KR20120122709A - Pharmaceutical composition containing extract of taro - Google Patents

Abstract

PURPOSE: A pharmaceutical composition and a health functional food containing taro extract for enhancing immune system are provided to be used as a therapeutic agent for anticancer and anti-metastasis. CONSTITUTION: A pharmaceutical composition for enhancing immune system contains polymer saccharides of chemical formula 1 as an active ingredient. A health functional food for enhancing immune system contains the polymer saccharides as an active ingredient. A pharmaceutical composition for anti-metastasis contains the polymer polysaccharides of chemical formula 1 as an active ingredient. A pharmaceutical composition for enhancing immune system contains taro exract as an active ingredient. The taro extract contains galactose and mannose. A health functional food for enhancing immune system contains the taro extract as an active ingredient.

Description

Pharmaceutical composition containing taro extract as active ingredient {PHARMACEUTICAL COMPOSITION CONTAINING EXTRACT OF TARO}

The present invention relates to a pharmaceutical composition and health functional food for enhancing immune system activity comprising taro extract as an active ingredient.

Recently, a new lifestyle of well-being has emerged, and it is becoming popular as an important social trend in the area of consumer life. This rapid rise in well-being is not only in Korea, but also in the world. It is expected that even individual lifestyles will change. In this social trend, the food industry of the world is paying the most attention and focusing on health functional foods, and in particular, much attention and research are focused on foods using natural materials. Among them, carbohydrates, which have only been functioning as energy sources in the past, and various polysaccharides derived from natural products have been reported to have very good immune stimulating activity. Polysaccharides isolated from natural products have been reported to enhance immunosuppressive activities such as anti-complementary, macrophage and anti-tumor activity. Attempts are gaining much attention.

Taro, the corms of Colocasia esculenta L. Scotte) is a perennial herb belonging to Araceae, which has a high value in use, and is distributed over 100 genus and 1,500 varieties worldwide, and only edible and medicinal plants are edible and medicinal. It is a crop used as a. The research on taro is declining due to deterioration of quality due to water loss after harvesting due to viscous flow observed in taro or browning caused by tissue damage in processing such as shear and peeling. There is a search for ways to secure safety and improve quality in a simpler and more economical way. In other words, studies on physiological changes, pathological decay and biochemical changes related to the storage and shelf life of taro, account for most of the taro (7, 18, 42, 44, 56). Except for browning and browning inhibition, there is no significant progress in research. Especially, studies on high value-adding of taro, ie, bioactivity and active ingredients, are very limited.

Therefore, the present inventors have come to the present invention to develop toran polysaccharides as functional materials by separating and purifying congenital immune system stimulating active polysaccharides from taro-derived viscous substances and identifying the structural characteristics of their immune activity and active ingredients.

It is an object of the present invention to extract a polysaccharide fraction from taro and to provide a pharmaceutical composition and health functional food for enhancing immune activity comprising the extract and an anti-metastatic pharmaceutical composition and health functional food for tumor cells comprising the extract.

In one embodiment of the present invention to achieve the above object, it provides a pharmaceutical composition for enhancing the activity of the immune system containing the polymer polysaccharide of Formula 1 as an active ingredient.

(1)

In another embodiment, the molecular weight of Formula 1 provides a pharmaceutical composition, characterized in that 5 to 500 kDa.

In one embodiment provides a functional food for enhancing immune system activity containing the polymer polysaccharide of Formula 1 as an active ingredient.

(1)

In another embodiment, the molecular weight of Formula 1 provides a dietary supplement comprising 5 to 500 kDa.

In one embodiment there is provided an anti-transduction pharmaceutical composition for tumor cells containing the polymer polysaccharide of Formula 1 as an active ingredient.

(1)

In another embodiment, the molecular weight of Formula 1 provides a pharmaceutical composition, characterized in that 5 to 500 kDa.

In one embodiment provides an anti-transition health functional food for tumor cells containing the polymer polysaccharide of Formula 1 as an active ingredient.

(1)

In another embodiment, the molecular weight of Formula 1 provides a dietary supplement comprising 5 to 500 kDa.

In one embodiment provides a pharmaceutical composition for enhancing immune system activity comprising the taro extract as an active ingredient. In another embodiment, the taro extract provides a pharmaceutical composition comprising galactose and mannose. In another embodiment, the pharmaceutical composition provides a pharmaceutical composition, characterized in that it has an anticomplement activating function. In another embodiment, the pharmaceutical composition provides a pharmaceutical composition, characterized in that it has an activating function of macrophages or natural killer cells.

In one embodiment, it provides a health functional food for enhancing immune system activity comprising the taro extract as an active ingredient. In another embodiment, the taro extract provides a health functional food comprising galactose and mannose. In another embodiment, the dietary supplement provides a dietary supplement characterized in that it has an anti-complement activating function. The health functional food provides a health functional food having an activation function of macrophages or natural killer cells.

In one embodiment, an anti-metastatic pharmaceutical composition for tumor cells comprising the taro extract as an active ingredient is provided. In another embodiment, the taro extract provides a pharmaceutical composition comprising galactose and mannose. In another embodiment, the pharmaceutical composition provides a pharmaceutical composition, characterized in that it has an anticomplement activating function. In another embodiment, the pharmaceutical composition provides a pharmaceutical composition, characterized in that it has an activating function of macrophages or natural killer cells.

In one embodiment, it provides a dietary supplement for tumor cells comprising the taro extract as an active ingredient. In another embodiment, the taro extract provides a health functional food comprising galactose and mannose. In another embodiment, the dietary supplement provides a dietary supplement characterized in that it has an anti-complement activating function. In another embodiment, the dietary supplement provides a dietary supplement characterized in that it has an activation function of macrophages or natural killer cells.

The composition of the present invention, the polymer polysaccharide or taro extract of Formula 1 based on the total weight of the composition 0.1 to 50% by weight. The composition comprising the polymer polysaccharide or the taro extract of Formula 1 of the present invention may further include a suitable carrier, excipient or diluent according to a conventional method. Examples of carriers, excipients and diluents that can be included in the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. The compositions according to the invention can be used in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral formulations, external preparations, suppositories or sterile injectable solutions, respectively, according to conventional methods. have. More specifically, when formulating the composition, it can be prepared using a diluent or an excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, and the like. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient such as starch, calcium carbonate (calcium) in the polymer polysaccharide or taro extract of Formula 1 carbonate, sucrose, lactose, gelatin and the like can be mixed. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, syrups and the like, and various excipients such as wetting agents, sweeteners, fragrances, preservatives, etc. in addition to commonly used diluents such as water and liquid paraffin . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations and suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. Examples of the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.

The present invention may vary depending on the age, sex and weight of the patient, but in general, an amount of 0.01 to 500 mg / kg, preferably 0.1 to 100 mg / kg, may be administered once to several times a day. . The dosage may also be increased or decreased depending on the route of administration, degree of disease, sex, weight, age, and the like. Thus, the dosage amounts are not intended to limit the scope of the invention in any manner.

The composition of the present invention can be administered to mammals such as rats, mice, livestock, humans, and the like in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections. The polymer polysaccharide or taro extract of Chemical Formula 1 of the present invention has little toxicity and side effects, and therefore can be used safely even when taken for long periods of time.

The present invention provides a health functional food comprising the polymer polysaccharide or taro extract of Formula 1 and a food supplement acceptable food supplement, various foods, for example, beverages, gums, tea, vitamin complexes, health supplements Foods and the like, and can be used in the form of pills, powders, granules, acupuncture, tablets, capsules or beverages. At this time, the amount of the polymer polysaccharide or taro extract of the formula (1) in the food or beverage can be generally added to 0.01 to 15% by weight of the total food weight for the health food composition of the present invention, 100 ml for the health beverage composition It can be added in a ratio of 0.02 to 10 g, preferably 0.3 to 1 g on the basis of.

Food supplementary additives as defined herein include food additives customary in the art, such as flavoring agents, flavoring agents, coloring agents, fillers, stabilizers, and the like. The health beverage composition according to the present invention is an essential ingredient in the indicated ratio, and there is no particular limitation on the ingredients added in addition to the polymer polysaccharide or taro extract of Chemical Formula 1, and additional ingredients such as various flavors or natural carbohydrates, such as ordinary drinks It may contain as. Examples of the natural carbohydrate include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; Polysaccharides such as dextrin, cyclodextrins; And sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (e.g., Rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. have. The ratio of the natural carbohydrate 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-mentioned composition, the composition of the present invention can be used as a flavoring agent such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and intermediates (cheese, chocolate etc.), pectic acid and its salts, Salts, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages and the like. The compositions of the present invention may also contain pulp for the production of natural fruit juices and fruit juice beverages and vegetable beverages. These components can be used independently or in combination. The proportion of such additives is not so 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.

Pharmaceutical composition and dietary supplement comprising the taro extract according to the present invention has an anti-complement activation function, the function of activating macrophages or natural killer cells to activate the immune system, and effectively reduce the metastasis of cancer cells, It can be used as a therapeutic for anti-metastasis.

1 is a graph showing the degree of anti-complement activity of the taro extract.
2 is a graph showing the effect of metal ions on the anti-complement activity of the taro extract.
Figure 3 is the result of observing the decomposition of the C ₃ factor after reacting the taro extract in the reaction system in which the basic reaction system and the specific metal ion is removed.
Figure 4 is a graph showing the ability to promote the production of taro extract IL-6, IL-12 and TNF-α.
5 is a graph showing the natural killer cell activation ability of the taro extract.
6 and 7 are graphs showing the results of analysis by GC-MS after methylation of the toran extract.
Figure 8 is the predicted overall structure of Taro-4-I.

Hereinafter, the present invention will be described in detail by the following examples. However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

Example

Example  1. Preparation of Taro Extract

1-1. Taro origin Crude  detach

After harvesting the taro harvested from Gokseong, Jeonnam, the shells were removed and sliced, and 10 L of water was added to 5 kg of taro, followed by cold water extraction at room temperature for 24 hours. The taro cold water extract was concentrated using a rotary vacuum evaporator (EYELA, Tokyo Rikakikai Co., Tokyo, Japan), to which 4-fold volume of ethanol was added and the polysaccharide was precipitated while stirring for one day. At this time, the precipitated polysaccharide was recovered using a centrifuge (6000 rpm, 30 min, 4 ° C.), dissolved in a small amount of water, and then dialyzed for 3 days using a dialysis membrane (MW cut-off 12,000, Sigma). The low molecular weight material was removed. The resultant was concentrated again using a rotary depressurizer, and then lyophilized (FreeZone 12 Liter, Labconco, MISSOVRI 64132, KANSAS CITY, USA) to obtain a crude polysaccharide Taro-0.

1-2. Purification of Taro-derived Polysaccharides

1-2-1. Anion exhange chromatography  ( AEC )

Crude Taro-0, isolated from taro, was dissolved in a small amount of distilled water, adsorbed onto a DEAE-Sepharose FF (Cl ^- form, 5.5 × 25 ㎝) column equilibrated with distilled water, and then distilled water was eluted to separate the nonadsorbed fraction. Then, the fractions were separated by eluting with 0.05 M ~ 2 M NaCl solution step by step. The fractions eluted from the anion exchange chromatography column were fractionated using a fraction collector (1200 series, EYELA, Tokyo, Japan), and the elution peaks were prepared by measuring the neutral sugar, acid sugar, protein and KDO content, respectively.

From the crude polysaccharide fraction Taro-0, one nonadsorbed fraction (Taro-1) and seven adsorption fractions (Taro-2 to 8) were obtained through the ion exchange resin. In addition, each fraction was concentrated, dialysis and lyophilized to be used in subsequent experiments.

1-2-2. Come permeation chromatography  ( GPC )

Sephadex G-100 equilibrated with 50 mM ammonium formate buffer (pH 5.5) after dissolving 0.5 g of Taro-4 fraction (0.2 M elution fraction), which had good yield and immunity during purification using ion exchange resin, in a small amount of water. Gel permeation chromatography (GPC) was performed using a column (4 × 120 cm). The eluate was fractionated into 100 fractions of 7 mL each, and each fraction was subjected to neutral sugar content analysis to obtain two fractions Taro-4-I and Taro-4-II with different molecular weights. Among them, Taro-4-I, a polymer polysaccharide, was excellent in purity, yield, and immune activity, and thus was used as a final fraction of taro-derived polysaccharide.

Example  2. Immune System Stimulation of Taro Extracts Bow performance  Measure

2-1. Of taro extract Complement  Activation

2-1-1. Anticomplement Bow performance

Blood from healthy adults was collected and left to coagulate for about 15 minutes at room temperature. The coagulated blood was cut and left at room temperature for about 5 minutes. The blood was left to stand again at 4 ° C. for about 20 minutes, and then centrifuged (2,200 rpm, 15 min, 4 ° C.) to separate serum, and then, 1 mL was dispensed into a small centrifuge tube and stored at -70 ° C. for freezing. Used for.

Anti-complement activity was measured by a complement fixation test method based on the level of erythrocyte hemolysis caused by the complement remaining after complement consumption by the sample using the Meyer method. Samples dissolved in distilled water at different concentrations were mixed with GVB ⁺⁺ (containing gelatin veronal buffer, pH 7.4, 0.1% gelatin, 0.15 mM Ca ⁺⁺ , 0.5 mM Mg ⁺⁺ ) and 50 μL of normal serum, respectively. First reaction was carried out for 30 minutes at. 350 μL of GVB ⁺⁺ was added to the reaction solution, and serial dilutions were made up to 10-160 fold, followed by 750 μL of GVB ⁺⁺ and positive erythrocytes (IgM-sensitizated sheep erythrocyte, EA cell, 1 × 10 ⁸ cells / 250 μL of mL) was added thereto, followed by secondary reaction at 37 ° C. for 60 minutes, and 2.5 mL of PBS (phosphate buffered saline, pH 7.4) was added to stop the reaction. The reaction solution was centrifuged at 2,000 rpm for 10 minutes, and the obtained supernatant was measured for absorbance at 412 nm to measure residual hemolytic activity. Anti-complement activity was inhibited by 50% total complement hemolysis (TCH ₅₀ ,%) of the negative control group that reacted only with normal serum and GVB ⁺⁺ and distilled water (inhibition of 50% total complement hemolysis, ITCH ₅₀ ,%). ). The positive control group was compared by using PSK (polysaccharide-K), an immune enhancer derived from fingering mushrooms.

As a result of confirming the activation ability of each sample with the ITCH ₅₀ 0% activation in the negative control group, it showed excellent anti-complement activity according to the positive control group with the same concentration at the concentration of 1,000 μg / mL of Taro-4-I ( 1). PSK, a positive control, is currently available as an anticancer drug, and is generally known as a polysaccharide that exhibits 50% or more anticomplementary activity at a concentration of 1,000 μg / mL. It was confirmed that the complementary system activation ability of I was excellent. In addition, it was found that the Taro-4-I activity was concentration dependent as a result of the difference in the concentration of the sample.

2-1-2. Complement  Review of Activation Path

The buffer and Ca ⁺⁺ and Mg ⁺⁺ ions to remove all EDTA-GVB ^{- -} In order to determine the active path of the complement system GVB ⁺⁺ buffer, Ca ⁺⁺ ions are selectively remove a Mg ^{++ -} EGTA-GVB buffer Was prepared and mixed with the sample and NHS, respectively, and reacted at 37 ° C for 30 minutes. Each reaction solution was activated again for 60 minutes at 37 ℃, 2.5 mL of PBS was added and centrifuged at 2,000 rpm for 10 minutes and the supernatant was measured for absorbance at 412 nm to compare the complement system activation ability by measuring the residual hemolytic activity .

As shown in FIG. 2, in the reaction system in which Ca ⁺⁺ was selectively removed, the activity was about 60% at 1,000 μg / mL compared to the basic reaction system in which both Ca ⁺⁺ and Mg ⁺⁺ existed, and less than 500 mg / mL. Showed a rapid decrease in activity, but a concentration-dependent trend was maintained. On the other hand, it was confirmed that almost no activity was lost in the reaction system in which both Ca ⁺⁺ and Mg ⁺⁺ were removed. This suggests that the activation of the complementary system of Taro-4-Ⅰ appears through both the classical and secondary paths.

By confirming the activation of C ₃ using two-dimensional immunoelectrophoresis, it is possible to confirm whether the anti-complement activity of the sample identified by the Meyer method is due to complement system activation or the result of complement inhibitor. ^{GVB ++ buffer, Mg ++ -EGTA-} GVB - buffer the GVB-EDTA ^-. Then buffer each normal human serum and the purified polysaccharide equal volume of the sample (50 μL by mixing for 30 minutes at 37 ℃ the reaction was cooled reaction 5 μL was loaded in a well of 1% agarose gel plate (5 × 5 cm) prepared by dissolving the solution in barbital buffer (pH 8.6), and primary electrophoresis (75 mA / plate) was performed at 4 ° C. for about 3 hours. Thereafter, agarose gel plate containing 1% anti-human C ₃ was subjected to secondary electrophoresis (25 mA / plate) for 4 hours at 4 ° C. The developed gel was bromophenol blue for about 10 minutes. After staining, decolorization was performed to confirm the precipitation line, and the activation of C ₃ was observed.

The reaction of Taro-4-I in the basic reaction system and the reaction system from which the specific metal ion was removed, respectively, and the degradation of the C ₃ factor were observed. In the normal reaction system containing both Ca ⁺⁺ and Mg ⁺⁺ , the first and second settler lines from the wells were found to be in similar proportions. Considering that the first settling line from the well is caused by C ₃ and the second settling line is caused by the degradation products C ₃ a and C ₃ b, the anti-complementary activity of Taro-4-I is due to complement inhibitors. I could prove no. On the other hand, in the reaction system where all metal ions were removed, only the first settler line was clearly visible and the second settler line was not observed. On the other hand, in the reaction system in which only Ca ⁺⁺ was selectively removed, the height of the second settler was relatively lower than that of the normal reaction system. This implies that C ₃ was activated only by the secondary path in the state where the classical path was inhibited. Therefore, the activation of the complement system by Taro-4-I was confirmed again through both the classical path and the secondary path. .

2-2. Macrophage ( macrophage ) Bow performance

To the polysaccharide sample solution prepared with PBS at a concentration of 0.5 μg / mL, RPMI 1640 was added, and serial dilutions were made in three folds at a concentration of 500 μg / mL to 0.2 μg / mL, and 100 μL aliquots were added to a flat bottomed 96-well microplate. Here, 1 mL of 5% thioglycollate medium was injected into the abdominal cavity of BALB / c (♀, 6 weeks) mouse, and the macrophage induced for 72 hours was recovered from the abdominal cavity and washed, followed by the number of cells (2.25 × 10 ⁵ cells / mL of RPMI). 1640) was adjusted to prepare a macrophage suspension, and 100 μL of this was added, followed by incubation for 24 hours in a 37 ° C., 5% CO ₂ incubator (22,23). After incubation, 150 μL of the cell culture solution was recovered by centrifugation at 1,500 rpm and 4 ° C. for 5 minutes, and the content of cytokine induced in the supernatant was measured.

The content of cytokine produced by macrophages was analyzed by sandwich ELISA (enzyme-linked immunosorbent assay). Each cytokine antibody was diluted in a coating buffer, coated on a flat-bottomed 96-well microplate, and left at 4 ° C. for 12 hours. The coated microplate was washed three times with washing buffer (PBS with 0.05% tween 20, PBST), and 200 μL of assay diluent (PBS with 10% FBS or 2% skim milk) was added and left for 1 hour. The non-stick well surface was blocked. After completion of blocking, each well was washed three times again using a washing buffer, and 50 μL of each of the serially diluted standards (recombinant mouse cytokine) and immune cell culture medium was dispensed into each well. The mixture was left at room temperature for 2 hours, washed with washing buffer, treated with 100 μL of detection antibody (in assay diluent), and left for 1 hour at room temperature, and washed again. After treatment with 100 μL of Enzyme reagent (avidin-horseradish peroxidase conjugate) for 30 minutes at room temperature, add 100 μL of substrate solution [tetramethylbenzidine (TMB) and hydrogen peroxide] and react for 30 minutes in the dark. 1 MH ₃ PO ₄ ) or (2 NH ₂ SO ₄ )] was measured for absorbance at 450 nm.

Results of cytokine production of macrophages by direct stimulation of taro derived polysaccharide sample was measured in in vitro, activated polysaccharide Taro-4-Ⅰ was confirmed that promote the production of IL-6, IL-12 and TNF-α (FIG. 4). For IL-6, concentration-dependent high yields were observed even at low concentrations above 1 μg / mL. Gradual concentration dependence was observed at each concentration of the sample, but production stimulatory activity of TNF-α was generally low. These results suggest that taro-4-I, a taro-derived polysaccharide, may have an effective activity in inflammatory immunity, and the concentration of the minimum sample for such activity may be several μg / mL. On the other hand, IL-12 showed a concentration-dependent increase in the concentration of Taro-4-I up to 19 μg / mL sample concentration, and at a higher concentration of IL-12, which was about 70% of the positive control LPS. However, at 500 μg / mL, the yield tended to decrease slightly. It is expected that taro-derived polysaccharides will be active in cell-mediated immunity such as activation of NK cells and activation of cytotoxic T lymphocytes (CTLs) through induction of Th1 type immune responses. The result was assumed to be nearby. In particular, since IL-12 is directly involved in the activation of NK cells that cause cancer cell death in the presence of cancer cells, it is recognized as an essential cytokine for inducing anticancer activity. Therefore, Taro-4-I was expected to be effective in cancer cells. . In this experiment, macrophages were stimulated by Taro-4-I for 24 hours, and the production and cellular immunity of TNF-α and IL-6, which are classified as inflammatory cytokines directly related to the homing of immune cells in the inflammatory site. It was confirmed that there is an activity that produces IL-12, which is directly related to the activation of, Taro-4-I from taro was found to have a function of activating (modulating) immune mechanisms that act on biological defense.

2-3. Natural killer cells ( NK cell Tumor cells by Killing ability

Taro-4-I polysaccharide samples purified from taro were injected intravenously into BALB / c (6 weeks, ♀) mice at different concentrations, and killed 3 days later by cervical dislocation. Spleen cells were obtained by grinding (100 mesh) and filtration (200 mesh) on PBS using a stainless steel mesh. 5 mL of 0.2% NaCl was added for 15-30 seconds to destroy the mixed erythrocytes, washed 2-3 times with serum-free medium, and adjusted to 1 × 10 ⁶ cells / mL and used as effector cells. Tumor cells with high susceptibility to natural killer cells, YAC-1, were used as target cells, and the ratio of effector cell and target cell (E / T ratio) was adjusted to 100, 50, 25 on a round-bottomed 96-well microplate. It was. The incidence of lactate dehydrogenase (LDH) liberated from the target cell by the killing ability of the effect cell after 6 hours of incubation at 37 ° C. in a 5% CO ₂ incubator was measured using Cytotox 96 (Promega, Madison, WI, USA). Tumor cell killing ability of natural killer cells was calculated by the following equation.

NK cell activity (%) = [(experimental--natural) / (maximum--natural)] × 100

The results of spontaneous killer cell stimulatory activity of taro-derived polysaccharide Taro-4-I were as shown in FIG. 5. It showed the ability to kill tumor cells depending on the ratio (E / T ratio) of effect cells (splenocytes) to target cells (YAC-1). As a result, the most pronounced activity was observed at E / T = 100. In particular, Taro-4-I was found to have higher activity than the control at 500 μg and 50 μg. In addition, the experimental results showed that the activity of about 250% was higher than that of normal cells at the low concentration of 50 μg rather than the high concentration of 500 μg. This suggests that taro-derived polysaccharide Taro-4-I contributes to the activation of natural killer cells that have the ability to kill tumor cells, and that they may have anticancer activity by natural killer cell stimulatory activity. The anticancer effect of Taro-4-I, a taro-derived polysaccharide, was expected to be strong at relatively low concentrations of 50 μg / mL.

Example  3. Antitransduction of Tumor Extracts Against Tumor Cells Bow performance

The anti-metastatic activity of the samples was determined using experimental animal tumor metastasis model using melanoma B16BL6, a highly metastatic tumor cell line for lungs. B16BL6 lung carcinoma (2.7 × 10 ⁴ ) was injected intravenously into BALB / c mice to observe the tumor metastasis effect of the samples. Samples were injected intravenously at each concentration 2 days before tumor inoculation. After 14 days of tumor inoculation, mice were killed and lungs, which are target organs of tumor cells, were extracted to fix metastasized tumors in Bouin's solution (Sigma), and the colony of metastasized tumors was counted. Antitumor metastasis effect by the sample was calculated in comparison with the control group inoculated only tumor.

As shown in Table 1, about 100 metastatic cancer colonies were identified in the control group that did not receive the sample, while in the experimental group administered Taro-4-I at the concentrations of 5 μg and 500 μg, about 22 and 18 As colony was found, excellent anti-tumor activity of 78.1% and 81.9% was confirmed, respectively.

Dose
(Μg / head) Number of lung metastasis Taro-4-l Mean ± SD Range Inhibition% Untreated 99.2 ± 13.2 86-112 0% 5 µg 21.8 ± 5.6 16-27 78.1% 50 µg 3.8 ± 1.3 2 to 5 96.8% 500 µg 18 ± 3.7 14-22 81.9%

On the other hand, when 50 μg was administered, about 4 metastasized colonies were identified, indicating the highest anti-transduction activity of 96.8%. The above results were judged to be a peculiar result that the administration of a lower concentration of 50 μg / mouse was effective in inhibiting tumor metastasis than in a high concentration of sample. This is the same result that Taro-4-Ⅰ showed the strongest stimulatory activity in the previous natural killer cell stimulation activity experiments. It is suggested that the effect of inhibiting tumor metastasis by Taro-4-I is mainly due to the activation of natural killer cells. Could be expected. Since natural killer cells are known to be activated mainly by IL-12, it was thought that they induced strong activation of natural killer cells by stimulating IL-12 production of macrophages by Taro-4-I.

Example  4. Structural analysis of taro extract

4-1. Determination of structure and binding position by methylation analysis

4-1-1. Methylsulfinyl carbanion Pharmacy

To prepare methylsulfinyl carbanion, 20 mL of anhydrous DMSO (dimethylsulfoxide) was added to 1.26 g of anhydrous NaH, and charged with nitrogen and reacted for about 10 to 15 minutes in a 90 ° C oil bath. The reaction was terminated at the point where the reaction solution became pale green, cooled to room temperature, and then centrifuged at 3,000 rpm and 30 ° C. The supernatant containing methylsulfinyl carbanion was replaced with nitrogen and dispensed in small amounts to prevent contact with air.

4-1-2. Methylation

Methylation to determine the binding site of the polysaccharide sample was carried out using the Hakomori method. 1 mL of anhydrous DMSO was added to each polysaccharide sample (0.5 mg) sufficiently dried in a desiccator for 1 to 2 days, stirred, and completely dissolved, followed by reaction for 4 hours by addition of 500 μL methylsulfinyl carbanion (MSCA). At this time, additional MSCA was added if necessary so that polysaccharide could be completely converted to polyalkoxide, and the presence of unreacted MSCA was confirmed by triphenylmethane. Polyalkoxide-converted samples were methylated by adding excess CH ₃ I. The remaining CH ₃ I was removed by N ₂ gas flushing and recovered using Sep-pak C ₁₈ cartridge.

4-1-3. Hydrolysis of methylated polysaccharides and Acetylation

The methylated sample was added with 1 mL of 2 M TFA, reacted at 121 DEG C for 1.5 hours, hydrolyzed and dried. The hydrolyzed sample was dissolved in ethanol to which 25% NH ₄ OH was added dropwise, and 10 mg of NaBH ₄ was added to ring-open and reduce for 4 hours. An appropriate amount of acetic acid was added to remove residual NaBH ₄ , and methanol was repeatedly dried. The excess acetic acid was then removed. After that, 1 mL of acetic anhydride was added and reacted at 121 ° C. for 3 hours to convert partially methylated alditol acetate, which was separated and extracted with a two-phase solvent system (chloroform, H ₂ O), and dissolved in acetone. Were analyzed by GC and GC-MS.

4-2. Partially methylated alditol acetate of GC  And GC - MS  analysis

GC analysis was carried out using the Young-Lin ACME-6100 GC equipped with SP-2380 capillary column (0.25 mm × 30 m, 0.2 mm film thickness, Supelco). [60 ℃ (1 min), 60 ℃ → 180 ℃ (30 ℃ / min), 180 ℃ → 250 ℃ (1.5 ℃ / min), 250 ℃ (5 min)] was analyzed by the splitless injection mode (1/20), wherein the flow rate of the carrier gas (N ₂ ) Was adjusted to 1.5 mL / min.

The GC-MS is equipped with an Agilent 6890N GC system equipped with SP-2380 capillary column (0.2 mm film, 0.25 mm id x 30 m, Supelco, Bellefonte, PA, USA) and 5973N mass spectrophotometer (Agilent Technologies, Palo Alto, CA, USA) using the splitless injection mode (He flow rate: 1.5 mL / min) under the same optimum temperature conditions as the GC analysis. The derivatives of methylated samples were identified by combining fragment ion analysis by GC-MS and relative retention time of GC. The molar% of each peak was converted from peak area and molecular response factor.

4-3. Taro By methylation analysis of -4-Ⅰ Sugar chain  Combined Form Analysis

6 and 7, after methylation, the total ion chromatogram analyzed by GC was analyzed to analyze sugar chain binding patterns of Taro-4-I, and each peak fragment ion was analyzed by GC-MS. In total, Table 2 summarizes the composition of the binding modalities per component.

As shown in Table 2, Taro-4-I had a total of 10 types of sugar chains involved in binding, and a high ratio of galactose binding and mannose binding was identified. In particular, galactose residues were detected at a high rate (48.4%) of terminal-Gal p, which is due to the high amount of galactose present at the non-reducing end, thus resulting in highly branched galactose and oligosaccharides in the side chain. It could be assumed that galactan is an outward form. 3-linked-Gal p and 2,3-branched-Gal p And side chains present in galactan form from the fact that 3,6-branched-Gal p is present at a high rate, form another non-reducing end by branching another galactose at the C2 or C6 position in the galactose core connected by 1 → 3 bond. Could be estimated.

Glycosyl residue
residue) Methyl group
location Decreased Glycoside Bonds
glycosidic linkage) Taro-4-l
(Molar%) ¹⁾ Arabinos
Galactose





Mannose

2,3,5
2,3,4,6
3,4,6
2,4,6
3,6
2,4
3,4
2,3,6
2,3
3 T-Ara f ^{2 )}
T-Gal p
2-Gal p
3-Gal p ^{3 )}
2,3-Gal p
3,6-Gal p
2,6-Gal p
4-Man p
4,6-Man p
2,4,6-Man p ^{4 )} 1.1
48.4
0.9
22.0
3.2
0.7
0.1
3.4
0.3
19.9 gun 100.0

¹⁾ Calculated from the peak area and molecular response factors of each partially methylated alditol acetates in GC and GC-MS.

²⁾ T-Ara f means non-reducing terminal arabinofuranoside.

³⁾ 3-Gal p means 3-linked galactopyranoside.

⁴⁾ 2,4,6-Man p means 2,4,6-branched mannopyranoside.

On the other hand, in the case of mannose residues, mannose at the non-reducing end was hardly detected while 4-linked Man p and 4,6-linked Man p and 2,4,6-linked Man p were detected at a high ratio. This fact suggests that the main chain of Taro-4-I is in the form of a mannan of (1 → 4) bonds, and at the C6 position of mannose, one side chain extends or at the C2 and C6 positions. At the same time, it was possible to estimate the exit of two strands. Especially Taro-4-I polysaccharide derived from taro is 2,4,6-linked Man p The unusually high proportion of residues was detected, which is a (1 → 4) mannose backbone with two galactan side chains, a highly branched form not found in other plant-derived polysaccharides. I could confirm that. In general, the high viscosity and physiological activity observed in taro are believed to be due to this highly branched galactomannan. However, even though polysaccharides are classified into the same type, the bioactivity of polysaccharides is known to show a great variety of microstructures for each plant, and accordingly, it is known that there is a difference in activity. have.

4-4. Taro Overall structure estimation of -4-I

The expected overall structure of Taro-4-I estimated based on the information obtained through the analysis of the sugar chain binding mode by methylation analysis of Taro-4-I immunoactive polysaccharide Taro-4-I and two enzymatic treatments is shown in FIG. 8. As shown in FIG. The molecular weight of Taro-4-I obtained in this experiment is about 200 kDa polysaccharide, which is composed of about 1,200 sugars in total. Therefore, it was reduced to about 1/30 because there was a limit in predicting their total microstructure. The structure was estimated and prepared. Taro-4-I of the taro-active polysaccharide Taro-4-I is composed of 1-mannan (1 → 4) -linked mannan, which forms the main chain, and most of the main chain is composed of one strand or C2 and At the C6 position, two side chains are simultaneously linked in a side chain structure. ② The side chain structure is composed of short galacto-oligosaccharides linked by α- (1 → 3) linkage, and some of them are inferred by the presence of galactose or arabinose residues linked at C2 or C6. Could. Therefore, the immune activity of the taro was found to be due to the highly branched galactomannan.

Example  5. In Vivo Administration of Taro Extracts

Acute toxicity test was performed using 6-week-old specific pathogen-free (SPF) SD rats from the Korea Experimental Supply Center. Two animals in each group were orally administered with the extract of Example 1 at a dose of 1 g / kg, and then examined for mortality, clinical symptoms, and weight changes of the animals, and hematological and blood biochemical tests were performed. Necropsy was performed to visually observe the abnormalities of organs and thoracic organs. As a result of the experiment, there were no clinical symptoms or dead animals in all animals treated with the test substance, and no toxic change was observed in weight change, blood test, blood biochemical test, and autopsy findings. As a result, the extract of the present invention did not show a change in toxicity even in rats up to 1 g / kg, respectively, it was confirmed that the minimum lethal dose (LD 50) is determined to be a safe substance more than 1 g / kg.

Examples of formulations for the composition of the present invention are illustrated below. However, the following formulation examples are merely illustrative of the present invention, and the content of the present invention is not limited by the following formulation examples.

Formulation example  One: Sanje  Produce

Taro Extract 300 mg

Lactose 100 mg

Talc 10 mg

The above components are mixed and filled in airtight bags to prepare powders.

Formulation example  2: Preparation of tablets

50 mg taro extract

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, tablets are prepared by tableting according to the usual preparation method of tablets.

Formulation example  3: Preparation of capsules

50 mg taro extract

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

The above components are mixed according to a conventional capsule preparation method and filled in gelatin capsules to prepare capsules.

Formulation example  4: Preparation of injection

50 mg taro extract

Sterile sterilized water for injection

pH adjuster

(2 ml) per 1 ampoule according to the usual injection preparation method.

Formulation example  5: Liquid  Produce

Taro Extract 100 mg

10 g per isomer

5 g of mannitol

Purified water

Each component was added to purified water in accordance with the usual liquid preparation method and dissolved, and the lemon flavor was added in an appropriate amount. Then, the above components were mixed, and purified water was added thereto. The whole was adjusted to 100 ml with purified water, And sterilized to prepare a liquid preparation.

Formulation example  6: Manufacture of health food

Taro extract 1000mg

Vitamin mixture proper amount

70 μg of Vitamin A Acetate

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

Vitamin B2 0.15 mg

Vitamin B6 0.5 mg

0.2 μg of vitamin B12

Vitamin C 10 mg

10 μg biotin

Nicotinic Acid 1.7 mg

50 μg folic acid

Calcium Pantothenate 0.5mg

Mineral mixture

Ferrous Sulfate 1.75 mg

Zinc Oxide 0.82 mg

Magnesium carbonate 25.3 mg

Potassium monophosphate 15 mg

Dibasic calcium phosphate 55 mg

Potassium Citrate 90 mg

Calcium Carbonate 100 mg

Magnesium chloride 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixture is comparatively mixed with a composition suitable for health food as a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional method for producing healthy foods , Granules can be prepared and used in the manufacture of health food compositions according to conventional methods.

Formulation example  7: Manufacture of health drinks

Taro extract 1000mg

Citric acid 1000 mg

100 g oligosaccharides

Plum concentrate 2 g

1 g of taurine

Add 900 ml of purified water

After mixing the above components according to a conventional healthy beverage production method, and then stirred and heated at 85 ℃ for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 l container, sealed sterilized and then refrigerated and stored in the present invention For the preparation of healthy beverage compositions.

Although the compositional ratio is relatively mixed with a component suitable for a favorite drink, it is also possible to arbitrarily modify the compounding ratio according to the regional or national preference such as the demand class, the demanding country, and the use purpose.

While the present invention has been described with reference to exemplary embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the scope of the invention. You will know. In addition, many modifications may be made to adapt a particular situation and material to the teachings of the invention without departing from the essential scope thereof. Accordingly, it is intended that the invention not be limited to the particular embodiment disclosed as the best mode contemplated for carrying out this invention, but that the invention be construed as including all embodiments falling within the scope of the appended claims.

Claims (24)

A pharmaceutical composition for enhancing immune system activity containing the polymer polysaccharide of Formula 1 as an active ingredient.
Formula 1:

The method of claim 1,
The pharmaceutical composition of claim 1 is a molecular weight of 5 to 500 kDa. A health functional food for enhancing immune system activity containing the polymer polysaccharide of Formula 1 as an active ingredient.
Formula 1:

The method of claim 3, wherein
Health functional food, characterized in that the molecular weight of Formula 1 is 5 to 500 kDa. An anti-metastatic pharmaceutical composition for tumor cells containing the polymer polysaccharide of Formula 1 as an active ingredient.
Formula 1:

6. The method of claim 5,
The pharmaceutical composition of claim 1 is a molecular weight of 5 to 500 kDa. Anti-transition health functional food for tumor cells containing the polymer polysaccharide of Formula 1 as an active ingredient.
Formula 1:

The method of claim 8,
Health functional food, characterized in that the molecular weight of Formula 1 is 5 to 500 kDa. Pharmaceutical composition for enhancing immune system activity comprising taro extract as an active ingredient. The method of claim 9,
The taro extract is a pharmaceutical composition, characterized in that it comprises galactose (galactose) and mannose (mannose). The method of claim 9,
The pharmaceutical composition is characterized in that it has an anti-complement activation function. The method of claim 9,
The pharmaceutical composition is a pharmaceutical composition, characterized in that it has an activation function of macrophages or natural killer cells. Health functional food for enhancing immune system activity, including taro extract as an active ingredient. The method of claim 13,
The taro extract is a dietary supplement characterized in that it contains galactose (galactose) and mannose (mannose). The method of claim 13,
The health functional food is a health functional food, characterized in that it has an anti-complement activation function. The method of claim 13,
The health functional food is a health functional food characterized in that it has an activation function of macrophages or natural killer cells. An anti-metastatic pharmaceutical composition against tumor cells comprising a taro extract as an active ingredient. 18. The method of claim 17,
The taro extract is a pharmaceutical composition, characterized in that it comprises galactose (galactose) and mannose (mannose). 18. The method of claim 17,
The pharmaceutical composition is characterized in that it has an anti-complement activation function. 18. The method of claim 17,
The pharmaceutical composition is a pharmaceutical composition, characterized in that it has an activation function of macrophages or natural killer cells. Anti-transition health functional food for tumor cells containing taro extract as an active ingredient. 22. The method of claim 21,
The taro extract is a dietary supplement characterized in that it contains galactose (galactose) and mannose (mannose). 22. The method of claim 21,
The health functional food is a health functional food, characterized in that it has an anti-complement activation function. 22. The method of claim 21,
The health functional food is a health functional food characterized in that it has an activation function of macrophages or natural killer cells.

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