KR101094733B1 - Composition for improving adultdisease or anti-aging comprising natokinase and royal jelly - Google Patents

Abstract

The present invention provides a composition comprising nattokinase and royal jelly. The composition of the present invention is excellent in antioxidant, anti-aging, and adult disease inhibiting effect, and specifically, has an effect of inhibiting the anti-aging, arteriosclerosis, hypertension, stroke, angina, and heart disease by the antioxidant, etc.

Natokinase, Royal Jelly, Anti-Aging, Adult Disease Suppression

Description

Composition for improving adultdisease or anti-aging comprising natokinase and royal jelly}

The present invention relates to a composition comprising nattokinase and royal jelly, and more particularly, to a composition having an antioxidant action, an anti-aging action and an adult disease suppression effect.

Aging is a gradual change in organs or organisms over time and can continue as long as life is alive. As human lifespan is extended, efforts are being made to slow down the aging process in terms of quality of life.

Free radicals are known as one of the causes of aging. In other words, oxygen free radicals target not only lipids of tissue cells but also proteins and DNA, resulting in lipid peroxide (LPO) and oxidized protein (OP) as a result of oxidative stress. (Freeman, BA and Crapo, JD Biology of disease: Free radical and tissue injury. Lav. Invest. 47: 412-426, 1982 .; Cross, CE, Halliwell, B., Borish, ET, Pryor, WA, Ames, BN, Saul, RA, McCord, JM and Harman, D. Oxygen radicals and human disease.Annn.Intern.Med. 107: 526-545, 1987 .; Southorn, PA and Powis, G Free radiocal s in medicine.II.Involvement in human disease.Mao Clin.Proc . 63: 390-408, 1988 .; Yu, BP Oxidative damage by free radicals and lipid peroxidation in aging.In: Yu, BP, ed. Free radicals In Aging.Boca Taton, CRC Press : 57 ~ 88, 1993.)

Therefore, in order to exhibit an anti-aging action, it is necessary to develop a substance or a composition excellent in antioxidant activity.

In addition, geriatric disease refers to diseases such as arteriosclerosis, hypertension, stroke, angina pectoris, and heart disease that occur as an adult, and adult diseases are the cause of death of most adults except in developing countries and developing countries.

Neutral lipid and cholesterol are known to affect the development of adult diseases such as atherosclerosis, myocardial infarction, hypertension, angina pectoris, stroke, and diabetes. Low density lipoprotein (LDL) -cholesterol and high density lipoprotein (HDL) -cholesterol. Among these cholesterols, cholesterol causing adult disease is LDL-cholesterol, whereas HDL-cholesterol is known as an anti-cholesterol factor that suppresses adult disease.

Therefore, there is a need for the development of substances or compositions that reduce the production of neutral lipids, LDL-cholesterol and the like, and activate the production of HDL-cholesterol in order to suppress adult diseases.

Natokinase is a type of serine proteolytic enzyme extracted from a traditional Japanese food called natto. It has strong thrombolytic ability and pro-urokinase activation. Recent studies have reported that nattokinase in Cheonggukjang is effective in cardiovascular diseases by breaking down blood clots. In addition, nattokinase has been studied as an effective thrombosis treatment for stroke, myocardial infarction, thrombosis, etc., and is known as a thrombolytic enzyme that dissolves fibrin.

Royal Jelly, also known as wangyu, is an oily substance that is secreted by the development of a lacquer as a carapaceous vessel only when worker bees raise larvae. The substance has an unusual scent, and when it comes into contact with air, the active ingredient is changed to decrease the efficacy. It contains 20-30% protein, 15% carbohydrate, 1-15% fat, 50-60% moisture, and it is rich in various kinds of vitamins.

However, until now, no studies on anti-aging and adult disease inhibition by the combined use of nattokinase and royal jelly have been found, and the present inventors have completed the present invention as a result of research on anti-aging and inhibiting adult disease.

The problem to be solved by the present invention is the anti-aging action and It is to provide a composition excellent in the effect of inhibiting adult disease.

The present invention provides a composition comprising nattokinase and royal jelly.

The composition of the present invention is an antioxidant and It has an anti-aging effect, and the anti-aging effect is due to antioxidant activity.

Therefore, the composition of the present invention is preferably an antioxidant composition or an anti-aging composition.

In addition, the composition of the present invention has an inhibitory effect on adult diseases such as atherosclerosis, hypertension, stroke, angina, or heart disease, the adult disease is a disease associated with triglycerides and cholesterol, the "adult disease suppression" is to improve, treat, Or prevention.

Therefore, the composition of the present invention is preferably a composition for inhibiting adult disease.

The composition of the present invention can reduce geriatric lipids, total cholesterol, and LDL-cholesterol levels and increase HDL-cholesterol levels associated with geriatric diseases, as in the examples described below, thereby inhibiting geriatric diseases.

Accordingly, the present invention provides an antioxidant method, an anti-aging method, comprising the use of an antioxidant for preparing the composition of the present invention, an anti-aging agent, or a preparation for an adult disease inhibitor, and administering the composition of the present invention to a mammal including a human. Or it provides a method for inhibiting adult disease.

Preferably, the nattokinase: royal jelly of the composition of the present invention is 1: 2 to 4 by weight.

The composition of the present invention in the weight ratio range is excellent in geriatric inhibitory effect, antioxidant effect and anti-aging effect.

The nattokinase may be prepared using a known fermentation method using Bacillus subtilis or the like, or extracted from NATO, or a commercially available one may be used. Commercially available products may be used in powder form.

The nattokinase can be used to have a variety of activities, such as 10000FU, or 20000FU depending on the purpose of use. “FU” stands for fibrinolytic unit.

The royal jelly can be taken directly from nature, or commercially available ones can be used, and powdered ones can be used.

The formulation of the composition of the present invention can be used as tablets, capsules, pills, powders, suspensions, solutions or granules.

The composition of the present invention can be used as a pharmaceutical composition or a food composition.

The food composition includes a health functional food composition.

The composition of the present invention may be formulated to include one or more pharmaceutically or food-pharmaceutically acceptable carriers in addition to the active ingredient for administration.

Pharmaceutically or foodly acceptable carriers may be used in combination with one or more of saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and these components, if necessary. Other conventional additives, such as a buffer and a preservative, can be added. Diluents, dispersants, surfactants, solvents, disintegrants, sweeteners, binders, coatings, swelling agents, lubricants, lubricants or flavoring agents may additionally be added. Furthermore, it may be preferably formulated according to each disease or component by any suitable method in the art or using methods disclosed in Remington's Pharmaceutical Science (Recent Edition), Mack Publishing Company, Easton PA.

Formulation forms of the pharmaceutical compositions of the present invention may be in the form of granules, powders, tablets, coated tablets, capsules, suppositories, solutions, syrups, juices, suspensions, emulsions, drops or injectable solutions, and sustained release formulations of the active compounds. Can be.

The pharmaceutical composition of the present invention can be administered in a conventional manner via oral or the like depending on the purpose.

The formulation of the food composition of the present invention may be prepared according to a conventional method and may be encapsulated after drying with a carrier or in the form of other tablets, granules, powders, beverages, porridges and the like.

In the composition of the present invention, the dosage of the active ingredient can be adjusted within the range of 1mg / kg ~ 100mg / kg per day weight 60kg adult basis, preferably 10mg / kg. However, the optimal dosage to be administered can be easily determined by those skilled in the art, and the type of disease, the severity of the disease, the amount of active ingredients and other ingredients contained in the composition, the type of formulation, and the age, weight, general health of the patient It may be adjusted according to various factors including the condition, sex and diet, time of administration, route of administration and the rate of secretion of the composition, the duration of treatment, and the drugs used simultaneously.

The content of the active ingredient in the composition of the present invention may be contained in an amount of 1 to 100% by weight based on the total weight of the composition.

The compositions of the present invention can be administered alone or in combination with natural products or compounds that have been found to have antioxidant, anti-aging or geriatric effects.

The composition of the present invention is excellent in anti-oxidation, anti-aging, adult disease suppression effect, specifically, anti-aging, arteriosclerosis, hypertension, anti-aging due to antioxidant, etc., excellent in inhibiting the adult disease, such as stroke, angina pectoris and heart disease.

Advantages and features of the present invention and methods for achieving them will be apparent with reference to the embodiments described below in detail. However, the present invention is not limited to the embodiments disclosed below, but may be implemented in various forms. It is provided to fully convey the scope of the invention to those skilled in the art, and the present invention is defined only by the scope of the claims.

Example 1 Preparation of Nattokinase and Royal Jelly Compositions

Natokinase powder (BIO SCIENCE LABORATORY CO., LTD; JAPAN) and royal jelly powder (Tuhsu Dongfang Imp. & Exp, Corp; China) were mixed in a weight ratio of 1: 3.

Comparative Example 1 Preparation of Nattokinase

The same powder as the nattokinase powder of Example 1 was prepared.

<Comparative Example 2> Preparation of Royal Jelly

The same powder as the royal jelly powder of Example 1 was prepared.

<Materials and Methods Used in Examples and Comparative Examples>

Materials and methods commonly used in the examples of the present invention are as follows.

1. Reagents and Other Materials

Natokinase and royal jelly were used in Example 1, starch and DL-methionine were purchased from Deoksan (Korea), casein and choline chloride were sigma. Purchased in the USA, lard was purchased from Samyang Corp., corn oil was purchased from Cheil Jedang (Korea), and cellulose was purchased from Daejeong (Korea). The vitamin and mineral mix (AIN-76) was purchased from ICN Co. (USA), and the cholesterol and sodium cholate (sodium cholate) were purchased from Sigma (USA). All other reagents used in the experiments were Sigma (US) reagents and special reagents.

2. Experimental Animals and Breeding Conditions

SPF SD rats (Sam taco, male, 220 ± 10 g) were pre-divided for two weeks in the animal feeding room and divided into four groups of 10 rats.

For each group, the experimental preparations each containing the composition of Example 1, the powder of Comparative Example 1, and the powder of Comparative Example 2 were administered, and only one preparation was administered to one group as a control. Natokinase and royal jelly powder 10.0 + 30.0 mg / day / object for the composition administration group of Example 1, Natokinase powder 10.0 mg / day / individual for the powder administration group of Comparative Example 1, Royal for the powder administration group of Comparative Example 2 Feed amount was adjusted to be administered to jerry powder 30.0mg / day / individual. The total administration period was 10 weeks.

The animal breeding room is automatically controlled with a 12 hour cycle (06:00 ~ 18:00) under constant temperature and humidity (22 ± 2 ℃, 65 ± 2% RH).

3. Experimental feed

The composition of the experimental feed is shown in Table 1 below, and cholesterol and sodium cholate in the feed were added to induce hypercholesterolemia in experimental animals.

Furtherance
(Weight ratio) Example 1
Administration group Comparative Example 1
Administration group Comparative Example 2
Administration group Control group Nattokinase 0.04 0.04 - - Royal Jelly 0.12 - 0.12 - carbohydrate
(Starch + sugar) 57.59
(45.0 + 12.59) 57.71
(45.0 + 12.71) 57.63
(45.0 + 12.63) 57.75
(45.0 + 12.75) Protein (casein) 18.0 18.0 18.0 18.0 Geology
(Rad: corn oil = 2: 1) 15.0 15.0 15.0 15.0 cellulose 3.0 3.0 3.0 3.0 Vitamin mix 1.0 1.0 1.0 1.0 Mineral mix 3.5 3.5 3.5 3.5 DL-Methionine 0.3 0.3 0.3 0.3 Choline chloride 0.2 0.2 0.2 0.2 cholesterol 1.0 1.0 1.0 1.0 Sodium cholate 0.25 0.25 0.25 0.25

4. Blood Collection and Separation

Treatment of experimental animals was performed by fasting 12 hours before the end of the experiment, leaving the blood collected by the soybeans at room temperature for 30 minutes, and centrifuging for 10 minutes at 700 g for sodium heparin (sigma, 100,000Units) per 1 ml of blood. In a complete blood cell count bottle (CBC, green cross) treated with 0.05ml, frozen and stored at -70 ℃.

5. Statistical Processing of Analysis Results

All experimental results were statistically calculated to calculate the mean and standard deviation, and the significance test between each group was performed by Student's test (Steel, R.G.D. and Torrie, J.H.Principles and procedures of statistics, McGrawhill. New York, 1960).

Example 2 Confirmation of Anti-Aging Activity-Confirmation of Inhibitory Effect of Superoxide Radical Generation in Serum

Free radicals are known to cause degradation of aging-related functions, such as inhibition of enzyme activity, errors in nucleic acid information, impairment of cell membrane function, and accumulation of aging pigments. By measuring the degree of inhibition of the production of superoxide radicals, one of the free radicals in the serum, the antioxidant activity of the test substance and its anti-aging activity can be measured.

In the present Example, in order to determine the inhibitory effect in the serum of the superoxide radicals represented by the composition of Example 1, the amount of superoxide radicals generated in blood for Example 1, Comparative Example 1, Comparative Example 2 administration group and control group Measured.

The production of superoxide radicals was measured by MaCord et al. (McCord, JM and Fridovch, I. Superoxide dismutase.An enzymic function for erythro cuprein (hemocuprein). JB Chem. 6055, 1969 .; Chan, PC and Bielski, BHJ Enzyme catalyzed free radical reactions with nicotinamide adenine nucleotide.According to JB Chem. 249 (4): 1317-1319, 1974.), it is possible to inhibit superoxide dismutase. The reduction rate of ferricytochrome C was measured. That is, 20 mM cyanide solution was added to 420 ul of a phosphate buffer solution (pH 7.8) containing 0.1 mM EDTA and heated at 37 ° C. for 10 minutes. 300ul of serum and 50ul of 0.1mM cytochrome C were added to the solution, and the absorbance was measured at 550 nm over time using a spectrophotometer. At this time, the amount of cytochrome C was calculated with a molecular absorption coefficient of 19500 M ⁻¹ cm ⁻¹ .

The results are shown in Table 2 below.

group Superoxy radical content (nmol / mg protein) Example 1 102.96 ± 4.89 ^** 84.1% ^a Comparative Example 1 117.15 ± 5.21 95.7% Comparative Example 2 115.59 ± 4.93 ^* 94.5% Control group 122.36 ± 7.26 ^b 100.0%

(a: ratio to control, b: mean ± standard deviation, *: p <0.05, **: p <0.01)

As shown in Table 2, the administration group of Example 1 was 84.1% compared to the superoxide radical generation amount of the control group, and it can be said that the effect of inhibiting the superoxide radical generation was 15.9%.

Comparative Example 1 administration group showed 4.3% production inhibitory effect, Comparative Example 2 administration group showed a 5.5% production inhibitory effect.

From the above results, it can be seen that the superoxyradical production inhibitory effect in the administration group of Example 1 was 3.7 times compared to Comparative Example 1 administration group and 2.9 times compared to Comparative Example 2 administration group. That is, when the royal jelly and nattokinase composition is administered, it can be seen that the effect of inhibiting superoxy radical production is superior to that of the single administration alone.

Therefore, it can be seen that the composition of the present invention has an excellent effect of inhibiting the production of superoxy radicals in the blood, and as a result, the composition of the present invention has an excellent antioxidant action, and thus exhibits an anti-aging action.

Example 3 Confirmation of Anti-Aging Activity-Confirmation of Inhibitory Effects of Hydroxy Radical Generation in Serum

Free radicals are known to cause degradation of aging-related functions, such as inhibition of enzyme activity, errors in nucleic acid information, impairment of cell membrane function, and accumulation of aging pigments. By measuring the degree of inhibition of the production of hydroxy radicals, one of the free radicals in the serum, the antioxidant activity of the test substance and its anti-aging effect can be measured.

In the present Example, in order to determine the inhibitory effect in the serum of the hydroxy radicals represented by the composition of Example 1, the amount of hydroxy radicals generated in blood for the administration groups of Example 1, Comparative Example 1, Comparative Example 2, and the control group was measured. .

Hydroxy radical generation is a method of measuring the degree of hydroxyl radical generation to the extent of deoxyribose. Deoxyribose is destroyed by reactive oxygen metabolites to generate aldehydes. Halliwell et al. (Halliwell, B. and Gutteridge, JMC Formation of a thiobarbituric acid-reactive substance from deoxyribose in the persence of iron salts.FEBS . Lett) , 128: 347-350, 1981.). Serum hydroxyl radicals were first measured with 0.1 M phosphate buffer (pH 7.4), 10 mM NaN ₃ , 7 mM deoxyribose, 5 mM ferrousammonium sulfate, 0.54 M NaCl. 33.3 μl of the reagents are added to each sample, and the sample group adds 15 μl of sample and 185 μl of distilled water to 200 μl, and warms the mixed solution in a 37 ° C. constant temperature water bath for 15 minutes. The control group skips the warming process and adds 30 μl of the control sample to the test tube. Add 185 μl of distilled water to all test tubes and mix well. Add 75 μl of 8.1% SDS solution, 500 μl of 20% acetic acid, and 25 μl of distilled water. Add 333 μl of 1.2% TBA solution and mix well. After boiling for 30 minutes and cooled at room temperature, the supernatant obtained by centrifugation at 800 x g for 5 minutes was measured using a spectrophotometer to measure the absorbance at 532 nm. The amount of hydroxy radicals (nmole / mg protein) was calculated.

The results are shown in Table 3 below.

group Hydroxy radical content (nmol / mg protein) Example 1 1.09 ± 0.10 ^* 81.3% ^a Comparative Example 1 1.28 ± 0.31 95.5% Comparative Example 2 1.27 ± 0.28 ^** 94.8% Control group 1.34 ± 0.09 ^b 100.0%

(a: ratio to control, b: mean ± standard deviation, *: p <0.05, **: p <0.01)

As shown in Table 3, the administration group of Example 1 was 81.3% compared to the amount of hydroxy radicals in the serum of the control group, and it can be said that the effect of inhibiting hydroxy radicals was 18.7%.

Comparative Example 1 administration group showed 4.5% production inhibitory effect, Comparative Example 2 administration group showed a 5.2% production inhibitory effect.

From the results, it can be seen that the hydroxy radical production inhibitory effect in the administration group of Example 1 was 4.2 times compared to Comparative Example 1 administration group, 3.6 times compared to Comparative Example 2 administration group. That is, it can be seen that the administration of the royal jelly and nattokinase composition is superior to the hydroxy radical production inhibitory effect as compared to the case of each administration alone.

Therefore, it can be seen that the composition of the present invention is excellent in inhibiting the production of hydroxy radicals in the blood, and as a result, the composition of the present invention has an excellent antioxidant action, thereby exhibiting an anti-aging action.

<Example 4> anti-aging action-inhibited lipid peroxide production in serum

In vivo lipids are cellular components that are susceptible to damage by oxidative stress in vivo, and the production of lipid peroxide is known to be associated with aging of tissues in vivo. Therefore, in this Example, the content of lipid peroxide in the serum of Example 1, Comparative Example 1, Comparative Example 2 administration group, and the control group to determine the anti-aging action of the composition of Example 1 was measured.

The content of lipid peroxide in serum was determined by Choi et al. (Choi, JH and Yu, BP Unsuitability of TBA test as a lipid peroxidation marker due to prostaglandin synthesis in the aging kideny.AGE , 13: 61-64, 1990.) According to the TBA method, malondialdehyde (MDA) content was measured. Take 20 μl of serum and 180 μl of distilled water in each test tube, add 200 μl of 8.1% SDS (sodium dodecyl sulfate) solution for about 5 seconds, add 1.5 ml of 20% acetic acid, and mix again for 5 seconds, 1.2% 1.0 ml of TBA (thiobarbituric acid) reagent was added, plugged with clean beads, and heated in a water bath for 30 minutes. The reaction solution was centrifuged at 800 x g for 10 minutes and the supernatant was measured for absorbance at 532 nm using a spectrophotometer to quantify the content of lipid peroxide (nmole / mg protein) according to a standard calibration curve.

The results are shown in Table 4 below.

group Lipid peroxide (nmol / mg protein) Example 1 1.43 ± 0.12 ^** 86.1% ^a Comparative Example 1 1.58 ± 0.29 95.2% Comparative Example 2 1.61 ± 0.35 ^* 97.0% Control group 1.66 ± 0.18 ^{b **} 100.0%

(a: ratio to control, b: mean ± standard deviation, *: p <0.05, **: p <0.01))

As shown in Table 4, the administration group of Example 1 was 86.3% compared to the amount of hydroxy radicals produced in the serum of the control group, and it can be said that the effect of inhibiting the production of lipid peroxide was 13.9%.

Comparative Example 1 administration group showed a 4.8% production inhibitory effect, Comparative Example 2 administration group showed a 3.0% production inhibitory effect.

It can be seen from the results that the inhibitory effect of lipid peroxide production in the administration group of Example 1 was 2.9 times compared to Comparative Example 1 administration group and 4.6 times compared to Comparative Example 2 administration group. That is, it can be seen that the administration of the royal jelly and nattokinase composition is superior to the lipid peroxide production inhibitory effect compared to the case of administration alone.

Accordingly, it can be seen that the composition of the present invention is excellent in inhibiting lipid peroxide production due to oxidative stress, and as a result, the composition of the present invention has an excellent antioxidative action and thus exhibits an anti-aging action.

<Example 5> anti-aging action-inhibited oxidized protein production in serum

Proteins are present in biological membranes along with lipids to protect cellular organelles, and oxidized amino acid residues are oxidized by free radicals, which are known to cause aging. In this Example, to determine the anti-aging action of the composition of Example 1, the content of oxidized protein in serum for Example 1, Comparative Example 1, Comparative Example 2 administration group, and the control group was measured.

The content of oxidized protein in serum was determined by Levine et al. (Levine, RL, Garland, D., Oliver, CN, Amici, A., Climent, I., Lenz, AG, Ahn, B., Shaltiel, S. and The production of carbonyl groups was measured according to Stadtman, ER Determination of carbonyl content in oxidatively modified proteins.Methods Enzymol . 1986: 464-478, 1990. Mix 0.5 ml of 30% trichloroacetic acid (TCA) in 0.1 ml of sample, centrifuge at 800 × g for 10 minutes, remove supernatant, add 0.5 ml of 10 mM DNPH (dinitrophenyl hydrazine) to residue, 15 Mixing every minute and allowed to stand at room temperature for 1 hour and centrifuged for 10 minutes at 800 × g. After removing the supernatant, 3.0 ml of ethanol / ethyl acetate (v / v 1: 1) was added to the residue, and the mixture was left at room temperature for 10 minutes. After centrifugation at 800 x g for 10 minutes to obtain a residue, 1.0 ml of 6.0 M guanidine (20 mM potassium phosphate buffer, pH 2.3) was added and mixed and warmed in a constant temperature water bath at 37 ° C for 15 minutes. The supernatant was obtained by centrifugation for a minute. The amount of carbonyl group was calculated using a molecular absorption coefficient (Ε = 22,000) at a wavelength between 360 nm and 370 nm.

The results are shown in Table 5 below.

group Oxidized protein content (nmol / mg protein) Example 1 4.83 ± 0.36 ^** 82.4% ^a Comparative Example 1 5.61 ± 0.40 ^* 95.7% Comparative Example 2 5.58 ± 0.34 ^** 95.2% Control group 5.86 ± 0.29 ^{b *} 100.0%

(a: ratio to control, b: mean ± standard deviation, *: p <0.05, **: p <0.01)

As shown in Table 5, the administration group of Example 1 was 82.3% compared to the amount of oxidized protein production in the serum of the control group, and it can be said that the effect of inhibiting the production of oxidized protein was 17.6%.

Comparative Example 1 administration group showed 4.3% production inhibitory effect, Comparative Example 2 administration group showed a production inhibitory effect of 4.8%.

It can be seen from the results that the inhibitory effect of oxidized protein production in the administration group of Example 1 was 4.1 times compared to Comparative Example 1 administration group and 3.7 times compared to Comparative Example 2 administration group. That is, it can be seen that the administration of the royal jelly and nattokinase composition is superior to the inhibitory effect on the production of oxidized protein as compared to the case of administration alone.

Therefore, the composition of the present invention is excellent in inhibiting the production of oxidized protein in the blood by oxidative stress, as a result it can be seen that the composition of the present invention has an excellent antioxidant action, thereby showing an anti-aging action.

Example 6 Anti-Aging Activity-Increasing Superoxide Dismutase Activity in Serum

Superoxide dismutase is a biodefense enzyme that removes superoxide radicals, and has an antioxidant effect due to increased activity of the enzyme, thereby exhibiting anti-aging effect.

In this example, to determine the anti-aging action of the composition of Example 1, the increase in superoxide dismutase activity in the serum of the administration group of Example 1, Comparative Example 1, and Comparative Example 2 was measured for the control group.

Superoxide dismutase (SOD) activity was determined by Oyanagui et al. (Oyanagui, Y. Reevaluation of assay methods and estabishment of Kit for superoxide dismutase activity.Anal . Biochem. 42: 290-296, 1984.) Followed. That is, in 0.1 ml of serum diluted 30 times with phosphate buffer solution (pH 8.2), 0.5 ml of distilled water, 0.2 ml of A reagent (52.125 mg of hydoxylamine + 102.1 mg of hypoxanthine / 250 ml DW), and B reagent (20 μl of xanthine). Add 0.2 ml of oxidase + 0.9939 mg EDTA / 26.7 ml phosphate buffer, pH 8.2) and warm for 40 minutes in a constant temperature water bath at 37 ° C, then C reagent (300 mg of sulfanilic acid + N-1-naphthylethyene diamine acid / 500 ml of 2.0 ml of 16.7% acetic acid) was added to the mixture and allowed to stand at room temperature for 20 minutes, and then absorbance was measured at 550 nm using a spectrophotometer to quantify superoxide dismutase (unit / mg protein) using a standard calibration curve.

The results are shown in Table 6 below.

group Superoxide Dismutase Activity (unit / mg Protein) Example 1 19.44 ± 1.22 ^*** 120.3% ^a Comparative Example 1 17.13 ± 1.37 106.0% Comparative Example 2 17.02 ± 0.28 ^** 105.3% Control group 16.16 ± 0.33 ^{b *} 100.0%

(a: ratio to control, b: mean ± standard deviation, *: p <0.05, **: p <0.01,

 ***: p <0.001)

As shown in Table 6, the composition administered in Example 1 was 120.1% compared to the superoxide dismutase activity in the serum of the control group, and it can be said that the effect of increasing the superoxide dismutase activity by 20.3%.

Comparative Example 1 administration group showed a 6.0% activity increase effect, Comparative Example 2 administration group showed a 5.3% activity increase effect.

From the above results, it can be seen that the superoxide dismutase activity increasing effect in the administration group of Example 1 was 3.4 times higher than that of Comparative Example 1, and 3.8 times higher than that of Comparative Example 2. That is, it can be seen that the superoxide dismutase activity increase effect is excellent when the royal jelly and nattokinase compositions are administered, respectively, compared to the single administration.

Therefore, the composition of the present invention can effectively inhibit superoxide radicals by increasing the activity of superoxide dismutase, thereby reducing oxidative damage by free radicals, and consequently appear to exhibit anti-aging action.

Example 7 Confirmation of Anti-Aging Activity-Effect of Increasing Glutathione Peroxidase Activity in Serum

Glutathione peroxidase is one of the active oxygen scavenging enzymes, and serves to protect the living body by decomposing hydrogen peroxide into alcohol and detoxifying it, thereby exhibiting anti-aging effect.

In this Example, the increase in glutathione peroxidase activity in the serum of the administration group of Example 1, Comparative Example 1, and Comparative Example 2 was measured to determine the anti-aging action of the composition of Example 1.

Glutathione peroxidase (GPx) activity was measured by Lawrence et al. (Lawrence, RA and Burk, RF Species, tissue and subcellular distribution of non Se-dependent glutathione peroxidase activity. Lipid . 19: 444-452, 1978. ) And a method of measuring the reduction of NADPH at 340 nm. That is, the glutathione peroxidase (GPx) activity in serum was measured by diluting the serum 10-fold with phosphate buffer solution (0.3 M / 4.0 mM EDTA). Phosphate buffer in a test tube (0.3 M phosphate buffer with 4.0 mM EDTA, pH 7.2) 0.1 ml, distilled water, 1.295 ml, 26.56 mM sodium azide (sodium azide) solution _{(86.33 mg of NaN 3/50} ml of DW) 0.5 ml, 60 μl of 294.37 mM GSH solution (452.34 mg glutathione / 5.0 ml 0.3M phosphate buffer (4.0 mM EDTA), 110 μl of 8.4 mM NADPH (35.0 mg NADPH / 5.0 ml 0.3M phosphate buffer (4.0 mM EDTA)), glutathione reductase (5 mg of GSH-Re / 1.0 ml 0.3M phosphate buffer (4.0 mM EDTA)) Add 5 μl, 320 μl of 1 mM hydroperoxide and 30 μl of diluted serum, mix well for 5 seconds, and then immediately The absorbance at 340 nm was measured for 2 minutes at 15 second intervals, and the activity of GPx (nmol / min / g protein) was calculated by the standard calibration curve.

The results are shown in Table 7 below.

group Glutathione peroxidase activity (nmol / mg protein / min) Example 1 34.94 ± 2.64 ^*** 122.5% ^a Comparative Example 1 30.34 ± 3.31 ^* 106.4% Comparative Example 2 30.08 ± 2.94 ^** 105.5% Control group 28.52 ± 3.18 ^b 100.0%

(a: ratio to control, b: mean ± standard deviation, *: p <0.05, **: p <0.01, ***: p <0.001)

As shown in Table 7, the composition-treated group of Example 1 was 122.5% compared to glutathione peroxidase activity in the serum of the control group, it can be said that the effect of increasing the glutathione peroxidase activity by 22.5%.

Comparative Example 1 administration group showed a 6.4% activity increase effect, Comparative Example 2 administration group showed a 5.5% activity increase effect.

From the results, it can be seen that the glutathione peroxidase activity increasing effect in the administration group of Example 1 is 3.5 times compared to Comparative Example 1 administration group, 4.1 times compared to Comparative Example 2 administration group. That is, it can be seen that the administration of the royal jelly and nattokinase composition is superior in the effect of increasing glutathione peroxidase activity compared to the case of administration alone.

Therefore, the composition of the present invention can inhibit oxidative damage by detoxifying hydrogen peroxide in active oxygen by increasing activity of glutathione peroxidase, and consequently appears to exhibit anti-aging action.

<Example 8> confirm the effect of inhibiting the adult disease-confirm the effect of reducing the neutral lipid content in the serum

Triglyceride (TG) is known to affect the development of geriatric disease, a chronic degenerative disease such as arteriosclerosis, myocardial infarction, hypertension, angina pectoris, stroke, and diabetes mellitus. It is expected to act to suppress.

In this example, to determine the geriatric inhibitory effect of the composition of Example 1, the degree of decrease of the neutral lipid content in the serum of the administration group of Example 1, Comparative Example 1, and Comparative Example 2 for the control group was measured.

The content of triglyceride (TG) as a neutral lipid in serum was measured using TG kit reagent (triglyceride-V, Eiken, Japan). First, 1.0ul of TG kit reagent is added to 10ul of serum, 10ul of standard solution (300 mg / dl) and 10ul of deionized water with a blank (blank), mixed well, and reacted for 5 minutes in a 37 ° C water bath. As a control, the absorbance was measured at 540 nm to quantify the content of TG (mg / dl serum).

The results are shown in Table 8 below.

group Triglyceride Content (mg / dl Serum) Example 1 174.66 ± 14.78 ^** 82.4% ^a Comparative Example 1 120.59 ± 19.57 ^* 93.8% Comparative Example 2 122.18 ± 18.23 95.1% Control group 28.52 ± 3.18 ^b 100.0%

(a: ratio to control, b: mean ± standard deviation, *: p <0.05, **: p <0.001)

As shown in Table 8, the composition administered in Example 1 was 82.4% compared to the triglyceride content in the serum of the control group, which can be said to have an effect of inhibiting the production of neutral lipids by 17.6%.

It can be said that the comparative example 1 administration group showed 6.2% inhibitory effect, and the comparative example 2 administration group showed 4.9% inhibitory effect.

From the above results, it can be seen that the inhibitory effect of neutral lipid production in the administration group of Example 1 was 2.9 times compared to Comparative Example 1 administration group and 3.6 times compared to Comparative Example 2 administration group. That is, it can be seen that the administration of royal jelly and nattokinase composition is superior to the neutral lipid production inhibitory effect compared to the case of administration alone.

Therefore, the composition of the present invention is expected to act to suppress the adult disease by the effect of inhibiting the production of triglycerides.

Example 9 Confirmation of Inhibitory Effect on Adult Disease-Confirmation of Total Cholesterol Content Reduction Effect in Serum

Cholesterol is known to affect the development of adult disease, a chronic degenerative disease such as arteriosclerosis, myocardial infarction, hypertension, angina pectoris, stroke and diabetes mellitus. It is expected to be inhibitory.

In this example, to determine the geriatric inhibitory effect of the composition of Example 1, the total cholesterol content in the serum of Example 1, Comparative Example 1, and Comparative Example 2 administration group for the control group was measured.

Total cholesterol content in the serum are methods such as Rudel: measured, o -phthaldehyde method according to (Rudel, LL and Morris, MD Determination of cholesterol using o-phthaldehyde J. Lipid Res 14 364 ~ 366, 1973....) It was. First, 0.1 ml of the sample is taken, 0.3 ml of 33% KOH solution and 3.0 ml of 95% ethanol are added and mixed well. The serum is then heated in a 60 ° C. water bath for 15 minutes and then cooled. 5.0 ml of nucleic acid is added thereto, mixed again, 3.0 ml of distilled water is added thereto, then mixed well for 1 minute, and the layers are separated to take 1.0 ml of nucleic acid layer. The nucleic acid layer is concentrated and dried with nitrogen, and 2.0 ml of o- phthaldehyde reagent is added and mixed well. After 10 minutes, 1.0 ml of concentrated sulfuric acid is added and mixed as a coloring reagent. Within 10 to 90 minutes after the addition of sulfuric acid, the absorbance was measured at 550 nm using a spectrophotometer, and the total cholesterol content (mg / dl serum) was quantified by a standard calibration curve.

The results are shown in Table 9 below.

group Total Cholesterol Content (mg / dl Serum) Example 1 179.47 ± 11.75 ^** 82.6% ^a Comparative Example 1 205.73 ± 12.87 ^* 94.7% Comparative Example 2 207.11 ± 11.24 ^* 95.3% Control group 217.28 ± 13.51 ^{b *} 100.0%

(a: ratio to control, b: mean ± standard deviation, *: p <0.05, **: p <0.01)

As shown in Table 9, the composition administered in Example 1 was 82.6% compared to the total cholesterol content in the serum of the control group, it can be said that the effect of reducing the total cholesterol by 17.4%.

Comparative Example 1 administration group can be said to have a 5.3% total cholesterol reduction effect, Comparative Example 2 administration group showed a 4.7% total cholesterol reduction effect.

It can be seen from the results that the total cholesterol reduction effect in the administration group of Example 1 is 3.3 times compared to Comparative Example 1 administration group, 3.7 times compared to Comparative Example 2 administration group. That is, when the royal jelly and nattokinase composition is administered, it can be seen that the total cholesterol reduction effect is superior to that of the single administration alone.

Therefore, the composition of the present invention is expected to act to suppress the adult disease by the effect of reducing the total cholesterol in the blood.

<Example 10> Confirmation of geriatric disease inhibitory effect-Confirmation of low density lipoprotein (LDL) -cholesterol content reduction effect in serum

Cholesterol is known to affect the development of geriatric disease, a chronic degenerative disease such as arteriosclerosis, myocardial infarction, hypertension, angina pectoris, stroke and diabetes mellitus.Low density lipoprotein (LDL)- There is cholesterol. The component having the effect of inhibiting the production of the low density lipoprotein (LDL) -cholesterol is expected to act to suppress the adult disease.

In this example, to determine the geriatric inhibitory effect of the composition of Example 1, the decrease of low density lipoprotein (LDL) -cholesterol content in the serum of Example 1, Comparative Example 1, and Comparative Example 2 administration group was measured.

LDL-cholesterol (BLF, Eiken, Japan) kit was used to measure the serum LDL-cholesterol content.

Add 0.1 ml of serum and 0.1 ml of standard serum to the test tube, add 4.0 ml of BLF kit reagents I and II, mix well for 5 seconds, and distilled water within 10 minutes after standing at room temperature (25 ± 3 ℃) for 25 minutes. As a control, the absorbance was measured at 650 nm to quantify the content of LDL-cholesterol (mg / dl serum).

The results are shown in Table 10 below.

group Low Density Lipoprotein-Cholesterol Content (mg / dl Serum) Example 1 109.33 ± 9.51 ^** 77.8% ^a Comparative Example 1 129.94 ± 9.51 92.4% Comparative Example 2 131.47 ± 9.51 ^* 93.5% Control group 140.61 ± 25.95 ^b 100.0%

(a: ratio to control, b: mean ± standard deviation, *: p <0.05, **: p <0.01)

As shown in Table 10, the composition administered in Example 1 was 77.8% compared to the low density lipoprotein-cholesterol content in the serum of the control group, and the low density lipoprotein-cholesterol showed an effect of reducing 22.2%.

Comparative Example 1 administration group can be said to have a low density lipoprotein-cholesterol content reduction effect of 7.6%, Comparative Example 2 administration group showed a 6.5% low density lipoprotein-cholesterol content reduction effect.

From the results, it can be seen that the low-density lipoprotein-cholesterol reduction effect in the administration group of Example 1 was 2.9 times compared to Comparative Example 1 administration group and 3.4 times compared to Comparative Example 2 administration group. That is, when the royal jelly and nattokinase compositions are administered, it can be seen that the low-density lipoprotein-cholesterol content reduction effect in blood is superior to that of the single administration alone.

Therefore, the composition of the present invention is expected to act to inhibit the adult disease by the effect of reducing the low density lipoprotein-cholesterol content in the blood.

<Example 11> Confirm the effect of inhibiting the adult disease-Confirmation of the effect of increasing the high density lipoprotein (HDL) -cholesterol content in the serum

Cholesterol affects the development of adult disease, a chronic degenerative disease such as arteriosclerosis, myocardial infarction, hypertension, angina pectoris, stroke, and diabetes, known as an anti-cholesterol factor that suppresses adult disease. lipoprotein; HDL) -cholesterol. Therefore, the component having the effect of promoting the production of the high density lipoprotein (HDL) -cholesterol is expected to act to suppress the adult disease.

In this example, to determine the geriatric inhibitory effect of the composition of Example 1, the density of HDL-cholesterol content in the serum of the administration groups of Example 1, Comparative Example 1, and Comparative Example 2 was measured.

Determination of HDL-cholesterol content in serum was performed using the kit HDL-cholesterol (HDL-C 555, Eiken, Japan).

0.3 ml of serum is added to the test tube, and 0.3 ml of the precipitation reagent is mixed well, and the mixture is left at room temperature for 10 minutes and centrifuged at 700 x g for 10 minutes. Then, add 50 ml of the supernatant, 50 µl of standard solution (100 mg / dl), and 50 µl of distilled water to a blank (blank), and add 3.0 ml of HDL coloring reagent, respectively, mix well, and warm for 5 minutes in a 37 ° C water bath. As a blank, the absorbance was measured at 555 nm to quantify the content of HDL-cholesterol (mg / dl serum).

The results are shown in Table 11 below.

group High Density Lipoprotein-Cholesterol Content (mg / dl Serum) Example 1 27.25 ± 0.96 ^* 132.4% ^a Comparative Example 1 22.67 ± 1.12 ^* 110.1% Comparative Example 2 22.34 ± 1.35 ^** 108.5% Control group 20.59 ± 0.96 ^b 100.0%

(a: ratio to control, b: mean ± standard deviation, *: p <0.05, **: p <0.01)

As shown in Table 11, the composition administered in Example 1 was 132.4% compared to the high density lipoprotein-cholesterol content in the serum of the control group, and the high density lipoprotein-cholesterol content was 32.4%.

Comparative Example 1 administration group can be said to increase the content of 10.1%, Comparative Example 2 administration group showed a content increase effect of 8.5%.

From the above results, it can be seen that the high-density lipoprotein-cholesterol content increasing effect in the administration group of Example 1 was 3.2 times compared to Comparative Example 1 administration group and 3.8 times compared to Comparative Example 2 administration group. That is, it can be seen that when the royal jelly and nattokinase composition were administered, the effect of increasing the high density lipoprotein-cholesterol content was higher than that of the single administration alone.

Therefore, the composition of the present invention is expected to suppress the adult disease by the effect of increasing the high density lipoprotein-cholesterol content.

<Example 12> confirm the effect of inhibiting adult diseases-confirming the effect of reducing arteriosclerosis index

Atherosclerosis index (AI) is used as an indicator of the onset of atherosclerosis, which is known as an early symptom of adult disease, and the component having an effect of reducing the atherosclerosis index is expected to act to suppress the adult disease. do.

In the present Example, the degree of reduction of the arteriosclerosis index of Example 1, Comparative Example 1, and Comparative Example 2 administration group to the control group to determine the geriatric inhibitory effect of the composition was measured.

Atherosclerotic index (AI) is determined by Haglund et al. and malondialdehyde in humans supplemented with vitamin E. J. Nutr. 121: 165 ~ 169, 1991 . That is, the total cholesterol content in serum was subtracted from the content of HDL-cholesterol in serum, and then calculated by dividing it by HDL-cholesterol in serum.

The total cholesterol content in the serum was used in the value obtained in Example 9, the HDL-cholesterol content in the serum was used in the value obtained in Example 11.

The results are shown in Table 12 below.

group Arteriosclerosis Index (AI) Example 1 5.54 ± 0.61 ^*** 57.9% ^a Comparative Example 1 8.43 ± 0.49 ^* 88.1% Comparative Example 2 8.58 ± 0.56 ^** 89.7% Control group 9.57 ± 0.71 ^b 100.0%

(a: ratio to control, b: mean ± standard deviation, *: p <0.05, **: p <0.01,

***: p <0.001)

As shown in Table 12, the composition-administered group of Example 1 had an arterial stiffness index of 57.9% and a 42.1% reduction in arterial stiffness index compared to the control group.

On the other hand, it can be said that the comparative example 1 administration group showed a reduction effect of 11.9%, the comparative example 2 administration group showed a reduction effect of 10.3%.

From the above results, it can be seen that the effect of reducing the arteriosclerosis index in the administration group of Example 1 is 3.5 times compared to Comparative Example 1 administration group, 4.1 times compared to Comparative Example 2 administration group. That is, when the royal jelly and nattokinase composition is administered, it can be seen that the effect of reducing the arteriosclerosis index is superior to that of each administration alone.

Therefore, the composition of the present invention is expected to exhibit an adult disease suppression effect by reducing the arteriosclerosis index.

Claims (9)

A food composition for improving or anti-aging of adult diseases comprising nattokinase and royal jelly as an active ingredient in a weight ratio of 1: 3. delete The composition of claim 1, wherein the formulation of the composition is one selected from tablets, capsules, pills, powders, and granules. delete delete delete The food composition of claim 1 or 3, wherein the adult disease is at least one selected from arteriosclerosis, hypertension, stroke, angina pectoris, and heart disease. delete delete