KR20100039104A - An anti-oxidant active composition containing compounds from ishige okamurae - Google Patents

Abstract

PURPOSE: Antioxidant active compositions containing compounds from Ishige okamurae is provided to offer novel anti-allergy activity to compounds from Ishige okamurae, and to prevent and cure diseases. CONSTITUTION: Antioxidant active compositions containing compounds from Ishige okamurae are used as chemicals for treating or preventing various ROS-related diseases. A fluoro tannin compound is selected from a group consisting of phloroglucinol, diphlorethohydroxycarmalol, or 6,6'- bieckol.

Description

An anti-oxidant active composition containing compounds from Ishige okamurae}

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.

A group of highly reactive molecules, reactive oxygen species (ROS), are free radicals such as superoxide anions (O ² .-), hydroxyls (HO., Peroxyl (ROO.), Alkoxyl ( RO ·), and nitric oxide, and 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. Despite their important role, they damage organisms when they are overproduced by attacking lipids, proteins, enzymes, carbohydrates and DNA, which can lead to oxidative stress and imbalance between ROS and defense and restorative antioxidant systems. .

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.

On the other hand, they are highly toxic and cytotoxic oxidants at the pathological level, resulting in cell damage and death. More than 100 diseases are associated with ROS. 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 are widely studied 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 seaweed with narrow leaves, thick skin layer, and sharp peaks, and belongs to the Ishigeaceae, which inhabits rocks in the upper and middle low tide areas of the rough open coast.

The antibacterial effect of the plaque extract on the dental caries cause has been reported, and the separation and purification of phospholipase A2 activity inhibitor from the shell extract.

To date, few studies have been conducted on the antioxidant activity of components derived from them except for some studies on biological characteristics such as the antifouling activity and the cytotoxic effect of the algae.

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.

Therefore, an object of the present invention is to isolate and purify the phlorotannin compound having various physiological activities from the brown seaweed ( Ishige okamurae ).

It is also an object of the present invention to provide a novel anti-allergic activity use of the phlorotannin compound derived from 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. DPPH), superoxide, hydroxyl, and free radical scavenging activity on alkyl radicals were analyzed 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.

The present invention provides an antioxidant active pharmaceutical composition comprising a phlorotannin compound derived from plaque (Ishige okamurae) as an active ingredient.

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).

<Formula 1>

Floroglucinol

<Formula 2>

Difluoroethoxy hydroxycarmalol

<Formula 3>

6,6'-bieckol

In the present invention, 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 nutrition products, beverages and alcohol, food additives, sweets or bread, etc. However, the present invention is not limited thereto, and may be used for various other foods.

In the present invention, 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.

In the present invention, 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 medicine 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. In addition, 6,6′-bieckol and difluoroethoxycarmalol show significant potential to scaveng hydroxyl radicals even at low concentrations, confirming that they are effective hydroxyl radical scavengers. In addition, 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. On the other hand, in the superoxide radical scavenging activity, phloroglucinol showed a strong scavenging activity with respect to alkyl and superoxide radicals, although it showed very inferior scavenging activity with respect to DPPH and hydroxyl radicals.

Previous studies suggest that the radical scavenging activity of polyphenols such as flavonoids is due to phenolic acid hydroxyl groups attached to the benzene ring, and the structure-activity relationship of flavonoids has been studied using various antioxidant assays. Although the structure-activity relationship of phlorotannin is not clear, it can be said that the phenolic acid hydroxyl group attached to the ecchol backbone plays an important role in radical scavenging activity. In the present invention, it has been found that 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.

On the other hand, as a result of analyzing the antioxidant activity in the cell system of the plaque tannin compound derived from the present invention, it 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.

These results, showing radical scavenging activity and antioxidant activity in the cellular system of phlorotannins isolated from plaques, suggest that marine sources-derived phlorotannins may be used for the prevention or treatment of functional foods, dietary supplements or various ROS-related diseases. It can be suggested as a drug.

Since the plaque-derived phlorotannin compound according to the present invention has excellent radical scavenging and antioxidant activity in a cellular system, marine source-derived phlorotannin is a functional food, dietary supplement or drug for the prevention or treatment of various ROS-related diseases. There is an effect available.

Hereinafter, preferred embodiments of the present invention will be described in more detail with reference to Examples. However, the scope of the present invention is not limited to these examples.

Example 1 Extraction and Separation of Phlorotannin from Plaque

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. Finally, the samples were air dried, ground in a coffee grinder and powdered and stored in a -20 freezer until use.

Silica gel 60 (0.036-0.2 mm, Merck), Sephadex LH-20 (GE Healthcare Bioscience AB, Uppsala, Sweden), and thin layer chromatography (TLC) plates (Kieselgel 60 F254 5715, Merck), respectively It was used for column chromatography and analytical TLC. 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.

The data obtained in the experiments were expressed as mean ± standard deviation of three replicate measurements, and the statistical comparison was based on the Student's t test, with a P of less than 0.05. Values were considered significant.

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). 5 L), dichloromethane (3 L), and dichloromethane and methanol mixtures (10: 1, 5: 1, 1: 1, v / v, 5 L each) in succession eluting with 29 Fractions were obtained (Fr. 1 to Fr. 29).

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). As shown in the following Chemical Formulas 1 to 3, 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.

Floroglucinol (Compound 1) . ¹ H NMR (400 MHz, DMSO- d 6): d 9.02 (s, 3, -1, 3, 5), 5.68 (s, 3, H-2, 4, 6). ¹³ C NMR (100 MHz, DMSO- d 6): d 159.8 (C-1, 3, 5), 95.4 (C-2, 4, 6). EI-MS m / z : 126 [M] + (100), 111 (9), 97 (20), 85 (30), 69 (41), 52 (26).

Difluoroethoxycamalol (Compound 2). ¹ H NMR (400 MHz, DMSO-d6): d 9.29-9.11 (m, 9, OH-1, 2, 6, 8, 2 ', 4', 6 ', 3'',5''), 6.09 (s, 1, H-4), 5.90 (br s, 2, H-3 ', 5'), 5.81 (t, 1, J ) 1.8 Hz, H-4 ''), 5.71 (s, 1, H-9), 5.70 (d, 2, J ) 1.8 Hz, H-2``, 6 ''). <sup>13</sup> C NMR (100 MHz, DMSO- d 6): d 160.2 (C-1``), 159.0 (C-3``, 5 '), 155.0 (C-4'), 151.4 (C-2 ', 6 '), 146.1 (C-3), 143.1 (C-7), 139.7 (C-1), 138.9 (C-4a), 135.2 (C-6), 133.9 (C-9a), 130.8 (C- 5a), 126.4 (C-2), 125.5 (C-8), 124.2 (C-10a), 122.9 (C-1 '), 96.2 (C-4''), 95.1 (C-3', 5 ' ), 94.4 (C-4), 93.8 (C-2``, 6 ''), 92.4 (C-9). FAB-MS m / z : 512 [M] &lt; + &gt;.

6,6'-bieckol (Compound 3). ¹ H NMR (400 MHz, DMSO- d 6): d 9.28 (s, 2, HO-9, 9 '), 9.16 (br s, 6, HO-2, 2', 3 '', 3 ''' , 5 '', 5 '''), 9.09 (s, 2, HO-4, 4'), 8.65 (s, 2, HO-7, 7 '), 6.08 (s, 2, H-3, 3 '), 6.04 (s, 2, H-8, 8'), 5.79 (t, 2, J ) 1.8 Hz, H-4``, 4 '''), 5.74 (d, 4, J ) 1.8 Hz , H-2 '', 2 ''',6'',6'''). <sup>13</sup> C NMR (100 MHz, DMSO- d 6): d 160.4 (C-1``, 1 '''), 158.7 (C-3``, 3''', 5 '', 5 ''') , 151.3 (C-7, 7 '), 145.4 (C-2, 2'), 144.4 (C-9, 9 '), 141.7 (C-4, 4'), 141.3 (C-5a, 5'a ), 137.1 (C-10a, 10'a), 123.5 (C-1, 1 '), 122.6 (C-9a, 9'a), 121.9 (C-4a, 4'a), 99.6 (C-6 '', 6 '''), 97.8 (C-8, 8'), 97.7 (C-3, 3 '), 96.1 (C-4'',4'''), 93.6 (C-2 '' , 2 '', 6 '', 6 '''). FAB-MS m / z : 742 [M] &lt; + &gt;.

Example 2 Determination of Free Radical Scavenging Activity of Phlorotannin by ESR

Currently, 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.

Example 2-1 Analysis of DPPH Radical Scavenging Activity

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 μL of phlorotannin solution (or ethanol itself as a control) was added to 30 μL of DPPH (60 μM) in ethanol solution. After vigorous mixing for 10 seconds, the solution was transferred to a 100 μL 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. Experimental conditions were as follows: magnetic field, 336.5 ± 5 mT; Power, 5 mW; Modulation frequency, 9.41 GHz; Amplitude, 1X000; Cleaning time, 30 s. DPPH radical scavenging ability was calculated according to the following formula.

DPPH Clear Activity (%)) (1- A / A 0) X100

Wherein 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 μM, 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 μM, respectively. In contrast, the EC ₅₀ of R-tocopherol, a positive control, was 45.3 μΜ. Although 6,6'-bieckol showed the strongest DPPH scavenging activity, the difference in scavenging activity between 6,6'-bieckol and difluoroetohydroxycarmalol was not significant ( p > 0.05). ).

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.

The iron-catalyzed Fenton Haber-Weiss reaction produced hydroxyl radicals, and the resulting hydroxyl radicals reacted rapidly with nitrone spin trap DMPO. The generated DMPO-OH byproduct was detected with an ESR spectrometer. The phlorotannin solution (20 μL) was mixed with DMPO (0.3 M, 20 μL), FeSO ₄ (10 mM, 20 μL), and H ₂ O ₂ (10 mM, 20 μL) in phosphate buffer solution (pH 7.4). , Transferred to 100 μL 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.

Scavenging activity (%)) (1- A / A 0) X 100,

Wherein 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.

The inhibitory ability of phlorotannin on the hydroxyl radicals produced by the Fenton method was measured and the results are shown in Figure 1B. 6,6′-bieckol and difluoroethoxycarmalol showed significant potential ( p <0.05) to extinguish hydroxyl radicals at low concentrations of 160 μM and 6,6′-bieckol and di The EC ₅₀ of floretohydroxycarmalol was 28.7 and 27.1 μM, respectively. 6,6' non ekkol and were the de Flor comparing EC ₅₀ of soil hydroxycarboxylic malrol and EC ₅₀ of EGCG (30.6 μM), which non-6,6' ekkol and sat hydroxycarboxylic malrol the de Flor the It is proposed to be an effective hydroxyl radical scavenger. On the other hand, phloroglucinol showed the weakest scavenging activity against hydroxyl radicals, and 800 μM of phloroglucinol only scavenged 67.5% of the hydroxyl radicals.

Example 2-3: Alkyl Radical Scavenging Activity Assay

Alkaline radicals were generated by AAPH according to the method of Hiramoto et al. Briefly, 20 μL of 40 mM AAPH was mixed with 20 μL of phosphate-buffered function (PBS) and 20 μL 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.

Scavenging activity (%)) (1- A / A 0) X 100,

Wherein 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 μM 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 μM, 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).

Example 2-4: Superoxide Radical Scavenging Activity Assay

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 μL quartz capillary tube on an ESR spectrometer for measurement. Experimental conditions were as follows: magnetic field, 336.5 ± 5 mT; Power, 10 mW; Modulation frequency, 9.41 GHz; Amplitude, 1 × 1000; Cleaning time, 1 min. Superoxide scavenging ability was calculated by the following equation.

Scavenging activity (%)) (1- A / A 0) X 100,

Wherein 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 μM, respectively. The superoxide radical scavenging activity of these phlorotannin and EGCG was in the order of EGCG>6,6'-bieckol>difluoroetohydroxycarmalol> phloroglucinol. Although phloroglucinol showed very poor scavenging activity against DPPH and hydroxyl radicals, it showed strong scavenging activity against alkyl and superoxide radicals.

TABLE 1

EC ₅₀ Values of Plaque-derived Phlorotannin Compounds

Example 3 Analysis of Phlorotannin Effect on Free Radical Oxidation in Cellular Systems

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.

In the present invention, 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.

Example 3-1 Cytotoxic Effects of Phlorotannin on MRC-5, RAW264.7, and HL-60 Cell Lines

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 μg / mL) in 37, 95% air and 5% CO ₂ environments. Was supplemented and the medium was changed daily. 3- (4,5-dimethylthiazol-2-yl) -2,5-dibenyl tetrazolium bromide (MTT) assay based on the conversion rate at which MTT is converted to formazan crystals by mitochondrial dehydrogenases Cell viability was measured using. Cells were cultured in microtiter 96-well plates (1.5 × 10 ⁵ cells / well) in serum-free medium and treated with different concentrations of phlorotannin for 24 hours. Phlorotannin was dissolved in 10% dimethyl sulfoxide (DMSO) and filtered through a 0.22 μM filter membrane to sterilize. The final DMSO concentration in the culture medium was not more than 0.1%. 20 μL of MTT staining solution was then added to each well. Four hours after incubation, 200 μL of DMSO solution was added to dissolve formazan crystals, and the absorbance was read at 450 nm using a Genios multifunction microplate reader (TECAN Austria Gmbh, Austria). It was.

As a result of the assay (see FIG. 2), no phlorotannin showed a cytotoxic effect on the cells under test concentration. Even at the highest concentration of 50 μM, only about 5% of cells decreased, and the cell viability of cells treated with varying concentrations of phlorotannin was not significantly different from the control ( p > 0.05).

Example 3-2 Measurement of Intracellular ROS by DCFH-DA

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.

To investigate whether plaque-derived phlorotannin affects intracellular production of ROS, RAW264.7 cells were pre-incubated with 5, 10, and 50 μM of phlorotannin for 1 hour and described above. One was labeled with the fluorescent probe DCFH-DA. When DCFH-DA enters viable cells, it is deacetylated by intracellular esterases to form 2 ', 7'-dichloro-dihydrofluorescein (DCFH), which quantitatively reacts with ROS in cells and fluoresces Adult 2 ', 7'-dichlorofluorescein (DCF) can be produced. As shown in FIG. 3, the fluorescence emitted from DCF following ROS-mediated oxidation of DCFH showed a time course increase up to 210 minutes.

Preculture with 6,6′-bieckol and difluoroetohydroxycarmalol was found to decrease DCF fluorescence dose- and time-dependently. Even after 30 minutes of incubation, 6,6′-bieckol and difluoroetohydroxycarmalol showed significant radical scavenging activity at a concentration of 5 μM. More specifically, at a concentration of 50 μM, 6,6′-bieckol and difluoroetohydroxycarmalol were able to significantly erase ROS throughout the incubation time. After 210 minutes of incubation, 6,6′-bieckol and difluoroetohydroxycarmalol reduced the production of ROS by 78.9% and 77.2% at 50 μM, respectively.

However, 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.

Example 3-3 Membrane Protein Oxidation Assay

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 μL of 0.1 M FeSO _{4 and} 100 μL of 2 mM H ₂ O ₂ were added and the mixture was incubated at 37 for 1 hour. After adding 400 μL of 20% trichloroacetic acid, the solubilized protein (1 mg) was precipitated by centrifugation. The supernatant was decanted and the pellet resuspended in 150 μL 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 μL of 6 N guanidine hydrochloride and incubated at 37 for 15 minutes. After centrifugation at 6000 g for 5 minutes, the absorbency of the supernatant was read for complementary blanks at 370 nm. 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. In the present invention, 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.

About 12 times more carbonyl groups were observed during the oxidative stress induced by the control compared to the blank group. The protein carbonyl groups in the 6,6′-bieckol-treated and difluoroetohydroxycarmalol-treated groups were less in the control.

Pretreatment with 6,6′-bieckol and difluoroetherhydroxycarmalol for 30 minutes inhibited the oxidation of membrane proteins. 6,6′-bieckol and difluoroetohydroxycarmalol can effectively inhibit protein oxidation to 51.8% and 59.1% of the control, respectively. On the other hand, phloroglucinol showed a very weak inhibitory effect only at the highest concentration. These results suggest that 6,6′-bieckol and difluoroetohydroxycarmalol can protect proteins in RAW264.7 cells from ROS-induced oxidation.

Example 3-4: MPO Activity Assay

Finally, the present invention focused on the study of the effect of phlorotannin on the activity of MPO. MPO is a leukocyte-induced heme peroxidase and has long been considered 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. .

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 μg / mL) for 37 to 30 minutes. Cells were then added an assay mixture containing 0.05 mL of TNF-R (0.05 μg / mL) in phosphate buffer (pH 6.0) and 0.05 mL of 0.02 M o -dianisidine (freshly prepared) in deionized water. The amount of MPO released at 460 nm was measured spectrophotometrically, and the inhibition rate of phlorotannin on MPO activity was calculated by the following formula.

% Inhibition = 100 x (1-(absorbance of sample-absorbance of blank) / (absorbance of control-absorbance of blank)

Cells in control and blank were cultured without phlorotannin, and blanks were prepared without TNF-R stimulation.

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. As shown in FIG. 5, 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 μM. , 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.

As a result, three phlorotannins, including phloroglucinol, 6,6'-bieckol, and difluoroethoxycarmalol, were separated from the plaques, which are brown algae, by column chromatography. The structure of phlorotannin was revealed based on NMR and MS data. Among the separated phlorotannins, difluoroethoxycarmalol and 6,6'-bieckol have strong free radical scavenging activity against DPPH, hydroxyl, alkyl, and superoxide radicals as measured by ESR. Indicated. 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. These results, showing radical scavenging activity and antioxidant activity in the cellular system of phlorotannins isolated from the plaque, indicate that marine sources-derived phlorotannin is a drug for the prevention or treatment of functional foods, dietary supplements or various ROS-related diseases. It can be suggested.

Abbreviation

ESR, electron spin resonance spectrometry;

DPPH, 2,2-diphenyl-1-picrylhydrazyl;

MRC-5, human fetal lung fibroblast cell line;

RAW264.7, mouse macrophage cell line;

HL-60, human leukemia cell line;

DCFH-DA, 2 ', 7'-dichlorofluorescein diacetate;

MPO, myeloperoxide;

ROS, reactive oxygen species;

BHA, Butylated Hydroxyanisole;

BHT, butylated hydroxytoluene;

TLC, Thin Layer Chromatography

DMPO, 5,5-dimethyl-1-pyridine N -oxide;

AAPH, 2,2-azobis- (2-amidinopropane) -hydrochloride;

4-POBN, R- (4-pyridyl-1-oxide) -N - tert -butylnitron;

EGCG, epigallocatechin gallate;

DMEM, Dulbecco's modified Eagle's medium;

RPMI, Roswell Park Memorial Institute medium;

MTT, 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide; DMSO, dimethyl sulfoxide;

HBSS, Hank's balanced salt solution;

PBS, phosphate-buffered saline.

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) 50μM, 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.

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 repeated experiments. *, P <0.05 compared to control; ** p <0.01.

Claims (4)

Antioxidant active food composition comprising a plarotannin compound derived from l (Ishige okamurae) as an active ingredient. Antioxidant active cosmetic composition comprising a plarotannin compound derived from Ishige okamurae as an active ingredient. Antioxidant active pharmaceutical composition comprising a plarotannin compound derived from l (Ishige okamurae) as an active ingredient. The composition of claim 1, wherein said phlorotannin compound is selected from the group consisting of phloroglucinol, difluoroethoxyhydroxycarmalol or 6,6'-bieckol.

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