KR20060035289A - Antitumor pharmaceutical composition containing red ginseng acid polysaccharide and shiitake mushroom - Google Patents

Abstract

The present invention relates to an anti-tumor pharmaceutical composition containing red ginseng acid polysaccharide and shiitake mushroom extract as an active ingredient. The pharmaceutical composition containing red ginseng acid polysaccharide and shiitake mushroom according to the present invention selectively increases the concentration of immunoglobulin IgM, thereby rapidly recovering immunity in vivo, increasing survival rate, maximizing life extension effect and anti-tumor effect, and thus being used for effective cancer treatment. Can be.

Red Ginseng Acid Polysaccharide, Shiitake, Antitumor, Immunoglobulin IgM, Selective Concentration

Description

Anti-tumor pharmaceutical composition containing red ginseng acid polysaccharide and shiitake mushrooms {PHARMACEUTICAL COMPOSITIONS, COMPRISING RED GINSENG ACID POLYSACCHAIDE AND LENTINUS EDODES, OF PREVENTING AND TREATING CANCER}

The present invention relates to an anti-tumor pharmaceutical composition that can be used for effective cancer treatment by increasing the selective concentration of immunoglobulin IgM, thereby rapidly recovering immunity in vivo to increase survival rate, maximizing lifespan and antitumor effects.

Anticancer drugs are generally classified into chemotherapy and biotherapy. Biotherapeutics are based on the prevention of cancer progression by indirectly weakening the activity of cancer cells, and there are cytokines, immunotherapeutics, gene therapy, and angiogenesis inhibitors. Immunotherapeutic agents, one of biotherapeutics, do not show direct anticancer activity, but rather prevent or reverse the progression of cancer cells by restoring or increasing the immune function of the body.

In recent years, anti-cancer research by biotherapy has been directed toward the development of cytostatic drugs with high selectivity in the development of cytotoxic drugs with low selectivity for cancer cells. The research is based on the research of natural products to develop anticancer active substances from substances.

Among the natural materials, red ginseng is manufactured as a unique technology of Korea, and is a globally recognized herbal processing material. It has various effects such as anti-cancer effect, anti-diabetic effect and immune regulation function. In particular, the red ginseng acid polysaccharide among the main components of the red ginseng was separated from the red ginseng in the Republic of Korea Patent Publication No. 2002-94725 by the present inventors to have an anti-cancer immunomodulatory effect, which is a macrophage which is a major cell of the innate immune system ( It acts on macrophage and natural killer cells (NK) to enhance cytokines, such as NO and TNF-α, or to activate the function of these cells. In this case, NO exhibits anti-cancer activity by destroying bacteria and cancer cells, regulating mitogen cell proliferation and T cell activity, and TNF-α (tumor necrosis factor-α) causes hemolytic necrosis of tumor cells, septic shock and local inflammation. It is mediated by pathophysiology in tissues to show anticancer activity.

Red ginseng acid polysaccharide (hereinafter referred to as 'RGAP') has the above anti-cancer immunomodulatory effect and thus increases the concentration of immunoglobulin (Ig), which plays a major role in humoral immunity. Can be predicted.

However, the increase in the concentration of red ginseng acid polysaccharide (RGAP), which is known by the present inventors, to the IgM in immunoglobulins, depending on the dosage, is a mechanism showing an anti-cancer immune effect.

Immunoglobulin IgM, unlike IgG, is made faster than other immunoglobulins (Ig) for antigen stimulation to the extent that it is also called an initial antibody, and is an immune factor that acts greatly on the lytic action of complement. And the ability to treat cancer cells with a rapid rate of proliferation early.

On the other hand, in the study to investigate the anticancer effect of the conventional red ginseng acid polysaccharide (RGAP), the survival rate was 57.1, 85.7% after RGAP 100, 300mg / kg 30 days after the administration of sarcoma 180 cancer cells to mice intraperitoneally transplanted In addition, anti-cancer activity of B16 melanima cancer cells was inhibited by 39% and 53%, respectively (Kim Young-sook, Park Kyung-mi, Shin Han-jae, Nam Ki-yeol, Park Jong-dae, RGAP's macrophage and natural killer cell activation. Journal, 46 (2), 113-119. 2002). In addition, according to the Republic of Korea Patent Publication No. 2002-94725, RGAP 30, 100, 300mg / kg administered to the mouse to examine the cancer cell killing ability of natural killer cells as a result of the dose of 9.9 to 22.0% compared to the control group, 30mg / kg, In the case of 100 mg / kg, it was 16.0 to 26.1%, and in the case of 300 mg / kg, 14.9 to 30.0%. In addition, as a result of investigation of cancer cell killing ability by macrophages, the dose of 100 mg / kg showed an increase of 36.3% cancer cell killing capacity at 14.3% and 300 mg / kg compared to the control group. It can be seen that the anticancer activity efficacy is associated with an increase in dosage.

However, as can be seen in the Republic of Korea Patent Publication No. 2002-94725, the red ginseng acid polysaccharide (RGAP) from the expensive red ginseng has a problem that only a small amount of extraction (yield 6.7%).

The present invention has been confirmed that shiitake mushroom is a cheaper, mass-produced anti-cancer activity substitute that has less side effects and toxicity and is equal to or higher in efficacy, such as red ginseng acid polysaccharide (RGAP).

Shiitake mushroom (Lentinus edodes) shows that LC-33, a polysaccharide isolated from its fruiting body, shows potent anti-tumor activity against sarcoma-180 and promotes cellular immune response (Chihara, G., Hamuro, T. and Maeda, Y., Fractionnation and purification of thew polysaccharide with marked antitumor activity especially lentinan from Lentinus edosed, Cancer Res., 30: 2776-2781, 1970. Maeda, Y. and Chihara, G., Lentinan a new immuno-accelerator of cell mediatrd responses.Nature 229: 634, 1971).

The anticancer activity of shiitake mushrooms is known to be due to protein-bound polysaccharide (PBP) and lentinan substances. PBP indirectly increases the growth of tumor cells by enhancing the intrinsic defense against tumors by promoting the production of intephenone. Inhibits (Tsunoda, A, A mushroom agents and their mechanisms lentinan, a T-cell oriented immunopotentiator, NY and Basel., Vol. 19, p.436, 1985), or plays an important role in directly killing cancer cells or pathogenic bacteria. Anti-cancer effect by increasing the number of macrophages (Takehara, A., Antiviral activity of virus -like particles from lentinus edodes, Shiitake. Arch Virol, 59: 269-280, 1979), isolated from the fruiting body of shiitake mushroom Lentinan, a polymer β-1,3 glucan, has anticancer effects by enhancing immunity (Suga, T., Dhiio, T., Maeda, Y, Y. and Chihara, G, Antitumor activity of lentinan in murine syngeneic and autotochthonous ho sts and its suppressive effection 3-methycholanthrene-induced carcinogenesis. Cancer Res., 44: 5132-5137, 1984).

The present inventors found that RGAP was specifically concentration dependent only on immunoglobulin IgM while studying alternatives of red ginseng acid polysaccharide (RGAP), and Shiitake mushroom extract was also selective on immunoglobulin IgM. By confirming that the concentration is increased, and by preparing a combination composition of RGAP and shiitake mushroom in the range of RGAP minimum dose as an RGAP substitute, the complex composition selectively generates immunoglobulin IgM to rapidly recover the immunity in vivo to increase survival rate It was confirmed that the life extension effect and anti-tumor effect can be maximized and completed the present invention.

An object of the present invention is to provide a red ginseng acid polysaccharide (RGAP) complex composition that can replace the red ginseng acid polysaccharide (RGAP) of the anti-tumor effect by a single administration.

In another aspect, the present invention is to provide a pharmaceutical composition that can quickly recover the immunity in vivo by increasing the selective concentration of immunoglobulin IgM to increase the survival rate and maximize the life extension effect and anti-tumor effect.

The present invention provides a complex composition consisting essentially of red ginseng acid polysaccharide (RGAP) and shiitake mushroom extract.

The red ginseng acid polysaccharide (RGAP) and shiitake mushroom extract used in the present invention may be administered in a range of significant minimum doses, wherein the red ginseng acid polysaccharide (RGAP) and shiitake mushroom composite compositions are administered more than each single administration. It has more than equivalent anticancer effect.

In addition, the composite composition according to the present invention selectively increases the concentration of immunoglobulin IgM, thereby rapidly recovering immunity in vivo, maintaining high survival rate, and maximizing life extension and antitumor effects.

In particular, the composite composition according to the present invention can promote the production of immunoglobulin IgM when red ginseng acid polysaccharide (RGAP) and shiitake mushroom extract are mixed in the same weight ratio, and can increase the life-extension effect antitumor effect.

Referring to the present invention in more detail as follows.

The red ginseng acid polysaccharide (RGAP) of the present invention is prepared by a method known from red ginseng, and preferably prepared by the method described in Korean Laid-Open Patent Publication No. 2002-94725.

Shiitake mushroom extract of the present invention can be prepared by a known method using water or a lower alcohol, it is preferable to use an extract of hot water treatment (90 ℃ or more) with water.

In addition, shiitake mushroom extract according to the present invention is a polysaccharide LC-33 isolated from the shiitake fruiting body described in the above-mentioned documents, lentinan or PBP (protein-bound polysaccharide) of the polymer β-1,3 glucan isolated from the fruiting body ) May contain one or more components.

In the present invention, red ginseng acid polysaccharide (RGAP) and shiitake mushroom extract may be mixed in an appropriate ratio, and preferably mixed in a weight ratio of 1: 1.

The pharmaceutical composition of the present invention increases the initial survival rate by rapidly recovering immunity in vivo by selectively increasing the concentration of immunoglobulin IgM, which is an immune factor unlike the conventionally known mechanisms for enhancing immunity due to macrophages and natural killer cell ability. In addition, it exhibits the synergistic effect of anti-tumor activity by maximizing the life-span extension and the inhibition of solid cancer development in animals with cancer.

In addition, in the pharmaceutical composition of the present invention, the dose of RGAP having an antitumor effect depending on the dose shows an antitumor effect equal to or higher than that of a significant RGAP alone at the minimum dose of which suspicion is significant. RGAP can be usefully used in economical, side effects and low toxicity anticancer drugs.

In addition, the composition of the present invention increases the selective concentration of immunoglobulin IgM in the composition of RGAP and shiitake extract in a weight ratio of 1: 1 compared to the case of administering more than about 2 weight ratios alone, and prolongs the lifespan of animals suffering from cancer. And it can be seen that the anti-cancer activity effect is maximized because the solid cancer inhibiting effect is excellent.

The pharmaceutical composition of the present invention may include one or more pharmaceutically acceptable carriers in addition to the RGAP and shiitake mushroom extract, and may be prepared by solid methods such as tablets, hard and soft capsules, pills, powders, and oral liquids by conventional methods. It may be formulated as a parenteral preparation such as oral preparations or injections, such as liquid preparations such as syrups and suspensions.

The daily dosage of the pharmaceutical composition according to the present invention to the human body may vary depending on various factors such as the type and severity of cancer, age, and patient's condition, but in general, the pharmaceutical composition containing RGAP and shiitake extract If the single dose is preferably 5mg / kg to 150mg / kg, more preferably 10mg / kg to 100mg / kg.

Hereinafter, the present invention will be described in detail by examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited by the examples.

Example 1 Immunoglobulin Concentration Effect Experiment

The concentration change of immunoglobulin IgG and IgM for the pharmaceutical composition of the present invention was observed.

end. Sample Preparation

RGAP was separately prepared by the method described in Korean Patent Application No. 2001-33115. More specifically, 10 g of red ginseng was added to 10% of 85% ethanol and extracted three times at 70 ° C., followed by centrifugation to recover the residue. The resultant was dried by ventilation and extracted four times with 10 L of water at 80 ° C. to obtain a water-soluble extract. The extract was dialyzed with distilled water for 4 days in a refrigerating chamber using a dialysis membrane having a molecular weight cut-off value of 12 kD or more to obtain a polymer dialysis solution of 12 kD or more, which was centrifuged at 8,000 rpm for 20 minutes to obtain a supernatant. Precipitated polysaccharide RGAP fraction was prepared by freezing and drying the precipitate obtained by centrifugation (8,000 rpm, 30 minutes) from the supernatant by adding 85% ethanol to 5 times.

Shiitake mushroom extract was prepared by extracting with hot water at high temperature (90 ℃) and then filtered.

Sample 1: Sample 1 was prepared by mixing the prepared RGAP and shiitake mushroom extract in the same weight ratio.

Sample 2: Sample 2 was prepared by mixing the prepared RGAP and shiitake extract in a weight ratio of 1: 2.

Sample 3: Sample 3 was prepared by mixing the prepared RGAP and shiitake extract in a weight ratio of 1: 3.

I. Experiment method

Cancer cells (Sarcoma 180) were diluted in physiological saline (1 × 10 ⁶ cells / ml) and injected into the abdominal cavity of mice (20-23 g, 4-5 weeks old ICR mice, 10 mice each). After 24 hours, the normal saline (control), RGAP, shiitake mushroom extract alone and the samples 1, 2, and 3 were administered for 1 day as shown in Table 1 below, and then blood was collected from the heart to separate serum. The concentrations of IgM and IgG, which are immune factors, were then measured from the serum. The normal group was measured the concentration of IgM and IgG after the administration of physiological saline to mice not administered cancer cells (Sarcoma 180) (Table 1).

All. Experiment result

Test group Single dose (mg / kg)   IgM (mg / dl) IgG (mg / dl)  Normal group (physiological saline) - 10.9 ± 3.9    11.6 ± 6.3  Control group (physiological saline) - 9.6 ± 4.2    10.7 ± 7.6  RGAP 50 24.8 ± 6.9 ^{*, #} 21.1 ± 7.4 ^{*, #}  RGAP 100 28.5 ± 7.5 ^{*, #} 17.1 ± 5.9 ^{*, #}  Shiitake Mushroom Extract 50 19.5 ± 8.1 ^{*, #} 15.2 ± 6.2 ^{*, #}  Shiitake Mushroom Extract 100 22.6 ± 7.5    16.5 ± 7.1  Shiitake Mushroom Extract 150 24.4 ± 8.0    17.5 ± 6.8  Sample 1 50 + 50 29.4 ± 9.8 ^{*, #} 17.5 ± 7.6 ^{*, #}  Sample 2 50 + 100 26.4 ± 9.1    17.9 ± 8.2  Sample 3 50 + 150 25.9 ± 9.5    18.4 ± 7.7  *: p <0.01 control vs. sample group, #: normal vs. sample group

As shown in Table 1, the concentration of IgM and IgG in all the sample administration group significantly increased compared to the control group, especially in the RGAP and shiitake mushroom extract alone group, the concentration of IgM compared to the control group of 9.6 ± 4.2 mg / dl, respectively It was increased to 24.8 ± 6.9, 28.5 ± 7.5 mg / dl and 19.5 ± 8.1, 22.6 ± 7.5, 24.4 ± 8.0 mg / dl. IgG showed little change in dose, but the concentration of IgM increased depending on the dose of RGAP and shiitake extract, respectively.

The groups administered with Samples 1, 2, and 3 were 29.4 ± 9.8, 26.4 ± 9.1, and 25.9 ± 9.5 mg / dl, which were higher than the group administered alone. The concentration increase rate compared to the control group was increased to 306.3%, 275.0%, and 269.8% of Samples 1, 2, and 3, respectively, and the group administered with Sample 1 showed the highest concentration increase rate. The group administered the composition of Sample 1 showed an increase rate of 296.9% or more in the RGAP 100 mg / kg administration group even when 50 mg / kg of RGAP was used.

In the pharmaceutical composition containing RGAP and shiitake extract, the concentration of immunoglobulin IgG and IgM was increased compared to the control group, but only the concentration of IgM was specifically increased for the administration group alone.

Therefore, the pharmaceutical composition of the present invention can initially treat cancer cells rapidly dividing and proliferating by rapidly restoring immunity in vivo by increasing the selective concentration of immunoglobulin IgM.

In addition, the pharmaceutical composition of the present invention exhibited a synergistic effect of antitumor activity due to the superior anti-cancer immunity effect in the minimum dose of the composite composition having a suspicion of significance compared to the RGAP monotherapy group having antitumor effect depending on the dose. In particular, the pharmaceutical composition of RGAP and 50 mg / kg dose of shiitake mushroom extract at a 1: 1 ratio is found to significantly increase the concentration of IgM, thereby maximizing antitumor activity.

Example 2 Life Extension Effect Experiment

The effect of extending the lifespan of animals suffering from cancer on the pharmaceutical composition of the present invention was confirmed.

end. Sample Preparation

Samples 1, 2 and 3 prepared in Example 1 were used for this experiment.

I. Experiment method

Cancer cells (Sarcoma 180) were diluted in physiological saline (1 × 10 ⁶ cells / ml) and injected into the abdominal cavity of mice (20-23 g, 4-5 week old ICR mice, 10 mice each). After 24 hours, physiological saline, RGAP or shiitake mushroom extract alone and the samples 1, 2, and 3 were administered once per day for 7 days as shown in Table 2 below, and then the survival rate of the mice was measured. The results are shown in Table 2 below. The survival rate was expressed by the method of calculating "survival rate = number of surviving cancer cell transplants / number of initial cancer cell transplants x 100" 30 days after administering a pharmaceutical composition to the cancerous mouse.

All. Experiment result

Test group Single dose (mg / kg) Average number of days living (day) Survival rate ¹⁾ (%)   Control group (physiological saline) -   15.3 ± 4.1 0   RGAP 50 19.8 ± 6.9 ^** 30   RGAP 100 25.4 ± 7.9 ^** 70   Shiitake Mushroom Extract 50 21.3 ± 6.9 ^** 30   Shiitake Mushroom Extract 100   22.1 ± 7.0 40   Shiitake Mushroom Extract 150   23.0 ± 5.8 50   Sample 1 50 + 50 25.5 ± 7.3 ^** 70   Sample 2 50 + 100   24.6 ± 6.5 60   Sample 3 50 + 150   24.8 ± 7.2 60 ** p <0.01 control vs administration group

As can be seen in Table 2, the survival rate of the RGAP (50, 100 mg / kg) or shiitake mushroom extract (50, 100, 150 mg / kg) alone-administered groups in the cancer cell (Sarcoma-180) transplanted mice was examined. , 70, 30, 40 and 50%. The survival rate of the groups administered with Samples 1, 2 and 3 was higher than that of the group administered alone. In particular, the survival rate of the group administered with Sample 1 was the highest as 70%, and the survival rate of the group administered with Samples 2 and 3 was 60%. In addition, the group administered with Sample 1 showed the same effect as the survival rate of 70% of the RGAP 100 mg / kg alone group even when using RGAP 50 mg / kg.

Therefore, it can be seen that the pharmaceutical composition of the present invention exhibits an effect of extending the lifespan of the equivalent in the minimum dose of the composite composition, which is suspected of significance, when RGAP alone is administered with RGAP, which shows anticancer activity effects depending on the dose.

Example 3 Solid Cancer Inhibition Experiment

Solid tumor model was used to specifically demonstrate the antitumor synergistic effect of the pharmaceutical composition according to the present invention. The solid cancer model confirmed the inhibitory effect by extracting solid cancer after a certain period of time after administering a sample to a mouse transplanted with B16 melanoma cells.

end. Sample Preparation

Samples 1, 2 and 3 prepared in Example 1 were used for this experiment.

I. Experiment method

Melanoma (B16 melanoma cells) was diluted to a constant concentration (1 × 10 ⁵ cells / ml) and then subcutaneously injected (100 μl) into the dorsal portion of mice (C57BL / 6 mice). 24 hours after the melanoma transplant, physiological saline, RGAP or shiitake mushroom extract alone and RGAP + shiitake mushroom extract mixed composition were administered once a day as shown in Table 2 once daily for 7 days. Twenty days from the day of administration, the amount of solid cancer produced by removing the solid cancer was measured to determine the degree of inhibition of solid cancer production (Table 3).

All. Experiment result

Test group Single dose (mg / kg) Solid cancer weight (mg) Solid cancer suppression rate (%)    Control group (physiological saline) - 2120 ± 870 -    RGAP 50 860 ± 300 ^** 59.4    RGAP 100 570 ± 250 ^** 73.1    Shiitake Mushroom Extract 50 1640 ± 660 22.6    Shiitake Mushroom Extract 100 1480 ± 420 30.2    Shiitake Mushroom Extract 150 850 ± 236 59.9    Sample 1 50 + 50 160 ± 60 ^** 92.5    Sample 2 50 + 100 360 ± 100 83.0    Sample 3 50 + 150 700 ± 220 67.0 ** statistically significant compared to control at p <0.01

As can be seen in Table 3, as a result of investigating the degree of inhibition of solid cancer production by administration of RGAP or shiitake mushroom extract alone, the samples 1, 2, and 3, all were significantly inhibited compared to the control group, RGAP (50, 100 mg / kg) and shiitake mushroom extracts (50, 100, 150 mg / kg) alone showed 59.4%, 73.1%, 22.6%, 30.2%, and 59.9% inhibition, respectively. Samples 1, 2, and 3 administration groups were 92.5%, 83.0%, 67.0% inhibited, respectively.

Therefore, the pharmaceutical composition containing the RGAP and shiitake mushroom extract according to the present invention has a solid cancer production inhibitory effect in the combination of RGAP having an anticancer activity depending on the dose of the RGAP alone in the minimum dose of the composite composition suspected of significant significance It was generally higher than the administration group. In particular, the sample containing RGAP and shiitake mushroom extract in a ratio of 1: 1 and 1: 2 has a significantly higher inhibition rate of solid cancer production than the group administered with RGAP in an amount of 2 weight fold than RGAP contained in the pharmaceutical composition of the present invention. The shiitake mushroom extract alone showed more significant differences than expected.

In addition, this experiment can confirm the significant increase in the effects of RGAP and shiitake extract.

The pharmaceutical composition of the present invention shows a better antitumor effect than RGAP having an anti-tumor effect depending on the dose of the composite composition of the minimum dose, which is suspected to be more significant than the RGAP alone dose, so that only a small amount is extracted from the expensive red ginseng. RGAP can be useful for anticancer drugs that are economical and have low side effects and low toxicity.

In addition, the composition of the present invention by selectively increasing the concentration of immunoglobulin IgM to quickly restore the immune system in vivo, to prolong the life of the animal with cancer, and to inhibit the formation of solid cancer to create a synergistic effect of anti-tumor to rapidly divide and Proliferating cancer cells can be effectively treated early on.

Claims (12)

Red ginseng acid polysaccharide and shiitake mushroom extract in a 1: 1 weight ratio, selectively increasing the concentration of immunoglobulin IgM and having antitumor activity. Red ginseng acid polysaccharide and shiitake mushroom extract in a 1: 2 weight ratio, selectively increasing the concentration of immunoglobulin IgM and having antitumor activity. Red ginseng acid polysaccharide and shiitake mushroom extract in a 1: 3 weight ratio, selectively increasing the concentration of immunoglobulin IgM and having an anti-tumor activity. An anti-tumor agent containing red ginseng acid polysaccharide (RGAP) and shiitake mushroom extract as an active ingredient and a pharmaceutically acceptable carrier. The anti-tumor agent according to claim 4, wherein the shiitake mushroom extract is selected from LC-33, a polysaccharide isolated from shiitake fruiting body, lentinan, a protein β-1,3 glucan isolated from fruiting body, or protein-bound polysaccharide (PBP). . The anti-tumor agent according to claim 4, wherein the red ginseng acid polysaccharide and shiitake extract are contained in a weight ratio of 1: 1 to 1: 3. The antitumor agent according to claim 4, wherein the tumor is solid cancer. An immunopotentiator containing red ginseng acid polysaccharide (RGAP) and shiitake mushroom extract, and comprising a pharmaceutically acceptable carrier. 9. The immunopotentiator according to claim 8, wherein the red ginseng acid polysaccharide and shiitake extract are contained in a weight ratio of 1: 1 to 1: 3. 10. The immunopotentiator according to claim 8 or 9, wherein the immunopotentiating effect selectively increases the concentration of immunoglobulin IgM. An anti-tumor pharmaceutical preparation containing the composition according to any one of claims 1 to 3 as an active ingredient and formulated in an oral or parenteral dosage form. A pharmaceutical preparation for immuno-enhancing, comprising the composition according to any one of claims 1 to 3 as an active ingredient and formulated in an oral or parenteral dosage form.