KR20190064315A - Anticancer or immunactive polysaccharide from residues of immature citrus extract or immature citrus peel and composition comprising the same as an active ingredient - Google Patents

Abstract

The present invention relates to a crude polysaccharide manufactured by hydrothermal extraction of Citrus nippokoreana extract residue or Citrus nippokoreana peels. Specifically, the present invention relates to anticancer or immune-enhancing polysaccharide, in which a constituent sugar constituting the crude polysaccharide comprises, based on 100 parts by mol of rhamnose, 650 to 700 parts by mol of arabinose, 300 to 360 parts by mol of galactose, and 2000 to 2200 parts by mol of galacturonic acid. Therefore, it is possible to enhance anticancer activity or immunological activity by increasing a content of functional ingredients such as galacturonic acid, compared with an existing citrus extract.

Description

TECHNICAL FIELD [0001] The present invention relates to an anticancer or immunosuppressive polysaccharide derived from a citrus fruit extract or a cheongyub, and a composition comprising the same as an active ingredient. [0002] The present invention relates to an anticancer or immunosuppressive polysaccharide

The present invention relates to an anticancer or immunosuppressive polysaccharide derived from a citrus fruit extract or a cheongyub, and a composition comprising the same as an active ingredient. The present invention relates to a polysaccharide having immunity and anticancer activity remarkably improved compared with ordinary citrus fruits, ≪ / RTI >

The first cause of death in Koreans is cancer, which traditionally uses surgery, radiation therapy, and drug therapy to overcome cancer, but it is still difficult to expect a complete treatment effect 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, persimmon leaves, and the like. 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 into various oligos 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.

On the other hand, citrus fruits such as mandarin orange and hara bong are fruits which contain health functional ingredients such as immunological activity and anti-cancer function, and are widely cultivated in southern part of Korea and Jeju Island and widely used for manufacturing drinks and health functional foods.

Korean Patent Registration No. 10-1530982

The object of the present invention is to provide a polysaccharide derived from a citrus extract or a cheongyang which has significantly improved contents of galacturonic acid, rhamnose, galactose and arabinose compared with polysaccharides extracted from citrus fruits, have.

Another object of the present invention is to provide a pharmaceutical composition or a food composition comprising as an active ingredient a polysaccharide derived from a citrus fruit extract or a cheongyang which has remarkably improved immunological activity and anticancer function.

According to an aspect of the present invention,

A blueberry extract powder or a blueberry extract; and the constituent sugar constituting the crude polysaccharide is 650 to 700 molar parts of arabinose, 100 to 650 molar parts of rhamnose, 300 to 360 parts by mole of galactose; And 2200 parts by weight of galacturonic acid 2000 antimicrobial agent.

Preferably, the constituent sugar constituting the crude polysaccharide is 5 to 10 molar parts of fucose with respect to 100 molar parts of the laminose; 90 to 110 molar parts of xylose; 100 to 130 moles of mannose; 200 to 230 moles of glucose; And 80 to 100 parts by mole of glucuronic acid.

According to another aspect of the present invention,

Wherein the constituent sugar constituting the crude polysaccharide is selected from the group consisting of 85 to 95 molar parts of arabinose to 100 molar parts of rhamnose; 65 to 75 molar parts of galactose; And 200 to 250 parts by mole of galacturonic acid.

Preferably,

The constituent sugar that constitutes the crude polysaccharide is 2 to 5 parts by weight of 2-methylfucose to 100 parts by mol of the above-mentioned rhamnose; 6 to 10 molar parts of fucose; 6 to 10 molar parts of xylose; 0.5 to 3 molar parts of apiose; 0.2 to 1 molar parts of aceric acid; Mannose 2 to 5 molar parts; 20 to 30 parts by mole of glucose; And 15 to 25 parts by mole of glucuronic acid.

According to another aspect of the present invention,

A purified polysaccharide having a molecular weight of 50 to 70 kD obtained by separating crude polysaccharide prepared by enzymatic treatment of citrus fruit extract or cheongyang by gel permeation chromatography, and the constituent sugar constituting the purified polysaccharide is laminose 35 to 45 molar parts of arabinose, relative to 100 molar parts of rhamnose; 90 to 100 molar parts of galactose; And 200 to 220 molar parts of galacturonic acid.

Preferably, the constituent sugar constituting the purified polysaccharide is 5 to 10 molar parts of fucose to 100 molar parts of the rhamnose; Xylose 14 to 19 molar parts; Mannose 2 to 5 molar parts; 2 to 6 molar parts of glucose; And 3 to 7 molar parts of glucuronic acid.

According to another aspect of the present invention,

A purified polysaccharide having a molecular weight of 10 to 20 kD obtained by separating crude polysaccharide prepared by enzymatic treatment of citrus fruit extract or cheongpy peel by gel permeation chromatography, and the constituent sugar constituting the purified polysaccharide is laminose 72 to 82 molar parts of arabinose, relative to 100 molar parts of rhamnose; 50 to 60 molar parts of galactose; And galacturonic acid in an amount of 350 to 400 parts by mol.

Preferably, the constituent sugar constituting the purified polysaccharide is 15 to 25 molar parts of 2-methylfucose to 100 molar parts of the rhamnose; 15 to 25 molar parts of fucose; 7 to 13 molar parts of 2-methylxylose; Xylose 15 to 22 molar parts; 10 to 15 molar parts of aceric acid; 0.5 to 3 moles of mannose; 2 to 6 molar parts of glucose; And 20 to 30 parts by mole of glucuronic acid.

According to another aspect of the present invention,

A purified polysaccharide having a molecular weight of 0.1 to 5 kD obtained by separating crude polysaccharide prepared by enzymatic treatment of citrus fruit extract or cheongyang by gel permeation chromatography, wherein the constituent sugar constituting the purified polysaccharide is laminose 5 to 10 molar parts of arabinose, relative to 100 molar parts of rhamnose; 10 to 16 molar parts of galactose; And 120 to 160 parts by mole of galacturonic acid.

Preferably, the constituent sugar constituting the purified polysaccharide is 0.2 to 1 part by mole of fucose, Xylose 3 to 8 molar parts; Mannose 1-4 moles; 12 to 17 molar parts of glucose; And 5 to 10 molar parts of glucuronic acid.

According to another aspect of the present invention,

An anticancer or immunostimulatory pharmaceutical composition comprising the polysaccharide as an active ingredient is provided.

According to another aspect of the present invention,

An anticancer or immunostimulatory food composition comprising the polysaccharide as an active ingredient is provided.

The polysaccharide derived from the citrus peel extract or the citrus fruit of the present invention and the pharmaceutical composition or the food composition comprising the citrus extract of the present invention have a significantly increased content of galacturonic acid, rhamnose, galactose and arabinose compared with polysaccharides derived from citrus fruits, .

Fig. 1 is an elution curve of fractionated polysaccharide prepared according to Example 4. Fig.
Fig. 2 shows the results of HPLC analysis according to Test Example 1. Fig.
Fig. 3 shows the results on the cytotoxicity and cytokine production on macrophages according to Test Example 2. Fig.
Fig. 4 shows the results on the cytotoxicity and cytokine production on macrophages according to Test Example 3. Fig.
FIG. 5 shows the results of NK cell stimulation activity of the enzyme-treated purified fraction CCE-I derived from citrus peel extract-like peptide according to Test Example 4. FIG.
6 shows the result of the antitumor activity test by intravenous administration of the purified polysaccharide CCE-I derived from citrus fruit extract extract according to Test Example 5. FIG.
FIGS. 7 to 9 show the results of the antitumor activity test of the enzyme-treated polysaccharide fractions derived from citrus peel extracts in a mouse in which NK cell function was controlled according to Test Example 6. FIG.
10 shows the results of analysis of NK cell activity of natural killer cells by oral administration of crude polysaccharide CCE-0 derived from citrus peel extracts according to Test Example 7.
Fig. 11 shows the results of the analysis of antioxidant activity by oral administration of an enzyme-treated crude polysaccharide CCE-0 derived from a citrus peel-derived foil according to Test Example 8. Fig.
12 is a GC-MS analysis result for estimating the total structure of CCE-I of Test Example 10. Fig.
13 is a schematic diagram of CCE-I.

BRIEF DESCRIPTION OF THE DRAWINGS The present invention is capable of various modifications and various embodiments, and specific embodiments are illustrated in the drawings and described in detail in the detailed description. It is to be understood, however, that the invention is not to be limited to the specific embodiments, but includes all modifications, equivalents, and alternatives falling within the spirit and scope of the invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described in detail with reference to the accompanying drawings.

Hereinafter, anti-cancer or immunostimulatory polysaccharides according to the first embodiment of the present invention will be described.

The anticancer or immunostimulatory polysaccharide of the present invention is a crude polysaccharide prepared by extracting citrus fruit or cheongyang by hydrothermal extraction. The constituent sugar constituting the crude polysaccharide is arabinose (100 moles) per 100 moles of rhamnose, 650 to 700 molar parts; 300 to 360 parts by mole of galactose; And 2200 parts by mol of galacturonic acid 2000.

Preferably, the constituent sugar constituting the crude polysaccharide is 5 to 10 molar parts of fucose with respect to 100 molar parts of the laminose; 90 to 110 molar parts of xylose; 100 to 130 moles of mannose; 200 to 230 moles of glucose; And 80 to 100 parts by mole of glucuronic acid.

The crude polysaccharide prepared by the hot water extraction can be prepared according to the following method.

Hereinafter, a method for producing anticancer or immunostimulatory polysaccharide according to one embodiment of the present invention will be described.

First, the dried powder of citrus fruit extract or chewing gum is extracted with hot water to prepare a hot water extract (step a).

It is preferable to prepare a suspension by mixing with water having a weight of 10 to 30 times the dry powder before the hot water extraction.

The hot water extraction is preferably carried out by hot water at 80 to 120 ° C, more preferably at 90 to 110 ° C.

Thereafter, the residue is removed from the hot-water extract, and an aqueous alcohol solution is added to precipitate the polysaccharide, thereby recovering the precipitate (step b).

At this time, the residue removal can be performed by centrifuging at 5000 to 700 rpm for 10 to 30 minutes.

The alcohol aqueous solution preferably has a volume ratio (v / v) of distilled water to alcohol of 1:10 to 1:30, more preferably 1:15 to 1:25, still more preferably 1:17 To 1:22.

The precipitate recovery can also be performed by centrifuging at 500 to 700 rpm for 20 to 40 minutes.

Finally, after the precipitate is dissolved in distilled water, low molecular impurities are removed by dialysis to obtain a hydrothermally extracted polysaccharide of citrus peel or chewing gum (step c).

The dialysis is preferably performed for 40 to 60 hours, more preferably 45 to 50 hours.

The hot-water-extracting polysaccharide may further be freeze-dried.

Hereinafter, the anticancer or immunostimulatory polysaccharide according to the second embodiment of the present invention will be described.

The anticancer or immunostimulatory polysaccharide of the present invention is a crude polysaccharide produced by the enzymatic treatment of the citrus extract or chewing gum, and the constituent sugar constituting the crude polysaccharide is arabinose (100 g / 100 g) per 100 moles of rhamnose ) 85 to 95 molar parts; 65 to 75 molar parts of galactose; And 200 to 250 parts by mole of galacturonic acid.

Preferably, the constituent sugar constituting the crude polysaccharide is 2 to 5 parts by weight of 2-methylfucose relative to 100 parts by mol of the rhamnose; 6 to 10 molar parts of fucose; 6 to 10 molar parts of xylose; 0.5 to 3 molar parts of apiose; 0.2 to 1 molar parts of aceric acid; Mannose 2 to 5 molar parts; 20 to 30 parts by mole of glucose; And 15 to 25 parts by mole of glucuronic acid.

The crude polysaccharide prepared by the above enzyme treatment can be produced according to the following method.

First, the dried powder of citrus peel or cheongpy peel is treated with an enzyme to prepare an enzyme-treated product (Step 1).

It is preferable to prepare a suspension by mixing with water having a weight of 10 to 30 times the dry powder before the enzyme treatment.

The enzyme treatment may be carried out by at least one enzyme selected from pectinase, cellulase, xylanase and mananase.

The enzymatic treatment may be carried out in the following order: Pectinex, Econitase, Rapidase, Viscozyme, Celluclast, Rohament, Ultraflo, (Cytolase). ≪ / RTI >

Preferably, the enzymatic treatment is carried out for 4 to 8 hours by adding 0.3 to 1.5 parts by weight of enzyme to 100 parts by weight of the dried powder of the citrus peel or chewing gum.

Thereafter, the residue is removed from the enzyme-treated product, and the precipitate is recovered by precipitating polysaccharide by adding an alcohol solvent (step 2).

At this time, the residue removal can be performed by centrifuging at 5000 to 700 rpm for 10 to 30 minutes.

The alcohol aqueous solution preferably has a volume ratio (v / v) of distilled water to alcohol of 1:10 to 1:30, more preferably 1:15 to 1:25, still more preferably 1:17 To 1:22.

The precipitate recovery can also be performed by centrifuging at 500 to 700 rpm for 20 to 40 minutes.

Next, the precipitate is dissolved in distilled water, and then low molecular impurities are removed by dialysis to obtain an enzyme-treated polysaccharide of a citrus peel or chewy gum (step 3).

The dialysis is preferably performed for 40 to 60 hours, more preferably 45 to 50 hours.

The enzyme-treated polysaccharide may further be dried.

The drying may be carried out by a method such as vacuum distillation, freeze drying or spray drying.

Hereinafter, the anti-cancer or immunostimulatory polysaccharide prepared according to the third embodiment of the present invention will be described.

The anti-cancer or immunostimulatory polysaccharide of the present invention is a purified polysaccharide having a molecular weight of 50 to 70 kD obtained by separating crude polysaccharide prepared by enzymatic treatment of citrus fruit extract or cheongapi by gel permeation chromatography, Is composed of 35 to 45 molar parts of arabinose, 100 molar parts of rhamnose, 90 to 100 molar parts of galactose; And 200 to 220 molar parts of galacturonic acid. Preferably, the constituent sugar constituting the crude polysaccharide comprises 5 to 10 molar parts of fucose to 100 molar parts of the laminose; Xylose 14 to 19 molar parts; Mannose 2 to 5 molar parts; 2 to 6 molar parts of glucose; And 3 to 7 molar parts of glucuronic acid.

Hereinafter, the anticancer or immunostimulatory polysaccharide produced according to the fourth embodiment of the present invention will be described.

The anticancer or immunostimulatory polysaccharide of the present invention is a purified polysaccharide having a molecular weight of 10 to 20 kD obtained by separating crude polysaccharide prepared by enzymatic treatment of an extract of mandarin orange extract or cheongapi by gel permeation chromatography, The constituent sugar constituting the polysaccharide is 72 to 82 molar parts of arabinose, 100 molar parts of rhamnose, 50 to 60 molar parts of galactose; And galacturonic acid in an amount of 350 to 400 parts by mol.

Preferably, the constituent sugar constituting the purified polysaccharide is 15 to 25 molar parts of 2-methylfucose to 100 molar parts of the rhamnose; 15 to 25 molar parts of fucose; 7 to 13 molar parts of 2-methylxylose; Xylose 15 to 22 molar parts; 10 to 15 molar parts of aceric acid; 0.5 to 3 moles of mannose; 2 to 6 molar parts of glucose; And 20 to 30 parts by mole of glucuronic acid.

Hereinafter, the anticancer or immunostimulatory polysaccharide produced according to the fifth embodiment of the present invention will be described.

The anticancer or immunostimulatory polysaccharide of the present invention is a purified polysaccharide having a molecular weight of 0.1 to 5 kD obtained by separating crude polysaccharide prepared by enzymatic treatment of citrus extract or chewing gum by gel permeation chromatography, Comprises 5 to 10 molar parts of arabinose per 100 molar parts of rhamnose; 10 to 16 molar parts of galactose; And 120 to 160 parts by mole of galacturonic acid.

Preferably, the constituent sugar constituting the purified polysaccharide is 0.2 to 1 part by mole of fucose, Xylose 3 to 8 molar parts; Mannose 1-4 moles; 12 to 17 molar parts of glucose; And 5 to 10 molar parts of glucuronic acid.

The purified polysaccharide separated according to the gel permeation chromatography according to the third to fourth embodiments can be produced according to the following method.

The crude polysaccharide produced in step (3) of the sugar treatment method can be further separated by gel permeation chromatography according to molecular weight to obtain purified polysaccharide (step 4 ').

Preferably, the purified polysaccharide obtained in step (4 ') is sequentially subjected to concentration, dialysis and drying processes.

The drying may be carried out by a method such as vacuum distillation, freeze drying or spray drying.

Accordingly, the purified tablet polysaccharide has a high molecular weight purified polysaccharide of 50 to 70 kDa (third embodiment), a medium molecular weight purified polysaccharide of 10 to 20 kDa (fourth embodiment), and a low molecular weight purified polysaccharide of 0.1 to 5 kDa Fifth embodiment). ≪ / RTI >

The polysaccharide derived from the citrus peel extract or the cheongyang of the present invention is different in composition from polysaccharides derived from citrus fruits such as hara bong, mandarin orange and the like, so that the anti-cancer and immune activity can be more useful. Specifically, pectin has a homogalacturonan covalently bound to rhamnogalacturonan (RG) -I and RG-II, and a part involved in the physiological activity of pectin is RG-I and RG- -II. Polysaccharides, which mainly contribute to the immunological activity and anticancer activity isolated from hyalabroma, are not well observed in nature, and 2-methyl fucose, 2-methyl xylose, apiose, 14 types of monosaccharides and 23 types of sugar chains, including dioxy-D-manno-2-octulosonic acid (KDO) and 3-deoxy-D-leuco-2-heptulosaric acid And it was confirmed that it is a typical structure of RG-II. On the contrary, in the case of citrus fruit extract, physiological activity was exhibited in the pectin polysaccharide similar to that of the hirabong skin, and it was confirmed that the RG-I structure rather than RG-II contributed to the activity. Immunologically active polysaccharides derived from citrus peel extracts were analyzed by constitutional sugar analysis to determine the structure of RG-I having a high content of arabinose, galactose, rhamnose and galacturonic acid and a relatively branched branch with a sugar chain binding assay .

The present invention provides anti-cancer or immunostimulatory polysaccharides prepared according to the above method.

The present invention also provides an anticancer or immunologically active pharmaceutical composition comprising the anti-cancer or immunosuppressive polysaccharide as an active ingredient.

In addition, the present invention provides an anti-cancer or immunostimulatory food composition comprising the anti-cancer or immunosuppressive polysaccharide as an active ingredient.

In the present specification, the term " comprising as an active ingredient " is meant to include an amount sufficient to achieve the efficacy or activity of a citrus extract powder or a chewing gum polysaccharide. In one embodiment of the invention, the polysaccharide of citrus fruit extract or chewing gum in the composition of the present invention has a concentration of 0.001 mg / kg or more, preferably 0.1 mg / kg or more, more preferably 10 mg / kg or more More preferably at least 100 mg / kg, even more preferably at least 250 mg / kg, most preferably at least 0.1 g / kg. The amount of the polysaccharide extracted from citrus peel or cheongpy peel is a natural product and there is no adverse effect on the human body even when administered in an excessive amount. Therefore, the quantitative upper limit of the polysaccharide contained in the composition of the present invention can be selected by a person skilled in the art within a suitable range.

The pharmaceutical composition of the present invention may be prepared by using pharmaceutically acceptable and physiologically acceptable adjuvants in addition to the above-mentioned active ingredients. Examples of the adjuvants include excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants, Or a flavoring agent may be used.

The pharmaceutical composition may be formulated into a pharmaceutical composition containing at least one pharmaceutically acceptable carrier in addition to the above-described active ingredients for administration.

The pharmaceutical form of the pharmaceutical composition may be a granule, a powder, a tablet, a coated tablet, a capsule, a suppository, a liquid, a syrup, a juice, a suspension, an emulsion, a drip agent or an injectable liquid agent. For example, for formulation into tablets or capsules, the active ingredient may be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. Also, if desired or necessary, suitable binders, lubricants, disintegrants and coloring agents may also be included as a mixture. Suitable binders include, but are not limited to, natural sugars such as starch, gelatin, glucose or beta-lactose, natural and synthetic gums such as corn sweeteners, acacia, tracker candles or sodium oleate, sodium stearate, magnesium stearate, sodium Benzoate, sodium acetate, sodium chloride, and the like. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum and the like.

Acceptable pharmaceutical carriers for compositions that are formulated into a liquid solution include sterile water and sterile water suitable for the living body such as saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, And other conventional additives such as an antioxidant, a buffer, and a bacteriostatic agent may be added as needed. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.

The pharmaceutical composition of the present invention can be administered orally or parenterally. In the case of parenteral administration, it can be administered by intravenous injection, subcutaneous injection, muscle injection, intraperitoneal injection, transdermal administration, etc., preferably oral administration.

The appropriate dosage of the pharmaceutical composition of the present invention varies depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, route of administration, excretion rate and responsiveness of the patient, , A skilled physician can readily determine and prescribe dosages that are effective for the desired treatment or prophylaxis. According to a preferred embodiment of the present invention, the daily dosage of the pharmaceutical composition of the present invention is 0.001-10 g / kg.

The pharmaceutical composition of the present invention may be prepared in a unit dose form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs Into a multi-dose container. The formulations may be in the form of solutions, suspensions or emulsions in oils or aqueous media, or in the form of excipients, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

Further, the present invention provides a food composition comprising as an active ingredient polysaccharide extracted from citrus fruit extract or cheongju. The description of the polysaccharide of citrus peel extract or cheongyang is the same as that described in the above-mentioned pharmaceutical composition, and therefore, the details will be referred to above.

The food composition according to the present invention can be used as a functional food or added to various foods. Foods to which the composition of the present invention can be added include, for example, beverages, alcoholic beverages, confectionery, diet bars, dairy products, meats, chocolates, pizza, breads, noodles, gums, ice creams, And food.

The food composition of the present invention may contain, as an active ingredient, not only a citrus extract powder or a polysaccharide of a cheongapi, but also a component which is ordinarily added during the manufacture of a food, for example, a protein, a carbohydrate, a fat, a nutrient, . Examples of the above-mentioned carbohydrates are monosaccharides such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose, oligosaccharides and the like; And polysaccharides such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. Natural flavorings such as tau martin and stevia extract (e.g., rebaudioside A and glycyrrhizin) and synthetic flavorings (saccharine, aspartame, etc.) can be used as flavorings. For example, when the food composition of the present invention is prepared from a drink and a beverage, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, juice, various plant extracts, Can be included.

The present invention provides a health functional food comprising a food composition comprising as an active ingredient polysaccharide extracted from citrus fruit extract or citrus fruit. The health functional food refers to a food prepared by adding a citrus extract powder or a polysaccharide of CheongPyu to a food material such as beverage, tea, spice, gum or confection, or encapsulated, powdered or suspended, But it has the advantage that there is no side effects that can occur when a drug is taken for a long time by using food as a raw material. The health functional food of the present invention thus obtained is very useful because it can be ingested routinely. The added amount of the citrus extract powder or chewing gum polysaccharide in such a health functional food can not be uniformly determined depending on the type of the health functional food to be added but may be added within a range that does not impair the original taste of the food, Is usually in the range of 0.01 to 50% by weight, preferably 0.1 to 20% by weight, based on the food. In the case of health functional foods in the form of pills, granules, tablets or capsules, they may be added usually in the range of 0.1 to 100% by weight, preferably 0.5 to 80% by weight. In one embodiment, the health functional food of the present invention may be in the form of a pill, tablet, capsule or beverage.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the present invention. Such variations and modifications are intended to be within the scope of the appended claims.

[Example]

Example  1: orange juice Extract foil Hot water extraction Group ( CCW -0) Manufacturing

100 g of dried citrus fruit extract powder was suspended in 2 L of DIW and heat-treated at 100 ° C to conduct hot water extraction until the volume became half of the initial volume. The extract was centrifuged at 6,000 rpm for 20 minutes to remove the residues, and multiparticulates were precipitated by adding thereto 4 times volume (v / v) of 95% ethanol and stirring for 24 hours. The resulting precipitate was centrifuged at 6,000 rpm for 30 minutes The precipitate was recovered by separation. The recovered precipitate was dissolved in distilled water, dialyzed for 2 days using dialysis tubing (MW cut-off 12,400), and lyophilized (FreeZone 12Liter, Labconco, Kansas City, Missouri, USA) A simple hydrothermal extraction crude polysaccharide fraction (CCW-0) was obtained.

Example  2: citrus Extract foil  Enzyme treatment Group ( CCE -0) Manufacturing

One kilogram of dried citrus fruit extract powder was suspended in 20 times of distilled water, and the commercially available commercial commercial enzyme Pectinex ultra SP-L (from Aspergillus aculeatus , 20 ℃, pH 3.5) (added with 0.5 ~ 1% of powder) was treated for 6 hours and then the enzyme activity was inactivated at 100 ℃ for 15 minutes. Then, to remove the enzyme protein, the citrus extract-treated enzymes were centrifuged at 6,000 rpm for 20 minutes to remove the residues, followed by addition of 4-fold (v / v) 95% ethanol and stirring for 24 hours to precipitate the polysaccharide After centrifugation, the supernatant was separated from the precipitate. Thereafter, the collected precipitates were redissolved in a small amount of distilled water, dialyzed for 2 days using dialysis tubing (MW cut-off 12,400), and lyophilized to obtain an enzyme-treated polysaccharide fraction CCE-0 .

Example  3: Blueberry Extract foil  Enzyme treatment Group ( CCE -0) Purification using alcohol

(DIW: EtOH, v / v, 1: 4, 1: 1, 1: 2, and 1: 4) mixed with a constant ratio of distilled water and ethanol was added to a predetermined amount of a dried sample of polysaccharide- 1: 3, 1: 2, 1: 1.5, 1: 1, 1: 0.5), and the precipitate and the supernatant were separated by centrifugation. Thereafter, the recovered precipitates were washed three times with the same volume of alcohol solution, and the precipitates and supernatant were recovered and lyophilized to obtain 12 solvent fractions by treatment alcohol concentration. Specifically, six precipitants CCE-4P, CCE-3P, CCE-2P, CCE-1.5P, CCE-1P, CCE- S, CCE-1S and CCE-0.5S. In this case, the yield of CCE-4P fraction washed with a solvent mixed with DIW: EtOH = 1: 4 was the highest at 67.5%, and the lower the alcohol concentration, the lower the yield.

Example  4: orange juice Extract foil  Enzyme treatment Group ( CCE -0) Purification using alcohol chromatography

The enzyme-treated crude polysaccharide (CCE-0) prepared according to Example 2 was dissolved in distilled water and developed on a Sephadex G-100 column (4 x 120 cm) equilibrated with 50 mM ammonium formate buffer (pH 5.5) Gel permeation chromatography (GPC) was performed. Each fraction sample was analyzed for neutral sugar, acidic sugar, protein and KDO contents to prepare a dissolution curve, which is shown in Fig. 1. Three fractions of different molecular weights and constituents, CCE-I, CCE-II and CCE-III After separation, each fraction was concentrated, dialyzed and lyophilized.

The yields of CCE-I, CCE-Ⅱ and CCE-Ⅲ were 1.42%, 2.02% and 0.51%, respectively.

[Test Example]

Test Example  1: Analysis of components of polysaccharide

The chemical composition and constituent sugars of simple hydrothermal extraction crude polysaccharide CCW-0, enzyme-treated crude polysaccharide CCE-0 and purified polysaccharide fractions CCE-I, CCE-II and CCE- And the results of HPLC analysis are shown in Fig.

Chemical composition (%) CCW-0 CCE-0 CCE-I CCE-II CCE-III Molecular weight (kDa) - - 62 16 0.19-4.5 Neutral sugar 40.4 ± 2.1 52.0 ± 1.4 52.1 ± 1.9 40.8 ± 1.6 47.6 ± 1.9 Uronic acid 56.9 ± 1.2 41.1 ± 2.0 42.0 + - 4.8 51.6 ± 5.1 48.0 ± 5.2 Protein 1.0 ± 0.1 3.3 ± 0.2 2.6 ± 0.8 1.4 ± 0.2 0.7 ± 0.5 Kdo-liked materials 0.0 ± 0.5 1.8 ± 0.2 2.2 ± 0.2 5.5 ± 0.3 2.5 ± 0.2 phenolic compounds 1.8 ± 0.5 1.8 ± 0.3 1.0 ± 0.5 0.7 ± 0.5 1.2 ± 0.5 Component sugar (mole%) CCW-0 CCE-0 CCE-I CCE-II CCE-III 2-Methyl fucose - 0.8 ± 0.1 - 2.4 ± 0.1 - Rhamnose 2.6 ± 0.2 16.7 ± 0.9 19.5 ± 0.4 12.8 ± 0.3 32.5 ± 0.6 Fucose 0.2 ± 0.0 1.3 ± 0.0 1.4 ± 0.1 2.4 ± 0.2 0.2 ± 0.1 2-methyl xylose - 0.7 ± 0.1 - 1.3 ± 1.4 - Arabinose 17.8 ± 0.5 14.6 ± 0.5 8.1 ± 0.4 9.9 ± 1.0 2.9 ± 0.1 Xylose 2.5 ± 0.1 1.3 ± 0.1 3.3 ± 0.0 2.4 ± 0.0 2.1 ± 0.3 Apiose 0.0 ± 0.0 0.2 ± 0.0 - - - Aceric acid - 0.1 ± 0.0 - 1.6 ± 0.1 - Mannose 3.0 ± 0.1 0.6 ± 0.0 0.6 ± 0.0 0.2 ± 0.0 0.9 ± 0.1 Galactose 8.6 ± 0.1 11.6 ± 0.1 18.5 ± 0.9 7.2 ± 0.2 4.3 ± 0.0 Glucose 5.5 ± 0.1 4.2 ± 0.0 0.7 ± 0.0 0.6 ± 0.1 4.8 ± 0.1 Galacturonic acid 54.5 ± 0.1 37.6 ± 0.6 41.0 ± 0.2 48.5 ± 0.4 45.7 ± 0.2 Glucuronic acid 2.4 ± 0.1 3.5 ± 0.6 1.0 ± 0.2 3.1 ± 0.4 2.3 ± 0.2

According to the analysis, chemical composition of CCW-0, a simple hydrothermal extraction crude polysaccharide derived from citrus peel extract, was 40.4% for neutral sugar, 56.9% for acid, and 1.0% and 1.8% for protein and polyphenol, respectively. Was not detected. (54.8%), arabinose (Ara) (17.8%) and galacrose (Gal) (8.6%) were identified in order of molecular weight after the derivatization with Alditol acetate. , And the elution pattern was confirmed by HPLC. As a result, it was confirmed that the molecular weight was large.

In addition, the enzyme-treated crude polysaccharide CCE-0 derived from citrus peel extracts showed 52.0% neutral sugar, 41.1% acidity, 3.3% protein, 1.8% polyphenol and 3-Deoxy-D-manno-oct-2-ulosonic (16.6%), arabinose (14.6%), and galacrose (Gal) (11.6%) were found to be the most abundant and 1.8% ). Some of the specific sugars found in pectin-derived rhamnogalacturonan II (RG-II) such as 2-methyl fucose, 2-methyl xylose, apiose and acetic acid were also detected. The results of the HPLC analysis were confirmed as a mixture of a polymer substance and a low molecular substance.

According to the results, the hydrolyzate group CCW-0 and the enzyme-treated group CCE-0 mainly contain neutral sugars and acid groups. Especially, the enzyme-treated group CCE-0 is more acid The lower content of catechins was detected by the treatment with pectinase enzyme, which suggests that the homogalacturonan (HG) domain derived from pectin was hydrolyzed and reduced by low molecular weight, which was due to dialysis. In addition, in the case of the enzyme-treated polysaccharide CCE-0, Kdo and specific sugars found only in the pectin-derived RG-Ⅱ structure were detected along with various constituent sugars. Therefore, it is presumed that the polysaccharide is an acidic polysaccharide.

On the other hand, the enzyme-treated purified polysaccharide CCE-I derived from citrus peel extract was composed of 52.1% of neutral sugar, 42.0% of acid, 2.6% of protein, 2.2% of Kdo and 1.0% of phenolic compounds. ), Rha (19.5%), Gal (18.5%) and Ara (8.1%), respectively. As a result of molecular weight determination, it was confirmed that polysaccharide having molecular weight of about 62 kDa was mainly present. It is suggested that CCE-I is a rhamnogalacturonan I (RG-I) structure derived from pectin mainly composed of rhamnose and galacturonic acid, considering that GalA, Rha, Gal and Ara are mainly present. Especially considering the high composition of arabinose and galactose, It is estimated that a high proportion of arabinogalactan in the RG-I structure will be present.

As a result of the constitutional analysis, GalA (48.5%) and Rha (12.8%) were found to be relatively low-molecular polysaccharide CCE-II, which consisted of 40.8% neutral sugar, 51.6% acidity, 1.4% protein, , And ara (9.9%). In the RG-II structure, 2-methyl fucose, 2-methyl xylose, apiose and acic acid were detected in large amounts. Purity and molecular weight were measured by HPLC. As a result, a purified fraction of 16 kDa showed excellent single fraction. It is estimated that CCE-II is an RG-II structure covalently bonded to the HG portion of pectin.

The low-molecular fraction, CCE-Ⅲ, consisted of 47.6% neutral sugar, 48.0% acid, 0.7% protein, 2.5% Kdo and 1.2% phenolic compounds and was composed mainly of GalA 45.7% and Rha 32.5% , It is presumed that the monomeric form of RG-Ⅱ is mixed with the low-molecular-weight decomposed by enzymatic hydrolysis.

Test Example  2: Hot water extraction Group CCW -0 and enzyme treatment Group CCE -0 macrophages ( macrophage ) And cytokines (< RTI ID = 0.0 > cytokione ) Impact on production

The results of the test according to Test Example 2 are shown in Fig. Where NC is the negative control and PC (LPS, 1 ug / mL) is the negative control.

The cytotoxicity of simple hydrothermal extracted crude polysaccharide CCW-0 and enzyme-treated crude polysaccharide CCE-0 derived from citrus peel extracts was measured using macrophages, which are normal cells separated from the abdominal cavity of mice, (A) of FIG.

CCW-0 and CCE-0 were added to normal cell lines adjusted to 1 × 10 ⁶ cells / mL at various concentrations ranging from 1 μg to 1000 μg respectively, and then viability of the cells was examined. As a result, , Neither cytotoxicity was observed in both samples, but rather the ability to proliferate to normal cells.

3 (b) to 3 (c), the production of cytokines of abdominal-derived macrophages was measured in vitro for simple hydrothermal extraction crude polysaccharide CCW-0 derived from citrus peel extract and enzyme-treated crude polysaccharide CCE-0 As a result, it was confirmed that CCE-0 promotes the production of cytokines such as IL-6, IL-12 and TNF-α only at a concentration of 1,000 μg / mL. -12, and TNF- [alpha].

In this test, stimulation of macrophages with CCE-0 enzyme-treated polysaccharide-derived polysaccharide-derived polysaccharide for 24 hours revealed that IL-6 and TNF-α, which are classified as inflammatory cytokines, α production and IL-12 activity, which is directly related to the activation of cellular immunity, was found to be significant. Therefore, the enzyme-treated polysaccharide-derived CCE-0 derived from citrus peel extracts was found to bind to the macrophage receptor It is thought to be involved in the function of activating the immune system by inducing cytokine in the nucleus by inducing intracellular signal transduction.

In particular, IL-12 is recognized as a cytokine essential for the induction of anticancer activity because it is directly involved in the activation of NK cells that are involved in cancer cell death in the presence of cancer cells. Therefore, the enzyme-treated group CK- .

Test Example  3: Blueberry Extract foil  Effect of Enzyme-Treated Purified Polysaccharide Fraction on Cytotoxicity and Production of Cytokines to Macrophages

The results of Test Example 3 are shown in Fig.

In the above test example, the enzyme-treated crude polysaccharide CCE-0 derived from citrus peel extract showed higher cytokine production ability than the simple hydrothermal extraction crude polysaccharide CCW-0. However, since it produced only at a high concentration, Three fractions CCE-I, CCE-Ⅱ and CCE-Ⅲ were obtained through the screening, and their cytokine production ability was confirmed.

First, the cytotoxicity of CCE-I, CCE-II, and CCE-III treated with the enzyme-treated purified fractions derived from citrus fruit extract was measured using macrophages, which are normal cells isolated from the abdominal cavity of mice, Respectively.

The cells were cultured in the presence of various concentrations ranging from 1 μg to 1000 μg of the purified fractions CCE-I, CCE-II and CCE-III in tumor and normal cell lines adjusted to 1 × 10 ⁶ cells / mL, As a result, no cytotoxicity was observed in the purified fraction samples of the macrophages isolated from the peritoneal cavity.

Test Example  4: orange juice Extract foil  Derived tablets polysaccharide CCE Performance of killing action by natural killer cells of -I NK cell activity ) analysis

The results of the NK cell stimulation activity of the enzyme-treated purified fraction CCE-I derived from the citrus peel extract-like peptide according to Test Example 4 are shown in FIG.

(Splenocytes, NK-cells) obtained by intravenous injection of CCE-I at 125, 250 and 500 μg / mouse, respectively, showed the ratio of effect cell (splenocytes) to target cell (YAC-1) / T ratio) showed the highest killability at E / T ratio of 100: 1, and especially at 250 ㎍ / mouse, NK cell activity was about 3 times higher than that of normal control (NC). It is confirmed that CCE-I, an enzyme-treated purified fraction derived from citrus peel extract, activates NK cells and acts mainly on the anticancer activity of citrus fruit extract.

Test Example  5: orange juice Extract foil  Derived tablets polysaccharide CCE Of intravenous administration of -I

The results of the previous experiment showed that the strong NK cell activating ability was confirmed, and to measure the antitumor activity of these NK cells, the Colon 26-M3.1 carcinoma cell was intravenously injected with the CCE-I enzyme-treated fraction- The antitumor activities were evaluated and the results are shown in Fig.

The antitumor activity of the enzyme-treated purified fraction CCE-I derived from the citrus fruit extract-treated leaves was examined by counting the number of tumors in the control group to which no sample was administered as 100%. As a result, the group treated with 125 μg and 250 μg of CCE- 73.8% and 72.8% of the metastatic lesions were inhibited, respectively. However, the inhibition of cancer metastasis by 28.2% was observed at 500 ㎍ concentration.

These results indicate that the antitumor effect of CCE-I is similar to that of the previous NK cell activity, and thus the inhibitory effect of CCE-I on tumor metastasis is due to the activation of NK cell .

Since NK cells are known to be activated by IL-12 produced by macrophages, the production of IL-12 by macrophages is induced by CCE-I and the strong antitumor effect by NK cells activated by these stimuli .

Test Example  6: NK cell  Function controlled mouse In oranges Extract foil  Enzyme-Treated Polysaccharide Fraction Having Antioxidant Activity

It is well known that antitumor activity by the administration of biological response modifier (BRM) in the experimental metastasis model is mainly activated by macrophage or NK cell activation. Therefore, in order to determine whether the antitumor activity of the enzyme-treated purified fraction CUE-I derived from the citrus peel extract-derived fraction was effected by NK cell stimulation, the NK cell function of the mouse was blocked using rabbit anti-asialo GM1 antibody Colon 26-M3.1 lung carcinoma cell, and the results are shown in FIG. 7 to FIG. 9 for each concentration of the administered sample.

As a result of administration of 125 ㎍, 250 ㎍ and 500 ㎍ of CCE-I to mice, 500 ㎍ of antiretroviral effect was observed, but especially, the antagonistic effect was observed at 125 ㎍ and 250 ㎍ concentrations. On the other hand, when CCE-I was administered at 125, 250, and 500 μg to the mice blocked with NK cell function, fewer colonies were observed than those treated with anti-asialo GM1 alone, .

These results suggest that the antitumor effect of enzyme-treated purified fraction CCE-I derived from citrus peel-derived starch is mainly due to the activity of NK cells, but other immune cells (for example, CTL, macrophages) It is believed that the antitumor will work on the effect.

Test Example  7: Orange Extract foil  origin Group CCE -0 on the tumor killing ability of natural killer cells by oral administration ( NK cell activity )

Previous animal studies have confirmed the fact that CCE-I, an enzyme-treated purified fraction derived from a citrus fruit extract, has been shown to exert an excellent effect on NK cell activation and antitumor activity by intravenous administration, The results are shown in Fig.

To investigate NK cell activity and antitumor effect by oral administration, the experiment was conducted using enzyme-treated crude polysaccharide CCE-0 derived from citrus peel extract. In order to reduce the cost and time, it is necessary to use the raw material as the raw material for the functional functional food.

The results of the oral administration of the enzyme-treated crude polysaccharide-derived CCE-0 sample (100, 500, and 1,000 ㎍ / mouse) to the mouse once a day for 14 days and the NK cell stimulating activity were measured A) and tumor cells, the higher the E / T ratio, the better the killing ability. In particular, 100 and 500 ㎍ / mouse showed excellent killability.

Test Example  8: Blueberry Extract foil  Derived enzyme treatment Group CCE -0 < / RTI >

The enzyme-treated crude polysaccharide-derived CCE-0 from citrus fruit extracts was found to exhibit excellent antitumor activity through intravenous administration. The antitumor activity of the antitumor activity was measured again by oral administration, After one oral administration, Colon26-M3.1 cells were inoculated, and 14 days later, the lungs were removed from the experimental animal model to measure the antitumor activity. The results are shown in FIG.

The number of tumor cells in the control group, which was not administered with the sample, was 100% transferred. As a result, CCE-0 was found to have antitumor activity of about 12.0% in the group administered 1,000 μg, 18.8% in the group administered 500 μg, And 22.0% of the antioxidant activity was observed in the culture media.

Based on the above results, it was confirmed that the enzyme-treated crude polysaccharide CCE-0 derived from the citrus peel-derived starch showed excellent antioxidant activity not only by intravenous administration but also by oral administration.

Based on the above results, if the daily dosage of oral administration of animals and humans is used to estimate the daily intake, if an adult male (60 kg) receives 243.3 mg per day for about 2 weeks, It can be estimated that it can exhibit a tumor metastasis suppressing effect of about 22.0%.

Test Example  9: orange juice Extract foil  Derived immune active polysaccharide CCE Structure analysis of -I

As described above, the crude polysaccharide CCE-0 isolated from the citrus fruit extract showed excellent immunoactivity and anticancer activity. The purified CCE-I, CCE-II and CCE-III fractions were obtained and administered intravenously and orally As a result of the antituberculous effect, the structure of CCE-I was analyzed by showing that the degree of contribution of CCE-I to the physiological activity of citrus fruit extract was high.

As shown in Test Example 1, the general chemical properties of the purified polysaccharide CCE-I derived from citrus peel-derived starch were 52.1% for neutral sugar, 42.0% for acid, 2.2% for Kdo, 2.6% for protein and 1.0% for polyphenol, Showed polysaccharides with a molecular weight of 62 kDa, consisting mainly of 41.1% GalA, 19.5% Rha, 18.5% Gal and 8.1% Ara. In addition, it is presumed that RG-I is a pectin-derived RG-I structure in which Rhamnose, galactose and arabinose are mainly present and RG-I is a galactose- In order to confirm whether the arabinogalactan in the structure is present in a high ratio and to confirm whether the arabimavalactan structure is I-type or II-type, the reactivity with glucosyl Yariv reagent, a selective chromogenic reagent of type II arabimavalactan , It was judged that there was no reactivity and arabino-β-4,6-galactan, a type I arabinogalactan, was present.

Test Example  10: methylation analysis Methylation 분석 )On by CCE -I Confirm binding style

In the structural analysis of polysaccharides, the binding pattern of sugar chains is the most important analysis in that it reveals the basic structure of polysaccharides. 1, 2, 1 → 3, 1 → 4, 1) between the constituent sugars, while phosphodiester bonds or peptide bonds (amide bonds) are connected between nucleotides and amino acids, → 6, etc., and it can be present in branched form in the carbon of each constitution. Therefore, structural analysis is difficult compared with other biomolecules, and therefore analysis of binding mode is the most important process in identification of the structure characteristic of polysaccharide .

Methylation analysis, which is often used for glycoconjugate analysis, methylates the hydroxyl attached to non-bonded carbon and hydrolyzes the partially methylated constituent sugar ring to convert the remaining hydroxyl to acetylation The fragment ion was analyzed using GC-MS to identify the structure.

In order to estimate the overall structure of CCE-I, methylation analysis was carried out and analyzed by GC-MS. The results are shown in FIG. In particular, terminal Gal (30.1%), 2,4-linked Rha (21.3%) and 4-linked Gal (14.1%) were found to participate in the binding of three constituents of arabinose, rhamnose and galactose. ) And 2-linked Rha (13.1%) were found to be present. Terminal arabinose (f) (8.8%) and 2-linked Ara (8.1%) were also partially detected. As a result, it was found that the immunoconjugated polysaccharide CCE-I derived from citrus peel extract had a large amount of 2,4-linked Rha and 2-linked Rha, indicating that GalA and Rha, which are components of RG-I main chain, We confirmed that the disaccharides of -GalA- (1,2) -α-L-Rha- (1) n were repeatedly linked to the backbone (rhamnogalacturonan core) The schematic diagram of CCE-I estimated based on the above results is shown in FIG.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, many modifications and changes may be made by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims. The present invention can be variously modified and changed by those skilled in the art, and it is also within the scope of the present invention.

Claims (12)

A crude polysaccharide prepared according to the hot water extraction of citrus extract or cheong pei,
The constituent sugar constituting the crude polysaccharide,
For rhamnose 100 molar parts,
650 to 700 molar parts of arabinose;
300 to 360 parts by mole of galactose; And
Galacturonic acid 2000 Antimicrobial 2200 moles of anti-cancer or immunostimulatory polysaccharide. The method according to claim 1,
The constituent sugar constituting the crude polysaccharide,
With respect to the molar part of rhamnose 100,
5 to 10 molar parts of fucose;
90 to 110 molar parts of xylose;
100 to 130 moles of mannose;
200 to 230 moles of glucose; And
Wherein the polysaccharide further comprises 80 to 100 parts by mol of glucuronic acid. The crude polysaccharide prepared according to the enzymatic treatment of citrus extract or cheongpy peel,
The constituent sugar constituting the crude polysaccharide,
For rhamnose 100 molar parts,
85 to 95 molar parts of arabinose;
65 to 75 molar parts of galactose; And
200 to 250 parts by mol of galacturonic acid. The method of claim 3,
The constituent sugar constituting the crude polysaccharide,
With respect to the molar part of rhamnose 100,
2 to 5 parts by weight of 2-methylfucose;
6 to 10 molar parts of fucose;
6 to 10 molar parts of xylose;
0.5 to 3 molar parts of apiose;
0.2 to 1 molar parts of aceric acid;
Mannose 2 to 5 molar parts;
20 to 30 parts by mole of glucose; And
Wherein the composition further comprises 15 to 25 parts by mole of glucuronic acid. A purified polysaccharide having a molecular weight of 50 to 70 kD obtained by separating gelatine polysaccharide prepared according to the enzyme treatment of citrus fruit extract or cheongyang of claim 3 by gel permeation chromatography,
The constituent sugar constituting the purified polysaccharide may be,
For rhamnose 100 molar parts,
35 to 45 molar parts of arabinose;
90 to 100 molar parts of galactose; And
200 to 220 moles of galacturonic acid. 6. The method of claim 5,
The constituent sugar constituting the purified polysaccharide may be,
With respect to the molar part of rhamnose 100,
5 to 10 molar parts of fucose;
Xylose 14 to 19 molar parts;
Mannose 2 to 5 molar parts;
2 to 6 molar parts of glucose; And
Wherein the polysaccharide further comprises 3 to 7 molar parts of glucuronic acid. A purified polysaccharide having a molecular weight of 10 to 20 kD obtained by separating crude polysaccharide prepared according to the enzyme treatment of citrus fruit extract or cheongyang of claim 3 by gel permeation chromatography,
The constituent sugar constituting the purified polysaccharide may be,
For rhamnose 100 molar parts,
72 to 82 molar parts of arabinose;
50 to 60 molar parts of galactose; And
350 to 400 molar parts of galacturonic acid. 8. The method of claim 7,
The constituent sugar constituting the purified polysaccharide may be,
With respect to the molar part of rhamnose 100,
15 to 25 molar parts of 2-methylfucose;
15 to 25 molar parts of fucose;
7 to 13 molar parts of 2-methylxylose;
Xylose 15 to 22 molar parts;
10 to 15 molar parts of aceric acid;
0.5 to 3 moles of mannose;
2 to 6 molar parts of glucose; And
Wherein the composition further comprises 20 to 30 parts by mole of glucuronic acid. A purified polysaccharide having a molecular weight of 0.1 to 5 kD obtained by separating crude polysaccharide prepared according to the enzyme treatment of citrus extract or chewing gum of claim 3 by gel permeation chromatography,
The constituent sugar constituting the purified polysaccharide may be,
For rhamnose 100 molar parts,
5 to 10 molar parts of arabinose;
10 to 16 molar parts of galactose; And
120 to 160 parts by mol of galacturonic acid. 10. The method of claim 9,
The constituent sugar constituting the purified polysaccharide may be,
With respect to the molar part of rhamnose 100,
0.2 to 1 molar fraction of fucose;
Xylose 3 to 8 molar parts;
Mannose 1-4 moles;
12 to 17 molar parts of glucose; And
And 5 to 10 molar parts of glucuronic acid. ≪ RTI ID = 0.0 > 5. < / RTI > 10. An anticancer or immunostimulatory pharmaceutical composition comprising the polysaccharide of any one of claims 1 to 10 as an active ingredient. An anticancer or immunostimulatory food composition comprising the polysaccharide of any one of claims 1 to 10 as an active ingredient.

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