KR20240011367A - Peanut sprout extract with enhanced immune function - Google Patents

Abstract

Disclosed is a peanut sprout extract with improved immune-boosting efficacy. The peanut sprout extract with improved immune-boosting efficacy according to the present invention is a peanut sprout extract extracted in hot water by enzymatic hydrolysis, by adding cellulase as a sole hydrolytic enzyme to the dried ground peanut sprout to hydrolyze the peanut sprout. By hot water extraction, the peanut sprout extract is characterized in that it has the effect of promoting the expression of cytokines IL-1β and IL-6, which are innate immune cell activators.

Description

Peanut sprout extract with improved immune-boosting efficacy {PEANUT SPROUT EXTRACT WITH ENHANCED IMMUNE FUNCTION}

The present invention relates to peanut sprout extract, and more specifically to peanut sprout extract with enhanced immune-boosting efficacy.

Peanut sprouts refer to peanuts that have sprouted by fertilizing and germinating them in a facility for about a week. Like bean sprouts, they are grown only with water. In the process of sprouting peanuts, fat and calories are lowered and vitamin C, which is not present in peanuts, is produced, while resveratrol, which exists in trace amounts in peanuts, increases in large quantities.

Resveratrol is a type of polyphenol compound that has anti-cancer, powerful antioxidant, anti-aging, anti-inflammatory, neuroprotective, antiviral, blood cholesterol-lowering effects, prevention of heart disease, diabetes, and obesity. It is known to do.

The invention of Patent Publication No. 10-2018-0098888 discloses a composition for enhancing immunity containing peanut sprout extract as an active ingredient. The invention of Patent Publication No. 10-2018-0098888 is an immunity-boosting composition containing 10 to 70% by weight of chlorella, 10 to 70% by weight of peanut sprout extract, 10 to 70% by weight of inulin, and 10 to 70% by weight of palatinose. Regarding this, the immunity-enhancing effect was confirmed by conducting in-vitro measurement of lymphocyte proliferation activity, in-vitro measurement of activity as a cytokine derivative, and clinical measurement of changes in the number of lymphocytes in the blood before and after administration.

Meanwhile, the invention of Korean Patent Publication No. 10-2018-0098888 only confirmed the immunity-enhancing effect of complexes such as chlorella, peanut sprout extract, inulin, and palatinose, but did not confirm the independent immune function-enhancing effect of peanut sprout extract. I can't do it.

In addition, the invention of Korean Patent Publication No. 10-2018-0098888 does not recognize that the immune-boosting effect of peanut sprout extract can vary greatly depending on the extraction method of the peanut sprout extract or the enzyme used.

The method of hydrolyzing plant materials using enzymes and extracting useful ingredients through this is already widely known. Korean Patent Publication No. 10-2014-0034981 refers to pulverizing hot-air dried peanut sprouts and enzymatically treating them with pectinase or cellulase, or enzymatically treating the liquid extract extracted from dried peanut sprouts. A method for manufacturing a beverage is proposed. In addition, Korean Patent Publication No. 10-2014-0023124 proposes a method of producing a functional beverage by drying and powdering peanut sprouts and then enzymatically treating them with pectinase or cellulase to extract the liquid. there is. In addition, Korean Patent No. 10-2053941 discloses that peanut sprouts contain celluclast, viscozyme, amyloglucosidase, promozyme, maltogenase, and dex. The reaction product is obtained by enzymatic reaction with a complex enzyme consisting of dextrozyme, termamyl, papain, protamex, neutrase, and flavourzyme, A method of extracting phenolic compounds, including resveratrol, from peanut sprouts is proposed through hot water extraction from the reaction product.

However, these inventions that propose methods for extracting active ingredients from peanut sprouts using single enzymes or complex enzymes do not recognize that the immune function enhancing effect of the extract varies depending on the type of enzyme used during extraction.

Korea Patent Publication No. 10-2018-0098888 (Publication date: 2018.09.05) Korean Patent Publication No. 10-2014-0034981 (Publication date: 2014.03.21) Korean Patent Publication No. 10-2014-0023124 (Publication date: 2014.02.26) Korean Patent No. 10-2053941 (registration date: 2019.12.03)

The present invention was made to solve the problems caused by the extraction method and sole use of the conventional peanut sprout extract described above. The problem to be solved by the present invention is to find the optimal extraction method and use it alone without combining it with other immune-enhancing raw materials. The goal is to provide a peanut sprout extract that can achieve sufficient immune-boosting effects alone.

The object of the present invention described above is to hot-water extract peanut sprout extract by enzymatic hydrolysis, by adding cellulase as a sole hydrolytic enzyme to the dried ground peanut sprout, to hydrolyze the peanut sprout, and to extract the peanut sprout. This can be solved by making the sprout extract effective in promoting the expression of cytokines IL-1β and IL-6, which are innate immune cell activators.

The addition ratio of cellulase as a hydrolytic enzyme compared to the dried ground peanut sprouts may be 0.8 to 1.2% by weight of the dried ground peanut sprouts.

Before the hydrolysis reaction, cellulase can be activated at 40°C for 80-100 minutes, then distilled water can be added 10 times the dry weight of dried ground peanut sprouts and reacted at 38-40°C for 4-8 hours.

The peanut sprout extract may further have the effect of increasing white blood cells.

The peanut sprout extract may further have the effect of increasing the number of activated NK cells.

According to the present invention, in an immunocompromised animal model experiment, the mRNA expression of IL-1β and IL-6, which are innate cell activators of bone marrow, were analyzed, and the results showed that compared to the case of peanut sprout hot water extract with other hydrolytic enzymes added. In particular, it showed a remarkable effect of promoting the expression of IL-1β and IL-6.

In addition, according to the present invention, as a result of analyzing the total white blood cell count using an immunosuppressed mouse model, the effect was shown to significantly increase the number of white blood cells decreased due to immunosuppression.

In addition, according to the present invention, as a result of analyzing the total number of NK cells using an immunosuppression mouse model, the effect of significantly restoring the number of activated NK cells decreased due to immunosuppression was shown.

Therefore, according to the present invention, a sufficient immune-enhancing effect can be obtained just by using it alone without combining it with other immune-enhancing raw materials.

Figure 1 is a chart measuring the effect of peanut sprout extract on NK cell activity.
Figures 2a, 2b, and 2c are charts measuring the effect of peanut sprout extract on macrophage activity (cell viability, NO and PGE ₂ ).
Figure 3 is a chart measuring the effect of peanut sprout extract on IL-12 secretion of spleen cells.
Figure 4 is a chart measuring the effect of peanut sprout extract on the secretion of IFN-gamma by spleen cells.
Figure 5 is a chart measuring the effect of peanut sprout extract on T cell and B cell proliferation ability of spleen cells.
Figure 6 is a chart measuring the effect of peanut sprout extract on the content of four inflammatory cytokines (TNF-α, IL-6, IL-1β, and MCP-1) in macrophages.
Figure 7 is a chart measuring the effect of peanut sprout extract on the spleen and thymus of CP-induced immunocompromised mice.
Figure 8 is a chart measuring the overall effect of peanut sprout extract on CP-induced immunocompromised mice (relative weight of spleen and thymus, IgG in serum, mRNA expression of IL-1β and IL-6 in bone marrow) am.
Figure 9 is a chart measuring the effect of peanut sprout extract-2 (cellulase hydrolyzed extract) according to the present invention on body weight change in immunocompromised mice induced by CP.
Figure 10 is a chart measuring the effect of peanut sprout extract-2 (cellulase hydrolyzed extract) according to the present invention on blood leukocytes in CP-induced immunocompromised mice.
Figure 11 is a chart measuring the effect of peanut sprout extract-2 (cellulase hydrolyzed extract) on cytokine production in the bone marrow of immunocompromised mice induced by CP.
Figure 12 is a chart measuring the effect of peanut sprout extract-2 (cellulase hydrolyzed extract) on NK cell activation in the spleen of immunocompromised mice induced by CP.
Figure 13 is a chart measuring the effect of peanut sprout extract-2 (cellulase hydrolyzed extract) on the immune activation signals iNOS, COX-2, and NF-kB.
Figure 14 is a chart measuring the effect of peanut sprout extract-2 (cellulase hydrolyzed extract) on NF-κB activation (NF-κB p65 trans-localization).
Figure 15 is a chart measuring the effect of peanut sprout extract-2 (cellulase hydrolyzed extract) on MAPKs, an immune activation signal.

Hereinafter, specific embodiments of the present invention will be described with reference to the attached drawings.

1. Preparation of peanut sprout extract

Peanut sprout extract was prepared through hot water extraction and enzymatic hydrolysis of dried ground peanut sprouts. Peanut sprout extract-1 was extracted by adding 1 L of distilled water to 100 g of dried ground peanut sprouts and refluxing at 100°C for 4 to 8 hours, then filtered using a 5um filter, concentrated to 40 brix, and used in subsequent experiments. Enzymes (cellulase and pactinase) used for enzymatic hydrolysis were purchased from Bision (Seongnam, Korea). The enzymatically hydrolyzed extracts, peanut sprout extract-2, peanut sprout extract-3, and peanut sprout extract-4, have enzyme-to-substrate enzyme ratios of cellulase 1%, pactinase 1%, and cellulase and pactinase mixed enzymes (0.5% + 0.5%), respectively. Hydrolyzed. Before the hydrolysis reaction, each enzyme was activated at 38-42 ℃ for 80-100 minutes, then distilled water was added 10 times the dry weight of dried ground peanut sprouts and reacted at 38-42 ℃ for 4-8 hours. Afterwards, the enzyme hydrolyzate was inactivated at 100°C for 20 minutes, filtered using a 5um filter, concentrated to 40 brix, and used in subsequent experiments.

2. Efficacy evaluation through cell experiments

1) Natural killer cell activity measurement and results using mouse spleen cells

(a) Experimental method

NK cells are the only immune cells involved in both innate and adaptive immunity. They are lymphocytes containing granules such as granzyme and perforin, and recognize and kill tumor cells or virus-infected cells. NK cells can be distinguished by a cell surface differentiation cluster (CD) unique to each cell, and can be co-cultured with target cells such as YAC-1 cancer cells to measure the amount of lactate dehydrogenase (LDH) secreted by the attacked cancer cells. This can measure the activity of NK cells.

A 5-week-old male BALB/c mouse was purchased from Orient Bio, allowed to acclimate for a week, then anesthetized with isoflurane and sacrificed to remove the spleen. The extracted spleen was placed on a Petri dish, and a small amount of 500 mL of RPMI1640 (LM011-51, Welgene) + 10% FBS (S1480, Biowest) + 1% penicillin-streptomycin (LS202-02, Welgene) culture medium was added. After grinding with a 3 mL syringe plunger, the cells were single-celled by passing them through a 70 μm cell strainer. The recovered cells were centrifuged (400 × g, 4 min, 4°C), and to remove red blood cells, Red blood cell lysis buffer was added and reacted in ICE for 3 minutes. Afterwards, the reaction was terminated and the number of cells in the cell suspension was measured after a washing process. Cells were distributed to a V-bottom ⁹⁶ well plate at 4 Yac-1 cells, the target cells of NK cells in the spleen, were mixed with 500 mL of RPMI1640 (LM 011-51, Welgene) + 10% FBS (S1480, Biowest) + 1% penicillin-streptomycin (LS202-02, Welgene). Culture was performed at 37°C and 5% CO2 using culture medium. The cultured Yac-1 cells were recovered, centrifuged (400 × g, 4 minutes, 20-25°C), and the number of cells was measured and used in the experiment. Yac-1 cells were diluted to 2 × 10 ⁵ cells/mL by re-counting the number of cells after CFSE staining according to the protocol of the CFSE Cell Division Tracker Kit (423801, Biolegend). Spleen cells cultured for 24 hours in a V-bottom 96 well plate were centrifuged (400 × g, 4 minutes) to remove the supernatant, and then 200 μL each was dispensed into the Yac-1 suspension and reacted with the spleen cells for 4 hours. After the reaction, the supernatant was removed by centrifugation (400 × g, 4 minutes, 20-25°C), and 200 μL of PBS containing 0.5% FBS was added. Afterwards, the extent to which CFSE strained Yac-1 cells were killed by natural killer cells was measured using flow cytometry.

(B)Experiment results

NK cells are one of the innate lymphocytes and play a key role in selectively eliminating cancer cells, cells infected with viruses, parasites, and various bacteria. NK cells are innate lymphocytes, but they are the only immune cells involved in both innate and adaptive immunity. They are lymphocytes containing granules such as granzyme and perforin, and recognize and kill tumor cells or virus-infected cells. In addition, since they are cells with a specific ability to respond to bacterial and viral infections, the activation of NK cells can be said to be a representative method of enhancing immunity in response to infections. In addition, since the enhancement of NK cell activity can be controlled by food and health functional foods, the invention of NK cell activity enhancement is important because enhancing the immunity of NK cells through food can prevent various diseases in advance. Accordingly, mouse spleen cells pre-treated with peanut sprout extract and activated were co-cultured with Yac-1 cells, the target cells of NK cells, and then NK cell activity was evaluated based on the degree of death of Yac-1 cells. Figure 1 shows the measurement results graphically. As can be seen with reference to Figure 1, it was confirmed that the peanut sprout extract significantly increased NK cell activity in a concentration-dependent manner.

2) Cell viability, NO and PGE ₂ analysis and results using Raw 264.7 macrophages

(a) Experimental method

RAW264.7 cells, a mouse macrophage cell line, were purchased from the Korean Cell Line Bank, and the cell culture medium was DMEM with 10% FBS and 100 Unit/mL streptomycin added. Cells were cultured under conditions of 5% CO ₂ and 37°C. All processing and sample preparation for cell culture were performed on a sterile table, and all experimental equipment used in the experiment was sterilized using a high-pressure sterilizer.

MTT assay was performed to measure cell survival rate. After dispensing ¹ After incubation, the supernatant was removed, DMSO was added to lyse the cells, and formazan (purple) produced by MTT reduction in the mitochondria within the cells was 540 mL using an ELISA microplate reader (Infinite®200 PRO, TECAN Group Ltd., Switzerland). Absorbance was measured in nm.

To ^analyze the effect of samples on Nitric Oxide (NO) production, Raw 264.7 cells were adjusted to 1 Afterwards, the samples were treated according to concentration and incubated for 24 hours. The amount of NO produced was determined by Griess reagent [1% (w/v) sulfanilamide, 0.1 % (w/v) naphylethylenediamine in 2.5% (v/v) phosphoric acid]. It was measured in the form of N0 ^2- present in the cell culture medium using . 100 μL of cell culture supernatant and 100 μL of Griess reagent were mixed and reacted for 10 minutes in 24 well plates. Absorbance was measured at 540 nm using a microplate reader. The amount of NO produced was measured using sodium nitrite (NaN0 ₂ ) as a standard. compared. In addition, the culture medium was obtained in the same way as the NO production amount analysis, and the expression level of PGE ₂ contained in the culture medium was measured using an ELISA kit (Enzo Life Sciences) according to the manufacturer's analysis method.

(B)Experiment results

Lipopolysaccharide (LPS) bound to the TLR4 receptor on the surface of macrophages and activated NF-kB and MAPK signaling pathways. This activated NF-kB and AP-1 transcription factors to produce inflammatory mediators nitric oxide (NO) and prostaglandin E2 (PGE ₂ ) and induced an inflammatory response, thereby improving the immune response. It was confirmed that RAW 264.7 macrophages were treated with LPS at a concentration of 1 μg/mL, and the NO and PGE ₂ contents were significantly increased. This LPS treatment can be seen as a reference point for inflammatory and immune-boosting reactions in the activation of immune cells, and was used as a positive control. The measurement results are shown in Figures 2A, 2B, and 2C. As can be seen in Figures 2a, 2b, and 2c, when the peanut sprout extract, which is the material for efficacy evaluation, was treated at each concentration, the peanut sprout extract (1, 2, 3, 4) was treated at a concentration of 25 μg/mL or higher. It was confirmed that the NO content increased significantly, and the PGE ₂ content significantly increased through treatment with peanut sprout extract-2, 3, and 4 compared to peanut sprout extract-1. (* p < 0.05, ** p < 0.01, *** p < 0.001 vs. nor group, one-way ANOVA by Tukey's test).

3) Th1, Th2 immune response evaluation and results using mouse spleen cells

(a) Experimental method

Spleen cells were single-celled in the same manner as described in 1) above, diluted to a concentration of 1 × 10 ⁷ cells/mL, and distributed in 200 μL each in 48 well plates. Afterwards, 200 μL of culture medium diluted according to the final treatment concentration of the peanut sprout extract was added to make a total of 400 μL, and then cultured for 3 days in an incubator at 37°C and 5% CO2. The recovered culture supernatant was then used to measure cytokines, and cell viability was measured through WST assay. To measure cell viability, cells were treated with a 1:10 mixture of culture medium and Water soluble tetrazolium salt (WST, Abfrontier) at a concentration of 300 μL/well, incubated in a 5% CO2 incubator at 37°C for 30 minutes, and then incubated at 450 nm. It was confirmed by measuring the absorbance at different wavelengths.

(B)Experiment results

As a method to evaluate the immune-enhancing activity in splenocytes, the concentration of IL-12 derived from antigen-presenting cells (macrophages, B cells, dendritic cells) and IFN-gamma (Th1-mediated T cells) by the produced IL-12 Response) Secretion amount was measured. The results are shown in Figures 3 and 4. As shown in Figure 3, it was confirmed that peanut sprout extract did not affect IL-12 production. On the other hand, as shown in Figure 4, upon treatment with peanut sprout extract, overall, IFN-gamma secretion increased. Therefore, the increase in the amount of IFN-gamma secretion is not the production of IFN-gamma in the IL-12-mediated Th1 response, and considering the possibility of production of IFN-gamma in innate cells such as NK cells, it is judged that there is a possibility of immune-enhancing activity. . Additionally, peanut sprout extract was expected to have an immune-boosting effect caused by innate immune cells rather than T cell immune response.

4) Evaluation and results of T Lymphocytes immune response using mouse spleen cells

(a) Experimental method

Spleen cells were single-celled in the same manner as described in 1) above, diluted to a concentration of 1 × 10 ⁷ cells/mL, and distributed at 400 μL each in 24 well plates. Afterwards, 400 μL of culture medium diluted to the final treatment concentration of 50 μg/mL of peanut sprout extract was added to make a total of 800 μL, and then cultured in a 5% CO2 incubator at 37°C for 24 hours. Afterwards, the recovered cells were suspended at a concentration of 1 × 10 ⁷ cells/mL for flow cytometry analysis and then distributed at 200 μg/mL to a V-bottom 96 well plate. Afterwards, to prevent non-specific antibody binding, the cells were treated with purified rat anti-mouse CD16/CD32 (mouse BD Fc Block) and incubated at 4°C for 15 minutes. Additionally, for surface antibody staining, Brilliant violet 510 anti-mouse CD3 antibody, PE anti-mouse CD4 antibody, APC/Cyanine7 anti-mouse CD8a antibody, and Alexa Fluor 488 anti-mouse CD19 antibody were used, and staining was performed for 30 minutes at room temperature. . After washing with PBS and blocking light for 10 minutes, 7-AAD staining was performed. Flow cytometry was performed using Cytoflex equipment from Beckman Coulter, and the ratios of CD4 T cells, CD8 T cells, and B cells were measured using the CytoExpert 2.4 program. Confirmed.

(B)Experiment results

Unlike the innate immune system cells such as NK cells and macrophages described above, T lymphocytes have the characteristic of reacting specifically to antigens. These characteristics allow naïve T cells that have not yet encountered antigens to grow and differentiate into functionally specialized T lymphocytes such as Helper T cells (CD4 ⁺ ) and Cytotoxic T cells (CD8 ⁺ ), and these various types of T lymphocytes directly interact with antigens. It played an important role in overseeing and regulating the immune system in various ways, such as killing or controlling the activation of other immune cells. Therefore, in the present invention, the cell proliferation ability of CD4 and CD8 of peanut sprout extract was evaluated using splenocytes isolated from the spleen of mice. The results are shown graphically in Figure 5. As shown in Figure 5, it was confirmed that the ratio of CD4+ T cells and B cells increased simultaneously in some peanut sprout extracts.

5) Pro-inflammatory cytokines analysis and results using Raw 264.7 macrophages

(a) Experimental method

Raw 264.7 cells were cultured in the same manner as described in 2) above, and for the experiment, they were distributed to a 24-well plate at 1 × 10 ⁵ cells/well and cultured for 24 hours. Samples were then treated by concentration for 1 hour. cultured for a while. Afterwards, it was treated with LPS (1 μg/mL) and cultured for 24 hours. After that, the culture medium was obtained, and the expression levels of TNF-α, IL-6, IL-1β, and MCP-1 contained in the culture medium were measured using each ELISA kit. (Enzo Life Sciences) was measured according to the manufacturer's analysis method.

(B)Experiment results

It was confirmed that 1 μg/mL of LPS significantly increased the content of four inflammatory cytokines (TNF-α, IL-6, IL-1β, and MCP-1) in RAW 264.7 macrophages. Therefore, to confirm whether peanut sprout extract-1, 2, 3, and 4 induce cytokine production like LPS, RAW 264.7 macrophages were treated at concentrations of 25, 100, and 200 μg/mL. Figure 6 shows the experimental results. As can be seen in Figure 6, all four extracts significantly increased the production of TNF-α, IL-1β, and IL-6 at concentrations of 100 and 200 μg/mL, and in the case of MCP-1, pectinase extract (YEM2 Except for -3), it was confirmed that the production amount significantly increased at concentrations of 100 and 200 μg/mL. (* p < 0.05, *** p < 0.001 vs. normal group, one-way ANOVA by Tukey's test)

2. Evaluation of efficacy in preventing immune decline through animal experiments

1) Experimental method

Five-week-old male BALB/c mice were purchased from Orient Bio and acclimatized for a week, then sorted into three groups per cage and grouped into groups of six (n=6). For two weeks before intraperitoneal administration of CP (Cyclophosphamide), a total of five materials, peanut sprout extract-1, 2, 3, and 4, were administered orally at a concentration of 200 mg/kg, 200 μL each, and the Naive and CP-only treatment groups received the same amount. PBS was administered. To induce an immune-lowering effect in mice, 150 and 100 mg/kg-BW were administered intraperitoneally over two days, respectively. One week after the first day of induction, mice were anesthetized with Isoflurane using an inhalation anesthetic machine and sacrificed to obtain thymus and spleen tissue. were extracted and the relative weight of each immune organ was measured. During the experiment period, the light cycle and dark cycle were adjusted at 12-hour intervals. Feed and drinking water were provided in the form of self-feeding, and animals were reared in an environment of 24°C and 60% humidity. Approval (KFRI-M-21014) was obtained from the Animal Testing Committee of the Korea Food Research Institute, and animal testing was conducted in accordance with the standard operating guidelines of the Korea Food Research Institute. was carried out.

2) Experiment results

Since it is difficult to evaluate the significant immune-enhancing activity of peanut sprout extract in a normal mouse model with a normal immune system, we used a mouse model with reduced immunity induced by Cyclophosphamide, a representative drug that reduces the immune function of mice, to evaluate the immunity of peanut sprout extract. An immunocompromised animal model was constructed to evaluate the enhancing activity. Representative tissues related to immune function include the spleen and thymus. The spleen was involved in the production and maturation of lymphocytes, and the thymus was involved in the differentiation and maturation of lymphocytes, especially T cells. In the case of animal experiments, the increase in the weight of the thymus and spleen after ingestion of the test substance reflects the physiological state in which appropriate production and maturation of immune cells can occur, and can be judged to have a favorable effect on immune function. Therefore, to evaluate the effectiveness of peanut sprout extract in preventing immune decline, it was administered orally 2 weeks before intraperitoneal administration of CP. Afterwards, 150 and 100 mg/kg of CP was intraperitoneally administered over two days to induce an immune-lowering effect in the mice, and then sacrificed one week later to measure the relative weights of Spleen and Thymus. The results are shown graphically in Figure 7. As can be seen in Figure 7, it was confirmed that the thymus and spleen were noticeably smaller in the CP group compared to the normal group. On the other hand, no improvement in the thymus or spleen was observed in the group administered orally with peanut sprout extract. Therefore, it was determined that peanut sprout extract had no effect in preventing decline in immunity in the immunosuppression model.

3. Evaluation of immune enhancement efficacy through animal experiments (immune-compromised mice induced by high-concentration CP)

1) Experimental method

As it was confirmed that there was no preventive effect of peanut sprout extract on immunodeterioration in immunosuppressed mice induced by cyclophosphamide (CP), 200 mg/day of peanut sprout extract was administered one week before and after CP induction to evaluate the improvement effect. 200 μL each was administered orally at a concentration of ml. In the meantime, 150 and 100 mg/kg of CP was administered intraperitoneally over two days to induce a decrease in the immunity of the mouse. One week later, the mouse was sacrificed, and the relative weight of Spleen and Thymus were measured, as well as IgG analysis in serum and bone marrow. We analyzed the mRNA expression of IL-1β and IL-6, which are innate cell activation factors.

2) Experiment results

The analysis results are shown in Figure 8. As can be seen in Figure 8, in the CP-induced immunosuppressed mouse model, an overall, although not significant, increase in spleen indicators was observed in the peanut sprout extract. As a result of analyzing total IgG in serum, a slightly increased trend was observed in peanut sprout extract-1/2/3. The mRNA expression levels of IL-1β and IL-6 analyzed in bone marrow were confirmed to be significantly increased in peanut sprout extract-2 (cellulase hydrolyzed extract). Therefore, the peanut sprout extract was confirmed to have a weak, but immune-boosting effect. Among them, peanut sprout extract-2 (cellulase hydrolyzed extract) was the most effective, and it was confirmed that it can fully demonstrate immune function-enhancing effects even when used alone without mixing with other immune function-enhancing ingredients.

4. Evaluation of the immune-enhancing efficacy of peanut sprout extract-2 (cellulase hydrolyzed extract) through animal experiments (immune-compromised mice induced by low-concentration CP)

1) Immunocompromised mice induced by low concentration CP

As we confirmed the immune-enhancing activity of peanut sprout extract-2 (cellulase hydrolyzed extract) in the above immunity-lowering improvement effect experiment, we used an immunocompromised animal model with a lower concentration of CP and used peanut sprout extract-2 (cellulase hydrolyzed extract). We sought to additionally evaluate the immune-enhancing activity of the extract).

Five-week-old male BALB/c mice were purchased from Orient Bio and acclimatized for one week, then sorted into three groups per cage and grouped into groups of six (n=6). To induce an immune-lowering effect in mice, CP was administered intraperitoneally twice at 50 mg/kg-BW at 3-day intervals. To observe the improvement effect, 200 μL of peanut sprout extract at concentrations of 200, 400, and 800 mg/kg were administered orally for one week before and after the initial induction, and the Naive and CP treatment groups alone were administered the same amount of PBS. One week after the first day of induction, the patient was anesthetized with isoflurane using an inhalation anesthesia machine and then sacrificed. Relevant biomarkers were confirmed through thymus and spleen removal and orbital blood collection. During the experiment period, the light cycle and dark cycle were adjusted at 12-hour intervals. Feed and drinking water were provided in the form of self-feeding, and the animals were raised in an environment of 24°C and 60% humidity. Approval (KFRI-M-21015) was obtained from the Animal Testing Committee of the Korea Food Research Institute, and animal testing was performed in accordance with the institute's standard operating guidelines. did.

2) Measurement and results of mouse body weight change and tissue weight change

(a) Experimental method

The thymus and spleen extracted from experimental animals that consumed peanut sprout extract were washed with physiological saline, moisture was removed with a filter paper, and the weight was measured. The measured weight is called absolute weight, and the tissue weight divided by body weight is called relative weight. In this experiment, relative weight was measured considering body weight. Relative weight is tissue weight (mg) divided by body weight (g).

BALB/c mice were orally administered peanut sprout extract-2 (cellulase hydrolyzed extract) at 200, 400, and 800 mg/kg body weight for 14 days. We attempted to induce immunosuppression through intraperitoneal administration of CP (50 mg/kg BW) on the 8th and 11th day of administration, and the peanut sprout extract-2 (cellulase hydrolyzed extract) sample was used to induce weight loss and weight loss due to immunosuppression. We wanted to determine whether tissue weight loss could be suppressed.

(B) Experiment results

The experimental results are shown graphically in Figure 9. As shown in Figure 9, intraperitoneal administration of CP increased mouse body weight (from 23.98 ± 0.64 to 21.69 ± 0.65 g, *** p < 0.001) and the weight of the spleen and thymus (spleen: from 3.63 ± 0.09 to 2.70 ± 0.14 mg/g, Thymus: 1.98 ± 0.19 to 0.94 ± 0.09 mg/g, ### p < 0.001) was significantly reduced and it was confirmed that immunosuppression was properly induced. In addition, treatment with 400 mg/kg BW of peanut sprout extract-2 (cellulase hydrolyzed extract) significantly inhibited CP-induced weight loss (from 21.69 ± 0.65 to 23.18 ± 0.47 g, * p < 0.05). was able to confirm.

3) Total white blood cell count analysis and results using immunosuppressed mouse model

(a) Experimental method

White blood cells include important immune cells in the blood, including granulocytes (neutrophils, eosinophils, basophils), natural killer cells, monocytes, macrophages, and lymphocytes, and a lack of white blood cells causes immunodeficiency. It is known that intraperitoneal administration of cyclophosphamide reduces the number of white blood cells and induces immunosuppression.

The immunocompromised mouse model according to the method in 1) was anesthetized using isoflurane and a respiratory anesthetic machine, and then blood was collected through orbital blood collection using a vacuum collection tube (BD microtainer®blood collection tubes, 36594). Afterwards, using a blood cell analyzer (HemaVet 950), white blood cell levels such as monocyte, neutrophil, basophil, eosinophil, and lymphocyte were measured.

(B) Experiment results

Figure 10 shows the experimental results graphically. As shown in Figure 10, the number of immune cells decreased due to Cyclophosphamide treatment, but was significantly decreased (total white blood cells (* p < 0.05), lymphocytes (* p < 0.05), and monocytes (*** p < 0.001)) were confirmed to have improved.

4) Measurement and results of inflammatory cytokine (IL-1β, IL-6) expression in bone marrow

(a) Experimental method

Bone marrow is known as a major immune organ that regulates immune responses by producing immune cells and blood cells and expressing cytokines such as interleukin-1β (IL-1β) and interleukin-6 (IL-6).

The immunocompromised mouse model was anesthetized using isoflurane and a respiratory anesthetic machine according to the method in 1), and then the femoral bone was excavated. Bone marrow cells were collected by cutting both ends of the deboned bone and injecting PBS through a syringe. Afterwards, the supernatant was removed by centrifugation (400 1 mL of lysis buffer was added and reacted in ICE for 3 min. To terminate the reaction, 9 mL of medium was dispensed and the supernatant was removed by centrifugation (400 RNeasy Mini Kit (250) was carried out according to Qiagen's protocol. The brief experimental method is as follows. After dispensing 350 μl of Buffer RLT Lysis Buffer and lysing the cells, 350 μl of RNase-free water and 70% Ethanol were added. After vortexing, 700 μl was dispensed into the RNeasy Mini Spin Column, centrifuged (10,000 rpm), and the liquid recovered at the bottom of the column was removed. 700 μl of RW1 buffer, 600 μl, and 500 μl of RPE buffer were also dispensed into the column, and after centrifugation, all of the column recovery liquid was removed in the same order. To recover RNA, transfer the column to a 1.5ml-EP tube, add 50μl of RNA-free water, and centrifuge at 10,000 rpm for 1 minute. To measure RNA concentration, use RNase-free water as a blank. And the concentration of extracted RNA was measured using the program Gen5-Take 3-Nucleic Acid Quantification. When synthesizing cDNA, PCR was performed using the Maxime RT preMix Kit. PCR was simple in two steps and was synthesized at 45°C for 60 minutes and 95°C for 5 minutes. 180 μl of water for RNA was added to 20 μl of synthesized cDNA and diluted to 1/10. Using cDNA diluted 1/10, qRT-PCR was performed using a Roter gnen Q gene amplifier with primers specific for IL-1β and IL-6, and the results of the analysis using the △△Ct method were confirmed.

(B) Experiment results

Figure 11 is a diagram showing the results of measuring IL-1β and IL-6 expression in the bone marrow of mice induced by immunosuppression with cyclophosphamide by RT-qPCR. As shown in Figure 11, it was confirmed that the expression was reduced by more than 50% in the Cyclophosphamide-treated group compared to the normal group, and treatment with peanut sprout extract-2 (cellulase hydrolyzed extract) at a concentration of 800 mg/kg BW reduced IL. It was confirmed that the expression of -1β (** p < 0.01) and IL-6 (*** p < 0.001) were both significantly increased.

5) Natural killer cell activity and cell number analysis and results

(a) Experimental method

To convert the extracted spleen into single cells, place it on a Petri dish, add a small amount of culture medium containing 500 mL of RPMI1640 + 10% FBS + 1% penicillin-streptomycin, grind it with a 3 mL syringe plunger, and filter it with a 70 μm cell strainer to remove single cells. He made me angry. Afterwards, the supernatant was removed by centrifugation (400 Then, to terminate the reaction, 9 mL of medium was dispensed and centrifuged (400 xg, 4 minutes) to remove the supernatant, and then 1 mL of culture medium was added and suspended. Afterwards, the number of cells in the suspension was measured using a cell counter, diluted to ² After removing the supernatant by centrifugation (400 Afterwards, 0.2 μL of APC anti-mouse CD49b Antibody, PE anti-mouse CD335 (NKp46) Antibody, Brilliant Violet 510™ anti-mouse CD3 Antibody, and BIoLegend antibodies were diluted and mixed in 20 μl of ebioscience flow cytometry staining buffer and then added at room temperature. The reaction was performed for 30 minutes while blocking light. Afterwards, the supernatant was removed by centrifugation (400 It was suspended in PBS containing 0.5% FBS and analyzed using a flow cytometer.

(B) Experiment results

To determine whether peanut sprout extract-2 (cellulase hydrolyzed extract) can help boost immunity by increasing NK cell activity and increasing the number of activated NK cells, NK cell cytotoxic activity on target cells was tested. , %) and the number of activated NK cells (NK cell population, labeled as CD3 ⁻ NKp46 ⁺ and CD49b ⁺ NKp46 ⁺ ) were measured.

The measurement results are shown graphically in Figure 12. As shown in Figure 12, intraperitoneal administration of Cyclophophamide not only significantly reduced NK cell activity by 53.78% (### p < 0.001 vs. normal) compared to the normal group, but also increased the number of activated NK cells by two markers. It was confirmed that immunosuppression was properly induced as all were significantly reduced (CD3 ^- NKp46 ⁺ : ## p < 0.01, CD49b ⁺ NKp46 ⁺ : # p < 0.05 vs. normal). Peanut sprout extract-2 (cellulase hydrolyzed extract) did not significantly increase the NK cell activity decreased due to immunosuppression, but when treated at a concentration of 800 mg/kg BW, it reduced the number of activated NK cells decreased due to immunosuppression. It was confirmed that both markers were significantly recovered (** p < 0.01 vs. CP group).

6. Evaluation of RAW 264.7 macrophage immune response stimulation and activation of peanut sprout extract-2 (cellulase hydrolyzed extract) by additional cell experiments

1) Measurement and results of iNOS, COX-2, NF- kB expression in RAW 264.7 macrophages

(a) Experimental method

Macrophages are activated by recognizing foreign substances such as lipopolysaccharide (LPS) through the cell surface TLR4 receptor. Activated macrophages regulate immune responses by inducing inflammatory responses through NF-κB and MAPK signaling pathways. To evaluate whether peanut sprout extract-2 (cellulase hydrolyzed extract) can stimulate immune responses, the protein expression levels of iNOS, COX-2, and NF-κB signaling pathways were measured by western blot using RAW 264.7 macrophages. did.

(B) Experiment results

The measurement results are shown in the diagram in FIG. 13. As shown in Figure 13, LPS used as a positive control significantly increased the expression of iNOS and COX-2 proteins in RAW 264.7 macrophages and the degree of phosphorylation of NF-κB p65 and IκB, an upper signaling pathway. Additionally, peanut sprout extract-2 (cellulase hydrolyzed extract) significantly increased the expression of iNOS and COX-2 at concentrations of 100 and 200 μg/mL, and significantly increased phosphorylation of NF-κB p65 at all concentrations. increased to

2) Measurement and results of nuclear translocation of NF- kB p65 in RAW 264.7 macrophages

(a) Experimental method

The translocation of NF-κB p65 into the nucleus was used as a measure of the degree of macrophage activation. Therefore, immunocytochemical detection method was used to determine whether peanut sprout extract-2 (cellulase hydrolyzed extract) increases the translocation of NF-κB p65 to the nucleus in RAW 264.7 macrophages.

(B) Experiment results

The measurement results are shown in the diagram in Figure 14. As shown in the diagram of Figure 14, it was confirmed that, like LPS used as a positive control, peanut sprout extract-2 (cellulase hydrolyzed extract) also induced the movement of NF-κB p65 present in the cytoplasm into the nucleus.

3) Measurement and results of MAPK expression in RAW 264.7 macrophages

(a) Experimental method

Mitogen-activated protein kinase (MAPK), including ERK, JNK, and p38, was activated through the TLR4 receptor on macrophages as a phosphorylation signal transduction pathway. The MAPK pathway is a major transcription factor that regulates the immune response to infection and regulates various cellular processes such as cell proliferation, survival, death, and differentiation through DNA transcription, inflammatory cytokine production, and extracellular signaling. To evaluate whether peanut sprout extract-2 (cellulase hydrolyzed extract) can stimulate immune responses, the protein expression level of the MAPK signaling pathway was measured by western blot using RAW 264.7 macrophages.

(B) Experiment results

The measurement results are shown in the diagram in Figure 15. As shown in Figure 15, the degree of phosphorylation of JNK, ERK, and p38, which constitute the MAPK signaling pathway, significantly increased due to peanut sprout extract-2 (cellulase hydrolyzed extract) at concentrations of 100 and 200 μg/mL. was confirmed.

7. Conclusion

Among peanut sprout extracts, cellulase hydrolyzed extract in particular has a significantly superior immune function-enhancing effect when compared to extracts using other extraction methods in various aspects. It can be usefully used as a functional food ingredient or pharmaceutical ingredient.

Claims (5)

In the peanut sprout extract extracted in hot water by enzymatic hydrolysis,
Cellulase was added as a sole hydrolytic enzyme to the dried ground peanut sprouts to hydrolyze the peanut sprouts and then extracted with hot water. The peanut sprout extract has the effect of promoting the expression of cytokines IL-1β and IL-6, which are innate immune cell activators. Peanut sprout extract with improved immune-boosting efficacy, characterized by having .
According to paragraph 1,
A peanut sprout extract with improved immune-boosting efficacy, characterized in that the addition ratio of cellulase as a hydrolytic enzyme compared to the dried ground peanut sprout is 0.8 to 1.2% by weight of the dried ground peanut sprout.
According to paragraph 1,
Before the hydrolysis reaction, cellulase was activated at 38-42 ℃ for 80-100 minutes, then distilled water was added 10 times the dry weight of dried ground peanut sprouts and reacted at 38-42 ℃ for 4-8 hours. Peanut sprout extract with improved immune-boosting efficacy.
According to paragraph 1,
A peanut sprout extract with improved immune-boosting efficacy, characterized in that the peanut sprout extract has an additional effect of increasing white blood cells.
According to paragraph 1,
A peanut sprout extract with improved immune-boosting efficacy, characterized in that the peanut sprout extract further has the effect of increasing the number of activated NK cells.

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