KR102022068B1 - Method for production of Malpighia emarginata fruit extract without mass destruction of effective compound and cosmetic composition with the extract - Google Patents

Abstract

The present invention relates to a method for producing acerola fruit extract with little destruction of useful ingredients and to a cosmetic composition containing the extract prepared as described above. In particular, it was confirmed that acerola fruit extract having excellent antioxidant power due to low destruction of vitamin C could be prepared. As a result, the acerola fruit extract according to the nitrogen injection extraction process of the present invention exhibits an excellent anti-aging effect, anti-wrinkle effect, whitening effect, and anti-inflammatory (skin irritation) effect.

Description

Method for production of Malpighia emarginata fruit extract without mass destruction of effective compound and cosmetic composition with the extract}

The present invention relates to a method for producing acerola fruit extract with little destruction of useful ingredients and to a cosmetic composition containing the extract thus prepared.

The skin of the human body is an organ that functions as a protective film to protect the living body from the external environment, and prevents the loss of biological components such as water and electrolyte in the human body, and at the same time serves to prevent the ingress of harmful substances from the outside.

However, in general, as the body ages, these functions are gradually weakened, and aging of the skin proceeds from the activation of free radicals due to ultraviolet rays, stress, disease state, environmental factors, and wounds. In particular, as the lipid composition and content of the lipid layer, which functions as a skin barrier, changes, the function decreases rapidly. When the moisture content of the skin decreases, the skin dries, wrinkles are formed, blemishes, freckles, pigmentation and Various skin lesions are caused.

If this condition is exacerbated, antioxidant defenses existing in vivo are destroyed, cells and tissues are damaged, which promotes adult disease and aging. In particular, the oxidation of proteins is caused by severe cuts in the skin's connective tissues such as collagen, hyaluronic acid, elastin, proteoglycans, and fibronectin, resulting in severe excessive inflammatory reactions and skin elasticity. It leads to the induction of the immune function.

Therefore, the use of cosmetics that can protect the skin from various external factors is required, and many kinds of functional cosmetics have been released and used according to these requirements. Functional cosmetics are largely contrasted with cosmetics using synthetic compounds and cosmetics using extracts derived from natural products. Cosmetics using a synthetic compound has the advantage that the efficacy is relatively clearly exhibited, there is a big side effect, a consumer's rejection due to the use of the synthetic compound has emerged as a big problem. On the other hand, the compound using the extract derived from natural products has the advantage of less side effects, less consumer's rejection, but there is a problem that the use of a lot of raw materials in order to exhibit the efficacy, the effect is insignificant. Therefore, when using the extract derived from natural products as a cosmetic raw material, it will be required to develop a technology that can maximize the efficacy.

Acerola ( Malpighia glabra ), a small shrub, grows 5 m in dry, deciduous forests and produces abundantly bright red fruits, about 1-2 cm in diameter. The fruit looks similar to the European cherry, and for this reason, acerola is called by Antilles, Barbados, Puerto Rican cherry and West Indian cherry.

Acerola is found growing wild in the sands of northeast Brazil, originating in South America, Central America and Jamaica. It survives temperatures above 28 ° F, withstands prolonged droughts, and favors a warm or hot climate of 85-90 ° F.

Acerola fruit is succulent, tender, with a sour taste and aroma, and many enjoy it. It is fresh and delicious for drinks, and it is used in South America as an additive to flavor and flavor ice cream and soda cocktails.

Korean Patent Registration No. 10-1563310 (Registration Date: 2015.10.20) describes the powder of acerola cherry fruit and its manufacturing method. Korean Patent Laid-Open No. 10-2017-0121033 (published date: 2017.11.01) discloses a marigold, acai berry, astragalus, barberry, chaga, plant-derived minerals, or the like on a substrate for a gel or cream type composition. A cosmetic composition for lips containing at least one of acerola is described.

The present invention is to develop and provide the optimal extraction conditions of acerola fruit extract for use as a cosmetic raw material, to provide a functional material that can be used in cosmetics by checking the various skin functionalities of the extracted extract.

The present invention provides a method for producing acerola extract, characterized in that the extraction is added to the freeze-dried powder of acerola fruit extract while purging nitrogen for 108-132 minutes at a temperature of 23 ~ 28 ℃.

In the method for producing acerola extract of the present invention, the acerola extract preferably has a solid content of 55 to 57% by weight and preferably contains 22 to 24% by weight of vitamin C.

The present invention was added to the freeze-dried powder of acerola fruit extracted with a solvent as an extraction solvent and purged with nitrogen at 108 ~ 132 minutes at a temperature of 23 ~ 28 ℃, the solid content is 55 to 57% by weight, vitamin C 22 It provides acerola extract of ˜24% by weight.

The present invention provides a cosmetic composition for preventing skin aging containing the acerola extract of the present invention.

The present invention provides a cosmetic composition for preventing skin wrinkles containing the acerola extract of the present invention.

The present invention provides a cosmetic composition for skin whitening containing the acerola extract of the present invention.

The present invention provides a cosmetic composition for alleviating skin irritation containing the acerola extract of the present invention.

According to the nitrogen-injected extraction process newly conceived in the present invention, it was confirmed that the acerola fruit extract having excellent antioxidant power may be prepared due to low destruction of useful components, particularly vitamin C, in the acerola fruit extract. As a result, the acerola fruit extract according to the nitrogen injection extraction process of the present invention exhibits an excellent anti-aging effect, anti-wrinkle effect, whitening effect, and anti-inflammatory (skin irritation) effect.

1 is a graph showing the change in solid yield with temperature and time when extracting acerola fruit lyophilized powder.
2 is a graph showing a change in extraction yield for each ethanol concentration during the extraction of acerola fruit lyophilized powder.
Figure 3 is a graph showing the change in solid yield with temperature and time in general extraction and nitrogen purge extraction of acerola fruit lyophilized powder.
Figure 4 is a graph showing the change of antioxidant activity with temperature, time in the general extraction and nitrogen purge extraction of acerola fruit lyophilized powder.
5 is a reaction surface analysis results for the yield of vitamin C according to the temperature, time of nitrogen purge extraction of acerola fruit freeze-dried powder.
6 is a graph showing the content of vitamin C according to the extraction time and extraction time of nitrogen when acerola fruit freeze-dried powder.
Figure 7 shows the results of DPPH experiment of acerola fruit extract (Sample).
8 is a result of measuring the reducing power of the acerola fruit extract.
9 is a measurement result of the nitrite scavenging ability of the acerola fruit extract.
10 is a result of measuring the inhibition of MMP-1 expression of acerola fruit extract.
Figure 11 is the result of measuring the collagen production of acerola fruit extract.
12 is a result of measuring the tyrosinase inhibitory activity of acerola fruit extract (Sample).
Figure 13 is the result of measuring the melanin inhibition of the acerola fruit extract.
It is a result of measuring the NO production of the acerola fruit extract of FIG.
FIG. 15A is a result of measuring the AP-1 gene by real-time PCR, FIG. 15B is a result of measuring the HMOX-1 gene by real-time PCR, and FIG. 15C is a real-time PCR of the TrxR gene. It is a result measured by time PCR, D of FIG. 15 is a result of measuring the GAPDH gene by real-time PCR, E of FIG. 15 is a result of measuring the Involucrin gene by real-time PCR.
16 is a result showing the hyaluronidase inhibitory activity of acerola extract.
17 is a result of measuring the in vitro tyrosinase inhibitory ability of the acerola nitrogen purge extract (Acerola extract A) and the general extract (Acerola extract B).
18 is a result of measuring the melanin inhibitory ability of the acerola nitrogen purge extract (Acerola extract A) and the general extract (Acerola extract B).

The present invention provides a method for producing acerola extract, characterized in that the extraction is added to the freeze-dried powder of acerola fruit extract while purging nitrogen for 108-132 minutes at a temperature of 23 ~ 28 ℃.

In the present invention, it was intended to establish the optimum extraction conditions of acerola extract, first to determine the extraction solvent. In the present invention, after obtaining the extract using a variety of solvents, and compared the solids extraction yield, when using purified water was able to confirm the highest extraction yield. From this, purified water was selected as the extraction solvent in the present invention.

On the other hand, vitamin C of the high functional active ingredient derived from acerola is very vulnerable to high temperature extraction method and various oxidative stress, it is required to develop an extraction method to minimize the destruction thereof, to be contained as much as possible. Introducing a nitrogen injection (purging) extraction process in the present invention, as a result, it was confirmed that less destruction of vitamin C than otherwise. From this, the present invention introduced a nitrogen injection extraction process.

Meanwhile, in the present invention, the optimum extraction temperature and extraction time were intended to be established through response surface methodology (RSM). RSM is used to analyze the experimental data obtained from the minimum number of experiments and the experimental data obtained by the experimental design to understand the performance of complex systems and to find and optimize significant factor effects affecting the response variables. .

It is difficult to find the optimal experimental condition by the one factor at a time method, which is a conventional method of changing and experimenting one by one. In addition, there is a problem in that the local optimum point is found because the interaction between important factors in the complex process is not considered and the entire experimental area cannot be judged in a balanced manner.

RSM is a typical optimization method that generally finds the optimal condition using several variables. By measuring the effects of one variable's interaction with other variables, the optimal values of the variables can be determined.

As a result of preparing the acerola extract by varying the temperature and extraction time by the RSM setting, the extraction for 120 minutes (2 hours) at 25 ° C. was confirmed as the optimal vitamin C extraction condition. Therefore, in this invention, the extraction time of 25 degreeC temperature and 120 minutes (2 hours) was judged as the optimal extraction condition intended by this invention. Nitrogen purging acerola extract for 2 hours at 25 ℃ above, the solid content is about 56% by weight, vitamin C is about It was found to be 23 wt%.

On the other hand, the ± 10% of the optimum conditions can be generally recognized as a family register condition, the temperature of 23 ~ 28 ℃ and nitrogen purge extraction conditions for 108 ~ 132 minutes can be established as a family register extraction condition of the acerola extract of the present invention there was.

On the other hand, the present invention is added to the freeze-dried powder of acerola fruit extracted with solvent as an extraction solvent and extracted with nitrogen purge for 108-132 minutes at a temperature of 23 ~ 28 ℃, solid content is 55 ~ 57% by weight, vitamin C It provides an acerola extract with a content of 22-24% by weight. The cosmetic composition containing the acerola extract of the present invention exhibits skin anti-aging effect, anti-wrinkle effect, skin whitening effect, skin inflammation alleviation (skin irritation) effect.

The cosmetic composition of the present invention is, for example, solution, suspension, emulsion, paste, lotion, gel, water-soluble liquid, cream, essence, surfactant-containing cleansing, oil, oil-in-water (O / W) type and water-in-oil ( A base cosmetic formulation of any one selected from W / O) type; skin; Lotion; Eye cream; Soothing Gel; Ointment; Formulation for mask pack; Body wash formulations; Peeling gel; Oil-in-water and water-in-oil makeup bases; foundation; Skin cover; Color cosmetic formulations selected from lipstick, lip gloss, face powder, two-way cake, eye shadow, cheek color and eyebrow pencils; Formulations for scalp; It may be any one selected from.

Meanwhile, the cosmetic composition of the present invention may include components commonly used, and may include, for example, conventional auxiliaries and carriers such as antioxidants, stabilizers, solubilizers, vitamins, pigments, and flavors.

On the other hand, in the cosmetic composition of the present invention, when the formulation is a paste, cream or gel, the carrier component is animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, Talc or zinc oxide may be used.

In addition, in the cosmetic composition of the present invention, when the formulation is a solution or an emulsion, a solvent, a solubilizer or an emulsifier is used as the carrier component. For example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, Fatty acid esters of benzyl benzoate, propylene glycol, 1,3-butylene glycol oil, glycerol aliphatic esters, polyethylene glycols or sorbitan may be used.

In the cosmetic composition of the present invention, when the dosage form is a suspension, a liquid diluent such as water, ethanol or propylene glycol, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, and polyoxyethylene sorbitan ester are used as carrier components. Suspending agents such as, microcrystalline cellulose, aluminum metahydroxy, bentonite, agar or tragacanth and the like can be used.

On the other hand, in the cosmetic composition of the present invention, when the formulation is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used, and particularly in the case of spray Propellants such as chlorofluorohydrocarbons, propane / butane or dimethyl ether.

On the other hand, in the cosmetic composition of the present invention, when the formulation is a surfactant-containing cleansing, the carrier component is an aliphatic alcohol sulfate, an aliphatic alcohol ether sulfate, a sulfosuccinic acid monoester, isethionate, an imidazolinium derivative, a sarcosinate, Fatty acid amide ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters and the like can be used.

In addition, in the cosmetic composition of the present invention, in the case of a surfactant-containing cleansing formulation or a surfactant-free cleansing formulation or soap, after applying to the skin, it may be wiped off, peeled off or washed with water. In one example, the soap is liquid soap, powdered soap, solid soap and oil soap, the surfactant-containing cleansing formulation is a cleansing foam, cleansing water, cleansing towel and cleansing pack, the surfactant-free cleansing formulation is a cleansing cream, Cleansing lotion, cleansing water and cleansing gel.

Moreover, the cosmetic composition of this invention can be used overlapping with other cosmetic compositions other than this invention. In addition, the cosmetic composition according to the present invention may be used according to a conventional method of use, and the number of times of use may vary depending on the skin condition or taste of the user.

Hereinafter, the content of the present invention will be described in more detail through the following examples and experimental examples. However, the scope of the present invention is not limited to the following Examples and Experimental Examples, and includes modifications of equivalent raw materials.

Example 1 Selection of Solvent for Optimal Extraction of Acerola Fruits

Extraction time, extraction temperature and extraction solvent are the most important factors in the development of natural extracts. Finding the optimal extraction conditions that can contain the most active ingredients through the most economical process is an important factor for industrialization.

First, it was confirmed that the solid yield change with temperature and time, as shown in Figure 1, it was confirmed that the yield increases as the temperature and time elapses.

On the other hand, to select the optimal extraction solvent, the polyphenol content in the fraction obtained through the fractionation process (hexane-> EA (ethyl acetate)-> ACN (acetonitrile)-> butanol-> ethanol-> purified water) was checked.

Polyphenol Content in Fractions of Acerola Fruits (Measured by Folin Denis Method. Standard Curve is y = 0.0309x + 0.0742, R ² = 0.9988) Raw material input amount (g) 250 Absorbance (765nm) Content (mg GAE / g) Hexane 0.067 2.18 E.A 0.051 14.18 ACN 0.114 13.66 Butanol 0.187 112.66 ethanol 0.341 459.5 Purified water 2.597 826.2

As a result of the fractionation test, as shown in Table 1, the polyphenol content was the highest in purified water and the second highest in ethanol. From this result, the solvent used in the development of the extraction process was to use purified water or ethanol.

Example 2: Extraction yield analysis for each ethanol concentration

In the experiment of Example 1, purified water was found to be suitable as an optimal solvent for obtaining acerola extract, but in this experiment, the solid content extraction yield was analyzed by varying the concentration of ethanol.

Experimental results are shown in FIG. As shown in Figure 2, the higher the ethanol content was confirmed that the extraction yield is lower. When ethanol was used as the extraction solvent, it was determined that 30% ethanol was the most preferable in terms of extraction yield.

The low ethanol content, on the contrary, means that the water content is high. In this experiment, when the ethanol aqueous solution having low ethanol content (ie, high water content) was used as the extraction solvent, the yield was high. When water is used as the extraction solvent in step 1, the extraction yield is the highest and the result is in line with the result.

Example 3: Development of Nitrogen Purging Extraction Process

The useful components of acerola fruit are mostly water-soluble substances and are sensitive to the severe physical and chemical stresses applied during the extraction process. Therefore, in the present invention, in order to develop an optimal extract process, it was intended to develop an extract process that is as compliant as possible, yet effective and economical. To this end, the nitrogen purging method was introduced during the extraction process, and the oxidation of useful components such as vitamin C was maximally suppressed.

Experimental results according to the introduction of the nitrogen purging method was as shown in Table 2 below.

Measurement result of DPPH change by extraction time when purging nitrogen Nitrogen injection No nitrogen injection Extraction time Concentration (ppm) Absorbance Radical scavenging ability Extraction time Concentration (ppm) Absorbance Radical scavenging ability 2 hours 10 0.447 5.99% 2 hours 10 0.424 5.24% 25 0.335 26.08% 25 0.171 21.33% 50 0.136 70.09% 50 0.185 63.25% 100 0.078 82.88% 100 0.149 74.94% 200 0.080 82.29% 200 0.149 78.32% 4 hours 10 0.455 5.32% 4 hours 10 0.341 4.88% 25 0.422 21.55% 25 0.279 17.43% 50 0.307 66.43% 50 0.307 61.51% 100 0.129 77.44% 100 0.129 72.33% 200 0.089 80.21% 200 0.089 76.58% 6 hours 10 0.208 5.11% 6 hours 10 0.429 4.53% 25 0.384 18.93% 25 0.428 16.80% 50 0.260 64.90% 50 0.205 61.40% 100 0.086 76.30% 100 0.103 71.30% 200 0.068 79.43% 200 0.062 75.50% 8 hours 10 0.559 5.09% 8 hours 10 0.503 4.43% 25 0.496 18.10% 25 0.391 16.30% 50 0.407 64.10% 50 0.209 60.60% 100 0.194 75.44% 100 0.066 71.20% 200 0.067 79.00% 200 0.067 74.50% 10 hours 10 0.568 4.98% 10 hours 10 0.494 4.33% 25 0.538 17.93% 25 0.380 15.88% 50 0.431 62.90% 50 0.218 60.20% 100 0.180 75.20% 100 0.074 70.70% 200 0.066 79.32% 200 0.079 73.90%

As shown in Table 2, the radical scavenging ability of the extract extracted through nitrogen purging at the same concentration was higher than in the other case. This resulted in less destruction of antioxidant-related substances when nitrogen purging was performed. Therefore, in the present invention, the nitrogen purging method will be introduced.

Example 4 Comparison of Solid Extraction Yields by General Extraction and Nitrogen Purging Extraction

In this example, we tried to compare the solids extraction yield with respect to the general extraction and nitrogen purge extraction. Experimental results are shown in FIG. As shown in Figure 3, the solids extraction yield of nitrogen purge extraction was higher than the solids extraction yield of the general extraction.

On the other hand, antioxidant activities were analyzed for general extraction and nitrogen purge extraction. Experimental results are shown in FIG. As shown in Figure 4, nitrogen purging extract was found to exhibit higher antioxidant activity.

From the above results, it can be seen that nitrogen purging extraction is a method of obtaining an extract with higher solids extraction yield and antioxidant activity than the general extraction.

Example 5 Establishment of Optimal Vitamin C Extraction Conditions from Acerola Fruits through Response Surface Methodology (RSM)

 (1) Overview

In this embodiment, vitamin C optimum for nitrogen purge extract Extraction conditions were explored with RSM.

 (2) Establish optimal conditions for vitamin C extraction

Analysis of variance Factor ¹⁾ DF Sum of squares F-value X1 One 44.872 157.79 *** ²⁾ X2 One 4.52 0.17

1) X1: Process time (hr), X2: Temperature (℃)

2) * Significant at p <0.05, ** Significant at p <0.01, *** Significant at p <0.001

Measurement of Regression Coefficients of Quadratic Polynomials Parameter DF Coefficient T-value P-value Intercept One 31.1422 8.076 0.000 X ₁ One -1.8522 -3.894 0.006 X ₂ One -0.2018 -1.199 0.270 X ₁ X ₁ One 0.0751 5.638 0.001 X ₂ X ₂ One 0.0023 1.064 0.323 X ₁ X ₂ One -0.0012 -0.098 0.925 R ² = 0.991

As a result of the analysis, as shown in Tables 3 to 4 and FIG. 5, the optimum conditions for extracting vitamin C were 2 h and 25 ° C., and the theoretical optimum yield was 24.0492%.

Meanwhile, the content of vitamin C in the acerola extract extracted at different temperatures at 25 ° C. is shown in FIG. 6. As shown in FIG. 6, the vitamin C content was higher than the extraction at other times when extracted for 2 hours. When extracted by injecting nitrogen when extracted for 2 hours at 25 ℃ was obtained acerola extract containing about 23% by weight of vitamin C in the extract.

Meanwhile, in the following, various skin functionalities were tested with the acerola extract obtained through the nitrogen injection extraction method established above. At this time, the extraction is performed for 2 hours at 25 ℃.

Experimental Example 1: Measurement of the Effect of Acerola Fruit Extract on Cytotoxicity

The acerola fruit extract extracted in the present invention was to determine whether there is cytotoxicity. Cytotoxicity was measured using an MTT assay. The cultured cells were mixed well in DMEM medium, adjusted to 1x10 ⁵ cells / mL, and then cultured for 3 to 4 days after adding 1 mL of prepared cells to a 24-well culture plate. When the cells have grown to some extent, each sample (0, 25, 50, 100, 300, 400, 500, 1000 μg / mL) is treated with a medium without fetal calf serum and antibiotics, and further incubated for 24 hours. The survival rate of the cultured cells was measured using the MTT reduction method. That is, 5 mg / mL of MTT solution was added to each well by one-tenth of the growth medium, followed by further incubation at 37 ° C for 4 hours to reduce MTT so that the formazan produced in the medium did not go out along the medium. Carefully removed. Thereafter, the solution was left at room temperature for 30 minutes to completely remove the remaining medium, and then the absorbance was measured at 570 nm for the sample dissolved using DMSO. The absorbance was measured by DMSO, and cell viability was calculated as follows.

Experimental results are shown in Table 5 below. As shown in Table 5, when compared to the control, the acerola extract of the present invention was shown to be non-toxic to cells.

Cytotoxicity Test of Acerola Fruit Extract Concentration
(w / v%)
Sample 0.005 0.01 Control 100 ± 4.5 Acerola Fruits extract 99.8 ± 1.7 99.1 ± 0.9

Experimental Example 2: Confirmation of DPPH Scavenging Activity of Acerola Fruit Extract

In this Experimental Example, DPPH scavenging ability was determined to measure the antioxidant power of the acerola fruit extract of the present invention.

2 ml of a solution of acerola extract diluted to an appropriate concentration in ethanol or methanol and 2 ml of 0.2 mM DPPH (1,1-dipenyl-2-picrylhydrazyl) were uniformly mixed. After leaving the mixed solution at room temperature for 30 minutes, the absorbance was measured at 517 nm. After absorbance measurement, DPPH radical scavenging ability was calculated according to Equation 2 below.

As shown in FIG. 7, DPPH scavenging ability of acerola fruit extract was confirmed as 95.34, 95.34 and 45.21% at concentrations of 1, 0.5 and 0.1 mg / mL, respectively. In addition, the DPPH comparative test of ascorbic acid and acerola fruit final extract showed a similar effect to ascorbic acid at a concentration of 5 times the final extract.

From the above results, it was confirmed that the acerola fruit extract of the present invention has excellent free radical (free radical) removal ability, it was confirmed that the antioxidant power.

Experimental Example 3: Confirmation of Total Polyphenol Content and Flavonoid Content of Acerola Fruit Extract

In this experiment, the total phenol content was measured by adding 1 mL of Folin-Ciocalteau reagent and 10% Na ² CO ³ solution to 1 mL of acerola fruit extract according to the Folin-Denis method. (UV 1600 PC, Shimadzu, Tokyo, Japan) was used to measure the absorbance at 700 nm. Caffeic acid (Sigma Co., USA) was prepared at a concentration of 0 ~ 100 ug / mL and the total phenol content of the sample extract was calculated from the standard calibration curve obtained by the same method as the sample.

Meanwhile, the total flavonoid content was mixed by adding 0.1 mL of 10% aluminum nitrate, 0.1 mL of 1 M potassium acetate and 4.3 mL of ethanol in order to 0.5 mL of extract according to the method of Moreno. After standing at room temperature for 40 minutes, the absorbance was measured at 415 nm. The total flavonoid content of the extract was calculated from a standard calibration curve obtained at a concentration range of 0-100 μg / mL using 'Quercetin (Sigma Co., USA)' as a standard.

As shown in Table 6 below, the total phenol content of the acerola fruit extract was 90.37 mg / g, and the total flavonoid content was 3.76 mg / g. Correlation between total phenol and flavonoid content and antioxidant activity of 20 kinds of medicinal plants showed similar results as higher polyphenol content than flavonoids.

Total Phenolic Content and Total Flavonoid Content of Acerola Fruit Extract items Results Total Phenolic Content (mg GAE / g) 90.37 ± 0.46 Total Flavonoid Content (mg RE / g) 3.76 ± 1.37

Experimental Example 4: Measurement of reducing power of acerola fruit extract

Reducing power of acerola fruit extract It was measured according to the method of Oyaizu. To 1 mL of the sample, 200 mM phosphate buffer of pH 6.6 and 1% potassium ferricyanide were added to each of 1 mL in turn, followed by stirring for 20 minutes in a 50 ° C water bath. 1 mL of 15% TCA (trichloroacetic acid) solution was added thereto, followed by centrifugation at 12,000 rpm for 15 minutes to obtain a supernatant. 1 mL of the supernatant thus obtained was mixed with 1 mL of distilled water and ferric chloride, and the absorbance was measured at 700 mm.

As a result, as shown in FIG. 8, concentration-dependently increased at concentrations of 0.1, 10, and 100 mg / mL. Therefore, the acerola fruit extract of the present invention was confirmed to have a reducing power.

Experimental Example 5: Confirmation of Nitrite Scavenging Ability of Acerola Fruit Extract

Nitrite scavenging activity was measured by the method of Gray and Dugan. That is, 1 mL of a certain concentration of sample was added to 1 mL of 1 mM NaNO ₂ solution, and the reaction solution was adjusted to pH 1.2 using 0.1 N HCl (pH 1.2) to make a total amount of 10 mL. After reacting this solution at 37 ° C for 1 hour, 1 mL of each reaction solution was added, 5 mL of 2% acetic acid solution and 0.4 mL of Greases reagent were added, mixed well, and left at room temperature for 15 minutes. Absorbance was measured at 520 nm using a 'spectrophotometer' to calculate the amount of residual nitrite. The blank test was made identical by adding 0.4 mL of distilled water in place of the Greries reagent.

Nitrite scavenging activity is represented by the following equation (3) as a nitrite percentage with and without the addition of the sample, the larger the value means that the nitrite scavenging action is greater.

Experimental results were as shown in Figure 9, it was confirmed that the nitrite scavenging ability increased depending on the concentration. Therefore, it could be confirmed that nitrite scavenging ability exists in the acerola fruit extract of the present invention.

Experimental Example 6: Measurement of inhibition of MMP-1 expression of acerola fruit extract]

In this experiment, we tried to determine whether Acerola fruit extract inhibits MMP-1 expression.

CCD-986sk was incubated in a 35 mm dish at a concentration of 1.5 × 10 ⁵ cells / ml until reaching 80% confluency. After removing the original medium before UV irradiation, washed with PBS to remove the serum components in the medium, UVA (Coralife, 35W, UV) was irradiated with UVA (6.3J / ㎠) for 24 hours using a filter. After UVA irradiation, the culture medium was incubated for 24 hours by administering the acerola fruit extract at a concentration of 0-1 mg / mL in DMEM medium without FBS.

MMP-1 expression level induced by UVA irradiation was carried out using the method used by Dunsmore et al. After irradiating the UVA, the sample was treated and cultured for 24 hours into a 96 well plate, and reacted overnight at 4 ℃. After washing three times with TBS (phosphate buffered saline + 0.05% Tween20) and blocking with 3% PBS for 1 hour at 37 ° C, a primary antibody (monoclonal anti-MMP-1 antibody) was used as a blocking buffer. The solution was diluted with 1: 3000 and reacted at 37 ° C for 90 minutes. After washing with PBS, the secondary antibody (alkaline phosphatase conjugated Goat anti-mouse IgG) was diluted 1: 3,000 in blocking buffer, treated with 90 minutes at 37 ° C, washed with PBS, and washed with alkaline phosphatase. (alkaline phosphatase) substrate solution (1 mg / ml, p-mitrophenylphosphate in diethanolamine buffer) was added and reacted at room temperature for 30 minutes. After the reaction was completely stopped with 3 N NaOH, the absorbance was measured at 405 nm using a 'microplate reader'.

As a result, as shown in Figure 10, it was confirmed that the expression of MMP-1 is suppressed concentration-dependent. From this, the present invention was confirmed to have the effect of suppressing the expression of MMP-1, ultimately it was confirmed that the effect of improving skin wrinkles.

Experimental Example 7: Collagen Production Effect of Acerola Fruit Extract]

In this experiment, the collagen production effect of acerola fruit extract was tested.

Fibroblasts were seeded at a density of 1 × 10 ⁴ cells / well in 48 well plates, incubated for 24 hours in DMEM medium containing 10% FBS, and further cultured in serum-free medium containing acerola fruit extract for 24 hours. It was. The amount of free collagen (procollagen) in the medium was measured using the 'procollagen type I C-peptide EIA kit (MK101, Takara, Japan)'.

As a result, as shown in Figure 11, the content of procollagen did not increase by treatment of the acerola fruit extract of the present invention.

Experimental Example 8: Tyrosinase Inhibitory Effect of Acerola Fruit Extract

As an experiment for measuring the whitening effect in the skin, the inhibitory effect of tyrosinase was measured using the dopachrome method. 150 μl of mushroom tyrosinase-150 unit, 225 μl of 2.5 mM L-tyrosine, 225 μl of 0.4 M hepes buffer (pH 6.8), and 300 μl of ethanol After mixing a solution or a sample (1 mg / ml) solution, the cells were incubated for 15 minutes before and after incubation, and the absorbance was measured at 475 nm, respectively.

As a result of measuring the tyrosinase inhibitory activity of the acerola fruit extract, as shown in Figure 12, the acerola fruit extract showed inhibition rates of 20.1, 31.5 and 79.5% at 0.1, 1.0 and 10 mg / mL, respectively. Therefore, it was confirmed that the acerola fruit extract of the present invention has excellent tyrosinase inhibitory activity, and it was confirmed that the skin whitening effect was present.

Experimental Example 9: Melanin Production Inhibitory Effect of Acerola Fruit Extract]

B-16 F1 cells, which are mouse-derived melanocytes, produce melanin. Melanin (Melanin) is biosynthesized in the melanocytes (melanosome) in the melanocytes (melanocyte) present in the basement layer of the epidermis, which can be compared by measuring the absorbance of the visible region.

Therefore, in the present invention, after treating the sample for 3 days after B-16 F1 cell inoculation, the medium is removed, the cells are washed with PBS, 1 ml of 1 N NaOH per well is added, followed by stirring to fuse the melanin (melanin). melanin) was dissolved. The amount produced was compared by measuring the absorbance at 400 nm, which is the shortest blue visible light region in the visible region.

     As a result, as shown in Figure 13, the acerola fruit extract of the present invention was confirmed to inhibit the production of melanin (melanin) in a concentration-dependent manner. From this, it could be confirmed once again that the acerola fruit extract of the present invention has a skin whitening effect.

Experimental Example 10 Determination of Anti-inflammatory Activity of Acerola Fruit Extract

 (1) Cell culture

Mouse-derived macrophage line J774.1 used RPMI1640 medium and Raw264.7 used DMEM medium. Cells were incubated in 10% FBS and RPMI 1640 medium or DMEM medium and placed in a 24 well plate at a concentration of 4.5 x 10 cells / well, and then the sample was added to and not added to both groups at 37 ° C and 5% CO. After 48 hours of incubation in ² incubators, LPS (lipipilysaccharide) was treated at a concentration of 200 ng / well to incubate the cells for a predetermined time. HaCaT cells, a human Keratinocyte cell line (ATCC, USA), were cultured in 10% FBS and DMEM medium and placed in a concentration of 4.5 x 10 cells / well in a 24 well plate.The sample was added and the two groups were not added. All were incubated for 48 hours at 37 ℃, 5% CO ² incubator.

 (2) NO production measurement of acerola fruit extract

100 μl of the cell culture supernatant cultured above was collected, and the same amount of the grease reagent was mixed and reacted for 15 minutes, and then the absorbance was measured at 540 nm using a 'microplate reader'. Sodium nitrite was used as a standard for nitrite, and the concentration was calculated by comparing with a standard curve obtained by diluting twice with RPMI 1640 medium from 32 μM to 0.25 μM.

Nitric oxide (NO) production amount was confirmed in order to confirm the inflammatory effect on the acerola extract, which is shown in FIG. 14. In the untreated group containing only no sample, 0.2 μM was produced, and when LPS was treated, the production of 35.0 μM showed that a large amount of NO was induced by endotoxin LPS. When treated with acerola extract after LPS treatment, it can be seen that the concentration of NO production decreases. Through this, the acerola extract of the present invention was confirmed to exhibit an anti-inflammatory effect. In general, skin irritation causes an inflammatory response. From the confirmation of the anti-inflammatory effect of the acerola extract of the present invention, it could be deduced that the skin irritation is effective.

 (3) Cytokine Production Measurement of Acerola Fruit Extract

Raw264.7 cells, a mouse macrophage line cultured above, were pretreated with acerola fruit extract, treated with 1 μg / ml LPS, an inflammation-causing substance, and secreted IL-6 and TNF in the medium after a certain time. The amount of -α was measured using the 'Elisa detection kit (Life Technologies, USA)'. The ELISA kits used were tested according to the manufacturer's recommendation. All experiments were repeated in duplicate and the concentration was measured at 450 nm using an 'ELISA microplate reader'.

The amount of TNF-α production is shown in Table 7 below. The control group treated only with medium showed 401.6 pg / ml production, the LPS treatment group showed 1154.2 pg / ml production, and in addition to LPS treatment, the sample treated with the present invention (1.0 mg / ml) was treated with TNF-. It was confirmed that the production amount of α was lowered to 986.3 pg / ml.

The production results of IL-6 are shown in Table 8. When the IL-6 production amount was confirmed, the control group treated with the normal medium showed a production amount of 45.3 pg / ml, and the LPS-only group showed a production amount of 1103.5 pg / ml, and the sample of the present invention was added to the LPS treatment. In the treated group (1.0 mg / ml) was confirmed to be lowered to 135.6 pg / ml.

Whether Acerola Fruit Extract Reduces TNF-α Sample 0.1 mg / ml 1.0 mg / ml 100 mg / ml TNF-α
(pg / ml)
Control 401.6 ± 15.4 LPS 1154.2 ± 29.4 SAMPLE 1013.1 ± 17.2 986.3 ± 21.5 453.2 ± 14.8

Reduction of IL-6 in Acerola Fruit Extract Sample 0.1 mg / ml 1.0 mg / ml 100 mg / ml IL-6
(pg / ml)
Control 45.3 ± 11.2 LPS 1103.5 ± 10.7 SAMPLE 580.2 ± 9.2 135.6 ± 17.2 51.2 ± 9.5

Experimental Example 11: Gene Expression Measurement by Real Time PCR

Raw 264.7 cells, a mouse macrophage line, were pretreated with acerola fruit extract, and then treated with 1 ㎍ / ml LPS, an inflammation-causing substance, and RNA was isolated from the cells after a certain time. RNA isolation was performed using the RNA isolation kit (Life Technologies, USA) according to the manufacturer's recommendations.

Using 2 μg of isolated RNA, prepare a c-DNA using the 'C-DNA Synthesis kit (Life Technologies, USA)' according to the manufacturer's recommendation, and use 'QuantiTest SYBR green PCR kit (Qiagen, Kore)'. Real-time PCR was performed. Primers specific for the selected gene were selected and used in the experiments. The base sequences of the primers were shown in Table 9 below.

Base sequence of primer Gene types sense primer anti-sense primer Cox2 5'-CAAAAGCTGGGAAGCCTTCT-3 ' 5'-CCATCCTTGAAAAGGCGCAG-3 ' iNOS 5'-GTTCTCAAGGCACAGGTCTC-3 ' 5'-GCAGGTCACTTATGTCACTTATC-3 ' c-Fos for AP-1 5'-AGAATCCGAAGGGAAAGGAA-3 ' 5'-CTTCTCCTTCAGCAG GTTGG-3 hHMOX1 5'-AAGACTGCGTTCCTGCTCAAC-3 ' 5-'AAAGCCCTACAGCAA CTGTCG-3 ' hTrxR1 5 -ATGGGCAATTTATTGGTCCTCAC-3 ' 5'-CCCAAGTAACGTGGTCTTTCAC-3 ' GAPDH 5 -CATGAGAAGTATGACAACAGCCT-3 ' 5'-AGTCCTTCCACGATACCAAAGT-3 ' Involucrin 5'-CAAAGAACCTGGAGCAGGAG-3 ' 5'-CAGGGCTGGTTGAATGTCTT-3 '

All real-time PCR uses 'ABI 7300 system (Applie Biosystems, USA)' and PCR amplification condition is 15 minutes at 95 ℃, initial denaturation process, 15 seconds at 95 ℃, 55 ℃ 15 cycles, the cycle of 30 seconds at 72 ℃ was repeated 40 cycles, the last extension (final extension) was maintained for 5 minutes at 72 ℃.

Experimental results, as shown in Figure 15, It was confirmed that the expression level of each gene increases in a concentration-dependent manner.

Experimental Example 12: Hyaluronidase Activity Inhibitory Effect of Acerola Fruit Extract]

In addition to the skin whitening effect, the hyaluronidase inhibitory activity was confirmed in order to examine the skin protection effect, which is one of the important factors of cosmetic activity. Hyaluronidase is a degrading enzyme of hyaluronic acid that contributes to skin moisturization and elasticity and is known to be involved in skin immunity. Therefore, the hyaluronidase inhibitory activity was measured to compare the hyaluronidase inhibitory activity of the acerola extract.

The inhibitory effect of hyaluronidase activity was determined by 50 μl of hyaluronidase (77900 unit / ml) dissolved in 0.1 M acetate buffer (pH 3.5). 20 μl each was added to make mg / ml, and 200 μl of 12.5 mM CaCl 2 was mixed for activation of the enzyme, followed by incubation for 20 minutes in a 37 ° C. water bath. As a control, DMSO solution was added and incubated for 20 minutes in a water bath. To the activated hyaluronidase solution, 250 μl of hyaluronic acid (12 mg / 5 ml) dissolved in 0.1 M acetate buffer (pH 3.5) was added and incubated for 40 minutes in a water bath. Then, 100 μl of 0.4 N NaOH solution and 100 μl of 0.4 M potassium tetraborate were added to the reaction mixture, followed by incubation in a boiling water bath for 3 minutes, followed by cooling. 3.28 ml of 'dimethyl aminobenzaldegye solution (4 g of p-dimethyl amino-benzaldehyde, 350 ml of 100% acetic acid, and 50 ml of 10 N HCl)' was added to the reaction mixture, followed by incubation for 20 minutes in a 37 ° C water bath. And absorbance at 585 nm. Inhibition rate was calculated as follows.

As a result, as shown in FIG. 16, the hyaluronidase inhibitory activity of the extract was the highest as 42.3% at the concentration of 100 mg / mL, and the concentration-dependent inhibition of hyaluronidase activity was increased. Indicated.

On the other hand, the following attempt to compare the functionality of the acerola extract obtained through the nitrogen injection extraction method established above and the acerola extract obtained through the general extraction method. At this time, the extraction of both methods is the extraction for 2 hours at 25 ℃. In the following, 'Acerola extract A' is an acerola extract obtained through a nitrogen purge extraction method, 'Acerola extract B' is an acerola extract obtained through a general extraction method.

Experimental Example 13: Cross-functional Comparison of General Extracts and Nitrogen Purging Extracts

 1. Comparison of whitening efficacy

  One) in vitro tyrosinase activity inhibition test

Acerola extracts A, and B were prepared by dissolving in sterile distilled water, respectively, and diluting them from the maximum concentration without cytotoxicity. 0.1 M phosphate buffer, sample, mushroom tyrosinase was added sequentially and reacted. Then, 1.5 mM tyrosine was added and reacted at 37 ° C. for 15 minutes, and the absorbance was measured at 490 nm using an 'ELISA reader'.

The tyrosinase activity inhibition test results of the acerola extract were as shown in FIG. 17. As shown in FIG. 17, the nitrogen purging extract (Acerola extract A in FIG. 17) of the present invention showed a higher inhibitory effect on tyrosinase activity than the general extract (Acerola extract B in FIG. 17). Meanwhile, in the case of acerola extract A, the IC50 concentration of inhibiting tyrosinase activity was 0.0835%, and in the case of acerola extract B, the IC50 concentration was 0.0967%, indicating that nitrogen purge extract (Acerola extract A) was more sensitive to tyrosinase activity. It was confirmed that there is an inhibitory effect.

 2) Melanin production inhibition test

B-16 F1, a mouse-derived melanoma cell, was divided into 6 well plates at a concentration of 1 x 10 ⁵ cells / well and incubated for 24 hours. After removing the culture solution, washed twice with PBS, and then treated with acerola extract A, and B each concentration in a complete medium and incubated for 72 hours. Cells attached to the bottom were washed twice with PBS, and then cell lysed using M-per cell lysis buffer, and cell lysate was collected and centrifuged at 3000 rpm for 10 minutes. The supernatant was recovered and the protein was measured using the 'Pierce BCA proteion assay kit'. Cell pellet dried in 70 ℃ oven dried melanocytes were extracted by adding 1N NaOH containing 10% DMSO in a 60 ℃ constant temperature water bath. The melanin amount measured using the melanin standard was corrected with the measured protein amount to yield a result.

Melanin production inhibition test results of the acerola extract was as shown in FIG. In the case of nitrogen purge extract (Acerola extract A in Fig. 18), statistically significant decrease in melanogenesis was observed at the applied concentrations of 0.01%, 0.05% and 0.1% (p <0.05), 0.05% (0.5 mg / mL) And at a concentration of 0.1% (1 mg / mL), a decrease in melanin production was found to be more than 30% compared to the control.

Claims (7)

Purified water is added to the freeze-dried powder of acerola fruit, and acerola extract is prepared by purging nitrogen at a temperature of 25 ° C. for 120 minutes.
The acerola extract is a solid content of 55 to 57% by weight, vitamin C content of 22 to 24% by weight of the production method of the acerola extract.
delete delete Adding purified water to the lyophilized powder of acerola fruit, and purifying nitrogen for 120 minutes at a temperature of 25 ° C .;
The acerola extract has a solid content of 55 to 57% by weight, vitamin C content of 22 to 24% by weight of the preparation method of the cosmetic composition for antioxidant.
Adding purified water to the lyophilized powder of acerola fruit, and purifying nitrogen for 120 minutes at a temperature of 25 ° C .;
The acerola extract has a solid content of 55 to 57% by weight, vitamin C content of 22 to 24% by weight of the method for producing a cosmetic composition for preventing skin wrinkles.
Adding purified water to the lyophilized powder of acerola fruit, and purifying nitrogen for 120 minutes at a temperature of 25 ° C .;
The acerola extract has a solid content of 55 to 57% by weight, the vitamin C content of 22 to 24% by weight of the method for producing a cosmetic composition for skin whitening.
Adding purified water to the lyophilized powder of acerola fruit, and purifying nitrogen for 120 minutes at a temperature of 25 ° C .;
The acerola extract has a solid content of 55 to 57% by weight, the vitamin C content of 22 to 24% by weight of the method for producing a cosmetic composition for skin irritation.

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