KR100969325B1 - Manufacturing method of non-toxic extracts with uv ray protective function from red algae and non-toxic sunscreen utilizing the same - Google Patents

Abstract

PURPOSE: A method for manufacturing nontoxic extract for blocking UV ray is provided to improve yield of extract for blocking UV containing mycosporine-like amino acids(MAA). CONSTITUTION: A method for manufacturing non-toxic extract for blocking UV comprises: a step of preparing red algae; a step of adding mixture solvent of alcohol of 1-5 carbon atoms and water; a step of isolating the red algae at 30-60°C to extract active ingredients; a step of filtering mixture solvent to separate supernatant and residual red algae; and a step of repeating the extraction steps once to three times.

Description

Manufacturing method of non-toxic extracts with UV ray protective function from red algae and non-toxic sunscreen utilizing the same}

The present invention relates to a method for producing a non-toxic sunscreen extract from red algae and a non-toxic sunscreen using the extract, more specifically, Mycosporine-like amino acids (MAA) content of red algae It relates to a method for producing a non-toxic sunscreen extract from relatively high laver or jindubal and a non-toxic sunscreen using the ultraviolet absorption properties of the non-toxic sunscreen extract.

Solar ultraviolet light not only causes acute skin reactions such as erythema, but also causes skin aging and skin cancer after prolonged exposure. Ultraviolet ray (UV ray) included in the sunlight reaching the earth is divided into two areas according to the wavelength. UV-A (320-400 nm), which is long wave, and UV-B (280-, which is medium wave) 320 nm).

Among these, UV-B, which is relatively energetic, is absorbed into the skin layer and is known to cause acute skin desorption, DNA mutation, or cancer.

On the other hand, although UV-A is less energy than UV-B, UV-A is known to penetrate deeper than UV-B to cause premature skin aging, and to generate free radicals or to produce IL-1β and TNF-α. It causes the same inflammatory symptoms and alters the expression of genes. It is reported that UV-A activates transcription factors such as NF-κB and AP-1. The activation of transcription factors by UV-A induces a series of collagen and elastin degrading enzymes called matrix metalloproteinases (MMP's), resulting in reduced collagen content and fibrin fragmentation ultimately leading to typical photoaging. do. In addition, UV-A is well absorbed by urocanic acid or DNA, which is a pigment of the skin, and as a result, free radicals are generated and react with important molecules such as proteins and lipids, thereby destroying important biomolecules or losing their function.

The amount of UV-B varies depending on the season, time of day, cloud presence, latitude, etc., whereas UV-A reaches relatively high levels throughout the year. Protection is more important. Sunscreens used to protect the skin from ultraviolet rays are largely divided into inorganic particle forms and organic compound forms. The sunscreen agent in the form of inorganic particles includes inorganic particles such as ultrafine mineral particles such as zinc oxide, titanium dioxide and iron dioxide, and the inorganic particles adhere to the skin surface and serve as a barrier for reflecting sunlight (ultraviolet rays). do. The sunscreen in the form of inorganic particles keeps the skin cool, provides natural sun (ultraviolet) protection, and is not absorbed by the skin, which does not cause skin problems, while giving a heavy feeling to the skin and residue on the skin. There is a problem that leaves. On the other hand, sunscreens in the form of organic compounds include active synthetic compounds that absorb ultraviolet light very well, such as parmeth (Dimethico-diethylbenzalmalonate, Parsol), benzophenone (Sodium Dihydroxy Dimethoxy Disulfobenzophenone), and paba (p-Aminobenzoic acid (PABA)). UV-A penetrates deep into the skin, causing aging in collagen fibers and other tissues, and preventing sunburn or skin cancer from being induced by UV-B. And the disadvantage that it can easily decompose or produce free radicals, and recently, it has been reported that the organic compounds can cause side effects including endocrine disorders.

Thus, natural materials for sunscreens without side effects such as irritation dermatitis, contact dermatitis, photoallergic or phototoxic dermatitis, and allergic reactions, and toxicity, while not giving the skin a heavy feeling or foreign body caused by residues. Development is urgently needed.

The present invention has been derived to solve the above problems, one object of the present invention is to produce a non-toxic sunscreen extract that can be used as a main component of the sunscreen with excellent UV absorption ability without phototoxicity or cytotoxicity, etc. To provide a method.

In addition, another object of the present invention to provide a non-toxic sunscreen using a non-toxic sunscreen extract.

The inventors of the present invention, through a myriad of experiments when extracting a sunscreen extract containing mycosporine-like amino acids (MAA) known as UV-A blocking material from seaweed and jindubal in red algae The present invention has been found to have a higher yield than when extracted from brown algae and red algae plants, and that the UV-protective extracts extracted from laver and laver are very low in phototoxicity and cytotoxicity.

In order to achieve the object of the present invention, the present invention comprises the steps of (a) preparing at least one red alga selected from laver or jindubal; (b) adding a mixed solvent having a volume ratio of 1 to 5 carbon atoms to water of 1: 9 to 9: 1 to the prepared red algae and maintaining the temperature at a temperature of 30 to 60 ° C. to extract the active ingredient; And (c) filtering the mixed solvent including the active ingredient and the remaining red algae solids into a supernatant and the remaining red algae solids. It provides a method for producing a non-toxic sunscreen extract comprising a.

The method for producing a non-toxic UV protection extract according to the present invention may preferably further comprise (d) removing impurities by passing the separated supernatant through a membrane filter.

In addition, the method for producing a non-toxic UV protection extract according to the present invention preferably comprises the steps of (e) concentrating and drying the separated supernatant or the supernatant from which impurities are removed to obtain a solidified crude extract; can do.

In addition, the method for preparing a non-toxic UV protection extract according to the present invention is preferably (f) dissolving the solidified crude extract in water when the solidified extract is not subjected to the step of removing impurities by a membrane filter. Passing through the membrane filter to remove impurities; may further include.

In addition, the method for producing a non-toxic UV protection extract according to the present invention is preferably (g) to the concentration gradient of water and alcohol having 1 to 5 carbon atoms in the crude extract aqueous solution from which impurities are removed by the step (f). Expanding and obtaining a crude extract fraction absorbing the ultraviolet wavelength of 280 ~ 400 nm; may further include. At this time, the UV detector is used to measure the absorption of the ultraviolet wavelength, and supplementary RI (refractive index) may be further used.

In addition, the method for producing a non-toxic sunscreen extract according to the present invention may preferably further comprise (h) passing the crude extract fraction through a hydrophobic mutual column to obtain a primary purified extract.

In addition, the method for producing a non-toxic UV protection extract according to the present invention preferably comprises the steps of (i) passing the first purified extract through a size exclusion column (Size Exclusion Column) to obtain a second purified extract; It may further include.

In addition, the method for producing a non-toxic sunscreen extract according to the present invention may preferably further comprise the step (j) to concentrate and dry the secondary tablet extract to obtain a solidified tablet extract.

In order to achieve the other object of the present invention, the present invention provides a non-toxic sunscreen comprising a non-toxic sunscreen extract prepared by the above method. At this time, the sunscreen extract contains Mycosporine-like amino acids (MAA) as an active ingredient, and as a kind of Mycosporine-like amino acids (MAA) contained, parine ( Palythine), Asterina-330, and Shinorine, and further include Porphyra-334. Mycosporine-like amino acids (MAAs) have a cyclohexanone or cyclohexenimine pigment structure that absorbs UV light well in the UV-A (320-400 nm) region. The extinction coefficient is similar to that of the artificial sunscreen (sunscreen).

Since the method for preparing the non-toxic sunscreen extract according to the present invention uses laver or jindubal as an extraction material, the yield of the sunscreen extract containing Mycosporine-like amino acids (MAA) is increased. It has a relatively significant advantage over extraction from other green algae, brown algae and red algae plants. In addition, the sunscreen extract extracted from laver or jindubal has very low phototoxicity and cytotoxicity, while at the same time giving irritating dermatitis, contact dermatitis, photoallergic or phototoxic dermatitis without giving heavy feeling to the skin or foreign substance caused by residue. It can be used as a major component of sunscreens without side effects such as skin irritation, allergic reactions, and toxicity.

Figure 1 shows the process of the method for producing a non-toxic anti-UV extract according to an embodiment of the present invention.
Figure 2 is a graph showing the phototoxicity results of the non-toxic UV protection crude extract (PE) and secondary purified extract (P7) extracted from seaweed according to the photohemolysis test using human red blood cells.
Figure 3 is a photograph showing the phototoxicity results according to the photohemolysis test using human erythrocytes when the concentration of non-toxic UV protection crude extract (PE) extracted from seaweed is 500㎍ / ㎖.
Figure 4 is a photograph showing the phototoxicity results according to the photohemolysis test using human erythrocytes when the concentration of the non-toxic UV protection secondary tablet extract (P7) extracted from seaweed is 250㎍ / ㎖.

One aspect of the present invention relates to a method for preparing a non-toxic sunscreen extract that can be used as a main component of a sunscreen because there is no phototoxicity or cytotoxicity and excellent UV absorption ability. Hereinafter, a method for producing a non-toxic UV protection extract according to the present invention will be described step by step through FIG. However, Figure 1 shows a process of a method for producing a non-toxic UV protection extract according to a preferred embodiment, the technical idea of the present invention is not limited or limited to this can be variously modified by those skilled in the art Of course.

Preparing the extract raw material ( S1 )

As the raw material for extraction, at least one selected from laver (Porphyra) or chindrus (Chondrus) is used. Laver or Jindubal is one of the red algae (Rhodophyta) plants, and according to the research of the present inventors, green algae, brown algae and red algae plants having different yields of sunscreen extracts containing Mycosporine-like amino acids (MAA) It has a relatively significant advantage over extraction from.

The laver or jindubal preferably uses dried ones, and more preferably, pulverized. Crushed laver or soybean hair has a very large surface area to improve extraction speed reading and extraction yield by the extraction solvent. Steaming or head can be crushed through a mortar or mortar or the like.

Extracting an active ingredient using an extraction solvent ( S2 )

The extraction solvent is added to the red algae such as crushed laver or jindubal and maintained at a predetermined temperature to extract the active ingredient. At this time, as the extraction solvent, water, alcohol, or a mixed solvent of water and alcohol is used. Preferably, the volume ratio of alcohol to water is 1: 9 to 9: 1 mixed solvent, and more preferably, the volume ratio of alcohol to water is 4: 1. It is characterized by that. If the volume ratio of alcohol to water is lower than 1: 9, the content of alcohol is too small to reduce the extraction rate and extraction yield. If the volume ratio of alcohol to water is greater than 9: 1, the extraction rate and the increase rate of extraction rate are increased with the increase of alcohol content. It is not large, so it is inferior in economy, and the extraction rate, extraction yield, and economy are optimal when the alcohol to water volume ratio is 4: 1.

Alcohols are those having 1 to 5 carbon atoms, preferably methanol or ethanol, more preferably methanol. When using methanol the extraction rate and extraction yield are higher than other alcohols.

The extraction temperature is not particularly limited in the range, but is preferably about 30 ~ 60 ℃. If the extraction temperature is less than 30 ℃ extraction rate and extraction yield is too low and if the extraction temperature exceeds 60 ℃ some of the alcohol is vaporized and not available for extraction.

Separating the supernatant from the remaining red algae solids by filtration ( S3 )

When the extraction of the active ingredient is completed by the extraction solvent, the mixed solvent including the active ingredient and the remaining red algae solid is filtered to separate the supernatant and the remaining red algae solids. The filter medium used for the filtration is not particularly limited as long as it can effectively separate the supernatant from the remaining red algae solids. Specifically, the filter medium or the filter cloth has a pore size of about 0.1 μm to 0.005. It has a range of mm.

On the other hand, the remaining red algae solids separated by filtration after one extraction have an active ingredient such as Mycosporine-like amino acids (MAA), which is not extracted. The process of separating the supernatant and the remaining red algae solids by addition of the solids to the mixed solvent and by filtration after extraction may be repeated one to three times.

In addition, the method for producing the extract for non-toxic UV protection according to the present invention, although not shown in Figure 1, may further comprise the step of removing impurities by passing the separated supernatant through a membrane filter (Membrane filter). . The membrane filter is a membrane having a nano-sized pore size (less than about 100 nm) and serves to remove impurities, contaminants, and the like contained in the filtered supernatant from the supernatant.

Solidified from supernatant Crude extract  Obtaining step ( S4 )

The crude extract is obtained by solidifying the filtration separation or membrane filtered supernatant by concentration and drying.

The concentration of the supernatant is preferably characterized by concentration under reduced pressure, and drying is characterized by freeze drying. When concentrated under reduced pressure, the extraction solvent can be easily evaporated at low temperature, and the extract solidifies at low temperature by lyophilization, thereby preventing the active ingredient of the extract from being denatured by heat.

Solidified Crude extract  Prepare an aqueous solution Membrane  Filtering step ( S5 )

Crude crude extract is dissolved in water to prepare an aqueous crude extract. On the other hand, when the crude extract of the crude extract aqueous solution is not subjected to the membrane filter treatment in the manufacturing process, the crude extract aqueous solution is passed through the membrane filter to remove impurities and the like. The crude extract aqueous solution from which impurities are removed is subjected to the fractionation and purification steps described below. In addition, although not shown in FIG. 1, in the step S3 of separating the supernatant and the remaining red algae solids by filtration, the supernatant after filtration and membrane filter treatment may be fractionated and purified.

Crude extract  Fractionating the aqueous solution ( S6 )

In the step (S3) of separating the supernatant and the remaining red algae solids by removing the impurities from the crude extract aqueous solution or the filtration, the supernatant subjected to the filtration and membrane filter treatment was developed with a water-alcohol concentration gradient, and 280- in the developed crude extract aqueous solution. A crude extract fraction is obtained which absorbs an ultraviolet wavelength of 400 nm. In this case, an alcohol having 1 to 5 carbon atoms is used, and preferably methanol. A specific example of the development of the water-alcohol concentration gradient is a method of sequentially developing water, an aqueous 50% alcohol solution. On the other hand, a UV detector is used to measure the fraction of absorbing the ultraviolet wavelength of 80 ~ 400 nm in the developed extraction extract aqueous solution or supernatant, supplementary RI (refractive index) may be further used.

Crude extraction Fractions  First purification with a hydrophobic cross column ( S7 )

The collected crude extract fractions are passed through a hydrophobic mutual column to obtain a primary purified extract. The type of hydrophobic cross-column used here is not limited, and specifically, a hydrophobic alkyl group, a hydrophobic aryl group, a hydrophobic cyano group, and a hydrophobic alkyl group on the surface of a base particle made of agarose gel, silica gel, or organic polymer resin, and One hydrophobic functional group selected from the group consisting of hydrophobic amine groups is combined. Hydrophobic alkyl groups include butyl, octyl, and octadecyl groups. Hydrophobic aryl groups include phenylethyl groups, hydrophobic cyano groups include cyanopropyl groups, and hydrophobic amine groups include aminopropyl groups. In addition, the hydrophobic cross column used in the present invention is characterized in that one hydrophobic functional group selected from the group consisting of hydrophobic alkyl groups, hydrophobic aryl groups, hydrophobic cyano groups, and hydrophobic amine groups is bonded to the silica gel surface, Such hydrophobic interaction columns include CAPCELL PAK C8, CAPCEll PAk C18, etc. manufactured by Shiseido, Japan. The CAPCELL PAK C18 is a single layer formed by depositing a silicon (Silicone) polymer on the surface of the gel-type silica gel to combine the octadecyl group on the single layer surface. In particular, when using CAPCELL PAK C18 UG120 as a hydrophobic cross column in the present invention, an aqueous acetic acid solution is used.

Secondary purification of the primary purified extract with a size exclusion column ( S8 )

The primary purified extract is passed through a size exclusion column to obtain a secondary purified extract. In this case, the size exclusion column to be used in the water-soluble system is not limited in kind, specifically, Shiseido's silica base water-based size exclusion column (KW-800 series, KW-400 series), polyhydroxy methacrylate Base size exclusion columns (OHpak series) and the like.

Obtaining a solidified tablet extract from the secondary tablet extract ( S9 )

The secondary purified extract is concentrated and dried to give a solidified purified extract. Concentration of the secondary purified extract is preferably characterized by concentration under reduced pressure, and drying is characterized by freeze drying. When concentrated under reduced pressure, solvents such as water can be easily evaporated at a low temperature, and when the freeze-dried solidifies the purified extract at a low temperature, the active ingredient of the purified extract can be prevented from being denatured by heat.

Another aspect of the present invention relates to a non-toxic sunscreen using a non-toxic sunscreen extract prepared by the production method according to the present invention.

The non-toxic sunscreen extract according to the present invention contains Mycosporine-like amino acids (MAA) as an active ingredient, and palytin (Mycosporine-like amino acids (MAA)). Palythine), Asterina-330 (Asterina-330), and Shinorine (Shinorine), and further includes Porphyra-334 (Porphyra-334), and excellent UV-A absorption capacity. In addition, the non-toxic sunscreen extract (particularly the secondary tablet extract) according to the present invention can be used as a main component of the non-toxic sunscreen is very low phototoxicity or cytotoxicity.

Looking at the specific composition of the sunscreen, as an active ingredient non-toxic sunscreen extract according to the present invention about 0.05 to 10% by weight, oil medium medium about 5 to 40% by weight, emulsifier about 1 to 10% by weight, trace amounts of auxiliary and residual amount Water phase media such as water.

Oily media include hydrocarbon oils such as paraffin and mineral oils, paraffin waxes, natural oils, silicone oils, fatty acid esters such as isopropyl palmitate, fatty acid alcohols such as stearyl alcohol, and oily media are water in water. It constitutes an oily component in oil or oil in water.

 Emulsifiers include sorbitan esters (trade name SPAN), ethoxylated sorbitan esters (Sorbitan eser, trade name Tween), silicone polyols, potassium stearate, ethoxylated fatty acid esters, and the like.

Adjuvants include emollients, humectants, antioxidants, emulsion stabilizers, thickeners, preservatives, fragrances, colorants, and the like, which are commonly included in sunscreens.

Hereinafter, the present invention will be described more clearly through specific examples. However, the protection scope of the present invention is not limited by the following examples.

1. Screening of Extracted Raw Materials

Take samples of green algae (CHLOROPHYTA), brown algae (PHAEOPHYTA), red algae (Red algae, RHODOPHYTA) present in domestic seawater, remove water, and then crush them in jujube to extract 100g of extracted raw material for each sample. 10 ml of an aqueous solution (mixed solvent consisting of methanol volume 80 and water volume 20) having a methanol concentration of 80% by volume was added to 100 g of the extraction raw material, and the active ingredient was extracted while stirring for 5 minutes with a mixer at about 45 ° C, followed by filter paper Filtration and scanning with ultraviolet light having a wavelength of about 280 ~ 400nm to detect the ultraviolet absorption capacity of the extract.

Table 1 shows the results of ultraviolet absorption of the extract extracted from the green algae sample, Table 2 shows the results of ultraviolet absorption of the extract extracted from the brown algae sample, Table 3 shows the results of ultraviolet absorption of the extract extracted from the red algae sample. As shown in Table 1, no absorption peak of ultraviolet light with wavelength of 280-400 nm was found in green algae, and as shown in Table 2, absorption of ultraviolet light with wavelength of 280-400 nm was observed in 5 of 16 species in brown algae. Although the peak was found, its height was lower than that of the absorption peak found in the red algae, showing a relatively low active ingredient content. In the case of red algae, the absorption peak of ultraviolet ray with wavelength of 280-400nm was found in 24 of 27 species, and the absorption peak height was increased in 26 species except Plocamium telfairiae. It was larger than the absorption peak shown, showing a relatively high active ingredient content.

Green algae Ultraviolet wavelength (nm) with absorption peak Ulva lactuca L. Not detected Ulva pertusa Kjellman Not detected Ulva conglobata Kjellman Not detected

Brown algae Ultraviolet wavelength (nm) with absorption peak Agarum cribrosom Bory Not detected Corda filum Lamouroux Not detected Eklonia stolonifera Not detected Kjellmaniela crassifolia Miyabe Not detected Hizikia fusiforme Not detected Laminaria japonica Not detected Sargassum confusum Agardh 335 Sargassum thunbergii  O. Kuntze Not detected Sargassum horneri  C. Agardh Not detected Sargassum fulvellum Agardh Not detected Sargassum tortile C. Agardh Not detected Spathoglossum pacificum Yendo 322 Scytosiphon lomentaria J. Agardh Not detected Pelvetia wrightii Yendo 332 Pardina aborescens Holmes 305 Undaria pinnatifida Suringar 337

Red algae Ultraviolet wavelength (nm) with absorption peak Acrosorium polyneurum Okamura Not detected Carpopeltis affinis Okamura 328, 334 Chondria crassicaulis Harvey 327, 329 Chondrus ocellatus Holmes 326 Corallina officinalis Linne 326 Carpopeltis affinis Okamura 305 Gelidium amansii Lamouroux 329, 333 Gracilaria textorii J.Agardh Not detected Grateloupia filicina J. Agardh 330 Grateloupia filicina C. Agardh 330 Grateloupia turuturu Yamada 330 Gymnogongrus flabelliformis Harvey 334 Gigartina tenella Harvey 326 Gloiopeltis complanata Yamada 320 Gloiopeltis furcata  J. Agardh 329, 332 Grateloupia prolongata  J. Agardh Not detected Hypnea saidana Holmes 332 Halymenia acuminata  J. Agardh 329 Hypnea charoides Lamouroux 320 Lomentaria catenata Harvey 330 Porphyra suborbiculata Kjellman 334 Porphyra yezoensis 337 Pachymeniopsis elliptica Yamada 328, 330, 334 Phacelocarpus japonicus Okamura 300 Pterocladia tenuis Okamura 334 Plocamium telfairiae Harvey 322 (trace) Schizymenia dubyi  J. Agardh 334

As most of the red algae extracts were examined for the presence of UV-absorbing substances, four mycosporine-like amino acids (MAA), parlythine and asterina-330, were identified for red algae extracts. (Asterina-330), Shinorine, Porphyra-334 (Porphyra-334) were used as a standard to analyze the content of mycosporin-like amino acid components in red algae extracts, the results are shown in Table 4. . Analysis was performed by high performance liquid chromatography.

Red algae Mycosporin-like amino acid extraction yield (% of extract raw material dry weight) Pallitin Asterina-330 Sinorin Porfira-334 All four mycosporin-like amino acids Porphyra yezoensis 0.225540 0.131668 0.228878 1.099902 1.685989 Chondrus ocellatus 1.058505 0.448038 0.173267 - 1.276576 Grateloupia filicina 0.43331 0.000412 0.315449 - 0.749172 Gloiopeltis furcata 0.342213 0.009644 0.030747 0.261463 0.644067 Pachymeniopsis elliptica 0.411870 0.006765 0.159964 - 0.578598 Carpopeltis affinis 0.29995 0.005995 0.090416 - 0.396367 Grateloupia turuturu 0.190443 0.002925 0.115745 - 0.309112 Gymnogongrus flabelliformis 0.000482 0.000161 0.297636 0.000097 0.298375 Gelidium amansii 0.024122 0.120514 0.123046 - 0.267681

As shown in Table 4, the yield based on the total content of the four Mycosporine-like amino acids (MAA) standards is about 1.7% in the Porphyra extract and about in the Chondrus extract. It was 1.3% higher than other red algae extracts (about 0.3 ~ 0.7%). In addition, the seaweed extract contained all four standard substances, and showed a very high content of porphyrin-334. In addition, the extract of Jindubal included three standard substances except porphyrin-334, and the content of palatin was found to be very high. Based on the results examined above, seaweed and jindubal were selected as candidates for the sunscreen extract.

2. Preparation of UV Protection Extract from Seaweed

(1) Preparation of crude extract (PE)

Dry steaming ( Porphyra 100 g of yezoensis ) was added to the vial, and 20 ml of 80% by volume aqueous methanol solution (mixed solvent consisting of 80 methanol and 20 water volumes) was added thereto, followed by 3 hours in a constant temperature bath maintained at 45 ° C. The active ingredient was extracted. The contents in the vial were then filtered through filter paper (whatman No. 2) to separate the supernatant from the remaining red algae solids. Extraction was carried out two more times by extraction and filtration from the remaining red algae solids. The separated supernatants were collected, concentrated in vacuo, concentrated and extracted twice over and over. The solution was then filtered (whatman No. 2) and the remaining solids were extracted twice in the same way. The extracts thus obtained were collected, concentrated in a vacuum evaporator and dried to give a solidified crude extract (Pophyra extract, PE).

(2) Preparation of Crude Extract Fractions

10 mL of the solidified crude extract was added to 90 mL of distilled water to dissolve to prepare an aqueous crude extract solution, which was then filtered through a membrane filter (Gelman, USA) having a pore size of 0.45 nm to remove impurities. The crude extract aqueous solution from which impurities are removed is composed of Prep LC system (dual pump (Koto, Japan), auto injector, UV / RI detector, and fraction collector) in water-methanol concentration gradient (first water, then 50% by volume methanol solution). The crude extract fraction absorbing the ultraviolet wavelength of 280-400 nm was obtained using UV detector and RI detector after development ().

(3) Preparation of the purified extract (P7)

The obtained crude extract fractions were developed at a rate of 10 ml / min with a 0.2% by volume aqueous acetic acid solution in a hydrophobic cross column (Capcellpak C18 UG120 semi-prepatative column; Shiseido, Japan) protected by a UG120 guard column (Shiseido, Japan). It was purified first. The first purified extract was purified on a size exclusion column (Ohpak 2002; Shiseido, Japan) with water at a rate of 2.5 ml / min for second purification. The secondary tablet extract was a bright yellow liquid, which was concentrated on a vacuum evaporator and lyophilized to give a solidified tablet extract (P7).

3. Toxicity test of sunscreen extract extracted from seaweed

(1) phototoxicity

The 3T3 neutral red uptake photoxicity test (3T3 NRU PT) and oxygen-dependent cell membrane damage, an assessment of OECD chemicals known to show good agreement with human phototoxicity results for extracts from PE and P7 The photohemolysis test (Photohemolysis Test), which is known as an effective method of screening, was applied to evaluate phototoxicity. Fibrosismolecule derived from mouse (NIH / 3T3) was used for the 3T3 NRU phototoxicity test, and human erythrocytes (Human Erythrocyte) were used for the photohemolysis test.

The 3T3 neutral red uptake photoxicity test (3T3 NRU PT) was adopted as an alternative to the evaluation of cosmetic phototoxicity in April 2004 as an OECD Toxicity Test standard. It is an evaluation method to classify the cytotoxicity between light irradiated and non-irradiated cells by using the NRU test to classify the phototoxic substance when the difference (PIF, photoirritation factor) becomes 5 times or more.

Table 5 shows the phototoxicity results of PE and P7 extracts prepared from laver by the 3T3 NRU phototoxicity assay.

As shown in Table 5, P7, a purified extract, has an average half lethal dose (IC ₅₀ , which is the amount of animals used in the experiment where a certain amount of a substance that is expected to be toxic is killed more than half of the animal). It was determined as a non-phototoxic substance with a PIF value of 1 exceeding 1000 mg / ml regardless of whether or not. In addition, the crude extract PE had a relatively low toxicity level than the P7 concentration, but the toxicity was relatively high, but the PIF value was less than 5, which was determined as a non-phototoxic material.

Extract Type 3T3 NRU Phototoxicity Test Results Average IC ₅₀ Concentration
(UV-, mg / ml) Average IC ₅₀ Concentration
(UV +, mg / ml) PIF
[IC ₅₀ (UV-) / IC ₅₀ (UV +)] PE 932.5 215.0 4.34 P7 > 1000 > 1000 One

Figure 2 is a graph showing the phototoxicity results of the non-toxic UV protection crude extract (PE) and secondary purified extract (P7) extracted from seaweed according to the photohemolysis test using human red blood cells. In addition, Figure 3 is a photograph showing the phototoxicity results according to the photohemolysis test using human erythrocytes when the concentration of non-toxic UV protection crude extract (PE) extracted from seaweed is 500㎍ / ㎖, Figure 4 It is a photograph showing the phototoxicity results according to the photohemolysis test using human erythrocytes when the concentration of the non-toxic UV blocking secondary tablet extract (P7) extracted from the 250 ㎍ / ㎖. The ultraviolet dose used in the photohemolysis test shown in FIGS. 2 to 4 was UVA 15J / cm 2.

The phototoxicity result by the photohemolysis test using human erythrocytes was almost the same as the phototoxicity test result by the 3T3 NRU phototoxicity test.

(2) Cytotoxicity

Cytotoxicity test was performed on HaCaT, a human keratinocyte, against PE and P7 extracts prepared from seaweed. The cytotoxicity was evaluated using the Neutral red assay.

Table 6 shows the results of cytotoxicity test on HaCaT, human keratinocytes of PE and P7, which are extracts prepared from 3 ginseng.

As shown in Table 6, both the half lethal concentrations of HaCaT of PE and P7 were more than 5 mg / ml, which is considered to be a non-cytotoxic substance.

Extract Type IC ₅₀ concentration (mg / ml) PE > 5 P7 > 5

As discussed above, both the crude extract and the purified extract prepared from laver were found to be non-toxic sunscreen extracts, and the extract may be used as a main component of a cosmetic composition such as a sunscreen. At this time, the cosmetic composition may be prepared in the form of cream, lotion, gel, tonic or mist, the content of the sunscreen injection may be appropriately selected in the range that does not cause toxicity to the skin.

In Figures 2 to 4, PE represents a non-toxic UV-blocking crude extract extracted from laver by the manufacturing method according to an embodiment of the present invention, P7 from the laver by the manufacturing method according to an embodiment of the present invention The extracted non-toxic UV blocking secondary tablet extract is shown, UV- represents the state not irradiated with UV, UV + represents the state irradiated with ultraviolet rays.

Claims (14)

(a) preparing one or more red algae selected from laver or edible hair;
(b) adding a mixed solvent having a volume ratio of 1 to 5 carbon atoms to water of 1: 9 to 9: 1 to the prepared red algae and maintaining the temperature at a temperature of 30 to 60 ° C. to extract the active ingredient; And
(c) filtering the mixed solvent including the active ingredient and the remaining red algae solids to separate the supernatant from the remaining red algae solids; Method for producing a non-toxic sunscreen extract comprising a.
According to claim 1, wherein the red alga of the step (a) is a method for producing a non-toxic sunscreen extract, characterized in that the cabbage.
According to claim 1, wherein the non-toxic extract for sun protection, characterized in that it further comprises the steps of (b) and (c) repeating steps 1 to 3 with the remaining red algae solid of step (c) How to make.
The method of claim 1,
(d) removing the impurities by passing the separated supernatant through a membrane filter.
The method of claim 1,
(e) concentrating and drying the separated supernatant to obtain a solidified crude extract.
6. The method of claim 5,
(f) dissolving the solidified crude extract in water and passing through a membrane filter to remove impurities; And
(g) developing the crude extract aqueous solution from which the impurities have been removed in a concentration gradient between water and an alcohol having 1 to 5 carbon atoms and obtaining a crude extract fraction absorbing an ultraviolet wavelength of 280 to 400 nm. How to prepare a non-toxic sunscreen extract.
The method of claim 6,
(h) passing the crude extract fraction through a hydrophobic mutual column to obtain a primary purified extract. The method of manufacturing a non-toxic sunscreen extract further comprising a.
The method of claim 7, wherein
The hydrophobic cross column is a method for producing a non-toxic sunscreen extract, characterized in that the hydrophobic alkyl group, a hydrophobic aryl group, a hydrophobic cyano group, and a hydrophobic functional group selected from the group consisting of a hydrophobic amine group bonded to the silica gel surface. .
The method of claim 7, wherein
(i) passing the first purified extract through a size exclusion column (Size Exclusion Column) to obtain a second purified extract; method for producing a non-toxic sunscreen extract, characterized in that it further comprises.
The method of claim 9,
(j) concentrating and drying the secondary tablet extract to obtain a solidified tablet extract; a method for manufacturing an extract for non-toxic UV protection, further comprising:
Non-toxic sunscreen comprising a non-toxic sunscreen extract prepared by the method of any one of claims 1 to 10.
12. The non-toxic sunscreen according to claim 11, wherein the non-toxic sunscreen extract contains Mycosporine-like amino acids (MAA) as an active ingredient.
The method of claim 12, wherein the non-toxic sunscreen extract is Mycosporine-like amino acids (MAA), parity (Palythine), Asterina-330 (Asterina-330), and cynoline ( Shinorine) non-toxic sunscreen.
14. The non-toxic sunscreen extract according to claim 13, wherein the non-toxic sunscreen extract further comprises Porphyra-334 as Mycosporine-like amino acids (MAA). .

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