KR20030028009A - Polysaccaride from the safflower having macrophage activity, the process for preparation thereof and a use thereof - Google Patents

Abstract

PURPOSE: An anticancer agent composition containing a water extract of Carthamus Tinctorius L. as an active ingredient is provided which can be used a medicament for immune disorders or cancer, or a functional food product. CONSTITUTION: Polysaccharides for activating macrophages are prepared by the steps of: removing a methanol soluble fraction from an water extract of Carthamus Tinctorius L. and then precipitating it in ethanol; ultrafiltering the precipitates and recovering a solution having a molecular weight of 300,000Da or more; loading the solution into anion exchange resin, followed by fractionation with 0.1 mol NaCl solution; and subjecting an active fraction to gel filtration chromatography having a separation capacity of 10,000 to 1,000,000.

Description

Polysaccaride from the safflower having macrophage activity, the process for preparations and and use using}

The present invention relates to a polysaccharide having macrophage activating ability isolated from safflower ( Carthamus tinctorius L.), a preparation method thereof and a use thereof, and more particularly, to purify the polymer polysaccharide from the cold water extract of safflower and to contain it as an active ingredient. The present invention relates to a composition for anti-cancer agents comprising a macrophage activator composition and water extract of safflower as an active ingredient.

In the immune system, macrophages are immune cells involved in both the innate and adaptive immunity (Playfair, JHL 1992. Immunology at a glance. 5th ed. Blackwell Scientific Publications. London). Nitric oxide (NO) with the ability to release antigens to lymphocytes through the breakdown and processing, secrete certain substances such as cytokines that can regulate the entire immune system, and kill antigens that enter the body It is an important immune cell of the cellular immune system that activates the immune system through mechanisms such as the production of the back.

On the other hand, macrophages can be divided into inflammatory macrophages and activated macrophages according to their activity when exposed to various stimulants or activated by external stimulation. Inflammatory macrophages refer to exposure to inflammatory mediators such as thioglycolate, which involves phagocytosis, surface adhesion, increased prostaglandin secretion, plasminogen activating enzymes, elastase, collagenase, etc. Functional changes of the cells and increase in cell size and various cell secretions (Hibbs, JB, Taintor, RR, Vavrin, I. and Rachlin, EM 1988. Nitric oxide: A cytotoxic activated macrophage effector molecule.Biochem. Biophys Res. Commun., 157 , 87).

Macrophages activated by cytokines such as interferon-gamma (IFN-γ) and Tumor Necrosis Factor-α and lipopolysaccharides of Gram-negative bacteria have anticancer and antimicrobial effects. Cytokines (cancer suicide-alpha, interleukin-1, interleukin-6, interleukin-8, interleukin-12) secreted by activated macrophages in the mechanism of cytotoxic action against cancer cells, hydrogen peroxide, Cytolytic lysates proteases and the like have been suggested as substances showing cytotoxicity against cancer cells (Thomas, PM and Edginton, S. 1984. Human monocyte-mediated tumor cytotoxicity. J. Immunol., 132 , 1980).

Macrophages activated by lectins or macromolecules of polysaccharides and polysaccharides have excellent ability to inhibit tumorigenesis in the body, inhibit metastasis of cancer cells and distinguish tumor cells from normal cells. Although the target structure recognized by macrophages is still unclear, the process of destroying tumor cells consists of dissolving target tumor cells by releasing lysosomal enzymes in contact with tumor cells, unlike phagocytosis against bacteria. However, inactivated macrophages have a weak cytotoxicity and must undergo an activation process, but have an increased killing capacity for tumor cells. This fact suggests that immunotherapy resulting in the activation of macrophages can be an effective treatment.

Studies on macrophage activating materials from natural products are being actively conducted, and they are mainly found to be active in polymer fractions rather than small molecules. Among macromolecular macrophage activators, many polysaccharides that have anti-cancer effects have been reported. The correlation between macrophage activation and anti-tumor activity by polysaccharides is associated with the recovery or enhancement of immune function impaired by tumor development due to the involvement of host-mediated immune responses. It is understood as a phenomenon (Chihara, G., Hamuro, J., Maeda, Y., Arai, Y., and Fukuok, F. 1970. Nature, 225, 7; Hamuro, J., Jadding, H. and Bitter -suermann, D. 1978. Immunol., 34, 695). For example, lentinan isolated from Lentinus deodes and krestin of cloud mushroom ( Coliolus versicolor ) are currently marketed as anticancer drugs in Japan, and chemotherapeutic agents have low specificity for cancer cells. On the other hand, these polysaccharides have been reported to have no side effects in the treatment of tumors.

Recently, the cell wall component of Gram-positive bacteria, peptidoglycan and its derivatives, has been reported to activate macrophages as well as to induce various immune regulatory actions and complement each other (Heumann, C., Barras, C). Severin, A., Glauser, MP and Tomasz, A. 1994. Gram-positive cell walls stimulate synthesis of tumor necrosis factor alpha and interleukin-6 by human monocytes.Infect.Immunol., 62, 2715) Streptococcus mutants Among the polysaccharides, the main component of activating monocytes to induce the secretion of cancer cell killer-alpha and interleukin-1 was Rhamnose and its receptor was also identified (Benabdelmoumene, S., Dumont, S., Petit, C., Poindron). , P., Wachsmann, D. and Klein, JP 1991.Activation of human monocytes by Streptococcus mutans serotype polysaccharide, Immunoglobulin G Fc receptor expression and tumor necrosis factor and interleukin-1 production.Infect. Immunol., 59, 3261). Polysaccharides containing mannose, beta-1,4-linked D-mannuronic acid, beta-1,3 glucan, dicarboxy Polysaccharides such as dicarboxy-glucomannan have been reported to promote anticancer activity and cytokine secretion of monocytes and macrophages (Otterlei, M., Sundan, A., Skjak-Brak, G., Ryan, L). and Smidsrod, O. 1993. Similar mechanisms of action of defined polysaccharides and lipopolysaccharides: Characterization of binding and tumor necrosis factor alpha induction.Infect.Imunmun., 61, 1917). Anticancer effects, antimicrobial activity, and macrophage activity of basidiomycetes, such as lentinan, have been reported. Also, immune-boosting main components such as immune suppression of docomari extract, and B lymphocyte proliferation effect by enhancing the immune system of Angelica extract This protein is known as polysaccharide or polysaccharide.

As described above, the polysaccharide having macrophage activating ability can be used as a material of functional food and adjuvant as an anticancer and immunostimulating agent.

Therefore, the inventors of the present invention, while studying the applicability of traditional tea and bamboo plant material used as a food material as a functional food, confirmed that there is a high macrophage activation ability in the cold water extract of safflower and having a macrophage activation ability from the cold water extract While purifying the polysaccharide, the present invention was completed by confirming that safflower crude polysaccharide had an effect of suppressing solid cancer.

It is therefore an object of the present invention to provide a method for extracting and purifying macrophage activated polysaccharides from safflower cold water extracts.

Another object of the present invention is to provide a polysaccharide purified from safflower cold water extract and its use as a macrophage activator and an anticancer agent.

The object of the present invention is the ultrafiltration of the safflower cold water extract after ethanol precipitation to obtain a polymer material having a molecular weight of 300,000 Da or more, the material is purified by anionic exchange resin chromatography, gel filtration chromatography to polymerize polysaccharides, The use of the purified polysaccharide as an adjuvant was ascertained and the anticancer activity of the ultrafiltration safflower water extract was achieved.

Hereinafter, the configuration and operation of the present invention.

1 is a result of anion exchange resin chromatography of 300,000 Da polymer material (CT-3) obtained by ethanol precipitation of safflower water extract of Example 1 and then ultrafiltration.

Figure 2 shows the result of gel filtration chromatography of the CT-3IIa-1 fraction having the highest macrophage activity among the fractions obtained by anion exchange resin chromatography.

Figure 3 is a result of measuring the blocking rate in Sarcoma-180 solid cancer model of 300,000 Da polymer material (CT-3) obtained by ethanol precipitation of safflower water extract and ultrafiltration.

In the present invention, the active body is a polymer polysaccharide by showing high macrophage activity in a fraction having a molecular weight of 300,000 or more as a result of separation using ultrafiltration membranes having different molecular weights to separate the active polysaccharides contained in the cold water extract of safflower. Confirmed.

In order to purify the polymer polysaccharide, a fraction having a molecular weight of 300,000 or more was dissolved in distilled water and then injected into anion exchange resin chromatography. It was confirmed that polysaccharide having macrophage activating ability was eluted in 0.1 molar concentration sodium chloride solution, and the fraction was subjected to gel filtration chromatography using a resin having a separation capacity of 1 million or less molecular weight to separate the activity from 0.2 molar concentration sodium chloride solution. The material was obtained. Gel filtration chromatography was performed using a resin having a resolution of 1 million or less substance. As a result, the polysaccharide separated by gel filtration chromatography was identified as a polymer polysaccharide having a molecular weight of about 380,000.

On the other hand, after ethanol precipitation of cold water extract of safflower, ultrafiltration was confirmed that the polymer material having a molecular weight of 300,000 Da or more has an effect of inhibiting solid cancer in experimental animals of the Sarcoma-180 solid cancer model.

As described above, the polysaccharide isolated from safflower has macrophage activating ability, and the ultra-filtered water extract of safflower has the effect of inhibiting solid cancer, and thus can be used as a material for functional food and adjuvant as an adjuvant and anticancer agent.

Polysaccharides isolated and purified from the water extract of safflower according to the present invention are formulated into unit dosage forms or multiple dose formulations, such as tablets, capsules, granules, suspensions, emulsions, etc. It can be used as a composition for.

In addition, the composition containing the polysaccharide as an active ingredient can be administered as several times per day divided into 10 mg to 30 mg per 1kg body weight per dose as desired.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

Example 1: Purification of safflower cold water extract and polysaccharide having macrophage activation ability therefrom

Step 1: Preparation of cold water extract of safflower

1 kg safflower was chopped, 20 L of distilled water was added, pulverized and stirred, followed by centrifugation (5000 x g , 30 minutes), and the supernatant was concentrated and lyophilized. The lyophilized sample was dissolved in distilled water, refluxed at 100 ° C. for 2 hours, and then concentrated and lyophilized by centrifugation (5000 × g , 30 minutes). The freeze-dried sample was again refluxed with methanol five times to remove the methanol soluble fraction, and the residue was redissolved in distilled water, and 75% ethanol was added to precipitate the precipitate. The ethanol soluble fraction was removed by centrifugation (5000 x g , 30 minutes), and the precipitate was recovered, redissolved in distilled water, and then the polymer fraction of cold water extraction was prepared by dialysis, concentration, and lyophilization.

Second Step: Purification of Polysaccharides by Anion Exchange Resin (DEAE-Toyopearl 650M) Column

To purify macrophage-activated polysaccharides from cold safflower extracts, precipitates obtained through 75% ethanol precipitation were dissolved in distilled water and polymers with a molecular weight of 300,000 or more using an ultrafiltration membrane (300,000 Da, Viskase Sales). The substance (hereinafter abbreviated as 'CT-3') was obtained. After dissolving it in distilled water, 1 mg of sample per 1 ml of wet gel was injected into an anion exchange resin (DEAE-Toyopearl 650M, Cl ^- form) column equilibrated with distilled water (4.0 × 33 cm), and distilled water at a flow rate of 3.0 ml per minute, 0.1 , 0.2, 0.3, 0.4, 0.5, 1.0, 2.0 molarity of the sodium chloride solution was eluted 8 ml per fraction, respectively (Fig. 1).

Step 3: Purification of Polysaccharides by Gel Filtration Chromatography

Among the fractions eluted in the second step, the material eluted in the 0.1 molar sodium chloride solution showed the highest macrophage activity, which was then dialyzed, concentrated under reduced pressure, and lyophilized, followed by gel filtration chromatography equilibrated with 0.2 molar sodium chloride solution (Sepharose). CL-6B) 0.1 mg of sample per ml of wet gel was injected into a column (2.5 × 85 cm), and 3 ml of each fraction was eluted with a 0.2 molar concentration of sodium chloride solution at a flow rate of 0.2 ml per minute. The second peak was separated from the eluted peak (FIG. 2).

Example 2: Determination of Macrophage Activation Capacity of Polysaccharides Isolated by Anion Exchange Resin Chromatography

The fractions eluted from distilled water among the fractions separated by the second anion exchange resin chromatography were 'CT-3I', the fraction eluted with 0.1M NaCl was extracted with 'CT-3IIa' and 0.2M NaCl. 'CT-3Ⅱb', fractions eluted in 0.3M NaCl, 'CT-3IIc', fractions eluted in 0.4M NaCl, 'CT-3IId', fractions eluted in 0.5M NaCl and 'CT-3IIe', 1M The fraction eluted in NaCl was named 'CT-3IIf' and the fraction eluted in 2M NaCl were named 'CT-3IIg', respectively, and macrophage activation capacity was measured.

Suzuki et al., As follows, to determine whether they have the macrophage activation ability of CT-3I, CT-3IIa, CT-3IIb, CT-3IIc, CT-3IId, CT-3IIe, CT-3IIf, CT-3IIg. Method (Suzuki, I., Tanaka, H., Kinoshita, A., Oikawa, S., Osawa, M. and Yadomae, T. 1990. Effect of orally administered β-glucan on macrophage function in mice.Int. Soc. Immunopharmac. 12 , 675).

First, it was carried out using the activity measurement of lysosomal phosphatase of macrophages, the method proceeded as follows. In other words, After injecting 1 ml of thioglycolate medium into the abdominal cavity of 5-10 week-old male ICR (5 week-old, Korea Experimental Animal Center) rats, the rats were washed with RPMI-1640 medium 48-72 hours later. Macrophages were then recovered from the abdominal cavity. The recovered macrophages were washed twice with RPMI-1640 medium (Gibco, U.S.A) and the cell number was 1 × 10.⁶Re-dispersed in RPMI-1640 medium to cells / ml. 180 μl of this dispersion was dispensed into each well of a 96-well plate, followed by incubation for 2 hours in an incubator (37 ° C., 5% carbon dioxide) to allow macrophage cells to adhere to the base wall of each well plate to form a monolayer. . After 2 hours, non-adsorbed cells were washed and removed, and 180 μl of RPMI-1640 medium containing 10% fetal bovine serum was added to each well, and 20 μl of sample was added. , 5% carbon dioxide) for 24 hours to activate macrophages. 0.1% Triton X-100 (25 μl) was added to the monolayer of the activated macrophages to lyse the cell membranes of the macrophages. At this time, 100 mM para-nitrophenyl phosphite (150 μl) and 0.1 molar citric acid buffer (50 μl) were mixed with the phosphatase of the secreted lysosome as a substrate and reacted in an acidic state for 1 hour, followed by 0.2 mole concentration. Borate buffer was added to stop the reaction, and the absorbance was measured at 405 nm using an ELISA reader. From the measured absorbance, the activity of phosphatase was converted by the following formula (I) and expressed as the activity of macrophages.

As shown in Table 1, the fraction eluted in 0.1M sodium chloride solution (CT-3IIa) showed 220% higher macrophage activation and 15.5% higher yield than the control at 1.0 mg / mL. It has high activity even at low tablets, suggesting the possibility of use as adjuvant.

Dash Cell Activation Capacity and Yield of Anion Exchange Resin Chromatography Fractions Fraction activation(%) Yield (%) CT-3I (0 M) 190 3.5 CT-3Ⅱa (0.1 M) 220 15.5 CT-3Ⅱb (0.2 M) 120 12.3 CT-3Ⅱc (0.3 M) 190 8.0 CT-3Ⅱd (0.4 M) 180 3.5 CT-3Ⅱe (0.5 M) 200 1.8 CT-3Ⅱf (1.0 M) 170 2.9 CT-3Ⅱg (2.0 M) 130 1.7

Example 3: Determination of Macrophage Activation Capacity of Polysaccharide Purified by Gel Filtration Chromatography

CT-3IIa, which showed the highest macrophage activation ability in Example 2, was subjected to gel filtration chromatography to separate three fractions, 'CT-3IIa-1-1', 'CT-3IIa-1-2' and 'CT. -3IIa-1-3 'was named, and macrophage activation ability and yield were measured in the same manner as in Example 2.

As a result, as shown in Table 2 below, CT-3IIa-1-2 in the three fractions showed 250% activity and 35.6% yield compared to the control at the concentration of 1 mg / ml.

Activity and Yield of Gel Filtration Chromatography Fractions activation(%) yield(%) CT-3Ⅱa-1-1 220 30.1 CT-3Ⅱa-1-2 250 35.6 CT-3Ⅱa-1-3 190 15.6

Example 4 Characterization of Sugars Isolated from Safflower

Dextran T-2000 (molecular weight: 2 x 10 ⁶ ), T-500 (molecular weight: 5 x 10 ⁵ ), T- as reference materials to determine the molecular weight of CT-3IIa-1-2 of Example 3 High performance liquid chromatography (Waters model 2690; Shodex OHpak KB-805 (0.8 × 30 cm) column and refractiveindex detector using 70 (molecular weight: 7 × 10 ⁴ ), T-10 (molecular weight: 1 × 10 ⁴ ) and galactose Adhesion). As a result, the purified polysaccharide was found to be a polysaccharide having a molecular weight of about 380,000 Da.

On the other hand, carbohydrate, uronic acid and protein contents of the CT-3IIa-1-1, CT-3IIa-1-2 and CT-3IIa-1-3 polysaccharide fractions were measured by the phenol sulfate method, the m-hydroxybiphenyl method, the Laurie method. The sugar composition was measured by the following method.

First, hydrolyze the sample (500 ㎍) for 1.5 hours at 121 ° C. with 2.0 M trifluoroacetic acid (TFA), reduce the neutral sugar to alditol using NaBH _4, and then adsorb ethanol Sep-pak C ₁₈ cartridge The methylated sample was recovered. Acidic sugars in the methylated polysaccharides were reduced to LiB (C ₂ H ₅ ) 3D (Super-Deutride, 1 mL, room temperature, 1 hour) dissolved in THF, and then recovered with Sep-pak C ₁₈ cartridge. This was hydrolyzed at 121 ° C. with 1.0 M TFA for 2 hours and then reduced to NaBD ₄ to carry out an acetylation reaction. Partially methylated alditol acetate was analyzed by GC-MS. Methylated aditol acetates were identified by fragment ion and relative retention time, and peaks were measured by peak areas and response factor of flame ionization detector (FID).

Analysis of Carbohydrate Uronic Acid Protein Content and Sugar Composition of Polysaccharides CT-3IIa-1-1 CT-3IIa-1-2 CT-3IIa-1-3 Yield (%) 6.4 6.5 4.8 content (%) carbohydrate 47.2 58.8 47.6 Uronic acid 6.4 6.5 4.8 protein 9.3 13.8 19.1 Sugar composition (mol.%) Rhamnos (Rha) 5.8 15.1 6.8 Fucose 1.0 2.2 2.8 Arabinos (Ara) 12.6 20.6 31.2 Xylose trace ^e) 2.5 4.7 Mannose 9.4 3.7 4.8 Galactose (Gal) 35.5 32.0 26.6 Glucose (Glc) 29.4 15.4 18.2 Galacturonic acid (GalA) 2.1 3.8 1.1 Glucuronic Acid (GlcA) 4.2 4.7 3.8

Example 5: Anticancer Activity of Polysaccharides Isolated from Safflower

The anticancer activity of 'CT-3' showing high macrophage activity prepared in Example 1 was carried out using Sarcoma-180 solid cancer model as follows.

Macrophage activating material (CT-3) from safflower was dissolved in saline at concentrations of 0 (saline), 5, 10, 50, and 100 mg / kg, and tumor cells were distributed from Korea Cell Line Bank (ICR mice) (5 weeks old). , Sarcoma-180 cells were transplanted at two week intervals in the abdominal cavity of the Korea Experimental Animal Center) and the sarcoma-180 cells cultured in rat abdominal cavity for two weeks were taken with ascites and isolated. The cells were washed several times with physiological saline for injection under ice cooling to separate and remove red blood cells. It was adjusted to 10 ⁶ cells / ㎖ 0.1ml per horse subcutaneously implanted in the right groin and the sample was measured by intraperitoneal administration once a day for 10 days from 24 hours later. Proliferation inhibition rate was calculated as the percentage of inhibition of solid cancer growth (IR,%) compared to the control group administered with saline, as shown in the following formula (II).

Experimental results As shown in FIG. 3, the anticancer effect was analyzed using the Sarcoma-180 solid cancer model (preparation site) and showed a 50% or more inhibition effect even at a dose of 10 mg / kg.

These results suggest that the macrophage-activated polysaccharide purified from safflower has a high molecular weight of 380,000 Da, which activates macrophages and has anticancer effects. It is expected to be very useful for use as a functional food or drug, as well as for cancer patients or immunocompromised patients.

As is apparent from the above Examples and Experimental Examples, the present invention provides a macrophage-activated polysaccharide isolated from safflower cold water extract and its purification method. The macrophage activated polysaccharide of the present invention is purely separated from safflower cold water extract by a series of processes through fractionation of ethanol 75%, anion exchange resin chromatography, gel filtration chromatography. The macrophage-activated polysaccharides isolated in this way are polysaccharides of high molecular weight and have anti-cancer effects as in solid cancer experiments.

Claims (3)

A composition for macrophage activator, characterized in that it contains a polysaccharide isolated from the water extract of safflower as an active ingredient. Anticancer composition, characterized in that it contains a polysaccharide isolated from the water extract of safflower as an active ingredient. a) removing methanol soluble fraction from safflower water extract followed by ethanol precipitation; b) obtaining a solution having a molecular weight of 300,000 or more through ultrafiltration of the precipitate obtained above; c) injecting the obtained solution into an anion exchange resin column and then eluting and fractionating with a 0.1 molar concentration of sodium chloride solution; d) A method for producing macrophage-activated polysaccharide from safflower cold water extract, comprising separating the active fractions by gel filtration chromatography having a molecular weight of 10,000 to 1 million.