KR20220052204A - A pharmaceutical composition for immunity enhancement comprising Quinizarin-O-glucoside - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for enhancing immunity comprising quinizarin-O-glucoside, and more specifically, to a composition with excellent activity for enhancing immunity, in which Quinizarin-O-glucoside promotes the secretion of TNF-α, a representative proinflammatory cytokine, and MIP-1α and RANTES, which are chemokines; increases an expression level of iNOS and IL-6, increases NO production, promotes a phagocytic activity of macrophages, activates a MAPKs signaling pathway (MEK, ERK, p38, JNK), remarkably increases the expression of cell cycle related factors Cyclin D1 and PCNA, and activates the proliferation of mouse-derived macrophages and spleen cells, thereby remarkably increasing an initial innate immune system.

Description

A pharmaceutical composition for immunity enhancement comprising Quinizarin-O-glucoside

The present invention relates to a pharmaceutical composition for enhancing immunity comprising quinizarin-O-glucoside, and more particularly, quinizarin-O-glucoside is a representative proinflammatory cytokine (proinflammatory cytokine). ) promotes secretion of TNF-α and chemokines MIP-1α and RANTES, increases the expression level of iNOS and IL-6, increases NO production, and phagocytic activity of macrophages , and significantly increases the activation of MAPKs signaling pathways (MEK, ERK, p38, JNK) and the expression of Cyclin D1 and PCNA, which are cell cycle-related factors, and activates the proliferative capacity of mouse-derived macrophages and splenocytes. It relates to a composition with excellent immune enhancing activity that significantly increases the initial innate immunity by using it.

As the world becomes global, infectious diseases are also occurring regardless of the distance at which they spread. Due to the recent outbreak of viruses such as Corona 19 (COVID-19) and MERS (MERS), interest in strengthening immunity at home and abroad is increasing. In particular, while the prolonged period of COVID-19 (COVID-19) is being maintained, in the absence of a treatment or vaccine, it is necessary to strengthen the immune system, the individual body defense system, through regular living. However, recommending the above solutions to busy modern people who suffer from lack of sleep, irregular meals, chronic fatigue, and stress in their daily life can be even more stressful. In this situation, maintaining immunity by using health functional foods, vitamins, and immune-enhancing injections that can help boost immunity can also be used as a method.

According to the statistics of the Ministry of Food and Drug Safety, sales of health functional food in Korea in 2018 increased to KRW 2.522 trillion, and the market size in 2018 was KRW 3.69 trillion, a 13.5% increase from the previous year. In addition, according to the statistics of the Korea Health Functional Food Association, in the 2019 health functional food market status and consumer survey, the highest goal for purchasing health functional food was to improve immunity, recording 59.7%. In particular, while red ginseng has ranked first in sales every year for the past five years, it has been investigated that immune functional ingredients such as probiotics and vitamins are attracting attention. In addition, due to the recent virus epidemic, interest in immune adjuvants for immune enhancement in the health functional food market is drawing attention.

Among the secondary metabolites of plants, anthraquinone-based compounds, which are one of the largest groups of natural products having a quinone structure, are abundantly present in mardi Gras and lilies. , rhein, chrysophanol, and quinizarin. They have been used as colorants in foods, drugs and cosmetics (Zengin and Locatelli et al., 2016). As an important intermediate for the production of antibiotics in plants, various physiological activities such as antibacterial, anticancer, antiviral, antiallergic, autoimmune diabetes and anti-inflammatory have recently been studied (Malik and Muller, 2016).

Accordingly, plant-based phenolic compounds such as anthraquinone have already been developed as an antithrombotic agent, an anti-gastric ulcer, an anti-gastric cancer specialty agent, and a preventive and therapeutic agent for dementia. As interest and demand for non-related compounds or their application continues to increase, the need for discovery and development of related substances is continuously required (Dixon and Steele, 1999; Bradley et al., 1999).

It is known that the glycosides of quinizarine are mainly present in the root of Rubia tinctorum (Mather), also known as maroon (Nguyen and Sin et al., 2020). The root of this plant is mainly used as a natural red dye and is used as a treatment for kidney stones and bladder stones. Esalat Nejad, 2013). Studies on derivatives of quinizarine glycosides are known to exhibit anticancer activity, but studies on quinizarine glucoside have not been conducted, and in particular, immune enhancing activity has not been studied or reported to date (Sun and Ji et al., 2008). ). Accordingly, the present inventors confirmed the immune enhancing effect of quinizarine-O-glucoside and completed the present invention.

Immunity is the body's defense system that protects itself from bacteria and viruses. This immune response can be divided into an innate immune response that is an initial immune response and an adaptive immunity that is a late immune response. In the initial immune response, the host is protected by suppressing pathogens by the activation of macrophages and natural killer cells (NK cells). NK cells produce and secrete a lot of perforin, an activation marker, to kill pathogen-infected cells. Subsequently, cytotoxic T lymphocytes, helper T lymphocytes, and B lymphocytes involved in adaptive immunity are activated to kill infected cells or create antibodies to protect the host (Murray and Wynn, 2011). Cytotoxic T lymphocytes, like NK cells, produce and secrete a lot of perforin to kill pathogen-infected cells, and B lymphocytes protect the host by making antibodies dependent or independent of helper T lymphocytes. The proinflammatory cytokines represented by IL-6 and TNF-α are substances that mediate the immune response, and are particularly deeply involved in the initial immune response, and are stimulated by these cytokines to induce lymphocytes, such as MIP-1α, It is known that chemokines such as RANTES are secreted (Commins and Borish et al., 2010).

In addition, when nitric oxide (NO) is produced from L-arginine by the nitric oxide synthases (NOS) enzyme group as a radical, it acts as an important secondary transporter of cells (Palmer and Rees et al., 1988). Inducible NOS (inducible NOS; iNOS) produces a large amount of NO during inflammation and generates reactive radicals such as peroxynitrite to exhibit anticancer, antibacterial and antiparasitic effects (Szabo, 1997). NO produced by activated macrophages has been identified as a major effective molecule involved in the destruction of tumor cells, and moreover, it non-specifically defends the host, macrophage-mediated killing, microorganisms and tumors in vitro and in vivo. It has been reported that NO is involved in cell proliferation inhibition. Therefore, it is very important to develop an effective immunomodulator that can activate macrophages in defense against bacterial or environmental pathogens (Wu-Hsieh and Chen et al., 1998).

Immunodeficiency disease is deeply involved in clinical immunological intractable diseases, cancer, diabetes, and male infertility. All of them can be said to be diseases related to immunosuppression. The bioimmune system is controlled by the autonomic nervous system and the endocrine system, and has the characteristic of maintaining homeostasis by excluding non-self that is invaded from the outside or generated from the inside through humoral and cellular immunity. However, if this system is abnormally disturbed by some cause, immune failure, immunodeficiency, etc. will be caused. Immune function enhancers, immunopotentiators, etc. are generally used to correct and restore the abnormally disturbed immune system and to revive damaged immune functions (Schijns and O'Hagan, 2016).

Immune function enhancers are drugs mainly used for congenital and acquired immunodeficiency syndrome, and in particular, are urgently required as part of drug therapy for tumors or acquired immunodeficiency syndrome (AIDS). Representative immunostimulants currently used include immunoglobulin and interferon (Odean and Frane et al., 1990). Among them, the concentrated and purified IgG, one of human immunoglobulin, is used for preventive treatment of measles, chickenpox, hepatitis B, etc., but anaphylaxis due to pain at the injection site and lowering of blood pressure There is a disadvantage that large doses are dangerous because of a positive reaction (Zdziarski and Gamian et al., 2017). In addition, interferon (INF) was discovered as an antiviral factor, but after that, cell proliferation inhibitory action and immune function regulation action were confirmed, and it is used as an antiviral agent and an antitumor agent, etc., but the β-type is fever, malaise, Side effects such as anorexia and alopecia are observed, and type α shows side effects such as a decrease in white blood cells due to temporary suppression of bone marrow function and an increase in GOT (Taylor, 2014)

Immunopotentiators improve the secondary immunodeficiency state caused by excessive use of radiotherapy and anti-tumor agents or adrenocortical steroids by increasing the body's immune response. CWS (Cell Wall Skelton) and WSA (Water Soluble Adjuvant), which have improved the side effects of the drug, are used. Lentinan extracted and purified from picibanil obtained from group A type 3 Su strain of Streptococcus pyogenes, crestin, a protein polysaccharide obtained from Ganoderma lucidum, and Lentinus edodes. (lentinan), etc., are recognized, but anti-tumor activity or anti-AIDS effect is difficult to expect (Schijns and O'Hagan, 2016).

On the other hand, the immune adjuvant is a kind of vaccine additive to amplify the efficacy of the administered vaccine, and currently approved in Europe and the United States, there are aluminum salts, MF59, AS03, AS04, and the like. The adjuvant administered together with the vaccine induces dendritic cells and macrophages to readily absorb the antigen of the vaccine, and at the same time promotes the secretion of several cytokines and NO (Lee, 2012). Currently, metal salt compounds, cytokines, attenuated cell components, and lipid particle-type immune adjuvants are under development, but with the exception of aluminum salt, there remain problems such as safety of the formulation, preservation of vaccine quality, control of half-life, and various side effects. (Sohn et al, 2004).

In addition, aluminum complex (alum), developed by Glenny in 1926, has been proven to be the most widely used adjuvant so far, and its stability has been proven, but it has low antibody activity enhancing efficacy and does not activate cell-mediated immune response. Due to the shortcomings of this, a new immune enhancer is required. In addition to inorganic salts such as aluminum calcium, many immune adjuvants using bacteria or derivatives including mycobacteria, gram-negative endotoxin, and muramyl dipeptide (MDP) have been researched and developed, but these also have problems such as toxicity, side effects, and cost. has not been used clinically (Bae and Cho et al., 2018).

Accordingly, in recent years, research has been conducted to develop immune enhancing agents using natural products including crude extracts, compounds isolated from plants, microorganisms-derived natural substances, flavonoids, anthraquinones, etc., which have minimal side effects to the human body (Siddiquee, 2014) ; Grigore, 2017). In the present invention, the immune enhancing activity of quinizarine-O-glucoside, which is one of the anthraquinone glycosides among natural products, was revealed and completed the present invention.

The present invention has been devised to solve the above problems, and the present invention is to provide a pharmaceutical composition or a health functional food composition for enhancing immunity comprising quinizarine-O-glucoside having an immune enhancing effect.

The present invention may provide a pharmaceutical composition for enhancing immunity comprising quinizarine-O-glucoside.

The pharmaceutical composition may include an expression level of IL-6; Alternatively, the secretion amount of RNATES, TNF-α or MIP-1α may be increased.

The pharmaceutical composition may increase the amount of expression of NO synthetase (iNOS) or the amount of generation of nitric oxide (NO).

The pharmaceutical composition may increase phosphorylation of ERK, p38 or JNK.

The pharmaceutical composition may increase the expression level of Cyclin D1 or PCNA.

The pharmaceutical composition may include quinizarine-O-glucoside at a concentration of 10 to 100 μM.

The pharmaceutical composition may activate the proliferative ability of splenocytes.

The pharmaceutical composition may activate phagocytic activity.

The pharmaceutical composition may be used for one or more diseases from the group consisting of intestinal function, gastrointestinal disease, constipation, gastric ulcer, gastritis, gastric cancer, respiratory infection, skin disease, bird flu, and various cancers.

The present invention may also provide a health functional food composition for enhancing immunity comprising quinizarine-O-glucoside.

The quinizarine-O-glucoside of the present invention increases the secretion of TNF-α and MIP-1α and RANTES, which are representative inflammatory cytokines for immune cells, particularly macrophages, and increases the expression levels of iNOS and IL-6 mediating immune responses and It has a high immune enhancing effect by increasing NO production. In addition, by increasing the phosphorylation expression of MAPKs (ERK, p38, JNK), the expression level of Cyclin D1 and PCNA, which are cell cycle-related factors, is increased to induce cell proliferation, thereby having an immune enhancing effect. The quinizarine-O-glucoside of the present invention has an immune enhancing effect by increasing the phagocytosis of immune cells, particularly macrophages, and increasing cell proliferation.

1 shows a graph confirming the cytotoxicity and cell proliferation of quinizarine-O-glucoside by treating and culturing mouse-derived macrophages (RAW264.7 cells), and MTT assay method.
2 is a result of measuring the concentration of TNF-α secreted into the medium by ELISA method after treatment with quinizarine-O-glucoside by concentration in mouse-derived macrophages (RAW264.7 cells) (FIG. 2A), Shows the results of measuring the change in the mRNA expression level of IL-6 (FIG. 2B) and a graph quantifying it using the RT-PCR method.
3 is a ELISA method showing the concentrations of MIP-1α ( FIG. 3A ) and RANTES ( FIG. 3B ) secreted into the medium after quinizarine-O-glucoside was treated with mouse-derived macrophages (RAW264.7 cells) by concentration. is the result of measurement.
Figure 4 shows the results of measuring the change in mRNA expression level of iNOS (Figure 4A) and a graph quantifying it after treatment with quinizarine-O-glucoside in mouse-derived macrophages (RAW264.7 cells). Shows a photograph measuring the amount of production by confocal microscopy (FIG. 4B).
FIG. 5 shows quinizarine-O-glucoside was treated with mouse-derived macrophages (RAW264.7 cells) by concentration, and then Phospho-MEK (FIG. 5A), Phospho-ERK ( Figure 5B), Phospho-p38 (Figure 5C), Phospho-JNK (Figure 5D), and Phospho-MEK after pretreatment with UO126, an inhibitor of Phospho-MEK, shows changes in Phospho-MEK upon treatment with quinizarine-O-glucoside ( 5F, 5G).
FIG. 6 shows quinizarine-O-glucoside was treated with mouse-derived macrophages (RAW264.7 cells) by concentration, and then Cyclin D1 (FIG. 6B) and PCNA (FIG. 6C) using a western blot method. represents the expression level of
7 shows activation of phagocytosis by visualizing quinizarine-O-glucoside in mouse-derived macrophages (RAW264.7 cells) by concentration, and then visualized through a confocal microscope.
8 shows the splenocyte proliferation ability of quinizarine-O-glucoside by CCK-8 assay after treatment with quinizarine-O-glucoside in mouse splenocytes.

Hereinafter, the present invention will be described in more detail.

As described above, drugs capable of enhancing or recovering immune dysfunction or immunodeficiency due to the destruction of the immune system are rare. There are problems that are difficult to use in practice.

The present inventors found that while searching for a substance for enhancing immunity, quinizarine-O-glucoside increased the secretion of TNF-α, a proinflammatory cytokine, and MIP-1α and RANTES, a chemokine, and IL-6 and iNOS expression, increase NO production, promote phagocytic activity of macrophages, activate MAPKs signaling pathways (MEK, ERK, p38, JNK, and cell cycle-related factors Cyclin D1 and Since it significantly increases the expression of PCNA and activates the proliferative capacity of mouse-derived macrophages and splenocytes, it was confirmed that the composition has excellent immune enhancing activity that significantly increases the initial innate immunity using this composition and completed the present invention.

The present invention may provide a pharmaceutical composition for enhancing immunity comprising quinizarine-O-glucoside.

The quinizarin-O-glucoside may be represented by Formula 1 below.

[Formula 1]

The quinizarine-O-glucoside is a glycoside of quinizarine. It is known that the glycosides of quinizarine are mainly present in the root of Rubia tinctorum (madther), also known as horsetail. The root of this plant is mainly used as a natural red dye and is used as a treatment for kidney stones and bladder stones. Studies on derivatives of quinizarine glycosides are known to exhibit anticancer activity, but studies on quinizarine glucoside have not been conducted, and in particular, immune enhancing activity has not been studied or reported to date. The quinizarin-O-glucoside may be preferably quinizarin-1-O-β-D-glucoside. .

As used herein, the term “immunomodulation” refers to an action that has no effect on the normal immune response by suppressing the enhanced immune response of the living body and enhancing the decreased immune response.

The pharmaceutical composition may include an expression level of IL-6; Alternatively, the secretion amount of RNATES, TNF-α or MIP-1α may be increased. Specifically, quinizarine-O-glucoside can enhance immune activity by increasing the secretion of RNATES, TNF-α or MIP-1α in a concentration-dependent manner. When quinizarine-O-glucoside was treated at concentrations of 10, 25, 50 and 100 μM, it was confirmed that the mRNA expression level of IL-6 increased in a concentration-dependent manner, and at a concentration of 100 μM of quinizarine-O-glucoside, Compared with the control group, the mRNA expression level of IL-6 increased by 2.9-fold ( FIG. 2B ). When quinizarine-O-glucoside was treated by concentration, the amount of TNF-α production increased significantly in a concentration-dependent manner, and at 100 μM, it increased about 10.1 times compared to the control (Con) that was not treated with anything ( FIG. 2A ). When quinizarine-O-glucoside was treated by concentration, the amount of chemokine production (MIP-1α and RANTES) increased in a concentration-dependent manner, and the amount of MIP-1α and RANTES at 100 μM was approximately 4 times that of the control group that was not treated with anything ( Fig. 3A), increased 1.6 times (Fig. 3B).

The pharmaceutical composition may increase the amount of expression of NO synthetase (iNOS) or the amount of generation of nitric oxide (NO). When quinizarine-O-glucoside was treated at concentrations of 10, 25, 50 and 100 μM, it was confirmed that the mRNA expression level of iNOS increased in a concentration-dependent manner. In comparison, the mRNA expression level of iNOS was increased by 2.6-fold, respectively (FIG. 4A). It was confirmed that the concentration of NO in the cell increased in a quinizarine-O-glucoside concentration-dependent manner ( FIG. 4B ). Through this, it was found that the quinizarine-O-glucoside of the present invention increases the amount of NO as the expression of iNOS increases, thereby giving an immune enhancing effect.

The pharmaceutical composition may increase phosphorylation of ERK, p38 or JNK. As a result of measuring phosphorylation of MEK, p38, JNK and ERK after treatment with quinizarine-O-glucoside at different concentrations, the phosphorylation of MEK, ERK, p38, and JNK was concentration-dependently higher than that of the control untreated. It was confirmed that the increase (FIG. 5). In addition, it was confirmed that phosphorylation was reduced due to the MEK inhibitor that inhibits the ERK pathway among the MAPKs signaling pathways ( FIG. 5 ). It was found that the immune enhancing activity of quinizarine-O-glucoside in mouse macrophages occurs by activating the MAPKs signaling pathway involved in the immune response.

The pharmaceutical composition may increase the expression level of Cyclin D1 or PCNA. After treatment with quinizarine-O-glucoside at different concentrations, the expression levels of Cyclin D1 and PCNA were measured. As a result, it was confirmed that the expression levels of Cyclin D1 and PCNA, which are factors related to cell proliferation, were increased ( FIG. 6 ).

The pharmaceutical composition may activate the proliferative ability of splenocytes. After treatment with quinizarine-O-glucoside by concentration, phagocytosis of apoptotic Jurkat T cells inside BMDM was imaged by fluorescence microscopy, and as a result, it was confirmed that phagocytosis increased 2.5-fold in a concentration-dependent manner ( FIG. 7 ). Through this, it was confirmed that the quinizarine-O-glucoside of the present invention has an immune enhancing effect by activating phagocytosis.

The pharmaceutical composition may activate phagocytic activity. When quinizarine-O-glucoside was prepared at a concentration of 10, 25, 50 and 100 μM, respectively, and treated with splenocytes, it was found that about 52% increased splenocyte activity. It was confirmed that quinizarine-O-glucoside showed an immune enhancing effect through activation of the proliferative ability of splenocytes ( FIG. 8 ).

The pharmaceutical composition may include quinizarine-O-glucoside at a concentration of 10 to 100 μM, preferably 20 to 100 μM. As a result of a cytotoxicity experiment using RAW264.7 cell, a mouse-derived macrophage, it was confirmed that there was no toxicity to cells up to a concentration of 100 μM of quinizarine-O-glucoside, but rather cell proliferation ( FIG. 1 ).

The pharmaceutical composition may be used for one or more diseases from the group consisting of intestinal function, gastrointestinal disease, constipation, gastric ulcer, gastritis, gastric cancer, respiratory infection, skin disease, bird flu, and various cancers.

Carriers, excipients and diluents that may be included in the pharmaceutical composition comprising quinizarine-O-glucoside include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate. , gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, it is prepared using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient in the extract, for example, starch, calcium carbonate, sucrose ( sucrose) or lactose, gelatin, etc. are mixed and prepared. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid formulations for oral use include suspensions, solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients, for example, wetting agents, sweeteners, fragrances, preservatives, etc. may be included. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like can be used.

The quinizarine-O-glucoside of the present invention may be administered to mammals such as rats, mice, livestock, and humans by various routes. Any mode of administration may be contemplated, for example oral, rectal or intravenous, intramuscular, subcutaneous, sublingual, intramedullary, intranasal, intrathecal or transdermal administration.

In addition, the dosage of the compound of the present invention to the human body may vary depending on the patient's age, weight, sex, dosage form, health condition, and disease level, and is generally based on an adult patient weighing 60 kg. 0.001 to 1,000 mg/day, preferably 0.01 to 500 mg/day, may be administered in divided doses once or several times a day at regular time intervals according to the judgment of a doctor or pharmacist.

The present invention may also provide a functional health food composition for enhancing immunity comprising quinizarine-O-glucoside. Specific details of the quinizarine-O-glucoside are the same as described above.

There is no particular limitation on the type of the health functional food. The type of health food to which the quinizarine-O-glucoside can be added is not particularly limited, and examples include meat, sausage, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, gum, and ice cream. dairy products, various soups, beverages, teas, drinks, alcoholic beverages, and vitamin complexes.

The health beverage composition of the present invention has no particular limitation on the liquid component except for containing the RPE-1 as an active material as an essential component in the indicated ratio, and contains various flavoring agents or natural carbohydrates as additional components like a conventional beverage. can do. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; disaccharides such as maltose, sucrose and the like; polysaccharides such as dextrins, cyclodextrins; and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (taumatine, stevia extract (eg rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. there is.

In addition to the above, the health functional food of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, colorants and thickeners (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. In addition, the health functional food of the present invention may contain natural fruit juice, fruit juice for the production of fruit juice beverages and vegetable beverages. These components may be used independently or in combination.

Hereinafter, the present invention will be described in detail through examples. However, these Examples are for explaining the present invention in more detail, and the scope of the present invention is not limited to these Examples.

Preparation Example 1. Method for preparing quinizarine-O-glucoside

Quinizarine was purchased from Tokyo Chemical Industry (Tokyo, Japan), and according to the method of Pandey et al. (Pandey et al., 2019), quinizarine-O-glucoside (quinizarine -1-O-β-D-glucoside) (Pandey et al., 2019, Organic letters, 21(19), 8058-8064.). The E. coli strain into which pET28a-YjiC was introduced was cultured in 5 ml of LB medium supplemented with 50 μg/ml of kanamycin for recombinant maintenance, and then cultured at 37 °C for 5-6 hours. After that, the E. coli BL-21 (DE3) strain was transferred to a large flask containing 500 ml LB medium and kanamycin and incubated at 37 °C until the value of the optical density of the cells was 0.6, followed by 0.5 mM isotope. Protein expression was induced by the addition of propyl β-D-1-thiogalactopyranoside (IPTG), followed by further incubation at 20 °C for 18 h. 0.2 mM quinizarine was added to the culture with 10% glucoside and incubated for 48 hours, all in vivo transformation cultures were harvested with a separatory funnel, ethyl acetate was added in double volume and shaken vertically for 30 minutes and then an aqueous layer and an organic layer were fixed for 30 minutes. Then, the organic layer of ethyl acetate was transferred to a rotary evaporator and evaporated. The final residual sample was dissolved in 1 ml of methanol. The structure of the final material was confirmed using HPLC-PDA and HQ-QTOF ESI /MS (Pandey et al., 2019). Quinizarine-O-glucoside (quinizarine-1-O-β-D-glucoside) used in the present invention was provided by Professor Jaekyung Song of Sunmoon University and used.

Example 1: Measurement of cytotoxicity (MTT assay)

RAW264.7 cells, a mouse-derived macrophage cell line, were treated with 10% fetal bovine serum (Gibco-BRL, USA) and 1% Penicillin-Streptomycin (+5,000 Units/ml Penicillin, +5,000 μg/μl Streptomycin, Gibco-BRL, USA). As the DMEM culture medium, 5% CO ₂ was supplied and cultured at 37° C. in an incubator. RAW264.7 cells were added at 5×10 ⁴ cells/ml to 96-well plates (SPL, Korea) by 100 μl and incubated in an incubator at 37° C., 5% CO ₂ for 24 hours, and then quinizarine-O-gluco The seeds were prepared at a concentration of 10, 25, 50 and 100 μM, respectively, and treated in RAW264.7 cells. After incubation for 24 hours, 20 μl of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT, 5 mg/ml, Roch, Germany) solution in PBS buffer was added to each well. Each was added and incubated for 4 hours again. After confirming the formation of Formazan, the medium was completely removed, and 100 μl of dimethyl sulfoxide (DMSO; dimethyl sulfoxide) was added to dissolve the non-aqueous formazan formed at the bottom of the well, and then a microplate reader (Molecular Devices, USA) ) was used to measure the absorbance at 570 nm. It was confirmed that quinizarine-O-glucoside was not toxic to cells up to a concentration of 100 μM, but rather cell proliferation. Therefore, in this experiment, it was decided to use quinizarine-O-glucoside up to a concentration of 100 μM ( FIG. 1 ).

Example 2: Cytokine and Chemokine Measurement

2-1. Cytokine measurement in RAW264.7 macrophages

The amount of cytokine production in macrophages following stimulation of quinizarine-O-glucoside was measured. Cytokines refer to all substances secreted by cells, and especially play an important role in immune and inflammatory responses including innate immunity, antigen presentation, myeloid cell differentiation, cell recruitment and activation, and cell adhesion molecule expression. RAW264.7 cells were added to 24 well plates at 1×10 ⁵ cells/ml, 1 ml each, and incubated in an incubator at 37° C., 5% CO ₂ for 24 hours, followed by 10, 25, 50 quinizarine-O-glucoside. It was prepared at a concentration of 100 μM and treated in RAW264.7 cells. After culturing for 24 hours under the same conditions as above, a cell culture medium was obtained, and the cytokine production amount was investigated using the TNF-α (Mouse TNF-α ELISA kit, R&D System, USA) ELISA kit, which is a cytokine contained in the culture medium. When quinizarine-O-glucoside was treated by concentration, the amount of cytokine production was significantly increased in a concentration-dependent manner, and at 100 μM, it increased about 10.1 times compared to the control (Con) that was not treated with anything. From this, it was found that quinizarine-O-glucoside induces macrophages to increase the secretion of TNF-α, an immune response-mediated cytokine, to bring about an immune enhancing effect (FIG. 2A).

2-2. Chemokine determination in RAW264.7 macrophages

The amount of chemokine production in macrophages following stimulation of quinizarine-O-glucoside was measured. Chemokines are small chemical molecules that induce chemotaxis in neutrophils, monocytes, lymphocytes, eosinophils, fibroblasts, and keratinocytes. Chemokines secreted by stimulation of pro-inflammatory cytokines induce lymphocytes, neutrophils and monocytes to the site of infection and initiate an immune response to treat inflammation. Raw264.7 cells were incubated in 12 well plates at a density of 1×10 ⁵ cells/ml at 37°C, 5% CO ₂ condition for 24 hours, and then quinizarine-O-glucoside was added with 10, 25, 50 100 μM concentration and incubated for 24 hours under the same conditions as above. After that, the culture medium for each concentration was recovered and the amount of chemokine production was investigated using an ELISA kit (murine RANTES ELISA kit, Peprotech, USA; murine MIP-1α ELISA kit, Peprotech, USA). When quinizarine-O-glucoside was treated by concentration, the amount of chemokine production increased in a concentration-dependent manner, and the amounts of MIP-1α and RANTES at 100 μM were approximately 4 times (Fig. 3A) and 1.6 times (Fig. 3A) and 1.6 times (Fig. 3B) increased.

Example 3: Measurement of expression levels of NO synthetase (iNOS) and IL-6 mRNA by RT-PCR

3-1. RNA Isolation from RAW264.7 Macrophages

RAW264.7 cells were cultured in 6 well plates at a density of 5×10 ⁵ cells/ml, and then treated with quinizarine-O-glucoside at 10, 25, 50 and 100 μM concentrations for each concentration. After washing with phosphate-buffered saline, 1 ml of Trizol (Trizol, Invitrogen, USA) was added to lyse the cells for 30 minutes at room temperature, and then transferred to an Eppendorf tube (EP tube, eppendorf tube). After that, add 200 μl of chloroform (chloroform, Sigma, USA) for RNA extraction (Total RNA Extraction Kit, Intron biotechnology, Korea) per 1 ml of Trizol, mix, and centrifuge (13,000 rpm, 10 minutes, 4℃) to the top RNA was recovered from the supernatant of 400 μl of Binding Buffer was added to the recovered RNA and mixed for about 1 minute. After removing chloroform using an RNA-only column and washing buffer 1 (700 μl) and 2 (700 μl) to recover only RNA, remove the remaining RNA column, transfer it to a new EP tube, and use the elution buffer ( Elution Buffer) was used to filter RNA under the column to recover only RNA.

3-2. cDNA synthesis in RAW264.7 macrophages

RNA recovered in Example 4-1 was quantified with a microplate reader (Bio-Tek, USA) to prepare quantified RNA, and diethyl pyrocarbonate water (DEPC water; dimethyl pyrocarbonate water) was prepared. After adding quantified RNA and DEPC water to a PCR tube, 1 μl of reverse transcriptase and Oligo (dT) ₁₅ were added. After that, the high capacity cDNA reverse transcription kit (Applied Biosystems, USA) is mixed well and cDNA is synthesized using a PCR machine.

3-3. Investigation of mRNA expression level in RAW264.7 macrophages using cDNA

1 μl of the cDNA synthesized in Example 3-2 and 2 μl of the primers shown in Table 1 below were put into a master mix solution (Master Mix Solution; iNtRON, i-StatTaq, Korea), and then 17 μl of DEPC water was added, followed by PCR. amplified. Thereafter, the PCR reaction product was electrophoresed on a 2% agarose gel and stained with ethidium bromide (EtBr; ethidium bromide, Sigma, USA), and confirmed in a UV lamp. After the PCR, the concentration of IL-6 and iNOS was confirmed by using the Gel Doc EZ imager (Bio-Rad, USA) device, and the expression intensity was derived through the imageJ (NIH, USA) program, and then the value was extracted as an Excel file. It was transferred and calculated and shown in Figures 3B and 5A, respectively. When quinizarine-O-glucoside was treated at concentrations of 10, 25, 50 and 100 μM, it was confirmed that the mRNA expression level of iNOS increased in a concentration-dependent manner. In comparison, it was confirmed that the mRNA expression levels of iNOS and IL-6 increased by 2.6-fold ( FIG. 4A ) and 2.9-fold ( FIG. 2B ), respectively. Through this, it was found that the quinizarine-O-glucoside of the present invention increases the expression of iNOS to bring about an immune enhancing effect.

Base sequence of primers used Sequence Forward Reverse iNOS 5'-CTGCAGCACTTGGATCAGGAACCTG-3' 5'-GGGAGTAGCCTGTGTGCACCTGGAA-3' IL-6 5’-GTTCTCTGGGAAATCGTGGA-3’ 5’-TGGTCTTGGTCCTTAGCCAC-3’ β-actin 5'-TGGAATCCTGTGGCATCCATGAAAC-3' 5'-TAAAACGCAGCTCAGTAACAGTCCG-3'

Example 4: Nitric oxide (NO) measurement

Raw264.7 cells were incubated in a confocal dish at a density of 5×10 ⁵ cells/ml at 37° C. and 5% CO ₂ for 24 hours, and then quinizarine-O-glucoside was added at a concentration of 10, 25, 50 and 100 μM. treated with and incubated for 24 hours under the same conditions as above. To measure the concentration of intracellular NO, 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM DA), a fluorescence used as an indicator of NO, was added at a concentration of 10 μM at 37°C for 1 hour. dyed. To image the degree of fluorescence emission, the fluorescence intensity was measured at 495 nm (excitation) and 515 nm (emission) using a Zeiss LSM 710 laser scanning confocal microscope (Carl Zeiss Microimaging, Jena, Germany) in a concentration-dependent manner. It was confirmed that the concentration of NO in the inside increases (Fig. 4B). Through this, it was found that the quinizarine-O-glucoside of the present invention increases the amount of NO as the expression of iNOS increases, thereby giving an immune enhancing effect.

Example 5: Investigation of immune enhancement mechanism through MAPK (mitogen activated protein kinases, ERK, p38 and JNK) signaling pathway

To investigate the mechanism of immune enhancement of quinizarine-O-glucoside against mouse macrophages (RAW264.7 cells), the mechanism of immune enhancement through the MAPK signaling pathway was investigated by Western blot method. MAPK (p38 and ERK) signaling pathways are known as representative signaling pathways involved in cell survival. Mouse macrophages (RAW264.7 cells) were cultured in 6 well plates for 24 hours, quinizarine-O-glucoside was prepared at a concentration of 10, 25, 50 and 100 μM, and the cells were treated for 30 minutes. After 30 minutes, PBS was washed and cells were harvested. In addition, in the case of blocking the MEK1/2/ERK signaling pathway, 10, 20, or 30 μM of U0126 (Sigma, USA), a type of MEK1/2 inhibitor, was pretreated 10 minutes before treatment with quinizarine-O-glucoside. did After adding 60 μl of each lysis buffer, the reaction was performed on ice for 20 minutes and centrifuged at 13,000 rpm for 10 minutes to harvest the protein. Proteins were quantified using the Bradford assay, sample loading buffer was added, denatured at 90°C or higher for 10 minutes, and then electrophoresed using SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis) gel. After transferring to PVDF (polyvinylidene fluoride) membrane, blocking with 5% skim milk powder at room temperature for 2 hours The reaction was carried out overnight. After washing 3 times every 10 minutes with TBST (TBS, 0.1% Tween 20), the HRP (horeseradish peroxidase)-conjugated secondary antibody was diluted 1:2000 in 5% skim milk powder and reacted at room temperature for 1 hour. After washing three times with TBST in the same way as above, ECL (Enhanced Chemiluminescence) substrate solution was reacted on the membrane for 1 minute, and then developed on an X-ray film. As a result, the phosphorylation of MEK, p38, JNK and ERK was measured after treatment with quinizarine-O-glucoside at different concentrations. As a result, phosphorylation of MEK, ERK, p38, and JNK was significantly higher than that of the control untreated. It was confirmed that the concentration-dependent increase (FIG. 5). Accordingly, it was confirmed that the expression levels of Cyclin D1 and PCNA, which are factors related to cell proliferation, were increased (FIG. 6). In addition, it was confirmed that phosphorylation was reduced due to the MEK inhibitor that inhibits the ERK pathway among the MAPKs signaling pathways (FIG. 5). Therefore, it was found that the immune enhancing activity of quinizarine-O-glucoside in mouse macrophages occurs by activating the MAPKs signaling pathway involved in the immune response.

Example 6: Immune enhancement activity investigation through phagocytic activity

To investigate the phagocytosis activity of quinizarine-O-glucoside using BMDM on mouse macrophages (RAW264.7 cells), BMDM was cultured at a density of 5×10 ⁴ cells/ml for 24 hours and then quinizarine-O- Glucoside was treated and incubated for an additional 48 hours. Jurkat T cells were labeled with CFSE and exposed to UV irradiation (254 nm) for 10 min to induce apoptosis, incubated at 37°C for 2 h, and then co-cultured with BMDM at a 4:1 ratio (target to BMDM). Phagocytosis was incubated at 37 °C for 90 min, washed 4 times with cold PBS to remove non-phagocytic apoptotic Jurkat T cells, and then cells were stained with rhodamine-labeled phalloidin according to the manufacturer's instructions. The phagocytic apoptotic Jurkat T cells inside BMDM were imaged with a fluorescence microscope (Carl Zeiss, German Magnification, X200), and it was confirmed that the phagocytosis increased 2.5-fold in a concentration-dependent manner ( FIG. 7 ). Through this, it was found that the quinizarine-O-glucoside of the present invention has an immune enhancing effect by activating phagocytosis.

Example 7: Measurement of proliferation capacity of splenocytes

In this experiment, 12-week-old male C57BL/6 mice were used for splenocyte isolation and culture. Mice were purchased from Orient Bio (Orient Co., Seongnam, Korea) and pre-bred for 1 week in an animal laboratory where a temperature of 20±2° C., a humidity of 50±10%, and a light-dark cycle of 12 hours were maintained, and then used for the experiment. The extracted spleen was sterilized in RPMI 1640 medium using stainless mesh (0.038 aperture, Sigma-Aldrich, Korea) to make a single cell suspension. The suspension was centrifuged to remove the supernatant, and RBC lysis buffer (Biolegend, USA) was added to hemolyze the red blood cells. The hemolysis layer was removed by centrifugation, washed with RPMI 1640 medium, and the splenocytes were resuspended in complete RPMI 1640 medium containing 10% FBS. Then, 100 μl of splenocytes were added to 96-well plates (SPL, Korea) at 1×10 ⁶ cells/ml and incubated in an incubator at 37° C., 5% CO ₂ for 24 hours, and then quinizarine-O-gluco Seeds were prepared at a concentration of 10, 25, 50 and 100 μM, respectively, and treated with splenocytes. After incubation for 72 hours, 10 μl of CCK-8 solution was added to each well and further incubated for 30 minutes. After the reaction, absorbance was measured at 450 nm using a microplate reader (Molecular Devices, USA). As a result, at a concentration of 100 μM of quinizarine-O-glucoside, splenocyte activity was increased by about 52% compared to the control group not treated with anything. Therefore, in this experiment, it was confirmed that quinizarine-O-glucoside showed an immune enhancing effect through activation of the proliferative capacity of splenocytes (FIG. 8).

As described above in detail a specific part of the present invention, for those of ordinary skill in the art, this specific description is only a preferred embodiment, and it is clear that the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (10)

A pharmaceutical composition for enhancing immunity, comprising quinizarine-O-glucoside.
According to claim 1, wherein the pharmaceutical composition increases the secretion of RNATES, TNF-α or MIP-1α, the pharmaceutical composition for enhancing immunity.
The pharmaceutical composition for enhancing immunity according to claim 1, wherein the pharmaceutical composition increases the expression level of NO synthetase (iNOS) or the amount of NO (Nitric Oxide) generation.
The pharmaceutical composition for enhancing immunity according to claim 1, wherein the pharmaceutical composition increases phosphorylation of ERK, p38 or JNK.
The pharmaceutical composition of claim 1, wherein the pharmaceutical composition increases the expression level of Cyclin D1 or PCNA.
The pharmaceutical composition for enhancing immunity according to claim 1, wherein the pharmaceutical composition activates the proliferative ability of splenocytes.
The pharmaceutical composition for enhancing immunity according to claim 1, wherein the pharmaceutical composition activates phagocytic activity.
The pharmaceutical composition for enhancing immunity according to claim 1, wherein the pharmaceutical composition contains quinizarine-O-glucoside in a concentration of 10 to 100 μM.
According to claim 1, wherein the pharmaceutical composition can be used for one or more diseases from the group consisting of intestinal function, gastrointestinal disease, constipation, gastric ulcer, gastritis, gastric cancer, respiratory infection, skin disease, bird flu and various cancers, increase immunity pharmaceutical composition for use.
A health functional food composition for enhancing immunity, comprising quinizarine-O-glucoside.

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