KR20090043656A - An antioxidant composition comprising extracts of hippocampus kuda - Google Patents

Abstract

The invention hippocampus ( Hippocampus kuda) that on the antioxidant composition containing the extract, the hippocampus (Hippocampus by preparing the extract from kuda) and measuring the DPPH radicals, hydroxyl radicals, superoxide radicals and hydroxyl radical scavenging activity of the extract of pepper hippocampus (Hippocampus kuda ) has an excellent effect of providing an antioxidant composition containing an extract.

Hippocampus, Hippocampus kuda, Hippocampus kuda, Radical oxide, Free radical species, ROS, Superoxide anion radical, Hydroxyl radical, Superoxide anion, Hydrogen peroxide

Description

An antioxidant composition comprising extracts of Hippocampus kuda}

The invention hippocampus ( Hippocampus kuda.) as that of the antioxidant composition containing the extract, more particularly the hippocampus (Hippocampus kuda ) relates to the use as an antioxidant that can be provided by separating the extract and examining the antioxidant activity of the extract.

The generation of reactive oxygen species in cells and damage to enzymes, nucleic acids, membranes, and proteins that are cellular components of reactive oxygen species (ROS) are associated with cytotoxicity and carcinogenicity of chemical carcinogens (Kehrer, 1993; Aruoma, 1994). ).

Free radicals and other active oxygen species such as superoxide anion radicals (O2 ·-), hydroxyl radicals (OH ·), superoxide anions (O2-˙), and activated oxygen for non-free radicals It has many forms and is often caused by biological reactions or external factors.

In vivo, some of these reactive oxygen species have positive functions such as energy production, phagocytosis, regulation of cell growth, and regulation of intracellular signals, but on the one hand damage a wide range of essential biomolecules (Farber, 1994).

Various endogenous antioxidant mechanisms play a major role in the peroxidation of reactive oxygen species and lipids and protect cells from the toxicity caused by peroxidation of reactive oxygen species and lipids (Halliwell, 1991).

These defense mechanisms are based on antioxidant enzymes such as superoxide dismutase (SOD), glutathione peroxidase, catalase, glutathione, vitamin C and vitamin E (Fridovich, 1999), but antioxidant systems are less effective at pathological conditions. Altered, resulting in inefficient removal of over-produced free radicals that injure tissue (Aruoma, 1994 Halliwell, 1994).

Artificially synthesized antioxidants have limited adverse effects as carcinogens, and natural antioxidants prevent the body from free radicals and slow the progression of many chronic diseases as well as the rancidity of lipids in food and drugs. Because it plays a major role (Zheng, and Wang, 2001; Kinsella et al., 1993; Lai et al., 2001), the use of naturally occurring antioxidants in food or pharmaceuticals as a replacement for artificially synthesized antioxidants in recent years. There is increasing interest in.

The hippocampus (Hippocampus kuda.) Is a tibial fish of the ocean, according to the Chinese medical book according to the Yin-Yang theory, the hippocampus is selected to strengthen the kidneys and supplement the yang, and the reinforcing function is essential for the control of the genitourinary system. And nerves, hormonal immune system is said to have a big relationship.

It is therefore an object of the present invention is to find a new use of the extract of the active oxygen species and removal of the radical to the hippocampal function (Hippocampus kuda.), Hippocampus (Hippocampus with antioxidant properties to provide an use of an extract of kuda .) as an antioxidant.

The object of the present invention is to obtain an extract from the hippocampus kuda., To investigate the reducing capacity of the extracts, to investigate the antioxidant activity, to remove the free radicals, the viability of the cell and the active oxygen species in the cell It was achieved by measuring the effect on the DNA damage prevention activity by the hydroxyl radical.

The present invention provides an antioxidant composition comprising an extract isolated from hippocampus kuda. As an active ingredient.

In particular, the extract is to provide an antioxidant composition, characterized in that any one selected from the group consisting of water extract, methanol extract, ethanol extract, propane extract and butanol extract.

The present invention, the antioxidant composition is characterized in that it is used for the prevention and treatment of wounds, arthritis, inflammation, wrinkles, whitening, diabetes, gastric ulcers, trauma, burns, periodontal disease or arteriosclerosis.

In another aspect, the present invention is to provide an antioxidant composition, characterized in that the powder, tablets, capsules, powders, ointment composition, solution, gel, paste, patch or granules.

Antioxidant composition of the present invention can be used in cosmetics, food, medicine.

The present invention provides an antioxidant for the composition containing the hippocampus (Hippocampus kuda.) To have on the antioxidant composition containing the extract isolated from, isolated from the hippocampus (Hippocampus kuda.) That has the ability to eliminate active oxygen species extract Has an excellent effect.

Hereinafter, the present invention will be described in detail by way of examples, but the scope of the present invention is not limited only to these examples.

Example 1 : Preparation of Water Extract, Alcohol Extract of Hippocampus and Determination of Phenolic Content in Extract

In September 2006, fresh seahorses were purchased from Zhushan, China, and RAW264.7, MRC-5, and U937 cell lines were obtained from the American Type of Culture Collection (Manassas, VA, USA).

DMEM medium (Dulbecco's Modified Eagle's Medium), RPMI 1640 medium, trypsin-EDTA, penicillin 10,000 U / ml, streptomycin 10,000 ug / ml, amphotericin 2,500 ug / ml and fetal bovine serum (FBS) were obtained from Gibco BRL.

MTT reagent (3- (4,5-dimethyl-2-yl) -2,5-diphenyltetrazolium bromide), phoroglucinol, DMPO reagent (5,5-dimethyl-1-pyrroline-N-oxide) 4 -POBN reagent (α- (4-pyridyl-1-oxide) -N-tert-butylnitrone), AAPH reagent (2,2`-azobis (2-amidinopropane) dihydrochloride), and Folin-Ciocalteu reagent are listed in Sigma Chemical Co. (St. Louis, MO, USA). DCFH-DA (2 ', 7'-dichlorofluorescin diacetate) and monobromoobimane were purchased from Molecular probes (Eugene, OR, USA). Other chemicals and reagents used were commercially available in analytical grade.

Alcohol extraction solvents that can be used for the extraction of hippocampus kuda. Include a lower alcohol solvent having 1 to 4 carbon atoms such as methanol, ethanol, propanol, butanol, and the like, and preferably ethanol is used.

In order to obtain a water extract, 50 g of dried hippocampus ( Hippocampus kuda.) Was put in a grinder, finely pulverized, and mixed with 500 ml of boiling water in a stirrer. The extract was filtered by Whatman No. 41, and the filtrate was lyophilized by freezing at minus 50 degrees.

The methanol and ethanol extract was obtained by first weighing 50 g of barley powder from which fat was removed and then putting it in a 500 ml bottle. 200 ml of methanol (v / v) and 95% ethanol (v / v) were added to each bottle. After 4 hours of extraction at 50 ° C., the supernatant and precipitate were separated by vacuum filtration. The residue was extracted again in the same manner as the above extraction and the resulting solution was dried at 40 ° C. in a vacuum evaporator. The weight and yield of the dried extracts were calculated, and the antioxidant extracts were stored in the dark at 4 ° C. for later analysis.

The phenolic component of the extract by lyophilization was determined by Folin-Ciocalteu's reagent (FCR), 0.5 ml of each sample was mixed with 2.5 ml Folin-Ciocalteau reagent (diluted 1:10, v / v), followed by Na ₂ Mix with CO ₃ (2.5%, v / v) solution. Absorbance was measured at 750 nm after incubation at 30 ° C. for 90 minutes and the results were expressed in gallium acid equivalents (mg gallium acid / g dried extract).

Yield (%) Total Phenolic Content (mg / g) Water extract (SHW) 16.2 ± 0.56 10.99 ± 1.25 Methanol Extract (SHM) 6.9-0.35 17.43 + 1.30 Ethanol Extract (SHE) 4.7-0.26 15.71 ± 1.27

The yield of extract was highest in water (16.2%, w / w), followed by methanol (6.9%, w / w) and ethanol (4.7%, w / w). The total phenolic content was the lowest at 10.99 mg / g in water extract, 17.43 mg / g in methanol and 15.71 mg / ml in ethanol.

Folin-Ciocalteu phenolic reagent was used to determine the amount of natural phenolic compounds present in the extract. The total amount of phenol obtained in the extract shows the high antioxidant activity of the extract. A large amount of phenolic compounds exhibits strong free radical scavenging activity, and this scavenging activity is expressed through chelate reactivity with hydrogen or electron donor material and metal ions.

Example  2: reduction capacity investigation

Reducing ability of seahorse extract was determined by Oyaizu method. That is, various extracts and standard compounds (Vitamin C and α-tocopherol) contained in 1 ml of distilled water were mixed with 2.5 ml of phosphate buffer solution (0.2 M, pH 6.6) and 2.5 ml of red blood salt. The mixture was stored at 50 for 20 minutes in water. And 2.5ml TCA solution (10%, w / v) was added. And the mixture was centrifuged for 10 minutes at 3000G. Aliquots of the upper layer of 2.5 ml were mixed with 2.5 ml of distilled water and 0.5 ml of ferric chloride solution, and the absorbance was measured at 700 nm. The increase in absorbance of the mixture was associated with strong reducing power.

Figure 1 shows the reduction ability of the hippocampus extract compared to vitamin C and α-tocopherol. Fe ^{3 +} -Fe ^{2 +} conversion was observed in the presence of hippocampus extract by Oyaizu method. In Figure 1, the reduction ability of the three hippocampus extracts (SHW, SHE, SHM) increased as the amount of the sample increased, methanol extract (SHM) of the hippocampus was higher than the other extracts. The reducing ability of seahorse extract and standard compound was as follows. Vitamin C> Methanol extract of hippocampus (SHM)>α-tocopherol> Ethanol extract of hippocampus (SHE)> Water extract of hippocampus (SHW).

The reducing capacity of the compound is indicative of the potential antioxidant capacity. However, the antioxidant capacity of antioxidants is due to various mechanisms, among them, which prevents chain start, prevents catalysis of metal ions, decomposes peroxide, inhibits continuous hydrogen separation, and reduces the ability to remove and radical. .

Example 3 : Measurement of Antioxidant Activity

Antioxidant activity was measured by a linoleic acid model system by Osawa and Namiki (1985) method. That is, 1.3 g of the sample was dissolved in 10 ml of 50 mM phosphate buffer (ph 7.0), and a solution containing 0.13 ml of linoleic acid solution and 10 ml of 99.5% ethanol was added thereto. The total volume was then adjusted to 25 ml with distilled water. The mixture in a closed capped flask was stored at 40 ± 1 ° C in a dark place and the degree of oxidation was measured by ferric thiocyanate. 100 μl of the cultured reaction solution of the linoleic acid model was mixed with 4.7 ml of 75% ethanol, 0.1 ml of 30% ammonium thiocyanate, and 0.1 ml of 2 × 10 ^-2 M ferrous chloride solution with 3.5% HCl. After 3 minutes, the value of thiocyanate was measured by reading the absorbance at 500 nm, and the color change of FeCl ₂ and thiocyanate was measured at different intervals during incubation at 40 ± 1 ° C.

Total antioxidant activity of the hippocampus with water, methanol and ethanol extracts was determined by the ferric thiocyanate (FTC) method, which was determined by measuring the peroxide level in the initial lipid oxidation state. Peroxide is produced during the oxidation of linoleic acid, the last ions are complexed with SCN ^- and the complex shows maximum absorbance at 500 nm.

The antioxidative activity of linoleic acid of various seahorse extracts is shown in FIG. 2. After a 7 day incubation period, the production of peroxide stopped. The intermediate also converted to a stable final product as the oxidation of iron stopped. Therefore, low absorbance indicates high antioxidant activity. The results indicate that extracts extracted from all other solvents have efficient antioxidant activity. After 7 days, methanol extract was 84.0% and ethanol extract was 80.3%, showing higher activity than α-tocopherol. And water extract showed a positive effect as 70.4%. In this model system, peroxyl radicals (ROO ') and alkoxy radicals (RO') derived from existing lipid peroxidation are directly involved in initiating lipid peroxidation in the emulsified linoleic acid system. Phenolic compounds provide the function of preventing peroxidation of lipids by stopping the chain reaction through the removal of lipid-derived radicals (R ', RO' or ROO ') from the heterogeneous lipid layer.

Example  4: ESR Measured by Free radical  Removal active

Spin addition product was recorded by JES-FA ESR spectrometer. Radical removal activity was calculated using the following equation.

1. DPPH radical scavenging activity

DPPH radical scavenging activity is described by Nanjo et al. It was measured by the method described by (1996). 30 μl of extract solution (30 μl of ethanol for control) was added to 30 μl of 60 μM DPPH in ethanol solution. After much mixing for 10 seconds, the solution was transferred to 100 μl capillary quartz tube. Inhibitory activity against DPPH radicals was measured by JES-FA ESR spectrometer (JEOL Ltd., Tokyo, Japan). The spin adduct was measured after 2 minutes by an ESR spectrometer. The magnetic field was measured at 336.5 ± 5 mT, power at 5 mW, modulation frequency at 9.41 GHz, amplitude at 1 x 1000, and sweep time of 30 seconds. Removal activity of the DPPH radical was calculated according to the above equation (1).

 DPPH is a stable free radical that accepts electrons or hydrogen radicals to form stable diamagnetic molecules. Thus, DPPH often acts as a substrate to measure the antioxidant activity of natural antioxidants. 3A shows the ESR spectrum of DPPH when treated with various concentrations of seahorse extract. In addition, the elimination activity of the hippocampus extract increases at a concentration of 10-600 ug / ml. Among the extracts, methanol extract (SHM) of hippocampus showed the highest DPPH removal activity.

Increasing phenolic compounds was found to increase radical scavenging activity. The previous salping studies showed a positive relationship between DPPH radical scavenging activity and total phenolic compounds. Phenolics are effective antioxidants for polyunsaturated fatty acids, which can easily convert hydrogen atoms to lipid peroxyl radicals and form aryloxyl. Aryloxyl, along with other radicals, inactivates radical chain carriers, preventing the radical reaction of radicals.

2. hydroxyl radical scavenging activity

The hydroxyl radicals are produced by the iron catalyzed Fenton Haber-Weiss reaction and the resulting hydroxyl radicals react rapidly with the nitrone spin trap DMPO. DMPO-OH adduct was measured by ESR spectrometer. 20 ul of the extract was mixed with 0.3 M 20 μl DMPO, 10 mM 20 μl FeSO _{4 ,} 10 mM 20 μl H ₂ O ₂ and phosphate buffer solution (pH 7.4) and then transferred to 100 μl capillary quartz tubes. After 2.5 minutes, the results were recorded using the ESR spectrum, and the experimental conditions were as follows. The magnetic field was 336.5 = 5 mT, power 1 mW, modulation frequency 9.41 GHz, amplitude 1> 200 and sweep time 4 minutes. The removal activity of the hydroxyl radical was calculated according to the above equation (1).

Hydroxyl radicals are important free radical species that can cause significant biological damage, which triggers peroxidation of lipids. The hydroxyl radicals generated from Fenton system _{^{(Fe 2 + / H 2 O}} 2) is trapped by the DMPO DMPO was added and spin to produce water and is observed by the ESR spectrometer. ESR signal is suppressed by OH remover and competes with DMPO for OH.

 As shown in Figure 3b, the concentration-dependent (10-400 ug / ml) showed hydroxyl radical scavenging activity, the hippocampal methanol extract (SHM) showed the highest scavenging activity compared to other extracts. These results demonstrate that hippocampal methanol extract (SHM) and hippocampal ethanol extract (SHE) have a removal activity of hydroxyl radicals and also chelate iron metal ions.

Hydroxyl radicals are extremely reactive oxygen species and have the ability to modify all molecules of living cells. Hydroxyl radicals cause significant damage to DNA and cause cancer formation, mutations, and cytotoxicity. Furthermore, hydroxyl radicals can quickly begin the onset of lipid peroxidation from unsaturated fatty acids, and Figure 6 shows the protective effect of methanol extract (SHM) and ethanol extract (SHE) of hippocampus against DNA damage. This higher activity is attributable to the Fe ^{2 +} chelation it via the Fenton reaction has the effect of delaying the generation of hydroxyl radicals.

3. Removal activity of superoxide anion radical

Superoxide anion radicals are produced by the UV irradiated riboflavin / EDTA system. A reaction mixture of 0.3 mM riboflavin 20ul, 1.6mM EDTA 20ul, 800mM DMPO 20ul, and 20ul extract was irradiated with a UV lamp of 365 nm wavelength for 1 minute. The reaction mixture was then transferred to 100 μl ESR spectrometer quartz capillary tube and the experimental conditions were as follows. Magnetic field 336.5 = 5 mT, power 10 mW, modulation frequency 9.41 GHz, amplitude1> 1,000, sweep time 1 minute. The removal activity of the superoxide anion radical was calculated using Equation 1 above.

Most hippocampus extracts in Figure 3c removed hydroxyl radicals. And the removal activity increased with increasing concentration of the extract.

Superoxide anion radicals are also known to be very detrimental to cell constituents and are known as precursors of reactive oxygen species. Singlet oxygen, with the potential to react with biological macromolecules, has therefore been associated with tissue damage and the onset of oxidation in the aging process.

4. Removal Activity of Peroxyl Radicals

Peroxyl radicals are described in Hiramoto et al. (1993) measured by method. 20 μl of 40 mM AAPH (2,2'-azobis (2-amidinopropane) dihydrochloride) is 20 μl of PBS (hosphate buffered-saline) and 20 μl of 40 mM 4-POBN (α- (4-pyridyl-1-oxide)- N- tert- butylnitrone) and 20 μl of hippocampus extract solution, which was then incubated at 37 ° C. for 30 minutes. The reaction mixture was then transferred to a sealed capillary and the spin adduct was recorded under controlled spectrometric conditions. Modulation frequency is 100 kHz; Microwave power 10 mW; Microwave frequency 9441 MHz, magnetic field 336.5 = 5 mT sweep time was 30 seconds. The ability to remove peroxyl radicals was calculated using Equation 1 above.

Peroxyl radical spin byproducts were observed when AAPH was incubated with spin trap 4-POBN for 30 minutes at 37 ° C. A decrease in ESR signal was observed with dose increment.

In FIG. 3 d, SHM in the extract was shown to have high alkyl radical removal activity. Radicals in the center of the carbon take the form of R, RO and ROO- and they have been removed from the extract of the hippocampus at different concentrations. These experimental results are in agreement with the results of the inhibition of fat peroxide by the removal of lipid-derived radicals.

Example  5: Determination of Cell Culture and Viability

The leukocyte cell lines RAW264.7 and MRC-5 contained 2 mM glutamine, 100 μg / ml penicillin / streptomycin, and 5% CO _2. In humid conditions at 37 ° C., each was grown in DMEM medium containing 10% fetal calf serum, and cytotoxicity was determined by MTT (3- (4,5-dimethyl-2-yl) -2,5-diphenyltetrazolium bromide) method. Was measured. Cells were grown to a density of 5> 10 ³ cells / well in 96-well plates and after 24 hours cells were washed with fresh stock and treated with different concentrations of solvent extracts. After 48 hours of incubation, the cells were washed again and 100 ul of MTT (1 mg / ml) was added and finally cultured for 4 hours.

150 ul of DMSO was added to dissolve the formazan salt and the formazan salt was measured at 540 nm using a GENios ^® microplate reader. The relative viability of the cells was determined from the measurement of the conversion of MTT to formazan salt. Surviving cells are expressed in percent in relation to the control and represented by the following equation.

 Data are listed as mean values from at least three experiments and considered significant at P <0.05.

The effect on the cytotoxicity of the hippocampus was evaluated using the rat leukocytes RAW 264.7 and MRC-5. As shown in Figure 4, the hippocampus extract showed some cytotoxic effects in MRC-5 and RAW264.7 cells. Seemed.

Example  6: DCFH - DA Cell activity by Oxygen species  decision

The formation of reactive oxygen species in erythrocytes was measured using DCFH-DA (2`, 7`-dichlorofluorescin diacetate), an oxidation-sensitive dye. RAW264.7 cells grown in fluorescent micro96-well plates were loaded in HBSS with 20 uM DCFH-DA and incubated in the dark for 20 minutes. Non-fluorescent DCFH-DA staining easily passed through the cells and was hydrolyzed by intracellular esterase to become DCFH (2 ′, 7′-dichlorofluorescin) and trapped within the cells. The cells were then treated with different concentrations of test chemicals and incubated for 1 hour. Cells were then washed three times with PBS, and 300 μM H ₂ O ₂ dissolved in HBSS was added to the cells.

Formation of DCF (2`, 7`-dichlorofluorescin) due to oxidation of DCFH due to the presence of reactive oxygen species was observed in active wavelengths using a microplate reader every 30 minutes at a 485 nm emission wavelength of 535 nm. Measured. The effect of amount and time dependence of the test group was prepared by comparing the fluorescence intensity of the control group. According to Fig. 5, the fluorescence by DCF results in DCFH time course increment of 20 minutes as a result of DCFH oxidation of reactive oxygen species. Fluorescence was reduced. Seahorse extract exhibited radical removal efficiency. After 30 minutes at a concentration of 10 μg / ml, the seahorse extract more clearly removed the radicals during the total incubation time at the concentration of 100 μg / ml. These results indicate that the extract of hippocampus has the function of preventing it from oxidative damage of cells by reactive oxygen species.

Example  7 Genomic DNA  Isolation and of Seahorse Extract Hydroxyl To radicals  by DNA  Prevention of damage

High molecular weight DNA of the genome was extracted from U937 cells by standard phenol / proteinase potassium process, cells cultured in 10 cm dishes were washed twice with PBS with 5 mM EDTA and after centrifugation of the cells. RNase 0.5 mg / ml, sodium acetate 0.2 M, proteinase K 10 mg / ml and SDS (10%), the mixture was incubated at 37 ° C. for 30 minutes and at 55 ° C. for 1 hour. Incubated. The conditions were as follows. Phenol: chloroform: isoamyl alcohol (25: 24: 1) was added at a ratio of 1: 1. The mixture was centrifuged at 4 ° C. for 5 minutes at 12,000 G. The supernatant was mixed with 100% ice cold ethanol at a ratio of 1: 1.5 and kept at -20 ° C for 15 minutes. Small aggregates were dissolved in TE buffer and purely isolated DNA was measured by spectrophotometer under conditions of 260/280 nm. Furthermore, the quality of the isolated DNA was evaluated by 1% agar electrophoresis in 0.04 M tri-acetate-0.001 M EDTA buffer. To study the protective activity against DNA damage by hydroxyl radicals of the extract, 3 μl of 50 mM phosphate buffer solution (pH 7.4) containing 0.5 μl of DNA and 3 μl of 2 mM FeSO ₄ The reaction was carried out in a total volume of 12 μl Eppendorf tube containing 2 μl of extracts of various concentrations. And after adding 4μl of 30% H ₂ O _{2 The} mixture was incubated for 30 minutes at 37 ° C. The mixture was subjected to 0.8% agar gel electrophoresis and DNA bands were stained with ethidium bromide.

All data were measured as mean ± standard deviation and the mean was calculated from independent experiments using fresh prepared reactants on at least three different days each.

From the above results, it is thought that the extract of Hippocampus kuda can be used as a potential candidate for preventing damage caused by reactive oxygen species as antioxidant.

As described above, the hippocampus of the present invention ( Hippocampus) as described above. Antioxidants, characterized in that the extract of kuda .) contains the active ingredient is a very useful invention in the food, cosmetics, and pharmaceutical industry because it provides a high antioxidant effect.

Figure 1 shows the reducing capacity of water extract (SHW), ethanol extract (SHE), methanol extract (SHM), vitamin C, α-tocopherol (50-200 ug / ml) isolated from the hippocampus.

2 is a diagram showing the antioxidant activity of the hippocampus extract by the ferricthiocyanate method in the linoleic acid emulsion and the antioxidant activity of α-tocopherol.

Figure 3 shows the removal activity of free radicals at various concentrations of seahorse extract. a is DPPH radical scavenging activity, b is hydroxyl radical scavenging activity by trapping of hydroxyl radical by DMPO according to Fenton reaction, c is superoxide radical scavenging activity by DMPO trap of superoxide radical , d is a diagram showing the removal activity by the trap by 4-POPN of the alkyl radical produced by AAPH.

Figure 4 shows the cell affinity (A) of the extract for CyRAW 264.7 cells and the viability (B) of the cells when treated with different concentrations of extract for 24 hours in the case of MRC-5 cells.

5 is a diagram showing the removal activity of the cell radical in 10 ug / ml extract (a). 20 ug / ml extract (b), 50 ug / ml extract (c) 100 ug / ml extract (d). RAW 264.7 cells were marked by nontoxic fluorescence staining (DCFH-DA) and treated with different concentrations of hippocampal extract. The fluorescence intensity by DCF according to the oxidation of DCFH by ROS produced by H ₂ O ₂ of cells was observed independent of time (λ _excitation = 485 nm and λ _excitation = 528 nm). The effect of ROS removal on the hippocampus extract was H ₂ O ₂ Three independent experiments were performed compared to the unstimulated blank and no treatment.

FIG. 6 shows the results of electrophoresis of DNA damaged by OH production by Fenton reaction in the presence of methanol extract (SHM) and ethanol extract (SHE) of hippocampus. 0.5 ul of DNA contains FeSO ₄ and 30% H ₂ O ₂ And incubated at 37 to 30 minutes with the addition of a pesticide compound at the back, and Line 1 was without any additives (DNA from U937 cells); Line 2 is added FeSO ₄ and H ₂ O ₂ (DNA damage control); Lines 3-5 show FeSO ₄ and H ₂ O ₂ additions at 1, 10 and 50 ug / ml methanol extracts from hippocampus, and Lines 6-8 show 1, 10 and 50 ug / ml ethanol extracts at hippocampus. The result at the time of addition of FeSO ₄ and H ₂ O ₂ is shown.

Claims (4)

Antioxidant composition characterized in that it contains an extract of hippocampus (Hippocampus kuda.) As an active ingredient. The composition of claim 1, wherein the extract is any one selected from the group consisting of water extract, methanol extract, ethanol extract, propane extract and butanol extract. The composition of claim 1, wherein the antioxidant composition is used for the prevention and treatment of wounds, arthritis, inflammation, wrinkles, whitening, diabetes, gastric ulcer, trauma, burns, periodontal disease or atherosclerosis. According to claim 1, wherein the antioxidant composition is an antioxidant composition, characterized in that powder, tablets, capsules, powders, ointment composition, solution, gel, paste, patch or granules.

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