KR20140121191A - Immuno-stimulating activities of polysaccharide fractions isolated from citrus peel, preparing method thereof and health functional food comprising the the same - Google Patents

Abstract

The present invention relates to a method for extracting immunostimulating polysaccharide having an immune boosting effect from by-products of citrus fruits such as a tangerine and a hallabong. The immunostimulating polysaccharide obtained by enzyme treatment according to the present invention has an excellent anticomplementary activity and superior capacities on proliferation of lymphocytes and stimulation of the production of macrophage cytokines (IL-6 and IL-12) compared to immunostimulating polysaccharide from citrus fruits obtained by hot water extraction, and a composition comprising the immunostimulating polysaccharide as an active ingredient can be used as an immune adjuvant.

Description

TECHNICAL FIELD [0001] The present invention relates to an immunosuppressive polysaccharide derived from citrus processing by-products, a method for producing the same, and a health functional food containing the polysaccharide fractions isolated from a citrus peel,

The present invention relates to a method for extracting active polysaccharide having immunity enhancing function from citrus processing by-products and a health functional food containing the same

The first cause of death in Koreans is cancer, which traditionally uses surgery, radiation therapy and pharmacotherapy to overcome cancer, but it is still difficult to expect complete therapeutic effects on metastatic tumor cells. Currently, most of the chemotherapy depends on the administration of anticancer drugs. However, since most of the anticancer drugs are synthetic chemicals, biohazard problems such as hematopoietic function and immune function abnormality are raised, I am interested.

Recently, active polysaccharides having immunity-enhancing functions have been reported in natural materials such as ginseng, mushroom, mold, bacteria, and persimmon leaves. T and B lymphocytes, macrophages , NK cells (natural killer cells), and lymphokine activated killer cells (LAK cells). Korean Patent No. 10-0594353 discloses an anticancer agent or anticancer adjuvant composition containing a polysaccharide component isolated from leaves and stems of ginseng as an active ingredient. In Korean Patent No. 10-0078107, Korean Patent No. 10-0491362 discloses a method for producing a polysaccharide containing arabinogalactan which decomposes a polysaccharide by treating the plant tissue with an enzyme solution and extracting the plant tissue with hot water or a base, Which is prepared by inoculating an immune activity-promoting complex polysaccharide.

Pectin is a homogalactouronan composed of a large number of whole molecules, and rhamnogalactouronan I (RG-I) and rhamnogalactouronan Ⅱ (RG-Ⅱ), which are branched by various oligo- and polysaccharides, are known [Pharmaceutical Chemistry Journal Volume 46, Number 4 219-221]. Studies on various physiological activities such as cytokine production stimulation, macrophage activation and NK cell stimulating activity, including immunological activity and anticancer activity, have been published in the RG category, but commercialization using this material as a material has been limited.

Citrus fruits such as mandarin orange and hirabong are widely cultivated in southern part of Korea and Jeju Island, and citrus fruit processing by-products such as mandarin orange and halabong from the process of making beverage are generated more than 50 thousand tons per year. Citrus processing by-products are used for the production of pectin and oil, but most of them are industrial wastes and are discarded without secondary use. Recently, it has been reported that hesperidin, which is an antioxidant, has been added to feed pigs to improve disease resistance. However, no technology has been reported to isolate polysaccharides having immunity enhancing function from citrus fruits such as Halabong.

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made to solve the above-mentioned problems, and it is a first object of the present invention to provide a method for producing an immunopotentiating polysaccharide having immunity enhancing function from citrus processing by-products of citrus.

A second object of the present invention is to provide a health functional food comprising the immunostimulatory polysaccharide prepared according to the present invention as an active ingredient.

In order to achieve the first object of the present invention,

Enzymatic treatment of citrus processing by-products in citrus; And adding a 30-90% by volume ethanol aqueous solution to the enzyme-treated citrus processing by-product to obtain a precipitate. The present invention also provides a method for producing an immunocompetent polysaccharide derived from citrus fruits.

According to an embodiment of the present invention, the enzyme treatment may be one or two selected from the group consisting of pectinase, cellulase, xylanase, and mananase, Or by mixing the above enzymes,

Preferably, the enzyme can be treated using a commercial enzyme. Examples of the enzyme include Econitase, Rapidase, Viscozyme, Celluclast, Pectinex, (Rohament), Ultraflo, and Cytolase, and may be a mixture of two or more selected from the group consisting of,

The enzyme treatment may be performed by adding 0.05 to 5 parts by weight of an enzyme to 100 parts by weight of the solids of the citrus processing by-product and treating the mixture for 6-96 hours,

The citrus fruits are not limited as long as they are citrus plants and may be at least one selected from the group consisting of citrus fruits, halobongs (blooming), chrysanthemums, bluegrass, kumquat and citron. Preferably, have.

In addition, the immunostimulatory polysaccharide prepared according to the present invention may have a complement activity at a concentration of 1,000 μg / ml of 60% or more, a production stimulating ability of Interleukin-6 (IL-6) of 600-800 pg / 12 (IL-12) may be 550-800 pg / mL and the molecular weight may be 5-300 Kda.

The immunologically active polysaccharide may be selected from the group consisting of 2-methylfucose, 2-methylxylose, apiose, aceric acid, 3-deoxy- 3-deoxy-D-manno-2-heptulosaric acid, DHA (3-deoxy-D-manno-octanoic acid, KDO) , Rhamnose, fucose, arabinose, xylose, mannose, galactose, glucose, galacturonic acid, and glue. And may be at least one selected from the group consisting of Glucuronic Acid.

In order to achieve the second object, the present invention provides a health functional food comprising the immunostimulatory polysaccharide prepared according to the present invention as an active ingredient.

The immunostimulatory polysaccharide can be used as a health functional food because it can enhance anti-complement activity, production of interleukin-6 (IL-6) and production of interleukin-12 (IL-12).

The citrus-derived immunopotentiating polysaccharide obtained by the enzyme treatment according to the present invention exhibited more than 60% of complement activity at a concentration of 1,000 / / ml and stimulated lymphocyte proliferation, production of macrophage cytokines (IL-6 and IL-12) The ability to activate natural killer cells is superior to the citrus-derived immunostimulatory polysaccharides obtained by hot water extraction, and a composition comprising the immunostimulatory polysaccharide as an active ingredient can be used as a health functional food since it has an immunity enhancing function.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a diagram illustrating a method of preparing an immunocompetent polysaccharide from citrus processing by-products according to an embodiment of the present invention. FIG.
FIG. 2 is an analysis of an immunocompetent polysaccharide prepared from a citrus processing by-product according to an embodiment of the present invention by gas chromatography. FIG. 2 (a) is a gas chromatographic result according to an embodiment of the present invention, and FIG. 2 (b) is a gas chromatographic result according to a comparative example of the present invention.
FIG. 3 is a graph comparing the anti-complement activity of the immunostimulatory polysaccharides prepared according to an embodiment of the present invention, according to concentration.
Figure 4 is a graph comparing cytotoxicity assays of immunostimulatory polysaccharides prepared according to one embodiment of the present invention. FIG. 4A is an assessment of cytotoxicity in macrophages recovered from the peritoneal cavity of mice, and FIG. 4B is a cytotoxicity assay of Colon 26-M3.1, a pulmonary metastatic tumor cell line.
FIG. 5 is a graph comparing the production stimulation of cytokines of macrophages of immunostimulatory polysaccharides prepared according to one embodiment of the present invention and a comparative example. Figure 5a is an assessment of production stimulation of IL-6, and Figure 5b is an evaluation of production stimulation of IL-12.

Hereinafter, the present invention will be described in detail.

According to one aspect of the present invention, there is provided a method for producing a citrus fruit, comprising: enzymatically treating a citrus processing by-product in citrus; And adding a 30-90 vol.% Ethanol aqueous solution to the enzyme-treated citrus processing by-product to obtain a precipitate. The present invention also provides a method for producing an immunoconactive polysaccharide derived from citrus fruits.

The by-product of citrus processing may be a citrus husk and a by-product obtained from the process of making a citrus product such as beverage, citrus chocolate, etc., and the by-product may be used directly or dried.

According to the present invention, the enzyme treatment can be used as it is without any reprocessing, but it is preferable to crush and use the citrus byproducts. Crushing and using the citrus byproducts increases the surface area of the citrus byproduct, It is preferable since the enzyme treatment time can be shortened and the hydrolysis phenomenon caused by the long term enzyme treatment can be prevented.

According to the present invention, the enzyme used in the enzyme treatment is not limited as long as it is an enzyme capable of separating the immunologically active polysaccharide present in the by-product of citrus fruits. Preferably, the enzyme is selected from the group consisting of pectinase, cellulase, (Xylanase) and mananase. Specific examples of the enzymes include econitase, Rapidase, and the like, which are commercially available enzymes. One or more selected from the group consisting of Viscozyme, Celluclast, Pectinex, Rohament, Ultraflo, and Cytolase, (Pectinex) and cyotrazine, which are used alone or in combination, may be used as a surface active agent having a high functionality It is possible to obtain a polysaccharide is more preferred.

The enzymatic treatment may be performed by adding 0.05-5 parts by weight of enzyme to 100 parts by weight of the solids of the citrus processing by-product. If the amount of the enzyme is less than 0.05 part by weight, the amount of enzyme is too small to separate the immunologically active polysaccharide present in the citrus by- When the amount is more than 5 parts by weight, the function of the immunogically active polysaccharide may be hydrolyzed by excessive enzymatic action to lower the efficacy of the immunostimulatory polysaccharide.

The reaction time of the enzyme treatment is preferably 6-96 hours, more preferably 12-72 hours, and the temperature and the pH during the enzyme treatment are preferably adjusted under optimal conditions for each enzyme to obtain an immunocompetent polysaccharide And is preferable. If the reaction time of the enzyme treatment is less than 6 hours, it is difficult to isolate the immunologically active polysaccharides present in the citrus fruits because the time for the citrus processing by-products to treat the enzyme is too short. If the enzyme treatment reaction time exceeds 96 hours, The functional group of the immunosuppressive polysaccharide may be hydrolyzed to lower the efficacy of the active polysaccharide.

According to the present invention, the immunopotentiating polysaccharide produced by the enzyme treatment can be purified by dialysis, column chromatography or membrane filtration, but it is more convenient and inexpensive in terms of facilities and process costs, It is preferable to be purified.

The purification can be performed by inducing precipitation of an immunostimulatory polysaccharide by adding 30-90% by volume of ethanol to a reaction solution containing an immunopotent polysaccharide obtained by an enzyme treatment, more preferably 40-80% by volume It is difficult to obtain immune-active polysaccharide crystals because ethanol does not precipitate at a concentration lower than 40% by volume. When ethanol at a concentration higher than 80% by volume is used, the manufacturing cost is increased and it is not economical. Which is undesirable.

According to the present invention, the citrus fruits are not limited as long as they are citrus plants, and may be at least one selected from the group consisting of citrus fruits, hala bongs, May be Halabong

According to the present invention, the immunopotentiating polysaccharide prepared by the above-mentioned method can be used as a complement fixation test method based on the degree of erythrocyte hemolysis due to complement remaining after complement consumption by the sample using the Mayer method Can have an ITCH ₅₀ value of 60% or more at a sample concentration of 1,000 / / ml,

In addition, the immunostimulatory polysaccharide has a production stimulating ability of Interleukin-6 (IL-6) of 600-800 pg / mL at a concentration of 1,000 μg / ml and a production stimulating ability of Interleukin-12 (IL- 800 pg / mL. ≪ / RTI >

According to the present invention, the immunostimulatory polysaccharide crystals may have a molecular weight of 5-300 Kda,

The immunologically active polysaccharide may be selected from the group consisting of 2-methylfucose, 2-methylxylose, apiose, aceric acid, 3-deoxy- 3-deoxy-D-manno-2-heptulosaric acid, DHA (3-deoxy-D-manno-octanoic acid, KDO) , Rhamnose, fucose, arabinose, xylose, mannose, galactose, glucose, galacturonic acid, and glue. May be at least one selected from the group consisting of Glucuronic Acid,

Preferably 0.1-3% of 3-deoxy-D-manno-2-oxulosonic acid (KDO), 3-deoxy-D-leuco-2- (DHA) 0.1-3%, 2-methyl fucose 0.1-5%, 2-methyl xylulose 0.1-5%, apiose 0.1-5%, and acetic acid 0.1-5%. But not limited to, 2.4% by weight of 2-methylfucose, 20.2% by weight of rhamnose, 4.8% by weight of fucose, 100% by weight of fumaric acid, 2.4 wt% of 2-methylxylose, 16.3 wt% of arabinose, 2.6 wt% of apiose, 1.4 wt% of aceric acid, 2.7 wt% of mannose, %, Galactose 7.3 wt.%, Glucose 5.9 wt.%, Galacturonic acid 29.3 wt.%, Glucuronic acid 3.1 wt.%, 3-deoxy- - 3-deoxy-D-manno-octulosonic 0.7% by weight of 3-deoxy-D-lyxo-2-heptulosaric acid (DHA) and 0.7% by weight of 3-deoxy-D-lyxo-2-heptulosaric acid But is not limited to.

The present invention also provides a health functional food comprising the immunologically active polysaccharide prepared according to the method for producing an immunocompetent polysaccharide derived from citrus as an active ingredient.

When the immunopotentiating polysaccharide according to the present invention is used as an active ingredient additive for a health functional food or a general food, the immunopotentiating polysaccharide according to the present invention can be added as it is or can be used together with other food or food ingredients, Can be used. The amount of the active ingredient to be mixed may be suitably determined according to each use purpose such as prevention, health, or treatment.

Generally, the immunostimulatory polysaccharide according to the present invention can be added in an amount of not more than 15 parts by weight, preferably not more than 10 parts by weight, based on the raw material. However, in the case of long-term consumption intended for health or hygiene purposes or for health control purposes, the amount may be less than the above range. Further, since the present invention uses an extract from a natural product, Or more.

There is no particular limitation on the type of the food, and examples of the food to which the above substance can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, , Various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and may include foods in a conventional sense.

The beverage food of the health functional food according to the present invention may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. The above-mentioned natural carbohydrates may be monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. Examples of sweeteners include natural sweeteners such as tau martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like. The ratio of the natural carbohydrate may be about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 mL of the functional food according to the present invention.

The health functional food containing the immunopotentiating polysaccharide according to the present invention as an active ingredient can be used as a nutrient, a vitamin, an electrolyte, a flavoring agent, a coloring agent, a pectic acid and its salt, an alginic acid and its salt, an organic acid, a protective colloid thickener, a pH adjusting agent, a stabilizer, a preservative, a glycerin, an alcohol, and a carbonating agent used in a carbonated beverage. These components may be used independently or in combination. The ratio of such additives is not limited, but is generally selected in the range of 0.01 to 0.1 parts by weight based on 100 parts by weight of the total composition of the present invention.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. The present invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

Example

Manufacturing example  One. cytolase Preparation of Immunostimulatory Polysaccharide

100 g of the dried hara bark peel powder was added to 1000 ml of 0.1 M citrate buffer (pH 5), and then the commercial enzyme cytolase was stirred at 50 ° C for 24 hours at 0.1% (v / w) based on the dry weight of the hyaline peel powder . After completion of the reaction, the supernatant was recovered by filtration, and the recovered supernatant was concentrated to ½. The concentrate was added to the concentrate so as to have a volume of 80% by volume based on 100% by volume of the total volume of the concentrate. The suspension was suspended at 4 ° C for 12 hours to induce precipitation. After precipitation, centrifugation and filtration were performed to remove the supernatant, and the precipitate was recovered and lyophilized to prepare an immunostimulatory polysaccharide (Preparation Example 1). The above procedure was illustrated in FIG.

Manufacturing example  2. Pectinex Preparation of Immunostimulatory Polysaccharide

An immunoconactive polysaccharide (Preparation Example 2) derived from hara bong was prepared by the method of Preparation Example 1, except that pectinex was used as the enzyme.

Comparative Example  One. Hot water treatment  Preparation of Immunologically Active Polysaccharide

100 g of dried hara bark peel powder was added to 1000 ml of distilled water and extracted at 95 ° C for 6 hours. All extracts were filtered to recover the supernatant, and the recovered supernatant was concentrated to give the same amount as in Preparation Example 1. The concentrate was suspended in a distilled water so that the final concentration of the concentrate was 80%, and then allowed to stand at 4 ° C for 12 hours to induce precipitation. After precipitation, centrifugation and filtration were performed to remove the supernatant, and the precipitate was recovered and lyophilized to prepare an immunostimulatory polysaccharide (Comparative Example 1), and the above procedure was illustrated in FIG.

Test Example  One. Hanbalong  Analysis of Components of Immunologically Active Polysaccharide Derived from

Test Example  1.1 General compositional analysis

Analysis of neutral sugar content of polysaccharide samples was carried out by using phenolsulfonic acid method using galactose as a standard substance. The acid content analysis was performed by m-hydroxybiphenyl method using D-galacturonic acid as a standard, and TBA- The content of TBA-positive material was analyzed by thiobarbituric acid method using 2-Keto-3-deoxy-D-manna-octulosonic acid (KDO) Albumin as a reference material, and quantitative analysis was carried out by the Bradford method. The results are shown in Table 1. < tb > < TABLE >

chemical property Production Example 1 (% by weight) Production Example 2 (% by weight) Comparative Example 1 (% by weight)  Neutral sugar 64.4 64.2 45.6  Uronic acid 31.7 32.1 5.4  KDO like materials 1.6 1.5 0.0  Protein 2.3 2.2 0.0

The KDO-like substance and the protein were detected in the immunopotentiating polysaccharide isolated by the enzymatic treatment according to Preparation Examples 1 and 2, but the KDO-like substance and the protein were not detected in the immunopotentiating polysaccharide isolated by the hot water extraction according to Comparative Example 1 .

Test Example  1.2. Per constitution  analysis

The constitutional analysis was carried out by gas chromatography using hydrolysis and alditol acetate derivatization of sugar components using the method of Albersheim. The mol% of each component was calculated by calculating the molecular sedaction factor for the peak area of each derivative, and the results are shown in Table 2 below.

component sugar Production Example 1 (mole%) Preparation Example 2 (mole%) Comparative Example 1 (mole%) 2-methylfucose 2.4 2.1 0.0 Rhamnose 20.2 20.9 2.1 fucose 4.8 4.3 0.4 2-methylxylose 2.4 2.5 0.0 arabinose 16.3 16.2 26.0 xylose 0.0 0.0 1.4 apiose 2.6 1.8 0.0 acic acid 1.4 1.7 0.0 mannose 2.7 2.8 1.8 가카성 7.3 8.1 10.4 glucose 5.9 5.4 3.5 가alatongonic acid 29.3 31.1 53.7 glucuronic acid 3.1 2.6 0.7 DHA 0.7 0.7 0.0 KDO 0.9 0.8 0.0

The immunologically active polysaccharide isolated by the enzymatic treatment according to Preparation Examples 1 and 2 contains a specific polysaccharide such as 2-methyl fucose, 2-methyl xylose, apiose, acetic acid, KDO and DHA rarely observed in nature The polysaccharides separated by hydrothermal method according to Comparative Example 1 accounted for about 80% of the total amount of arabinose and galacturonic acid, and the polysaccharide diversity tended to be significantly lowered.

The immune-active polysaccharide was analyzed using gas chromatography (GC), which is shown in FIG.

Test Example  2. Hanbalong  Of the resulting immunologically active polysaccharide Anti-complement activity  evaluation

The complement fixation test method based on the degree of erythrocyte hemolysis due to the residual complement after complement consumption by the Mayer method was used to measure the complement activity. As a positive control, a commercially available immunopotentiating polysaccharide PSK (polysaccharide-K) was used, which is shown in FIG.

At the sample concentration of 100 / / ml, the complement activity (ITCH ₅₀ ) of Production Example 1 was 33.7%, and the complement activity of Production Example 2 was 33.6%, which was similar to the complement activity of the control PSK of 36.3% , And the complement activity of Comparative Example 1 was 8.1%, indicating that the activity was inferior. In addition, at the concentration of 1,000 占 퐂 / ml, the complement activity of Preparation Example 1 was 62.3%, and the complement activity of Preparation Example 2 was 62.1%, which was similar to the anti-complement activity of the control PSK of 60.6% The anti-complement activity was about 30.2%, which was relatively low, and was half of that of Preparation Examples 1 and 2 and the control group.

Generally, it is known that polysaccharides exhibiting an anti-complement activity of 60% or more at a concentration of 1,000 μg / ml are generally recognized to have pharmacological properties. Production Examples 1 and 2 obtained by enzyme treatment according to the present invention have excellent complement system activating ability can do. In addition, as a result of confirming the activation ability by concentration, it was confirmed that the complement system activity of the activated oligosaccharide-derived polysaccharide sample by enzyme treatment was concentration-dependent, as shown in Table 3 below.

Positive control (%) Production Example 1 (%) Production Example 2 (%) Comparative Example 1 (%)
density
100 [mu] g / ml 36.3 33.7 33.6 8.1 500 / / ml 56.6 53.9 53.7 21.0 1000 / / ml 60.0 62.3 62.1 30.2

Test Example  3. Hanbalong  Derived immunosuppressive polysaccharide of  Cytotoxicity Assessment

Cytotoxicity of the active polysaccharide samples on tumor cells and normal cells was carried out using macrophages recovered from the peritoneal cavity of mice and Colon 26-M3.1 tumor-derived tumor cell line, which are shown in Tables 4, 5 and 4 .

Macrophages Production Example 1 Production Example 2 Comparative Example Concentration (/ / ml) Average Standard Deviation Average Standard Deviation Average Standard Deviation 1000 1.152 0.167 1.215 0.112 1.104 0.001 200 1.021 0.094 1.125 0.037 1.113 0.007 40 1.030 0.062 1.115 0.057 1.087 0.014 8 0.993 0.074 1.135 0.012 1.079 0.012 1.6 0.981 0.013 0.987 0.123 1.143 0.000

Colon 26 M3.1 Production Example 1 Production Example 2 Comparative Example Concentration (/ / ml) Average Standard Deviation Average Standard Deviation Average Standard Deviation 1000 0.338 0.026 0.313 0.014 0.348 0.006 200 0.364 0.005 0.337 0.002 0.353 0.007 40 0.353 0.003 0.345 0.007 0.365 0.010 8 0.351 0.002 0.368 0.005 0.378 0.004 1.6 0.352 0.006 0.331 0.012 0.380 0.008

Cytotoxicity Test Results Using Peritoneal Macrophage of BALB / c Mice and Colon 26-M3.1 Tumor Cell Lines Both preparation examples 1 and 2 and comparative example 1 showed significant cytotoxicity up to a concentration of 1.6 ug / ml to 1000 ug / ml .

Test Example  4. Hanbalong  Derived immunosuppressive polysaccharide of  Assessment of cytokine production capacity

In general, IL-1, IL-6 and TNF-alpha are representative cytokines induced by macrophages, which play a pivotal role in the inflammatory response caused by bacterial infection and are known to be increased in inflammatory lesions. In addition, IL-12 is a cytokine that induces NK cell activation and Th1-type immune response, and it is known that IL-12 enhances the reactivity to extracellular substances such as cancer cells.

Immunostimulatory polysaccharides prepared according to Preparation Examples 1 and 2 and Comparative Example 1 of the present invention were tested for their ability to inhibit cytokine production by using sandwich ELISA using macrophages obtained from abdominal cavity of BALB / c (female, 6 weeks old) (cytokine production stimulation), which are shown in Tables 5, 6 and 5 below. Negative control was measured without any addition, and lipopolysaccharide (LPS) was added as a comparative group to measure cytokine production stimulation. In the case of the immunostimulatory polysaccharide of Comparative Example 1, it was confirmed that the stimulating ability of interleukin-6 (IL-6) and interleukin-12 (IL-12) cytokine production was remarkably low, whereas the immunostimulatory polysaccharides of Preparation Examples 1 and 2 IL-6 and IL-12, and that the production stimulation effect was also dose-dependent.

IL-6 (pg / ml) Production Example 1 Production Example 2 Comparative Example 1 Control group Comparative group density
(占 퐂 / ml) Average Standard
Deviation Average Standard
Deviation Average Standard
Deviation Average Standard
Deviation Average Standard
Deviation 1000 710.00 23.57 708.23 21.47 365.00 49.50 116.67 23.33 873.33 36.67 200 626.67 9.43 615.14 14.53 135.00 58.93 - - - - 40 558.33 2.36 542.34 7.64 63.33 28.28 - - - - 8 378.33 11.79 366.89 10.92 55.00 49.50 - - - - 1.6 286.67 28.28 281.28 28.49 46.67 51.85 - - - -

IL-6 (pg / ml) Production Example 1 Production Example 2 Comparative Example 1 Control group Comparative group density
(占 퐂 / ml) Average Standard
Deviation Average Standard
Deviation Average Standard
Deviation Average Standard
Deviation Average Standard
Deviation 1000 655.00 21.21 657.08 32.50 323.33 14.14 116.67 23.33 873.33 36.67 200 561.67 11.79 555.27 21.59 210.00 18.86 - - - - 40 436.67 0.00 432.56 11.24 130.00 9.43 - - - - 8 320.00 47.14 316.21 35.43 76.67 23.57 - - - - 1.6 225.00 2.36 221.46 3.85 6.67 0.00 - - - -

As can be seen from the above results, the immunopotentiating polysaccharide according to the present invention is useful for the activation of IL-6 cytokine and NK cell and the induction of Th1 type immune response, which have an effective activity on inflammatory immunity and cellular extracellular substances such as cancer cells 12 cytokines that have activity in cell-mediated immunity, such as the activation of cytotoxic T lymphocytes (CTLs).

In particular, IL-12 is known to be a cytokine essential for the induction of anticancer activity by directly participating in the activation of NK cells which are lethal to cancer cells in the presence of cancer cells. The immunopotentiating polysaccharide prepared according to the preparation method of the present invention is also effective for cancer cells Can be applied.

Hereinafter, formulation examples of the composition containing the extract of the present invention will be described, but the present invention is not intended to be limited thereto but is specifically described.

Formulation example  One. Sanje  Produce

20 mg of the immunostimulatory polysaccharide powder of Preparation Example 1

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

10 mg of the immunostimulatory polysaccharide powder of Preparation Example 1

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

10 mg of the immunostimulatory polysaccharide powder of Preparation Example 1

Crystalline cellulose 3 mg

Lactose 14.8 mg

Magnesium stearate 0.2 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 injections

10 mg of the immunostimulatory polysaccharide powder of Preparation Example 1

180 mg mannitol

Sterile sterilized water for injection 2974 mg

Na ₂ HPO ₄ , 12H ₂ O 26 mg

It is prepared by the above-mentioned component content per ampoule according to the usual injection preparation method.

Formulation example  5. Liquid  Produce

20 mg of the immunostimulatory polysaccharide powder of Preparation Example 1

10 g per isomer

5 g mannitol

Purified water quantity

Each component was added to purified water in accordance with the conventional 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 added with purified water and adjusted to 100 as a whole. To prepare a liquid agent.

Formulation example  6. Manufacture of health functional foods

1,000 mg of the immunostimulatory polysaccharide powder of Preparation Example 1

Vitamin mixture quantity

70 [mu] g of vitamin A acetate

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

0.15 mg of vitamin B2

Vitamin B6 0.5 mg

0.2 [mu] g vitamin B12

Vitamin C 10 mg

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

Potassium monophosphate 15 mg

Secondary calcium phosphate 55 mg

Potassium citrate 90 mg

Calcium carbonate 100 mg

Magnesium chloride 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixture is comparatively mixed with a component suitable for a health functional food as a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above components may be mixed , Granules may be prepared and used in the manufacture of a health functional food composition according to a conventional method.

Formulation example  7. Manufacture of functional beverages

1,000 mg of the immunostimulatory polysaccharide powder of Preparation Example 1

Citric acid 1,000 mg

100 g of oligosaccharide

Plum concentrate 2 g

Taurine 1 g

Purified water was added to the flask to obtain 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 solution thus prepared was filtered and sterilized in a sterilized 2 L container, It is used in the production of the functional beverage composition of the invention.

Although the composition ratio is a mixture of the components suitable for the preferred beverage as a preferred embodiment, the blending ratio may be arbitrarily varied according to the regional and national preferences such as the demand level, the demanding country, and the intended use.

Claims (14)

Enzymatic treatment of citrus processing by-products in citrus; And adding a 30-90 vol% ethanol aqueous solution to the enzyme-treated citrus processing by-product to obtain a precipitate. The method according to claim 1, wherein the enzyme is one or more mixed enzymes selected from the group consisting of pectinase, cellulase, xylanase, and mananase ≪ RTI ID = 0.0 > 1, < / RTI > The method according to claim 1, wherein the enzymatic treatment is carried out using enzymes such as Econitase, Rapidase, Viscozyme, Celluclast, Pectinex, Rohament, , Ultraflo, and Cytolase. The method of producing a citrus-derived immunologically active polysaccharide of the present invention is characterized in that the method is carried out by using one or more selected from the group consisting of ultrafiltration, ultraflo, and cytolase. [Claim 2] The method according to claim 1, wherein the enzyme treatment is performed by adding 0.05-5 parts by weight of enzyme to 100 parts by weight of the solids of the citrus processing by-product and treating the mixture for 6-96 hours. [Claim 3] The method according to claim 1, wherein the citrus fruits are at least one selected from the group consisting of citrus fruits, hala bongs (bushing), chrysanthemum bushes, blue crabs, kumquat and citron. An immunologically active polysaccharide prepared according to any one of claims 1 to 5. [Claim 7] The immunostimulatory polysaccharide according to claim 6, wherein the immune-active polysaccharide has an anti-complement activity at a concentration of 1,000 [mu] g / ml of 60% or more. The method according to claim 6, wherein the immunostimulatory polysaccharide has a production stimulating ability of Interleukin-6 (IL-6) of 600-800 pg / mL at a concentration of 1,000 μg / ml, Lt; RTI ID = 0.0 > pg / mL. ≪ / RTI > 7. The immunologically active polysaccharide according to claim 6, wherein the immunogically active polysaccharide has a molecular weight of 5-300 Kda. 7. The composition of claim 6, wherein the immunostimulatory polysaccharide is selected from the group consisting of 2-methylfucose, 2-methylxylose, apiose, aceric acid, 3-deoxy-D-manno-octolosonic acid (KDO), 3-deoxy-D-lyxo- 2-heptulosaric acid, DHA, rhamnose, fucose, arabinose, xylose, mannose, galactose, glucose, galacturonic acid (Galacturonic Acid) and glucuronic acid (Glucuronic Acid). 7. The method of claim 6, wherein 0.1 to 3% by weight of 3-deoxy-D-manno-2-oxulosonic acid (KDO) is added to 100% 0.1 to 3 wt% of heptulosaric acid (DHA), 0.1 to 5 wt% of 2-methyl fucose, 0.1 to 5 wt% of 2-methyl xylose, 0.1 to 5 wt% of apiose, % ≪ / RTI > of the immunostimulatory polysaccharide. 7. The composition of claim 6, wherein the immunopotentiating polysaccharide comprises 2.4% by weight of 2-methylfucose, 20.2% by weight of rhamnose, 4.8% by weight of fucose, 2- 2.4% by weight of 2-methylxylose, 16.3% by weight of arabinose, 2.6% by weight of apiose, 1.4% by weight of aceric acid, 2.7% by weight of mannose, 7.3% by weight of galactose, 5.9% by weight of glucose, 29.3% by weight of Galacturonic Acid, 3.1% by weight of Glucuronic Acid, 3-deoxy-D- 0.7 weight% of 3-deoxy-D-manno-octulosonic acid (KDO) and 3-deoxy-D-lyxo-2-heptulosaric acid (DHA) 0.9% < / RTI > by weight of the immunostimulatory polysaccharide. A health functional food comprising an immunologically active polysaccharide prepared according to any one of claims 1 to 5 as an active ingredient. 14. The health functional food according to claim 13, wherein said immunologically active polysaccharide enhances anti-complement activity, production of interleukin-6 (IL-6) and production of interleukin-12 (IL-12).

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