KR20240039939A - A composition comprising ginsenoside showing anti-cancer activity against breast cancer as an active ingredient - Google Patents

Abstract

The present invention relates to a composition containing ginsenoside, which has anticancer activity against breast cancer, as an active ingredient. In particular, it is based on an experiment that revealed the type, mixing ratio, and concentration of ginsenosides that have anticancer activity against breast cancer by inducing the death of breast cancer cells.

Description

A composition comprising ginsenoside showing anti-cancer activity against breast cancer as an active ingredient}

The present invention relates to a composition containing ginsenoside, which has anticancer activity against breast cancer, as an active ingredient. In particular, it is based on an experiment that revealed the type, mixing ratio, and concentration of ginsenosides that have anticancer activity against breast cancer by inducing the death of breast cancer cells.

Ginseng is a type of ginseng (Panax ginseng C. A. Meyer) from the aralia family. The effects of ginseng include promoting metabolism, lowering blood sugar, lowering blood pressure, improving immunity, suppressing cancer cells, relieving fatigue, and anti-aging.

This is an effect caused by ginsenoside, an active ingredient present in ginseng. Depending on the structure, ginsenoside is divided into Protopanaxadiol-type, Protopanaxatriol-type, and Olea. It is classified as Oleanane-type.

Ginsenosides in ginseng are known to have excellent immunity-boosting effects, but their absorption rate in the body is low. In general, many glycoside compounds that exist in nature tend to increase their physiological activity when the sugar is decomposed into a non-saccharide form rather than by itself. In the case of ginsenosides, ginsenosides Rb1 and Rb2 with three or more sugars bonded to each other. , Rd, and Re, ginsenosides Rg3, Rh1, Rh2, F2, CY, and CK, which are produced by hydrolyzing some sugars, are known to have much better effects in terms of absorption into the body and physiological activity. .

In other words, it is known that the amount of ginseng saponin, which is highly bound to sugar, is absorbed into our body within the small intestine is very small. As a result of testing the hydrolyzing ability of ginsenoside Rb1 of intestinal microorganisms extracted from human excrement, the 21% were found to have no decomposition ability, and it was confirmed that about 70% of the intestinal microorganisms with decomposition ability had significant differences in their ability to decompose ginseng saponin. In addition, according to data from the Ministry of Food and Drug Safety that measured the absorption rate of ginsenoside, 25% of test subjects were unable to fully absorb ginsenoside into the blood due to enzyme inactivation of intestinal microorganisms. In addition, even if ginsenosides were absorbed, there were individual differences in efficacy and did not appear to be related to gender or age. This leads to the conclusion that the reason the efficacy of ginseng varies from person to person is because of intestinal microorganisms.

In the end, ginsenosides in their natural state must be converted into substances that can be absorbed in the body by intestinal microorganisms to show their efficacy. These substances are Compound K and Compound Y, which are the final metabolites and bioactive substances of ginsenosides. (Compound Y). In other words, saponin in its natural state, which is bound to several sugar molecules, must be converted into Compound K and Compound Y, which have fewer sugar molecules bound together, by the decomposition enzymes of intestinal microorganisms in order for it to be properly absorbed into the body and to obtain bioactive effects.

Accordingly, it is desirable to convert the protopanaxadiol-based ginsenosides Rb1, Rb2, Rd, Rc, Rg3, and Rh2 into compound K and compound Y to improve absorption in the body and maximize bioactivity. In addition, it is desirable to maintain functionality by including the protopanaxatriol-based ginsenosides Re, Rf, Rg1, Rg2, and Rh1 as is. For example, Rg2 has an anti-thrombotic effect in inhibiting platelet aggregation and an effect in suppressing the secretion of stress hormones such as catecholamines from the adrenal cortex.

The present applicant applied for an invention that can simultaneously increase the content of compound K and Y, and this application was published as Patent Publication No. 10-2022-0009830. Looking at Table 2, it can be seen that the contents of compounds K and Y are simultaneously increased and the protopanaxatriol-based ginsenosides are maintained. In particular, it can be seen that the contents of Compound K and Compound Y are contained in a ratio of 2.29 to 2.65:1.

Meanwhile, looking at patents related to anti-cancer compositions for breast cancer containing ginseng ginsenoside as an active ingredient, Patent Registration No. 10-1237428 describes “Pharmaceutical composition for treating breast cancer containing processed red ginseng extract.” In Experimental Example 2, 100 mg/kg of Rg3-RGP, including ginsenosides Rg1, Re, Rh1, Rb1, Rc, Rb2, Rd, Rg3, Rb3, Rg2 and Rh2, did not suppress the tumor size of liver cancer. However, the tumor size of lung cancer was suppressed. Of note, compared to the RGP component, the Rb1, Rc, Rb2, Rd, and Rg3 of the Rg3-RGP component were significantly increased. (See Table 2, identification numbers [0046]~[0065])

Additionally, “Ginsenoside composition for treating lung cancer” is described in Registered Patent Publication 10-0892764. Looking at Example 2-2, it is described that the active ingredient H-2 has an anticancer effect on breast cancer cells NCI-H23. (Refer to identification numbers <41>~<50>) Active ingredient H-2 has Rb1 16-23%, Rg1 19-25%, Rd 4-6.3%, Re 2-3.8%, Rg3 5-7.5%, Rh1 2 It contains -3.8%, Rh2 7-11.5%, and PPT 0.8-1.5%. (See claim 1)

Republic of Korea Patent Publication 10-2022-0009830 (published on January 25, 2022) Republic of Korea Patent Publication No. 10-1237428 (registered on February 20, 2013) Republic of Korea Patent Publication No. 10-0892764 (registered on April 2, 2009)

The problem is to provide a composition containing ginsenoside, which has anticancer activity against breast cancer, as an active ingredient.

The challenge is to find out the type, mixing ratio, and concentration of ginsenosides that have anticancer activity against breast cancer by inducing the death of breast cancer cells, and to provide a composition reflecting this.

The solution is a health functional food composition for preventing or improving breast cancer, which contains 100 parts by weight of Compound Y and 200 to 400 parts by weight of Compound K as active ingredients.

The solution is, in the above,

Characterized in that 5 to 11 parts by weight of ginsenoside Rg1+Re, 7 to 13 parts by weight of Rf, 0.3 to 0.9 parts by weight of Rg2, 20 to 60 parts by weight of Rh1 and 3 to 9 parts by weight of F2 are additionally included in the above active ingredients. It is a health functional food composition for preventing or improving breast cancer.

The solution is, in the above,

It is a health functional food composition for preventing or improving breast cancer, characterized in that the concentration of the active ingredient is 62.5 to 500 μg/mL.

The solution is a pharmaceutical composition for treating breast cancer containing 100 parts by weight of Compound Y and 200 to 400 parts by weight of Compound K as active ingredients.

The composition according to the present invention possesses anti-cancer activity against breast cancer by inducing the death of breast cancer cells. This is based on the expression of factors related to breast cancer cell death, the cleavage phenomenon of breast cancer cell DNA, and the results of morphological analysis of breast cancer cells.

Figure 1 is a graph showing the results of analyzing the content of the ginsenoside active ingredient included in the composition of the present invention.
Figure 2 is a graph showing the results of normal cell survival rate analysis of the ginsenoside active ingredient included in the composition of the present invention, and Figure 3 is a graph showing the results of breast cancer cell survival rate analysis.
Figures 4 and 5 are graphs showing the results of analysis of the expression of proteins related to breast cancer cell activity.
Figures 6 and 7 are graphs showing the results of analyzing the mechanism of suppressing breast cancer cells.
Figure 8 is a photograph of the results of electrophoresis analysis of DNA from breast cancer cells.
Figures 9 and 10 are photographs showing the results of morphological confirmation of breast cancer cell apoptosis.

The terms and words used in this specification and claims are consistent with the technical idea of the present invention based on the “principle that the inventor can appropriately define the concept of the term in order to explain his or her invention in the best way.” It should be interpreted with the same meaning and concept, and should not be interpreted limited to the usual or dictionary meaning.

Therefore, the embodiments described in this specification and the configurations shown in the drawings are only the most preferred embodiments of the present invention, and do not represent the entire technical idea of the present invention, so various equivalents that can replace them at the time of filing the present application It should be understood that there may be variations.

Example 1. Preparation of active ingredients that can prevent, improve or treat breast cancer

The active ingredient according to the present invention can be manufactured using the prior application technology for simultaneous enhancement of compounds K and Y owned by the present applicant. However, the active ingredient manufacturing process according to the present invention is not limited to the technology of the preceding application, and it is natural that any process that satisfies the content ratio of ginsenosides contained in the active ingredient can be adopted.

Additionally, the ginsenoside included in the active ingredient according to the present invention may be a ginsenoside manufactured by processing ginseng, such as ginseng or red ginseng, through processes such as steaming or fermentation.

Ginsenosides as active ingredients included in the composition of the present invention include Rg1, Re, Rf, Rg2, Rh1, F2, Compound K and Compound Y. In particular, the content ratio of Compound K (C-K) and Compound Y (C-Y) should be maintained at 2 to 4:1, or 2.5 to 3.5:1.

The active ingredient included in the composition of the present invention has the following content. Based on Table 2 in the following experimental examples.

ingredient Rg1+Re Rf Rg2 Rh1 F2 C-Y C-K weight part 5~11 7~13 0.3~0.9 20~60 3~9 100 200~400

In summary, the active ingredients of the composition of the present invention include 5 to 11 parts by weight of ginsenoside Rg1+Re, 7 to 13 parts by weight of Rf, 0.3 to 0.9 parts by weight of Rg2, 20 to 60 parts by weight of Rh1, and 3 to 9 parts by weight of F2. , 100 parts by weight of compound Y and 200 to 400 parts by weight of compound K. Additionally, the composition of the present invention contains the active ingredient at a concentration of 62.5 to 500 μg/mL. This is based on the following experimental example.

Experimental Example 1. Efficacy in preventing, improving or treating breast cancer

The present applicant conducted an experiment naming the active ingredient as CKY, and accordingly used the symbol CKY as an indication of the active ingredient in the drawings.

Apoptosis refers to the active death of cells that occurs by regulating the expression and activity of various genes and proteins according to signals programmed inside the cell. Apoptosomes produced through this process are removed by phagocytosis of surrounding cells or macrophages, so they do not cause inflammation.

Cell death not only plays an important role in maintaining various physiological functions of living organisms, but is also closely related to the pathogenesis of various diseases.

Morphological and physiological characteristics of cell death include cytoplasm shrinkage, membrane blebbing, chromatin condensation, DNA fragmentation, and loss of phosphatidylserine, a cell membrane lipid, outside the cell. Exposure to toxins and formation of apoptosomes have been reported.

Unlike apoptosis, cell necrosis is a passive death that occurs rapidly due to changes in the external environment, and goes through the processes of irregular clumping of chromatin and swelling of cytoplasm, and ultimately leads to cell decomposition. Through this, cell debris is created, which causes inflammation.

1-1. Ginsenoside content of active ingredient

Figure 1 is a graph showing the results of analyzing the content of the ginsenoside active ingredient included in the composition of the present invention. This quantitative analysis is shown in Table 2 below.

ingredient Rg1+Re Rf Rg2 Rh1 F2 C-Y C-K content
(mg/g) 1.41 1.68 0.11 7.71 1.00 17.54 52.10 % 1.729 2.060 0.135 9.454 1.226 21.508 63.887

(% calculations are rounded to the fourth decimal place based on the total of 81.55 mg/g)

Looking at it, you can see that the combined ratio of Compound K and Compound Y is over 85%.

1-2. Normal and breast cancer cell survival rates

Figure 2 is a graph showing the results of normal cell survival rate analysis of the ginsenoside active ingredient included in the composition of the present invention, and Figure 3 is a graph showing the results of breast cancer cell survival rate analysis.

As a result of the experimental analysis, cytotoxicity was confirmed from the active ingredient concentration of 0.5mg/mL in normal cells (3T3-L1 cells), and cytotoxicity was confirmed at about 62% at 0.063mg/mL in breast cancer cells (MCF-7 cells). Accordingly, the concentration to confirm self-death in breast cancer cells was determined in the future.

1-3. Expression analysis of breast cancer cell activity-related proteins

Figures 4 and 5 are graphs showing the results of analysis of the expression of proteins related to breast cancer cell activity.

As a result of the experimental analysis, the expression of the anti-apoptotic Bcl-2 gene decreased, and the expression of the pro-apoptotic Bax gene significantly increased from 62.5 ug/ml.

In other words, cell death is confirmed through intrinsic pathways generated from the mitochondrial mechanism, and death due to DNA damage caused by cellular stress caused by the active ingredient (CKY) can be confirmed by a decrease in PARP protein expression. In addition, the effect of the active ingredient targeting breast cancer cells can be confirmed due to the increased absorption of the active ingredient into the cell, and strong breast cancer cell death occurred due to the pro-apoptotic effect.

1-4. Analysis of breast cancer cell inhibition mechanism

Figures 6 and 7 are graphs showing the results of analyzing the mechanism of suppressing breast cancer cells.

As a result of the experimental analysis, the PI3K-AKT pathway was confirmed through caspase gene activity. In other words, an increase in Caspase-3 and Caspase-9 gene expression was confirmed due to intracellular DNA damage in mitochondria through the endogenous pathway of breast cancer cell death mechanism.

1-5. Breast cancer cell DNA analysis

Figure 8 is a photograph of the results of electrophoresis analysis of DNA from breast cancer cells. DNA was extracted from cells treated with the active ingredient, and DNA fragmentation was confirmed by agarose electrophoresis.

As a result of the experimental analysis, the phenomenon of causing DNA breakage was clearly observed at 62.5 and 125 μg/mL of the active ingredient, confirming the phenomenon of breast cancer cell death. In other words, DNA fragmentation occurs during cell death, and is a phenomenon in which the DNA within the cell is accurately cut to a size of about 180 bp. At concentrations of 62.5 and 125 μg/mL of the active ingredient, a white band appeared as a result of DNA splitting.

1-6. Confirmation of breast cancer cell apoptosis phenomenon

Figures 9 and 10 are photographs showing the results of morphological confirmation of breast cancer cell apoptosis. The principle of the DAPI staining experiment is used to confirm the cell shape of the nucleus and chromatin by staining DNA, or to compare based on the intensity of light transmitted into the cell nucleus.

As a result of morphological observation of the effect of the active ingredient on breast cancer cell death, it can be seen that only the nuclei of living cells are stained and appear blue. Differences in the distribution of nuclei through DNA in stained cells can be confirmed compared to the normal group (Control). As the cell membrane was torn and membrane permeability increased, a larger amount of DAPI penetrated the nucleus and connected with DNA compared to other normal cells.

In particular, the phenomenon of cell death was clearly confirmed in the group treated with concentrations of 62.5 and 125 μg/mL of the active ingredient. Cells were morphologically damaged as evidenced by stained nuclei.

(Arrow sign: A phenomenon in which the nucleus bursts or the cell membrane is torn and separated into various forms. If you look inside the red dotted line, you can see the cell nucleus becoming punctured and condensed.)

Example 2. Composition for preventing, improving or treating breast cancer containing the active ingredient of Example 1

The active ingredients according to Example 1 are ginsenoside Rg1+Re 5-11 parts by weight, Rf 7-13 parts by weight, Rg2 0.3-0.9 parts by weight, Rh1 20-60 parts by weight, F2 3-9 parts by weight, compound Y Contains 100 parts by weight and 200 to 400 parts by weight of compound K. Additionally, the composition of the present invention contains the active ingredient at a concentration of 62.5 to 500 μg/mL.

The present invention can provide a preparation for the prevention, improvement or treatment of breast cancer that contains the above active ingredients and is formulated in a pharmaceutical unit dosage form by adding a pharmaceutically acceptable carrier, excipient or diluent. Here, carriers, excipients, and diluents include sorbitol, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, and not yet determined. Examples include quality cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

In addition, the pharmaceutical dosage form may be used in the form of a pharmaceutically acceptable salt, and may be used alone or in combination with other pharmaceutically active compounds, as well as in an appropriate combination. Additionally, when formulating the active ingredient, it may be prepared using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants. In addition, the above pharmaceutical dosage forms can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and sterile injection solutions according to conventional methods. You can.

The solid preparation for oral administration may be prepared by mixing the composition with at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used.

Preparations for parenteral administration may include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. The non-aqueous solvent and suspension may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate.

As a base for suppositories, witepsol, macrogol, tween 61, cacao, laurin, glycerogeratin, etc. can be used.

The preferred dosage of the composition of the present invention varies depending on the patient's condition and weight, degree of disease, age, gender, drug form, administration route and period, but can be appropriately selected by a person skilled in the art. However, for a desirable effect, the composition of the present invention is preferably administered at 0.001 to 300 mg/kg, and may be administered once a day or in divided doses. The above dosage does not limit the scope of the present invention in any way.

The composition of the present invention can be administered to mammals such as rats, mice, livestock, and humans through various routes. All modes of administration are contemplated, for example, it may be administered orally, rectally or by intravenous, intramuscular, or subcutaneous injection.

A health functional food composition that prevents or improves breast cancer can be provided by adding a food supplement to the active ingredient of the present invention.

Foods to which the above active ingredients can be added include, for example, various foods, beverages, gum, tea, vitamin complexes, health functional foods, etc.

The amount of the active ingredient in the food or beverage may be 0.01 to 20% by weight of the total weight of the food or beverage, and the health drink composition may be added at a rate of 0.02 to 5 g, preferably 0.3 to 1 g, based on 100 ml. there is.

The health functional beverage composition of the present invention has no particular restrictions on other ingredients other than those containing the composition, and may contain various flavoring agents or natural carbohydrates as additional ingredients like ordinary beverages. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose; Common sugars such as disaccharides, such as maltose, sucrose, etc., and polysaccharides, such as dextrin, cyclodextrin, etc., and sugar alcohols such as xylitol, sorbitol, and erythritol. In addition to the above-described flavoring agents, natural flavoring agents (thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of natural carbohydrates is generally about 1 to 20 g, preferably about 5 to 12 g, per 100 ml of the composition of the present invention.

In addition to the above, the composition of the present invention contains various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, colorants and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, organic acids, and protection. It may contain colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, and carbonating agents used in carbonated beverages.

In addition, the composition of the present invention may contain pulp for the production of natural fruit juice and fruit juice beverages and vegetable beverages. These ingredients can be used independently or in combination. The proportion of these additives is not critical, but is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

So far, the present invention has been examined with a focus on preferred embodiments.

The embodiments described in this specification and the configurations shown in the drawings are related to one of the most preferred embodiments of the present invention, and do not represent the entire technical idea of the present invention, so various equivalents and modifications that can replace them are provided. It should be understood that there may be examples.

Therefore, the present invention is not limited to the presented embodiments, and is within the equivalent scope of the technical idea of the present invention and the technical idea described in the patent claims below, as can be understood by those skilled in the art to which the present invention pertains. There may be embodiments in which various modifications and changes are possible.

Claims (4)

A health functional food composition for preventing or improving breast cancer, comprising 100 parts by weight of Compound Y and 200 to 400 parts by weight of Compound K as active ingredients. In claim 1,
Ginsenoside Rg1+Re 5-11 parts by weight, Rf 7-13 parts by weight, Rg2 0.3-0.9 parts by weight, Rh1 20-60 parts by weight and F2 3-9 parts by weight are additionally included in the above active ingredients. A health functional food composition for preventing or improving breast cancer. In claim 1 or 2,
A health functional food composition for preventing or improving breast cancer, characterized in that the concentration of the active ingredient is 62.5 to 500 μg/mL. A pharmaceutical composition for treating breast cancer, comprising 100 parts by weight of Compound Y and 200 to 400 parts by weight of Compound K as active ingredients.