KR102636921B1 - A composite for health functional food hanving excellent anti-inflammatory and anti-oxidant, and health functional food - Google Patents

Abstract

The present invention relates to a health functional food composition that mixes red ginseng concentrate, black garlic concentrate, and pomegranate concentrate in an appropriate ratio, and has excellent anti-inflammatory and antioxidant effects while having excellent preference due to low intensity of bitterness, and a health functional food containing the same.

Description

Health functional food composition with excellent anti-inflammatory and antioxidant effects and health functional food containing the same {A COMPOSITE FOR HEALTH FUNCTIONAL FOOD HANVING EXCELLENT ANTI-INFLAMMATORY AND ANTI-OXIDANT, AND HEALTH FUNCTIONAL FOOD}

The present invention relates to a health functional food composition with excellent anti-inflammatory and antioxidant effects and a health functional food containing the same.

Health functional foods derived from ginseng are being actively developed into various formulations such as snack products, extracts, concentrates, and beverages in which the active ingredients and functionality of processed ginseng have been evaluated due to the increasing interest in health, including immunity, among modern consumers.

Meanwhile, as the demand for various raw materials such as black garlic, black ginseng, and pomegranate in addition to ginseng as health functional foods has recently increased, there is a demand for the development of new products that combine processed ginseng with other raw materials.

Meanwhile, Republic of Korea Patent Publication No. 2003-0037380 discloses a health supplement using red ginseng concentrate, but when it contains only red ginseng concentrate, it has a strong bitter taste and thus lowers preference.

Republic of Korea Patent Publication No. 2003-0037380 (May 14, 2003)

The present invention provides a health functional food composition with excellent anti-inflammatory and antioxidant effects, including red ginseng concentrate, black garlic concentrate, and pomegranate concentrate, which has low bitterness, has excellent palatability, low cytotoxicity, and exhibits both excellent anti-inflammatory and antioxidant effects, and a health functional food composition containing red ginseng concentrate, black garlic concentrate, and pomegranate concentrate. We aim to provide health functional foods that contain

The health functional food composition with excellent anti-inflammatory and antioxidant effects of the present invention to solve the above problems may include the red ginseng concentrate, black garlic concentrate, and pomegranate concentrate in a weight ratio of 1: 1 to 20: 1 to 20.

In a preferred embodiment of the present invention, the health functional food composition may include 5 to 150 μg/ml of the red ginseng concentrate.

In a preferred embodiment of the present invention, the red ginseng concentrate, black garlic concentrate, and pomegranate concentrate may be included in a weight ratio of 1:5 to 15:5 to 15.

For another purpose of the present invention, a health functional food containing the above health functional food composition can be manufactured.

In a preferred embodiment of the present invention, when the health functional food composition contains the health functional food composition at a concentration of 1,000 μg/ml, the NO (nitrite) removal rate measured based on Equation 1 below may be 60.0% or more.

[Equation 1]

NO removal rate (%) = {1-(NO concentration in inflammation-induced cells treated with health functional food/NO concentration in inflammation-induced cells)}×100%

In Equation 1, the concentration of NO in inflammation-induced cells is the concentration of NO in RAW264.7 cells (μM) measured 24 hours after treatment of RAW264.7 cells with 100 ng/ml induced inflammatory cells (LPS), The concentration of NO in inflammation-induced cells treated with health functional food was measured 24 hours after treatment with induced inflammatory cells (LPS) in RAW264.7 cells treated with health functional food at a concentration of 200 μg/ml. This is the concentration of NO in one cell (μM).

In a preferred embodiment of the present invention, when the health functional food contains the health functional food composition at a concentration of 1,000 ㎍/㎖, the ROS (reactive oxygen species) removal rate measured based on Equation 2 below is 40.0. It may be more than %.

[Equation 2]

ROS removal rate (%) = 100% - (ROS concentration in inflammation-induced cells treated with health functional food/ROS concentration in inflammation-induced cells) × 100%

In Equation 2, the ROS concentration in inflammation-induced cells is the ROS concentration (%) relative to LPS in RAW264.7 cells measured 24 hours after treatment of RAW264.7 cells with 100 ng/ml induced inflammatory cells (LPS). ), and the ROS concentration (%) of LPS in inflammation-induced cells treated with health functional food is 24 hours after treating RAW264.7 cells with health functional food at a concentration of 200 μg/ml 24 hours after treatment with LPS. After doing so, the concentration of ROS (%) relative to LPS in the measured cells is determined.

In a preferred embodiment of the present invention, the health functional food may be packaged in a stick container.

In a preferred embodiment of the present invention, the health functional food includes the health functional food composition and purified water, and may include the health functional food composition at a concentration of 300 μg/ml or more.

Through the present invention, the intensity of bitterness is low, so the preference is excellent, but cytotoxicity is low, and NO (Nitrite) and intracellular reactive oxygen species (RCO) are detected at low levels, thereby expressing both anti-inflammatory and antioxidant effects. Excellent health with anti-inflammatory and antioxidant effects. Functional food compositions and health functional foods containing them can be provided.

Figure 1 is an analysis diagram of the main ginsenosides of red ginseng concentrate according to an embodiment of the present invention,
Figures 2a to 2c are cell viability (cytotoxicity) analysis graphs of red ginseng concentrate, black garlic concentrate, and pomegranate concentrate according to an embodiment of the present invention.
3 a to c are graphs of cell viability (cytotoxicity) analysis after inducing inflammation of red ginseng concentrate, black garlic concentrate, and pomegranate concentrate according to an embodiment of the present invention;
4 a to c are NO (Nitrite) detection analysis graphs of red ginseng concentrate, black garlic concentrate, and pomegranate concentrate according to an embodiment of the present invention;
5 a to c are reactive oxygen species (ROS) detection analysis graphs of red ginseng concentrate, black garlic concentrate, and pomegranate concentrate according to an embodiment of the present invention;
6A to 6D are graphs showing cell viability (cytotoxicity) analysis of a health functional food composition according to an embodiment of the present invention.
7 a to d are graphs of cell viability (cytotoxicity) analysis after inducing inflammation of a health functional food composition according to an embodiment of the present invention;
8 a to d are NO (Nitrite) detection analysis graphs of health functional food compositions according to an embodiment of the present invention.
9A to 9D are reactive oxygen species (ROS) detection analysis graphs of health functional food compositions according to an embodiment of the present invention;
Figure 10 is a NO (Nitrite) detection analysis graph and a reactive oxygen species (ROS) detection analysis graph of a health functional food composition, red ginseng concentrate, black garlic concentrate, pomegranate concentrate, and lipopolysaccharide (LPS) according to an embodiment of the present invention;
Figure 11 is a graph showing the inhibition of inflammatory factor expression of a health functional food composition, red ginseng concentrate, black garlic concentrate, pomegranate concentrate, and lipopolysaccharide (LPS) according to an embodiment of the present invention.

The inventors of the present invention properly mixed red ginseng concentrate, black ginseng concentrate, and pomegranate concentrate to have excellent cytotoxicity, suppress NO (Nitrite) production, have an anti-inflammatory effect, and suppress the production of intracellular reactive oxygen species (ROS). The optimal mixing ratio with excellent antioxidant effect was derived.

Below, first, a health functional food composition with excellent anti-inflammatory and antioxidant effects according to an embodiment of the present invention will be described in detail.

The health functional food composition with excellent anti-inflammatory and antioxidant effects of the present invention may include red ginseng concentrate, black garlic concentrate, and pomegranate concentrate.

Among the health functional food compositions, the red ginseng concentrate used in the present invention will be described in detail as follows.

First, the red ginseng concentrate includes a first step of preparing an alcohol extract by adding alcohol to red ginseng, extracting and filtering; Step 2 of obtaining a purified water extract from the red ginseng used in the alcohol extraction; Step 3 of preparing a mixture of the alcohol extract and purified water extract; And step 4 of concentrating the mixture to prepare red ginseng concentrate.

At this time, the alcohol in the first stage may contain alcohol at a concentration of 50 to 90%, and preferably may contain alcohol at a concentration of 55 to 85%.

Additionally, the extraction can be performed at 50 to 80°C for 6 to 10 hours, and preferably at 60 to 70°C for 7 to 9 hours.

Next, the purified water extract of the second step can be obtained by filtering the alcohol extract, adding purified water to the filtered red ginseng, extracting and filtering.

At this time, the extraction can be performed at 70 to 100°C for 6 to 10 hours, and preferably at 80 to 90°C for 7 to 9 hours.

Next, the three steps of mixing can be performed by mixing the alcohol extract and purified water extract.

Next, the fourth step of concentration can be performed on the mixture at 70 to 100°C, preferably at 80 to 90°C, and the concentration is performed so that the sweetness of the red ginseng concentrate is 50 to 75 Brix. It can be performed until the sugar content of the red ginseng concentrate reaches 60 to 65 Brix.

Next, step 5 of drying the red ginseng concentrate through a freeze dryer after step 4 to obtain a powder-type red ginseng concentrate may be further included.

Meanwhile, the health functional food composition with excellent anti-inflammatory and antioxidant effects may include red ginseng concentrate, black garlic concentrate, and pomegranate concentrate in a weight ratio of 1:1 to 20:1 to 20, preferably 1:5 to 15:5. It can be included at a weight ratio of ~ 15, and more preferably at a weight ratio of 1:8 ~ 13: 8 ~ 13.

If the amount of the black garlic concentrate is less than 1 time, the problem of not showing anti-inflammatory/antioxidant activity may occur, and if it is included in more than 20 times, the problem of causing cytotoxicity may occur.

In addition, if the pomegranate concentrate is included in less than 1 time, the problem of not showing anti-inflammatory/antioxidant activity may occur, and if it is included in more than 20 times, the problem of causing cytotoxicity may occur.

More specifically, the red ginseng concentrate may be included in the composition at 5 to 150 μg/ml, preferably at 10 to 130 μg/ml, and more preferably at 25 to 100 μg/ml. It may be included, and more preferably, it may be included at 30 to 100 μg/ml.

At this time, the weight ratio may be a concentration ratio.

For another purpose of the present invention, a health functional food can be manufactured including the above-described health functional food composition with excellent anti-inflammatory and antioxidant effects.

The health functional food can be manufactured by mixing (or mixing) the health functional food composition and then diluting it in purified water.

On the other hand, when the health functional food contains the health functional food composition at a concentration of 1,000 ㎍ / ㎖, the NO (nitrite) removal rate measured according to the following equation 1 may be 60.0% or more, preferably 64% or more. It can be, and more preferably, it can be 75% or more.

[Equation 1]

NO removal rate (%) = {1-(NO concentration in inflammation-induced cells treated with health functional food/NO concentration in inflammation-induced cells)}×100%

In Equation 1, the concentration of NO in inflammation-induced cells is the concentration of NO in RAW264.7 cells (μM) measured 24 hours after treatment of RAW264.7 cells with 100 ng/ml induced inflammatory cells (LPS), The concentration of NO in inflammation-induced cells treated with health functional food was measured 24 hours after treatment with induced inflammatory cells (LPS) in RAW264.7 cells treated with health functional food at a concentration of 200 μg/ml. This is the concentration of NO in one cell (μM).

On the other hand, when the health functional food contains the health functional food composition at a concentration of 1,000 ㎍ / ㎖, the ROS (reactive oxygen species) removal rate measured according to Equation 2 below may be 40.0% or more, preferably It may be 45.0% or more, and more preferably 50% or more.

[Equation 2]

ROS removal rate (%) = 100% - (ROS concentration in inflammation-induced cells treated with health functional food/ROS concentration in inflammation-induced cells) × 100%

In Equation 2, the ROS concentration in inflammation-induced cells is the ROS concentration (%) relative to LPS in RAW264.7 cells measured 24 hours after treatment of RAW264.7 cells with 100 ng/ml induced inflammatory cells (LPS). ), and the ROS concentration (%) of LPS in inflammation-induced cells treated with health functional food is 24 hours after treating RAW264.7 cells with health functional food at a concentration of 200 μg/ml 24 hours after treatment with LPS. After doing so, the concentration of ROS (%) relative to LPS in the measured cells is determined.

At this time, the formulation can be performed by any method of simple mixing at the optimal concentration ratio with anti-inflammatory/antioxidant activity.

Meanwhile, the health functional food composition may contain more than 300 μg/ml, preferably more than 400 μg/ml, and more preferably more than 500 μg/ml among the health functional foods. You can.

If the health functional food composition contains less than 300 ㎍/㎖, the concentration may be too diluted and there may be a problem of not showing anti-inflammatory/antioxidant activity.

Meanwhile, the health functional food may be packaged in a stick container, but is not particularly limited thereto.

Hereinafter, the present invention will be explained through the following examples. At this time, the following examples are provided only to illustrate the invention, and the scope of the present invention is not limited by the following examples.

[Example]

Preparation Example 1-1: Preparation of red ginseng concentrate

Red ginseng and 70% concentration alcohol were added at a weight ratio of 1:10 and extracted for 7 hours at 65°C to prepare a red ginseng extract.

Next, the red ginseng extract was filtered to obtain an alcohol extract. Separately, purified water was added to the red ginseng filtered through the filtration, and then extracted and filtered at 85° C. for 7 hours to obtain a purified water extract.

Next, a mixture was prepared by mixing the alcohol extract and purified water extract.

Then, the mixture was concentrated under reduced pressure at 55°C until the sugar content reached 62.5 (±2.5) Brix to obtain a red ginseng concentrate.

Preparation Example 2-1: Black garlic concentrate

Black garlic concentrate from Shinwoo Corporation was prepared.

Preparation Example 3-1: Pomegranate concentrate

Pomegranate concentrate from Haechan Sol Food was prepared.

Experimental Example 1: Analysis of major ginsenosides in red ginseng concentrate

Ginsenosides in the red ginseng concentrate prepared in Preparation Example 1-1 were analyzed for organic compounds by component through high-performance liquid chromatography (HPLC), and are shown in FIG. 1.

Looking at Figure 1, the red ginseng concentrate according to an embodiment of the present invention was found to have a high content of Rb1 and Rg1, which have high intensity of bitterness.

Example 1-1: Production of health functional food

(1) Manufacturing of health functional food composition

The red ginseng concentrate prepared in Preparation Example 1-1, the black garlic concentrate prepared in Preparation Example 2-1, and the pomegranate concentrate prepared in Preparation Example 3-1 were prepared.

In addition, red ginseng concentrate, black garlic concentrate, and pomegranate concentrate were included in a weight ratio of 1:10:10.

At this time, the red ginseng concentrate, black garlic concentrate, and pomegranate concentrate were each 50 μg/ml. Concentrations of 500 μg/ml and 500 μg/ml were included.

(2)Manufacture of health functional foods

After mixing the health functional food composition, a health functional food (MIX-A) was prepared by adding the health functional food composition to purified water at a concentration of 1,000 μg/ml.

Example 1-2 to Example 1-4: Production of health functional food

Health functional foods were prepared in the same manner as in Example 1-1, except that the health functional food composition among the health functional foods was 100 μg/ml each. Examples 1-2 to 1-4 were performed including concentrations of 250 μg/ml and 500 μg/ml.

Example 2-1: Production of health functional food

(1) Manufacturing of health functional food composition

The red ginseng concentrate prepared in Preparation Example 1-1, the black garlic concentrate prepared in Preparation Example 2-1, and the pomegranate concentrate prepared in Preparation Example 3-1 were prepared.

Also, red ginseng concentrate, black garlic concentrate, and pomegranate concentrate were mixed in a weight ratio of 1:5:5.

At this time, the red ginseng concentrate, black garlic concentrate, and pomegranate concentrate were each 50 μg/ml. Concentrations of 250 μg/ml and 250 μg/ml were included.

(2)Manufacture of health functional foods

After mixing the health functional food composition with purified water, a health functional food (MIX-B) containing it at a concentration of 1,000 μg/ml was prepared.

Example 2-2 to Example 2-4: Production of health functional food

Health functional foods were prepared in the same manner as in Example 2-1, except that the health functional food compositions among the health functional foods were each 100 μg/ml. Examples 2-2 to 2-4 were performed including concentrations of 250 μg/ml and 500 μg/ml.

Example 3-1: Production of health functional food

(1) Manufacturing of health functional food composition

The red ginseng concentrate prepared in Preparation Example 1-1, the black garlic concentrate prepared in Preparation Example 2-1, and the pomegranate concentrate prepared in Preparation Example 3-1 were prepared.

In addition, red ginseng concentrate, black garlic concentrate, and pomegranate concentrate were included in a weight ratio of 1:20:20.

At this time, the red ginseng concentrate, black garlic concentrate, and pomegranate concentrate were each 25㎍/㎖. Concentrations of 500 μg/ml and 500 μg/ml were included.

(2)Manufacture of health functional foods

The health functional food composition (MIX-C) was prepared by mixing the health functional food composition and adding it to purified water at a concentration of 1,000 μg/ml.

Example 3-2 to Example 3-4: Production of health functional food

Health functional foods were prepared in the same manner as in Example 3-1, except that the health functional food composition among the health functional foods was 100 μg/ml each. Examples 3-2 to 3-4 were performed including concentrations of 250 μg/ml and 500 μg/ml.

Example 4-1: Production of health functional food

(1) Health functional food composition

The red ginseng concentrate prepared in Preparation Example 1-1, the black garlic concentrate prepared in Preparation Example 2-1, and the pomegranate concentrate prepared in Preparation Example 3-1 were prepared.

In addition, red ginseng concentrate, black garlic concentrate, and pomegranate concentrate were included in a weight ratio of 1:10:10. (MIX-D) was prepared.

At this time, the red ginseng concentrate, black garlic concentrate, and pomegranate concentrate were each 25㎍/㎖. Concentrations of 250 μg/ml and 250 μg/ml were included.

(2)Manufacture of health functional foods

After mixing the health functional food composition, a health functional food (MIX-D) was prepared by adding the health functional food composition to purified water at a concentration of 1,000 μg/ml.

Example 4-2 to Example 4-4: Production of health functional food

Health functional foods were prepared in the same manner as in Example 4-1, except that the health functional food composition among the health functional foods was 100 μg/ml each. Examples 4-2 to 4-4 were performed including concentrations of 250 μg/ml and 500 μg/ml.

Examples 5 to 6 and Comparative Examples 1 to 6: Preparation of health functional food

It was prepared in the same manner as Example 1-1, but Examples 5 to 6 and Comparative Examples 1 to 6 were performed under the conditions shown in Tables 5 and 6 below.

Experimental Example 2: Cytotoxicity evaluation

Preparation Example 1-1, Preparation Example 2-1, Preparation Example 3-1, Example 1-1 to Example 1-4, Example 2-1 to Example 2-4, Example 3-1 to Example The cytotoxicity of 3-4, Example 4-1 to Example 4-4, Example 5 to Example 6 and Comparative Example 1 to Comparative Example 6 was evaluated by the following method, and the results are shown in Table 1 below. Shown in Table 6, Figures 2 and 6.

Mouse macrophages (RAW264.7) from American Type Culture Collection were distributed in a 96-well plate at a concentration of 5 × 10 ⁴ cells/well and stabilized in DMEM containing 10% FBS and 1% penicillin-streptomycin for 24 hours.

Then, the lysates of the raw materials from Preparation Example 1-1 to Preparation Example 3-1 were treated in DMEM-high medium at a concentration of 0 (Cont), 50, 100, 250, 500, and 1000㎍/㎖, or treated in the Examples and Comparative Examples above. Each was treated and cultured in an incubator at 5% CO ₂ and 37°C for 24 hours.

Then, after incubation, 50㎕ of 2mg MTT solution {3-(4,5-dimethylthiazol-2yl)-2,5-dipenyl-2H-tetrazolium bromide} was added and incubated in a 5% CO ₂ , 37°C incubator for 2 hours. After reacting for a while, the medium was removed, and dimethyl sulfoxide (DMSO) was added in an amount of 150 ㎕ and reacted at room temperature for 20 minutes to dissolve the resulting insoluble formazan crystals.

Then, cytotoxicity was evaluated (cell viability measurement) by measuring absorbance at 540 nm using a microplate reader (BIO-RAD 450), and the results are shown in Figures 2 and 6.

At this time, as a control, LPS (induced inflammatory cells) were prepared and evaluated.

First, looking at Figure 2, while the cytotoxicity of Preparation Example 2-1 and Preparation Example 3-1 does not decrease even as the concentration increases, the cytotoxicity of Preparation Example 1-1 tends to improve as the concentration increases. In particular, it was confirmed that when administered at 250㎍/㎖, 500㎍/㎖, and 1,000㎍/㎖, it showed the lowest cytotoxicity and excellent physical properties.

In addition, looking at Figure 6, it was confirmed that Example 1-1 containing 1,000 μg/ml of MIX-A had the most notable decrease in cytotoxicity.

Experimental Example 3: Evaluation of cytotoxicity after inducing inflammation

Preparation Example 1-1, Preparation Example 2-1, Preparation Example 3-1, Example 1-1 to Example 1-4, Example 2-1 to Example 2-4, Example 3-1 to Example 3-4, Example 4-1 to Example 4-4, Example 5 to Example 6, and Comparative Example 1 to Comparative Example 6, the cytotoxicity was evaluated by the following method and the results were as follows. Shown in Tables 1 to 6, Figures 3 and 7.

Mouse macrophages (RAW264.7) from American Type Culture Collection were distributed in a 96-well plate at a concentration of 5 × 10 ⁴ cells/well and added with 10% fetal bovine serum (FBS) and 1% penicillin. -It was stabilized in DMEM (Dulbecco's modified eagle's medium) containing streptomycin (penicillin-streptomycin) for 24 hours.

Then, the lysates of the raw materials from Preparation Example 1-1 to Preparation Example 3-1 were treated in DMEM-high medium at concentrations of 0 (Cont), 50, 100, 250, 500, and 1000㎍/㎖ or Example 1-1 to After processing each of Examples 1-4, Examples 2-1 to 2-4, Examples 3-1 to 3-4, and Examples 4-1 to 4-4, DMEM-high After adding LPS (inflammatory factor) at a final concentration of 100ng/ml to the medium (or H ₂ O ₂ 50μM/ml), the culture was cultured in a 5% CO ₂ , 37°C incubator for 24 hours.

Then, after incubation, 50㎕ of 2㎎ MTT solution {3-(4,5-dimethylthiazol-2yl)-2,5-dipenyl-2H-tetrazolium bromide} was added and incubated in an incubator at 5% CO ₂ and 37°C for 2 hours. After reacting for an hour, the medium was removed, and dimethyl sulfoxide (DMSO) was added at a volume of 150 μl and reacted at room temperature for 20 minutes to dissolve the resulting insoluble formazan crystals.

Then, cytotoxicity (measurement of cell viability) was evaluated by measuring absorbance at 540 nm using a microplate reader (BIO-RAD 450), and the results are shown in Figures 3 and 7.

At this time, as a control, LPS-induced inflammatory cells were prepared and evaluated.

First, looking at Figure 3, Preparation Example 2-1 and Preparation Example 3-1 showed high cytotoxicity even as the concentration increased, while Preparation Example 1-1 showed a tendency for cytotoxicity to decrease as the concentration increased, especially It was confirmed that when administered at 250㎍/㎖, 500㎍/㎖, and 1,000㎍/㎖, cytotoxicity was lowest and excellent physical properties were observed.

In addition, looking at Figure 7, it can be seen that Examples 1-1 to 1-4 (Mix-A) had the highest cytotoxicity reduction rate, and in particular, Example 1-1 had the most outstanding cytotoxicity. there was.

Experimental Example 4: Anti-inflammatory evaluation (NO (Nitrite) detection)

Preparation Example 1-1, Preparation Example 2-1, Preparation Example 3-1, Example 1-1 to Example 1-4, Example 2-1 to Example 2-4, Example 3-1 to Example To evaluate the anti-inflammatory efficacy of 3-4, Examples 4-1 to 4-4, Examples 5 to 6, and Comparative Examples 1 to 6,

After culturing mouse macrophages in the same manner as in Experimental Example 2, LPS-induced inflammatory cells were prepared and evaluated as a control.

After taking 80 μl/ml of the supernatant, 40 μl/ml of substrate solution (N1 buffer) was added and reacted at room temperature for 10 minutes. Then, 40 μl/ml of color development solution (N ₂ buffer) was added and reacted at room temperature for 10 minutes. Then, the absorbance was measured at 540 nm using a microplate reader (BIO-RAD 450), and the NO (nitrite) removal rate was measured using Equation 1 below, and the results are shown in Figures 4, 8, and 10. and are shown in Tables 1 to 6 below.

[Equation 1]

NO removal rate (%) = {1-(NO concentration in inflammation-induced cells treated with health functional food/NO concentration in inflammation-induced cells)}×100%

In Equation 1, the concentration of NO in inflammation-induced cells is the concentration of NO in RAW264.7 cells (μM) measured 24 hours after treatment of RAW264.7 cells with 100 ng/ml of inflammatory substance (LPS), The concentration of NO in inflammation-induced cells treated with health functional food was measured 24 hours after treatment of RAW264.7 cells with 200μg/ml concentration of health functional food 24 hours after treatment with inflammatory substance (LPS). This is the concentration of NO in one cell (μM).

First, looking at Figure 4, the decrease in the amount of NO (Nitrite) detected in Preparation Example 1-1 was the largest as the concentration increased, especially when the concentration was 100 μg/ml, 250 μg/ml, 500 μg/ml, and 1,000 μg/ml. The most noticeable effect appeared when In addition, Preparation Example 2-1 showed a dull decrease compared to Preparation Example 1-1, but it was found that the decrease in NO (Nitrite) detection amount was most noticeable, especially when the concentration was 500 ㎍/㎖ or more. In addition, it was found that Preparation Example 3-1 did not significantly reduce NO (Nitrite) when used alone.

In addition, looking at Figure 8, the amount of NO (Nitrite) detected tended to decrease as the concentration of the health functional food composition increased, and it was confirmed that the decrease in NO (Nitrite) detection was most noticeable in Mix-A. .

In addition, looking at FIG. 10, Examples 1-1 to 4-4 obtained by mixing each of Preparation Examples 1-1 to 3-1 are NO ( Nitrite) was detected at a lower level.

In particular, a lower concentration of NO (Nitrite) was detected in Mix-A (Examples 1-1 to 1-4) than when 50 μg/ml of Preparation Example 1-1 (red ginseng concentrate) was used alone. , it was found that mixing red ginseng concentrate, black garlic concentrate, and pomegranate concentrate as in the present invention showed a better NO (Nitrite) inhibition rate than using the red ginseng concentrate alone.

Experimental Example 5: Antioxidant evaluation (ROS measurement)

Preparation Example 1-1, Preparation Example 2-1, Preparation Example 3-1, Example 1-1 to Example 1-4, Example 2-1 to Example 2-4, Example 3-1 to Example To evaluate the antioxidant efficacy of 3-4, Examples 4-1 to 4-4, Examples 5 to 6, and Comparative Examples 1 to 6,

It was stabilized for 24 hours in DMEM (Dulbecco's modified eagle's medium) containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin.

In addition, the melts of raw materials from Preparation Example 1-1 to Preparation Example 3-1 were treated at concentrations of 0 (Cont), 50, 100, 250, 500, and 1000㎍/㎖ or treated with each of the above Examples and Comparisons. Then, cultured in 5% _CO2 , 37℃ incubator for 24 hours, reacted with 10μM DCF-DA (2', 7'-dichlorofluorescin diacetate) in 37℃ incubator for 40 minutes, and then reacted with 50μM H2O2 in 37℃ incubator for 60 minutes. , Absorbance was measured at Ex 485, Em 530 nm, and ROS (reactive oxygen species) removal rate was measured using Equation 2 below, and the results are shown in Figures 5, 9, 10 and Tables 1 to 6 below. It was.

[Equation 2]

ROS removal rate (%) = 100% - (ROS concentration in inflammation-induced cells treated with health functional food/ROS concentration in inflammation-induced cells) × 100%

In Equation 2, the ROS concentration in inflammation-induced cells is the ROS concentration (%) relative to LPS in RAW264.7 cells measured 24 hours after treatment of RAW264.7 cells with 100 ng/ml inflammatory substance (LPS). ), and the ROS concentration (%) of LPS in inflammation-induced cells treated with health functional food is 24 hours after treating RAW264.7 cells with health functional food at a concentration of 200 μg/ml 24 hours after treatment with LPS. After doing so, the concentration of ROS (%) relative to LPS in the measured cells is determined.

First, looking at Figure 5, Preparation Example 1-1 shows the lowest level of intracellular reactive oxygen species when administered at 50 μg/ml, 100 μg/ml, 250 μg/ml, 500 μg/ml, and 1,000 μg/ml. It was confirmed that (ROS) was detected.

In addition, Preparation Example 2-1 and Preparation Example 3-1 were also found to show a tendency for intracellular reactive oxygen species (ROS) to decrease as the concentration increased.

In addition, looking at Figure 9, Mix-A (Examples 1-1 to 1-4) showed the highest reduction rate of reactive oxygen species, and Mix-B (Examples 2-1 to 2-4) showed the next highest reduction rate of reactive oxygen species.

In addition, looking at Figure 10, Mix-A (Examples 1-1 to 1-4) has a lower concentration of active oxygen than when 50 μg/ml of Preparation Example 1-1 (red ginseng concentrate) was used alone. species were detected, and it was found that lower reactive oxygen species were detected when mixing red ginseng concentrate, black garlic concentrate, and pomegranate concentrate as in the present invention, rather than using the red ginseng concentrate alone.

Mix-A Example
1-1 Example
1-2 Example
1-3 Example
1-4 health function
food
composition red ginseng
concentrate
(One) division Preparation example
1-1 Preparation example
1-1 Preparation example
1-1 Preparation example
1-1 Concentration (㎍/㎖) 50 50 50 50 Black Garlic Concentrate
(2) division Preparation example
2-1 Preparation example
2-1 Preparation example
2-1 Preparation example
2-1 Concentration (㎍/㎖) 500 500 500 500 Pomegranate
concentrate
(3) division Preparation example
3-1 Preparation example
3-1 Preparation example
3-1 Preparation example
3-1 Concentration (㎍/㎖) 500 500 500 500 (1):(2):(3) Weight ratio 1:10:10 1:10:10 1:10:10 1:10:10 health function
food Composition concentration (㎍/㎖) 1,000 100 250 500 menstruum Purified water Purified water Purified water Purified water Cytotoxicity (%) 95.3 - - - Cytotoxicity after inducing inflammation (%) 90.2 - - - NO(Nitrite) Concentration (nM) 2.50 20.13 14.57 8.19 Removal rate (%) 88.92 10.74 35.37 63.67 reactive oxygen species (% of LPS) 25.43 90.09 78.71 61.71 Removal rate (%) 74.57 9.01 21.29 38.29

Mix-B Example
2-1 Example
2-2 Example
2-3 Example
2-4 health function
food
composition red ginseng
concentrate
(One) division Preparation example
1-1 Preparation example
1-1 Preparation example
1-1 Preparation example
1-1 Concentration (㎍/㎖) 50 50 50 50 black garlic
concentrate
(2) division Preparation example
2-1 Preparation example
2-1 Preparation example
2-1 Preparation example
2-1 Concentration (㎍/㎖) 250 250 250 250 Pomegranate
concentrate
(3) division Preparation example
3-1 Preparation example
3-1 Preparation example
3-1 Preparation example
3-1 Concentration (㎍/㎖) 250 250 250 250 (1):(2):(3) Weight ratio 1:5:5 1:5:5 1:5:5 1:5:5 health function
food Composition concentration (㎍/㎖) 1,000 100 250 500 menstruum Purified water Purified water Purified water Purified water Cytotoxicity (%) 97.5 - - - Cytotoxicity after inducing inflammation (%) 98.1 - 89.8 - NO(Nitrite) Concentration (nM) 3.98 19.14 16.38 11.44 Removal rate (%) 82.37 15.14 27.38 49.28 reactive oxygen species (% of LPS) 37.20 88.37 81.70 67.70 Removal rate (%) 62.80 11.63 18.30 32.30

Mix-C Example
3-1 Example
3-2 Example
3-3 Example
3-4 health function
food
composition red ginseng
concentrate
(One) division Preparation example
1-1 Preparation example
1-1 Preparation example
1-1 Preparation example
1-1 Concentration (㎍/㎖) 25 25 25 25 black garlic
concentrate
(2) division Preparation example
2-1 Preparation example
2-1 Preparation example
2-1 Preparation example
2-1 Concentration (㎍/㎖) 500 500 500 500 Pomegranate
concentrate
(3) division Preparation example
3-1 Preparation example
3-1 Preparation example
3-1 Preparation example
3-1 Concentration (㎍/㎖) 500 500 500 500 (1):(2):(3) Weight ratio 1:20:20 1:15:15 1:15:15 1:15:15 health function
food Composition concentration (㎍/㎖) 1,000 100 250 500 menstruum Purified water Purified water Purified water Purified water Cytotoxicity (% of LPS) 97.9 - - - Cytotoxicity after inducing inflammation (% of LPS) 98.6 - - - NO(Nitrite) Concentration (nM) 4.67 21.15 17.59 12.38 Removal rate (%) 79.45 6.98 22.63 45.55 reactive oxygen species (% of LPS) 47.47 91.14 83.39 72.70

Mix-D Example
4-1 Example
4-2 Example
4-3 Example
4-4 health function
food
composition red ginseng
concentrate
(One) division Preparation example
1-1 Preparation example
1-1 Preparation example
1-1 Preparation example
1-1 Concentration (㎍/㎖) 25 25 25 25 black garlic
concentrate
(2) division Preparation example
2-1 Preparation example
2-1 Preparation example
2-1 Preparation example
2-1 Concentration (㎍/㎖) 250 250 250 250 Pomegranate
concentrate
(3) division Preparation example
3-1 Preparation example
3-1 Preparation example
3-1 Preparation example
3-1 Concentration (㎍/㎖) 250 250 250 250 (1):(2):(3) Weight ratio 1:10:10 1:10:10 1:10:10 1:10:10 health function
food Composition concentration (㎍/㎖) 1,000 100 250 500 menstruum Purified water Purified water Purified water Purified water Cytotoxicity (% of LPS) 98.2 - - - Cytotoxicity after inducing inflammation (% of LPS) 98.9 - - - NO(Nitrite) Concentration (nM) 7.95 21.01 18.62 15.23 Removal rate (%) 65.00 7.58 18.09 33.00 reactive oxygen species (% of LPS) 59.62 93.34 81.93 77.43 Removal rate (%) 40.38 6.67 18.07 22.57

Example 5 Example 6 Comparative Example 1 Comparative example 2 health function
food
composition red ginseng
concentrate
(One) division Preparation example
1-1 Preparation example
1-1 Preparation example
1-1 Preparation example
1-1 Concentration (㎍/㎖) 130 30 50 50 black garlic
concentrate
(2) division Preparation example
2-1 Preparation example
2-1 Preparation example
2-1 Preparation example
2-1 Concentration (㎍/㎖) 650 390 - 500 Pomegranate
concentrate
(3) division Preparation example
3-1 Preparation example
3-1 Preparation example
3-1 Preparation example
3-1 Concentration (㎍/㎖) 650 390 500 - (1):(2):(3) Weight ratio 1:5:5 1:13:13 1:0:10 1:10:0 health function
food Composition concentration (㎍/㎖) 1,000 1,000 1,000 1,000 menstruum Purified water Purified water Purified water Purified water Cytotoxicity (% of LPS) 92.4 96.7 96.5 96.0 Cytotoxicity after inducing inflammation (% of LPS) 88.6 96.0 96.1 95.4 NO(Nitrite) Concentration (nM) 4.98 7.15 18.8 16.92 Removal rate (%) 78.06 68.5 17.18 25.46 reactive oxygen species (% of LPS) 47.48 39.54 78.82 79.53 Removal rate (%) 52.52 60.46 21.18 20.47

Comparative example 3 Comparative example 4 Comparative Example 5 Comparative Example 6 health function
food
composition red ginseng
concentrate
(One) division Preparation example
1-1 Preparation example
1-1 Preparation example
1-1 Preparation example
1-1 Concentration (㎍/㎖) 50 50 50 50 black garlic
concentrate
(2) division Preparation example
2-1 Preparation example
2-1 Preparation example
2-1 Preparation example
2-1 Concentration (㎍/㎖) 30 1,150 500 500 Pomegranate
concentrate
(3) division Preparation example
3-1 Preparation example
3-1 Preparation example
3-1 Preparation example
3-1 Concentration (㎍/㎖) 500 500 30 1,150 (1):(2):(3) Weight ratio 1:0.6:10 1:23:10 1:10:0.6 1:10:23 health function
food Composition concentration (㎍/㎖) 1,000 1,000 1,000 100 menstruum Purified water Purified water Purified water Purified water Cytotoxicity (% of LPS) 95.5 99.7 95.6 99.5 Cytotoxicity after inducing inflammation (% of LPS) 94.8 99.5 94.9 99.0 NO(Nitrite) Concentration (nM) 12.79 2.1 14.51 2.3 Removal rate (%) 43.65 90.75 36.08 89.87 reactive oxygen species (% of LPS) 64.77 24.29 65.84 25.06 Removal rate (%) 35.23 75.71 34.16 74.94

Looking at Tables 1 to 6 above, it was confirmed that the Example had low cytotoxicity, high cell survival rate, and low NO and reactive oxygen species generation.

On the other hand, it was confirmed that Comparative Examples 1 to 2, which did not contain black garlic concentrate or pomegranate concentrate, significantly increased the amount of NO and reactive oxygen species generated, showing poor anti-inflammatory and antioxidant efficacy.

In addition, in Comparative Examples 3 and 5, in which the weight ratio of the black garlic concentrate was less than 1 weight ratio or the weight ratio of the pomegranate concentrate was less than 1 weight ratio, the amount of NO and reactive oxygen species generated was significantly increased, and it was confirmed that the anti-inflammatory and antioxidant efficacy was poor.

In addition, Comparative Examples 4 and 6, in which the weight ratio of black garlic concentrate exceeds 20 or the weight ratio of pomegranate concentrate exceeds 20, have significantly increased cytotoxicity and low cell viability, making it difficult to apply them as health foods. I was able to confirm.

Experimental Example 6: Evaluation of inhibition of inflammatory factor expression

To evaluate the efficacy of suppressing the expression of inflammatory factors, lipopolysaccharide (LPS), black garlic concentrate of Preparation Example 2-1 500㎍/㎖, red ginseng concentrate of Preparation Example 1-1 50㎍/㎖, pomegranate concentrate 500㎍/㎖ and Mix-A to Mix-D (Examples 1-1 to 4-4), respectively, were reacted with mouse macrophages (RAW264.7), and the cells were lysed with cold lysis buffer (Pro-PREP ^TM , iNiRON). ) 400 ㎕ was added and frozen (-20°C) for 30 minutes, centrifuged (4°C, 13,000 rpm, 10 min conditions), the supernatant was taken, and the protein was quantified using the BCA method.

Then, 50 μg/ml of the quantified protein was mixed with sample buffer (60 mM Tris-Cl; pH 6.8, 2% SDS, 25% glycerol, 14.4 mM 2-mercaptaethanol, 0.1% bromphenol blue) and incubated at 100°C for 5 minutes. After boiling and rapid cooling, each lane was subjected to electrophoresis at 120 V for 1 hour and 20 minutes, and then the protein was attached to a PVDF membrane.

Then, blocking was performed with 5% bovine serum albumin, and primary antibodies including COX-2, iNOS, and β-actin were reacted at 4°C for 16 hours, respectively, and then reacted with secondary antibody, HRP-conjugated anti-rabbit. Treated at room temperature for 2 hours.

Then, to identify the target protein, ECL (enhanced chemiluminescence, GE, Healthcare, Piscataway, NJ, USA) was used as a substrate, and the membrane was exposed to an X-ray film using a LAS3,000 image analyzer (Fujifilm Life The band was developed and quantified using (Science, Tokyo, Japan), and the results are shown in Figure 11.

Looking at Figure 11, it was confirmed that Example 1-1 suppressed the expression of inflammatory factors the best.

Although an embodiment of the present invention has been described above, the spirit of the present invention is not limited to the embodiment presented in the present specification, and those skilled in the art who understand the spirit of the present invention can add components within the scope of the same spirit. , other embodiments can be easily proposed by change, deletion, addition, etc., but this will also be said to be within the scope of the present invention.

Claims (8)

delete delete delete Contains health functional food composition and purified water,
Containing the health functional food composition at a concentration of 400㎍/㎖ or more,
The health functional food composition is a health functional food comprising red ginseng concentrate, black garlic concentrate, and pomegranate concentrate with a sugar content of 50 to 75 brix in a weight ratio of 1:1 to 20:1 to 20.
According to paragraph 4,
When containing the health functional food composition at a concentration of 1,000 ㎍/㎖, a health functional food with excellent anti-inflammatory and antioxidant effects, characterized in that the NO (nitrite) removal rate is 60.0% or more as measured according to Equation 1 below:
[Equation 1]
NO removal rate (%) = {1-(NO concentration in inflammation-induced cells treated with health functional food/NO concentration in inflammation-induced cells)}×100%
In Equation 1, the concentration of NO in inflammation-induced cells is the concentration of NO in RAW264.7 cells (μM) measured 24 hours after treatment of RAW264.7 cells with 100 ng/ml induced inflammatory cells (LPS), NO concentration in inflammation-induced cells treated with health functional food was measured 24 hours after treatment with induced inflammatory cells (LPS) in RAW264.7 cells treated with health functional food at a concentration of 200 μg/ml. This is the concentration of NO in one cell (μM).
According to paragraph 4,
A health functional food with excellent anti-inflammatory and antioxidant effects, characterized by a ROS (reactive oxygen species) removal rate of 40.0% or more as measured according to Equation 2 below when the health functional food composition is included at a concentration of 1,000㎍/㎖ :
[Equation 2]
ROS removal rate (%) = 100% - (ROS concentration in inflammation-induced cells treated with health functional food/ROS concentration in inflammation-induced cells) × 100%
In Equation 2, the ROS concentration in inflammation-induced cells is the ROS concentration (%) relative to LPS in RAW264.7 cells measured 24 hours after treatment of RAW264.7 cells with 100 ng/ml induced inflammatory cells (LPS). ), and the ROS concentration (%) of LPS in inflammation-induced cells treated with health functional food is 24 hours after treating RAW264.7 cells with health functional food at a concentration of 200 μg/ml 24 hours after treatment with LPS. After doing so, the concentration of ROS (%) relative to LPS in the measured cells is determined.
According to paragraph 4,
The health functional food is a health functional food characterized in that it is packaged in a stick container.
delete