KR101136520B1 - Effect of wrinkless and UV protection from water soluble extract of Tetraselmis suecica - Google Patents

Abstract

 The present invention relates to a water-soluble extract having an anti-wrinkle effect and a UV-blocking effect using a microalga Tetraselmis suecica, and a method for preparing the same, in detail, a water-soluble extract was prepared using Tetraselmis suecica which is a kind of microalgae. In addition, this water-soluble extract has a skin wrinkle reduction and UV-blocking effect, and is used to provide a health food material, a functional food material, a functional cosmetic material, and a medical drug material by using a water-soluble extract of Tetraselmis suecica, a kind of microalgae. The present invention relates to Tetraselmis suecica water soluble extract of micro green algae and its manufacturing method to develop new materials and increase the utility value of micro green algae through high value-added products.

Tetraselmis suecica, wrinkle improvement, sun protection

Description

Water-soluble extract having a skin algae reduction and UV-blocking effect using a microalgae Tetraselmis suesica {Effect of wrinkless and UV protection from water soluble extract of Tetraselmis suecica}

The present invention relates to a water-soluble extract having an anti-wrinkle effect and a UV-blocking effect using a microalga Tetraselmis suecica, and a method for preparing the same, in detail, a water-soluble extract was prepared using Tetraselmis suecica which is a kind of microalgae. In addition, this water-soluble extract has a skin wrinkle reduction and UV-blocking effect, and is used to provide a health food material, a functional food material, a functional cosmetic material, and a medical drug material by using a water-soluble extract of Tetraselmis suecica, a kind of microalgae. The present invention relates to Tetraselmis suecica water soluble extract of micro green algae and its manufacturing method to develop new materials and increase the utility value of micro green algae through high value-added products.

The ocean accounts for 71% of the earth's surface and is a treasure trove of more than 10 million species, 80% of the world's life. Marine organisms that produce a variety of products can produce many functional substances that underlie the bioindustry.

In addition, the global market of marine biotechnology is increasing several times every year, and the success rate of commercialization of marine materials is twice as efficient as that of terrestrial biomaterials.

There are tens of thousands of microalgae, phytoplankton that produce organic matter from minerals in the ocean using solar energy, and feed from these as well as primary metabolites such as proteins, fats and carbohydrates. Secondary metabolites and their biological activities are secondary, depending on the chain. These microalgae have explosive growth power according to environmental conditions, and because they can be cultured, they can be used for various aspects such as producing useful materials or solving environmental problems without damaging the existing ecosystem.

Industrial applications of microalgae have broadened their applications to alternative energy sources, foods, dietary supplements, aquaculture feeds, pharmaceutical raw materials and biochemicals. In addition, the utilization of microalgae, which is a representative marine material in the cosmetics industry, is used as a raw material for removing keratin by separating diatomaceous earth from carcasses or as a raw material for cosmetics to block UV rays by extracting marine DNA enzymes from surface plankton. . Therefore, the industrial sector applying phytoplankton is extremely limited in size and diversity compared to the rich species, and thus, the development of these fields is urgently needed.

The extract using the organic solvent using the microalgae as a raw material has problems such as a difficulty in using cosmetic raw materials due to the coloring of the pigments of the existing species.

In order to improve the problem of the extraction process, the microalgae as a raw material to obtain a water-soluble extract using the water-soluble extraction method using the enzyme treatment, and showed a lot of differences in the color of the raw material after extraction. In other words, in the case of Tetraselmis suecica, the green color was almost removed when the enzyme extraction was carried out.

In the present invention, Tetraselmis suecica, which is a kind of microalgae, is an extract using organic solvent, which is extracted with pigments of existing species, and is expected to be somewhat difficult in the use of cosmetic raw materials due to pigments to improve these extraction processes. Aqueous extraction method was used, and the color of the raw material was different after extraction. That is, in the case of Tetraselmis suecica, it was found that the water-soluble extract was almost eliminated when the green color was extracted by enzyme extraction. As its function, it has wrinkle improvement effect and UV blocking effect, so that each of these functional properties is used to It is to develop new materials to be used for cosmetics, functional foods and medicines, and to provide cosmetics, foods and pharmaceutical materials necessary for the industry.

The present invention is to examine the function of the water-soluble extract of Tetraselmis suecica, a kind of microalgae, to reduce the skin wrinkles and UV-blocking effect, by using the water-soluble extract of Tetraselmis suecica, a kind of microalgae, health food material, functional food material It is to provide functional cosmetic materials and medical drug materials, and to develop new materials and increase the utility value of microalgae through high value-added products to provide the effect of expanding the range of industrial use.

The present invention relates to a water-soluble extract having an anti-wrinkle effect and a UV-blocking effect using a microalga Tetraselmis suecica, and a method for preparing the same, in detail, a water-soluble extract was prepared using Tetraselmis suecica, which is a kind of microalgae. In addition, this water-soluble extract has a skin wrinkle reduction and UV-blocking effect, and is used to provide a health food material, a functional food material, a functional cosmetic material, and a medical drug material by using a water-soluble extract of Tetraselmis suecica, a kind of microalgae. The present invention relates to a microalgae Tetraselmis suecica water-soluble extract and its manufacturing method to develop new materials and increase the utility value of microalgae algae through high value-added products.

The present invention will be described in more detail with reference to the following Examples and Experimental Examples. However, the examples are not intended to limit the present invention only to illustrate the invention, and various modifications are possible.

 Example 1 Water-Soluble Extract Raw Material Process of Microalgae

Figure 1 is a photograph showing the form of Tetraselmis suecica, Figure 2 shows a flow chart of a water-soluble extract raw material of Tetraselmis suecica, when described in more detail step by step,

First, 20L culture medium in which Tetraselmis suecica, a kind of microalgae, was centrifuged at 3,000 rpm for 15 minutes to collect cell pellets, washed three times with 100 ml of tertiary distilled water, and the final pellet was suspended in 10 ml and then lyophilized. Dry powder process (1) recovering the dry powder,

20 minutes (v / w) of sterile distilled water was added to the dry powder sample of Tetraselmis suecica, stirred for 1 hour, and then crushed with an ultrasonic crusher for 30 minutes (2),

After tertiary distilled water was added to twice the volume (v / w) in the crushed sample, and cooled sufficiently at 50 ° C. while stirring in a 90 ° C. thermostat, and then 1% (weight) of Protamex enzyme compared to the dry powder sample. ) And the enzyme reaction process (3) to react by stirring at 50 ° C. for 3 hours;

After the enzyme reaction solution in the enzyme reaction process (3) was put into L-tartaric acid (L-tartaric acid) in the supernatant recovered using a centrifuge, it took several hours or more until the supernatant became transparent. And refrigerated storage process (4) to prevent further enzymatic reactions,

The supernatant collected by centrifugation of the refrigerated sample was treated with calcium carbonate, adjusted to pH 6, and the supernatant was filtered with a 5 μm filter paper, and activated carbon was added to the filtrate with 2% of the dry powder sample weight. Weight)), and then the sample solution was filtered twice with 5 μm filter paper. Lyophilization process (5) for recovering the filtrate and freeze-drying;

The final lyophilized raw material was dissolved in a small amount of sterilized distilled water, centrifuged to obtain a supernatant, filtered through a 0.45 μm MCE filter, and autoclaved at 121 ° C. for 30 minutes by filling distilled water to 80% of the final volume. 6);

For preservation, add 20% of the final volume of propylene glycol (PG) to the clean bottle, place the bottle in a water bath and weigh methylparaben (MP) at 0.25% of the final volume. A water-soluble extract of microalgae is prepared through the antiseptic process (7), which is carried out, reacted in a container, and then subjected to antiseptic treatment.

The extraction yield was calculated by lyophilization after raw material extraction.

Example 2 Determination of Nitrogen Content in Water-Soluble Extract Raw Materials of Microalgae

The total nitrogen content was determined from the sample collected from the microalgae Tetraselmis suecica aqueous extract through the enzyme extraction process. Therefore, decomposition accelerator (CuSO ₄ : K ₂ SO ₄ mixed components) and sulfuric acid were added to the separated aqueous extract to decompose at 450 ° C. for 1 hour to 2 hours. After the decomposition, the Kjeldahl flask was sufficiently cooled, steam distilled with 30% to 32% NaOH for 5 minutes, and then neutralized with 2% Boric acid. A Brunch Week solution was added to the neutralized solution, and titrated with 0.1N HCl solution. Total nitrogen was calculated as follows.

* Total nitrogen = {(Sample titration-blank titration) / sample volume} × 0.001401 × 0.1N HCl factor × 100

The average total nitrogen content of the water-soluble extracts contained 1-1.5%.

Example 3 Physical Properties of Water-Soluble Extracts (Raw Materials) of Microalgae

 1. Stability test of raw materials: Changes in appearance were observed at room temperature, 40 ℃, 50 ℃, refrigerated for the raw material stability test for the microalgae water-soluble extract.

 2. Safety test of raw materials: Skin irritation test was performed through human patch test to confirm whether water-soluble extract of microalgae has human irritation. The subjects were selected from 20 cosmetics consumers, and they were attached to the inside of the right (left) arm in the Haye's test chamber using 0.2 g of the raw material in a closed state for 24 hours, then peeled off and blank skin reactions (erythema, edema, etc.) were removed. ) Condition was observed and evaluated.

3. Microbiological testing of raw materials: In order to carry out microbiological testing of microalgae water-soluble extracts, it was conducted according to the Food and Drug Administration microbial limit standards and test method guidelines.

4. Heavy metal analysis: For the detection of heavy metals in microalgae soluble extracts, the lead and arsenic cases were analyzed by the Korea Food and Drug Administration standards and test methods.

5. Other measurements: The pH values, Specific Gravity ( d ²⁵ ₂₅ ) and Refractive index (25) for microalgae aqueous extracts were analyzed using a measuring instrument.

Example 4 Evaluation of Efficacy of Water-Soluble Extracts (Raw Materials) of Microalgae

1. Reduces wrinkles of skin

In vitro evaluation was performed to evaluate the skin wrinkle improvement effect from the microalgae water-soluble extract as a sample. First, human dermal fibroblast was isolated from human skin tissue. In order to confirm the cytotoxicity of these samples prior to the efficacy evaluation, normal dermal cells, human dermal fibroblast cells were incubated for 24 hours in DMEM medium (Dulbecco's modified Eagle's medium) with 10% FBS, and then per well in a 96 well plate. 100 ul of a cell suspension containing 1 × 10 ⁵ / ml of cells was added and incubated for 24 hours. After incubation, the medium was removed, and the sample prepared by diluting the medium and the concentration of serum-free was prepared to be 200 μl, added to each well, incubated for 24 hours, and further incubated for 4 hours by adding MTT reagent. 100 μl acid iso-propanol (0.04N HCl in iso-propanol) was added to each well, stirred for 30 minutes, and the absorbance was measured at 570 nm using an ELISA reader to set the IC ₅₀ value of each sample.

 To evaluate the effect of skin wrinkle reduction on human dermal fibroblast cells based on the highest concentration, the cells were washed with PBS and stabilized for 6 hours by replacing the medium with serum free medium. After treating the extract sample by concentration for 24 hours, washed twice with PBS, and then irradiated with UV-B ultraviolet light, washed twice with PBS and incubated in serum free medium for 24 hours, cells were collected. RNA was isolated from these cells using a Trizol kit and cDNA synthesis was performed. The expression of human MMP1 and procollagen type I genes related to the formation of wrinkles on skin was analyzed by real-time PCR. Antioxidant enzymes, superoxide radical deoxygenase (SOD) activity, reduced glutathione (GSH) activity and malondialdehyde (MDA) related to lipid peroxidation were measured from the treated cells.

2. UV protection

In order to measure the UV blocking ability was carried out through the evaluation of the ultraviolet absorption capacity of the microalgae water-soluble extract. The effective concentration of the material was determined, the material was dissolved in a solvent, and the form of the absorption spectrum formed within the ultraviolet wavelength of 280-420 nm was analyzed using a spectrophotometer. In particular, the absorption capacity of UVA (320-400 nm) and UVB (290-320 nm) wavelengths affecting wrinkle formation and aging were evaluated. For the evaluation of UV protection using animals, the extract of the hairless mouse was divided into two parts, and an extract sample was applied on one side and only an excipient was applied on the other side. After adjusting the height to 1MED using a UVB measuring instrument was irradiated with UV light for a certain time, it was determined whether the UVB blocking 48 hours after the UV irradiation.

   The following describes the results of various functional experiments using microalgae water-soluble extracts.

Experimental Example 1 Process Using Water-Soluble Extract of Microalgae

First, Tetraselmis suecica, a kind of microalgae, was compared with the recovery rate of the sample obtained by the 80% methanol extraction process using the organic solvent and the water-soluble extract using the enzyme treatment, as shown in Table 1 in the sample recovery rate. Suecica showed almost 10 times higher sample recovery rate of water-soluble extract using enzyme treatment than that of 80% methanol extraction using organic solvent. Three kinds of PG (propylene glycol), BG (Butylene glycol), and MP (Methyl paraben) were treated with these water-soluble extracts.

As shown in Table 2, when the final concentration of Tetraselmis suecica was added to 20% PG (propylene glycol) and 0.2% MP (Methyl paraben), it was confirmed that bacteria were not detected because it was suitable as a preservative. In addition, as a result of confirming the presence of color, odor, and precipitation after preservative treatment, it was confirmed that the precipitation hardly occurs in the state in which the translucent odor was removed under the above conditions, and it could be used as a cosmetic raw material (FIG. 3).

Experimental Example 2 Stability and Safety Test of the Water-Soluble Extract of Microalgae

end. Stability Test of Tetraselmis suecica Water Soluble Extract

 Tetraselmis suecica water-soluble extracts were observed at room temperature, 40 ℃, 50 ℃, refrigerated to observe the change in appearance, the results are shown in Table 3. In general, cosmetic raw materials (beauty ingredients) should be kept at room temperature (1 ~ 30 ℃) or room temperature (15 ~ 25 ℃) for one year without any change in appearance, even if the stability of room temperature or room temperature is low. Under the conditions, it should be maintained for at least 6 months to 1 year, but as a raw material, Tetraselmis suecica water-soluble extract does not seem to have a significant problem in stability.

I. Safety Test of Tetraselmis suecica Water Soluble Extract

Tests were conducted primarily to check the safety of Tetraselmis suecica water-soluble extracts. In this experiment, 20 subjects were patched for 24 hours, and the criterion was judged based on Draize's PII method. The determination method was the first judgment at 30 minutes after patch removal and 2 after patch removal. After 2 days, after the patch removal, 1 day after removing the patch, 3 days after the removal of the patch, and 4 days after the removal of the patch, the test position was made by wrapping the patch in the inner part of the left arm. Run (Haye's test Chamber).

As a result of the human skin compatibility test, the extract was tested twice, and all 20 patients were confirmed to have no irritation.

All. Microbiological Testing of Water-Soluble Extracts from Tetraselmis suecica

In order to detect microorganisms in raw materials after preservative treatment of Tetraselmis suecica water-soluble extracts, tests were conducted in accordance with the Korea Food and Drug Administration's microbial limit criteria and test method guideline. As a result, the total viable cell count ≤ 100 cfu / g or less in all extracts, Was not detected.

la. Heavy Metal Analysis of Tetraselmis suecica Water Soluble Extract

The analysis of heavy metals in water-soluble extracts of Tetraselmis suecica was conducted according to the Korea Food and Drug Administration's standards and test methods.

Experimental Example 3 Skin Wrinkle Reduction Capability Using Microalgae Water Soluble Extract

In order to confirm the ability to reduce skin wrinkles using water-soluble extracts using Tetraselmis suecica, the expression levels of human MMP-1 and procollagen genes, which are involved in the formation of wrinkles, were compared using fibroblast cells. In addition, superoxide radical deoxygenase (SOD) activity related to anti-oxidant, reduced glutathione (GSH) activity, and malondialdehyde (MDA) related to lipid peroxidation were measured using each kit.

end. In vitro skin wrinkle improvement using Fibroblast cells

1) Comparison of Gene Expression

To evaluate the ability to reduce wrinkles in vitro, the expression of MMP-1 and procollagen in relation to the induction of wrinkles was determined by irradiating ultraviolet (UV) light using fibroblast cells directly isolated from human skin tissues from water-soluble extracts using micro green alga Tetraselmis suecica. Measured by real-time PCR. First, MTT assay was performed to determine the toxicity and IC ₅₀ concentration.

As shown in Table 4, the IC ₅₀ concentration of these extracts showed almost no cytotoxicity as in melan-a cells above 1000 ug / ml concentration. In addition, the UVB-treated fibroblast cells were treated with four concentrations of these extracts diluted 1000 aug / ml to azeotropic 10 at the highest concentration, and then analyzed for expression of MMP-1 and procollagen genes. In general, it is known that the expression of MMP-1 increases and the expression of procallagen gene decreases when the UV rays are irradiated.

As shown in Table 5, as the concentration of the water-soluble extract of the micro green alga Tetraselmis suecica increased, the amount of MMP-1 expression was decreased (Fig. 4), the amount of procollagen expression was increased (Fig. 5).

2) Determination of Antioxidant-related Enzyme Activity

Superoxide radicals were directly extracted from fibroblast cells harvested from the microbromocytic Tetraselmis suecica water-soluble extract using the same procedure as the gene expression test to confirm the association with antioxidant enzymes in addition to the genes related to skin wrinkle formation. Deoxygenase (SOD) activity, reduced glutathione (GSH) activity, and malondialdehyde (MDA) related to lipid peroxidation were measured using the respective kits.

In general, exposure to ultraviolet light (UV) decreases the intracellular GSH and increases the MDA. As shown in Table 6, in the water-soluble extract of Tetraselmis suecica microalgae, the GSH content increased with increasing concentration (Fig. 6), and the MDA decreased (Fig. 7). appear. In addition, the superoxide anione (O ^{2 −} ) scavening acitivity showed excellent activity in Tetraselmis suecica water-soluble extract (FIG. 8). All measurements represent the mean of three measurements.

I. UV protection

Ultraviolet ray blocking ability according to UV exposure was confirmed using water-soluble extract using Tetraselmis suecica. The water extract of Tetraselmis suecica microalgae showed UV absorption at UVB wavelength (290-320 nm) and conducted animal experiments using hairless mice (FIGS. 9 and 10). As a result, erythema was not observed at all of the areas where the water-soluble extract of Tetraselmis suecica was applied after 24 hours after applying the test substance to the back area and exposing the ultraviolet ray to the erythema. Therefore, Tetraselmis suecica water-soluble extracts are expected to contain useful ingredients with UV-protective properties.

Therefore, the characteristics of the water-soluble extract of Tetraselmis suecica, which is a kind of microalgae, have the effect of reducing skin wrinkles and UV rays, and by using the water-soluble extract of Tetraselmis suecica, which is a type of micro green algae, using health food material, functional food material, It is characterized in that it can be used as a functional cosmetic material, a medical drug material.

While the invention has been shown and described with reference to certain exemplary embodiments thereof, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention as defined by the appended claims.

The present invention is to examine the function of the water-soluble extract of Tetraselmis suecica, a kind of microalgae, to reduce the skin wrinkles and UV-blocking effect, by using the water-soluble extract of Tetraselmis suecica, a kind of the micro green algae, health food material, functional food material It is to provide functional cosmetic materials and medical materials, and to develop new materials and increase the utility value of microalgae through high value-added products to provide industrial expansion effect.

1 is a photograph showing the form of Tetraselmis suecica

Figure 2 is a flow chart of a water-soluble extract raw material of Tetraselmis suecica

3 is a sample color of Tetraselmis suecica extract

Figure 4 MMP-1 gene expression by water soluble extract of Tetraselmis suecica

Figure 5 procallgen gene by water-soluble extract of Tetraselmis suecica

Figure 6 GSH content change by the water-soluble extract of Tetraselmis suecica

7 is a change in MDA content by the water-soluble extract of Tetraselmis suecica

Figure 8 SOD activity by Tetraselmis suecica water-soluble extract

9 is ultraviolet absorbing ability of Tetraselmis suecica water-soluble extract

10 is a UV protection ability using a hairless mouse of Tetraselmis suecica water-soluble extract

Claims (4)

 Centrifuge the 20L culture medium of Tetraselmis suecica, a kind of microalgae, at 3,000 rpm for 15 minutes to collect cell pellets, and then wash three times with 100 ml of tertiary distilled water. The final cells were suspended in 10 ml. Next, the dry powder process (1) to recover the dry powder by lyophilization,  Ultrasonic crushing process (2) to put the sterile distilled water of 20 times (v / w) volume in the dry powder sample of Tetraselmis suecica, and stirred for 1 hour and then crush the cells by ultrasonic crusher for 30 minutes; After tertiary distilled water was added to twice the volume (v / w) in the crushed sample, and cooled sufficiently at 50 ° C. while stirring in a 90 ° C. thermostat, and then 1% of the Protamex enzyme was added to the weight of the dry powder sample. An enzyme reaction process (3) which is added so as to stir and reacted at 50 ° C. for 3 hours; The enzyme reaction solution in the enzyme reaction process (3) was put into L-tartaric acid (L-tartaric acid) in the supernatant recovered using a centrifuge to adjust the pH to 3 so that the enzyme reaction does not occur until the supernatant becomes clear. Cold storage process (4), The supernatant collected by centrifugation of the refrigerated sample was treated with calcium carbonate, adjusted to pH 6, and the supernatant was filtered with a 5 μm filter paper, and activated carbon was added to the filtrate with 2% of the dry powder sample weight. After the mixture was stirred, the sample solution was filtered twice with 5 μm filter paper. Lyophilization process (5) for recovering the filtrate and freeze-drying; The lyophilized raw material was dissolved in sterile distilled water and then centrifuged to obtain a supernatant and filtered through a 0.45 μm MCE filter. The volume of the filtered raw material was 20%, and the distilled water was changed to 80% of the volume of distilled water. Autoclave sterilization (6) to sterilize for 30 minutes at 121 ℃ filled ; In a clean bottle for preservation, propylene glycol was added to a volume of 80% of the raw material subjected to autoclaving so that the volume of propylene glycol was 20% for the raw material. Was added to a constant temperature water bath and weighed 0.25% of methyl paraben to the final volume of 20% of propylene glycol in 80% of the raw material, and further added methyl paraben to the container and reacted. Method of producing a water-soluble extract of Tetraselmis suecica, characterized in that the antiseptic process (7) to be treated. Aqueous extract of Tetraselmis suecica, a kind of microalgae, characterized by being extracted by the method of claim 1 The water-soluble extract of Tetraselmis suecica according to claim 2, wherein the water-soluble extract of Tetraselmis suecica, which is a kind of microalgae, has the effect of reducing skin wrinkles and sunscreen. The method of claim 2, wherein the water-soluble extract of Tetraselmis suecica, a kind of microalgae, can be used as one or more materials selected from health food materials, functional food materials, functional cosmetic materials, and medical drug materials. Water soluble extract

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