JP5829866B2 - Ultraviolet radiation damage protective agent containing chlorotanine-derived fluorotannins as an active ingredient and method for producing the same - Google Patents

Description

The present invention relates to an ultraviolet radiation damage protective agent comprising fluorochrome tannin derived from chrome as an active ingredient and a method for producing the same , for example, a protective agent for preventing sunburn caused by sunlight irradiation and reducing skin damage, and production thereof. The present invention relates to methods, cosmetics, pharmaceuticals, quasi drugs and foods containing natural ingredients extracted from seaweeds.

Conventionally, extracts from seaweed have physiological activity and have been proposed for use as useful components for various medical and food uses. For example, as an angiogenesis inhibitor that can suppress angiogenesis more effectively and has improved safety for living organisms, it has the following characteristics: brown algae, cucurbitaceae ( Laminaria japonica ), arame ( Eisenia bicyclis ), sagarame ( Eisenia arborea ) ), Ecklonia stolonifera (Ecklonia stolonifera), chromate (Ecklonia kurome), beforehand (Ecklonia cava), brown algae kelp eyes Chigaiso Department of wakame (Undaria pinnatifida), Aowakame (Undaria petersenian a), spread (Undaria undarioides), Ainu wakame ( Alaria praelonga ), and Tigaiso ( Alaria crassifolia ); brown algae slats and scallops of the genus Strawberry ( Sargassum f ulvellum ), hijiki ( Hizikia fusiformis ), and red algae ( Sargassu m horneri ); Mozuku ( Nemacystus decipiens ), brown alga An angiogenic preparation (Patent Document 1) containing, as an active ingredient, an extract derived from at least one seaweed selected from the group consisting of: Cladosiphon okamuranus ; In addition, aldose reductase inhibitors (Patent Document 2), AGE production inhibitors (Patent Document 3), and the like have been proposed.

As one of such physiologically active substances derived from seaweeds, fluorotannins have attracted attention. For example, a β-glucuronidase inhibitor, which is safe and easy to apply to food and cosmetics, is an extract derived from seaweed consisting of Ecklonia kurome and Ecklonia cava. (Patent Document 4), as a lipid metabolism improving substance effective in improving lipid metabolism, it has an excellent lipid metabolism improving effect by ingesting seaweed, particularly chlorotannin derived from chrome, and is effective for foods and beverages and pharmaceuticals. By using it as a component, it can be applied to the prevention and treatment of diseases caused by abnormal lipid metabolism (Patent Document 5), and as a hepatitis therapeutic agent or hepatitis preventive agent, containing phlorotannin (Patent Document) 6) An antibacterial agent mainly composed of fluorotannins, including phloroglucinol, eckol, and florofcofurecoal A An antibacterial agent (Patent Document 7) that is one or a combination of two or more selected from (phlorofucofuroeckol A), dieckol, and 8,8′-bieckol has been proposed.

JP 2006-22033 A JP 2008-214245 A JP 2008-214246 A JP 2010-189439 A JP 2006-328010 A JP 2010-30917 A JP 2003-277203 A

J. Dermatol. Sci., 55, 161-169 (2009) J. Korean Med. Sci., 25, 930-937 (2010) Biol. Pharm. Bull., 28 (7), 1244-1248 (2005) Biol. Phram. Bull., 31 (2), 284-289 (2008) Exp. Mol. Med., 35 (4), 263-268 (2003)

  Many compounds having an ultraviolet irradiation protection action are known and used as sunscreens, etc., but the present invention has newly found a naturally-occurring substance having an ultraviolet irradiation damage protection action and is derived from chrome. It is intended to provide an ultraviolet radiation damage protective agent comprising the above fluorotannins as an active ingredient.

The gist of the present invention is the ultraviolet radiation damage protective agent of the following (1) to ( 6 ).
(1) The following chemical structural formula:
A protective agent for ultraviolet radiation damage characterized by comprising, as an active ingredient, a chrome-derived fluorotannin containing a chrome polyphenol selected from:
(2) The ultraviolet radiation damage protective agent according to the above (1), wherein the chlorotannins derived from chrome are ethanol aqueous extracts of chrome.
( 3 ) The ultraviolet radiation damage protective agent according to the above (1) or (2) , which has a function of protecting the skin from ultraviolet radiation damage.
( 4 ) The ultraviolet radiation damage protective agent according to the above ( 3 ), wherein the skin is dermal fibroblasts.

Further, the external preparation for skin of the present invention the following (5), and the gist of the method for producing the ultraviolet radiation fault protection agent (6) and cosmetics (7), and (8) to (10).
( 5 ) A skin external preparation characterized by containing the ultraviolet radiation damage protective agent according to any one of (1) to ( 4 ) above.
( 6 ) A cosmetic comprising the ultraviolet radiation damage protective agent according to any one of (1) to ( 4 ) above.
( 7 ) The cosmetic according to ( 6 ), wherein the cosmetic is a sunscreen material.
(8) The method for producing an ultraviolet radiation damage protective agent according to (1) or (2) above, wherein chrome is extracted with an aqueous solution having an ethanol concentration of 40 to 80 (v / v)%.
(9) The method for producing the ultraviolet radiation damage protective agent according to (8), wherein the column fractionation is further performed using an aqueous ethanol solution as an eluent.
(10) The method for producing the ultraviolet radiation damage protective agent according to the above (9), wherein the aqueous ethanol solution as an eluent is an aqueous solution having an ethanol concentration of 20 to 60 (v / v)%.

  The ultraviolet irradiation protection agent containing fluorochrome tannins derived from chrome of the present invention exhibits an effect superior to that of a commercially available protection agent as an ultraviolet irradiation protection agent. It is possible to protect the skin and skin fibroblasts caused by damage.

The structural formula of each fluorotannin derived from chrome is shown. The structural formula of each fluorotannin derived from chrome is shown. The structural formula of each fluorotannin derived from chrome is shown. The dry weight of the extract obtained by extracting chrome with 70 (v / v)% EtOH for 1 to 24 hours is shown. The total polyphenol content of the extract obtained by extracting chrome with 70 (v / v)% EtOH for 1 to 24 hours is shown. The relationship between the EtOH density | concentration which extracted chrome and the dry weight of an extract is shown. The relationship between the EtOH density | concentration which extracted chrome and the total polyphenol amount of an extract is shown. The relationship between the elution ethanol density | concentration at the time of fractionating the extract which extracted chrome with 70 (v / v)% EtOH with a Diaion (trademark) HP20 column, and the dry weight of the eluate of each fraction is shown. The extract obtained by extracting chrome with 70 (v / v)% EtOH is Diaion (registered trademark). The relationship between the elution ethanol density | concentration at the time of fractionating with HP20 column and the total polyphenol amount of each fraction is shown. The protective effect of the black fraction on UVA irradiation damage is shown by animal test data based on skin thickness changes in hairless mice. The protective effect of the black fraction on UVB irradiation damage is shown by animal test data based on skin thickness changes in hairless mice. The protective effect of the black fraction on UVB irradiation damage is shown by animal test data based on the skin color change of hairless mice. The protective effect against UVB irradiation damage by the concentration of the chrome fraction is shown by animal test data based on the skin thickness of hairless mice. The protective effect against UVB irradiation damage of each fraction obtained by fractionating an extract obtained by extracting chrome with 70 (v / v)% EtOH with a Diaion (registered trademark) HP20 column is shown in animal test data based on the skin thickness of hairless mice. Show. The UVB irradiation protective effect of each fraction obtained by fractionating an extract obtained by extracting chrome with 70 (v / v)% EtOH using a Diaion (registered trademark) HP20 column is shown by test data in human skin fibroblasts. Extracts obtained by extracting chrome with 70 (v / v)% EtOH were fractionated with a Diaion (registered trademark) HP20 column and separated with 40 (v / v)% EtOH. The protective effect of the compound on UVB irradiation damage is shown by test data in human skin fibroblasts. The protective effect against UVB irradiation damage of the chrome fraction is shown by animal test data based on skin thickness change of hairless mice applied for a long time. The protective effect of the black fraction on UVB irradiation damage is shown by animal test data based on changes in skin water content of hairless mice applied for a long time.

  The present invention relates to a UV radiation damage protective agent comprising, as an active ingredient, fluorotannins, an extract derived from chrome, for example, having an effect of protecting from skin damage and skin fibroblast damage caused by UV light. It is. Further, the protective agent of the present invention is effective for promoting the proliferation of fibroblasts of human skin as an external preparation for skin, and is a constituent material for wound healing agents, cosmetics, bath preparations, fibroblast culture media, foods and the like. Can be used as

[UV]
Ultraviolet rays emitted from the sun are classified as UVA, UVB, and UVC from the viewpoints of human health and environmental impacts. However, UVC is absorbed by the atmosphere and does not reach the ground. From the main, protection from UVA and UVB is important.
These ultraviolet rays will be described.
UVA is a kind of ultraviolet rays that reach the earth's surface and refers to ultraviolet rays having a wavelength of 320 to 400 nm. This ultraviolet ray reaches deep into the dermis and promotes aging of the skin such as reduced elasticity, wrinkles and sagging. In addition, UVA is related to the reaction (immediate blackening) in which skin becomes dark immediately after exposure to strong sunlight and disappears in 30 minutes to several hours. This is a phenomenon in which light melanin pigment in the skin epidermis changes to dark melanin pigment and turns black.
UVB is a kind of ultraviolet rays that reach the earth's surface and refers to ultraviolet rays having a wavelength of 290 to 320 nm. Mostly absorbed by the epidermis, causing inflammation (redness, swelling, skin cancer, etc.) in the skin in a short time. A few days after the inflammation subsides, the melanin pigment in the skin increases and turns black (delayed blackening). The blackened epidermis peels off by metabolism in about one month and returns to the original skin color, but repeated UVB promotes skin aging such as wrinkles and blemishes. It can also induce skin cancer.

[Kurome]
Kurome is a kind of brown algae belonging to the genus Kombuaceae, and is a seaweed that grows by adhering to rocks near the tidal zone. The size of alga bodies is usually 40-50 cm, sometimes over 1 m, and the alga bodies in winter are edible. Its form resembles that of the same genus, but it can be distinguished from the fact that wrinkles are formed in the leaf-shaped part and the overall size is small. Also, unlike arame, the stem does not bisect. In the waters near Japan, it is distributed from the middle south of Honshu Pacific coast to Kyushu, the Seto Inland Sea, and the Sea of Japan. Large and perennial. Individuals that begin to sprout are single leaves on short stems.

[Fluorotannins]
As the fluorotannins contained in seaweed, No. 1 shown in FIG. 7 = equol, no. 3 = Dieckol, no. 2 = 8,8'-bieckol, no. 15 = 6,8'-bieckol, no. 6 = 2-phloroeckol, no. 5 = phlorofucofuroeckol-A, No. 5 4 = Dioxinodehydroeckol is known. The total amount of phlorotannins is shown by the total amount of polyphenols measured by “Folin-Ciocalteu method using phloroglucinol as a standard product” (measurement wavelength is 720 nm).

[Extraction of fluorotannins from chrome]
A method for extracting fluorotannins from chrome is described below.
In the present invention, an ethanol aqueous solution, which is a solvent used in the food industry, is used to extract fluorotannins from chrome in consideration of the influence on the human body and use in foods, pharmaceuticals, and the like. In the extraction test, water, ethanol, and an aqueous solution with an ethanol concentration of 20 to 80 (v / v)% were used, and the extraction was promoted by mixing with the raw material chrome and stirring. As can be seen from FIG. 3, the stirring time and the total polyphenol extraction amount are considered to increase as the stirring time becomes longer, and the whole amount is extracted after 24 hours. Regarding the ethanol concentration of the solvent for extraction, polyphenol is extracted when the ethanol concentration is 40 (v / v)% or more, particularly preferably in the range of 40 to 80 (v / v)%, more preferably The range of ethanol concentration 50-70 (v / v)% can be mentioned.

Subsequent extraction conditions mainly consisted of “70 (v / v)% EtOH extraction”. This indicates that the polarity of 70 (v / v)% concentration EtOH is suitable for the extraction of fluorotannins. The possibility of microbial contamination when the temperature is high due to high EtOH concentration can be avoided. After extraction, a styrene-divinylbenzene synthetic adsorbent [for example, Diaion (registered trademark) manufactured by Mitsubishi Chemical Corporation] HP20] or a styrene-based synthetic adsorbent (for example, Amberlite XAD (registered trademark) 2 or Amberlite XAD (registered trademark) 4 manufactured by Dow Chemical Co., Ltd.) is easily concentrated in the pretreatment for fractionation. by. The ratio of chrome and extraction solvent in the extraction can be changed according to the extraction conditions such as the stirrer and the capacity of the container, but the ratio was 1:10. As the extraction temperature is higher, the extraction rate is improved, but substances other than the target substance are extracted, and the viscosity of the extract may be increased. Therefore, extraction at room temperature (water temperature 20 to 25 ° C.) is preferable.
In the following description, “ethanol” may be abbreviated as “EtOH”, and “ethanol (** (v / v)% aqueous solution)” may be abbreviated as “** (v / v)% EtOH”.

[Column fraction]
Extraction with 70 (v / v)% and 50 (v / v)% ethanol from chrome was separated by a column packed with Diaion (registered trademark) HP20 [Mitsubishi Chemical Corporation] by the above extraction method. I drew it. When water, an aqueous solution having an ethanol concentration of 20 to 80 (v / v)%, and 100 (v / v)% ethanol were used as an eluent, the total amount of polyphenols was 60 (v / v)% ethanol concentration from water. The fraction was eluted in a large amount. When the liquid eluted at each concentration was analyzed by NMR, the composition of each fraction was as follows, and it was found that fluorotannins elute into ethanol fractions of 20-60 (v / v)%. did.
The eluate of each fraction is described next.
“Water-eluting” fraction: almost polysaccharides, excluding inorganic salts.
“20 (v / v)% EtOH elution” fraction: Fluorotannins start to appear. Some contamination with unsaturated fatty acids is observed.
“40 (v / v)% EtOH elution” fraction: the mainstream fraction of the discharge of phlorotannins. Some contamination with unsaturated fatty acids is observed.
“60 (v / v)% EtOH elution” fraction: mainly unsaturated fatty acids, but residual fluorotannins. The absolute amount of fluorotannins is estimated to be about 5 to 10% of the total amount of the “20 (v / v)% EtOH” fraction and the “40 (v / v)% EtOH” fraction.
"80 (v / v)% EtOH elution" fraction: Almost only unsaturated fatty acids, and no fluorotannins are observed.
"100 (v / v)% EtOH elution" fraction: almost only unsaturated fatty acids, and no fluorotannins are observed.

In addition, as a result of the previous tests, the following matters were also clarified.
(1) The fraction that is detected by the foreign thiocult method (Folin-Ciocalteu method) but does not show the peak of fluorotannins by NMR is presumed to contain a reducing substance different from fluorotannins. Is done.
(2) “70 (v / v)% EtOH” is the basis of extraction, but when the concentration of extracted EtOH is compared, “50 to 70 (v / v)% EtOH” is polyphenol (fluorotannins). It turned out to be suitable for extraction.
(3) As a result of “fractionation of column of 70 (v / v)% EtOH extract”, the target fluorotannins were “20 (v / v)% EtOH elution fraction” to “60 (v / v )% EtOH elution fraction ".
(4) As a result of “diaion (registered trademark) HP20 column fractionation of 50 (v / v)% EtOH extract”, the target fluorotannins are “20 (v / v)% EtOH elution fraction” to “ 60 (v / v)%
It was found that "EtOH elution fraction" was eluted at a high rate.

[Protective effect of chrome fraction on UVA or UVB irradiation damage (animal experiment)]
In order to demonstrate the protective effect of the chrome fraction of the present invention against UVA irradiation damage, the fraction was applied to the skin of hairless mice together with 8-methoxypsoralen, a UVA sensitizer, and irradiated with UVA. The degree of damage afterwards was judged by skin thickness and color tone. The change in skin thickness was greatly reduced compared to control (ethanol aqueous solution) and the same effect as positive control (commercially available sunscreen) was recognized It was. In addition, the change in color tone after 72 hours of irradiation showed that the chromate fraction of the present invention had an excellent effect with a smaller amount of change than the control group. When the same animal experiment was conducted by changing the concentration of the chromate fraction using UVB as the light source, an effect of suppressing skin thickening was observed in the concentration range of 1% to 0.25%.
The fraction containing chrome polyphenol (fluorotannins) eluted with 20-60 (v / v)% ethanol aqueous solution has an inhibitory effect on skin thickening due to UVB, and the chrome fraction of the present invention contains UVA and UVB. The protective effect against radiation damage was shown.

[Effect of chrome extract on damage of human skin fibroblast TIG-114 by UVB irradiation]
The UVB protective effect of the chromate HP20 column fraction was examined. Human skin fibroblast TIG
-114 was irradiated with UVB, and the number of cells after culturing was measured to determine the protective effect. As a result, the fraction eluted with 20-60 (v / v)% EtOH showed human skin fibroblasts by UVB irradiation. The inhibitory effect was confirmed. From the results of the above animal experiments and cell experiments, fluorotannins extracted from seaweeds such as chrome are effective for promoting the growth of fibroblasts in human skin as a skin external preparation, and are used as wound healing agents and cosmetics. It has been found that it is a useful substance that can be used as a constituent material for bath preparations, fibroblast culture media, foods and the like.
Next, the present invention will be described with specific examples, but the present invention is not limited to the following examples.

[Production conditions and evaluation method for fluorotannin-containing extracts]
The outline | summary of the extraction apparatus and evaluation method of fluoro tannins from the chrome in the following examples is shown.
1. Extraction device Beaker used: 3L (diameter 130mm)
Equipment used: Stirring with stirring blades (φ100mm, 6-blade snow crystal type) using Three One Motor BLh300P (Shinto Kagaku).
Raw material: Chromome 2010 crops cultivated in the Oniike area of the Amakusa Fisheries Cooperative Kumamoto Prefecture (water content = 9.9%, solid content = 90.1%, polyphenol content (vs solid content) = 3 .41%)
Raw material state = Coarsely pulverized product (state crushed with garden shredder)
Filtration conditions after extraction = water-flow suction filtration, No. 2 filter paper of 150 mmφ (1) Extraction solvent = EtOH concentration 0 to 100% aqueous solution (2) Chrome: solvent ratio = 1: 10
(3) Extraction conditions = 7 days standing extraction or stirring for 1 to 24 hours (4) Extraction temperature = 20 ° C

2. Evaluation Method (1) The extraction efficiency was judged by “dry weight”.
(2) The polyphenol content was determined at a measurement wavelength of 720 nm using a “foreign thiocult method using phloroglucinol as a standard product”.
(3) The composition was confirmed by NMR analysis according to the following method.
1) The EtOH extract was dried and dissolved in an ethanol-water mixture.
2) After removing the solvent with a rotary evaporator, dry in vacuum overnight.
3) Dissolve in deuterated methanol (CD ₃ OD).
4) Measured with Bruker AVANCE III500 ( ¹ H resonance frequency = 500 MHz) manufactured by Bruker BioSpin Corporation.
5) About the NMR chart of each extract, the composition was confirmed by the integrated intensity (area intensity) ratio in the following two ranges. The “CH signal of the polysaccharide” contained in a large amount was set to “100.0”. This value does not directly indicate the molar ratio or weight ratio between the compounds (group), but is a measure of the relative quantitative ratio.
a) Signal of δ 3.6 to 4.0 ppm Polysaccharide b) δ 5.8 to 6.9 ppm Aromatic H signal of fluorotannins

[Examination of separation conditions by column]
Outline of conditions for concentrating ethanol extract of chrome and adsorbing it on a column packed with Diaion (registered trademark) HP20 manufactured by Mitsubishi Chemical Corporation, which is an example of a styrene-divinylbenzene synthetic adsorbent, followed by fractionation with ethanol Indicates.
1. Method for measuring the dry weight of the extract;
A part (100 ml) of the extract was sampled, the extract was evaporated with a rotary evaporator, dried with a vacuum pump for 24 hours or more, and weighed to determine the dry weight.
2. Diaion (registered trademark) Fractionation by HP20 column;
(1) The ethanol extract from chrome was fractionated on a column packed with Diaion (registered trademark) HP20. Concentrate 50 or 70 (v / v)% EtOH extract with a rotary evaporator, and use a φ38 mm x 600 mm column (resin length 110 mm) to fractionate the concentrate with Diaion (registered trademark) HP20 resin. did.
(2) The dry weight was determined from the results of weighing the fractionated liquid after it was distilled off with a rotary evaporator and then dried with a vacuum pump for 24 hours or more.
(3) A 0-100 (v / v)% aqueous ethanol solution was used for fractionation.

[Extraction time]
In this example, the optimum extraction time was examined.
The sample was 200 g of chrome (solid content: 180.2 g), the dried concentration of the extract after stirring for a predetermined time at a ratio of chrome: 70 (v / v)% EtOH = 1: 10, and total polyphenols contained in the extract The amount was measured. At the same time, a stationary extraction test was conducted for 7 days. The dry weight is shown in FIG. 2, and the total polyphenol content is shown in FIG.
As a result, it is estimated that almost the entire amount of polyphenols was extracted under “7-day standing conditions” and “24-hour stirring conditions”, compared with the polyphenol content (3.41% solid content) of the raw chrome. . It was confirmed by NMR that each extract contained polyphenol. As a stirring extraction condition, it was found that an extraction time of “at least 1 hour or more, preferably 3 hours or more” is required.

[Concentration of extracted EtOH]
In this example, the optimum EtOH concentration for extraction was examined. As a sample, 200 g of chrome (solid content = 180.2 g) was stirred for a predetermined time at a ratio of chrome: EtOH solution = 1: 10, and the dry concentration of the extract and the total amount of polyphenol contained in the extract were measured. In the extraction of “0 (v / v)% EtOH” and “30 (v / v)% EtOH”, the extract was too viscous to be filtered by filter paper, but could be filtered by other means. It is judged that there is. The dry weight is shown in FIG. 4, and the total polyphenol content is shown in FIG. As is clear from FIGS. 4 and 5, “50 (v / v)% EtOH extraction” was able to extract more polyphenol than “70 (v / v)% EtOH extraction”.

[Diaion (registered trademark) Adsorption fraction by HP20 column]
The sample “70 (v / v)% EtOH extract from chrome” [equivalent to 216.7 g of chrome (solid content = 195.2 g)] was concentrated with a rotary evaporator, and this concentrated solution was Diaion (registered trademark). It was made to adsorb | suck to HP20 column and fractionated with 0-100 (v / v)% EtOH aqueous solution. The dry weight of each fraction is shown in FIG. The total amount of polyphenols in each fraction was measured and shown in FIG. The “water-eluted fraction” has a low polyphenol content but a large dry weight (a lot of distribution), so the total amount of polyphenol is large.
In the “80 (v / v)% EtOH elution fraction” and “100 (v / v)% EtOH elution fraction”, a small amount of polyphenol was detected by the foreign thiocult method. Since the signal of aromatic H is not recognized, it is presumed that another reducing substance is contained. The total amount of polyphenol detected by the foreign thiocult method is “2.15%”, which is a recovery rate of about 63% compared to the polyphenol content of raw material chrome (solid content: 3.41%).
In addition, the recovery rate is about 74% compared to the “recovery rate = 2.9%” under the “6 hr stirring extraction” condition under which the conditions were examined. "20 (v / v)% EtOH elution fraction", "40 (v / v)% EtOH elution fraction", "60 (v / v)% EtOH elution estimated to contain phlorotannins The total polyphenol content of “fraction” is “2871.5 mg” and the ratio of “raw material solids” to “1.47%”. The recovery rate is about 43% compared to the polyphenol content of raw material chrome (3.41% solid content). In addition, the recovery rate is about 51% compared with the “recovery rate = 2.9%” under the “6 hr stirring extraction” condition in which the conditions were examined.

[Diaion (registered trademark) Composition of product fractionated by HP20 column]
The composition of each fraction (Example 5) obtained by fractionating the extract obtained by extracting chrome with 70 (v / v)% EtOH was compared and examined with the next peak of the NMR chart.
(1) δ 3.6 to 4.0 ppm: CH signal of polysaccharide (2) δ 5.3 to 5.5 ppm: signal of olefin H of unsaturated fatty acid (3) δ 5.8 to 6.9 ppm: of fluorotannins Aromatic H signal The analysis results of the NMR chart of each fraction are as follows.
“Water-eluting” fraction: Mostly polysaccharides, excluding inorganic salts.
“20 (v / v)% EtOH elution” fraction: Fluorotannins start to appear. Some contamination with unsaturated fatty acids is observed.
“40 (v / v)% EtOH elution” fraction: the mainstream fraction of the discharge of phlorotannins. Some contamination with unsaturated fatty acids is observed.
“60 (v / v)% EtOH elution” fraction: mainly unsaturated fatty acids, but residual fluorotannins. The absolute amount of fluorotannins is estimated to be about 5 to 10% of the total amount of the “20 (v / v)% EtOH” fraction and the “40 (v / v)% EtOH” fraction.
"80 (v / v)% EtOH elution" fraction: Almost only unsaturated fatty acids, and no fluorotannins are observed.
"100 (v / v)% EtOH elution" fraction: almost only unsaturated fatty acids, and no fluorotannins are observed.

  The same tests as in Examples 5 and 6 were performed on Diaion (registered trademark) HP20 column fractions of 50 (v / v)% EtOH extract, and the same results as in Example 6 were obtained.

[Summary of the animal test of the protective effect of the chromate fraction against UVA irradiation damage]
In this example, for a 40 (v / v)% EtOH eluate obtained by fractionating a chrome 70 (v / v)% EtOH extract with a Diaion (registered trademark) HP20 column, UVA irradiation failure was observed in a UVA irradiation short-term model. The outline of the test conditions for confirming the protective effect was shown (references: non-patent documents 1 and 2).
1. Reagent / Specimen (1) Chromome extract: Extract extracted from 30 kg of dried chrome and left standing for 7 days with 120 L of 70 (v / v)% EtOH.
(2) Chromome fraction: A fraction obtained by adsorbing a chromate extract onto a Diaion (registered trademark) HP20 column and then eluting with "40 (v / v)% EtOH".
(3) Solarene solution: 8-methoxypsoralen [8-MOPS: manufactured by Tokyo Chemical Industry Co., Ltd.] was dissolved in EtOH at 0.1%. Used as a sensitizer for amplifying damage caused by UVA.
(4) Control solution: 60 (v / v)% EtOH.
(5) KP40 solution: 40 (v / v)% EtOH Dissolve the eluted fraction in 60 (v / v)% EtOH to 2.5%.
(6) Sunscreen agent: Commercially available sunscreen (SPF 25, PA ++). It has the effect of blocking ultraviolet rays that hit the skin. Used as a positive control.
(7) UVA: Sankyo Electric GL-20BLB x 5 pieces are arranged in parallel and irradiated.
2. Test group (1) Control group: A control solution was used.
(2) Positive control group: Sunscreen agent is used.
(3) KP-40 group: KP-40 solution is used.

3. Test method
(1) Prepare 18 hairless mice.
(2) All mice are labeled with an ear punch and the back skin thickness is measured with a micrometer.
(3) Divide into three groups so that the average skin thickness is uniform (control group, positive control group, KP-40 group)
(4) Gently strip the back of the mouse 5 times with cello tape (registered trademark).
(5) Drop 50 microL of 0.1% 8-MOP / EtOH on the back of the mouse and dry with a dryer.
(6) Repeat (4)-(5) twice.
(7) After 30 minutes, mark the back of the control group mouse with eyelets (15 mm diameter) and vermilion, drop 25 microL of 60 (v / v)% EtOH into the mark, and dry with a drier.
(8) In the same way, mark the mice in the positive control group with eyelets and apply a commercial sunscreen inside (approx. 1 cup of microspatel).
(9) Mark KP-40 group mice with eyelets, drop 25 microL of KP40 in the mark, spread, and dry with a dryer.
(10) The mouse tail is fixed to the cage with gum tape, and the buttocks including the hind limbs and the back of the head including the fore limbs are fixed with vinyl tape. Place 4 heads x 3 rows = 12 heads in a plastic cage.
(11) Irradiate with UVA for 2 hours.
(12) Immediately after UV irradiation and after 20 hours, the skin thickness in the mark on the back is measured with a micrometer.
(13) Perform (4)-(10) again, this time for 4 hours.
(14) Immediately after UV irradiation and 20 hours later, the skin thickness in the mark on the back is measured with a micrometer.

4). Test results
The result of the animal test of the protective effect against UVA irradiation damage by KP-40 is shown in FIG.
In the control group, the skin thickness was increased by UVA irradiation, and skin damage due to UVA irradiation was shown. The positive control group was found to suppress skin thickness thickening by UVA. The KP-40 group was found to suppress UVA skin thickening more strongly than the positive control group. Since the 40 (v / v)% EtOH eluate is mainly composed of polyphenol by NMR analysis, it is presumed that the UVA irradiation protection effect is due to chrome polyphenol (fluorotannins).

[Summary of the animal test of the protective effect of the black fraction on UVB irradiation damage]
In this example, a chromate 70 (v / v)% EtOH extract was fractionated on a Diaion (registered trademark) HP20 column, and 40 (v / v)% EtOH eluate was irradiated with UVB irradiation in a short-term UVB irradiation model. The outline of the test conditions for confirming the obstacle protection effect was shown (references: Non-Patent Documents 1 and 2).
1. Reagent / Specimen (1) Chromome extract: Extract extracted from 30 kg of dried chrome and left standing for 7 days with 120 L of 70 (v / v)% EtOH.
(2) Chromome fraction: Diatom (registered trademark) A fraction eluted with “40 (v / v)% EtOH” after being adsorbed on an HP20 column.
(3) Hydrophilic ointment: Pharmacopoeia hydrophilic ointment “Whey” (Nipro Pharma)
(4) Control ointment: Water was added to 4 times the amount of hydrophilic ointment and stirred well in a mortar.
(5) KP-40 ointment: 40 (v / v)% EtOH elution fraction was dissolved in 60 (v / v)% EtOH so as to be 10%, added to 4 times amount of hydrophilic ointment, and mortar Stir well (final concentration = 2% KP-40).
(6) Sunscreen agent: Commercially available sunscreen (SPF 25, PA ++). It has the effect of blocking ultraviolet rays that hit the skin. Used as a positive control.
(7) UVB: Sankyo Electric GL-20SE x 4 pieces are irradiated with a glass filter equivalent to SCHOTT WG295 with low wavelength components cut.
2. Test group
(1) Control group: Use control ointment.
(2) Positive control group: Sunscreen agent was used.
(3) KP-40 group: KP-40 ointment is used.

3. Prepare 18 ares mice HOS: HR-1 (female) (8 weeks old) to test method (1).
(2) The color tone of the back skin is measured with a spectrocolorimeter (Konica Minolta CR-400), and divided into 3 groups (6 heads per group) so that the a * value (redness) is uniform.
(3) Apply about 10 mg / cm ^{2 of} control ointment, sunscreen agent and KP-40 ointment to the back of each group.
(4) Irradiate UVB with 200 mJ / cm ² .
(5) Reapply each group of ointments immediately after irradiation.
(6) At 48 hours and 72 hours after irradiation, measure the skin thickness with a micrometer and use it as an index of edema.
(7) 72 hours after irradiation, measure the color tone of the back skin with a spectrocolorimeter and calculate the a * value.

4). Test results
The result of the animal test of the protective effect with respect to UVB irradiation damage by KP-40 is shown in FIG. 9 and FIG. In the control group, the skin thickness increased by UVB irradiation, and at the same time the redness increased,
Skin damage due to UVB irradiation was shown. The positive control group was found to suppress UVB damage in both skin thickness and color tone. In the 2.0% concentration group of KP-40 ointment, the same disorder inhibiting effect as that in the positive control group was observed.
Furthermore, the active ingredient concentration of KP-40 ointment is 1.0%, 0.25%, 0.1%,
When the skin thickening was measured 24 to 72 hours after UVB irradiation while changing to 0.025% and 0.01%, the skin thickness showed the maximum value 48 hours after irradiation in all groups. When the skin thickness after 48 hours was compared, as shown in FIG. 11, the “1% group” and “0.25% group” of “KP-40” showed a skin thickening suppressing action.
In addition, when comparing the skin thickening after 48 hours, which is usually used for determining the UV radiation damage protective effect, the “20 (v / v)% EtOH elution fraction” and “40 (v / v)% EtOH elution fraction” Min ”and“ 60 (v / v)% EtOH-eluting fraction ”ointment group were found to significantly suppress the increase in skin thickness (edema) compared to the“ UV (+) control ”group. (FIG. 12). Since these fractions are recognized to contain polyphenols, it is presumed that the UVB radiation damage protection effect is due to chrome polyphenols (fluorotannins).

[Effect of chromate HP-20 fraction on human skin fibroblast TIG-114 injury by UVB irradiation]
About each fraction which fractionated chrome 70 (v / v)% EtOH extract with HP-20 column, the protection effect with respect to the damage by UVB irradiation of human fibroblast TIG-114 was examined (references: nonpatent) Literature 3-5).
1. Reagent / Specimen (1) Chromome extract: 30 kg to 70 (v / v)% EtOH with stirring and extraction for 6 hours. (2) Chromome fraction: Chromome extract was adsorbed to HP-20 column. Later, “water”, “20 (v / v)% EtOH”, “40% (v / v) EtOH”, “60 (v / v)% EtOH”, “80 (v / v)% EtOH”, Fraction eluted with "100 (v / v)% EtOH".
(3) Human fibroblast TIG-114: Obtained from Human Science Research Resource Bank (JCRB0534) and cultured in DMEM medium supplemented with 10% fetal bovine serum.
(4) UVB: Sankyo Electric GL-15SE x 3 pieces are arranged in parallel and irradiated.
2. Test group (1) Control group: Mixed in PBS medium containing neither chrome extract nor fraction.
(2) Chromome fraction group: “Water”, “20 (v / v)% EtOH”, “40 (v / v)% EtOH”, “60 (v / v)% EtOH” fractions are “ 200 μg / ml, mixed in PBS medium containing 400 μg / ml ”. “80% EtOH” and “100 (v / v)% EtOH” fractions were mixed in PBS medium containing “100 μg / ml, 200 μg / ml”.

3. Test method (1) The cell concentration is adjusted to 1 × 10 ⁵ cells / ml.
(2) 100 μl / well (10,000 cells / well) in a 96-well microbrate
well)
(3) Incubate at 37 ° C. for 24 hours. (DMEM medium supplemented with 10% fetal bovine serum)
(4) Replace the medium with 100 μl of PBS (−) medium containing the specimens of each test group, and irradiate with UVB at 400 mJ / cm ² .
(5) After 72 hours, 10% Cell Counting Kit-8 (manufactured by Wako Pure Chemical Industries, Ltd.) was added and incubated at 37 degrees, 5% CO ² incubator for 1.5 hours, and the absorbance at 450 nm was measured. To measure the number of fibroblasts.

4). Results The number of cells after culturing for 72 hours is expressed as a ratio (UVB (+) / UVB (−)) to “number of cells at ∪VB (−)”, and the results are shown in FIG.
Among the chrome HP-20 fractions, “20 (v / v)% EtOH eluate”, “40 (v / v)% EtOH eluate”, “60% (v / v) EtOH eluate low In the “concentration group”, “protective action against damage of human skin fibroblasts caused by VB irradiation” was observed. These fractions were fractions with a high polyphenol content, and the same results as those of animal tests were obtained.
Among the chromate Diaion (registered trademark) HP 20 fractions, “water eluate”, “80 (v / v)% EtOH eluate”, and “100 (v / v)% EtOH eluate” include “ “Protective action against damage of human dermal fibroblasts by UVB irradiation” was not observed. In “60 (v / v)% EtOH eluate”, “80 (v / v)% EtOH eluate”, and “100 (v / v)% EtOH eluate”, the number of cells tends to decrease as the concentration increases. It is done. Since these fractions contain fatty acids, there may be cell damage caused by fatty acids.

[Diaion (registered trademark) Compound alone contained in 40 (v / v)% EtOH fraction obtained by fractionation with HP20 column]
In the next step, 19 kinds of known and novel chrome 70 (v / v)% EtOH extracts were obtained from 40 (v / v)% EtOH fractions obtained by fractionation using Diaion (registered trademark) HP20 column. The compound simple substance was obtained.

The Sephadex LH-20 column chromatography (GE Healthcare Japan Co., Ltd., Φ5 × 22 cm) was performed in four portions on 40 (v / v)% EtOH fraction (63.4 g). Elution and separation with methanol, methanol / isopropanol 5/1 solution, 4/1 solution, 2/1 solution, isopropanol, ethyl acetate, and dimethyl sulfoxide in order, (1) 9 fractions, (2) 8 fractions, (3 ) 8 fractions and (4) 9 fractions were obtained. NMR similar (1) 4th fraction, (2) 4th fraction, (3) 6th fraction, (4) 5th fraction together (5.96 g), and Cosmosil 75C18-OPN ODS silica gel column chromatography was performed using a column (Nacalai Tex Co., Ltd., Φ5 × 15 cm). 9 fractions were obtained by separating methanol / water with 500 mL of 15/85 solution, 400 mL of 2/8 solution, 400 mL of 3/7 solution, 400 mL of 4/6 solution, 400 mL of 5/5 solution, and 400 mL of methanol. The obtained first fraction (187.6 mg) was again subjected to ODS silica gel column chromatography using a Cosmo Seal 75C18-OPN column (Φ2 × 15 cm). Methanol / water was separated by 60 mL of 1/9 liquid, 10 mL of 15/85 liquid, 10 mL of 3/7 liquid, 30 mL of 4/6 liquid, and 50 mL of methanol to obtain 6 fractions.
The obtained second fraction (23.8 mg) was subjected to high performance liquid chromatography using a Unison UK-C18 column (manufactured by Intact Co., Ltd., 10 × 250 mm). Water / acetonitrile 85/15 solution was used as a solvent at a flow rate of 2 mL / min, and the eluate was detected by UV 270 nm absorption. By one purification, “8,8′-bieckol-7-sulfate, elution time 12.8 minutes” (No. 1 in FIG. 1-1) is 10 .3 g was obtained.
The physicochemical properties are shown below.
1) Molecular formula: C ₃₆ H ₂₁ O ₂₁ SNa (Measured by high resolution mass spectrum (HRMS) method)
2) Molecular weight: 844.59 (determined by ESI-MS method (MH) ⁻ = 821.0296)
3) Hydrogen nuclear magnetic resonance spectrum (400MHz, solvent CD ₃ OD):
(Ppm): 6.95 (s, 1H), 6.20 (s, 1H), 6.17 (s, 1H), 6.14 (s, 1H), 5.98 (d, J = 1.84Hz, 2H), 5.97 (d, J = 1.84Hz, 2H), 5.95 (t, J = 1.84Hz, 1H), 5.95 (t, J = 1.84Hz, 1H)
4) Carbon nuclear magnetic resonance spectrum (100 MHz, solvent DMSO-d ₆ ):
(Ppm): 160.3, 160.3, 158.7 (2C), 158.7 (2C), 151.3, 147.2, 146.0, 145.7, 144.3, 144.0, 141.8, 141.7, 141.4, 141.0, 137.1, 136.9, 126.6, 124.0, 123.3, 123.1, 122.5, 122.5, 109.9, 105.3, 99.4, 98.5, 98.1, 96.3, 96.3, 94.9, 93.9 (2C), 93.9 (2C)
5) Color reaction: Positive for anisaldehyde-sulfuric acid.

The 40% ethanol extract (63.4 g) was subjected to Sephadex LH-20 column chromatography (Φ5 × 22 cm) in four portions. Elution and separation with methanol, methanol / isopropanol 5/1 solution, 4/1 solution, 2/1 solution, isopropanol, ethyl acetate, and dimethyl sulfoxide in order, (1) 9 fractions, (2) 8 fractions, (3 ) 8 fractions and (4) 9 fractions were obtained. NMR similar (1) 3rd fraction, (2) 5th fraction, (3) 5th fraction, (4) 4th fraction together (2.15 g), and Cosmosil 75C18-OPN ODS silica gel column chromatography was performed using a column (Φ4 × 15 cm). Methanol / water separated by 2/8 liquid 200mL, 25/75 liquid 200mL, 3/7 liquid 200mL, 4/6 liquid 200mL, 5/5 liquid 200mL, 6/4 liquid 200mL, 7/3 liquid 200mL, methanol 200mL 13 fractions were obtained. The obtained second fraction (126.6 mg) was subjected to ODS silica gel column chromatography again using a Cosmo Seal 75C18-OPN column (Φ1.5 × 11.5 cm). Methanol / water was separated with 15/85 liquid 30 mL, 2/8 liquid 30 mL, 3/7 liquid 30 mL, and methanol 30 mL to obtain 8 fractions.
The obtained seventh fraction (48.8 mg) was subjected to high performance liquid chromatography using a Unison UK-phenyl column (10 × 250 mm) column. Water / acetonitrile (8/2) solution was used as a solvent at a flow rate of 2 mL / min, and the eluate was detected by UV 270 nm absorption. One purification yielded 33.5 mg of “8,8′-bieckol, elution time 28.8 minutes” (No. 2 in FIG. 1-1).
The physicochemical properties are shown below.
1) Molecular formula: C ₃₆ H ₂₂ O ₁₈ (measured by high-resolution mass spectrum (HRMS) method)
2) Molecular weight: 742.55 (determined by ESI-MS method (MH) ⁻ = 741.0722)
3) Hydrogen nuclear magnetic resonance spectrum (400MHz, solvent CD ₃ OD):
(Ppm): 6.19 (s, 2H), 6.14 (s, 2H), 5.98 (d, J = 1.88Hz, 4H), 5.96 (t, J = 1.88Hz, 2H)
4) Carbon nuclear magnetic resonance spectrum (100 MHz, solvent DMSO-d ₆ ):
(Ppm): 160.4 (2C), 158.8 (4C), 151.8 (2C), 145.9 (2C), 144.6 (2C), 141.9 (2C), 141.3 (2C), 137.3 (2C), 123.4 (2C), 123.1 (2C), 122.5 (2C), 104.4 (2C), 98.2 (2C), 96.3 (2C), 94.0 (4C), 93.9 (2C)
5) Color reaction: Positive for anisaldehyde-sulfuric acid.

Sephadex LH-20 (Φ5 cm × 21 cm) column chromatography was performed on 40% ethanol extract (15 g). Methanol 700mL, then methanol / 2-propanol was eluted and separated sequentially with 5/1 solution 560mL, 4/1 solution 560mL, 2/1 solution 560mL, 2-propanol 560mL, ethyl acetate 500mL, dimethyl sulfoxide 400mL. Obtained. This operation was repeated 5 more times.
ODS silica gel column chromatography using a Cosmo Seal 75C18-OPN column (Φ5 cm × 12 cm) was performed on the third eluted fraction (3 g). Methanol / water was separated by 250 mL of 1/9 liquid, 700 mL of 2/8 liquid, 700 mL of 3/7 liquid, 300 mL of 4/6 liquid, and 500 mL of methanol to obtain 5 fractions. “Dieckol” (No. 3 in FIG. 1-1) was obtained in the methanol fraction (468 mg).
The physicochemical properties are shown below.
1) Molecular formula: C ₃₆ H ₂₂ O ₁₈ (measured by high-resolution mass spectrum (HRMS) method)
2) Molecular weight: 742.55 (measured by ESI-MS method (M + Na) ⁺ = 765.0704)
3) Hydrogen nuclear magnetic resonance spectrum (400 MHz (solvent CD ₃ OD)):
(Ppm): 6.21 (s, 1H), 6.19 (s, 1H), 6.15 (s, 2H), 6.12 (d, J = 2.9 Hz, 1H), 6.11 (d, J = 2.9 Hz, 1H), 6.04 (D, J = 2.7 Hz, 1H), 6.01 (d, J = 2.7 Hz, 1H), 5.98 (s, 3H)
4) Carbon nuclear magnetic resonance spectrum (125 MHz (solvent DMSO-d ₆ )):
(Ppm): 160.3, 158.8 (2C), 155.9, 154.2, 153.0, 151.1 (2C), 146.1, 146.0, 145.9, 145.9, 142.6, 142.4, 141.9, 141.8, 137.2, 137.0, 124.2, 124.0, 123.2, 123.1, 122.6, 122.3, 122.2, 98.5, 98.3, 98.2, 98.0, 96.2, 94.5 (2C), 93.8, 93.6 (2C), 93.5
5) Color reaction: positive for anisaldehyde-sulfuric acid

A 40% ethanol extract (15 g) was subjected to Sephadex LH-20 column chromatography (Φ5 cm × 21 cm). Methanol 700mL, then methanol / 2-propanol was eluted and separated sequentially with 5/1 solution 560mL, 4/1 solution 560mL, 2/1 solution 560mL, 2-propanol 560mL, ethyl acetate 500mL, dimethyl sulfoxide 400mL. Obtained. This operation was repeated 5 more times.
The first elution fraction (10 g) was subjected to ODS silica gel column chromatography using a Cosmo Seal 75C18-OPN column (Φ5 cm × 13 cm). Methanol / water was separated by 1/9 liquid 650 mL, 2/8 liquid 600 mL, 3/7 liquid 600 mL, 4/6 liquid 600 mL, and methanol 500 mL to obtain 5 fractions. The obtained fourth elution fraction (102 mg) was further separated by high performance liquid chromatography using a Unison US-C18 column (250 × 20 mm). A solution of acetonitrile / water = 15/85 was used as a solvent at a flow rate of 10.0 mL / min, and the eluate was detected by absorption at UV 270 nm. The purification operation was performed once to obtain 36.8 mg of “dioxinodehydroeckol, elution time 32.8 minutes” (No. 4 in FIG. 1-1).
The physicochemical properties are shown below.
1) Molecular formula: C ₁₈ H ₁₀ O ₉ (measured by high-resolution mass spectrum (HRMS) method)
2) Molecular weight: 370.27 (measured by ESI-MS method (M + Na) ⁺ = 393.0229)
3) Hydrogen nuclear magnetic resonance spectrum (400 MHz (solvent CD ₃ OD)):
(Ppm): 6.19 (s, 1H), 6.06 (d, J = 2.8 Hz, 1H), 6.05 (d, J = 2.8 Hz, 1H), 6.03 (d, J = 2.8 Hz, 1H), 6.00 (d , J = 2.8 Hz, 1H)
4) Carbon nuclear magnetic resonance spectrum (100 MHz (solvent DMSO-d ₆ )):
(Ppm): 153.3, 153.0, 146.1, 145.9, 142.1, 141.7, 140.1, 137.2, 131.6, 125.9, 122.7, 122.5, 122.3, 98.9, 98.8, 97.6, 93.9, 93.9
5) Color reaction: Positive for anisaldehyde-sulfuric acid.

A 40% ethanol extract (15 g) was subjected to Sephadex LH-20 column chromatography (Φ5 cm × 21 cm). Methanol 700mL, then methanol / 2-propanol was eluted and separated sequentially with 5/1 solution 560mL, 4/1 solution 560mL, 2/1 solution 560mL, 2-propanol 560mL, ethyl acetate 500mL, dimethyl sulfoxide 400mL. Obtained. This operation was repeated 5 more times.
ODS silica gel column chromatography using a Cosmo Seal 75C18-OPN column (Φ5 cm × 12 cm) was performed on the third eluted fraction (3 g). Methanol / water was separated by 250 mL of 1/9 liquid, 700 mL of 2/8 liquid, 700 mL of 3/7 liquid, 300 mL of 4/6 liquid, and 500 mL of methanol to obtain 5 fractions. The obtained fourth elution fraction (828 mg) was further subjected to ODS silica gel column chromatography using a Cosmosil 75C18-OPN column (Φ3 cm × 13 cm). Methanol / water was separated by 1/9 liquid 180 mL, 2/8 liquid 180 mL, 3/7 liquid 420 mL, 4/6 liquid 150 mL, 5/5 liquid 150 mL, and methanol 500 mL to obtain 6 fractions. The obtained third elution fraction (80 mg) was separated by high performance liquid chromatography using a Unison US-C18 column (250 × 20 mm). A solution of acetonitrile / water = 25/75 was used as a solvent at a flow rate of 10.0 mL / min, and the eluate was detected by absorption at UV 270 nm. The purification operation was performed once to obtain 7.9 mg of “phlorofucofuroeckol-A, elution time 58.4 minutes” (No. 5 in FIG. 1-1).
The physicochemical properties are shown below.
1) Molecular formula: C ₃₀ H ₁₈ O ₁₄ (measured by high-resolution mass spectrum (HRMS) method)
2) Molecular weight: 602.46 (measured by ESI-MS method (M−H) ⁻ = 601.0615)
3) Hydrogen nuclear magnetic resonance spectrum (500 MHz (solvent CD ₃ OD)):
(Ppm): 6.68 (s, 1H), 6.45 (s, 1H), 6.31 (s, 1H), 6.01 (d, J = 2.2 Hz, 2H), 5.99 (t, J = 2.2 Hz, 1H), 5.97 (T, J = 2.2 Hz, 1H), 5.93 (d, J = 2.2 Hz, 2H)
4) Carbon nuclear magnetic resonance spectrum (125 MHz (solvent DMSO-d ₆ )):
(Ppm): 160.3, 160.1, 159.1 (2C), 158.9 (2C), 150.9, 150.5, 149.6, 147.0, 146.6, 144.8, 142.1, 136.9, 134.1, 126.5, 122.7, 122.6, 120.2, 103.5, 103.3, 99.2, 98.4, 96.6, 96.5, 94.9, 93.8 (2C), 93.6 (2C)
5) Color reaction: Positive for anisaldehyde-sulfuric acid.

A 40% ethanol extract (15 g) was subjected to Sephadex LH-20 column chromatography (Φ5 cm × 21 cm). Methanol 700mL, then methanol / 2-propanol was eluted and separated sequentially with 5/1 solution 560mL, 4/1 solution 560mL, 2/1 solution 560mL, 2-propanol 560mL, ethyl acetate 500mL, dimethyl sulfoxide 400mL. Obtained. This operation was repeated 5 more times.
The first elution fraction (10 g) was subjected to ODS silica gel column chromatography using a Cosmo Seal 75C18-OPN column (Φ5 cm × 13 cm). Methanol / water was separated by 1/9 liquid 650 mL, 2/8 liquid 600 mL, 3/7 liquid 600 mL, 4/6 liquid 600 mL, and methanol 500 mL to obtain 5 fractions. The obtained first elution fraction (2.9 g) was further subjected to ODS silica gel column chromatography using a Cosmo Seal 75C18-OPN column (Φ4 cm × 12 cm). Separation was carried out with 600 mL of water, 350 mL of 1/9 liquid, 350 mL of 2/8 liquid, 300 mL of 3/7 liquid, 300 mL of 5/5 liquid, 300 mL of methanol, and 6 fractions. The obtained second elution fraction (565 mg) was further subjected to ODS silica gel column chromatography using a Cosmosil 75C18-OPN column (Φ3 cm × 13 cm). 300 mL water, 90 mL methanol / water 90 mL, 1/9 solution 90 mL, 15/85 solution 90 mL, 2/8 solution 60 mL, 3/7 solution 90 mL, 35/65 solution 90 mL, 4/6 solution 90 mL, 2 Nine fractions were obtained by separation with 60 mL of / 8 liquid. The obtained second elution fraction (122 mg) was separated by high performance liquid chromatography using a Unison US-C18 column (250 × 20 mm). A solution of acetonitrile / water = 15/85 was used as a solvent at a flow rate of 9.0 mL / min, and the eluate was detected by absorption at UV 270 nm. The purification operation was carried out once to obtain 37.2 mg of “2-phloroeckol, elution time 41.6 minutes” (No. 6 in FIG. 1-1).
The physicochemical properties are shown below.
1) Molecular formula: C ₂₄ H ₁₆ O ₁₂ (measured by high resolution mass spectrum (HRMS) method)
2) Molecular weight: 496.38 (measured by ESI-MS method (M + Na) ⁺ = 519.0374)
3) Hydrogen nuclear magnetic resonance spectrum (500 MHz (solvent CD ₃ OD)):
(Ppm): 6.12 (d, J = 2.2 Hz, 2H), 6.02 (t, d = 2.2 Hz, 1H), 6.01 (d, J = 2.5 Hz, 1H), 6.00 (d, J = 2.5 Hz, 2H) ), 5.95 (s, 2H)
4) Carbon nuclear magnetic resonance spectrum (100 MHz (solvent DMSO-d ₆ )):
(Ppm): 160.4, 158.6 (2C), 154.7, 153.0, 151.0 (2C), 147.6, 146.0, 142.0, 141.5, 137.3, 124.9, 122.9, 122.7, 121.8, 98.7, 96.4, 96.2, 94.9 (2C), 94.2 (2C), 93.9
5) Color reaction: Positive for anisaldehyde-sulfuric acid.

A 40% ethanol extract (15 g) was subjected to Sephadex LH-20 column chromatography (Φ5 cm × 21 cm). Methanol 700mL, then methanol / 2-propanol was eluted and separated sequentially with 5/1 solution 560mL, 4/1 solution 560mL, 2/1 solution 560mL, 2-propanol 560mL, ethyl acetate 500mL, dimethyl sulfoxide 400mL. Obtained. This operation was repeated 5 more times.
The first elution fraction (10 g) was subjected to ODS silica gel column chromatography using a Cosmo Seal 75C18-OPN column (Φ5 cm × 13 cm). Methanol / water was separated by 1/9 liquid 650 mL, 2/8 liquid 600 mL, 3/7 liquid 600 mL, 4/6 liquid 600 mL, and methanol 500 mL to obtain 5 fractions. The obtained first elution fraction (2.9 g) was further subjected to ODS silica gel column chromatography using a Cosmo Seal 75C18-OPN column (Φ4 cm × 12 cm). Separation was carried out with 600 mL of water, 350 mL of 1/9 liquid, 350 mL of 2/8 liquid, 300 mL of 3/7 liquid, 300 mL of 5/5 liquid, 300 mL of methanol, and 6 fractions. The obtained second elution fraction (565 mg) was further subjected to ODS silica gel column chromatography using a Cosmosil 75C18-OPN column (Φ3 cm × 13 cm). 300 mL water, 90 mL methanol / water 90 mL, 1/9 solution 90 mL, 15/85 solution 90 mL, 2/8 solution 60 mL, 3/7 solution 90 mL, 35/65 solution 90 mL, 4/6 solution 90 mL, 2 Nine fractions were obtained by separation with 60 mL of / 8 liquid. The obtained second elution fraction (122 mg) was separated by high performance liquid chromatography using a Unison US-C18 column (250 × 20 mm). A solution of acetonitrile / water = 15/85 was used as a solvent at a flow rate of 9.0 mL / min, and the eluate was detected by absorption at UV 270 nm. The purification operation was performed once to obtain 18.0 mg of “equol, elution time 36.0 minutes” (No. 7 in FIG. 1-1).
The physicochemical properties are shown below.
1) Molecular formula: C ₁₈ H ₁₂ O ₉ (measured by high-resolution mass spectrum (HRMS) method)
2) Molecular weight: 372.28 (measured by ESI-MS method (M−H) ⁻ = 371.0410)
3) Hydrogen nuclear magnetic resonance spectrum (500 MHz (solvent CD ₃ OD)):
(Ppm): 6.20 (s, 1H), 6.01 (s, 2H), 6.00 (s, 3H)
4) Carbon nuclear magnetic resonance spectrum (125 MHz (solvent DMSO-d ₆ )):
(Ppm): 160.5, 158.9 (2C), 153.1, 146.2, 146.1, 142.7, 142.0, 137.3, 123.3, 122.8, 122.4, 98.6, 98.3, 96.4, 94.0, 93.9 (2C)
5) Color reaction: Positive for anisaldehyde-sulfuric acid.

A 40% ethanol extract (63.4 g) was subjected to Sephadex LH-20 column chromatography (Φ5 cm × 22 cm) in four portions. Elution and separation with methanol, methanol / isopropanol 5/1 solution, 4/1 solution, 2/1 solution, isopropanol, ethyl acetate, and dimethyl sulfoxide in order, (1) 9 fractions, (2) 8 fractions, (3 ) 8 fractions and (4) 9 fractions were obtained. The fourth fraction (3.97 g) was subjected to ODS silica gel column chromatography using a Cosmo Seal 75C18-OPN column (Φ5 cm × 11 cm). The methanol / water was separated by 1/9 liquid 400 mL, 2/8 liquid 300 mL, 3/7 liquid 300 mL, 4/6 liquid 300 mL, 5/5 liquid 240 mL, and methanol 300 mL to obtain 12 fractions.
The obtained second fraction (105.8 mg) was subjected to high performance liquid chromatography using a Unison US-C18 column (20 × 250 mm). Water / acetonitrile (88/12) solution was used as a solvent at a flow rate of 10 mL / min, and the eluate was detected by UV 270 nm absorption. By one purification, 3.9 mg of “fucodiphlorethol G, elution time 15.6 minutes” (No. 8 in FIG. 1-1) was obtained.
The physicochemical properties are shown below.
1) Molecular formula: C ₂₄ H ₁₈ O ₁₂ (measured by high-resolution mass spectrum (HRMS) method)
2) Molecular weight: 498.39 (determined by ESI-MS method (MH) ⁻ = 497.0726)
3) Hydrogen nuclear magnetic resonance spectrum (400MHz, solvent CD ₃ OD):
(Ppm): 6,16 (d, J = 2.32Hz, 1H), 6.10 (s, 2H), 6.06 (d, J = 2.32Hz, 1H), 6.05 (d, J = 2.76Hz, 1H), 5.95 (S, 2H), 5.73 (d, J = 2.76Hz, 1H)
4) Carbon nuclear magnetic resonance spectrum (100 MHz, solvent DMSO-d ₆ ):
(Ppm): 157.8, 157.7, 157.2, 157.1 (2C), 156.5, 154.6, 154.5, 153.0, 151.1 (2C), 150.3, 123.3, 121.9, 101.6, 101.0, 96.8, 95.7, 94.7 (2C), 94.7 (2C) ), 93.3, 92.3
5) Color reaction: Positive for anisaldehyde-sulfuric acid.

A 40% ethanol extract (15 g) was subjected to Sephadex LH-20 column chromatography (Φ5 cm × 21 cm). Methanol 700mL, then methanol / 2-propanol was eluted and separated sequentially with 5/1 solution 560mL, 4/1 solution 560mL, 2/1 solution 560mL, 2-propanol 560mL, ethyl acetate 500mL, dimethyl sulfoxide 400mL. Obtained. This operation was repeated 5 more times.
ODS silica gel column chromatography using a Cosmo Seal 75C18-OPN column (Φ5 cm × 12 cm) was performed on the third eluted fraction (3 g). Methanol / water was separated by 250 mL of 1/9 liquid, 700 mL of 2/8 liquid, 700 mL of 3/7 liquid, 300 mL of 4/6 liquid, and 500 mL of methanol to obtain 5 fractions. The obtained fourth elution fraction (828 mg) was further subjected to ODS silica gel column chromatography using a Cosmosil 75C18-OPN column (Φ3 cm × 13 cm). Methanol / water was separated by 1/9 liquid 180 mL, 2/8 liquid 180 mL, 3/7 liquid 420 mL, 4/6 liquid 150 mL, 5/5 liquid 150 mL, and methanol 500 mL to obtain 6 fractions. The obtained third eluted fraction (80 mg) was separated by high performance liquid chromatography using a Unison US-C18 column (250 × 20 mm). A solution of acetonitrile / water = 25/75 was used as a solvent at a flow rate of 10.0 mL / min, and the eluate was detected by absorption at UV 270 nm. The purification operation was performed once to obtain 7.9 mg of “phlorofucofuroeckol-B, elution time 52.0 minutes” (No. 9 in FIG. 1-2).
The physicochemical properties are shown below.
1) Molecular formula: C ₃₀ H ₁₈ O ₁₄ (measured by high-resolution mass spectrum (HRMS) method)
2) Molecular weight: 602.46 (measured by ESI-MS method (M−H) ⁻ = 601.0618)
3) Hydrogen nuclear magnetic resonance spectrum (500 MHz (solvent CD ₃ OD)):
(Ppm): 6.71 (s, 1H), 6.42 (s, 1H), 6.21 (s, 1H), 6.02 (d, J = 2.2 Hz, 2H), 5.99 (t, J = 2.2 Hz, 1H), 5.97 (T, J = 2.2 Hz, 1H), 5.92 (d, J = 2.2 Hz, 2H)
4) Carbon nuclear magnetic resonance spectrum (125 MHz (solvent CD ₃ OD)):
(Ppm): 162.0, 161.8, 160.2 (2C), 160.1 (2C), 152.6, 151.5, 150.9, 147.6, 147.6, 143.4, 143.2, 138.7, 138.3, 127.8, 125.5, 124.6, 122.5, 109.8, 106.9, 99.7, 99.3, 97.7, 97.6, 95.5 (2C), 95.3 (2C), 92.6
5) Color reaction: Positive for anisaldehyde-sulfuric acid.

A 40% ethanol extract (15 g) was subjected to Sephadex LH-20 column chromatography (Φ5 cm × 21 cm). Methanol 700mL, then methanol / 2-propanol was eluted and separated sequentially with 5/1 solution 560mL, 4/1 solution 560mL, 2/1 solution 560mL, 2-propanol 560mL, ethyl acetate 500mL, dimethyl sulfoxide 400mL. Obtained. This operation was repeated 5 more times.
The sixth elution fraction (725 mg) was subjected to ODS silica gel column chromatography using a Cosmo Seal 75C18-OPN column (Φ3 cm × 12 cm). Methanol / water was separated by 2/8 liquid 300 mL, 3/7 liquid 600 mL, 5/5 liquid 360 mL, and methanol 500 mL to obtain 4 fractions. The obtained third elution fraction (96 mg) was separated by high performance liquid chromatography using a Unison US-C18 column (250 × 20 mm). A solution of acetonitrile / water = 25/75 was used as a solvent at a flow rate of 10.0 mL / min, and the eluate was detected by absorption at UV 270 nm. The purification operation was performed once to obtain 18.8 mg of “No. 10 (a new compound whose compound name was not determined), elution time 69.6 minutes” (No. 10 in FIG. 1-2).
The physicochemical properties are shown below.
1) Molecular formula: C ₄₈ H ₃₀ O ₂₃ (measured by high-resolution mass spectrum (HRMS) method)
2) Molecular weight: 974.74 (measured by ESI-MS method (M + Na) ⁺ = 997.1077)
3) Hydrogen nuclear magnetic resonance spectrum (500 MHz (solvent CD ₃ OD)):
(Ppm): 6.68 (s, 1H), 6.45 (s, 1H), 6.30 (s, 1H), 6.25 (d, J = 2.3 Hz, 1H), 6.23 (d, J = 2.2 Hz, 1H), 6.17 (D, J = 2.2 Hz, 1H), 6.09 (d, J = 2.8 Hz, 1H), 5.98 (s, 2H), 5.96 (t, J = 2.0 Hz, 1H), 5.95 (d, J = 2.3 Hz , 1H), 5.93 (d, J = 2.0 Hz, 2H) 5.79 (d, J = 2.8 Hz, 1H)
4) Carbon nuclear magnetic resonance spectrum (125 MHz (solvent CD ₃ OD)):
(Ppm): 161.7, 160.3 (2C), 159.5, 159.5, 159.0, 159.0, 157.5, 156.9, 156.4, 156.4, 153.7, 153.3, 152.1 (2C), 151.8, 151.6, 151.1, 148.1, 147.6, 145.7, 144.2, 138.0, 135.1, 127.6, 125.0, 124.9, 124.3, 124.2, 122.3, 105.2, 105.2, 102.6, 102.4, 99.9, 99.3, 98.7, 98.4, 97.7, 97.6, 96.4 (2C), 96.0, 95.3 (2C), 95.2, 94.4, 94.2
5) Color reaction: Positive for anisaldehyde-sulfuric acid.

A 40% ethanol extract (15 g) was subjected to Sephadex LH-20 column chromatography (Φ5 cm × 21 cm). Methanol 700mL, then methanol / 2-propanol was eluted and separated sequentially with 5/1 solution 560mL, 4/1 solution 560mL, 2/1 solution 560mL, 2-propanol 560mL, ethyl acetate 500mL, dimethyl sulfoxide 400mL. Obtained. This operation was repeated 5 more times.
The sixth elution fraction (725 mg) was subjected to ODS silica gel column chromatography using a Cosmo Seal 75C18-OPN column (Φ3 cm × 12 cm). Methanol / water was separated by 2/8 liquid 300 mL, 3/7 liquid 600 mL, 5/5 liquid 360 mL, and methanol 500 mL to obtain 4 fractions. The obtained third elution fraction (96 mg) was separated by high performance liquid chromatography using a Unison US-C18 column (250 × 20 mm). A solution of acetonitrile / water = 25/75 was used as a solvent at a flow rate of 10.0 mL / min, and the eluate was detected by absorption at UV 270 nm. The purification operation was performed once to obtain 47.3 mg of “No. 11 (a new compound whose compound name was not determined), elution time of 56.0 minutes” (No. 11 in FIG. 1-2).
The physicochemical properties are shown below.
1) Molecular formula: C ₄₈ H ₃₀ O ₂₃ (measured by high-resolution mass spectrum (HRMS) method)
2) Molecular weight: 974.74 (measured by ESI-MS method (M + Na) ⁺ = 997.1077)
3) Hydrogen nuclear magnetic resonance spectrum (500 MHz (solvent CD ₃ OD)):
(Ppm): 6.73 (s, 1H), 6.43 (s, 1H), 6.27 (d, J = 2.5 Hz, 1H), 6.25 (d, J = 2.2 Hz, 1H), 6.22 (s, 1H), 6.20 (D, J = 2.2 Hz, 1H), 6.11 (d, J = 2.7 Hz, 1H), 6.05 (s, 2H), 5.96 (t, J = 2.0 Hz, 1H), 5.95 (d, J = 2.5 Hz , 1H), 5.93 (d, J = 2.0 Hz, 2H) 5.81 (d, J = 2.7 Hz, 1H)
4) Carbon nuclear magnetic resonance spectrum (125 MHz (solvent CD ₃ OD)):
(Ppm): 161.8, 160.0 (2C), 159.6, 159.5, 159.1, 158.9, 157.4, 156.8, 156.5, 156.3, 153.7, 152.6, 152.1 (2C), 151.9, 151.5, 150.8, 148.2, 146.9, 143.7, 142.8, 138.0, 137.9, 127.3, 125.3, 124.7, 124.0 (2C), 122.2, 109.8, 106.3, 102.6, 102.5, 99.6, 99.5, 98.6, 98.4, 97.7, 97.6, 96.5 (2C), 95.3 (2C), 95.1, 94.3 (2C), 92.8
5) Color reaction: Positive for anisaldehyde-sulfuric acid.

A 40% ethanol extract (63.4 g) was subjected to Sephadex LH-20 column chromatography (Φ5 cm × 22 cm) in four portions. Elution and separation with methanol, methanol / isopropanol 5/1 solution, 4/1 solution, 2/1 solution, isopropanol, ethyl acetate, and dimethyl sulfoxide in order, (1) 9 fractions, (2) 8 fractions, (3 ) 8 fractions and (4) 9 fractions were obtained. The first fraction (5.74 g) was subjected to ODS silica gel column chromatography using a Cosmo Seal 75C18-OPN column (Φ5 cm × 10 cm). Seven fractions were obtained by separating methanol / water with 1/9 liquid 300 mL, 2/8 liquid 200 mL, 3/7 liquid 400 mL, 5/5 liquid 400 mL, and methanol 400 mL. The obtained first fraction (932.8 mg) was subjected to ODS silica gel column chromatography using a Cosmosil 75C18-OPN column (Φ3.5 × 11 cm). Methanol / water was separated by 300 ml of 1/9 liquid, 200 ml of 13/87 liquid, 100 ml of 3/7 liquid, 100 ml of 4/6 liquid, and 100 ml of methanol to obtain 7 fractions.
The obtained second fraction (63.4 mg) was subjected to high performance liquid chromatography using a Unison US-C18 column (20 × 250 mm). Water / acetonitrile (9/1) solution was used as a solvent at a flow rate of 10 mL / min, and the eluate was detected by UV 270 nm absorption. 3.8 mg of “Triphlorethol A, elution time 20.4 minutes” (No. 12 in FIG. 1-2) was obtained by one purification.
The physicochemical properties are shown below.
1) Molecular formula: C ₁₈ H ₁₄ O ₉ (measured by high-resolution mass spectrum (HRMS) method)
2) Molecular weight: 374.30 (determined by ESI-MS method (MH) ⁻ = 373.0573)
3) Hydrogen nuclear magnetic resonance spectrum (500MHz, solvent CD ₃ OD):
(Ppm): 6.10 (d, J = 2.76Hz, 1H), 6.05 (d, J = 2.13Hz, 2H), 5.98 (t, J = 2.13Hz, 1H), 5.94 (s, 2H), 5.80 (d , J = 2.76Hz, 1H)
4) Carbon nuclear magnetic resonance spectrum (125 MHz, solvent DMSO-d ₆ ):
(Ppm): 160.7, 158.6 (2C), 154.5, 154.2, 153.0, 151.1, 151.1 (2C), 123.2, 122.2, 96.1, 95.7, 94.7 (2C), 94.0 (2C), 92.7
5) Color reaction: Positive for anisaldehyde-sulfuric acid.

A 40% ethanol extract (63.4 g) was subjected to Sephadex LH-20 column chromatography (Φ5 cm × 22 cm) in four portions. Elution and separation with methanol, methanol / isopropanol 5/1 solution, 4/1 solution, 2/1 solution, isopropanol, ethyl acetate, and dimethyl sulfoxide in order, (1) 9 fractions, (2) 8 fractions, (3 ) 8 fractions and (4) 9 fractions were obtained. The third fraction (779.2 mg) of the third round was subjected to ODS silica gel column chromatography using a Cosmosil 75C18-OPN column (Φ2.5 cm × 12 cm). Methanol / water was separated by 1/9 liquid 200 mL, 2/8 liquid 100 mL, 3/7 liquid 100 mL, 4/6 liquid 100 mL, and methanol 100 mL to obtain 11 fractions.
The obtained ninth fraction (93.6 mg) was subjected to high performance liquid chromatography using a Unison US-C18 column (10 × 250 mm). Water / acetonitrile (85/15) was used as a solvent at a flow rate of 2 mL / min, and the eluate was detected by UV 270 nm absorption. One purification yielded 14.0 mg of “dioxinodehydroeckol-11-sulfate, elution time 48.0 minutes” (No. 13 in FIG. 1-2).
The physicochemical properties are shown below.
1) Molecular _{formula: C 18 H 9 O 12 SNa} - measured by (high resolution mass spectrum (HRMS) method)
2) Molecular weight: 472.31 (measured by ESI-MS method (MH) ⁻ = 448.9801)
3) Hydrogen nuclear magnetic resonance spectrum (400MHz, solvent CD ₃ OD):
(Ppm): 6.57 (d, J = 2.68Hz, 1H), 6.52 (d, J = 2.68Hz, 1H), 6.21 (s, 1H), 6.05 (d, J = 2.74Hz, 1H), 6.03 (d , J = 2.74Hz, 1H)
4) Carbon nuclear magnetic resonance spectrum (125 MHz, solvent DMSO-d ₆ ):
(Ppm): 153.0, 149.0, 145.9, 145.4, 141.6, 141.3, 140.1, 137.2, 131.3, 125.9, 125.2, 122.6, 122.4, 104.1, 99.2, 98.8, 97.9, 93.9
5) Color reaction: Positive for anisaldehyde-sulfuric acid.

A 40% ethanol extract (63.4 g) was subjected to Sephadex LH-20 column chromatography (Φ5 cm × 22 cm) in four portions. Elution and separation with methanol, methanol / isopropanol 5/1 solution, 4/1 solution, 2/1 solution, isopropanol, ethyl acetate, and dimethyl sulfoxide in order, (1) 9 fractions, (2) 8 fractions, (3 ) 8 fractions and (4) 9 fractions were obtained. NMR similar (1) 3rd fraction, (2) 5th fraction, (3) 5th fraction, (4) 4th fraction together (2.15 g), and Cosmosil 75C18-OPN ODS silica gel column chromatography was performed using a column (Φ4 cm × 15 cm). Methanol / water 2/8 liquid 200 mL, 2.5 / 7.5 liquid 200 mL, 3/7 liquid 200 mL, 4/6 liquid 200 mL, 5/5 liquid 200 mL, 6/4 liquid 200 mL, 7/3 liquid 200 mL, Separation with 200 mL of methanol gave 13 fractions.
The obtained ninth fraction (12.1 mg) was subjected to high performance liquid chromatography using a Unison UK-phenyl column (10 × 250 mm). Water / acetonitrile (7/3) solution was used as a solvent at a flow rate of 2 mL / min, and the eluate was detected by UV 270 nm absorption. By purification once, 1.6 mg of “No. 14 (a new compound whose compound name has not been determined) and an elution time of 30.4 minutes” (No. 14 in FIG. 1-2) was obtained.
The physicochemical properties are shown below.
1) Molecular formula: C ₂₄ H ₁₄ O ₁₁ (measured by high-resolution mass spectrum (HRMS) method)
2) Molecular weight: 478.36 (determined by ESI-MS method (MH) ⁻ = 477.0445)
3) Hydrogen nuclear magnetic resonance spectrum (400MHz, solvent CD ₃ OD):
(Ppm): 6.68 (s, 1H), 6.49 (d, J = 1.88Hz, 1H), 6.31 (d, overlapped, 1H), 6.30 (s, 1H), 6.01 (d, J = 2.07Hz, 2H) 5.99 (t, J = 2.07Hz, 1H)
4) Carbon nuclear magnetic resonance spectrum (100 MHz, solvent DMSO-d ₆ ):
(Ppm): 160.2, 158.8 (2C), 158.2, 157.7, 150.4, 150.2, 146.9, 144.3, 141.9, 136.9, 133.5, 126.1, 122.6, 122.4, 103.1, 102.3, 98.2, 98.0, 96.3, 94.6, 93.7 (2C ), 90.5
5) Color reaction: Positive for anisaldehyde-sulfuric acid.

A 40% ethanol extract (63.4 g) was subjected to Sephadex LH-20 column chromatography (Φ5 cm × 22 cm) in four portions. Elution and separation with methanol, methanol / isopropanol 5/1 solution, 4/1 solution, 2/1 solution, isopropanol, ethyl acetate, and dimethyl sulfoxide in order, (1) 9 fractions, (2) 8 fractions, (3 ) 8 fractions and (4) 9 fractions were obtained. NMR similar (1) 3rd fraction, (2) 5th fraction, (3) 5th fraction, (4) 4th fraction together (2.15 g), and Cosmosil 75C18-OPN ODS silica gel column chromatography was performed using a column (Φ4 cm × 15 cm). Methanol / water 2/8 liquid 200 mL, 2.5 / 7.5 liquid 200 mL, 3/7 liquid 200 mL, 4/6 liquid 200 mL, 5/5 liquid 200 mL, 6/4 liquid 200 mL, 7/3 liquid 200 mL, Separation with 200 mL of methanol gave 13 fractions.
The obtained first fraction (21.3 mg) was subjected to high performance liquid chromatography using a Unison UK-C18 column (10 × 250 mm). Water / acetonitrile (containing 85/15 0.1% trifluoroacetic acid) as a solvent was used at a flow rate of 2 mL / min, and the eluate was detected by absorption at UV 270 nm. One purification yielded 4.9 mg of “6,8′-bieckol, elution time 86.4 minutes” (No. 15 in FIG. 1-2).
The physicochemical properties are shown below.
1) Molecular formula: C ₃₆ H ₂₂ O ₁₈ (measured by high-resolution mass spectrum (HRMS) method)
2) Molecular weight: 742.55 (determined by ESI-MS method (MH) ⁻ = 741.0737)
3) Hydrogen nuclear magnetic resonance spectrum (400MHz, solvent CD ₃ OD):
(Ppm): 6.21 (s, 1H), 6.19 (s, 1H), 6.12 (s, 2H), 5.98 (d, overlapped, 2H), 5.98 (d, overlapped, 2H), 5.97 (t, overlapped, 1H ), 5.97 (t, overlapped, 1H)
4) Carbon nuclear magnetic resonance spectrum (100 MHz, solvent DMSO-d ₆ ):
(Ppm): 160.4, 160.4, 158.7 (2C), 158.7 (2C), 151.5, 151.5, 145.8, 145.4, 144.8, 144.3, 141.9, 141.8, 141.6, 140.9, 137.3, 137.2, 123.6, 123.4, 123.0, 122.9, 122.4, 122.0, 104.5, 99.5, 98.2, 97.9, 97.9, 96.3, 96.2, 93.9 (2C), 93.7 (2C), 93.6
5) Color reaction: Positive for anisaldehyde-sulfuric acid.

A 40% ethanol extract (63.4 g) was subjected to Sephadex LH-20 column chromatography (Φ5 cm × 22 cm) in four portions. Elution and separation with methanol, methanol / isopropanol 5/1 solution, 4/1 solution, 2/1 solution, isopropanol, ethyl acetate, and dimethyl sulfoxide in order, (1) 9 fractions, (2) 8 fractions, (3 ) 8 fractions and (4) 9 fractions were obtained. NMR similar (1) 4th fraction, (2) 4th fraction, (3) 6th fraction, (4) 5th fraction together (5.96 g), and Cosmosil 75C18-OPN ODS silica gel column chromatography was performed using a column (Φ5 cm × 15 cm). 9 fractions were obtained by separating methanol / water with 15/85 liquid 500 mL, 2/8 liquid 400 mL, 3/7 liquid 400 mL, 4/6 liquid 400 mL, 5/5 liquid 400 mL, and methanol 400 mL. The obtained first fraction (187.6 mg) was again subjected to ODS silica gel column chromatography using a Cosmo Seal 75C18-OPN column (Φ2 cm × 15 cm). Methanol / water was separated by 60 mL of 1/9 liquid, 10 mL of 15/85 liquid, 3 mL of 10/7 liquid, 30 mL of 4/6 liquid, and 50 mL of methanol to obtain 6 fractions.
The obtained third fraction (34.3 mg) was subjected to high performance liquid chromatography using a Unison UK-C18 column (10 × 250 mm). Water / acetonitrile (9/1) solution was used as a solvent at a flow rate of 2 mL / min, and the eluate was detected by UV 270 nm absorption. By purification once, “6,8′-bieckol-7′-sulfate, elution time 24.8 minutes” (No. 16 in FIG. 1-2). 16.2 mg was obtained.
The physicochemical properties are shown below.
1) Molecular _{formula: C 36 H 21 O 21 SNa} - measured by (high resolution mass spectrum (HRMS) method)
2) Molecular weight: 844.59 (determined by ESI-MS method (MH) ⁻ = 821.0296)
3) Hydrogen nuclear magnetic resonance spectrum (400MHz, solvent CD ₃ OD):
(Ppm): 7.00 (s, 1H), 6.21 (s, 1H), 6.10 (s, 1H), 6.08 (s, 1H), 6.00 (d, J = 2.07Hz, 2H), 6.00 (d, J = 2.07Hz, 2H), 5.96 (t, J = 2.07Hz, 1H), 5.96 (t, J = 2.07Hz, 1H)
4) Carbon nuclear magnetic resonance spectrum (125 MHz, solvent DMSO-d ₆ )
(Ppm): 160.3, 160.3, 158.8 (2C), 158.7 (2C), 151.1, 147.5, 145.9, 145.5, 145.0, 143.6, 141.9, 141.4, 140.9, 140.6, 137.0, 136.7, 125.9, 123.9, 123.4, 122.8, 122.4, 122.3, 108.4, 99.3, 98.8, 98.5, 98.4, 98.0, 96.2, 96.1, 93.8 (2C), 93.6 (2C)
5) Color reaction: Positive for anisaldehyde-sulfuric acid.

A 40% ethanol extract (63.4 g) was subjected to Sephadex LH-20 column chromatography (Φ5 cm × 22 cm) in four portions. Elution and separation with methanol, methanol / isopropanol 5/1 solution, 4/1 solution, 2/1 solution, isopropanol, ethyl acetate, and dimethyl sulfoxide in order, (1) 9 fractions, (2) 8 fractions, (3 ) 8 fractions and (4) 9 fractions were obtained. The fourth fraction (3.97 g) was subjected to ODS silica gel column chromatography using a Cosmosil 75C18-OPN column (Φ5 cm × 11 cm). The methanol / water was separated by 1/9 liquid 400 mL, 2/8 liquid 300 mL, 3/7 liquid 300 mL, 4/6 liquid 300 mL, 5/5 liquid 240 mL, and methanol 300 mL to obtain 12 fractions. The obtained eighth fraction (453.2 mg) was again subjected to ODS silica gel column chromatography using a Cosmo Seal 75C18-OPN column (Φ3.5 cm × 13 cm). The methanol / water was separated by 15/85 liquid 200 mL, 2/8 liquid 200 mL, 25/75 liquid 400 mL, 3/7 liquid 200 mL, 4/6 liquid 200 mL, and methanol 200 mL to obtain 7 fractions.
The obtained fifth fraction (60.0 mg) was subjected to high performance liquid chromatography using a Unison US-C18 column (20 × 250 mm). Water / acetonitrile (75/25) solution was used as a solvent at a flow rate of 10 mL / min, and the eluate was detected by UV 270 nm absorption. By purification once, 20.1 mg of “No. 17 (a new compound whose compound name was not determined), elution time 18.0 minutes” (No. 17 in FIG. 1-3) was obtained.
The physicochemical properties are shown below.
1) Molecular formula: C ₃₆ H ₂₄ O ₁₈ (measured by high resolution mass spectrum (HRMS) method)
2) Molecular weight: 744.56 (measured by ESI-MS method (M + Na) ⁺ = 767.0836)
3) Hydrogen nuclear magnetic resonance spectrum (400MHz, solvent CD ₃ OD):
(Ppm): 6.24 (d, J = 2.26Hz, 1H), 6.22 (d, J = 2.26Hz, 1H), 6.17 (s, 1H), 6.16 (d, J = 2.26Hz, 1H), 6.09 (d , J = 2.76Hz, 1H), 5.99 (d, J = 2.76Hz, 1H), 5.98 (s, 2H), 5.97 (d, J = 2.76Hz, 1H), 5.92 (d, J = 2.26Hz, 1H) ), 5.79 (d, J = 2.76Hz, 1H)
4) Carbon nuclear magnetic resonance spectrum (125 MHz, solvent CD ₃ OD)
(Ppm): 159.6, 159.5, 159.2, 159.1, 157.5, 156.9, 156.6, 156.5, 154.7, 153.9, 152.3 (2C), 151.9, 147.0, 146.7, 144.2, 143.8, 138.4, 125.5, 124.8, 124.4, 124.1, 124.0 , 102.6, 102.5, 99.6, 99.2, 98.6,
98.4, 97.6, 96.4 (2C), 95.7, 95.3, 94.4, 94.3
5) Color reaction: Positive for anisaldehyde-sulfuric acid.

A 40% ethanol extract (63.4 g) was subjected to Sephadex LH-20 column chromatography (Φ5 cm × 22 cm) in four portions. Elution and separation with methanol, methanol / isopropanol 5/1 solution, 4/1 solution, 2/1 solution, isopropanol, ethyl acetate, and dimethyl sulfoxide in order, (1) 9 fractions, (2) 8 fractions, (3 ) 8 fractions and (4) 9 fractions were obtained. The third fraction (779.2 mg) of the third round was subjected to ODS silica gel column chromatography using a Cosmosil 75C18-OPN column (Φ2.5 cm × 12 cm). Methanol / water was separated by 1/9 liquid 200 mL, 2/8 liquid 100 mL, 3/7 liquid 100 mL, 4/6 liquid 100 mL, and methanol 100 mL to obtain 11 fractions.
The obtained sixth fraction (83.9 mg) was subjected to high performance liquid chromatography using a Unison US-C18 column (20 × 250 mm). Using A: water / acetonitrile (95/5) solution and B: water / acetonitrile (5/5) solution as solvents, gradient elution was performed at 0-45 min; 15-60% B. The flow rate was 5 mL / min, and the eluate was detected by UV 270 nm absorption. One purification yielded 10.5 mg of “6,6′-bieckol elution time 44.0 minutes” (No. 18 in FIG. 1-3).
The physicochemical properties are shown below.
1) Molecular formula: C ₃₆ H ₂₂ O ₁₈ (measured by high-resolution mass spectrum (HRMS) method)
2) Molecular weight: 742.55 (determined by ESI-MS method (MH) ⁻ = 741.0717)
3) Hydrogen nuclear magnetic resonance spectrum (500MHz, solvent CD ₃ OD):
(Ppm): 6.18 (s, 2H), 6.12 (s, 2H), 6.02 (d, J = 2.05Hz, 4H), 5.99 (t, J = 2.05Hz, 2H)
4) Carbon nuclear magnetic resonance spectrum (100 MHz, solvent DMSO-d ₆ ):
(Ppm): 160.4 (2C), 158.8 (4C), 151.3 (2C), 145.4 (2C), 144.5 (2C), 141.9 (2C), 141.4 (2C), 137.2 (2C), 123.6 (2C), 122.7 (2C), 122.0 (2C), 99.7 (2C), 97.9 (2C), 97.8 (2C), 96.2 (2C), 93.7 (4C)
5) Color reaction: Positive for anisaldehyde-sulfuric acid.

From the fraction in Sephadex LH-20 column chromatography [manufactured by GE Healthcare Japan Co., Ltd.], “Phloroglucinol” (No. 19 in FIGS. 1-3) was obtained.
The physicochemical properties of commercially available reagents are shown below.
Reagent name: phloroglucinol, dehydrate
Manufacturer: Tokyo Chemical Industry Co., Ltd. Product Number: P1376
1) Molecular formula: C ₆ H ₆ O ₃
2) Molecular weight: 162.14
3) Hydrogen nuclear magnetic resonance spectrum (300 MHz, solvent CDCl ₃ + DMSO-d ₆ ):
(Ppm): 5.92 (s)
4) Carbon nuclear magnetic resonance spectrum (60 MHz, solvent CDCl ₃ + DMSO-d ₆ ):
(Ppm): 158.8, 94.2

[Effect of each compound in the chromate HP-20 fraction on the damage of human skin fibroblast TIG-114 caused by UVB irradiation]
19 kinds of known and novel compounds contained in 40 (v / v)% EtOH fraction obtained by fractionating chrome 70 (v / v)% EtOH extract with Diaion (registered trademark) HP20 column The effect of protecting human fibroblasts TIG-114 against damage caused by UVB irradiation was examined (reference: non-patent documents 3 to 5).
1. Reagents / Samples (1) Chromome extract: Extract extracted by stirring for 6 hours from 30 kg of dried chromate to 70 (v / v)% EtOH (2) Chromome fraction: Diaion (registered trademark) HP20 column from chromate extract Fraction fraction eluted with “water”, “20 (v / v)% EtOH” followed by “40% (v / v) EtOH”.
(3) Human fibroblast TIG-114: Obtained from Human Science Research Resource Bank (JCRB0534) and cultured in DMEM medium supplemented with 10% fetal bovine serum.
(4) UVB: Sankyo Electric GL-15SE x 3 pieces are arranged in parallel and irradiated.
2. Test group (1) Control group: Mixed in PBS medium containing neither chrome extract nor fraction.
(2) Positive control group: Mixed in PBS medium containing vitamin C at a concentration of 50 μM.
(3) Chromome compound group: Mixed in PBS medium containing 19 kinds of compounds obtained in Example 11 at a concentration of 50 μg / ml.
(4) KP-40 fraction group: For comparison, mixed with PBS medium containing “40% (v / v) EtOH” fraction at a concentration of 50 μg / ml.

3. Test method (1) The cell concentration is adjusted to 1 × 10 ⁵ cells / ml.
(2) 50 μl / well (5,000 cells / well) is seeded in a 96-well microbrate.
(3) Incubate at 37 ° C. for 24 hours. (DMEM medium supplemented with 10% fetal bovine serum)
(4) The medium is replaced with 100 μl of PBS (−) medium containing the specimens of each test group, and UVB is irradiated at 400 mJ / cm ² .
(5) 48 hours later, 10% Cell Counting Kit-8 (manufactured by Wako Pure Chemical Industries, Ltd.) is added and incubated at 37 ° C. and 1.5% CO ² incubator for 1.5 hours, and the absorbance at 450 nm is measured. By measuring the number of fibroblasts.

4). Results The cell viability (%) of the sample group after 48 hours of culture was determined by the following formula, and the results are shown in FIG.
Cell viability (%) = (number of cells in sample group−number of cells in control group) /
(Number of cells not irradiated with UV-number of cells in control group) × 100
The fraction KP-40 group showed the same survival rate as the positive control group vitamin C group.
Among each compound, No. which is a novel compound. No. 10 which is a known substance but is not known to be contained in chrome. No. 8 fucodiphlorethol G, no. No. 9, which is known to be contained in phlorofucofuroeckol-B and chrome. The four compounds of dioxinodehydroeckol (4) tended to have a higher cell viability than the vitamin C group as the positive control group. These compounds are presumed to have an effect of protecting cells from UVB irradiation damage.

[Overview of the long-term application animal test of the protective effect of the black fraction on UVB irradiation damage]
In this example, a 40B / v) EtOH eluate obtained by fractionating a chrome 70 (v / v)% EtOH extract on a Diaion (registered trademark) HP20 column was used as a UVB irradiation long-term model for UVB irradiation damage. The outline of the test conditions for confirming the protective effect was shown (references: non-patent documents 1 and 2).
1. Reagent / Specimen (1) Chromome extract: Extract extracted from 30 kg of dried chrome and left standing for 7 days with 120 L of 70 (v / v)% EtOH.
(2) Chromome fraction: A fraction obtained by adsorbing a chromate extract to a Diaion (registered trademark) HP20 column and then eluting with "40 (v / v)% EtOH".
(3) Hydrophilic ointment: Pharmacopoeia hydrophilic ointment “Whey” (Nipro Pharma)
(4) Control ointment: 60% (v / v) EtOH was added to 4 times the amount of hydrophilic ointment and stirred well in a mortar.
(5) 0.025% ointment: 40 (v / v)% EtOH Dissolve the elution fraction in 60 (v / v)% EtOH to 0.125% and add to 4 times the amount of hydrophilic ointment And stirred well in a mortar (final concentration = 0.025% KP-40).
(6) 0.25% ointment: 40 (v / v)% EtOH Dissolve the elution fraction in 60 (v / v)% EtOH to 1.25% and add to 4 times the amount of hydrophilic ointment And stirred well in a mortar (final concentration = 0.25% KP-40).
(7) UVB: Sankyo Electric GL-20SE x 5 pieces are irradiated with a low-wavelength component cut with a glass filter equivalent to SCHOTT WG295.
2. Test group
(1) UV (-) control group: Use control ointment. No UVB irradiation.
(2) UV (+) control group: use control ointment. Irradiate with UVB.
(3) 0.025% KP-40 group: 0.025% KP-40 ointment was used. UVB irradiation is performed.
(4) 0.25% KP-40 group: 0.25% KP-40 ointment was used. UVB irradiation is performed.

3. Prepare 24 Ares mice HOS: HR-1 (female) (7 weeks old) to test method (1).
(2) After 3 days of preliminary rearing, the groups are divided into 4 groups so that the average weight of each group is equal.
(3) Apply about 10 mg / cm ^{2 of} control ointment, 0.025% KP-40 ointment, and 0.25% KP-40 ointment to the back of each group.
(4) For groups other than the UV (−) control group, the back is irradiated with UVB three times a week. The irradiation dose is increased to 54, 108, 162, 216, 216, 216 mJ / cm ² every week.
(5) Reapply each group of ointments immediately after irradiation.
(6) Perform each measurement after 7 weeks of irradiation / coating test. The skin thickness is measured with a micrometer and used as an index of edema. Measure the color tone of the three places on the back skin with a spectrophotometer and calculate the L ^* value, a ^* value, and b ^* value. Measure skin moisture. Measure transdermal moisture transpiration (TEWL).

4). Test results Fig. 15 shows the results of a long-term application animal test of the protective effect against UVB irradiation damage by KP-40.
And shown in FIG. After the 7-week irradiation / application test, the UV (+) control group increased skin thickness due to UVB irradiation, and at the same time decreased skin moisture, indicating skin damage due to UVB irradiation.
In the group to which 0.025% KP-40 ointment was applied, a decrease in skin water amount due to UVB irradiation for 7 weeks was significantly suppressed.
Moreover, in the group which apply | coated 0.25% KP-40 ointment, it was recognized that the raise (edema) of the skin thickness by UVB irradiation for 7 weeks was suppressed significantly.
From these results, the continuous application of the ointment is expected to protect the skin from damage caused by daily ultraviolet rays. Moreover, since the content of polyphenol is recognized in this fraction, it is speculated that the UVB irradiation protection effect is due to chrome polyphenol (fluorotannins).

The present invention relates to an ultraviolet radiation damage protective agent comprising fluorotannins extracted from chrome as an active ingredient, and extracts a substance having an effect equivalent to or better than that of a conventional sunscreen agent from chrome seaweed produced in nature. The present invention provides an excellent ultraviolet radiation damage protective agent. Since the fluorotannins of the present invention use natural products as raw materials, they can provide a UV radiation damage protective agent that is less harmful to the human body. For example, as a skin external preparation, human skin fibroblasts It can be used as a constituent material for wound healing agents, cosmetics, foods, bath preparations, fibroblast culture media and the like.

Claims (10)

The following chemical structural formula:
A protective agent for ultraviolet radiation damage characterized by comprising, as an active ingredient, a chrome-derived fluorotannin containing a chrome polyphenol selected from:   The protective agent for ultraviolet radiation damage according to claim 1, wherein the chlorotannin derived from chrome is an aqueous ethanol extract of chrome. The ultraviolet-irradiation-protection agent according to claim 1 or 2 , which has a function of protecting the skin from ultraviolet irradiation injury. 4. The ultraviolet radiation damage protective agent according to claim 3 , wherein the skin is dermal fibroblasts. An external preparation for skin comprising the ultraviolet radiation damage protective agent according to any one of claims 1 to 4 . A cosmetic comprising the ultraviolet radiation damage protective agent according to any one of claims 1 to 4 . The cosmetic according to claim 6 , wherein the cosmetic is a sunscreen material. The method for producing an ultraviolet radiation damage protective agent according to claim 1 or 2, wherein chrome is extracted with an aqueous solution having an ethanol concentration of 40 to 80 (v / v)%. Furthermore, the method of manufacturing the ultraviolet irradiation obstacle protective agent of Claim 8 which fractionates a column using ethanol aqueous solution as an eluent. The method for producing an ultraviolet radiation damage protective agent according to claim 9, wherein the ethanol aqueous solution as an eluent is an aqueous solution having an ethanol concentration of 20 to 60 (v / v)%.