KR102353048B1 - Black garlic extract, black garlic drink comprising it and composition for improving male health comprising it - Google Patents

Abstract

The present invention relates to a black garlic extract, a black garlic drink containing the same, and a composition for promoting men's health, and more particularly, to a black garlic extract having a high content of total phenolic compounds and SAC, a black garlic drink containing the same, and a black ginseng extract to the black garlic extract Or, by mixing black ginseng powder, a composition for promoting men's health having a higher activity than that of each is disclosed.

Description

Black garlic extract, black garlic drink comprising the same, and a composition for improving men's health {Black garlic extract, black garlic drink comprising it and composition for improving male health comprising it}

The present invention relates to a black garlic extract, a black garlic drink containing the same, and a composition for promoting men's health, and more particularly, to a black garlic extract having a high content of total phenolic compounds and SAC, a black garlic drink containing the same, and a black ginseng extract to the black garlic extract Or by mixing black ginseng powder, it relates to a composition for promoting men's health having a higher activity than the activity each has.

Black garlic appears dark black due to a non-enzymatic browning reaction during the long-term aging of raw garlic at a temperature of 80~90℃. It is due to the movement of protons from the free electron band of nitrogen atoms, and when exposed to high temperatures for a long time, the brown substances produced by the Maillard reaction gather and appear black.

Black garlic has a sweet and sour taste in harmony with organic acids as the strong taste and irritating odor unique to garlic are removed, sugars are decomposed, and it has a texture similar to jelly, so it has advantages in consumption compared to raw garlic. In addition, during the aging process, the content of polyphenol compounds, a representative functional component present in plants, is increased, and S-allylcysteine (SAC), a water-soluble sulfur compound, has functions such as antioxidant, inhibition of cancer cell proliferation, improvement of cognitive function, and liver protection. . Therefore, black garlic is known to have functionalities derived from phenolic compounds and SAC, as well as functions such as improvement of blood lipid concentration, reduction of blood cholesterol, recovery from fatigue, and improvement of endurance, which are the intrinsic activities of garlic.

Black ginseng has undergone several steaming and drying processes to change the color of ginseng to black. Basically, it is known that the saponin present in ginseng is not lost, and the content of ginseng saponin present only in processed ginseng is further increased. Among them, ginsenoside Rg3, a representative component, is known as a component having many physiological activities such as brain neuroprotection, anticancer, immune enhancement, and obesity suppression, and its content is increased compared to fresh ginseng or red ginseng.

Although black garlic and black ginseng have differences in the manufacturing process, they are representative black food that turns black by the reaction of amino acids and sugars contained in its own ingredients through the same heat treatment process to produce a large amount of browning substances. It has already been proven through several studies that all of these are foods with various functions.

However, black garlic has a unique flavor of garlic, and there are many sugars in its ingredients, which causes it to harden during storage, so there is a limit to using it as a 100% powder.

Black ginseng retains its unique flavor and has a strong bitter taste, making it difficult to drink when extracted alone. The higher the concentration, the stronger the bitter taste. In addition, when used as a powder, the bitter taste is not removed, and it has a relatively low sugar content compared to black garlic, so powdering is advantageous, but it has a disadvantage in that the unit price is high because it goes through more processes than red ginseng.

Therefore, it can be seen that both of these materials have limitations in processing, although they are widely known for their excellent functionality. As one method to compensate for this disadvantage, a method of mixing black garlic with strong sugar and black ginseng with strong bitter taste can be proposed.

As prior inventions in which an attempt was made to mix black garlic and black (red) ginseng, Korean Patent Publication No. 10-2009-0068960 proposes a method for preparing a health supplement for antioxidants containing black garlic and red ginseng mixed extract, raw garlic, black garlic and After pulverizing each red ginseng, add about 5 times of water to the freeze-dried powder, mix at room temperature in a shaker incubator for 14 hours, and then centrifuge to obtain an extract with increased antioxidant activity by taking only the supernatant.

In the method for producing a black garlic extract containing raw material (Patent Publication No. 10-2018-0136680), three times the amount of water is added to black garlic and the first extraction is performed at 85-95° C. for 5-10 hours, and then the residue is extracted again, and then in the residue. By mixing peony, mulberry, and dandelion ginseng and extracting at 80~95℃ for 3~6 hours to prepare an extract mixed with herbal medicine, improvement activity in relation to blood lipid components and lipid metabolism such as inhibition of thrombosis, hyperlipidemia, hypercholesterolemia, hypertension, etc. A method for producing a black garlic extract composition is proposed.

Patent Registration No. 10-1191980 proposes a method for producing a black garlic extract with a high content of active ingredients and excellent decomposition extraction efficiency while minimizing heat loss by using an enzyme of black garlic. In this invention, after mixing black garlic and purified water, the enzyme is added, decomposed at 55 ° C. for 1 hour 30 minutes to 3 hours, and heat-treated at 95 ° C. for 10 minutes to inactivate the enzyme, then juiced, filtered, and concentrated again at low temperature. It is manufactured through a process, but three commercially available enzymes are used, and there is no example of an increase in active ingredient or extraction efficiency. In this method, the production cost increases due to the enzyme added by 0.5%, and there is a high possibility that the process will not be smooth due to the clogging of the filter during the extraction process after enzyme decomposition. .

Patent Registration No. 10-1105471 proposes a method for improving functionality and enhancing sensory characteristics by adding plum concentrate and agaricus mushroom mycelium extract in the preparation of black garlic extract. In the patent for conger eel drink with black garlic, black ginseng, and black jujube added, and its manufacturing method (Registration Patent No. 10-11181920), black ginseng is prepared in the same manner as black garlic, and then added to conger eel and extracted to improve sensory in the manufacture of conger eel drink. , discloses a method for enhancing functionality.

In this way, attempts have been made to enhance the various functions of black garlic and improve the unique taste of black garlic by mixing sub-materials. More development is needed.

Therefore, in the present invention, black garlic extract can be obtained by heating by adding an appropriate amount of water according to a conventional method. However, when extracted under the conditions presented in the present invention, the content of total phenolic compounds and SAC, which are representative active ingredients of black garlic extract, is conventional. An object of the present invention is to provide an extract higher than the extract and a black garlic beverage comprising the same.

In addition, an object of the present invention is to provide a composition for promoting men's health with enhanced sensory characteristics and functionality by mixing black garlic extract and black ginseng.

One aspect of the present invention for solving the above problems provides a black garlic extract obtained by adding 3 to 4 times the weight of black garlic and extracting it at 80 to 100° C. for 400 to 800 minutes.

Another aspect of the present invention provides a black garlic beverage comprising the black garlic extract.

Another aspect of the present invention provides a composition for promoting male health comprising the black garlic extract and black ginseng extract or black ginseng powder. Preferably, the black garlic extract is 93 to 97% by weight, the black ginseng powder is 3 to 7% by weight, and the concentration of the black garlic extract is 30 brix.

The composition for promoting male health may further include honey. Preferably, the black garlic extract is 78 to 82% by weight, the honey is 12 to 17% by weight, the black ginseng powder is 1 to 5% by weight, and the concentration of the black garlic extract is 30 brix.

According to the present invention, it is possible to provide a black garlic extract having a high content of total phenolic compounds and SAC as active ingredients, and a black garlic beverage containing the same.

In addition, according to the present invention, by mixing black garlic extract and black ginseng, etc., the content of active ingredients, increase in anti-inflammatory and antioxidant activity, increase in secretion of testosterone, and increase in factors related to maintenance of erection are also helpful in promoting male health. A composition comprising black garlic extract may be provided.

1 is a graph showing the evaluation of the viability of RAW 264.7 cells of 5 kinds of extracts by concentration according to an embodiment of the present invention.
Figure 2 is a graph showing the NO production inhibitory activity evaluation results for RAW 264.7 cells of 5 kinds of extracts by concentration according to an embodiment of the present invention.
3 is a graph showing the results of evaluation of ROS production inhibitory activity on RAW 264.7 cells of 5 kinds of extracts by concentration according to an embodiment of the present invention.
4 is a graph showing the evaluation results of Phosphodiesterase (PDE) inhibitory activity of 5 extracts by concentration according to an embodiment of the present invention.
5 is a graph showing the evaluation results of the viability of 5 types of TM3 cells of the extract according to the concentration according to an embodiment of the present invention.
6 is a graph showing the survival effect in TM3 cells for the treatment with 5 kinds of hydrogen peroxide extracts by concentration according to an embodiment of the present invention.
7 is a graph showing the amount of testosterone secretion in TM3 cells treated with 5 types of extracts by concentration according to an embodiment of the present invention.

One aspect of the present invention provides a black garlic extract obtained by adding 3 to 4 times the weight of black garlic and extracting it at 80 to 100° C. for 400 to 800 minutes.

Another aspect of the present invention provides a black garlic beverage comprising the black garlic extract.

Another aspect of the present invention provides a composition for promoting male health comprising the black garlic extract and black ginseng extract or black ginseng powder. Preferably, the black garlic extract is 93 to 97% by weight, the black ginseng powder is 3 to 7% by weight, and the concentration of the black garlic extract is 30 brix.

The composition for promoting male health may further include honey. Preferably, the black garlic extract is 78 to 82% by weight, the honey is 12 to 17% by weight, the black ginseng powder is 1 to 5% by weight, and the concentration of the black garlic extract is 30 brix.

Hereinafter, the present invention will be described in more detail with reference to examples of the present invention. The following examples are merely examples for explaining the present invention, and the scope of the present invention is not limited to these examples.

[ Example 1] According to the central synthesis plan black garlic Optimization of extraction conditions

In order to optimize the extraction conditions of black garlic, extraction was carried out for 360 to 668 minutes at 80-93° C., which is used for conventional extraction conditions of black garlic. At this time, it was confirmed that the amount of water had a relatively low effect on the properties of the extract compared to temperature or time in the results of previous studies. Therefore, the amount of water was usually carried out in the range of 3 to 4 times applied during extraction. Experimental conditions for optimization of extraction conditions are shown in Table 1 below. In the preparation of samples under various conditions, a response surface analysis method based on the central synthesis programming method, which is a statistical method, was applied.

[Table 1]

(1) Physicochemical analysis of black garlic extract by central synthesis scheme

According to the conditions in Table 1, 11 types of samples with different temperature and time conditions were prepared, respectively, and an extract filtered with a filter paper was used as a sample. The solid content of the extract was measured using an automatic refraction saccharometer and expressed as brix. The content of total phenolic compounds in the extract was quantified using the Foiln-Denis method, which is the principle that a phenolic substance reacts with phosphomolybdic acid to produce a blue color. To 1 mL of the sample solution, 1 mL each of Foline-Ciocalteau reagent (Sigma-Aldrich Co., St. Louis, MO, USA) and 10% _{NaCO 3} solution (Daejung, Siheung, Gyenggi, Korea) were sequentially added, followed by 1 mL at room temperature. After standing for time, absorbance was measured at 760 nm with a spectrophotometer (Libra S 35, Biochrom, Cambridge, England). Using gallic acid (Sigma-Aldrich Co., St, Louis, MO, USA) as a standard material, the content of total phenolic compounds was calculated from the calibration curve obtained by analyzing in the same manner as the sample.

The S-allyl-cysteine (SAC) content was analyzed by filtration with a 0.22 μm syringe filter and HPLC-PDA-MS/MS (TSQ Quantum LC-MS/MS, Thermo scientific, Waltham, MA, USA). Agilent Zorbax SB-C ₁₈ (4.6×250 mm, 5 μm) was used as the analytical column, and mobile phase A (0.1% formic acid containing water) and B (0.1% formic acid containing acetonitrile) were used as the mobile phase solvent in positive mode. The analysis was performed while varying the mixing ratio according to time. The speed of the mobile phase was 0.7 mL/min, the sample injection amount was 10 μL, and the scan type was SRM mode. The content of SAC in the extract was quantified by analyzing the standard material for each concentration under the same conditions as the sample. The analysis results are shown in Table 2 below.

[Table 2]

As shown in Table 2, the solid content was in the range of 9.3 to 14.70 brix, the total phenolic compound content was 80.89 to 125.60 mg/100g, and the SAC content, one of the main active ingredients of the black garlic extract, was 10.91 to 30.53 mg/100g. was the range. The analyzed physicochemical properties were higher with higher temperature and longer extraction time.

(2) Polynomial based on black garlic extraction conditions and analysis results

From the physicochemical analysis results in Table 2 above, the variables corresponding to the extraction conditions, X ₁ (extraction temperature, ℃), X ₂ (extraction time, minutes), and the analysis results, Y ₁ (brix), Y ₂ (total phenol), A reaction equation based on the correlation between Y ₃ (SAC) is shown in Table 3.

[Table 3]

From Table 3 above, it can be seen that there is a correlation between the extraction conditions and the result value given that the R ² value is 80% or more for all of the solid content, total phenolic compounds, and SAC.

(3) Results of analysis of commercially available black garlic and expected under optimal extraction conditions

The above analysis results confirmed the optimal conditions for preparing the black garlic extract through comparison with the commercially available black garlic extract. The average value was obtained by analyzing 6 commercially available black garlic extracts under the same method and conditions as those of this extract, and the results of optimizing the production conditions for the black garlic extract to be developed in the present invention are shown in Table 4 below.

[Table 4]

In Table 4, extraction condition 1 is a value obtained when extraction is performed at 86.6° C. for 720 minutes, and extraction condition 2 is a value obtained by extraction at 92.7° C. for 412 minutes. Under both extraction conditions, an extract with an increased SAC content by 20% compared to the commercial product can be obtained. .

In addition, if the total phenolic compound content is maintained the same as that of the commercial product, but the solid content is to be increased by about 1.7 times compared to the commercial product, it can be extracted according to extraction condition 2.

In addition, in both conditions, when the extraction temperature is relatively low as low as 87 ℃, the time should be maintained long, but the optimal time can be confirmed. It can be seen that it can be eluted.

The liquid extracted under the above conditions is similar to or thicker than the commercially available black garlic extract, so it can be prepared as a beverage only with the extract. In addition, in order to develop into another formulation, it can be concentrated and used to increase the concentration.

[ Example 2] black garlic Preparation of a composition based on the extract

In order to enhance the functional and sensory properties of black garlic extract, a composition with additional ingredients was prepared, and a blending ratio was set for using it as a beverage. Functionality can be enhanced by adding various sub-materials, but in the present invention, by proposing a composition serving as a base to which other sub-materials can be additionally added, it was intended to provide the possibility of developing additional products.

Black ginseng and honey were used as mixing materials for the base composition. Black ginseng may be used as an extract, or dried and powdered. In the present invention, powder was used to mix with black garlic extract to prepare a highly concentrated stick drink. Table 5 shows the composition base, which is a mixture of black garlic extract concentrate with a solid content of 30 brix, black ginseng powder, and honey. Formulation 1 is a control to confirm the effect of black garlic extract, Formulation 2 is one of the basic compounding ratios of the composition to be proposed in the present invention, and Formulation 3 is one of the controls to confirm the effect of adding black ginseng. In the table below, the proportions are % by weight.

[Table 5]

(1) Total phenolic compound content and cholesterol adsorption activity of black garlic extract and black ginseng powder mixture composition

The total phenolic compound content was analyzed according to the same method as the total phenolic compound content analysis method by taking 1 mL of the composition. Cholesterol adsorption activity was measured with a kit reagent (AM 202-k, Asan Pharm., Seoul, Korea). Add 50 µL of cholesterol (300 mg/dL) to 1 mL of the sample solution, react at 25°C for 20 minutes, and then add 50 µL of 0.1 M hexadecyl-trimethylammonium bromide (Sigma Co., St Louis, MO, USA) to 25,000×g. was centrifuged for 15 min. After taking 200 μL of the supernatant, adding 1.5 mL of the enzyme solution, mixing, and reacting at 37°C for 5 minutes, the absorbance was measured at 500 nm. it was

The results of analysis of total phenolic compounds and cholesterol adsorption activity are shown in Table 6 below.

[Table 6]

From Table 6, the total phenolic compound content of the formulation 1 sample without the addition of black garlic extract was 51.18 μ, the formulation 3 without the addition of black ginseng was 50.26 μ, which did not show a significant difference, whereas the formulation 2 in which the two samples were mixed was 139.33 μ. It increased about 2.7 times compared to formulations 1 and 2. The fact that the two samples showed a synergistic effect on the content is thought to indicate that the phenolic compounds present in trace amounts were mixed with each other and quantification became possible as the amount increased, resulting in an increased content.

Cholesterol adsorption activity was 24.75% for Formulation 1 and 22.9% for Formulation 3, whereas Formulation 2 was 52.62%, which was approximately 2.1 times higher than that of Formulations 1 and 2, which was consistent with the analysis results of total phenolic compounds.

(2) ABTS and DPPH radical antioxidant activity of black garlic extract and black ginseng powder mixture composition

Among the antioxidant activity, DPPH (1,1-Diphenyl-2-picrylhydrazyl) radical scavenging activity is expressed as electron donating activity for DPPH. After mixing the sample solution and DPPH solution (5 mg/100 mL methanol) in equal amounts, the After reacting for a minute, absorbance was measured at 525 nm using a spectrophotometer.

The radical scavenging activity of ABTS (2,2-azinobis-(3-ethylbenzo-thiazoline-6-sulphonate) was determined by adding potassium persulfate (Sigma-Aldrich Co., St. Louis, MO, USA) to 7 mM ABTS solution at 2.4 mM. After dissolving as much as possible, and reacting in the dark for 12 to 16 hours, an ABTS solution adjusted with distilled water so that the absorbance at 415 nm becomes 1.5 was mixed 50 μL of the sample solution was mixed with 150 μL of ABTS solution, reacted at room temperature, followed by spectroscopy Absorbance was measured at 415 nm using a photometer.

The DPPH and ABTS radical scavenging activities were calculated as the absorbance ratio of the sample-added group to the non-sample-added group and expressed in %, and the results are shown in Table 7 below.

[Table 7]

Referring to Table 7, the antioxidant activity according to the blending ratio was different in activity depending on the blending ratio, and the activity of blend 2 was higher than blends 1 and 3 in which only black garlic and black ginseng were added. At a concentration of 100 mg/g, the ABTS radical scavenging activity of Formulation 1 was 54.02%, whereas in Formulation 2, in which the two samples were mixed, it increased more than twice to 97.11%. In addition, formulation 3 containing only black ginseng was 53.76% lower than the sample in which the two samples were mixed.

From the above results, compared to the case of using black garlic extract or black ginseng alone, it was confirmed that the content of active ingredients such as phenolic compounds or SAC increased due to the mixing of the two components, which resulted in increased antioxidant activity. In most of the results, if the content or activity of the two samples is large, mixing the two components increases the activity of the sample with a lower content and lower activity, but increases the content or activity of the active ingredient. The activity of the excellent sample is rather lowered, and the content or activity of the active ingredient reaches an average value.

However, in the present invention, a synergistic effect was exerted by mixing ingredients with no significant difference in activity, thereby showing a positive result in which the active ingredients or activities of the two components were further increased.

[ Example 3] black garlic Preparation of samples for verification of activity at the cellular level of compositions based on extracts

To verify the physiological activity of black garlic and black ginseng and their composition at the cellular level, black garlic extract and black ginseng extract extracted under the same conditions as black garlic were each freeze-dried and then powdered. According to the weight ratio of freeze-dried black garlic extract powder (B. garlic) and black ginseng extract powder (B. ginseng), respectively, 97:3 (Mix 1), 95:5 (Mix 2), 93:7 (Mix 3) Three types of compositions mixed in a ratio were prepared as samples, and purified water was added to make a constant concentration and used for cell experiments.

(1) Cytotoxicity evaluation for verification of activity against RAW 264.7 macrophages

The RAW 264.7 cells used in the experiment were purchased from the Korea Cell Line Bank (KCLB, Korea), and for cell culture, DMEM containing 10% fetal bovine serum (FBS, Hyclone, Loran, UT, USA) and 1% penicillin-streptomycin (dulbecco's modied eagle's medium) medium was used.

In order to set the concentration of the sample effective for the experiment, the toxicity to the cells was first confirmed. For cytotoxicity evaluation, RAW 264.7 cells were cultured in a CO ₂ incubator (37℃, 5% CO ₂ ), and the cell toxicity of the extract was measured with 3-(4,5-dimethylthiazole-2-yl)-2,5- Diphenyltetrazolium bromide (MTT, Gibco, Waltham, MA, USA) was measured using the reduction method. ^{Cells were aliquoted to 5×10 4} cells per well in a 96 well-plate and adhered for 24 hours. The extracts were diluted to a constant concentration and treated with lipopolysaccharide (LPS, Sigma) at a concentration of 1 μg/mL after 30 minutes. -Aldrich Co., St. Louis, MO, USA) was treated and cultured for 24 hours. After removing the medium containing the sample, serum-free medium and MTT solution at a concentration of 5 mg/mL were added, and incubated at 37°C for 2 hours, then DMSO was dispensed, sonicated, and the absorbance was measured at 570 nm after stirring for 10 minutes. was measured to determine the cell viability. Cell viability was expressed as a percentage of the untreated group.

As a result of processing RAW 264.7 cells with black garlic, black ginseng, and three types of mixed extracts at each concentration (0, 100, 200, 400 and 800 μg/mL) for 24 hours (Fig. 1), all extracts up to 800 μg/mL concentration range It was confirmed that the sample did not induce cytotoxicity compared to the control. Therefore, the treatment concentration of the highest sample for the experiment was set to 800 μg/mL or less.

A graph showing the evaluation of the viability of RAW 264.7 cells of 5 extracts by concentration is shown in FIG. 1 . In FIG. 1, all sample values were expressed as mean±deviation values for the results of three repeated experiments under the same conditions.

(2) Evaluation of anti-inflammatory activity using RAW 264.7 macrophages

Anti-inflammatory activity was evaluated through the inhibition rate of nitric oxide (NO) production in RAW 264.7 macrophages. RAW 264.7 cells, a mouse macrophage cell line, ^{were dispensed in a 24-well plate at a concentration of 5×10 5} cells/well and cultured for 24 hours, and then each extract was treated by concentration. After incubating this for 30 minutes, it was treated with LPS (1 μg/mL) and cultured for 20 hours or 24 hours, and then the cell supernatant was recovered. After centrifugation (1,000 rpm, 10min, 4℃) of the recovered cell culture supernatant, take 50 μL, mix with 50 μL of sulfanilamide solution, block light for 5 minutes, react, and mix with 50 μL of NED solution at room temperature for 10 minutes. After reacting for minutes, absorbance was measured at 540 nm using an ELISA reader (Epoch, Bioteck, Winooski, VT, Germany), and it was expressed as a relative production rate for the LPS-only treated cell group.

The results of measuring the nitric oxide (NO) production inhibitory activity of black garlic, black ginseng and three types of mixed extracts in RAW 264.7 cells treated with LPS are shown in FIG. 2 . RAW 264.7 cells treated with LPS increased NO production compared to the control group, and when each of the five extracts was treated, the amount of NO production significantly decreased as the concentration of the sample increased at a concentration of 100 to 800 μg/mL (p <0.05). The NO production inhibitory effect of each extract was highest in the black ginseng extract at the concentration of 800 μg/mL, and the remaining extracts were in a similar range.

A graph for the evaluation of NO production inhibitory activity against RAW 264.7 cells of 5 types of extracts by concentration is shown in FIG. 2 . All sample values were expressed as mean±deviation values for the results of three repeated experiments under the same conditions. (*, p<0.05; **, p<0.01; ***, p<0.001).

NO (nitric oxide) is known as an inflammatory response mediator secreted when cells stimulated by inflammatory substances such as LPS or TNF-α mainly induce inflammation. Such NO is produced through the L-argininenitric oxide pathway in which the guanidino nitrogen of L-arginine is converted to L-citrulline by oxidation. It is known that a large amount of NO is produced by the activity of the inducible nitric oxide synthase (iNOS) enzyme in epithelial cells and increases the inflammatory response.

(3) Evaluation of antioxidant activity using RAW 264.7 macrophages

Antioxidant activity was evaluated at the cellular level by measuring the intracellular reactive oxygen species (ROS) of the extract. ^{5×10 4} cells/well of RAW 264.7 cells were aliquoted in a 96-well black plate and cultured, followed by exchange with serum-free DMEM medium. After culturing the cells for 24 hours, the samples were treated by concentration and cultured for 24 hours at 37° C., 5% CO ₂ condition. After washing 3 times with PBS (pH 7.4), add 100 μL of 1X dichloro-dihydro-fluorescein diacetate (DCFH-DA) to the medium, incubate at 37°C, 5% CO ₂ for 1 hour, and then wash 3 times with PBS again paid After adding and mixing 100 μL of lysis buffer, fluorescence was measured at excitation 485 nm and emisssion 535 nm using a fluorescence microplate reader, and the relative inhibition of ROS generation compared to the LPS-only treatment group was shown.

The ROS inhibitory activity by the five extracts used the principle of green fluorescence observed when ROS increased in cells reacted with DCF-DA. The results of confirming the inhibition of ROS production by black garlic, black ginseng, and three types of mixed extracts after induced oxidative stress by treating RAW 264.7 cells with LPS are shown in FIG. 3 . In FIG. 3 , all sample values were expressed as mean±deviation values for the results of three repeated experiments under the same conditions. (*, p<0.05; **, p<0.01; ***, p<0.001)

Referring to FIG. 3 , as a result of analyzing cells stained with DCF-DA using a fluorescence microplate reader, all five samples significantly reduced ROS generation at a concentration of 800 μg/mL (p<0.05). As in the previous inhibition of NO production, the ROS production inhibition rate of black ginseng extract was the highest at 800 μg/mL concentration, followed by Mix 3.

(4) Phosphodiesterase (PDE) inhibitory effect measurement

The PDE inhibitory effect was measured using a cyclic nucleotide phosphodiesterase assay kit. Using 3,5-cGMP as a substrate (20 μL), 5 μL of PDE (4 mU/μL) and 10 μL of each extract were added and reacted, then treated with 10 μL of 5-nucleotidase and mixed. 100 μL of BIOMOL GREEN reagent was treated and reacted at room temperature for 30 minutes, and then absorbance was measured at 620 nm. 5-GMP was expressed as PDE inhibition (%) using a standard curve.

When the penis is erect, cGMP acts to dilate blood vessels, which is broken down by PDE. Therefore, it is known that erectile function is improved only when the concentration of cGMP is increased by inhibiting the activity of PDE. From this point of view, the results of evaluating PDE inhibitory activity in order to evaluate the effect of black garlic black ginseng extract and three mixtures thereof on maintaining erection are shown in FIG. 4 . In FIG. 4 , all sample values were expressed as mean±deviation values for the results of three repeated experiments under the same conditions. (*P < 0.05, **P < 0.01 and ***P < 0.001 compared to control)

Referring to FIG. 4 , when the extracts were treated at a concentration of 800 μg/mL, the inhibition rate of PDE was increased by 95.54% in the case of black garlic extract powder compared to the control group, and by 70.81% in the case of black ginseng. Among the three mixtures, Mix 1, which had the highest content ratio of black garlic, had a high PDE inhibition rate of 73.06%, and other mixtures were also confirmed to have similar activity to the group treated with black ginseng extract alone.

(5) Cytotoxicity to TM3 cell proliferation

TM3 cells are cells that produce testosteron, a representative male hormone in the testis. Cytotoxicity was first evaluated to analyze the effect of black garlic extract, which is the main raw material of the present invention, black ginseng extract, which is a subsidiary material, and their composition on male hormone secretion.

Leydig cells, TM3 cells, were maintained _{at 37°C and 5% CO 2} in DMEM medium supplemented with 10% (v/v) fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (100 μg/mL). while culturing. Cells were aliquoted at a cell concentration of 2.5 × 10 ⁵ cells/mL in a 96-well plate, and then cultured for 24 hours for cell adhesion. Samples were treated by concentration to confirm cell cytotoxicity. Cell viability was measured using MTT assay. Absorbance was measured at 570 nm, and the measurement result was expressed as a percentage compared to the cell viability of the untreated group.

In order to confirm the cell viability of black garlic, black ginseng, and Mix 3, the results of MTT assay on TM3 cells are shown in FIG. 5 . All the tested samples did not cause a significant decrease in survival rate compared to the untreated control up to the highest concentration of 800 μg/mL, so the following experiments were conducted at concentrations below this level. The viability evaluation results for the TM3 cells of 5 types of extracts by concentration are shown in FIG. 5 , where all sample values are expressed as mean±deviation values for the results of three repeated experiments under the same conditions.

(6) Cytoprotective effect of TM3 cells and intracellular testosterone content

To measure TM3 cell protection, oxidative stress was applied to TM3 cells with hydrogen peroxide to measure the protective effect of the extract. ^{TM3 cells were cultured for 24 hours by dispensing 1×10 5} cells/well cells in a 48 wells plate, removing the medium, and dispensing 0.5 mL of a medium containing 3% serum in which the extract was dissolved and cultured for 24 hours. After removal of the culture medium, a culture solution containing 300 μM hydrogen peroxide and extract was dispensed and cultured for 24 hours, after which the medium was removed. The cytoprotective effect was measured using the MTT assay. Absorbance was measured at 570 nm, and the measurement result was expressed as a percentage compared to the cell viability of the 300 μM hydrogen peroxide treatment group.

Testosterone content in TM3 cells was measured with TM3 cell culture medium. ^{TM3 cells were cultured for 24 hours by dispensing 1×10 5} cells/well of cells in a 48 wells plate, removing the medium, and dispensing 0.5 mL of medium treated with extracts by concentration, and incubating for 3 days. After the culture was completed, the testosterone content of the medium was measured using an assay kit. Transfer 100 μL of medium to goat anti-mouse IgG microtiter plate, add 50 μL of testosterone EIA antibody, incubate for 1 hour, add 50 μL of conjugate, incubate for 1 hour, and wash with washing solution. 200 μL of pNpp substrate solution was added to each, and after reaction for 1 hour, 50 μL of stop solution was added to stop the reaction, and absorbance was measured at 405 nm to calculate the content using a testosterone standard curve.

Five types of extracts were treated in TM3 cells at concentrations of 100, 200, 400 and 800 μg/mL, respectively, and after culturing for 3 days, the content of testosterone secreted from the cells into the culture was measured ( FIG. 7 ). As a result, the testosterone content in the untreated group was 0.86 ng/mL, whereas in the case of 800 μg/mL black ginseng extract, it increased 3.59 times to 3.09 ng/mL. And as the ratio of black ginseng increased in the three types of mixture, the content of testosterone increased.

The results of evaluating the effects of the five extracts on the cytoprotective ability against hydrogen peroxide and the testosterone content in TM3 cells to determine whether the five extracts can alleviate the symptoms of decreased testosterone production and the protective effect against oxidative stress, respectively. 6 and 7 are shown.

In FIG. 6 , all sample values were expressed as mean±deviation values for the results of three repeated experiments under the same conditions. * indicates that there is a significant difference in the range of P < 0.05 for the control group, and in FIG. 7 , all sample values are expressed as the mean±deviation value for the results of three repeated experiments under the same conditions, and compared with the control group, * indicates P In < 0.05, ** marks have a significant difference in the range of P < 0.01.

Referring to FIG. 6 , after inducing oxidative stress by treating TM3 cells with 0.3 mM hydrogen peroxide, the five extracts were treated at concentrations of 100, 200, 400, and 800 μg/mL. As a result, the group treated with only hydrogen peroxide was the untreated group. The cell viability was reduced by 32.47%. On the other hand, the case where each of the five extracts was treated at 800 μg/mL showed a relatively high cytoprotective ability compared to the group treated with only hydrogen peroxide.

Referring to FIG. 7 , TM3 cells are cells that synthesize cholesterol into testosterone in the interstitium of the testis, and testosterone reduction and cell viability are reduced by stimulation of reactive oxygen species. This decrease in testosterone and cell viability leads to decreased erectile function.

Since the five extracts have a cytoprotective effect against the oxidative stress of TM3 cells caused by hydrogen peroxide, it is judged that the result of increasing the testosterone content was shown. These results are consistent with the study that oxidative stress-induced cell damage and testosterone levels were improved when the extract was treated, and that the extracts suppressed oxidative stress in testicular cells and increased intracellular hormone synthesis. Therefore, it is judged that side effects such as erectile dysfunction and symptoms caused by a decrease in testosterone content can be alleviated when finasteride, which is used as a treatment for benign prostatic hyperplasia, is taken.

The above description is merely illustrative of the present invention, and various modifications and variations will be possible without departing from the essential characteristics of the present invention by those skilled in the art to which the present invention pertains. Therefore, the embodiments disclosed in the present invention are not intended to limit the technical spirit of the present invention, but to explain, and the scope of the technical spirit of the present invention is not limited by these embodiments. The protection scope of the present invention should be construed by the following claims, and all technical ideas that are equivalent to or within the equivalent range should be construed as being included in the scope of the present invention.

Claims (6)

It consists of black garlic extract, honey and black ginseng powder.
A composition for promoting male health with a total phenolic compound content of 130 μg/g or more and a cholesterol adsorption activity of 50% or more,
The weight ratio of the black garlic extract: honey: black ginseng powder is 80:15:5,
The black garlic extract was concentrated to 30 brix of 20 brix of black garlic extract obtained by adding 3 to 4 times the weight of black garlic, followed by extraction at 90 to 95 ° C. for 400 to 430 minutes,
The composition for men's health promotion, characterized in that the total phenolic compounds contained in the 20 brix black garlic extract is 99 mg/100g or more, and the S-allyl-cysteine is 12 mg/100g or more. The method of claim 1,
The black garlic extract is a composition for men's health promotion, characterized in that it is extracted at 92.7 ℃ for 412 minutes. delete delete delete delete

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