KR101414141B1 - Pharmaceutical composition containing extract of taro - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for enhancing the immune system activity and a health functional food comprising the taro extract as an active ingredient, wherein the pharmaceutical composition comprising the taro extract according to the present invention and the health functional food have anticoagulant activity, macrophage or natural killer It has an activating function of cells and activates the immune system and effectively alleviates the cancer cell metastasis, so that it can be used as an anticancer agent and antitumor agent.

Description

[0001] PHARMACEUTICAL COMPOSITION CONTAINING EXTRACT OF TARO [0002]

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

In recent years, a new lifestyle of well-being has emerged, and it is becoming an important social tide in the consumer life. The rapid rise of well-being is not only a change in consumer culture, a huge market formation of new industries and products, It is expected that even the individual's lifestyle will change. In this societal trend, the food industry in the world is concentrating the most attention on health functional foods, and especially, there is a lot of interest and research on foods using natural materials. Among them, it has been reported that carbohydrates that have been highlighted as functions of energy sources in the past, among them various polysaccharides derived from natural products, have excellent immunostimulatory activity. Polysaccharides isolated from natural materials have been reported to have immuno-enhancing activities such as anti-complementary, macrophage activity, and anti-tumor activity. Therefore, Attempts have attracted much attention.

Taro (the corms of Colocasia esculenta L. Scotte) is a perennial herb that belongs to the genus Araceae. It is distributed over 100 genera and 1,500 varieties worldwide. It is a crop that mostly uses only vegetables or medicinal plants. The study on taro has been accompanied by a deterioration in quality due to the loss of water after harvest due to the viscous flow observed in the taro, browning due to tissue damage caused by processing such as shearing and peeling, A method of securing safety and improving quality in a simpler and more economical direction has been sought. In other words, studies on physiological changes, pathological corruption and biochemical changes related to the storage and shelf life of taro accounted for most of the studies of the taro (7,18,42,44,56), and the changes in physicochemical composition during storage , And browning inhibition. In particular, studies on the high value-added of taro, ie, studies on physiological activity and active ingredients, are extremely limited.

Accordingly, the present inventors have accomplished the present invention in order to separate and purify the innate immune system-stimulating polysaccharide from the talc-derived viscous substance and to identify the immunological activity and the structural characteristic of the active ingredient of them, thereby developing the talan polysaccharide as a functional material.

It is an object of the present invention to provide a pharmaceutical composition for enhancing immunity and a health functional food comprising the extract of polysaccharide extracted from the tar, and an antitumor pharmaceutical composition and a health functional food for tumor cells comprising the extract.

In order to accomplish the above object, the present invention provides a pharmaceutical composition for enhancing the immune system activity, which comprises, as an active ingredient, a polymeric polysaccharide represented by the following formula 1:

  (1)

In another embodiment, the present invention provides a pharmaceutical composition, wherein the molecular weight of the compound of Formula 1 is 5 to 500 kDa.

In one embodiment, there is provided a health functional food for enhancing the immune system activity, which comprises a polysaccharide of the following formula (1) as an active ingredient.

  (1)

In another embodiment, the present invention provides a health functional food characterized in that the molecular weight of Formula 1 is 5 to 500 kDa.

In one embodiment, there is provided an antitumor pharmaceutical composition for tumor cells, which comprises, as an active ingredient, a polymeric polysaccharide represented by the following formula (1).

  (1)

In another embodiment, the present invention provides a pharmaceutical composition, wherein the molecular weight of Formula 1 is 5 to 500 kDa.

(1)

In another embodiment, the present invention provides a health functional food characterized in that the molecular weight of Formula 1 is 5 to 500 kDa.

In one embodiment, there is provided a pharmaceutical composition for enhancing an immune system activity comprising a taro extract as an active ingredient. In another embodiment, the taro extract comprises galactose and mannose. In another embodiment, the pharmaceutical composition is characterized by having anti-complement activation activity. In another embodiment, the pharmaceutical composition provides a pharmaceutical composition characterized by having the function of activating macrophages or natural killer cells.

In one embodiment, there is provided a health functional food for enhancing immune system activity comprising taro extract as an active ingredient. In another embodiment, the Taran Extract comprises a galactose and mannose. In another embodiment, the health functional food has a complement activating function. Wherein the health functional food has an activating function of macrophages or natural killer cells.

In one embodiment, there is provided an antidepressive pharmaceutical composition for tumor cells comprising the taro extract as an active ingredient. In another embodiment, the taro extract comprises galactose and mannose. In another embodiment, the pharmaceutical composition is characterized by having anti-complement activation activity. In another embodiment, the pharmaceutical composition provides a pharmaceutical composition characterized by having the function of activating macrophages or natural killer cells.

In one embodiment, an anticoagulant functional food for tumor cells comprising taro extract as an active ingredient is provided. In another embodiment, the Taran Extract comprises a galactose and mannose. In another embodiment, the health functional food has a complement activating function. In another embodiment, the health functional food has a function of activating macrophages or natural killer cells.

The composition of the present invention comprises 0.1 to 50% by weight of the polymeric polysaccharide or the taranic extract of the formula (1) based on the total weight of the composition. The composition comprising the polymeric polysaccharide or the taran extract of Formula 1 of the present invention may further comprise an appropriate 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 composition according to the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols or the like, oral preparations, suppositories or sterilized injection solutions according to a conventional method 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. The solid preparation for oral administration includes tablets, pills, powders, granules, capsules and the like. The solid preparation is prepared by mixing at least one excipient such as starch, calcium carbonate (calcium carbonate, sucrose, lactose, gelatin, and the like. 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. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.

The present invention may be varied depending on the age, sex and body weight of the patient, but it is generally administered in an amount of 0.01 to 500 mg / kg, preferably 0.1 to 100 mg / kg, once to several times per 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, intrauterine or intracerebroventricular injection. The polysaccharide or taran extract of formula (1) of the present invention has little toxicity and side effects, so that it can be safely used for prolonged use even for prophylactic purposes.

The present invention provides a health functional food comprising the polymeric polysaccharide or the taranic extract of the above formula 1 and a pharmaceutically acceptable food supplementary additive and can be used in various food products such as drinks, gums, tea, vitamins, Etc., and can be used in the form of pills, powders, granules, infusions, tablets, capsules or beverages. In this case, the amount of the polysaccharide or the taran extract of Formula 1 in the food or beverage may be generally 0.01 to 15% by weight of the total food weight of the health food composition of the present invention, and 100 ml , Preferably from 0.3 to 1 g.

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 not particularly limited as long as it is an essential component in addition to the polymeric polysaccharide or the taranic extract of the above formula (1) in a prescribed ratio, and may contain various flavors or natural carbohydrates, . 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 natural flavors other than those described above, natural flavors (such as tau martin, stevia extract (e.g., rebaudioside A, glycyrrhizin)) and synthetic flavors (saccharin, aspartame, etc.) 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. In addition, the compositions of the present invention may contain flesh for the production of natural fruit juices and fruit juice drinks and vegetable drinks. These components may 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.

The pharmaceutical composition and the health functional food including the taro extract according to the present invention have anti-complement activity, macrophage or natural killer cell activation function, thereby activating the immune system and effectively alleviating the cancer cell metastasis, It can be used as an antitussive agent.

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

Hereinafter, the present invention will be described in detail with reference to 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.

Production of taro extract

1-1. Separation of Toran-derived Jodadang

The taro harvested from Jeonnam Kukseong was removed, and the shells were removed and sectioned. Then, 10 L of water was added to 5 kg of taro, followed by cold water extraction at room temperature for 24 hours. The water extract of turmeric 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 with stirring for one day. The precipitated polysaccharide was recovered by centrifugation (6000 rpm, 30 min, 4 ° C), dissolved in a small amount of water and dialyzed for 3 days using a dialysis membrane (MW cut-off 12,000, Sigma) And the low molecular weight material was removed. After concentrating again using a rotary vacuum concentrator, Taro-0, a crude polysaccharide fraction, was obtained by freeze-drying (FreeZone 12 Liter, Labconco, MISSO VRI 64132, KANSAS CITY, USA).

1-2. Purification of polysaccharide derived from taro

1-2-1 Anion exchange chromatography (AEC)

Taro-0 was dissolved in a small amount of distilled water and adsorbed on a DEAE-Sepharose FF (Cl form, 5.5 × 25 cm) column equilibrated with distilled water. Eluted distilled water was eluted to separate non-adsorbed fractions Then, the adsorbed fractions were separated by elution with 0.05 M to 2 M NaCl solution. The fractions eluted from the anion exchange chromatography column were fractionated using a fraction collector (1200series, EYELA, Tokyo, Japan), and the elution peaks were determined by measuring neutral sugars, acid groups, proteins and KDO contents.

One non-adsorbed fraction (Taro-1) and seven adsorbed fractions (Taro-2 ~ 8) were obtained from the crude polysaccharide fraction Taro-0 through this ion exchange resin. Each of the fractions was concentrated, dialyzed and lyophilized and used in subsequent experiments.

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

0.5 g of the Taro-4 fraction (0.2 M elution fraction), which had a good yield and immunoreactivity, was dissolved in a small amount of water and purified with a Sephadex G-100 equilibrated with 50 mM ammonium formate buffer (pH 5.5) gel permeation chromatography (GPC) was performed using a column (4 x 120 cm). The eluate was divided into 100 fractions by 7 mL. Each fraction was analyzed for neutral sugar content and two fractions, Taro-4-Ⅰ and Taro-4-Ⅱ, with different molecular weights were obtained. Among them, taro-4-Ⅰ, a polymeric polysaccharide, was used as a final fraction of tarosan-derived polysaccharide and used for the subsequent experiments since it was excellent in purification, yield and immunological activity.

Measurement of Immune System Stimulating Activity of Taran Extract

2-1. Complement activation of taro extract

2-1-1. Anti-complement action performance

Blood from healthy adults was collected and allowed to stand at room temperature for about 15 minutes. After coagulation, the coagulated blood was cut and left at room temperature for about 5 minutes. The serum was separated by centrifugation (2,200 rpm, 15 min, 4 ° C) at 4 ° C for about 20 minutes, and then 1 ml was added to the microfuge tube. Lt; / RTI >

Antibody activity was measured by the complement fixation test method based on the degree of erythrocyte hemolysis due to the complement after residual complement consumption by the Meyer method. Samples dissolved in distilled water at various concentrations were mixed with 50 μL each of GVB ⁺⁺ (containing gelatin veronal buffer, pH 7.4, 0.1% gelatin, 0.15 mM Ca ⁺⁺ , 0.5 mM Mg ⁺⁺ ) For 30 minutes. To the reaction mixture, 350 μL of GVB ⁺⁺ was added, and the dilution was continuously diluted to 10 to 160 times. Then, 750 μL of GVB ⁺⁺ and IgM-sensitized sheep erythrocyte (EA cell, 1 × 10 ⁸ cells / mL) was added to the reaction mixture at 37 ° C for 60 minutes, and 2.5 mL of PBS (phosphate buffered saline, pH 7.4) was added to terminate the reaction. The reaction solution was centrifuged at 2,000 rpm for 10 minutes, and the absorbance of the supernatant was measured at 412 nm to measure the residual hemolytic activity. The anti-complement activity was inhibition of 50% total complement hemolysis (ITCH50,%) against the total complement hemolysis (TCH50,%) of the negative control group reacted only with normal serum of the GVB ⁺⁺ and distilled water Respectively. The positive control group was compared with PSK (polysaccharide-K), an immunostimulant derived from chestnut mushroom.

The activity of each sample was determined by ITCH ₅₀ 0% in the negative control group. As a result, Taro-4-Ⅰ showed excellent anti-complement activity comparable to that of the positive control group at the concentration of 1,000 μg / mL 1). PSK, a positive control, is an immunologically active polysaccharide currently marketed as an anticancer drug. Generally, polysaccharides exhibiting more than 50% anti-complement activity at a concentration of 1,000 μg / mL are known to have pharmacological properties. Therefore, Taro-4- I was found to be excellent in the ability to activate the complement system. In addition, it was found that the activity of Taro-4-Ⅰ was dependent on the concentration of the sample.

2-1-2. Review of complement system activation path

In order to determine the active path of the complement system GVB ⁺⁺ buffer, Ca ⁺⁺ ions are the selective removal of Mg ⁺⁺ -EGTA-GVB ^- buffer and Ca ⁺⁺ and Mg ⁺⁺ ions are all removed GVB-EDTA-buffer Were prepared and mixed with the sample and NHS, respectively, and reacted at 37 DEG C for 30 minutes. Each reaction solution was reactivated at 37 ° C for 60 minutes, 2.5 ml of PBS was added, and centrifugation was performed at 2,000 rpm for 10 minutes. Absorbance of the supernatant was measured at 412 nm to measure the residual hemolytic activity, .

In the reaction system is also a Ca ⁺⁺ selectively removed, as shown in 2, 1,000 μg / mL compared to the primary reaction system in the presence of both Ca ⁺⁺ and Mg ⁺⁺ showed about 60% active, 500 mg / mL or less , The concentration-dependent tendency was maintained. On the other hand, almost no activity was observed in the reaction system in which both Ca ++ and Mg ++ were removed. This indicates that the complement system activation of Taro-4-Ⅰ appears via both the classical pathway and the subpopulation.

By confirming the activation of C ₃ by two-dimensional immunoelectrophoresis, it can be confirmed whether the complement activity of the sample confirmed by the Meyer method is due to the complement system activation or by the 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) into a 1% agarose gel plate (5 × 5 cm) made by dissolving the solution in a barbital buffer (pH 8.6), and subjected to a first electrophoresis (75 mA / plate) at 4 ° C. for about 3 hours The gels were then stained with bromophenol blue for about 10 minutes at 25 ° C for 15 h at 4 ° C on an agarose gel plate containing 1% anti-human C ₃ . The presence of C ₃ was observed by decolorizing and identifying the precipitation line.

The reaction of Taro-4-Ⅰ in the basic reaction system and the reaction system in which the specific metal ion was removed was observed, and the decomposition of C ₃ factor was observed. It was confirmed that the first sedimentation line from the well and the second sedimentation line appeared at a similar rate in the normal system in which both Ca ⁺⁺ and Mg ⁺⁺ exist. Considering that the first sedimentation line from the well is caused by C ₃ and the second sedimentation line is due to decomposition products C _3a and C _3b , the anticoagulant activity of Taro-4-Ⅰ is not due to the complement inhibitor I could prove it. On the other hand, in the reaction system in which all the metal ions were removed, only the first settling line appeared and no second settling line was observed. On the other hand, the height of the second settling line was relatively lower in the reaction system in which only Ca ⁺⁺ was selectively removed than in the normal system. This suggests that C ₃ activation occurs only in the subpaths with the classical pathway inhibited. Thus, activation of the complement system by Taro-4-Ⅰ reaffirms that it appears via both classical pathways and subpathways .

2-2. 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. In this study, 1 mL of 5% thioglycollate medium was injected into the peritoneal cavity of BALB / c (female, 6 weeks) mice, and the induced macrophages were collected from the abdominal cavity for 72 hours. After washing, the number of cells was 2.25 × 10 ⁵ cells / mL of RPMI 1640) was prepared to prepare macrophage suspension. 100 μL of the suspension was cultured in a 5% CO ₂ incubator at 37 ° C for 24 hours (22, 23). After completion of the culture, 150 μL of the cell culture was recovered by centrifugation at 1,500 rpm and 4 ° C. for 5 minutes, and the amount of cytokine induced in the supernatant was measured.

The content of cytokines 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 allowed to stand at 4 ° C for 12 hours. The coated microplates were washed three times with PBS (0.05% Tween 20, PBST) and 200 μL of assay diluent (PBS with 10% FBS or 2% skim milk) The unbound well surface was blocked. After blocking, each well was washed three times with washing buffer, and 50 μL of recombinant mouse cytokine and immune cell culture were dispensed into each well. After incubation at room temperature for 2 hours, the cells were washed with washing buffer, and 100 μL of detection antibody (in assay diluent) was treated. After incubation at room temperature for 1 hour, the cells were washed again. After 100 μL of the enzyme reaction (avidin-horseradish peroxidase conjugate) was added to 100 μL of substrate solution [tetramethylbenzidine (TMB) and hydrogen peroxide] for 30 min at room temperature, 1 MH ₃ PO ₄ ) or (2 NH ₂ SO ₄ )], and the absorbance at 450 nm was measured.

In vitro measurement of macrophage cytokine production by direct stimulation of tarosan-derived polysaccharide samples confirmed that the active polysaccharide Taro-4-I promotes the production of IL-6, IL-12 and TNF-α (Fig. 4). In the case of IL-6, a concentration-dependent increase in production was observed even at concentrations as low as 1 μg / mL or more. The concentrations of TNF-α were gradually decreased at each concentration of the sample, but the production-stimulating activity of TNF-α was generally low. These results suggest that Taro-4-Ⅰ, a taro-derived polysaccharide, has an effective activity for inflammatory immunity, and the minimum sample concentration for this activity is estimated to be several μg / mL. In the case of IL-12, Taro-4-Ⅰ showed a concentration-dependent uptake up to the concentration of 19 μg / mL, and at higher concentrations, IL-12 was found to produce about 70% of the positive control LPS. However, at 500 μg / mL concentration, the yield tended to decrease slightly. It is anticipated that the taro-derived polysaccharide will have activity in cell-mediated immunity such as activation of NK cell and activation of cytotoxic T lymphocyte (CTL) through induction of Th1-type immune response, It is thought that it is near. In particular, IL-12 is considered to be a cytokine essential for the induction of anticancer activity since it is directly involved in the NK cell activation that carries the cancer cell lethal effect in the presence of cancer cells. Therefore, Taro-4-I is expected to act efficiently on cancer cells . In this experiment, the stimulation of macrophages with Taro-4-I for 24 hours resulted in the production of TNF-α and IL-6, which are classified as inflammatory cytokines which are directly related to the immune cell necrosis, And Taro-4-Ⅰ, which is derived from taro-derived polysaccharide, has a function to activate (regulate) the immune system functioning in vivo defense.

2-3. The ability of tumor cells to kill by natural killer cells (NK cells)

Taro-4-Ⅰ polysaccharide purified from taro was intravenously injected intraperitoneally into BALB / c (6 weeks, female) mice and after 3 days, the spleen was aseptically removed by cervical dislocation. Spleen cells were obtained by milling (100 mesh) and filtering (200 mesh) on PBS using a stainless steel mesh. 5 mL of 0.2% NaCl was added for 15-30 seconds to destroy the erythrocytes and the cells were washed 2 to 3 times with serum-free medium. The cells were adjusted to 1 × 10 ⁶ cells / mL and used as effector cells. The effector cell and target cell ratio (E / T ratio) were adjusted to 100, 50, and 25 in a round-bottomed 96-well microplate using YAC-1, which is highly sensitive to natural killer cells, as a target cell. Respectively. The amount of lactate dehydrogenase (LDH) liberated from the target cell by the ability to kill effect cells after 6 hours incubation at 37 ° C in a 5% CO ₂ incubator was measured using Cytotox 96 (Promega, Madison, WI, USA). The tumor cell killing ability of natural killer cells was calculated by the following equation.

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

The result of the natural killer cell stimulation activity of taro-4-Ⅰ derived from taro derived polysaccharide was as shown in Fig. (E / T ratio) of the effect cell (splenocytes) to the target cell (YAC-1). As a result, the most prominent activity was observed at E / T = 100, and Taro-4-Ⅰ showed higher activity than 500 μg and 50 μg of the control group. In addition, the results of the experiment showed that at a low concentration of 50 μg, about 250% higher activity than the normal cells was observed at a high concentration of 500 μg. Thus, Taro-4-Ⅰ derived from taro-derived polysaccharide was found to contribute to the activation of natural killer cells capable of killing tumor cells, and they were thought to have anticancer activity by natural killer cell stimulating activity. The anticancer effect of Taro-4-Ⅰ, a taran-derived polysaccharide, can be assumed to be strong at a relatively low concentration of 50 μg / mL.

Antioxidant activity of taro extract on tumor cells

The antitumor activity of the sample was determined using an animal tumor metastasis model using melanoma B16BL6, a classical tumor cell line for lung. B16BL6 lung carcinoma (2.7 × 10 ⁴ ) was intravenously injected into BALB / c mice to examine the tumor metastasis effect. Samples were intravenously injected at each concentration two days before tumor inoculation. After 14 days of tumor inoculation, the mice were sacrificed and the lungs of target cells of the tumor cells were excised and the metastasized tumors were fixed in Bouin's solution (Sigma) and the colony counts of metastatic tumors were counted. The antitumor effect of the samples was compared with the control group inoculated with the tumor alone.

As shown in Table 1, about 100 metastatic cancer colonies were observed in the control group without the sample, whereas in the test group administered with Taro-4-I at a concentration of 5 μg and 500 μg, about 22 and 18 and 78.1% and 81.9%, respectively, of antioxidant activity.

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

On the other hand, when 50 μg was administered, about 4 transfected colonies were identified, showing 96.8% of the highest antitumor activity. These results suggest that the low dose of 50 μg / mouse is effective in inhibiting metastasis of tumor. This result is equivalent to the concentration at which Taro-4-Ⅰ showed the strongest stimulating activity in the natural killer cell stimulating activity experiment, and the effect of Taro-4-Ⅰ on tumor metastasis was mainly due to activation of natural killer cells I could have expected. Since natural killer cells are known to be mainly activated by IL-12, it is thought that Taro-4-Ⅰ induces activation of natural killer cells by strong stimulation of IL-12 production by macrophages and thus has a strong antitumor effect.

Structure analysis of taro extract

4-1. Determination of Structure and Binding Position by Methylation Analysis

4-1-1. Preparation of Methylsulfinyl carbanion

To prepare methylsulfinyl carbanion, 20 mL of anhydrous DMSO (dimethylsulfoxide) was added to 1.26 g of anhydrous NaH, and the mixture was filled with nitrogen and reacted for about 10 to 15 minutes under an oil bath at 90 ° C. The reaction was terminated at the point when the reaction solution became pale green, and the reaction solution was cooled to room temperature and then centrifuged at 3,000 rpm at 30 ° C. The supernatant containing Methylsulfinyl carbanion was replaced with nitrogen to avoid contact with air, followed by small batches and then kept frozen.

4-1-2. Methylation

The methylation to determine the binding site of the polysaccharide sample was carried out using the Hakomori method. 1 mg of anhydrous DMSO was added to each polysaccharide sample (0.5 mg) which had been thoroughly dried for 1-2 days in a desiccator, and completely dissolved by stirring, followed by reaction with 500 μL of methylsulfinyl carbanion (MSCA) for 4 hours. MSCA was additionally added if necessary so that polysaccharide could be completely converted to polyalkoxide, and the remaining unreacted MSCA was identified as triphenylmethane. The polyalkoxide-converted samples were methylated by adding excess CH3I, and residual CH3I was removed by N2 gas flushing and recovered using Sep-pak C18 cartridge.

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

The methylated sample was hydrolyzed by reacting with 1 mL of 2 M TFA at 121 ° C for 1.5 hours and then dried. The hydrolyzed samples were dissolved in ethanol containing 25% NH ₄ OH and added with 10 mg of NaBH 4, followed by ring opening and reduction for 4 hours. The appropriate amount of acetic acid was added to remove the remaining NaBH 4, To remove the added acetic acid. Then, 1 mL of acetic anhydride was added and the mixture was reacted at 121 ° C for 3 hours to convert it to partially methylated alditol acetate. This was separated and extracted with chloroform (H2O) And analyzed by GC-MS.

4-2. GC and GC-MS analysis of partially methylated alditol acetate

GC analysis was carried out using a Young-Lin ACME-6100 GC equipped with a SP-2380 capillary column (0.25 mm × 30 m, 0.2 mm film thickness, Supelco) The flow rate of the carrier gas (N2) was analyzed by splitless injection mode (1/20) at 180 ° C → 250 ° C (1.5 ° C / min) and 250 ° C (5 min) 1.5 mL / min.

The GC-MS was analyzed using an Agilent 6890N GC system and a 5973N mass spectrophotometer (Agilent Technologies, Palo Alto, CA, USA) equipped with a SP-2380 capillary column (0.2 mm film, 0.25 mm id × 30 m, Supelco, Bellefonte, USA) and analyzed by splitless injection mode (He flow rate: 1.5 mL / min) at the same optimum temperature conditions as GC analysis. The methylated samples were identified by a combination of 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. Analysis of sugar chain binding pattern by methylation analysis of Taro-4-I

As shown in FIG. 6 and FIG. 7, the total ion chromatogram of the Taro-4-I analyzed by GC was analyzed after methylation and the peak fragment ions were analyzed by GC-MS. Table 2 summarizes the composition of binding mode per constitution.

As shown in Table 2, Taro-4-I contained 10 kinds of sugar chains and galactose bond and mannose bond were found at a high ratio. In particular, the presence of galactose residues in the galactose residue was detected in a high proportion (48.4%) of terminal-Galp, and galactosyltransferase residues in galactomannan It can be assumed that it is a stretched form. From the fact that 3-linked-Galp and 2,3-branched-Galp and 3,6-branched-Galp are present in a high ratio, the side chain existing in the form of galactan has C2 or C6 position in the galactose core connected by 1 → 3 bond It was assumed that another galactose was branched to constitute a non-reducing end.

Glycosyl
The residue (Glycosyl
residue) Methyl group
location Reduced glycoside
Deduced
glycosidic linkage) Taro-4-l
(Molar%) ¹⁾ Arabinose
Galactose





Mannos 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-Araf means non-reducing terminal arabinofuranoside.

³⁾ 3-Galp means 3-linked galactopyranoside.

⁴⁾ 2,4,6-Manp means 2,4,6-branched mannopyranoside.

On the other hand, in the case of mannose residues, non-reducing terminal mannose was hardly detected, whereas 4-linked Manp and 4,6-linked Manp and 2,4,6-linked Manp were detected in a high ratio. The main chain of -I exists in the mannan form of (1 → 4) bond, and one strand of the side chain is extended again at the C6 position of mannose, or at the C2 and C6 positions, It is possible to estimate the outcome. In particular, the Taro-4-I polysaccharide derived from taro showed a particularly high detection rate for the 2,4,6-linked Manp residues. This fact indicates that the (1 → 4) galactan side chain, and it was confirmed that it was a highly branched form not found in polysaccharides derived from other plants. In general, the high viscosity and physiological activity observed in the taro are attributed to this highly branched galactomannan. However, even if polysaccharides are classified into the same kind of physiological activity by polysaccharides, it is known that microstructures exhibit a considerable diversity in each plant and thus show a difference in activity. Therefore, it is necessary to study the microstructure afterwards have.

4-4. Estimation of total structure of Taro-4-I

The predicted overall structure of Taro-4-I, based on the analysis of glycoconjugation pattern by methylation analysis of the immunologically active polysaccharide Taro-4-I of the taro and the information obtained through two enzymatic treatments, 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 has a limitation of predicting the total microstructure of these Taro-4-I, And the structure was estimated and prepared. Taro-4-I, an immunologically active polysaccharide of taro, is composed of mannan linked with (1 → 4) mannose as the main chain, and most of the main chain consists of one strand at the C6 position of mannose, At the C6 position, two strands of the side chain are present in a side chain structure. ② The branched structure consists of short galacto-oligosaccharides linked by α- (1 → 3) bonds. Some of these residues are inferred to exist in structures with galactose or arabinose residues linked at C2 or C6 positions I could. Therefore, it was found that the immunological activity of taro is due to highly branched galactomannan.

In vivo administration of taro extract

The acute toxicity test was carried out as follows using a specific pathogen free (SPF) SD rats of 6 weeks old, supplied from the experimental supply center of Korea. Two animals per group were orally administered once at a dose of 1 g / kg of the extract of Example 1, followed by observation of animal mortality, clinical symptoms and weight change, and hematological and blood biochemical tests , And autopsy was performed to observe whether or not the organs and thoracic organs were abnormal. 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, it was confirmed that the extract of the present invention did not exhibit any toxicity at 1 g / kg or more in rats, and that the oral LDL was at least 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

Taran Extract 50 mg

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

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

Formulation example  3: Preparation of capsules

Taran Extract 50 mg

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

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

Taran Extract 50 mg

Sterile sterilized water for injection

pH adjuster

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

Formulation example  5: Liquid  Produce

Taran Extract 100 mg

10 g per isomer

5 g mannitol

Purified water quantity

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 1000 mg

Vitamin mixture quantity

70 [mu] g of vitamin A acetate

Vitamin E 1.0 mg

0.13 mg vitamin B1

0.15 mg of vitamin B2

0.5 mg vitamin B6

0.2 [mu] g vitamin B12

10 mg vitamin C

Biotin 10 μg

Nicotinic acid amide 1.7 mg

50 ㎍ of folic acid

Calcium pantothenate 0.5 mg

Mineral mixture quantity

1.75 mg of ferrous sulfate

0.82 mg of zinc oxide

Magnesium carbonate 25.3 mg

15 mg of potassium phosphate monobasic

Secondary calcium phosphate 55 mg

Potassium citrate 90 mg

100 mg of calcium carbonate

24.8 mg of magnesium chloride

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 1000 mg

Citric acid 1000 mg

100 g of oligosaccharide

Plum concentrate 2 g

Taurine 1 g

Purified water was added to a total of 900 ml

The above components were mixed according to a conventional health drink manufacturing method, and the mixture was stirred and heated at 85 DEG C for about 1 hour. The resulting solution was filtered and sterilized in a sterilized 2 liter container, ≪ / RTI >

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 (12)

A pharmaceutical composition for preventing or treating the metastasis of tumor cells, which comprises the polymeric polysaccharide of the formula (1) as an active ingredient.
(1)

The method according to claim 1,
Wherein the molecular weight of Formula 1 is 5 to 500 kDa.
A health functional food for preventing or alleviating metastasis of tumor cells containing an effective amount of a polysaccharide of the formula (1) as an active ingredient.
(1)

The method of claim 3,
Wherein the molecular weight of the formula (1) is 5 to 500 kDa.
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