KR100889973B1 - Effect of antioxdant, wrinkles protection, antibacterial, anti-inflammatory and whitening from Tetraselmis suecica extract - Google Patents

Abstract

The present invention relates to a functional extract having anti-oxidation, anti-wrinkle, anti-acne, anti-inflammatory and whitening efficacy using a micro green alga ( Tetraselmis suecica ), and a method for preparing the same, in detail, the micro green alga (Tetraselmis suecica) As a species belonging to phytoplankton Prasinophyceae, it is a high-functional useful food organism and contains high nutrients such as EPA, AA, DHA, etc. As it is prepared, it is easy to develop new functional cosmetic materials using high nutrients and bioactive substances contained in microalgae cells, and it is possible to grasp the characteristics of microalgae (Tetraselmis suecica) and to use as raw materials for functional cosmetics, functional foods and pharmaceuticals. Antioxidant and Wrinkles Using Micro Green Algae ( Tetraselmis suecica ) The present invention relates to a functional extract having a zen, anti-acne, anti-inflammatory and whitening effect, and a preparation method thereof.

Tetraselmis suecica, antioxidant, anti-wrinkle, anti-acne, anti-inflammatory, whitening

Description

Functional extract with antioxidant, anti-wrinkle, anti-acne, anti-inflammatory and whitening effect using fine green algae and preparation method thereof {Effect of antioxdant, wrinkles protection, antibacterial, anti-inflammatory and whitening from Tetraselmis suecica extract}

The present invention is a micro green algae ( Tetraselmis suecica ) is a species belonging to phytoplankton Prasinophyceae, and contains high-nutrition substances such as EPA, AA, DHA, as a highly functional useful food organism, and has a high nutritional status by developing phytoplankton mass culture technology. With the provision of a large amount of living organisms, it is easy to develop new functional cosmetic materials using high nutrients and physiologically active substances contained in microalgal cells, and identify the characteristics of Tetraselmis suecica. It relates to a method to utilize as a raw material of functional cosmetics, functional foods and pharmaceuticals.

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 in marine materials is twice as efficient as that of terrestrial biomaterials.

 In general, the industrial use of phytoplankton has been widely applied to alternative energy sources, foods, dietary supplements, aquaculture feeds, pharmaceutical raw materials, biochemicals, and the like. Although chlorella and spirulina have been mainly developed in the fields utilizing microalgae's physiological activity, chlorella has a poor nutritional value as freshwater and spirulina is cultivated at high temperature, which is not suitable for Korea. In the cosmetics industry, microalgae is used as a raw material for removing keratin by separating diatomaceous earth from carcasses or as a raw material for removing ultraviolet DNA enzymes from surface plankton to block UV rays. 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.

In the field of utilizing microalgae physiological activity, chlorella and spirulina have been developed mainly, but chlorella has a lot of nutritional value as fresh water and spirulina is cultured at high temperature.

Therefore, according to the present invention, the development of phytoplankton mass culture technology provides a high nutritional status and a condition for receiving a large amount of living organisms, thereby providing a new functional cosmetic material using high nutrients and bioactive substances contained in microalgal cells. Development will be easy. In addition, the fine green algae ( Tetraselmis suecica ) is a species belonging to phytoplankton Prasinophyceae, and is known to contain high-nutrition substances such as EPA, AA, and DHA, as a highly functional useful food organism. There is.

The present invention provides an in-depth fractionation of extracts from Tetraselmis suecica and search for various actions on the skin to develop new functional cosmetic materials and find ways to utilize them as high value products. The effect of increasing the utility value of Tetraselmis suecica may be simultaneously pursued.

The present invention relates to a functional extract having antioxidant, anti-wrinkle, anti-acne, anti-inflammatory and whitening efficacy using a micro green alga ( Tetraselmis suecica ) and a method for preparing the same, in detail, the collected micro green alga ( Tetraselmis suecica ) By dividing and extracting extracts from in-depth, searching for antioxidant effect, anti-wrinkle effect, whitening effect, anti-inflammatory, anti-acne effect on skin, develop new functional cosmetic materials and utilize them as high value-added products. The present invention was completed in order to use as a material of functional products, which is useful for antioxidant, anti-wrinkle, anti-acne, anti-inflammatory and whitening efficacy using Tetraselmis suecica . It will be possible to pursue the effect of increasing the same time.

 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 Extraction Method of Micro Green Algae (Tetraselmis suecica) Extract

In order to isolate and purify the bioactive substance from the microalgae of the present invention, the microalgal algae collected from the south coast were washed three times by centrifugation at 10,000 rpm for 20 minutes at 4 ° C. with sterile purified water, and then the pellets were collected. Thereafter, 5 ml of sterile purified water was added to 1 g of the collected microalgal algae cells, and the suspension was sufficiently suspended, followed by ultrasonic disruption for 30 minutes under cell optimal crushing conditions.

 Example 2 Fractionation Process of Tetraselmis suecica Extract

First, in order to establish the crude extraction conditions of the micro green algae ( Tetraselmis suecica ), ethanol is added to the prepared micro green algae at 90, 80, 70, 60, and 50%, respectively, using a vacuum concentrator at 40 ° C. Each sample was lyophilized by recovering the extract sample. Experimental results using the ethanol 90, 80, 70, 60, 50% of the extract according to the conditions of the respective evaluation results, it was determined that the crude extract of 60% ethanol fine green algae is most suitable.

Example 3 Cytotoxicity of Extracts of Tetraselmis suecica

In order to confirm the cytotoxicity from crude extracts of the condition of the green algae (Tetraselmis suecica), MTT (methylthiazol-2-yl-2.5 diphenyl tetrazolium bromide) analysis was performed on various cell lines, mouse macrophage lines (RAW264.3) and human normal skin. The dermal cells isolated from tissues (Human Dermal Fibroblast) were used to determine the IC ₅₀ value to be used for anti-inflammatory experiments.

The results were obtained using the cytotoxicity test ( IC ₅₀ concentration) using the mouse macrophage line (RAW264.3) from the crude ethanol crude extracts of the microgreen algae in Table 1, and the human Dermal Fibroblast from the 60% ethanol crude extracts of the microalgae in Table 2 As shown in the cytotoxicity test ( IC ₅₀ concentration), the IC ₅₀ value of the mouse macrophage (RAW264.3) in the micro green algae 60% ethanol crude extract was confirmed to be 70.8 μg / ml (Table 1). According to the results, in the cytotoxicity test using the dermal cells (Human Dermal Fibroblast) IC ₅₀ value was determined using only 60% ethanol crude extract. In this case, the IC ₅₀ value surviving 50% through the cell survival rate according to the concentration of 60% ethanol crude extract (Table 2) is shown without the results of each crude extract (Table 2). Fibroblast), the IC ₅₀ value was determined to be 1,715 μg / ml (Table 2).

 The following describes the results of various functional experiments using crude green algae ethanol crude extract.

Example 4 Antioxidant (Aging, Wrinkle Improvement) Efficacy of Fine Green Algae Extracts

Experiment 1.DPPH (1,1-diphenyl-2-picrylhydrazyl radical) scavenging

Figure 1 shows the results of measuring DPPH scavenging ability according to the ethanol extraction conditions, DPPH scavenging ability showed the highest scavenging ability in 60% ethanol crude extract. In the experimental results for determining DPPH scavenging ability according to the change in concentration of the 60% ethanol crude extract having excellent scavenging ability, as shown in FIG. 2, the radical scavenging ability also increased with increasing concentration of 60% ethanol crude extract.

 60% ethanol crude extract was obtained by measuring DPPH (1,1-diphenyl-2-picrylhydrazyl radical) scavenging ability using crude extracts of five ethanol samples (90-50%) according to ethanol extraction conditions from microalgae. The highest scavenging activity and the concentration scavenging activity increased with increasing the concentration of crude extract.

Experiment 2. Inhibition of Superoxide Radicals (NBT / XO assay)

From the results of confirming the superoxide radical scavenging ability using the crude extract according to the five ethanol conditions (ethanol 90, 80, 70, 60, 50% extract) from the fine green algae, Figure 3 is according to the ethanol extraction conditions Superoxide radical scavenging ability according to NBT / XO analysis was shown, showing the highest scavenging ability in 60% ethanol crude extract. In the experimental results for determining the scavenging ability of the superoxide radical according to the concentration change of the 60% ethanol crude extract excellent in scavenging ability, as shown in Figure 4, superoxide according to the increase in the concentration of 60% ethanol crude extract Superoxide radical quenching ability was also increased.

 Experimental results on the inhibition of superoxide radical showed the highest scavenging capacity and concentration dependence of 60% ethanol crude extract from microalgae.

Experiment 3. Inhibition of Peroxyl Radicals

From the results of confirming the peroxyl radical scavenging ability using the crude extract according to the five ethanol conditions (extract of ethanol 90, 80, 70, 60, 50%) from the microalgae, Figure 5 is ethanol extraction The results of the peroxyl radical scavenging ability under the conditions, the highest scavenging ability in 60% ethanol crude extract. In the experimental results for determining the peroxyl radical scavenging ability according to the concentration change of the 60% ethanol crude extract excellent in scavenging ability, as shown in Figure 6, peroxyl radical according to the increase in the concentration of 60% ethanol crude extract Peroxyl radical scavenging activity also increased.

 Experimental results on the peroxyl radical scavenging ability showed the highest scavenging capacity and concentration dependence of 60% ethanol crude extract from microalgae.

Experiment 4. UV irradiation test; Measures the effect of reducing skin wrinkles

(Human Dermal Fibroblast)

In the result of confirming the ultraviolet irradiation test using the crude extract according to the five ethanol conditions (ethanol 90, 80, 70, 60, 50% extract) from the micro-algae, Figure 7 shows the ultraviolet-ray by the micro-algae 60% ethanol crude extract (UVB 290-320 nm) due to the analysis of the expression of MMP-1 and procollagen genes related to the formation of wrinkles, and the results are the group irradiated with ultraviolet light (UVB 290-320 nm) only in 60% ethanol extract of microalgae Compared with (UV + / negative), the expression level of MMP-1 gene was decreased and the expression level of procollagen gene was increased. In addition, these extracts showed similar activity compared with ascorbic acid, which generally exhibits anti-wrinkle effect.

 After treatment with 100 μg / ml and 10 μg / ml, respectively, concentrations that showed no cytotoxicity in the 60% ethanol extract were irradiated with ultraviolet light (UVB 290-320 nm) and MMP-1, a gene related to wrinkle induction, (Matrix metallopeptidase 1) and procollagen (procollagen) expression was measured by real-time PCR, it was shown to induce a decrease in MMP-1 gene expression and increased expression of procollagen (procollagen) gene (Fig. 8).

Example 5 Immune Activity Effect of Micro Green Algae Extract (Anti-inflammatory Effect)

Experiment 1.Inhibition of macrophage nitric oxide (NO) secretion (In vitro experiment)

As a result of confirming the inhibition of nitric oxide (NO) secretion of macrophages using the crude extract according to five ethanol conditions (ethanol 90, 80, 70, 60, 50% extract) from the microalgae, as shown in FIG. As a result, the nitric oxide (NO) secretion inhibition ability according to the ethanol extraction conditions, showed the highest activity in 60% ethanol crude extract. In the experimental results for determining the inhibitory ability of nitric oxide (NO) secretion according to the concentration change of the 60% ethanol crude extract with excellent scavenging ability, as shown in FIG. 10, nitric oxide (NO) with increasing concentration of 60% ethanol crude extract ) Secretion inhibition was also increased.

 Therefore, the experimental results on the inhibition of nitric oxide (NO) secretion showed the highest scavenging activity and concentration dependence of crude extract of 60% ethanol from green algae.

Experiment 2. Inhibition of Inflammatory Cytokine Secretion in Macrophages (In vitro Experiment)

As a result of confirming the inhibitory ability of inflammatory cytokines according to the concentration using the crude extract of 60% ethanol, as shown in FIG. 11, the secretion amount of IL-6 inflammatory cytokine was decreased as the concentration of 60% ethanol crude extract was increased, The difference was not significant.

 12 shows the secretion of TNF-α inflammatory cytokine secreted by increasing the concentration of 60% ethanol crude extract, the secretion of TNF-α inflammatory cytokine decreased with increasing concentration of 60% ethanol crude extract, The difference was not significant.

Experiment 3. Suppression of ear edema (In vivo animal experiment)

FIG. 13 shows the result of measuring the thickness of the ear after applying the micro green algae 60% ethanol crude extract to the mouse ear, and then applying the Arachidonic acid, which is an inflammation-causing substance, respectively. After application for 1 hour and 24 hours after application of Arachidonic acid, an inflammation-causing substance, the thickness of the ear was measured, and 60% ethanol crude extract of microalgae was slightly lower than positive control (Nimesulide), but ear edema was somewhat reduced. It was confirmed that it has an inflammation inhibitory effect.

Example 6 Whitening Effect of Micro Green Algae Extract

1) Inhibition of Tyrosinase Activity

As shown in FIG. 14, as a result of confirming the inhibition of tyrosinase activity according to the ethanol extraction conditions, it showed the highest activity in the crude extract of 60% ethanol. Figure 15 shows the inhibitory activity of tyrosinase activity with increasing concentration of 60% ethanol crude extract, tyrosinase activity was also increased with increasing concentration of 60% ethanol crude extract.

 The results showed a concentration dependency showing the highest inhibitory activity of tyrosinase activity in the 60% ethanol crude extract and 60% ethanol crude extract from the microalgae, and the tyrosinase activity inhibitory activity.

2) inhibition of DOPA activity

Figure 16 shows the ability to inhibit DOPA activity according to the increase in the concentration of 60% ethanol crude extract, showing the DOPA activity inhibitory ability and concentration dependence according to the increase in the concentration of 60% ethanol crude extract.

3) Inhibition of melanin secretion using Melan-α cells

FIG. 17 shows melanin secretion inhibitory ability as the concentration of 60% ethanol crude extract increased, and melanin secretion inhibitory activity and concentration dependence was shown as 60% ethanol crude extract increased.

Example 7 Anti-acne Effect of Fine Green Algae Extract

Antimicrobial activity (MIC) against standard strains of P. acune and S. epidermis, the acne- causing bacteria, was found in Propionibacterium acnes among 60% crude extracts and 90% crude extracts of acne. The antimicrobial activity was weak for (Table 3).

 Putting the above experimental results together,

1. In the experiment of ethanol extract to determine the antioxidant (aging, wrinkle improvement) efficacy of the micro green algae extract,

 1) As a result of measuring DPPH scavenging ability, 60% ethanol crude extract showed the highest scavenging ability and concentration dependence of radical scavenging ability with increasing concentration of crude extract.

 2) Experimental results on the inhibition of superoxide radical showed the highest scavenging capacity and concentration dependence of 60% ethanol crude extract from microalgae.

 3) The results of peroxyl radical quenching showed the highest scavenging activity and concentration dependence of 60% ethanol extract from microalgae.

 4) Analysis of the expression of MMP-1 and procollagen genes related to the formation of wrinkles due to ultraviolet rays (UVB 290-320 nm), and the result is the ultraviolet (UVB 290-320 nm) in 60% ethanol extract of microalgae ) Compared with the group irradiated only (UV + / negative), the expression level of the MMP-1 gene was reduced, the expression level of the procollagen (procollagen) gene was confirmed to increase. In addition, these extracts showed similar activity compared with ascorbic acid, which generally exhibits anti-wrinkle effect.

2. In the experiment of the ethanol extract to determine the anti-inflammatory effect of the microalgae extract,

 1) Experimental results on nitric oxide (NO) secretion of macrophages showed the highest scavenging activity and concentration dependence of 60% ethanol crude extract from microalgae.

 2) As the concentration of 60% ethanol crude extract increased, the secretion of TNF-α inflammatory cytokines decreased, but there was no significant difference according to the concentration.

 3) After the 60% ethanol crude extract of microalgae was applied to the mouse ears, Arachidonic acid, an inflammation-inducing substance, was applied, and the thickness of the ear was measured. The 60% ethanol extract of the microalgae was a positive control ( Nimesulide) was slightly lower, but ear edema was somewhat reduced, indicating an inhibitory effect.

3. In the experiment of ethanol extract to determine the whitening effect of microalgae extract,

 1) The highest tyrosinase activity inhibitory activity and concentration dependence were observed in 60% ethanol crude extract from microalgae.

 2) Inhibition of DOPA activity and concentration dependence of 60% ethanol crude extracts of green algae were shown.

 3) The melanin secretion inhibitory activity was shown to be dependent on the concentration of 60% ethanol crude extract of microalgae.

4. In the experiment of the ethanol extract to examine the anti-acne effect of the microalgae extract, it showed the antimicrobial activity (MIC) against the acne- causing bacteria P. acune, S. epidermis standard strains, 60% crude algae ethanol extract and In 90% crude extract, the antibacterial activity against Propionibacterium acnes was weak among acne-causing bacteria.

 Therefore, looking at the characteristics of the functional extract in the 60% ethanol crude extract from the microalgae, it was found that it has antioxidant efficacy, anti-wrinkle effect, anti-acne effect, anti-inflammatory and whitening effect.

Although the present invention has been described in detail only with respect to the described embodiments, it will be apparent to those skilled in the art that various changes and modifications can be made within the spirit of the present invention, and such modifications and modifications belong to the appended claims .

The present invention is the development of new functional cosmetic materials utilizing high nutrients and physiologically active substances contained in microalgae cells as the conditions for high nutrient status and a large amount of living organisms are provided by the development of phytoplankton mass culture technology. This will be easy. In addition, the fine green algae (Tetraselmis suecica) belongs to the phytoplankton Prasinophyceae species, and is known to contain high-nutrition substances such as EPA, AA, and DHA, which are useful foods of high performance. There is.

1 is a diagram showing the results of measuring DPPH scavenging ability according to ethanol extraction conditions.

Figure 2 shows the radical scavenging ability according to the increase in concentration of 60% ethanol crude extract.

Figure 3 shows the superoxide radical scavenging ability according to NBT / XO analysis according to the ethanol extraction conditions.

4 shows that the superoxide radical scavenging ability increased with increasing concentration of 60% ethanol crude extract.

Figure 5 shows the results of the peroxyl radical scavenging ability in 60% ethanol crude extract extraction conditions.

Figure 6 shows the increase in the peroxyl radical scavenging ability as the concentration of 60% ethanol crude extract increased.

Figure 7 analyzed the expression of MMP-1 expression gene related to wrinkle formation due to ultraviolet (UV) by microalgae 60% ethanol crude extract.

Figure 8 analyzed the expression level of procollagen gene related to wrinkle formation by ultraviolet (UV) by microalgae 60% ethanol extract.

9 shows the results of confirming the inhibition of nitric oxide (NO) secretion according to the ethanol extraction conditions.

FIG. 10 also shows that nitric oxide (NO) secretion inhibitory ability increased with increasing concentration of 60% ethanol crude extract.

Figure 11 shows the secretion amount of IL-6 inflammatory cytokine according to the increase in the concentration of 60% ethanol crude extract.

Figure 12 shows the secretion amount of TNF-α inflammatory cytokine according to the increase in concentration of 60% ethanol crude extract.

Figure 13 shows the result of measuring the thickness of the ear after applying the micro green algae 60% ethanol crude extract to the mouse ear after applying Arachidonic acid, an inflammation-causing substance.

14 shows the results of confirming the tyrosinase activity inhibitory ability according to the ethanol extraction conditions.

15 shows the inhibitory effect of tyrosinase activity with increasing concentration of 60% ethanol crude extract.

Figure 16 shows the ability to inhibit DOPA activity according to the increase in concentration of 60% ethanol crude extract.

Figure 17 shows the melanin activity inhibitory effect with increasing concentration of 60% ethanol crude extract.

Claims (4)

In the separation and purification of physiologically active substances from Tetraselmis suecica, a kind of microalgae , the collected Tetraselmis suecica cultures were washed three times by centrifugation at 10,000 rpm for 20 minutes at 4 ° C with sterile purified water. pellets), 5 ml of sterile purified water was added to 1 g of collected Tetraselmis suecica cells, and the suspension was sufficiently suspended. Ultrasonic crushing was performed under cell crushing conditions for 30 minutes, and 60% ethanol was added thereto under reduced pressure at 40 ° C. The extract sample was collected using a concentrator and lyophilized to obtain 60% extract of ethanol, which was lyophilized . Antioxidation, wrinkle improvement, anti acne, anti-inflammatory and whitening using Tetraselmis suecica , a kind of microalgae , was obtained. Method of producing a functional extract having efficacy. A functional extract having anti-oxidation, anti-wrinkle, anti-acne, anti-inflammatory and whitening effects using Tetraselmis suecica , a kind of microalgae, which is extracted by the extraction method of claim 1. The method of claim 2, wherein the functional extract using Tetraselmis suecica , a kind of microalgae , is effective for anti-wrinkle and anti-aging, anti-inflammatory and anti-inflammatory activity, whitening, antibacterial and anti-acne. Functional extract with antioxidant, anti-wrinkle, anti-acne, anti-inflammatory and whitening effect using Tetraselmis suecica , a kind of microalgae . The functional extract having antioxidant, anti-wrinkle, anti-acne, anti-inflammatory and whitening effects using Tetraselmis suecica , a kind of microalgae , is selected from among health food materials, functional food materials, functional cosmetic materials, and medical drug materials. Functional extract with antioxidant, anti-wrinkle, anti-acne, anti-inflammatory and whitening effect using Tetraselmis suecica , a kind of microalgae , characterized by being available in one or more selected materials.

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