KR102546187B1 - Cosmetic composition for wrinkle improvement including fucoxanthin derived from diatoms - Google Patents

Abstract

The present invention relates to a cosmetic composition for improving wrinkles containing fucoxanthin, and more particularly, to an invention in which fucoxanthin is separated and purified from adherent diatoms taken together with Jeju lava seawater and used as a cosmetic composition for wrinkle improvement. . The present invention, which relates to a cosmetic composition containing fucoxanthin as an active ingredient, is related to the cosmetic composition containing fucoxanthin as an active ingredient in that high-purity fucoxanthin, separated and purified in an optimal way from adherent diatoms taken from Jeju lava seawater in Korea, has an effect of improving skin wrinkles. It can be used in various industrial fields such as beauty field.

Description

Cosmetic composition for wrinkle improvement including fucoxanthin derived from diatoms}

The present invention relates to a cosmetic composition for improving wrinkles containing fucoxanthin, and more particularly, to an invention in which fucoxanthin is separated and purified from adherent diatoms taken together with Jeju lava seawater and used as a cosmetic composition for wrinkle improvement. .

Lava seawater is seawater that penetrates the basalt layer of Jeju Island and flows underground. Lava seawater is found mainly in the western and eastern parts of Jeju Island and contains a high concentration of minerals. In addition, general seawater is exposed to unstable environments such as domestic sewage, industrial wastewater, and port pollution, requiring a lot of cost for industrial material processing, while lava seawater is naturally purified and filtered by volcanic bedrock, adsorbing heavy metals and blocking harmful substances. Therefore, it secures safety, stability, and economic feasibility, and the cost of water intake is incomparably cheaper than that of deep sea water taken from the deep sea.

The inventor of the present application confirmed that adherent diatoms were taken in together with lava seawater in the process of aquaculture using Jeju lava seawater, and established a method for mass-cultivating these adherent diatoms. Adherent diatoms can be used as a resource with high value as an alternative energy source for feed in the aquaculture industry or as a basic material in various industries such as medicine, environment, and bioindustry.

On the other hand, the carotenoid market is classified into synthetic carotenoids and natural carotenoids, and is achieving steady growth. The global carotenoid market recorded $1.5 billion in 2014 and is expected to increase to about $1.8 billion in 2019. In particular, the value of xanthophyll-based carotenoids such as lutein, canthaxanthin, and astaxanthin is increasing, and are mainly used as natural pigments, antioxidants, vitamin A precursors, and aquaculture feed additives.

Fucoxanthin refers to a kind of carotenoid pigment mainly present in brown algae such as wakame, kelp, kelp, and fusiformis. The brown color of brown algae is because the unique photosynthetic pigment called fucoxanthin is relatively more than chlorophyll. It assists in photosynthesis by capturing light energy and delivering about 80% of it to chlorophyll. The captured light energy absorbs light with a wavelength of 450 to 540 nm on the spectrum, and absorbs the most light with a wavelength of 510 to 525 nm, and is known to have various effects such as anti-obesity and anti-inflammatory.

On the other hand, as the physiological changes of the skin due to aging, the thickness of the epidermis, dermis, and subcutaneous tissue, which are components of the skin, becomes thinner, and the lipid composition and content of the lipid barrier, which is in charge of the skin barrier function, changes, resulting in a change in its function. As this rapidly decreases, the moisture content of the skin decreases, the skin becomes dry, and various changes such as spots, freckles, and pigmentation appear. Recently, among these aging processes, various studies have been conducted on changes in extracellular matrix (ECM) components and collagen, a major component of ECM, which are deeply related to wrinkles and elasticity of the skin. Collagen accounts for 70-80% of ECM components, and its production rapidly decreases with age, and is known to cause wrinkles. In addition, deterioration in function due to oxidation of elastin, proteoglycan, glucosaminoglycan, laminin, and fibronectin, which constitute skin connective tissue in addition to collagen, is known and studied as the main cause of reduced skin elasticity and wrinkle formation.

Various cosmetic compositions are being studied to solve the problem of skin aging, but there is no example in which fucoxanthin is used as a cosmetic composition for reducing skin elasticity and preventing wrinkle formation.

Provided is a functional cosmetic composition for improving skin elasticity and wrinkles containing fucoxanthin separated from adherent diatoms taken from Jeju lava seawater in Korea.

In order to solve the above problems, the present invention provides a cosmetic composition for improving wrinkles containing Fucoxanthin.

In the present invention, fucoxanthin may be separated and purified from diatoms.

Also, in the present invention, diatoms may be adherent diatoms that are taken together with Jeju lava seawater.

In addition, in the present invention, the adherent diatoms are Melosira nummuloides, Achnanthes brevipes var. intermedia, Achnanthes sancti-pauli, Achnanthes brevipes or Melosira octogona.

Also, in the present invention, diatoms may be diatoms collected from bottom diatom culture.

In addition, in the present invention, fucoxanthin may be included in 2% by weight compared to the total cosmetic composition.

In addition, in the present invention, the cosmetic composition may further include a stabilizer, a solubilizer, vitamins, pigments, or fragrances.

In the present invention, the formulation of the cosmetic composition may be a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, cleansing, oil, powder foundation, emulsion foundation, wax foundation, or spray. .

In addition, the present invention provides a cosmetic comprising the cosmetic composition for improving wrinkles as an active ingredient.

In the present invention, cosmetics are at least one from the group consisting of softening lotion, astringent lotion, nutrient lotion, hair growth nutrient, eye cream, nutrient cream, massage cream, cleansing cream, cleansing foam, cleansing water, powder, essence, serum and pack can be chosen

The present invention, which relates to a cosmetic composition containing fucoxanthin as an active ingredient, is related to the cosmetic composition containing fucoxanthin as an active ingredient in that high-purity fucoxanthin, separated and purified in an optimal way from adherent diatoms taken from Jeju lava seawater in Korea, has an effect of improving skin wrinkles. It can be used in various industrial fields such as beauty field.

1 is a standard solution 1 in which coxanthin is dissolved in methanol after stirring raw diatoms in alcohol, loading and filtering on a filter bed precoated with diatomaceous earth, and separating and refining by silica gel column chromatography It shows the HPLC analysis for.
Figure 2 is HPLC for the mother liquor of standard solution 2 in which the raw material of diatomite was stirred in alcohol, loaded on a filter bed precoated with diatomaceous earth, filtered, and stored at -20 ° C for two days for recrystallization. analysis is shown.
Figure 3 is HPLC for the crystal of standard solution 2 in which the raw material of diatomite was stirred in alcohol, loaded on a filter bed precoated with diatomaceous earth, filtered, and stored at -20 ° C for two days for recrystallization. analysis is shown.
Figure 4 shows the HPLC analysis of standard solution 3 prepared in the same way as standard solution 1, but without desalination.
Figure 5 shows the HPLC analysis of Standard Solution 3 prepared in the same way as Standard Solution 1, but subjected to desalination.
Figure 6 shows the HPLC analysis of standard solution 4 prepared in the same way as standard solution 1, but exposed to sunlight.
7 shows HPLC quantitative analysis of Sample No 4 in Table 1.
8 shows the HPLC quantitative analysis of Sample No 5 in Table 1.
9 shows the HPLC quantitative analysis of Sample No 6 in Table 1.
10 shows a schematic diagram of a coxanthin separation and purification process.
11 shows a process for separating and purifying fucoxanthin by silica gel column chromatography.
12 shows the TLC (Thin Layer Chromatography) results of the fraction containing fucoxanthin.
Figure 13 shows the concentration process of coxanthin.
Figure 14 shows the preparation of a standard calibration curve using standard postcoxanthin (purchased from Sigma Aldrich).
15 shows HPLC quantitative analysis of 60% purity postcoxanthin finally obtained through fractionation/concentration from 70% alcohol extract of diatom powder.
16 shows the diatomaceous earth precoat filtration and recrystallization process.
17 shows micrographs (×400) of filtration and recrystallization.
18 shows HPLC quantitative analysis of coxanthin after recrystallization.
19 shows HPLC quantitative analysis of Sample No 1 in Table 3.
20 shows HPLC quantitative analysis of Sample No 3 in Table 3.
21 shows HPLC quantitative analysis of Sample No 15 in Table 3.
22 shows HPLC quantitative analysis of Sample No 16 in Table 3.
23 shows a powder formulation of fucoxanthin.
24 shows HPLC quantitative analysis of Sample No 8 in Table 3.
25 shows HPLC quantitative analysis of Sample No 9 in Table 3.
26 shows HPLC quantitative analysis of Sample No 10 in Table 3.
27 shows HPLC quantitative analysis of Sample No 11 in Table 3.
28 to 47 show HPLC quantitative analysis of samples 1 to 20 in Table 6.
48 shows a coxanthin sample subjected to the collagenase activity inhibition test.
Figure 49 shows the cell viability according to the administration concentration of fucoxanthin.
50 shows the inhibition rate of intracellular collagenase activity of coxanthin.
51 and 52 are graphs showing the anti-wrinkle effect of fucoxanthin.

In one embodiment according to the present invention, a cosmetic composition for improving wrinkles containing fucoxanthin is provided.

In another embodiment according to the present invention, fucoxanthin may be separated and purified from diatoms.

In another embodiment according to the present invention, the diatoms may be adherent diatoms that are taken together with Jeju lava seawater.

In another embodiment according to the present invention, the adherent diatoms are Melosira nummuloides, Achnanthes brevipes var. intermedia, Achnanthes sancti-pauli, Achnanthes brevipes or Melosira octogona.

In another embodiment according to the present invention, the diatoms may be diatoms harvested from a bottom diatom culture.

In another embodiment according to the present invention, fucoxanthin may be included in 2% by weight compared to the total cosmetic composition.

In another embodiment according to the present invention, the cosmetic composition may further include a stabilizer, a solubilizer, vitamins, pigments or fragrances.

In another embodiment according to the present invention, the formulation of the cosmetic composition is a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, cleansing, oil, powder foundation, emulsion foundation, wax foundation, or spray may be

In addition, as an embodiment according to the present invention, a cosmetic comprising a cosmetic composition for improving wrinkles as an active ingredient is provided.

In another embodiment according to the present invention, cosmetics consist of softening lotion, astringent lotion, nutrient lotion, hair growth nutrient, eye cream, nutrient cream, massage cream, cleansing cream, cleansing foam, cleansing water, powder, essence, serum and pack One or more may be selected from the group.

Hereinafter, the present invention will be described in more detail through specific examples. These examples are only for exemplifying the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example 1. Optimization of fucoxanthin separation and purification method

The separation and purification method was optimized by measuring the change in the content of coxanthin according to the separation and purification method. To this end, 50 g of wet diatoms were first dried in a vacuum dryer for 48 hours, and then the content change of coxanthin by various processes was measured using HPLC.

First, standard solution 1 was stirred in 70% alcohol for 2 hours, loaded with diatomaceous earth on a filter bed, filtered, and concentrated under reduced pressure. Accordingly, 1.2 g of the sample was weighed with an electronic balance and subjected to silica gel column chromatography (Φ 3.5 cm x 15 cm), and N-Hexane: Acetone = 70: 30 was used as the elution solvent. After separation and purification, coxanthin was dissolved in methanol to prepare standard solution 1 having a concentration of 0.1%. Thereafter, standard solution 1 was dispensed into a transparent container and the content of coxanthin was measured by HPLC method. There was no color change, and the postcoxanthin content was calculated based on the peak area value. When compared with the standard solution 2 below, the content of fucoxanthin was found to be high, and other components were removed, resulting in a purity of 90% (FIG. 1).

Next, standard solution 2 was prepared through a recrystallization process. When a solute is dissolved in a solvent at a high temperature, more solute can be dissolved than at a lower temperature. In this dissolved state, if the temperature is lowered slowly again, the solubility decreases, so the solute precipitates again and crystallizes, which is called recrystallization. In the present invention, standard solution 2 was prepared by stirring and filtering 5 g of raw diatoms in 70% alcohol for 2 hours, and then storing the filtered sample at -20 ° C for two days for recrystallization experiments. Thereafter, standard solution 2 was dispensed into a transparent container and the content of coxanthin was measured by HPLC method. There was no color change, and the postcoxanthin content was calculated based on the peak area value. Compared to standard solution 1, the chlorophyll component was not removed, so the crystal sample had a purity of 75% and the mother liquor sample had a purity of 61% (FIGS. 2 and 3).

Standard solution 3 was carried out in the same way as standard solution 1, except that the desalination process for raw diatoms was added, and the effect of the desalination process was verified. Standard solution 3 was dispensed into a transparent container and the content of coxanthin was measured by HPLC method. There was no color change, and the postcoxanthin content was calculated based on the peak area value. The observed coxanthin content was lowered to 21% compared to standard solution 1 when desalting was performed, and lowered to 17% when desalting was not performed (FIGS. 4 and 5).

In the following standard solution 4, the stability of fucoxanthin was verified through the content change when fucoxanthin was exposed to sunlight. Standard solution 4 was dispensed into a transparent container, and the content of coxanthin was measured by HPLC method. There was no color change, and the postcoxanthin content was calculated based on the peak area value. As a result of observing the content and purity of coxanthin after being left in sunlight for a week, the content decreased by about 82%, and the purity decreased from 90% to 55% (FIG. 6).

In conclusion, as a result of HPLC analysis of the fucoxanthin preparation dissolved in methanol at a concentration of 0.1%, standard solution 1 was found to be excellent in terms of content and purity, and separation and purification were not performed properly when silica gel column chromatography was not performed. As a result, it was found that the content and purity of fucoxanthin were lowered. In addition, it was confirmed that the desalting process did not significantly affect the content and purity of coxanthin, and the recrystallization process also showed a slightly lower purity and content of coxanthin compared to standard solution 1. Furthermore, it was confirmed in standard solution 4 that fucoxanthin acts sensitively to sunlight exposure.

Example 2. Determination of Titration Solvent in Fucoxanthin Extraction

In the separation of fucoxanthin, solvent extraction of the same kind of microalgae powder showed the highest extraction yield when alcohol was used, and other extraction methods (Sohxlet method, ultrasonic extraction method, subcritical extraction method, etc.) when alcohol was used as the solvent 70% alcohol was used as an extraction solvent in consideration of the results that were not significantly different from those used and the fact that it could be used for food.

However, since the amount of extraction solvent used is an important factor in determining the production cost in the future industrialization process, an appropriate solvent ratio compared to the original diatom material was tested. 100 g of diatom powder (sample Nos. 1, 2, 3) was stirred with 10 times 70% alcohol, and 10 times, 20 times, and 30 times 70% alcohol were stirred with 50 g (No. 4, 5, 6), respectively. Extraction efficiency was compared and shown (Table 1, Figures 7 to 9)

sample name Ingredient content
(μg/mL (ppm)) s.d.
[standard deviation] note
(total content) Sample No 1 240.40 2.27 900 mL of filtrate x 240 = 216 mg Sample No 2 335.00 3.54 900 mL of filtrate x 335 = 301 mg Sample No 3 366.30 3.89 900 mL of filtrate x 366 = 329 mg Sample No. 4 418.53 4.54 600 mL of filtrate x 418 = 250 mg Sample No. 5 289.48 2.36 950 mL of filtrate x 289 = 274 mg Sample No. 6 186.64 0.29 Filtrate 1400mLx 186 = 260mg Positive control STDs 158.71 0.46

(※ No.1-3; 70% alcohol 10x filtrate, No.4; 70% alcohol 10x filtrate, No.5; 70% alcohol 20x filtrate, No. 6; 70% alcohol 30x filtrate)

A total of 250 mg was extracted from the section extracted with 10-fold volume of alcohol, 274 mg from 20-fold volume, and 260 mg of fucoxanthin from 30-fold volume. The amount of solvent was based on 10 times 70% alcohol. However, additional experiments were conducted to determine more accurate solvent conditions, and the results are shown in Example 9 below.

Example 3. Separation and purification of coxanthin

Silica gel chromatography is used for the separation and purification of functional substances such as fucoxanthin. will be. In the present invention, silica gel chromatography was performed under various conditions in order to efficiently separate and purify the target substance, fucoxanthin.

In the entire process, according to FIG. 10, 70% alcohol in a 10-fold volume was added to diatom powder, stirred for 2 hours, filtered, and each extract was obtained, followed by fractionation and concentration, and then separated by silica gel chromatography.

Specifically, first, the extract extracted from 100 g of diatom powder with 70% alcohol at 10 times the capacity was filtered with a 5um filter paper, and the primary alcohol extract obtained was separated into n-Hexane: 70% alcohol (1:1) to obtain 70% alcohol in the lower layer. fractions were obtained. In order to separate fucoxanthin from the fractions, a column was filled with silica to load the sample, and a gradient (Hexane: Acetone = 7:3 → 6:4 → 5:5 → Acetone) separation process was performed to obtain final fractions. . Thereafter, a fraction expected to contain fucoxanthin (dark orange layer) was secured and TLC (Thin Layer Chromatography) was performed (FIGS. 11 and 12), and as a result, fractions from Fraction No. 1 to No. 9 After confirming that coxanthin was separated, the concentration process was performed using a high-pressure vacuum concentrator (FIG. 13).

Example 4. Quantitative analysis of coxanthin using HPLC

First, an HPLC standard calibration curve was prepared by diluting by concentration using a standard material, coxanthin (purchased from Sigma Aldrich) (FIG. 14). For HPLC analysis, Waters 2695 HPLC equipment was used, the column used was Kromasil 100-5-C18 (4.6x250mm, 5um), the detection wavelength was 450nm, and the solvent was Acetonitrile / water (Table 2).

Control Factor Conditions Injection volume 10 uL Column Kromasil 100-5-C18
(4.6×250mm, 5um) Mobile phase A: 0.1% FA in ACN, B: 0.1% FA in H ₂ O Flow rate 0.7mL/min Column Temperature 35℃ Wavelength 450 nm Detector Waters 2998 PDA (Waters, USA) Separation Module Waters 2695 (Waters, USA) Time(min) Flow (mL/min) A (0.1% FA in ACN) B (0.1% FA in H ₂ O) 0 0.7 10 90 10 0.7 60 40 17 0.7 100 0 30 0.7 100 0 35 0.7 10 90 40 0.7 10 90

As a result of HPLC quantitative analysis, 8.7 g of fucoxanthin with a purity of 60% was finally obtained through fractionation / concentration from 70% alcohol extract of 100 g of diatom powder, resulting in a yield of 5.22% (FIG. 15).

Example 5. Usability test of recrystallization method

In order to improve the degree of purification and yield of fucoxanthin, the usefulness of recrystallization of the concentrate was examined. Specifically, the primary extract obtained by stirring 100 g of diatom powder with 70% alcohol at 10 times the capacity for 2 hours was loaded onto a filter bed coated with 50 g of diatomaceous earth to obtain an extract filtrate. The finally obtained concentrate was dissolved in acetone, and the fucoxanthin fraction was sampled by silica gel chromatography and checked by TLC (Thin Layer Chromatography) to confirm that fucoxanthin was separated (FIG. 16).

Recrystallization was attempted from the postcoxanthin fraction, and the recrystallization process is as follows. The sample was mixed with the same amount of water at 50 ° C, stirred at 40 ° C for 2 hours, stored overnight at -20 ° C, and recrystallized through filtration. The crystal of coxanthin was confirmed through microscopic observation (400 magnification) (FIG. 17), and HPLC quantitative analysis was performed under the above conditions (FIG. 18). As a result, although postcoxanthin concentration was possible through recrystallization, it was confirmed that the usefulness was not great for separating the desired high-purity postcoxanthin.

Example 6. Comparison of fucoxanthin content in various culture/environmental conditions

Changes in postcoxanthin content in diatoms according to diatom culture environment, sea salt removal, drying type, drying temperature, etc. were compared and analyzed (Figs. 19 to 22, Table 3).

sample name Ingredient content
(μg/mL (ppm)) s.d.
[standard deviation] note Sample No 1 51.03 2.13 Dissolve at 50 mg/mL and assay Sample No 2 20.52 1.12 Dissolve at 50 mg/mL and assay Sample No 3 26.65 1.28 Dissolve at 50 mg/mL and assay Sample No. 4 27.44 1.27 Dissolve at 50 mg/mL and assay Sample No. 5 56.38 2.03 Dissolve at 50 mg/mL and assay Sample No. 6 67.58 2.42 Dissolve at 50 mg/mL and assay Sample No. 7 49.66 1.86 Dissolve at 50 mg/mL and assay Sample No. 8 316.99 8.91 Dissolve at 50 mg/mL and assay Sample No 9 61.22 2.16 Dissolve at 50 mg/mL and assay Sample No. 10 48.69 1.83 Dissolve at 50 mg/mL and assay Sample No. 11 43.68 1.73 Dissolve at 50 mg/mL and assay Sample No. 12 43.92 1.81 900 mL of filtrate x 43 = 38 mg Sample No. 13 61.36 2.24 600 mL of filtrate x 61 = 36 mg Sample No. 14 47.43 1.94 900 mL of filtrate x 47 = 42 mg Sample No. 15 272.26 7.96 700 mL of filtrate x 272 = 190 mg Sample No. 16 242.30 7.12 900 mL of filtrate x 242 = 217 mg

(※ No.1; bottom diatom/desalination X/natural drying, No.2; bottom diatom/desalination/drying at 60℃, No.3; floating diatom/desalination X/freezing and thawing/natural drying, No.4; floating diatom /Desalination/60℃ drying, No.5 Bottom diatoms/Desalination 2/Natural drying, No.7 Floating diatoms/40℃ hot air drying, No.8-11: Beta cyclodextrin formulation powder, No.12-14: Drying Powder/1,3BG Extract/10x,7x,10x, No.15; Fresh Diatom/1,3BG Extract 10x, No. 16; Fresh Diatom/Alcohol Extract 10x)

HPLC analysis was analyzed under the same conditions as in the above example, and diatom dry powder (No.1 to No.11) was dissolved in methanol at a concentration of 50 mg/mL, treated with ultrasonic waves for 30 minutes, and used for analysis after filtering. Three replicates were performed. As a result, in the diatom growth environment, it was found that bottom diatoms had higher fucoxanthin content than floating diatoms (Table 3, No.1 & No.3), and the presence or absence of desalination did not significantly affect the change in content. appear.

On the other hand, as a result of analyzing the change in fucoxanthin content according to the drying temperature, it was found that the fucoxanthin content significantly decreased under the condition of a drying temperature of 50 ° C or higher compared to natural drying (Table 3, No.1 to No.5). This result is consistent with the previous research results that fucoxanthin has low thermal stability, and suggests that it is important to set the environment and extraction conditions below 50 ℃ when industrially producing fucoxanthin.

Among the analyzed samples, the highest content of postcoxanthin was the undried diatom sample, and this result indicates that part of the postcoxanthin in the diatom was decomposed during the process of collecting and drying (natural drying or hot air drying) the diatom culture ( No. 15, 16). In conclusion, in the harvesting/processing step of diatom, which is the first step in establishing the mass extraction process of fucoxanthin, it is the most efficient process to harvest the raw diatom itself without going through a drying process and directly enter the extraction process.

Example 7. Formulation of Fucoxanthin

The same amount of betacyclodextrin and an increased amount of 30% were added to the concentrate obtained by preparing the primary alcohol extract, filtering, and concentration, and then freeze-drying was performed for 72 hours. The lyophilized samples were pulverized to prepare a powder formulation, and the content of fucoxanthin in each sample was analyzed (Figs. 23 to 27, Tables 3 No. 8 to 11). As a result of the analysis, there was no significant difference in the content of fucoxanthin according to the amount of dextrin added, and in the sample (No.8) in which the same amount of dextrin was added to the concentrate 5 times more concentrated than other samples, the highest was 316ppm (0.6%). However, it showed problems in color and flavor compared to other samples. In conclusion, it can be confirmed that the most efficient method is to prepare a formulation for food under the condition of adding the least amount of betacyclodextrin as an excipient, that is, by adding the same amount (1:1) of dextrin as the extract concentrate.

Example 8. Analysis of coxanthin extraction efficiency after plasma treatment

In extracting postcoxanthin from fresh diatom, the optimal extraction conditions were determined by comparatively analyzing the extraction efficiency of postcoxanthin with and without plasma treatment.

First, extracts were prepared from biological diatoms treated with plasma and samples without treatment, and the extraction yields of coxanthin were compared. After adding 20 mL of 70% alcohol to 5 g of biological diatoms, treating the plasma for 25 minutes, extracting at 40 ° C for 3 days, centrifuging the extract at 3500 rpm for 10 minutes to remove the extraction residue, and HPLC analysis using the upper extract. As a control, a sample without plasma treatment was set as a control under the same extraction conditions as above. Since the extract was small, the extract was obtained after removing the residue using centrifugation without going through a filtration process (Table 4), and the post-coxanthin extraction efficiency according to the plasma treatment was examined through HPLC analysis (Table 5).

No. sample name Initial volume (mL) Filtrate volume (mL) Remarks (residue removal) 19 NP (No Pla) 20 15 3500rpm, 10min 20 Pla-25min 20 17 3500rpm, 10min

* NP: non-plasma treatment, Pla: plasma treatment

No. sample name Fx content 19 NP (No Pla) 1.08 20 Pla-25min 1.22

According to the above table, 1.08 mg was extracted from the sample not treated with plasma, whereas 1.22 mg was extracted from the sample treated with plasma, confirming that the plasma treatment increased the extraction efficiency by about 13%. This suggests that the plasma treatment of the sample resulted in a change in the physical properties of the diatom cell wall components, and thus the intracellular components were easily eluted.

Example 9. Optimization of fucoxanthin extraction efficiency

In extracting fucoxanthin from fresh diatoms, the optimal extraction conditions were determined by comparatively analyzing the extraction efficiency according to the type of solvent used, the amount of solvent, and the extraction time.

First, in order to determine the conditions according to the amount and time of the extraction solvent, the sample amount was 50 g of biological diatoms (No.1-18), and 70% alcohol or 1,3BG (Butylene Glycol) was used as the extraction solvent at 40 ° C. , extracted by setting the amount of solvent 3 times, 5 times, and 7 times the sample amount (v / v), respectively (sample name = alcohol solvent: A3, A5, A7. BG solvent: B3, B5, B7), here Extraction was performed by setting the extraction time to 1 day, 3 days, and 5 days, respectively (sample name = 1 day: 1 d, 3 days: 3 d, 5 days: 5 d).

The filtration and centrifugation process is as follows. Each extracted sample was filtered with a filter paper (Hyundai No.21: 8-12um, 90mm) and a vacuum pump to remove residues, and then used for analysis (Table 6, Figs. 28-47). However, since the BG extract has a very high viscosity, it takes more than 24 hours for filtration, which causes fluctuations in the extraction time conditions. Therefore, samples extracted for 3 days and 5 days were centrifuged at 3500 rpm for 10 minutes to obtain an extract (No.10 to 12 , No. 16 to 18).

No. sample name Initial volume (mL) Filtrate volume (mL) note One A3-1d 150 125 2 A5-1d 250 225 3 A7-1d 350 350 4 B3-1d 150 100 5 B5-1d 250 170 6 B7-1d 350 270 2d with filtering time delay 7 A3-3d 150 120 8 A5-3d 250 225 9 A7-3d 350 320 10 B3-3d 150 120 3500rpm, 10min 11 B5-3d 250 210 3500rpm, 10min 12 B7-3d 350 300 3500rpm, 10min 13 A3-5d 150 130 14 A5-5d 250 220 15 A7-5d 350 310 16 B3-5d 150 130 3500rpm, 10min 17 B5-5d 250 210 3500rpm, 10min 18 B7-5d 350 290 3500rpm, 10min 19 NP (No Pla) 20 15 3500rpm, 10min 20 Pla-25min 20 17 3500rpm, 10min

(* A: 70% alcohol, B: 1,3BG, 3/5/7: 3x,5x,7x, d: day, NP: Plasma untreated, Pla: Plasma treated)

amount of solvent extraction time 1d 3d 5d A 3 13 9 8 A 5 29 23 19 A7 43 30 22

(Unit: mg)

First, as a result of analyzing the Fx extraction efficiency according to the extraction time using 70% alcohol as a solvent, 70% alcohol 3 times (150mL) extraction 1 day 13mg, 3 days 9mg, 5 days 8mg, 5 times (250mL) extraction 1 29mg per day, 23mg on 3rd day, 19mg on 5th day, 13mg per day, 9mg on 3rd day, and 8mg on 5th day when extracting 7 times (350mL), the Fx extraction efficiency was found to decrease over time (Table 7). From this, it can be seen that in the 70% alcohol extraction condition, extraction for one day with an amount of alcohol 7 times (v / v) compared to the raw material is most effective.

amount of solvent extraction time 1d 3d 5d B3 18 24 28 B 5 32 38 38 B7 43 45 45

(Unit: mg)

Next, as a result of analyzing the Fx extraction efficiency according to the extraction time with 1,3BG as a solvent, when extracting 1,3BG 3 times (150mL), 18mg on 1 day, 24mg on 3rd day, 28mg on 5th day, 1 when extracting 5 times (250mL) 32mg per day, 38mg per day, 38mg per day, 38mg per day, 7 times (350mL), 43mg per day, 45mg per day, 45mg per day, 45mg per day, 45mg per day. From this result, it can be seen that in the 1,3BG extraction condition, extracting for 3 or 5 days with an amount of 1,3BG 7 times (v / v) compared to the raw material is effective. However, since the difference between 1 day, 3 days, and 5 days at 7 times the amount is 43 mg and 45 mg, it can be said that it is efficient to extract for 1 day at 1,3 BG at 7 times (v/v) compared to the original material, considering the time efficiency. there is.

Example 10. Inhibition of Collagenase Activity of Fucoxanthin

After extracting by the above method, a collagenase activity inhibition test was performed using the coxanthin sample (FIG. 48). First, after calculating the purity of the sample, it was diluted to the corresponding concentration with the culture medium and used. CCD986SK, a human fibroblast cell line used as a cell line, was purchased from ATCC (accession number: CRL-1947) and used. Next, the composition of the medium of the cell culture medium is shown in Tables 9 and 10 below, and the culture conditions were temperature (37 ± 1) ° C, CO ₂ concentration 5%, humidity was humidified, and CO ₂ cell culture was used as the incubator.

designation Composition (mL) Dulbecco's Modified Eagle Medium (DMEM) 445 Heat Inactivated Fetal Bovine Serum (HI FBS) 50 Penicillin-Streptomycin 5 Total volume 500

(composition of complete medium)

designation Composition (mL) Dulbecco's Modified Eagle Medium (DMEM) 99 Heat Inactivated Fetal Bovine Serum (HI FBS) One Total volume 100

(Serum-free medium composition)

Example 10-1. Cytotoxicity test of fucoxanthin

As shown in Table 11 below, the cytotoxicity test was performed by diluting the sample to 5 concentrations.

test group Substance name Dosage concentration (ppm) G1 negative control group - - G2 Test substance group Fucoxanthin 6.25 G3 12.5 G4 25 G5 50 G6 100

First, 3 × 10 ⁴ (200 μl / well) cells were dispensed into a 96 well plate and cultured for 24 hours in a 37 ° C, 5% CO ₂ incubator, and then replaced with a serum-free medium containing samples at different concentrations and maintained at 37 ° C , and cultured again for 24 hours in a 5% CO ₂ incubator. Then, for the MTT assay to measure cell viability, 200 μl/well of MTT solution was dispensed and cultured for 3 hours in a 37 °C, 5% CO ₂ incubator. Minutes of extraction followed by ELISA measurement at a wavelength of 570 nm.

As a result, absorbance was measured at 570 nm using a Microplate reader, and cell viability was calculated according to Equation 1 below, and the results are shown in Table 12 and FIG. 49.

[Calculation 1]

Group Substance Dose(ppm) OD Value
(570 nm) % of control G1 Control - Mean 0.851 100.00 S.D. 0.034 4.00 N 9 9 G2 Fucoxanthin 6.25 Mean 0.907 106.65 S.D. 0.053 6.23 N 9 9 G3 12.5 Mean 0.924 108.59 S.D. 0.053 6.25 N 9 9 G4 25 Mean 0.346 40.69 S.D. 0.067 7.87 N 9 9 G5 50 Mean 0.088 10.36 S.D. 0.002 0.22 N 9 9 G6 100 Mean 0.089 10.42 S.D. 0.003 0.35 N 9 9

N: Number of well, S.D.: Standard deviation

According to Table 12 and FIG. 49, as a result of treatment with fucoxanthin at concentrations of 6.25 ppm, 12.5 ppm, 25 ppm, 50 ppm, and 100 ppm, 106.65 ± 6.23%, 108.59 ± 6.25%, 40.69 ± 7.87 at each concentration %, 10.36 ± 0.22%, 10.42 ± 0.35% of cell viability was observed, and it was confirmed that the cell viability was highest when fucoxanthin was treated at concentrations of 6.25 ppm and 12.5 ppm. As a result, coxanthin showed a cell viability of 70% or more at a concentration of 12.5 ppm or less, and the highest concentration of the sample used in the intracellular collagenase activity inhibition test using CCD-986SK cells was selected as 12.5 ppm.

Example 10-2. Inhibition test of intracellular collagenase activity of fucoxanthin

As shown in Table 13 below, the test was conducted by diluting the sample to three concentrations.

test group Substance name Dosage concentration G1 negative control group - - G2 positive control TGF-b1 60 ng/mL G3 Test substance group Fucoxanthin 3.13 ppm G4 6.25 ppm G5 12.5 ppm

First, cells were dispensed into 5×10 ⁴ (1 mL/well) pieces in a 48-well plate and incubated for 24 hours in a 37 °C, 5% CO ₂ incubator. ℃, 5% CO ₂ It was cultured again for 48 hours in an incubator. Then, for protein quantification, the cell lysate was collected and performed according to the BSA protein assay reagent kit (Thermo scientific PIERCE, USA), and the protein content was measured according to the BSA standard curve. For the quantification of collagenase, the cell lysate was After the collection was performed according to the Human MMP-1 ELISA Kit (SIGMA), the collagenase content was measured according to the collagenase standard curve.

Intracellular collagenase activity inhibition rate was converted into collagenase content for protein content, and was calculated according to the following formula 2, and the results are shown in Table 14 and FIG. 50.

[Calculation 2]

Group Substance Dose Human MMP-1
Conc. (pg/mL) Protein Conc.
(μg/mL) Human MMP-1
/Protein(%) G1 - - Mean 1167.981 93.464 100.00 S.D. 40.042 9.385 8.14 N 3 3 3 G2 TGF-β1 60 ng/mL Mean 797.919 95.013 66.79 S.D. 66.044 5.318 2.05 N 3 3 3 G3 Fucoxanthin 3.13 ppm Mean 1542.041 97.795 125.93 S.D. 24.274 6.212 9.64 N 3 3 3 G4 6.25 ppm Mean 1379.241 100.296 109.63 S.D. 40.817 5.212 5.34 N 3 3 3 G5 12.5 ppm Mean 964.579 102.092 75.36 S.D. 53.024 4.655 6.28 N 3 3 3

N: Number of well, S.D.: Standard deviation, Conc.: Concentration

As a result of treatment with coxanthin at concentrations of 3.13 ppm, 6.25 ppm, and 12.5 ppm, inhibition of collagenase activity was measured at a concentration of 12.5 ppm. Specifically, as a result of treatment with fucoxanthin at concentrations of 3.13 ppm, 6.25 ppm, and 12.5 ppm, 125.96 ± 9.64%, 109.63 ± 5.34%, and 75.36 ± 6.28% were measured at each concentration, and collagenase at a concentration of 12.5 ppm Inhibition of activity was measured. On the other hand, when treated with 60 ng/mL of TGF-β1 as a positive control, it was confirmed that intracellular collagenase activity was inhibited by 66.79 ± 2.05% compared to the control group.

Example 11. Human application test for wrinkle improvement effect of fucoxanthin

Example 11-1. test subject

This human application test was conducted on 22 ordinary people at the request of the Korea Testing & Research Institute. The sexes were all female, the average age was 42.7 years old, and the standard deviation was 6.6. ), 10 people (45.5%) in their 40s and 4 people (18.2%) in their 50s, and the general skin conditions and characteristics of the participating test subjects are shown in Table 15 below.

skin type average percentage(%) inattention 7 31.8 Intelligence 0 0 neutrality 3 13.6 Complexity (medium) 4 18.2 Combination (moderate to dry) 8 36.4

Example 11-2. Methods

First, the composition ratio of the samples used to prove the wrinkle improvement effect of fucoxanthin is shown in Table 16 below.

raw material name composition ratio Purified water 57.4 glycerin 3 zinc oxide 10 titanium dioxide 6.6 Ethylhexylmethoxycinnamate 7.5 Hydroxyethyl Acrylate/Sodium Acryloyl Dimethyl Taurate Copolymer 3 Caprylic/Capric Triglyceride 3 1,2-Hexanediol 2 fucoxanthin 2 dimethicone 2 cyclomethicone 2 biosilica One Spices 0.5 total 100

The test was conducted under constant temperature and humidity conditions in which a constant temperature (20.2 - 22.2 ° C) and humidity (44.0 - 57.0 % R.H.) were maintained. The subject's face was used as the measurement site, and upon visitation, the test site was washed with the same detergent as the center, and then rested for 30 minutes under constant temperature and humidity conditions. For objective measurement, each measurement was performed by the same tester.

The compliance evaluation was conducted by distributing the product and compliance diary to the test subjects during the test period, and the test subjects directly filled in whether or not they were used, and evaluated according to the following formula 3.

[Calculation 3]

As shown in Table 17 below, all test subjects who completed the test had a compliance rate of 80% or more using the test product, and all data were analyzed as results.

total number of uses 28.00 times Average number of uses 27.59 times average compliance 98.54%

Example 11-3. Test result

Antera 3D CS (Miravex, Ireland) was used as a skin wrinkle measuring device, and the Wrinkles medium value representing skin wrinkles was analyzed after photographing the test area, and the unit was expressed as A.U., an arbitrary unit. As the result value decreases, it means improvement.

Table 18 and FIGS. 51 and 52 show the results of measuring wrinkles around the eyes before, after 2 weeks and 4 weeks of use of products using Antera.

before use after 2 weeks of use after 4 weeks of use average 13.98 13.00 12.74 Standard Deviation 3.03 2.38 2.37 Improvement rate (%) 7.01 8.87

significance probability after 2 weeks of use after 4 weeks of use Comparison within the military 0.222 0.037 ^*

Repeated measures ANOVA (*: P<0.05)

As a result of testing with Repeated measures ANOVA, which is a parametric method according to the normality test, the wrinkles around the eyes were statistically significantly reduced after 4 weeks of use compared to before use. Specifically, as a result of checking skin wrinkles, the value of wrinkles around the eyes showed an improvement rate of 7.01% and 8.87% after 2 weeks and 4 weeks of use, respectively, compared to before use, and a statistically significant decrease after 4 weeks of use (P<0.05 ) was confirmed.

Furthermore, as a result of the safety evaluation, after using the test product in the test subjects, no adverse reactions to allergic contact dermatitis or irritant contact dermatitis were observed. No specific symptoms were reported.

Claims (10)

A cosmetic composition for improving wrinkles containing fucoxanthin,
The fucoxanthin is
(S1) Plasma treatment for 25 minutes after stirring the diatoms collected by mass culture in 70% alcohol;
(S2) loading and filtering on a filter bed pre-coated with diatomaceous earth;
(S3) fractionating and concentrating the filtered extract;
(S4) separating and purifying postcoxanthin from the concentrated extract; and
(S5) A cosmetic composition for improving wrinkles characterized by being extracted through the step of storing the separated and purified coxanthin in a dark room,
The fucoxanthin is a cosmetic composition for improving wrinkles, characterized in that contained in a concentration of 12.5ppm.
delete The cosmetic composition for improving wrinkles according to claim 1, wherein the diatoms are adherent diatoms that are taken in together with Jeju lava seawater.
The method of claim 3, wherein the adherent diatoms are Melosira nummuloides, Achnanthes brevipes var. A cosmetic composition for improving wrinkles, characterized in that at least one species selected from the group consisting of intermedia, Achnanthes sancti-pauli, Achnanthes brevipes or Melosira octogona.
The cosmetic composition for improving wrinkles according to claim 1, wherein the diatoms are diatoms collected from bottom diatom culture.
The cosmetic composition for improving wrinkles according to claim 1, wherein the fucoxanthin is contained in an amount of 2% by weight relative to the total cosmetic composition.
The cosmetic composition for improving wrinkles according to any one of claims 1 and 3 to 6, wherein the cosmetic composition further comprises a stabilizer, a solubilizer, vitamins, pigments or fragrances.
The method of any one of claims 1 and 3 to 6, wherein the formulation of the cosmetic composition is a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, cleansing, oil, powder foundation , Emulsion foundation, wax foundation, or a cosmetic composition for improving wrinkles, characterized in that the spray.
A cosmetic comprising the cosmetic composition for improving wrinkles according to any one of claims 1 and 3 to 6 as an active ingredient.
10. The method of claim 9, wherein the cosmetics are from the group consisting of softening lotion, astringent lotion, nutrient lotion, hair growth nutrient, eye cream, nutrient cream, massage cream, cleansing cream, cleansing foam, cleansing water, powder, essence, serum and pack Cosmetics, characterized in that one or more are selected.

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