JP2008001623A - Vascularization inhibitor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a safe and excellent vascularization inhibitor. <P>SOLUTION: The vascularization inhibitor and the medicinal composition contain fucoxanthin and/or fucoxanthinol. The cosmetic composition contains the fucoxanthinol. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to an angiogenesis inhibitor containing fucoxanthin and / or fucoxanthinol, and a cosmetic composition containing fucoxanthinol.

  When a new blood vessel is generated from an existing blood vessel (capillary blood vessel or the like) and is elongated, it is called angiogenesis. Angiogenesis is deeply related to pathological deterioration such as diabetic retinopathy and atherosclerosis, and in recent years, treatment and prevention of these diseases with angiogenesis inhibitors have been recommended. Although development of drugs for the purpose of inhibiting angiogenesis has been actively conducted, there are concerns about unexpected side effects and the like with synthesized drugs, and there are problems in terms of safety (for example, Non-Patent Document 1). .

From such a background, a substance derived from a natural product and having an excellent anti-angiogenic action has been demanded.
Cell Engineering Vol. 14, no. 4, 1995, pp. 426-431.

  The main object of the present invention is to provide a safe and effective angiogenesis inhibitor, and a pharmaceutical composition and a cosmetic containing the angiogenesis inhibitor.

  As a result of intensive studies, the present inventors have found that fucoxanthin and its metabolite fucoxanthinol have an anti-angiogenic action. Furthermore, the present inventors have found that fucoxanthinol can exert an excellent anti-angiogenic effect even at an extremely low concentration. The present invention has been completed as a result of further research based on these findings.

The present invention provides the following angiogenesis inhibitors, pharmaceutical compositions and cosmetic compositions.
Item 1. An angiogenesis inhibitor comprising fucoxanthin and / or fucoxanthinol.
Item 2. Containing fucoxanthin and / or fucoxanthinol, diabetic retinopathy, atherosclerosis, obesity, type II diabetes, rheumatoid arthritis, Graves' disease, Kaposi's sarcoma, Alzheimer's disease, periodontal disease, scleroderma, A pharmaceutical composition for treating or preventing at least one disease selected from the group consisting of glaucoma, psoriasis, and age-related macular degeneration.
Item 3. A cosmetic composition containing fucoxanthinol.
Item 4. Item 4. The cosmetic composition according to Item 3, comprising fucoxanthinol in an amount of 0.000001 to 10% by weight.
Item 5. Item 5. The cosmetic composition according to Item 3 or 4, which is used for wrinkle improvement.

  Both fucoxanthin and fucoxanthinol used as active ingredients in the present invention are components contained in algae and the like. That is, fucoxanthin and fucoxanthinol are types of natural ingredients that have been ingested as foods in the past, and their safety to the human body has been confirmed. Therefore, according to the present invention, a safe and excellent angiogenesis inhibitor can be provided.

  In particular, fucoxanthinol exhibits an excellent anti-angiogenic action even at an extremely low concentration, and an excellent effect for improving skin wrinkles can be expected. Therefore, the cosmetic composition containing fucoxanthinol of the present invention has high safety and is excellent in wrinkle improving action. Further, when fucoxanthinol is blended in a large amount in a cosmetic composition or the like, there is a problem of coloring or the like, but fucoxanthinol can exhibit the above excellent effect at an extremely low concentration. There is no problem. Furthermore, according to the cosmetic composition containing fucoxanthinol of the present invention, not only the improvement of wrinkles but also the effect of enhancing the firmness and gloss of the skin can be expected.

Hereinafter, the angiogenesis inhibitor and the cosmetic composition of the present invention will be described.
(1) Angiogenesis inhibitor The angiogenesis inhibitor of the present invention contains fucoxanthin and / or fucoxanthinol as an active ingredient.

  The fucoxanthin used in the present invention is desirably derived from a natural product, and it is preferable to use algae such as brown algae and microalgae that are rich in fucoxanthin. The type of brown algae and microalgae is not particularly limited as long as fucoxanthin is contained in the plate body or filamentous body. Examples of such algae include, for example, Laminariaceae, Brown algae belonging to the order of Laminariales, such as Alariaceae; The algae plate-like body or filamentous body is preferred. Among the brown algae plate-like bodies or filamentous bodies, the leaf-like bodies of the clamaceae Okinawa mozuku (Cladosiphon okamuranus) or the mosquito family Mozuku (Nemacytus decipiens), the comb-like bodies of the kombu family Kombu The lamellar body or filamentous body of diatomaceae, diatoms and the like are preferred. In this invention, these plants can be used individually by 1 type or in combination of 2 or more types. Moreover, although these plants containing fucoxanthin can be used as they are, the extract can be used as an active ingredient of the present invention. Plants containing fucoxanthin may be subjected to treatments such as drying, shredding, crushing, and grinding.

  The extract containing fucoxanthin is obtained by using the above-mentioned plant (or animal) as an extraction raw material, as it is or as needed, dried, shredded, crushed, crushed, pressed, boiled or fermented with a solvent. It can be obtained by performing an extraction process.

  Examples of the solvent used for the extraction treatment of the plant (or animal) extract containing fucoxanthin include polar solvents such as a polar organic solvent and a mixture of water and a polar organic solvent. Examples of the polar organic solvent include lower alcohols having 1 to 5 carbon atoms such as methanol, ethanol, butanol and isopropanol; propylene glycol; acetone; ethyl acetate; hexane; dichloromethane; chloroform alone or a combination of two or more. Can be mentioned. Among the above polar solvents, from the viewpoint of safety to the human body and handleability, preferably a lower alcohol having 2 to 4 carbon atoms such as ethanol, propanol, butanol, etc .; acetone; hexane; or a mixture thereof. More preferably, ethanol, acetone, hexane, or a mixture thereof is used.

  Fucoxanthin is fat soluble. Therefore, for example, when a lower alcohol having 1 to 5 carbon atoms and water are used as an extraction solvent, the content ratio of the lower alcohol having 1 to 5 carbon atoms with respect to the total weight of the mixed solution is 50 to 99.9 wt. %, Preferably about 70 to 99.9% by weight, more preferably about 90 to 99.9% by weight, but is not particularly limited as long as fucoxanthin is extracted.

  An extraction process is performed using about 5-100 weight part of extraction solvents with respect to 1 weight part of extraction raw materials, such as a brown algae, for example. Further, as the extraction treatment method, a conventionally used extraction method for plant extracts can be adopted, and specifically, immersion methods such as immersion and digestion; low temperature, room temperature or high temperature. Examples of the stirring method include a percolation method. As a specific example of the extraction process of a plant extract containing fucoxanthin, for example, a dry brown algae extraction raw material is pulverized, and about 5 to 100 parts by weight of an extraction solvent is added to 1 part by weight of the dry pulverized product. Examples of the method of obtaining the solution fraction by performing extraction for 1 to 5 hours while stirring at room temperature and then removing the solid content by a conventional solid-liquid separation means such as filtration or centrifugation.

  The extract containing fucoxanthin obtained by the above method is in a liquid state, and in the present invention, the extract can be used as it is. However, when an organic solvent is contained, It is preferred to remove the solvent. Further, if necessary, it can be used as a dried product by removing moisture by subjecting it to a drying treatment such as reduced pressure drying, freeze drying, spray drying or the like. Moreover, what can be obtained commercially can also be used simply as an extract containing the said fucoxanthin.

  Fucoxanthinol is obtained by hydrolyzing fucoxanthin. Therefore, the fucoxanthinol used in the present invention can be obtained by hydrolyzing the fucoxanthin obtained by the above method according to a conventionally known method. For example, decomposing fucoxanthinol into fucoxanthin by a lipolytic enzyme such as lipase (J. Nutr. 132, 946-951, 2002) or cholesterol esterase (Drug Metab. Dispos. 32, 205-211, 2004). Can do.

  Fucoxanthin and fucoxanthinol are also present in protozoa such as sea squirts; molluscs such as shellfish; and echinoderms such as sea cucumbers. In the present invention, fucoxanthin or fucoxanthinol obtained by the above extraction method using these animals as raw materials can also be used.

  As an active ingredient of the angiogenesis inhibitor of the present invention, either fucoxanthin or fucoxanthinol can be used, or these can be used in combination.

  The angiogenesis inhibitor of the present invention may be composed of the above active ingredients, but it is prepared in various dosage forms in combination with pharmaceutically acceptable conventionally known carriers, diluents, excipients and the like. You can also.

  The total amount of active ingredients in the angiogenesis inhibitor of the present invention is about 0.01 to 99% by weight, preferably about 0.1 to 99% by weight, more preferably about 1 to 99% by weight.

  The angiogenesis inhibitor of the present invention can be used for the treatment or prevention of diseases caused by angiogenesis, and the dosage varies depending on the age, sex, type of disease, degree of symptoms, etc. The total amount of active ingredients per day can be appropriately set in the range of about 0.001 to 1,000 mg / kg, preferably 0.01 to 1,000 mg / kg, more preferably 0.1 to 1,000 mg / kg. .

  Since the angiogenesis inhibitor of the present invention has an excellent angiogenesis inhibitory action, it can also be used as a pharmaceutical composition for treatment or prevention of a disease caused by angiogenesis or a disease whose state deteriorates due to angiogenesis. For example, in diabetic retinopathy, it is known that angiogenesis is caused by retinal ischemia. In addition, angiogenesis is also considered to play an important role in the onset or deterioration of these diseases in rheumatoid arthritis, Kaposi's sarcoma, psoriasis, and Graves' disease (Hiroshi Takagi, Cell Engineering Vol. 19, No. 8, 2000; Protein Nucleic Acid Enzyme Vol.45, No.6 p.1182-1187).

  Angiogenesis of the arterial endothelium is thought to contribute to atherosclerosis and cause atherosclerosis, indicating that angiogenesis inhibition is effective in the treatment of atherosclerosis. (K. S. Multon et al., PNAS, Vol. 100, No. 8, p. 4736-4741).

  Moreover, it has been shown that the growth of adipose tissue depends on angiogenesis, and it is known that inhibiting angiogenesis is effective in preventing obesity and the like (E. Brakenheim et al., Circulation Research). , June 25, 2004).

  In Alzheimer's disease, brain endothelial cells are activated by angiogenesis, and β-amyloid precursors and neurotoxin peptides are secreted to selectively cause cortical neuron cell death. It is said that it is effective for prevention and treatment of diseases (AH Vagnucci Jr. et al., The Lancet, Vol. 361, Feb. 15, 2003).

  Therefore, the pharmaceutical composition of the present invention having an excellent angiogenesis inhibitory effect can be suitably used for the purpose of preventing or treating these diseases.

  In addition to the above, the pharmaceutical composition of the present invention is expected to be effective for the treatment or prevention of diseases associated with angiogenesis such as periodontal disease, scleroderma, glaucoma, age-related macular degeneration, and type II diabetes. The

  The pharmaceutical composition of the present invention can be prepared in various preparation forms regardless of whether it is oral or parenteral, but is preferably an oral preparation. Examples of the dosage form of the pharmaceutical composition of the present invention include liquid preparations (including syrups), drops, injections, eye drops and the like; tablets, pills, powders, granules, capsules (soft capsules) A solid preparation).

  When the pharmaceutical composition of the present invention is a liquid preparation, it can be stored frozen, or it may be stored after removing moisture by lyophilization or the like. Freeze-dried preparations, dry syrups and the like are used by dissolving again by adding distilled water for injection, sterilized water or the like at the time of use.

  For example, when the pharmaceutical compositions of the present invention are prepared as injections, infusions, etc., they are preferably sterilized and isotonic with blood. When preparing such a dosage form, for example, water, ethyl alcohol, macrogol, propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitan fatty acid esters, etc. should be used as diluents. Can do. In this case, the pharmaceutical composition of the present invention may contain a sufficient amount of sodium chloride, glucose or glycerin to adjust a solution that is isotonic with the body fluid. Moreover, you may add the solubilizing agent, buffering agent, soothing agent, etc. which are generally used in this field | area.

  When the pharmaceutical composition of the present invention is prepared as a solid agent, for example, in the case of a tablet, various carriers well known in the art can be widely used as carriers. Examples thereof include excipients such as lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, silicic acid, water, ethanol, propanol, simple syrup, glucose solution, starch solution, Gelatin solution, binders such as carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, polyvinylpyrrolidone, dry starch, sodium alginate, agar powder, laminaran powder, sodium bicarbonate, calcium carbonate, polyoxyethylene sorbitan fatty acid esters, lauryl sulfate Disintegrators such as sodium, stearic acid monoglyceride, starch, lactose, disintegration inhibitors such as sucrose, stearin, cocoa butter, hydrogenated oil, absorption accelerators such as quaternary ammonium base, sodium lauryl sulfate, glyce Emissions, moisturizing agents such as starch, starch, lactose, kaolin, bentonite, adsorbent such as colloidal silicic acid, purified talc, stearates, boric acid powder, a lubricant such as polyethylene glycol can be used. Further, the tablets can be made into tablets with ordinary coatings as necessary, for example, sugar-coated tablets, gelatin-encapsulated tablets, enteric-coated tablets, film-coated tablets, double tablets, and multilayer tablets.

  Moreover, when preparing in the form of a pill, a conventionally well-known thing can be widely used as a support | carrier in this field | area. Examples include excipients such as glucose, lactose, starch, cacao butter, hydrogenated vegetable oil, kaolin and talc, binders such as gum arabic powder, tragacanth powder, gelatin, ethanol, and disintegrants such as laminaran and agar. Can be used.

  In addition to the above, additives include, for example, binders, surfactants, absorption promoters, humectants, adsorbents, lubricants, fillers, extenders, moisturizers, preservatives, stabilizers, emulsifiers, A solubilizer and a salt for adjusting osmotic pressure can be appropriately selected and used depending on the dosage unit form of the resulting preparation. Moreover, you may contain other active ingredients (for example, vitamins, inorganic salts, etc.). Furthermore, you may use in combination with another medicinal component. Moreover, in the pharmaceutical composition of this invention, a coloring agent, a preservative, a fragrance | flavor, a flavoring agent, a sweetening agent, etc. can also be mix | blended and prepared as needed.

  The blending amount and dosage of fucoxanthin and / or fucoxanthinol in the pharmaceutical composition of the present invention can be appropriately set based on the blending amount in the angiogenesis inhibitor.

(2) Cosmetic composition It has been reported that angiogenesis is induced in the skin (dermis) by stimulation by ultraviolet irradiation or the like, which is one cause of wrinkles (Japan Cosmetic Engineers Association 23rd) Abstracts of IFSCC Orlando Conference Paper Report Meeting). Also, in Yano et al., The Journal of Investigative Dermatology, Vol 118, No 5, May 2002, an endogenous angiogenesis inhibitor (Thrombospondin-1) is caused by vascularization of the skin caused by UV-B irradiation. (Vascularization) and suppression of wrinkle formation were confirmed. From such knowledge, it is known that suppression of angiogenesis is important in preventing skin aging such as wrinkles.

  Since fucoxanthinol can exhibit an excellent anti-angiogenic action when applied transdermally, effects such as improvement of wrinkles can be expected. Therefore, fucoxanthinol can be mixed with a conventionally known base material and carrier that are cosmetically acceptable to prepare a cosmetic composition, which can be suitably used for the purpose of improving wrinkles. . That is, the cosmetic composition of the present invention can be used as a wrinkle improving cosmetic. As fucoxanthinol, those described in (1) above may be used. The amount of fucoxanthinol in the cosmetic composition of the present invention varies depending on the dosage form, but is, for example, about 0.000001 to 10% by weight, preferably about 0.00001 to 5% by weight, more preferably The amount is about 0.00001 to 2% by weight, more preferably about 0.00001 to 0.1% by weight, and more preferably about 0.00001 to 0.0005% by weight. Alternatively, in the cosmetic composition of the present invention, fucoxanthinol is about 0.01 to 24 μM, preferably about 0.01 to 20 μM, preferably about 0.01 to 15 μM, preferably about 0.01 to 10 μM. It is desirable to blend about 0.01 to 5 μM, more preferably about 0.01 to 2 μM. Since fucoxanthinol can exhibit an excellent anti-angiogenic action even at extremely low concentrations, an excellent wrinkle improving effect is expected even at about 0.01 to 2 μM.

  The form of the cosmetic is not particularly limited, and examples thereof include cleansing agents, skin cleansing agents, massage agents, ointments, creams, lotions, oils, packs, face wash, lotion, milky lotion, jelly and the like.

  The preparation method in preparing the dosage form is not particularly limited, and may be a method known in the art as long as the effects of the present invention are not impaired.

  The application amount of the cosmetic composition of the present invention is not particularly limited. With reference to the dose of fucoxanthinol described in (1) above, an appropriate amount is applied to the wrinkle-prone part and the wrinkle-prone part. What is necessary is just to apply according to a dosage form. Moreover, by applying the cosmetic composition of the present invention to the skin, in addition to improving wrinkles, the effect of improving skin elasticity, gloss and the like is expected.

EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example and a test example, this invention is not limited to these.
(1) Extraction of fucoxanthin and fucoxanthinol from wakame 100 g of ethanol was added to 2 g of pulverized commercially available dried wakame with a blender and stirred with a stirrer for 3 hours. When the ethanol extract obtained by filtration was analyzed by HPLC, it contained 390 μg of fucoxanthin and 9 μg of fucoxanthinol.

(2) Purification of fucoxanthin and fucoxanthinol Nutr. Fucoxanthin was obtained according to the method of 131,2921-2927,2001.

  1L of acetone: methanol (7: 3, v / v) was added in several batches to commercially available dried seaweed powder (dry weight about 100g) that was pulverized with a pulverizer. A fraction was obtained. After removing the solvent with an evaporator and dissolving in 100 mL of methanol, 100 mL of hexane and 10 mL of water were added, and liquid-liquid partition was performed using a separatory funnel.

  The upper layer of the obtained solution was removed, and the lower layer was washed twice with 100 mL of hexane. Thereafter, 31 mL of water was added to the lower layer to make the ratio of methanol 70%, and then 100 mL of hexane was added for distribution. Furthermore, after concentrating the lower layer with an evaporator until the volume became 1/3, 150 mL of diethyl ether and 100 mL of water were added and distributed.

The upper layer (ether layer) was collected, the solvent was removed by an evaporator, and the obtained extract was subjected to thin layer chromatography (developing solvent = hexane: acetone (6: 4, v / v); PLC plate (15 PLC plates 20 × 20 cm, Silica gel 60 F254, 1 mm, Merck Ltd., Japan)).
The separated carotenoid fraction was scraped off from the plate and extracted again to obtain a carotenoid extract.

The carotenoid extract obtained above is subjected to HPLC, a fraction of the peak portion of fucoxanthin appearing at a measurement wavelength of 444 nm and a retention time of 8.7 minutes is fractionated, and the fraction thus fractioned with dichloromethane and Extraction with water was purified. The HPLC conditions are as shown below. When fucoxanthin was dissolved in acetone and calculated using specific absorbance (E1% 1 cm = 1660, Acetone) as a concentration, 11.3 mg of fucoxanthin was obtained.
(HPLC preparative conditions)
・ Flow rate 1.0mL / min
・ Column temperature 40 ℃
Mobile phase (A) acetonitrile: methanol: water = 75: 15: 10 (v / v / v, containing 0.1% ammonium acetate) and (B) ethyl acetate: methanol = 30: 70 (v / v, 2-component gradient with 0.1% ammonium acetate)

  The fucoxanthin obtained as described above was prepared as follows. Asai et al. , Drug Metabolism and Dipositio, Vol. 32, no. 2, p. In accordance with the method described in 205-211, enzymatic degradation was performed using cholesterol esterase to obtain fucoxanthinol.

  In the following Test Examples 1 to 3, fucoxanthin and fucoxanthinol obtained by the method described in (2) above were used.

Test Example 1 Angiogenesis suppression experiment Wistar male rats (6-8 weeks old, manufactured by CLEA Japan, Inc.) were anesthetized with diethyl ether and bleeded by cutting the right femoral artery. Cut to length. The arterial piece was transferred to a 6-well culture plate, embedded in a collagen gel solution (Cellmatrix type Ia, Nitta Gelatin Co., Ltd.), and then gelled at 37 ° C.

Thereafter, 2 ml of RPMI 1640 medium containing 1% ITS + (Becton Dickinson Bioscience) was added, and fucoxanthinol was added to a concentration of 1 μM or 25 μM, followed by culturing in a CO ₂ incubator. In addition, an arterial piece cultured without adding anything was used as a control.

  On day 7 of culture, microvascular hairs produced from the arterial piece were photographed under an inverted microscope, and after taking the image into a computer, the length of the microvessel was measured. The collagen gel solution was a collagen stock solution (Cellmatrix type Ia, Nitta Gelatin Co., Ltd.), 10 × Eagle's MEM medium (Gibco), and a reconstitution buffer (Nitta Gelatin Co., Ltd.) in a volume ratio of 8: A mixture of 1: 1 and stored at 4 ° C. was used.

  Based on the measured length of the microvessel, the relative value of the vessel length at each concentration when the control was 100% was determined. The results are shown in Table 1. Further, FIG. 1 shows an image of microvascular hairs generated from an arterial piece when fucoxanthinol 25 μM is added to the medium or when fucoxanthinol is not added (control).

  From the results shown in Table 1, fucoxanthinol was found to have an anti-angiogenic effect even at very low concentrations.

Test Example 2 Human Umbilical Vein Endothelial Cell (HUVEC) Growth Inhibition Experiment HUVEC was prepared to 1.5 × 10 ⁴ cells / ml in a special medium (HuMedia EG-2, Kurabo Industries), and 100 μl was seeded on a 96-well culture plate. did. After culturing overnight in a CO ₂ incubator to attach HUVEC to the plate, the medium was changed to a dedicated medium containing fucoxanthin at a concentration of 10 to 100 μM, and cultured in a CO ₂ incubator for 3 days. 2 hours after the medium was replaced with 100 μl of a special medium not added with fucoxanthin, 10 μl of Cell Counting Kit reagent (manufactured by Dojindo Laboratories) was added, and the absorbance at 450 nm was measured with a microplate reader, thereby affecting the cell proliferation It was investigated. Moreover, the cell cultured without adding anything was used as control. Based on the measured number of cells, the relative value of the number of cells at each concentration when the control was 100% was determined. The results are shown in Table 2.

  From the results shown in Table 2, it was confirmed that fucoxanthin has an inhibitory effect on the proliferation of vascular endothelial cells.

Test Example 3 Experiment on suppression of luminal formation of human umbilical vein endothelial cells (HUVEC) 50 μl of Matrigel (Becton Dickinson Bioscience) was added to a 96-well culture plate and gelled in a CO ₂ incubator at 37 ° C. for 1 hour. HUVEC was suspended in a dedicated medium (HuMedia EG-2, Kurabo Industries) to 1.5 × 10 ⁵ cells / ml. 100 μl (1.5 × 10 ⁴ cells) of HUVEC suspension was added onto the gelled Matrigel. Then, after adding such fucoxanthin a concentration of 10 or 25 [mu] M, and 12-hour culture in a CO ₂ incubator. The formed luminal structure was photographed under an inverted microscope, the image was taken into a computer, and the length of the luminal structure was measured. Moreover, the cell cultured without adding anything was used as control. Based on the length of the obtained blood vessel cavity, the lumen length (relative value) at each concentration when the control was 100% was determined. The results are shown in Table 3.

  From the results shown in Table 3, it was confirmed that fucoxanthin has a lumen formation inhibitory action.

The result in Test Example 1 is shown. That is, the results of an angiogenesis inhibition experiment using rat arterial pieces (addition of control or fucoxanthinol 25 μM) are shown by photographs.

Claims (5)

  An angiogenesis inhibitor comprising fucoxanthin and / or fucoxanthinol.   Containing fucoxanthin and / or fucoxanthinol, diabetic retinopathy, atherosclerosis, obesity, type II diabetes, rheumatoid arthritis, Graves' disease, Kaposi's sarcoma, Alzheimer's disease, periodontal disease, scleroderma, A pharmaceutical composition for treating or preventing at least one disease selected from the group consisting of glaucoma, psoriasis, and age-related macular degeneration.   A cosmetic composition containing fucoxanthinol.   The cosmetic composition according to claim 3, comprising 0.000001 to 10% by weight of fucoxanthinol. The cosmetic composition according to claim 3 or 4, which is used for wrinkle improvement.

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