KR20080093488A - An extract from bupleuri radix having improved immunity capability of animals and its use - Google Patents

Abstract

An extract of Bupleuri Radix is provided to increase various activities of main immunocytes of an animal such as granulocyte and macrophage and induce strong defence capability on attach inoculation of pathogenic bacteria. A composition comprising the same extract is provided to be used as an animal medicine such as an animal immuno-enhancing agent and a vaccine adjuvant and a feed additive for treating and preventing animal diseases efficiently. A method for purifying an extract of Bupleuri Radix comprises the steps of: (a) heat-treating the Bupleuri Radix with hot water at a temperature of 80-120 deg.C for 2-3 hours to obtain a crude extract; (b) concentrating the crude extract; and (c) filtering the concentrate to obtain a polysaccharide fraction by filtering paper and microorganism filtering. An extract of Bupleuri Radix purified by the method is a Bupleuri Radix polysaccharide, of which sugar is a glycoprotein including a protein. A composition for increasing immunity comprises the extract of Bupleuri Radix as an active ingredient and a pharmaceutically acceptable carrier or an excipient. An animal immunity enhancing agent, a vaccine adjuvant, or a feed additive for an animal comprises the composition for increasing immunity as an active ingredient.

Description

An extract from Bupleuri Radix having improved immunity capability of animals and its use}

The present invention relates to novel Bupleuri Radix extracts and their use to enhance the immune capacity of animals. More specifically, the present invention enhances various activities of neutrophils and macrophages, which are the main immune cells of animals, and extracts the extract of Shiho, which induces a strong defense against the inoculation of pathogens, as an active ingredient to efficiently contain animal diseases. The present invention relates to animal medicines such as treatment, prevention of animal immunopotentiators, adjuvants to improve the efficacy of vaccines, and adjuvant treatments to enhance therapeutic efficacy, and feed additives.

In animals, the primary resistance to various pathogenic microorganism invasion is neutrophils, macrophages, and lymphocytes, which are the main immune cells in the body. Microorganisms infect and multiply and develop. Immunopotentiators enhance resistance to disease infection by enhancing the function of various immune cells, which play a major role in defense. These immune enhancing substances are known to exist in various organisms such as animals, plants, and microorganisms, and many substances extracted therefrom have been developed and used as immune enhancers for humans and animals.

Conventional techniques related to immune-enhancing substances extracted from nature include ethanol precipitation fraction of 30-75% from thyme hydrothermal extract and molecular weight of 300,000 or more in Korean Patent Publication No. 2001-0037497 (Thyme extract having complementary system activation function and preparation method thereof). Ultrafiltration, anion exchange chromatography, hydrophobic chromatography, and gel chromatography provide complement-activated polysaccharides to find immune-boosting effects of thyme extract, which can be useful in functional foods or drugs for cancer patients or immunocompromised patients. have.

In addition, Korean Patent Publication No. 2002-0004083 (processed food containing peony or nutmeg extract) uses peony and / or nutmeg extract, which are natural herbal extracts, not artificial preservatives, to improve water retention and suppress pH drop, Disclosed is a processed food having a high food preservation effect.

Furthermore, in Korean Patent Laid-Open Publication No. 2003-0091665 (a new ginseng leaf and stem polysaccharide, a method for preparing the same and an anticancer and anticancer adjuvant composition containing the same as an active ingredient), the polysaccharide component isolated from the ginseng leaf and / or stem is an active ingredient. An anticancer agent, a cancer metastasis inhibitor and / or an anticancer agent or an anticancer adjuvant composition excellent in hematopoietic promotion, bone marrow defense, and radiation sensitivity are disclosed.

In general, the main ingredient of Shiho is known as saikosaponin and its derivatives, and antipyretic, analgesic, antitussive, chronic hepatitis treatment, and pruritus are used as main drugs in oriental medicine. Also in the liver due to the effective T peuseu vaccine, ethanol, or the like, but used as organophosphate hanyakje used in one shot, and extract useful materials from the plant Bupleurum, in vitro from these (in in vitro and in experimental animals ( in In vivo , there have been no reports on the efficacy of immunopotentiation.

Therefore, the present inventors extracted a new useful substance from the natural material Shiho by physical method, and then tested the effect of enhancing the immune cell activity of the extract and the non-specific defense against the pathogenic microorganism inoculation in the experimental animal. As a result, it confirmed the immunopotentiation activity of Shiho extract and came to complete this invention.

In order to solve the conventional problems as described above, an object of the present invention is to provide a novel method for purifying a novel extract of Shiho extract having excellent immuno-enhancing activity as part of the research on the development of immune-enhancing substances using natural substances.

In addition, another object of the present invention is to provide a novel use of the purified Shiho extract as an animal enhancer, vaccine adjuvant and adjuvant, feed additives and the like.

In order to achieve the above object, Shiho extract purification method of the present invention is characterized in that the heat treatment, concentration from Shiho. In particular, in the heat treatment, performing hot water extraction is preferable in view of preventing the destruction of components.

In addition, Shiho extract of the present invention confirms the juvenile ability to the neutrophils and macrophages of the main immune cells of animals, mouse spleen cell proliferation ability, cytokine acid production and mouse defense effect against the pathogenic microorganisms challenge and extracts immune enhancing substances It is characterized by demonstrating efficacy as an active ingredient, and providing a composition comprising the same as an active ingredient, an animal adjuvant, a vaccine adjuvant and a therapeutic adjuvant, and a feed additive.

Hereinafter, the technical method of the purification method and the use of the extract of Shiho extract of the present invention will be described in detail.

In the present invention, 3-10 times of water is added to the siho, and heat extraction is performed 2-5 times for 2-3 hours, and the obtained extracts are collected. At this time, the heating conditions are preferably performed at 80-120 ℃, more preferably around 100 ℃.

The seahawk is more preferably used lightly washed seahawk extract to remove dust and the like.

In this case, the extract and the residue are separated using a filter paper to remove the residue in the extract, and more preferably, a microfiltration process is carried out to remove even the small particles suspended, and sterilization by secondary filtration using 0.45㎛ bacterial filter paper. It is most desirable because it can be performed together.

Or in general, the hydrothermal extract of natural products is a mixture of low-molecular substances such as alkaloids, flavonoids, terpenoids and high-molecular substances such as polysaccharides, proteins, tannins, etc., 3 to 4 times in the extract to recover the polysaccharides By adding ethanol to the extent, the polysaccharide precipitates and the crude polysaccharide fraction can be obtained by recovering the precipitate.

The collected hot water extracts are then mixed and lyophilized. At this time, the drying method is selected from the general hot air, vacuum drying, freeze drying, spray drying. Depending on the formulation or use desired, more effective and additional separation or purification methods may be employed.

The obtained extract was concentrated in concentration of 100 ml, dried amount of 1.49 g / 20 ml, and extraction efficiency of 7.45 g / 100 g (%). In addition, the extract is Shiho polysaccharide, the sugar of the polysaccharide is characterized in that it comprises a protein polysaccharide containing protein, in particular, the protein content is characterized in that consisting of 0.0001 to 2%.

Specifically, the sugar composition of the polysaccharide is characterized by including rhamnose, fucose, arabinose, xylose, mannose, glucose, galactose, furactose, galacturonic acid, glucuronic acid and KDO, molecular weight of the polysaccharide Has a range of 250,000 Da to 6,000 Da, and a main peak is detected at 140,000 Da. In addition, when analyzed by ion chromatography, it contains 2.85 mg / ml or more of glucose which is monosaccharide, and 2.38 mg / ml or more of furactose.

With respect to the extract obtained as described above through the various experiments to determine the immune enhancing action:

One. Goat random blood neutrophils (neutrophil) migration capacity (Random migration) Looking at the enhancement effect, the area of the migration of neutrophils treated with Bupleurum extract each ² 8.25mm, 7.14mm ^2, 16.02mm 13.66mm ^{2 2} significant compared to the control group (p <0.05) showed difference. However, 1 mg and 100 ㎍ treatment showed lower random jujube than control group.

2. In the mouse peritoneal macrophages nitric oxide (NO) production ability, nitric oxide was detected in all mouse abdominal macrophage cultures treated with Shiho extract at different concentrations and treated with 1mg and 100㎍ / ml. 0.78 and 0.39 micro-moles, respectively, were detected in macrophages.

3. As a result of examining the effect on the mouse splenocyte proliferation ability, it was confirmed that the proliferation capacity of splenocytes treated with 100 μg, 10 μg / ml concentration of Shiho extract was significantly higher (p <0.01 ~ 0.05) than the control group. It was.

4. In the mouse blood cytokine production, gamma interferon was detected in 62.5 picograms (pg / ml) and interleukin-2 was detected in 15.6 picograms in control mice with little detected gamma interferon and interleukin-2. In comparison, the major cytokine production ability in the blood is greatly improved.

5. As a result of mouse defense against pathogenic staphylococcal challenge, mice inoculated with S. aureus survived in pathogenic staphylococcus vaccination and showed 100% protection rate. Nonspecific defense ability could be confirmed.

As a result, according to the purification process consisting of hot water, concentration according to the present invention, it is possible to obtain a seaweed extract that enhances the activity of various immune cells of the animal and improves the non-specific defense against pathogenic microorganisms.

Shiho extract thus obtained can be utilized in the manufacture of a variety of veterinary medicine. At this time, the veterinary medicine can be used as an animal immunopotentiator that can effectively treat and prevent animal diseases, as a supplement for improving the efficacy of vaccines, as an adjuvant that can enhance the therapeutic efficacy, and as a feed additive. .

For this purpose, the composition of the present invention may be used on its own or in combination with a carrier commonly used in the pharmaceutical field for oral administration of conventional agents in the pharmaceutical field, such as tablets, capsules, solutions, suspensions and the like. It may be formulated into various preparations, such as preparations, injectable preparations or suspensions, feed additives and the like. In addition, in order to prevent the decomposition of the drug by gastric acid during oral administration, an antacid may be used in combination or a solid preparation for oral administration such as tablets may be formulated into a formulation coated with enteric skin.

The dosage of the polysaccharide fraction of Shiho extract according to the present invention to the human body should be suitably used according to the absorbency, inactivation rate and excretion rate of the active ingredient in the body of the animal, the age, sex and condition of the animal, but generally 1 kg An amount of 100-1000 mg / day, preferably 2-5 mg / day, is to be administered.

Specifically, in the animal immune enhancer comprising Shiho extract obtained by the present invention as an active ingredient, the extract is composed of a polysaccharide comprising a protein polysaccharide, prepared in a lyophilized state and then diluted in sterile distilled water upon use for injection. It can be prepared as an animal immune enhancer.

In addition, in the agent for improving the efficacy of a vaccine comprising the extract of Shiho obtained by the present invention as an active ingredient, the extract is composed of a polysaccharide including a protein polysaccharide, and is used as an antigen of an inactivated vaccine or as a counseling agent of a vaccine. Vaccine supplements can be prepared by injection into gels or oil-based oil supplements. In this case, as the gel used as a backing agent of the vaccine, it is not limited to this, but a commercially available aluminum hydroxide gel or the like is sufficient.

In addition, in the adjuvant therapeutic agent for enhancing the therapeutic efficacy of an animal comprising Shiho extract obtained by the present invention as an active ingredient, the extract is composed of a polysaccharide comprising a protein polysaccharide, produced in a lyophilized state and then used in sterile distilled water. An adjuvant therapeutic agent for enhancing the therapeutic efficacy of an animal injected by dilution can be prepared.

Furthermore, in the animal feed additive comprising Shiho extract obtained by the present invention as an active ingredient, the extract is composed of a polysaccharide comprising a protein polysaccharide, produced in the form of a powder for animal feed further included in the animal feed Additives can be prepared. In this case, the animal feed may be selected from the group consisting of animal feed, calf substitute oil or granulated feed of piglets, and the content of the powdered extract may be within the range of 100-1000mg (0.1% level) per kg of feed. Do.

Hereinafter, one or more exemplary embodiments will be described in detail in order to show the structure and effects of the present invention.

<Example>

Example  One: Shiho Hot Spring  Extract manufacturer

100 g of the lake was washed with water to remove dust. At this time, since most of the drugs are water-soluble, the active ingredient of the drug is washed out for a long time, so it was lightly washed as much as possible.

Shiho washed the medicinal herbs in a white bag for boiling water, put 1000ml of distilled water and soaked for about 20-30 minutes to soak the medicine and collected the extract of shou extract decoction for 150 minutes. Again, 800 ml of distilled water was added and the extract of Shiho extract extract decocted for 150 minutes was collected. Then, 700 ml of distilled water was added thereto and the extract of Shiho extract decoction decoction was collected for 150 minutes.

Collect the extract extracted three times and concentrate to 100ml. At this time, pay attention not to burn the concentrate, if burnt, discarded and extracted again.

The extract concentrated to 100 ml was lyophilized and the dried extract was weighed. The content of Shiho extract extracted by the above extraction method is as follows: Sample concentration; 100 ml, dry weight; 1.49 g / 20 ml, extraction efficiency; 7.45g / 100g (%). In addition, when analyzed by ion chromatography, it contained 2.85mg / ml of monosaccharide glucose and 2.38mg / ml of furactose, and it was confirmed that the protein content as protein polysaccharide was 0.0001 to 2%.

Example  2: Goat blood neutrophils neutrophil ) Random Yoo Ju  ( Random migration Augmentation effect

1) Isolation of Goat Blood Neutrophil and Mixed Treatment with Seahorse Extract

Blood was collected from the jugular vein of goats and mixed with anticoagulant ACD (ACD, sodium citrate, citric acid, dextrose) and centrifuged at 2,500 xg for 20 minutes to remove the buff coat and hemolyzed red blood cells. minutes, separated by a number of neutrophils after collecting neutrophils rpm children (RPMI1640) medium were centrifuged and used in the test so that the floating 1x10 ⁷ / ml. In order to mix neutrophils with Shiho hot water extract, the dried extracts stored in refrigeration were suspended in AlpM medium at 1mg / ml concentration, diluted 10 times (100㎍ / ml), 100 times (10㎍ / ml) and the same amount. (30 μl) of neutrophil suspension (1 × 10 ⁷ / ml) was mixed and treated at 37 ^° C. for 1 hour in a CO ₂ incubator.

2) random Yoo Ju  ( Directional migration Augmentation effect

10 μl each of the neutrophils mixed with the core lake extract in Example 2-1) was placed in a hole made 3 mm in diameter on an agarose plate, and then applied at 37 ^° C. in a carbon dioxide incubator for 8 hours. After fixing with gurualdehyde solution, the gel was removed, washed and dried. The dried plate was stained with a start stain (Stat stain, Volu-Sol, Inc.) to measure the distance of neutrophil drift under a microscope (MC-50T, MEIJI / Japan), and the area of neutrophils in Equation 1 below. Calculated based on.

Perimeter area = πr ² -π (hole radius) ²

The effects of random neutrophils on goat blood neutrophils treated with Shiho extract by dilution multiples are summarized in Table 1 below.

Effect of random neutrophils on goat blood neutrophils treated with Shiho extract     division  Treatment concentration (/ ml) Slope area (mm ² )   Remarks  Shiho extract   1mg     8.25 ± 0.07   100 µg     7.14 ± 0.54   10 µg     16.02 ± 1.02 *    Control     -     13.66 ± 3.28

   As shown in Table 1, the effects on the random chemotactic capacity of neutrophils in goat blood treated by dilution fold with Shiho extract are shown in Table 1. The neutrophils treated with neutrophils at concentrations of 1 mg, 100 μg and 10 μg / ml were 8.25, 7.14 and 16.02, respectively. <0.05) difference. However, 1 mg and 100 ㎍ treated concentration showed lower random jujube than control group.

Example 3: mouse peritoneal macrophages nitric oxide of Shiho extract nitric oxide , NO ) Birth

1) Separation of mouse abdominal macrophage

Treatment of 25g of Icyal (ICR, ♂) mice with cervical distal bone method and infusion of 5ml of phosphate buffered saline (PBS, pH 7.2) into the abdominal cavity to collect intraperitoneal macrophages and washing twice with phosphate buffered saline Suspended in AlMPM medium to 2x10 ⁶ / ml and aliquoted 200μL to the crude culture microplate and incubated for 2 hours at 37 ^° C in a carbon dioxide incubator. After incubation, the plate was washed three times with warm ALPM medium to remove cells that did not adhere to the plate and macrophages attached to the bottom were used for the test.

2) Nitric Oxide (NO) Acid Production

Dilute Siho extract to 1mg, 100µg, 10µg / ml in each hole of microplates with macrophages so as to disperse each 50µL holes in concentration and 37 ^o C in CO ₂ incubator Incubated for 48 hours. A standard curve of sodium nitrite used as a standard solution is obtained by mixing 100 μl of the supernatant of each hole with 50 μl of Gries' reagent, and reading the absorbance at an absorbing wavelength of 540 nm using an ELISA reader (Titertec multiscan). The amount of nitric oxide produced was measured.

The effects on nitric oxide acid production of mouse abdominal macrophages treated with Shiho extract by dilution multiples are summarized in Table 2 below.

Nitric Oxide Production of Mouse Peritoneal Macrophages Treated with Shiho Extract       division  Treatment concentration (ml)  Optical Density (O.D)  Nitric Oxide Concentration (micro-M)  Remarks   Shiho extract  1mg    0.1550    0.78  100 µg    0.1415    0.39  10 µg    0.1293    0.19   Control    -    0.1273    <0.19    Standard Nitric Oxide    -    0.1593 0.1347 0.1290     0.78 0.39 0.19

As shown in Table 2, nitric oxide was detected in all mouse intraperitoneal macrophage cultures treated with Shiho extract 1 mg, 100 μg, and 10 μg / ml, and 0.78 and 0.39 micromol, respectively, in macrophages treated with 1 mg and 100 μg / ml. 0.19 micromole was detected in macrophage cultures treated with (micro-mole) at 10 μg / ml.

Example 4: Mouse Spleen Cell Enhancement of Shiho Extract (proliferation)

1) Spleen Cell Isolation

 Twenty five grams of ISI (ICR, ♂) mice were treated with cervical dislocation, the abdominal wall was dissected to separate the spleen, and the spleen cells were collected by a homogeneous cast method.

Erythrocytes mixed in the spleen cells were removed by hemolysis, washed twice with phosphate buffered saline, and suspended in AlfM medium to give a cell number of 2 × 10 ⁶ / ml, and 100 μl were dispensed into each hole of the microculture plate.

2) Proliferation investigation

To each hole containing mouse spleen cells, 50 μl of Seaho extract and Con A (Con A, 15 μg / ml) diluted to 1 mg, 100 μg, and 10 μg / ml with AlMPE medium were added to each hole, Incubated for 48 hours at 37 ^° C in a gas incubator.

50 μl of empty reagent (MTT, 4mg / ml) was added to each plate of incubated plate, and then reacted for 4 hours at 37 ^o C in a carbon dioxide incubator. The supernatant was removed and 200 μl of DMS solution (DMSO: EtOH = 1 : 1) 200 μl and 50 μl of Sorenson's buffer were added to dissolve the formazan produced in the reaction.

The absorbance was read at an absorbing wavelength of 560 nm with an ELISA reader (Titertec multiscan) to measure germination, and the effects on the growth of mouse spleen cells treated by dilution multiples of Shiho extract were summarized in Table 3 below. .

Effect on Growth Potency of Mouse Spleen Cells Treated with Seahawk Extract      division  Treatment concentration (ml)  Optical Density (O.D)  Remarks  Shiho extract  1mg   0.1687 ± 0.03  100 µg   0.2867 ± 0.025 **  10 µg   0.2287 ± 0.013 *   Control     -   0.2157 ± 0.011

As shown in Table 3, the growth capacity of splenocytes treated with 100 g, 10 µg / ml concentrations of Shiho extract was significantly (p <0.01 ~ 0.05) higher than that of the control group, but was increased when treated with a high concentration of 1 mg. This was lower than the control.

Example 5 Shiho Extract Inoculated Mouse Blood Cytokine (Interleukin-2 and Gamma Interferon) Production

1) Mouse Inoculation and Blood Collection of Windproof Extracts

 Shiho extract was prepared in sterile distilled water at a concentration of 100 ㎍ / ml, inoculated with 0.2 g of 2 water with 25 g of Icyal (ICR, ♂) mouse intraperitoneally, and collected from the orbital artery after 3 days, and then frozen and stored immediately. The amount of cytokines in the serum was tested.

2) Interleukin-2 and Gamma Interferon Amount  Research

 Interleukin-2 and gamma interferon production in the mouse serum was investigated using the instant eliza kit (Bender MedSystems, Austria).

That is, 50 μl of serum diluted twice with diluent and 100 μl of distilled water were added to the microplate holes of the cytokine kits of each type, and reacted for 3 hours at room temperature, followed by washing six times with a wash buffer. 100 μl of TMB substrate solution was added thereto, followed by 10 minutes of color development at room temperature. Then, 100 μl of stop solution was added and the absorbance was absorbed at an absorption wavelength of 450 nm (ELISA reader, Titertec multiscan). The interleukin-2 and gamma interferon production were measured using the standard curve of the standard product.

 Cytokine production in mice inoculated with Shiho extract was summarized in Table 4 below.

Serum Cytokine Production in Mice Extract Inoculated Mice  Substance  Inoculation concentration   Interleukin-2 (IL-2) Gamma Interferon (IFN-gamma)    Remarks   Optical density (O.D)  Production amount (pg / ml)  Optical density (O.D)   Production amount (pg / ml) Shiho extract 100 µg / ml   0.0900   15.6   0.1515    62.5  Control     _   0.0685   <7.81   0.0790    <7.81

As shown in Table 4, 62.5 picograms (pg / ml) of gamma interferon was detected and 15.6 picograms of interleukin-2 were detected, resulting in major cytokine production in the blood compared to control mice in which gamma interferon and interleukin-2 were rarely detected. This has been greatly improved, especially for gamma interferon.

Example 6 Protective Effect of Shiho Extract Against Pathogenic Staphylococcus aureus Inoculation

Inoculation and Aggression of Seahawk Extracts

 Shiho extract was prepared in sterile distilled water at a concentration of 100 ㎍ / ml, inoculated with 0.2 g of 4 water with 25 g of Icyal (ICR, ♂) mouse intraperitoneally and after 3 days, the minimum lethal dose with Staphylococcus aureus (strain 289). 10 times (10MLD / 0.3ml) was inoculated intraperitoneally.

The results of examining the protective effect at 5 days survival rate are summarized in Table 5 below.

Protective Effect of Shiho Extract Against Pathogenic Streptococcus Inoculation  Substance    Inoculation (0.2ml, abdominal cavity)    Attack      Protective effect (%)    Remarks  Shiho Extract   100 µg / ml    0.3 ml (10 mld) abdominal cavity       0/4 (100)  Control      -        “       4/4 (0)

As shown in Table 5, the mice inoculated with Shiho extract survived all of the pathogenic staphylococcus challenge and exhibited a 100% protection rate, and all of the control mice died to confirm the excellent nonspecific defense ability of Shiho extract.

Preparation Example 1 Animal Immune Enhancer

For use as an animal immune enhancer, the extract of Example 1 was prepared in a glass bottle in a lyophilized state and made available for injection. For future use, it is sufficient to dilute it properly with sterile distilled water.

Preparation Example 2 Vaccine Adjuvant

Improve the efficacy of the vaccine by injecting the extract of Example 1 with the injection of the extract of Example 1 into the antigen of the inactivated vaccine or the gel (aluminum hydroxide gel) or oily assistant (oils) used as the agent for the vaccine. You can.

Preparation Example 3 Adjuvant

In order to use when treating diseases of animals with antibiotics or the like, the extract of Example 1 was prepared in a glass bottle in a lyophilized state and made available for injection. For future use, it is sufficient to dilute it properly with sterile distilled water.

Preparation Example 4 Animal Feed Additive

Animal feed, calf substitute oil, granulated feed of piglets, etc. The extract of Example 1 produced in the form of powder was added to the 0.1% level (100㎍-1000mg / kg) was prepared by feeding.

Shiho extract according to the present invention is a novel substance which is not yet known for its immune enhancing effect at home and abroad, so it is a plant widely distributed in Korea. It is expected to improve the efficacy of nutrients, which can greatly reduce the economic damage caused by livestock diseases of livestock farmers, and has the advantage of localizing veterinary medicines that depend on foreign imports.

In addition, the present invention has a useful effect in terms of the development of additional income sources of farmhouses because the cultivation of the Shiho is possible in the country.

Claims (18)

Sea Lake ( Bupleuri Heat treating Radix ) to obtain a crude extract; Concentrating the extract; And Filtering the concentrate to obtain a polysaccharide fraction; The method of claim 1, wherein the heat treatment is a method of purifying Shiho extract, characterized in that the hot water extracted for 2-3 hours at 80-120 ℃ The method of claim 1, wherein the filtration is a method for purifying Shiho extract, characterized in that using a filter paper and bacterial filtration sequentially. Shiho extract purified by any one of claims 1 to 3 [Claim 5] The extract of claim 4, wherein the extract is a polysaccharide of Siho, and the extract of the polysaccharide comprises a protein polysaccharide including a protein. [Claim 6] The extract according to claim 5, wherein the protein content is comprised from 0.0001 to 2% and contains as a monosaccharide at least 2.85 mg / ml of glucose and at least 2.38 mg / ml of fructose. 6. The extract of claim 5, wherein the sugar composition of the polysaccharide comprises rhamnose, fucose, arabinose, xylose, mannose, glucose, galactose, furactose, galacturonic acid, glucuronic acid and KDO. The extract of claim 5, wherein the polysaccharide has a molecular weight of 250,000 Da to 6,000 Da. Immunity-enhancing composition comprising Shiho extract according to any one of claims 4 to 8 as an active ingredient 10. A composition according to claim 9 comprising a pharmaceutically acceptable carrier or excipient. The composition of claim 10, further comprising an antacid. 10. The composition of claim 9, further comprising a pharmaceutically acceptable carrier or excipient in the form of an edible beverage or food. An animal comprising the composition according to any one of claims 9 to 12 as an active ingredient, the composition consisting of a polysaccharide comprising a protein polysaccharide, prepared in a lyophilized state and then diluted and injected into sterile distilled water for use. Immune enhancers Claim 1 to claim 12, wherein the composition according to any one of the active ingredient, the composition is composed of a polysaccharide comprising a protein polysaccharide, oil or oil of gels or oils to be used as an antigen of the inactivated vaccine or as a supplement for the vaccine Vaccine backing agents injected in addition to backing agents An animal comprising the composition according to any one of claims 9 to 12 as an active ingredient, the composition consisting of a polysaccharide comprising a protein polysaccharide, prepared in a lyophilized state, and then diluted with sterile distilled water and used for injection of the animal. Supplementary treatment for enhancing therapeutic efficacy An animal feed additive comprising the composition according to any one of claims 9 to 12 as an active ingredient, the composition consisting of a polysaccharide comprising protein polysaccharide and made into a powder form and further included in the animal feed. The animal feed additive according to claim 16, wherein the animal feed is selected from the group consisting of animal powder feed, calf substitute oil or granulated feed of piglets. 18. The animal feed additive according to claim 17, wherein the content of the powdered active ingredient is in the range of 100-1000 mg per kg of feed.