Pharmaceutical composition for prevention or alleviation of inflammatory bowel disease, comprising ginseng-derived low-molecular-weight glucan polysaccharide l-gps

Abstract

The present invention reveals that a low-molecular-weight polysaccharide prepared from a ginseng-derived water-soluble high-molecular-weight polysaccharide by means of enzymatic degradation is a low-molecular-weight ginseng glucan polysaccharide (L-GPS) which is a type of glucan composed of 96% or more of glucose, has a backbone structure of α-1,4-linked glucose, and has a molecular weight of 3,400 Da. In addition, the present invention confirms a novel functionality related to an inflammatory bowel disease (IBD) alleviation effect of this L-GPS.

Description

Pharmaceutical composition for preventing or improving inflammatory bowel disease comprising ginseng-derived low-molecular-weight glucan polysaccharide L-GPS

[0001] The present invention discloses that a low-molecular weight polysaccharide produced by enzymatic decomposition from a water-soluble high-molecular weight polysaccharide derived from ginseng is a type of glucan composed of 96% or more of glucose, and has a backbone structure composed of α-1,4 bonds of glucose and a molecular weight of 3,400 Da, and that the L-GPS has an effect of improving inflammatory bowel disease (IBD), thereby suggesting a novel use thereof.

[0002] In the present invention, low molecular weight ginseng glucan polysaccharide (L-GPS) is a human colorectal epithelial adenocarcinoma cell line model for human colorectal inflammatory bowel disease (IBD) in which inflammation and apoptosis are induced by TNF-α treatment. In this case, L-GPS treatment inhibits the expression of pro-inflammatory cytokines TNF-α and IL-6, inhibits phosphorylation of inflammatory protein ERK, significantly reduces mitochondrial dysfuction induced by inflammation, inhibits the activation of caspase 3, caspase 9 and PARP, which are factors related to inducing apoptosis, and increases the expression of Bcl-2, which is a factor related to inhibiting apoptosis. It effectively inhibited cell death (inflammatory apoptosis) caused by mitochondrial dysfunction, and it was confirmed that it has the activity of inhibiting intracellular reactive oxygen species (ROS) generated during an inflammatory response. This suggests that this low-molecular-weight ginseng glucan polysaccharide (L-GPS) can improve colonic inflammation by removing inflamed cells through apoptosis induction when inflammation occurs in epithelial cells in the colon. In addition, it was confirmed that this low-molecular-weight ginseng glucan polysaccharide (L-GPS) up-regulates autophagy decreased due to inflammation by increasing the expression of LC-3, a marker protein of autophagy decreased due to inflammation.This confirms that L-GPS can improve inflammatory diseases in the colon through two simultaneous (dual function) mechanisms: promoting apoptosis of inflammatory colon cells induced by inflammation, while upregulating autophagy reduced by inflammation.

[0003] In addition, L-GPS restored the length of the colon, which was significantly reduced in a mouse animal model induced with inflammatory bowel disease, to a length similar to that of the normal control group, and maintained the villi of the colon damaged due to inflammatory bowel disease at a level similar to that of the normal control group, thereby completing this invention by identifying a novel pharmacologically active material that exhibits protective and therapeutic effects against inflammatory bowel disease at the cellular and animal levels.

[0004] This invention was carried out with the support of the Ministry of Education's Research and Development (R&D) project, "Research on the treatment of inflammatory bowel disease using naturally derived sugar materials" (Project ID: 1345303909, Project ID: 2016R1A6A3A11933923, Implementing organization: Catholic University of Korea, Research period: 2016.11.01 ~ 2019.10.31).

[0005] Inflammatory bowel disease (IBD) is a general term for diseases characterized by persistent or recurrent inflammation of the colon and small intestine, including ulcerative colitis and Crohn's disease. IBD is a global disease with a rapidly increasing incidence, posing a serious threat to human health and imposing a significant financial burden on individuals, families, and society (Cai et al., Front Med (Lausanne), 8: 765474, 2021). While the exact cause of IBD remains unclear, multifactorial factors, including genetic predisposition, mucosal barrier dysfunction, disruption of the gastrointestinal microbiota, dysregulation of the intestinal immune system, and environmental and lifestyle factors, are considered (Ocansey et al., Biol Rev Camb Philos Soc. 95:1287-307, 2020).

[0006] Treatment of inflammatory bowel disease (IBD) has primarily focused on nonspecifically suppressing inflammation and immune responses. Anti-inflammatory drugs such as sulfasalazine and mesalamine, as well as steroids, are commonly used in the early stages of the disease. While anti-inflammatory drugs have relatively few side effects even with long-term use, their efficacy declines in patients with moderate to severe ulcerative colitis. Steroids, while capable of dramatically reducing mortality in patients with moderate ulcerative colitis, are associated with systemic side effects and lack the efficacy to maintain long-term remission (Ford et al., Am Gastroenterol. 106 (4):601, 2011; Jeen et al., Korean J Med 76:654-660, 2009). Immunomodulators such as azathioprine, 6-MP, cyclosporine A (CsA), and methotrexate (MTX) have been proposed as complementary treatments. These drugs are known to be effective and promote mucosal healing, and their use is increasingly occurring earlier. However, there were problems such as causing bone marrow damage, pneumonia, liver damage, etc. due to the patient's immunity being lowered too much, and the treatment effect was slow to appear. Since then, using molecular biological technology, new biological treatments that can suppress cytokines such as TNF-α, IL-12, and IL-23 that are significantly involved in the pathogenesis of inflammatory bowel disease have been developed, and many studies have been conducted and are still actively in progress. Among them, the most widely used are anti-TNF-α antibody protein treatments, which are TNF antagonists, such as adalimumab (Humira) and infliximab (Remicade), which show therapeutic effects by suppressing TNF-α, which causes chronic inflammation and tissue damage.The use of anti-TNF-α antibodies reduced the colectomy rate by 7% (Sandborn et al, Gastroenterology, 137:1250-60, 2009), and it is approved for use in patients with severe inflammatory bowel disease who do not respond to existing treatments (Adegbola et al, Int J Mol Sci., 19:2244, 2018). However, 40% of patients with inflammatory bowel disease do not respond to anti-TNF-α antibodies, and 23-46% of patients who received TNF-α inhibitors experienced relapse (Ben-Horin et al, Autoimmun Rev, 13:24-30, 2014). In the case of antibody drugs, the possibility of developing resistant patients increases with long-term administration, so the development of additional drugs for these patients is required.

[0007] With recent active research into identifying biological markers for inflammatory bowel disease, there is growing anticipation that gut microbiota-based drugs will become a key player in the treatment of inflammatory bowel disease. Identifying microbial metabolites that play a key role in anti-inflammatory effects in patients with inflammatory bowel disease and elucidating the interactions between these metabolites and receptors on patients' immune cells could lead to the development of drugs that operate in a similar manner. Most of these microbial metabolites are carbohydrates, such as polysaccharides, and their immunomodulatory properties have been previously reported in numerous studies. Marine polysaccharides such as alginate, porphyran, fucoidan, chitin, and chitin derivatives have been demonstrated to suppress allergic reactions, and sulfated polysaccharides from algae have been shown to exhibit anti-inflammatory effects in cellular (in vitro) and animal model (in vivo) studies due to their structure and physicochemical properties.

[0008] Among the representative natural products, ginseng has long been recognized as a very good medicinal herb, especially in Korea, China, and Japan, and its medicinal efficacy has been highly evaluated and used as a medicinal herb in many countries (Hakchangsa, Modern Pharmacognosy, Pharmacognosy Research Society, 251-297, 1997).

[0009] The saponin component, which represents the main efficacy of ginseng, is found in the roots, stems, and leaves of ginseng, and is composed of a total of about 30 types of ginsenosides. Ginsenosides are known to have various health-promoting effects, such as anti-cancer, anti-aging, prevention of adult diseases, liver protection, anti-inflammation, and nerve protection.

[0010] However, ginseng residue, which is the residue left after extracting soluble ginseng components from ginseng roots, is mostly composed of polysaccharides and insoluble dietary fiber, so despite the expectation of various physiological activities, its industrial use is very limited, as it is simply used as feed or discarded.

[0011] In the present invention, a low-molecular weight polysaccharide prepared by enzymatic decomposition from a water-soluble high-molecular weight polysaccharide isolated from ginseng marc is a type of glucan composed of 96% or more of glucose, and has a backbone structure composed of α-1,4 bonds of glucose and a molecular weight of 1,000 to 5,000 Da or 2,000 to 4,000 Da (preferably 3,400 Da), and it has been confirmed that it has a novel effect of improving inflammatory bowel disease (IBD) at the cellular level (in vitro) and in an animal model (in vivo).

[0012] In the study for the present invention, HT-29 cells, a human colon cancer cell line, were treated with L-GPS at various concentrations (0, 25, 50, 100, 250, 500 μg / ml) and human TNF-α was treated to evaluate the protective efficacy of L-GPS against TNF-α-induced inflammation and inflammatory cell death of colon cells. As a result, the mechanisms of L-GPS inducing inhibition of apoptosis due to inflammation, upregulation of autophagy decreased due to inflammation, reduction of inflammatory proteins, and inhibition of inflammatory cell death were confirmed, and these were verified in an animal model.

[0013] Numerous papers and patents are referenced and cited throughout this specification. The disclosures of these cited papers and patents are incorporated herein by reference in their entirety to provide a clearer understanding of the state of the art and the scope of the invention.

[0014] The purpose of the present invention is to elucidate the structure of a low-molecular weight polysaccharide prepared by enzymatically decomposing a water-soluble high-molecular weight polysaccharide isolated from ginseng marc, through component analysis and structural analysis of the low-molecular weight polysaccharide, and to elucidate its novel physiological activity. The purpose of the present invention is to provide a composition having an inflammatory bowel disease (IBD) prevention and improvement effect, which contains L-GPS, a low-molecular weight polysaccharide material derived from ginseng, as an active ingredient.

[0015] Other objects and advantages of the present invention will become more apparent from the detailed description, claims and drawings below.

[0016] According to one aspect of the present invention, the present invention provides a composition of a low-molecular weight polysaccharide prepared by enzymatically decomposing a water-soluble high-molecular weight polysaccharide isolated from ginseng marc (Fig. 1). In addition, it has been confirmed that the low-molecular weight polysaccharide, L-GPS, has a novel effect of improving inflammatory bowel disease (IBD) at the cellular level (in vitro) and in an animal model (in vivo), and thus a composition having an inflammatory bowel disease prevention and improvement effect, which contains the L-GPS as an active ingredient, is provided.

[0017] The present inventors attempted to elucidate the composition and structure of the low-molecular-weight polysaccharide L-GPS. As a result, the composition, molecular weight, and sugar linkage structure of L-GPS were elucidated. According to one aspect of the present invention, the composition, molecular weight, and sugar bond structure of a low-molecular weight polysaccharide prepared by enzymatically decomposing a water-soluble high-molecular weight polysaccharide separated from ginseng marc were determined, and the ginseng-derived low-molecular weight polysaccharide was found to be a type of glucan in which glucose constitutes 96% or more of the sugar component (Fig. 3). Through methylation, GC-MS analysis (Fig. 4), and NMR analysis (Fig. 5), the low-molecular weight polysaccharide was found to be a low-molecular weight ginseng glucan polysaccharide (L-GPS) having a molecular weight of 3,400 Da (Fig. 2) and a backbone structure composed of α-1,4 bonds of glucose.

[0018] The present inventors sought to elucidate the novel physiological activity of the low-molecular-weight ginseng glucan polysaccharide L-GPS. To this end, we established inflammatory bowel disease model cell lines and animal models, evaluated the inflammatory bowel disease-modifying efficacy of L-GPS treatment at the cellular level (in vitro) and in animal models (in vivo), and elucidated its mechanism of action.

[0019] In addition, it was confirmed that L-GPS has the effect of suppressing inflammatory bowel disease as a major physiological activity. More specifically, it effectively inhibited cell death (inflammatory apoptosis) caused by mitochondrial dysfunction by suppressing the expression of pro-inflammatory cytokines TNF-α and IL-6 in the human colon cell line HT-29 (Fig. 10B), inhibiting the phosphorylation of inflammatory protein ERK, significantly reducing mitochondrial dysfuction induced by inflammation (Fig. 6), inhibiting the activation of caspase 3, caspase 9, and cleaved PARP, which are factors related to inducing apoptosis (Fig. 9 A, B), and increasing the expression of Bcl-2, which is a factor related to inhibiting apoptosis (Fig. 9B), and inhibiting intracellular reactive oxygen species (ROS) generated during an inflammatory response (Fig. 5). It was confirmed that there was (Fig. 10C). This suggests that the low-molecular-weight ginseng glucan polysaccharide (L-GPS) can improve colonic inflammation by removing inflamed cells through apoptosis induction when inflammation occurs in epithelial cells in the colon. In addition, it was confirmed that the low-molecular-weight ginseng glucan polysaccharide (L-GPS) up-regulated autophagy decreased due to inflammation by increasing the expression of LC-3, a marker protein of autophagy decreased due to inflammation (Figs. 8, 9C). Therefore, it can be seen that the anti-inflammatory effect of L-GPS is useful for preventing diseases caused by inflammatory bowel disease.

[0020] The low molecular weight ginseng glucan polysaccharide (L-GPS) of the present invention may have a molecular weight of 3,400 Da.

[0021] The low molecular weight ginseng glucan polysaccharide (L-GPS) of the present invention may have a structure including a backbone structure in which glucose is formed by α-1,4 bonds.

[0022] The inflammatory bowel disease of the present invention may be a disease induced by TNF-α overexpression.

[0023] The inflammatory bowel disease of the present invention may be a disease induced by ulcerative colitis.

[0024] The present invention confirmed that L-GPS reduces inflammatory cell death caused by mitochondrial dysfunction and inflammatory apoptosis induced by inflammation induced by TNF-α, a proinflammatory cytokine, and it can be estimated that it is a natural material capable of preventing and improving inflammatory bowel disease.

[0025] According to another aspect of the present invention, the present invention provides a food composition, a functional food composition and a pharmaceutical composition for improving inflammatory bowel disease, which contain L-GPS as an active ingredient.

[0026] Specifically, the pharmacological composition containing L-GPS according to the present invention can be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and sterile injection solutions, respectively, according to conventional methods.

[0027] Carriers, excipients, and diluents that may be included in a pharmaceutical composition containing L-GPS 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, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. When formulated, the composition is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations are prepared by mixing the extract with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, syrups, etc., and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized 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. Suppository bases include witepsol, macrogol, Tween 61, cocoa butter, laurin, and glycerogelatin.

[0028] The pharmaceutical composition containing the L-GPS of the present invention as an active ingredient can be administered to mammals such as rats, mice, livestock, and humans through various routes including orally.

[0029] In the present invention, the description of pharmaceutical compositions primarily relates to pharmaceutical compositions intended for administration to humans. However, those skilled in the art will appreciate that such compositions are generally suitable for administration to all types of animals (particularly mammals other than humans). A skilled veterinarian or pharmacologist, with a keen understanding of the various modifications of pharmaceutical compositions intended for administration to various animals, can design and / or implement such modifications, if necessary, simply through routine experimentation.

[0030] In one aspect of the present invention, a use of low-molecular-weight glucan polysaccharide (L-GPS) for the prevention, improvement, or treatment of inflammatory bowel disease is provided. Accordingly, the present invention provides a pharmaceutical composition for the prevention or treatment of inflammatory bowel disease comprising the above-described low-molecular-weight glucan polysaccharide (L-GPS) or a pharmaceutically acceptable salt thereof as an active ingredient, and / or a method for the prevention or treatment of inflammatory bowel disease comprising administering the above-described low-molecular-weight glucan polysaccharide (L-GPS) or a pharmaceutically acceptable salt thereof to a subject in need thereof.

[0031] Matters mentioned in the pharmaceutical composition, health functional food composition, use, and prevention or treatment method of the present invention are equally applicable unless they are contradictory.

[0032] The features and advantages of the present invention are summarized as follows:

[0033] (a) The present invention provides a low molecular weight ginseng glucan polysaccharide (L-GPS) having a molecular weight of 3,400 Da and comprising a 1,4-α-glucan backbone structure in which glucose is linked by an α-1,4- bond.

[0034] (b) The present invention provides a composition for improving inflammatory bowel disease, which comprises L-GPS as an active ingredient.

[0035] (c) The L-GPS of the present invention has an effect of preventing or treating inflammatory bowel disease by confirming a mechanism of reducing inflammatory apoptosis and downregulating increased inflammatory factors in an inflammatory bowel disease model cell line induced by TNF-α, a pro-inflammatory cytokine, and increasing autophagy reduced due to inflammation, and verifying this in an animal model.

[0036] (d) The L-GPS of the present invention has a therapeutic effect of improving inflammatory bowel disease (IBD) by restoring the length of the large intestine, which is significantly reduced due to the induction of inflammatory bowel disease in a mouse animal model, to a length similar to that of a normal control group that is not induced with inflammatory bowel disease.

[0037] (e) The L-GPS of the present invention has a therapeutic effect of improving inflammatory bowel disease (IBD) by maintaining the villi of the colon damaged by inflammatory bowel disease induction in a mouse animal model at a level similar to that of the control group in which inflammatory bowel disease was not induced.

[0038] Figure 1 is a flow chart showing a manufacturing process of a low-molecular weight ginseng glucan polysaccharide (L-GPS) according to the present invention. Figure 1 shows a process of enzymatically decomposing and separating a water-soluble high-molecular weight polysaccharide isolated from ginseng marc and manufacturing a low-molecular weight polysaccharide.

[0039] Figure 2 shows the results of measuring the molecular weight of L-GPS obtained according to the present invention using HPLC.

[0040] Figure 3 is a result of analyzing the monosaccharide composition of L-GPS obtained according to the present invention using GC (Gas chromatography) analysis technique.

[0041] Figure 4 shows the results of investigating the binding structure of each component of L-GPS using methlylation and GC-MS analysis.

[0042] Figure 5 shows the results of analyzing and confirming the structure of L-GPS using NMR. Figure 5a shows the structure of L-GPS. 1 H and 13 This is a table of C NMR analysis results, and FIGS. 5ba and 5bb show the NMR analysis results of L-GPS (A, Proton NMR (1H NMR); B, Carbon NMR (13C NMR); C, COSY; D, TOCSY; E & F, HSQC; G, HMBC spectra of L-GPS), and FIG. 5c is an image showing a schematic binding structure of L-GPS.

[0043] Figure 6 shows the results of measuring the protective effect against inflammation-induced apoptosis by treating HT-29 cells with L-GPS and then treating them with TNF-α, as measured by changes in cell viability. Figure 6 confirms the inhibitory effect of L-GPS on inflammatory apoptosis induced by TNF-α treatment in a human colon epithelial cell line (HT-29).

[0044] Figure 7 shows the results of confirming the effect of L-GPS on the mitochondrial membrane potential of inflammatory bowel disease cell lines. After L-GPS was treated on HT-29 cells, the protective effect against mitochondrial dysfunction induced by inflammation by treating with TNF-α was measured using JC-1 staining.

[0045] Figure 8 shows the results of confirming the apoptosis inhibition ability of L-GPS of the present invention in inflammatory bowel disease cell lines. After L-GPS was treated on HT-29 cells, the protective effect against apoptosis induced by inflammation was measured using TUNEL staining by treating with TNF-α.

[0046] Figure 9 shows the results of IHC analysis to determine the cytoprotective effect of L-GPS through increased autophagy in inflammatory bowel disease cell lines. After L-GPS was treated in HT-29 cells, the effect of upregulating the decrease in autophagy induced by inflammation by treating with TNF-α was measured.

[0047] Figure 10 shows the results of confirming the apoptosis inhibition mechanism of L-GPS in inflammatory bowel disease cell lines. In Figure 10, after L-GPS was treated in HT-29 cells, the efficacy of L-GPS in regulating inflammation-induced apoptosis-related and inflammatory protein mechanisms by treating with TNF-α was measured using Western blot.

[0048] Figure 11 shows the results of confirming the inflammatory factor (p-ERK, TNF-α, IL-6, ROS) inhibition effect of L-GPS in an inflammatory bowel disease cell line model. In Figure 11, after L-GPS was treated to HT-29 cells, the changes in the gene expression of proinflammatory cytokines TNF-α and IL-6 and intracellular ROS induced by inflammation by TNF-α treatment were analyzed using RT-PCR and ROS fluorescent staining (DCF-DA), respectively.

[0049] Figure 12 shows the results of confirming the protective effect of L-GPS on inflammatory bowel disease by measuring colon length and H&E staining in an animal model.

[0050] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples, in accordance with the gist of the present invention.

[0051]

[0052] [Manufacturing example]

[0053] Manufacturing process of low-molecular weight ginseng glucan polysaccharide (L-GPS) derived from ginseng

[0054] In the present invention, low-molecular weight polysaccharides (L-GPS) were obtained by enzymatic hydrolysis from high-molecular weight polysaccharides extracted from ginseng marc according to the process diagram shown in Fig. 1. First, 15 L of distilled water was added to 3 kg of ginseng marc powder, stirred for 3 hours, and then centrifuged to obtain a supernatant. Then, ethanol (EtOH) was treated in an amount three times the volume of the obtained supernatant. Then, centrifuged once more to obtain a precipitate, freeze-dried, and the dried product was enzymatically hydrolyzed with pectinase (Pectinex ultra pulp, Novozymes, Denmark) at 40°C for 30 minutes. Afterwards, the enzymatic hydrolyzate was obtained by centrifugation and passed through a 10 kDa dialysis membrane (RC Tubing MWCO: 10 kD, Spectra / Por ® , USA) was used to obtain fractions. L-GPS was separated from the obtained fractions through ion exchange chromatography using DEAE-Sepharose Fast flow (cytiva, USA) resin (Fig. 1).

[0055]

[0056] [Example 1]

[0057] Molecular weight measurement of L-GPS

[0058] The molecular weight of L-GPS was measured using HPLC. The HPLC used for molecular weight measurement was a product of Waters, USA (Waters Alliance HPLC 2695). The column used was an SB-Showdex 805 HQ column, and 3-distilled water was used as the mobile phase. The mobile phase speed during analysis was 0.7 ml / min, and pullulan (342, 1320, 6200, and 9900 Da) was used as the standard. The molecular weight of L-GPS was confirmed to be 3,400 Da (Fig. 2).

[0059]

[0060] [Example 2]

[0061] Analysis of monosaccharide composition through GC (Gas chromatography) analysis of L-GPS

[0062] To analyze the monosaccharide composition of L-GPS, L-GPS was derivatized after acid hydrolysis and analyzed by gas chromatography (GC). 10 mg of L-GPS was hydrolyzed with 2 M trifluoroacetic acid (TFA) at 120°C for 1 h. The hydrolyzed monosaccharide mixture was reduced with sodium borodeuteride for 3 h and then with acetic acid, followed by acetylation using 3 ml of acetic anhydride and 1 ml of pyridine. Afterwards, impurities were removed using distilled water and chloroform, dried, and dissolved in methanol. The monosaccharide composition was measured using Trace GC Ultra (Thermo Scientific, USA). The results showed that glucose accounted for the majority (96.5%), confirming that L-GPS is a type of glucan polysaccharide (Fig. 3). In addition to glucose, trace amounts of arabinose (2.1%), mannose (0.8%), galactose (0.4%), and xylose (0.4%) were found.

[0063]

[0064] [Example 3]

[0065] Methylation and GC-MS analysis to determine the sugar linkage structure of L-GPS

[0066] To determine the linkage type of L-GPS, methylation analysis was performed using the Ciucanu and Purdie method. 5 mg of L-GPS was dissolved in 1 ml of DMSO and reacted with 0.5 ml of methyl iodide for methylation. The resulting permethylated L-GPS was separated from the reaction mixture using chloroform and dried using sodium sulfate. Next, the mixture was pretreated with 90% formic acid at 100°C for 1 h, the formic acid was evaporated, and the mixture was hydrolyzed with 2 M trifluoroacetic acid (TFA) at 120°C for 1 h. The mixture of hydrolyzed monosaccharides was reduced with sodium borodeuteride and acetylated to obtain methylated alditol acetates. The sugar linkage structure of the resulting methylated alditol acetates was determined using a Trace GC Ultra (Thermo Scientific, USA) equipped with a mass spectrometer (TSQ Quantum XLS, Thermo Scientific, USA). GC-MS analysis conditions were 80℃ (12 min) - 160℃ (8℃ / min) - 250℃ (4℃ / min, 25 min at 250℃) - 265℃ (20℃ / min, 10 min at 265℃) using a Restek RT- 2330-NB column (0.32 mm × 105 m), helium was used as the mobile phase, and the mobile phase speed was 1 ml / min. The methylation analysis results of L-GPS revealed that the structure contained 74.9% of the 1,4-glucose type binding structure, 14.2% of 1,4,6-glucose, and 4.2% of 1,3,4-glucose (Fig. 4).

[0067]

[0068] [Example 4]

[0069] NMR structural analysis of L-GPS

[0070] NMR spectroscopy analysis was performed to determine the structure of L-GPS. Proton (1H) NMR and 13C APT NMR spectra were measured using a Bruker Avance 600 and Bruker Avance 500 NMR spectroscopy (Bruker, USA), respectively. Measurements were performed after dissolving L-GPS in D2O at 80°C, and the operating frequencies were 600.1 MHz and 499.8 MHz for 1H and 150.9 MHz and 125.7 MHz for 13C, respectively. Proton, 13C, COSY, TOCSY, HSQC, and HMBC were analyzed using MestReNova 10.0 software (Mestrelab Research, Spain). The analysis results showed that the main carbohydrate unit was glucose, which formed a 1,4-α-glucopyranosyl backbone structure, and that 1,3,4-α-D-Glcp, 1,6-α-D-Glcp, and 1,3-β-D-Glcp were present (Fig. 5a). The HMBC analysis results revealed that the two signals at δ 4.87 / 77.5 (C1 / A3) and δ 4.12 / 102.46 (A3 / C1) were signals of the structure of 1,3-β-D-Glcp bound to the 1,4-α-glucopyranosyl backbone (Fig. 5ba, 5bb). These results were combined to confirm that L-GPS is a type of glucan with a 1,4-α-D-glucopyranoside backbone with branches at the O-3 and O-6 positions (Fig. 5c).

[0071]

[0072] [Example 5]

[0073] Investigation of the ability of L-GPS to alleviate apoptosis in inflammatory bowel disease cell lines

[0074] Human colonic epithelial cells (HT-29 cells) were added to 48-well culture plates (SPL, USA) at 1 × 105 cells / ml (500 ㎕) and cultured for 24 hours at 37℃ in a 5% CO2 incubator. Before treating with L-GPS, the growth medium was replaced with fresh growth medium (RPMI1640 + 10% fetal bovine serum). L-GPS was prepared at concentrations of 25, 50, 100, 250, and 500 ㎍ / ㎖, respectively, and treated to HT-29 cells. After culturing for 48 hours, each well was treated with Recombinant TNF-α (R&D, USA) at a concentration of 20 ng / ml for 48 hours. After 48 hours, 100 μl of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT, 5 mg / ml, ROTH, Germany) solution dissolved in PBS buffer was added and incubated for another 4 hours. After confirming formazan formation, the medium was completely removed, and 500 μl of DMSO (dimethyl sulfoxide) was added to dissolve the formazan formed at the bottom of the well, and then the absorbance was measured at 570 nm using a Microplate reader (Molecular Devices, USA). As a result of measuring cell viability, it was confirmed that cell viability, which was reduced by about 26% compared to the control when treated with TNF-α, was recovered in a concentration-dependent manner when treated with L-GPS (Fig. 6).

[0075]

[0076] [Example 6]

[0077] Investigation of the effect of L-GPS on mitochondrial membrane potential

[0078] To determine the effect of L-GPS on TNF-α-induced inflammatory apoptosis in human colon epithelial cells (HT-29 cells), mitochondrial membrane potential depolarization was examined using the JC-1 staining method. Human colon cancer cells (HT-29) were cultured in 6-well plates for 24 h, and L-GPS was prepared at concentrations of 50, 100, 250, and 500 mg / ml, respectively, and treated with the cells for 48 h. Afterwards, the cells were treated with recombinant TNF-α (R&D, USA) at a concentration of 20 ng / ml per well for 48 h. After washing with PBS, the cells were stained using the JC-1 Mitochondrial Membrane Potential Detection Kit (Biotium, USA) according to the manufacturer's instructions, and the mitochondrial membrane potential was measured using a fluorescence spectrophotometer at wavelengths of 550 / 600 nm; 485 / 535 nm. Through this, it was confirmed that treatment with L-GPS showed a protective effect against inflammatory cell death by suppressing the increase in mitochondrial membrane potential depolarization caused by inflammatory cell death (Fig. 7).

[0079]

[0080] [Example 7]

[0081] Confirmation of the protective efficacy of L-GPS through inhibition of apoptosis in inflammatory bowel disease cell line models.

[0082] To determine the effect of L-GPS on TNF-α-induced inflammatory apoptosis in human colonic epithelial cells (HT-29 cells), DNA fragmentation was examined using the terminal transferase-mediated dUTP-fluorescein nick end labeling (TUNEL) assay. The TUNEL assay uses TUNEL (green) and PI fluorescence (red) together to distinguish between living cells and dying or already dead cells (apoptotic cells).

[0083] Human colonic epithelial cells (HT-29 cells) were cultured in 8-chamber slides for 24 h, and L-GPS was prepared at concentrations of 250 and 500 mg / ml, respectively, and treated with the cells for 48 h. Afterwards, recombinant TNF-α (R&D, USA) was treated with each well at a concentration of 20 ng / ml for 48 h, and the cells were washed with PBS. After staining using the ApoDIRECT In Situ DNA fragmentation assay kit (Biovision Research Products, Mountain View, CA, USA) according to the manufacturer's instructions, the fragmentation was measured using a fluorescence microscope. Through this, it was confirmed that L-GPS treatment has a protective effect against inflammatory apoptosis by reducing the increased DNA fragmentation caused by TNF-α treatment (Fig. 8).

[0084]

[0085] [Example 8]

[0086] Confirmation of the protective efficacy of L-GPS through increased autophagy in inflammatory bowel disease cell line models.

[0087] To determine the effect of L-GPS on autophagy, which is important for maintaining cellular homeostasis in an inflammatory bowel disease cell line model, immunocytochemistry was used. Human colonic epithelial cells (HT-29) were cultured on 8-chamber slides for 24 h, and L-GPS was prepared at a concentration of 500 μg / ml and treated with the cells for 48 h. After treatment with recombinant TNF-α (R&D, USA) at a concentration of 20 ng / ml per well for 48 h, the cells were washed with PBS, fixed with 4% paraformaldehyde, blocked with 5% BSA, and stained for LC-3B (Green), a marker of autophagy, using a primary antibody against LC-3B and an Alexa Fluor 488-conjugated secondary antibody. Afterwards, nuclei and lysosomes were stained with DAPI (Blue) and Lysotracker (Red), respectively, and the degree of autophagy was confirmed using a confocal microscope (Zeiss, Germany). As a result, the degree of autophagy decreased by TNF-α was maintained when L-GPS was treated, confirming the protective effect against TNF-α-induced inflammation (Fig. 9).

[0088]

[0089] [Example 9]

[0090] Identification of the inflammatory cell death and inflammatory protein suppression mechanisms of L-GPS in inflammatory bowel disease cell line models.

[0091] To determine the effect of L-GPS on the inflammatory apoptosis pathway in an inflammatory bowel disease cell line model, the expression levels of Caspase 3, Caspase 9, PARP, BCl-2, and LC-3 proteins were examined by Western blotting. Human colon cancer cells (HT-29) were cultured in 6-well plates for 24 h, and L-GPS was prepared at concentrations of 25, 50, 100, 250, and 500 μg / ml, respectively, and treated with the cells for 48 h. Afterwards, recombinant TNF-α (R&D, USA) was treated to each well at a concentration of 20 ng / ml for 48 h, and the cells were washed with PBS. 60 μl of RIPA buffer was added to each well. The cells were incubated on ice for 30 min and centrifuged at 13,000 rpm for 15 min to obtain proteins. Proteins were quantified using the Bradford assay, denatured at 95°C for 10 minutes with sample loading buffer, and then electrophoresed using a 12% SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis) gel. After transferring to a nitrocellulose (NC) membrane, the membrane was blocked with 5% skim milk powder for 1 hour at room temperature, and then primary anti-cleaved caspase 3 antibody (1:2,000), anti-cleaved caspase 9 antibody (1:2,000), anti-cleaved PARP antibody (1:2,000), anti-LC-3 antibody (1:2,000), anti-Bcl-2 antibody (1:2,000), anti-p-ERK antibody (1:2,000), anti-t-ERK antibody (1:2,000), and anti-beta actin antibody (1:5,000) were diluted in 5% skim milk powder at the respective ratios and reacted overnight at 4°C.The membrane was washed three times with TBST (TBS, 1% Tween 20) every 10 minutes, and HRP (horseradish peroxidase)-conjugated secondary antibody was diluted 1:2000 in 5% skim milk powder and reacted for 2 hours at room temperature. After washing three times with TBST in the same manner as above, the membrane was reacted with ECL (Enhanced Chemiluminescence) substrate solution for 1 minute and then developed on X-ray film.

[0092] Caspase 3 and 9 are proteins that exist in the cytoplasm and are cleaved when the apoptosis pathway is activated, and they play a role in transmitting an apoptosis signal. PARP is a protein that repairs cleaved DNA fragments. When PARP is cleaved, the cleaved DNA cannot be repaired, leading to apoptosis. Bcl-2 protein exists in the outer mitochondrial membrane, binds to Bax / Bak proteins, inhibits apoptosis, and dissociates from Bax / Bak when the apoptosis pathway is activated, leading to apoptosis. LC-3 is a marker of autophagy, and the expression level of LC3-II, a lipidated form of LC-3 that increases during autophagosome formation, was confirmed. Western blot experiments showed that as the concentration of L-GPS treated to the cells increased, the expression levels of cleaved caspase 3, 9, and PARP proteins induced by TNF-α decreased, and the decrease in Bcl-2 increased in a concentration-dependent manner. This suggests that L-GPS treatment has a protective effect against inflammation-induced apoptosis in an inflammatory bowel disease cell line model (Fig. 10A, B). Subsequently, it was confirmed that as the concentration of L-GPS treated to the cells increased, LC3-II reduced by TNF-α increased in a concentration-dependent manner. This suggests that inflammation-induced inhibition of autophagy is related to damage to colonic epithelial cells, and that pretreatment with L-GPS protected colonic epithelial cells by maintaining autophagy. This suggests that L-GPS treatment has the effect of protecting colonic epithelial cells from inflammation by preventing the inhibition of autophagy induced by inflammation in an inflammatory bowel disease cell line model (Fig. 10C).

[0093]

[0094] [Example 10]

[0095] Confirmation of the inhibitory effect of L-GPS on inflammatory factors (p-ERK, TNF-α, IL-6, and ROS) in inflammatory bowel disease cell line models.

[0096] To determine the effect of L-GPS on the inflammatory response pathway in an inflammatory bowel disease cell line model, p-ERK and t-ERK protein expression levels were examined by Western blotting. Human colonic epithelial cells (HT-29) were cultured in 6-well plates for 24 h, and L-GPS was prepared at concentrations of 25, 50, 100, 250, and 500 μg / ml and treated with the cells for 48 h. After treatment with recombinant TNF-α (R&D, USA) at a concentration of 20 ng / ml to each well for 48 h, the cells were washed with PBS, and 60 μl of RIPA buffer was added to each well. The cells were incubated on ice for 30 min and centrifuged at 13,000 rpm for 15 min to obtain proteins. Proteins were quantified using the Bradford assay, and the sample loading buffer was added, denatured at 95°C for 10 minutes, and electrophoresed using a 12% SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis) gel. The membrane was transferred to a nitrocellulose (NC) membrane and blocked with 5% skim milk powder for 1 hour at room temperature. Then, primary anti-p-ERK antibody (1:2,000), anti-t-ERK antibody (1:2,000), and anti-beta actin antibody (1:5,000) were diluted in 5% skim milk powder at the respective ratios and reacted overnight at 4°C. The membrane was washed three times with TBST (TBS, 1% Tween 20) every 10 minutes, and HRP (horseradish peroxidase)-conjugated secondary antibody was diluted 1:2000 in 5% skim milk powder and reacted for 2 hours at room temperature. After washing three times with TBST in the same manner as above, the membrane was reacted with ECL (Enhanced Chemiluminescence) substrate solution for 1 minute and then developed on X-ray film.

[0097] ERK is a protein that is involved in cell survival and differentiation and is activated by phosphorylation during inflammation. As the amount of p-ERK protein expression increased by TNF-α treatment in an inflammatory bowel disease cell line model was decreased by L-GPS treatment, it was confirmed that L-GPS treatment had an effect on suppressing the expression of inflammatory proteins (Fig. 11A).

[0098] To determine the effect of L-GPS on inflammatory genes in an inflammatory bowel disease cell line model, RT-PCR was performed. Human colonic epithelial cells (HT-29) were cultured in 6-well plates for 24 h, and L-GPS was prepared at concentrations of 25, 50, 100, 250, and 500 μg / ml, respectively, and treated with the cells for 48 h. Recombinant TNF-α (R&D, USA) was then treated with each well at a concentration of 20 ng / ml for 48 h. The culture supernatant was removed, washed three times with PBS, and the cells were lysed to extract RNA. cDNA was then synthesized using a cDNA synthesis kit. Primers corresponding to TNF-α and IL-6 were added, and the cells were amplified by PCR. The mRNA expression levels were measured by electrophoresis on a 1.5% agarose gel containing ethidium bromide. As a result, L-GPS concentration-dependently reduced the increase in TNF-α and IL-6 genes caused by TNF-α (Fig. 11B).

[0099] To determine the effect of L-GPS on ROS production, an inflammatory factor, in an inflammatory bowel disease cell line model, intracellular ROS was stained using DCF-DA. Human colonic epithelial cells (HT-29) were cultured in 96-well plates for 24 h, and L-GPS was prepared at concentrations of 25, 50, 100, 250, and 500 μg / ml, respectively, and treated with the cells for 48 h. Recombinant TNF-α (R&D, USA) was then treated to each well at a concentration of 20 ng / ml for 48 h. The culture supernatant was removed, washed three times with PBS, and stained with DCF-DA (Invitrogen, USA) diluted in buffer for 45 min. After washing three times with PBS, the cells were measured at Ex / Em 495 / 527 wavelength using a fluorescence spectrophotometer. As a result, L-GPS concentration-dependently reduced the increase in ROS caused by TNF-α (Fig. 11C).

[0100]

[0101] [Example 11]

[0102] Verification of the efficacy of L-GPS in improving inflammatory bowel disease in an animal model of inflammatory bowel disease (in vivo).

[0103] To verify the protective efficacy of L-GPS against inflammatory bowel disease in vivo, Balb / c mice were fed L-GPS at low concentrations (200 mg / kg) and high concentrations (400 mg / kg) for 2 weeks, and then dextran sulfate sodium (DSS) was administered for 5 days to induce inflammatory bowel disease. The body weight changes and colon length of the mice were measured, and the pathological phenotype of the colon tissue was confirmed using H&E staining. As a result, it was confirmed that the colon length of the group fed L-GPS was maintained similar to that of the control group that did not induce inflammatory bowel disease, compared to the group in which inflammatory bowel disease was induced by DSS, which was significantly reduced (Fig. 12A, B). As shown in the results of H&E staining, in the group in which inflammatory bowel disease was induced with DSS, the villi of the colon were damaged, whereas in the group that consumed L-GPS, the villi morphology was maintained at a level similar to the control group (Con). In addition, it was confirmed that the villi morphology was maintained in the group that consumed the existing treatment, CsA (Fig. 12C).

[0104]

[0105] Although the embodiments of the present invention have been described above, those skilled in the art will understand that the present invention can be implemented in other specific forms without altering the technical concept or essential characteristics thereof. Therefore, the embodiments described above should be understood to be illustrative in all respects and not restrictive.

Claims

1. A pharmaceutical composition for preventing or treating inflammatory bowel disease, comprising low molecular weight glucan polysaccharide (L-GPS) as an active ingredient.

2. A pharmaceutical composition according to claim 1, wherein the L-GPS is separated from ginseng extract through enzymatic decomposition.

3. A pharmaceutical composition according to claim 1, wherein the L-GPS has a molecular weight of 1000 Da to 5000 Da.

4. A pharmaceutical composition according to claim 1, wherein the L-GPS is a low-molecular weight glucan polysaccharide containing glucose at 96% or more as a component.

5. A pharmaceutical composition according to claim 1, wherein the binding structure of the L-GPS comprises a skeleton structure (1,4-α-glucan backbone) in which glucose is formed by α-1,4 bonds.

6. A pharmaceutical composition according to claim 1, wherein the L-GPS exhibits a protective effect against inflammatory cell death caused by inflammation induction in a human colon cell line.

7. A pharmaceutical composition according to claim 1, wherein the L-GPS is contained in the composition at a concentration of 25 to 500 μg / ml.

8. A pharmaceutical composition according to claim 1, wherein the L-GPS is characterized by an efficacy in inhibiting inflammatory cell apoptosis caused by inflammation induction in a human colon cell line.

9. A pharmaceutical composition according to claim 1, wherein the L-GPS promotes the recovery of autophagy of cells decreased due to inflammation induction in a human colon cell line.

10. A pharmaceutical composition according to claim 1, wherein the L-GPS inhibits the production of inflammatory cytokines (TNF-α, IL-6) and ROS in an inflammatory bowel disease cell line model.

11. In paragraph 1, The above L-GPS is a pharmaceutical composition that improves inflammatory bowel disease (IBD) by restoring the length of the colon reduced in patients with inflammatory bowel disease to a length similar to that of a normal group and maintaining the villi of the colon damaged by inflammatory bowel disease to a level similar to that of a normal group.

12. A health functional food composition for preventing or improving inflammatory bowel disease, comprising low molecular weight glucan polysaccharide (L-GPS) as an active ingredient.

13. In paragraph 12, The above L-GPS is a health functional food composition separated from ginseng extract through enzymatic decomposition.

14. In paragraph 12, The above L-GPS is a health functional food composition having a molecular weight of 1000 Da to 5000 Da.

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