KR102218600B1 - A preparation method of immunity enhancing and anti-cancer composition comprising polysaccharide derived from fermented ginseng leaves - Google Patents

Abstract

Inoculating and culturing a strain of Cordyceps sinensis on ginseng leaves to prepare a ginseng leaf fermentation product; After heating the ginseng leaf fermented product, ethanol is added to prepare a precipitate; And to obtain a polysaccharide by centrifuging the precipitate and dialysis, it relates to a method for preparing a composition for enhancing immunity. In addition, the present invention comprises the steps of preparing a ginseng leaf fermented product by inoculating and culturing a strain of Cordyceps sinensis on ginseng leaves; After heating the ginseng leaf fermentation product, adding ethanol to prepare a precipitate; And, a pharmaceutical composition for preventing or treating cancer and/or cancer comprising the step of obtaining a polysaccharide by centrifuging the precipitate and dialysis It relates to a method of manufacturing a food composition for prevention or improvement.

Description

TECHNICAL FIELD A PREPARATION METHOD OF IMMUNITY ENHANCING AND ANTI-CANCER COMPOSITION COMPRISING POLYSACCHARIDE DERIVED FROM FERMENTED GINSENG LEAVES containing polysaccharide derived from fermented ginseng leaves

The present invention relates to a method for preparing an immunity enhancing and anticancer composition comprising a polysaccharide derived from fermented ginseng leaf.

Korean ginseng is called Korean ginseng worldwide. It is a perennial herbaceous plant belonging to the genus Panax of the Araliaceae family, and its scientific name is Panax ginseng C. A. Meyer. Ginseng has been used as a traditional medicine in East Asia for more than 2000 years, and pharmaceutical and clinical studies conducted over the past 40 years have focused on the biological activities of ginseng such as radiation protection, antitumor and antiviral. The main ingredients of ginseng, known to regulate the immune system, are ginsenosides and polysaccharides, which are mostly obtained through simple water extraction.

Ginseng mainly uses the root, and recently, studies to study and use other parts of ginseng, which have been underutilized, are being conducted. For example, Korean Patent Publication No. 10-2011-0078362 proposes an anti-dandruff hair cosmetic composition using ginseng fruit extract, and Korean Patent Publication No. 10-2009-0032501 discloses a food composition for improving atopic dermatitis containing ginseng fruit extract. Are presented.

While studying physiologically active substances using ginseng leaves, the present inventors confirmed that polysaccharides isolated from fermented ginseng leaves fermented with specific bacteria have enhanced immune activity and inhibited cancer metastasis, and completed the present invention.

An object of the present invention is to provide a composition for enhancing immunity, a pharmaceutical composition for preventing or treating cancer, and a food composition for preventing or improving cancer by using ginseng leaves.

In order to achieve the above object, the present invention,

Inoculating and culturing a strain of Cordyceps sinensis on ginseng leaves to prepare a ginseng leaf fermentation product;

After heating the ginseng leaf fermented product, adding ethanol to prepare a precipitate; And

Comprising the step of centrifuging the precipitate and dialysis to obtain a polysaccharide

It provides a method of preparing a composition for enhancing immunity.

In addition, the present invention,

Inoculating and culturing a strain of Cordyceps sinensis on ginseng leaves to prepare a ginseng leaf fermentation product;

After heating the ginseng leaf fermented product, adding ethanol to prepare a precipitate; And

Comprising the step of centrifuging the precipitate and dialysis to obtain a polysaccharide

It provides a method of preparing a pharmaceutical composition for preventing or treating cancer.

In addition, the present invention,

Inoculating and culturing a strain of Cordyceps sinensis on ginseng leaves to prepare a ginseng leaf fermentation product;

After heating the ginseng leaf fermented product, adding ethanol to prepare a precipitate; And

Comprising the step of centrifuging the precipitate and dialysis to obtain a polysaccharide

It provides a method of preparing a food composition for preventing or improving cancer.

The composition of the present invention has an immunity-enhancing effect and an effect of preventing, treating or improving cancer, particularly an inhibitory effect of cancer metastasis.

1 is a schematic diagram showing a method of separating crude polysaccharide fermented with Cordyceps sinensis (GLF-0) and hot water extracted crude polysaccharide (GLW-0) from ginseng leaves.
Figure 2 shows the elution patterns of GLW-0 (A) and GLF-0 (B) derived from ginseng leaves on a size exclusion HPLC. The HPLC was equipped with Superdex 75 GL 10/300 columns.
Figure 3 shows the anti-complement activity of GLW-0 (A) and GLF-0 (B) derived from ginseng leaves. Anticomplement activity was expressed as inhibition of 50% total complement hemolysis by the Mayer method. PSK, a known immunoactive polysaccharide derived from Coriolus versicolor, was used as a positive control. Means denoted by different superscript characters (ae) indicate that they have a significant difference of p<0.05 by Duncan's multiple range test.
Figure 4 is in vitro for peritoneal macrophages (A), RAW 264.7 cells (B), Colon26-M3.1 cells (C) and B16BL6 melanoma cells (D) of GLW-0 and GLF-0 derived from ginseng leaves. Shows the cytotoxic effect of Each of the cells (2.0 × 10 ⁵ cells/well) was treated in 96-well plates with various concentrations (0.06 to 1000 μg/mL) GLW-0 and GLF-0 for 24 hours, and the cytotoxicity was CCK. It was determined through an -8-based colorimetric analysis. NC is a negative control. Data are expressed as the mean ± standard deviation of three independent experiments.
Figure 5 shows the effects of GLW-0 and GLF-0 on the production of cytokines such as IL-6(A), IL-12(B) and TNF-α(C) by murine peritoneal macrophages. NC: medium used as a negative control, PC: μg/mL lipopolysaccharide (LPS) as a positive control. Means denoted by different superscript characters (aj) indicate significant differences of p<0.05 by Duncan's multiple range test.
6 shows the effect of GLW-0 and GLF-0 on the cytolytic activity of NK cells in vivo. NK cells were incubated with YAC-I target in the presence or absence of GLW-0 and GLF-0 for 6 hours in a 37° C. CO2 incubator. Means denoted by other superscript characters (ab, AB, i-ii) indicate significant differences of p<0.05 by Duncan's multiple range test.
7 shows the inhibitory effect of GLF-0 derived from ginseng leaves on lung metastasis produced by iv inoculation of Colon26-M3.1 carcinoma cells. (A) 5 BALB/c mice per group ^{were iv inoculated with 4×10 4} Colon26-M3.1 carcinoma cells and iv injected with the indicated dose of GLF-0 suspended in PBS 2 days before tumor inoculation. . Mice were sacrificed 14 days after tumor inoculation for tumor evaluation. (B) Scan images of resected mouse lungs after iv inoculation of Colon 26-M3.1 carcinoma cells.
8 shows the principle of analysis for confirming the role of NK cells in anti-metastatic activity.
9 shows the effect of inhibition of NK cell function on the anti-metastatic activity of GLF-O derived from fermented ginseng leaves.
10 shows the effect of crude polysaccharide GLF-0 derived from fermented ginseng leaf on the activity of cytotoxic T-lymphocytes in ex vivo. Mice were administered iv with the indicated dose of GLF-0 for 2 days and then ^{iv inoculated with 4×10 4} Colon26-M3.1 carcinoma cells. Splenocytes were harvested and cultured with Colon26-M3.1 cells. CTL activity was measured using an LDH kit after 6 hours incubation.
11 shows the effect of oral administration of GLF-0 on the cytolytic activity of NK cells in ex vivo. After oral administration of GLF-0 for 14 days, NK cells were incubated with YAC-I target in the presence or absence of GLF-0 for 6 hours in a CO2 incubator at 37°C. Means denoted by different superscript characters (ab, AB, I-III) indicate significant differences of p<0.05 by Duncan's multiple range test.
12 shows the effect of oral administration of GLF-0 on lung metastasis produced by inoculation of Colon26-M3.1 carcinoma cells. Five BALB/c mice per group were orally administered for 14 days at the indicated dose of GLF-0, followed by iv inoculation with 4×10⁴Colon26-M3.1 carcinoma cells. Mice were sacrificed 14 days after tumor inoculation for tumor evaluation. Means denoted by other superscript characters (ab) indicate significant differences of p<0.05 by Duncan's multiple range test.
13 shows the effect of GLF-0 derived from ginseng leaf on cytotoxic T-lymphocytes by inoculation of Colon 26-M3.1 carcinoma cells.

The present invention,

Inoculating and culturing a strain of Cordyceps sinensis on ginseng leaves to prepare a ginseng leaf fermentation product;

After heating the ginseng leaf fermented product, adding ethanol to prepare a precipitate; And

Comprising the step of centrifuging the precipitate and dialysis to obtain a polysaccharide

It relates to a method for preparing a composition for enhancing immunity.

In addition, the present invention,

Inoculating and culturing a strain of Cordyceps sinensis on ginseng leaves to prepare a ginseng leaf fermentation product;

After heating the ginseng leaf fermented product, adding ethanol to prepare a precipitate; And

Comprising the step of centrifuging the precipitate and dialysis to obtain a polysaccharide

It relates to a method for preparing a pharmaceutical composition for preventing or treating cancer.

In addition, the present invention,

Inoculating and culturing a strain of Cordyceps sinensis on ginseng leaves to prepare a ginseng leaf fermentation product;

After heating the ginseng leaf fermented product, adding ethanol to prepare a precipitate; And

Comprising the step of centrifuging the precipitate and dialysis to obtain a polysaccharide

It relates to a method for preparing a food composition for preventing or improving cancer.

Hereinafter, the present invention will be described in detail.

Step of preparing fermented ginseng leaf

The method for preparing the composition of the present invention includes the step of inoculating and culturing a strain of Cordyceps sinensis to a ginseng leaf to prepare a ginseng leaf fermentation product. The cordyceps strain is Cordyceps sinensis. The step of preparing the fermented ginseng leaf is preferably performed by adding water to the ginseng leaf, inoculating the cordyceps strain thereto, and performing liquid culture.

Preparing a precipitate

The method for preparing the composition of the present invention includes heating the ginseng leaf fermented product, and then adding ethanol to prepare a precipitate.

Steps to obtain polysaccharide

The method for preparing the composition of the present invention includes the step of centrifuging the precipitate and dialysis to obtain a polysaccharide. The polysaccharide may have a neutral sugar content of 50 to 65% by weight. The polysaccharide may have a uronic acid content of 30 to 43% by weight. The polysaccharide may have a protein content of 0.4 to 0.9% by weight. The polysaccharide may contain 0.1 to 2.5% by weight of a phenolic compound.

The polysaccharide may contain 2.9 to 3.5 mol% of mannose in the constituent sugar. The polysaccharide may contain 4.3 to 6.5 mol% of rhamnose among the constituent sugars. The polysaccharide may contain 1.5 to 2.8 mol% of glucuronic acid among the constituent sugars. The polysaccharide may contain 25 to 43 mol% of galacturonic acid among the constituent sugars. The polysaccharide may contain 15 to 35 mol% of glucose among the constituent sugars. The polysaccharide may contain 8 to 18 mol% galactose among the constituent sugars. The polysaccharide may contain 0.2 to 1.1 mol% of xylose in the constituent sugar. The polysaccharide may contain 4 to 14 mol% of arabinose among the constituent sugars. The polysaccharide may have a fucose of 0.2 to 0.8 mol% of the constituent sugar. The polysaccharide may have a glucose content greater than that of galactose among constituent sugars. In addition, the content of glucose among the constituent sugars of the polysaccharide may be higher than that of arabinose.

Composition for enhancing immunity

The composition for enhancing immunity of the present invention may be a food composition or a pharmaceutical composition.

Pharmaceutical composition

The pharmaceutical composition of the present invention may be a pharmaceutical composition for enhancing immunity or a pharmaceutical composition for preventing or treating cancer.

The present invention relates to a method of preparing a pharmaceutical composition for enhancing immunity or a pharmaceutical composition for preventing or treating cancer. It also relates to a method for enhancing immunity or a method for preventing or treating cancer, comprising administering the pharmaceutical composition of the present invention to a subject. The subject may be a mammal, including a human, who has symptoms due to decreased immunity, is diagnosed with a related disease, or is likely to develop a related disease. Further, the subject may be a mammal, including a human at risk of developing cancer or diagnosed with cancer.

The pharmaceutical composition of the present invention may contain 0.01 to 80% by weight of the polysaccharide, preferably 0.02 to 65% by weight. However, this can be increased or decreased according to the needs of the patient, and can be appropriately increased or decreased according to circumstances such as the age, diet, nutritional status, and progression of the disease.

The pharmaceutical composition of the present invention can be administered orally or parenterally, and can be used in the form of a general pharmaceutical formulation. Preferred pharmaceutical preparations include preparations for oral administration such as tablets, hard or soft capsules, solutions, suspensions, and the like, and these pharmaceutical preparations are conventional pharmaceutically acceptable carriers, for example, excipients for preparations for oral administration, It can be prepared using a binder, disintegrant, lubricant, solubilizer, suspending agent, preservative or extender.

The dosage of the pharmaceutical composition containing the polysaccharide of the present invention may be determined by an expert according to various factors such as the patient's condition, age, sex, and complications, but is generally 0.1 mg to 10 g per 1 kg of an adult, preferably It can be administered in a dose of 10 mg to 5 g. In addition, a daily dose of the pharmaceutical composition or a dose of 1/2, 1/3 or 1/4 thereof is contained per unit dosage form, and may be administered 1 to 6 times a day. However, in the case of long-term intake for the purpose of health and hygiene or for the purpose of health control, the amount may be less than the above range, and the active ingredient may be used in an amount above the above range because there is no problem in terms of safety.

Food composition

The food composition of the present invention may be a composition for enhancing immunity or a food composition for preventing or improving cancer.

The food of the present invention may be a health supplement food, a health functional food, a functional food, etc., but is not limited thereto, and includes a polysaccharide of the present invention, such as natural foods, processed foods, and general food materials.

The food composition of the present invention may be added as it is to the polysaccharide of the present invention, or may be used with other foods or food compositions, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined depending on the purpose of use (prevention, improvement or therapeutic treatment). In general, the polysaccharide of the present invention may be added in an amount of 0.1 to 5% by weight, preferably 2 to 3% by weight, based on 100% by weight of raw materials for food or beverage when preparing food or beverage. The effective dose of the polysaccharide of the present invention may be used in accordance with the effective dose of the pharmaceutical composition, but in the case of long-term intake for the purpose of health and hygiene or health control, it may be less than the above range, and is effective Since the component has no problem in terms of safety, it can be used in an amount beyond the above range.

There is no particular limitation on the type of food. The food composition may be used in the form of preparations for oral administration such as tablets, hard or soft capsules, solutions, suspensions, etc., and these preparations are acceptable conventional carriers, for example, excipients in the case of preparations for oral administration, It can be prepared using a binder, disintegrant, lubricant, solubilizer, suspending agent, preservative or extender.

Examples of foods to which the mixed composition of the present invention can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, Tea, drinks, alcoholic beverages and vitamin complexes, and other nutritional supplements, but are not limited to these types of foods.

Feed composition

The present invention comprises the steps of preparing a ginseng leaf fermentation product by inoculating and culturing a strain of Cordyceps sinensis to a ginseng leaf; After heating the ginseng leaf fermentation product, adding ethanol to prepare a precipitate; And to a feed composition comprising the step of centrifuging the precipitate and dialysis to obtain a polysaccharide.

The feed composition of the present invention contains 0.01 to 5.00% by weight of the polysaccharide of the present invention on a dry matter basis, preferably 0.03 to 3.00% by weight, more preferably 0.05 to 0.10% by weight.

The feed composition of the present invention may be a feed composition such as vertebrate animals such as cows and pigs, poultry such as chickens and ducks, fish, and crustaceans, but is not limited thereto. The feed composition of the present invention can be used regardless of the blending ratio in the feed composition. For example, there is no problem in using the present invention even for feed with high protein content.

Advantages and features of the present invention, and a method of achieving them will become apparent with reference to embodiments described below in detail. However, the present invention is not limited to the embodiments disclosed below, but will be implemented in a variety of different forms, only these embodiments make the disclosure of the present invention complete, and common knowledge in the technical field to which the present invention pertains. It is provided to completely inform the scope of the invention to those who have, and the invention is only defined by the scope of the claims.

<Materials and methods>

Ginseng leaves were harvested in Geumsan-gun, Chungcheongnam-do, and commercially available mature ginseng leaves of 8-10 years were purchased and used.

Cordyceps sinensis was isolated from domestic cordyceps and cultured before use.

<Experimental Example 1> Extraction and separation of polysaccharides derived from ginseng leaves

Two methods were used in this experiment to separate highly immunoactive polysaccharides from ginseng leaves.

100 g of pulverized ginseng leaves were suspended in 20 times the volume of water, and hot water extraction was performed at 100°C until half of the initial volume. Polysaccharide was precipitated while stirring at 4° C. for 24 hours by adding 4 times the volume (v/v) of 95% ethanol to the extract. The precipitate generated at this time was centrifuged at 6,000 rpm for 15 minutes to recover the precipitate, dissolved in a small amount of distilled water, and dialysis was performed for 3-4 days using dialysis tubing (MW cut-off 12,400). Thereafter, freeze-drying (FreeZone 12Liter, Labconco, Kansas City, Missouri. USA) was performed to obtain a simple hot water extract crude polysaccharide GLW (hereinafter referred to as “GLW-0”) (hot water crude polysaccharide of ginseng leaf) derived from ginseng leaf. (Fig. 1).

Separately, 600 g of pulverized ginseng leaves were suspended in 20-fold volume of water, and 500 mL of Cordyceps sinensis, a representative strain of Cordyceps sinensis, was liquid-cultured and cultured at 25° C. for 5 days. After fermentation was completed and hot water extraction was performed at 100° C. for 2 hours, polysaccharide was precipitated while stirring at 4° C. for 24 hours by adding 3 times the volume (v/v) of 95% ethanol. The precipitate generated at this time was centrifuged at 6,000 rpm for 15 minutes to recover the precipitate, dissolved in a small amount of distilled water, and dialysis was performed for 3-4 days using dialysis tubing (MW cut-off 12,400). Thereafter, freeze-dried (FreeZone 12Liter, Labconco, Kansas City, Missouri. USA) to obtain crude polysaccharide GLF derived from fermented ginseng leaves using Cordyceps sinensis (hereinafter referred to as “GLF-0” (crude polysaccharide fermented with Cordyceps sinensis)). (Fig. 1).

After fermentation using hot water extraction and Cordyceps sinensis as described above, polysaccharides are precipitated in the extract using 3-4 times 95% ethanol, and then dialysis and freeze-drying are performed, followed by simple hot water extraction from ginseng leaves crude polysaccharide GLW-0 And crude polysaccharide GLF-0 derived from fermented ginseng leaves were obtained. The yield of crude polysaccharide GLW-0 obtained by simple hot water extraction was 2.48%, and the yield of crude polysaccharide GLF-0 from fermented ginseng leaves was 6.08%, which was about 2.5 times higher than that of the GLW-0 sample.

Subsequently, the evaluation of general chemical properties, immune activity and anti-metastatic activity in vitro and in vivo were compared using samples of crude polysaccharide GLW-0 extracted from ginseng leaves and crude polysaccharide GLF-0 from fermented ginseng leaves.

<Experimental Example 2> Analysis of general chemical properties of crude polysaccharide

<2-1> General analysis

To confirm the general analysis of the simple hot-water extracted crude polysaccharide GLW-0 from ginseng leaves and the crude polysaccharide GLF-0 derived from fermented ginseng leaves, the content of neutral sugar is phenol-sulfuric acid using galactose as a standard, and the content of acidic sugar is galacturonic. acid as a standard material by m-hydroxybiphenyl method, the content of TBA-positive material by thiobarbituric acid method with 2-keto-3-deoxy-D-manno-octulosonic acid (KDO) as a standard material, protein content is Bovine serum albumin (BSA) was used as a standard substance by Bradford method, and phenolic compounds content was quantitatively analyzed by Folin-Ciocalteu (FC) method using gallic acid as standard substance.

<2-2> Purification and purity measurement using HPLC

HPLC equipped with Superdex 75 GL column (GE healthcare) equilibrated with 50 mM ammonium formate buffer to measure the purity and purity of simple hot water extracted crude polysaccharide GLW-0 from ginseng leaves and crude polysaccharide GLF-0 from fermented ginseng leaves. The analysis was performed using -RID (High performance liquid chromatography-Refractive index detector).

<2-3> Analysis per composition

Honda et al. (Honda S, Akao E, Suzuki S, Okuda M, Kakehi K, Nakamuraa J.) to analyze the constituents of the simple hot water extracted crude polysaccharide GLW-0 derived from ginseng leaves and the crude polysaccharide GLF-0 derived from fermented ginseng leaves. High-performance liquid chromatography of reducing carbohydrates as strongly ultraviolet-absorbing and electrochemically sensitive 1-phenyl-3-methyl5-pyrazolone derivatives.Anal.Biochem. 180: 351-357 (1989)). . After hydrolysis with 2M TFA, 1-phenyl-3-methyl-5-pyrazolone (PMP) was used to derivatize each component, and after phase separation using DIW and choloroform, only the aqueous layer was recovered and filtered. In order to proceed with the analysis, it was measured at 254 nm using an HPLC-UVD equipped with an Acclaim C18 column equilibrated with a solvent of 0.1M phosphate buffer (pH 6.7):acetonitrile=82:18. The mole% of each component was calculated by calculating the peak area, molecular weight, and molecular response factor for the UV detector of each derivative.

<2-4> General chemical properties of crude polysaccharide sample

As a result of analyzing the general chemical properties of crude polysaccharide GLW-0 derived from simple hot water extract from ginseng leaf and crude polysaccharide GLF-0 derived from fermented ginseng leaf, simple hot water extract crude polysaccharide GLW-0 was 44.1% neutral sugar and 47.3% acidic sugar. It was composed, and a trace amount of 4.4% KDO was detected. In addition, protein and polyphenol were contained in trace amounts of 0.8% and 3.4%, respectively.

On the other hand, in the case of crude polysaccharide GLF-0 derived from fermented ginseng leaves, it was composed of 58.5% neutral sugar and 36.9% acidic sugar, and trace amounts of 2.9% KDO were detected, and protein and polyphenols contained in trace amounts of 0.6% and 1.1%, respectively. Was done (Table 1).

General Chemical Properties of GLW-0 and GLF-0 Isolated from Ginseng Leaves

As a result of analyzing the constituent sugars of the two samples, GLW-0, a crude polysaccharide extracted from ginseng leaves, mainly consisted of 49.9% galacturnic acid, 19.4% galactose, and 16.3% arabinose. In the case of crude polysaccharide GLF-0 derived from fermented ginseng leaves, 32.5% of galacturnic acid, 28.0% of glucose, 15.8% of galactose and 11.9% of arabinose consisted of similar monosaccharides, but the content of glucose in GLF-0 Was detected quite high. It was presumed that the glucan form derived from Cordyceps sinensis appeared high during the fermentation process.

In addition, in order to measure the purity of crude polysaccharide GLW-0 extracted from ginseng leaves and crude polysaccharide GLF-0 derived from fermented ginseng leaves, each sample was equilibrated with 50 mM ammonium formate buffer (pH 5.5) using a Superdex 75 GL column. Then, HPLC-RID was performed. As a result, both samples showed similar patterns, but in the case of the GLF-0 peak pattern detected in the 20-35 minute retention time range, it was confirmed that the peak in the low molecular weight range increased by a large amount as the peak was divided into two. Overall, it was confirmed that polysaccharides of various molecular weights of 1 kDa to 100 kDa exist in both crude polysaccharides (FIG. 2).

<Experimental Example 3> Measurement of immune activity by sample

<3-1> Measurement of anti-complement activity

The complement system is an enzyme reaction system composed of about 20 kinds of circulating proteins in the blood, including active proteins and regulators of C1~C9, through complement receptors of red blood cells, leukocytes, reticuloendothelial cells, and other tissue cells. It is a major defense mechanism of the living body that non-specifically removes invasion factors such as external infectious pathogens in the presence or absence of antibodies, and it is possible to transfer a series of information between complement system proteins and these immune cells.

It is known that when the complement system is activated, complement proteins are decomposed into active molecules through a continuous cascade reaction, and these are attached to the surface of invasion factors to finally form a membrane attack complex (MAC) to remove infectious pathogens. In addition, it is known that several complement degradation products produced during the complement activation process mediate various physiological reactions, and in particular, it is reported that there is a close correlation with the activation of macrophages and lymphocytes, and immunity enhancement.

In this experiment, simple hot water extract crude polysaccharide GLW-0 derived from ginseng leaf and crude polysaccharide GLF-0 derived from fermented ginseng leaf for the complement system that plays an important role in the initial immune response of the human body before adaptive immunity to foreign substances occurs. Anticomplement activity was measured according to the Mayer method in order to determine whether or not to activate.

Blood from healthy adults is collected and allowed to stand at room temperature for about 15 minutes to coagulate. The coagulated blood is cut and centrifuged (2,000 rpm, 15 min, 4 ℃) to separate the serum, and then 1 mL into a microcentrifuge tube. It was dispensed each and used in the experiment while frozen at -70 °C. Complement activity was measured by a complement fixation test method based on the degree of red blood cell hemolysis by remaining complement after complement consumption by the sample using the Meyer method. Samples dissolved in distilled water at various concentrations were mixed with GVB++ (containing gelatin veronal buffer, pH 7.4, 0.1% gelatin, 0.15 mM Ca++, 0.5 mM Mg++) and serum of normal people, respectively, and reacted for 30 minutes at 37°C. Made it. To the reaction solution, 350 μL of GVB++ was added, serially diluted 10 to 160 times, and then 750 μL of GVB++ and positive sensitized red blood cells (IgM-sensitizated sheep erythrocyte, EA cell, 1×10 ⁸ cells/mL) were added to 250 µL was added, followed by a second reaction at 37°C for 60 minutes, and 2.5 mL of PBS (pH 7.4) was added to stop the reaction. The reaction solution was centrifuged at 2,000 rpm for 10 minutes, and the resulting supernatant was measured for absorbance at 412 nm to measure the residual hemolytic activity, and calculated through Equation 1 below.

The anticomplement activity was expressed as the inhibition of 50% total complement hemolysis (ITCH50, %) for the negative control group reacted with serum of normal subjects only with GVB++ and distilled water (50% total complement hemolysis, TCH50, %). . As a positive control, polysaccharide K (PSK), an immunity enhancing agent derived from Coriolus versicolor, was used as a negative control, and the degree of activation in the negative control was set to 0% ITCH50 using distilled water without adding a sample. The activation ability was confirmed.

<Equation 1>

As a result, both samples of crude polysaccharide GLW-0 derived from ginseng leaves and crude polysaccharide GLF-0 derived from fermented ginseng leaves showed concentration-dependent anti-complement activity. In general, polysaccharides exhibiting anticomplement activity of 60% or more compared to the positive control at a concentration of 1000 μg/mL of PSK are generally recognized for their pharmacological activity, so simple hot water extract crude polysaccharide GLW-0 derived from ginseng leaves and crude polysaccharide derived from fermented ginseng leaves Both samples of GLF-0 were judged to exhibit excellent anticomplement activity at a concentration of 1000 μg/mL. In addition, it was confirmed that the GLF-0 sample showed more excellent anti-complement activity than GLW-0 (Fig. 3).

<3-2> Measurement of cytotoxicity against normal cells and tumor cells

For samples of simple hot-water extracted crude polysaccharide GLW-0 from ginseng leaf and crude polysaccharide GLF-0 derived from fermented ginseng leaf, normal cells, mouse peritoneal macrophages and Raw264.7 cells, and tumor cell lines, Colon26-M3.1 carcinoma cells and B16BL6 Direct cytotoxicity to melanoma cells was measured.

In order to measure the cell viability of the sample against normal cell macrophages, 1 mL of 5% thioglycollate (TG) was intraperitoneally injected into Balb/c mice, and 4 days later, the mice were sacrificed by cervical dislocation, and phosphate buffered seline ( PBS) 10 mL was injected to collect peritoneal exudative cells (PEC). The collected PEC was dispensed into a 96 well culture plate at ^{a concentration of 2.5×10 5} cells/well and cultured for 2 hours to attach macrophages to the plate, and then samples were added at various concentrations and cultured for 24 hours. .

In order to measure the cell viability of the sample for the normal cell line Raw264.7 cells, the sample is dispensed in a 96 well culture plate at ^{a concentration of 1.5×10 5} cells/well, incubated for 2 hours, and the cells are attached to the plate. It was added at various concentrations and incubated for 24 hours.

To check the cytotoxicity of the sample against the cancer cell lines Colon26-M3.1 carcinoma cells and B16BL6 melanoma cells, the cancer cells were ^{dispensed in a 96 well culture plate at a concentration of 1×10 4} cells/well, and cultured for 2 hours. After attaching the cells to the plate, samples were added at various concentrations and incubated for 24 hours.

For the effect of cytotoxicity according to the concentration of each sample, dilute EZ-cytox (Dogen, Seoul, Korea) 10 times, add 100 μL per well, and react for 30 to 60 minutes in a 5% CO2 incubator at 37° C. and absorbance at 450 nm. Was evaluated by measuring.

Simple hot water extract from ginseng leaves using macrophage, normal cell line Raw264.7 cell and tumor cell line Colon26-M3.1 carcinoma cell and B16BL6 melanoma cell isolated from mouse abdominal cavity, crude polysaccharide GLW-0 and fermented ginseng leaf Direct cell viability and cytotoxicity by crude polysaccharide GLF-0 were measured.

After culturing the cells adjusted to 2×10 ⁶ cells/mL by adding GLW-0 and GLF-0 samples at various concentrations from 0.06 to 1000 μg/mL, the cells were confirmed to survive. No toxicity was observed at all concentrations, and no apparent cell proliferation ability was observed (Fig. 4(A)).

In addition, as a result of measuring the cell viability of the normal cell line Raw264.7, in the case of GLF-0, toxicity was not observed at all concentrations, but in the case of GLW-0, a little toxicity was exhibited with increasing concentration (FIG. 4(B)).

As a result of confirming the cytotoxicity of the tumor cell lines Colon26-M3.1 and B16BL6 melanoma cells, both GLW-0 and GLF-0 samples did not show cytotoxicity at all concentrations of 0.06 to 1000 μg/mL. It was estimated that there was no anticancer activity due to cytotoxicity (FIGS. 4(C) and 4(D)).

<3-3> Measurement of cytokine production activity

Macrophages are cells that secrete various cytokines in the process of phagocytosis and removal of bacteria or foreign substances to regulate immune phenomena and play a pivotal role in the immune function against antigens. It is related to the immune function, and shows direct injury to tumor cells. In addition, substances (LPS or natural products) that respond to TLR (toll-like receptor) activate macrophages to proliferate T cells and B cells, activate macrophages for phagocytosis, and protect against microbial infections. It is known to produce cytokines such as IL-1, IL-6, IL-10, IL-12 and TNF-α that can modulate the response. IL-6 and TNF-α are representative cytokines induced by macrophages and play a pivotal role in the inflammatory response following bacterial infection.In particular, IL-6 cooperatively with IL-1 to act as T cells and B It is reported that it is involved in the differentiation of cells and has anti-cancer effects. IL-12 is a cytokine that activates NK cells and induces Th1 type immune responses and increases the reactivity to cellular foreign substances such as cancer cells. It is known to do. It is thought that the activation of macrophages by polysaccharides derived from natural products is mediated by the recognition of polysaccharide molecules by specific receptors of macrophages. These receptors are cell membrane-binding proteins known as pattern recognition molecules, and are molecules that can recognize ligands from outside at the beginning of the immune response. In particular, macrophages are known to be capable of binding to polysaccharides or glycoproteins of plant origin through toll-like receptor (TLR), CD14, complement receptor 3 (CR3), scavenger receptor, dectin-1 and mannose receptors. Activation of these receptors induces a series of continuous intracellular signaling, resulting in the activation of transcription of specific genes involved in the production of iNOS (inducible nitric oxide synthase) and ROS in the nucleus and the production of various cytokines. In addition, the induction of these various active glycoproteins also induces increased phagocytosis and proliferation of cells.

In this experiment, cytokine production of macrophages was measured in vitro by direct stimulation of crude polysaccharide GLW-0 derived from ginseng leaves and crude polysaccharide GLF-0 from fermented ginseng leaves. BALB/c (female, 6 weeks) Inject 1 mL of 5% TG into the peritoneal cavity of the mouse, and ^{add 100 μL of recovered macrophages (2.5×10 6} cells/mL of DMEM) by induction for 96 hours at 37°C, 5 Incubated for 2 hours in% CO2 incubator. Here, samples diluted to various concentrations were dispensed into a flat-bottomed 96-well microplate at 100 μL and incubated for 24 hours in a 37°C, 5% CO2 incubator. After the culture was completed, 150 μL of the cell culture solution was collected by centrifugation at 900 rpm for 5 minutes at 4° C., and the content of induced cytokines in the supernatant was measured.

The content of cytokines produced by macrophages was analyzed by sandwich ELISA (enzymelinked immunosorbent assay). Each cytokine was measured using interleukin (IL)-6, IL-12 kit (BD Biosciences, San Diego, CA, USA) and tumor necrosis factor (TNF)-α kit (eBioscience, San Diego, CA, USA). I did. It was diluted in coating buffer, coated on an immuno 96-well microplate, and left at 4°C for 12 hours. The coated microplate is washed 3 times with washing buffer (PBS with 0.05% tween 20, PBST), and 200 μL of assay diluent (PBS with 2% skim milk or Diluent buffer) is dispensed, and then left for 1 hour. The well surface to which the antibody did not adhere was blocked. After blocking was completed, each well was washed three times again using a washing buffer, and 50 μL of each of the serially diluted standard (recombinant mouse cytokines) and the immune cell culture solution were dispensed into each well. This was allowed to stand at room temperature for 2 hours, washed with a washing buffer, and treated with 50 μL of detection antibody (in assay diluent), left at room temperature for 1 hour, and washed again. Treat 50 μL of enzyme reagent (avidinhorseradish peroxidase conjugate) and combine at room temperature for 30 minutes, add 50 μL of substrate solution [tetramethylbenzidine (TMB) and hydrogen peroxide] and react in the dark for 30 minutes, and 50 μL of stop solution (2 N H2SO4) ) Was treated to measure absorbance at 450 nm.

As a result of in vitro measurement of cytokine production in macrophages by direct stimulation of crude polysaccharide GLW-0 derived from ginseng leaf and crude polysaccharide GLF-0 derived from fermented ginseng leaf, both samples were IL-6 and IL- It was confirmed that the production of 12 and TNF-α was promoted, and a higher cytokine production ability was confirmed in GLF-0 than in GLW-0 (Figs. 5 (A), (B) and (C)).

In particular, in the case of IL-6 production ability for crude polysaccharide GLF-0 sample derived from fermented ginseng leaf, it showed high production ability at concentrations of 3.9 to 1000 μg/mL, and production equivalent to positive control LPS at concentrations above 250 μg/mL Showed ability (Fig. 5(A)).

On the other hand, in the case of IL-12, both samples showed the best production capacity at a concentration of 62.5 μg/mL, but the production capacity decreased with increasing concentration. Like IL-6, GLF-0 sample showed better production capacity. , This showed a higher production capacity than the positive control LPS (Fig. 5(B)).

In the case of TNF-α, the simple hot water extract crude polysaccharide GLW-0 sample from ginseng leaves at a concentration of 62.5 μg/mL, whereas the crude polysaccharide GLF-0 sample from fermented ginseng leaves is the best at a low concentration of 3.9 μg/mL. Was shown, and both samples exhibited similar activity at a concentration of 15.6 μg/mL or higher (FIG. 5(C)).

From the above results, both samples of crude polysaccharide GLW-0 derived from ginseng leaves and crude polysaccharide GLF-0 derived from fermented ginseng leaves promote the production of representative cytokines IL-6, IL-12 and TNF-α, which are involved in immune activity. However, it was confirmed that the production capacity of crude polysaccharide GLF-0 derived from ginseng leaves fermented with Cordyceps sinensis was much better than GLW-0. Therefore, crude polysaccharide GLF-0 derived from fermented ginseng leaves was considered to have activity in cell-mediated immunity such as activation of cytotoxic T lymphocytes (CTL) through activation of NK cells and induction of Th1 type immune responses. In particular, IL-12 is recognized as an essential cytokine for inducing anticancer activity because it is directly involved in the activation of NK cells that kill cancer cells in the presence of cancer cells.Therefore, crude polysaccharide GLF-0 derived from fermented ginseng leaves also has a function to activate NK cells. Was expected. In addition, the production of IL-6 and TNF-α, which are classified as inflammatory cytokines that are directly related to the homing of immune cells in the inflammatory site, and the activity of producing IL-12, which is directly related to the activation of cellular immunity, are significant. Since it was confirmed that there was a simple hot water extraction, ginseng leaves fermented with Cordyceps sinensis were more effective in activating (regulating) immune mechanisms acting on bioprotection.

<Experimental Example 4> Evaluation of immune activity and anti-metastatic efficacy of samples through intravenous administration

<4-1> Evaluation of tumor cell killing ability by natural killer cells

NK cells are known to be excellent in removing cancer cells, and as large granular lymphocytes that account for 5-10% of lymphocytes in peripheral blood, NK cells are cells with natural immune function that destroy virus-infected cells or cancer cells without prior detection of specific antigens. In particular, it is known that the number of NK cells is extremely reduced in cancer patients. NK cells are known to work by the following two mechanisms of action. First, cells whose class I HLA (human leukocyte antigen) is expressed on the cell surface, that is, autologous cells, do not kill, but are infected with viruses or class I like cancer cells. Second, NK cells secrete cytokines such as interleukin, which directly attack and remove cells with reduced HLA, and thereby Tc lymphocytes (Cytotoxic T cells, cell killer T lymphocytes), macrophages (macrophages) and Likewise, it is known to activate immune cells that have direct attack ability against infected cells and cancer cells. Therefore, it can be said that the activating stimulating effect of NK cells is in an extension line similar to the increase in body immunity in addition to the expectation of simple anticancer effect. In addition, it distinguishes whether it is a normal cell through the relationship between the intrinsic receptor of the NK cell and the ligand of the target cell, and when it is not a normal cell, it secretes perforin to create a complex in the cell membrane of the target cell to make a hole. After that, granzyme enzyme is secreted to dissolve the inner cell, and when NK cells are activated, cytokines such as IFN-γ are secreted to activate the adaptive immune system, thereby activating macrophages and cytotoxic T lymphocytes (CTL). It is known to induce

In Experimental Example 3, both samples of simple hot water extracted crude polysaccharide GLW-0 derived from ginseng leaf and crude polysaccharide GLF-0 derived from fermented ginseng leaf showed excellent cytokine production ability against mouse peritoneal macrophages in in vitro, but GLF- It was confirmed that the 0 sample showed better production performance. In this experiment, GLW-0 and GLF-0 samples were injected intravenously at 10, 100 and 1000 μg/mouse, respectively, to BALB/c (6 weeks, female) mice, and killed 1 day later by cervical dislocation, resulting in aseptic conditions. The spleen was excised from and confirmed the cytolytic activity of NK cells. The specific method is as follows.

Simple hot water extract from ginseng leaves and crude polysaccharide GLF-0 samples from fermented ginseng leaves were administered intravenously to BALB/c (6 weeks, female) mice 3 days before the experiment and 1 day before the experiment. Then, the spleen was removed aseptically by killing by cervical dislocation. Using stainless steel mesh, splenocytes were obtained by grinding (100 mesh) and filtration (200 mesh) on PBS. NK cells were recovered using a Natural killer (NK) cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's instructions, and the number of cells was 1.0 × 10 ⁷ cells/mL, 5 × 10 ⁶ cells/mL and 2.5 It was adjusted to be ×10 ⁶ cells/mL and used as an effector cell. ^{Adjust to 1×10 5} cells/mL using tumor cell YAC-1, which is highly susceptible to natural killer (NK) cells, as a target cell, and finally the ratio of effector cells and target cells to a round-bottomed 96-well microplate. (E/T ratio) was adjusted to be 100, 50 and 25:1. After 6 hours incubation in a 37 ℃, 5% CO2 incubator, the amount of lactate dehydrogenase (LDH) released from the target cell by the killing ability of the effect cell was measured using EZ-LDH (Daeil lab service), and cytokine was measured. For 24 hours, it was incubated in a 5% CO2 incubator. As NK cell activity, LDH generated when killing tumor cells of NK cells was calculated, which was calculated using Equation 2 below.

<Equation 2>

NK cell activity (%) = [(Experimental secretion amount-natural secretion amount)/(maximum secretion amount-natural secretion amount)]×100

The results of NK cell stimulating activity of crude polysaccharide GLW-0 derived from ginseng leaf and crude polysaccharide GLF-0 derived from fermented ginseng leaf were all 25, 50 and 100:1 of effect cells (splenocytes) against target cell (YAC1). In terms of the ratio (E/T ratio), crude polysaccharide GLF-0 derived from fermented ginseng leaf showed higher killing ability against tumor cells than simple hot water extracted crude polysaccharide GLW-0 derived from ginseng leaf. In particular, at 100:1 E/T ratio. The GLW-0 sample showed about twice the negative control (NC) compared to about 2 times, the GLF-0 sample showed about 4 times the cancer cell killing ability compared to NC (Figs. 6 A and B).

Summarizing the above results, similar to the results of cytokine production in vitro, NK cell activity results in vivo have better anticancer activity in crude polysaccharide GLF-0 derived from fermented ginseng leaves than crude polysaccharide GLW-0 derived from simple hot water extract from ginseng leaves. Appeared to represent. As a result, it was confirmed that crude polysaccharide extracted from ginseng leaves fermented through Cordyceps sinensis was superior to the crude polysaccharide extracted from simple ginseng leaves.

<4-2> Evaluation of anti-metastatic activity against tumor cells through intravenous administration

As a result of the experiment in <4-1> above, the strong NK cell activity stimulating ability of crude polysaccharide GLF-0 derived from fermented ginseng leaves was confirmed. In order to measure its anti-metastatic activity, a sample was injected intravenously by concentration, followed by Colon 26M3.1 carcinoma. Cells were administered intravenously to evaluate anti-metastatic activity. The specific method is as follows.

Anti-metastatic activity against crude polysaccharide GLF-0 samples derived from fermented ginseng leaves was evaluated using an experimental animal tumor metastasis model using Colon26-M3.1 cells, a highly metastatic tumor cell line for the lung. In order to observe the tumor metastasis effect by the sample, GLF-0 samples at each concentration (10, 100, 1,000 μg/mouse) 3 days and 1 day before the injection of the tumor cell line, as a positive control, as a commercial anticancer agent derived from fingering mushrooms. The polysaccharide-K (PSK) used was intravenously administered at 1000 μg/mouse, and then Colon26-M3.1 cells (4×10 ⁴ cells/mouse) were inoculated through the vein of BALB/c mice. 14 days after tumor inoculation, the mice were killed, lungs, which are the target organs of tumor cells, were excised, and the metastasized tumors were fixed in Bouin's solution (Sigma), and then the colony of the metastasized tumors was counted. The anti-metastatic effect by the sample was calculated by comparing with the control group inoculated with only tumors, and the number of tumors in the control group not administered with the sample was 100% metastasized to evaluate the anti-metastatic activity of crude polysaccharide GLF-0 derived from fermented ginseng leaves. I did.

As a result, in the group administered with 10 μg, 100 μg, and 1000 μg of GLF-0, an anti-metastatic effect was found in which metastasis was inhibited by 68.9%, 87.7%, and 79.5%, respectively. Rather than a high concentration of 1000 μg, 100 μg It showed significantly the best anti-metastatic effect in concentration. It was confirmed that the anti-metastatic activity was far superior to the anti-metastatic activity (72.5%) at the concentration of 1000 μg of PSK, which is used as a commercially available anticancer agent derived from Unji mushroom. In addition, it was predicted that the effect of inhibiting tumor metastasis by crude polysaccharide GLF-0 derived from fermented ginseng leaves may be partly due to activation of NK cells. Since NK cells are known to be activated by IL12 produced by macrophages, the crude polysaccharide GLF-0 derived from fermented ginseng leaves induces the production of IL12 by macrophages at low concentrations rather than at high concentrations. It was determined to induce a strong anti-metastatic effect by the cell (Fig. 7).

<4-3> Evaluation of anti-metastatic activity of GLF-0 in NK cell function-controlled mice

It is known that the antitumor activity by administration of BRM (biological response modifier) substances in the pre-experimental model is mainly activated by macrophage or NK cell activation. Therefore, in this experiment, in order to determine whether the anti-transferrative activity of crude polysaccharide GLF-0 derived from fermented ginseng leaves is the effect of NK cell stimulation, rabbit antiasialo GM1 antibody was used to block the NK cell function of the mouse and colon 26M3.1. An experiment to measure anti-metastatic activity was performed using lung carcinoma cells (FIG. 8). The specific method is as follows. A rabbit anti-asialo GM1 antibody, which inhibits NK cells, was administered intravenously to an experimental animal tumor metastasis model using Colon26-M3.1 cells, a classically metastatic tumor cell line for lungs. A crude polysaccharide GLF-0 sample derived from fermented ginseng leaves, which was previously confirmed to have excellent anti-metastatic activity, was administered intravenously at a concentration of 1000 μg/mouse 3 days and 1 day before tumor cell line inoculation. In addition, rabbit anti-asialo GM1 antibody (Wako Pure Chemicals industries, Ltd., Japan) was injected intravenously 2 days before inoculation according to the manufacturer's manual. Thereafter, Colon26-M3.1 cells (4×10 ⁴ cells/mouse) were inoculated into the caudal vein of BALB/c mice. 14 days after tumor inoculation, the mice were killed, lungs, which are the target organs of tumor cells, were excised, and the metastasized tumors were fixed with Bouin's solution (Sigma), and then the colony of the metastasized tumors was counted.

As a result of each administration of 1000 μg of GLF-0 sample to mice, it showed excellent anti-metastatic effect. When GLF-0 was administered at a concentration of 1000 μg to mice that blocked NK cell function, colony compared to the group treated with only antiasialo GM1. It was confirmed that the number was reduced by about 2/3 (FIG. 9). From these results, it was determined that the anti-metastatic effect of crude polysaccharide GLF-0 derived from fermented ginseng leaves would act by immune cells other than NK cells (eg, CTL, macrophages). In several experimental reports, it is reported that the antitumor metastasis effect by natural products in the experimental animal metastasis model is mainly induced by tumoricidal macrophages and NK cells, so the antitumor effect of crude polysaccharide GLF-0 derived from fermented ginseng leaves is mainly NK cell. In addition to this, immune cells such as macrophages and T cells were also speculated to contribute to some antitumor metastasis effects.

<4-4> Evaluation of tumor cell killing ability by cytotoxic T lymphocytes

When the human body is infected with non-magnetic cells, innate immune cells such as macrophages, NK cells, and complement systems are activated, causing an immune response to remove non-magnetic cells, and when macrophages recognize non-magnetic cells, and when predated, proteolytic enzymes in the cytoplasm It produces and kills the phagocytic cells, and at the same time, peptides generated from microbial proteins present antigens to T lymphocytes to trigger an adaptive immune response. Then, T lymphocytes that have recognized the antigen are differentiated into cytotoxic T lymphocytes, auxiliary T lymphocytes, and memory T lymphocytes to activate adaptive immunity.

In <4-3>, in the anti-metastatic effect confirmed by intravenously injecting crude polysaccharide GLF-0 derived from fermented ginseng leaves into mice whose function of NK cells was controlled, colon26-M3.1 cells were administered, other immune cells other than NK cells. It was found that some of the anti-metastatic activity was observed. To confirm this, the spleen of the mouse was excised and re-sensitized with Colon26-M3.1 cells to measure the activation of immune cells. The specific method is as follows.

Cytotoxic T lymphocyte (CTL) activity on crude polysaccharide GLF-0 samples derived from fermented ginseng leaves was obtained by recovering splenocytes in an experimental animal tumor metastasis model using Colon26-M3.1 cell, a highly metastatic tumor cell line for lung. When co-cultured with the inoculated tumor metastasis cells, the activity against CTL, a kind of adaptive immune cells, was measured and confirmed. In order to observe the effect of CTL by the sample, the sample was administered intravenously at 10, 100 and 1000 μg/mouse 3 days and 1 day before the injection of the tumor cell line. Thereafter, Colon26-M3.1 cells (4×10 ⁴ cells/mouse) were inoculated into the veins of BALB/c mice. The mice were killed 14 days after tumor inoculation, and then, the spleen was aseptically removed by killing by cervical dislocation. Using stainless steel mesh, splenocytes were obtained by grinding (100 mesh) and filtration (200 mesh) on PBS. CTL is recovered using a CTL isolation kit (Miltenyi Biotec) according to the manufacturer's instructions, and the number of cells is ^{adjusted to 1.0×10 7} cells/mL, 5×10 ⁶ cells/mL, and 2.5×10 ⁶ cells/mL. It was used as an effector cell. To confirm the adaptive immunity, use the inoculated tumor cell line, Colon26-M3.1 cell as a target cell, ^{adjust it to 1×10 5} cells/mL, and finally place the effector cell and target cell in a round-bottomed 96-well microplate. The ratio of (E/T ratio) was adjusted to be 100, 50, 25:1. After 6 hours incubation in a 37 ℃, 5% CO2 incubator, the amount of LDH released from the target cell by the killing ability of the effect cell was measured using EZ-LDH (Daeil lab service), and an indicator of the tumor cell killing ability of CTL The material LDH was calculated according to the following equation 3.

<Equation 3>

Cytotoxic T lymphocyte activity (%) = [(Experimental secretion volume-natural secretion volume)/(maximum secretion volume-natural secretion volume)]×100

As a result, in the case of crude polysaccharide GLF-0 derived from fermented ginseng leaves, which was predicted that other immune cells other than NK cells would be involved in the anti-metastatic effect, cytotoxic T compared to the normal control (NC) group without sample administration at 100:1 ratio. It was confirmed that the activity of lymphocytes showed a significant difference and increased in a concentration-dependent manner (FIG. 10). Taken together, these results show that crude polysaccharide GLF-0 derived from fermented ginseng leaves partially increases the activity of cytotoxic T lymphocytes, which are adaptive immune cells, which together with innate immune cells such as macrophages and NK cells increase the adaptive immunity of the host. It has been suggested that it can enhance immunity.

<Experimental Example 5> Evaluation of immune activity and anti-metastatic activity of samples through oral administration

<5-1> Activation of natural killer cells through oral administration

In Experimental Example 4, the crude polysaccharide GLF-0 sample derived from fermented ginseng leaves, which showed excellent results in the NK cell activation and anti-metastatic activity experiments by intravenous administration, was orally administered to mice once a day for 14 days by concentration, and NK cell stimulating activity was observed. Measured. The specific method is as follows.

Crude polysaccharide GLF-0 derived from fermented ginseng leaves was administered to BALB/c (6 weeks, ♀) mice once a day for 14 days for each concentration, and then killed by cervical dislocation and spleen aseptically removed. Using a stainless steel mesh, splenocytes were obtained by grinding (100 mesh) and filtering (200 mesh) on PBS. NK cells were recovered using a Natural killer (NK) cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's instructions, and the number of cells was 1.0×10 ⁶ cells/mL, 5×10 ⁵ cells/mL and 2.5 It was adjusted to be ×10 ⁵ cells/mL and used as an effector cell. ^{Adjust to 1×10 5} cells/mL using tumor cell YAC-1, which is highly susceptible to natural killer (NK) cells, as a target cell, and finally the ratio of effector cells and target cells to a round-bottomed 96-well microplate. (E/T ratio) was adjusted to be 10, 5 and 2.5:1. After 6 hours incubation in a 37 ℃, 5% CO2 incubator, the amount of lactate dehydrogenase (LDH) released from the target cell by the killing ability of the effect cell was measured using EZ-LDH (Daeil lab service), and cytokine was measured. For 24 hours, incubated in a 5% CO2 incubator. The results were calculated using Equation 2 below.

<Equation 2>

NK cell activity (%) = [(Experimental secretion amount-natural secretion amount)/(maximum secretion amount-natural secretion amount)]×100

As a result, crude polysaccharide GLF-0 derived from fermented ginseng leaves showed excellent NK cell activity at 100 μg rather than at a high concentration of 1000 μg, contrary to the results of NK cell activity in vivo. It was confirmed that the killing ability appeared (FIG. 11)

<5-2> Evaluation of anti-metastatic activity against tumor cells through oral administration

In a previous experiment, it was confirmed that crude polysaccharide GLF-0 derived from fermented ginseng leaves exhibited excellent anti-metastatic activity through intravenous administration, and the anti-metastatic activity was re-measured through oral administration. The specific method is as follows. Anti-metastatic activity against crude polysaccharide GLF-0 derived from fermented ginseng leaves was evaluated using an experimental animal tumor metastasis model using Colon26-M3.1 cells, a classically metastatic tumor cell line for lungs. In order to observe the tumor metastasis effect by the sample, the GLF-0 sample was orally administered once a day for 14 days at each concentration (100, 1000 μg/mouse). Colon26-M3.1 cells (4×10 ⁴ cells/mouse) were inoculated into the veins of BALB/c mice, and 14 days after tumor inoculation, the mice were killed, and the lungs, which are the target organs of tumor cells, were excised and Bouin's solution (Sigma) After fixing the metastatic tumor at, the colony of the metastatic tumor was counted. The anti-metastatic effect of the sample was calculated by comparing with the control group inoculated with only the tumor.

As a result of confirming the anti-metastatic activity of GLF-0 by setting the number of tumors in the tumor control group to which the sample was not administered as 100% metastasis, about 35.7% of the anti-metastatic activity was confirmed in the group administered with 100 μg of the GLF-0 sample. In the -0 sample 1000 μg administration group, about 84% of excellent anti-metastatic activity was exhibited (FIG. 12). Based on the above results, it was again confirmed that crude polysaccharide GLF-0 derived from fermented ginseng leaves exhibited excellent anti-metastatic activity not only in intravenous administration but also in oral administration, and the NK cell stimulation activity test and dosage were compared through previous oral administration. Although the results were different from those of the time, it could be expected that the inhibitory effect of tumor metastasis by GLF-0 was mainly due to the activation of NK cells. In addition, if the daily intake is estimated using the daily dosage of oral administration between animals and humans based on the above results, 243.3 mg per day based on adult males (60 kg) is approximately 1 as shown in Equation 4 below. If consumed for a month, it could be estimated that about 84% of tumor metastasis inhibitory effects would be achieved.

<Equation 4>

<Experimental Example 6> Measurement of killing ability of tumor metastases by cytotoxic T lymphocytes

In order to determine whether the anti-metastatic activity of crude polysaccharide GLF-0 derived from fermented ginseng leaves, which showed excellent anti-metastatic effect through oral administration, was due to NK cells or other immune cells, the spleen of mice was removed after oral administration and Colon26. -M3.1 cells were resensitized to measure the activation of immune cells.

As a result, superior activity of cytotoxic T lymphocytes was confirmed at a concentration of 1000 μg compared to the normal control (NC) group without sample administration at 100:1 ratio. Taken together, these results show that crude polysaccharide GLF-0 derived from fermented ginseng leaves partially increases the activity of cytotoxic T lymphocytes, which are adaptive immune cells, which, together with innate immune cells such as macrophages and NK cells, increase the adaptive immunity of the host. It was determined that it can improve (Fig. 13).

Claims (10)

Adding water to ginseng leaves, inoculating a Cordyceps sinensis strain thereto, and liquid culturing to prepare a ginseng leaf fermentation product;
Heating the ginseng leaf fermented product to extract hot water, and then adding ethanol to prepare a precipitate; And
The sediment was centrifuged to recover the sediment, the recovered sediment was dissolved in distilled water, dialyzed, and then freeze-dried to contain 2.9 to 3.5 mol% of mannose, 4.3 to 6.5 mol% of rhamnose, and glue. Curonic acid is 1.5 to 2.8 mol%, galacturonic acid is 25 to 43 mol%, glucose is 15 to 35 mol%, galactose is 8 to 18 mol%, xylose is 0.2 to 1.1 mol%, arabinose is 4 to 14 Mole%, and a fucose comprising the step of obtaining a polysaccharide of 0.2 to 0.8 mol%
A method for preparing a composition for enhancing immunity,
The polysaccharide has a glucose content greater than galactose content, and a glucose content greater than arabinose content,
The polysaccharide has higher production capacity of one or more cytokines selected from the group consisting of IL-6, IL-12 and TNF-α than polysaccharide obtained by hot water extraction of ginseng leaves without a fermentation process using Cordyceps sinensis,
Method for producing a composition for enhancing immunity.
The method of claim 1,
The cordyceps strain is a method for producing a composition for enhancing immunity, characterized in that the cordyceps sinensis.
delete The method of claim 1,
The polysaccharide is a method for producing a composition for enhancing immunity, characterized in that the neutral sugar content is 50 to 65% by weight.
The method of claim 1,
The polysaccharide is a method for producing a composition for enhancing immunity, characterized in that the uronic acid content is 30 to 43% by weight.
The method of claim 1,
The composition for enhancing immunity is a method for producing a composition for enhancing immunity, wherein the composition is a feed composition or a food composition.
delete Adding water to ginseng leaves, inoculating a Cordyceps sinensis strain thereto, and liquid culturing to prepare a ginseng leaf fermentation product;
Heating the ginseng leaf fermented product to heat extraction, and then adding ethanol to prepare a precipitate; And
The sediment was centrifuged to recover the sediment, the recovered sediment was dissolved in distilled water, dialyzed, and then freeze-dried to contain 2.9 to 3.5 mol% of mannose, 4.3 to 6.5 mol% of rhamnose, and glue. Curonic acid is 1.5 to 2.8 mol%, galacturonic acid is 25 to 43 mol%, glucose is 15 to 35 mol%, galactose is 8 to 18 mol%, xylose is 0.2 to 1.1 mol%, arabinose is 4 to 14 Mole%, and a fucose comprising the step of obtaining a polysaccharide of 0.2 to 0.8 mol%
As a method for preparing a pharmaceutical composition for preventing or treating cancer,
The polysaccharide has a glucose content greater than galactose content, and a glucose content greater than arabinose content,
The polysaccharide has higher production capacity of one or more cytokines selected from the group consisting of IL-6, IL-12 and TNF-α than polysaccharide obtained by hot water extraction of ginseng leaves without a fermentation process using Cordyceps sinensis,
A method of preparing a pharmaceutical composition for preventing or treating cancer.
Adding water to ginseng leaves, inoculating a Cordyceps sinensis strain thereto, and liquid culturing to prepare a ginseng leaf fermentation product;
Heating the ginseng leaf fermented product to extract hot water, and then adding ethanol to prepare a precipitate; And
The sediment was centrifuged to recover the sediment, the recovered sediment was dissolved in distilled water, dialyzed, and then freeze-dried to contain 2.9 to 3.5 mol% of mannose, 4.3 to 6.5 mol% of rhamnose, and glue. Curonic acid is 1.5 to 2.8 mol%, galacturonic acid is 25 to 43 mol%, glucose is 15 to 35 mol%, galactose is 8 to 18 mol%, xylose is 0.2 to 1.1 mol%, arabinose is 4 to 14 Mole%, and a fucose comprising the step of obtaining a polysaccharide of 0.2 to 0.8 mol%
As a method for preparing a food composition for preventing or improving cancer,
The polysaccharide has a glucose content greater than galactose content, and a glucose content greater than arabinose content,
The polysaccharide has higher production capacity of one or more cytokines selected from the group consisting of IL-6, IL-12 and TNF-α than polysaccharide obtained by hot water extraction of ginseng leaves without a fermentation process using Cordyceps sinensis,
A method of preparing a food composition for preventing or improving cancer.
The method of claim 9,
The food composition for preventing or improving cancer, characterized in that it has an inhibitory activity of cancer metastasis.

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