KR20040087407A - Immune stimulative constituents of ginseng saponins - Google Patents

Abstract

PURPOSE: Provided is a composition containing ginsenosides Rh2 and Rg3 to facilitate the generation of TNF-alpha, thereby displaying immune-stimulating properties. Therefore, the composition is used for medicaments and foods for prevention and treatment of diseases. CONSTITUTION: The composition having immune-stimulating properties is characterized by containing ginsenoside Rh2 and Rg3 represented by the formulae(2) and (1), respectively in a ratio of 1:1 to 10:1. The composition is applied to the food or medicament and is then used as an immune-stimulating agent.

Description

Immune stimulative constituents of ginseng saponins}

The present invention relates to a composition exhibiting an immunostimulating effect containing ginsenoside Rh ₂ and ginsenoside Rg ₃ of the following structural formula (1). More specifically, the present invention relates to a composition exhibiting an immune enhancing effect, wherein ginsenoside Rh ₂ and ginsenoside Rg ₃ are contained in a ratio of 1: 1 to 10: 1. The composition containing ginsenoside Rh ₂ and ginsenoside Rg ₃ of the present invention in a ratio of 1: 1 to 10: 1 can be used as an adjuvant by preparing and taking food or medicine.

[Formula 1]

[Formula 2]

The present invention relates to the use of ginsenosides having a specific composition ratio as a TNF-α activity inducing agent.

Breckmann, a Russian pharmacologist, suggested earlier that it acts as a so-called "adaptogen" that enhances nonspecific bio-resistance against external harmful factors of ginseng and normalizes the body against physical, chemical and biological changes (Brekmann). 1969) suggests that ginseng may act to boost immune function. Recently, the effectiveness of ginseng is gradually revealed as a result of examining changes in various immune function indices of saponin and polysaccharide components including extracts (fractions) of ginseng.

Natural killer cells (NK cells) have the ability to destroy cells or tumor cells infected with viruses without prior sense of the antigen in the living body. The activity of natural killer cells is changed by a number of factors, interferon (interferon), interferon-inducing substances and interleukin-2 (Interleukin-2), etc. are known to have the effect of enhancing the killing ability of NK cells. Total saponins isolated from red ginseng (6 years old) and PPD and PPT-based saponins have the effect of enhancing NK cell activity in vitro (Kim et al. 1989). Ginseng extract has been reported to enhance the proliferation of lymphocytes, increase the activity of NK cells and ADCC (antibody dependent cytotoxic) and increase interferon production in human peripheral blood lymphocytes (Gupta et al. 1980). . Animal and in vitro studies have reported that ginseng extract promotes NK cell activation of NK cells in splenocytes and significantly promotes immune interferon production, particularly when combined with interferon inducers (6-MFA) (Jie et al 1984, Sigh et al. 1984).

As a result of administering 70% ethanol extract of Korean red ginseng (50, 100mg / kg, intraperitoneally) to mice (9B6C3F1), it enhanced the activity of natural killer cells (NK cells) and lowered immune function induced by anti-cancer cyclophosphamide treatment. It has the effect of promoting recovery of NK cell function in mice and has a protective effect against infection with Listeria monocytogenes (bacteria causing meningitis encephalomyelitis) (Kim et al. 1990).

The extract (ethanol insoluble fraction) of Korean red ginseng (6 years old) induced the proliferation of splenocytes, promoted production of interleukin-2 (IL-2) from splenocytes, and activated natural killer cells. These fractions also inhibited the development of pulmonary adenoma induced by benzopyrene (B (a) P) administration in newborn mice. (Yun et al. 1993).

Recently, red ginseng extract was found to increase the ratio of helper T cell / suppressor T cell and NK cell activity in T lymphocytes of spleen cells (Jang et al. 1994).

G-Rh ₂ , a major saponin component of Korean ginseng, increased the number of plaque-forming cells and the number of T-cells in spleen, increased the activity of spontaneous killer cells, and the total number of T-cells. There is a report to increase the ratio of the number of T-helper cells. In addition, G-Rh ₂ expressed mitosis promoting effect of cultured thymic cells and induced an increase in the production of interleukin-1 (IL-1) of macrophages, in particular damaged humoral (humoral) and treatment by the anti-cancer drug cyclophosphamide It has been reported to have the effect of partially restoring the cell-mediated immune response (kenarova et al 1990).

Anticancer drugs are cytotoxic to normal cells as well as cancer cells, and one of the main side effects of anticancer drugs is a severe immunosuppressive action. Therefore, since anticancer drugs are limited in clinical applications, immunotherapy that promotes reduced immune function is becoming very important. As a result of animal experiments, ginseng ethanol extract, butanol fraction and ether extract have the effect of revitalizing humoral and cellular immune responses lowered by the administration of mitomycin C, an anticancer agent with bone marrow toxicity, kidney disorder and immunotoxicity. It has been reported that there is an effect of reviving or enhancing the activation of NK cells, sex and cellular immunity (Ahn et al. 1987).

In addition, the combination of red ginseng saponin fraction and anticancer drugs adriamycin and vicristine has been shown to restore the suppression of the killing ability of sarcoma-180 cells of celiac macrophages caused by the administration of these anticancer drugs (Lee et al. 1988). ).

It has been reported that saponin (G-Rh ₂ ) and polysaccharide components of Korean ginseng have the effect of restoring impaired immune function, which is one of the major side effects of chemical anticancer therapies (cisplatin, mitomycin C, cyclophosphamide, etc.). (Ahn et al. 1997, Wang et al. 1985, Kim et al 1990, 1991, 1995).

Physical or psychological stress inhibits immune function, and administration of Korean red ginseng (0.2-1% to rat feed, 8 weeks of addition) inhibits lymphocyte weakening response of spleen cells induced by foot shock. It has been suggested to have an effect of improving the reduced immune function, such as lowering the activity of natural killer cells.

On the other hand, administration of LPS (lipopolysaccharide), a toxin, to BALB / C mice causes weight loss of the thymus, an organ responsible for immune function, and apoptosis, the thymus cell death. Using these experimental models, the effects of red ginseng extracts on LPS-induced apoptosis of thymus cells were evaluated by weight of thymus, cell viability, DNA fragmentation, morphological changes, and Bax protein. Expression was examined as an indicator. As a result, the treatment of ginseng extract (GS) 1 hour before LPS treatment increased the weight of thymus, survival of thymic cells and suppressed thymus apoptosis by LPS. As a result of immunostaining, excessive expression of Bax protein induced by LPS was reduced (Wei et al. 1996).

Based on the results of the ginseng test on the immune function, it is suggested that the ginseng component acts as a so-called biological response modifier (BRM) that enhances and regulates the immune function of the host.

However, since ginsenoside Rh ₂ is obtained in extremely small amounts only in red ginseng, it is difficult to be used clinically and at the same time, the mechanism of immunological effects has not been fully studied.

The inventors who have studied natural products having the effect of promoting the growth of tumor necrosis factor α have established the optimal composition ratio of Rh ₂ and Rg ₃ of ginseng saponin, maximize the immune enhancing effect in ginseng, and trace amounts of ginseng saponin Overcoming the limitations of the present invention has been found to promote the production of TNF-α, an immunoactivity indicator, and to complete the present invention.

That is, it is an object of the present invention to provide a TNF-α activity inducible composition which is an immunoactive component.

An object of the present invention is to provide a composition for maximizing the immune enhancing effect containing ginsenosides as an active ingredient that can be obtained more easily and inexpensively.

TNF-α is a cytokine produced by T cells and macrophages activated in excessive immune responses. TNF-α is a name that causes necrosis of some tumors. Genetic recombination technology has been introduced to produce large amounts of TNF-α, but serious side effects such as hypotension, hepatotoxicity, and weight loss have been reported.

Therefore, research is being conducted toward remodeling sites that have negative effects while leaving the inherent properties of TNF-α attacking tumors, and at the same time, gene therapy to induce injected cells to produce TNF-α is studied. However, there are no reports of effective TNF derivatives (Decision Resources, Commericialiation of cytokine and anti-cytokine therapies, 1995).

Lipopolysaccharide (LPS), known as one of the TNF-α production promoters, stimulates immune cells to produce TNF-α when administered in vivo. However, washing NPS-treated immune cells and contacting LPS again is known to inhibit TNF-α production. On the other hand, immune cells in contact with ginsenosides Rh ₂ and Rg ₃ of the present invention continue to produce TNF-α when contacted with ginsenosides after washing, thereby producing TNF-α from immune cells by a mechanism different from that of LPS. It could be seen that it promotes.

However, ginsenoside Rh ₂ has a problem in that its immune enhancing effect is not so remarkable that its immunopotentiating effect is insufficient when used alone for the purpose of immune enhancing. Furthermore, ginsenoside Rh ₂ is white ginseng is not present (Kitagawa et al., Japanese Pharmacology magazines, 103, 612, 1983), red ginseng is 0.001%, ginseng is contained by 0.004%, only trace amount it Jean a large amount of ginsenoside Rh ₂ There is also a limit to increase the insufficient immune boosting effect.

In addition, ginsenoside Rg ₃ is known to have cancer cell metastasis suppression, platelet aggregation inhibitory action, antithrombotic action, liver disorder inhibitory action, vascular relaxation action and anti-cancer drug inhibitory action. However, ginsenoside Rg ₃ has a problem that immunity enhancement effects are insignificant as in ginsenoside Rh _2, and that its components are present in ginseng or red ginseng in extremely small amounts, and thus it is difficult to enhance immune effects through large intake.

Accordingly, the present inventors invented medicines and food compositions for the purpose of immuno-enhancing by using ingredients such as ginseng or red ginseng by improving the effects of ginsenoside Rh ₂ and ginsenoside Rg ₃ , which have no side effects but lack immuno-enhancing effects. As a result of a long study, it was found that when the ginsenoside Rh ₂ and the ginsenoside Rg ₃ is administered in a ratio of 1: 1 to 10: 1, excellent immuno-enhancing effects are shown, thereby completing the present invention.

The composition containing ginsenoside Rh ₂ and ginsenoside Rg ₃ of the present invention in a ratio of 1: 1 to 10: 1 is superior to the case where ginsenoside Rh ₂ or ginsenoside Rg ₃ is administered alone. Effect.

Therefore, the present invention is superior to ginsenoside Rh ₂ or ginsenoside Rg ₃ which lacks an immune enhancing effect as a single substance, and has no side effects, ginsenoside Rh ₂ and ginsenoside Rg ₃ are 1: 1 to 10: It is an object to provide a composition contained in 1.

Another object of the present invention is to provide a drink, tablets, pills, capsules and injections using a composition which continuously improves immunity by taking a dose and exhibits the excellent immuno-enhancing effect.

Hereinafter, the present invention will be described in detail.

Ginsenoside Rh ₂ and ginsenoside Rg ₃ as an active ingredient according to the present invention is characterized in that the composition containing a ratio of 1: 1 to 10: 1 exhibits an excellent immune enhancing effect to prevent cancer or cancer treatment and It may be usefully used in combination administration.

Especially preferably, ginsenoside Rh ₂ and ginsenoside Rg ₃ are contained in a ratio of 3: 1 to 5: 1.

When the ratio of ginsenoside Rh ₂ and ginsenoside Rg ₃ is 1: 1 or less, or 10: 1 or more, the effect is decreased or no synergistic effect is shown.

The composition of the present invention is generally a composition of ginsenoside Rh ₂ and ginsenoside Rg ₃ is administered to the human body in terms of the absorption rate, inactivation rate and excretion rate of the active ingredient in the body, the age, sex and condition of the patient, Although appropriately selected depending on the progress of the cancer, etc., it is generally preferable to administer the total amount to an adult at 10 to 20 mg, preferably 10 to 15 mg per day. If you take less than 1mg per day, the immune boosting effect is insufficient, if you take more than 20mg per day, the immune boosting effect is no longer increased.

The composition of the present invention may be prepared by food or medicine and divided into one to three times a day in accordance with the daily dose.

The composition of the present invention may be prepared orally in the form of a drink, tablet, pill or capsule in a conventional manner, or may be prepared and administered as an injection. At this time, it is preferable to prepare each formulation to contain 3 to 7 mg in the total amount of ginsenoside Rh ₂ and ginsenoside Rg ₃ in accordance with the number of daily administration.

When preparing the composition of the present invention as a pharmaceutical, carriers which can be used in the composition of the present invention are conventional in the pharmaceutical field, for example, in the case of oral preparations, binders, suspending agents, disintegrants, excipients, solubles There are an agent, a dispersant, a stabilizer, a suspending agent, a pigment, a fragrance, and the like, and in the case of an injection, there are a preservative, a painless agent, a solubilizer, and a stabilizer. The pharmaceutical preparations thus prepared may be administered orally or prepared by injection. In addition, in order to prevent the decomposition of the drug by gastric acid during oral administration, an antacid may be used in combination, or a solid oral dosage form such as a tablet may be formulated into a formulation coated with enteric skin.

When ginsenoside Rh ₂ and ginsenoside Rg ₃ , which are active ingredients of the composition according to the present invention, are prepared in a ratio of 1: 1 to 10: 1, the effect is not only excellent, but also from the experimental results described below. As demonstrated, it has no side effects and is not acutely toxic to laboratory animals and can be used safely.

In the present invention, the effect of generating TNF-α from immune cells is a method of quantifying TNF-α secreted into the medium by adding an inducer to macrophage RAW264.7 (Beutler et al., Science, 225, 869, 1985).

The present invention is described in more detail by the following examples and experimental examples, but the present invention is not limited in any way by these.

Example 1 Preparation of Ginsenoside Rh2

Bacillus bacteria (麴菌, yeast) were added to the medium containing 3% veins and 1% ginseng powder, and the cells were removed by centrifugation for 60-80 hours at 28-30 ℃. The enzyme protein (酵素 蛋白) was precipitated with an ethyl alcohol solution, and the protein was collected, dissolved in 1/10 volume of sodium acetate solution (0.02M, pH5.0) in the culture solution, and centrifuged to remove impurities to obtain an enzyme solution. have.

3 g of ginsenoside Rd is dissolved in 100 ml of acetic acid solution (0.02 M, pH 5.0) and 50 ml of the enzyme solution is added thereto. Reaction at 40 ° C. for 6-24 hours results in ginsenoside F2. Then, 1/3 volume of butanol was added, and saponin was extracted three times, and evaporated to dryness under reduced pressure (減壓). Organic acids (such as nitric acid, acetic acid, hydrochloric acid, sulfuric acid, and the like) hydrolyze the carboxylic acid at the 20th carbon position of the ginsenoside F 2 molecule to convert it into ginsenoside Rh ₂ . When this is separated by silica gel column separation, 0.5-1.4 g of ginsenoside Rh ₂ may be prepared.

Example 2 Preparation of Ginsenoside Rg ₃

6 g of ginseng irradiated ponin was dissolved in 20 ml of 50% acetic acid and heated with stirring at 70 ° C. for 2 hours, and then the reaction mixture was cooled to room temperature. The reaction solution is diluted with 200 ml of distilled water and extracted three times with 100 ml of n-butanol. The combined extracts were washed twice with saturated sodium bicarbonate solution and the n-butanol layer was concentrated under reduced pressure. The reaction mixture was separated by silica gel column chromatography (chloroform: ethanol: water = 100: 30: 10, lower layer) to obtain 2.25 g of ginsenoside Rg ₃ .

Experimental Example 1 In-vitro Immune Enhancement of Ginsenoside Rh ₂ and Ginsenoside Rg ₃

1. Experimental method

RAW 264 was pre-incubated and then trypsinized to collect the cells, prepared in test medium at 1.5 × 10 5 cells / ml, inoculated at 1.5 × 10 6 cells / 100 μl on 96 well plates, and then incubated at 37 ° C. and 5% CO 2 for 4 hours. . After dissolving the test medium, 100 μl of the sample (DMSO final concentration 0.5%) was added thereto, followed by incubation for 20 hours, and 170 μl of the culture supernatant was carefully taken with a micropipette, followed by centrifugation at 3000 rpm, 10 minutes, and 4 ° C. 150 µl of the supernatant is collected, stored at -20 ° C, and used as a sample for measuring the amount of TNF-α. Using the mouse TNF-α ELISA system, the absolute amount of TNF-α in the culture supernatant sample is determined from the calibration curve of the TNF-α absolute amount. From the comparison and comparison of these values, the immunoreactivity is assessed using the TNF-α content of the test sample as an indicator. In addition, the sample for measuring the amount of TNF-α of each sample (culture supernatant) is measured by Duplicate.

2. Experimental Results

Aid group Amount control = 1 Rh ₂ 50 ppm200 ppm 4.632.0 * Rg ₃ 50 ppm200 ppm 0.52.9 Rh ₂ : Rg ₃ = 1: 1 50 ppm200 ppm 34.8 * 44.3 * Rh ₂ : Rg ₃ = 5: 1 50 ppm200 ppm 44.8 * 64.3 * Rh ₂ : Rg ₃ = 10: 1 50 ppm200 ppm 33.5 * 41.7 * Rh ₂ : Rg ₃ = 1: 5 50 ppm200 ppm 3.14.5 Rh ₂ : Rg ₃ = 1: 10 50 ppm200 ppm 3.64.9

Statistical significance from control

(student's T-test, * = p <0.05)

According to the above result table, ginsenoside Rh ₂ alone treated group showed significantly increased TNF-α activity in the 200ppm treated group compared to the control group, but the ginsenoside Rg ₃ alone treated group showed no increase in activity. On the other hand, ginsenosides Rh ₂ and Rg ₃ of 1; 1, 5: 1, 10; 1 all significantly increased the TNF-α activity compared to the single treatment group showed that the immune enhancing effect is excellent Able to know. In addition, it can be seen that there is a very strong immune enhancing effect in the composition ratio of 5: 1 of ginsenosides Rh ₂ and Rg ₃ .

Experimental Example 4 Acute Toxicity Test of Ginsenoside Rh ₂ and Ginsenoside Rg ₃

1) Test substance: Ginsenoside Rh ₂ and Ginsenoside Rg ₃

Test animals: Male rats 6 weeks old and male mice 5 weeks old (10 animals / group)

3) Preparation of test substance: The composition was prepared by completely suspending the composition in 1% CMC before administration on the test day.

4) Test Method: After fasting the test animals for 6 hours, the drug was administered orally into the stomach of the test animals using a mouse and rat bulge. The dose was administered based on the weight of the body immediately before administration, and the death of the agent was observed for 14 days.

5) Test Results: As a result of acute toxicity test in rats and mice, LD ₅₀ of the present pharmaceutical composition in rats and mice was more than 3g / ㎏.

Acute Toxicity Test Results in Rats and Mice Medication Test animals Test concentration (mg / kg) Number of animals Dead animals % Mortality LD ₅₀ Rh ₂ : Rg ₃ = 1: 1 Rat 01883757501,5003,000 101010101010 000000 000000 > 3g / kg mouse 01883757501,5003,000 101010101010 000000 000000 > 3g / kg Rh ₂ : Rg ₃ = 10: 1 Rat 01883757501,5003,000 101010101010 000000 000000 > 3g / kg mouse 01883757501,5003,000 101010101010 000000 000000 > 3g / kg

Ginsenosides Rh ₂ and ginsenosides Rg ₃ of the present invention, an immuno-enhancing composition may be formulated according to the following examples.

[Production Example 1]

Ginsenoside Rh ₂ 7mg

Ginsenoside Rg ₃ 7mg

Chitooligosaccharide 5 mg

Pear Juice 200 mg

Vitamin C 10 mg

800 mg liquid fructose

Honey 500 mg

Citric acid 120 mg

100 mg herbal flavor

Purified water was added to the components of the composition to prepare a drink having a final volume of 100 ml.

[Production Example 2]

Corn Starch 44g

40g of crystalline cellulose

Carboxymethylcellulose 5g

Magnesium Stearate 1g

Ginsenoside Rh ₂ 540mg

Ginsenoside Rg ₃ 540mg

91.8 g of total

According to the prescription described above, each component was uniformly mixed and then compression-molded in a tableting machine to prepare a tablet of 500 mg. This tablet contains 14 mg of ginsenoside Rh ₂ and ginsenoside Rg ₃ . Adults take 1-3 tablets several times a day.

[Production Example 3]

440 g of crystalline cellulose

Magnesium Stearate 10g

Ginsenoside Rh ₂ 3g

Ginsenoside Rg ₃ 3g

456 g in total

According to the prescription described above, all the ingredients were mixed uniformly, assembled using a granulator according to a conventional method, and filled into a charger to prepare 500 mg of capsules per capsule. One capsule of this capsule contains 14 mg of ginsenoside Rh ₂ and ginsenoside Rg ₃ . Adults take 1-3 capsules several times a day.

[Production Example 4]

86.5 g of distilled water for injection

5 g of ethanol

Soy Phospholipid 2.5g

5 g of glycerin

Ginsenoside Rh ₂ 540mg

Ginsenoside Rg ₃ 540mg

Total 100.08 g

Ginsenosides Rh ₂ and Ginsenosides Rg ₃ were dissolved in ethanol and soybean phospholipids according to the above-described prescription, and then injected with distilled water and glycerin solution for injection to prepare an injection.

Ginsenosides Rh ₂ and ginsenosides Rg ₃ of the active ingredient according to the present invention contains a 1: 1 to 10: 1 composition exhibits an immune boosting effect than ginsenoside Rh ₂ and ginsenoside Rg ₃ alone In addition, it has no side effects and can be effectively used for cancer prevention and cancer treatment when combined with chemotherapy.

Claims (7)

Ginsenoside Rh ₂ and Ginsenoside Rg ₃ is 1: 1 to 10: 1, characterized in that the immunopotentiating composition. The immunopotentiating composition according to claim 1, which contains one or two pharmaceutically acceptable carriers and additives. 3. The immunopotentiating composition according to claim 2, wherein the carrier is selected from diluents, lubricants, binders, disintegrants, sweeteners, stabilizers, and preservatives. The immuno-enhancing composition according to claim 2, wherein the additive is one or two or more selected from fragrances and vitamin B groups. 2. The immunopotentiating composition according to claim 1, wherein the daily oral dose is 10 mg to 20 mg in the total amount of ginsenoside Rh ₂ and ginsenoside Rg ₃ . 3. The immunopotentiating composition according to claim 2, wherein the formulation is selected from drink, tablet, pill, capsule or injection. 7. The immunopotentiating composition according to claim 6, wherein the total amount of ginsenoside Rh ₂ and ginsenoside Rg ₃ is contained in an amount of ₃ mg to 7 mg per each formulation.