KR20210024726A - 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, and a black garlic drink and a male health enhancing composition containing the same. More specifically, disclosed in the present invention are: a black garlic extract having a high content of total phenolic compounds and SAC; a black garlic drink containing the same; and a male health enhancing composition having a higher activity compared to each activity of the black garlic extract, and a black ginseng extract or black ginseng powder by mixing the black ginseng extract or the black ginseng powder with the black garlic extract.

Description

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 beverage containing the same, and a composition for promoting male health, and more particularly, a black garlic extract having a high content of total phenolic compounds and SAC, a black garlic beverage containing the same, and a black garlic extract containing the black garlic extract. Or, by mixing black ginseng powder, it relates to a composition for promoting male health that has a higher activity than that of each.

Black garlic appears dark black due to a non-enzymatic browning reaction during the process of aging raw garlic at a temperature of 80~90℃ for a long time. This mechanism is due to the removal of free radicals and nitrogen-containing compounds due to the reducing component of melanoidins, a brown substance. It is due to the movement of protons from the glass electron band of the nitrogen atom, and the brown substances generated by the Myyal reaction after exposure to high temperatures for a long time are gathered and become black.

Black garlic's strong taste and irritating odor are removed, sugars are decomposed, and it has a sweet and sour taste by harmonizing with organic acids. It has a texture similar to jelly, which is advantageous in consumption compared to raw garlic. In addition, the content of polyphenol compounds, which is a representative functional component present in plants, increases during the ripening process, and S-allylcysteine (SAC), a water-soluble sulfur compound, has functions such as antioxidant, cancer cell proliferation, cognitive enhancement, and liver protection. . Therefore, black garlic is known to have functions derived from phenolic compounds and SAC, as well as functionalities such as improving blood lipid concentration, reducing blood cholesterol, recovering from fatigue, and enhancing endurance, which are unique activities of garlic.

In black ginseng, the color of ginseng has turned black through several steaming and drying processes, and 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 cranial nerve protection, anti-cancer, immune enhancing action, and obesity suppression, and its content is increased compared to fresh ginseng or red ginseng.

Black garlic and black ginseng have differences in manufacturing process, but they are representative black foods whose color has turned black by reacting amino acids and sugars contained in their own ingredients to produce a large amount of browning substances through the same heat treatment process. All of these foods have been proven to have many functionalities through several studies.

However, black garlic has its own characteristic scent, and it hardens during storage due to a large amount of sugars in its own ingredients.Therefore, it is limited to use as a 100% powder, so it is mainly consumed as black garlic itself or in the form of an extract.

Black ginseng retains its peculiar aroma and has a strong bitter taste, making it difficult to drink when extracted alone, and the higher the concentration, the stronger the bitter taste. In addition, when used as a powder, the bitter taste is not removed, and powdering is advantageous because it contains relatively less sugar than black garlic, but there is a disadvantage in that the unit cost is high due to more processes than red ginseng.

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

As prior inventions in which attempts were made to mix black garlic and black (red) ginseng, Korean Patent Publication No. 10-2009-0068960 proposes a method for producing antioxidant health supplements containing black garlic and red ginseng extract. After pulverizing each red ginseng, 5 times more water is added to the lyophilized powder, mixed at room temperature in a shaking incubator for 14 hours, and then centrifuged to obtain an extract with increased antioxidant activity by taking only the supernatant.

In the manufacturing method of black garlic extract containing raw material (Patent Publication No. 10-2018-0136680), three times the amount of water is added to black garlic, the first extraction is performed at 85 to 95°C for 5 to 10 hours, and the residue is extracted again, and then the residue is added to the black garlic. By mixing peony, prickly pear, and sweet ginseng for 3 to 6 hours at 80-95℃ to prepare an extract mixed with herbal substances, it has improved activity in relation to lipid components or lipid metabolism in blood such as blood clot formation inhibition, hyperlipidemia, hypercholesterolemia, and hypertension. It is proposed a method of manufacturing black garlic extract composition.

Registration Patent No. 10-1191980 proposes a method for preparing black garlic extract with high content of active ingredients and excellent decomposition extraction efficiency while minimizing heat loss by using enzymes of black garlic. In this invention, after mixing black garlic and purified water, enzyme is added, decomposed at 55°C for 1 hour 30 minutes to 3 hours, heat treated at 95°C for 10 minutes to deactivate the enzyme, then juiced, filtered and concentrated again at low temperature. It is manufactured through a process, but uses three commercially available enzymes, and does not present an example of an increase in the active ingredient or extraction efficiency. In this method, production costs increase due to the enzyme added by 0.5% increments, and it is highly likely that the process is not smooth due to clogging of the filter during the juice process after enzymatic decomposition. .

Registered Patent No. 10-1105471 proposes a method of improving functionality and enhancing sensory properties by adding plum concentrate and agaricus mushroom mycelium extract in preparing black garlic extract. Conger eel drink with black garlic, black ginseng, and black jujube added, and its manufacturing method (Registration Patent 10-1181920), in the patent, extracting black ginseng in the same manner as black garlic and then adding it to conger eel to improve sensory performance , Discloses a method of enhancing functionality.

By mixing the subsidiary materials in this way, attempts have been made to enhance the various functions of the existing black garlic and to improve the unique taste of black garlic, but a study on the black garlic extract with an increased content of the active ingredient and a composition for promoting male health using the same Further 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 suggested in the present invention, the total phenolic compounds and SACs, which are representative active ingredients of the black garlic extract, are conventional. It is an object to provide a higher extract than the extract and a black garlic beverage comprising the same.

In addition, it is an object of the present invention to provide a composition for promoting male health in which sensory properties and functionality are enhanced 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 water 3 to 4 times the weight of black garlic and extracting for 400 to 800 minutes at 80 to 100°C.

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 comprising the same.

In addition, according to the present invention, by mixing black garlic extract and black ginseng, etc., it is helpful in promoting male health by inducing an increase in the content of active ingredients, an increase in anti-inflammatory and antioxidant activity, and an increase in the secretion of testosterone and factors related to maintaining erection. It is possible to provide a composition comprising a black garlic extract.

1 is a graph showing the survival rate evaluation for RAW 264.7 cells of five 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 five kinds of extracts according to an embodiment of the present invention.
Figure 3 is a graph showing the ROS production inhibitory activity evaluation results for RAW 264.7 cells of 5 kinds of extracts according to an embodiment of the present invention.
4 is a graph showing the results of evaluating the inhibitory activity of Phosphodiesterase (PDE) of five extracts by concentration according to an embodiment of the present invention.
5 is a graph showing the results of evaluating the viability of TM3 cells of five types of extracts according to concentrations according to an embodiment of the present invention.
6 is a graph showing the survival effect in TM3 cells for treatment with hydrogen peroxide of five kinds of extracts according to concentrations according to an embodiment of the present invention.
7 is a graph showing the amount of testosterone secretion in TM3 cells treated with five extracts by concentration according to an embodiment of the present invention.

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

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 by way of examples of the present invention. The following examples are only 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 performed for 360-668 minutes at 80-93°C, which is used in conventional black garlic extraction conditions. At this time, it was confirmed that the water content had a relatively low effect on the properties of the extract compared to the temperature or time in the previous study results. Therefore, the amount of water was usually carried out in the range of 3 to 4 times applied during extraction. Experimental conditions for optimizing the extraction conditions are shown in Table 1 below. In preparing samples under various conditions, the response surface analysis method based on the central synthesis planning method, which is a statistical method, was applied.

[Table 1]

(1) Physicochemical Analysis of Black Garlic Extract by Central Synthesis Plan

Each of 11 samples having different temperature and time conditions according to the conditions in Table 1 were prepared, and then the extract filtered with a filter paper was used as a sample. As for the extract, the solid content was measured using an automatic refracting sugar meter 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 give a blue color. To 1 mL of the sample solution, 1 mL of Foline- Ciocalteau reagent (Sigma-Aldrich Co., St. Louis, MO, USA) and 10% _{NaCO 3} solution (Daejung, Siheung, Gyenggi, Korea) were sequentially added, and then 1 mL at room temperature. After standing for a period of time, the 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 total phenolic compound content was calculated from the calibration curve obtained by analyzing in the same manner as the sample.

S-allyl-cysteine (SAC) content was analyzed by HPLC-PDA-MS/MS (TSQ Quantum LC-MS/MS, Thermo scientific, Waltham, MA, USA) by filtering the sample with a 0.22 μm syringe filter. 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 in positive mode. Analysis was performed while varying the mixing ratio according to time. The mobile phase speed was 0.7 mL/min, the sample injection volume was 10 μL, and the scan type was analyzed in SRM mode. The content of SAC in the extract was quantified by analyzing the standard substance 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 ranged from 9.3 to 14.70 brix, and 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. It was a 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, variables corresponding to extraction conditions, X ₁ (extraction temperature, ℃), X ₂ (extraction time, minutes), and analysis results, Y ₁ (brix), Y ₂ (total phenol), The _{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 the solid content, total phenolic compounds, and SAC have R ² values of 80% or more, and there is a correlation between the given extraction conditions and the result values.

(3) Commercial black garlic analysis results and predicted values under optimal extraction conditions

The above analysis results confirmed the optimum conditions for preparing the black garlic extract through comparison with the commercially available black garlic extract. Six commercially available black garlic extracts were analyzed in the same method and conditions as the present extract to obtain an average value, and the results of optimizing the production conditions of 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 by extracting at 86.6°C for 720 minutes, and extraction condition 2 is a value obtained by extraction at 92.7°C for 412 minutes. In both extraction conditions, an extract with an increased SAC content of 20% compared to a commercial product can be obtained, and if it has the same concentration as the commercial product, but wants to increase the total phenolic compound content, it can be extracted according to extraction condition 1. .

In addition, the content of total phenolic compounds is maintained the same as that of commercial products, but if you want to increase the content of solids by about 1.7 times higher than that of commercial products, extraction can be performed according to extraction condition 2.

In addition, under the two conditions, if the extraction temperature is relatively low at about 87°C, the time should be maintained for a long time, but the optimum time can be checked. When the temperature is increased to about 93°C, all the desired components can be extracted for about 7 hours. It can be seen that it can be eluted.

The liquid extracted under the above conditions can be prepared as a beverage with only the extract because the concentration is similar or thick to the black garlic extract on the market. In addition, in order to develop into other formulations, it can be concentrated and used to increase the concentration.

[ Example 2] Black garlic Preparation of composition based on extract

In order to enhance the functionality and organoleptic properties of the black garlic extract, a composition containing additives was prepared, and a blending ratio for using it as a beverage was set. Although it is possible to enhance functionality by adding various subsidiary materials, in the present invention, by proposing a composition as a base to which other subsidiary materials can be added additionally, it is intended to provide the possibility of developing additional products.

Black ginseng and honey were used as a mixed subsidiary material for the composition as a base. Black ginseng may be used as an extract, or dried as a powder. In the present invention, powder was used in order to prepare a highly concentrated stick drink by mixing it with black garlic extract. Table 5 shows the composition base obtained by mixing black garlic extract concentrate, black ginseng powder, and honey with a solid content of 30 brix. Formulation 1 is a control for confirming the effect of the black garlic extract, Formulation 2 is one of the basic mixing ratios of the composition proposed in the present invention, and Formulation 3 is one of the control groups for confirming the effect of adding black ginseng. In the table below, the ratio is in weight percent.

[Table 5]

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

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 kit reagent (AM 202-k, AsanPharm., Seoul, Korea). 50 μL of cholesterol (300 mg/dL) was added to 1 mL of the sample solution, reacted at 25°C for 20 minutes, and 50 μL of 0.1 M hexadecyl-trimethylammonium bromide (Sigma Co., St Louis, MO, USA) was added to 25,000×g. Centrifuged at 15 minutes. Take 200 μL of the supernatant, add 1.5 mL of enzyme solution, mix, react at 37°C for 5 minutes, and then measure the absorbance at 500 nm, and show the cholesterol adsorption activity (%) as the absorbance ratio of the sample addition group to the sample-free group. I got it.

The results of analyzing the 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 in which the black garlic extract was not added was 51.18 μ, the formulation 3 in which the black ginseng was not added 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. Compared to Formulations 1 and 2, the increase was about 2.7 times. As the two samples were mixed, the synergistic effect on the content was seen as a result of a further increase in the content as phenolic compounds, which were present in trace amounts, were mixed with each other and quantified as the amount increased.

Cholesterol adsorption activity was 24.75% in Formulation 1 and 22.9% in Formulation 3, whereas Formulation 2 was 52.62%, which was about 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 mixed composition of black garlic extract and black ginseng powder

Among the antioxidant activities, DPPH (1,1-Diphenyl-2-picrylhydrazyl) radical scavenging activity is indicated by electron donating activity for DPPH. After mixing the sample solution and DPPH solution (5 mg/100 mL methanol) in equal amounts, 20 at room temperature. After reacting for a minute, absorbance was measured at 525 nm using a spectrophotometer.

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

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

[Table 7]

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

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

However, in the present invention, a synergistic effect was exerted by mixing components that do not have a significant difference in activity, thereby showing a positive result of further increasing the active ingredient or activity of the two components.

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

In order 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 lyophilized and powdered. Freeze-dried black garlic extract powder (B. garlic) and black ginseng extract powder (B. ginseng) were prepared in 97:3 (Mix 1), 95:5 (Mix 2), 93: 7 (Mix 3), respectively, depending on the weight ratio. Three kinds of compositions mixed at the ratio were prepared as samples, and purified water was added to make them at a constant concentration and used for cell experiments.

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

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

In order to set the concentration of the sample effective in 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°C, 5% CO ₂ ), and the toxicity of the extract to cells was 3-(4,5-dimethylthiazole-2-yl)-2,5- It was measured using diphenyltetrazolium bromide (MTT, Gibco, Waltham, MA, USA) reduction method. After dispensing the cells into a 96 well-plate ^{so as to be 5×10 4} cells per well and attaching them for 24 hours, the extracts were diluted to a certain concentration and treated on the cells. After 30 minutes, 1 μg/mL of lipopolysaccharide (LPS, Sigma -Aldrich Co., St. Louis, MO, USA) was treated and incubated for 24 hours. After removing the medium containing the sample, add serum-free medium and 5 mg/mL MTT solution, incubate at 37°C for 2 more hours, dispense DMSO, sonicate, and stir for 10 minutes, and then adjust the absorbance at 570 nm. The cell viability was determined by measurement. Cell viability was expressed as a percentage of the untreated group.

RAW 264.7 cells were treated with black garlic, black ginseng, and three kinds of mixed extracts at each concentration (0, 100, 200, 400 and 800 μg/mL) for 24 hours (Fig. 1), all up to 800 μg/mL concentration range. It was confirmed that the sample did not cause cytotoxicity compared to the control group. 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 survival rate for RAW 264.7 cells of 5 types of extracts by concentration is shown in FIG. 1. In FIG. 1, all sample values are expressed as mean±deviation values for the results of repeated experiments three times 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 into a 24-well plate at a concentration of 5×10 5} cells/well, cultured for 24 hours, and each extract was treated by concentration. After incubating for 30 minutes, LPS (1 μg/mL) was treated and cultured for 20 or 24 hours, and then the cell supernatant was recovered. After centrifuging the recovered cell culture supernatant (1,000 rpm, 10min, 4℃), take 50 μL and mix it with 50 μL of sulfanilamide solution, block the light for 5 minutes, react, and mix with 50 μL of NED solution at room temperature. After reacting for minutes, the absorbance was measured at 540 nm using an ELISA reader (Epoch, Bioteck, Winooski, VT, Germany), and it was expressed as a relative production rate to the LPS alone-treated cell group.

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

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

NO (nitric oxide) is known as a mediator of the inflammatory response secreted when cells stimulated by inflammatory substances such as LPS or TNF-α induce inflammation. The production of NO is produced through the L-argininenitric oxide pathway in which guanidino nitrogen of L-arginine is converted into L-citrulline by oxidation.Immune cells such as macrophages and neutrophils, fibroblasts, smooth muscles of blood vessels, and airways It is known that a large amount of NO is produced by the activity of the enzyme inducible nitric oxide synthase (iNOS) 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 intracellular reactive oxygen species (ROS) of the extract. ^{After dispensing and culturing RAW 264.7 cells of 5×10 4} cells/well in a 96 well black plate, the cells were exchanged with serum free DMEM medium. After the cells were cultured again for 24 hours, the samples were treated by concentration and cultured for 24 hours at 37°C and 5% CO ₂ . After washing 3 times with PBS (pH 7.4), 100 μL of 1X dichloro-dihydro-fluorescein diacetate (DCFH-DA) was added to the medium and _{incubated for 1 hour at 37°C and 5% CO 2} and then washed 3 times with PBS. I put it out. After mixing by adding 100 μL of lysis buffer, fluorescence was measured at excitation 485 nm and emisssion 535 nm using a fluorescence microplate reader, and the relative ROS generation inhibition rate for the LPS alone treatment group was compared and shown.

The ROS inhibitory activity by the five extracts used the principle that when the increased ROS in cells reacts with DCF-DA, it is observed as green fluorescence. After inducing oxidative stress by treating RAW 264.7 cells with LPS, the results of confirming the inhibition of ROS production by black garlic, black ginseng, and three mixed extracts are shown in FIG. 3. In FIG. 3, all sample values are expressed as mean ± deviation values for the results of repeated experiments three times 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 the production of ROS at a concentration of 800 μg/mL (p<0.05). As in the previous inhibition of NO production, the rate of inhibition of ROS production of black ginseng extract was 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 of the extracts were added and reacted, followed by treatment 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 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 are broken down by PDE. Therefore, it is known that the foot 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 the PDE inhibitory activity to evaluate the effect of the black garlic extract and three mixtures thereof on the maintenance of erectile power are shown in FIG. 4. In FIG. 4, all sample values are expressed as mean ± deviation values for the results of repeated experiments three times under the same conditions. (Compared to the control group *P <0.05, **P <0.01 and ***P <0.001)

Referring to FIG. 4, when the extracts were treated at a concentration of 800 μg/mL, the inhibition rate of PDE increased by 95.54% compared to the control in the case of black garlic extract powder and 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 found to have similar activities to those treated with black ginseng extract alone.

(5) Cytotoxicity against 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 the black garlic extract, the main raw material of the present invention, the black ginseng extract, and the composition thereof, on the secretion of male hormones.

Leydig cells, TM3 cells, were maintained _{at 37℃ 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 incubating. After dispensing at a cell concentration of 2.5×10 ⁵ cells/mL in a 96 well plate, culture was performed for 24 hours for cell attachment, and the samples were treated by concentration to confirm cytotoxicity of cells. Cell viability was measured using the MTT assay. Absorbance was measured at 570 nm, and the measurement result was expressed as a percentage compared with the cell viability of the untreated group.

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

(6) TM3 cell cytoprotective effect and intracellular testosterone content

TM3 cytoprotective ability was measured by applying oxidative stress to TM3 cells with hydrogen peroxide to measure the protective effect of the extract. For TM3 cells, 1×10 ⁵ cells/well of cells were dispensed into a 48 wells plate and cultured for 24 hours, and then the medium was removed, and 0.5 mL of a medium containing 3% serum in which the extract was dissolved was dispensed and cultured for 24 hours. After removal of the culture medium, the culture medium containing 300 μM hydrogen peroxide and the extract was dispensed, cultured for 24 hours, and then 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 with the cell viability of the 300 μM hydrogen peroxide-treated group.

The content of testosterone in TM3 cells was measured with TM3 cell culture. For TM3 cells, 1×10 ⁵ cells/well of cells were dispensed into a 48 wells plate and cultured for 24 hours, and then the medium was removed, and 0.5 mL of the extract-treated medium was dispensed and cultured for 3 days. After the culture was completed, the testosterone content was measured for the medium using an assay kit. After transferring 100 μL of the medium to a goat anti-mouse IgG microtiter plate, 50 μL of testosterone EIA antibody was added to incubate for 1 hour, and 50 μL of conjugate was added to incubate for 1 hour, followed by washing with a washing solution. 200 μL of pNpp substrate solution was added to each and reacted for 1 hour, and then 50 μL of stop solution was added to stop the reaction. Absorbance was measured at 405 nm and the content was calculated using a testosterone standard curve.

TM3 cells were treated with 5 kinds of extracts at concentrations of 100, 200, 400 and 800 μg/mL, respectively, and cultured for 3 days, and then the content of testosterone secreted from the cells in the culture medium was measured (FIG. 7). As a result, in the untreated group, the testosterone content was 0.86 ng/mL, whereas when 800 μg/mL black ginseng extract was treated, it increased 3.59 times to 3.09 ng/mL. And the content of testosterone increased as the ratio of black ginseng increased in the three mixtures.

In order to find out whether the five extracts can alleviate the symptoms resulting from decreased testosterone production and the protective effect against oxidative stress, the results of evaluating how they affect the cytoprotective ability against hydrogen peroxide and testosterone content in TM3 cells were obtained. It is shown in Fig. 6 and Fig. 7.

In FIG. 6, all sample values are expressed as mean ± deviation values for the results of repeated experiments three times under the same conditions. * The mark 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 result of repeated experiments three times under the same conditions, and * mark is P compared to the control group. There is a significant difference between <0.05 and **marks in the range of P <0.01.

Referring to FIG. 6, TM3 cells were treated with 0.3 mM hydrogen peroxide to induce oxidative stress, and then the five extracts were treated at a concentration of 100, 200, 400, 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, when each of the five extracts was treated with 800 μg/mL, it showed a relatively high cell protection 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 interstitial of the testis, and testosterone decreases and cell viability decreases by stimulation of free radicals. This decrease in testosterone and cell viability leads to poor paw function.

Since the five extracts have cytoprotective effects against the oxidative stress of TM3 cells caused by hydrogen peroxide, it is judged that the results of increasing the content of testosterone were found. These results are consistent with studies showing that cell damage induced by oxidative stress and decrease in testosterone levels were improved during extract treatment, and that extracts inhibited the oxidative stress of cells in the testis, thereby increasing intracellular hormone synthesis. Therefore, it is believed that finasteride, which is used as a treatment for prostatic hyperplasia, can alleviate side effects such as erectile dysfunction and symptoms resulting from decreased testosterone content.

The above description is merely illustrative of the present invention, and those of ordinary skill in the art to which the present invention pertains will be able to make various modifications and variations without departing from the essential characteristics of the present invention. Accordingly, the embodiments disclosed in the present invention are not intended to limit the technical idea of the present invention, but to explain the technical idea, and the scope of the technical idea of the present invention is not limited by these embodiments. The scope of protection of the present invention should be construed by the following claims, and all technical thoughts within the scope equivalent or equivalent thereto should be construed as being included in the scope of the present invention.

Claims (6)

Black garlic extract obtained by extracting for 400 to 800 minutes at 80 to 100°C by adding water 3 to 4 times the weight of black garlic. A black garlic beverage comprising the black garlic extract of claim 1. A composition for promoting male health comprising the black garlic extract of claim 1 and black ginseng extract or black ginseng powder. The method of claim 3,
A composition for promoting male health further comprising honey. The method of claim 3,
The black garlic extract is 93 to 97% by weight, the black ginseng powder is 3 to 7% by weight,
Men's health promotion composition, characterized in that the concentration of the black garlic extract is 30 brix. The method of claim 4,
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,
Men's health promotion composition, characterized in that the concentration of the black garlic extract is 30 brix.

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