Composition for enhancing immunity comprising polysaccharide enzyme-treated product or purified fraction thereof

Abstract

The present invention relates to a composition for enhancing immunity, containing, as an active ingredient, a polysaccharide enzyme-treated product derived from a mixed extract of Angelica gigas, Cnidium officinale, and Paeonia lactiflora, or a novel purified polysaccharide fraction thereof. The composition for enhancing immunity according to the present invention, comprising the polysaccharide enzyme-treated product of the mixed extract of Angelica gigas, Cnidium officinale, and Paeonia lactiflora or the novel purified polysaccharide fraction thereof, is composed of specific types and contents of sugars and significantly increases the activity of macrophages, splenocytes, and NK cells upon application thereto, thereby being applicable as an immunopotentiator or a composition for enhancing immunity.

Description

Immunostimulating composition comprising a polysaccharide enzyme-treated product or a purified fraction thereof

[0001] The present invention relates to an immune-enhancing composition comprising, as an active ingredient, an enzyme-treated polysaccharide derived from a mixed extract of Angelica gigas, Cnidium officinale, and Paeonia lactiflora, or a purified fraction thereof.

[0002] The immune response is a self-defense mechanism designed to protect our bodies. It involves the process of eliminating or neutralizing various antigens introduced from the outside and unnecessary metabolic products within the body, and plays a role in suppressing the occurrence of disease by maintaining homeostasis. The body's immune response is broadly divided into innate immunity, which is possessed from birth, and acquired immunity, which is obtained through adaptation to life and other factors.

[0003] Innate immunity, also known as natural immunity, responds non-specifically to antigens and does not exhibit any special memory function. In fact, this innate immunity defends against most infections. Innate immune systems include skin and mucous tissues that block the invasion of antigens, highly acidic stomach acid, and complement present in the blood. Cells include macrophages, polymorphonuclear leukocytes, and NK cells responsible for phagocytosis.

[0004] Acquired immunity, also known as adaptive immunity, allows the body to remember the antigen that first invaded and react specifically upon re-invasion to effectively eliminate the antigen. This acquired immunity is divided into humoral immunity and cellular immunity. Humoral immunity involves B lymphocytes recognizing an antigen, differentiating, and secreting antibodies, which primarily eliminate infected bacteria. Cellular immunity involves T lymphocytes (T cells) originating from the thymus recognizing antigens and either secreting lymphokines or directly killing infected cells; this cellular immunity primarily functions to eliminate cells infected by viruses or bacteria capable of growing within the cell.

[0005] Among the cells involved in the immune response, macrophages regulate immune phenomena by secreting various cytokines during the process of phagocytizing and eliminating bacteria or foreign substances. As cells that play a central role in immune action against antigens, they are involved in antigen presentation and the non-specific immune response of lymphocytes, and exhibit direct detrimental activity against tumor cells. Furthermore, it is known that substances reacting with TLRs (tool-like receptors) (LPS or natural products) activate macrophages to produce cytokines such as IL-1, IL-6, IL-10, IL-12, and TNF-α, which can regulate secondary immune responses such as the proliferation of T and B cells, phagocytosis, and defense against microbial infections. IL-1, IL-6, and TNF-α are representative cytokines induced by macrophages; they play a pivotal role in inflammatory responses caused by bacterial infections and are known to increase in amounts at inflammatory sites. IL-6 has been reported to act synergistically with IL-1 to be involved in the differentiation of T cells and B cells, and TNF-α is known to have antiviral effects and play an important role in various biological reactions. Additionally, IL-12 is known to act as a cytokine that activates NK cells and induces Th1-type immune responses, thereby increasing responsiveness to cellular foreign substances.

[0006] Natural killer (NK) cells play a crucial role in innate immune responses, which involve directly attacking host-infected cells or tumor cells, and adaptive immune responses, which involve activating cytotoxic T cells or B cells through the secretion of cytokines (Advances in Immunology, 2014, 137-170). NK cells are activated by cytokines derived from IFN or macrophages, and activated NK cells secrete cytokines such as IFN-γ and TNF-α to promote apoptosis in tumor cells or contribute to the activation of other immune cells. The activity of NK cells is regulated by a signaling balance involving the presence of various activating and inhibitory receptors; through this regulation, they eliminate infected cells without attacking normal cells (Cancer Immunol. Immunother. 2014; 63(6): 571-580).

[0007] It is known that the regulation of NK cell activity is associated with various diseases such as cancer or immune diseases, and there is a growing need for efficient immune enhancement methods using NK cells.

[0008] Meanwhile, natural products contain a diverse mixture of low-molecular-weight substances such as alkaloids, flavonoids, terpenoids, and saponins, as well as high-molecular-weight substances such as polysaccharides, proteins, and tannins, and have low toxicity. Consequently, there is a growing trend of research into various pharmaceuticals and functional foods utilizing polysaccharides derived from these products, but research is still needed regarding the pharmacological activity and immune-enhancing activity of high-molecular-weight carbohydrates.

[0009] Against this background, the inventors confirmed that when a polysaccharide enzyme-treated product derived from a mixed extract of Angelica gigas, Cnidium officinale, and Paeonia lactiflora composed of specific types and amounts of sugars, or a purified fraction thereof, is treated to macrophages, splenocytes, and NK cells, their activity is significantly increased, and newly identified that this can be utilized as an immune enhancer or an anticancer immunomodulator, thereby completing the present invention.

[0010] The object of the present invention is to provide a composition for immune enhancement or anticancer immune modulation comprising, as an active ingredient, a polysaccharide enzyme-treated product or a fraction thereof of a mixed extract of Angelica gigas, Cnidium officinale, and Paeonia lactiflora.

[0011] Another objective of the present invention is to provide a food composition for immune enhancement or anticancer immune modulation, comprising as an active ingredient a polysaccharide enzyme-treated product or a fraction thereof of a mixed extract of Angelica gigas, Cnidium officinale, and Paeonia lactiflora.

[0012] Another objective of the present invention is to provide a method for activating cells in vitro, ex vivo, or both, comprising the step of treating macrophages, splenocytes, or NK cells with a polysaccharide enzyme-treated product of a mixed extract of Angelica gigas, Cnidium officinale, and Paeonia lactiflora, or a purified fraction thereof.

[0013] Another objective of the present invention is to provide an immune-enhancing or anticancer immune-modulating use of a composition comprising, as an active ingredient, a polysaccharide enzyme-treated product of a mixed extract of Angelica gigas, Cnidium officinale, and Paeonia lactiflora or a purified fraction thereof.

[0014] Another objective of the present invention is to provide a method for immune enhancement or anticancer immune modulation, comprising the step of administering to an individual a composition comprising, as an active ingredient, a polysaccharide enzyme-treated product of a mixed extract of Angelica gigas, Cnidium officinale, and Paeonia lactiflora or a purified fraction thereof.

[0015] In order to solve the above problem,

[0016] An embodiment of the present invention is a composition for immune enhancement or anticancer immune modulation comprising, as an active ingredient, a polysaccharide enzyme-treated product or a fraction thereof of a mixed extract of Angelica gigas, Cnidium officinale, and Paeonia lactiflora.

[0017] As one specific example, the immunity is characterized as being innate immunity, adaptive immunity, or a combination thereof.

[0018] In another specific example, the mixed extract is characterized by being extracted with a solvent selected from the group consisting of water, straight-chain or branched alcohols having 1 to 6 carbon atoms, organic solvents, and mixed solvents thereof.

[0019] As one of the above embodiments, the mixed extract is characterized by containing Angelica gigas, Cnidium officinale, and Paeonia lactiflora in a weight ratio of 1 to 5:1 to 5:1 to 5.

[0020] As one of the above embodiments, the mixed extract is characterized as being a mixed extract of Angelica gigas, Cnidium officinale, and Paeonia lactiflora.

[0021] As one of the above embodiments, the polysaccharide is characterized as being an ethanol polysaccharide.

[0022] As one of the above embodiments, the enzyme is characterized by including pectinase.

[0023] As an embodiment of any one of the above embodiments, the polysaccharide enzyme-treated product or the purified fraction thereof is characterized by comprising a neutral sugar, an acidic sugar, a protein, a KDO-like substance, and a polyphenol.

[0024] As an embodiment of any one of the above embodiments, the polysaccharide enzyme-treated product is characterized by comprising 71.2 to 75.4% neutral sugar, 17.2 to 17.6% acidic sugar, 5.1 to 6.1% protein, 3.1 to 3.3% KDO-like substance, and 0.1 to 0.5% polyphenol.

[0025] As an embodiment of any one of the above embodiments, the purified fraction is characterized by comprising 72.7 to 75.5% neutral sugar, 18.8 to 20.8% acidic sugar, 1.7 to 2.5% protein, 3.4 to 3.6% KDO-like substance, and 0.3 to 0.5% polyphenol.

[0026] As an embodiment of any one of the above embodiments, the polysaccharide enzyme-treated product or the purified fraction thereof is characterized by comprising one or more selected from the group consisting of mannose, rhamnose, arabinose, xylose, galactose, glucose, and galacturonic acid.

[0027] As an embodiment of any one of the above embodiments, the polysaccharide enzyme-treated product is characterized by comprising 0.7 to 3.1 mol% rhamnose, 6.3 to 18.7 mol% arabinose, 4.8 to 10.6 mol% galactose, 54 to 72.8 mol% glucose, and 13.6 to 15.4 mol% galacturonic acid.

[0028] As an embodiment of any one of the above embodiments, the purified fraction is characterized by comprising 5.2 to 7.8 mol% rhamnose, 34.8 to 35.2 mol% arabinose, 18.6 to 19.6 mol% galactose, 22 to 23.8 mol% glucose, and 16.5 to 16.7 mol% galacturonic acid.

[0029] As an embodiment of any one of the above embodiments, the polysaccharide enzyme-treated product or its purified fraction is characterized by activating macrophages, spleen cells, or natural killer cells.

[0030] As an embodiment of any one of the above embodiments, the polysaccharide enzyme-treated product or the purified fraction thereof is characterized by an increase in immune-related cytokines.

[0031] As an embodiment of any one of the above embodiments, the cytokine is characterized by being one or more selected from the group consisting of IL-2, IFN-γ, IL-4, IL-10, TNF-α, IL-12, and IL-6.

[0032] As an embodiment of any one of the above embodiments, the composition is characterized by further comprising a mixed extract of Angelica gigas, Cnidium officinale, and Paeonia lactiflora.

[0033] As an embodiment of any one of the above embodiments, the mixed extract and the polysaccharide enzyme-treated product or the purified fraction thereof are mixed in a weight ratio of 60-70:30-40.

[0034] As an embodiment of any one of the above embodiments, the polysaccharide enzyme-treated product or the purified fraction thereof is characterized by being included in an amount of 0.001 to 99 weight% based on the total weight of the composition.

[0035] As one of the above embodiments, the composition is characterized as being a food composition.

[0036] As an embodiment of any one of the above embodiments, the food composition is characterized by being prepared as a powder, granule, tablet, capsule, syrup, or beverage.

[0037] Another aspect of the present invention embodying the present invention is a health supplement comprising a composition for immune enhancement or anticancer immune modulation comprising a polysaccharide enzyme-treated product of a mixed extract of Angelica gigas, Cnidium officinale, and Paeonia lactiflora, or a purified fraction thereof.

[0038] Another aspect of the present invention embodying the present invention is a method for activating cells in vitro, ex vivo, or both, comprising the step of treating cells with a polysaccharide enzyme-treated product of a mixed extract of Angelica gigas, Cnidium officinale, and Paeonia lactiflora, or a purified fraction thereof.

[0039] As one specific example, the cell is characterized as being a macrophage, a splenocyte, an NK cell, or a combination thereof.

[0040]

[0041] Another aspect of the present invention embodying the present invention is the use of a composition comprising, as an active ingredient, a polysaccharide enzyme-treated product of a mixed extract of Angelica gigas, Cnidium officinale, and Paeonia lactiflora or a purified fraction thereof for immune enhancement or anticancer immune modulation.

[0042] Another aspect of the present invention embodying the present invention is a method for immune enhancement or anticancer immune modulation comprising the step of administering to an individual a composition comprising, as an active ingredient, a polysaccharide enzyme-treated product of a mixed extract of Angelica gigas, Cnidium officinale, and Paeonia lactiflora or a purified fraction thereof.

[0043]

[0044] An immune-enhancing composition comprising a polysaccharide enzyme-treated product of a mixed extract of Angelica gigas, Cnidium officinale, and Paeonia lactiflora according to the present invention, or a novel purified fraction thereof, as an active ingredient is composed of a specific type and amount of sugar, and when treated with macrophages, splenocytes, and NK cells, it significantly increases their activity, so it can be applied as an immune enhancer or a food composition for immune enhancement.

[0045] Figure 1 is the result of confirming the purity of an ethanol polysaccharide and an enzyme-treated product thereof according to one embodiment of the present invention by size exclusion chromatography.

[0046] Figure 2 is the result of confirming the degree of purification using an open column of a purified fraction according to one embodiment of the present invention.

[0047] Figure 3 is the result of confirming the macrophage activation effect of a polysaccharide enzyme-treated product or a purified fraction thereof according to one embodiment of the present invention.

[0048] Figure 4 is the result of confirming the macrophage activation effect of a 4-type polysaccharide enzyme-treated product according to one embodiment of the invention.

[0049] Figure 5 is the result of confirming the spleen cell activation effect of a polysaccharide enzyme-treated product or a purified fraction thereof according to one embodiment of the present invention.

[0050] Figure 6 shows the results of confirming the tumor cell killing effect of natural killer cells of the purified fraction HHE-I according to one embodiment of the present invention. Here, the E / T ratio is the ratio of NK cells (effector cells, E) isolated from the spleen to tumor cells Yac-1 (target cells, T) known to be NK cell sensitive.

[0051] Each description and embodiment disclosed herein may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed herein fall within the scope of the invention. Furthermore, the scope of the invention is not to be limited by the specific descriptions provided below.

[0052] Furthermore, a person skilled in the art can recognize or identify a number of equivalents to the specific embodiments of the invention described in this application using only ordinary experiments. Furthermore, such equivalents are intended to be included in the invention.

[0053] Furthermore, throughout the entire specification of this application, when a part is described as "including" a certain component, this means that, unless specifically stated otherwise, it does not exclude other components but may include additional components.

[0054] The present invention will be described in more detail below.

[0055] The present invention is based on the novel confirmation that when a polysaccharide enzyme-treated product derived from a mixed extract of Angelica gigas, Cnidium officinale, and Paeonia lactiflora composed of specific types and amounts of sugars, or a purified fraction thereof, is treated to macrophages, splenocytes, and NK cells, their activity is significantly increased, and the discovery that this can be utilized as an immune enhancer or an anticancer immunomodulator.

[0056] An embodiment of the present invention for achieving the above objective provides a composition for immune enhancement or anticancer immune modulation comprising, as an active ingredient, a polysaccharide enzyme-treated product of a mixed extract of Angelica gigas, Cnidium officinale, and Paeonia lactiflora, or a purified fraction thereof.

[0057] In the present invention, the term “Dang-gui” refers to a perennial aromatic herb belonging to the Apiaceae family that has been used medicinally in East Asia for a long time, and in particular, the root portion of Dang-gui is generally used. Accordingly, the dried root of the above-mentioned Dang-gui is also referred to interchangeably as “Dang-gui.”

[0058] Specifically, in the present invention, the Angelica is *Angelica gigas Nakai* from the Korean family, which is distinguished from the roots of *Angelica sinensis (Oliv.) Diels*, *Angelica acutiloba (Siebold. & Zucc.) Kitag.* from the Japanese family, or *Angelica acutiloba (Siebold. & Zucc.) Kitag. var. sugiyamae Hikino*. Angelica made from the roots of *Angelica gigas Nakai* has excellent blood-activating effects that promote smooth blood circulation rather than blood-tonifying effects, and has strong anticancer and blood pressure-lowering effects. However, unlike *Angelica gigas Nakai*, *Angelica sinensis* has a warm energy and a sweet yet pungent taste; generally, compared to *Angelica gigas Nakai*, it has a stronger sweet taste and a weaker pungent taste. The efficacy of Chinese Angelica is its blood-replenishing effect, which generates blood when there is a blood deficiency. Angelica made from the roots of Chinese Angelica has excellent blood-replenishing properties, but its effect is lower than that of Korean Angelica. Pharmacologically, Angelica is known to promote coronary artery blood flow and stimulate the vigorous production of red blood cells.

[0059] In the present invention, the term “Cheongung” refers to a perennial plant of the Apiaceae family, order Apiaceae, class Dicotyledonous plants, distributed in Korea, Japan, and other regions. Cheongung has sedative, analgesic, and tonic effects and is used to treat headaches, anemia, gynecological diseases, etc. The rhizomes are dug up from September to November, the leaves and stems are removed, and after drying in the sun, they are decocted and consumed, or used in the form of pills or powder. Specifically, in the present invention, Cheongung may be Cheongung with the scientific name Cnidium officinale, which is distinguished from Chinese Cheongung (Ligusticum chuanxiongHort.).

[0060] In the present invention, the term “peony (Paeonia lactiflora Pallas)” refers to a perennial plant of the genus Paeonia in the family Paeoniaceae that grows in mountainous areas and is used for horticulture due to its beautiful flowers. Additionally, its roots are used as a medicinal herb for pain relief, abdominal pain, menstrual pain, amenorrhea, hemoptysis, anemia, bruises, etc. In China, it was already cultivated as an ornamental plant during the Qin and Ming dynasties, and its cultivation history is older than that of the peony. Through the Song dynasty and into the Qing dynasty, dozens of varieties were recorded, and it is distributed in Korea, Mongolia, and Eastern Siberia.

[0061] In this specification, the peony may comprise one or more selected from Paeonia lactiflora, Paeonia Japonica Miyabe & Takeda, Paeonia lactiflora Pall., Paeonia lactiflora Pall. var. hirta Regel, and Paeonia lactiflora Pall. var. trichocarpa (Bunge) Stern, and may be used without limitation regardless of the processing method.

[0062] The above-mentioned Angelica gigas, Cnidium officinale, and Paeonia lactiflora may be purchased commercially, or used without restriction if harvested from nature or cultivated.

[0063] In the present invention, the term "extract" refers to a mixed extract containing the above-mentioned Angelica gigas, Cnidium officinale, and Paeonia lactiflora, wherein the mixed extract of Angelica gigas, Cnidium officinale, and Paeonia lactiflora can be extracted from various organs of natural, hybrid, or variant plants, and specifically, can be extracted from roots, above-ground parts, stems, leaves, flowers, fruit bodies, fruit peels, as well as plant tissue cultures.

[0064] The above "mixed extract" is a mixed extract comprising Angelica gigas, Cnidium officinale, and Paeonia lactiflora. It may be an extract prepared by first mixing the three substances and then extracting them together, or an extract prepared by individually extracting each substance and then mixing the extracts. Additionally, any mixed extract prepared by any known method of preparing a mixed extract is included without limitation.

[0065] The above extract may be obtained by extracting the ground material of each component with a solvent selected from the group consisting of water, straight-chain or branched alcohols having 1 to 6 carbon atoms, organic solvents, and mixed solvents thereof, but is not limited thereto. Additionally, the extraction temperature may be in the range of 20°C to 100°C, and the extraction period may be about 1 hour to 10 days using extraction methods such as hot water extraction, cold maceration extraction, reflux cooling extraction, or ultrasonic extraction, but is not limited to the above extraction temperature, extraction period, or extraction method.

[0066] If the solvent of the above extract is water, it may include a cold water extract or a hot water extract.

[0067] The above organic solvent is not particularly limited and may be polyhydric alcohols such as glycerol, ethylene glycol, propylene glycol, 1,3-butylene glycol, hydrocarbon solvents such as methyl acetate, ethyl acetate, benzene, n-hexane, diethyl ether, dichloromethane, chloroform, and non-polar organic solvents such as petroleum ether, methyl acetate, benzene, hexane, chloroform, methylene chloride, dimethyl ether, and ethyl acetate.

[0068] The above solvent may also include an aqueous solution of an organic solvent, and its concentration is not particularly limited but may be 1 to 99% (v / v), specifically 1 to 90% (v / v), more specifically 60 to 98% (v / v), even more specifically 80 to 95% (v / v), 85 to 95% (v / v), and even more specifically 90 to 95% (v / v).

[0069] The above extract may include an extract, a diluted or concentrated extract, a dried product obtained by drying the extract, or all of these modified or purified products.

[0070] The above extract may contain Angelica gigas, Cnidium officinale, and Paeonia lactiflora in a weight ratio of 1 to 100:1 to 100:1 to 100. Specifically, the Angelica gigas, Paeonia lactiflora, and Cnidium officinale may be contained in a weight ratio of 1 to 30:1 to 30:1 to 30, more specifically in a weight ratio of 1 to 10:1 to 10:1 to 10, even more specifically in a weight ratio of 1 to 5:1 to 5:1 to 5, and even more specifically in a weight ratio of 0.8 to 1.2:0.8 to 1.2:0.8 to 1.2. As one embodiment, the above mixed extract may be a mixed extract containing Angelica gigas, Cnidium officinale, and Paeonia lactiflora in a weight ratio of 1:1:1.

[0071] In the extract of the present invention, extracts of known natural substances or known ingredients that promote or strengthen immunity may be optionally added.

[0072] In the present invention, the term "polysaccharide" refers to a mixture of Angelica gigas, Cnidium officinale, and Paeonia lactiflora extracts precipitated by adding a solvent, wherein the polysaccharide is also called a polysaccharide and may contain one or more polysaccharides.

[0073] In the present invention, the polysaccharide refers to a polysaccharide obtained by treating an extract with a solvent to precipitate it without purifying it itself, and may be used in combination with crude polysaccharide or GHHW-P.

[0074] The method for obtaining the polysaccharide in the present invention is not particularly limited and may be carried out according to methods commonly used in the art. Non-limiting examples of the above method include polysaccharides obtained from extracts of Angelica gigas, Cnidium officinale, and Paeonia lactiflora of the present invention by treating said extracts with a predetermined solvent, and specifically, polysaccharides produced by adding an organic solvent to the extracts of Angelica gigas, Cnidium officinale, and Paeonia lactiflora of the present invention, but are not limited thereto.

[0075] In the present invention, the type of solvent used to obtain the polysaccharide is not particularly limited, and any solvent known in the art may be used. Non-limiting examples of the solvent may include water, straight-chain or branched alcohols having 1 to 6 carbon atoms, organic solvents, or mixtures thereof, which may be used alone or in combination of one or more, but are not limited thereto. Additionally, the organic solvent may be one or more selected from the group consisting of dichloromethane, diethyl ether, chloroform, and ethyl acetate, but are not limited thereto.

[0076] Specifically, the above polysaccharide may be an ethanol polysaccharide, and more specifically, may be a 90-95% ethanol polysaccharide.

[0077] In the present invention, the term “polysaccharide enzyme-treated product” means a polysaccharide obtained by treating an extract with a solvent to precipitate it, and then treating the polysaccharide with an enzyme, wherein the enzyme may include pectinase.

[0078] For example, the polysaccharide enzyme-treated product of the present invention comprises an enzyme-treated product in which pectinase is applied to a polysaccharide precipitated by treating a mixed hot water extract of Angelica gigas, Cnidium officinale, and Paeonia lactiflora with ethanol. The product may be characterized in that, even if the polysaccharide is the same, the enzyme-treated product treated with other enzymes excluding pectinase, such as cellulase, protease, or amylase, has minimal immune-enhancing activity and therefore does not include other enzyme-treated products.

[0079] The above-mentioned polysaccharide enzyme-treated product is characterized by increased immune-enhancing activity through the removal of the inactive fraction, the HG region, by treating the ethanol polysaccharide with an enzyme.

[0080] The above term, “purified fraction,” refers to a fraction obtained through purification of a polysaccharide enzyme-treated product obtained by treating a polysaccharide obtained by precipitating an extract with a solvent with an enzyme, and refers to a fraction purified according to the molecular weight of the constituent sugars contained therein. The purified fraction may be used interchangeably with polysaccharide fraction, enzyme-treated product fraction, polysaccharide enzyme-treated product fraction, purified polysaccharide fraction, purified polysaccharide fraction, etc. Specifically, it may include HHE-I, HHE-II, and HHE-III, and more specifically, it may include HHE-I.

[0081] The above purified fractions HHE-I, HHE-II, and HHE-III are fractions obtained by purifying an enzyme-treated product obtained by treating a polysaccharide obtained by precipitating an extract with a solvent, and can be fractionated according to molecular weight.

[0082] Specifically, the purified fraction HHE-I is a fraction with a higher molecular weight compared to HHE-II and HHE-III, and HHE-II is a fraction with a lower molecular weight compared to HHE-I and a higher molecular weight compared to HHE-III. HHE-III is the fraction with the lowest molecular weight and is characterized by being composed of small forms of glucan, low-molecular-weight HG, proteins, polyphenols, etc.

[0083] The above polysaccharide enzyme-treated product and purified fractions HHE-I, HHE-II, and HHE-III are obtained from the same polysaccharide, but the type or content of the sugars constituting them may differ. The polysaccharide enzyme-treated product of the present invention may include neutral sugars, acidic sugars, proteins, KDO-like substances, and polyphenols, and specifically may be HHE-0, and may further include 71.2 to 75.4% neutral sugars, 17.2 to 17.6% acidic sugars, 5.1 to 6.1% proteins, 3.1 to 3.3% KDO-like substances, and 0.1 to 0.5% polyphenols.

[0084] The purified fraction of the present invention may comprise neutral sugars, acidic sugars, proteins, KDO-like substances, and polyphenols, specifically may be HHE-I, and more specifically may comprise 70 to 77% neutral sugars, 18 to 22% acidic sugars, 1.5 to 3% proteins, 3 to 3.8% KDO-like substances, and 0.3 to 0.6% polyphenols.

[0085] For example, the purified fraction HHE-I may contain 72.7 to 75.5% neutral sugars, 18.8 to 20.8% acidic sugars, 1.7 to 2.5% protein, 3.4 to 3.6% KDO-like substances, and 0.3 to 0.5% polyphenols.

[0086] The above term neutral sugar refers to a sugar having neutral properties and may be included in an amount of 70 to 77%, specifically 70 to 76%, 72 to 76%, 72.7 to 75.5%, 73 to 75%, or 73.5 to 75%, more specifically 74 to 74.5%, and even more specifically 74.1%.

[0087] The above term uronic acid, also known as sugar acid, refers to a monosaccharide in which one or both ends are substituted with a carboxyl group. In the present invention, the uronic acid may be included in an amount of 17 to 22%, specifically in an amount of 18 to 20%, 18.8 to 20.8%, or 19 to 20%, more specifically in an amount of 19.5 to 20%, and even more specifically in an amount of 19.8%.

[0088] The content of the above protein may be included in an amount of 1.5 to 3%, specifically 1.5 to 7% or 1.5 to 6.8%, specifically 1.7 to 2.5% or 2 to 2.5%, and more specifically 2.1%.

[0089] The above term KDO (ketodeoxy-octonate)-like material refers to any substance corresponding to ketodeoxy-octonate, and may include, for example, 2-keto-3-deoxy-D-manno-octulosonic acid. Here, KDO is an 8-carbon sugar and is one of the components constituting LPS (Lipopolysaccharide) of G(-) bacteria. By measuring KDO, it is possible to determine the extent to which immune activity can be produced. Specifically, the content of KDO may be included in an amount of 3 to 4% or 3 to 3.8%, specifically in an amount of 3 to 3.7%, 3 to 3.6%, or 3.4 to 3.6%, and more specifically in an amount of 3.5%.

[0090] The term polyphenol is a type of aromatic alcohol compound found in plants, characterized by having multiple hydroxyl groups as a functional group having two or more phenol groups in a single molecule. The polyphenol may be included in the composition of the present invention in an amount of 0.1 to 1% or 0.3 to 0.6%, specifically in an amount of 0.3 to 0.5% or 0.35 to 0.45%, and more specifically in an amount of 0.4%.

[0091] The neutral sugars, acidic sugars, proteins, KDO-like substances, and polyphenols contained in the above-mentioned purified fraction can be analyzed using the Phenol-H2SO4 analysis method, Uronic acid analysis method, KDO analysis method, Bradford analysis method, polyphenol analysis method, etc., and any known method can be analyzed without limitation using such method.

[0092] The polysaccharide enzyme-treated product of the present invention or the purified fraction thereof may include one or more selected from the group consisting of mannose, rhamnose, arabinose, xylose, galactose, glucose, and galacturonic acid.

[0093] The above polysaccharide enzyme-treated product may contain 0.7 to 3.1 mol% rhamnose, 6.3 to 18.7 mol% arabinose, 4.8 to 10.6 mol% galactose, 54 to 72.8 mol% glucose, and 13.6 to 15.4 mol% galacturonic acid.

[0094] The above purified fraction may contain 5 to 8 mol% rhamnose, 25 to 40 mol% arabinose, 15 to 25 mol% galactose, 20 to 25 mol% glucose, and 15 to 20 mol% galacturonic acid.

[0095] For example, the above-mentioned purified fraction may comprise 5.2 to 7.8 mol% rhamnose, 34.8 to 35.2 mol% arabinose, 18.6 to 19.6 mol% galactose, 22.0 to 23.8 mol% glucose, and 16.5 to 16.7 mol% galacturonic acid.

[0096] The constituent sugars included in the above polysaccharide enzyme-treated product or purified fraction can be analyzed, for example, by the PMP component sugar method, and can be analyzed by any known method without limitation.

[0097] The polysaccharide enzyme-treated product or purified fraction thereof of the present invention may be characterized by activating macrophages, spleen cells, or natural killer cells.

[0098] The aforementioned term, macrophage, refers to a cell that regulates immune phenomena by secreting various cytokines during the process of phagocytizing and eliminating bacteria or foreign substances. As a cell playing a central role in immune action against antigens, it is involved in antigen presentation and the non-specific immune action of lymphocytes, and exhibits direct toxic activity against tumor cells. Furthermore, it is known to produce cytokines such as IL-1, IL-6, IL-10, IL-12, and TNF-α, which can regulate secondary immune responses. Among these, IL-12 is known to act as a cytokine that activates NK cells and induces Th1-type immune responses, thereby increasing responsiveness to cellular foreign substances.

[0099] The term NK cell (natural killer cell) refers to a cell that plays a crucial role in the innate immune response by directly attacking host-infected cells or tumor cells. It is activated by cytokines derived from IFN or macrophages, and activated NK cells secrete cytokines such as IFN-γ and TNF-α to promote apoptosis in tumor cells or contribute to the activation of other immune cells. In particular, it is known that the regulation of NK cell activity is associated with various diseases, such as cancer or immune diseases.

[0100] The polysaccharide enzyme-treated product or its purified fraction according to the present invention can enhance the secretion of immune-related cytokines, specifically, it can enhance the secretion ability of one or more selected from the group consisting of IL-2, IFN-γ, IL-4, IL-10, TNF-α, IL-12, and IL-6, and more specifically, the secretion ability of the cytokine IL-10 or IFN-γ may be increased relative to the polysaccharide.

[0101] In one embodiment of the present invention, it was confirmed that an enzyme-treated polysaccharide containing 0.7 to 3.1 mol% rhamnose, 6.3 to 18.7 mol% arabinose, 4.8 to 10.6 mol% galactose, 54 to 72.8 mol% glucose, and 13.6 to 15.4 mol% galacturonic acid has superior immune-enhancing activity compared to polysaccharides containing different types or contents of constituent sugars.

[0102] In particular, it was confirmed that purified fraction HHE-I, containing 5.2 to 7.8 mol% rhamnose, 34.8 to 35.2 mol% arabinose, 18.6 to 19.6 mol% galactose, 22.0 to 23.8 mol% glucose, and 16.5 to 16.7 mol% galacturonic acid, has significantly superior IL-10 and IFN-γ secretion ability compared to polysaccharides containing different types or contents of the constituent sugars, polysaccharide enzyme-treated products, and purified fractions HHE-II and HHE-III, and possesses very excellent immune-enhancing activity and anticancer immune-modulating ability.

[0103] The composition of the present invention may further include a mixed extract of Angelica gigas, Cnidium officinale, and Paeonia lactiflora in a polysaccharide enzyme-treated product derived from a mixed extract of Angelica gigas, Cnidium officinale, and Paeonia lactiflora, or in a purified fraction thereof.

[0104] In the present invention, the term "mixture" refers to mixing the extracts of Angelica gigas, Cnidium officinale, and Paeonia lactiflora prepared in the present invention with a polysaccharide enzyme-treated product obtained by treating each of the said extracts with a predetermined solvent and then treating the product with an enzyme, or additionally purifying the product according to molecular weight, or a purified fraction thereof, and the mixing ratio is not particularly limited.

[0105] The above composition can achieve significantly superior effects compared to cases where only the extract or polysaccharide of a single plant is included, or where the extract contains a polysaccharide, by combining the extract of each of the above plants with the enzyme-treated polysaccharide or the purified fraction thereof. In addition, the above composition can exhibit significantly superior immune-enhancing or strengthening effects compared to cases where only the extract or polysaccharide of a plant other than Angelica gigas, Cnidium officinale, and Paeonia lactiflora is included, or where the extract contains only a polysaccharide.

[0106] When the above mixture is a mixture of an extract and a polysaccharide enzyme-treated product or a purified fraction thereof, the extract and the polysaccharide enzyme-treated product or the purified fraction thereof may be included in a weight ratio of about 40 to 80:20 to 60, specifically in a weight ratio of 45 to 75:25 to 55, more specifically in a weight ratio of 50 to 70:30 to 50, even more specifically in a weight ratio of 60 to 70:30 to 40, and even more specifically in a weight ratio of 65:35, but is not limited thereto.

[0107] The polysaccharide enzyme-treated product derived from the extracts of Angelica gigas, Cnidium officinale, and Paeonia lactiflora, or the purified fraction thereof, or a composition containing the same may be included in an amount of 0.0001 to 99 weight% based on the total weight of the composition, specifically in an amount of 30 to 80 weight% based on the total weight of the composition, more specifically in an amount of 35 to 70 weight%, and even more specifically in an amount of 40 to 65 weight%, but is not limited thereto.

[0108] In the present invention, the term “improvement” means applying the polysaccharide enzyme-treated product derived from the extracts of Angelica gigas, Cnidium officinale, and Paeonia lactiflora of the present invention, or a purified fraction thereof, to a composition to promote, enhance, or strengthen immunity.

[0109] In the present invention, the term “anti-cancer immune modulation” refers to anti-cancer immune modulation through NK cell activation against tumor cells.

[0110] The term cancer refers to a disease caused by the excessive proliferation of cells when the balance of normal cell death is disrupted. These abnormally overproliferated cells may, in some cases, invade surrounding tissues and organs to form tumors, destroying or deforming normal structures within the body; this condition is called cancer.

[0111] The above types of cancer include, but are not limited to, brain and spinal cord tumors, brain tumors, head and neck cancers, lung cancer, breast cancer, thymoma, mesocystoma, esophageal cancer, colorectal cancer, liver cancer, stomach cancer, pancreatic cancer, biliary tract cancer, kidney cancer, bladder cancer, prostate cancer, testicular cancer, germ cell tumors, ovarian cancer, cervical cancer, endometrial cancer, colorectal cancer, lymphoma, acute leukemia, chronic leukemia, multiple myeloma, sarcoma, malignant melanoma, and skin cancer.

[0112] NK cells can distinguish between cancer cells and normal cells through various immune receptors present on their surface, and thus exhibit selective cytotoxicity against cancer cells. Many studies have identified that NK cells play a key role in eliminating cancer cells, and it has been reported that cancer patients have defects in the number or activity of NK cells (Hanyang Med Rev 2013: 59-64). Therefore, increased activity of NK cells can lead to the prevention, improvement, or treatment of cancer through the elimination of cancer cells.

[0113] In a specific embodiment of the present invention, it has been confirmed that the polysaccharide enzyme-treated product of the present invention or its purified fraction has an NK cell activation effect against tumor cells, suggesting that a composition containing the polysaccharide enzyme-treated product or its purified fraction as an active ingredient can be usefully used for anticancer immune modulation.

[0114] The above composition may be added to a food composition for the purpose of promoting, improving, enhancing, strengthening, or regulating anticancer immunity using an enzyme-treated polysaccharide derived from extracts of Angelica gigas, Cnidium officinale, and Paeonia lactiflora, or a purified fraction thereof, and the food composition may be a health functional food composition.

[0115] The above food composition may include a food-grade acceptable carrier.

[0116] The food composition of the present invention includes all forms such as functional food, nutritional supplement, health food, and food additives, and said types of food compositions can be prepared in various forms according to conventional methods known in the art.

[0117] When using polysaccharide enzyme-treated extracts of Angelica gigas, Cnidium officinale, and Paeonia lactiflora, or purified fractions thereof as food additives, the polysaccharide enzyme-treated extracts of Angelica gigas, Cnidium officinale, and Paeonia lactiflora, or purified fractions thereof, may be added as is or used in combination with other foods or food ingredients, and may be used appropriately according to conventional methods. The amount of the active ingredient mixture may be appropriately determined according to the purpose of use (prevention, health, or therapeutic treatment). Generally, when manufacturing food or beverages, the polysaccharides of the extracts of Angelica gigas, Cnidium officinale, and Paeonia lactiflora are added in an amount of 0.0001 to 90 weight%, preferably 0.001 to 50 weight%, of the raw material composition. However, in the case of long-term consumption for the purpose of health and hygiene or health control, the above amount may be used in an amount less than the above range.

[0118] There are no special restrictions on the types of the above-mentioned foods. Examples of foods to which the above-mentioned substance may be added include meat, sausage, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and may include all health foods in the conventional sense.

[0119] The health beverage composition of the present invention may contain various flavoring agents or natural carbohydrates as additional ingredients, as in conventional beverages. The natural carbohydrates mentioned above are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As sweeteners, natural sweeteners such as taumatin and stevia extract, or synthetic sweeteners such as saccharin and aspartame may be used. The proportion of the natural carbohydrates may generally be about 0.001 to 50 parts by weight, specifically about 0.01 to 30 parts by weight, per 100 parts by weight of the composition of the present invention.

[0120] In addition to the above, the composition of the present invention may contain various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. Although the proportion of these additives is not critical, they are generally selected in the range of 0.01 to 30 parts by weight per 100 parts by weight of the composition of the present invention. Furthermore, the composition of the present invention may contain fruit pulp for the production of natural fruit juices, fruit juice beverages, and vegetable beverages. Although the proportion of such fruit pulp is not critical, it is generally selected in the range of 0.01 to 30 parts by weight per 100 parts by weight of the composition of the present invention, and these components may be used independently or in combination.

[0121] Another aspect of the present invention for achieving the above objective provides a health supplement comprising a composition for immune enhancement or anticancer immune modulation, comprising a polysaccharide enzyme-treated product of a mixed extract of Angelica gigas, Cnidium officinale, and Paeonia lactiflora, or a purified fraction thereof, as an active ingredient.

[0122] The above terms, “Dangui,” “Cheongung,” “Paeonia,” “mixed extract,” “polysaccharide,” “enzyme-treated product,” “purified fraction,” “immune enhancement,” and “anticancer immune modulation” are as described above.

[0123] The above term “health supplement” may include all known ranges of substances that play an auxiliary role for health.

[0124] Another aspect of the present invention for achieving the above objective provides a method for activating cells in vitro, ex vivo, or both, comprising the step of treating cells with a polysaccharide enzyme-treated product of a mixed extract of Angelica gigas, Cnidium officinale, and Paeonia lactiflora, or a purified fraction thereof.

[0125] The above terms, “Dangui,” “Cheongung,” “Paeonia,” “mixed extract,” “polysaccharide,” “enzyme-treated product,” “purified fraction,” “immune enhancement,” and “anticancer immune modulation” are as described above.

[0126] The above term, cell, may be a macrophage, splenocyte, NK cell, or a combination thereof.

[0127] In the present invention, the treatment of the polysaccharide enzyme-treated product or the purified fraction thereof may be carried out according to methods conventional in the art, and the treatment concentration of the polysaccharide enzyme-treated product or the purified fraction thereof may be 1 to 100 μm, 1 to 90 μm, 1 to 80 μm, 1 to 70 μm, or 1 to 60 μm, but is not limited thereto.

[0128] The "in vitro" of the present invention refers to an experimental process performed in a controllable environment, such as a test tube, rather than inside a living organism. In the environment inside the test tube, various variables can be easily controlled, allowing for efficient verification of results.

[0129] The term “ex vivo” in the present invention refers to the process of conducting experiments on cells or tissues isolated from a living organism in an in vitro environment. Through the above ex vivo experiment, precise manipulation is possible while maintaining reactions similar to those in the body.

[0130] In a specific embodiment of the present invention, it was confirmed that the polysaccharide enzyme-treated product or its purified fraction was treated to macrophages, splenocytes, and NK cells and increased the secretion of various cytokines associated with said cells, which suggests that the polysaccharide enzyme-treated product or its purified fraction of the present invention can activate macrophages, splenocytes, and NK cells in vitro or ex vivo.

[0131]

[0132] Another aspect of the present invention for achieving the above objective provides an immune-enhancing or anticancer immune-modulating use of a composition comprising, as an active ingredient, a polysaccharide enzyme-treated product of a mixed extract of Angelica gigas, Cnidium officinale, and Paeonia lactiflora or a purified fraction thereof.

[0133]

[0134] Another aspect of the present invention for achieving the above objective provides a method for immune enhancement or anticancer immune modulation, comprising the step of administering to an individual a composition comprising, as an active ingredient, a polysaccharide enzyme-treated product of a mixed extract of Angelica gigas, Cnidium officinale, and Paeonia lactiflora or a purified fraction thereof.

[0135] The above mixed extract, polysaccharide enzyme-treated product, and purified fraction are as described above.

[0136] In the present invention, the term "administration" refers to introducing a composition of the present invention to a subject by any appropriate method, and the route of administration may be administered via various oral or parenteral routes as long as it can reach the target tissue.

[0137] In the present invention, the individual may include humans or non-human species, and more specifically, may include mammals including humans, and even more specifically, may include humans, dogs, rats, rabbits, cats, horses, or cattle, but may include any individual capable of enhancing immunity or anticancer immune regulation by administering the composition of the present invention without limitation.

[0138]

[0139] The present invention will be explained in more detail below by way of examples. However, these examples are intended to illustrate the present invention and the scope of the present invention is not limited by these examples, and it will be obvious to those skilled in the art to which the present invention pertains.

[0140]

[0141] Example 1: Preparation of mixed extract of Angelica gigas, Cnidium officinale, and Paeonia lactiflora, its polysaccharides, polysaccharide enzyme-treated products, and purified fractions thereof

[0142] 1-1: Preparation of Mixed Extract of Angelica gigas, Cnidium officinale, and Paeonia lactiflora

[0143] The roots of *Angelica gigas*, the rhizomes of *Cnidium officinale*, and the roots of *Paeonia lactiflora* were each air-dried, chopped, and mixed. Then, distilled water equivalent to 10 times the total weight of each herbal medicine was added, and the mixture was hot-water extracted at 95°C for 4 hours to obtain an extract. At this time, the *Angelica gigas*, *Cnidium officinale*, and *Paeonia lactiflora* were mixed in a weight ratio of 1:1:1. The hot-water extract was then concentrated under reduced pressure and filtered to obtain a mixed extract of *Angelica gigas*, *Cnidium officinale*, and *Paeonia lactiflora*.

[0144] 1-2: Preparation of Ethanol Polysaccharides from Mixed Extracts

[0145] An ethanol polysaccharide (HHW-P) of the mixed extract prepared in Example 1-1 above was prepared. The ethanol polysaccharide HHW-P is a crude polysaccharide obtained directly without purification from the polysaccharide obtained by precipitating with ethanol.

[0146] Specifically, the mixed hot water extract prepared in Example 1-1 above was taken, 95% ethanol was added thereto, and after standing at 25°C or lower for 16 hours, the precipitated ethanol polysaccharide was obtained by centrifugation.

[0147] 1-3: Polysaccharide enzyme-treated product from ethanol polysaccharide and purification of three types of its purified fractions

[0148] 1-3-1: Verification of Ethanol Polysaccharide Purity

[0149] The purity of the ethanol polysaccharide (HHW-P) obtained in Examples 1-2 above was checked to determine if there were any substances mixed within the ethanol polysaccharide.

[0150] Specifically, size exclusion chromatography was performed using a refractive index detector-HPLC equipped with a Superdex 75 GL column pre-packed with a dextran porous resin.

[0151] As a result, as can be seen in Figure 1a, the peak corresponding to a relatively high molecular weight was detected first at the 14-minute mark, followed by the peak corresponding to a relatively low molecular weight after 30 minutes.

[0152] These results indicate that the ethanol polysaccharide HHW-P from the mixed extract of Angelica gigas, Cnidium officinale, and Paeonia lactiflora is in a crude state containing various substances; therefore, it is necessary to identify the active moiety that actually possesses activity within the ethanol polysaccharide of the said mixed extract. To this end, an enzyme-treated product obtained by treating the ethanol polysaccharide obtained above with an enzyme, and a purified fraction obtained by purifying the same were each acquired.

[0153] 1-3-2: Obtaining Enzyme-Treated Polysaccharides

[0154] The ethanol polysaccharide HHW-P of the mixed extract obtained in Example 1-3-1 was deesterified using NaOH, and then adjusted to pH 6, the optimal reaction pH for polygalacturonase (a pectinase), using HCl. Subsequently, polygalacturonase was added and reacted at 50°C for 72 hours, after which the enzymatic reaction was terminated by heating. The precipitated enzyme and reaction solution were separated by centrifugation. Then, the low molecular weight fragments generated by the enzymatic reaction were removed by dialysis using a dialysis tubing bag (MW-cut off: 14,000 Da) for 3 days, and the polysaccharide enzyme-treated product HHE-0 was obtained through concentration and freeze-drying. Next, the types of enzymes were changed to cellulase, alpha-amylase, and protease, respectively, and polysaccharide enzyme-treated products HHE-C (HHW-P + cellulase), HHE-α (HHW-P + α-Amylase), and HHE-pro (HHW-P + Protease) were obtained in the same way.

[0155] To confirm whether the enzyme treatment worked properly, the elution pattern of the polysaccharide enzyme-treated product was examined using size exclusion chromatography with HPLC. As a result, as can be seen in Figure 1b, it was confirmed that the low molecular weight fraction of HHE-0 increased relatively compared to before enzyme treatment. These results indicate that the enzyme treatment of the polysaccharide worked properly.

[0156] 1-3-3: Three types of purified fractions obtained

[0157] We intended to obtain purified fractions through a purification process using the polysaccharide enzyme-treated product HHE-0 obtained in Example 1-3-2 above. Specifically, the swollen Sephadex G-75 resin was packed into an open column (diameter: 2.5 cm, length: 90 cm) and the resin was equilibrated using 50 mM ammonium formate buffer (pH 5.5). Then, the previously obtained ethanol polysaccharide enzyme-treated product HHE-0 was loaded and separated into 100 fractions. These fractions were then separated into three types of purified fractions with different molecular weights by analyzing their chemical properties, such as neutral sugars, acidic sugars, proteins, and KDO.

[0158] The three types of purified fractions mentioned above were named HHE-I, HHE-II, and HHE-III, respectively, and are shown in Fig. 2.

[0159] As a result, as can be seen in Figure 2, considering that in the case of GPC, high molecular weight substances appear earlier and low molecular weight substances appear later, looking at the peak patterns of the three purified fractions, it can be confirmed that the main peak moves toward the lower molecular weight side as one moves from the high molecular weight fraction HHE-I to the low molecular weight fraction HHE-III. Through this, it can be confirmed that HHE-1, HHE-2, and HHE-3 were well fractionated according to molecular weight. In addition, it was confirmed that HHE-I exhibits a sharp single peak with left-right symmetry as its main peak. These results indicate that among the three purified fractions, HHE-I has the best degree of purification compared to HHE-II and HHE-III and exhibits a peak corresponding to the high molecular weight.

[0160] Subsequently, the above process was repeated 10 or more times to obtain an amount for confirming the various characteristics and immune-enhancing activity of the three purified fractions.

[0161] Example 2: Chemical and constituent sugar composition analysis of five types of polysaccharides, enzymatically treated polysaccharides, and purified fractions

[0162] For a total of five types (HHW-P, HHE-0, HHE-I, HHE-II, HHE-III), including the polysaccharide isolated or purified in Example 1, the polysaccharide enzyme-treated product, and the purified fraction thereof, the chemical composition and constituent sugar composition were each confirmed and are shown in Tables 1 and 2.

[0163] 2-1. Chemical Composition Analysis

[0164] The chemical composition was confirmed to consist of neutral sugars, acidic sugars, KDO-like substances, proteins, and polyphenols, and the specific method for confirming the chemical composition is as follows.

[0165] Specifically, for the analysis of the general components of polysaccharides, the content of neutral sugars was measured using the phenol-sulfuric method with galactose as the standard (see Dubois, M., Gilles, KA., Hamilton, JK., Rebers, PA., Smith FAJN.(1951). A colorimetric method for the determination of sugars. Nature, 168(4265): 167.), and acidic sugars were measured using the m-hydroxydiphenyl method with galacturonic acid as the standard (see Blumenkrantz N, Gustav AH. New method for quantitative determination of uronic acids. Analytical Biochemistry 54 (1973): 484-489.).

[0166] The content of 2-keto-3-deoxy-D-manno-octulosonic acid (KDO) was analyzed using the thiobarbituric acid method. Specifically, because KDO has very weak bonds, the sugar was hydrolyzed by adding 0.4N dilute sulfuric acid and heating, and 0.04M HIO₄ was added to reduce the hydroxyl(-OH) group and open the ring, thereby forming a malondialdehyde group. Subsequently, 2% Na₂SO₃ / 0.5M HCl was added for decolorization, and TBA (2-thiobarbituric acid) was added to combine with malondialdehyde to form a red chromogen. Then, DMSO was added to stabilize the colorimetric reaction of the chromogen, thereby confirming the KDO analogue.

[0167] Proteins were analyzed using the Bradford method with Bovine serum albumin (BSA) as a standard. Specifically, the Coomassie dye ligand was identified by binding to the positively charged portions (Arginine, Lysine, Histidine) of the protein to produce a blue color.

[0168] Finally, the phenolic compound content was quantified and analyzed using the Folin-Ciocalteu method with gallic acid as the standard. Specifically, standards and samples were prepared separately. Standards were pre-prepared in ep. tubes at concentrations of 0, 200, 400, 600, 800, and 1000 μg / mL. Three replicates were prepared for both the STD and Sample ep. tubes. 790 μL of IW was added to all ep. tubes, followed by 10 μL of the STD and Sample. Subsequently, 500 μL of Folin-Ciocalteu reagent was added to all ep. tubes and left to stand for 1 minute. Afterward, 150 μL of 20% sodium carbonate was added; the lids were opened immediately after vortexing, and the tubes were left in a dark room for 2 hours. Afterward, 200 μL samples were taken and measured at a wavelength of 750 nm to confirm the presence of polyphenols.

[0169] The results are as shown in Table 1 below.

[0170]

[0171] ChemicalProperties (%)HHW-PHHE-0HHE-IHHE-IIHHE-IIINeutral sugar77.9±1.973.3±2.174.1±1.464.1±2.329.6±1.0Uronic acid14.4±4.617.4±0.219.8±1.016.2±0.217.5±0.4Protein2.3±0.75.6±0.02.1±0 .415.0±0.143.1±2.4Polyphenol0.7±0.10.4±0.00.4±0.10.6±0.01.8±0.1KDO-like material4.7±0.13.2±0.13.5±0.14.1±0.08.1±1.7

[0172]

[0173] As a result, as can be seen in Table 1 above, it was confirmed that the ethanol polysaccharide HHW-P consists of 77.9±1.9% neutral sugars and 14.4±4.6% acidic sugars, indicating that it is mostly composed of sugars. For comparison, the chemical composition of one type of polysaccharide enzyme-treated product and three types of purified fractions was examined. As shown in Table 1, the chemical composition of HHE-0 consisted of 73.3±2.1% neutral sugars, 17.4±0.2% acidic sugars, 5.6% protein, 0.4% polyphenols, and 3.2±0.1% KDO-like substances, confirming that its distribution was distinctly different from that of the polysaccharide.

[0174] In addition, in the purified fractions, neutral sugars decreased compared to polysaccharides, with HHE-I at 74.1±1.4%, HHE-II at 64.1±2.3%, and HHE-III at 29.6±1.0%. Acid sugars increased compared to polysaccharides in all purified fractions, with HHE-I at 19.8±1.0%, HHE-II at 16.2±0.2%, and HHE-III at 17.5±0.4%. It was confirmed that HHE-I and HHE-III showed more increases than HHE-II. Notably, regarding protein and polyphenols, in the case of HHE-I, they did not increase to a similar degree as polysaccharides but rather decreased, whereas in HHE-III, protein and polyphenols increased significantly compared to polysaccharides. This confirmed that even though HHE-I, HHE-II, and HHE-III are purified fractions of ethanol polysaccharides, their chemical composition is completely different from that of the polysaccharides.

[0175] 2-2 Analysis of Constituent Sugar Composition

[0176] Next, the composition of constituent sugars was also confirmed using HPLC analysis with PMP (1-Phenyl3-methyl-5-pyrazolone) labeling, and the constituent sugars were identified as rhamnose, fructose, arabinose, xylose, mannose, galactose, glucose, glucuronic acid, and galaturonic acid. The specific verification method is as follows.

[0177] Specifically, after dissolving the sample to 1 mg / mL, 500 μL was transferred to a capped tube and dried by N2 flushing. Then, 1 mL of 2 M trifluoroacetic acid (TFA) was added, the tube was wrapped with Teflon tape, and the mixture was reacted at 121°C for 90 minutes. After cooling the sample, it was dried by N2 flushing, followed by the addition of 200 μL of methanol and drying by N2 flushing. Based on 10 samples, 100 μL of 0.3 M NaOH + 120 μL of 0.5 M PMP (in MeOH) was prepared to a ratio of 130 mg / 1.5 mL (MW 174.2). The mixture was reacted at 70°C for 100 minutes, cooled sufficiently, and neutralized with 100 μL of 0.3 M HCl. Subsequently, after N2 flushing, 0.5 mL each of water and chloroform were added and the mixture was shaken. The aqueous layer (top) was recovered by centrifugation at 2500 rpm for 5 minutes and transferred to a 2 mL ep-tube. Chloroform was added, and centrifugation was repeated 3 times, followed by filtration through a 0.45 μm filter. Finally, each constituent sugar was measured by HPLC, and the results are shown in Table 2 below.

[0178]

[0179] Chemicalproperties(%)HHW-PHHE-0HHE-IHHE-IIHHE-IIIComponent sugar(PMP)(Mole%)Mannose----3.2±0.4Rhamnose2.1±1.41.9±1.26.5±1.32.8±0.48.2±0.0Glucuronic acid-----Galacturonic acid13.9±2.014.5±0.916.6±0.114.9±0.125.8±1.4Glucose68.2±4.063.4±9.422.9±0.947.9±0.027.2±0.6Galactose2.7±3.4 7.7±2.919.1±0.56.9±0.119.7±0.3

[0180]

[0181] As a result, as can be seen in Table 2 above, the analysis of the constituent sugar composition revealed that the ethanol polysaccharide HHW-P contained 68.2±4.0% glucose, 13.9±2.0% galacturonic acid, 13.2±8.0% arabinose, and 2.1±1.4% rhamnose, while containing less galactose at 2.7±3.4% and xylose at 2.4±0.3%.

[0182] On the other hand, regarding the composition of constituent sugars, it was confirmed that HHE-0 contains 1.9±1.2 mol% rhamnose, 14.5±0.9 mol% glucuronic acid, 63.4±9.4 mol% glucose, 7.7±2.9 mol% galactose, and 12.5±6.2 mol% arabinose, confirming that the composition is very different from that of the polysaccharide HHW-P.

[0183] In addition, in the purified fraction, HHE-I contained 35.0±0.2 mol% arabinose, 22.9±0.9 mol% glucose, 19.1±0.5 mol% galactose, 16.6±0.1 mol% galacturonic acid, and 6.5±1.3 mol% rhamnose. It was confirmed that the glucose content was significantly lower compared to the polysaccharide, while the content of rhamnose, galacturonic acid, galactose, and arabinose was significantly increased, confirming that the composition of the polysaccharide and the constituent sugars in the purified fraction was also completely different.

[0184] In particular, while the types of constituent sugars contained in the purified fractions HHE-II and HHE-III were not significantly different from those in HHE-I, there were differences in their content. Specifically, compared to HHE-I, HHE-II contained a rhamnose content that was as low as that of a polysaccharide, and contained small amounts of galacturonic acid, galactose, and arabinose. In contrast, glucose was significantly higher than that in HHE-I. In the case of HHE-III, compared to HHE-I, the rhamnose content was higher, and the levels of galacturonic acid, galactose, and glucose were similar. On the other hand, the arabinose content was very low, at about 50% of that in HHE-I. Through these results, it was confirmed that even when obtained through a purification process from polysaccharide enzyme-treated products obtained from the same mixed extract, there were differences in the types and content of constituent sugars contained; among them, it was confirmed that the HHE-I fraction contained a large number of constituent sugars with higher molecular weights compared to the HHE-II and HHE-III fractions.

[0185] Example 3: Confirmation of Immuno-enhancing and Anticancer Immunomodulatory Effects of 5 Types of Polysaccharides, Enzyme-treated Polysaccharides, and Their Purified Fractions

[0186] In order to confirm the immune-enhancing effects of the five types of ethanol polysaccharides obtained in Example 1 above, the enzyme-treated polysaccharides, and their purified fractions, the degree of activation of macrophages, splenocytes, and NK-cells was confirmed and compared.

[0187] 3-1: Confirmation of Macrophage Activation

[0188] Macrophage activation of the above five types of polysaccharides, polysaccharide enzyme-treated products, or purified fractions (HHW-P, HHE-0, HHE-I, HHE-II, HHE-III) was confirmed by the cytokine IL-6, IL-12, and TNF-α secretion ability.

[0189] Specifically, after recovering peritoneal-derived macrophages from BALB / c mice, the above five types lysed at various concentrations were each treated with the macrophages, and the cytokines secreted in the supernatant for 24 hours were measured using an ELISA kit.

[0190] As a result, as can be seen in Figure 3, no significant toxicological differences were found in the five types of polysaccharides, the polysaccharide enzyme-treated product, or the purified fraction between concentrations of 1 and 1000 μg / mL.

[0191] Next, when the cytokines secreted in the culture supernatant were measured using an ELISA kit, it was confirmed that the ethanol polysaccharide HHW-P had no effect on the secretion of IL-6, IL-12, and TNF-α. On the other hand, the polysaccharide enzyme-treated product HHE-0, which was obtained by enzyme treatment of the ethanol polysaccharide, and the three purified fractions HHE-I, HHE-II, and HHE-III obtained by purifying the said enzyme-treated product all showed the secretion effects of the three cytokines starting from a low concentration of 10 μg / mL or higher, confirming that macrophage activation occurred even at very low concentrations, unlike the ethanol polysaccharide which had almost no activity.

[0192] In particular, among the purified polysaccharide fractions, HHE-I showed secretion ability for all three types of cytokines from a concentration of 10 μg / mL or higher compared to HHE-II and HHE-III, and unlike HHE-II or HHE-III which were ineffective at some concentrations, it was confirmed that it had excellent secretion ability for all three types of cytokines at concentrations of 10 to 1,000 μg / mL.

[0193] Through this, it can be confirmed that, unlike ethanol polysaccharides which exhibit a crude form due to the mixture of various substances, the enzyme-treated product or the purified fraction obtained by purifying it effectively exhibits immune-enhancing activity even at very low concentrations, and among them, the purified fraction HHE-I is superior to other purified fractions in immune-enhancing activity.

[0194] Next, macrophage activation and cytokine production capacity were confirmed and compared when different types of enzymes were used in the polysaccharide enzyme-treated products.

[0195] After recovering peritoneal-derived macrophages from BALB / c mice, the ethanol polysaccharide HHW-P and the four types of polysaccharide enzyme-treated products prepared in Example 1-3-2, HHE-0, HHE-C, HHE-α, and HHE-pro, were treated with the macrophages at concentrations of 1 μg / mL, 10 μg / mL, and 100 μg / mL, respectively, and the cytokines secreted in the supernatant for 24 hours were measured using an ELISA kit.

[0196] As a result, no significant toxicological differences were observed between the ethanol polysaccharide and the four types of polysaccharide enzyme-treated products. In addition, cytokines secreted into the culture supernatant were measured using an ELISA kit, and it was confirmed that HHE-0 had a significantly superior IL-6 secretion effect across the entire concentration range compared to the ethanol polysaccharide HHW-P and other enzyme-treated products (Fig. 4).

[0197] 3-2: Confirmation of splenocyte proliferation and cytokine expression ability

[0198] Next, splenocyte activation of the above five types of polysaccharides, polysaccharide enzyme-treated products, or purified fractions (HHW-P, HHE-0, HHE-I, HHE-II, HHE-III) was confirmed by splenocyte proliferation ability and the expression levels of IL-10 and IFN-γ cytokines.

[0199] Specifically, after recovering splenocytes from BALB / c mice, the five types of the above dissolved at various concentrations were each treated together with the splenocytes, and the cytokines secreted in the supernatant for 96 hours were measured using an ELISA kit.

[0200] As a result, as can be seen in Figure 5, no significant toxicological differences were found in any of the five types of polysaccharides, polysaccharide enzyme-treated products, or purified fractions between concentrations of 10 and 1000 μg / mL.

[0201] In the measurement of cytokines, it was confirmed that HHE-I exhibited distinct secretory activity for IL-10 and IFN-γ compared to the polysaccharide and the purified fractions HHE-II and HHE-III. Unlike the crude polysaccharide, HHE-II, and HHE-III, which all had secretory activity of less than 50 pg / mL or no secretory activity at all, HHE-I had a secretory activity of about 150 pg / mL for IL-10, which was about 100–150% higher than that of the polysaccharide or other purified fractions. Similarly, it was confirmed that HHE-I had a secretory activity of 300 pg / mL for IFN-γ, which was about 150–300% higher than that of the crude polysaccharide or other purified fractions.

[0202] These results indicate that when the mixed hot water extract of Angelica gigas, Cnidium officinale, and Paeonia lactiflora is treated with pectinase enzyme, the immune-activating efficacy is enhanced. In particular, it was confirmed that the active body of the polysaccharide derived from the mixed hot water extract of Angelica gigas, Cnidium officinale, and Paeonia lactiflora is the purified fraction HHE-I.

[0203] Accordingly, additionally, the natural killer cell activation effect of HHE-I, which exhibited excellent immune-activating efficacy, was confirmed.

[0204] 3-3: Confirmation of Natural Killer Cells' Tumor Cell Killing Effect

[0205] The tumor cell killing effect of natural killer cells was confirmed for the purified fraction of the active body HHE-I, which was identified through the previous results regarding macrophage and splenocyte activation.

[0206] Specifically, the induction was induced by administering 0.5, 5, and 50 mg / kg of the sample into the tail vein of BALB / c mice, and the purified fraction of HHE-I was intravenously injected twice, 3 days and 1 day before cell recovery. On the day of the experiment, mice were sacrificed to obtain cells from the spleen, which were then cultured with YAC-1 cells, a blood cancer cell line lacking MHC class I molecules, to compare and analyze the hemolytic effects of natural killer cells.

[0207] As a result, as can be seen in Figure 6, the concentration-dependent natural killer cell activation effect of the purified HHE-I fraction was confirmed. In particular, the experimental groups administered 5 and 50 mg / kg showed an effect approximately twice as strong as the negative control group administered only PBS. Here, the E / T ratio is the ratio of NK cells (effector cells, E) isolated from the spleen to Yac-1 tumor cells (target cells, T) known for NK cell sensitivity. Since NK cell activity may or may not be well-represented depending on the specific E / T ratio, the effect was confirmed by examining two different ratios.

[0208] Through this, it was confirmed that the purified fraction HHE-I not only induces the secretion of cytokines representing the immune activity of macrophages and splenocytes, but also enhances the cancer cell damage effect of natural killer cells, which are known to exhibit specific activity against tumor cells upon in vivo administration.

[0209]

[0210] Thus, it was confirmed that HHE-I, a purified fraction consisting of constituent sugars of specific types and amounts obtained by enzymatically treating the ethanol polysaccharides of the mixed extract of Angelica gigas, Cnidium officinale, and Paeonia lactiflora of the present invention, can be utilized as an immune enhancer including innate immunity, and in particular, possesses anticancer immune modulating ability against tumor cells.

[0211] From the foregoing description, those skilled in the art to which the present invention pertains will understand that the present invention may be implemented in other specific forms without altering its technical concept or essential features. In this regard, the embodiments described above should be understood as illustrative in all respects and not restrictive. The scope of the present invention should be interpreted as including all modifications or variations derived from the meaning and scope of the claims set forth below and their equivalents, rather than from the detailed description above.

Claims

1. An immune-enhancing composition comprising, as an active ingredient, a polysaccharide enzyme-treated product of a mixed extract of Angelica gigas, Cnidium officinale, and Paeonia lactiflora, or a purified fraction thereof.

2. An immune-enhancing composition according to claim 1, wherein the immune is innate immunity, adaptive immunity, or a combination thereof.

3. In Paragraph 1, An immune-enhancing composition, wherein the above mixed extract is extracted with a solvent selected from the group consisting of water, straight-chain or branched alcohols having 1 to 6 carbon atoms, organic solvents, and mixed solvents thereof.

4. In Paragraph 1, An immune-enhancing composition comprising the above mixed extract in a weight ratio of 1 to 5:1 to 5:1 to 5, comprising Angelica gigas, Cnidium officinale, and Paeonia lactiflora.

5. In Paragraph 1, An immune-enhancing composition wherein the above mixed extract is a mixed extract of Angelica gigas, Cnidium officinale, and Paeonia lactiflora.

6. In Paragraph 1, An immune-enhancing composition in which the above-mentioned polysaccharide is an ethanol polysaccharide.

7. In Paragraph 1, An immune-enhancing composition comprising the enzyme pectinase.

8. In Paragraph 1, An immune-enhancing composition wherein the above-mentioned polysaccharide enzyme-treated product or its purified fraction comprises neutral sugars, acidic sugars, proteins, KDO-like substances, and polyphenols.

9. In Paragraph 8, The above polysaccharide enzyme-treated product comprises 71.2 to 75.4% neutral sugar, 17.2 to 17.6% acidic sugar, 5.1 to 6.1% protein, 3.1 to 3.3% KDO-like substance, and 0.1 to 0.5% polyphenol, for an immune-enhancing composition.

10. In Paragraph 8, An immune-enhancing composition comprising the above purified fraction 72.7 to 75.5% neutral sugars, 18.8 to 20.8% acidic sugars, 1.7 to 2.5% protein, 3.4 to 3.6% KDO-like substances, and 0.3 to 0.5% polyphenols.

11. In Paragraph 1, An immune-enhancing composition wherein the above-mentioned polysaccharide enzyme-treated product or its purified fraction comprises one or more selected from the group consisting of mannose, rhamnose, arabinose, xylose, galactose, glucose, and galacturonic acid.

12. In Paragraph 11, An immune-enhancing composition comprising the above polysaccharide enzyme-treated product containing 0.7 to 3.1 mol% rhamnose, 6.3 to 18.7 mol% arabinose, 4.8 to 10.6 mol% galactose, 54 to 72.8 mol% glucose, and 13.6 to 15.4 mol% galacturonic acid.

13. In Paragraph 11, An immune-enhancing composition comprising the above purified fraction 5.2 to 7.8 mol% rhamnose, 34.8 to 35.2 mol% arabinose, 18.6 to 19.6 mol% galactose, 22 to 23.8 mol% glucose, and 16.5 to 16.7 mol% galacturonic acid.

14. In Paragraph 1, An immune-enhancing composition characterized in that the enzyme-treated product or its purified fraction activates macrophages, spleen cells, or natural killer cells.

15. In Paragraph 1, An immune-enhancing composition characterized in that the enzyme-treated product or its purified fraction increases immune-related cytokines.

16. In Paragraph 15, An immune-enhancing composition wherein the above cytokine is one or more selected from the group consisting of IL-2, IFN-γ, IL-4, IL-10, TNF-α, IL-12, and IL-6.

17. In Paragraph 1, The above composition is an immune-enhancing composition that further comprises a mixed extract of Angelica gigas, Cnidium officinale, and Paeonia lactiflora.

18. In Paragraph 17, An immune-enhancing composition wherein the above mixed extract and the polysaccharide enzyme-treated product or the purified fraction thereof are mixed in a weight ratio of 60-70:30-40.

19. In Paragraph 1, An immune-enhancing composition comprising the above-mentioned polysaccharide enzyme-treated product or its purified fraction in an amount of 0.001 to 99 weight% based on the total weight of the composition.

20. In Paragraph 1, The above composition is a food composition, an immune-enhancing composition.

21. In Paragraph 20, The above food composition is a composition for immune enhancement or anticancer immune modulation, which is manufactured as a powder, granule, tablet, capsule, syrup, or beverage.

22. A health supplement comprising the composition of any one of claims 1 to 21.

23. A method for activating cells in vitro, ex vivo, or both, comprising the step of treating cells with a polysaccharide enzyme-treated product of a mixed extract of Angelica gigas, Cnidium officinale, and Paeonia lactiflora, or a purified fraction thereof.

24. In Paragraph 23, A method for activating cells in vitro, ex vivo, or both, wherein the cells are macrophages, splenocytes, NK cells, or a combination thereof.

25. Immunostimulation use of a composition comprising, as an active ingredient, a polysaccharide enzyme-treated product of a mixed extract of Angelica gigas, Cnidium officinale, and Paeonia lactiflora or a purified fraction thereof.

26. A method for enhancing immunity comprising the step of administering to an individual a composition containing, as an active ingredient, a polysaccharide enzyme-treated product of a mixed extract of Angelica gigas, Cnidium officinale, and Paeonia lactiflora or a purified fraction thereof.

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