KR20220133454A - A composition comprising fermented Stichopus japonicus extract and fermented Black ginseng extract for improvement of immunity - Google Patents

Abstract

The present invention relates to an immunity enhancing composition containing a fermented sea cucumber extract and a fermented black ginseng extract as active ingredients. The composition of the present invention increases cytokine (TNF-α, IL-6, IL-1β) secretion ability and nitric oxide secretion ability without increasing reactive oxygen species and induces phosphorylation of MAPKs, expression of NF-κB, and expression of cell surface activators.

Description

Immunity enhancement composition comprising fermented sea cucumber extract and fermented black ginseng extract as active ingredients {A composition comprising fermented Stichopus japonicus extract and fermented Black ginseng extract for improvement of immunity}

The present invention relates to an immunity enhancing composition comprising a fermented sea cucumber extract and a fermented black ginseng extract as active ingredients.

The present invention is a result derived through the following research project.

[Name of department: Seocheon-gun, Chungcheongnam-do / Name of task management agency: Chungnam Technopark

Research project name: 2020 marine bio strategic material and commercialization process development support project

Research project name: Manufacture of materials for enhancing immune regulation using sea cucumber complex (SJBG-F) with enhanced polysaccharide components, development and industrialization of applied products

Research period: 2020.5.11~2021.4.10]

The major physiologically active substances of ginseng have been known, such as ginsenoside, polyacetylene, polysaccharide, ginseng protein, and phenolic substances.

Ginsenosides are known to be involved in the regulation of immune and inflammatory responses. In addition, polysaccharides have been reported to have effects such as immunostimulatory, antitumor, and antioxidant.

Sea cucumber (Stichopus japonicus) means 'ginseng of the sea'. Sea cucumber actually contains saponins, and there are reports that the saponins of sea cucumber are effective in obesity, hepatic steatosis, and glucose hypersensitivity in mice induced with a high-fat diet. .

The general component of sea cucumber has a protein content of 2-5% and fat is 0-0.4%, which is very low. As for minerals, calcium and sodium are 10 times more than oysters and scallops, and potassium, zinc, copper, iron It contains 2.6~3.2% of chondroitin sulfate (Chs), a glycoprotein found in intercellular tissues, nerve tissues and cartilage tissues such as animals.

As a study on the functionality of sea cucumber, the protective effect of sea cucumber saponin against obesity and hepatic steatosis in mice fed a high-fat diet was reported. It is known that it significantly inhibits the growth of pancreatic cancer by 82~95%, and there are reports that frondanol-A5 extracted from sea cucumber inhibits growth by causing cell cycle arrest and apoptosis of pancreatic cancer cells. Sea cucumber has been eaten for a long time in Korea, but studies on its usefulness have not been conducted much.

Therefore, the present applicant fermented sea cucumber and black ginseng with white-koji mold to develop immune-related health food using sea cucumber and black ginseng as a food using the functionalities of acidic polysaccharide, and tested the change in physiological activity of the present invention. was completed.

Looking at the prior art literature, Korean Patent Publication No. 10-1712565 describes 'a method for manufacturing steamed and dried sea cucumber and a method for manufacturing a health food containing sea cucumber component using the steamed and dried sea cucumber', and Korean Patent Publication No. 10-2013-0081009 'Method for manufacturing dried sea ginseng mixed red ginseng pills and dried sea ginseng mixed red ginseng manufactured according to the manufacturing method' is described in Korean Patent Publication No. 10-1708167, 'Food composition including hydrolyzed raw materials of red ginseng and sea cucumber and manufacturing method thereof' This is described. However, these documents do not describe fermented black ginseng and fermented sea cucumber.

Republic of Korea Patent Publication No. 10-1712565 (registered on February 27, 2017) Korean Patent Publication No. 10-2013-0081009 (published on July 16, 2013) Republic of Korea Patent Publication No. 10-1708167 (Registered on Feb. 14, 2017)

An object to be solved is to provide an immunity enhancing composition containing a fermented sea cucumber extract and a fermented black ginseng extract as an active ingredient.

The solution is an immunity enhancing composition containing at least one of a fermented sea cucumber extract or a fermented black ginseng extract as an active ingredient.

In the above, the fermented strain is characterized in that the white chrysanthemum.

In the above, when the fermented sea cucumber is used, the fermented sea cucumber is characterized in that it is fermented for 60 hours by adding 1% of white porcini bacteria to the dried sea cucumber.

In the above, when the fermented sea cucumber extract and the fermented black ginseng extract are mixed,

It is characterized in that the fermented sea cucumber extract and the fermented black ginseng extract are mixed in a weight ratio of 3:7.

The composition is

Without increasing reactive oxygen species, cytokine (TNF-α, IL-6, IL-1β) secretion and nitric oxide secretion, MAPKs phosphorylation, NF-κB expression, and cell surface activator expression induction characterized in that

The composition according to the present invention has the effect thereof by using fermented black ginseng with an increased acidic polysaccharide content of 21-25% by fermenting black ginseng, and Rg3, Rg5, and Rk1, which are ginsenosides characteristic of black ginseng. .

In addition, by fermenting the sea cucumber, it has the corresponding effect by using the fermented sea cucumber with the acidic polysaccharide increased to 26.14-74.21% depending on the bacterial inoculation amount and fermentation time.

In addition, without increasing reactive oxygen species, it improves cytokine (TNF-α, IL-6, IL-1β) secretion ability, improves nitric oxide secretion, induces phosphorylation of MAPKs and expression of NF-κB, By inducing the expression of cell surface activator, it has a remarkable effect of enhancing immune activity.

1 is a graph of the analysis result of acidic polysaccharide content of fermented black ginseng extract.
Figure 2 is a graph of the analysis result of the acid polysaccharide content of the fermented sea cucumber extract.
Figure 3 is a ginsenoside content change analysis result table of fermented black ginseng extract, Figure 4 is a chromatogram.
5 is a graph showing the results of cell viability analysis.
6 is a graph showing apoptosis analysis results.
7 is a graph showing the evaluation result of reactive oxygen species.
8 is a graph showing the results of cytokine secretion analysis.
9 is a graph showing the results of analysis of nitric oxide secretion.
10 is a graph showing the results of MAPKs phosphorylation and NF-κB expression analysis.

The terms or words used in the present specification and claims conform to the technical idea of the present invention based on the "principle that the inventor can appropriately define the concept of a term in order to best describe his invention" It should be interpreted as the meaning and concept that

Therefore, the embodiments described in this specification and the configurations shown in the drawings are only the most preferred embodiments of the present invention, and do not represent all of the technical spirit of the present invention, so various equivalents that can replace them at the time of the present application It should be understood that there may be water and variations.

Experimental Example 1. Materials and Methods

1. Manufacture of fermented black ginseng

Five-year-old ginseng (Panax ginseng C. A. Meyer) grown in Geumsan was purchased and used at the Geumsan Fresh Ginseng Market in Geumsan, Chungcheongnam-do. Ginseng was placed in a screw washing machine (Cleaning Machines, Sambo, Pocheon, Korea) and washed three times for 10 minutes in total, and washed so as not to damage the skin during the washing process. After washing, weight, moisture content, total sugar, and crude protein were analyzed, and ginseng meeting the average standard was selected and used as raw material for processing black ginseng.

Washed ginseng was dried at 50°C for 24 hours using a dryer (KL-290, WooSung Election Company, Andong, Korea) to prepare ginseng with the surface dried. Eumseong, Korea) was used for steaming at 95°C for 6 hours, and then dried at 60°C to a moisture content of 15%.

Fermented ginseng was prepared by inoculating the dried ginseng with moisture and white koji mold (Aspergillus awamori var. It was dried as much as possible to prepare fermented black ginseng .

The prepared black ginseng was added to 70% (V/V) ethanol in a ratio of 1:10 and extracted twice at 80°C for 8 hours each . After extraction, 5㎛ qualitative filter paper (No.1, Advantec MFS Inc., Dubiln, CA) , USA) was used for filtration. The filtered extract was concentrated using a reduced pressure concentrator and then used as a material.

2. Manufacture of fermented sea cucumber

Sea cucumber (Stichopus japonicus) was purchased from Boryeong-si and Taean-gun, Chungcheongnam-do, and dried, self-fermented and fermented to produce fermented sea cucumber, extracted and concentrated. Dried sea cucumber (DS) was rehydrated, and raw sea cucumber was steamed (SS) and self-fertilized (BS), and three samples were used in this experiment.

Sea cucumber was fermented using white-koji mold. After adjusting the moisture content of the raw material to 60% and sterilizing it in a sterilizer, 1, 2, 3% of white-koji mold of the raw material weight is added, respectively, and 0, 24, 36, 48, 60, 72 at 30℃ Fermentation conditions were determined by measuring the acid polysaccharide content by time fermentation.

The fermented sea cucumber was extracted at 60°C by adding 10 times of sterilized water, and the extract was concentrated under reduced pressure to prepare a fermented concentrate and used in the experiment.

3. Measurement of acid polysaccharides

The acidic polysaccharide content of fermented sea cucumber (SJF) and fermented black ginseng (BGF) was measured by the 3-phenylphenol method (Choi et al., 2014). 10 mL of distilled water was added to 0.1 g of the extract, followed by centrifugation at 7,000 rpm for 15 minutes using an ultra-high speed centrifuge, followed by filtration with filter paper (Whatman No. 1.). After adding 5 mL of 0.0125 M sodium tetraborate/H2SO4 to 1 g of the extract, it was heated in a constant temperature water bath at 100°C for 5 minutes and cooled for 15 minutes. After adding 1 mL of 0.15% 3-phenyl-phenol/0.5% NaOH, it was measured at 520 nm using a spectrophotometer. The standard curve was measured at 0-100 μg/g using galacturonic acid.

4. Ginsenoside Analysis

Weigh 1.0 g of the sample into a concentration flask, add 50 mL of 70% methanol, extract for 1 hour in a reflux condenser at 80 °C, cool for 10 minutes, filter the extract, add the same amount of solvent to the residue, and repeat twice extracted. The extracts were collected in a concentration flask and concentrated in vacuo using a rotary evaporator in a water bath at 45°C. After concentration, the residue in the flask was dissolved in 10 mL distilled water and filtered through a 0.45 μm membrane filter. Ginsenoside content was measured using HPLC (Agilent 1260, Agilent Technologies, USA). For HPLC analysis, an Agilent poroshell 120EC-C18 (3.0 mm × 50 mm, 2.7 μm) column was used. As shown in the table, the flow rate and column temperature of the mobile phase were 0.8 ml/min and 35°C, respectively, and the detection wavelength of the UV detector was 203 Analyzed in nm. (Table 1: HPLC mobile phase conditions)

Time (min) Acetonitrile (%) Water (%) 0 5 95 2 5 95 4 11 89 9 13 57 19 21 79 26 28 72 28 28 72 32 32 68 34 32 68 40 45 55 42 45 55 50 55 45 55 55 45 62 5 95

5. Macrophage Culture

The mouse-derived macrophage cell line, Raw264.7 cell, was purchased from the Korea Cell Line Bank (Seoul, Korea) and used for cell culture by Dulbecco's containing 100 unit/mL penicillin and 100 unit/mL streptomycin and 10% fetal bovine serum. Modified Eagle's medium (Lonza, Walkersville, MD, USA) was used, and cells were incubated at 37°C and 5% CO2 incubator (Thermo, Carlsbad, CA, USA).

6. Assessment of macrophage viability

To measure the effect of treatment with sea cucumber composite (SJBG-F) on macrophage viability, macrophages were aliquoted in a 96-well plate at a concentration of 1×10 ⁴ cells/well and placed at 37°C, 5% CO2 incubator for 15 minutes. After the cells were completely attached while culturing for an hour, sea cucumber, black ginseng, and sea cucumber composite were treated according to the dilution ratio, respectively, and then cultured for 24 hours in a 5% CO2 cell incubator maintained at 37 ° C. (4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT; Sigma-Aldrich Co., St. Louis, MO, USA) was measured by the method. MTT reagent was dissolved in PBS at a concentration of 5 mg/mL, 30 μL per well was added, and after reacting for 2 hours, the culture supernatant was removed and the formazan crystal generated in the cell was dimethyl sulfoxide (DMSO; Sigma-Aldrich Co.) The absorbance was measured at 570 nm.

7. Macrophage Cytotoxicity Assessment

To analyze apoptosis caused by sea cucumber composite (SJBG-F), it was measured using FITC Annexin V Apoptosis Detection Kit I (BD Biosciences, San Diego, CA, USA). After washing the cells with PBS, the cells were suspended at a concentration of 5×10 ⁵ cells/mL using Annexin V binding buffer. 100 μL (1×10 ⁵ cells) of cells are collected from this solution, aliquoted into a 5 mL culture tube, and stained at room temperature for 20 minutes by adding Annexin V-FITC (5 μL) and PI-PE (5 μL). carry out After adding Annexin V binding buffer (400 μL) to the stained mixture, flow cytometer (BD FACSVerse™; BD Biosciences) analysis was performed. Data obtained after analysis were evaluated using FlowJo software (V10, Tree Star, Ashland, OR, USA).

8. Measurement of macrophage reactive oxygen species (ROS)

Intracellular reactive oxygen species were identified by staining the cells with 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA; Molecular Probes, Eugene, OR, USA). When H2DCFDA penetrates into the cell, it is converted to dichlorofluorescein diacetate by hydrolysis and reacts with reactive oxygen species to be oxidized to dichlorofluorescein and emit green fluorescence. Cells were stained with 10 µM of H2DCFDA at 37 °C for 30 minutes, washed with PBS, and then analyzed by flow cytometer (BD FACSVerse™; BD Biosciences). Data obtained after analysis were evaluated using FlowJo software.

9. Measurement of macrophage cytokine secretion

To measure macrophage cytokine secretion by sea cucumber complex (SJBG-F), macrophages were aliquoted into 1×10 ⁵ per well in a 24 well plate and cultured for 15 hours, followed by fermented sea cucumber (SJF) and fermented black ginseng. (BGF) and sea cucumber composite material (SJBG-F) were treated according to the dilution ratio, respectively, and then incubated for 24 hours at 37°C, 5% CO2 incubator, and cytokines (TNF-α, IL -6 and IL-1β) were measured. Cytokines were measured using an enzyme linked immunosorbent assay (ELISA) kit (BD Biosciences).

10. Measurement of nitric oxide (NO) secretion ability in macrophages

100 μL of the separated culture supernatant was treated with the same amount of Griess (Sigma-Aldrich) reagent and reacted for 10 minutes, and then absorbance was measured at 540 nm using a microplate reader (BioTek). The concentration of nitric oxide was calculated by comparing it with a standard straight line obtained using sodium nitrite (NaNO2, Sigma-Aldrich).

11. Western blot analysis

Macrophage Raw264.7 was dispensed into a 6-well plate at a concentration of 1×10 ⁶ cells/well, completely adhered for 15 hours, and fermented sea cucumber (SJF), fermented black ginseng (BGF) and sea cucumber composite material (SJBG-F) each was treated. After culturing cells were collected, washed 3 times with PBS, NP40 cell lysis buffer (Biosource, Seoul, Korea) was added, and then centrifuged at 13,000 rpm for 15 minutes to separate cell lysis. To isolate proteins in the nucleus, hypotonic buffer solution (10 mM HEPES, pH 7.9, 1.5 mM magnesium chloride (MgCl2), 10 mM potassium chloride (KCl), 0.5 mM dithiothreitol (DTT), 1 μM leupeptin) in the collected cells. and 0.2 mM phenyl methyl sulfonyl fluoride (PMSF)) for 20 minutes, centrifugation at 12,000 rpm for 1 minute to separate the cytoplasm and nucleus, and the separated nucleus in hypertonic buffer (20 mM HEPES, pH 7.9, 25% glycerol) , 420 mM ethylene diaminetetraacetic acid (EDTA), 0.5 mM DTT, 1 μM leupeptin, 0.2 mM PMSF) and centrifuged at 12,000 rpm for 20 minutes to extract nuclear proteins. The separated cell lysate was subjected to protein orientation using the BCA protein detection kit (Thermo scientific, Rockford, IL, USA), and 10 μg of cell lysate per well was loaded into polyacrylamide gel, respectively, and denatured separation was performed by SDS-PAGE. did. Then, it was transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore Merck KGaA, Darmstadt, Germany), and the membrane was left in a blocking solution (5% skim milk solution) for 1 hour to prevent non-specific binding of the antibody. Then, after washing 3 times with TBST (20 mM Tris-HCl, 150 mM NaCl, 0.1% Tween-20, pH 7.5) for 10 min each, Cox-2, iNOS, p-ERK1/2, p-p38, p-IκBα And in order to measure the expression level of NF-κB, the primary antibody (Cell signaling, Danvers, MN, USA) was diluted 1:2,000 and reacted at 4 °C for 15 hours, and then 3 to 5 times with TBST for 10 minutes each. After washing, the secondary antibody (goat-anti rabbit IgG; Calbiochem, La Jolla, CA, USA) was diluted 1:5,000 and reacted for 1 hour. Then, it was washed 3 to 5 times for 10 minutes with TBST, and then printed using an electrochemiluminescence (ECL; Millipore Merck KGaA) reagent for development.

12. Confirmation of cell surface marker expression

To measure the effect of the treatment of sea cucumber composite (SJBG-F) on the expression of cell surface activators of macrophages, macrophages were aliquoted into 2×10 ⁵ per well in a 12-well plate, and then fermented sea cucumber (SJF) , fermented black ginseng (BGF) and sea cucumber composite material (SJBG-F) were treated and reacted for 24 hours, and each cell was recovered. In order to prevent non-specific binding of the antibody, each recovered cell was treated with 1 μg/mL Fcγ I/III (BD Biosciences) and reacted at 4 °C for 20 minutes, and then anti-CD80 for cell surface activation factor analysis. -PE, anti-CD86-APC, anti-MHC-I-PE, and anti-MHC-II-PE (BD Biosciences), respectively, diluted 1,000-fold with cell surface antibodies, treated with each cell, and reacted for 30 minutes. Analysis was performed using a flow cytometer (BD FACSVerse™; BD Biosciences). Data obtained after analysis were evaluated using FlowJo software.

13. Statistical processing

The results obtained in the above experiments were analyzed by one-way ANOVA test followed by Tukey's multiple comparison test using statistical software (GraphPad Prism Software, version 4.03; GraphPad Software, San Diego, CA, USA). Comparisons were made at the levels of P<0.05, P<0.01 and P<0.001 by multiple range test.

Experimental Example 2. Experimental results and analysis

1. Acidic polysaccharide content of fermented black ginseng (BGF)

The efficacy of ginseng for enhancing immunity by saponins and polysaccharides among ginseng components has also been reported in many studies. As a result of animal testing, it is known that red ginseng acid polysaccharide isolated from red ginseng enhances the immune system of the body.

As a result of the experiment, the acid polysaccharide content of the extracts of white ginseng (W.G), red ginseng (R.G), and black ginseng (B.G) before fermentation was 15.0% of white ginseng, 15.2% of red ginseng, and 17.3% of black ginseng. F.R.G) was analyzed to increase by 19.2% and black ginseng (F.B.G) by 21.3%.

Through fermentation, it was possible to see the effect of increasing the extraction rate of acidic polysaccharides by up to 21-25% from black ginseng, and reducing sugars were also fermented at the yields of 11.9%, 14.8%, and 13.2% of white ginseng before fermentation, white ginseng 22.3%, red ginseng 26.8%, and black ginseng. It showed an increase of 28.1%, and it was possible to see the effect of increasing reducing sugar by up to 80-110% in black ginseng (see Fig. 1).

2. Acidic polysaccharide content of fermented sea cucumber (SJF)

The acidic polysaccharide of sea cucumber was measured in the range of 26.14-74.21% according to the bacterial inoculation amount and fermentation time (see FIG. 2). Acidic polysaccharide content was measured to be high in white-koji mold fermentation, and a pattern of increase and decrease was observed depending on the bacterial inoculation amount and fermentation time.

In this experiment, when fermented for 60 hours in the group with 1% white-koji mold for dried sea cucumber, the highest acidic polysaccharide content was exhibited, and the acidic polysaccharide content was increased by fermentation.

3. Changes in ginsenoside content of fermented black ginseng

In fermented black ginseng using white-koji mold, the contents of Rg3, Rg5, and Rk1, which are ginsenosides unique to black ginseng, were increased .

Through white-koji mold fermentation, the content of indicator substances Rg3, Rg5, and Rk1 in white ginseng is 0 mg/g, in red ginseng 1.3 mg/g, in black ginseng 1.9 mg/g, and in fermented black ginseng 4.13 mg/g increased results could be seen. (See Figs. 3 and 4)

4. Assessing Cell Viability

Fermented sea cucumber (SJF, fermented sea cucumber extract, dried sea cucumber with 1% added white koji bacteria extracted at 60℃ after fermentation for 60 hours), fermented black ginseng (BGF, fermented black ginseng extract, white group bacteria fermentation 70% (V/V) ethanol extraction) and sea cucumber composite material (SJBG-F, fermented sea cucumber extract: fermented black ginseng extract = mixed in a weight ratio of 3:7) were cytotoxic to macrophage Raw264.7 cells by dilution factor (200X, 400X, 800X, 1600X, 3200X, 6400X) and the cell viability was evaluated through the MTT method.

As a result, fermented sea cucumber showed cytotoxicity at dilutions of 200X and 400X, but did not show cytotoxicity from 800X (Fig. 5A). did not appear (Fig. 5B, C).

Therefore, for the comparative evaluation of immune activity of fermented sea cucumber (SJF), fermented black ginseng (BGF) and sea cucumber composite material (SJBG-F) later, it was fixed at a dilution factor of 3200X that does not show cytotoxicity and tested.

5. Assessment of apoptosis

Fermented sea cucumber (SJF) at a dilution factor of 3200X in macrophage Raw264.7 cells to check whether apoptosis caused by fermented sea cucumber, fermented black ginseng and sea cucumber composite (SJBG-F) at a fixed dilution factor of 3200X Black ginseng (BGF) and sea cucumber composite material (SJBG-F) were respectively treated and evaluated through Annexin V/PI staining analysis (see FIG. 6 ).

As a result, fermented sea cucumber (SJF), fermented black ginseng (BGF), and sea cucumber composite (SJBG-F) at a dilution factor of 3200X did not cause apoptosis, and it was confirmed that cytotoxicity was not observed at the dilution factor 3200X.

6. Reactive oxygen species (ROS) evaluation

Reactive oxygen species synthesize oxidative production in the living body, and the oxidized substances produced in this way are known to cause fatal toxicity to the living body (Rao & Balachandran, 2002), so in this experiment, fermented sea cucumber (SJF), fermented black ginseng (BGF) and Fermented sea cucumber (SJF), fermented black ginseng (BGF), and sea cucumber composite material (SJBG-F) at a dilution factor of 3200X in macrophage Raw264.7 cells to check whether reactive oxygen species are generated by the composite material (SJBG-F). After each treatment, H2DCFDA was measured by staining the cells (see FIG. 7).

As a result, it was confirmed that active oxygen species did not increase in all of fermented sea cucumber (SJF), fermented black ginseng (BGF) and sea cucumber composite material (SJBG-F).

7. Comparison of cytokine secretion

Cytokines are important immunomodulators for signal transduction by mediating interactions between immune cells (Byun & Byun, 2015), and representative cytokines of macrophages include TNF-α, IL-6 and IL-1β.

In this experiment, in order to compare and evaluate the effects of fermented sea cucumber (SJF), fermented black ginseng (BGF) and sea cucumber composite material (SJBG-F) on macrophage immune activity, fermented sea cucumber with a dilution factor of 3200X in macrophage Raw264.7 cells. (SJF), fermented black ginseng (BGF) and sea cucumber composite material (SJBG-F) were treated, respectively, and the secretion ability of the cytokines TNF-α, IL-6 and IL-1β was measured (see FIG. 8).

As a result, fermented sea cucumber (SJF), fermented black ginseng (BGF), and sea cucumber complex (SJBG-F) all showed increased secretion of cytokines TNF-α, IL-6 and IL-1β, and in particular, sea cucumber complex (SJBG) -F) increased cytokine secretion significantly. Therefore, it was confirmed that the cytokine secretion ability due to the immune activity of the sea cucumber complex (SJBG-F) was higher than that of the fermented sea cucumber (SJF) and the fermented black ginseng (BGF) .

8. Comparison of nitric oxide (NO) secretion in relation to Cox-2 and iNOS

Since nitric oxide is used as an important biomarker for macrophage activation (Flurkey, 1991; Gao et al., 2000; Jung & Park, 2005), fermented sea cucumber (SJF), fermented black ginseng (BGF) and sea cucumber composite (SJBG- F) induced nitric oxide secretion and expression of nitric oxide-related proteins (Cox-2 and iNOS) were confirmed.

As a result, similar to the previous cytokine results, fermented sea cucumber (SJF), fermented black ginseng (BGF) and sea cucumber composite (SJBG-F) all increased the secretion of nitric oxide, and in particular, oxidation of sea cucumber composite (SJBG-F) Nitrogen secretion was significantly increased (see FIG. 9A).

In addition, the expression of nitric oxide-related proteins Cox-2 and iNOS were also confirmed in fermented sea cucumber (SJF), fermented black ginseng (BGF) and sea cucumber composite (SJBG-F), and fermented sea cucumber (SJF), fermented black ginseng (BGF) and It was confirmed that the expression of Cox-2 and iNOS was the highest in the sea cucumber composite (SJBG-F) (see FIG. 9B).

As a result, it was confirmed that the sea cucumber composite material (SJBG-F) had higher immune activity than fermented sea cucumber (SJF) and fermented black ginseng (BGF), and thus also had higher nitric oxide secretion .

9. Comparison of MAPKs Phosphorylation and NF-κB Expression

Mitogen-activated protein kinases (MAPKs) and nuclear factor (NF)-κB are representative signaling systems mediating immune activity in macrophages. The secretion of cytokines and nitric oxide, which are immune mediators, is promoted (Brewer et al., 1992).

Representative MAPKs proteins include extracellular regulated protein kinase (ERK) and serine/threonine protein kinase (p38), and their activity is determined according to the presence or absence of phosphorylation of each protein. (Cobb & Goldsmith, 2000).

In this experiment, fermented sea cucumber (SJF), fermented black ginseng (BGF) and sea cucumber composite material were used to compare and analyze the immune response mechanism caused by fermented sea cucumber (SJF), fermented black ginseng (BGF) and sea cucumber complex (SJBG-F). (SJBG-F) Phosphorylation of MAPKs (ERK1/2 and p38) and activation of NF-κB in each treated cell were confirmed.

Macrophage Raw264.7 cells were treated with fermented sea cucumber (SJF), fermented black ginseng (BGF) and sea cucumber composite (SJBG-F) at a dilution factor of 3200X, respectively, and then phosphorylation of MAPKs in macrophages and expression of NF-κB in the nucleus were observed. As a result of observation, it was observed that phosphorylation of ERK1/2 and p38 significantly increased in all of fermented sea cucumber (SJF), fermented black ginseng (BGF), and sea cucumber composite (SJBG-F), and in particular, in sea cucumber composite (SJBG-F) ), phosphorylation of ERK1/2 and p38 was observed to be higher than that of fermented sea cucumber (SJF) and fermented black ginseng (BGF) (see FIG. 10A).

In addition, it was observed that the NF-κB expression in the nucleus was increased similarly to the MAPKs result, and the NF-κB expression was also higher in sea cucumber composite (SJBG-F) than in fermented sea cucumber (SJF) and fermented black ginseng (BGF). (See Fig. 10B).

Therefore , fermented sea cucumber (SJF), fermented black ginseng (BGF) and sea cucumber complex (SJBG-F) induce immune activity due to phosphorylation of MAPKs and expression of NF-κB, among which sea cucumber complex (SJBG-F) is It was confirmed that it induces the highest immune activity .

10. Comparison of cell surface marker expression

Since the expression of cell surface activating factors (CD80, CD86, MHC class I and II) is closely related to the activation of macrophages (Mo et al., 2017), fermented sea cucumber (SJF), fermented black ginseng (BGF) and sea cucumber The effect of the composite material (SJBG-F) on the expression level of the cell surface activator of macrophages was confirmed using flow cytometry.

Macrophage Raw264.7 cells were treated with 3200X dilution factor of fermented sea cucumber (SJF), fermented black ginseng (BGF) and sea cucumber composite (SJBG-F), respectively, and then macrophage cell surface activation factors (CD80, CD86, MHC class) As a result of observing the expression of I and II) (see Fig. 11), the expression of CD80, CD86, and MHC class I and II was increased in all of fermented sea cucumber (SJF), fermented black ginseng (BGF) and sea cucumber composite (SJBG-F). Similar to the previous results, it was observed that the sea cucumber composite material (SJBG-F) was expressed higher than that of the fermented sea cucumber (SJF) and the fermented black ginseng (BGF).

Based on these results, it was confirmed that the sea cucumber composite material (SJBG-F) had higher cell surface activator expression than fermented sea cucumber (SJF) and fermented black ginseng (BGF) and thus had higher immune activity.`

11. Summative Analysis

In the extracts of white ginseng, red ginseng, and black ginseng before fermentation, the acid polysaccharide content was analyzed to be 15.0% white ginseng, 15.2% red ginseng, and 17.3% black ginseng. Therefore, it was possible to see the effect of increasing the acidic polysaccharide by up to 21-25% in fermented black ginseng (BGF) through fermentation.

The acidic polysaccharide content of sea cucumber was measured in the range of 26.14-74.21% depending on the bacterial inoculation amount and fermentation time.

In fermented black ginseng using white-koji mold, it was seen that the contents of Rg3, Rg5, and Rk1, which are ginsenosides unique to black ginseng, were increased.

In the measurement of reactive oxygen species using macrophages, it was confirmed that active oxygen species did not increase in all of fermented sea cucumber (SJF), fermented black ginseng (BGF) and sea cucumber composite (SJBG-F). It was confirmed that the cytokine (TNF-α, IL-6, IL-1β) secretion ability due to immune activity was higher than that of fermented sea cucumber (SJF) and fermented black ginseng (BGF).

In addition, the expression of nitric oxide-related proteins Cox-2 and iNOS were also confirmed in fermented sea cucumber (SJF), fermented black ginseng (BGF) and sea cucumber composite (SJBG-F), and fermented sea cucumber (SJF), fermented black ginseng (BGF) and It was confirmed that the expression of Cox-2 and iNOS was the highest in the sea cucumber composite (SJBG-F) . As a result, it was confirmed that the sea cucumber composite material (SJBG-F) had higher immune activity than fermented sea cucumber (SJF) and fermented black ginseng (BGF), and thus also had higher nitric oxide secretion.

Phosphorylation of ERK1/2 and p38 due to sea cucumber composite (SJBG-F) was observed to be higher than that of fermented sea cucumber (SJF) and fermented black ginseng (BGF), and NF-κB expression in the nucleus was observed to increase similarly to MAPKs results. and NF-κB expression was observed to be higher in sea cucumber composite (SJBG-F) than in fermented sea cucumber (SJF) and fermented black ginseng (BGF). Therefore, fermented sea cucumber (SJF), fermented black ginseng (BGF) and sea cucumber complex (SJBG-F) induce immune activity due to phosphorylation of MAPKs and expression of NF-κB, among which sea cucumber complex (SJBG-F) is It was confirmed that it induces the highest immune activity .

Fermented sea cucumber (SJF), fermented black ginseng (BGF) and sea cucumber composite (SJBG-F) all showed increased expression of CD80, CD86, and MHC class I and II, and similarly to the previous results, sea cucumber composite (SJBG-F) F) was observed to be expressed higher than that of fermented sea cucumber (SJF) and fermented black ginseng (BGF). From these results, it was confirmed that the sea cucumber composite (SJBG-F) had higher cell surface activator expression than fermented sea cucumber (SJF) and fermented black ginseng (BGF), and thus had higher immune activity.

Example 1. Immunity enhancing composition containing fermented sea cucumber extract and/or fermented black heart extract as an active ingredient and formulation using the same

By using the fermented sea cucumber extract and/or the fermented black ginseng extract prepared according to Experimental Examples 1 and 2, an immunity enhancing composition and a formulation using the same can be carried out.

As can be seen from Experimental Examples 1 and 2, by fermenting black ginseng and sea cucumber, the acidic polysaccharide of each material is enhanced, and the unique ginsenosides of black ginseng are increased, and both the fermented black ginseng extract and the fermented sea cucumber extract exhibited immune activity. because it has In addition, this is because the mixture of the fermented sea cucumber extract and the fermented black ginseng extract in a 3:7 weight ratio also has immune activity.

The composition according to this embodiment may further include a pharmaceutically acceptable additive or a food acceptable additive. In addition, formulations for various formulations may be additionally included and implemented in the form of pills, tablets, powders, injections, powders, and the like.

So far, the present invention has been focused on preferred embodiments.

The embodiments described in this specification and the configurations shown in the drawings relate to one most preferred embodiment of the present invention, and do not represent all of the technical spirit of the present invention, so various equivalents and modifications that can be substituted for them are It should be understood that there may be examples.

Therefore, the present invention is not limited to the presented embodiment, and within the equivalent scope of the technical spirit of the present invention and the technical spirit described in the claims to be described below by those of ordinary skill in the technical field to which the present invention belongs. There may be embodiments in which various modifications and changes are possible.

Claims (5)

An immunity enhancing composition comprising at least one of fermented sea cucumber extract or fermented black ginseng extract as an active ingredient. The method according to claim 1,
The fermented strain is an immunity enhancing composition, characterized in that the white chrysanthemum. The method according to claim 1,
When fermented sea cucumber is used, the fermented sea cucumber is an immunity enhancing composition, characterized in that the dried sea cucumber is fermented for 60 hours by adding 1% of Baekkuk bacteria. 4. The method according to any one of claims 1 to 3,
When fermented sea cucumber extract and fermented black ginseng extract are mixed,
Immunity enhancement composition, characterized in that the fermented sea cucumber extract and the fermented black ginseng extract are mixed in a weight ratio of 3:7. The method according to claim 1,
The composition is
Without increasing reactive oxygen species, cytokine (TNF-α, IL-6, IL-1β) secretion and nitric oxide secretion, MAPKs phosphorylation, NF-κB expression, and cell surface activator expression induction Characterized in that, immunity enhancing composition.

Images