KR20230099528A - Composition for inhancing immune function containing extract of Protaetia brevitarsis seulensis larvae and Method of producing the composition - Google Patents

Abstract

The present invention relates to a composition for enhancing immune functions containing an extract of Protaetia brevitarsis larvae as an active component. According to the present invention, provided are the composition for enhancing immune functions that can improve immune function by promoting the production of NO, PGE2, IL-6, and IL-12 in macrophages using Protaetia brevitarsis larvae, and a method for manufacturing the composition for enhancing immune functions using the extract of Protaetia brevitarsis larvae.

Description

Composition for inhancing immune function containing extract of Protaetia brevitarsis seulensis larvae and Method of producing the composition}

The present invention relates to a composition for strengthening immune function containing an extract of larvae of white-spotted radishes and a method for preparing the composition for enhancing immune function, and more particularly, the extract of larvae of white-spotted radishes, also called slugs, is used to treat macrophages of mice It promotes the activation and NO production and at the same time promotes the production of PGE2, IL-6, IL-12, and exhibits excellent effects in enhancing immunity through TLR-4, thereby enhancing immune function with immune activation and enhancing effects. It relates to compositions and methods of making the compositions.

Among the human immune systems, the innate immune system has a major function of phagocytosis against pathogens, and includes macrophages, monocytes, and neutrophils. They have a memory function against invading pathogens and play a very important role in the immune system. In particular, macrophages are the most important cells of the innate immune system and play an essential role in the immune response. Through phagocytosis on infectious agents, it produces immune mediators such as nitric oxide (NO) and prostagladin E ₂ (PGE ₂ ), and also produces cytokines such as interleukin (IL)-6 and IL-12, which are inflammatory response substances. . These reactions are known to be mainly regulated by nuclear factor-κB (NF-κB), and the synthesis of various immune mediators and inflammatory response substances is activated by NF-κB. NO is synthesized by inducible NO synthase (iNOS), and PGE ₂ is synthesized by cyclooxygenase-2 (COX-2), which is regulated by NF-κB. Therefore, NF-κB is a very important transcription factor in the immune response.

Insect protein is attracting attention as an alternative food resource to solve the global food crisis. Currently, the global insect industry is worth 38 trillion won, and in Europe, 10 types of insects are licensed for food, and they are gaining economic effects by preoccupying the edible insect market, such as developing hamburger patties. In Korea, the Ministry of Agriculture, Food and Rural Affairs registers and manages 14 species of insects as axes through the ‘Announcement on Enforcement Regulations of the Livestock Act’, and pays attention to the insect industry continuously by announcing a comprehensive plan to foster the insect industry.

Slugs, the larvae of the white-spotted flower, have been used as a special medicine for diseases originating from the liver (liver cancer, liver cirrhosis, hepatitis, and fatigue). In addition, there are records that it is effective in treating breast cancer, dysmenorrhea, loss of vision, cataract, tetanus, and various adult diseases. However, studies on the pharmacological efficacy and mechanism of slugs have been very insufficient to date. Recent studies have reported that slugs contain 58% of crude protein, 17% of crude fat, 11% of carbohydrates and other fibers, and various studies are being conducted to develop them as food materials. In particular, research on hepatoprotective function and anticancer efficacy is the main focus. However, there is no study on the improvement of the immune function of slugs so far, and there is no report on a clear immune function enhancement mechanism.

Korean Patent Registration No. 0377252

The present invention was made to solve the above problems, and production of NO, PGE2, IL-6, and IL-12 in macrophages using white-spotted flower radish larvae, which have been used as medicines for various diseases from the past. Its object is to provide a composition for enhancing immune function that can enhance immune function by promoting.

Another object of the present invention is to provide a method for preparing a composition for enhancing immune function using an extract of the larvae of the white-spotted flower radish.

The present invention has been made to achieve the above object, and provides a composition for enhancing immune function comprising an extract from the larvae of white-spotted radishes as an active ingredient.

In addition, the white-spotted flower radish larvae extract is characterized in that the hot water extract.

In addition, the hot water extract is characterized in that it is obtained by drying and pulverizing white-spotted flower larvae, extracting them using distilled water, and then freeze-drying them.

In addition, the white-spotted radish larvae extract is characterized in that it has an effect of promoting the activity of macrophages and NO production, and at the same time promoting the production of PGE2, IL-6, and IL-12.

The present invention is to achieve the above object, (a) drying the white-spotted flower iris larvae; (b) grinding the dried white-spotted flower larvae; (c) immersing the pulverized white-spotted flower larvae in distilled water; (d) removing the solvent from the solution using a vacuum filter; and (e) freeze-drying the solution (d) to obtain an extract of the white-spotted radishes larvae.

According to the present invention, a composition for enhancing immune function capable of enhancing immune function by promoting the production of NO, PGE2, IL-6, and IL-12 in macrophages using white-spotted flower larvae and white-spotted flower larvae It is possible to provide a method for preparing a composition for enhancing immune function using an extract of.

1 is a photographic image and a graph showing the results of cell shape and viability experiments using a composition according to an embodiment of the present invention.
Figure 2 is an image photograph and graph showing the results of NO and PGE ₂ production experiments using a composition according to an embodiment of the present invention.
Figure 3 is a photographic image and graph showing the results of IL-6 and IL-12 production experiments using the composition according to an embodiment of the present invention.
Figure 4 is a photographic image showing the results of an NF-κB activity experiment using a composition according to an embodiment of the present invention.
Figure 5 is a photographic image showing the results of the immune function enhancement mechanism test using a composition according to an embodiment of the present invention.

The present invention relates to a composition for enhancing immune function comprising an extract from the larvae of white-spotted radishes as an active ingredient.

Hereinafter, the present invention will be described in detail with reference to the accompanying drawings.

The present invention confirms the enhancement of immune function of slugs, which are _white -spotted flower larvae, and identifies the mechanism. It was confirmed that the effect was through the lower signaling pathway through stimulation. During the immune response, macrophages play a very important role in initial infection. Through phagocytosis on the infectious agent, immune factors against the infectious agent are produced and released. The released factors induce activation of the innate immune system, and macrophages endow specific infectious agent recognition functions through antigen expression. Through MHCII, CD80, and CD86 molecules, specific molecular patterns of infectious agents are recognized and an immune response is induced through receptor stimulation such as TLR. The present invention confirmed that the slug extract induced a strong immune response by stimulating macrophages and inducing stimulation of TLR4.

The white-spotted flower radish larvae extract according to one embodiment of the present invention is characterized in that it is a hot water extract. The white-spotted flower larvae were dried and then pulverized, and can be used by immersing them in distilled water and removing the solvent.

The white-spotted radish larvae extract according to the present invention is characterized in that it promotes macrophage activity and NO production, and at the same time promotes the production of PGE2, IL-6, and IL-12.

The method for preparing a composition for enhancing immune function according to one aspect of the present invention is composed of the following steps:

(a) drying the white-spotted flower larvae

(b) grinding the dried white-spotted flower larvae

(c) immersing the pulverized white-spotted flower larvae in distilled water

(d) removing the solvent from the solution using a vacuum filter

(e) freeze-drying the solution (d) to obtain an extract of the white-spotted flower radish larvae

Hereinafter, the present invention will be described in more detail through examples. However, these examples are only for illustrating the present invention, and the scope of the present invention will not be construed as being limited by these examples, and those skilled in the art will not be construed as limiting the technical spirit of the present invention. It will be understood that it is possible to substitute and modify parts of the embodiments within the range not departing from the above.

[Example]

Manufacturing slug hot water extract

The slug samples were supplied from the Hope Insect Farm and used, dried and then pulverized to prepare the extract. It was immersed for 1 hour using 1L of distilled water per 100g of sample. The solvent was removed through a vacuum filter, and freeze-dried at -100 ° C to obtain a final extract. The extraction yield was 23%.

[Experimental example]

go. immune cell culture

The experiment was conducted using RAW 264.7 cell line, which is a mouse macrophage, and the cell culture conditions were cultured in a 37°C, CO ₂ 5% environment by adding 5% FBS and 0.1% antibiotics to DMEM medium.

me. Cell viability experiment

The MTT assay was used to evaluate cell viability. The immune cells were cultured at a density of 1×10 ⁵ cell/mL, and the slug extract was treated at a concentration of 0 to 800 μg/mL. LPS was used as a positive control and treated at a concentration of 500 ng/mL. MTT test was performed after 24 hours of treatment with slug extract and LPS, and formazan molecules generated after 30 minutes of treatment with 0.5 mg/mL of MTT were dissolved in DMSO. The amount of dissolved formazan was measured for absorbance at 540 nm using an ELISA microplate reader. Observation through cell morphology was performed using an objective microscope.

all. NO production measurement

The production of NO by immune cells was measured using the Grisee test method. The experiment was performed 24 hours after treatment by culturing immune cells at a density of 1×10 ⁵ cell/mL and treating the slug extract at a concentration of 0 to 800 μg/mL and LPS at a concentration of 500 ng/mL. After 15 minutes of collecting the cell culture medium and mixing the same volume of Griess reagent, the absorbance was measured at 590 nm using an ELISA microplate reader. NO production was calculated based on the concentration of NaNO ₂ .

la. PGE ₂ , IL-6, IL-12 production measurement

The immune mediator PGE ₂ . An ELISA test method was used to measure the amount of production of IL-6 and IL-12. The experiment was performed 48 hours after treatment by culturing immune cells at a density of 1×10 ⁵ cell/mL and treating the slug extract at a concentration of 0 to 800 μg/mL and LPS at a concentration of 500 ng/mL. The cell culture fluid was collected and the PGE ₂ contained therein. The concentrations of IL-6 and IL-12 were measured for absorbance at 450 nm using an ELISA microplate reader.

mind. Protein expression experiments

Total protein contained in immune cells was extracted using RIPA lysis buffer. RAW 264.7 cells were put in lysis buffer and lysed at 4°C for 30 minutes. Thereafter, the protein was separated by centrifugation for 10 minutes at a pressure of 14,000 × g and 4 ° C. For separation and extraction of nuclear and cytoplasmic proteins, NE-PER nuclear and cytosolic extraction reagent was used. Protein concentration was measured using the Bio-Rad protein assay kit. And protein expression experiments were performed using a western blot test method. Proteins were separated through SDS-PAGE and attached to a polyvinylidene difluoride membrane. The attached protein is reacted with a primary antibody after passing through a blocking step using skim milk. After that, after reacting with the secondary antibody once again, the degree of luminescence is confirmed.

bar. mRNA expression experiment

Total RNA contained in immune cells was extracted using easy-BLUE reagent. cDNA was synthesized using reverse transcriptase MMLV, and PCR was performed using the primer sequences in Table 1. iNOS, IL-6, IL-12, and GAPDH were subjected to 25 cycles of PCR, and the annealing temperature was 55°C. COX-2 performed 32 cycles of PCR and the annealing temperature was 57°C.

iNOS 199 bp sense 5`-CCT CCT CCA CCC TAC CAA GT-3` anti-sense 5`-CAC CCA AAG TGC TTC AGT CA-3` COX-2 141 bp sense 5`-TGC TGT ACC AGC AGT GGC AA-3` anti-sense 5`-GCA GCC ATT TCC TTC TCT CC-3` IL-6 141 bp sense 5`-AAG TGC ATC ATC GTT GTT TTC A-3` anti-sense 5`-GAG GAT ACC ACT CCC AAC AG-3` IL-12 334 bp sense 5`-TCT AAC TTC AGC GCA GTG GA-3` anti-sense 5`-TGC GGT GGT GTA GTG AGT G-3` GAPDH 123 bp sense 5`-AGG TCG GTG TGA ACG GAT TTG-3` anti-sense 5`-TGT AGA CCA TGT AGT TGA GGT CA-3`

buy. Fluorescence staining experiment

In order to confirm the activity of the p65 protein and the TLR4 receptor, protein expression experiments were performed through fluorescent staining. In the experiment, immune cells were cultured on a gelatin-coated cover glass at a density of 1×10 ⁴ cell/mL. The slug extract was treated at a concentration of 0 ~ 800 μg/mL and fixed with 4% paraformaldehyde after 1 hour. The fixed cells were treated with 0.1% Tritox-X100 for 10 minutes and washed with cold PBST for 5 minutes. Thereafter, blocking is performed using 10% donkey serum, and p65 and TLR4 primary antibodies are treated at 4° C. for 12 hours. Finally, the fluorescent secondary antibody is treated at room temperature for 2 hours. For nuclear staining, DAPI was treated at a concentration of 300 nM for 10 minutes. Observe the fluorescence-treated sample through a fluorescence microscope and confirm the degree of protein expression.

[Experiment result]

go. Cytotoxicity evaluation of slug hot water extract and cell shape observation

To evaluate the toxicity of the slug extract, mouse macrophage RAW 264.7 cells were treated with the slug extract at various concentrations, and cell viability analysis and cell shape observation were performed. As a result of observing changes in cell shape, cells with a dead form could not be observed. However, it was observed that the macrophages, which had a circular shape before treatment, changed to an elongated shape after treatment with the slug extract (FIG. 1A). This leads us to speculate that high concentrations of slug extract induce activation of RAW 264.7 cells. Referring to B of FIG. 1, as a result of the MTT experiment, it was confirmed that the slug extract did not have cytotoxicity. When the results of FIG. 1A and FIG. 1B are taken together, it can be seen that the slug extract induces cell activation without having cytotoxicity.

me. Evaluation of NO and PGE2 production and evaluation of iNOS and COX-2 expression

It was confirmed whether the slug extract induces a change in the immune function by inducing a change in cell shape. First, as a result of comparing the expression changes of COX-2, a synthetase of iNOS and PGE ₂ , a synthase of NO, which is a major immune mediator, the results of FIG. It was confirmed that the concentration-dependent increase was evident. In C and D of FIG. 2 , it was confirmed that the expression of COX-2 was increased. When comparing the expression with LPS, a strong immune response substance, it was found that the slug extract induced very strong expression of iNOS and COX-2. It was also confirmed whether it substantially affected the production of NO and PGE ₂ . Looking at E and F of FIG. 2 , it was found that the slug extract at a high concentration very strongly increased the production of NO and PGE ₂ . However, a low concentration (100 μg/mL) of slug extract did not affect NO and PGE2 production. Summarizing the results of FIG. 2 , it can be seen that the high-concentration slug extract induces an increase in the production of NO and PGE ₂ , which are immune mediators, by increasing the expression of iNOS and COX-2.

all. Evaluation of expression and production of IL-6 and IL-12

As the slug extract induced the expression of major immune mediators, changes in the expression of IL-6 and IL-12 were confirmed to confirm changes in other immune-inducing factors. 3A and B, it can be seen that treatment with a high concentration (800 μg/mL) of slug extract very strongly induced the mRNA expression of IL-6 and IL-12 in the mouse macrophage RAW 264.7 cell line. These results were strong enough to be comparable to the mRNA expression of macrophages stimulated by LPS. Substantially comparing the production of IL-6 and IL-12 (FIG. 3 C and D), it was confirmed that a high concentration of slug extract induces very strong inflammatory factor production. However, it was confirmed that a low concentration (100 μg/mL) of slug extract could not affect the expression of IL-6 and IL-12. Summarizing the experimental results, low concentration of slug extract did not affect the immune response, but high concentration of slug extract increased the production of IL-6 and IL-12 by inducing mRNA expression of IL-6 and IL-12. It has been confirmed to induce immune activity of phagocytes.

la. Confirmation of changes in NF-κB activity and corresponding changes in immune mediator expression

NF-κB is a key transcription factor that regulates immune stimulation and inflammation-inducing factors, and regulates the expression of most immune mediators. Therefore, an experiment was conducted to promote the production of immune mediators and change the activity of NF-κB in the slug extract. When p65, an accessory protein of NF-κB, was stained with red fluorescence and the nucleus was stained with blue, it was observed that p65 in the cytoplasm moved into the nucleus (FIG. 4A). As a result of examining NF-κB expression by extracting nuclear proteins, it was also confirmed that the expression of p65 and p50 proteins in the nucleus was increased (FIG. 4B). Therefore, it was thought that the activity of NF-κB would affect the expression of iNOS, COX-2, IL-6, and IL-12, and the expression was compared by treatment with an NF-κB inhibitor (FIG. 4C). As a result, the NF-κB inhibitor suppressed the expression of iNOS, COX-2, IL-6, and IL-12, which means that the slug extract induces NF-κB activity and increases the production of immune mediators.

mind. Identification of the immune function enhancement mechanism of slug extract

It is known that most immune responses start with stimulation of TLR4 receptors, and TLR4 receptors induce NF-κB activity through the lower MyD88 and IRAK4 protein activation pathways. Therefore, in order to identify the mechanism of the immune function enhancement effect of the slug extract, the expression of TLR4 through fluorescent staining (A in FIG. 5) and the expression of MyD88 and IRAK4 proteins (FIG. 5 B) were confirmed. As a result, it can be confirmed that the expression of TLR4 stained with green fluorescence is increased by the slug extract, which was observed to show a similar degree of expression to LPS stimulation. The expression of MyD88 and IRAK4 protein was also strongly increased, and this was also similar to the level of expression by LPS stimulation. In conclusion, it was confirmed that the slug extract has an effect of enhancing immune function by inducing translocation (translocation into the nucleus) of NF-κB by activating MyD88 and IRAK4 proteins through TLR4 stimulation.

Claims (5)

A composition for strengthening immune function comprising an extract from the larvae of white-spotted radishes as an active ingredient.
According to claim 1,
The white-spotted flower radish larvae extract is a composition for strengthening immune function, characterized in that the hot water extract.
According to claim 2,
The hot-water extract is a composition for enhancing immune function, characterized in that obtained by drying and pulverizing the larvae of the white-spotted flower radish, extracting them using distilled water, and then freeze-drying them.
According to any one of claims 1 to 3,
The composition for enhancing immune function, characterized in that the extract of the white-spotted radish larvae promotes macrophage activity and NO production, and at the same time promotes the production of PGE2, IL-6, and IL-12.
(a) drying the white-spotted flower larvae;
(b) grinding the dried white-spotted flower larvae;
(c) immersing the pulverized white-spotted flower larvae in distilled water;
(d) removing the solvent from the solution using a vacuum filter; and
(e) freeze-drying the solution (d) to obtain an extract of the white-spotted flower radish larvae
Method for producing a composition for enhancing immune function comprising a.

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