KR101289432B1 - Extract containing polyssacharide derived from lippia citriodora and cosmetic composition containing the same - Google Patents

Abstract

PURPOSE: A Lippia citriodora extract containing polysaccharides is provided to be used as a cosmetic composition with excellent antioxidative, whitening, anti-wrinkling, anti-inflammatory, anti-bacterial, and atopic dermatitis therapeutic effects. CONSTITUTION: A method for preparing a Lippia citriodora extract containing polysaccharides comprises the steps of: adding ethanol to Lippia citriodora and eluting pigments and impurities; obtaining and drying Lippia citriodora cake; adding water to the dried Lippia citriodora cake and obtaining a polysaccharide fraction; removing remaining pigments of the polysaccharide fraction; adding ethanol and precipitating the extract; and spray-drying and decompression-drying the prepared pellet and obtaining the Lippia citriodora extract of a powder type containing polysaccharides. A cosmetic composition contains the Lippia citriodora extract as an active ingredient. [Reference numerals] (AA) Primary extraction step of eluting impurities from Lippia citriodora; (BB) Step of drying eluted cake by reduced pressure; (CC) Secondary extraction step of extracting remaining cake; (DD) Precipitation step of adding ethanol to an extracted liquid; (EE) Centrifuging step of isolating precipitated pellet; (FF) Step of collecting the pellet

Description

Extract extract containing lemon verbena-derived polysaccharide and cosmetic composition comprising same {Extract Containing Polyssacharide Derived from Lippia citriodora and Cosmetic Composition Containing the Same}

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

Human skin has various physicochemical changes in the aging process, and the causes are largely divided into internal aging and photo-aging. Free radicals may be activated by UV rays, stress, disease conditions, environmental factors, wounds, and age. If this condition is exacerbated, it destroys the antioxidant defenses in vivo and damages cells and tissues. 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. In particular, the oxidation of proteins is caused by severe cuts in the skin's connective tissues such as collagen, hyaluronic acid, elastin, proteoglycan and fibronectin, resulting in severe over-inflammatory reactions and skin elasticity. If this is exacerbated, mutations in the DNA can lead to mutations, cancer and reduced immune function. 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.

 Aging involves not only free radicals, but also an enzyme called MMP. In vivo, the synthesis and degradation of extracellular substrates such as collagen is properly regulated, but the synthesis decreases as aging progresses, and the substrate metalloproteinase, an enzyme that degrades collagen ( MMP) expression is promoted, the elasticity of the skin is reduced, wrinkles are formed. Ultraviolet irradiation also activates these degrading enzymes.

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 more than the effective concentration in cosmetics or pharmaceuticals in terms of safety, the possibility of discoloration stability, and does not produce a satisfactory effect.

Polysaccharides (polyssachride) is a state in which not only glucose but also galactose, mannose, rhamnose, aribinose, and xylose are combined with proteins, and antitumor activity, cancer metastasis inhibitory effect and the effect of promoting the activity of the medicinal system Kim Hwan-Mook et al ., Archives of Pharmacol Research , 23 (3): 240, 2000) Unlike polyimmune anticancer drugs, polysaccharides are characterized by physiological activity by enhancing immune activity rather than directly attacking cancer cells. Polysaccharides produced to date are commonly extracted from plants such as barley, ginseng, mugwort and ginkgo, chlorella and some seaweeds, and most of the polysaccharides currently used are extracted and produced from edible or medicinal mushrooms. The method of extracting polysaccharides is obtained by hydrothermal extraction of fruiting bodies or mycelium. This technical method has problems of supply and demand of raw materials due to the limitation of the production of fruiting bodies, and it is difficult to obtain a large amount when extracting polysaccharides from mycelium. There is this.

Polysaccharide is used as a cosmetic composition because of its anti-aging, moisturizing, antioxidant and anti-inflammatory effects, as well as immunomodulatory effects. Disclosed is a description of a cosmetic composition containing polysaccharides isolated from various plants. (Korean Unexamined Patent Publication Nos. 2011-0060003, 2009-0098083, 2010-0010132, 2012-0058798, and Korean Registered Patent No. 0407455).

On the other hand, lemon verbena ( lippia citriodora ), also called deodorant (防臭 木), is a deciduous herb of the dicotyledon moth plant, native to South America. 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. 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. Biological activity has been reported (Masateru Ono meat al ., J Nat Med , 62: 101, 2008; Esam Qnais et al ., Jordan Journal of Biological Sciences , 2 (4): 167, 2009; Lorena Funes et al., Eur. J. Appl. Physiol., 111: 695, 2011), none of the prior art discloses lemon verbena extracts containing polysaccharides using lemon verbena and 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 polysaccharide and the extract has an antioxidant effect, The present invention was completed by confirming that it has an effect such as a whitening effect, an anti-wrinkle effect, an anti-inflammatory effect, an antibacterial effect, an atopy improvement effect, and an ultraviolet protection effect.

SUMMARY OF THE INVENTION An object of the present invention is to provide a lemon verbena extract containing a polysaccharide excellent in antioxidant effect, whitening effect, wrinkle improvement effect, anti-inflammatory effect, antibacterial effect, atopy improvement effect and UV protection effect, and a cosmetic composition comprising the same There is.

In order to achieve the above object, the present invention comprises the steps of (a) adding a solvent to the lemon verbena, eluting the pigment and impurities, filtering, and then obtaining and drying the remaining lemon verbena foil; (b) adding at least one solvent selected from the group consisting of water, ethanol, methanol, butanol, ethyl acetate, ethyl ether, chloroform and hexane to the dried lemon verbena foil of step (a) to obtain a polysaccharide fraction Making; (c) removing residual pigments of the polysaccharide fraction obtained in step (b); And (d) adding ethanol to the extract from which the residual pigment was removed to precipitate the polysaccharide, and then spraying or drying the resulting pellet to obtain a lemon verbena extract containing the polysaccharide in powder form. It provides a method of producing a lemon verbena extract containing a polysaccharide.

The present invention also provides a lemon verbena extract containing polysaccharide prepared by the above method.

The present invention also provides a cosmetic composition and a topical skin composition comprising the lemon bevena extract containing the polysaccharide as an active ingredient.

Lemon verbena extract containing the polysaccharide of the present invention can be prepared and applied as a cosmetic composition excellent in antioxidant, whitening, anti-wrinkle, anti-inflammatory, antibacterial and atopic improvement.

1 is a flowchart illustrating a method of preparing a lemon verbena extract containing polysaccharide (Flow chart).
Figure 2 is a data analysis of the content of the residual organic acid and impurities using the lemon verbena extract containing polysaccharide using high pressure liquid chromatography (HPLC).

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 accordance with the present invention, the method comprises the steps of: (a) adding a solvent to the lemon verbena, eluting the pigments and impurities, and then filtering and obtaining the remaining lemon verbena foil; (b) adding at least one solvent selected from the group consisting of water, ethanol, methanol, butanol, ethyl acetate, ethyl ether, chloroform and hexane to the dried lemon verbena foil of step (a) to obtain a polysaccharide fraction Making; (c) removing residual pigments of the polysaccharide fraction obtained in step (b); And (d) adding ethanol to the extract from which the residual pigment was removed to precipitate the polysaccharide, and then spraying or drying the resulting pellet to obtain a lemon verbena extract containing the polysaccharide in powder form. It relates to a method for producing lemon verbena extract containing polysaccharide.

In the present invention, the solvent of step (a) may be characterized in that at least one solvent selected from the group consisting of water, ethanol, methanol, butanol, ethyl acetate, ethyl ether, chloroform and hexane.

In the present invention, the removal of the residual pigment in the step (c) may be preferably characterized in that using a column, the column is Amberlite XAD resin, Dow X resin, Superlite DAX resin, Diaion HP-10 It may be characterized in that it comprises at least one resin selected from the group consisting of, HP-20, HP-30, HP-40 and HP-50 resin.

In one embodiment of the present invention, the solvent is added to the lemon verbena through the extraction step to elute the pigments and impurities, water, ethanol, methanol, butanol, ethyl acetate, ethyl ether, chloroform in the proportion of 20 to 99.5% And it adds to solvents, such as hexane, and extracts for 0.5 to 5 hours. The lemon verbena foil in which the pigment and impurities are eluted through the extraction step is subjected to a drying process, and may be applied in various ways such as heat drying, reduced pressure drying, spray drying, and freeze drying.

The lemon verbena foil obtained through the drying process undergoes a secondary extraction step to extract polysaccharides, and the extraction process according to the present invention comprises one or two selected from the group consisting of water, ethanol, methanol and butanol and the like. More than one solvent is used. Lemon verbena extract, which has undergone the second extraction step, uses a column to remove residual pigment, and a resin column using an adsorptive resin is suitable. The resin may be Amberlite XAD resin, Dow X resin, Superlite DAX resin, Diaion HP-10, HP-20, HP-30, HP-40 and HP-50 resin.

In the present invention, the step (d) of the precipitation step may be characterized in that 1 to 10 times (w / w) of ethanol is added to precipitate for 1 to 24 hours at -80 ℃ ~ 25 ℃.

After the column process is subjected to the precipitation process of precipitating the polysaccharide using ethanol, the precipitation process according to the present invention is added 1 ~ 10 times (w / w) of ethanol 1 ~ 24 at -80 ℃ ~ 25 ℃ The precipitation process is carried out for a time. The precipitated polysaccharide is subjected to a step of obtaining a lemon verbena extract containing a polysaccharide in a powder form by spray drying or vacuum drying after the separation process through centrifugation. And it can be used by concentrating by vacuum concentration method, it is possible to obtain a lemon verbena extract containing a powdery high content of polysaccharide by using a spray dryer and lyophilization.

In one embodiment of the present invention, after adding lemon verbena at 10% by weight to 99% ethanol aqueous solution extracted in a reflux extractor equipped with a cooling capacitor for 2 hours at a temperature of 80 ℃, the remaining foil filtered by 80 mesh filter and 1.2㎛ filter Got. The obtained foil was dried at 80 ° C. for 10 hours, distilled water was added at 10% by weight to the obtained powdery dried foil, and extracted at a temperature of 80 ° C. for 2 hours in a reflux extractor equipped with a cooling capacitor. Thereafter, the filtrate was obtained by filtration with an 80 mesh filter and a 1.2 μm filter, and then an HP-20 column was used to remove residual pigments and impurities. A total of 10 fractions were eluted with HP-20 column using distilled water, and fractions 3 to 7 with residual pigment removed were obtained. 99% ethanol was added to the obtained filtrate four times (w / w), and precipitated at -20 ° C for 5 hours. The precipitated pellet was obtained by centrifugation at 8000 rpm for 20 minutes, and then using a vacuum concentrator. Concentration for 1 hour at 65 ° C. and 20 hPa conditions yielded a lemon verbena extract powder containing polysaccharide.

In order to confirm the purity of the lemon verbena extract containing polysaccharide of the present invention, as a result of the detection test of residual organic acid and impurities using HPLC, the lemon verbena extract containing polysaccharide of the present invention is organic acid and other It was confirmed that no impurities were detected (Table 1). In addition, as a result of performing the phenol-sulfate method to measure the total sugar content of the lemon verbena extract containing polysaccharide, the high sugar content of the total sugar content of more than 99% by the manufacturing method of the present invention. It was confirmed that lemon verbena extract containing polysaccharide could be prepared (Table 2).

In another aspect, the present invention relates to a lemon verbena extract containing polysaccharide prepared by the above method.

In the present invention, lemon verbena extract containing polysaccharide has the use selected from the group consisting of antioxidants, wrinkle improvement, whitening, skin damage inhibition or irritation by ultraviolet rays, anti-inflammatory, antibacterial and atopic improvement can do.

As a result of performing a free radical scavenging test and an active oxygen scavenging test in order to confirm the antioxidant effect by the skin aging improvement effect assay test of the lemon saccharide extract containing polysaccharide of the present invention, the lemon verbena extract containing polysaccharide was The antioxidant activity was very similar to that of L-ascorbic acid, and the free radical scavenging rate SC ₅₀ and free radical scavenging rate IC ₅₀ were 8 µg / ml and 10 µg / ml, respectively. It was confirmed that the antioxidant efficacy is excellent (Table 3).

In order to test the whitening effect on the lemon verbena extract containing polysaccharide of the present invention, as a result of evaluating the degree of inhibition of the function of an enzyme called tyrosinase, the lemon verbena extract containing polysaccharide was The whitening effect was almost similar to that of arbutin, which is known to have excellent whitening effect. The concentration of 50% of tyrosinase inhibitory rate was 0.035 mg / ml, and it was confirmed that the whitening effect was excellent at the small amount (Table 4). ). In addition, as a result of assaying the whitening effect of the extract by measuring the degree of inhibition of melanin production against B16F1 melanocytes, the IC ₅₀ of the lemon verbena extract containing polysaccharide was 0.11% compared to the conventional whitening agent arbutin and useful licorice extract. A similar or superior effect was confirmed (Table 5).

In order to test the anti-wrinkle and improvement effect of the lemon verbena extract containing polysaccharide of the present invention, the results of the elastase inhibition activity test (elastase inhibition activity test), the lemon verbena extract containing polysaccharide is wrinkled Compared with retinol, which is known to have an excellent improvement effect, it showed a similar elastase inhibitory effect, and thus an excellent wrinkle improvement effect of lemon verbena extract containing polysaccharide was confirmed (Table 6). In addition, as a result of measuring the collagen synthesis ability to test the skin wrinkle improvement effect of the polysaccharide-containing lemon verbena extract, the lemon verbena extract containing polysaccharide was ascorbic acid (vitamin-C) excellent in collagen synthesis effect Collagen synthesis similar to), it was confirmed that the excellent skin wrinkle suppression and improvement effect of lemon verbena extract containing polysaccharide prepared by the method of the present invention (Table 7).

As a result of performing enzyme-immunoassay (ELISA) to measure MMP-1 concentration after UV irradiation and sample addition of lemon verbena extract containing polysaccharide of the present invention, the lemon verbena extract containing polysaccharide was exposed to ultraviolet radiation. The MMP-1 expression-induced expression showed more than 67% inhibition rate compared to the control group was not treated, it was confirmed to be superior to the inhibition rate of the retinol used as a positive control (Table 8). In addition, as a result of confirming the cytotoxic mitigation effect of the lemon verbena extract containing polysaccharide by UV irradiation, the lemon verbena extract containing polysaccharide showed a 42% cytotoxic alleviation rate at 0.1% concentration. It was found that it effectively prevents cytotoxicity (Table 9), and the results of inhibiting the expression of inflammatory cytokines expressed by ultraviolet irradiation of lemon verbena extract containing polysaccharide were found to be lemon verbena containing polysaccharide. The extract inhibited 55% production of IL-1α, an inflammatory cytokine caused by UV light, at a concentration of 0.1%, and effectively prevented UV-induced inflammation even at low concentrations (Table 10).

The antibacterial effect assay for the lemon verbena extract containing the polysaccharide of the present invention was carried out by agar serial dilution method and paper disc method. As a result, it was confirmed that lemon verbena extract containing polysaccharide exhibited superior antimicrobial activity against four kinds of bacteria, yeast and filamentous fungus than methylparaben, which is a powerful synthetic preservative (Table 11 and Table 12). In addition, as a UV protection test assay for lemon verbena extract containing polysaccharide in As a result of measuring the sunscreen effect using the in vitro SPF analyzer (Optometrics SPF-290S), lemon verbena extract containing polysaccharides has SPF 7, OMC is SPF 12, which is lower than OMC, which is known as a powerful synthetic organic sunscreen. Blocking capacity (SPF value) was shown, but it was confirmed that there is a UV blocking effect (Table 13).

In another aspect, the present invention relates to a cosmetic composition comprising a lemon verbena extract containing polysaccharide as an active ingredient.

In the present invention, the cosmetic composition comprising a lemon verbena extract containing polysaccharide as an active ingredient in the group consisting of antioxidant, wrinkle improvement, whitening, skin damage inhibition or irritation by ultraviolet rays, anti-inflammatory, antibacterial and atopic improvement It may be characterized by having a selected use.

In one embodiment of the present invention, a nutrition cream comprising a lemon verbena extract containing the polysaccharide of the present invention was prepared (Table 14). In order to confirm the wrinkle improvement and atopy improvement effect of the nourishing cream containing the lemon verbena extract containing the polysaccharide, the experiment was performed in comparison with the nutrition cream containing no lemon verbena extract containing the polysaccharide It was. As a result, it was confirmed that the excellent wrinkle improvement effect and atopy improvement effect in the nutrition cream to which the lemon verbena extract containing polysaccharide was added (Tables 15, 16 and 17).

In the cosmetic composition of the present invention, the content of the lemon verbena extract containing the polysaccharide may be characterized in that 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.

The cosmetic composition may contain at least one active ingredient exhibiting the same or similar function in addition to the lemon verbena extract containing polysaccharide.

In another aspect, the present invention relates to an external skin composition comprising a lemon verbena extract containing polysaccharide as an active ingredient.

In the present invention, the external composition for skin containing lemon verbena extract containing polysaccharide as an active ingredient is a group consisting of antioxidant, anti-wrinkle, whitening, inhibition of skin damage or alleviation of irritation by UV rays, anti-inflammatory, antibacterial and atopic improvement It may be characterized by having the use selected from.

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

The external preparation composition for skin may include one or more active ingredients exhibiting the same or similar functions in addition to the lemon verbena extract containing polysaccharide.

Lemon verbena extract containing polysaccharide prepared by the method of the present invention, and cosmetic composition and external skin composition comprising the same, antioxidant effect, wrinkle improvement effect, whitening effect, collagen synthesis promoting effect, UV damage inhibition or stimulation It has excellent effects such as mitigating effect, anti-inflammatory effect, antibacterial effect and atopy improvement effect. In addition, the cosmetic composition comprising a lemon verbena extract containing polysaccharide may be prepared in various formulations, such as cosmetics, essences, creams, packs, patches, skin adhesive gels, foundations, makeup bases ( Tables 18 to 22) 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 Polysaccharide  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 at a temperature of 80 ° C. for 2 hours, followed by filtering with an 80 mesh filter and a 1.2 μm filter to obtain the remaining foil. The obtained foil was dried at 80 ° C. for 10 hours, distilled water was added at 10% by weight to the obtained powdery dried foil, and extracted at a temperature of 80 ° C. for 2 hours in a reflux extractor equipped with a cooling capacitor. Thereafter, the filtrate was obtained by filtration with an 80 mesh filter and a 1.2 μm filter, and then an HP-20 column was used to remove residual pigments and impurities. A total of 10 fractions were eluted with HP-20 column using distilled water, and fractions 3 to 7 with residual pigment removed were obtained. 99% ethanol was added to the obtained filtrate four times (w / w), and precipitated at -20 ° C for 5 hours. The precipitated pellet was obtained by centrifugation at 8000 rpm for 20 minutes, and then using a vacuum concentrator. Concentration was carried out for 1 hour at 65 ℃, 20 hPa conditions to obtain a concentrated powder. This thus obtained was referred to as "sample 1" and was used in the following test example.

Test Example  One: HPLC Analysis of Residual Organic Acid and Impurity Content Using

In order to confirm the purity of the lemon verbena extract containing the polysaccharide of the present invention, detection tests for residual organic acids and impurities were performed using HPLC.

Analysis was performed using high pressure liquid chromatography (HPLC) for qualitative and quantitative analysis of the impurity components of the lemon verbena extract (sample 1) containing the polysaccharide prepared through the above example.

Samples were prepared in a suitable concentration with a solvent, filtered through a 0.2 μm filter, and injected into the apparatus after pretreatment. 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. The solvent used in the analysis was all HPLC grade reagents, the standard curve was calculated and quantitative analysis was performed (Table 1).

Analysis of Residual Organic Acid and Impurity Content by HPLC Name of sample Organic acid content (%) Other Impurity Flavonoid Content (%) Sample 1 N.D. N.D.

As a result, it was confirmed that the lemon verbena extract containing the polysaccharide of the present invention did not detect organic acids and other impurities.

Test Example  2: Per total  Content measurement

In order to measure the total sugar content of the lemon verbena extract containing the polysaccharide of the present invention, a phenol-sulfate method was performed. The phenol-cephade method is reduced by treating the reducing sugar with concentrated sulfuric acid to produce furfural or a derivative thereof. The method of measuring the absorbance of the derivative at 490 nm is as follows.

First, 0.5 ml of 5% phenol was added to 0.5 ml of lemon verbena extract (sample 1) containing polysaccharide, and then 2.5 ml of sulfuric acid was added. After vortexing for 30 seconds using a vortex mixer, the mixture was left at 25 ° C. for 20 minutes. Thereafter, the absorbance was measured at 490 nm wavelength using a microplate reader to prepare a calibration curve and to quantify the total sugar content (Table 2). As a calibration standard, glucose standard (Glucose standard, sigma, USA) was used.

 Total Sugar Content of Lemon Verbena Extract Containing Polysaccharides Name of sample Total sugar content (%) Sample 1 99.5 ± 1.2

As a result, it was confirmed that the lemon verbena extract containing a high content of polysaccharide with a total sugar content of more than 99% can be produced by the method of the present invention.

Test Example  3: Antioxidant effect measurement

In order to confirm the antioxidant effect in the skin aging improvement effect assay test for lemon verbena extract containing polysaccharide of the present invention, free radical elimination test and active oxygen elimination test were performed. The sample used in the test was Sample 1 obtained in Example 1, and ascorbic acid (L-ascorbic acid) having excellent antioxidant effect was used as a positive control to compare the antioxidant effect.

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 left for 15 minutes at 37 ℃, the absorbance at 540nm wavelength was measured using a microplate reader (UVT-06685, Thermo max, USA) (Table 3).

In the free radical scavenging test, the control group was measured in the same manner by adding 1 ml of DPPH and 1 ml of methanol, and 1 ml of methanol and 1 ml of sample were added to obtain respective color correction values for the sample and the control group. Equation 1 was used to calculate the free radical elimination rate, SC ₅₀ is the concentration of the sample required to eliminate 50% free radicals, the smaller the value means that the antioxidant activity is higher.

[Equation 1]

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

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.

2.4 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), NBT 0.1 ㎖ and 0.1ml of sample was added to a vortex mixer (Vortex mixer, Type 37600 Mixer, Mini mix, USA), and then left at 25 ° C for 10 minutes after voltexing. Then, 0.1ml of xanthine oxidase was added and 25 ° C. After reacting for 20 minutes at 6 mM CuCl ₂ , the reaction was stopped and the absorbance was measured at 540 nm using a microplate reader (Table 3).

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. .

Equation 2 was used to calculate the active oxygen removal rate, IC ₅₀ is the concentration of the sample required to remove 50% of the active oxygen, the smaller the value means that the antioxidant activity is higher.

&Quot; (2) "

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

Antioxidant Effect of Lemon Verbena Extract Containing Polysaccharides Name of sample Antioxidative effect Free radical scavenging rate SC ₅₀ (ug / ml) The active oxygen scavenging rate IC ₅₀ (ug / ml) Sample 1 8 10 Ascorbic acid
(L-ascorbic acid) 5 7

As a result, the lemon verbena extract containing the polysaccharide of the present invention showed an antioxidant effect similar to that of the excellent antioxidant L-ascorbic acid, and the free radical scavenging ratio SC ₅₀ and the active oxygen scavenging ratio IC ₅₀ value. Was 8 ㎍ / ㎖ and 10 ㎍ / ㎖, respectively, it was confirmed that the antioxidant efficacy was excellent at a small concentration.

Test Example  4: Tairosineiz  Active inhibition test

In order to assay the whitening effect on the lemon verbena extract containing the polysaccharide 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 4).

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 used for the test was Sample 1 obtained in Example 1, and arbutin synthetic (Sigma), which was known to have excellent whitening effect, was used as a comparison group. The tyrosinase inhibition rate was calculated numerically using Equation 3, and 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.

&Quot; (3) "

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

Inhibition of Tyrosinase Activity of Lemon Verbena Extract Containing Polysaccharides Name of sample Tyrosinase activity inhibition rate, IC ₅₀ (mg / ml) Sample 1 0.035 Arbutin 0.032

As a result, lemon verbena extract containing polysaccharide 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 0.035 mg / ml. It was confirmed that the whitening effect is excellent at the concentration.

Test Example  5: B16F1 Melanocyte  Experiment to measure melanin production inhibitory effect

In order to assay the whitening effect of lemon verbena extract containing polysaccharide of the present invention, the degree of inhibition of melanin production against B16F1 melanosite was measured as follows.

The B16F1 melanocytes used are cell lines derived from mice and are cells that secrete a black 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 (%) was measured (Table 5). 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%.

&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 Effects of Lemon Verbena Extract Containing Polysaccharides on Melanin Production Name of sample Treatment concentration (%) Melanin formation inhibitory effect (IC ₅₀ ) Sample 1 0.1 0.11% Arbutin 0.1 0.21% Usefulness Licorice extract 0.1 0.09%

As a result of testing the melanin production inhibitory effect of B16F1 melanocytes, the IC ₅₀ of lemon verbena extract containing polysaccharide was 0.11%, which showed similar or superior effect to the conventional whitening agent arbutin and oil-soluble licorice extract. .

Test Example  6: Ella Stays  Active inhibition test

In order to test the anti-wrinkle and improvement effect of the lemon verbena extract containing polysaccharide of the present invention, an elastase inhibition activity test was performed.

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, and 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 1 (sample Lemon Verbena extract containing polysaccharide obtained in 1) 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. 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.

&Quot; (5) "

Elastay activity inhibition rate (%) = 100-((absorbance of sample) / absorbance of control) x100)

Inhibition of Elastase Activity of Lemon Verbena Extract Containing Polysaccharides Name of sample Elastase activity inhibition rate, IC ₅₀ (mg / ml) Sample 1 0.42 Retinol 0.38

As a result, the lemon verbena extract containing polysaccharide showed a similar elastase inhibitory effect as compared to retinol, which is known to be excellent in anti-wrinkle effect. Thus, the anti-wrinkle effect of lemon verbena extract containing polysaccharide was confirmed. .

Test Example  7: Collagen Synthesis Effect Test

Collagen synthesis ability was measured to assay the anti-wrinkle effect of lemon verbena extract containing the polysaccharide 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 dermal fibroblasts were seeded in 24-well microplates (1 × 10 ⁵ cells / well) and incubated for 24 hours in 37 ° C., 5% CO ₂ incubator. Each sample was incubated for 24 hours in DMEM medium containing no added serum by concentration, collagen synthesis was performed using enzyme immunoassay.

  Cultures incubated for 24 hours were dispensed into 96-well microplates and coated overnight at 4 ° C. Washed three times with washing buffer (PBS-T; 0.05% Tween 20 in phosphate buffered saline) and blocking solution (5% skin milk, Fluka) was added for 1 hour at 37 ° C. The blocking solution was discarded and washed three times with a washing buffer, and the primary antibody (rabbit anti-collagen type I, Sigma) was diluted in PBS-T, and added to 100 µl and reacted at 37 ° C for 2 hours. After washing three times with a washing buffer, the secondary antibody (anti-rabbit IgG alkaline phosphatase conjugate, Sigma) was diluted in PBS-T and added to 100μL and reacted for 2 hours at 37 ℃. After washing three times with the washing buffer, 100 μl of an alkaline phosphatase substrate solution (Sigma) was added thereto and developed at a constant temperature of 25 ° C., and then the absorbance was measured at 405 nm using a microplate reader (Table 7).

The control group for measuring the collagen synthesis effect is the reaction absorbance of the cell culture without the sample, and to determine the collagen synthesis effect, ascorbic acid was set as a comparison group, which was performed three times each to obtain an average value. The collagen synthesis effect was numerically analyzed using Equation 6.

&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 Polysaccharides The concentration of the sample (占 퐂 / ml) 0 One 10 100 Sample 1 100.00 110.14 125.31 138.66 Ascorbic acid (vitamin-C) 100.00 108.45 123.57 131.58

As a result, it was confirmed that lemon verbena extract containing polysaccharide showed a collagen synthesis effect similar to ascorbic acid (vitamin-C), which has excellent collagen synthesis effect.

Test Example  6: after UV irradiation MMP -1 expression inhibition evaluation

The enzyme immunoassay (ELISA) was performed to measure the concentration of MMP-1 after UV irradiation and sample addition of the lemon verbena extract containing the polysaccharide 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, and the secondary antibody, anti mouse IgG (whole mouse, alkaline) After reacting the phosphatase conjugated again for about 60 minutes, the alkaline phosphatase substrate solution (1 mg / ml-nitrophenyl phosphate in diethanolamine buffer solution) was reacted at room temperature for 30 minutes, and the absorbance was measured at 405 nm with a microplate reader. 8). As a control group, a sample to which no sample was added was used.

Inhibition of MMP-1 Expression in Lemon Verbena Extract Containing Polysaccharides Name of sample Treatment concentration (%) MMP-1 expression inhibition rate (%) Sample 1 0.1 67 Retinol 0.1 43

As a result, the lemon verbena extract containing the polysaccharide of the present invention showed an inhibition rate of 67% or more compared to the control group without the sample for MMP-1, which is expressed during UV irradiation, and used as a positive control retinol It confirmed that it was superior to the inhibition rate of.

Test Example  9: Cytotoxic Alleviation Effect by UV Irradiation

In order to evaluate the alleviation effect of cytotoxicity by ultraviolet irradiation of lemon verbena extract containing polysaccharide of the present invention, the experiment was conducted as follows.

Fibroblasts (fibroblasts) were placed in a 24-well test plate 1 x 10 ⁵ and attached 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. After shaking for 5 minutes, the cells were lysed and 100 μl of the supernatant was transferred to a 96-well test plate, and the absorbance was measured at 565 nm with a microplate reader. Cell survival rate (%) was measured using Equation 7, and cytotoxic alleviation rate by UV light was measured using Equation 8.

[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 Polysaccharides Name of sample Treatment concentration (%) Cytotoxic Relaxation Rate (%) Sample 1 0.1 42

As a result, the lemon verbena extract containing polysaccharides showed a cytotoxic alleviation rate of 42% at a concentration of 0.1%, which effectively prevented cytotoxicity caused by ultraviolet rays.

Test Example  10: Inhibitory effect of inflammatory cytokine expression by UV irradiation

In order to evaluate the inhibitory effect of inflammatory cytokine expression expressed by UV irradiation of lemon verbena extract containing polysaccharide of the present invention, fibroblasts isolated from epidermal tissues of humans were placed on a 5-well 10-well test plate. ^{Four were} added 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 polysaccharide to be evaluated therein was incubated for 5 hours. 150 μl of the culture supernatant was taken to quantify IL-1α to determine the inhibitory effect of inflammatory cytokine expression of Sample 1. 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).

&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 in Lemon Verbena Extract Containing Polysaccharides Sample treatment concentration (%) Inhibitory rate of inflammatory cytokine expression (%) 0.05 32 0.1 55

As a result, lemon verbena extract containing polysaccharide inhibited the production of inflammatory cytokine IL-1α by UV rays at 0.1% and 55%, and effectively prevented the inflammation caused by UV rays even at low concentrations. there was.

Test Example  11: antibacterial effect

The antibacterial effect assay for the lemon verbena extract containing the polysaccharide of the present invention was carried out by agar serial dilution method and paper disc method. The experimental strain was distributed by the Korea Research Institute of Bioscience and Biotechnology, and it was Gram-positive bacteria as Staphylococcus aureus KCTC 6910 and Gram-negative bacteria 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 determine the antimicrobial activity of the lemon verbena extract containing the polysaccharide, the minimum inhibitory concentration was measured 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 and colony formation was observed in each compartment (Table 11). The minimum sample concentration of the ungrown plate is referred to as the minimum inhibitory concentration (MIC), and the smaller the mean, the higher the antimicrobial effect.

Antibacterial Effect of Lemon Verbena Extract Containing Polysaccharides Strain Extraction method Minimum inhibitory concentration (MIC, 占 퐂 / ml) S. aureus Sample 1 150 P. aeruginosa Sample 1 210 E. coli Sample 1 250 C. albicans Sample 1 260 A. niger Sample 1 320

As a result, lemon verbena extract containing polysaccharides showed excellent antimicrobial activity against all four kinds of bacteria, yeast and filamentous fungi.

In addition, in order to test the antimicrobial effect of the lemon verbena extract containing the polysaccharide obtained in Example 1 (Sample 1) was carried out as follows using a circular filter paper. 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) and Escherichia coli (E. Coli) at 37 ℃, the type of fungi Candida yeast (Candida albicans ) and filamentous fungi ( Aspergillus niger ) were incubated at 27 ° C. for 24 hours and 120 hours, respectively, to measure the diameter of the transparent zone around the disc (Table 12). 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.

Antibacterial Effect of Lemon Verbena Extract Containing Polysaccharides Strain Extraction method Transparent zone diameter (mm) S. aureus Sample 1 10 Methylparaben 11 P. aeruginosa Sample 1 17 Methylparaben 12 E. coli Sample 1 20 Methylparaben 18 C. albicans Sample 1 20 Methylparaben 13 A. niger Sample 1 27 Methylparaben 24

As a result, it was confirmed that lemon verbena extract containing polysaccharide exhibited superior antimicrobial activity against four kinds of bacteria, yeast and filamentous fungus than methylparaben, which is known as a powerful synthetic preservative.

Test Example  11: sunscreen effect

As a sunscreen test assay for lemon verbena extract containing polysaccharide 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. It carried out in the same manner as in Table 13.

In of Lemon Verbena Extract Containing Polysaccharides method for measuring SPF in vitro 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

As a result, Lemon Verbena extract containing polysaccharides showed SPF 7, OMC showed SPF value lower than OMC, which is well known as a strong synthetic organic sunscreen as SPF 12, but it has UV blocking effect. .

Formulation Example  1 and formulation Comparative example  One: Polysaccharides  Containing Lemon Verbena  Ingredient Composition of Nutritional Cream Including Extract

Component composition of the nutrition cream containing the lemon verbena extract containing the polysaccharide according to Example 1 (sample 1) was prepared as shown in Table 14.

Nutritional Cream Containing Lemon Verbena Extract Containing Polysaccharides 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 1 1.0 - 17 Distilled water Balance Balance 18 Preservative, fragrance, pigment a very small amount a very small amount

Of the components composed of the composition of Table 14, first, the aqueous components of the raw materials 12 to 15 and 17 were completely dissolved by heating to 80 ° C., and then the raw materials 1 to 11 and 16 were heated to 80 ° C. to prepare the raw materials 12 to 15 and 17. The solution was added to the solution and emulsified at 3,000 rpm for 15 minutes using a Homo Mixer Mark II, Primix, Japan. Then, the raw material 18 was added and stirred for 5 minutes, and then cooled to room temperature.

Test Example  13: Determination 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. Thirty-five and 25-year-old women with blemishes, freckles, and pigmentation were treated with nutrient creams of Formulation Example 1 and Comparative Example 1 for 12 weeks and then the color change of the skin was determined by the chromometer (CR-410, Minolta). , Japan) was used to measure the change in brightness of color (ΔL) (Table 15). The average value of 30 persons was calculated. The higher the brightness change value, the higher the whitening effect.

Measuring whitening effect of formulation medium Formulation Example 1 Formulation Comparative Example 1 Skin color brightness change (L) 6.81 0.52

As a result, it was confirmed that the nutrition cream of Formulation Example 1 to which the lemon verbena extract containing the polysaccharide of the present invention was added showed an excellent whitening effect when compared to the nutrition cream of Formulation Comparative Example 1 not added.

Test Example  14: Wrinkle improvement effect measured by formulation

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. For 10 women over 30 years old who are undergoing skin aging, the nourishing cream of Formulation Example 1 and Comparative Example 1 is applied to both crow feet for 12 weeks. At intervals, the wrinkles around the eyes (replica) were measured using a skin visiometer (SV-600, C + K, Germany) by image analysis to measure the depth (μm) of the wrinkles (Table 16).

Wrinkle improvement effect of formulation medium Formulation Example 1 Formulation Comparative Example 1 Before application 331 ± 10 326 ± 12 12 weeks after application 245 ± 24 320 ± 22 Wrinkle depth reduction rate (%) 28.5 1.8

As a result, it was confirmed that the nutrition cream of Formulation Example 1 to which the lemon verbena extract containing the polysaccharide of the present invention was added showed an excellent wrinkle improvement effect when compared to the nutrition cream of Formulation Comparative Example 1 not added.

Test Example  15: Determination of atopic dermatitis improvement effect

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, the degree of TEWL was reduced by measuring the degree of TEWL reduction by measuring the extent to which the skin barrier function was restored (Table 17). The recovery rate used for the efficacy evaluation was calculated using Equation 10.

&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) 53 12

As a result, the nutritional cream of Formulation Example 1 to which the lemon verbena extract containing the polysaccharide of the present invention was added was superior to the nutritional cream of Formulation Comparative Example 1, which did not contain the skin recovery effect after skin barrier damage. Confirmed.

Formulation Example  2: Polysaccharides  Containing Lemon Verbena  Components of Cosmetic Compositions Containing Extracts

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 1 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 1 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 1 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 1 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 1 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 polysaccharide comprising the following steps:
(a) adding ethanol to lemon verbena, eluting the pigments and impurities, filtering, and then obtaining and drying the remaining lemon verbena foil;
(b) adding water to the dried lemon verbena foil of step (a) to obtain a polysaccharide fraction;
(c) removing residual pigments of the polysaccharide fraction obtained in step (b); And
(d) adding ethanol to the extract from which the residual pigment was removed to precipitate the polysaccharide, and then spraying or drying the resulting pellet to obtain a lemon verbena extract containing polysaccharide in powder form.
delete The method of claim 1, wherein the removing of the residual pigments in the step (c) is characterized in that using a column.
4. The column of claim 3 wherein the column is selected from the group consisting of Amberlite XAD Resin, Dow X Resin, Superlite DAX Resin, Diaion HP-10, HP-20, HP-30, HP-40 and HP-50 Resin. A method comprising at least one resin.
The method of claim 1, wherein in step (d), 1 to 10 times (w / w) of ethanol is added to precipitate for 1 to 24 hours at -80 ℃ to 25 ℃.
Lemon verbena extract containing polysaccharide prepared by the method of any one of claims 1 and 3 to 5.
The lemon verbena containing polysaccharide according to claim 6, which has a use selected from the group consisting of antioxidants, wrinkle improvement, whitening, skin damage inhibition or irritation by ultraviolet rays, anti-inflammatory, antibacterial and atopic improvement. extract.
A cosmetic composition comprising a lemon verbena extract containing polysaccharide prepared by the method of any one of claims 1 and 3 to 5 as an active ingredient. delete

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