lemon verbena extract containing a high content of actioside obtained in Example (Sample 5) of the present invention, the degree of inhibition of the function of an enzyme called tyrosinase was evaluated. Tyrosinase is an enzyme that helps the production of melanin by promoting the oxidation process of tyrosine in vivo. Melanin is a black polymer, which melanin is formed in the body by oxidation as described above. When melanin is formed in the upper part of the skin, the skin turns black and produces spots and freckles. (Pomerantz S. H., J. Biochem., 24: 161-168, 1966) was used to measure the degree of inhibition of the action of tyrosinase and to test the whitening effect.

0.9 ml of sample, 1.0 ml of 0.1 M phosphate buffer (pH 6.8) and 1.0 ml of 1.5 mM L-tyrosine solution were added, and the mixture was maintained at 37 ° C. for 10 minutes. After 0.1 ml of mushroom tyrosinase (1,500 units / ml) was added and reacted at 37 ° C. for 10 minutes, the absorbance was measured at 475 nm using a UV-bisspectrophotometer (Smartspec Plus, Biorad, USA). Inhibition rate for cinease was measured (Table 3).

The control group for the tyrosinase activity inhibition test was measured in the same manner by adding a buffer solution instead of the sample solution, and was set to the case where each color correction value was obtained for the sample and the control group by adding the buffer solution instead of the tyrosinase. , The sample was set as a comparison group lemon arvena extract containing a high content of actioside obtained in Example 5 (sample 5) and arbutin (arbutin synthetic, Sigma) known to have excellent whitening effect, The tyrosinase inhibition rate was numerically calculated using. IC ₅₀ is the concentration of the sample required to inhibit tyrosinase activity by 50%, and the smaller the value, the higher the inhibition rate.

As a result, the obtained lemon verbena extract containing high content of actioside showed a similar whitening effect to arbutin, which is known to have excellent whitening effect, and the concentration of 50% of tyrosinase activity inhibition rate was 0.031 mg. It was confirmed that the whitening effect was excellent at a trace concentration of / ml.

&Quot; (3) "

Inhibition rate (%) = 100 - ((absorbance of sample / absorbance of control group) x 100)

Inhibition of Tyrosinase Activity of Lemon Verbena Extract Containing High Content of Actioside Name of sample Tyrosinase activity inhibition rate, IC ₅₀ (mg / ml) Sample 5 0.031 Arbutin 0.032

Test Example  4: B16F1 Melanocyte  Experiment to measure the inhibitory effect of melanin formation

In order to assay the whitening effect of lemon verbena extract containing a high content of actioside obtained in Example 5 (Sample 5) of the present invention, the degree of inhibition of melanin production against B16F1 melanosite was measured as follows.

The B16F1 melanocyte used is a cell strain derived from a mouse, and is a cell that secretes a melanin pigment called melanin. These cells were treated with a sample to compare the degree of decrease of melanin pigment. The B16F1 melanocyte used in this test example was purchased from ATCC (American Type Culture Collection, Accession No. 6323).

The melanin biosynthesis inhibitory effect of B16F1 melanocyte was measured as follows. B16F1 melanocytes were dispensed in a ⁶ -well plate at a concentration of 2x10 < ⁶ > cells per well. After attaching the cells, the samples were treated at a concentration not causing toxicity and cultured for 72 hours. After 72 hours of incubation, the cells were separated with trypsin-EDTA, the number of cells was measured, and the cells were recovered by centrifugation. The intracellular melanin was quantitatively determined by Lotan: Cancer. Res . , 40: 3345-3350, 1980). The cell pellet was washed once with PBS, and 1 ml of a homogenization buffer solution (50 mM sodium phosphate, pH 6.8, 1% Triton X-100, 2 mM PMSF) was added and vortexed for 5 minutes to disrupt the cells. After dissolving the extracted melanin by adding 10% DMSO sodium hydroxide (1N NaOH) to the cell filtrate obtained by centrifugation (3,000 rpm, 10 minutes), the absorbance of melanin was measured at 405 nm using a microplate reader, and the melanin was quantified. Melanin production inhibition rate of (%) was measured. Melanogenesis inhibition rate (%) of B16F1 melanoma site was calculated by the following equation 6, IC ₅₀ value is the concentration of a substance that inhibits melanin production by 50%.

As a result of testing the melanin production inhibitory effect of B16F1 melanocytes, the IC ₅₀ of Example 5 (Sample 5) was 0.08%, it was confirmed that exhibits a similar or superior effect to the conventional whitening agent arbutin, oil-soluble licorice extract and the like.

&Quot; (4) "

Inhibition rate (%) = [(A-B) / A] x 100

A: Amount of melanin in wells to which no sample was added

B: Amount of melanin in the well to which the sample was added

Inhibitory Effect of Lemon Verbena Extract Containing High Content of Actioside on Melanin Production Name of sample Treatment concentration (%) Melanin formation inhibitory effect (IC ₅₀ ) Sample 5 0.1 0.08% Arbutin 0.1 0.21% Usefulness Licorice extract 0.1 0.09%

Test Example  5: Ella Stays  Active inhibition test

In order to test the skin wrinkle inhibition and improvement effect of lemon verbena extract containing a high content of actioside obtained in Example 5 (Sample 5) of the present invention, an elastase inhibition activity test was conducted. It was. Elastin is a constituent of the matrix layer in the dermis of the skin. As the skin ages, elastin breaks down and the matrix layer in the dermis breaks down and wrinkles occur. (Ala) 3 p-nitroaniline (N-succinyl- (Ala)), which is an elastase substrate, is used as a method for measuring the activity of Elastase, an enzyme that decomposes elastin, 3 p-nitroaniline, Sigma) was used to measure the change in color caused by decomposition of p-nitroaniline by measuring absorbance at a wavelength of 405 nm.

The control group for measuring the elastase activity was measured in the same manner by adding the third distilled water instead of the sample, the case was obtained by adding the third distilled water instead of the enzyme solution to obtain a color correction value for each, the sample is Example 5 (sample Lemon Verbena extract containing a high content of actioside obtained in 5) and retinol (retinol,> 99%, Sigma, USA), which are known to have excellent anti-wrinkle effect, were compared and tested as a positive control group. Elastase inhibition rate was calculated numerically using 5. The IC ₅₀ is the concentration of the sample required to inhibit the enzyme activity by 50%, and the lower the value, the higher the inhibition rate.

As a result, the concentration of 50% of the elastase activity inhibition rate of the lemon verbena extract containing the high content of actioside was 0.20 mg / ml, which inhibited elastase more than 0.38 mg / ml of retinol, which is known to be excellent in wrinkle improvement. Highly effective, it was confirmed that the excellent wrinkle improvement effect of lemon verbena extract containing a high content of actioside.

&Quot; (5) "

Elastase activity inhibition rate (%) = 100 - ((absorbance of sample) / absorbance of control group) x 100)

Inhibition of Elastase Activity of Lemon Verbena Extract Containing High Content of Actioside Name of sample Elastase activity inhibition rate, IC ₅₀ (mg / ml) Sample 5 0.20 Retinol 0.38

Test Example  6: Collagen synthesis effect test

Collagen synthesis ability was measured to assay the anti-wrinkle effect of lemon verbena extract containing a high content of actioside obtained in Example 5 (Sample 5) of the present invention. Collagen is a constituent component of the dermal matrix layer in the skin together with the elastin, and the skin gradually ages, and the components constituting the matrix layer in the dermis are decomposed to form wrinkles. The collagen is a constituent of the dermal matrix layer By confirming the synthesis effect, the effect of improving the skin wrinkles can be confirmed.

Human normal dermal fibroblasts were inoculated into 24-well microplates (1 x 10 ⁵ cells / well) and incubated for 24 hours at 37 ° C in a 5% CO ₂ incubator. Each sample was incubated in serum-free DMEM medium for 24 hours, and collagen synthesis was performed by enzyme immunoassay.

  The medium cultured for 24 hours was dispensed in a 96-well microplate and coated overnight at 4 占 폚. After washing three times with wash buffer (PBS-T; 0.05% Tween 20 in phosphate buffered saline), blocking solution (5% skin milk, Fluka) was added and blocked for 1 hour at 37 ° C. The blocking solution was discarded and washed three times with washing buffer. The primary antibody (rabbit anti-collagen type I, Sigma) was diluted in PBS-T and added at 100 μl each. After washing three times with washing buffer, 100 μl of secondary antibody (anti-rabbit IgG alkaline phosphatase conjugate, Sigma) was diluted in PBS-T and reacted at 37 ° C for 2 hours. After washing three times with washing buffer, 100 μl of alkaline phosphatase substrate solution (Sigma) was added, and the solution was developed at 25 ° C. The absorbance was measured at 405 nm using a microplate reader (Table 6).

The control group for measuring the collagen synthesis effect was a reaction absorbance of a cell culture solution not treated with the sample. To determine the collagen synthesis effect, ascorbic acid was set as a comparative group, and the results were averaged three times. The collagen synthesis effect was numerically analyzed using the following equation (4). As a result, it was confirmed that the lemon verbena extract containing the high content of actioside obtained in Example 5 exhibited an excellent effect similar to ascorbic acid (vitamin-C) having excellent collagen synthesis effect.

&Quot; (6) "

Collagen synthesis effect (%) = 100 + ((absorbance of control group - absorbance of cell culture liquid of sample) / absorbance of control group) x100

Collagen Synthesis Effect of Lemon Verbena Extract Containing High Content of Actioside The concentration of the sample (占 퐂 / ml) 0 One 10 100 Sample 5 100.00 111.59 122.84 133.21 Ascorbic acid (vitamin-C) 100.00 108.45 123.57 131.58

Test Example  7: After UV irradiation MMP -1 expression inhibition evaluation

The enzyme immunoassay (ELISA) was performed to measure the MMP-1 concentration after UV irradiation and sample addition of the lemon verbena extract containing the high content of actioside obtained in Example 5 (Sample 5) of the present invention.

UVA is irradiated to human dermal fibroblasts at an energy of 5 J / cm < ² > using a UV chamber. Ultraviolet irradiation dose and incubation time were established through the preliminary experiment to maximize the amount of MMP expression in fibroblasts. Negative control group was wrapped in silver foil and kept in UVA environment for the same time. UVA emission was measured using a UV radiometer. Cells under UVA irradiation were irradiated with UVA and then replaced with a medium containing the sample. The cells were cultured for 24 hours, and the medium was recovered and coated on a 96-well microplate. The primary antibody (MMP-1 (Ab-5) monoclonal antibody, MMP-2 (Ab-3) monoclonal antibody) was treated and reacted at 37 ° C for 60 minutes. The secondary antibody, anti mouse IgG phosphatase conjugated) was reacted again for 60 minutes. Then, the alkaline phosphatase substrate solution (1 mg / ml-nitrophenyl phosphate in diethanolamine buffer solution) was reacted at room temperature for 30 minutes and absorbance was measured at 405 nm with a microplate reader. As a control group, a sample to which no sample was added was used.

Example 5 (Sample 5) showed more than 48% inhibition rate compared to the control group did not process the MMP-1 expression induced during UV irradiation, which was superior to the inhibition rate of the retinol used as a control.

Inhibition of MMP-1 Expression in Lemon Verbena Extracts Containing High Content of Actiosides Name of sample Treatment concentration (%) MMP-1 expression inhibition rate (%) Sample 5 0.1 48 Retinol 0.1 28

Test Example  8: Cytotoxicity mitigation effect by ultraviolet irradiation

In order to evaluate the alleviation effect of cytotoxicity by ultraviolet irradiation of the lemon verbena extract containing a high content of actioside obtained in Example 5 (Sample 5) of the present invention, the experiment was carried out as follows.

Fibroblasts were placed in 24-well test plates at 1 × 10 ⁵ cells and adhered for 24 hours. Each well was washed once with PBS and 500 占 퐇 of PBS was added to each well. The fibroblasts were irradiated with UV 10mJ / cm 2 using an ultraviolet B (UVB) lamp (Model: F15T8, UV B 15W, Sankyo Dennki, Japan), after which PBS was removed and cell culture medium (10% FBS was added to DMEM). 1 ml) was added thereto, followed by treatment with lemon verbena extract containing actioside, followed by incubation for 24 hours. After 24 hours, the medium was removed, and cell culture medium (500 μl) and MTT solution (2.5 mg / ml) (60 μl) were added to each well, followed by incubation for 2 hours at 37 ° C in a CO ₂ incubator. The medium was removed and 500 μl of isopropanol-HCl (0.04 N) was added. The cells were shaken for 5 minutes to dissolve the cells. 100 μl of the supernatant was transferred to a 96-well test plate, absorbance was measured at 565 nm by a microplate reader, and cell viability (%) and cytotoxic relaxation rate by ultraviolet were measured. As a result, the lemon verbena extract containing actioside showed a cytotoxic alleviation rate of 41% at a concentration of 0.1%.

[Equation 7]

Cell survival rate (%) = [(St-Bo) / (Bt-Bo)] X 100

Bo: Absorbance at 565 nm of the well of the cell culture medium alone

Bt: Absorbance at 565 nm of a well that underwent chromogenic reaction in a sample not treated with the sample

St: absorbance at 565 nm of wells treated with the sample

[Equation 8]

(%) = [1- (St-Bo) / (Bt-Bo)] X 100

Bo: Cell survival rate of wells not irradiated with ultraviolet light and not treated with sample

Bt: Cell viability of wells irradiated with ultraviolet light and not treated with the sample

St: Cell viability of well treated with ultraviolet light and treated with sample

Cytotoxicity Reduction of Lemon Verbena Extract Containing High Content of Actioside Name of sample Treatment concentration (%) Cytotoxic Relaxation Rate (%) Sample 5 0.1 41

Test Example  9: Inhibitory effect of inflammatory cytokine expression by ultraviolet irradiation

In order to evaluate the inhibitory effect of inflammatory cytokine expression expressed by ultraviolet irradiation of lemon verbena extract containing a high content of actioside obtained in Example 5 (Sample 5) of the present invention, it was isolated from human epidermal tissue. Fibroblasts (fibroblasts) were placed in a 24-well test plate each 5x10 ⁴ and attached for 24 hours. Each well was washed once with PBS and 500 占 퐇 of PBS was added to each well. After irradiating the fibroblasts with 10 mJ / cm ² of ultraviolet light using an ultraviolet B (UVB) lamp (Model: F15T8, UVB 15W, Sankyo Dennki, Japan), PBS was removed and the cell culture medium ≪ / RTI > medium). After treatment with lemon verbena extract containing a high content of actioside to be evaluated and incubated for 5 hours. 150 [mu] l of the culture supernatant was taken to quantitate IL-l [alpha] to determine the effect of inhibiting the inflammatory cytokine expression of the sample. The amount of IL-1 alpha was quantitated using an enzyme-linked immunosorbent assay, and the production rate of IL-1 alpha was calculated by the following equation (9). As a result, Example 5 (Sample 5) inhibited the production of inflammatory cytokines, IL-1α, by UV at 41% at 41% concentration, thereby effectively preventing the inflammation caused by UV at low concentrations.

&Quot; (9) "

Inhibitory rate of inflammatory cytokine expression (%) = [1- (St-Bo) / (Bt-Bo)] X 100

Bo: IL-1? Production in wells without UV irradiation

Bt: IL-1? Production in wells irradiated with ultraviolet light and not treated with the sample

St: IL-1α production in wells irradiated with ultraviolet light and treated with the sample

Inhibitory Rate of Inflammatory Cytokine Expression of Lemon Verbena Extract Containing High Content of Actioside Sample treatment concentration (%) Inhibitory rate of inflammatory cytokine expression (%) 0.05 25 0.1 41

Test Example  10: Antibacterial effect

As a test for antimicrobial activity against lemon verbena extract containing a high content of actioside obtained in Example 5 (Sample 5) of the present invention, agar serial dilution method and paper disc method Antibacterial test was carried out by. The test strains were distributed from the Korea Research Institute of Bioscience and Biotechnology. Staphylococcus aureus KCTC 6910), gram-negative bacteria such as Pseudomonas aeruginosa KCTC 1637), Escherichia coli (E. coli KCTC 1039), a yeast Candida yeast (Candida albicans KCTC 7965), black Aspergillus (Aspergillus fungi as niger KCTC 6910) were used.

In order to measure the antimicrobial activity of the lemon verbena extract containing the actioside was measured the minimum inhibitory concentration using a solid culture dilution method (Table 10).

Inoculated with tryptic bacteria in culture medium at 37 ℃ for 24 hours pre-incubation, in the case of yeast, inoculated bacteria in potato dextrose medium and pre-cultured at 25 ℃ for 2 days, filamentous fungus was in potato dextrose agar medium After inoculating the bacteria and pre-cultivation in a 25 ℃ incubator for 7 days, using a smear rod, the spores of filamentous fungi formed on the surface of the medium was recovered and diluted with sterile saline.

2 ml of each extract diluted in appropriate concentration in 5% dimethyl sulfoxide (DMSO) saline solution for each sample and experimental strain was added to the sterilized petri dish, and the control group diluted to the appropriate concentration in 5% saline solution. 2 ml of each sample was added, and 2 ml of 5% DMSO physiological saline solution was added to the control group, and 18 ml of agar medium and potato dextrose agar medium were sterilized in each petri dish and cooled to 48 ° C. After stirring, it is left to solidify.

Each of the pre-cultured bacteria was applied to each petri dish at a final concentration of about 1 × 10 ⁶ CFU / ml in the case of bacteria, 1 × 10 ⁵ CFU / ml for yeast, about 1 Spread each petri dish at a bacterial concentration of X 10 ⁴ CFU / ml. Each petri dish was incubated for 24 hours at 37 ° C, yeast for 3 days at 25 ° C, and filamentous fungi for 7 days at 25 ° C incubator, followed by colony formation in each compartment. The sample concentration is the minimum inhibitory concentration (MIC), and the smaller the concentration, the higher the antimicrobial effect. As a result, lemon verbena extract containing actioside showed excellent antimicrobial effect against all four kinds of bacteria, yeast and filamentous fungi.

In addition, in order to assay the antimicrobial effect of the lemon verbena extract containing a high content of actioside obtained in Example 5 (Sample 5) was carried out by the circular filter method as follows. One liter of each strain cultured on a plate medium was inoculated into 10 ml of the liquid medium and incubated for 6 hours in a liquid medium of 10 ml, followed by incubation for 6 hours. ⁷ cfu / ml and sterilized with a sterilized donor rod. Sterilized paper discs (6 mm, Satorius, Germany) were placed on a solid plate medium, and the samples dissolved in the solvent were absorbed to 0.05 ml / disk and cultured. Staphylococcus aureus ), gram-negative bacteria such as Pseudomonas aeruginosa), Escherichia coli (E. Coli) is 37 ℃, the type of fungi Candida yeast (Candida albicans ). Aspergillus niger ( Aspergillus niger ) was cultured at 27 ℃ for 24 hours and 120 hours, respectively, and the diameter of the transparent zone around the disc was measured. Methylparaben, a strong antimicrobial active antibiotic, was used as a comparative group. The transparent zone around the disc was an indicator of the degree of proliferation inhibition of the strain. The larger the diameter, the higher the antimicrobial activity against the strain do. As a result, it was confirmed that lemon verbena extract containing actioside showed superior antimicrobial activity against four kinds of bacteria, yeast and filamentous fungus than methylparaben, which is known as a powerful synthetic preservative.

Antibacterial Effect of Lemon Verbena Extract Containing High Content of Actioside Strain Extraction method Minimum inhibitory concentration (MIC, 占 퐂 / ml) S. aureus Sample 5 50 P. aeruginosa Sample 5 20 E. coli Sample 5 10 C. albicans Sample 5 110 A. niger Sample 5 140

Antibacterial Effect of Lemon Verbena Extract Containing High Content of Actioside Strain Extraction method Transparent zone diameter (mm) S. aureus Sample 5 13 Methylparaben 11 P. aeruginosa Sample 5 15 Methylparaben 12 E. coli Sample 5 24 Methylparaben 18 C. albicans Sample 5 17 Methylparaben 13 A. niger Sample 5 25 Methylparaben 24

Test Example  10: UV protection effect

UV protection effect assay test for lemon verbena extract containing a high content of actioside obtained in Example 5 (Sample 5) of the present invention in UV spectrophotometer was measured using an in vitro SPF analyzer (Optometrics SPF-290S). To evaluate the positive control group octyl methoxycinnamate (OMC), an organic sunscreen most commonly used in sunscreen cosmetics. The results are shown in Table 12 (FIG. 4). As a result, the lemon verbena extract containing a high content of actioside was able to confirm the excellent UV blocking ability (SPF, PA value) than OMC, a powerful synthetic organic sunscreen.

In vitro SPF Measurement Method of Lemon Verbena Extract Containing High Content of Actioside from Lemon Verbena system SPF-290S Analyzer (Optomerics, USA) temperament Transpore tape (3M, USA) Dimensions US FDA Wavelength range 290 to 400 nm Sample loading 2 mg / cm 2 Number of scans 6, Legacy

Formulation Example 1 and Formulation Comparative Example 1: A high content of Lemon Verbena side containing thiosulfate solution Ingredient Composition of Nutritional Cream Including Extract

Component composition of the nutrition cream containing lemon verbena extract containing a high content of actioside according to Example 5 (Sample 5) was prepared as shown in Table 13.

Nutritional Cream Containing Lemon Verbena Extract Containing High Content Actioside number ingredient Formulation Example 1 Formulation Comparative Example 1 One Pro-type glycerin monostearate 2.0 2.0 2 Stearic acid 1.5 1.5 3 Cetearyl alcohol 2.2 2.2 4 Wax 1.0 1.0 5 Squalane 3.0 3.0 6 Hardened vegetable oil 1.0 1.0 7 Sorbitan stearate 0.6 0.6 8 Mineral oil 5.0 5.0 9 Polysorbate 60 1.5 1.5 10 Dimethicone 1.0 1.0 11 Trioctanoin 5.0 5.0 12 Betaine 3.0 3.0 13 Triethanolamine 1.0 1.0 14 glycerin 5.0 5.0 15 Sodium hyaluronate 3.0 3.0 16 Sample 5 1.0 - 17 Distilled water Balance Balance 18 Preservative, fragrance, pigment a very small amount a very small amount

The aqueous components of the raw materials 12 to 15 and 17 were completely dissolved by heating to 80 DEG C and then the raw materials 1 to 11 and 16 were heated to 80 DEG C to obtain the raw materials 12 to 15 and 17 (Homo Mixer Mark II, Primix, Japan) and emulsified at 3,000 rpm for 15 minutes. Thereafter, the raw material 18 was added, stirred for 5 minutes, and then cooled to room temperature.

Test Example  11: Application of whitening effect of formulation

The whitening effect of the nutritional cream prepared in Formulation Example 1 and Comparative Formulation Example 1 was measured by the following method. 30 women aged 25 years or older who had spots, freckles and pigmentosis were allowed to use the nutritional creams of Formulation Example 1 and Comparative Example 1 for 12 weeks. Changes in skin color were measured with a chroma meter (CR-410, Minolta , Japan) was used to measure the change in color brightness (? L) (Table 14). The average value of 30 persons was calculated. The higher the brightness change value, the higher the whitening effect. As a result, the nutrition cream of Formulation Example 1 to which the lemon verbena extract containing the high content of actioside was added was compared with the nutrition cream of Formulation Comparative Example 1 to which no addition was added, and showed an excellent whitening effect.

Measuring whitening effect of formulation medium Formulation Example 1 Formulation Comparative Example 1 Skin color brightness change (L) 5.74 0.22

Test Example  12: Measurement of wrinkle improvement effect

The wrinkle-reducing effect of the nutritional cream prepared in Formulation Example 1 and Formulation Comparative Example 1 of Formulation Example 1 was measured by the following method. Ten women aged 30 or older who had undergone skin aging were allowed to use the nutritional cream of Formulation Example 1 and Comparative Example 1 for 12 weeks on the crow feets region, and after 4 weeks, 8 weeks, 12 weeks The depth of the wrinkles (㎛) was measured using a skin visometer (SV-600, C + K, Germany) with an image analysis of the eye wrinkle template at intervals. As a result, the nutrition cream of Formulation Example 1 to which the lemon verbena extract containing a high content of actioside was added was compared with the nutrition cream of Formulation Comparative Example 1 without the addition, and confirmed an excellent wrinkle improvement effect.

Measuring wrinkle improvement effect of formulation medium Formulation Example 1 Formulation Comparative Example 1 Before application 337 ± 10 326 ± 12 12 weeks after application 260 ± 11 320 ± 22 Wrinkle depth reduction rate (%) 22.8 1.8

Test Example  13: Application of atopy

In order to measure the atopic improvement effect of the nutritional cream prepared in Formulation Example 1 and Comparative Formulation Example 1, acetone was applied as a skin barrier damaging method. When the transepidermal water loss (TEWL) reached 4.0 g / m ² / h by dispensing acetone into the aliquots of 8-12 week old reared mice, the nutritional cream of Formulation Example 1 and Comparative Example 1 was applied Respectively. TEWL was measured with a Tewameter 210 evaporator from C + K (Cologne, Germany). After 6 hours of application, TEWL was measured and the degree of reduction of TEWL was measured to evaluate the degree of reduction of TEWL. As a result, the nutritional cream of Formulation Example 1 to which the lemon verbena extract containing a high content of actioside was added was increased compared to the nutritional cream of Formulation Comparative Example 1, which did not contain the effect of recovery after skin barrier damage. It was confirmed.

&Quot; (10) "

B ( _t ) = (1- (B _{t = 6} - B _{t = 0} ) / (B _{t = d} - B _{t = 0} )

B _{t = 6} : TEWL measurement after 6 hours after skin barrier damage

B _{t = 0} : TEWL measured before skin barrier damage

B _{t = d} : TEWL measured immediately after skin barrier damage

Atopy improvement effect analysis
Recovery rate (%) medium Formulation Example 1 Formulation Comparative Example 1 6 hour recovery rate
(Based on initial TEWL) 43 12

Formulation Example 2: high content of The liquid constituents of the screen enteric composition comprising the lemon verbena extract containing the thio side

2-1: Softening longevity

Components of softening longevity number ingredient content(%) One glycerin 5.00 2 1,3-butylene glycol 3.00 3 PEG 1500 1.00 4 Allantoin 0.10 5 DL-Panthenol 0.30 6 EDTA-2Na 0.02 7 Benzophenone-9 0.04 8 ethanol 10.00 9 Octidodeces-16 0.20 10 Polysorbate 20 0.20 11 Sample 5 5.0 12 Preservative, fragrance, pigment a very small amount 13 Distilled water Balance

2-2: convergent lotion

Ingredients of convergent lotion number ingredient content(%) One glycerin 2.00 2 1,3-butylene glycol 2.00 3 Allantoin 0.10 4 DL-Panthenol 0.30 5 EDTA-2Na 0.02 6 Benzophenone-9 0.04 7 ethanol 15.00 8 Polysorbate 20 0.20 9 Sample 5 1.0 10 Citric acid a very small amount 11 Preservative, fragrance, pigment a very small amount 12 Distilled water Balance

2-3: Nourishing Lotion

Ingredients of nutrition lotion number ingredient content(%) One Cetearyl alcohol 1.00 2 Glyceryl stearate 0.50 3 Polysorbate 60 1.00 4 Sorbitan sesquioleate 0.30 5 Cetyl octanoate 6.00 6 Squalane 4.00 7 Shapower Oil 4.00 8 Butylene glycol 4.00 9 glycerin 4.00 10 Carbomer 0.10 11 Triethanolamine 0.10 12 Sample 5 1.00 13 Preservative, fragrance, pigment a very small amount 14 Distilled water Balance

2-4: Essence

Ingredients of Essence number ingredient content(%) One glycerin 10.00 2 Betaine 5.00 3 PEG 1500 2.00 4 Allantoin 0.10 5 DL-Panthenol 0.30 6 EDTA-2Na 0.02 7 Benzophenone-9 0.04 8 Hydroxyethylcellulose 0.10 9 Carboxyvinyl polymer 0.20 10 Triethanolamine 0.18 11 Octyldodecanol 0.30 12 Octyldodec-16 0.40 13 ethanol 6.00 14 Sample 5 1.00 15 Preservative, fragrance, pigment a very small amount 16 Distilled water Balance

2-5: Pack

Components of the pack number ingredient content(%) One Polyvinyl alcohol 15.00 2 Cellulose sword 0.15 3 glycerin 3.00 4 PEG 1500 2.00 5 Sheik Destrin 0.15 6 DL-Panthenol 0.30 7 Allantoin 0.10 8 Monoammonium glycyrrhizin acid 0.20 9 Nicotinamide 0.40 10 ethanol 5.00 11 PEG 40 hardened castor oil 0.30 12 Sample 5 1.00 13 Preservative, fragrance, pigment a very small amount 14 Distilled water Balance

As described above, specific parts of the present disclosure have been described in detail, and for those skilled in the art, these specific descriptions are merely preferred embodiments, and the scope of the present disclosure is not limited thereto. Will be obvious. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Claims (9)

Method for preparing lemon verbena extract containing actiosides comprising the following steps:
(a) adding a solvent to lemon verbena to extract actioside, and then concentrating;
(b) adding at least two solvents selected from the group consisting of water, ethanol, methanol, butanol, ethyl acetate, ethyl ether, chloroform and hexane to the concentrated extract of step (a) to obtain an actioside fraction; ;
(c) removing the organic solvent of step (b) and secondarily concentrating the organic solvent to prepare a fractionally concentrated powder;
(d) solubilizing the fractional concentrated powder of step (c) in a solvent, and then removing the pigment and chlorophyll using a column;
(e) recrystallizing by adjusting the pressure and temperature to the extractant from which the dye and chlorophyll have been removed, and
(f) spray drying or lyophilizing the pellet precipitated through the recrystallization step to obtain a powdered actioside.
The method of claim 1, wherein the solvent of step (a) is at least one solvent selected from the group consisting of water, ethanol, methanol, butanol, ethyl acetate, ethyl ether, chloroform and hexane.
The method of claim 1, wherein the fraction comprising the actioside obtained in step (b) is an ethyl acetate fraction.
2. The method of claim 1, wherein the column of step (d) is selected from the group consisting of Amberlite XAD resin, Dowx resin, Suferite DAX resin, Diaion HP-10, HP-20, HP-30, HP- ≪ / RTI > wherein the resin comprises at least one resin selected from the group consisting of: < RTI ID = 0.0 >
The method of claim 1, wherein the step (e) is characterized in that the recrystallization at the conditions of -300 ~ 25 ℃ and 0.5 ~ 800MPa.
It is prepared by the method of any one of claims 1 to 5, characterized in that it has an use selected from the group consisting of antioxidant, wrinkle improvement, whitening, skin damage inhibition or irritation by ultraviolet rays, anti-inflammatory, antibacterial and atopic improvement Lemon verbena extract containing actioside.
A functional cosmetic comprising a lemon verbena extract containing the actioside of claim 6 as an active ingredient and selected from the group consisting of anti-aging, whitening and anti-irritant for skin damage or stimulation. Composition.
The functional cosmetic composition according to claim 7, wherein the functional cosmetic composition is selected from the group consisting of lotion, gel, water-soluble liquid, cream, essence, oil-in-water (O / W) type and water-in-oil (W / O) type.
A skin external composition comprising the lemon verbena extract containing the actioside of claim 6 as an active ingredient, and having a use selected from the group consisting of anti-inflammatory, antibacterial and atopy improvement.

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