KR102140702B1 - Galactouronoglucooligosaccharide Derived from Ginseng cake and Use Thereof - Google Patents

Abstract

The present invention relates to a method for producing galacturonoglucooligosaccharides (GuG-OS) derived from ginseng cake (ginseng cake) and uses thereof, and the gacto-urono glucooligosaccharide derived from ginseng cake of the present invention is ginseng Galactouronoglucopolysaccharide (Galacturonoglucopolysaccharide; GuG-PS) isolated from foil was prepared by enzymatic decomposition, and the low-molecular-galactouronoglucooligosaccharide prepared by the above method increases the proliferation rate of macrophages and increases TNF-α and It increases the production rate of IL-6, has a remarkable apoptosis effect on cancer cells, and is not toxic to normal cells, so it can be usefully used in drugs for immune enhancement or anticancer drugs and health functional foods.

Description

Galactouronoglucooligosaccharides derived from ginseng bak and its use{Galactouronoglucooligosaccharide Derived from Ginseng cake and Use Thereof}

The present invention relates to a galacturonoglucooligosaccharide (GuG-OS) derived from ginseng cake (ginseng cake) and uses thereof, and more specifically, a galactouronoglucan type polysaccharide separated from ginseng foil (Galacturonoglucopolysaccharide; GuG-PS) is a low molecular weight galacto-uro gluco-oligosaccharide prepared by enzymatic decomposition and relates to a composition for enhancing or anti-cancer comprising the same as an active ingredient.

Immunity refers to a mechanism for physiologically recognizing and removing foreign and endogenous foreign substances in humans and animals, and maintaining homeostasis. It refers to a series of biodefense reactions that occur in order to exclude a substance other than its own component from breaking the homeostasis of the body or threatening it, and non-specific immunity (congenital immunity) involving macrophages and T lymphocytes and B Lymphocytes can be classified as related specific immunity (acquired immunity) (Seo SY et al., Korean J Oriental Physiol Pathol, 23:1385-1391, 2009).

Macrophages are distributed in all tissues of the body, and primarily have infectious pathogens such as bacteria and viruses, as well as protective ability to cause phagocytosis and remove aging normal cells and cancer cells. Antigen-presenting action and interleukin It secretes various cytokines and bioactive substances such as TNF-α and acts as an important mediator to maximize the immune response. When macrophages are activated, they secrete cytokines such as NO and TNF-α, which are known as representative substances showing toxicity to cancer cells (Hibbs JB Jr et al., Biochem Biophys Res Commun, 157:87-94, 1998 ; Nathan CF, J Clin Invest, 79:319-326, 1987).

Immunodeficiency disease is deeply involved in clinical immunological refractory diseases, cancer, diabetes, and male infertility. In addition, AIDS, various pathogenic viral disease chance infections, parasitic infections, etc. are all involved. It can be said to be a disease related to immune failure.

Recently, in the case of a cancer patient, since immunity falls on its own, it may cause complications, and the therapeutic effect may be reduced, so strengthening immunity is very important. Macrophages (Macrophages) are known to play an essential role in biodefense according to a previously published paper (Shin JY et al., Immunopharmacology and Immunotoxicology, 24;469-482, 2002).Activation of macrophages inhibits tumors It was reported to be related to (Coha ZA, J Immunol, 121;813-816, 1978).

In the case of cancer, according to the World Health Organization, the spread of Western eating habits and current smoking trends predict that cancer diagnosis will increase by more than 50% to 15 million by 2020. In addition, according to the statistical data of the cause of death in 2013 by the Korea National Statistical Office, the number of deaths from lung cancer increased by 7.8% in 2013 compared to 2003. For this reason, as various immunity enhancers, anticancer agents and cancer treatment methods for strengthening immunity and suppressing cancer are continuously developed, treatment for cancer or prolongation of life is expected.

Immune function enhancers are drugs that are mainly used for congenital and acquired immunodeficiency syndrome, and are in urgent need, especially as part of drug therapy for tumors or acquired immunodeficiency syndrome (AIDS). Representative immunological enhancers currently in use include immunoglobulins and inteferons. Among them, a concentrated and purified preparation of one IgG of human immunoglobulin is used for the prevention and treatment of measles, chickenpox, hepatitis B, mumps, etc., but a large amount due to anaphylaxis positive reaction due to pain at the injection site and blood pressure drop The disadvantage is that administration is dangerous. In addition, in the case of interferon, it was found as an antiviral factor, but after that, cell proliferation inhibitory action, immune function regulation action, etc. were confirmed, and it is used as an antiviral agent and an anti-tumor agent, but β-type is fever, boredom, anorexia, injection Side effects such as topical pain and alopecia are observed, and the α-type exhibits side effects such as decreased white blood cells due to temporary suppression of bone marrow function and elevated GOT.

In the case of anticancer drugs, many parts of drug resistance and selectivity with normal cells are still unresolved, and the biggest problem with anticancer drugs used in clinical trials is that the inability to selectively distinguish cancer cells from normal cells is the biggest problem. have. In order to solve this problem, intensive studies on the characteristics of cancer at the molecular level, such as cell cancer, drug resistance or metastasis, have been conducted in recent years.

Particularly, in recent years, interest in research to enhance the immune function as a natural substance has been highlighted, and in particular, an immune enhancer derived from a natural substance is expected to enhance the immune response or restore the reduced immune function. (Kim HS and Kang JS, J Korean Soc Food Sci Nutr, 37:109-116, 2008).

Among them, ginseng has been recognized as a very good medicine for a long time, mainly in Korea, China, and Japan, and has been highly regarded for its effectiveness and has been used as a medicine in many countries (Hakchangsa, Modern Pharmacy, Research Institute of Pharmacy, 251-297 , 1997). The components of saponin that show the main efficacy of ginseng are in the roots, stems, and leaves of ginseng, and are composed of a total of 30 types of ginsenosides, which are anti-cancer, anti-aging, adult disease prevention, liver protection, anti-inflammatory, It is known to have various health promoting effects such as nerve protection.

However, in the case of ginseng foil, which is the residue left after extracting the soluble ginseng component, it is mostly composed of polysaccharides, and although various physiological activities are expected, industrial utilization such as simply feed or disposal is very insufficient.

Accordingly, the present inventors have tried hard to select a substance that does not have side effects and has an immune-enhancing and anti-cancer effect by using a substance derived from nature, and the result is a ginseng gourd extract galacturonoglucopolysaccharide (Galacturonoglucopolysaccharide, GuG) -PS) Galacturonoglucooligosaccharide (GuG-OS) prepared by treating enzymes increases the proliferation rate of macrophages, increases the production rate of TNF-α and IL-6, and inhibits cell viability for lung cancer cells It confirmed that it had, and completed this invention.

The present invention has been devised to solve the above problems, and the first object of the present invention is to provide a method for producing galacturonoglucooligosaccharides (GaG-OS) derived from ginseng leaves.

The second problem to be solved of the present invention is to provide a composition for enhancing immune function, or preventing or treating cancer, comprising the galacto-uronoglucooligosaccharide.

In order to solve the first problem of the present invention described above, the present invention, (a) galacto-uro-no-glucan polysaccharide (Galacturonoglucopolysaccharide) obtained from ginseng cake (ginseng cake) is enzymatically decomposed, followed by centrifugation to obtain a supernatant step; (b) filtering the obtained supernatant to obtain a polysaccharide having a molecular weight of 1 to 10 kDa; And (c) dialysis of the obtained polysaccharide to obtain galacturonoglucooligosaccharides (Galacturonoglucooligosaccharide).

According to a preferred embodiment of the present invention, the galacto-uronoglucan polysaccharide of step (a) is mixed by adding a solvent to ginseng foil, followed by centrifugation to obtain a supernatant; (Ii) adding a solvent to the obtained supernatant and mixing, followed by centrifugation to obtain a pellet; (Iii) treating the solvent with the obtained precipitate again, pulverizing, and then dialysis to obtain a polysaccharide having a molecular weight of 4,000,000 Da to 7,000,000 Da;

According to another preferred embodiment of the present invention, the solvent of step (i) to (i) is selected from the group consisting of water, ethanol, methanol, butanol, ethyl acetate, ethyl ether, chloroform, hexane and mixtures thereof. It may be one or more solvents.

According to another preferred embodiment of the present invention, the enzymatic decomposition of the step (a) is (p) adding pectinase to the galacto-uroglucan polysaccharide, followed by 45 to 55 to 30 to 90 minutes. While stirring; And (ii) inactivating the enzyme by heating at 90 to 110 for 10 to 20 minutes.

According to another preferred embodiment of the present invention, the molecular weight of the galacto-uronoglucooligosaccharide obtained in step (c) may be 1800 to 2600 Da.

According to another preferred embodiment of the present invention, the content of galacturonic acid obtained in step (c) is 2 to 3 times that of galacturonic acid compared to glucuronic acid. It can be high.

The present invention also includes galacto-uronoglucooligosaccharides prepared by the above method.

In order to solve the second problem of the present invention, the present invention includes a pharmaceutical composition for strengthening immune function, or preventing or treating cancer, comprising ginseng gourd-derived galacto-uro glucooligosaccharide as an active ingredient.

The present invention also includes a composition for health functional food for strengthening immune function, or preventing or treating cancer, containing ginseng pak derived galacto-uro glucooligosaccharide as an active ingredient.

According to a preferred embodiment of the present invention, the galacto-uro-no-gluco-oligosaccharide may be included in an amount of 0.01 to 10 parts by weight based on 100 parts by weight of the total composition, and galacto-uro-no-glucose prepared by the method for producing the galacto-uro-no-gluco-oligosaccharide. Glucooligosaccharides.

According to another preferred embodiment of the present invention, the galacto-uronoglucooligosaccharide improves the survival rate of macrophages, or tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL -6; interleukin-6) can enhance immune function by increasing the expression of any one or more cytokines selected from the group consisting of interleukin-6).

According to another preferred embodiment of the present invention, the cancer may be at least one selected from the group consisting of lung cancer, stomach cancer, colorectal cancer, breast cancer, cervical cancer, and liver cancer, and the galacto-uronoglucooligosaccharide is a cancer cell. Can induce apoptosis.

The present invention ginseng cake (ginseng cake)-derived galacto-uronoglucooligosaccharides (Galacturonoglucooligosaccharides; GuG-OS) was prepared by enzymatic digestion of galacturonoglucopolysaccharides (Galacturonoglucopolysaccharides; GuG-PS) isolated from ginseng foil. As such, the galacto-uro-no-glucooligosaccharide increases the proliferation rate of macrophages, increases the production rate of TNF-α and IL-6, has a remarkable apoptosis effect on cancer cells, and is not toxic to normal cells, thereby enhancing immunity or anticancer It can be usefully used in medicines and health functional foods.

Figure 1a is a schematic diagram showing the manufacturing process of galacturonoglucopolysaccharide (Galacturonoglucopolysaccharide; GuG-PS) isolated from ginseng leaves, Figure 1b is a galactosyl through the enzymatic decomposition of polysaccharides of galacturouronoglucan type It is a schematic diagram showing the manufacturing process of nogluco-oligosaccharide (Galacturonoglucooligosaccharide; GuG-OS).
Figure 2a is the generated GuG-PS, Figure 2b is a result of analyzing GuG-OS by HPLC (High Performance Liquid Chromatography).
3 is a result of analyzing the generated neutrophils of GuG-PS and GuG-OS using HPEAC-PAD (High Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection).
4 is a result of analyzing the generated GuG-PS, GuG-OS uronic acid using HPEAC-PAD (High Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection, Dionex, USA).
5 is a result of analyzing the change in cell viability after treating GuG-OS to RAW 264.7 cells.
6 is a result of analyzing the change in the expression level of TNF-α, one of cytokines, after treating GuG-OS with RAW 264.7 cells.
7 is a result of analyzing the change in expression level of IL-6, one of the cytokine, after treating GuG-OS with RAW 264.7 cells.
Figure 8 is the result of confirming the morphological changes of cells when treated with GuG-PS and GuG-OS A549 cells, a lung cancer cell line.
9 is a result of analyzing the change in cell viability through MTT assay after treating GuG-PS and GuG-OS to A549 cells, a lung cancer cell line.

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

As described above, drugs and anti-cancer agents used as immunity-boosting agents have a problem in that various side effects occur, and efforts have been made to continuously select immune-enhancing or anti-cancer substances derived from natural substances to minimize side effects.

The present invention seeks to solve the above-mentioned problems by providing a method for producing galacturonoglucooligosaccharides (GuG-OS) derived from ginseng cake (ginseng cake) and uses thereof. Through this, it provides a galacto-uro-no-gluco-oligosaccharide prepared by treating an enzyme with galacto-uronoglucan-type polysaccharide (Galacturonoglucopolysaccharide, GuG-PS) which is a ginseng extract, increases the proliferation rate of the macrophages and increases TNF-α and It increases the production rate of IL-6, has a remarkable apoptosis effect on cancer cells, and thus can provide a composition for enhancing or anti-cancer.

Therefore, the present invention is (a) enzymatic decomposition of galactouronoglucopolysaccharide (Galacturonoglucopolysaccharide) obtained from ginseng cake (ginseng cake), followed by centrifugation to obtain a supernatant; (b) filtering the obtained supernatant to obtain a polysaccharide having a molecular weight of 1 to 10 kDa; And (c) dialysis the obtained polysaccharide to obtain galacto-uronogluco-oligosaccharides; a method for producing galacto-uro-gluco-oligosaccharides (Galacturonoglucooligosaccharide).

When explaining in detail the method of preparing the galacto-uro-no-gluco-oligosaccharide (Galacturonoglucooligosaccharide; hereinafter referred to as'GuG-OS'),

First, step (a) is a step of enzymatic decomposition of galactouronoglucopolysaccharide (Galacturonoglucopolysaccharide; hereinafter referred to as'GuG-PS') obtained from ginseng foil, followed by centrifugation to obtain a supernatant.

The'bak (cake, cake)' refers to the fork, debris,'ginseng foil (人蔘粕)' refers to the remaining residue after extracting the soluble ginseng component, water, ethanol, methanol, butanol, ethyl acetate , Ethyl ether, chloroform, hexane and mixtures thereof, and may be residues extracted from any one or more solvents selected from the group.

Figure 1a is a schematic diagram showing the production process of a galacto-uronoglucan type polysaccharide separated from ginseng foil, and more specifically, the galacto-uronoglucan polysaccharide of step (a) is (i) a solvent in ginseng foil. Adding and mixing, followed by centrifugation to obtain a supernatant; (Ii) adding a solvent to the obtained supernatant and mixing, followed by centrifugation to obtain a pellet; (Iii) treating the solvent with the obtained precipitate again, pulverizing, and then dialysis to obtain a polysaccharide having a molecular weight of 4,000,000 Da to 7,000,000 Da;

The solvent of step (i) to (i) may be any one or more solvents selected from the group consisting of water, ethanol, methanol, butanol, ethyl acetate, ethyl ether, chloroform, hexane and mixtures thereof. Preferably, in step (i), distilled water was added to be 10 times the weight of ginseng foil powder, and in step (ii), ethanol was precipitated by adding ethanol (ethanol) in a volume three times the obtained supernatant, (i) In the step, distilled water was added to the obtained precipitate and crushed.

In addition, the dialysis in the step (i) may be performed using a method known in the art, and a material of a desired size may be used with different types of dialysis membranes to obtain, and preferably 12 to 14 in the present invention. Fractions were obtained using a kDa dialysis membrane.

In the present invention, the enzymatic decomposition of the step (a) is (i) adding pectinase to the galacto-uronoglucan polysaccharide, followed by stirring at 45 to 55 for 30 to 90 minutes; And (ii) inactivating the enzyme by heating at 90 to 110 for 10 to 20 minutes.

Figure 1b is a schematic diagram of the production process of galacto-uro-no-glucan polysaccharide through enzymatic decomposition of galacto-uro-no-glucan polysaccharide, and in the present invention, pectinase is preferably used to separate the galacto-uro-no-glucan polysaccharide.

The'pectinase' is an enzyme that hydrolyzes α-1,4-galacturonic acid (α-1,4-galacturonic acid) bonds such as pectin and pectic acid. It is also called galacturonase (polygal-acturonase, EC 3.2.1.15.), polymethylgalactronase. It is a kind of pectin hydrolase, but it can also be used generically. It can be classified into endo-type pectinase, which produces oligosaccharides by randomly decomposing from the inside of α-1,4 bonds in the form of enzyme action, and exo-type pectinase, which sequentially decomposes at the end groups to produce monosaccharides. The former is liquefied and the latter belongs to glycated pectin hydrolase.

Next, step (b) is a step of filtering the obtained supernatant to obtain a polysaccharide having a molecular weight of 1 to 10 kDa, and step (c) is a step of dialysis the obtained polysaccharide to obtain a galacto-uronoglucooligosaccharide. .

In one embodiment of the present invention, a sample having a molecular weight of 1 to 10 kDa was obtained by using the obtained supernatant as an ultrafilter, and the obtained sample was then fractionated with galactouronoglucooligosaccharide using a 1 kDa dialysis membrane. Was obtained.

The molecular weight of the galacto-uronogluco-oligosaccharide prepared by the method of the present invention may be characterized in that it is 1800 to 2600 Da, preferably 2000 to 2400 Da, more preferably 2400 Da, and the obtained galacto-urono Glucooligosaccharides may be characterized in that the content of galacturonic acid is 2 to 3 times, preferably 2.4 to 2.8 times higher than that of glucuronic acid.

In the present invention, as a result of measuring the molecular weight of GuG-PS and GuG-OS prepared above by HPLC (High Performance Liquid Chromatography), as shown in Figure 2, the molecular weight of GuG-PS is in the order of PS1, PS2, PS3, PS4 605000, 513000, 470000, 439000 Da, and the molecular weight of GuG-OS was confirmed to be 2200 Da.

As a result of measuring the neutrophils of GuG-PS and GuG-OS in one embodiment of the present invention, as shown in FIG. 3, in the case of GuG-PS, the content of glucose was the highest, galactose, arabino Neutral sugars are included in the order of arabinose and rhamnose, and in the case of GuG-OS, the content of glucose is increased, while the content of galactose and arabinose is reduced. (rhamnose) was confirmed to be reduced to a similar level.

In addition, as a result of measuring the uronic acid of GuG-PS and GuG-OS, the content of galacturonic acid is about 10.3 times as compared with glucuronic acid in the case of GuG-PS, as shown in FIG. 4. It was confirmed that it was high, and that it was about 2.6 times higher in GuG-OS.

Based on the above results, the present inventors have not only GuG-OS, but also GuG-PS, the composition per component of which is a conventional pectin form, Homogalacturonan, Rhamnogalacturonan I, and Rhamnogalacturonan I. (Rhamnogalacturonan Ⅱ) was identified as a new polysaccharide structure.

That is, GuG-PS is mainly composed of glucose, and the composition ratio of glucose: arabinose: galactose: galacturonic acid is 10:1:1:1. It was confirmed that the composition was different from the existing pectin polysaccharide structure.

In addition, the present invention, ginseng-derived galacto-uro-no-gluco-oligosaccharides to strengthen the immune function containing as an active ingredient, or cancer prevention or treatment pharmaceutical composition; Or a composition for strengthening immune function, or for preventing or improving cancer, functional food for health.

The galacto-uro gluco-oligosaccharide may preferably use galacto-uro-no-gluco-oligosaccharide prepared from the gacto-derived galacto-uro glucopolysaccharide, 0.01 to 10 parts by weight based on 100 parts by weight of the total composition, preferably It may be included as 0.01 to 5 parts by weight, more specifically 100 to 1000 ㎍ / ㎖ concentration may be included.

The solvent of the composition is not particularly limited as long as it does not interfere with the enhancement of the immune function or anti-cancer effect of galacto-uronogluco-oligosaccharides, and more preferably water, ethanol, propyl alcohol, DMSO, and the like. When the concentration of the galacto-uro glucooligosaccharide is less than 0.01 part by weight, the immune-enhancing or anti-cancer effect may be deteriorated. In addition, when it exceeds 10 parts by weight, there may be a problem in that efficiency is reduced due to less improvement in efficacy due to an increase in concentration.

In the present invention, the galacto-uro glucooligosaccharide improves the proliferation rate of macrophages, or tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6; interleukin-6) ) To increase the expression of any one or more cytokines (cytokine) selected from the group to strengthen the immune function.

In one embodiment of the present invention, the survival rate of RAW264.7 cells, which are macrophages by galacto-uro-no-gluco-oligosaccharide treatment, was measured, and as a result, as shown in FIG. 5, galacto-uro-no-gluco-oligosaccharide was treated. In the group, it was confirmed that the proliferation of macrophages was improved, which also means that galacto-uronoglucooligosaccharides did not show toxicity to normal cells.

In addition, in another embodiment of the present invention, as a result of confirming the degree of cytokine expression in macrophages treated with galacto-uro gluco-oligosaccharide treatment, as shown in FIGS. It was confirmed that the production of -α and IL-6 increased.

The cancer may be at least one selected from the group consisting of lung cancer, stomach cancer, colorectal cancer, breast cancer, cervical cancer, and liver cancer, and the galacto-uronoglucooligosaccharide can induce apoptosis of cancer cells.

In one embodiment of the present invention, as a result of observing the morphological changes of cancer cells according to the treatment of galacto-uronoglucan polysaccharide (GuG-PS) and galacto-uronogluco-oligosaccharide (GuG-OS), as shown in FIG. , GuG-PS was confirmed to have no effect on cancer cells, while GuG-OS was confirmed to increase the cell death pattern such as cell body condensation, neurite outgrowth, segmentation, and cell membrane blistering, depending on the concentration.

In addition, as a result of confirming the effect of inhibiting cancer cell growth according to the concentration of GuG-PS and GuG-OS treatment, as shown in FIG. 9, it was confirmed that in the case of GuG-PS, rather, the proliferation of cancer cells was improved, while GuG-OS was concentration-dependent. As a result, it was confirmed that the survival rate of cancer cells was significantly reduced, and when treatment with GuG-OS at a concentration of 1000 µg/ml showed a cancer cell growth inhibitory effect of about 50% or more.

In the present invention, lung cancer-derived cells were used to confirm the anti-cancer effect, but the present invention is not limited thereto, and it is possible to induce apoptosis of various cancers described above, and the galacto-uronoglucooligosaccharide of the present invention activates macrophages Therefore, it is possible to induce toxicity to cancer cells due to macrophage activation.

In addition, when looking at the results of FIGS. 8 and 9, the gacto-uronoglucan polysaccharide (GuG-PS) derived from ginseng leaves has no anti-cancer effect on cancer cells, and rather, when treated at a high concentration, proliferation of cancer cells It can not be seen that most of the ginseng-derived substances have anti-cancer activity, and the immune-enhancing and anti-cancer activity can be seen as a unique function of the galacto-uro gluco-oligosaccharide (GuG-OS) prepared according to the present invention.

Therefore, the immune function enhancing or anti-cancer composition comprising the galacto-uro-no-gluco-oligosaccharide of the present invention as an active ingredient has no side effects that have appeared in the conventional pharmaceutical composition, effectively increasing the proliferation rate of macrophages and TNF-α and IL- It increases the production rate of 6 and can induce the death of cancer cells, so it can be used as an immune function enhancing or anti-cancer drug or health functional food.

The pharmaceutical composition for enhancing immune function or preventing and treating cancer comprising galacto-uronoglucooligosaccharide according to the present invention as an active ingredient may further include other natural substances or compounds having immune-enhancing or anti-cancer activity, , The pharmaceutical composition of the present invention can be administered through various routes including oral, transdermal, subcutaneous, intravenous or intramuscular to mammals such as rats, mice, livestock, and humans. In addition, a pharmaceutical composition for enhancing immune function or preventing and treating cancer according to the present invention may be formulated into various dosage forms. In the case of formulation, it may be formulated using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and these solid preparations include at least one excipient (eg, starch, sucrose, lactose, and the like) of galactouronoglucooligosaccharides. Gelatin) and the like. In addition, lubricants may be used in addition to simple excipients. Liquid preparations for oral administration include suspending agents, intravenous solutions, emulsions, and syrups. In addition to water and liquid paraffin, which are commonly used diluents, various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, etc. Can be included. Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations and suppositories. As the water-insoluble solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used. As a base for suppositories, glycerol and gelatin may be used.

The dosage of the pharmaceutical composition may be applied differently depending on the age, sex, condition, absorption of the active ingredient in the body, inactivation rate and excretion rate, and drugs used in combination.

When the pharmaceutical composition for strengthening the immune function of the present invention or preventing or treating cancer is administered to the human body, since it is a natural extract, there is less fear of side effects compared to other synthetic medicines, so it can be used with confidence.

In addition, the present invention provides a health functional food composition for strengthening immune function, or preventing and improving cancer, comprising galacto-uronoglucooligosaccharide as an active ingredient. The type of health functional food containing the health functional food composition for strengthening or preventing and improving the immune function of the present invention is not particularly limited, for example, meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, It may be ramen, other noodles, gum products, dairy products including ice cream, various soups, beverages, teas, drinks, alcoholic beverages, and vitamin complexes.

The health food is used in conjunction with other foods or food additives other than galacto-uronogluco-oligosaccharides, and may be appropriately used according to conventional methods. For example, the beverage for strengthening immune function, or for preventing or improving cancer, which contains the galacto-uro-no-gluco-oligosaccharide as an active ingredient, calcium, thorny-organic concentrate, liquid fructose in addition to containing the galacto-uro-no-gluco-oligosaccharide as an active ingredient , Purified water and the like are added and mixed into a drink bottle, sterilized, and cooled to room temperature to produce a beverage. In addition, the health supplement for enhancing immune function or preventing and improving cancer containing the galacto-uronogluco-oligosaccharide as an active ingredient is a nutritional supplement component (vitamins B1, B2, B5, B6, E and Acetic acid ester, nicotinic acid amide), oligosaccharide, 50% ethanol, and purified water were added and mixed to form granules, dried in a vacuum dryer, and then passed through 12-14 mesh to uniformly prepare granules and extruded by an appropriate amount. It can be made into a hard capsule product by filling it into a tablet or powder or filling it into a hard capsule.

The effective dose of the galacto-uronoglucooligosaccharide contained in the health food can be used in accordance with the effective dose of the pharmaceutical composition, and the amount of the active ingredient mixed can be appropriately determined according to the purpose of use such as prevention or therapeutic treatment. have. It may be below the above range for long-term intake for health and hygiene purposes or for health control purposes.

Hereinafter, the present invention will be described in detail with reference to preferred embodiments so that those skilled in the art to which the present invention pertains can easily practice. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein.

Example One. Galacto-uronoglucan Polysaccharide (Galacturonoglucopolysaccharide; GuG-PS) manufacturing process

In the present invention, in order to obtain galacto-uro glucan polysaccharide from ginseng foil, galacto-uro glucan polysaccharide was obtained according to the process shown in FIG. 1A.

First, 600 ml of distilled water was added to 60 g of ginseng foil powder, stirred for 5 hours, and then centrifuged to obtain a supernatant, and then treated with 3 times the volume of ethanol (ethanol; EtOH). Then, once again, the precipitate was obtained by centrifugation, and then pulverized using a homogenizer, and the pulverized product was used with a dialysis membrane (Part No. 132576, SPECTRUM LABS, USA) of 12 to 14 kDa. Fractions were obtained. The obtained fraction was dried to obtain a polysaccharide in the form of powder.

Example 2. Galacto-uronoglucan From polysaccharides Galacto-uronogluco-oligosaccharides (Galacturonoglucooligosaccharide; GuG -OS) Manufacturing process

In the present invention, according to the process shown in Figure 1b, galacto-uronoglucooligosaccharide was prepared from GuG-PS prepared in Example 1.

First, it was prepared by dissolving in 0.1 M sodium acetate (0.1 M sodium acetate, pH 4.0) so that the GuG-PS in the powder form obtained in the example was 1%, and then hydrolytic enzyme pectinase (No. 17389, Sigma) , USA) was prepared by dissolving in 0.1 M sodium acetate (pH 4.0) to be 10%. Then, 1% GuG-PS and 10% pectinase were mixed 1:1 and stirred at 50 for 1 hour. The sample was heated at 100 for 15 minutes to inactivate the enzyme, followed by centrifugation to obtain a supernatant, and the obtained supernatant was subjected to an ultrafiltration (ultrafiltrator; No. 17521-105, Sartorius, Germany) to have a molecular weight of 1 to 10 kDa. A sample was obtained. Thereafter, the obtained sample was dried using a 1 kDa dialysis membrane (Part Number. 132640, SPECTRUM LABS, USA), and then the fraction was dried to obtain a powder form of galacturouronoglucooligosaccharide.

Example 3. GuG -PS and GuG -Molecular weight measurement of OS

The molecular weights of GuG-PS obtained in Example 1 of the present invention and GuG-OS obtained in Example 2 were performed using HPLC (High Performance Liquid Chromatography), and HPLC was performed by Waters Alliance HPLC 2695 of the United States. The product was used.

SB-showdex 804 HQ column was used as the column, and tertiary distilled water was used as the mobile phase. The analysis samples GuG-PS and GuG-OS were each injected to be 10 µl, and the speed of the mobile phase during the analysis was analyzed at 0.7 ml/min. Pullulan (P-1320, P-5900, P-11800, P-212000, P-404000; Fluka, Germany) was used as a standard.

As a result, as shown in Figure 2a, the molecular weight of GuG-PS was found to be 605000, 513000, 470000, 439000 Da in the order of PS1, PS2, PS3, PS4, and the molecular weight of GuG-OS was 2200Da as shown in Figure 2b. It was confirmed that.

Example 4. GuG -PS and GuG -Measurement of triglyceride in OS

In the present invention, the neutral sugars constituting the GuG-PS and GuG-OS obtained in Examples 1 and 2 were measured.

First, 100 μg of each of GuG-PS and GuG-OS was dissolved in 340 μl of distilled water, and then 60 μl of trifluoroacetic acid (TFA) was added, followed by heating at 100 for 4 hours to hydrolyze. Then, moisture and solvent in the sample were removed using SpeedVac.

Standards of monosaccharides (Fuc; fucose, Rha; rhamnose, Ara; arabinose, Gal; galactose; Glc; glucose, Man; mannose, Xyl; xylose) were used after the filter was made with 1 M and used for the measurement of neutral sugar. LC was a product of Dionex (DX 500 Chromatography system, Dionex Co, USA). The column was Carbo-Pak PA1 (Dionex Co, USA), and the detector was ED 50 (Dionex Co, USA).

Each sample was injected 10 µl and eluted at 0.8 ml/min using Buffer A (dH ₂ O) and Buffer B (200 mM NaOH).

As a result of the analysis of neutrophils of GuG-PS and GuG-OS, as shown in FIG. 3, the content of glucose was the highest in GuG-PS, galactose, arabinose, and rhamnose. ) Contains neutrophils in order, and in the case of GuG-OS, the content of glucose increased, whereas the content of galactose and arabinose decreased to a level similar to rhamnose. Was confirmed.

Example 5. GuG -PS and GuG -OS Uronic acid Measure

In the present invention, the content of uronic acid constituting GuG-PS and GuG-OS obtained in Examples 1 and 2 was measured.

First, 100 μg of each of GuG-PS and GuG-OS was dissolved in 340 μl of distilled water, and then 60 μl of trifluoroacetic acid (TFA) was added, followed by heating at 100 for 4 hours to hydrolyze. Then, moisture and solvent in the sample were removed using SpeedVac.

A standard product of uronic acid (GalA; galacturonic acid, GlcA; glucuronic acid) was made of 1 M and used after filtering. Bioion-LC used for the measurement of uronic acid was a product of Dionex (DX 500 Chromatography system, Dionex Co, USA). The column was Carbo-Pak PA100 (Dionex Co, USA) and the detector was ED 50 (Dionex Co, USA).

Each sample was injected 10 µl and eluted at 0.8 ml/min using Buffer A (dH ₂ O) and Buffer B (200 mM NaOH).

As a result of measuring the uronic acid of GuG-PS and GuG-OS, as shown in FIG. 4, in the case of GuG-PS, the content of galacturonic acid is about 10.3 times higher than that of glucuronic acid. It was confirmed that the GuG-OS was about 2.6 times higher.

Example 6. GuG -Confirmation of survival rate of RAW 264.7 cells according to OS treatment

In the present invention, the survival rate of RAW264.7 cells, macrophages according to the concentration of galacto-uro-no-gluco-oligosaccharides, was measured in order to confirm the potency of enhancing the immune function of galacto-uro-gluco-oligosaccharide.

First, 500 µl of each well of a 48-well culture plate (SPL, Korea) to make RAW264.7 cells (No. TIB-71 ^TM , ATCC, USA) 1×10 ⁵ cells/ml It was added and incubated in a 37, 5% CO ₂ incubator for 24 hours, and then exchanged with fresh DMEM medium (10% FBS, 1% penicillin-streptomycine) before treating GuG-OS. Then, GuG-OS was prepared at concentrations of 100, 250, 500, and 1000 μg/ml, respectively, and treated with RAW264.7 cells.

After incubation for 24 hours, cell viability was measured using the MTT assay method, and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide dissolved in PBS buffer in each well ( MTT, 5 mg/ml, ROTH, Germany) solution was added in 100 µl increments and incubated for another 4 hours.

After confirming the formation of Formazan, the medium was completely removed, and then 500 μl of DMSO (dimethyl sulfoxide) was added to dissolve the formazan formed at the bottom of each well, and absorbance was measured at 570 nm using an ELISA reader.

As a result, as shown in FIG. 5, it was confirmed that the proliferation of macrophages was improved in the group treated with galacto-uro-no-glucooligosaccharides compared to the control group. It also means.

Example 7. GuG Measurement of cytokine production in RAW 264.7 cells according to OS treatment

In the present invention, in order to confirm the degree of cytokine expression in macrophages by galacto-uronogluco-oligosaccharide treatment, tumor necrosis factor-alpha (TNF-α) according to the concentration of galacto-uronogluco-oligosaccharide treatment and The degree of production of interleukin-6 (IL-6) was measured.

First, 1 ml of RAW264.7 cells was added to each well of a 6-well culture plates (SPL, Korea) to be 1×10 ⁵ cells/ml and incubated with 37, 5% CO ₂ for 24 hours. After cultivation in, it was exchanged with fresh DMEM medium (10% FBS, 1% penicillin-streptomycine) prior to treatment with GuG-OS. Then, GuG-OS was prepared at concentrations of 100, 250, 500, and 1000 μg/ml, respectively, and treated with RAW264.7 cells.

After culturing for 24 hours, cell culture was obtained, and then cytokines contained in the culture were measured using an ELISA kit (Quantikine Immunoassay Mouse TNF-α, Quantikine Immunoassay Mouse IL-6, R&D systems, USA).

As a result, as shown in FIG. 6, the concentrations of TNF-α production were 8.96, 534.94, 1801.77, 2045.96, 2031.59, respectively, depending on the concentration of GuG-OS (0, 100, 250, 500, 1000 µg/ml) compared to the control group. It was confirmed that the increase to pg / ㎖, as shown in Figure 7, IL-6 production concentration was confirmed to increase to 2.645, 6.228, 18.272, 75.484, 570.974pg / ㎖, respectively.

Example 8. GuG -PS and GuG -Observation of cancer cell morphology according to OS treatment

In the present invention, the morphological changes of cancer cells according to the treatment of galacto-uronoglucan polysaccharide (GuG-PS) and galacto-uronoglucooligosaccharide (GuG-OS) were observed with an optical microscope.

First, 1 ml of each well of a 6-well culture plate (SPL, Korea) so that the human lung cancer cell line A549 cells (KCLB No. 10185, Korea Cell Line Bank) is 1×10 ⁵ cells/ml. It was added and cultured in 5% CO ₂ incubator at 37 conditions for 24 hours in RPMI 1640 medium (10% FBS, 1% penicillin-streptomycine), and GuG-PS and GuG-OS were 0, 125, 250, 500, 750 respectively. Prepared at a concentration of 1000 µg/ml, treated with cells, and then incubated for 96 hours, to observe the morphology of cells with an optical microscope (×200).

As a result, as shown in FIG. 8, it was confirmed that GuG-PS had no effect on cancer cells, while GuG-OS increased the concentration of apoptosis such as cell body condensation, loss of neurites, segmentation, and cell membrane blistering in a concentration-dependent manner. Was confirmed.

Example 9. GuG -PS and GuG -Confirmation of cancer cell growth inhibition effect by OS treatment

In the present invention, the growth inhibitory effect of cancer cells according to the treatment of galacto-uronoglucan polysaccharide (GuG-PS) and galacto-uronoglucooligosaccharide (GuG-OS) was measured using an MTT assay.

First, 200 μl was added to each well of a 96-well culture plates (SPL, Korea) so that the human lung cancer cell line A549 cells were 1×10 ⁵ cells/ml and 37, 5% CO for 24 hours. _{2 After} incubation in the incubator, GuG-PS and GuG-OS were cultured for 2 hours by exchanging with serum-free RPMI 1640 medium before treatment.

Subsequently, GuG-PS and GuG-OS were prepared at concentrations of 0, 125, 250, 500, 750, and 1000 μg/ml, respectively, and treated with cells to incubate for 96 hours, followed by MTT in the same manner as in Example 6. Assays were performed.

As a result, as shown in Figure 9, in the case of GuG-PS was confirmed that rather improves the proliferation of cancer cells, GuG-OS was confirmed to significantly reduce the survival rate of cancer cells in a concentration-dependent manner, the concentration of 1000 ㎍ / ㎖ When treated with GuG-OS, it was confirmed that the cancer cell growth inhibitory effect was at least about 50%.

Claims (16)

delete delete delete delete delete delete Pectinase in galactouronlucopolysaccharide having a composition ratio of 10:1:1:1 in the composition of glucose: arabinose: galactose: galacturonic acid And then stirring for 30 to 90 minutes at 45 to 55;
(Ii) heating at 90 to 110 for 10 to 20 minutes to inactivate the enzyme, followed by centrifugation to obtain a supernatant;
(iii) filtering the obtained supernatant to obtain a polysaccharide having a molecular weight of 1 to 10 kDa; And
(iv) obtaining the galactouronoglucooligosaccharide by dialysis of the obtained polysaccharide; containing ginseng bacterium-derived galactouronoglucooligosaccharide as an active ingredient;
The molecular weight of the galacto-uro glucooligosaccharide derived from ginseng foil is 2200 Da,
The ginseng-derived galacto-uronogluco-oligosaccharide comprises glucose, galactose, arabinose, and rhamnose, a pharmaceutical composition for preventing or treating lung cancer.
delete The method of claim 7,
The galacto-uro glucooligosaccharide improves the survival rate of macrophages, or is a group consisting of tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6). A pharmaceutical composition for preventing or treating lung cancer, which is characterized by enhancing the immune function by increasing the expression of any one or more cytokines selected from.
delete The method of claim 7,
The galacto-uro glucooligosaccharide is characterized in that it induces apoptosis (apoptosis) of cancer cells, lung cancer prevention or treatment pharmaceutical composition.
Pectinase in galactouronlucopolysaccharide having a composition ratio of 10:1:1:1 in the composition of glucose: arabinose: galactose: galacturonic acid And then stirring for 30 to 90 minutes at 45 to 55;
(Ii) heating at 90 to 110 for 10 to 20 minutes to inactivate the enzyme, followed by centrifugation to obtain a supernatant;
(iii) filtering the obtained supernatant to obtain a polysaccharide having a molecular weight of 1 to 10 kDa; And
(iv) obtaining the galactouronoglucooligosaccharide by dialysis of the obtained polysaccharide; containing ginseng bacterium-derived galactouronoglucooligosaccharide as an active ingredient;
The molecular weight of the galacto-uro glucooligosaccharide derived from ginseng pak is 2200 Da,
The ginseng-derived galacto-uro glucooligosaccharide comprises glucose, galactose, arabinose, and rhamnose, a health functional food composition for preventing or improving lung cancer.
delete delete delete The method of claim 12, wherein the galacto-uro gluco-oligosaccharide is characterized in that it induces apoptosis (apoptosis) of cancer cells, lung cancer prevention or improvement health functional food composition.

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