KR100986880B1 - Composition for improving male regenerative function - Google Patents

Abstract

The present invention provides a composition for improving male reproductive function, including nattokinase and royal jelly. The composition of the present invention is excellent in the effect of improving male reproductive function, specifically, the effect of improving male foot function, preventing testicular tissue damage, preventing and improving sperm count.

Nattokinase, royal jelly, male reproductive function

Description

Composition for improving male regenerative function

The present invention relates to a composition for improving male reproductive function.

The reproductive function of modern men is declining due to overwork, drinking, stress, environmental pollution, and the like, and diseases due to deterioration of various male reproductive functions such as insomnia, infertility, and sexual dysfunction are spreading.

The causes of male infertility include idiopathic disorders, accounting for more than half of all male infertility, spermatogenesis disorders 22%, sperm transport obstruction 35%, semen abnormalities 7%, intercourse abnormalities 1%, sperm maturity 3% And endocrine abnormalities account for 2%. In particular, the reduction of sperm count due to internal accumulation of environmental pollutants is the biggest problem, which is caused by anxiety, stress, fatigue, marital frequency, smoking, and environmental pollution due to changes in social structure and lifestyle. Is getting. Toxic free radicals appear to be a decrease in fertility in reproductive organs such as testicular tissues, and oxidative damage has been reported to play an important role in this decline (Kang, HK, NO, JK, Soung, DY). , Kim, ND, Lee, KH , Kim, KW, Choi, WC, Lim, WK, Yu, BP and Ching, HY Effect of aging and dietary restriction on free radical generation and GSH / GSSH level in rat testis. Kor. J .. Gerontol 7 (3): 92 ~ 97, 1997.).

Male sexual dysfunction is erectile dysfunction, ejaculation disorders, lowering libido, etc. Among them, erectile dysfunction is defined as a lack of force for sexual intercourse in men, and includes a condition that can not penis erection or ejaculation. Specifically, erectile dysfunction or erectile dysfunction is a case in which the erection is not enough to maintain sexual activity, or even if the erection is not maintained, 2-7% of the male population is known to have these symptoms.

Recent studies have shown that the mechanism of erection relaxes the corpus cavernosum smooth muscle by the action of NO and cGMP through NANC (nonadrenergic-noncholinergic) neurotransmission mechanisms in the corpus cavernosal smooth muscle. Increased bleeding and blockage of venous blood resulting in erection (Bush, PA, Aronson, WJ, Rajfer, J. and Ignarro, LJ The L-arginine-nitric oxide-cyclic GMP pathway mediaters inhibitory nonadrenergic-noncholinergic neurotransmission in the corpus cavernosum of human and rabbit.Circulation . 87: 30-32, 1993.). The erectile corpus cavernosum is interrupted by the breakdown of cGMP under the action of phosphodiesterase (PDE). Therefore, it is necessary to increase the concentration of NO, cGMP, cGMP in order to maintain an erection.

Nitrogen monoxide (NO) in the body are nitric oxide synthase: Since the biosynthesis by the action of (nitric oxide synthase NOS) (Bredt , DS, Ferris, CD and Snyder, SH nitric oxide shnthase regulatory setes J Biol. Chem . 267 (16): 1976 ~ 1981, 1992.), erection can be maintained by maintaining or increasing the activity of nitric oxide synthase in the corpus cavernosum smooth muscle.

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 excreted by the development of a beetle when the 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.

The present inventors have completed the present invention by discovering that a composition comprising nattokinase and royal jelly can effectively improve male reproductive function.

The problem to be solved by the present invention is to provide a composition for improving male reproductive function.

The present invention provides a composition for improving male reproductive function, including nattokinase and royal jelly.

Improving male reproductive function includes improving sexual function, and specifically includes improving foot function, preventing and improving testicular tissue damage, preventing and improving sperm count.

Formulations of the compositions of the invention may be tablets, capsules, pills, powders, suspensions, solutions or / and granules.

The composition of the present invention may be a pharmaceutical composition or a food composition.

The food composition is meant to include a health food composition, functional food composition, or health functional food composition.

The pharmaceutical composition of the present invention can be used for the prevention and treatment of diseases caused by the degradation of various male reproductive functions, such as adolescents, infertility, sexual dysfunction associated with male reproductive dysfunction.

Accordingly, the present invention is intended for the prevention and treatment of diseases caused by a decrease in male reproductive function of the pharmaceutical composition of the present invention and a disease due to a decrease in male reproductive function, comprising administering the pharmaceutical composition of the present invention to a mammal, including a human. To prevent and treat

In addition, the food composition of the present invention can be used to improve male reproductive function and prevent decline.

Accordingly, the present invention provides a method for preventing and improving male reproductive function of the male composition of the present invention, and a method for preventing and improving male reproductive function, comprising the step of ingesting the food composition of the present invention to a mammal including a human. .

Natokinase: royal jelly of the composition of the present invention may be 1: 2 to 4.

It is excellent in the effect of improving male sexual function of the composition of the present invention in the weight ratio range.

The nattokinase can be prepared by using a known fermentation method using Bacillus subtilis or the like, or extracting from NATO, or a commercially available one. A commercial item can use a powder form.

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

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

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.

Pharmaceutical or food acceptable carriers are sterile and biocompatible when formulated as liquid solutions and include saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, and / or ethanol. It can be used, and other conventional additives such as antioxidants, buffers, bacteriostatics can be added as necessary. Diluents, dispersants, surfactants, solvents, disintegrants, sweeteners, binders, coatings, swelling agents, lubricants, lubricants and / or flavoring agents may additionally be added. Furthermore, it may be preferably formulated according to the purpose or component by a suitable method in the art or using a method disclosed in Remington's Pharmaceutical Science (Recent Edition), Mack Publishing Company, Easton PA.

Formulation forms of the compositions of the present invention may be granules, powders, tablets, coated tablets, capsules, suppositories, solutions, syrups, juices, suspensions, emulsions, sustained release formulations of the active compounds, 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 optimum 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.

In addition, the present invention provides a method for improving sexual function comprising the step of administering the composition and the sexual function of the composition.

In addition, the present invention provides a method for producing a therapeutic agent for improving sexual function of the composition.

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 may be formulated in the form of other tablets, granules, powders, beverages, porridges, etc. It is possible to manufacture.

In addition, the present invention provides a method for improving sexual function comprising the step of administering the food composition and the sexual function of the food composition.

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.

Compositions of the present invention, either alone or papaverine, phentolamine, prostagladin E1, trazodone, yohimbin, sildenafil, which have been found to have an effect on improving male reproductive function It can be administered in parallel.

The composition of the present invention is excellent in the effect of improving male reproductive function, specifically, the effect of improving male foot function, preventing testicular tissue damage, preventing and improving sperm count.

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 embodied in various different forms, but the present embodiments only make the disclosure of the present invention complete and common knowledge in the technical field to which the present invention belongs. It is provided to fully inform the person having the scope of the invention, which is defined only by the scope of the claims.

<Examples 1 to 4>

Natokinase powder (BIO SCIENCE LABORATORY CO., LTD; JAPAN) and royal jelly powder (Tuhsu Dongfang Imp. & Exp, Corp; China) were mixed to have the composition of Table 1 below. The nattokinase powder had an activity of 20,000 FU.

division Natokinase Powder (mg) Royal Jelly Powder (mg) Example 1 2.5 7.5 Example 2 5.0 15.0 Example 3 10 30.0 Example 4 20 60.0

<Comparative Examples 1 to 4>

Powders identical to those of the nattokinase powders of Examples 1 to 4 were prepared in the compositions shown in Table 2 below.

division Natokinase Powder (mg) Comparative Example 1 2.5 Comparative Example 2 5.0 Comparative Example 3 10.0 Comparative Example 4 20.0

<Comparative Examples 5 to 6>

The same powder as the royal jelly powder of Examples 1 to 4 was prepared in the composition of Table 3 below.

division Royal Jelly Powder (mg) Comparative Example 5 30 Comparative Example 6 60

Example 5 Confirmation of Male Reproductive Function Enhancement Activity-Activity Evaluation of NO Synthetase (NOS)

1) Experimental Animals and Breeding Conditions

SPF SD rats (Sam taco, male, 220 ± 10g) were pre-divided for 2 weeks in the animal feeding room and divided into 13 groups of 10 animals.

Groups 1 to 4 were fed on a daily basis by adding the composition of Examples 1 to 4 feed and raised for a total of 6 weeks, groups 5 to 10 were administered on a daily basis to feed containing the composition of Comparative Examples 1-6 A total of six weeks of breeding. In addition, for groups 11 and 12, the feed containing the composition of Example 2 or Example 3, respectively, was administered on a daily basis and raised for a total of 10 weeks. The groups 1 to 12 used the same feed except that the compositions of Examples 1 to 4 and Comparative Examples 1 to 6 were used in the feed, except that the group 13 did not use royal jelly and nattokinase as controls. The animals were fed for 6 weeks using the same feed as in groups 1-12.

2) fraction of testicular tissue

Fraction of testicular tissue is Choi (1989) used this method (Choi, JH and Yu, BP The effect of food restriction on kideny membrane structures of aging rats AGE, 12:. 133 ~ 136, 1989.) buffer in accordance with the jeoonsil Using a solution (1.15% KCl / 10 mM phosphate buffer + 5 mM EDTA, pH 7.4), homogenize at 3000 rpm using a tissue homogenizer with 10 times (w / v) of buffer solution per weight of testes, then centrifuge at 700 g for 10 minutes. The supernatant obtained was used for the experiment. Supernatants were analyzed while stored at -70 ° C until the experiment.

3) Measurement of NO Synthetase (NOS)

Nitric oxide synthase (NOS), a synthase of nitric oxide, a promoter of erectile function, was measured using a measurement kit (sigma, catalog NO. FCANOS-1). Using a 96well plate, add 50 μl reaction buffer to each sample and mix by adding 100 μl of reaction mix 1. Add 50 μl NADPH diluent to the next step and add 100 μl of reaction mix 2, mix, leave for 2 hours at room temperature with light blocking and excitation with a 96 well spectrofluorometer It was measured at 492 nm and emission at 515 nm.

4) Statistical processing of experimental results

Statistically processing the results was calculated the average and standard deviation, and significance tests of each group are Student's t-test (Steel, RGD and Torrie, JH Principles and procedures of statistics. McGrawhill. New York . 1960.).

The NOS measurement results are shown in Table 4 below.

group NO synthase activity (cpm) 1 (Example 1) 2648.0 ± 31.1 103.2% ^b 2 (Example 2) 2659.5 ± 43.1 103.7% 3 (Example 3) 2755.0 ± 31.8 107.4% 4 (Example 4) 2818.3 ± 140.8 109.9% 5 (Comparative Example 1) 2600.0 ± 41.5 101.4% 6 (comparative example 2) 2637.5 ± 36.1 102.8% 7 (Comparative Example 3) 2653.3 ± 50.8 103.4% 8 (Comparative Example 4) 2720.7 ± 35.3 106.1% 9 (comparative example 5) 2649.0 ± 52.0 103.3% 10 (comparative example 6) 2739.5 ± 50.2 106.8% 11 (Example 2) 2872.5 ± 23.3 ^* 112.0% 12 (Example 3) 2958.3 ± 66.7 ^** 115.3% 13 (control) 2565.3 ± 110.9 ^a 100.0%

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

As can be seen from the results of Table 4, the results were significant in groups 11 and 12. Groups 11 and 12, in which the nattokinase and royal jelly mixtures of Examples 2 and 3 were administered for 10 weeks, respectively, were 112.0% and 115.3% compared to the NOS activity of the control group, respectively, with 12% and 15% significant increase in NOS activity. Appeared. In addition, although the value is not significant, the group administered with the nattokinase and royal jelly mixture of Examples 1 to 4 for 6 weeks is the group administered with Comparative Examples 1 to 4 or Comparative Examples 5 to 6 administered only nattokinase or royal jelly. NOS activity was increased compared to Therefore, administration of a mixture of nattokinase and royal jelly increases NOS activity, and as a result, it is expected to promote the synthesis of NO.

Example 6 Confirmation of Male Reproductive Function Improvement Activity-Synthesis Evaluation of Erectile Promoting Factor NO

Experimental animals and breeding conditions, fractions of testicular tissue, and statistical processing of the experimental results were carried out in the same manner as in Example 5.

Methods, such as the erection promoting factor NO content in testis tissue Yu (Yu, L., Gengaro, PE , Burke, TJ and Schrier, RW Nitric oxide:.... A mediator in rat tubular hypoxia / reoxygenation injury Proc Natl Acad Sci , 91: 1691-1695. 1994.) 0.1 g of testicular tissue was homogenized with 1 ml of 0.1 M potassium phosphate buffer (pH 7.5), centrifuged at 600 × g for 5 minutes, and 200 μl of the supernatant 100 μl of 90munit nitrate reductase. 100 μl of 0.28 mM NADPH, 100 μl of 3.5 uM FAD, and 200 μl of 0.1 M potassium phosphate buffer (pH 7.5) were added and incubated at 25 ° C. for 1 hour. Boiling for 3 minutes to stop the reaction, Griss reagent (2% sulfanilic acid / 5% H ₃ PO ₄ : 0.2% N- (1-naphtyl) ethylendiamine / water. 1: 1, v / v) 700 μl was added. After warming again at 60 ° C. for 10 minutes, the absorbance was measured at 546 nm and quantified by a standard calibration curve (nmol / mg protein) by sodium nitrate.

The results are shown in Table 5 below.

group NO content (nmol / mg protein) 1 (Example 1) 14.38 ± 0.31 103.7% ^b 2 (Example 2) 14.80 ± 0.08 107.4% 3 (Example 3) 15.32 ± 0.21 ^* 110.5% 4 (Example 4) 15.60 ± 0.34 ^* 112.5% 5 (Comparative Example 1) 14.05 ± 0.65 101.3% 6 (comparative example 2) 14.53 ± 0.75 104.7% 7 (Comparative Example 3) 14.81 ± 0.36 106.7% 8 (Comparative Example 4) 14.80 ± 0.69 106.8% 9 (comparative example 5) 14.63 ± 0.50 105.5% 10 (comparative example 6) 15.31 ± 1.13 ^* 110.3% 11 (Example 2) 15.84 ± 1.83 ^* 114.2% 12 (Example 3) 16.46 ± 0.03 ^** 118.7% 13 (control) 13.87 ± 1.50 ^a 100.0%

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

As shown in Table 5, the results of Group 3, Group 4, Group 10, Group 11, and Group 12 were significant. Judging from the value of the significant value only, compared to 10 groups administered only 60 mg / day of royal jelly, group 3 administered 10 mg / day of nattokinase at 30 mg / day of royal jelly and 20 mg / day of nattokinase at 60 mg / day of royal jelly It can be seen that the NO content of the four groups administered at one dose significantly increased. In particular, in the case of Group 3, the amount of NO was increased even though the amount of Royal Jelly was 1/2 of the Royal Jelly-only group.

In addition, it can be confirmed that the NO content of the groups 11 and 12 administered royal jelly and natokinase significantly increased for 10 weeks.

  Therefore, administration of a mixture of nattokinase and royal jelly effectively promotes NO synthesis, which is found to be an stimulatory factor in testicular tissue, and thus is believed to improve male foot function.

Example 7 Confirmation of Male Reproductive Function Improvement Activity-Evaluation of Production of cGMP

Experimental animals and breeding conditions, fractions of testicular tissue, and statistical processing of the experimental results were carried out in the same manner as in Example 5.

The measurement of cGMP produced by NO activation was measured using Kit reagent (Amersham pharmacia, Code NO RPA525 cGMP [125 I] assay). A small amount of 125 I was labeled at the binding site of the cGMP-specific antibody and the amount of cGMP, which was known, was compared with that of the unlabeled cGMP. Testicular tissue was homogenized with 6% TCA solution, and the supernatant was used as a sample to label cGMP-specific antibody with a small amount of 125 I at the binding site of cGMP-specific antibody.

Place the sample in a glass tube (12 × 75 mm) and add 25 μl of an acetylation reagent (acetic anhydride: triethylamine = 1: 2). After acetylation, take 100 μl in pairs, transfer to polyproplyene assay tube, add 100 μl antiserum, mix well, and leave at room temperature for 1 hour. [ ¹²⁵ I] Add 100 μl of cGMP, mix, and stand at 2-8 ° C. for 15-18 hours. Next, 500 μl of Amerlex-M second antibody was added to each tube and centrifuged to completely remove the supernatant, followed by a 60 second gamma-sintillation counter. Radiation was measured.

The results are shown in Table 6 below.

group cGMP content (cpm) 1 (Example 1) 2771.2 ± 82.0 103.7% ^b 2 (Example 2) 2815.4 ± 116.8 105.3% 3 (Example 3) 2907.2 ± 121.5 108.7% 4 (Example 4) 2951.8 ± 66.5 ^* 110.4% 5 (Comparative Example 1) 2663.8 ± 93.1 99.6% 6 (comparative example 2) 2765.0 ± 147.4 103.4% 7 (Comparative Example 3) 2771.2 ± 130.7 103.7% 8 (Comparative Example 4) 2830.6 ± 67.0 105.9% 9 (comparative example 5) 2789.6 ± 21.5 104.3% 10 (comparative example 6) 2932.6 ± 167.7 109.7% 11 (Example 2) 3035.9 ± 170.1 ^* 113.6% 12 (Example 3) 3099.9 ± 61.9 ^** 116.0% 13 (control) 2673.4 ± 90.8 ^a 100.0%

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

The results shown in Table 6 are the results for the 4, 11, 12 groups administered the compositions of Examples 4, 2, 3, all three cases showed that the cGMP content is significantly increased compared to the control group have. Since cGMP directly affects penile cavernous relaxation before erection, an increase in the content of cGMP (cyclic guanosine monophosphate) in testicular tissue means that the composition of the present invention has an effect on improving foot function.

<Example 8> Male reproductive function check action-sperm count evaluation

Experimental animals and breeding conditions, and statistical processing of the experimental results were carried out in the same manner as in Example 5.

Methods such as sperm can measure Morrissey (Morrissey, RE, Schwetz, SJ, Ross, MD, Teague, JL and Morris, RW Evaluation of rodent sperm, vaginal cytology and reproductive organ weight data from national toxicity program 13-week studies. Fundam . Appl Toxicol, 11:.. was measured in accordance with the epididymis 343-358, 1988). The left epididymis of the rat was extracted and stored at -40 ° C until measurement. After dissolving at room temperature, HBSS solution was added to the epididymis and homogenized. After diluting the homogenate 40 times with HBSS solution, a certain amount was taken and sperm count was measured by an inverted microscope using a hemocytometer.

The results are shown in Table 7 below.

group Sperm (× 10 ⁶ / g epididymal) 1 (Example 1) 40.00 ± 2.95 101.6% ^b 2 (Example 2) 41.35 ± 4.02 105.0% 3 (Example 3) 42.70 ± 4.27 108.5% 4 (Example 4) 43.38 ± 3.65 ^* 110.2% 5 (Comparative Example 1) 39.86 ± 3.60 101.2% 6 (comparative example 2) 40.00 ± 3.35 101.6% 7 (Comparative Example 3) 40.23 ± 0.54 102.2% 8 (Comparative Example 4) 40.53 ± 4.82 103.0% 9 (comparative example 5) 40.49 ± 3.59 102.9% 10 (comparative example 6) 42.29 ± 1.98 107.4% 11 (Example 2) 43.25 ± 3.77 109.9% 12 (Example 3) 44.86 ± 2.11 ^* 113.9% 13 (control) 39.37 ± 2.39 ^a 100.0%

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

Significant results in Table 7 were found in groups 4 and 12, and it was confirmed that the number of sperm increased significantly in each group administered with Examples 4 and 3 compared to the control group. In addition, although the value is not significant, the sperm count was increased when the royal jelly and nattokinase composition were administered as compared with the administration of each alone.

Therefore, administration of the composition of the present invention shows an effect of increasing the number of sperm, as a result it can be seen that the composition of the present invention is effective in improving the reproductive function of men.

Example 9 Confirmation of Male Reproductive Function Improvement-Evaluation of Superoxide Radical Content in Testicular Tissue

Statistical treatment of experimental animals and breeding conditions, testicular tissue fractions and experimental results was carried out in the same manner as in Example 5.

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 . 244 (22): 6049 ~ 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. 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 testicular tissue homogenate 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 8 below.

group Superoxy radical content (nmol / mg protein) 1 (Example 1) 36.85 ± 3.68 101.3% ^b 2 (Example 2) 34.19 ± 4.07 93.9% 3 (Example 3) 31.72 ± 1.93 ^* 87.2% 4 (Example 4) 30.15 ± 2.60 ^** 82.8% 5 (Comparative Example 1) 37.10 ± 3.22 101.9% 6 (comparative example 2) 32.55 ± 0.53 ^* 89.5% 7 (Comparative Example 3) 31.86 ± 2.97 ^* 87.6% 8 (Comparative Example 4) 33.34 ± 1.56 91.6% 9 (comparative example 5) 38.21 ± 3.30 105.0% 10 (comparative example 6) 34.43 ± 0.89 94.6% 11 (Example 2) 30.67 ± 2.05 ^** 84.3% 12 (Example 3) 29.31 ± 0.56 ^** 80.5% 13 (control) 36.39 ± 2.72 ^a 100.0%

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

Significant results in Table 8 were found in groups 3, 4, 6, 7, 7, 11, and 12, and royal jelly to nattokinase of the same content compared to group 7 administered with nattokinase 10 mg / day alone. In the case of adding group 3, it can be seen that the superoxy radical content decreases. In addition, the hydroxy radical content in the groups 11 and 12 administered with a mixture of nattokinase and royal jelly for 10 weeks can be seen to decrease by 20% compared to the control. In addition, the evaluation including the results other than the significant value, the royal jelly and nattokinase composition administration was found to decrease the superoxy radical content in comparison with the single administration as a whole.

Therefore, the superoxy radical content of the testicular tissue is reduced when the composition of the present invention is administered, and as a result, the composition of the present invention can be seen to be effective in improving the reproductive function of men. It is known that oxidative damage plays an important role in the reduction of fertility in the reproductive organs, such as testicular tissues, and therefore, it is believed that the male fertility will be improved by inhibiting the production of superoxy radicals by administering the composition of the present invention.

<Example 10> Confirmation of male reproductive function improvement-Evaluation of hydroxy radical content of testicular tissue

Statistical treatment of experimental animals and breeding conditions, testicular tissue fractions and experimental results was carried out in the same manner as in Example 5.

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. methods, such as thio Bobby tube acid (thiobabituric acid) and using Halliwell in that the color developing reaction in solution (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.). The measurement of hydroxy radicals of testicular tissue homogenates was carried out first by 0.1 M phosphate buffer solution (pH 7.4), 10 mM NaN ₃ , 7 mM deoxyribose, 5 mM ferrousammonium sulfate, 0.54. 33.3 μl of M NaCl reagent 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 9 below.

group Hydroxy radical content (nmol / mg protein) 1 (Example 1) 0.33 ± 0.01 94.6% ^b 2 (Example 2) 0.32 ± 0.04 91.6% 3 (Example 3) 0.29 ± 0.02 ^** 84.3% 4 (Example 4) 0.29 ± 0.01 ^** 82.7% 5 (Comparative Example 1) 0.34 ± 0.04 97.2% 6 (comparative example 2) 0.33 ± 0.06 95.0% 7 (Comparative Example 3) 0.32 ± 0.03 92.8% 8 (Comparative Example 4) 0.31 ± 0.06 ^* 89.8% 9 (comparative example 5) 0.33 ± 0.01 95.3% 10 (comparative example 6) 0.32 ± 0.03 91.0% 11 (Example 2) 0.29 ± 0.03 ^** 83.3% 12 (Example 3) 0.28 ± 0.05 ^** 80.8% 13 (control) 0.35 ± 0.04 ^a 100.0%

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

Significant results in Table 9 were found in Groups 3, 4, 8, 11, and 12, and the hydroxy radical content was found in the control groups in groups 11 and 12 treated with a mixture of nattokinase and royal jelly for 10 weeks. It can be seen that the decrease by nearly 20%. In addition, the hydroxy radical content of group 4, which was administered by mixing royal jelly 60 mg / day with nattokinase 20 mg / day, was reduced, compared to group 8, which was administered with nattokinase 20 mg / day alone. In the case of group 3 (natokinase 10 mg / day + royal jelly 30 mg / day) administered half of the kinase, it was confirmed that the hydroxy radicals were significantly reduced as compared to the nattokinase alone group. Judging from the results including other significant results, the hydroxy radical content of the royal jelly and nattokinase compositions was decreased as compared with the administration of each alone.

Therefore, the administration of the composition of the present invention can reduce the oxidative damage of testicular tissues by the strongest hydroxy radicals among the active oxygen, and as a result it can be seen that the composition of the present invention is effective in improving the reproductive function of men. That is, since oxidative damage is known to play an important role in the reproductive organs, such as testicular tissues, it is believed that by suppressing the production of hydroxy radicals by administering the composition of the present invention, male fertility is improved.

<Example 11> Confirmation of male reproductive function improvement-Evaluation of the inhibitory effect of lipid peroxide production in testicular tissue

Statistical treatment of experimental animals and breeding conditions, testicular tissue fractions and experimental results was carried out in the same manner as in Example 5.

The method using the content of lipid peroxide in the testicular tissue is such Choi (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 testicular tissue homogenate and 180 μl of distilled water in each test tube, add 200 μl of 8.1% sodium dodecyl sulfate (SDS) solution for about 5 seconds, add 1.5 ml of 20% acetic acid, and mix again for 5 seconds, 1.0 ml of a 1.2% TBA (thiobarbituric acid) reagent was added thereto, 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 10 below.

group Lipid peroxide (nmol / mg protein) 1 (Example 1) 0.99 ± 0.05 92.5% ^b 2 (Example 2) 0.95 ± 0.14 ^* 88.7% 3 (Example 3) 0.91 ± 0.20 ^** 84.7% 4 (Example 4) 0.88 ± 0.14 ^** 82.3% 5 (Comparative Example 1) 1.11 ± 0.05 103.7% 6 (comparative example 2) 1.08 ± 0.10 100.9% 7 (Comparative Example 3) 1.02 ± 0.09 94.8% 8 (Comparative Example 4) 0.99 ± 0.06 92.4% 9 (comparative example 5) 1.06 ± 0.11 98.4% 10 (comparative example 6) 0.99 ± 0.08 92.3% 11 (Example 2) 0.93 ± 0.21 ^* 86.8% 12 (Example 3) 0.87 ± 0.08 ^** 81.3% 13 (control) 1.07 ± 0.17 ^a 100.0%

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

Significant results in Table 10 were found in Groups 2, 3, 4, 11, and 12, and the lipid peroxide content was found in the control group in groups 11 and 12 after 10 weeks of administration of a mixture of nattokinase and royal jelly. It can be seen that the decrease of 13% and 18%, respectively. In addition, it can be seen that the lipid peroxide content was significantly reduced even in the 2-4 group administered with nattokinase and royal jelly mixture for 6 weeks. Judging from the results including other significant results, it was shown that the lipid peroxide content decreased when the royal jelly and nattokinase compositions were administered as a whole.

Therefore, it can be seen that the administration of the composition of the present invention has an excellent inhibitory effect on the production of lipid peroxide by oxidative stress in testicular tissue, and as a result, the composition of the present invention is effective in improving the reproductive function of men. That is, since oxidative damage is known to play an important role in the reproductive organs, such as testicular tissues, it is believed that by suppressing the production of hydroxy radicals by administering the composition of the present invention, male fertility is improved.

<Example 12> Confirmation of male reproductive function improvement-Evaluation of oxidized protein content of testicular tissue

Statistical treatment of experimental animals and breeding conditions, testicular tissue fractions and experimental results was carried out in the same manner as in Example 5.

The content of oxidized protein in testicular tissue was determined by Levine et al. (Levine, RL, Garland, D., Oliver, CN, Amici, A., Climent, I., Lenz, AG, Ahn, B., Shaltiel, S. and 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 11 below.

group Oxidized protein content (nmol / mg protein) 1 (Example 1) 0.62 ± 0.11 96.4% ^b 2 (Example 2) 0.58 ± 0.06 90.7% 3 (Example 3) 0.57 ± 0.06 ^* 88.8% 4 (Example 4) 0.54 ± 0.07 ^** 84.9% 5 (Comparative Example 1) 0.64 ± 0.06 99.6% 6 (comparative example 2) 0.64 ± 0.03 99.5% 7 (Comparative Example 3) 0.63 ± 0.05 98.8% 8 (Comparative Example 4) 0.57 ± 0.04 ^* 89.9% 9 (comparative example 5) 0.62 ± 0.02 96.6% 10 (comparative example 6) 0.59 ± 0.02 92.0% 11 (Example 2) 0.52 ± 0.12 ^** 81.1% 12 (Example 3) 0.52 ± 0.04 ^** 82.1% 13 (control) 0.64 ± 0.15 ^a 100.0%

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

Significant results in Table 11 were found in Groups 3, 4, 8, 11, and 12, and the oxidized protein content was found in the control group in Groups 11 and 12 after 10 weeks of administration of a mixture of nattokinase and royal jelly. It can be seen that the decrease by nearly 20%. In addition, the oxidized protein content of group 4, which was administered by mixing royal jelly 60 mg / day with nattokinase 20 mg / day, was significantly lower than that of group 8, which was administered nattokinase 20 mg / day alone. In the third group (half of kinase 10 mg / day + royal jelly 30 mg / day), the amount of oxidized protein was significantly reduced. Judging from the results including other significant results, the oxidized protein content of the royal jelly and nattokinase composition was decreased as compared with the single administration as a whole.

Therefore, when the composition of the present invention is administered effectively prevents the production of oxidized protein by oxidation of the protein in testicular tissue, it is possible to reduce the oxidative damage of testicular tissue by oxidative stress, consequently the composition of the present invention It can be seen that effective in improving the reproductive function of the. That is, since oxidative damage is known to play an important role in the reproductive system in the reproductive organs, such as testicular tissue, it is believed that by administering the composition of the present invention to effectively prevent the production of oxidized protein, male fertility is improved.

Example 13 Confirmation of Male Reproductive Function Improvement Activity-Evaluation of Superoxide Dismutase Activity in Testicular Tissue

Statistical treatment of experimental animals and breeding conditions, testicular tissue fractions and experimental results was carried out in the same manner as in Example 5.

On: (290 ~ 296, 1984. Oyanagui , Y. Reevaluation of assay methods and estabishment of Kit for superoxide dismutase activity Anal Biochem 42...): Super-oxide discharge Muta dehydratase (superoxide dismutase SOD) activity, methods such as Oyanagui Followed. In other words, test solution tissue homogenate was diluted 30-fold with phosphate buffer solution (pH 8.2), 0.5 ml of distilled water, A reagent (52.125 mg of hydoxylamine + 102.1 mg of hypoxanthine / 250 ml DW), 0.2 ml, and B reagent ( 20 μl of xanthine oxidase + 0.9939 mg EDTA / 26.7 ml phosphate buffer, pH 8.2) is added to the mixture and warmed in a constant temperature water bath at 37 ° C for 40 minutes, followed by C reagent (300 mg of sulfanilic acid + N-1-naphthylethyene diamine acid / Add 2.0 ml of 500 ml of 16.7% acetic acid) and leave at room temperature for 20 minutes. Measure the absorbance at 550 nm using a spectrophotometer and measure superoxide dismutase (unit / mg protein) by standard calibration curve. Quantification

The results are shown in Table 12 below.

group Superoxide Dismutase Activity (unit / mg Protein) 1 (Example 1) 15.88 ± 0.88 107.0% ^b 2 (Example 2) 16.34 ± 0.75 ^* 110.2% 3 (Example 3) 16.92 ± 0.64 ^* 114.1% 4 (Example 4) 16.85 ± 1.35 ^* 113.6% 5 (Comparative Example 1) 15.80 ± 1.81 106.6% 6 (comparative example 2) 15.97 ± 0.74 107.5% 7 (Comparative Example 3) 16.37 ± 0.18 110.4% 8 (Comparative Example 4) 16.52 ± 0.98 ^* 111.4% 9 (comparative example 5) 16.08 ± 0.58 108.4% 10 (comparative example 6) 16.44 ± 0.63 ^* 110.9% 11 (Example 2) 17.71 ± 0.77 ^** 119.4% 12 (Example 3) 18.34 ± 1.20 ^*** 123.7% 13 (control) 14.83 ± 0.83 ^a 100.0%

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

Significant results in Table 12 were found in Group 2, Group 3, Group 4, Group 8, Group 10, Group 11, and Group 12, and in Groups 11 and 12 treated with a mixture of nattokinase and royal jelly for 10 weeks. It can be seen that superoxide dismutase activity is increased by about 20% compared to the control. In addition, the superoxide dismutase activity of the four groups administered with a mixture of 60 mg / day of royal jelly in 20 mg / day of nattokinase 20 mg / day was significantly increased, compared to the eight groups in which nattokinase 20 mg / day was administered alone. In the third group (half of nattokinase 10 mg / day + royal jelly 30 mg / day) administered with half the nattokinase, the superoxide dismutase activity was also significantly increased. Superoxide dismutase activity was significantly increased in four groups administered with Naturkinase (20 mg / day dose) and Royal Jelly (60 mg / day dose) compared to 10 groups that received only 60 mg / day royal jelly. . Judging from the results including the results other than the results, the superoxide dismutase activity was increased when the royal jelly and the nattokinase composition were administered as a whole.

Therefore, during the administration of the composition of the present invention, the superoxide radicals initially generated in the testicular tissues are effectively inhibited by increasing the activity of superoxide dismutase, thereby oxidative damage of testicular tissues by reactive oxygen. It can be seen that as a result, the composition of the present invention is effective in improving the reproductive function of men. That is, since oxidative damage is known to play an important role in the reproductive system in the reproductive organs, such as testicular tissue, it is believed that by administering the composition of the present invention to effectively prevent the production of oxidized protein, male fertility is improved.

Example 14 Confirmation of male reproductive function improvement-Evaluation of glutathione peroxidase activity of testicular tissue

Statistical treatment of experimental animals and breeding conditions, testicular tissue fractions and experimental results was carried out in the same manner as in Example 5.

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. In other words, glutathione peroxidase (GPx) activity in testicular tissue was measured by diluting the testicular homogenate 10 times with phosphate buffer (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)) 5 μl, 1 μM hydroperoxide 320 μl and 30 μl of diluted testicular tissue homogenate were added and mixed well for 5 seconds. Immediately using a spectrophotometer, absorbance at 340 nm was measured for 2 minutes at 15 second intervals, and the activity of GPx (umol / min / g protein) was calculated by standard calibration curve.

The results are shown in Table 13 below.

group Glutathione peroxidase activity (nmol / mg protein / min cpm) 1 (Example 1) 4.06 ± 1.52 104.0% ^b 2 (Example 2) 4.25 ± 0.16 109.0% 3 (Example 3) 4.41 ± 0.82 ^* 113.0% 4 (Example 4) 4.45 ± 0.62 ^* 113.9% 5 (Comparative Example 1) 3.90 ± 0.44 100.1% 6 (comparative example 2) 4.21 ± 0.98 107.9% 7 (Comparative Example 3) 4.23 ± 0.36 108.3% 8 (Comparative Example 4) 4.24 ± 0.56 108.6% 9 (comparative example 5) 4.07 ± 0.36 104.4% 10 (comparative example 6) 4.37 ± 0.54 ^* 111.9% 11 (Example 2) 4.67 ± 0.63 ^** 119.8% 12 (Example 3) 4.70 ± 0.67 ^*** 120.6% 13 (control) 3.90 ± 0.38 ^a 100.0%

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

Significant results in Table 13 were found in Group 3, Group 4, Group 10, Group 11, and Group 12. Glutathione peroxidase activity was significantly increased in Groups 11 and 12 treated with a mixture of nattokinase and royal jelly for 10 weeks. It can be seen that the increase is about 20%. In addition, the glutathione peroxidase activity of the four groups administered by mixing royal jelly 60 mg / day to 20 mg / day of nattokinase was significantly increased as compared to the 10 group administered only 60 mg / day of royal jelly alone, but also administered to group 10 In the third group (half of nattokinase 10 mg / day + royal jelly 30 mg / day), one group of royal jelly was also found to significantly increase glutathione peroxidase activity. Judging from the results including the results other than the results, the glutathione peroxidase activity was increased when the royal jelly and the nattokinase composition were administered as compared to the administration of each alone.

Therefore, since administration of the composition of the present invention has an effect of increasing the active oxygen scavenger glutathione peroxidase activity, detoxification of hydrogen peroxide (hydrogen peroxide) in the active oxygen can reduce the oxidative damage of testicular tissue, as a result It can be seen that the composition of the present invention is effective in improving the reproductive function of men. That is, since oxidative damage is known to play an important role in the reproductive organs, such as testicular tissue, it is believed that the administration of the composition of the present invention to detoxify hydrogen peroxide, thereby improving male fertility.

Claims (5)

Food composition for improving male reproductive function, including nattokinase and royal jelly. According to claim 1, Nato kinase: Royal jelly based on the natto kinase having 20,000 FU activity, male food reproduction composition for improving male reproductive function 1: 1: 2 to 4. The food composition of claim 1, wherein the formulation of the composition is one selected from tablets, capsules, pills, powders, and granules. The food composition according to any one of claims 1 to 3, wherein the composition is a health functional food composition. delete