Houttuynia cordata-derived polysaccharide fraction or houttuynia cordata-derived polysaccharide, composition for immune-stimulation comprising same, and method for preparing same

Abstract

The present invention relates to a specific Houttuynia cordata-derived polysaccharide fraction or polysaccharide having an immune-stimulation effect and / or an anticancer effect. The present invention also relates to a method for preparing a Houttuynia cordata-derived polysaccharide fraction or polysaccharide having an excellent immune-stimulation effect and / or anticancer effect.

Description

Polysaccharide fraction derived from Houttuynia cordata or polysaccharide derived from Houttuynia cordata, composition for enhancing immunity comprising the same, and method for producing the same

[0001] This application claims priority to Korean Patent Application No. 10-2023-0194103, filed December 28, 2023, the entire disclosure of which is incorporated herein by reference.

[0002] The present invention relates to a specific polysaccharide fraction of a plant extract that exhibits excellent effects in terms of immune enhancement and / or anticancer effects, a specific polysaccharide, a composition comprising the same, a method for producing the same, and a pharmaceutical use thereof.

[0003] Recently, the use of immunostimulators, which induce activation of the immune system, the body's defense mechanism, has been proposed. Immunostimulators do not directly attack cancer cells. Rather, they are immunotherapy, which activates the body's immune system to treat and prevent diseases and cancer cells without causing toxicity to normal cells. With the recent demonstration of the therapeutic efficacy of newly developed immunotherapy for cancer, which utilizes the human immune system, a paradigm shift is underway in cancer treatment from conventional chemotherapy and targeted therapy to immunotherapy for cancer, which utilizes immunotherapy.

[0004] Immunotherapy is a treatment method that activates the body's immune system, enhancing autoimmunity and enabling immune cells to attack cancer cells. In other words, immunotherapy drugs focus on the function of immune cells, unlocking their potential to attack cancer cells. While conventional anticancer drugs target cancer cells themselves, immunotherapy drugs target not only cancer cells themselves, but also the cells surrounding the cancer and the tumor microenvironment. This difference in mechanism of action leads to differences in therapeutic efficacy. Unlike conventional anticancer drugs, immunotherapy drugs are long-lasting, with an indeterminate response time. This long-term, sustainable efficacy is due to the memory capacity of immune cells. Another reason for this is the reduced risk of resistance to immunotherapy drugs and the associated fewer side effects. These advantages allow immunotherapy drugs to significantly extend survival and enhance patients' quality of life.

[0005] It is known that the immune-enhancing activity, anticancer activity, and antibacterial activity of these immunostimulants are caused by activating the complement system, macrophages, natural killer cells (NK cells), and T cells among the immune system cells of the human body (Yoon TJ et al., (2003) Antitumor activity of the Korean mistletoe lectin is attributed to activation of macrophages and NK cells. Arch. Pharm. Res. 26: 861-7; Mueller EA, Anderer FA (1990) Synergistic action of a plant rhamno-galacturonan enhancing antitumor cytotoxicity of human natural killer and lymphokine-activated killer cells: chemical specificity of target cell recognition. Cancer Res., 50: 3646-3651; and K gi D et al., (1994) Cytotoxicity mediated by T cells and natural killer cells is greatly impaired in perforin-deficient mice. Nature, 368: 31-37). NK cells, immune cells of the innate immune system, account for approximately 5-10% of the body's lymphocytes and play a role in T cells in the adaptive immune system. cUnlike NK cells, they are known to have the ability to naturally and innately kill viruses, non-self cells, and tumor cells. (Oh MY.(2005). Infection and innate immunity.Korean J. Pediatr., 48: 1153-1161). NK cells play a pivotal role in immune regulation because they secrete a number of immunologically important cytokines. In particular, IFN-γ, one of the cytokines they secrete, induces the participation and activation of macrophages in the innate immune system by activating the phagocytic activity and bactericidal ability of macrophages. Many researchers have reported that NK cell activity in cancer patients is significantly reduced compared to that in normal people, which suggests that NK cells are significantly involved in immune regulation and anticancer effects (Riley EM, Allen SJ, Wheeler JG, Blackman MJ, Bennett S, Takacs B, Greenwood BM.(1992). Naturally acquired cellular and humoral immune responses to the major merozoite surface antigen (Pf MSP1) of Plasmodium falciparum are associated with reduced malaria morbidity. Parasite Immunol., 14(3): 321-337.). In other words, NK cell activation by administration of active substances, i.e., enhancement of cell killing ability, is pointed out as an important factor in cancer prevention and treatment.

[0006] Cells involved in the immune response form a tissue or organ called the lymphoid system to perform their functions effectively, and are broadly classified into the primary lymphoid system and the secondary lymphoid system. Among these mucosal lymphoid organs, gut associated lymphoid tissue (GALT) is the largest lymphoid tissue in the body and is located in the intestinal mucosa. Approximately 70% of the body's immune cells are concentrated there, and it is known to play an important role in the body's immune response (Mowat AM, Viney JL. (1997). The anatomical basis of intestinal immunity. Immunol. Rev., 156(1): 145-166). These are composed of Peyer's patches, laminar propria, and mesenteric lymph nodes (MLN), and are known as inductive sites for secreting IgA on the mucosal surface (Sanderson, IR and Walker, WA (1994) Mucosal Barrier. In Handbook of Mucosal Immunology, eds. Orga, PL, Mestecky, J., Elamm, M., Strober, W., McGhee, JR and Bienenstock, p 41-49, Academic press, San Diego. USA). Among these, Peyer's patches are lymphocyte aggregates existing on the antimesentric side of the ileum and colon. Their distribution and number vary depending on the individual or species, and they are known to be most distinct in the ileum, and it is reported that their number and size gradually decrease with age (Kato, T. and Owen, RL(1994) Structure and function of intestinal mucosal epithelium. In Handbook of Mucosal Immunology, eds. Orga, PL, Mestecky, J., Elamm, M., Strober, W., McGhee, JR and Bienenstock, p 11-26, Academic press, San Diego. USA). In addition to being a central immune organ in the intestine, the lumen dome is lined with a flattered epithelium composed of M cells specialized for antigen uptake (Trier J. (1991). Structure and function of intestinal M cells. Gastroenterol. Clin. North Am., 20: 531-547). These M cells not only contribute to the activation of immune cells by engulfing soluble antigens, bacteria, and viruses from the lumen through phagocytosis or pinocytosis and delivering them to lymphocytes, but also serve as a passage for high molecular weight active substances such as polysaccharides to enter and exit the body (Bockman DE, Cooper MD.(1973). Pinocytosis by epithelium associated with lymphoid follicles in the bursa of labricus, appendix, and Peyer's patches: An electron microscopic study. Am. J. Anat., 136(4): 455-478).Antigens (or active macromolecules) captured by M cells activate phagocytes in Peyer's patches and also play a role in presenting antigens to lymphocytes. The primed lymphocytes in Peyer's patches differentiate and mature in the germinal center and migrate to other mucosal tissues through the mesenteric lymph nodes, where they can circulate in the body. In addition, some activated lymphocytes in Peyer's patches return to the lamina propria to differentiate into plasma cells that produce secretory IgA (sIgA), which neutralizes toxins and viruses and regulates bacterial adhesion to epithelial cells (Gregory, RL 1994) The biological role and clinical implications of IgA. Laboratory Medicine 25: 724-728). In other words, the intestinal immune system, including Peyer's patches, is thought to be able to effectively suppress diseases such as cancer, allergies, and autoimmune diseases by not only contributing to the local immunological defense system of the intestines but also enhancing the systemic immune system.

[0007] Recently, research results have been reported that macrophages are activated when the specific structure of plant-derived high molecular weight polysaccharides is recognized by the specific receptor of macrophages (Shin KS. (2012). Immunostimulating plant polysaccharides: macrophage immunomodulation and its possible mechanism. Food Sci. Ind., 45(1): 12-22.). Macrophage cell membranes contain complement receptor 3 (CR3; CD11b / CD18, Mac-1, etc.), CD14, toll-like receptor-4 (TLR4), mannose receptor (MR), dectin-1, and scavenger receptor (SR), and it has been revealed that these receptors can recognize plant-derived high-molecular-weight polysaccharides (ice PJ, Kelley JL, Kogan G, Ensley HE, Kalbfleisch JH, Browder IW, Williams DL. (2002). Human monocyte scavenger receptors are pattern recognition receptors for (1→3)-β-D-glucans. J. Leukoc. Biol., 72(1): 140-146.). In particular, signaling pathways through CR3 and SR activate phosphoinositide-2-kinase (PL3K) and protein kinase C (PKC), which phosphorylates mitogen-activated protein kinases (MAPKs) and nuclear factor-κB (NF-κB) pathways, which then induce gene transcription. MR activates intracellular signaling factors of macrophage phagocytosis, endocytosis, reactive oxygen species, and NF-κB pathways.In addition, TLR4 is known to activate myeloid differentiation protein 88 (MyD88), which in turn activates IL-1R-associated kinase (IRAK), TNF receptor-associated factor 6 (TRAF6), MAPKs, and NF-κB pathways. In summary, activation of intracellular signaling factors and transcriptional pathways is known to induce the expression of pro-inflammatory cytokines and inducible nitric oxide synthase (iNOS) (Janeway CA. (1968). Progress in immunology. Syndromes of diminished resistance to infection. J. Pediatr., 72(6): 885-903).

[0008] Until recently, carbohydrates, including polysaccharides, have been focused only on their role as structural components and energy sources in plants. However, as mentioned above, interest in the functionality of plant-derived high-molecular-weight polysaccharides has been rapidly increasing as it has been known to exhibit pharmacological effects on cell differentiation, information transmission, infection, and cancer metastasis by binding to various receptors expressed on the surface of immune cell membranes (Ruoslahti E. (1989). Proteoglycans in cell regulation. J. Biol. Chem., 264: 13369-13372.). In addition, complement system activation (Zhu H, Zhang Y, Zhang J, Chen D.(2008). Isolation and characterization of an anti-complementary protein-bound polysaccharide from the stem barks of Eucommia ulmoides. Int. Immunopharmacol., 8(9): 1222-1230.), lymphocyte proliferation (Bao X, Wang Z, Fang J, Li X.(2002). Structural Features of an Immunostimulating and Antioxidant Acidic Polysaccharide from the Seeds of Cuscuta chinensis. Planta Med., 68(3): 237-243.), and macrophage proliferation (Shin KS, Kiyohara H, Matsumoto T, Yamada H.(1997). Rhamnogalacturonan II from the leaves of Panax ginseng) were observed in high molecular weight polysaccharides derived from plants and herbal medicines that have been traditionally used as food or medicine. CA Meyer as a macrophage Fc receptor expression-enhancing polysaccharide.Carbohydr. Res., 300(3): 239-249..), anti-metastasis activity (Lee EH, Park HR, Shin MS, Cho SY, Choi HJ, Shin KS.(2014). Antitumor metastasis activity of pectic polysaccharide purified from the peels of KoreanCitrusHallabong.Carbohydr.Polym., 111: 72-79.), etc., and the role of plant-derived high molecular weight polysaccharides in activating the body's immune system is receiving significant attention.

[0009] Meanwhile, Houttuynia cordata (Thunb.) is a perennial herb belonging to the Saururaceae family, with slender stems and heart-shaped leaves. Houttuynia cordata has been reported to possess various physiological activities, including anti-inflammatory, immune-stimulating, antibacterial, and antioxidant properties. However, most of these activities are believed to originate from low-molecular-weight substances present in Houttuynia cordata. Furthermore, the aforementioned effects of Houttuynia cordata extract have not yet been sufficient for use as a therapeutic agent.

[0010] Therefore, the problem to be solved by the present invention is to provide a composition having beneficial activities such as immune enhancement, anticancer, and antitumor activities using Eoseongcho.

[0011] Another problem to be solved by the present invention is to provide a polysaccharide (fraction) having beneficial activities such as immune enhancement, anticancer, and antitumor activities from the plant and a method for producing the same.

[0012] Another problem to be solved by the present invention is to provide a pharmaceutical use of the composition and polysaccharide (fraction) according to the present invention.

[0013] In order to solve the above problem, one aspect of the present invention provides a method for producing a polysaccharide fraction of Houttuynia cordata, including the steps of (S1) adding water to Houttuynia cordata and then heating to extract it, (S2) adding ethanol or an aqueous ethanol solution to the supernatant obtained after the extraction in step S1 to cause precipitation and isolate the precipitate, (S3) purifying the precipitate obtained in step S2, (S4) adding a pectin hydrolyzing enzyme (preferably pectinase) to the purified product of step S3 to enzymatically treat it, and (S5) separating the resultant product of step S4 according to molecular weight.

[0014] Preferably, one aspect of the present invention provides a manufacturing method, characterized in that step S1 of the manufacturing method comprises adding water in an amount of 10-70 times (more preferably, 10-30 times) the volume of the perilla leaves to the perilla leaves and extracting at 90-110°C.

[0015] Preferably, one aspect of the present invention provides a manufacturing method characterized in that step S2 of the manufacturing method comprises concentrating the supernatant to 5-20 brix (preferably 10-20 brix, more preferably 13-17 brix), adding ethanol or 90-99 wt% ethanol aqueous solution in a volume of 2-10 times the concentrated volume to cause precipitation, and recovering the precipitate.

[0016] Preferably, one aspect of the present invention provides a manufacturing method characterized in that the purification of the S3 step among the manufacturing methods comprises the steps of (S3-1) adding an aqueous ethanol solution (preferably 1.5-5:1 volume ratio) and centrifuging to remove the supernatant to remove low-molecular substances and pigment substances, (S3-2) adding water to the S3-1 resultant to recover only the soluble fraction and remove the precipitate, and (S3-3) selectively removing low-molecular substances (for example, using a dialysis membrane having a cut-off of 10,000-20,000 Da).

[0017] Preferably, in the manufacturing method of the present invention, the pectin hydrolyzing enzyme may be pectinase, polygalacturonanase, pectin methylesterase, or pectin lyase, which may be used alone or in combination. More preferably, the pectin hydrolyzing enzyme according to the present invention is pectinase. For example, commercially available industrial enzymes such as Pectinex® Ultra SPL, Viscozyme L, Rapidase C80 MAX, Rapidase C80 CPO, and Pectinex 5XL may be used alone or in combination.

[0018] Preferably, one aspect of the present invention provides a manufacturing method characterized in that the step S4 of the manufacturing method includes a step of removing an ester group (e.g., methyl ester, acetyl ester) before treatment with a pectin hydrolyzing enzyme.

[0019] Preferably, one aspect of the present invention provides a manufacturing method characterized in that the step S4 of the manufacturing method further includes a step of removing low molecular weight substances after pectin hydrolysis enzyme treatment (for example, using a dialysis membrane having a cut-off of 10,000-20,000 Da).

[0020] Preferably, one aspect of the present invention provides a manufacturing method characterized in that step S5 of the manufacturing method comprises dividing the molecular weight into three categories according to size to obtain the highest molecular weight fraction. This classification according to molecular weight can preferably be performed using gel permeation chromatography (GPC).

[0021] One aspect of the present invention provides a polysaccharide fraction derived from Eoseongcho produced by the aforementioned production method.

[0022]

[0023] One aspect of the present invention also provides a polysaccharide fraction derived from Houttuynia cordata; when the content of neutral sugars is measured by the phenol-sulfuric acid method using galactose as a standard and the content of acidic sugars is measured by the m-hydroxybiphenyl method using galacturonic acid as a standard, the content of neutral sugars and acidic sugars is 60 to 80 wt% (neutral sugars) and 15 to 35 wt% (acidic sugars), respectively, based on the total weight of the polysaccharide fraction; and when analyzing the constituent sugars, the content of rhamnose is 8 to 18 wt%, the content of galactose is 36 to 56 wt%, the content of arabinose is 7 to 27 wt%, the content of glucuronic acid is 3 to 13 wt%, and the content of galacturonic acid is 4 to 14 wt% based on the total content of sugar components.

[0024] More preferably, the polysaccharide fraction derived from the above-mentioned perilla leaves shows, when analyzing the constituent sugars, contents of mannose, rhamnose, glucose, galactose, arabinose, fucose, glucuronic acid, and galacturonic acid of 0.1-2, 8-18, 0.1-2, 36-56, 7-27, 0.1-2, 3-13, and 4-14 wt%, respectively, relative to the total content of sugar components.

[0025]

[0026] One embodiment of the present invention is also a polysaccharide derived from Houttuynia cordata; when the content of neutral sugar is measured by the phenol-sulfuric acid method using galactose as a standard substance, and the content of acid sugar is measured by the m-hydroxybiphenyl method using galacturonic acid as a standard substance, the content of neutral sugar and acid sugar is 60 to 80 wt% (neutral sugar) and 15 to 35 wt% (acid sugar), respectively, based on the weight of the polysaccharide; and includes rhamnogalacturonan (RG)-I, which is an active polysaccharide, wherein RG-I has a main chain composed of rhamnose and galacturonic acid, and the main chain has a homogalacturonan portion composed of an α-(1→4) bond of galacturonic acid and a portion in which rhamnose and galacturonic acid are mixed; Provided is a polysaccharide derived from Houttuynia cordata, characterized in that α-(1→5)-arabinan, arabino-β-(3,6)-galactan, β-(1→4)-galactan, and β-(1→4)-glucuronan exist as side chains of the main chain; and arabino-β-(3,6)-galactan, which is a type II arabinogalactan, accounts for 40-60 wt% of the total weight of the polysaccharide.

[0027] The polysaccharide derived from the lactic acid bacteria according to the present invention may have a structure as shown in FIG. 3, for example, but is not limited to the exemplary structure of FIG. 3 of the present invention.

[0028] In one aspect of the present invention, HCPE-I, which corresponds to the preferred polysaccharide of the present invention, has a molecular weight of 100-150 kDa, preferably 110-140 kDa.

[0029] A polysaccharide fraction or polysaccharide derived from the plant having a very high immune-enhancing activity and having such a specific sugar ratio can be produced using the production methods according to the present invention as mentioned above.

[0030] The purity of the polysaccharide derived from the perilla leaves of the present invention may be, for example, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more based on the total weight of the polysaccharide. Preferably, the purity of the polysaccharide derived from the perilla leaves of the present invention is 90% or 95% or more. Such purity may be measured using, for example, HPLC, LC / MS, etc.

[0031]

[0032] One aspect of the present invention also provides a pharmaceutical or health functional food composition comprising, as an active ingredient, a polysaccharide fraction derived from Houttuynia cordata or a polysaccharide derived from Houttuynia cordata produced by the manufacturing method according to the present invention described above. The pharmaceutical or health functional food composition according to one aspect of the present invention has an excellent immune-boosting effect.

[0033] The above-mentioned immune-boosting effects include the prevention or treatment of diseases caused by a weakened immune system, such as anticancer effects, antibacterial effects, antiviral effects including colds, and chronic fatigue relief effects. In particular, the pharmaceutical or health functional food composition according to the present invention is useful for the treatment or prevention of cancer through immune-boosting effects.

[0034] That is, one aspect of the present invention provides a method for enhancing the immunity of a subject, comprising administering to a subject in need of treatment or prevention a therapeutically or prophylactically effective amount of a polysaccharide fraction derived from Houttuynia cordata or a polysaccharide derived from Houttuynia cordata produced by a method according to the present invention. This method is useful for preventing or treating diseases caused by a decrease in immunity, such as anticancer effects, antibacterial effects, antiviral effects including colds, and the like, and for improving chronic fatigue by enhancing immunity.

[0035] The food composition according to the present invention includes all forms, including functional foods, nutritional supplements, health foods, and food additives. The above types of food compositions can be prepared in various forms using conventional methods known in the art.

[0036] For example, as a health functional food, the polysaccharide fraction of the present invention or the polysaccharide itself can be manufactured into the form of tea, juice, or drink for consumption, or can be granulated, encapsulated, or powdered for consumption. In addition, the polysaccharide fraction of the present invention or the polysaccharide can be mixed with a known substance or active ingredient known to have an immune-boosting effect or a cancer-preventing or -treating effect, and can be manufactured in the form of a composition.

[0037] In addition, in order to use the polysaccharide fraction or polysaccharide of the present invention in the form of a food additive, it can be manufactured and used in the form of a powder or concentrate.

[0038] Pharmaceutically acceptable carriers may further include, for example, carriers for oral administration or carriers for parenteral administration. Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. Carriers for parenteral administration may also include water, suitable oils, saline solutions, aqueous glucose, and glycols, and may further include stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite, or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol.

[0039] The pharmaceutical composition according to the present invention can be administered to mammals, including humans, by any method. For example, it can be administered orally or parenterally. Parenteral administration methods include, but are not limited to, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal administration. In one embodiment of the present invention, the pharmaceutical composition of the present invention can be administered transdermally.

[0040] The pharmaceutical composition of the present invention can be formulated as a preparation for oral administration or parenteral administration according to the administration route described above.

[0041] In the case of preparations for oral administration, the composition of the present invention can be formulated into powders, granules, tablets, pills, sugar-coated tablets, capsules, liquids, gels, syrups, slurries, suspensions, etc. using methods known in the art. For example, oral preparations can be obtained by mixing the active ingredient with a solid excipient, grinding the mixture, adding a suitable auxiliary agent, and then processing it into a granule mixture to obtain tablets or sugar-coated tablets.

[0042] For example, in order to formulate into dosage forms such as tablets and capsules, excipients such as lactose, saccharose, sorbitol, mannitol, starch, milk sugar, microcrystalline cellulose, etc., binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose, etc., disintegrants such as dicalcium phosphate, crospovidone, etc., lubricants such as magnesium stearate, calcium stearate, sodium stearyl fumarate, polyethylene glycol wax, etc. may be contained. In the case of capsule dosage forms, in addition to the substances mentioned above, a liquid carrier such as fatty oil may be contained.

[0043] In the case of preparations for parenteral administration, they can be formulated in the form of injections, creams, lotions, ointments for external use, oils, moisturizers, gels, aerosols, and nasal inhalers using methods known in the art.

[0044] The total effective amount of the polysaccharide fraction or polysaccharide of the present invention can be administered to a patient as a single dose, or can be administered by a fractionated treatment protocol in which multiple doses are administered over a long period of time. The pharmaceutical composition of the present invention can vary the content of the effective ingredient depending on the severity of the disease. Preferably, the preferred total dose of the polysaccharide fraction of the present invention can be about 0.01 μg to 1,000 mg per 1 kg of patient body weight per day, more preferably 0.1 μg to 100 mg, and most preferably 1 μg to 10 mg. However, since the dosage of the polysaccharide fraction of the present invention is determined as an effective dosage for a patient by taking into consideration various factors such as the route of administration and number of treatments of the pharmaceutical composition as well as the patient's age, weight, health status, sex, severity of disease, diet, and excretion rate, taking these into consideration, a person having ordinary skill in the art will be able to determine an appropriate effective dosage for the polysaccharide fraction of the present invention according to a specific use as an immune enhancer. The pharmaceutical composition according to the present invention is not particularly limited in its formulation, route of administration, and method of administration as long as it exhibits the effects of the present invention.

[0045] One aspect of the present invention provides a polysaccharide fraction or polysaccharide of Houttuynia cordata having an excellent immune-enhancing effect, and the medicinal use of such components. One aspect of the present invention also provides a method for producing a polysaccharide fraction or polysaccharide of Houttuynia cordata having an excellent immune-enhancing effect. The polysaccharide fraction or polysaccharide of Houttuynia cordata according to the present invention promotes the production of, for example, IL-6, IL-12, and TNF-α, thereby enhancing immune function, has no significant toxicity toward macrophages, and increases the activity of natural killer cells (NK cells). The polysaccharide fraction or polysaccharide of Houttuynia cordata according to the present invention also exhibits an effect of inhibiting the metastasis of cancer cells.

[0046] The following drawings attached to this specification illustrate preferred embodiments of the present invention and, together with the contents of the invention described above, serve to further understand the technical idea of ​​the present invention, and therefore, the present invention should not be interpreted as being limited to matters described in such drawings.

[0047] Figure 1 is a flow chart showing a process for producing a polysaccharide fraction from Houttuynia cordata Thunberg, which is one embodiment of the present invention.

[0048] Figure 2 is a chromatogram for evaluating the molecular weight distribution and purification degree of polysaccharides derived from Eoseongcho evaluated by HPLC. Figure 2a shows the evaluation results of HCP, Figure 2b shows the evaluation results of HCPE, and Figure 2c shows the evaluation results of HCPE-I, HCPE-II, and HCPE-III.

[0049] Figure 3 is a diagram showing the estimated structure of HCPE-I, an immune-enhancing polysaccharide isolated from Eoseongcho.

[0050] Figure 4 shows the results of toxicity evaluation of HCP, HCPE, HCPE-I, HCPE-II, and HCPE-III isolated from Eoseongcho.

[0051] Figures 5 and 6 are graphs evaluating the effects of HCP and HCPE isolated from Eoseongcho on the secretion of cytokines (Figure 5) and bone marrow cell proliferation (Figure 6) via Peyer's patch cells. NC (medium) and PC (LPS, 1 μg / mL) were used as negative and positive controls, respectively. All data are expressed as the mean ± standard deviation (n = 3). Different letters indicate statistical significance (P < 0.05).

[0052] Figures 7 and 8 are graphs evaluating the effects of HCPE-I, -II, and -III, fractions isolated from Eoseongcho, on the secretion of cytokines (Figure 7) and bone marrow cell proliferation (Figure 8) through Peyer's patch cells. NC (medium) and PC (LPS, 1 μg / mL) were used as negative and positive controls, respectively. All data are expressed as the mean ± standard deviation (n = 3). Different letters indicate statistical significance (P < 0.05).

[0053] Figure 9 shows the results of evaluating the effects of oral administration of HCPE fractions on cytokine production and bone marrow proliferation via Peyer's patch cells. HCPE was administered at various concentrations, including 500, 1,000, and 2,000 μg / mouse. NC (distilled water) was used as a negative control. All data are expressed as the mean ± standard deviation (n = 3). Different letters indicate statistical significance (P < 0.05).

[0054] Figure 10 shows the results of evaluating the effects of oral administration of HCPE fractions on cytokine production and bone marrow proliferation via Peyer's patch cells. HCPE was administered at various concentrations, including 500, 1,000, and 2,000 μg / mouse. NC (distilled water) was used as a negative control. All data are expressed as the mean ± standard deviation (n = 3). Different letters indicate statistical significance (P < 0.05).

[0055] Figure 11 is a graph showing changes in serum TGF-β and IgA levels over the HCPE administration period. Serum TGF-β and IgA concentrations were measured using ELISA kits. HCPE was administered at various concentrations, including 500, 1000, and 2000 μg / mouse. NC (distilled water) was used as a negative control. All data are expressed as the mean ± standard deviation (n = 10), and different letters indicate statistical significance (P < 0.05).

[0056] Figure 12 is a graph showing changes in stool IgA levels over the HCPE administration period. Stool IgA concentrations were measured using an ELISA kit. HCPE was administered at various concentrations, including 500, 1,000, and 2,000 μg / mouse. NC (distilled water) was used as a negative control. All data are expressed as the mean ± standard deviation (n = 10), and different letters indicate statistical significance (P < 0.05).

[0057] Figures 13 and 14 are graphs showing the effects of isolated polysaccharide fractions of H. japonica L. on cytokine secretion from murine peritoneal macrophages. Figure 13 shows the results using HCP and HCPE, and Figure 14 shows the results using HCPE-I, II, and III. NC (distilled water) and PC (LPS) served as negative and positive controls, respectively. All data are expressed as the mean ± standard deviation (n = 3). Different letters indicate statistical significance (P < 0.05).

[0058] Figure 15 is a graph evaluating the effect of intravenous administration of the extracts of the present invention on the cytolytic activity of NK cells. Polysaccharide-administered NK cells were co-cultured with YAC-1 lymphoma cells in a CO2 incubator for 6 hours. NC (saline) represents the negative control. All data are expressed as the mean ± standard deviation (n = 3). Different letters indicate statistical significance (P < 0.05).

[0059] Figure 16 shows the results of evaluating the reactivity of purified polysaccharide HCPE-I derived from Eoseongcho to β-Glucosyl Yariv reagent and the content of type II arabinogalactan.

[0060] Hereinafter, the present invention will be described in detail, using examples and the like, to aid understanding. However, the examples according to the present invention may be modified in various different forms, and the scope of the present invention should not be construed as being limited to the following examples. The examples of the present invention are provided to more fully explain the present invention to those of average skill in the art.

[0061]

[0062] Example 1: Preparation of polysaccharide fraction of Eoseongcho

[0063] First, various polysaccharide fractions of the perilla leaves were prepared as shown in Fig. 1.

[0064]

[0065] Example 1-1: Preparation of polymer fraction (HC) extracted from hot water of Eoseongcho

[0066] To isolate a fraction enriched in polymer substances from the aerial parts of the plant (excluding the roots), the material was added to 20 times the volume of distilled water and extracted at 100℃ for 2 hours. After recovering the extract, the residue was removed through centrifugation (3,000 rpm, 15 min) and the supernatant was recovered. The recovered supernatant was concentrated to approximately 15 brix, and 4 times the volume of 95% EtOH was added twice. After cooling to 20℃ or below, it was stirred for 1 hour and centrifuged (6,000 rpm, 25 min). The precipitate was recovered and the dried powder through vacuum drying was named HC.

[0067]

[0068] Example 1-2: Production of crude polysaccharide (HCP) derived from Eoseongcho

[0069] First, a solution of ethanol and distilled water in a 2.5:1 ratio was added to the vacuum-dried HC powder and stirred for 1 hour. The supernatant was then removed by centrifugation (6,0000 rpm, 30 min, 4℃). By repeating the above process three times, low-molecular-weight substances and pigment substances present in the HC were removed. The step of adding distilled water to the final precipitate to recover only the soluble fraction was repeated three times. Subsequently, the remaining low-molecular-weight substances were removed using a dialysis tubing with a cut-off of 14,000 Da, and then the crude polysaccharide HCP derived from Houttuynia cordata was obtained through concentration and lyophilization.

[0070]

[0071] Example 1-3: Preparation of enzyme-treated active fractions (HCPE, HCPE-I, II, III) derived from Eoseongcho

[0072] Prior to purification of the active polysaccharide, HCP was dissolved in 0.2 M NaOH to preferentially remove esters such as methyl or acetyl groups. The pH was then adjusted with 1 M HCl and treated with commercial pectinase. The resulting product was dialyzed using dialysis tubing with a cut-off of 14,000 Da, concentrated, and lyophilized to obtain the enzyme-treated crude polysaccharide derived from Houttuynia cordata, which was designated HCPE.

[0073] Afterwards, HCPE was dissolved in a small amount of distilled water and gel permeation chromatography (GPC) was performed using a Sephadex G-100 column (2.5 × 100 cm) equilibrated with 50 mM ammonium formate buffer (pH 5.5). This process was repeated until a sufficient amount of sample was obtained. All eluted fractions were separated into three purified polysaccharide fractions with different molecular weights through analysis of neutral sugar, acid sugar, KDO, and protein contents. The samples obtained through dialysis, concentration, and lyophilization were designated HCPE-I, -II, and -III.

[0074]

[0075] Example 2: General chemical properties and structural analysis of the active polysaccharide fraction of Eoseongcho

[0076] Example 2-1: General analysis of polysaccharide fractions of Eoseongcho

[0077] Crude polysaccharides (HCPs) and fractions (HCPE-I, II, III) derived from Eoseongcho were analyzed. The content of neutral sugars was analyzed by the phenol-sulfuric acid method using galactose as a standard. The content of acidic sugars was analyzed by the m-hydroxybiphenyl method using galacturonic acid as a standard. The content of TBA-positive material was analyzed by the thiobarbituric acid method using 2-keto-3-deoxy-D-manno-octulosonic acid (KDO) as a standard. The content of protein was analyzed by the Bradford method using bovine serum albumin (BSA) as a standard. The content of phenolic compounds was quantitatively analyzed by the Folin-Ciocalteu (FC) method using gallic acid as a standard.

[0078]

[0079] Example 2-2: Analysis by composition

[0080] The constituent sugars of the sample were identified as follows. 1 mL of 2 M trifluoroacetic acid (TFA) was added to the sample and reacted at 120°C for 1 hour and 30 minutes to hydrolyze it into monosaccharides. The mixture was then dried using a heating mantle (50°C). The sample was redissolved in 0.3 M NaOH, and 120 μL of 0.5 M 1-phenyl-3-methyl-5-pyrazolone (PMP) (dissolved in methanol) was added under basic conditions and reacted at 70°C for 60 minutes. The mixture was then neutralized with 0.3 M HCl and completely dried. The resulting mixture was separated using a two-phase solvent system of chloroform / H2O, and only the H2O layer was recovered, filtered, and analyzed by HPLC. The detailed HPLC analysis conditions are shown in the table below, and the mole% of each constituent sugar was calculated by converting the peak area and molecular weight of the sample into those of the internal standard of the derivative.

[0081] HPLC analysis conditions for component sugar analysisPumpLiquid Chromatography LC-20AD (Shimadzu Corporation, Kyoto, Japan)DetectorUV / VIS detector (Shimadzu Corporation, Kyoto, Japan)ColumnAcclaim TM 120 C18 (Thermo Scientific, Sunnyvale, USA)Column size4.6×250 mmColumn temp.30 ℃Flow rate1 mL / minEluent0.1 M sodium phosphate buffer(pH 6.7): Acetonitrile (82:18)Injection vol.10 μLIntegratorShimadzu data module (Shimadzu Corporation, Kyoto, Japan)

[0082]

[0083] The chemical and sugar compositions resulting from the analysis are summarized in Table 2 (HCP and HCPE) and Table 3 (HCPE-I, -II, and -III).

[0084] Chemical properties (%)HCPHCPENeutral sugar29.5±0.657.1±1.3Uronic acid69.8±6.942.2±2.2Protein0.7±0.00.7±0.0Polyphenol--KDO-like material--Component sugar (Mole %)HCPHCPEMannose1.9±0.72.5±0.3Rhamnose3.1±1.012.1±0.1Glucose1.2±0.63.8±0.0Galactose5.6±2.125.2±0.0Xylose--Arabinose2.1±1.210.1±0.2Fucose-0.9±0.1Glucuronic acid-3.51±.1Galacturonic acid81.1±5.635.4±1.4

[0085] KDO: 2-keto-3-deoxy-D-manno-octulosonic acid

[0086] Chemical properties (%)HCPE-ⅠHCPE-ⅡHCPE-ⅢNeutral sugar72.6±2.552.3±5.843.6±2.08Uronic acid26.8±1.446.9±1.055.4±2.3Protein0.6±0.10.8±0.11.0±0.2Polyphenol---KDO-like material---Component sugar (Mole %)HCPE-ⅠHCPE-ⅡHCPE-ⅢMannose0.8±0.14.6±1.02.2±0.1Rhamnose12.8±0.214.3±0.210.9±0.2Glucose1.1±0.04.2±0.13.6±0.1G alactose46.5±0.114.7±0.310.1±0.2Xylose---Arabinose17.3±0.39.3±0.16.4±0.1Fucose0.8±0.22.4±0.52.6±0.1Glucuronic acid8.0±0.53.5±1.22.6±0.0Galacturonic acid9.1±0.142.4±1.455.0±0.6

[0087]

[0088] Analysis of the constituent sugar composition of HCP using the PMP derivatization method revealed that it was composed of 81.1% GalA, 5.6% galactose (Gal), 3.1% rhamnose (Rha), and 2.1% arabinose (Ara) (Table 2). This confirmed that HCP had a constituent sugar composition in the form of a typical pectin-type polysaccharide, and in particular, given the detection of a large amount of GalA, it could be inferred that the homogalacturonan (HG) region occupied most of the sample, with a small amount of rhamnogalacturonan (RG) present.

[0089] The results of the composition analysis confirmed that HCPE is composed of GalA (35.4%), Rha (12.1%), Gal (25.2%), and Ara (10.1%), which are typical constituent sugars of rhamnogalacturonan I (RG-I) (Table 2). Meanwhile, the backbone of RG-I is known to have a 1:1 ratio of GalA and Rha, and HCPE was found to contain a large amount of GalA compared to Rha. This is presumed to be due to the detection of the HG region, which was not yet decomposed by the enzyme treatment, together with the RG region. Nevertheless, given the dramatic decrease in GalA content, it was determined that the enzyme treatment performed for the purpose of removing HG and separating RG-I and RG-II was performed normally, and accordingly, the intestinal immune activities of HCP and HCPE were compared as described below.

[0090] Among HCPE-I, HCPE-II, and HCPE-III, the highest molecular weight fraction, HCPE-I, had a GalA (9.1%) and Rha (12.8%) content close to 1:1, and a large amount of Gal (46.5%) and Ara (17.3%) was detected, suggesting that it was a typical RG-I type polysaccharide (Table 3). On the other hand, HCPE-II and -III, which correspond to relatively low molecular weight fractions, were confirmed to contain relatively large amounts of GalA.

[0091] Meanwhile, when only the crude polysaccharide HCP from Eoseongcho was separated, it was confirmed that most of the sample was a high molecular weight polysaccharide composed of sugars, as HCP was composed of 29.5% neutral sugars and 69.8% acidic sugars (Table 2). HCPE-I was composed of 72.6% neutral sugars and 26.8% acidic sugars, HCPE-II was composed of 52.3% neutral sugars and 46.9% acidic sugars, and HCPE-III was composed of 43.6% neutral sugars and 55.4% acidic sugars, which again confirmed that most of the sample in all three fractions was composed of sugars (Table 3).

[0092]

[0093] Example 2-3: Measurement of the molecular weight of polysaccharides from Eoseongcho

[0094] The molecular weight distribution and purification degree of polysaccharides derived from Eoseongcho were confirmed. Samples prepared at a concentration of 10 mg / mL were filtered through a 2 μm membrane filter and analyzed using a refractive index detector of HPLC (Agilent 1260 Infinity) equipped with a Superdex 75 GL column (GE Healthcare) equilibrated with 50 mM ammonium formate (pH 5.5). Detailed analysis conditions are listed in Table 4, and the molecular weights of the samples were determined using the pullulan series (P-800, 400, 200, 100, 50, 20, 10, and 5) as standards. The molecular weight measurement results of each polysaccharide are shown in Figure 2.

[0095] The crude polysaccharide derived from Eoseongcho had a main peak with a molecular weight of approximately 130 kDa, and it was estimated that the crude polysaccharide was composed of polysaccharides of various molecular weights, as it showed a long tailing in the main peak (see Fig. 2a). Meanwhile, when the molecular weight of the enzyme-treated crude polysaccharide was measured as HCPE, three peaks with molecular weights of approximately 128 kDa, 4 kDa, and 0.9 kDa were detected. Therefore, it was estimated that the HCP, which existed as a single substance in the form of pectin, was decomposed into HG corresponding to the smooth region by enzyme treatment, and relatively low-molecular-weight RG-I and RG-II were detected (see Fig. 2b).

[0096] High performance liquid chromatography (HPLC) analysis conditions for determining polysaccharide molecular weight

[0097] Pump1260 infinity G1310B (Agilent Technologies Co., Ltd., Palo Alto, CA, USA)DetectorRefractive index (1260 infinity G1362A, Agilent Technologies, USA)ColumnSuperdex 75 GL columnColumn size10×300 mmColumn temp.25 ℃Flow rate0.5 mL / minEluent50 mM ammonium formate buffer (pH 5.5)Injection vol.20 μLIntegratorAgilent data module (Agilent Technologies, USA)

[0098] Kav = (Ve - Vo) / (Vt - Vo)

[0099] Vt: total volume

[0100] Vo: void volume

[0101] Ve: elution volume of sample

[0102]

[0103] The molecular weight, chemical composition, and constituent sugar composition of HCPE-I, -II, and -III purified from enzyme-treated crude polysaccharide HCPE were confirmed. As a result, single peaks corresponding to 122 kDa, 4 kDa, and 3 kDa were detected, respectively. This confirms that the three peaks detected in the existing HCPE were completely separated and purified through the purification process (see Fig. 2c).

[0104]

[0105] Example 2-4: Qualitative and quantitative analysis of type II arabinogalactan among the active polysaccharides of Eoseongcho

[0106] Since HCPE-I, the active substance of the purified polysaccharide derived from the aforementioned Eoseongcho, was presumed to be a polysaccharide in the form of rhamnogalacturonan I (RG-I), the presence and content of type II arabinogalactan in it were investigated. The quality and quantity of type II arabinogalactan were confirmed by observing the reactivity with β-glucosyl Yariv reagent, which shows a specific reaction with type II arabinogalactan. β-glucosyl Yariv reagent is known to react specifically with type II arabinogalactan to form a red precipitate.

[0107] To qualitatively and quantitatively determine type II arabinogalactan (arabino-β-3,6-galactan) in polysaccharide fractions derived from Houttuynia cordata, the reactivity with β-glucosyl Yariv reagent was confirmed by the single radial gel diffusion method. 10 μg / mL of β-Glucosyl Yariv reagent was prepared with 0.15 M NaCl agarose and dispensed onto a plate, and wells with a diameter of 2.5 mm were created. Then, solutions containing 5 μg of the sample and the standard substance gum arabic prepared at various concentrations were dispensed into each well. The plate was left to react for 12 hours at room temperature and under humid conditions, and the presence or absence of type II arabinogalactan in the sample was confirmed by measuring the diameter of the red precipitate formed. The diameter of the precipitate formed was calculated compared to that of the standard substance gum arabic and quantified.

[0108] Through this, the single radial gel diffusion of HCPE-I was compared and analyzed with that of gum arabic, a standard material manufactured by concentration, and a relatively small amount of type II arabinogalactan was detected in HCPE (18.4%), but HCPE-I, from which only RG-I was recovered through the purification process, was confirmed to contain approximately 62.7% type II arabinogalactan in the sample. Based on the previous results that HCPE-I is composed of approximately 46.5% Gal and 17.3% Ara and the results in Figure 16, it was expected that most of the Gal and Ara present in HCPE-I would constitute type II arabinogalactan. In addition, the qualitative result of type II arabinogalactan, known as a representative side chain of RG-I, is thought to support the previous expectation that HCPE-I would have a typical RG-I form.

[0109]

[0110] Example 2-5: Analysis of binding patterns of active polysaccharides from Eoseongcho

[0111] The molecular weight, constituent sugars, and reactivity with β-glucosyl Yariv reagent of HCPE-I previously suggested that HCPE-I was a typical RG-I type polysaccharide. Therefore, to elucidate the sugar chain linkage pattern, which is known to be the most important in the structural analysis of polysaccharides, HCPE-I was derivatized into the PMAA form and analyzed by GC-MS.

[0112] First, the methylation method to determine the bonding position of the polysaccharide sample was performed using the Hakomori method. To prepare methylsulfinyl carbanion (MSCA), 1.26 g of anhydrous NaH (sodium hydride) and 20 mL of anhydrous DMSO (dimethylsulfoxide) were added under N2 purging, and the reaction was performed in an oil bath at 90 °C for approximately 15 minutes. The reaction was terminated when the reaction solution turned light green, and the mixture was cooled to room temperature and centrifuged (3,000 rpm, 10 min, 25 °C). Only the supernatant was collected, dispensed into glass tubes in small amounts under N2 purging, and stored at -70 °C for use.

[0113] The detailed method for derivatization of polysaccharides with PMAA was as follows. 20 μL of glycerol was added to 1 mL of a 1 mg / mL polysaccharide sample, which was then completely dried by N2 flushing. 1 mL of anhydrous DMSO was added, sealed tightly, and stirred at 50°C for 14 days to ensure complete dissolution. Then, 500 μL of the previously prepared MSCA was added and reacted for 4 hours. MSCA was added until the sample was completely converted to polyalkoxide, and the presence of unreacted MSCA was confirmed by observing the red color of the reaction with triphenylmethane. For the polyalkoxide-converted sample, an excess of CH3I (iodomethane) was added, and the hydroxyl (-OH) groups of the polysaccharide not involved in the bond were methylated by stirring at 50°C for 4 hours. The remaining CH3I was completely removed by N2 flushing, and the partially methylated polysaccharide was recovered using a Seppak C18 cartridge (Waters).

[0114] Partially, the sample was hydrolyzed by adding 1 mL of 2 M TFA and reacting at 121 °C for 1 hour and 30 minutes. After adding a small amount of absolute ethanol to the hydrolyzed sample, 10 mg of NaBH4 in 1 M NH4OH was added and reacted and reduced for 4 hours to open the sugar ring. After that, an appropriate amount of acetic acid was added to remove the residual NaBH4, and methanol was added and repeatedly dried to remove the excess acetic acid. After that, 1 mL of acetic anhydride was added and reacted at 121 °C for 3 hours, the hydroxyl groups of the polysaccharide that were not methylated because they were participating in bonds or forming rings were acetylated and converted to PMAA. This was separated with a two-phase solvent system of chloroform / H2O, and the derivative eluted in the chloroform layer was recovered and dried, and then a small amount of acetone was added and analyzed by GC-MS. The fragment ions obtained from this were analyzed by the finger printing method, and the mole % of the binding pattern corresponding to each monosaccharide was calculated from the peak area to determine the sugar chain binding pattern of the sample.

[0115] The detailed GC-MS analysis method for methylation alditol acetate analysis is described in the table below.

[0116] Apparatus: Agilent 6890N GC (Agilent Technologies Co., Ltd., Palo Alto, CA, USA) Detector: Agilent 5975 MSD (Agilent Technologies Co., Ltd., Palo Alto, CA, USA) Column: SP-2380 capillary column (Supelco, Bellefonte, PA, USA) Column size: 30 m × 0.25 mm, 0.2 μm film thickness Oven temperature: Maintain at 60°C for 1 minute, then increase to 150°C at a rate of 30°C, then increase to 180°C at a rate of 1°C, then increase to 231°C at a rate of 1.5°C, then increase to 250°C at a rate of 30°C, and then maintain at 250°C for 10 minutes. Injector temperature: 250°C Detector temperature: 280°C Carrier gas: N2 (1.0 mL / min)

[0117]

[0118] The results of the evaluation of the glycosidic linkages composition of HCPE-I are summarized in the table below and Fig. 3.

[0119] Glycosyl residueDeduced linkageHCPE-I (Mole %)RhamnoseTerminal2.72-linked9.12,4-linked6.1ArabinoseTerminal (p)1.1Terminal (f)12.35-linked (f)5.0FucoseTerminal1.0GalactoseTerminal7.73-linked5.24-linked3.96-linked9.13,6-linked16.14,6-linked1.6Galacturonic acid4-linked10.3Glucose2-linked0.34-linked1.84,6-linked0.7Glucuronic acid4-linked5.9

[0120] Specifically, HCPE-I was confirmed to have a total of 18 types of sugar chain linkages. In particular, 4-linked GalpA (10%), 2,4-linked Rhap (6.1%), terminal Araf (12.3%), 5-linked Araf (5.0%), and 3,6-linked Galp (16.1%), which are typical sugar chain linkage types of RG-I polysaccharides, were detected, confirming that HCPE-I, the active body of the polysaccharide derived from Eoseongcho, is a typical RG-I type polysaccharide as expected. In particular, the presence of a large amount of type II arabinogalactan in HCPE-I was expected from the previous reactivity examination with β-glucosyl Yariv reagent. Considering that the sum of the sugar chain bonding patterns associated with type II arabinogalactan, such as 3,6-linked Galp (16.1%), 3-linked Galp (5.2%), 6-linked Galp (9.1%), terminal Galp (7.7%), terminal Araf (12.3%), and terminal Fucp (1.0%), was approximately 51.4%, it was confirmed again that type II arabinogalactan present in HCPE-I occupies more than half of the entire RG-I structure.

[0121] The results obtained through the previous experiments strongly suggest that HCPE-I has a typical RG-I structure, and when combined, it was finally determined that HCPE-I has a characteristic RG-I structure in which GalA and Rha form a main chain in alternating patterns, side chains such as relatively short α-(1→5)-arabinan, β-(1→4)-galactan, and β-(1→>4)-glucuronan, and type II arabinogalactan accounts for more than half of the sample (Fig. 3).

[0122]

[0123] Experimental Example 1: Toxicity Evaluation of Polysaccharide Fractions from Eoseongcho

[0124] The toxicity of the sample to normal cells was examined. BALB / c (female, 6 weeks) mice were intraperitoneally injected with 1 mL of 5% thioglycollate medium (TG; Sigma) for 96 hours, and then macrophages were harvested. The harvested non-tumorous macrophages were incubated at 2X10 6 The cell number was adjusted to cells / mL, and 100 μL was added to a 96-well plate, and cultured for 2 hours in a 37°C, 5% CO2 incubator. The crude polysaccharide HCP derived from Eoseongcho, enzyme-treated polysaccharide HCPE, and polysaccharide fractions HCPE-I, HCPE-II, and HCPE-III purified from HCPE were dissolved in DMEM at a concentration of 2,000 μg / mL, serially diluted 10-fold, and 100 μL was added to a 96-well plate cultured for 2 hours to make the final concentration 1,000 μg / mL to 1 μg / mL, and cultured for 24 hours in a 37°C, 5% CO2 incubator. After the culture was completed, 170 μL of cell culture supernatant was recovered by centrifugation (900 rpm, 5 min, 4 °C) and the remaining supernatant was removed. To measure cytotoxicity, EZ-cytox (Dogen) was diluted 10-fold in PBS and added 100 μL per well. The cells were incubated for 30 minutes in a 37 °C, 5% CO2 incubator and the absorbance at 450 nm was measured using a spectrophotometer. The results are shown in Fig. 4.

[0125] As shown in Fig. 4, when the polysaccharide HCP derived from Eoseongcho, enzyme-treated polysaccharide HCPE, and polysaccharide fractions HCPE-I, HCPE-II, and HCPE-III purified from HCPE were added at concentrations of 1-1,000 μg / mL and the survival of the cells was confirmed after culturing, no cytotoxicity was observed in any sample for macrophages isolated from the peritoneal cavity, and rather, a slight proliferative activity was observed as the concentration increased.

[0126]

[0127] Experimental Example 2: Evaluation of intestinal immune activity (in vitro)

[0128] The intestinal immune activity of crude polysaccharide HCP and enzyme-treated crude polysaccharide HCPE isolated from Eoseongcho was investigated in vitro. The samples were applied to Peyer's patches isolated from C3H / HeN mice, and the cytokine content secreted in the supernatant was determined using ELISA. The experimental results are summarized in Figure 5.

[0129] The experimental group treated with HCPE showed a higher secretion of interleukin (IL)-6 and granulocyte / monocyte-colony stimulating factor (GM-CSF) compared to the experimental group treated with HCP (Fig. 5). Furthermore, the effect of HCPE was observed to be concentration-dependent. IL-6 showed an effect similar to that of LPS, which was used as a positive control (PC), and GM-CSF was observed to have an effect half that of PC.

[0130]

[0131] IL-6 and GM-CSF perform various functions in the body, but are known as representative stimulators of hematopoietic stem cells. Therefore, by treating bone marrow cells recovered from the femurs of mice of the same age as those from which Peyer's patches were recovered with Peyer's patch culture supernatant, we confirmed the effect of the sample on bone marrow cell proliferation via Peyer's patches. The experimental results are shown in Figure 6.

[0132] As with cytokine secretion, the experimental group treated with HCPE-Peyer's patch culture supernatant showed a higher bone marrow cell proliferation effect compared to the experimental group treated with HCP-Peyer's patch culture supernatant. These bone marrow cells are known to be a very important indicator for evaluating immune activity because they can differentiate and proliferate into various lymphocytes. Therefore, it is thought that the bone marrow cell proliferation effect by the sample will lead to the activation of the immune system and contribute to the maintenance of body homeostasis.

[0133] In summary, the above results confirmed that HCPE, in which most of the HG region was removed by pectinase, had superior intestinal immune activity compared to HCP. This result is thought to be due to the removal of the inactive region (HG region) present in HCP by enzyme treatment, resulting in HCPE having a relatively high proportion of RG. Therefore, it was judged that the result reaffirmed that the physiological activity of pectin is derived from RG. However, the present invention is not limited to this theoretical speculation.

[0134]

[0135] Meanwhile, the activities of three purified polysaccharides, HCPE-I, -II, and -III, isolated through the purification process were evaluated. The in vitro effects on Peyer's patch cells recovered from C3H / HeN were confirmed. The experimental results are summarized and presented in Figure 7.

[0136] The experimental results showed that the experimental groups treated with HCPE-II and -III, which are relatively low-molecular fractions, secreted very small amounts of IL-6 and GM-CSF compared to PC. On the other hand, the experimental group treated with HCPE-I, which is the highest-molecular fraction, secreted more explosive amounts of IL-6 than PC (left graph in Fig. 7), and the secretion of GM-CSF was also confirmed to be similar to that of PC (right graph in Fig. 7).

[0137] In addition, the effect of bone marrow cell proliferation was confirmed again using Peyer's patch culture supernatant treated with purified polysaccharide derived from Eoseongcho, and the results are shown in Figure 8.

[0138] The experimental results confirmed that the experimental group treated with HCPE-I induced the highest bone marrow cell proliferation ability (Fig. 8). Therefore, among the three types of purified polysaccharides obtained from Eoseongcho, HCPE-I was confirmed to have the best intestinal immune activity, and this allowed us to determine that HCPE-I is the active substance of the Eoseongcho-derived polysaccharide.

[0139]

[0140] Experimental Example 3: Effect of Oral Administration of Polysaccharides from Eoseongcho on Peyer's Patch Activation (in vivo)

[0141] The effects on Peyer's patches, which play a pivotal role in the intestinal immune system, were investigated. HCPE fractions were orally administered once daily for 28 days to 6-week-old C3H / HeN female mice at concentrations of 500, 1000, and 2000 μg / mouse (HCPE-L, HCPE-M, and HCPE-H). Cytokines secreted by Peyer's patch cells recovered from C3H / HeN mice were identified using ELISA. The results are shown in Fig. 9.

[0142] IL-6 and GM-CSF secreted in the culture supernatant of Peyer's patches isolated after oral administration showed a concentration-dependent increase as the administered concentration of HCPE increased. (Upper graph of Fig. 9) In addition, when bone marrow cells collected from the femurs of mice orally administered HCPE were cultured and the degree of proliferation was confirmed, proliferation was confirmed to increase in a concentration-dependent manner (lower left graph of Fig. 9). On the other hand, when bone marrow cells collected from the femurs of mice of the same age that were not administered any sample were treated with the culture supernatant of Peyer's patch cells obtained previously and proliferation was confirmed, a tendency for proliferation to increase as the concentration of the administered sample increased was observed (lower right graph of Fig. 9).

[0143]

[0144] Experimental Example 4: Production of IL-4, IL-10, and TGF-β (IgA-producing cytokines) by Peyer's patch cells by oral administration of polysaccharides derived from Eoseongcho

[0145] The contents of IL-4, IL-10, and TGF-β were analyzed using the culture supernatant of Peyer's patches recovered in Experimental Example 3. The supernatant was evaluated using a sandwich ELISA (Enzyme-linked immunosorbent assay). Antibodies against each cytokine were diluted in coating buffer, 50 μL each was coated on a 96-well plate, and incubated at 4°C for 12 hours. Afterwards, the plates were washed three times with washing buffer (PBS with 0.05% Tween 20, PBS-T), and 200 μL each of assay diluent (PBS with 2% skim milk or diluent buffer) was added and incubated at room temperature for 1 hour to block the well surfaces not bound by antibodies. Afterwards, each well was washed three times with washing buffer. Afterwards, 50 μL each of serially diluted standard substances (recombinant mouse cytokines) and cell culture supernatant were dispensed into each well. After leaving it at room temperature for 2 hours, it was washed with washing buffer and treated with 50 μL of detection antibody (in assay diluent), left at room temperature for 1 hour, and washed again with washing buffer. After that, it was treated with 50 μL of enzyme reagent (avidin-horseradish peroxidase conjugate) and left at room temperature for 30 minutes. Then, 50 μL of substrate solution [tetramethylbenzidine (TMB) and hydrogen peroxide] was added and reacted in a dark room for 5 to 20 minutes. After that, 50 μL of stop solution (2 N H2SO4) was treated and the absorbance was measured at 450 nm.

[0146] The results are summarized and shown in Fig. 10. As shown in Fig. 10, the contents of IL-4, IL-10, and TGF-β, which are known to contribute to the conversion of B cells to IgA antibody production, were analyzed using Peyer's patch supernatant, and it was observed that the secretion of each cytokine increased in a dose-dependent manner as the sample was administered.

[0147]

[0148] Experimental Example 5: Evaluation of the content of cytokines related to IgA production, TGF-β, and IgA in the serum of mice orally administered polysaccharides derived from Eoseongcho

[0149] Serum was collected from the orbital vein of mice once every two days during the oral administration experiment (28 days) of polysaccharide samples, and the whole blood was centrifuged to obtain serum. The results are shown in Fig. 11. HCPE was administered at concentrations of 500, 1000, and 2000 μg / mouse, and was designated as HCPE-L, HCPE-M, and HCPE-H, respectively.

[0150] Neither TGF-β nor IgA showed any significant differences between the experimental groups at week 0, but the TGF-β content in the serum collected at week 2 was found to have increased significantly compared to NC, and although IgA increased slightly, it was observed to be insignificant compared to the increase in TGF-β. On the other hand, the IgA content in the serum collected at week 4 showed an explosive increase, suggesting that the TGF-β content, which had increased significantly in advance at week 2, promoted IgA production.

[0151]

[0152] Experimental Example 6: Analysis of the IgA content in feces recovered from mice orally administered polysaccharides derived from Eoseongcho

[0153] The IgA content in feces collected from mice every two weeks during the oral administration period of the entire polysaccharide sample was analyzed. The experimental results are shown in Fig. 12. HCPE was administered at concentrations of 500, 1000, and 2000 μg / mouse, and are designated as HCPE-L, HCPE-M, and HCPE-H, respectively.

[0154] As a result of the experiment, no significant difference in the fecal IgA content was observed in the 0th week before sample administration, but the content of IgA in the feces was observed to increase as the sample was administered. In particular, in the 2nd week, IgA production was induced only in the HCPE-M and HCPE-H groups, but in the 4th week, an average higher IgA secretion than the negative control group was confirmed in all sample administration groups. Therefore, it was determined that the enzyme-treated crude polysaccharide HPCE derived from Eoseongcho activates immune cells present in Peyer's patches and further effectively induces IgA production.

[0155]

[0156] Experimental Example 7: Effect of Polysaccharide from Eoseongcho on Stimulation of Macrophage Cytokine Secretion

[0157] Macrophage activation was examined using crude polysaccharides and enzyme-treated crude polysaccharides HCP and HCPE, and the results are shown in Figures 13 and 14.

[0158] Experimental results showed that HCPE exhibited a slight proliferative effect compared to the negative control at the highest concentration of 1000 μg / mL. However, both HCP and HCPE showed similar trends in cytokine secretion, inducing cytokine secretion at a level similar to that of LPS, used as a positive control (Fig. 13).

[0159] In addition, when macrophage activation was examined using purified polysaccharides derived from Houttuynia cordata, HCPE-I, -II, and -III (Fig. 14), all three purified polysaccharides were confirmed to have macrophage proliferation ability. In addition, all three purified polysaccharides were confirmed to target cytokine secretion by macrophages in a concentration-dependent manner, suggesting that Houttuynia cordata-derived polysaccharides effectively induce macrophage activation, which plays an important role in innate and adaptive immunity in addition to intestinal immunity.

[0160]

[0161] Experimental Example 8: Effect of polysaccharide derived from Eoseongcho on enhancing NK cell activity

[0162] To activate NK cells, samples were intravenously injected at various concentrations. Only NK cells residing in the spleen were selectively isolated using an isolation kit and co-cultured with tumor cells for 6 hours. The degree of NK cell activation in the samples was then assessed by measuring the amount of LDH released from the target cells into the culture supernatant by NK cells. The experimental results are shown in Figure 15.

[0163] The experimental results confirmed that HCP, HCPE, and HCPE-I all dramatically increased the tumor cell killing effect of NK cells. In particular, the experimental group treated with HCPE-I at a concentration of 1000 μg / mouse was observed to have approximately 16.5 times the NK cell killing effect compared to NC. These results suggest that polysaccharides derived from Eoseongcho can contribute to the enhancement of immune function in the body by activating various immune cells such as macrophages and NK cells.

Claims

1. (S1) Step of adding water to the sesame oil and heating it to extract it. (S2) A step of adding ethanol or an ethanol aqueous solution to the supernatant obtained after extraction in step S1 to cause precipitation and separate the precipitate. (S3) A step of purifying the precipitate obtained in step S2; (S4) Step of enzymatic treatment by adding pectin hydrolyzing enzyme to the purified product of S3. (S5) A step of separating the result of step S4 according to molecular weight, Method for producing a polysaccharide fraction of Eoseongcho.

2. A manufacturing method in the first paragraph, characterized in that step S1 comprises adding water 10-70 times the volume of the perilla leaves to the perilla leaves and extracting at 90-110°C.

3. A manufacturing method in the first paragraph, wherein the S2 step comprises concentrating the supernatant to 5-20 brix, adding ethanol or a 90-99 wt% ethanol aqueous solution in a volume 2-10 times the concentrated volume to cause precipitation, and recovering the precipitate.

4. In the first paragraph, the purification of the S3 stage (S3-1) Step of adding an ethanol solution, centrifuging, and removing the supernatant to remove low molecular weight substances and pigment substances. (S3-2) A step of adding water to the S3-1 result to recover only the available fraction and remove the sediment, and (S3-3) A step for selectively removing low molecular weight substances. A manufacturing method characterized by comprising:

5. A manufacturing method according to claim 1, characterized in that the step S4 comprises a step of removing an ester group prior to treatment with a pectin hydrolyzing enzyme.

6. A manufacturing method according to claim 1, characterized in that the step S4 further comprises a step of removing low-molecular-weight substances after treatment with a pectin hydrolyzing enzyme.

7. A manufacturing method in the first paragraph, characterized in that the S5 step divides the molecular weight into three categories according to size to obtain the highest molecular weight fraction.

8. A manufacturing method according to claim 7, characterized in that the method is performed using gel permeation chromatography (GPC).

9. It is a polysaccharide fraction derived from Eoseongcho. When the content of neutral sugar is measured by the phenol-sulfuric acid method using galactose as a standard substance and the content of acid sugar is measured by the m-hydroxybiphenyl method using galacturonic acid as a standard substance, the content of neutral sugar and acid sugar is 60 to 80 wt% (neutral sugar) and 15 to 35 wt% (acid sugar), respectively, relative to the total weight of the polysaccharide fraction. When analyzing the composition of the sugar, the content of Rhamnose is 8-18 wt%, the content of Galactose is 36-56 wt%, the content of Arabinose is 7-27 wt%, the content of Glucuronic acid is 3-13 wt%, and the content of Galacturonic acid is 4-14 wt% compared to the total content of sugar components. Polysaccharide fraction derived from Eoseongcho.

10. In paragraph 9, the polysaccharide fraction derived from the lactic acid bacteria A polysaccharide fraction derived from Houttuynia cordata, wherein the contents of mannose, rhamnose, glucose, galactose, arabinose, fucose, glucuronic acid, and galacturonic acid are 0.1-2, 8-18, 0.1-2, 36-56, 7-27, 0.1-2, 3-13, and 4-14 wt%, respectively, relative to the total content of sugar components when analyzing the constituent sugars.

11. It is a polysaccharide derived from Eoseongcho. When the content of neutral sugar is measured by the phenol-sulfuric acid method using galactose as a standard substance and the content of acid sugar is measured by the m-hydroxybiphenyl method using galacturonic acid as a standard substance, the content of neutral sugar and acid sugar is 60 to 80 wt% (neutral sugar) and 15 to 35 wt% (acid sugar), respectively, relative to the weight of the polysaccharide. It contains an active polysaccharide, rhamnogalacturonan (RG)-I, in which the main chain of RG-I is composed of rhamnose and galacturonic acid, and the main chain contains a homogalacturonan portion composed of α-(1→4) bonds of galacturonic acid and a portion in which rhamnose and galacturonic acid are mixed. α-(1→5)-arabinan, arabino-β-(3,6)-galactan, β-(1→)-galactan, and β-(1→4)-glucuronan exist as side chains of the main chain. Characterized in that arabino-β-(3,6)-galactan, which is type II arabinogalactan, accounts for 40-60 wt% of the total weight of the polysaccharide. A polysaccharide derived from the plant.

12. A pharmaceutical composition comprising, as an active ingredient, a polysaccharide fraction derived from Houttuynia cordata produced by the manufacturing method of any one of claims 1 to 8, a polysaccharide fraction derived from Houttuynia cordata of claim 9 or 10, or a polysaccharide derived from Houttuynia cordata of claim 11.

13. A health functional food composition comprising, as an active ingredient, a polysaccharide fraction derived from Houttuynia cordata produced by the manufacturing method of any one of claims 1 to 8, a polysaccharide fraction derived from Houttuynia cordata of claim 9 or 10, or a polysaccharide derived from Houttuynia cordata of claim 11.

14. An immune-enhancing composition comprising, as an active ingredient, a polysaccharide fraction derived from Houttuynia cordata produced by the production method of any one of claims 1 to 8, a polysaccharide fraction derived from Houttuynia cordata of claim 9 or 10, or a polysaccharide derived from Houttuynia cordata of claim 11.

15. A composition for treating or preventing cancer through immune enhancement, comprising as an active ingredient a polysaccharide fraction derived from Houttuynia cordata produced by the manufacturing method of any one of claims 1 to 8, a polysaccharide fraction derived from Houttuynia cordata of claim 9 or 10, or a polysaccharide derived from Houttuynia cordata of claim 11.

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