WO2010041832A2 - Composition antioxydante active contenant un composé dériivé d'ishige okamurae - Google Patents

Composition antioxydante active contenant un composé dériivé d'ishige okamurae Download PDF

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WO2010041832A2
WO2010041832A2 PCT/KR2009/005353 KR2009005353W WO2010041832A2 WO 2010041832 A2 WO2010041832 A2 WO 2010041832A2 KR 2009005353 W KR2009005353 W KR 2009005353W WO 2010041832 A2 WO2010041832 A2 WO 2010041832A2
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phlorotannin
activity
bieckol
ros
scavenging activity
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WO2010041832A3 (fr
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김문무
이상훈
김세권
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부경대학교 산학협력단
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L19/00—Products from fruits or vegetables; Preparation or treatment thereof
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Definitions

  • the present invention relates to an antioxidant active composition containing a compound derived from l ( Ishige okamurae ), and relates to a food, cosmetic or pharmaceutical composition having an antioxidant activity containing a phlorotannin compound isolated from the l extract as an active ingredient.
  • ROS reactive oxygen species
  • free radicals such as superoxide anions (O 2 .-), hydroxyls (HO., Peroxyl (ROO.), Alkoxyl ( RO ⁇ ), and nitric oxide
  • non-free radicals such as singlet oxygen ( 1 O 2 ) and hydrogen peroxide (H 2 O 2 ), wherein the ROS are involved in the origin of life and in the evolution of living things.
  • ROS reactive oxygen species
  • ROS are endogenous causes, such as normal aerobic respiration, promoted polymorphonuclear leukocytes, macrophages and peroxysomes, exogenous causes, such as smoking, ionizing radiation, certain contaminants, organic solvents, pesticides and high multiplexing. Unsaturated fatty acids are produced by diet.
  • the beneficial effect of ROS in biological systems is that they contribute to energy conservation, phagocytosis, cell growth and intracellular signal regulation, and the synthesis of biologically important compounds at the physiological level.
  • ROS is involved in cellular aging, mutations, carcinogenesis, coronary artery disease, diabetes, muscular dystrophy and neurodegeneration. Nevertheless, all aerobic organisms have evolved antioxidant defense systems to resist oxidative stress from ROS. These systems include some antioxidants produced in the body and other antioxidants obtained from the diet. If the antioxidants produced in the body are insufficient to defend against oxidative stress, it is necessary to reduce the accumulation of oxidative stress during life by ingesting dietary antioxidants. Recently, there has been a growing interest in dietary intake of antioxidant-rich foods, oxidative stress and age-dependent diseases.
  • Polyphenols are common secondary metabolites and abundant in land plants such as fruits, vegetables and herbs, and marine plants such as seaweed. These substances have received the utmost attention, and they have been studied extensively because they are free radical scavengers, have little toxicity, and have fewer side effects than synthetic antioxidants such as BHA and BHT. Marine brown algae derived polyphenols are called phlorotannins and are structurally different from those obtained from above-ground plants, which are strictly limited to polymers of polyglucinol. Phlorotannin has an action that includes antioxidant, antiallergic, antibacterial and anticancer activity, and research on phlorotannin is of great interest among researchers.
  • the plaque ( Ishige okamurae ) is a kind of brown algae with narrow leaves, thick skin layers and pointed peaks, belonging to the Ishigeaceae family, and inhabiting rocks in the upper and middle low tide areas of the rough open coast.
  • the present inventors have made intensive studies to overcome the above-mentioned conventional problems, and as a result, isolate the phlorotannin compound from the plaque and investigate the antioxidant activity thereof, thereby leading to the present invention.
  • an object of the present invention is to isolate and purify the phlorotannin compound having various physiological activities from l seaweed ( Ishige okamurae ).
  • the object of the present invention as described above is to separate three phlorotannins from the plaque by column chromatography, and 2,2-diphenyl-1-picrylhydrazil by electron spin resonance (ESR) spectroscopy.
  • ESR electron spin resonance
  • DPPH superoxide, hydroxyl, and alkyl radicals were analyzed for free radical scavenging activity and those by ROS in cell systems including 2 ', 7'-dichlorofluorescein diacetate (DCFH-DA) fluorescent probes. It was achieved by confirming the inhibitory effect on the antioxidant activity of myeloperoxide (MPO) activity and membrane protein oxidation.
  • MPO myeloperoxide
  • the present invention provides an antioxidant active pharmaceutical composition
  • the plaque-derived phlorotannin compound according to the present invention is characterized in that it is selected from the group consisting of phloroglucinol, difluoroethoxy hydroxycarmalol or 6,6'-bieckol of the following formulas (1) to (3).
  • the food composition as a plaque derived from the plaque tannin compound according to the present invention as an active ingredient may be added to give antioxidant activity to health supplements, special nutritional products, beverages and alcohol, food additives, sweets or bread, etc.
  • the present invention is not limited thereto, and may be used for various other foods.
  • the cosmetic composition containing the plaque derived from the plaque according to the present invention as an active ingredient may vary depending on the type of cosmetic.
  • Cosmetic composition according to the present invention means a cosmetic composition that exhibits antioxidant activity, including a plaotannin compound derived from the plaque, and may be in various forms, for example, liquid, cream, paste or solid phase, The present invention is not limited thereto and may be used in various other forms of cosmetics.
  • the term "active ingredient” refers to a substance or a group of substances (a medicinal agent whose pharmacologically active ingredient is not known, etc.) which is expected to express the efficacy or effect of the drug directly or indirectly by inherent pharmacological action. It means containing a main component).
  • the plaquetannin compound derived from the plaque according to the present invention exhibited scavenging activity against DPPH radicals in a dose dependent manner, and 6,6′-bieckol showed the strongest DPPH scavenging activity.
  • 6,6′-bieckol and difluoroethoxycarmalol show significant potential to scaveng hydroxyl radicals even at low concentrations, confirming that they are effective hydroxyl radical scavengers.
  • all of the phlorotannin compounds according to the present invention showed an alkyl radical scavenging activity, and the scavenging activities were 6,6′-bieckol> difluoroethoxycarmalol> fluoroglucinol.
  • the radical scavenging activity of 6,6'-bieckol and difluoroethoxycarmalol is significantly greater than that of phloroglucinol, which indicates that the amount of hydroxyl groups attached to the benzene ring It actually contributed to the radical scavenging activity. Since the radical scavenging activity between 6,6'-bieckol and difluoroethoxycarmalol is not very different, more phlorotannins with different amounts of benzene rings and hydroxyl groups were analyzed by various antioxidant assays. It would be necessary to study the globule activity of phlorotannin.
  • the antioxidant activity in the cell system of the plaque tannin compound derived from the present invention did not show any toxic effect on the MRC-5, RAW264.7, and HL-60 cell line. It was found that the antioxidant activity of tannins is due to the direct elimination of cellular ROS, and thus they can be potent antioxidant compounds that can inhibit cellular ROS formation, effectively inhibiting the oxidation of membrane proteins, It was also found that it could protect proteins in RAW264.7 cells from induced oxidation and also dose-dependently inhibited MPO activity.
  • plaque-derived phlorotannin compound according to the present invention has excellent radical scavenging activity and antioxidant activity in cellular systems, marine source-derived phlorotannin is used as a preventive or therapeutic drug for functional food, dietary supplement or various ROS-related diseases. There is an effect available.
  • FIG. 1 is a graph showing free radical scavenging activity of a plaque tannin compound derived from the plaque according to the present invention by an ESR spectrometer, (A) DPPH scavenging activity, (B) hydroxyl radical scavenging activity, (C) alkyl radical scavenging activity , (D) superoxide scavenging activity, and each value was expressed as mean ⁇ SD in three replicate experiments.
  • Figure 2 is a graph showing the cytotoxic effect of the plaque derived from the plaque according to the present invention (A) MRC-5 cells, (B) RAW246.7 cells, (C) HL-60 cells, each value is Three replicates were shown as mean ⁇ SD.
  • Figure 3 is a graph showing the intracellular radical scavenging activity of L-derived phlorotannin compound according to the present invention for RAW264.7 cells at concentrations of (A) 5, (B) 10, (C) 50 ⁇ M, ROS level is Expressed as DCF fluorescence, each value is expressed as mean ⁇ SD in 3 replicates.
  • Figure 4 is a graph showing the inhibitory effect of the plaque-derived phlorotannin compound according to the present invention on cell membrane protein oxidation in RAW246.7 cells, each value is represented by the mean ⁇ SD in three replicates. *, P ⁇ 0.05 compared to control; ** p ⁇ 0.01.
  • FIG. 5 is a graph showing the inhibitory effect of the plaque-derived phlorotannin compound according to the present invention on the MPO activity in HL-60 cells, each value is represented by the mean ⁇ SD in three replicates. *, P ⁇ 0.05 compared to control; ** p ⁇ 0.01.
  • the l I. okamurae was collected in October 2007 on the coast of Busan, South Korea.
  • the sample was washed three times with tap water to remove salts, parasitic discharges, and sand from the surface of the sample.
  • the samples were air dried, ground in a coffee grinder and powdered and stored in a -20 freezer until use.
  • DPPH 5,5-Dimethyl-1-pyrroline- N -oxide (DMPO), FeSO 4 , H 2 O 2 , 2,2-azobis- (2-amidinopropane) -hydrochloride (AAPH), R -(4-pyridyl-1-oxide) -N - tert -butylnitron (4-POBN), DCFH-DA, R-tocopherol, and epicalocatechin gallate (EGCG) are available from Sigma Chemical Co. ; St. Louis, Mo.).
  • Human fetal lung fibroblast line MRC-5, mouse macrophage cell line RAW264.7, and human leukemia cell line HL-60 were obtained from the American Type Culture Collection (Manassas, VA). Cell culture medium and other materials required for the culture were obtained from Gibco BRL, Life Technologies (USA). All other reagents used the highest grades commercially available.
  • Plaque powder 500 g was extracted three times with 2 L of methanol at room temperature. After evaporating off the solvent, the extract (96 g) was resuspended in H 2 O and subsequently partitioned with hexane, dichloromethane, EtOAc, and n- BuOH. EtOAC-soluble fraction (22 g) was column chromatographed on silica gel to give hexane (4 L), a mixture of hexane and ethyl acetate (20: 1, 10: 1, 5: 1, 1: 1, v / v, each).
  • Fraction 17 of the fractions was further chromatographed on silica gel and Sephadex LH-20 to give Compound 1 (142 mg).
  • Fraction 20 was separated on silica gel and eluted with chloroform-methanol-water (6: 1: 0.1 v / v / v) to give five subfractions (Fr.20.A to Fr.20.E).
  • Fraction 20.C was further purified on Sephadex LH-20 to give Compound 2 (24 mg).
  • Compound 3 was obtained by further purifying fraction 25 on silica gel and Sephadex LH-20.
  • each compound was characterized by 1 H NMR, 13 C NMR, and mass spectrometry (MS).
  • MS mass spectrometry
  • the structure of the phlorotannin according to the present invention is phloroglucinol (compound 1), difluoroethoxy hydroxycarmalol (compound 2) and 6,6'-bieckol ( Compounds 3) and their spectral data were similar to those previously reported. 6,6'-bieckol was best isolated from the plaque, a brown algae.
  • ESR is known to be the most useful spectrometer that can accurately measure the level of radicals remaining in the reactants. ESR is widely used as the most useful method for measuring radical species due to its convenience, high sensitivity and short term consumption. Therefore, the antioxidant activity of phlorotannin was first measured by free radical scavenging including DPPH, superoxide, hydroxyl, and alkyl radicals using an ESR spectrometer. The radical scavenging activity of phlorotannin is shown in FIG. 1. The EC 50 of phlorotannin is defined as the required concentration at which 50% of the radicals produced by the reaction system are scavenged, summarized in Table 1.
  • DPPH radicals are stable organic free radicals, which can be reduced to non-radical form DPPH by reaction when accepting electrons or hydrogen in the presence of a hydrogen-donating antioxidant. It is widely used for screening anti-radical activity because it can prepare a large number of samples in a short time and is sensitive enough to detect the active ingredient even at low concentration.
  • DPPH radical scavenging activity was measured using the method of Nanjo et al. 30 ⁇ L of phlorotannin solution (or ethanol itself as a control) was added to 30 ⁇ L of DPPH (60 ⁇ M) in ethanol solution. After vigorous mixing for 10 seconds, the solution was transferred to a 100 ⁇ L quartz capillary tube and the scavenging activity of phlorotannin on DPPH radicals was measured using a JESFA ESR spectrometer (JEOL, Tokyo, Japan). After exactly two minutes the spin adduct was measured on an ESR spectrometer.
  • A is the relative peak height of the radical signal using the sample
  • a 0 is the relative peak height of the radical signal without using the sample.
  • Percent scavenging activity was plotted against sample concentration to obtain EC 50 values.
  • DPPH scavenging activity of the three phlorotannins obtained by the above method is shown in Figure 1A. All phlorotannins showed a dose-dependent manner in eliminating DPPH radicals. As expected, however, florglucinol exhibited no scavenging activity against DPPH radicals at low concentrations and only scavenged 42.9% of DPPH radicals even at concentrations of 400 ⁇ M, which scavenging activity was statistically significant compared to the control. ( P > 0.05). The EC 50 of 6,6′-bieckol and difluoroetohydroxycarmalol was 9.1 and 10.5 ⁇ M, respectively.
  • Example 2-2 Analysis of hydroxyl radical scavenging activity
  • the hydroxyl radical (HO.) Is the most reactive ROS, which can react quickly with most biological molecules and may be involved in the pathology of many human diseases. Hydrogen radicals were generated in the Fenton system and HO was identified because of the ability to form nitroxide byproducts from commonly used DMPO spin traps. The spin trap has greater oxygen-centric radical trapping capability than other nitron spin traps. By-product DMPO-OH radicals exhibit a characteristic ESR reaction, which can be detected by an ESR spectrophotometer.
  • Iron-catalyzed Fenton Haber-Weiss reaction produced hydroxyl radicals, and the resulting hydroxyl radicals were reacted rapidly with nitrone spin trap DMPO.
  • the generated DMPO-OH byproduct was detected with an ESR spectrometer.
  • the phlorotannin solution (20 ⁇ L) was mixed with DMPO (0.3 M, 20 ⁇ L), FeSO 4 (10 mM, 20 ⁇ L), and H 2 O 2 (10 mM, 20 ⁇ L) in phosphate buffer solution (pH 7.4). , Transferred to 100 ⁇ L quartz capillary tube. After 2.5 minutes, ESR spectra were recorded using an ESR spectrometer.
  • Experimental conditions were as follows: magnetic field, 336.5 ⁇ 5 mT; Power, 1 mW; Modulation frequency, 9.41 GHz; Amplitude, 1 ⁇ 1000; Cleaning time, 4 min. Scavenging activity was calculated by the following formula.
  • A is the relative peak height of the radical signal using the sample
  • a 0 is the relative peak height of the radical signal without using the sample.
  • Alkaline radicals were generated by AAPH according to the method of Hiramoto et al. Briefly, 20 ⁇ L of 40 mM AAPH was mixed with 20 ⁇ L of phosphate-buffered function (PBS) and 20 ⁇ L of test sample at the indicated concentration. The mixture was vortexed and incubated at 37 to 30 minutes. The reaction mixture was then transferred to a sealed capillary tube and the spin byproducts were recorded under controlled spectroscopic conditions: modulation frequency, 100 kHz; Microwave power, 10 mW; Pyrowave frequency, 9441 MHz; Magnetic field, 336.5 ⁇ 5 mT; Cleaning time, 30 s. Alkyl radical scavenging ability was calculated by the following formula.
  • A is the relative peak height of the radical signal using the sample
  • a 0 is the relative peak height of the radical signal without using the sample.
  • Alkyl radicals were formed by decomposing AAPH, a water soluble peroxyl radical inhibitor, which was then trapped with 4-POBN and the spin adduct formed with ESR was measured according to the method described above. All test samples were observed for scavenging activity in a dose-dependent manner (FIG. 1C). All phlorotannins showed alkyl radical scavenging activity, and the scavenging abilities of 6,6'-bieckol, difluoroethoxycarmalol and phloroglucinol were 88.1%, 84.6%, and 42.6% at 80 ⁇ M concentrations, respectively. It was.
  • the EC 50 of 6,6′-bieckol, difluoroethoxycarmalol, phloroglucinol and EGCG were 17.3, 20.4, 20.8, and 110.8 ⁇ M, respectively.
  • the alkyl radical scavenging ability of these phlorotannins was in the order of 6,6'-bieckol>difluoroethoxycarmalol>EGCG> phloroglucinol, but 6,6'-bieckol, difluoroethoxy
  • the difference between carmalol and EGCG was not significant ( p > 0.05).
  • Superoxide radicals are formed early in the oxidation reactions of Chene and, despite being weak oxidants, they can be converted into strong and dangerous hydroxyl radicals in the presence of metals such as iron and copper, leading to cell damage and ROS-related diseases. have.
  • Superoxide radicals were generated by the UV-irradiated riboflavin / EDTA system.
  • the reaction mixture containing 0.3 mM riboflavin, 1.6 mM EDTA, 800 mM DMPO, and the test sample at the indicated concentrations was irradiated for 365 minutes at 365 nm under a UV lamp.
  • the reaction mixture was transferred to a 100 ⁇ L quartz capillary tube on an ESR spectrometer for measurement.
  • A is the relative peak height of the radical signal using the sample
  • a 0 is the relative peak height of the radical signal without using the sample.
  • 1D shows the superoxide radical scavenging activity of various concentrations of phlorotannin and EGCG. All of these showed strong superoxide radical scavenging activity in a dose-dependent manner, but EGCG used as a positive control showed the largest superoxide radical scavenging activity. The results were found to be statistically significant compared to the control group ( p ⁇ 0.05).
  • the EC 50 of 6,6′-bieckol, difluoroethoxycarmalol, EGCG, and phloroglucinol were 14.7, 16.2, 9.5, and 128.6 ⁇ M, respectively.
  • the superoxide radical scavenging activity of these phlorotannins and EGCG was in the order of EGCG>6,6'-bieckol>difluoroethoxycarmalol> phloroglucinol.
  • phloroglucinol showed very poor scavenging activity against DPPH and hydroxyl radicals, it showed strong scavenging activity against alkyl and superoxide radicals.
  • Phlorotannins mainly derived from brown, have various biological activities such as antioxidant, antiallergic, antitumor, antibacterial and enzyme inhibition. Although the antioxidant activity of phlorotannin has been widely studied, to the best of our knowledge, there has been little research on the antioxidant activity of phlorotannin in cellular systems.
  • the antioxidant activity of plaque derived phlorotannin was measured in the cellular system to investigate whether phlorotannin affects free radical-mediated oxidation in the cellular system.
  • RAW 264.7 was used to study the direct ROS scavenging effects of two cell lines, phlorotannin, and membrane protein oxidation inhibition, and HL-60 was used to study the MPO inhibitory effects of phlorotannin. These cells are commonly used to study ROS-mediated cell phenomena because they can produce large amounts of ROS after stimulation.
  • MRC-5 and RAW264.7 cells two anchor-dependent cell lines, respectively, were cultured in Dulbecco's modified Eagle's medium (DMEM), and HL-60 cells, suspension-dependent cell lines were prepared using RPMI medium (Roswell). Park Memorial Institute medium), 10% heat-unsulfated fetal calf serum, penicillin (100 U / mL), and streptomycin (100 ⁇ g / mL) in 37, 95% air and 5% CO 2 environments. was supplemented and the medium was changed daily.
  • DMEM Dulbecco's modified Eagle's medium
  • HL-60 cells suspension-dependent cell lines were prepared using RPMI medium (Roswell). Park Memorial Institute medium), 10% heat-unsulfated fetal calf serum, penicillin (100 U / mL), and streptomycin (100 ⁇ g / mL) in 37, 95% air and 5% CO 2 environments. was supplemented and the medium was changed daily.
  • Intracellular ROS production was measured using the DCFH-DA method.
  • RAW264.7 cells incubated in fluorescent microtiter 96-well plates were labeled with 20 mM DCFH-DA in Hank balanced salt solution (HBSS) for 20 minutes in the dark. Cells were then treated with different concentrations of phlorotannin and incubated for an additional hour, cells were washed three times with PBS and then 500 mM H 2 O 2 was added.
  • a Genios fluorescence microplate reader was used to detect fluorescence signal intensity in a time-dependent manner with an excitation wavelength of 485 nm and emission wavelength of 530 nm. Treatment effects were plotted and compared to the fluorescence intensities of the control and blank spheres.
  • phloroglucinol did not reduce ROS production at all test concentrations. Since the cells were preincubated with 6,6′-bieckol and difluoroetohydroxycarmalol and removed after incubation, their activity can only occur intracellularly, and the result of the present invention is that these phlorotannins Has been shown to be due to direct elimination of cellular ROS, and thus they can be potent antioxidant compounds that can inhibit cellular ROS formation.
  • the conventional method was improved to analyze the amount of membrane protein carbonyl groups.
  • RAW264.7 cells collected by centrifugation were washed with PBS and eluted with 20 mL of elution buffer (25 mM Tris-HCl pH 7.8, 2 mM EDTA, 180 mM NaCl, 1% Triton X-100) without reducing agent. I was. Eluates were aliquoted into microtubes (0.5 mL) and incubated for 37 to 30 minutes with samples of the indicated concentrations. Then 100 ⁇ L of 0.1 M FeSO 4 and 100 ⁇ L of 2 mM H 2 O 2 were added and the mixture was incubated at 37 for 1 hour.
  • the solubilized protein (1 mg) was precipitated by centrifugation. The supernatant was decanted and the pellet resuspended in 150 ⁇ L of 0.2% 2,4-dinitrophenyl hydrazine in 2 mM HCl and left at room temperature for 40 minutes. The reaction mixture was vortexed every 10 minutes to facilitate reaction with the protein. The protein was precipitated again with 20% trichloroacetic acid and the pellet washed three times with ethanol / ethyl acetate (1: 1 v / v) solution. The pellet was then dissolved in 500 ⁇ L of 6 N guanidine hydrochloride and incubated at 37 for 15 minutes.
  • Blanks were prepared by a parallel procedure using 2 mM HCl alone instead of 2,4-dinitrophenyl hydrazine agonist.
  • the carbonyl group content is expressed in nmol (mg / mg of protein) per mg of protein using a molar absorption coefficient of 22 000 M ⁇ 1 cm ⁇ 1 .
  • Protein oxidation by ROS plays an important role in the pathological mechanisms of various diseases. Attacks of ROS on proteins result in modifications of amino acid side chains such as lysine, arginine, proline and histidine, which then produce carbonyl residues (primarily aldehydes and ketones), identified as initial marker concentrations for protein oxidation and protein damage Used to measure Oxidative damage of the membrane also increases membrane fluidity, impairs intact appearance, and inactivates protein-binding receptors and enzymes.
  • the inhibitory effect of phlorotannin on RAW264.7 cell membrane protein oxidation was investigated as described above. As shown in FIG. 5, when the mouse macrophage membranes were exposed to HO. Produced by the Fe 2+ -H 2 O 2 Fenton reaction, the degree of membrane protein oxidation, as indicated by the increase in carbonyl group content, was observed. Is increased.
  • MPO is a leukocyte-induced heme peroxidase and has long been considered as a microbial enzyme that is centrally bound to a nonspecific immune defense system. MPO plays an important role in oxidant production by polymorphonuclear neutrophils (PMNs). As the most powerful oxidant, hydrogen peroxide ((H 2 O 2 ) and chloride were used to catalyze the production of hypochlorous acid (HOCl), which contributes to the killing of microorganisms and subsequent oxidative damage to host tissues, triggering serious inflammatory diseases. Therefore, the search for a compound for inhibiting MPO activity is an important attempt to regulate the ROS-mediated oxidation of biomolecules in neutrophils. .
  • PMNs polymorphonuclear neutrophils
  • Myeloperoxidase a marker enzyme of leukocytes, is believed to indicate the migration of leukocytes to damaged cells.
  • the amount of MPO released from neutrophils stimulated by the improved o -diinishin method was measured.
  • HL-60 cells were suspended in RPMI without phenol red and FBS and seeded in 96-well plates. Cells were preincubated with samples of varying concentrations for 30 minutes and then stimulated with TNF-R (0.05 ⁇ g / mL) for 37 to 30 minutes.
  • the absorbency of the control at 460 nm was very high compared to the group pretreated with 6,6′-bieckol and difluoroetohydroxycarmalol, indicating that the control's MPO activity was increased.
  • MPO activity was dose-dependently inhibited by pretreatment with 6,6′-bieckol and difluoroetohydroxycarmalol and only 55.3% of the control at concentrations of 10 and 50 ⁇ M. , 57.4% and 32.8%, 34.1% were observed, from which it can be assumed to act in an indirect manner as an intracellular antioxidant.
  • the inhibitory effect on MPO activity between 6,6′-bieckol and difluoroetohydroxycarmalol was nearly identical ( p ⁇ 0.05). However, phloroglucinol showed very weak activity against the inhibition of MPO compared to the control.
  • Difluoroethoxycarmalol and 6,6'-bieckol directly inhibited intracellular ROS as evident by DCFH-DA analysis.
  • Membrane protein oxidation and MPO activity were dose-dependently inhibited by pretreatment with difluoroethoxycarmalol and 6,6′-bieckol.
  • MRC-5 human fetal lung fibroblast cell line
  • RAW264.7 mouse macrophage cell line
  • HL-60 human leukemia cell line
  • MPO myeloperoxide
  • ROS reactive oxygen species
  • DMEM Dulbecco's modified Eagle's medium
  • PBS phosphate-buffered saline

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Abstract

La présente invention concerne des compositions antioxydantes alimentaires, cosmétiques ou pharmaceutiques contenant un composé dériivé d'Ishige okamurae comme principe actif. Etant donné que le composé phlorotannine dériivé d'Ishige okamurae présente une excellente activité d'interception radicalaire et une excellente activité antioxydante dans un système cellulaire, la phlorotannine d'origine marine peut être utilisée comme aliment fonctionnel, complément alimentaire, ou médicament destinés à prévenir ou traiter des maladies liées à ROS.
PCT/KR2009/005353 2008-10-07 2009-09-21 Composition antioxydante active contenant un composé dériivé d'ishige okamurae WO2010041832A2 (fr)

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KR10-2008-0098328 2008-10-07
KR1020080098328A KR20100039104A (ko) 2008-10-07 2008-10-07 패 유래 화합물 함유 항산화 활성 조성물

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