Compound hexadecaphlorethol isolated from <i>Ishige okamurae </i>and use thereof

Abstract

Disclosed herein are hexadecaphlorethol (HdP), which is a novel compound isolated from Ishige okamurae, and the use thereof in anti-diabetic treatment and exercise performance improvement. The novel compound HdP has the activity of inhibiting -glucosidase, inducing glucose uptake into differentiated myoblast C2C12 cells, reducing blood glucose levels in zebrafish which have reduced insulin secretion through treatment with alloxan, increasing an intracellular level of calcium ions necessary for muscle contraction and exercise performance in zebrafish, and inducing uptake of glucose, an energy source necessary for muscle contraction, into cells.

Claims

1. A method for treating diabetes, comprising: administering to a patient in need thereof a therapeutically effective amount of a compound of Chemical Formula 1, a hydrate thereof, or a solvate thereof: ##STR00002##

2. The method of claim 1, wherein the compound is administered in a form of a pharmaceutical composition.

3. The method of claim 2, wherein the composition comprises at least one excipient.

4. The method of claim 2, wherein the pharmaceutical composition further comprises at least one selected from the group consisting of maca gelatinized powder, creatine, Hovenia dulcis pedicle extract powder, and a vegetable worm fermentation extract; a fermented amino acid complex, Hovenia dulcis pedicle extract powder, and a Rhodiola sachalinensis extract; and L-arabinose, a nopal extract, cinnamon extract powder, a guava leaf extract, digestion-resistant maltodextrin, lyophilized silkworm powder, a Dioscorea batatas alcohol extract, a banana leaf extract, and a mulberry leaf extract.

5. The method of claim 1, the compound is administered in a form of a food composition.

6. The method of claim 5, wherein the food composition is in a form selected from the group consisting of tea, juice, carbonated beverage, ion beverage milk, yogurt, gum, rice cake, traditional Korean cookies, breads, confectionery, noodles, granules, liquids, powders, flakes, pastes, syrups, gels, jellies, and bars.

7. The method of claim 5, wherein the food composition further comprises at least one selected from the group consisting of sweeteners, flavoring agents, preservatives, emulsifiers, acidifiers, and thickeners.

8. The method of claim 5, wherein the food composition further comprises at least one selected from the group consisting of maca gelatinized powder, creatine, Hovenia dulcis pedicle extract powder, a vegetable worm fermentation extract; a fermented amino acid complex, Hovenia dulcis pedicle extract powder, a Rhodiola sachalinensis extract; L-arabinose, a nopal extract, cinnamon extract powder, a guava leaf extract, digestion-resistant maltodextrin, lyophilized silkworm powder, a Dioscorea batatas alcohol extract, a banana leaf extract, and a mulberry leaf extract.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The above and other objects, features and advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which:

(2) FIG. 1 schematically illustrate an HdP isolation process;

(3) FIG. 2 is an HPLC chromatogram of HdP;

(4) FIG. 3 is 1H and 13C NMR spectral data of HdP;

(5) FIGS. 4a and 4b shows two-dimensional NMR spectral HMQC (a) and HMBC (b) data of HdP;

(6) FIG. 5 is an LC-MS-MS spectral data of HdP;

(7) FIG. 6 shows results indicating that HdP induces glucose uptake into differentiated myoblast C2C12 cells;

(8) FIG. 7 shows toxicity test results of HdP in zebrafish embryos;

(9) FIG. 8 shows a result illustrating that HdP reduces a blood glucose level in adult zebrafishes;

(10) FIG. 9 shows a result illustrating that HdP increases an intracellular calcium ion (Ca2+) level in zebrafishes; and

(11) FIG. 10 shows a result illustrating that HdP induces glucose influx into zebrafish myocytes.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

(12) As illustrated in the following Examples and Experimental Examples, the novel material HdP of the following Chemical Formula 1, isolated and identified from Ishige okamurae, was found to inhibit the activity of -glucosidase and to induce glucose uptake in C2C12, which is a differentiated myoblast cell line. As shown in zebrafish experiments, the novel material was observed to reduce blood glucose levels in zebrafish which was rendered to secrete a reduced level of insulin by treatment with alloxan, increase an intracellular concentration of calcium ions necessary for muscle contraction and motility enhancement, and induce intracellular uptake of glucose, which is an energy source necessary for muscle contraction.

(13) ##STR00001##

(14) With the above-mentioned experimental results taken into consideration, one aspect of the present disclosure contemplates the compound represented by Chemical Formula 1, a prodrug thereof, a hydrate thereof, or a solvate thereof. Another aspect of the present disclosure contemplates an anti-diabetic composition comprising the compound of Chemical Formula 1, a prodrug thereof, a hydrate thereof, or a solvate thereof as an effective ingredient. A further aspect of the present disclosure contemplates a composition comprising the compound of Chemical Formula 1, a prodrug thereof, a hydrate thereof, or a solvate thereof as an effective ingredient for improving exercise performance.

(15) As used herein, the term prodrug refers to a drug that has a physically or chemically controlled property by chemically modifying a target drug and is itself biologically inactive, but is converted to a bioactive drug by chemical or enzymatic action in vivo after administration.

(16) As used herein, hydrate means a compound that is associated with water, without chemical bonds between water and the compound.

(17) As used herein, solvate means a compound formed between molecules or ions of a solute and molecules or ions of a solvent.

(18) The term anti-diabetic or anti-diabetes, as used herein, means alleviation, prevention, or treatment of a symptom of diabetes.

(19) The term diabetes, as used herein, means insulin-dependent diabetes mellitus (type 1 diabetes mellitus) and non-insulin dependent diabetes mellitus (type 2 diabetes mellitus) and is further intended to encompass diabetes resulting from pancreatic damage caused by a different disorder, for example, diabetes caused by hyperthyroidism, hyperadrenocorticism, hypersecretion of growth hormone, or hypersecretion of catecholamine, and gestational diabetes.

(20) The term effective ingredient, as used herein, means an ingredient that can exhibit a desired activity by itself or in combination with a carrier which is itself not active.

(21) So long as the effective ingredient exhibits an effect of improving exercise performance, a certain amount (effective amount) thereof may be contained in the composition of the present disclosure and may vary, depending on use, formulation, etc. Typically, the effective ingredient may be determined within the range of 0.001% by weight to 15% by weight. Here, the term effective amount refers to an amount of an effective ingredient contained in the composition of the present disclosure which is capable of producing a desirable medical and pharmaceutical effect such as exercise performance improvement, etc., when the composition is administered to a subject, such as a mammal, particularly a human, for a period of administration according to the suggestion of a medical expert. Such an effective amount may be empirically determined within the typical ability of a person skilled in the art.

(22) In addition to the effective ingredient, the composition of the present disclosure may further comprise any compound or natural extract that has been verified for safety and known to have corresponding activities in order to synergistically reinforce an anti-diabetic effect or an exercise performance improvement effect or to increase the convenience of administration or intake through addition of similar activities such as body fat reduction, fatigue alleviation, etc.

(23) Such compounds or extracts may be compounds or extracts described in the official compendium of each country, such as pharmacopoeia (e.g., Korean Pharmacopoeia in Korea), health functional food standards codex (e.g., Health Function Food Standards and Specifications notified by the Korean Ministry of Food and Drug Safety in Korea), compounds or extracts approved according to instructions of each country which regulate production and sales of drug products (the Pharmaceutical Affairs Act in Korea), and compounds or extracts which are approved for functionality according to instructions in each country which regulate production and sales of health functional foods (e.g., the Health Functional Foods Act in Korea). For example, maca gelatinized powder, creatine, Hovenia dulcis pedicle extract powder, and a vegetable worm fermentation extract; a fermented amino acid complex, Hovenia dulcis pedicle extract powder, and a Rhodiola sachalinensis extract; and L-arabinose, a nopal extract, cinnamon extract powder, a guava leaf extract, digestion-resistant maltodextrin, lyophilized silkworm powder, a Dioscorea batatas alcohol extract, a banana leaf extract, and a mulberry leaf extract, which are recognized as having respective functions of exercise performance improvement, fatigue alleviation, and blood sugar control, according to the Health Functional Foods Act in Korea, may fall within the scope of such compounds or extracts.

(24) At least one of such compounds or natural extracts may be contained in combination with the effective ingredient in the composition of the present invention.

(25) According to a concrete embodiment, the composition of the present disclosure may be regarded as a food.

(26) The food composition of the present disclosure may be prepared into any form, for example, beverages such as tea, juice, a carbonated beverage, an ion beverage, etc.; processed milk products, such as milk, yogurt, etc.; foods, such as gum, rice cake, traditional Korean sweets and cookies, breads, confectionery, noodles, etc.; and agents for health functional foods, such as tablets, capsules, pills, granules, liquids, powders, flakes, pastes, syrups, gels, jellies, bars, etc. In addition, the food composition of the present invention may be in the form of any product complying with legal and functional classifications according to an enforcement at the time of production and distribution. For example, the composition may be a health functional food according to the Health Functional Foods Act of Korea or may be confectionery, beans, teas, beverages, or special purpose foods according to the food types stipulated by the Korean Food Standards Codex (Health Function Food Standards and Specifications notified by the Korean Ministry of Food and Drug Safety in Korea) of the Food Sanitation Act in Korea.

(27) The food composition of the present disclosure may include a food additive in addition to the effective ingredient. A food additive may be understood as a substance that can be added to, mixed with, or precipitated in a food during food preparation, processing, or preservation. A food additive should be guaranteed safety because it may be ingested together with food every day and for a long period of time. Safety-guaranteed, food additives are limitedly stipulated in terms of components or functions by a food additives codex according to an act in each country (Food Sanitation Act in Korea) which regulates the production and distribution of foods. The Korean Food Additives Codex (Food Additive Standards and Specifications published by the Korean ministry of Food and Drug Safety) stipulates food additives in terms of components into categories of chemical synthetic products, natural additives, and mixed agents. Such food additives may be divided into sweeteners, flavoring agents, preservatives, emulsifiers, acidifiers, thickeners, etc. in view of functions.

(28) A sweetener, which is to provide a food with a sweet taste, may be used in the food composition of the present invention irrespective of whether natural or synthetic. Preferable is a natural sweetener. Examples of the natural sweetener include sugar sweeteners, such as a corn syrup solid, honey, sucrose, fructose, lactose, maltose, etc.

(29) A flavoring agent, which is to make a taste or a smell better, may be used irrespective of whether natural or synthetic. Preferable is a natural flavoring agent. A natural flavoring agent may be used for nutrition as well as enhancing flavor. Natural flavoring agents may be obtained from apples, lemons, tangerine, grapes, strawberries, peaches, etc. or from green tea leaves, Solomon's seals, bamboo leaves, cinnamon, chrysanthemum, jasmine, etc. In addition, a natural flavoring agent obtained from ginseng (red ginseng), bamboo sprouts, aloe vera, ginkgo nuts, etc. may be used. The natural flavoring agent may be a concentrated liquid or an extract of a solid phase. According to circumstances, a synthetic flavoring agent, such as ester, alcohol, aldehyde, terpene, etc. may be used.

(30) An available preservative may be exemplified by calcium sorbate, sodium sorbate, potassium sorbate, calcium benzoate, sodium benzoate, potassium benzoate, and EDTA (ethylenediaminetetraacetic acid). As an emulsifier, acacia gum, carboxymethylcellulose, xanthan gum, pectin, etc. may be used. Examples of acidifiers include citric acid, malic acid, fumaric acid, adipic acid, phosphoric acid, gluconic acid, tartaric acid, ascorbic acid, acetic acid. An acidifier may be added to adjust a pH of the food composition with the aim of restraining microbial growth as well as improving a taste. Available for use as a thickener are a suspending agent, a precipitating agent, a gelling agent, and a swelling agent.

(31) In addition to the above-mentioned food additives, the food composition of the present disclosure may further comprise bioactive substances or minerals which are known in the art and guaranteed safety as food additives in order to supplement functionality and nutrition.

(32) Such bioactive substances include catechins such as those contained in green tea; vitamins such as vitamin B1, vitamin C, vitamin E, vitamin B12, etc.; tocopherol; dibenzoyl thiamine, etc. Examples of available minerals include calcium agents such as calcium citrate; magnesium agents such as magnesium stearate; iron agents such as iron citrate; chrome chloride; potassium iodide; selenium; germanium; vanadium; and zinc.

(33) The food composition of the present disclosure may contain the above-mentioned food additives in respective appropriate amounts enough to achieve the purposes of addition according to types of the product.

(34) With regard to other food additives available for the food composition of the present disclosure, reference may be made to a food codex or food additive codex according to instruction of each country.

(35) In another embodiment, the composition of the present disclosure may be understood as a pharmaceutical composition.

(36) The pharmaceutical composition of the present disclosure may comprise a pharmaceutically acceptable carrier in addition to the effective ingredient and may be prepared into an oral or parenteral formulation according to administration routes in a typical manner. Here, the term pharmaceutically acceptable means retaining no more toxicity than is too high for an application (treatment) subject to adapt without limiting the activity of the effective ingredient.

(37) For oral formulations, the pharmaceutical composition of the present disclosure may be prepared, together with a suitable carrier, into a dosage form, such as a powder, a granule, a tablet, a pill, a sugar-coated pill, a capsule, a liquid, a gel, a syrup, a suspension, a wafer, etc., according to a method known in the art. In this context, examples of pharmaceutically acceptable carriers include: sugars, such as lactose, glucose, sucrose, dextrose, sorbitol, mannitol, xylitol; starches, such as corn starch, potato starch, wheat starch, etc.; celluloses such as cellulose, methyl cellulose, ethyl cellulose, sodium carboxymethyl cellulose, hydroxypropylmethyl cellulose, etc.; polyvinyl pyrrolidone; water; methylhydroxy benzoate; propylhydroxy benzoate; magnesium stearate; mineral oil; malt; gelatin; talc; polyol; and vegetable oil. If necessary, the composition may be formulated, together with a diluent and/or an excipient, such as a filler, a thickener, a binder, a humectant, a disintegrant, etc.

(38) For parenteral formulations, the pharmaceutical composition of the present disclosure may be prepared, together with a suitable carrier, into a dosage form, such as an eye drop, an injection, a transdermal agent, a nasal inhaler, a suppository, etc. A carrier suitable for use in an eye drop may be exemplified by an isotonic solution such as sterile water, a saline, and 5% dextrose. Optionally, benzalkonium chloride, methyl paraben, ethyl paraben, etc. may be used for preservation. When prepared into an injection, the composition may include a carrier such as sterile water, ethanol, polyol, e.g., glycerol or propylene glycol, or a combination thereof. Preferably, a Ringer's solution, triethanol amine-containing phosphate buffered saline (PBS) or sterile water for injection, an isotonic solution such as 5% dextrose, etc. may be used. A transdermal agent may be in the form of an ointment, a cream, a lotion, a gel, an external use liquid, a paste, a liniment, an aerosol, etc. A nasal inhaler may be prepared into an aerosol spray in which a suitable propellant such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, etc. may be used. For a suppository, Witepsol, Tween 61, polyethylene glycols, cacao butter, laurin butter, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates, sorbitan fatty acid esters, etc. may be used as a base.

(39) Concrete formulations of pharmaceutical compositions are known in the art. For example, reference may be made to document [Remington's Pharmaceutical Sciences (19th Ed., 1995)], which is incorporated herein by reference.

(40) The preferable dose of the pharmaceutical composition in accordance with the present disclosure may vary, depending on various factors including the patient's health state, weight, sex, and age, the severity of disease, and the route of administration. For a preferred effect, the effective amount of the pharmaceutical composition of the present invention may range in a daily dose from 0.001 mg/kg to 10 g/kg, and more preferably from 0.001 mg/kg to 1 g/kg. The composition may be administered in a single dose, or may be divided into multiple doses per day. Such doses should be construed to limit the scope of the present disclosure in no way.

(41) A better understanding of the present invention may be obtained through the following examples which are set forth to illustrate, but are not to be construed as the limit of the present invention.

EXAMPLE

Isolation and Identification of HdP from Ishige okamurae Extract

Example 1: Isolation of HdP

(42) Hdp was isolated from an Ishige okamurae extract as follows, and a schematic view of the isolation process thereof is depicted in FIG. 1.

(43) Ishige okamurae was gathered in an area of Sungsan, Jeju Island, Korea, desalted by washing with fresh water twice, and naturally dried. The dried Ishige okamurae was ground into powder in a blender and 100 g of the powder was immersed in 2 L of 50% ethanol for 24 hours at room temperature. Following filtration, concentration at a reduced pressure removed the solvent to leave an extract in a powder phase.

(44) In 5 L of distilled water was suspended 10 g of the extract powder to which 5 L of ethyl acetate was then added to give fractions. Removal of the solvent by concentration at room temperature afforded an ethyl acetate fraction.

(45) For use in centrifugal partition chromatography (CPC), a solvent system consisting of n-hexane-ethylacetate-methanol-water was preferentially selected through a preparative experiment. Investigation was made into an optimal partition coefficient K while component ratios in the solvent system were changed. As a result, a K value of 0.5 was identified to be optimal when n-hexane, ethylacetate, methanol, and water in the solvent system were present at a volume ratio of 1:9:4.5:6.5.

(46) The solvent system was put in a separatory funnel and thoroughly equilibrated by being vigorously shaken at room temperature to separate an upper organic layer and a lower phase which were used as a stationary phase and a mobile phase, respectively.

(47) In order to perform CPC, the CPC column was filled with the upper organic phase (stationary phase). While the column was then rotated at 1,000 rpm, the lower phase was injected at a flow rate of 2 ml/min to the column until the pump pressure was constantly maintained. After the pressure reached a constant value, 60 ml of a sample (a solution of 500 mg of the ethylacetate fraction in 6 mL of a mixture of 1:1 of water and methanol) was injected. While the effluent was monitored at 230 nm, fractions were collected. Relatively non-polar fractions were separated at 70 min to 120 min after the sample injection.

(48) Then, the CPC fractions thus obtained were subjected to HPLC, using a semi-preparative HPLC column (YMC-Pack ODS-A, 10250 mm, 5 m particle size, YMC Co Ltd, Tokyo, Japan). In brief, 32% acetonitrile containing 0.1% formic acid and 68% water containing 0.1% formic acid were used as a mobile phase which was allowed to flow at a flow rate of 2 mL/min. The effluent was monitored with a UV detector at 230 nm and a compound having the second highest intensity, detected at 17.302 min, was isolated. An HPLC chromatogram accounting for the compound is depicted in FIG. 2.

Example 2: Identification of the Compound

(49) For structural identification of the isolated compound, 1H NMR, and 13C NMR spectra were measured. HMQC and HMBC spectra, which are two-dimensional (2D) NMR spectra, were measured to analyze correlation between hydrogen atoms themselves and between hydrogen and carbon atoms. The molecular weight was determined using LC-MS-MS. The results are shown in FIGS. 3 to 5, respectively. Comparison of the data with those in related documents concluded the identification of HdP, a novel compound represented by Chemical Formula 1.

Experiment Examples: Assay of HdP for Anti-Diabetic Activity and Exercise Performance Improvement

Experiment Example 1: Anti-Diabetic Activity of HdP

(50) 1.1 Assay of Inhibitory Activity Against -Glucosidase

(51) Inhibitory activity against -glucosidase was evaluated using a yeast enzyme according to the method of Watanabe et al. (Watanabe, Kawabata, Kurihara, & Niki, 1997). In the reaction condition of pH 7.0 and 37 C., 5 mM p-nitrophenyl -D-glucopyranoside (PNP-G) serving as a substrate was reacted with a dilution of 32 mU/ml enzyme (yeast -glucosidase, Sigma) in 100 mM phosphate buffer and inhibitory activity was measured using spectrophotometry. The sample was reacted with PNP-G for 5 min and the absorbance of 4-nitrophenol released upon the hydrolysis of PNP-G was read at 405 nm. Subsequently, the same amount of the substrate solution was added and reacted at room temperature for an additional five min., after which the reaction was stopped by adding 0.5 M Na.sub.2CO.sub.3. A change in absorbance at 405 nm was measured.

(52) Inhibitory activity (%) against -glucosidase was calculated according to the formula (1-absorbance of sample-added group/absorbance of solvent-added group)100).

(53) Acabose, which is an oral hypoglycemic agent inhibitory of -glucosidase, was used as a positive control.

(54) 1.2 Measurement of Glucose Uptake of C2C12 Cell

(55) 1.2.1 C2C12 Cell Culture and Differentiation

(56) The mouse-derived myoblast C2C12 cells (ATCC, Manassas, Va., USA) were suspended in DMEM (Dulbecco's Modified Eagle's Media) medium supplemented with 10% FBS and an antibiotic and cultured at 37 C. under a 5% CO.sub.2 atmosphere in an incubator. Upon 80% confluency, the C2C12 cells subjected to differentiation in a DMEM medium containing 2% horse serum and a low concentration of glucose for 5 days, with the medium freshly changed every three days. The differentiated cells were serum starved for 12 hours in serum free DMEM containing a low concentration of glucose, washed with PBS, and then reacted for 24 hours with the sample (HdP) in a fresh serum free DMEM.

(57) 1.2.2 Measurement of Glucose Uptake of C2C12 Cell Using Flow Cytometry (FACS)

(58) Glucose uptake of mouse-derived myoblast C2C12 cells was measured using flow cytometry (FACS) according to the (2-(N-(7-nitrobenz-2oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG) assay method (Cell Death Dis. 2017 Oct. 5. 8 (10):e3078). The cultured cells were incubated with 10 M 2-NBDG at 37 C. for 24 hours and then analyzed using BD FACS Cantoll (BD Biosciences, NJ, USA), with the fluorescence of 2-NBDG detected in a FITC channel.

(59) 1.3 Animal Test

(60) For use in experiments, adult zebrafish were purchased from a market (Jeju Aquarium, Jeju Island, Korea). They were acclimated to a condition of a temperature of 28.51 C. and a 14/10 h light-dark cycle in a 3.5 L acryl tank and fed twice a day (Tetra GmgH D-49304 Melle, made in Germany). Experiments with zebrafish were approved by the Institutional Animal Care and Use Committee of Jeju University.

(61) 1.3.1 Test of HdP Toxicity in Zebrafish Embryo

(62) Four days after fertilization, each of the embryos (n=15) was transferred to 12-well plates, each containing 950 l of an embryo medium (distilled water containing 60 ppm salt) and 50 l of HdP (0.01, 0.1, 1, and 10 g/ml) and the viability of zebrafish embryos exposed to HdP for 168 hours after fertilization were measured.

(63) 1.3.2 Measurement of Blood Sugar Level in Zebrafish

(64) Wild-type adult zebrafish were treated for 1 hour with 2 mg/ml alloxan and then for 1 hour with 1% glucose in the absence of insulin. Subsequently, the reaction solution was exchanged with water in which the zebrafish were then left for 1 hour before treatment with HdP, metformin, or BAPTA-AM for 90 min. Thus, the experiment groups included a non-treated group, an alloxan-treated group (control), a group treated with alloxan and HdP (0.3 g/g body weight), a group treated with alloxan and metformin (5 g/g body weight), a group treated with alloxan and BAPTA-AM (3 g/g body weight), and a group treated with alloxan, BAPTA-AM (3 g/g body weight), and HdP (0.3 g/g body weight).

(65) 2. Test Results

(66) 2.1 Measurement Result of Inhibitory Activity Against -Glucosidase

(67) Concentrations of the samples which were necessary for inhibiting the activity of -glucosidase by 50% (IC.sub.50) are given in Table 1, below.

(68) TABLE-US-00001 TABLE 1 Sample IC.sub.50 (M) Hexadecaphlorethol(HdP) 54.97 Acarbose 1050.23

(69) As is understood from the data of Table 1, HdP according to the present disclosure exhibited higher inhibitory activity against -glucosidase than acarbose did.

(70) 2.2 Measurement Result of Glucose Uptake of C2C12 Cell

(71) Glucose uptake by HdP in differentiated C2C12 cells was measured and the results are depicted in FIG. 7. FACS is an instrument isolating fluorescence-marked cells from a heterogeneous cell mixture. In C2C12 cells which were treated with the fluorescence-marked glucose analogue 2-NBDG, fluorescence intensity became stronger with the detection of more 2-NBDG through a FITC filter. Thus, the detected cell populations migrated right in the graph. With reference to FIG. 6, glucose uptake into the differentiated C2C12 cells treated with HdP for 24 hours is shown to significantly increase in a dependent manner on the dose of HdP, indicating that HdP can lower a blood glucose level. Glucose uptake into differentiated C2C12 cells is known as an index accounting for a reduction in blood glucose level (Diabetes 30 (1981) 1000-1007; Biochemical and Biophysical Research Communications 420 (2012) 576-581; Nat. Rev. Mol. Cell Biol. 7 (2006) 85-96; Eur. J. Cell Biol. 87 (2008) 337-351)

(72) 2.3 Test Result of HdP Toxicity in Zebrafish Embryo

(73) In order to examine a toxicity-free concentration of HdP at which no toxicity occurs, survival rates of zebrafish embryos were evaluated. As shown in FIG. 7, the embryos were observed to survive 1 g/ml or less HdP at a rate of about 90% or higher.

(74) 2.4 Measurement Result of Blood Glucose Level of Zebrafish under Control of HdP

(75) Examination was made to see whether HdP controls blood sugar in an animal model. In this regard, adult zebrafish which had been treated with alloxan to release a reduced level of insulin were injected with glucose and monitored for blood glucose levels.

(76) Results are depicted in FIG. 8. With reference to FIG. 8, HdP was observed to significantly reduce blood glucose levels of alloxan-treated adult zebrafishes in a dose-dependent manner. This effect was similar to that of metformin, which is commercially used to control blood glucose. Particularly, when account is taken of the fact that HdP was used at a concentration of 0.3 g/g in contrast to 5 g/g metformin, a similar glucose uptake effect is expected even with a concentration of HdP which is 16.7-fold lower than that of metformin.

Experiment Example 2: Activity of HdP to Improve Exercise Performance

(77) 1. Experiment Method

(78) 1.1 Preparation of Zebrafish

(79) For use in experiments, adult zebrafish were purchased from a market (Jeju Aquarium, Jeju Island, Korea). They were acclimated to a condition of a temperature of 28.51 C. and a 14/10 h light-dark cycle in a 3.5 L acryl tank and fed twice a day (Tetra GmgH D-49304 Melle, made in Germany). Experiments with zebrafish were approved by the Institutional Animal Care and Use Committee of Jeju University.

(80) 1.2 Measurement of Intracellular Calcium Level in Zebrafish Embryo Using Fluo-4

(81) Intracellular calcium levels of zebrafish embryos were measured using a calcium-sensitive Fluo-4 probe. Embryos were incubated with Fluo-4 at 28.51 C. for 30 min before treatment with HdP (0.6, 3, and 6 g/ml). In order to block the entry of calcium into cells, the embryos were reacted with the calcium chelator BAPTA-AM 0.1 mM for 1 hour before incubation with Fluo-4. The experiment groups included a non-treated group (N), a group treated with Fluo-4 alone (F), a group treated with HdP alone (0.6, 3, and 6 g/ml), a group treated with Fluo-4 and HdP (0.6, 3, and 6 g/ml), and a group treated with Fluo-4, BAPTA-AM, and HdP (6 g/ml).

(82) 1.3 Measurement of Blood Glucose Level of Zebrafish

(83) Wild-type adult zebrafish were treated for 1 hour with 2 mg/ml alloxan and then for 1 hour with 1% glucose in the absence of insulin. Subsequently, the reaction solution was exchanged with water in which the zebrafish were then left for 1 hour before treatment with HdP, metformin, or BAPTA-AM for 90 min. Thus, the experiment groups included a non-treated group, an alloxan-treated group (control), a group treated with alloxan and HdP (0.3 g/g body weight), a group treated with alloxan and metformin (5 g/g body weight), a group treated with alloxan and BAPTA-AM (3 g/g body weight), and a group treated with alloxan, BAPTA-AM (3 g/g body weight), and HdP (0.3 g/g body weight).

(84) 2. Test Result

(85) 2.1 Measurement Result of Intracellular Calcium Level Change by HdP in Zebrafish

(86) Intracellular calcium level changes by HdP in zebrafish embryo cells were observed with Fluo-4. Referring to FIG. 9, a significant difference appeared around the abdomen between the non-treated group (N) and the Fluo-4-treated group (F). The group treated with HdP alone did not undergo a special phosphorescence change, compared to the non-treated group (N) or the Fluo-4-treated group (F) (left panel in FIG. 9), indicating no HdP-induced phosphorescence change. A significant increase in phosphorescence was observed in the group treated with HdP and Fluo-4 in combination, compared to the Fluo-4-treated group (F), indicating that treatment with HdP increases intracellular Ca.sup.2+ levels. A phosphorescence decrease in the group treated with the calcium chelator BAPTA-AM in combination with HdP identified the intracellular influx of Ca.sup.2+ (right panel in FIG. 9).

(87) 2.2 Measurement Result of Blood Glucose Level Change by HdP in Zebrafish

(88) Examination was made to see whether HdP induces the uptake of glucose necessary for muscle contraction with the increase of intracellular Ca.sup.2+ levels. In this regard, adult zebrafish which had reduced insulin secretion by treatment with alloxan were injected with glucose to monitor HdP-induce glucose influx into myocytes in terms of blood glucose levels. The results are shown in FIG. 10. A reduced level of the energy source glucose in blood is known as an index accounting for glucose influx into tissues, such as muscles, etc. (Diabetes 30 (1981) 1000-1007; Biochemical and Biophysical Research Communications 420 (2012) 576-581; Nat. Rev. Mol. Cell Biol. 7 (2006) 85-96; Eur. J. Cell Biol. 87 (2008) 337-351).

(89) Blood glucose levels in the alloxan-treated adult zebrafishes were significantly reduced by HdP. This effect was similar to that of metformin, which is commercially used to control blood glucose. Particularly, when account is taken of the fact that HdP was used at a concentration of 0.3 g/g in contrast to 5 g/g metformin, a similar glucose uptake effect is expected even with a concentration of HdP which is 16.7-fold lower than that of metformin.

(90) A relation between an effect of HdP on glucose influx and an increase of intracellular calcium levels was investigated. For this, zebrafish were treated with the calcium chelator BAPTA-AM in combination with alloxan or HdP. No significant differences in blood glucose level were observed between the groups treated with alloxan plus BAPTA-AM and with HdP plus BAPTA-AM, indicating that glucose uptake in adult zebrafish increases with the HdP-induced increase of intracellular calcium levels.

(91) Provided according to the present disclosure is a composition for improving exercise performance, using a novel compound HdP isolated from Ishige okamurae, as described hitherto. The composition of the present invention may be prepared into a food or drug product.

(92) Although the preferred embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.