Marker for discriminating ginseng species and method for preparing ginseng extract with increased immune activity

Abstract

The present invention relates to a marker for discriminating ginseng species and a method for preparing a ginseng extract with increased immune activity. According to the present invention, it was confirmed that when unhusked ginseng and husked fine ginseng of white ginseng, which are ginseng, are mixed and subjected to multimodal extraction, the extraction yield is increased. It was confirmed that the white ginseng extract extracted by the multimodal extraction has an excellent effect of increasing T cell proliferation, and the content ratio in the white ginseng extract extracted by multimodal extraction exhibited an excellent proliferative effect on T cells. In addition, extracts from a 7:3 mixture of the torso and roots (fine roots) of each of Korean ginseng and American ginseng were analyzed using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF / MS) and multivariate statistical analysis method to identify 10 kinds of ginsenosides specifically included in Korean ginseng and 9 kinds of ginsenosides specifically included in American ginseng, whereby metabolite markers differentially included according to the origin of ginseng were discovered.

Description

Ginseng species identification marker and method for producing ginseng extract with enhanced immune activity

[0001] The present invention relates to a ginseng species identification marker and a method for producing a ginseng extract with increased immune activity.

[0002] Immunity is a self-defense system that exists in the body. It is a process in which the body recognizes various substances or organisms that invade from outside as foreign substances and eliminates and metabolizes them.

[0003] While it protects against damage from external stimuli or invasion by pathogenic microorganisms, it can also cause damage to one's own tissues, such as through inflammatory responses. Immune function suppression and immune function enhancement are categorized as actions that regulate changes in immune function, restoring it to normal or reducing the extent of changes. Alleviating hypersensitivity refers to suppressing undesirable, heightened immune responses, such as allergic reactions resulting from adverse reactions to external substances or reactions to self-antigens or altered self-antigens.

[0004] To overcome the problem of such immune response, various immune enhancers and treatments are being used, but they have side effects, and those sold mainly are taken for treatment rather than prevention. There is a need for an immune enhancer composition that has no side effects, can be taken for a long time, and is also suitable for prevention.

[0005] Ginseng is a plant whose root is mainly used for medicinal purposes in oriental medicine, and is called Korean ginseng (Panax ginseng C. A. Meyer). The components, chemical and pharmacological properties of ginseng have been revealed to contain various components such as saponin (ginsenoside), phenol compounds, polyacetylene, and polysaccharide. In particular, ginseng contains a large amount of components that are effective in regulating body functions, represented by saponin components. Saponin (ginsenoside) components are known to have beneficial effects in immune function, anticancer action, antioxidant activity, improving neurological disorders, and preventing and improving symptoms or treating adult diseases such as diabetes and hypertension. Many research results have been reported on the physiological activity of ginseng or red ginseng related to cancer, and ginsenoside-Rb2, ginsenoside-Rg3, Rh1, and Rh2 are known to have high activity. In particular, non-saponin compounds (polysaccharides and polypeptides) have recently been reported to contribute to the regulation of immune function and the suppression of tumors.

[0006] In addition, ginseng has been cultivated in large quantities in Korea and has been widely used as a traditional folk remedy for strengthening the constitution and immunity. Recently, the saponin component (ginsenoside) extracted from ginseng has been widely used as a material for medicinal purposes or as a pharmaceutical raw material in Korea, Japan, and China. The main component that determines the physiological activity of ginseng is saponin (ginsenoside), and the efficacy varies depending on the content and type of saponin. As an attempt to increase the physiological activity of ginseng, a method of fumigating ginseng by applying heat to it induces chemical changes and concentration of the saponin component, which makes it possible to strengthen the physiological activity as well as create new physiological activities.

[0007] Meanwhile, many herbal medicines produced and distributed domestically are being mixed and misused due to inaccurate origins or similar names. Furthermore, countries that use herbal medicines, such as Korea, China, and Japan, define the origins of medicinal plants differently. Korea, which relies on imports for over 80% of its products, is directly exposed to such mixing and misuse. Traditionally, methods for identifying and identifying mixed and misused herbal medicines and their origins have included sensory evaluations based on morphology, physicochemical tests, cytological tests, and physiological tests. However, because herbal medicines are natural products, ensuring consistent quality is difficult. This is because the composition of each individual plant varies significantly, and significant differences can occur depending on factors such as breeding method, harvest time, growing environment, and the part of the plant. Recently, two of the most controversial herbs in the herbal medicine distribution market due to mixing and misuse related to origin are Korean ginseng (Panax ginseng C. A. Meyer) and American ginseng (Panax quinquefolius).

[0008] The purpose of the present invention is to provide an immune-enhancing food composition containing a mixed extract of the trunk and fine roots of ginseng (Panax ginseng C. A. Meyer) as an active ingredient.

[0009] Another object of the present invention is to provide a pharmaceutical composition for enhancing immunity, which comprises a mixed extract of the trunk and fine roots of ginseng (Panax ginseng C. A. Meyer) as an active ingredient.

[0010] Another object of the present invention is to provide a method for producing a ginseng mixture by mixing the trunk and fine roots of ginseng (Panax ginseng C. A. Meyer);

[0011] A step of preparing a first extract using the above mixed ginseng mixture as a first solvent;

[0012] A step of filtering the above primary extract and extracting the filtered residue with a secondary solvent to prepare a secondary extract;

[0013] A step of filtering the secondary extract and extracting the filtered residue with a tertiary solvent to prepare a tertiary extract; and

[0014] A method for producing a ginseng multi-extract is provided, comprising the steps of mixing the first extract, the second extract, and the third extract and concentrating them.

[0015] Another object of the present invention is to provide a step for producing a ginseng mixture by mixing the trunk and fine roots of Korean ginseng or American ginseng;

[0016] A step of extracting the above ginseng mixture to prepare an extract; and

[0017] The present invention provides a method for determining the origin of Korean ginseng (Panax ginseng C. A. Meyer) and American ginseng (Panax quinquefolius), including a step of analyzing the above extract using high-pressure resolution liquid chromatography-quadratic time-of-flight mass spectrometry (UPLC-QTOF / MS) and multivariate statistical analysis.

[0018] Another object of the present invention is to provide a step of extracting fine roots of Korean ginseng or American ginseng to produce an extract; and

[0019] The present invention provides a method for determining the origin of Korean ginseng (Panax ginseng C. A. Meyer) and American ginseng (Panax quinquefolius), including a step of analyzing the above extract using high-pressure resolution liquid chromatography-quadratic time-of-flight mass spectrometry (UPLC-QTOF / MS) and multivariate statistical analysis.

[0020] Another object of the present invention is to provide an immune enhancement method comprising the step of administering to a subject a pharmaceutically effective amount of a mixed extract of the trunk and fine roots of ginseng (Panax ginseng C. A. Meyer).

[0021] In order to achieve the above purpose, the present invention provides an immune-enhancing food composition comprising a mixed extract of the trunk and fine roots of ginseng (Panax ginseng C. A. Meyer) as an effective ingredient.

[0022] In addition, the present invention provides a pharmaceutical composition for enhancing immunity, which comprises a mixed extract of the trunk and fine roots of ginseng (Panax ginseng C. A. Meyer) as an active ingredient.

[0023] In addition, the present invention comprises a step of preparing a ginseng mixture by mixing the trunk and fine roots of ginseng (Panax ginseng C. A. Meyer);

[0024] A step of preparing a first extract using the above mixed ginseng mixture as a first solvent;

[0025] A step of filtering the above primary extract and extracting the filtered residue with a secondary solvent to prepare a secondary extract;

[0026] A step of filtering the secondary extract and extracting the filtered residue with a tertiary solvent to prepare a tertiary extract; and

[0027] A method for producing a ginseng multi-extract is provided, comprising the steps of mixing the first extract, the second extract, and the third extract and concentrating them.

[0028] In addition, the present invention comprises a step of preparing a ginseng mixture by mixing the trunk and fine roots of Korean ginseng or American ginseng;

[0029] A step of extracting the above ginseng mixture to prepare an extract; and

[0030] A method for determining the origin of Korean ginseng (Panax ginseng C. A. Meyer) and American ginseng (Panax quinquefolius) is provided, comprising the step of analyzing the above extract using high-pressure resolution liquid chromatography-quadratic time-of-flight mass spectrometry (UPLC-QTOF / MS) and multivariate statistical analysis.

[0031] In addition, the present invention comprises a step of extracting fine roots of Korean ginseng or American ginseng to prepare an extract; and

[0032] A method for determining the origin of Korean ginseng (Panax ginseng C. A. Meyer) and American ginseng (Panax quinquefolius) is provided, comprising the step of analyzing the above extract using high-pressure resolution liquid chromatography-quadratic time-of-flight mass spectrometry (UPLC-QTOF / MS) and multivariate statistical analysis.

[0033] In addition, the present invention provides a method for enhancing immunity, comprising the step of administering to a subject a pharmaceutically effective amount of a mixed extract of the trunk and fine roots of ginseng (Panax ginseng C. A. Meyer).

[0034] The present invention confirmed that when the skin of white ginseng and white ginseng were mixed and multiple extractions were performed, the extraction yield increased, and it was confirmed that the white ginseng extract extracted through multiple extractions had an excellent effect of increasing T cell proliferation, and it was confirmed that the ginsenoside content ratio of the white ginseng extract extracted through multiple extractions had an excellent effect of increasing T cell proliferation. In addition, using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF / MS) and multivariate statistical analysis, 10 kinds of ginsenosides specifically contained in Korean ginseng and 9 kinds of ginsenosides specifically contained in American ginseng were identified, thereby identifying metabolite markers differentially contained depending on the origin of ginseng, which can be usefully utilized in related industries.

[0035] Figure 1 quantifies the yield of ginseng extract extracted using the multiple extraction method of the present invention.

[0036] Figure 2 shows the quantification of the T cell proliferation effect of ginseng extract extracted using the multiple extraction method of the present invention.

[0037] Figure 3 shows the quantification of the ginsenoside content of ginseng extract and American ginseng extract extracted using the multiple extraction method of the present invention.

[0038] Figure 4 is a diagram comparing the T cell proliferation effect according to the extraction method and ginsenoside content ratio.

[0039] Figure 5 is a diagram showing the analysis of ginsenosides in the Korean ginseng extract of the present invention using UPLC analysis.

[0040] Figure 6 is a diagram showing the analysis of ginsenosides in American ginseng extract using UPLC analysis.

[0041] Figure 7 shows the score plots of PC1 and PC2 obtained by analyzing the metabolites of Korean ginseng extract and American ginseng extract using principal component analysis (PCA) based on UPLC-QTOP / MS.

[0042] Figure 8 shows the metabolites of Korean ginseng extract and American ginseng extract analyzed using orthogonal partial least squares discriminant analysis (OPLS-DA) based on UPLC-QTOP / MS.

[0043] Figure 9 shows the score plot of the metabolites of Korean ginseng extract and American ginseng extract analyzed by OPLS-DA based on UPLC-QTOP / MS.

[0044] Figure 10 is a diagram showing the metabolites of Korean ginseng root extract and American ginseng root extract analyzed by OPLS-DA based on UPLC-QTOP / MS.

[0045] Figure 11 shows the score plot of the metabolites of Korean ginseng root extract and American ginseng root extract analyzed by OPLS-DA based on UPLC-QTOP / MS.

[0046] Hereinafter, embodiments of the present invention will be described in detail with reference to the attached drawings. In the following description, detailed descriptions of well-known technologies to those skilled in the art may be omitted. Furthermore, in describing the present invention, detailed descriptions of related known functions or configurations may be omitted if it is determined that such detailed descriptions may unnecessarily obscure the gist of the present invention. Furthermore, the terminology used in this specification is intended to appropriately express preferred embodiments of the present invention, and may vary depending on the intentions of the user or operator, or the customs of the field to which the present invention pertains.

[0047] Therefore, definitions of these terms should be based on the overall content of this specification. Throughout this specification, whenever a part is said to "include" a component, this does not exclude other components, but rather implies the inclusion of additional components, unless otherwise specifically stated.

[0048] The present invention provides an immune-enhancing food composition comprising a mixed extract of the trunk and fine roots of ginseng (Panax ginseng C. A. Meyer) as an active ingredient.

[0049] The term “prevention” used in the present invention means any act of suppressing symptoms of a specific disease or delaying its progression by administering the composition of the present invention.

[0050] The term “improvement” as used herein means any action that at least reduces a parameter related to the condition being treated, for example, the severity of a symptom.

[0051] The food composition of the present invention may contain, in addition to containing the effective ingredient of the present invention, various flavoring agents or natural carbohydrates as additional ingredients, like conventional food compositions.

[0052] Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose, etc.; disaccharides such as maltose, sucrose, etc.; and polysaccharides such as dextrin, cyclodextrin, etc., and common sugars, and sugar alcohols such as xylitol, sorbitol, erythritol, etc. As the above-mentioned flavoring agent, natural flavoring agent (thaumatin), stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agent (saccharin, aspartame, etc.) can be advantageously used. The food composition of the present invention can be formulated in the same manner as the pharmaceutical composition and used as a functional food or added to various foods. Foods to which the composition of the present invention can be added include, for example, beverages, meat, chocolate, foods, confectionery, pizza, ramen, other noodles, gum, candy, ice cream, alcoholic beverages, vitamin complexes, and health supplements.

[0053] In addition, the food composition may contain, in addition to the extract as an active ingredient, various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, the food composition of the present invention may contain fruit pulp for producing natural fruit juice, fruit juice drinks, and vegetable drinks.

[0054] The functional food composition of the present invention can be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. for the purpose of enhancing immunity. The term "health functional food composition" in the present invention refers to a food manufactured and processed using raw materials or ingredients having functionality useful to the human body according to Act No. 6727 on Health Functional Foods, and means to be consumed for the purpose of obtaining a useful effect for health purposes such as regulating nutrients for the structure and function of the human body or physiological effects. The health functional food of the present invention may include conventional food additives, and whether it is suitable as a food additive is determined by the specifications and standards for the relevant item according to the general provisions and general test methods of the Food Additives Codex approved by the Ministry of Food and Drug Safety, unless otherwise specified. Items listed in the "Food Additives Codex" include, for example, chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; Examples thereof include natural additives such as persimmon pigment, licorice extract, crystalline cellulose, high-molecular weight pigment, and guar gum; mixed preparations such as sodium L-glutamate preparations, noodle additive alkaline agents, preservative preparations, and tar color preparations. For example, a health functional food in tablet form can be prepared by mixing the active ingredient of the present invention with excipients, binders, disintegrants, and other additives, granulating the mixture using a conventional method, and then adding a lubricant, etc. to compress and molding, or directly compressing and molding the mixture. In addition, the health functional food in tablet form can contain a maturing agent, etc., if necessary. Among health functional foods in capsule form, hard capsules can be prepared by filling a mixture of the active ingredient of the present invention with additives such as excipients into a conventional hard capsule, and soft capsules can be prepared by filling a mixture of the active ingredient of the present invention with additives such as excipients into a capsule base such as gelatin. The above soft capsule may contain a plasticizer such as glycerin or sorbitol, a coloring agent, a preservative, etc., as needed.The ring-shaped health functional food can be prepared by molding a mixture of the active ingredient of the present invention with excipients, binders, disintegrants, etc. using a conventionally known method, and, if necessary, can be coated with white sugar or other coating agents, or the surface can be coated with a substance such as starch or talc. The granular health functional food can be manufactured into a granular form using a mixture of the active ingredient of the present invention with excipients, binders, disintegrants, etc. using a conventionally known method, and, if necessary, can contain a flavoring agent, a flavoring agent, etc.

[0055] According to one embodiment of the present invention, the trunk of the ginseng may be a skin-like ginseng, and the skin-like ginseng may be a dried ginseng raw material.

[0056] The "skin-dried ginseng" of the present invention is ginseng that has been dried in the sun or with hot air without peeling the skin, and is also called white ginseng. It refers to ginseng that has been processed to have a moisture content of 15% or less and is kept in its original state.

[0057] According to one embodiment of the present invention, the fine roots may be white ginseng.

[0058] The "white ginseng" of the present invention refers to ginseng by-product, fine roots, dried in their original form without any processing.

[0059] According to one embodiment of the present invention, the trunk and fine roots of the ginseng may be mixed in a weight ratio of 3 to 9:1 to 7, preferably 4 to 8:2 to 6, and more preferably 5:5, 6:4 and 7:3, but is not limited thereto.

[0060] According to one embodiment of the present invention, the mixed extract may be extracted with a solvent selected from the group consisting of water, C1 to C4 lower alcohol, lower alcohol aqueous solution, hexane, chloroform and acetone, and preferably water or lower alcohol aqueous solution, but is not limited thereto.

[0061] According to one embodiment of the present invention, the mixed extract may be obtained by filtering the residue after extraction, and the residue may further include additional extraction with a solvent, and may be obtained by mixing all of the additionally extracted filtrates.

[0062] According to one embodiment of the present invention, the immune enhancement may be to increase the proliferation of immune cells, and the immune cells may be T cells.

[0063] According to one embodiment of the present invention, the mixed extract may include ginsenosides Rg1, Rf and Rb, and the ginsenosides Rg1, Rf and Rb may be included in a weight ratio of 8 to 10:4 to 6:18.5 to 22.5, preferably 9:4:19 or 9:5:22, but is not limited thereto.

[0064] The weight ratio of Rg1, Rf and Rb of the present invention refers to the content of Rg1, Rf and Rb contained in the Korean ginseng multi-extract, and when the weight ratio (content ratio) of Rg1, Rf and Rb is 9:4:19 or 9:5:22, it may increase T cell proliferation compared to the reference value of the control group.

[0065] In addition, the present invention provides a pharmaceutical composition for enhancing immunity, which comprises a mixed extract of the trunk and fine roots of ginseng (Panax ginseng C. A. Meyer) as an active ingredient.

[0066] The term “treatment” used in the present invention means any act of improving or beneficially altering the symptoms of a specific disease by administering the composition of the present invention.

[0067] The pharmaceutical composition of the present invention may further include an adjuvant in addition to the active ingredient. Any adjuvant known in the art may be used without limitation. However, for example, Freund's complete adjuvant or incomplete adjuvant may be further included to enhance its effectiveness.

[0068] The pharmaceutical composition according to the present invention can be prepared in a form in which the active ingredient is mixed with a pharmaceutically acceptable carrier. Here, the pharmaceutically acceptable carrier includes carriers, excipients, and diluents commonly used in the pharmaceutical field. Pharmaceutically acceptable carriers that can be used in the pharmaceutical composition of the present invention include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0069] The pharmaceutical composition of the present invention can be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, or sterile injection solutions, each according to a conventional method.

[0070] When formulated, it can be prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrating agents, and surfactants that are commonly used. Solid preparations for oral administration include tablets, pills, powders, granules, and capsules, and such solid preparations can be prepared by mixing the active ingredient with at least one excipient, such as starch, calcium carbonate, sucrose, lactose, and gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups, and in addition to commonly used diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives can be included. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases include witepsol, Tween 61, cocoa butter, laurin, and glycerogelatin.

[0071] The pharmaceutical composition according to the present invention can be administered to a subject via various routes. All modes of administration are contemplated, including oral, intravenous, intramuscular, subcutaneous, and intraperitoneal injection.

[0072] The dosage of the pharmaceutical composition according to the present invention is selected in consideration of the age, weight, sex, physical condition, etc. of the subject. It is obvious that the concentration of the active ingredient included in the pharmaceutical composition can be selected in various ways depending on the subject, and it is preferably included in the pharmaceutical composition at a concentration of 0.01 to 5,000 μg / ml. If the concentration is less than 0.01 μg / ml, pharmaceutical activity may not be observed, and if it exceeds 5,000 μg / ml, it may be toxic to the human body.

[0073] In addition, the present invention comprises a step of preparing a ginseng mixture by mixing the trunk and fine roots of ginseng (Panax ginseng C. A. Meyer);

[0074] A step of preparing a first extract using the above mixed ginseng mixture as a first solvent;

[0075] A step of filtering the above primary extract and extracting the filtered residue with a secondary solvent to prepare a secondary extract;

[0076] A step of filtering the secondary extract and extracting the filtered residue with a tertiary solvent to prepare a tertiary extract; and

[0077] A method for producing a ginseng multi-extract is provided, comprising the steps of mixing the first extract, the second extract, and the third extract and concentrating them.

[0078] In addition, the present invention comprises a step of preparing a ginseng mixture by mixing the trunk and fine roots of Korean ginseng or American ginseng;

[0079] A step of extracting the above ginseng mixture to prepare an extract; and

[0080] A method for determining the origin of Korean ginseng (Panax ginseng C. A. Meyer) and American ginseng (Panax quinquefolius) is provided, comprising the step of analyzing the above extract using high-pressure resolution liquid chromatography-quadratic time-of-flight mass spectrometry (UPLC-QTOF / MS) and multivariate statistical analysis.

[0081] The term “high-pressure resolution liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF / MS)” used in the present invention refers to a high-pressure resolution liquid chromatography (UPLC; Ultra performance liquid chromatography) device and a quadrupole time-of-flight mass spectrometer (QTOF / MS; Quadrupole time-of-flight Mass Spectrometer) device connected.

[0082] The term “gradient elution” used in the present invention refers to a method of eluting (column liquid chromatography) or developing (thin layer chromatography) while continuously changing the elution region (composition) of the mobile phase. It is also called gradient elution, gradient elution, or gradation elution.

[0083] The term "multivariate statistical analysis" used in this invention refers to a statistical analysis method used to analyze the data characteristics of multiple correlated variables (multivariates) and to express their comprehensive characteristics. It analyzes two or more variables. Examples include factor analysis, principal component analysis, and crystallographic analysis.

[0084] “Principal component analysis (PCA),” a term used in the present invention, is a statistical technique that explains the changes among multiple variables in the form of a mixture of a small number of independent factors, called principal components.

[0085] The term “orthogonal partial least squares discriminant analysis (OPLS-DA)” used in the present invention is an analysis method that rotates the image to derive hidden variables that contribute to class distinction, and derives biomarkers, which are the most influential variables separated. The OPLS-DA analysis method is designed to handle deviations of X that are orthogonal to Y.

[0086] According to one embodiment of the present invention, in the high-resolution liquid chromatography-4x time-of-flight mass spectrometry, the liquid chromatography may be performed using 0.1% formic acid (v / v) in water and 0.1% formic acid (v / v) in acetonitrile as a mobile phase, and using a gradient elution method.

[0087] According to one embodiment of the present invention, in the high-resolution liquid chromatography-4x time-of-flight mass spectrometry, mass analysis may be performed using a negative ion electrospray method.

[0088] According to one embodiment of the present invention, the origin of the Korean ginseng and American ginseng can be determined by analyzing the main metabolites of the sample using high-pressure resolution liquid chromatography-quadratic time-of-flight mass spectrometry (UPLC-QTOF / MS) and performing a multivariate statistical analysis on the types and amounts of the metabolites.

[0089] According to one embodiment of the present invention, the multivariate statistical analysis method may be a multivariate statistical analysis method using data from principal component analysis (PCA) or orthogonal partial least squares discriminant analysis (OPLS-DA).

[0090] According to one embodiment of the present invention, the Korean ginseng may include a metabolite selected from the group consisting of ginsenoside Rf, Rb2, malonyl-Rb2, Rg1, malonyl-Rc, 20-O-glucoginsenoside Rf, Rg2, and Rc.

[0091] According to one embodiment of the present invention, the American ginseng may include a metabolite selected from the group consisting of ginsenoside Gypenoside XVII, Re, malonyl-Rb1, pseudo-ginsenoside F11, and Rb1.

[0092] In addition, the present invention comprises a step of extracting fine roots of Korean ginseng or American ginseng to prepare an extract; and

[0093] A method for determining the origin of Korean ginseng (Panax ginseng C. A. Meyer) and American ginseng (Panax quinquefolius) is provided, comprising the step of analyzing the above extract using high-pressure resolution liquid chromatography-quadratic time-of-flight mass spectrometry (UPLC-QTOF / MS) and multivariate statistical analysis.

[0094] According to one embodiment of the present invention, the Korean ginseng may include a metabolite selected from the group consisting of 20-O-glucoginsenoside Rf, Ra2, Ra1, Rf, Notoginsenoside R2, Rh1, and Rg1.

[0095] According to one embodiment of the present invention, the American ginseng may include a metabolite selected from the group consisting of vinaginsenoside R4, notoginsenoside R1, notoginsenoside Fe, Re, compound O, pseudo-ginsenoside F11 and F2.

[0096] In addition, the present invention provides a method for enhancing immunity, comprising the step of administering to a subject a pharmaceutically effective amount of a mixed extract of the trunk and fine roots of ginseng (Panax ginseng C. A. Meyer).

[0097] The method for enhancing immunity of the present invention comprises administering to a subject a therapeutically effective amount of a mixed extract of the trunk and fine roots of ginseng. The specific therapeutically effective amount for a specific subject is preferably applied differently depending on various factors such as the type and degree of response to be achieved, the specific composition including whether other preparations are used in some cases, the age, body weight, general health, sex, and diet of the subject, the time of administration, the route of administration, and the secretion rate of the composition, the treatment period, drugs used together or simultaneously with the specific composition, and similar factors well known in the medical field. The daily dosage is 0.0001 to 100 mg / kg, preferably 0.01 to 100 mg / kg, based on the amount of the pharmaceutical composition of the present invention, and can be administered 1 to 6 times a day. However, it is obvious to those skilled in the art that the dosage or administration of each active ingredient should be such that the content of each active ingredient is not excessively high and does not cause side effects. Therefore, the effective amount of a composition suitable for the purpose of the present invention is preferably determined in consideration of the aforementioned factors.

[0098] The above object is applicable to any mammal, which includes not only humans and primates, but also livestock such as cows, pigs, sheep, horses, dogs and cats.

[0099] The active ingredient of the present invention can be administered to mammals such as rats, mice, livestock, and humans via various routes. All modes of administration are conceivable, and for example, administration can be by oral, rectal, intravenous, intramuscular, subcutaneous, intrauterine, or intracerebroventricular injection.

[0100] Hereinafter, the present invention will be described in more detail with reference to examples. These examples are intended merely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

[0101] <Example 1> Preparation of ginseng extract using multiple extraction methods

[0102] The ginseng extract of the present invention, which can determine the origin of ginseng and has increased immune-boosting activity, was extracted using multiple extraction methods. Specifically, Korean ginseng (Panax ginseng C. A. Meyer) was mixed with the skin and white ginseng in weight ratios of 5:5, 6:4, and 7:3, respectively, to prepare a white ginseng mixture. Then, for the first extraction, 60% ethanol was added in a volume 8 times the weight of the white ginseng mixture, and reflux extraction was performed at 70-90°C for 8 hours. Thereafter, the extract was filtered, and the filtered residue was added to 30% ethanol in the same solvent mixing ratio as above, and a second extraction was performed at 70-90°C for 8 hours. Thereafter, for the third extraction, the extract was filtered, and the remaining residue was immersed in distilled water in the same mixing ratio as above, and reflux extraction was performed in hot water at 70-90°C for 8 hours. After that, all the obtained extracts (filtrate of the first extract, filtrate of the second extract, and filtrate of the third extract) were mixed, concentrated under reduced pressure, and then freeze-dried to prepare a ginseng multi-ext. As a control, an extract obtained by refluxing a white ginseng mixture with 50% ethanol (50% ext.) or 70% ethanol (70% ext.) under the same temperature and time conditions as above was used. The sample names, abbreviations, and extraction yields of the ginseng extracts of the present invention are shown in Table 1 and Figure 1 below.

[0103] In addition, in order to determine whether the origin of American ginseng (Panax quinquefolius) could be determined, American ginseng skin ginseng (white ginseng, Panax ginseng C. A. Meyer) and white ginseng were mixed in weight ratios of 5:5, 6:4 and 7:3, respectively, and prepared by reflux extraction with 70% ethanol.

[0104] [Table 1]

[0105]

[0106] As a result, as shown in Fig. 1, it was confirmed that the yield of the ginseng extract extracted by the multiple extraction method was significantly increased compared to the yield of the 50% or 70% alcohol extract.

[0107] <Example 2> Confirmation of the immune-boosting effect of white ginseng extract

[0108] The immune-enhancing effect of ginseng extracts extracted using the multiple extraction method of the present invention was confirmed. Specifically, mice were humanely sacrificed, and spleen tissues were obtained. The obtained spleen tissues were crushed with a sterile glass rod and isolated. The isolated cell suspension was then passed through a 200 mesh stainless steel sieve in RPMI 1640 medium (Hyclone, Logan, UT, USA) to isolate the cells. The isolated cell suspensions were then passed through 100 μm cell strainers (BD Falcon, Palo Alto, CA, USA), washed twice with culture medium, centrifuged at 3,000 rpm for 10 minutes, and treated with red blood cell lysing buffer (Hybri-MaxTM, SigmaAldrich) for 5 minutes to remove red blood cells. The spleen cells from which red blood cells were removed were dispersed again in RPMI 1640 culture medium, stained with trypan blue solution, and the cell number was measured using a hemocytometer. After that, 1X10 of the spleen cells from which red blood cells were removed were seeded in a 96-well plate. 6 After dividing cells / well, each group was treated with each extract of Example 1 at a concentration of 100 or 200 μg / ml. After culturing for 24 hours in an incubator at 37°C and 5% CO2, WST-1 ⓡ(Daeil Lap Science, Seoul, Korea) 10 μL of the solution was added to each well, reacted for 2 hours, and the absorbance was measured at 450 nm. An untreated control group (Control) was used as the control, and Con A (concanavalin A, 2 μg / mL), which induces T cell proliferation, was used as the positive control. The measured absorbance values ​​were used to quantify the spleen cell proliferation capacity using the following mathematical equation (1).

[0109] [Mathematical Formula 1]

[0110]

[0111] As a result, as shown in Fig. 2, compared to the Control group, T cell proliferation increased in the 50% ext. group and the 70% ext. group, but compared to the positive control group, Con A group, T cell proliferation was significantly lower. However, the Multi-ext. group using the multiple extraction method showed a concentration-dependent increase in T cell proliferation ability, and in particular, when treated with a concentration of 200 μg / ml, there was no significant difference from the positive control group, Con A group, confirming that the T cell proliferation ability was excellent.

[0112] <Example 3> Comparison of differences in ginsenoside content and immune-boosting effects by ginseng origin

[0113] <3-1> Comparison of differences in ginsenoside content by country of origin

[0114] It was confirmed whether the index components of white ginseng extracts extracted using the multiple extraction method of the present invention differ from those of American ginseng depending on the extraction method. Specifically, the contents of ginsenosides Rg1, Rf, and Rb1 contained in the Korean ginseng multiple extracts (7:3 and 6:4 mult-extracts) of Example 1, the 70% Korean ginseng mixture ethanol extracts (7:3 and 6:4), and the 70% American ginseng mixture ethanol extracts (7:3 and 6:4) were analyzed by high performance liquid chromatography (HPLC). The results are shown in Table 2 and Fig. 3 below.

[0115] [Table 2]

[0116]

[0117] As a result, as shown in Fig. 3, Rf was detected in all Korean ginsengs compared to American ginseng.

[0118] <3-2> Confirmation of immune enhancement effect according to ginsenoside content ratio by origin

[0119] The immune enhancement effect according to the content ratio of the index components of the white ginseng extract extracted by the multiple extraction method of the present invention was confirmed. Specifically, T cell proliferation in spleen cells was evaluated in the same manner as in Example 2, and since the content ratios of the index components Rg1, Rf, and Rb1 in the extracts in Example 3-1 were all different, the immune cell proliferation ability according to each ratio was evaluated. The contents of Rg1, Rf, and Rb1 in each extract are shown in Table 3 below, and the immune enhancement effect at a concentration of 50 μg / ml of each extract was compared.

[0120] [Table 3]

[0121]

[0122] As a result, as shown in Fig. 4, compared to the positive control group, Con A group, the T cell proliferation ability was significantly lower in the American ginseng extract group with a content ratio of Rg1 and Rb1 of 1:22 and 1:23, but compared to the American ginseng extract group, the T cell proliferation ability was significantly increased in the Korean ginseng extract group. In particular, in the Korean ginseng multiple extract group with a content ratio of Rg1, Rf, and Rb1 of 9:4:19 and 9:5:22, there was no significant difference in T cell proliferation ability compared to the positive control group, confirming that the content ratio of the above indicator components was excellent in T cell proliferation ability.

[0123] <Example 4> Preparation for identification of metabolite markers for determining the origin of Korean ginseng and American ginseng

[0124] <4-1> Preparation of samples for determining the origin of Korean ginseng and American ginseng

[0125] In order to identify metabolite markers that can determine the origin of Korean ginseng and American ginseng in the extracts of the mixture of Korean ginseng and American ginseng of the present invention, samples for UPLC-QTOF / MS were prepared. Specifically, a mixture of Korean ginseng trunks and roots in a 7:3 ratio and a mixture of American ginseng prepared in the same manner were ground in a mixer, and 50 mg were used for metabolite extraction. 10 ml of 70% methanol was added to each ground substance, and ultrasonic extraction was performed at room temperature for 60 minutes. Each extract was then filtered to remove impurities, and the final analytical concentration was standardized to 5,000 ppm, and used for high-pressure resolution liquid chromatography-quaternary time-of-flight mass spectrometry (UPLC-QTOF / MS).

[0126] In addition, using the same method as above, the fine roots (roots) of Korean ginseng and American ginseng were extracted and used for UPLC-QTOF / MS analysis.

[0127] <4-2> UPLC analysis

[0128] UPLC analysis was performed using Waters ACQUITY UPLC TM The chromatography was performed using a system (Waters Corp., MA, USA). A Thermo Hypersil Gold (2.1 x 100 mm, 1.9 um) column was used, and water (A) containing 0.1% formic acid and acetonitrile (B) containing 0.1% formic acid were used as mobile phases. The column oven was maintained at 40°C, and the mobile phase consisted of solvent A (0.1% formic acid in water, v / v) and solvent B (0.1% formic acid in acetonitrile, v / v). The optimal LC elution conditions were applied as follows: 0-0.5 min, 15% B; 0.5-1 min, 15-20% B; 1-6 min, 20-20% B; 6-13 min, 20-30% B; 13-23 min, 30-35% B; 23-24 min, 35-38% B; 24-27 min, 38-60% B; 27-31 min, 60-90% B, and the flow rate was 0.5 mL / min. 2 μL aliquots were injected onto the column using an autosampler.

[0129] <4-3> Establishment of Q-TOF / MS conditions

[0130] Q-TOF / MS analysis was performed using a Q-TOF Micro mass detector (Waters, Manchester, UK) in negative and positive modes to establish optimal conditions for metabolite analysis of Korean ginseng and American ginseng, respectively. Comparison results showed that various metabolites were effectively analyzed in the negative mode, and the analytical conditions were as follows: capillary voltage 2.3 kV, cone voltage 40 V, low energy 6 V, high energy 20-45 V, desolvation temperature 550°C, source temperature 120°C, cone gas flow 30 L / h, and desolvation gas flow 800 L / h. Precursor ion information was collected between 100 and 2,000 m / z.

[0131] <Example 5> Identification of metabolite markers for determining the origin of Korean and American ginseng.

[0132] <5-1> UPLC-QTOF / MS results

[0133] UPLC-QTOF / MS spectra were recorded three times for each of 12 samples prepared by pooling 3 to 5 samples of Korean ginseng and American ginseng. UPLC combined with QTOF / MS, which enables accurate mass measurements of various metabolites, was performed for 30 minutes. Under optimal analytical conditions, the metabolites of Korean and American ginseng showed excellent resolution. In addition, isocratic elution did not show clear separation due to the similar polarity and structure of the metabolites, but gradient elution achieved better separation. The electrospray ionization device was tested in positive and negative ion modes, but the negative ion mode was more sensitive to all components. Each ginsenoside peak could be identified in the total ion chromatogram by matching the molecular weight or fragment ion in the mass spectrum.

[0134] The results of analyzing Korean ginseng and American ginseng, respectively, under the UPLC and Q-TOF / MS conditions described above are shown in Figures 5 and 6. Figures 5 and 6 show UPLC and Q-TOF / MS chromatograms of one representative sample of Korean ginseng and American ginseng, respectively. Each chromatogram shows the time from 0.1 to 30.0 minutes. The y-axis of the chromatograms (BPI counters) represents the amount of ion values ​​of individual compounds. These chromatogram data were used in the multivariate statistical analysis performed subsequently.

[0135] <5-2> Multivariate statistical analysis based on UPLC-QTOF / MS data - Principal component analysis (PCA)

[0136] Metabolite profiles obtained from UPLC-QTOF / MS of Korean and American ginseng were used for PCA analysis, which statistically analyzes the two sample groups without any additional information. It was confirmed that the left side of the y-axis was accurately identified as Korean ginseng, and the right side as American ginseng (Fig. 7).

[0137] <5-3> Multivariate statistical analysis based on UPLC-QTOF / MS data - S-Plot based on orthogonal partial least squares discriminant analysis (OPLS-DA)

[0138] The metabolite profiles obtained from UPLC-QTOF / MS of Korean and American ginseng were provided to OPLS-DA analysis, which maximizes the two sample groups and performs statistical processing. OPLS-DA was performed using SIMCA P15 (Umetrics, Umea, Sweden). Based on the OPLS-DA analysis, an S-plot was created. The S-plot provides information on which signals determine the group distinction in the OPLS-DA analysis method, and shows the S-plot for the total mass value (m / z).

[0139] As a result, as shown in Figure 8, it was confirmed that the OPLS-DA analysis accurately identified the left side of the y-axis as Korean ginseng and the right side as American ginseng.

[0140] In addition, as shown in Fig. 9, the S-Plot analysis results based on OPLS-DA analysis confirmed that the representative mass values ​​shown below the x-axis represent markers that exhibit characteristics in Korean ginseng samples, and the representative mass values ​​shown above represent markers that exhibit characteristics in American ginseng samples. This can be judged to be the compound that contributes the most to distinguishing OPLS-DA.

[0141] <5-4> OPLS-DA and S-Plot Analysis Based on UPLC-QTOF / MS Data of Root Extracts

[0142] Based on the UPLC-QTOF / MS data of the root extracts of Korean ginseng and American ginseng, OPLS-DA and S-Plot analyses were performed in the same manner as in Example 5-3.

[0143] As a result, as shown in Fig. 10, it was confirmed that the left side of the OPLS-DA y-axis was accurately identified as Korean ginseng and the right side as American ginseng.

[0144] In addition, based on the OPLS-DA analysis, the S-Plot analysis results confirmed that the representative mass values ​​shown below the x-axis represent markers that exhibit characteristics in the Korean ginseng sample, and the representative mass values ​​shown above represent markers that exhibit characteristics in the American ginseng sample (Fig. 11).

[0145] Therefore, the present invention confirmed that when the skin of white ginseng and white ginseng were mixed and multiple extractions were performed, the extraction yield increased, and the white ginseng extract extracted through multiple extractions was confirmed to have an excellent effect of increasing T cell proliferation, and the ginsenoside content ratio of the white ginseng extract extracted through multiple extractions was confirmed to have an excellent effect of increasing T cell proliferation. In addition, using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF / MS) and multivariate statistical analysis, 10 kinds of ginsenosides specifically contained in Korean ginseng and 9 kinds of ginsenosides specifically contained in American ginseng were identified, and metabolite markers differentially contained depending on the origin of ginseng were identified.

Claims

1. A food composition for enhancing immunity, comprising a mixed extract of the trunk and fine roots of ginseng (Panax ginseng C. A. Meyer) as an active ingredient.

2. In paragraph 1, A food composition wherein the body of the above ginseng is a skin-like ginseng.

3. In paragraph 2, The above-mentioned skin-dried ginseng is a food composition made by drying raw ginseng.

4. In paragraph 1, A food composition wherein the above-mentioned root is white ginseng.

5. In paragraph 1, A food composition wherein the trunk and fine roots of the above ginseng are mixed in a weight ratio of 3 to 9: 1 to 7.

6. In paragraph 1, A food composition wherein the above mixed extract is extracted with a solvent selected from the group consisting of water, lower alcohols of C1 to C4, lower alcohol aqueous solutions, hexane, chloroform and acetone.

7. In paragraph 6, A food composition wherein the above mixed extract is obtained by filtering the residue after extraction.

8. In paragraph 7, A food composition further comprising the residue being further extracted with a solvent.

9. In paragraph 1, The above-mentioned immune enhancement food composition increases the proliferation of immune cells.

10. In paragraph 9, A food composition wherein the above immune cells are T cells.

11. In paragraph 1, A food composition wherein the above mixed extract comprises ginsenosides Rg1, Rf and Rb.

12. In paragraph 11, A food composition wherein the above ginsenosides Rg1, Rf and Rb are included in a weight ratio of 8 to 10: 4 to 6: 18.5 to 22.

5.

13. In paragraph 12, A food composition that increases T cell proliferation compared to the reference value of the control group when the weight ratio of Rg1, Rf and Rb is 8 to 10:4 to 6:18.5 to 22.

5.

14. A pharmaceutical composition for enhancing immunity, comprising a mixed extract of the trunk and fine roots of ginseng (Panax ginseng C. A. Meyer) as an active ingredient.

15. A step of preparing a ginseng mixture by mixing the trunk and fine roots of ginseng (Panax ginseng C. A. Meyer); A step of preparing a first extract using the above mixed ginseng mixture as a first solvent; A step of filtering the above primary extract and extracting the filtered residue with a secondary solvent to prepare a secondary extract; A step of filtering the secondary extract and extracting the filtered residue with a tertiary solvent to prepare a tertiary extract; and A method for producing a ginseng multi-extract, comprising the steps of mixing the first extract, the second extract, and the third extract and concentrating them.

16. In paragraph 15, The above ginseng mixture is a method in which the ginseng trunk and fine roots are mixed in a weight ratio of 3 to 9: 1 to 7.

17. In paragraph 15, A method wherein the above ginseng multiple extract comprises ginsenosides Rg1, Rf and Rb.

18. In paragraph 17, A method wherein the above ginsenosides Rg1, Rf and Rb are included in a weight ratio of 8 to 10: 4 to 6: 18.5 to 22.

5.

19. A step of preparing a ginseng mixture by mixing the trunk and fine roots of Korean ginseng or American ginseng; A step of extracting the above ginseng mixture to prepare an extract; and A method for determining the origin of Korean ginseng (Panax ginseng C. A. Meyer) and American ginseng (Panax quinquefolius), comprising the step of analyzing the above extract using high-pressure resolution liquid chromatography-quadratic time-of-flight mass spectrometry (UPLC-QTOF / MS) and multivariate statistical analysis.

20. In paragraph 19, A method wherein the above body and fine roots are mixed in a weight ratio of 3 to 9: 1 to 7.

21. In paragraph 19, A method in which the liquid chromatography in the high-resolution liquid chromatography-4x time-of-flight mass spectrometry method uses 0.1% formic acid (v / v) in water and 0.1% formic acid (v / v) in acetonitrile as the mobile phase, and is performed using a gradient elution method.

22. In paragraph 19, A method wherein mass analysis in the high-resolution liquid chromatography-quadratic time-of-flight mass spectrometry is performed using a negative ion electrospray method.

23. In paragraph 19, The method for determining the origin of the above Korean ginseng and American ginseng is to analyze the main metabolites of the sample by high-pressure resolution liquid chromatography-quadratic time-of-flight mass spectrometry (UPLC-QTOF / MS) and to perform multivariate statistical analysis on the types and amounts of the metabolites.

24. In paragraph 19, The above multivariate statistical analysis method is a multivariate statistical analysis method using data from principal component analysis (PCA) or orthogonal partial least squares discriminant analysis (OPLS-DA).

25. In paragraph 19, A method wherein the above Korean ginseng comprises a metabolite selected from the group consisting of ginsenoside Rf, Rb2, malonyl-Rb2, Rg1, malonyl-Rc, 20-O-glucoginsenoside Rf, Rg2 and Rc.

26. In paragraph 19, A method wherein the American ginseng comprises a metabolite selected from the group consisting of ginsenoside Gypenoside XVII, Re, malonyl-Rb1, pseudo-ginsenoside F11 and Rb1.

27. A step of extracting fine roots of Korean ginseng or American ginseng to produce an extract; and A method for determining the origin of Korean ginseng (Panax ginseng C. A. Meyer) and American ginseng (Panax quinquefolius), comprising the step of analyzing the above extract using high-pressure resolution liquid chromatography-quadratic time-of-flight mass spectrometry (UPLC-QTOF / MS) and multivariate statistical analysis.

28. In paragraph 27, A method wherein the above Korean ginseng comprises a metabolite selected from the group consisting of 20-O-glucoginsenoside Rf, Ra2, Ra1, Rf, Notoginsenoside R2, Rh1 and Rg1.

29. In paragraph 27, A method wherein the American ginseng comprises a metabolite selected from the group consisting of vinaginsenoside R4, notoginsenoside R1, notoginsenoside Fe, Re, compound O, pseudo-ginsenoside F11 and F2.

30. A method for enhancing immunity, comprising the step of administering to a subject a pharmaceutically effective amount of a mixed extract of the trunk and fine roots of ginseng (Panax ginseng C. A. Meyer).

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