KR100657847B1 - Food and Food additive Comprising an Extract of Polysaccharides Having Immune Activity - Google Patents

Abstract

The present invention relates to a food additive and a food containing the polysaccharide spinach extract as an active ingredient, and more particularly, after hot water extract of spinach, the methanol is added to the hot water extract to obtain a methanol-insoluble residue, which is distilled water. After dissolving, ethanol is added to obtain a precipitate, and a crude polysaccharide fraction is prepared from the precipitate and relates to a food additive and a food containing the polysaccharide extract, characterized in that an active fraction is obtained using sodium chloride.

Prickly tortoise polysaccharide extract according to the present invention exhibits a mucosal immune enhancing effect, hematopoietic cell proliferative activity effect, tumor metastasis inhibitory effect and allergic immune response inhibitory effect to promote health, food and food additives containing the prickly omega polysaccharide extract It is effective to provide.

Thorns, polysaccharides, food additives, immune boosters, mucosal immunity

Description

Food and Food additive Comprising an Extract of Polysaccharides Having Immune Activity

Figure 1 is a schematic diagram showing the manufacturing process of prickly gingko polysaccharide extract according to the present invention.

Figure 2 shows the hematopoietic cell proliferation activity by direct stimulation in vitro of the polysaccharide extract of prickly gingiva according to the present invention.

Figure 3 shows the activity of IgA production by oral immunity of Ogapi polysaccharide extract and OVA (obalbumin) according to the present invention.

Figure 4 shows the effect of oral immunity of Ogapi polysaccharide extract and OVA according to the present invention on the production of serum IgG and IgE.

5 shows cell proliferation activity by restimulation of antigen OVA in Peyer's patch cells.

The present invention relates to a food additive and a food containing the polysaccharide spinach extract as an active ingredient, and more particularly, after hot water extract of spinach, the methanol is added to the hot water extract to obtain a methanol-insoluble residue, which is distilled water. After dissolving, ethanol is added to obtain a precipitate, and a crude polysaccharide fraction is prepared from the precipitate and relates to a food additive and a food containing the polysaccharide extract, characterized in that an active fraction is obtained using sodium chloride.

Acanthopanax Senticosus is a perennial shrub belonging to Raliaceae. It grows in Korea, Siberia and China, and its roots, stems, leaves and fruits have been used as oriental medicine in the East.

Siberian ginseng, called Siberian Ginseng, is used for the purpose of strengthening the body's vitality, strengthening the spleen and kidneys, and has been proven to be effective in relieving sedation, analgesia, loss of appetite or fatigue. In addition, there is a sedation in the central nervous system, and according to experiments, it shows both sides of sedation and excitement.

Until now, the application for thorny oocytes is mainly the cultivation method of thorny oocytes, and the mass production method is mainly the mass production method of thorny oak seedlings by biotechnological technology (Korean Patent Laid-Open Publication No. 1999-0064391), and thorny oocytes using somatic embryos. (Republic of Korea Patent Publication No. 10-0257991), the method of cultivating the thorny ogapi larvae by the biological phase culture method and its use (Korea Patent Publication No. 2001-0010217), and the like, and the polysaccharide thorny opi extract Researches or patent applications applied to foods or pharmaceuticals that can be used include immunopotentiating compositions, barley extracts, protein extracts and crude protein polysaccharide extracts, which are extracted from Korean barberry extracts, and food or food additives containing the immunopotentiating composition. And pharmaceutical compositions (Patent No. 10-0466735) are disclosed. The.

The application according to the registered Patent Publication No. 10-0466735 relates to a method for preparing a prickly pear extract and an immuno-enhancing composition containing the prickly pear extract, according to the present invention, the prickly pear extract and an immuno-enhancing composition using the same as an active ingredient Except for the direct stimulation of macrophages to derivatives of the cytokine, a bioactive substance, and the effect of activating natural killer cells. There is a drawback of inducing an abnormal immune response, such as an inflammatory response, and does not function to increase the differentiation of immune cells by activating the proliferative capacity of hematopoietic cells.

However, mucosal immunization by oral administration or non-administration has been known to be of great safety to the living body, such as suppressing the induction of the abnormal immune response, compared to systemic immunization, and has received a lot of attention in recent years. Since mucosal immunity is much exposed to various pathogens compared to systemic immunity, there is an advantage to increase the defense locally or systemically against various pathogens. In other words, the enhancement of mucosal immunity can include several pathogens in the digestive system, including Helicobacter pylori, Vibrio cholera, Rotavirus, and pathogens that cause infections through the respiratory system, including pathogens such as mycoplasma, influenza viruses, and HIV. Provides defense against disease.

To date, the mechanism of action of the host against such pathogens has been described as a defense mechanism by secreted immunoglobulins secreted from mucosal organs. In recent studies, however, the protective mechanism of activation of mucosal CD8 + toxic T lymphocytes, CD4 + helper T cells, and natural killer cells, which constitute the mucosal immune system, has been described. That is, oral immunization of the vaccine has been shown to induce typical acquired immune systems such as antigen-specific immunopotentiating effects of the local mucosal system, activation of the systemic immune system, and induction of memory cells' immune capacity.

Mucosal immunity has the function of inhibiting delayed and hypersensitivity reactions, which are T cell-mediated immune responses in peripheral tissues. It also has the property of inhibiting DTH or other allergic reactions in proteins or various antigens. In most cases when orally administered, immune tolerance to the antigen is induced, but in some cases, even if there is an immune tolerance at the peripheral site, an immune response is induced that produces IgA against the antigen locally. That is, cytokines involved in the induction of mucosal immunity, such as TGF-b and IL-4, result in the production of secretory IgA from the surface of the mucosa. These IgAs can be effectively used for the removal of antigens that invade the respiratory, digestive and genitourinary organs. Ultimately, IgA does not end with an immune response that is specific to mucosal tissues, but more important by inducing a systemic immune response to antigens. It is becoming.

Despite the many advantages of mucosal immunity, the development of mucosal vaccines is difficult because of the difficulty in producing specific IgA antibodies that can effectively remove orally administered antigens.

What is currently to be overcome for the development of oral vaccines is an antigen delivery method that can induce an effective immune response to antigens in the mucosal immune system. The most important immunoassay that can effectively deliver antigens and obtain defense against mucosal immune systems is the development of oral adjuvants.

Mucosal immune enhancers (adjuvant) currently being used or studied most actively include bacterial toxins and derivatives thereof, CpG DNA, cytokines, or viral particles as chemokines and antigen transporters. However, since these side effects have been reported to induce a strong inflammatory response in the systemic immune system, it is urgent to develop oral adjuvant, food additives and foods that can be applied to humans.

Therefore, the present inventors have made diligent efforts to develop more effective and safe immune enhancers, and when the polysaccharide isolated from thorns is administered orally or nasally to the mucosa, it stimulates immune cells constituting the mucosal immune system, and abnormal immunity such as an inflammatory response. Induces defense against specific antigens by inhibiting the induction of reactions, activating proliferative ability of hematopoietic cells to differentiate into immune cells with a safe immune response of the body, and increasing the ability of IgA antibodies to produce specific antigens In addition, by demonstrating the activity of IgG to induce a systemic immune response to the antigen, it was confirmed the usefulness as a food additive and food containing the polysaccharide extract of the bark and completed the present invention.

After all, the main object of the present invention is to provide food additives and foods containing the extract of prickly rot polysaccharide having mucosal immune enhancing effect.

In order to achieve the above object, the present invention is a food containing a barb of polysaccharide extract containing an arabinose, galactose, galactose, rhamnose, and galacturonic acid as an active ingredient, and Provide food additives.

In the present invention, the molar ratio of arabinose: galactose: rhamnose: galactonic acid may be 0.2 to 0.3: 0.2 to 0.3: 0.1 to 0.2: 0.9 to 1.1.

In the present invention, the pterygium polysaccharide extract may be characterized in that it is prepared through the following steps:

(a) Distilled water was added to the visible sample, and the mixture was distilled and separated into filtered primary filtrate and residue. The filtrate obtained by repeating the above process was separated from the separated residue with primary filtrate and centrifuged. Removing the insoluble material and then freeze drying to prepare a hot water extract;

(b) adding methanol to the hot water extract to reflux, separating the methanol soluble liquid, and adding methanol to the residue to separate the methanol-soluble fraction and the methanol-insoluble residue;

(c) dissolving the separated methanol-insoluble residue in distilled water, and then adding ethanol and stirring to separate the precipitate and the ethanol soluble fraction;

(d) dissolving the ethanol precipitate in distilled water, dialysis and centrifuging to separate the ethanol-insoluble material, followed by freeze drying to prepare crude polysaccharide fraction;

(e) charging the crude polysaccharide fraction into a column equilibrated with distilled water and eluting with distilled water to obtain a non-adsorbed fraction, followed by eluting sodium chloride, dialysis and lyophilization to obtain an adsorbed fraction; And

(f) eluting sodium chloride after filling the adsorbed fraction to obtain an active fraction.

Hereinafter, the present invention will be described in detail.

Ogapi polysaccharide extract of the present invention can be prepared as follows.

After freeze-drying, distilled water is added to the shredded bark, and the filtrate is separated until the amount of water is half of the amount added, and then the filtrate and the residue are separated. After distilled water was added to the residue, the filtrate obtained by repeating the above process was first mixed with the filtrate and centrifuged to completely remove the insoluble material, and then hot water extracts may be prepared by lyophilization. Methanol was added to the hot water extract again to reflux for about 1 hour, and the methanol soluble liquid was separated, and the residue was separated into methanol-soluble fraction and methanol-insoluble residue by adding methanol again. The separated methanol-insoluble residue was dissolved in distilled water, and then stirred for about 12 hours by adding 4 times of ethanol to separate the precipitate and the ethanol soluble fraction. The ethanol precipitate is again dissolved in distilled water, then dialyzed, the insoluble material is separated by centrifugation, and the crude polysaccharide fraction can be obtained through lyophilization.

The crude polysaccharide fraction is charged into a column equilibrated with distilled water and eluted with distilled water to obtain a non-adsorbed fraction. Next, 0.1, 0.2, 0.3, 0.4, 0.5, 1.0 and 2.0 M NaCl were eluted sequentially, and the seven adsorption fractions were obtained by dialysis and lyophilization in 0.1, 0.2, 0.3, 0.4, 0.5, 1.0 and 2.0 M NaCl elution fractions. Get Then, after filling the column of 0.5M NaCl of the seven fractions in the column and eluting 0.2M NaCl to be separated into an inactive fraction and an active fraction, it is possible to obtain the active extract polysaccharide extract.

By orally administering the polysaccharide extract of the present invention obtained by the above-mentioned method, non-specific activation ability and activation ability of hematopoietic cells of mucosal immune cells, Firespatch cells were confirmed, and tumor suppression effect by oral administration was confirmed. In addition, when oral immunity with the antigen, it was confirmed that there is a local and systemic stimulation of immune cells.

When the oral immunity with the antigen at the same time oral immunity with the antigen of the present invention, the antigen-specific IgA production capacity and IgG antibody production capacity was increased to obtain a result that it was confirmed that the systemic immunity is enhanced by oral administration as well as mucosal immunity. As evidence of this, it was confirmed that the activation of antigen-specific immune cells upon re-stimulation of the firepatch cells with the antigen.

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

Example 1 Preparation of Polysaccharide EN3.5-2 from Prickly Pear

After freeze-drying, 2 L of distilled water was added to 100 g of the shredded thorny skin samples, and the mixture was distilled until the amount of water became half the amount added, and then the filtrate and the residue were separated with a metal. Again, 2 L of distilled water was added to the residue, and the filtrate obtained by repeating this process was first mixed with the filtrate and centrifuged to completely remove the insoluble material, followed by lyophilization, to prepare a hot water extract (hereinafter referred to as EN-0). . Methanol-soluble fraction was prepared by refluxing EN-0 thus obtained by adding 5 L of methanol and refluxing for about 1 hour, then separating the methanol soluble liquid and adding methanol to the residue (hereinafter referred to as EN-1). The separated methanol-insoluble residue was dissolved in distilled water, added 4 times of ethanol, and stirred for about 12 hours to separate the precipitate and the ethanol soluble fraction (hereinafter referred to as EN-2). The ethanol precipitate was again dissolved in distilled water, dialyzed, insoluble matters were separated by centrifugation, and lyophilized to prepare crude polysaccharide fraction (hereinafter referred to as EN-3).

That is, 1 g of crude polysaccharide fraction is charged into a DEAE-Sepharose CL-6B (Cl-type, 4.0 × 40 cm, Pharmacia) column equilibrated with distilled water and eluted with distilled water to obtain a non-adsorbed fraction. (EN-3I, yield; 2.2% of EN-3). Next, 0.1, 0.2, 0.3, 0.4, 0.5, 1.0 and 2.0 M NaCl were eluted sequentially to allow dialysis and lyophilization at 0.1, 0.2, 0.3, 0.4, 0.5, 1.0 and 2.0 M NaCl eluted fractions. Seven adsorption fractions up to .7 were obtained. Afterwards, EN3.5, which showed various immunity, was filled in Sephacryl S-300 (3.0 × 90 cm, Pharmacia) column, followed by elution of 0.2 M NaCl to EN3.5-1 and active fraction near inactive fraction void volume. Fractionated with EN3.5-2.

In order to investigate the constituent sugars of the polysaccharide extracts of the present invention obtained by the above-mentioned method, the alditol acetate derivatives were converted and analyzed by GC. GC used Hewlett-Packard 6890 II equipped with SP-2380 capillary column (0.2 μm film, 0.25 mm i.d. × 30 m, Supelco) and temperature change was 60 ° C. for 1 minute; 60 → 220 ° C. (30 ° C./min); 220 ° C., 8 minutes; 220 ° C. → 250 ° C. (8 ° C./min); It progressed to 250 degreeC and 15 minutes. The molar ratio per composition was calculated from peak area and response factor using FID.

EN3.5-2, which is separated into fractions with mucosal immunity enhancing activity, is composed of 62.4% acidic sugar, 27.6% neutral sugar and 6.6% protein as main constituents. It can be seen that the acidic sugar is commonly contained (Table 1). As the sugar content is relatively high in the active fractions, the composition sugars of the active fractions were compared and the results showed that arabinose, galactose, rhamnose and galacturonic acid (molar ratio; 0.25: 0.26: 0.11: 1.00) Could know.

Example 2 Proliferation Activity of Hematopoietic Cells by Stimulation

Sterilize the hematopoietic cells from the mouse thighs, add 5 x 10 6 cells to each well of a 96-well culture plate and add EN3.5-2 adjusted to various concentrations as a sample for 5 days. Incubated. After completion of the culture, the proliferative activity of the bone marrow cells by the sample was XTT {sodium 3 '-[1-[(phenylamino) -carbonyl] -3,4-tetrazolium] -bis (4-methoxy-6-nitro). Measured by adding benzene-sulfonic acid hydrate (sodium 3 '-[1-[(phenylamino) -carbonyl] -3,4-tetrazolium] -bis (4-methoxy-6-nitro) benzene-sulfonic acid hydrate) It was. That is, 6 hours before completion of culture, 20 ml of XTT solution was added to each well, and bone marrow cells measured the degree of water-soluble formazan produced by the reaction of respiratory enzyme and XTT at 450 nm. Presented together. As a result, EN3.5-2 of the present invention had an activity of significantly increasing the proliferation of hematopoietic cells in vitro, and its activity was about 300 times higher than that of crude extract and more than 2 times higher than EN3.5. Showed activity.

Example 3 Proliferative Activity of Hematopoietic Cells Via Firepatch Cells of EN3.5-2

In this study, the fractional components from EN3 were applied to the experiment to investigate the induction capacity of bone marrow cell proliferation activity through Peyer's patch cells. Experiments to investigate the proliferative activity of myeloid cells were carried out as follows. In other words, the preparation of the Peyer's patch cell culture supernatant was collected from the small intestine of L3 independent C3H / He mice, the Peyer's patch of the small intestine wall was cut and transferred to a petri dish containing cold RPMI-1640 medium, and the Peyer's patch was crushed to release the cells. After washing with medium three times. Adjust the cell concentration to 2 × 106 cells / mL RPMI-1640, divide 200 μl into 96 well plates, adjust each sample to a concentration of 10 mg / ml, and add 5 mL of CO2 incubator at 37 ° C for 5 days. The supernatant was recovered by culturing, and the supernatant was used as a cell culture supernatant for measuring bone marrow cell proliferation activity. On the other hand, the preparation of bone marrow cells, while injecting the RPMI-1640 medium from the shin bone of the same mouse, the bone marrow cells were recovered, filtered and washed as described above and adjusted to 2.5 × 105 cells / mL RPMI-1640. Bone marrow cell proliferation was measured using Alamar Blue reduction assay. After dispensing 100 μl of the adjusted bone marrow cell solution into a 96 well plate, 50 μl of cell culture supernatant and RPMI-1640 medium obtained from Peyer's patch cells were used for 6 days at 37 ° C. Incubated. 20 μl of Alamar Blue solution was added 5 hours before the end of the incubation. Fluoroskan II was used to measure the fluorescence intensity at excitation 544 nm and emission 590 nm. The bone marrow cell proliferation promoting activity of the sample quantified the proliferation of bone marrow cells from the difference from the control. Experimental results showed that EN3.5 showed the highest activity, and EN3.5-2 isolated from EN3.5 showed significantly higher bone marrow cell proliferation activity (1.9 times control). The remaining fraction of EN3.5-1 showed weak activity (1.3-fold). Table 2

Example 4 Tumor Metastasis Inhibition Activity by EN3.5-2 Oral Administration

Investigation of nonspecific immunostimulatory activity by oral administration of EN3.5-2 of the present invention measured antitumor activity in experimental animal tumor metastasis model using colon 26-M3.1 carcinoma strain. Balb / c mice were used as experimental animals, and the inoculation for tumor metastasis was intravenously injected (i.v.) of 2.7 × 10 4 colon 26-M3.1 carcinoma cells. After 14 days, the mice were sacrificed and the lung, which is the target organ of the tumor, was extracted to fix the metastasized tumor in Bouin's solution, and the number of tumors was measured. In order to investigate the antitumor metastasis effect of the present invention, 1 mg of EN3, EN3.5-1 and EN3.5-2 were orally administered 5, 3 and 1 day before tumor inoculation. As a result, in the case of EN-3 and EN3.5-2, significant anti-tumor effects of 35% and 46% were recognized, but in the case of EN3.5-1, no significant results were found. In the case of 2, it was judged that nonspecific immunostimulatory ability was systemically implemented by oral administration from EN3 (Table 3).

Example 5 Preparation of IgA of Intestinal Content by Oral Immunity of EN3.5-2 and Ovalbumin

Animal testing was performed to determine whether mucosal immunity is enhanced for oral administration of EN3.5-2 with antigen. Experimental animals used in the experiment was a 6-week-old balb / c mouse, the obalbumin (OVA) was used as the antigen. That is, 100 ug of OVA and 1 mg of EN3.5-2 were dissolved in 0.2 ml of physiological saline (PBS) and immunized to 5 mice per group. OVA and EN3.5-2 alone were administered orally as a control and normal mice were used. Immunization for antibody production was immunized three times at weekly intervals and antibody titers were measured after 3 days of final immunization. Enhancement of mucosal immunity by oral administration of EN3.5-2 was measured by antibody-linked immunosorbent assay (ELISA) of the antibody titer of IgA present in the small intestine contents and serum. Experimental results, as shown in Figure 3, when immunized with the antigen OVA and EN3.5-2 at the same time compared to the control results in a significantly higher antibody production results, oral administration of EN3.5-2 is weak immunity of mucosal organs It confirmed that it has the production promoting activity of the instrument IgA.

Example 6 Preparation of IgG in Blood by Oral Immunity of EN3.5-2 and Ovalbumin

The OVA-specific antibody titer was examined from the serum of the mouse of Example 5 using a 500-fold diluted serum, and showed the same IgG production ability as the result of Example 5 (see FIG. 3). OVA, the antigen applied in this experiment, is known as a strong allergen. Therefore, the effects on the production of antibody IgE related to allergy induction were examined by EN3.5-2 immunized with antigen. As a result, as shown in Figure 4, the production of IgE to OVA in serum significantly decreased.

Example 7 Proliferation Activity of Firepatch Cells by Oral Immunity of EN3.5-2 and Ovalbumin

Peyer'patch cells were taken from the mice subjected to the experiment of Example 5, and 2.5 × 10 6 cells were added to each well of a 96-well culture plate and re-stimulated with OVA adjusted to various concentrations for 3 days. . 20 μl of Alamar Blue solution was added 5 hours before the end of the culture, and the proliferation activity of the cells was measured by excitation 544 nm and emission 590 nm. The control group was stimulated with 5 mg / ml LPS. As shown in FIG. 5, the group of cells re-stimulated with antigen OVA occurred only in the group immunized simultaneously with antigen OVA and EN3.5-2, and the degree of re-stimulation adjuvant to enhance mucosal immunity. Phosphorus showed a result dependent on the concentration of EN3.5-2. Therefore, oral administration of EN3.5-2 showed the result of enhancing the cell proliferation activity against the antigen, and supported the result that the immune response of the mucosal system, which is the immune response, and the IgG antibody production capacity of the systemic system.

As described above in detail a specific part of the content of the present invention, for those skilled in the art, such a specific description is only a preferred embodiment, which does not limit the scope of the present invention by Will be obvious. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

As described in detail above, the present invention has an effect of providing a food additive and a food containing an effective amount of the thorny ogapi polysaccharide extract containing arabinose, galactose, rhamnose and galactonic acid. Pseudomonas polysaccharide extract prepared according to the present invention inhibits the induction of abnormal inflammatory response by nasal administration, mucosal immune enhancing effect, hematopoietic cell proliferative activity effect, tumor metastatic activity inhibitory effect and allergic immune response suppression according to IgE In addition to the effects IgA of the mucosal immune system, as well as enhance the IgG antibody production capacity of systemic immunity can also be useful as a food additive and food that is beneficial to health by enhancing systemic immunity.

Claims (6)

Contains arabinose, galactose, galactose, rhamnose, and galacturonic acid. 0.2 ~ 0.3: 0.1 ~ 0.2: 0.9 ~ 1.1, characterized in that the food additive containing the barb polysaccharide extract as an active ingredient. Contains arabinose, galactose, galactose, rhamnose, and galacturonic acid. Food containing a barb of polysaccharide extract as an active ingredient, characterized in that 0.2 to 0.3: 0.1 to 0.2: 0.9 to 1.1. delete delete The food additive according to claim 1, wherein the extract of prickly pear skin is prepared through the following steps. (a) Distilled water was added to the visible sample, and the mixture was distilled and separated into filtered primary filtrate and residue. The filtrate obtained by repeating the above process was separated from the separated residue with primary filtrate and centrifuged. Removing the insoluble material and then freeze drying to prepare a hot water extract; (b) adding methanol to the hot water extract to reflux, separating the methanol soluble liquid, and adding methanol to the residue to separate the methanol-soluble fraction and the methanol-insoluble residue; (c) dissolving the separated methanol-insoluble residue in distilled water, and then adding ethanol and stirring to separate the precipitate and the ethanol soluble fraction; (d) dissolving the ethanol precipitate in distilled water, dialysis and centrifuging to separate the ethanol-insoluble material, followed by freeze drying to prepare crude polysaccharide fraction; (e) charging the crude polysaccharide fraction into a column equilibrated with distilled water and eluting with distilled water to obtain a non-adsorbed fraction, followed by eluting sodium chloride, dialysis and lyophilization to obtain an adsorbed fraction; And (f) eluting sodium chloride after filling the adsorbed fraction to obtain an active fraction. The food product according to claim 2, wherein the thorny ogapi extract is prepared through the following steps. (a) Distilled water was added to the visible sample, and the mixture was distilled and separated into filtered primary filtrate and residue. The filtrate obtained by repeating the above process was separated from the separated residue with primary filtrate and centrifuged. Removing the insoluble material and then freeze drying to prepare a hot water extract; (b) adding methanol to the hot water extract to reflux, separating the methanol soluble liquid, and adding methanol to the residue to separate the methanol-soluble fraction and the methanol-insoluble residue; (c) dissolving the separated methanol-insoluble residue in distilled water, and then adding ethanol and stirring to separate the precipitate and the ethanol soluble fraction; (d) dissolving the ethanol precipitate in distilled water, dialysis and centrifuging to separate the ethanol-insoluble material, followed by freeze drying to prepare crude polysaccharide fraction; (e) charging the crude polysaccharide fraction into a column equilibrated with distilled water and eluting with distilled water to obtain a non-adsorbed fraction, followed by eluting sodium chloride, dialysis and lyophilization to obtain an adsorbed fraction; And (f) eluting sodium chloride after filling the adsorbed fraction to obtain an active fraction.

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