KR101530982B1 - Preparing method of immuno-stimulating activities of polysaccharide fractions isolated and aglycon flavonoid from citrus peel - Google Patents

Abstract

The present invention relates to a process for the enzymatic treatment of citrus processing by-products in citrus; And a step of centrifuging the enzymatically treated citrus processing by-products, wherein the immunosuppressed polysaccharide is prepared from the centrifuged supernatant, and the non-glycosylated flavonoid is produced from the centrifuged precipitate. And an unglycosylated flavonoid. The immunopotentic polysaccharide prepared according to the present invention exhibits more than 45% of complement activity at a concentration of 1,000 μg / ml and is useful for stimulating lymphocyte proliferation, production of macrophage cytokines (IL-6 and IL-12) The ability is excellent.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for preparing immunosuppressive polysaccharides and non-glycosylated flavonoids from citrus processing by-

The present invention relates to a method for simultaneous extraction of immune-active polysaccharides and non-glycosylated flavonoids having immunoenhancing function from citrus processing by-products using an enzyme.

Recently, the improvement of living standards due to the economic development has improved the hygiene environment, and the caloric intake is rapidly increasing due to the rich diet, westernized dietary life and instant food consumption. However, while the amount of calories consumed as food increased, the amount of calories consumed due to lack of exercise tended to increase. Obesity has been reported to cause breast, uterine, and colorectal cancer as well as adult diseases such as hypertension, diabetes, hyperlipidemia, coronary artery disease and the like, and is now treated as one of the deadly diseases (nature, 404, 652-660).

Obesity is a disease caused by a metabolic abnormality due to an imbalance of energy metabolism, and it has been developed for the treatment of obesity since it is shown that surplus energy consumed in excess is stored in fatty tissue in the form of triglyceride and other fat metabolism. There is a strong interest in new treatment methods with less adverse effects, higher anti-obesity effects, and more side effects than the effects.

Domestic dietary functional food market is worth KRW 1,500-200 billion in 2009. Imported raw materials such as Conjugated Linoleic Acid (CLA) and hydroxyl citric acid (HCA) are dominant in the market. Except for the diet materials that have an impact on the market is not released. Therefore, it is time to develop individual health-functional food ingredients using natural products such as domestic agricultural products.

On the other hand, 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. to be. Currently, most of the chemotherapy is dependent on the administration of anticancer drugs, but the majority of anticancer drugs are synthetic chemicals, and biohazard problems such as hematopoietic function and immune function abnormality are raised, and a lot of research has been focused on immune response modifiers extracted from natural products .

There are about 340 kinds of citrus cultivated in Korea. The most cultivated varieties are mandarin oranges, producing about 600,000 tons per year. Citrus fruits such as mandarin orange and hirabong are widely cultivated in the southern part of Korea and Jeju Island. Citrus fruits have a yield of fruit juice only half of fruit weight, .

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 in the by-product of tangerine peel, but no technology has been reported to simultaneously produce polysaccharides and non-glycosylated flavonoids having immunoenhancing function in citrus .

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made to solve the above-mentioned problems, and it is an object of the present invention to provide a method for simultaneously producing an immune-active polysaccharide and an unglycosylated flavonoid having an immunostimulating function from citrus processing by- .

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

(1) enzymatic treatment of citrus processing by-products in citrus by adding pectinase; And (2) centrifuging the enzymatically treated citrus processing by-products,

The present invention also provides a method for simultaneously producing an immune-active polysaccharide derived from citrus fruits and a non-glycosylated flavonoid, which comprises preparing immunosuppressed polysaccharide from the centrifuged supernatant and producing non-glycosylated flavonoids from the centrifuged precipitate.

According to another embodiment of the present invention, the enzyme treatment may be performed using a mixed enzyme prepared by further containing at least one selected from hesperidinase and naringinase in addition to the pectinase , ≪ / RTI >

Preferably, the enzyme may be treated using a commercial enzyme. The mixed enzyme may be a commercially available enzyme such as Pectinex or Cytolase. The pectinex and cyotrazase may be used alone or in combination , ≪ / RTI >

0.01 to 1 part by weight of an enzyme may be added to 100 parts by weight of the solids of the citrus processing by-product and the mixture may be treated for 6-96 hours.

According to an embodiment of the present invention, the immunopotentiating polysaccharide may be prepared by concentrating the centrifuged supernatant, adding ethanol thereto, and allowing it to stand at -10 to 20 ° C for 3 to 24 hours to induce precipitation; Separating the precipitated ethanol mixture into a liquid phase and a precipitate; And lyophilizing the precipitate to prepare an immunologically active polysaccharide,

The non-glycosylated flavonoids are prepared by a) adding ethanol to the centrifuged precipitate and refluxing the mixture at 45-80 ° C for 6-48 hours to extract the flavonoid component; b) separating the ethanol extract into an ethanol-soluble material and an ethanol-insoluble material; And c) concentrating the ethanolic solubles and crystallizing at -10-20 占 폚 to produce an ungaclycated flavonoid,

Mixing the liquid phase layer generated in the process of manufacturing the immunopotentiating active polysaccharide with the ethanol soluble substance in the step b) may improve the yield of the non-glycosylated flavonoid,

The process further comprises washing the non-glycosylated flavonoid obtained by crystallization in step c) at 0-10 ° C with distilled water for 1-3 times, thereby obtaining an unglycosylated flavonoid having an improved yield of flavonoids converted to the non-glycosides.

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

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 Halabong (Bulgogi), Chrysanthemum, Citrus, Bluegrass, Kumquat and Citron, have.

According to another embodiment of the present invention, the conversion from the glycoside flavonoid to the non-glycosyl flavonoid may be 40-70% by weight.

The immunopotentic polysaccharide prepared according to the present invention may have an anticoagulant activity of 45% or more at a concentration of 1,000 μg / ml, a production stimulating ability of Interleukin-6 (IL-6) of 600-800 pg / (IL-12) may be 550-800 pg / mL,

The non-glycosylated flavonoids prepared according to the present invention are at least one selected from among naringenin and histoperitone.

Enzyme treatment of the present invention can be used to simultaneously produce immunostimulatory polysaccharides and non-glycosylated flavonoids. The immunostimulatory polysaccharides prepared according to the present invention generally exhibit concentration-dependent anti-complement activity and exhibit an activity of 45% or more at a concentration of 1,000 μg / ml. In addition, the immunopotentic polysaccharide is superior to the citrus-derived immunostimulatory polysaccharide obtained by hot-water extraction with the ability to stimulate lymphocyte proliferation, macrophage cytokine (IL-6 and IL-12) production and natural killer cells.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic view illustrating a method for simultaneously producing citrus-derived immunologically active polysaccharides and non-glycosylated flavonoids according to an embodiment of the present invention.
FIG. 2 is a graph showing the results of HPLC analysis of citrus-derived non-glycosylated flavonoids according to one embodiment of the present invention and a comparative example. FIG. 2A is an HPLC chart of a flavonoid extract extracted by simple extraction using ethanol, and FIG. 2B is an HPLC chart of a flavonoid extract prepared according to the present invention.

Citrus fruits such as mandarin orange and hirabong are widely cultivated in the southern part of Korea and Jeju Island. In the process of making citrus fruits such as beverages and citrus chocolates, processing by-products such as citrus husks and fruit juices are generated in excess of 50,000 tons per year, , And these citrus processing by-products are known to be rich in flavonoid components, pectin and oil.

Most of the flavonoids contained in citrus are present in the form of glycosides. Recently, research on the in vivo efficacy of flavonoids has been actively conducted. As a result, flavonoids in an aglycone form rather than glycoside forms And it is reported that it shows higher physiological effect. Recently, the development of non - glycosylated flavonoids has been actively developed by bioconversion of microorganisms or enzymatic reactions through structural characterization of flavonoids and characterization of metabolism.

In addition, pectin is composed of homogalactouronan in a large part of the whole molecule, and rhamnogalactouronan I (RG-I) and rhamnogalactouronan II (RG-II) are known to be branched into various oligos and polysaccharides [Pharmaceutical chemistry journal Volume 46 , ≪ / RTI > 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 actively conducted on RG species.

Accordingly, the inventors of the present invention have developed a method for simultaneously producing non-glycosylated flavonoids and immunosuppressive polysaccharides from citrus by-products through biotransformation using an enzyme, thereby completing the present invention.

Hereinafter, the present invention will be described in detail.

According to an aspect of the present invention, there is provided a method for producing a citrus fruit, comprising: (1) enzymatic treatment of citrus processing by-product in citrus with addition of pectinase; And

(2) centrifuging the enzymatically treated citrus processing by-products,

The present invention also provides a method for simultaneously producing an immune-active polysaccharide derived from citrus fruits and a non-glycosylated flavonoid, which comprises preparing immunosuppressed polysaccharide from the centrifuged supernatant and producing non-glycosylated flavonoids from the centrifuged precipitate.

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 immunoselective polysaccharide present in citrus by-products and an enzyme capable of converting glycosyl form of flavonoid into non-glycosyl flavonoid,

It may be treated with a mixed enzyme prepared by further containing at least one selected from hesperidinase and naringinase in addition to the pectinase.

Preferably, the enzyme may be treated using a commercial enzyme. The mixed enzyme may be a commercially available enzyme such as Pectinex or Cytolase. The pectinex and cyotrazase may be used alone or in combination An immunocompetent polysaccharide with high functionality can be obtained, and a flavonoid having a high conversion rate to an unglycosylated form can be obtained at the same time.

The enzyme may be added to 0.01 to 1 part by weight of the enzyme per 100 parts by weight of the solids of the citrus processing by-product and treated for 6-96 hours. If the enzyme is less than 0.01 part by weight, the amount of the enzyme is too low, Or it is difficult to convert the glycoside-type flavonoid into the non-glycoside form. If the amount exceeds 1 part by weight, hydrolysis of the functional group of the immunostimulatory polysaccharide may be undesirably caused 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 12-24 hours, and the temperature and the pH during the enzyme treatment may be suitably adjusted depending on the enzymes, A flavonoid converted into an unglycosylated form can be obtained. When the reaction time of the enzyme treatment is less than 6 hours, it is difficult to separate the immune-active polysaccharide present in the citrus fruits because the processing time of the citrus processing by-products is too short, and it is difficult for the flavonoids in glycoside form to be sufficiently converted into non- If the reaction time exceeds 96 hours, excessive enzyme treatment may hydrolyze the functional group of the immunoglobulin-active polysaccharide to lower the efficacy of the active polysaccharide, which is not preferable.

The immunopotent polysaccharide may be purified using dialysis, column chromatography or membrane filtration. However, it is preferable that the immobilized polysaccharide is purified using a precipitation method using ethanol in a simple and inexpensive manner in terms of facilities and process costs.

According to the present invention, the immunopotentiating polysaccharide may be prepared by concentrating the centrifuged supernatant, adding ethanol thereto, and allowing it to stand at -10 to 20 ° C for 3 to 24 hours to induce precipitation; Separating the precipitated ethanol mixture into a liquid phase and a precipitate; And lyophilizing the precipitate to prepare an immunostimulatory polysaccharide,

The supernatant is concentrated to 15-25% by volume based on 100% by volume of the centrifuged supernatant. The supernatant is diluted with 3-5 volumes of ethanol to induce precipitation of the immunopotentiating polysaccharide, and the precipitate is lyophilized to be purified .

If the amount of the ethanol is less than the above range, precipitation does not occur and it is difficult to obtain an immunopotent polysaccharide. If the amount exceeds the above range, the content of the impurities may increase, the manufacturing cost increases, This is undesirable because of increased risk.

The immunoprecipitated polysaccharide precipitate should be dried since it contains a large amount of solvents such as ethanol and water used in the production process. The solvent such as water is difficult to remove by natural drying and can be removed at a high temperature and a reduced pressure but it is not preferable because the function of the immunoglobulin-active polysaccharide is hydrolyzed upon heating at a high temperature to lower the efficacy of the immunopotentiating polysaccharide, Preferably by lyophilization.

According to the present invention, the immunoperatively active polysaccharide prepared by the above-described method can be used as a complement fixation test method based on the degree of erythrocyte hemolysis due to the complement remaining after complement consumption by the sample using the Mayer method Can have an ITCH ₅₀ value of 45% or more at a sample concentration of 1,000 / / ml,

In addition, the immunoactive polysaccharide has a production stimulating ability of Interleukin-6 (IL-6) of 600-800 pg / mL at the 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-octulosonic acid (KDO), 3-deoxy-D-leuco-2-heptulosaric acid (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 is not limited to.

According to the present invention, the non-glycosylated flavonoid may be prepared by a) adding ethanol to the centrifugal precipitate and refluxing the mixture at 45-80 ° C for 6-48 hours to extract a flavonoid component; b) separating the ethanol extract into an ethanol-soluble material and an ethanol-insoluble material; And c) concentrating the ethanolic solubles and crystallizing at -10-20 占 폚 to produce an ungaclycated flavonoid,

The flavonoid component may be extracted by adding 10-20 parts by weight of a 50-70 vol% ethanol aqueous solution to 1 part by weight of the centrifugal precipitate and refluxing.

In addition, mixing the liquid phase layer generated in the production of the immunopotentiating polysaccharide with the ethanol soluble substance in step b) may further improve the yield of the non-glycosylated flavonoid,

A small amount of hexane or ether may be added to induce crystal nucleation so that the non-glycosylated flavonoid crystallizes well.

According to the present invention, the non-glycosylated flavonoid obtained by crystallization in the step c) may further be washed with 0-10 ° C distilled water or treated with acetone or chloroform to remove impurities. The loss of glycosyl flavonoid is minimized, and the process is simple and economical.

By washing with distilled water 1-3 times, impurities can be removed and the content of flavonoid in the final product can be improved. The purity can be increased by 40-80% with respect to the initial purity according to the number of times of washing.

According to the present invention, the flavonoid component prepared by an enzyme-free method contains 0.01 to 0.03% by weight of non-glycosylated pravoloids per 100% by weight of the entire flavonoid component, It is possible to convert the glycoside-type flavonoids into the aglycon-type flavonoids to obtain the non-glycosylated flavonoids having a conversion of 40-70 wt%.

According to the present invention, the non-glycosylated flavonoid is any one or more selected from naringenin and histoperitone.

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

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 Halabong (Bulgogi), Chrysanthemum, Citrus, Bluegrass, Kumquat and Citron, have.

When the immunostimulatory polysaccharide or non-glycosylated flavonoid according to the present invention is used as an active ingredient additive of a health functional food or a general food, the immunostimulatory polysaccharide according to the present invention can be directly added or used together with other food or food ingredients, It can be used appropriately according to the method. 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 or non-glycosylated flavonoid according to the present invention may be added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less, based on the raw material in the production of food or beverage. 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 immunologically active polysaccharide or non-glycoside flavonoid according to the present invention as an active ingredient can be used as various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, A stabilizer, a preservative, a glycerin, an alcohol, a carbonating agent used in a carbonated beverage, and 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

Example  One. Halabong Pectinex treatment

Example 1.1

1,000 ml of distilled water was added to 100 g of the hara bark peel powder, and 0.5% (v / w) of the commercial enzyme pectinex was added to the hara bark peel powder, and the mixture was stirred for 14 hours. After completion of the reaction, the reaction mixture was centrifuged to separate the supernatant and the precipitate, and the supernatant was used for the preparation of the immunosorbent polysaccharide and the precipitate was used for the preparation of the non-glycosylated flavonoid.

Example 1.2

The supernatant obtained by the enzyme treatment of Example 1.1 was concentrated to 1/5 of the initial volume, and 4 parts of skin ethanol was added to 1 part of the concentrate. The mixture was allowed to stand at 4 ° C for 12 hours to induce precipitation of immunoselective polysaccharide Respectively. The precipitated mixture was centrifuged and separated into supernatant and precipitate, and the precipitate was lyophilized to obtain immunopotent polysaccharide.

Example 1.3

15 parts by weight of a 60% ethanol aqueous solution was added to 1 part by weight of the precipitate obtained by the enzyme treatment of Example 1.1, and the mixture was stirred at 60 DEG C for 24 hours to extract the active ingredient. After completion of the extraction, the extract supernatant was recovered by centrifugation, and the precipitate was discarded. The recovered extract supernatant was mixed with the supernatant obtained by centrifuging the mixture of the mixture in Example 1.2, and the mixture was concentrated to 1/15 volume of the skin dash of 1 part, and then allowed to stand at 4 ° C for 12 hours to precipitate the non-glycosyl flavonoid Respectively. The precipitated crystal components were recovered by centrifugation and washed with 1 part of concentrate and 1 part of distilled water at a temperature of 4 캜 with respect to the skin.

The obtained precipitate was dried at 50 ° C using a low temperature drier to prepare an unglycosylated flavonoid.

Example  2. Halabong Cyotrazine  process

Example 2.1

The enzyme treatment and centrifugation were carried out in the same manner as in Example 1.1 except that cyotrase was used instead of pectinex to obtain a supernatant and a precipitate.

Example 2.2

Immunoreactive polysaccharide was prepared by the method of Example 1.2 except that the supernatant of Example 2.1 was used instead of the supernatant of Example 1.1.

Example 2.3

An ungical saccharide flavonoid was prepared by the method of Example 1.3 except that the precipitate of Example 2.1 was used in place of the precipitate of Example 1.1.

Example  3. Chunhyang's Pectinex  process

Example 3.1

Enzyme treatment and centrifugation were carried out by the method of Example 1.1, except that Chungbyeol was used instead of Halabung, to obtain a supernatant and a precipitate.

Example 3.2

Immunoreactive polysaccharide was prepared according to the method of Example 1.2, except that Chungbyeol was used instead of Halabong.

Example 3.3

The non-glycosylated flavonoids were prepared according to the method of Example 1.3, except that Chung Hye-yang was used instead of Halabong.

Example  4. Chunhyang's Cyotrazine  process

Example 4.1

The supernatant and precipitate were obtained by enzyme treatment and centrifugation in the same manner as in Example 2.1,

Example 4.2

Immunoreactive polysaccharide was prepared according to the method of Example 2.2, except that Chun Hye-yang was used instead of Halabong.

Example 4.3

The non-glycosylated flavonoids were prepared by the method of Example 2.3, except that Chung Hye-yang was used instead of Halabong.

Example  5. Of citrus Pectinex  process

Example 5.1

Enzyme treatment and centrifugation were carried out in the same manner as in Example 1.1 except that citrus peel was used instead of halobenzene to obtain a supernatant and a precipitate.

Example 5.2

Immunoreactive polysaccharide was prepared according to the method of Example 1.2, except that the hull was replaced with citrus.

Example 5.3

The non-glycosylated flavonoid was prepared according to the method of Example 1.3, except that citron was used instead of halobar.

Example  6. Citrus Cyotrazine  process

Example 6.1

Enzyme treatment and centrifugation were carried out by the method of Example 2.1, except that citron was used instead of halobenzene to obtain a supernatant and a precipitate.

Example 6.2

Immunoreactive polysaccharide was prepared by the method of Example 2.2, except that citron was used instead of halobar.

Example 6.3

The non-glycosylated flavonoids were prepared according to the procedure of Example 2.3, except that citronelles were used in place of hanabong.

Comparative Example  One. Halabong  Cellulase KN  process

Comparative Example 1.1

The enzyme treatment and centrifugation were carried out in the same manner as in Example 1.1 except that the cellulase KN was used instead of pectinex to obtain a supernatant and a precipitate.

Comparative Example 1.2

Immunoreactive polysaccharide was prepared by the method of Example 1.2 except that the supernatant of Comparative Example 1.1 was used instead of the supernatant of Example 1.1.

Comparative Example 1.3

The non-glycosylated flavonoid was prepared by the method of Example 1.3 except that the precipitate of Comparative Example 1.1 was used in place of the precipitate of Example 1.1.

Comparative Example  2. Halabong Econita  process

Comparative Example 2.1

The enzyme treatment and centrifugation were carried out in the same manner as in Example 1.1 except that ectonine was used instead of pectinex to obtain a supernatant and a precipitate.

Comparative Example 2.2

Immunoreactive polysaccharide was prepared by the method of Example 1.2 except that the supernatant of Comparative Example 2.1 was used instead of the supernatant of Example 1.1.

Comparative Example 2.3

An ungical saccharide flavonoid was prepared by the method of Example 1.3, except that the precipitate of Comparative Example 2.1 was used in place of the precipitate of Example 1.1.

Comparative Example  3. Halabong Rapidadze  process

Comparative Example 3.1

Except that lapidase was used instead of pectinex, followed by centrifugation to obtain a supernatant and a precipitate.

Comparative Example 3.2

Immunoreactive polysaccharide was prepared by the method of Example 1.2 except that the supernatant of Comparative Example 3.1 was used instead of the supernatant of Example 1.1.

Comparative Example 3.3

The non-glycosylated flavonoid was prepared by the method of Example 1.3 except that the precipitate of Comparative Example 3.1 was used in place of the precipitate of Example 1.1.

Comparative Example  4. Halabong Viscoenzyme  process

Comparative Example 4.1

The supernatant and the precipitate were obtained by enzyme treatment and centrifugation in the same manner as in Example 1.1, except that visconoenzyme was used instead of pectinex.

Comparative Example 4.2

Immunoreactive polysaccharide was prepared by the method of Example 1.2 except that the supernatant of Comparative Example 4.1 was used instead of the supernatant of Example 1.1.

Comparative Example 4.3

The non-glycosylated flavonoid was prepared by the method of Example 1.3 except that the precipitate of Comparative Example 4.1 was used in place of the precipitate of Example 1.1.

Comparative Example  5. Halabong Celluclast  process

Comparative Example 5.1

The enzyme treatment and centrifugation were carried out in the same manner as in Example 1.1 except that Celluclast was used in place of pectinex to obtain a supernatant and a precipitate.

Comparative Example 5.2

Immunoreactive polysaccharide was prepared by the method of Example 1.2 except that the supernatant of Comparative Example 5.1 was used instead of the supernatant of Example 1.1.

Comparative Example 5.3

An ungical saccharide flavonoid was prepared by the method of Example 1.3 except that the precipitate of Comparative Example 5.1 was used in place of the precipitate of Example 1.1.

Comparative Example  6. Halabong Rohmement  process

Comparative Example 6.1

The enzyme treatment and centrifugation were carried out in the same manner as in Example 1.1 except that the rohundent was used instead of pectinex to obtain a supernatant and a precipitate.

Comparative Example 6.2

Immunoreactive polysaccharide was prepared by the method of Example 1.2 except that the supernatant of Comparative Example 6.1 was used in place of the supernatant of Example 1.1.

Comparative Example 6.3

The non-glycosylated flavonoid was prepared by the method of Example 1.3 except that the precipitate of Comparative Example 6.1 was used in place of the precipitate of Example 1.1.

Comparative Example  7. Halabong Ultraflow  process

Comparative Example 7.1

The supernatant and the precipitate were obtained by enzyme treatment and centrifugation in the same manner as in Example 1.1 except that Ultraflow was used instead of pectinex.

Comparative Example 7.2

Immunoreactive polysaccharide was prepared by the method of Example 1.2 except that the supernatant of Comparative Example 7.1 was used instead of the supernatant of Example 1.1.

Comparative Example 7.3

The non-glycosylated flavonoid was prepared by the method of Example 1.3 except that the precipitate of Comparative Example 7.1 was used in place of the precipitate of Example 1.1.

Comparative Example  8. Halabong Pectinase  process

Comparative Example 8.1

Except that pectinase was used instead of pectinex, followed by centrifugation to obtain a supernatant and a precipitate.

Comparative Example 8.2

Immunoreactive polysaccharide was prepared by the method of Example 1.2 except that the supernatant of Comparative Example 8.1 was used in place of the supernatant of Example 1.1.

Comparative Example 8.3

An ungical saccharide flavonoid was prepared by the method of Example 1.3, except that the precipitate of Comparative Example 8.1 was used in place of the precipitate of Example 1.1.

Comparative Example  9. Chunhyang's  Cellulase KN  process

Comparative Example 9.1

The supernatant and the precipitate were obtained by enzyme treatment and centrifugation in the same manner as in Example 1.1, except that Chun Hye-yang was used instead of Halabung, and the cellulase KN was used instead of Pectinex.

Comparative Example 9.2

Immunoreactive polysaccharide was prepared by the method of Example 1.2 except that the supernatant of Comparative Example 9.1 was used instead of the supernatant of Example 1.1.

Comparative Example 9.3

The non-glycosylated flavonoid was prepared by the method of Example 1.3, except that the precipitate of Comparative Example 9.1 was used in place of the precipitate of Example 1.1.

Comparative Example  10. Chunhyang's Econidase  process

Comparative Example 10.1

The supernatant and the precipitate were obtained by enzyme treatment and centrifugation in the same manner as in Example 1.1, except that Enalidase was used instead of Pectinex.

Comparative Example 10.2

Immunoreactive polysaccharide was prepared by the method of Example 1.2 except that the supernatant of Comparative Example 10.1 was used instead of the supernatant of Example 1.1.

Comparative Example 10.3

The non-glycosylated flavonoids were prepared by the method of Example 1.3 except that the precipitate of Comparative Example 10.1 was used in place of the precipitate of Example 1.1.

Comparative Example  11. Chunhyang's Lapidase  process

Comparative Example 11.1

The supernatant and the precipitate were obtained by enzyme treatment and centrifugation in the same manner as in Example 1.1, except that Chun Hye-yang was used instead of Halabung and lepidase was used instead of pectinex.

Comparative Example 11.2

Immunoreactive polysaccharide was prepared by the method of Example 1.2 except that the supernatant of Comparative Example 11.1 was used instead of the supernatant of Example 1.1.

Comparative Example 11.3

An ungical saccharide flavonoid was prepared by the method of Example 1.3 except that the precipitate of Comparative Example 11.1 was used in place of the precipitate of Example 1.1.

Comparative Example  12. Chunhyang's Viscoenzyme  process

Comparative Example 12.1

The supernatant and precipitate were obtained by enzymatic treatment and centrifugation in the same manner as in Example 1.1, except that Chun Hye-yang was used instead of Halabung, and Visconoenzyme was used instead of Pectinex.

Comparative Example 12.2

Immunoreactive polysaccharide was prepared by the method of Example 1.2 except that the supernatant of Comparative Example 12.1 was used instead of the supernatant of Example 1.1.

Comparative Example 12.3

The non-glycosylated flavonoids were prepared by the method of Example 1.3 except that the precipitate of Comparative Example 12.1 was used in place of the precipitate of Example 1.1.

Comparative Example  13. Chunhyang's Celluclast  process

Comparative Example 13.1

The supernatant and precipitate were obtained by enzymatic treatment and centrifugation in the same manner as in Example 1.1 except that Chun Hye-yang was used instead of Halabung and Celluclast was used in place of Pectinex.

Comparative Example 13.2

Immunoreactive polysaccharide was prepared by the method of Example 1.2 except that the supernatant of Comparative Example 13.1 was used instead of the supernatant of Example 1.1.

Comparative Example 13.3

The non-glycosylated flavonoid was prepared by the method of Example 1.3, except that the precipitate of Comparative Example 13.1 was used in place of the precipitate of Example 1.1.

Comparative Example  14. Chunhyang's Rohmement  process

Comparative Example 14.1

The supernatant and precipitate were obtained by enzymatic treatment and centrifugation in the same manner as in Example 1.1 except that Chun Hye-yang was used instead of Halabung and Lohaft was used instead of Pectinex.

Comparative Example 14.2

Immunoreactive polysaccharide was prepared by the method of Example 1.2 except that the supernatant of Comparative Example 14.1 was used instead of the supernatant of Example 1.1.

Comparative Example 14.3

The non-glycosylated flavonoid was prepared by the method of Example 1.3, except that the precipitate of Comparative Example 14.1 was used in place of the precipitate of Example 1.1.

Comparative Example  15. Chunhyang's Ultraflow  process

Comparative Example 15.1

The supernatant and precipitate were obtained by enzymatic treatment and centrifugation in the same manner as in Example 1.1, except that Ultrablow was used in place of pectinex, instead of Halabong.

Comparative Example 15.2

Immunoreactive polysaccharide was prepared by the method of Example 1.2 except that the supernatant of Comparative Example 15.1 was used instead of the supernatant of Example 1.1.

Comparative Example 15.3

An ungical saccharide flavonoid was prepared by the method of Example 1.3, except that the precipitate of Comparative Example 15.1 was used in place of the precipitate of Example 1.1.

Comparative Example  16. Chunhyang's Rohmement  process

Comparative Example 16.1

The supernatant and precipitate were obtained by enzymatic treatment and centrifugation in the same manner as in Example 1.1, except that pectinex was used instead of pectinex, which was used instead of Halabong.

Comparative Example 16.2

Immunoreactive polysaccharide was prepared by the method of Example 1.2 except that the supernatant of Comparative Example 16.1 was used instead of the supernatant of Example 1.1.

Comparative Example 16.3

The non-glycosylated flavonoid was prepared by the method of Example 1.3 except that the precipitate of Comparative Example 16.1 was used in place of the precipitate of Example 1.1.

Comparative Example  17. Cellulase of citrus KN   process

Comparative Example 17.1

Enzyme treatment and centrifugation were carried out in the same manner as in Example 1.1, except that citron was used instead of halobar and cellulase KN was used instead of pectinex, to obtain a supernatant and a precipitate.

Comparative Example 17.2

Immunoreactive polysaccharide was prepared by the method of Example 1.2 except that the supernatant of Comparative Example 17.1 was used instead of the supernatant of Example 1.1.

Comparative Example 17.3

The non-glycosylated flavonoid was prepared by the method of Example 1.3 except that the precipitate of Comparative Example 17.1 was used in place of the precipitate of Example 1.1.

Comparative Example  18. Citrus Econidase  process

Comparative Example 18.1

Enzyme treatment and centrifugation were carried out in the same manner as in Example 1.1, except that citron was used instead of halobar and ectonase was used instead of pectinex, to obtain a supernatant and a precipitate.

Comparative Example 18.2

Immunoreactive polysaccharide was prepared by the method of Example 1.2 except that the supernatant of Comparative Example 18.1 was used instead of the supernatant of Example 1.1.

Comparative Example 18.3

An ungical saccharide flavonoid was prepared by the method of Example 1.3 except that the precipitate of Comparative Example 18.1 was used in place of the precipitate of Example 1.1.

Comparative Example  19. Of citrus Lapidase  process

Comparative Example 19.1

The supernatant and the precipitate were obtained by enzyme treatment and centrifugation in the same manner as in Example 1.1, except that citron was used instead of halobar and raffidase was used instead of pectinex.

Comparative Example 19.2

Immunoreactive polysaccharide was prepared by the method of Example 1.2 except that the supernatant of Comparative Example 19.1 was used instead of the supernatant of Example 1.1.

Comparative Example 19.3

An ungical saccharide flavonoid was prepared by the method of Example 1.3 except that the precipitate of Comparative Example 18.1 was used in place of the precipitate of Example 1.1.

Comparative Example  20. Citrus Viscoenzyme  process

Comparative Example 20.1

The supernatant and precipitate were obtained by enzymatic treatment and centrifugation in the same manner as in Example 1.1, except that citron was used instead of halobar and visconoenzyme was used instead of pectinex.

Comparative Example 20.2

Immunoreactive polysaccharide was prepared by the method of Example 1.2 except that the supernatant of Comparative Example 20.1 was used instead of the supernatant of Example 1.1.

Comparative Example 20.3

The non-glycosylated flavonoids were prepared by the method of Example 1.3 except that the precipitate of Comparative Example 20.1 was used in place of the precipitate of Example 1.1.

Comparative Example  21. Of citrus Celluclast  process

Comparative Example 21.1

The supernatant and the precipitate were obtained by enzymatic treatment and centrifugation in the same manner as in Example 1.1, except that hull substitutes were used instead of hala bong, and cellulose was used in place of pectinex.

Comparative Example 21.2

Immunoreactive polysaccharide was prepared by the method of Example 1.2 except that the supernatant of Comparative Example 21.1 was used instead of the supernatant of Example 1.1.

Comparative Example 21.3

The non-glycosylated flavonoid was prepared by the method of Example 1.3 except that the precipitate of Comparative Example 21.1 was used in place of the precipitate of Example 1.1.

Comparative Example  22. Of citrus Rohmement  process

Comparative Example 22.1

The supernatant and precipitate were obtained by enzyme treatment and centrifugation in the same manner as in Example 1.1, except that citron was used instead of halobenzene and roformance was used instead of pectinex.

Comparative Example 22.2

Immunoreactive polysaccharide was prepared by the method of Example 1.2 except that the supernatant of Comparative Example 22.1 was used instead of the supernatant of Example 1.1.

Comparative Example 22.3

The non-glycosylated flavonoid was prepared by the method of Example 1.3 except that the precipitate of Comparative Example 22.1 was used in place of the precipitate of Example 1.1.

Comparative Example  23. Of citrus Ultraflow  process

Comparative Example 23.1

The supernatant and the precipitate were obtained by enzymatic treatment and centrifugation in the same manner as in Example 1.1, except that hull substitute was used instead of halobar and ultraflow was used instead of pectinex.

Comparative Example 23.2

Immunoreactive polysaccharide was prepared by the method of Example 1.2 except that the supernatant of Comparative Example 23.1 was used instead of the supernatant of Example 1.1.

Comparative Example 23.3

An ungical saccharide flavonoid was prepared by the method of Example 1.3 except that the precipitate of Comparative Example 23.1 was used in place of the precipitate of Example 1.1.

Comparative Example  24. Of the citrus Rohmement  process

Comparative Example 24.1

The supernatant and the precipitate were obtained by enzyme treatment and centrifugation in the same manner as in Example 1.1 except that citron was used in place of haplobin and pectinase was used instead of pectinex.

Comparative Example 24.2

Immunoreactive polysaccharide was prepared by the method of Example 1.2 except that the supernatant of Comparative Example 24.1 was used instead of the supernatant of Example 1.1.

Comparative Example 24.3

An ungical saccharide flavonoid was prepared by the method of Example 1.3, except that the precipitate of Comparative Example 24.1 was used in place of the precipitate of Example 1.1.

General Control 1

The non-glycosylated flavonoid and the immunosuperactive polysaccharide were prepared by the same method as in Example 1, except that 100 g of dried hara bark peel powder instead of enzyme treatment was added to 1000 ml of distilled water and the mixture was extracted at 95 캜 for 6 hours.

General Control 2

The non-glycosylated flavonoids and the immunosuppressed polysaccharides were prepared by the same method as in Example 1, except that 100 g of the dried ginseng sheath powder instead of the enzyme treatment was added to 1000 ml of distilled water and the mixture was extracted at 95 ° C for 6 hours.

General control group 3

The non-glycosylated flavonoids and the immunosuperactive polysaccharides were prepared according to the method of Example 1, except that 100 g of dried citrus peel powder was added to 1000 ml of distilled water instead of the enzyme treatment and the mixture was extracted at 95 캜 for 6 hours.

Test Example  1. Analysis of flavonoid components and Biological conversion rate

The contents of citron - derived flavonoids were analyzed by HPLC using DMSO as a solvent. The standard components were narirutin, naringin, hesperidin, naringenin and hesperitin, and each standard product was purchased from Sigma. FIG. 2 (a) is a graph showing analysis of flavonoids obtained by simple extraction in order to compare with the present invention, and FIG. 2 (b) is a graph of flavonoids obtained using a cytochrome enzyme.

The conversion rates of the prepared flavonoids were calculated by comparing the content of naringenin and hesperitin with respect to the total content of five indicator compounds including narirutin. The formula is as follows.

[Conversion rate (%) = (hesperitin + naringenin) x 100 / (narirutin + hesperidin + naringenin + hesperidin + naringin)]

Analysis of Halafung Flavonoid Purification (%) Name of sample enzyme Narirutin Naringin Hesperidin Naringenin Hesperitine sum Conversion Rate General Control 1 - 3.38 0.01 19.97 0.02 0.05 23.43 0.3 Example 1.2 Pectinex 0.92 0.00 14.87 5.57 5.84 27.20 41.9 Example 2.2 Cytolase 1.59 0.00 15.12 6.14 6.49 29.34 43.0 Comparative Example 1.2 Cellulase KN 1.67 0.00 14.74 5.22 5.25 26.88 39.0 Comparative Example 2.2 Econitase 2.81 0.00 21.54 0.01 0.05 24.41 0.2 Comparative Example 3.2 Rapidase 3.13 0.00 20.33 0.02 0.06 23.54 0.3 Comparative Example 4.2 Viscozyme 2.98 0.00 19.84 0.02 0.06 22.90 0.3 Comparative Example 5.2 Celluclast 3.05 0.01 20.38 0.02 0.05 23.51 0.3 Comparative Example 6.2 Rohament 2.81 0.00 21.54 0.01 0.05 24.41 0.2 Comparative Example 7.2 UltrafloL 2.81 0.00 21.54 0.01 0.05 24.41 0.2 Comparative Example 8.2 Pectinase 2.99 0.00 18.21 0.01 0.04 21.25 0.2

Analysis of Chung Hye Yang Plantaoid Purified Water (%) Name of sample enzyme Narirutin Naringin Hesperidin Naringenin Hesperitine sum Conversion Rate General Control 2 - 2.15 0.01 18.45 0.01 0.02 20.64 0.3 Example 3.2 Pectinex 0.88 0.00 13.54 3.84 4.68 22.94 37.1 Example 4.2 Cytolase 0.75 0.00 12.34 6.44 5.12 24.65 46.9 Comparative Example 9.2 Cellulase KN 0.84 0.00 13.28  4.01 4.14 22.27 36.6 Comparative Example 10.2 Econitase 2.21 0.00 19.15 0.01 0.03 21.40 0.2 Comparative Example 11.2 Rapidase 2.36 0.00 19.66 0.02 0.04 22.08 0.3 Comparative Example 12.2 Viscozyme 2.56 0.00 18.98 0.01 0.03 21.58 0.2 Comparative Example 13.2 Celluclast 2.42 0.01 18.86 0.01 0.03 21.33 0.2 Comparative Example 14.2 Rohament 2.28 0.01 18.92 0.00 0.06 21.27 0.3 Comparative Example 15.2 UltrafloL 2.34 0.00 18.97 0.00 0.04 21.35 0.2 Comparative Example 16.2 Pectinase 2.99 0.00 18.21 0.01 0.04 21.25 0.2

Analysis of Citrus Planoid Purified Water (%) Name of sample enzyme Narirutin Naringin Hesperidin Naringenin Hesperitine sum Conversion Rate General control group 3 - 3.13 0.00 15.84 0.01 0.03 19.01 0.21 Example 5.2 Pectinex 1.03 0.00 14.63 5.22 5.24 26.12 40.05 Example 6.2 Cytolase 1.27 0.00 14.08 5.85 5.78 26.98 43.11 Comparative Example 17.2 Cellulase KN 1.55 0.00 14.27 5.06 5.18 26.06 39.29 Comparative Example 18.2 Econitase 3.21 0.00 19.89 0.01 0.04 23.15 0.22 Comparative Example 19.2 Rapidase 3.02 0.00 20.03 0.01 0.04 23.10 0.22 Comparative Example 20.2 Viscozyme 2.88 0.00 19.94 0.01 0.04 22.7 0.22 Comparative Example 21.2 Celluclast 2.81 0.00 19.49 0.01 0.04 22.35 0.22 Comparative Example 22.2 Rohament 2.95 0.00 20.32 0.01 0.04 23.32 0.21 Comparative Example 23.2 UltrafloL 2.86 0.00 21.28 0.01 0.04 24.20 0.25 Comparative Example 24.2 Pectinase 3.08 0.00 17.68 0.01 0.04 20.81 0.24

HPLC analysis of the purified flavonoids obtained by each enzyme treatment revealed that the flavonoids (Naringin, Hesperidin) in the glycerol form decreased and the non-glycosylated flavonoids (Naringenin, Hesperitin ), And it was judged that the comparative example had no ability to convert to non-glycosides.

The conversion rates of each experimental group were 50% at the first 0.03% level by the treatment with Pectinex and Cytolase enzyme in the haraabong (Examples 1 and 2), Chunhyanghyang (Examples 3 and 4) and citrus (Examples 5 and 6) As shown in FIG.

Test Example  2. Flavonoid wash count and purity

In order to purify the flavonoid component obtained by treating Pectinex with Hara Bae, 1 ~ 3 times with distilled water. The results are shown in Table 3 below.

% Name of sample Number of floods Narirutin Naringin Hesperidin Naringenin Hesperitine sum Conversion Rate Example 2.2  One 1.59 0.00 15.12 6.14 6.49 29.34 43.0  3 1.84 0.00 17.54 11.76 11.82 42.96 54.9

It was confirmed that the purity of the purified product increased from 29.34% to 42.96% as the number of washings increased from 1 to 3 times.

Test Example  3. Citrus-derived immunostimulatory polysaccharides Anti-complement activity  evaluation

Based on the degree of red blood cell hemolysis due to the complement of remaining complement after sample supplementation using the Mayer method to confirm the activation of the complement system, which plays an important role in protecting the initial infection of the human body, , And PSK (polysaccharide-K), a commercially available immunostimulatory polysaccharide, was used as a positive control.

As a positive control, PSK (polysaccharide K), an immunologically active polysaccharide derived from coriolus versicolor, was used. Distilled water without addition of a sample was used as a negative control. The degree of activation in the negative control group was defined as ITCH50 0% The activity was confirmed and shown in Tables 5 to 7 below.

Sulfate Activity of Immunoreactive Polysaccharides Derived from Hallabong division enzyme ingredient Anti-complement activity Positive control group PSK - 53.4 ± 2.9 Negative control group - - 22.4 ± 1.3 Example 1.3 Pectinex pectinase 47.9 ± 2.9 Example 2.3 Cytolase pectinase 50.1 ± 1.8 Comparative Example 1.3 Cellulase KN cellulase 18.6 ± 1.8 Comparative Example 2.3 Econase cellulase 23.1 ± 4.2
Comparative Example 3.3
Rapidase pectinase, hemicellulase, cellulase
48.2 ± 2.9

Comparative Example 4.3

Viscozyme arabinase, cellulase,
β-glucanase, hemicellulase, xylanase

32.4 ± 1.9 Comparative Example 5.3 Celluclast cellulase 28.3 ± 1.8 Comparative Example 6.3 Rohament cellulase 19.4 ± 2.4 Comparative Example 7.3 Ultraflo β-glucanase 21.6 ± 1.1 Comparative Example 8.3 Pectinase pectinase 55.3 ± 2.5

Anti-complement activity of immune-activated polysaccharide division enzyme chief ingredient Anti-complement activity Positive control group PSK - 54.3 ± 3.8 Negative control group - - 16.8 ± 2.3 Example 3.3 Pectinex pectinase 21.5 ± 2.8 Example 4.3 Cytolase pectinase 19.3 ± 1.3 Comparative Example 9.3 Cellulase KN cellulase 20.5 ± 2.2 Comparative Example 10.3 Econase cellulase 12.8 ± 1.0
Comparative Example 11.3
Rapidase pectinase, hemicellulase, cellulase
21.4 ± 1.8

Comparative Example 12.3
Viscozyme arabinase, cellulase,
β-glucanase, hemicellulase, xylanase

13.2 ± 2.1 Comparative Example 13.3 Celluclast cellulase 10.1 ± 0.8 Comparative Example 14.3 Rohament cellulase 11.3 ± 2.3 Comparative Example 15.3 Ultraflo β-glucanase 5.6 ± 3.1 Comparative Example 16.3 Pectinase pectinase 22.5 ± 2.5

Anti-complement activity of immune-activated polysaccharides derived from citrus division enzyme ingredient Anti-complement activity Positive control group PSK - 53.4 ± 2.9 Negative control group - - 22.4 ± 1.3 Example 5.3 Pectinex pectinase 44.3 ± 1.5 Example 6.3 Cytolase pectinase 48.2 ± 1.3 Comparative Example 17.3 Cellulase KN cellulase 26.7 ± 3.4 Comparative Example 18.3 Econase cellulase 24.2 ± 2.6
Comparative Example 19.3
Rapidase pectinase, hemicellulase, cellulase
47.5 ± 2.2

Comparative Example 20.3
Viscozyme arabinase, cellulase,
β-glucanase, hemicellulase, xylanase

20.2 ± 1.6 Comparative Example 21.3 Celluclast cellulase 23.6 ± 1.4 Comparative Example 22.3 Rohament cellulase 21.4 ± 1.8 Comparative Example 23.3 Ultraflo β-glucanase 25.3 ± 1.4 Comparative Example 24.3 Pectinase pectinase 52.0 ± 2.8

As a result of the complement system activation experiment using the immunoperatively active polysaccharides obtained in the examples and the comparative examples of the present invention, it was found that in case of the test group obtained with hanrabong and citrus as a raw material, the commercial enzymes lapidase, pectinex, Immunopolysaccharides obtained from pectinase were found to have anti - complement activity similar to that of positive control PSK. However, there was no test group with high complement system activation ability.

However, in the case of raffidase and pectinase, as shown in Test Example 1-2, non-glycosylated flavonoids could not be prepared, and cellulase KN showed less anti-complement activity. Based on the above results, it has been confirmed that a commercial enzyme capable of simultaneously producing non-glycosylated flavonoids and immunostimulatory polysaccharides is cyotrazase or pectinex, which contains both pectinase and hesperidinase or naringinase as main components .

Test Example  4. Assessment of cytokine production capacity of immune-activated polysaccharides

Cytokine production stimulation was evaluated using a sandwich ELISA method using macrophages obtained from peritoneal cavity of BALB / c (female, 6 weeks old) mice. 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 1.3 treated with the cellulase KN, the ability to stimulate the production of interleukin-6 (IL-6) and interleukin-12 (IL-12) cytokines was found to be significantly lower, whereas pectinex and cyto- The immunostimulatory polysaccharides prepared by the enzyme-catalyzed enzymes were evaluated to have excellent production stimulating ability of IL-6 and IL-12, and the production stimulating effect was also found to be concentration-dependent.

IL-6 (pg / ml) Concentration (/ / ml) Example 1.3 Example 2.3 Comparative Example 1.3 Negative control group Positive control group 1000 711.20 ± 19.17 714.42 ± 14.34 341.12 ± 24.50 115.66 13.99 869.01 + - 31.01 200 621.68 + - 9.43 609.32 + - 14.26 119.17 ± 31.65 - - 40 546.11 + 13.31 544.25 + - 5.91 58.39 ± 19.46 - - 8 376.14 + 14.38 324.49 ± 11.15 51.09 ± 23.11 - - 1.6 277.61 + - 24.37 291.17 ± 21.37 39.24 + - 22.03 - -

IL-12 (pg / ml) Concentration (/ / ml) Example 1.3 Example 2.3 Comparative Example 1.3 Negative control group Positive control group 1000 654.15 ± 11.25 658.12 + - 22.05 318.28 ± 13.25 115.12 ± 15.24 869.71 + - 34.21 200 554.15 + - 11.95 547.26 + - 22.48 209.48 ± 14.58 - - 40 431.21 + - 5.48 425.15 + 13.22 129.44 ± 3.84 - - 8 321.12 + - 38.12 309.08 ± 29.15 75.86 + - 11.23 - - 1.6 221.53 + - 2.58 219.15 ± 3.48 5.99 ± 1.02 - -

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).

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 Example 1.2

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 Example 1.2

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 Example 1.2

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 Example 1.2

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 Example 1.2

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

1000 mg of the immunostimulatory polysaccharide powder of Example 1.2

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

1000 mg of the immunostimulatory polysaccharide powder of Example 1.2

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.

The immunostimulatory polysaccharide powders of Examples 2.2 to 6.2 or the unfractionated flavonoids of Examples 1.3 to 6.3 may be added instead of the immunostimulatory polysaccharide powder of Example 1.2 above.

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 (13)

(1) a method for producing a citrus fruit, which comprises (i) at least one enzyme selected from the group consisting of hesperidinase and naringinase and (ii) an enzyme having both pectinase activity Adding 0.01-1 parts by weight of a complex enzyme, pectinex or cytolase, for 6-96 hours; And
(2) centrifuging the enzymatically treated citrus processing by-products,
(3) a) concentrating the supernatant of supernatant, adding ethanol and allowing it to settle at -10-20 ° C for 3-24 hours; b) separating the precipitated ethanol mixture into a liquid phase and a precipitate; And c) lyophilizing the precipitate to produce an immunostimulatory polysaccharide, wherein the immunostimulatory polysaccharide is prepared from the centrifugation supernatant,
(4) a) adding an aqueous ethanol solution to the centrifugal precipitate and refluxing the mixture at 45-80 占 폚 for 6-48 hours to extract a flavonoid component; b) separating the ethanol extract into an ethanol-soluble material and an ethanol-insoluble material; c) mixing the liquid phase layer of step (b) of step (3) and the ethanol solubles, concentrating and crystallizing at -10 to 20 ° C to prepare an unglycosylated flavonoid; And d) washing the crystallized non-glycosylated flavonoid with 0-10 ° C distilled water for 1-3 times to prepare an unglycosylated flavonoid in the centrifuged precipitate,
In step (a) of (3), the concentrated supernatant is concentrated to a volume of 15-25% by volume of 100 vol% of the supernatant, and ethanol is added in an amount of 3-5 times that of the concentrated concentrate.
In step (a) of (4), 10 to 20 parts by weight of an aqueous solution of 50 to 70% by volume of ethanol is added to 1 part by weight of the centrifugal precipitate,
A method for simultaneous production of citrus-derived immunostimulatory polysaccharides and non-glycoside flavonoids characterized in that the conversion of glycosylated flavonoids to non-glycosylated flavonoids is 40-70% by weight. delete delete delete delete delete delete delete delete The method according to claim 1, wherein the citrus fruits are selected from the group consisting of Halabong, Chunhyang, and citrus. delete delete delete

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