KR20040036092A - Anticarcinogenic constituents of ginsenoside Rh2 and Rg3 - Google Patents

Abstract

PURPOSE: Provided an anticancer composition of ginsenoside Rh2 and Rg3 which has improved anticancer effect without adverse side effect, thereby producing remedies or foods for the prevention and treatment of cancer using ginseng or red ginseng. CONSTITUTION: An anticancer composition of ginsenoside Rh2 and Rg3 is characterized by containing ginsenoside Rh2 and Rg3 in a ratio of 1:1 to 10:1. It further contains at least one or two pharmaceutically acceptable carriers selected from diluents, lubricants, disintegrant, binding agents, sweeteners, stabilizers and preservatives, or additives selected from flavorings, vitamin B group. The ginsenoside Rh2 and Rg3 are represented by the formula(1) and (2), respectively.

Description

Ginsenoside Rh2 and Rg3 anticancer composition {Anticarcinogenic constituents of ginsenoside Rh2 and Rg3}

The present invention relates to a composition exhibiting an anticancer effect containing ginsenoside Rh2 of the following structural formula (1) and ginsenoside Rg3 of the following structural formula (2). More specifically, the present invention relates to a composition exhibiting an anticancer effect, wherein ginsenoside Rh2 and ginsenoside Rg3 are contained in a ratio of 1: 1 to 10: 1. The composition containing ginsenoside Rh2 and ginsenoside Rg3 of the present invention in a ratio of 1: 1 to 10: 1 can be used for treating or preventing cancer by preparing and taking food or medicine.

Structural Formula 1

Structural Formula 2

In general, anticancer drugs not only act selectively on cancer cells but also damage normal cells, especially tissue cells with active cell division, resulting in side effects such as bone marrow dysfunction, gastric mucosal damage, and hair loss. That is, since the general anticancer drugs have a disadvantage in that side effects increase as the effect is excellent, the development of anticancer drugs without side effects is urgently emerging.

Many studies have been attempted to develop anti-cancer drugs without such side effects, and in particular, studies to develop anti-cancer drugs using natural foods, such as ginseng or red ginseng, have been widely used.

Ginseng and red ginseng processed ginseng contains a unique saponin existing only in the plant of the ginseng, showing a unique pharmacological activity, 34 such saponins are known until now. Ginseng saponins are classified into ProtoPanaxadiol Ginsenoside, ProtoPanaxatriol Ginsenoside, and Oleanoic Acid Ginsenoside according to the structure of aglycone. Pharmacological efficacy differs depending on the number or location of binding. In particular, compounds in which saccharides such as glucose, rhamnose, arabinose or xylose are bound to the protoparnaxadiol and the protoparnaxatriol, which are triterpenoids of the dammaran series, exhibit unique pharmacological activity.

Various studies have published that ginsenoside Rh2 has an anticancer effect among the 34 ginseng saponins. For example, ginsenoside Rh2 induces regeneration of F9 teratocarcinoma (teratocarcinoma) (Lee et al., Proc. 6th Intl. Ginseng symp., 127, 1993), and Morris liver cancer cells (Odashiam et al., Europ. J. Cancer , 15, 885, 1979) and B16 melanoma (black tumor cells), MK-1 (gastric cancer cells) (Matsunaga et al., Chem. Pharm. Bull., 38, 3480, 1990) and HRA (ovarian cancer cells) (Kikuchi et al., Anticancer Drugs.England, 2, 63, 1991). In addition, in vivo testing of transplanted ovarian cancer cells (HRA) is known to have little toxicity or side effects when used in combination with an anticancer drug cisplatin or when administered alone (Tode et al., J. Cancer Res, Clin. Oncol., 120, 24, 1993), inhibiting ovarian cancer and prolonging survival were similar to cisplatin (Kikuchi et al., Anticancer Drugs, England, 2, 63, 1991).

However, ginsenoside Rh2 has a problem that its anticancer effect is not so remarkable that the cancer treatment effect is insufficient when used alone for cancer treatment. Moreover, ginsenoside Rh2 does not exist in white ginseng (Kitagawa et al., 103, 612, 1983), and red ginseng contains only 0.001% and wild ginseng contains only 0.004%. There is also a limit to increase the anti-cancer effect.

In addition, ginsenoside Rg3 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 Rg3, like ginsenoside Rh2, is insignificant in anticancer effects and is present in trace amounts in 0.003% in 0.0003% red ginseng, which is difficult to enhance the anticancer effect through ingestion.

Therefore, conventional anti-cancer foods or anti-cancer drugs using ginseng or red ginseng, which have been widely used as nutrient tonics, have no side effects, but they are not used alone as anti-cancer drugs due to their lack of effects, and thus may be used as adjuvant drugs of other anti-cancer drugs. The disadvantage is that there is no choice.

Accordingly, the present inventors have long researched to invent pharmaceutical products and food compositions that can prevent or treat cancer using ginseng or red ginseng by improving the effects of ginsenoside Rh2 and ginsenoside Rg3, which have no side effects but lack anticancer effects. As a result, when ginsenoside Rh2 and ginsenoside Rg3 is administered in a ratio of 1: 1 to 10: 1, it was found that excellent anticancer effect appeared, thus completing the present invention.

The composition containing ginsenoside Rh2 and ginsenoside Rg3 of the present invention at a ratio of 1: 1 to 10: 1 may be obtained when ginsenoside Rh2 or ginsenoside Rg3 is administered alone and red ginseng (with ginsenoside Rh2). Ginsenoside Rg3 contained in a ratio of 1: 30) shows an excellent anticancer effect.

Therefore, the present invention provides a composition containing ginsenoside Rh2 and ginsenoside Rg3 in a ratio of 1: 1 to 10: 1, which is superior to ginsenoside Rh2 or ginsenoside Rg3, which lacks an anticancer effect, and has no side effects. The purpose is.

Still another object of the present invention is to provide a drink, tablets, pills, capsules and injections using the composition exhibiting excellent anticancer effects, which can be prevented or treated by continuous administration.

Hereinafter, the present invention will be described in detail.

The composition comprising ginsenoside Rh2 and ginsenoside Rg3 in a ratio of 1: 1 to 10: 1 as an active ingredient according to the present invention exhibits an excellent anticancer effect and thus is useful for preventing or treating cancer. Can be.

Especially preferably, ginsenoside Rh2 and ginsenoside Rg3 are contained in a ratio of 2.5: 1 to 7.5: 1, and more preferably, ginsenoside Rh2 and ginsenoside Rg3 are contained in a ratio of 5: 1. will be.

When the ratio of ginsenoside Rh2 and ginsenoside Rg3 was less than 1: 1, or more than 10: 1, the effect was remarkably decreased or the anticancer effect was not sufficient.

The composition of the present invention is generally administered to the human body of the composition of ginsenoside Rh2 and ginsenoside Rg3, the absorption rate, inactivation rate and excretion rate of the active ingredient in the body, the age, sex and condition of the patient, cancer progression Although appropriately selected depending on the conditions and the like, in general, it is preferable that the adult is administered in a total amount of 1 to 15 mg, preferably 5 to 10 mg per day. If you take less than 1mg per day, the anti-cancer effect is insufficient, and if you take more than 15mg per day, the anti-cancer effect no longer increases.

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 5 to 15 mg in the total amount of ginsenoside Rh2 and ginsenoside Rg3 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 preparation for oral administration such as tablets may be formulated into a formulation coated with enteric skin.

When ginsenoside Rh2 and ginsenoside Rg3, which are the 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 proved from the experimental results described below. As shown, it has no side effects and does not show acute toxicity to experimental animals and can be used safely.

The present invention is explained 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

(1) Preparation of Glycolytic Enzyme Liquid

After incubation at 30 ° C for 80 hours by adding Korean bacteria (Nucillus bacteria) to a medium containing 3% wheat flour and 1% ginseng powder, the cells were removed by centrifugation, and the enzyme protein was precipitated with 70% ethyl alcohol solution. The protein was collected, dissolved in a 1/10 volume of sodium acetate solution (0.02M, pH5.0) of the culture solution, and impurities were removed by centrifugation to obtain a glycolysis enzyme solution.

(2) Preparation of Ginsenoside Rh2

After dissolving 3 g of ginsenoside Rd in 100 ml of acetic acid solution (0.02 M, pH 5.0), 50 ml of the above (1) enzyme solution was added thereto and reacted at 40 ° C. for 20 hours to prepare ginsenoside F2. Then, 1/3 volume of butanol was added, and saponin was extracted three times, and evaporated to dryness under reduced pressure. Acetic acid was converted into ginsenoside Rh2 by hydrolysis of the pull at the 20th carbon position of the ginsenoside F2 molecule with acetic acid. This was separated by silica gel column separation to prepare 1.2 g of ginsenoside Rh2.

Example 2 Preparation of Ginsenoside Rg3

6 g of ginseng irradiated with 20% of 50% acetic acid was prepared using a conventional method of separating the extracted extract using alcohol as an extraction solvent from ginseng or red ginseng, heated with stirring at 70 ° C. for 2 hours, and then the reaction mixture was Cooled down to room temperature. The reaction solution was 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 Rg3.

Experimental Example 1 In-vitro Anticancer Activity of Ginsenoside Rh2 and Ginsenoside Rg3

The anticancer effect of ginsenoside Rh2 and ginsenoside Rg3 according to the present invention was evaluated by the thymidine introduction measurement method as follows.

Dissolve 13.8 g of DMEM (Dulbecco's Modified Eagle's Medium, manufactured by Gibco) in 1 l of deionized water, adjust the pH to 7.4 using sodium carbonate and hydrochloric acid solution, and add 0.21 μm of insulin 1 107M and gentamicin 50 mg / l. After sterilization by filtration, calf serum was added to 5% and used as a culture solution. Human liver cancer cell line SK-HEP-1 received from Seoul National University Cancer Research Institute was inoculated at a rate of 1 106 cells per 25 cm ² of T flask and incubated in a 37 ° C. incubator maintaining 5% carbon dioxide gas for 48 hours. The culture was transferred to a 24-well culture vessel and passaged for 1 day, and then the samples were added to 0.1 to 50 M each. The control group was treated with the same amount of 70% ethanol as a solvent instead of the compound. Twelve hours after the compound treatment was treated with 3H-labeled thymidine at a concentration of 1 Ci / ml, and after 12 hours, the medium was removed from each well, cells were fixed with methanol and washed with PBS. Wash twice with 10% trichloroacetic acid to remove unreacted radioactive thymidine. The cells were lysed with 0.2 M sodium hydroxide-0.5% sodium dodesylsulfate (SDS) and neutralized with hydrochloric acid, and then radioactivity introduced into the DNA was measured by scintillation counter. The results are shown in Table 1.

Effect of thymidine introduced amount (unit:%) Concentration of compound Control 0.1 μm 1 μm 5㎛ 10 μm 25 μm 50 μm Rh2 100 88.5 84.3 81.7 78.1 76.2 75.6 Rg3 100 92.4 90.8 89.6 87.7 85.1 82.3 Rh2: Rg3 = 15: 1 100 74.2 70.2 66.3 67.4 56.8 50.6 Rh2: Rg3 = 10: 1 100 71.4 65.8 57.7 50.9 44.7 38.3 Rh2: Rg3 = 7.5: 1 100 67.2 58.4 50.2 43.9 37.1 33.8 Rh2: Rg3 = 5: 1 100 61.2 52.6 46.8 33.4 24.6 22.5 Rh2: Rg3 = 2.5: 1 100 64.8 54.9 48.4 37.9 33.5 30.2 Rh2: Rg3 = 1: 1 100 76.4 70.3 62.6 55.5 49.1 43.4 Rh2: Rg3 = 1: 2.5 100 77.4 69.5 68.1 53.8 48.4 42.1 Rh2: Rg3 = 1: 5 100 75.4 68.2 50.4 52.5 46.2 40.9 Rh2: Rg3 = 1: 10 100 75.2 73.5 70.1 56.8 50.6 45.6 Rh2: Rg3 = 1: 20 100 77.1 72.4 69.6 72.2 67.1 50.4 Rh2: Rg3 = 1: 30 100 77.2 73.5 70.8 71.5 67.8 53.9

As shown in Table 1, in the case of ginsenosides Rh2 and Rg3 alone, the thymidine incorporation effect was up to 75.6% and 82.3%, and when ginsenosides Rh2 and Rg3 were mixed in 1: 1 or 10: 1 The anticancer effect was significantly higher when combined doses of 43.3% and 38.3% of ginsenosides Rh2 and Rg3 were used, and ginsenosides Rh2 and Rg3 were 2.5: 1, respectively. When mixed to 7.5: 1 it can be seen that the thymidine introduction amount effect is 30.2% to 33.8% very good anticancer effect. However, when ginsenosides Rh2 and Rg3 are contained in a ratio of 15: 1 or 1: 2.5, 1: 5, 1:10, 1:20, and 1:30, the thymidine introduction effect is 40.9% or more. The anti-cancer effect was not so excellent.

Experimental Example 2 In-vivo Anticancer Effect of Ginsenoside Rh2 and Ginsenoside Rg3

In order to confirm the concentration showing the optimal effect using the composition containing the ginsenoside Rh2 and Rg3 5: 1 from the Experimental Example 1 using a mouse transplanted with a human tumor cell WiDr cell line as shown below Anticancer effect experiment was performed.

(1) experimental animals

WiBr transplantation of human tumor cells was performed using BALB / Cr SIc mice of 6-week-old males purchased from clear kk, Japan. Feed and water were sterilized and fed freely during breeding.

(2) transplantation method

WiDr cell lines were incubated at 100% EagleMEM medium (containing 10% FBS) in a 150 cm 2 plastic culture flask at 5% CO ₂ , 37 ° C. The cells were washed with PBS (-) solution, detached from the flask with EDTA solution containing 0.05% trypsin, and prepared by floating the cells by mixing Hanks' solution and PBS (-) solution in a 1: 1 ratio. Cell suspensions were implanted at 2 10 cells (0.2 ml) per animal under the abdominal skin of 5 nude mice. Tumors were removed from the mice with tumor necrosis, the membranes and necrosis were removed, streptomycin and penicillin were added, washed in Han's solution, and evenly cut to 2-3 cm in size. Implantation was done using trocar.

(3) Administration method of drug

The composition containing ginsenoside Rh2 and ginsenoside Rg3 at 5: 1 was diluted in physiological saline for injection to prepare 10, 50, 100, 300, and 500 µg / ml, and orally administered 0.1 ml per 10 g of mouse. . In the control group, physiological saline was administered intraperitoneally with 0.1 ml per 10 g of mouse body weight. Ginsenoside Rh2 and Ginsenoside Rg3 were divided into groups of mouse tumors, and then orally administered 6 times a week for 3-4 weeks.

(4) measuring method

Measure the maximum diameter (L) and the thickness (W) at right angles using a burner caliper and substitute them in the V = 1/2 LW equation. The experiment was divided. At the end of the experiment, tumor diameter was measured twice a week until 3-4 weeks after the start of administration.

(5) results

The composition containing ginsenoside Rh2 and ginsenoside Rg3 as 5: 1 showed an excellent anticancer effect when 100 μg or more per 10 g of mouse body was administered. That is, as can be seen in Table 2, the incidence rate of 60.7% in the 500 µg / ml administration group, 63.9% in the 1000 µg / ml administration group, and 62.2% in the 1500 µg / ml administration group was shown. These results show that the composition containing ginsenoside Rh2 and ginsenoside Rg3 of the present invention shows excellent anticancer effect even in in vivo anticancer experiments. It can be seen that taking from 15mg shows the best anticancer effect. If you take less than 5mg anticancer effect is weak, and if you take more than 15mg does not increase the anticancer effect.

Experimental Example 3 Cytotoxicity Test of Ginsenoside Rh2 and Ginsenoside Rg3

Toxicity of ginsenoside Rh2 and ginsenoside Rg3 according to the present invention was to be evaluated by the efflux test of platelet enzyme (LDH) as follows.

Samples were added to platelets isolated from rats so as to have a concentration of 1000 µg / ml and incubated for 30, 60, 90, and 120 minutes at 37 ° C (sample administration group). Separately, only the solvent in which the sample was dissolved was added to the negative control, and the menadione, which was known to be toxic, was administered to a concentration of 250 M and then cultured under the same conditions as the sample administration group. 100 μl of the culture solution was taken at each time period, followed by centrifugation at 17000 rpm for 2 minutes, and 25 μl of the supernatant was taken. 1.0 ml of Tris-EDTA-NADH buffer solution (pH 7.4) was added thereto and incubated at 37 ° for 10 minutes. 0.1ml of 14mM pyruvate, which had been incubated at 37 ° C, was added thereto, and the degree of platelet enzyme leakage was measured by measuring the decrease in absorbance at 339 nm. The results are shown in Table 3.

Cytotoxicity Measurement Results (Unit:%) Minutes 0 30 60 90 120 Negative Control 0.0 0.7 1.2 1.5 1.7 Menadione 0.0 1.8 22.2 63.2 66.3 Rh2 administration group 0.0 0.4 1.5 1.7 1.9 Rg3 administration group 0.0 0.5 1.8 1.7 2.0 Rh2: Rg3 = 1: 1 0.0 0.8 1.5 1.9 2.1 Rh2: Rg3 = 5: 1 0.0 0.3 1.2 1.5 1.9 Rh2: Rg3 = 10: 1 0.0 0.7 1.0 1.3 1.8

As shown in Table 2, the negative control showed little change with the incubation time, and the menadione administration group showed a marked increase in toxicity with the incubation time. The sample administration group does not show a significant difference from the negative control even at a very high concentration of 1000 ㎍ / ㎖ can be seen that the compositions of the present invention is a very safe composition.

Experimental Example 4 Acute Toxicity Test of Ginsenoside Rh2 and Ginsenoside Rg3

1) Test substance: Ginsenoside Rh2 and Ginsenoside Rg3

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 observed for 14 days.

5) Test Results: As a result of acute toxicity test in rats and mice, the LD50 of this composition in rats and mice was 3g / kg or more.

Acute Toxicity Test Results in Rats and Mice Medication Test animals Test concentration (mg / kg) Number of animals Dead animals % Mortality LD50 Rh2: Rg3 = 1: 1 Rat 01883757501,5003,000 101010101010 000000 000000 > 3g / kg mouse 01883757501,5003,000 101010101010 000000 000000 > 3g / kg Rh2: Rg3 = 5: 1 Rat 01883757501,5003,000 101010101010 000000 000000 > 3g / kg mouse 01883757501,5003,000 101010101010 000000 000000 > 3g / kg

The anticancer composition mainly comprising ginsenoside Rh2 and ginsenoside Rg3 of the present invention may be formulated according to the following example.

[Production Example 1]

Ginsenoside Rh2 5mg

Ginsenoside Rg3 5mg

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 at a final volume of 100 ml.

[Production Example 2]

Corn Starch 44g

40g of crystalline cellulose

Carboxymethylcellulose 5g

Magnesium Stearate 1g

Ginsenoside Rh2 900mg

Ginsenoside Rg3 900mg

90 g in 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 10 mg of ginsenoside Rh2 and ginsenoside Rg3. Adults take 1-3 tablets several times a day.

[Production Example 3]

440 g of crystalline cellulose

Magnesium Stearate 60g

Ginsenoside Rh2 5g

Ginsenoside Rg3 5g

500 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. This capsule contains 10 mg of ginsenoside Rh2 and ginsenoside Rg3. Adults take 1-3 capsules several times a day.

[Production Example 4]

87.5 g of distilled water for injection

5 g of ethanol

Soy Phospholipid 2.5g

glycerin

5 g

Ginsenoside Rh2 1g

Ginsenoside Rg3 1g

100 g in total

Ginsenosides Rh2 and Ginsenosides Rg3 were dissolved in ethanol and soybean phospholipids according to the above-described prescription, and then injected distilled water and glycerin solution were added thereto to prepare an injection.

Ginsenoside Rh2 and ginsenoside Rg3 of the active ingredient according to the present invention containing 1: 1 to 10: 1 composition not only shows an excellent anticancer effect than ginsenoside Rh2 and ginsenoside Rg3 alone, but also has side effects. It can be effectively used for cancer prevention and cancer treatment. In particular, red ginseng contains about 0.001% of ginsenoside Rh2 and about 0.029% of ginsenoside Rg3. That is, there is no synergistic effect because it contains about 1:30 of ginsenoside Rh2 and ginsenoside Rg3. Ginsenoside Rh2 and Ginsenoside Rg3 of the present invention when used in a composition containing 1: 1 to 10: 1 shows an excellent anti-cancer effect by a synergistic effect can be very useful in the prevention or treatment of cancer.

Claims (7)

An anticancer composition comprising ginsenoside Rh2 and ginsenoside Rg3 in a range of 1: 1 to 10: 1. The anticancer composition according to claim 1, which contains one or two or more pharmaceutically acceptable carriers and additives. The anticancer composition according to claim 2, wherein the carrier is selected from diluents, lubricants, binders, disintegrants, sweeteners, stabilizers, and preservatives. The anticancer composition according to claim 2, wherein the additive is selected from fragrances or vitamin B groups. 2. The anticancer composition according to claim 1, wherein the daily oral dose is 5 mg to 15 mg in the total amount of ginsenoside Rh2 and ginsenoside Rg3. The anticancer composition according to claim 2, wherein the dosage form is selected from drink, tablet, pill, capsule or injection. The anticancer composition according to claim 6, wherein the total amount of ginsenoside Rh2 and ginsenoside Rg3 is contained in an amount of 5 mg to 15 mg per each formulation.