KR20080114231A - An extract from zanthoxylli fructus crust having improved immunity capability of animals, a method for preparing thereof, and its use - Google Patents

Abstract

A method for manufacturing an immune enhancing composition using Zanthoxylli Fructus crust Fizeau polysaccharide extracts as an active ingredient is provided to have a superior immunostimulating activity, to enhance an immunogenicity of animal and to be used as an immunoadjuvant for animal, a vaccine throne and an adjuvant treating agent. A method for manufacturing an immune enhancing composition using Zanthoxylli Fructus crust Fizeau polysaccharide extracts as an active ingredient comprises steps of: obtaining crude extracts by heating Zanthoxylli Fructus crust; concentrating the extracts; and obtaining the polysaccharide fraction by filtering the condensed products.

Description

An extract from Zanthoxylli Fructus crust having improved immunity capability of animals, a method for preparing approximately, and its use}

The present invention relates to a novel Zanthoxylli Fructus crust extract, its preparation and use to enhance the immune capacity of animals. More specifically, the present invention enhances various activities of neutrophils, macrophages and lymphocytes, which are the main immune cells of animals, and extracts of Sanchopi, which induces a strong defense against the inoculation of pathogenic bacteria, by containing them as an active ingredient in diseases of animals The present invention relates to an animal drug such as an animal adjuvant that can effectively treat and prevent the adjuvant, an adjuvants that can improve the efficacy of the vaccine, and an adjuvant that can enhance the therapeutic efficacy.

In animals, the primary immune cells that can resist various invasion of pathogenic microorganisms are neutrophils and macrophages, which are the main immune cells in the body. Secondary immune cells that lead to specific immunity are lymphocytes. , T / B cells), and when their activity is abnormal, pathogenic microorganisms develop and infect. Immunopotentiators enhance the function of various immune cells, which play a major role in defense against diseases, thereby increasing specific and nonspecific resistance to disease infection. 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, an anticancer agent, and / or 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 components of Sanchopi are piperitol and xanthoxylol, which are herbal medicines used in Chinese medicine for the treatment of aesthetic dryness, intestinal parasite control, tonic action and dermatological diseases.

Therefore, the present inventors extracted a new useful substance by physical method from Sanchopi which is a natural substance, and then tested the effect of enhancing the immune cell activity of the extract and nonspecific defense against the pathogenic microorganism challenge in experimental animals. As a result, the immunopotentiating activity of the extract of Sancho skin was confirmed and the present invention was completed.

In order to solve the conventional problems as described above, an object of the present invention is to provide a novel method for purifying the extract of Sancho skin, particularly as part of the research on the development of immune enhancing substances using natural substances.

Another object of the present invention is to provide a novel use of purified sanchocho extract as an animal adjuvant, vaccine adjuvant and adjuvant therapy.

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

In addition, the extract of Sanchopi of the present invention confirmed the mouse defense effect against neutrophils, which are the main immune cells of animals, mouse splenocyte growth ability, cytokine acid productivity improvement and pathogenic microbial challenge and the extract as an immune enhancing substance It is characterized by demonstrating efficacy and providing a composition comprising the same as an active ingredient for the use of an animal adjuvant, vaccine adjuvant and therapeutic adjuvant, respectively.

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

In the present invention, 3-10 times of water is added to the acidic bark 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 acid chopi is more preferably used by washing lightly 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 1.05 g / 5 ml, and extraction efficiency of 21.0 g / 100 g (21%). In addition, the extract is an acid sheath polysaccharide, and the sugar of the polysaccharide contains 0.24 mg / ml or more of a monosaccharide glucose when analyzed by ion chromatography.

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

One. The random migrating effect of neutrophils in goat blood was examined when random doubling area of neutrophils treated with Sanchopi extract was diluted 10 times (21mg / ml) of each stock solution (210mg / ml). 18.35mm ² , which has the largest basal area, showed a significant (p <0.05) difference compared to 17.43mm ² of the untreated control group.

2. peroxide in the activation (superoxide O ₂ ^-) in goats blood neutrophils acid saengneung Referring to improvement, when the stock solution was diluted 1000-fold processing optical density (OD) is significantly higher than the optical density of the untreated control group to 0.4720 0.5320 High (p <0.05).

3. Nitric oxide (NO) production of mouse peritoneal macrophages, Nitrogen oxide production used as an indicator of macrophage activation was 1.6 micromoles of oxidized cells in 100-fold dilution of the extract of Sanchopi extract. Nitrogen was detected and below 0.39 micromolar in untreated macrophages.

4. As a result of examining the effect on the mouse splenocyte proliferation ability, the number of splenocytes activated and proliferated by Sancho skin extract was tested by the MTT method. The density (OD) of 0.3177 was found to be significantly higher (p <0.05) than the optical density of 0.2369 of untreated splenocytes.

5. As a result of examining cytokine acid production in mouse, 31.25 pg / ml of cytokine interleukin-2, which is used as an indicator of cellular immune activity, was detected in the blood of mice inoculated with Sanchopi extract and almost no interleukin-2 was detected. Compared to the untreated control group, the major cytokine production in blood was greatly improved.

6. As a result of the mouse defense against pathogenic staphylococcal challenge, mice inoculated with 10 times dilution of the extract of Sanchopi extract showed 75% protection rate with 3 out of 4 survival in pathogenic staphylococcus challenge. All the control mice died and confirmed excellent non-specific defense ability of the extract of Sanchopi.

As a result, according to the purification process consisting of hydrothermal, concentrated according to the present invention, it is possible to obtain an extract of Sanchopi to enhance the activity of various immune cells of the animal and improve the non-specific defense ability against the pathogenic microorganisms.

Sanchopi extract thus obtained can be utilized in the manufacture of a variety of animal medicines. 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 Sanchopi extract according to the present invention to the human body should be appropriately 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. A dose of 100-400 mg / day, per kg, should be administered.

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

In addition, in the agent for improving the efficacy of a vaccine comprising an extract of Sanchopi obtained by the present invention as an active ingredient, the extract is composed of polysaccharides, and is used in oil-based supplements of gels or oils that are used as agents for inactivating vaccines. Vaccine assistants can be prepared by addition. 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 therapy for enhancing the therapeutic efficacy of an animal comprising an extract of Sanchopi obtained by the present invention as an active ingredient, the extract is composed of polysaccharides, prepared in a lyophilized state, and then diluted and injected into sterile distilled water upon use. Supplementary therapeutic agents for enhancing the therapeutic efficacy of animals can be prepared.

Furthermore, in the animal feed additive comprising the extract of Sanchopi obtained by the present invention as an active ingredient, the extract is composed of a polysaccharide, produced in a powder form to produce an animal feed additive further comprising in the animal feed Can be. At this time, 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 is sufficient if it is within the range of 1-2g (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: Sanchopi  Boiling water extract manufacturer

100 g of Sanchopi 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.

Put the medicinal herbs in a white bag for boiling water, put 1000ml of distilled water, soak for about 20-30 minutes, soak the medicine and collect the extract extracted from the extracts for 150 minutes. The extract was added to extract the extract extracted with 800ml distilled water and then extracted with 150ml decoction of medicinal herb 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 Sanchopi extract extracted by the above extraction method is as follows: Sample concentration; 100 ml, dry weight; 1.05 g / 5 ml, extraction efficiency; 21.0 g / 100 g (21%). Moreover, when this was analyzed by ion chromatography, it confirmed that it contained 0.24 mg / ml of glucose which is a monosaccharide.

Example  2: Sanchopi  Goat blood neutrophils of boiling water extract ( neutrophil Random randomness Random migration Augmentation effect

1) Isolation of Goat Blood Neutrophils and Sanchopi  Mixed treatment with 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 Sanchopi hot water extract, lyophilized extract (5ml / bottle) in refrigerated suspension was suspended in 5ml of AlfM medium to make stock solution, 10 times (21mg / ml), 100 times (2.1mg / ml), diluted to 1000-fold (0.21mg / ml) and mixed with the same amount (30μl) of neutrophil suspension (1x10 ⁷ / ml) and mixed at 37 ^° C, 1 hour in a CO ₂ incubator. .

2) random Yoo Ju  ( Directional migration Augmentation effect

10 μl each of the neutrophils mixed with the acidic blood extract in Example 2-1) was placed in a hole made 3 mm in diameter in an agarose plate, and then applied at 37 ^° C. for 18 hours in a carbon dioxide incubator. It was fixed with% gurualdehyde and 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 on the random chemotaxis of goat blood neutrophils treated with Sanchopi extract by dilution multiples are summarized in Table 1 below.

Effect of Sanchopi Extract on Random Journey of Goat Blood Neutrophils division Treatment concentration (/ ml) Slope area (mm ² ) Remarks                  Sanchopi extract   10x dilution (21mg)     18.35 ± 1.19   100 times dilution (2.1mg)     17.61 ± 1.63   1000x Heeseok (0.21ml)     17.83 ± 0.86 Control     -     17.43 ± 2.85

As shown in Table 1, the effect on the random chemotactic ability of neutrophils in goat blood treated with dilution multiples with Sanchopi extract was 18.35, 17.61, respectively. The neutrophils, which were diluted 10-fold compared to 17.43 of the untreated control group, were 17.83, which was not statistically significant but was found to be high.

Example  3: Sanchopi  Of extract Goat blood  Neutrophils ( neutrophil ) Antimicrobial  Peroxide ( superoxide , O ₂ ^- ) Acid production  Research

In order to sterilize neutrophils invading pathogenic pathogens, the microbial oxygen metabolite superoxide (O ₂ ⁻ ) acid production was tested by the NBT method to respiratory burst.

50 µl of neutrophils (2x10 ⁶ / ml) mixed with Sanchopi extract and 25 µl of each suspension of Zymosan suspension treated with Earls balanced salt solution (EBSS) and opsonized with chlorine complement Three times in a hole of a crude culture microplate (Costar, 96 wells), and the reaction was performed at 37 ° C. for 1 hour in a carbon dioxide incubator, and 100 μl of NBT (nitroblue tetrazolium) solution was added to each hole. (4mg / ml) was added and reacted for 3 hours in a carbon dioxide gas incubator. After dissolving purple formazan particles formed in proportion to the amount of superoxide produced by adding 100µl of DMSO, absorption wavelength was 560nm. Absorbance at was read with an ELISA reader (Titertec multiscan) to determine the amount of peroxide produced.

The effects on the peroxide acid production of goat neutrophils in goat blood treated with distilled fold extracts are summarized in Table 2 below.

Effects of Peroxide Acid Production on Serum Neutrophils in Goat Blood Treated with division Treatment concentration (/ ml) Optical Density (O.D.) Remarks                             Sanchopi extract   10x dilution (21mg)     - 10 times optical density could not be measured due to the color development of the sample itself.   100 times dilution (2.1mg)     0.4417 ± 0.086   1000x Heeseok (0.21ml)     0.5320 ± 0.069 *    Control     -     0.4720 ± 0.074

As shown in Table 2, the optical density (O.D.) value according to the peroxide acid production of neutrophils, which was diluted 1000 times, was significantly (p <0.05) higher than that of 0.4720 of the control group.

Example  4: Sanchopi  Mouse of extract Abdominal family  ( peritoneal macrophages Nitric Oxide nitric oxide , NO ) Birth

In this example, the nitric oxide production was used as a measure of the activity of macrophages that play a pivotal role in specific and nonspecific immune responses in the body.

1) mouse Abdominal family  detach

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 in a crude microplate for incubation at 37 ^o C, 2 hours 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  Research

Dilute 10 times, 100 times, and 1000 times of Sanchopi extract with AlMPM medium in each hole of microplates with macrophages. Dispense 50μL into each hole in each concentration. 37 ^o C, 48 in CO ₂ incubator. Incubated for 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 peritoneal macrophages treated with extracts of dichotomy diarrhea were summarized in Table 3 below.

Nitric Oxide Production of Mouse Peritoneal Macrophages Treated with Sanchopi Extract division  Treatment concentration (ml)  Optical Density (O.D)  Nitric Oxide Concentration (micro-M) Remarks                                Sanchopi extract  10x (21mg)    0.1123    <0.19  100 times (2.1mg)    0.1543    1.60  1000x (0.21mg)    0.1133    <0.19 Control    -    0.1273    <0.19 Standard Nitric Oxide    -    0.1980 0.1450 0.1367     3.12 1.60 0.78

As shown in Table 3, it was confirmed that 1.6 micromole was detected in macrophages treated with 100-fold in mouse celiac macrophage culture diluted 10 times, 100 times, and 1000 times.

Example  5: Sanchopi  Mouse spleen cells of extract ( spleen cell ) Hypertrophy ( proliferation )

In this example, the activity of lymphocytes, which are the main immune cells of specific immune responses, such as humoral and cellular immune responses, will be disclosed by mouse spleen cells and tested by the MTT method.

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) Hypertrophy  Research

To each hole containing mouse spleen cells, 50 μl of Concho (Con A, 15 µg / ml) diluted 10 times, 100 times, and 1000 times with AlMPI medium was added to each hole. Incubated at 37 ^° C. for 48 hours.

50 μl of empty reagent (MTT, 4mg / ml) was added to each plate of the finished plate, and reacted for 4 hours at 37 ^o C in a carbon dioxide incubator, and then 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 purple formazan particles produced in the reaction.

The absorbance was measured by an ELISA reader (Titertec multiscan) at an absorption wavelength of 560 nm to measure germination, and the effects on the growth of mouse spleen cells treated by dilution multiples of Sanchopi extract were summarized in Table 4 below. It was.

Effect on the Spleen Capacity of Mouse Spleen Cells Treated with division  Treatment concentration (ml)  Optical Density (O.D)  Remarks                                Sanchopi extract  10x (21mg)   0.1763 ± 0.042  100 times (2.1mg)   0.3177 ± 0.061 *  1000x (0.21mg)   0.2617 ± 0.037 Control     -   0.2360 ± 0.050

As shown in Table 4, the optical densities of the 100- and 1000-fold dilutions were 0.3177 and 0.2617, respectively, higher than 0.2360 of the untreated control group, especially when treated at 100-fold (p <0.05). The difference is shown in the control group.

Example  6: Sanchopi  Inoculated Mouse Blood Cytokines (Interleukin-2 and Gamma Interferon) Production

In the present Example, the effect of Sanchopi extract on the cellular immunity was inoculated with Sanchopi extract by the production of interleukin 2 (IL-2) and gamma interferon (IFN-γ), the main cytokines of cellular immunity. I want to test it.

One) Sanchopi  Mice Inoculation and Blood Collection

Sanchopi extract undiluted solution was prepared in sterile distilled water at a concentration of 100 ㎍ / ml, inoculated with 0.2 ml of two waters with 25 g of Icyal (ICR, ♂) mouse intraperitoneally, and after 3 days, blood was collected from the orbital artery. During storage, the amount of cytokines in 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.

 The blood cytokine production in mice inoculated with Sanchopi extract was summarized in Table 5 below.

Blood Cytokine Production in Mice Inoculated with Sanchopi Extract                 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) Sanchopi extract 100 µg / ml   0.0899   31.25   0.1154    <7.81   The test serum was diluted 2x and converted to twice the standard O.D.value cytokine.                 Standard     _   0.1064 0.0874 0.0770   31.25 15.62 7.81   0.1319    7.81

As shown in Table 5, 31.25 pg of interleukin-2 was detected but no gamma interferon was detected.

Example  7: Sanchopi  Pathogenic staphylococci of extracts ( pathogenic Staphylococcus  aureus) for challenge vaccination Defense  Enhancement effect

This example is to challenge the pathogenic Staphylococcus aureus and test the mouse's defense against the pathogenic microorganisms of the extract of Sanchopi extract.

One) Sanchopi  Inoculation and Attack Vaccination of Extracts

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

The results of examining the protective effect at 5 days survival rate are summarized in Table 6 below.

Protective Effect of Sanchopi Extract Against Pathogenic Streptococcus Inoculation Substance Inoculation (0.2ml, abdominal cavity) Attack Protective effect (%) Remarks  Shiho Extract  100 times (2.1mg / ml)    0.3 ml (10 mld) abdominal cavity       1/4 * (75)  Control      -        “       4/4 (0)

* Number of our mice / vaccinated mice x 100

As shown in Table 6, the mice inoculated with Sanchopi extract showed 75% protection rate by surviving 3 out of 4 numbers in the pathogenic staphylococcus challenge, and all the control mice died and showed excellent nonspecific defense ability of the Sanchopi extract. Could confirm.

Production 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.

Production Example  2: vaccine Throne

The efficacy of the vaccine can be improved by injecting 100 μg −1 mg / ml of the extract of Example 1 into a gel (aluminum hydroxide gel) or oily assistant (oils) used as a supplement for an inactivated vaccine.

Production 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. In case of future use with antibacterial agent, it is sufficient to dilute properly in sterile distilled water.

Production Example  4: Animal Feed Additives

Animal feed, calf substitute oil, granulated feed of piglets, etc. The extract of Example 1 produced in powder form was added to the 0.1% level (1-2 g / kg) was prepared by feeding.

Sancho bark extract according to the present invention is a novel substance that is not yet known for its immune enhancing effect at home and abroad, so it is easy to obtain a large amount of extracts and is confirmed to enhance various immunity of animals. It is expected to improve the efficacy of the vaccine, which can greatly reduce the economic damage caused by livestock diseases in 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 development of additional income sources of farmhouses because the cultivation of the sancho bark is possible in the country.

Claims (14)

Heat treating Zanthoxylli Fructus crust to obtain a crude extract; Concentrating the extract; And Purifying the concentrate to obtain a polysaccharide fraction; Purifying method of Sanchopi extract comprising The method of claim 1, wherein the heat treatment is a hot water extract for 2-3 hours at 80-120 ℃ The method of claim 1, wherein the filtration is a method for purifying Sanchopi extract, characterized in that using a filter paper and bacterial filtration sequentially. Sanchopi extract purified by any one of claims 1 to 3 The extract of claim 4, wherein the extract is an acid sheath polysaccharide, and the sugar of the polysaccharide extract contains 0.24 mg / ml or more of glucose. Immunity enhancing composition comprising the extract of Sanchopi of claim 4 or 5 as an active ingredient 7. A composition according to claim 6 comprising a pharmaceutically acceptable carrier or excipient. 8. A composition according to claim 7, further comprising a pharmaceutically acceptable carrier or excipient in the form of an edible beverage or food. An animal immune enhancer comprising the composition of claim 7 or 8 as an active ingredient, the composition consisting of a polysaccharide, lyophilized, and then diluted and injected into sterile distilled water for use. A vaccine backing agent comprising the composition of claim 7 or 8 as an active ingredient, which is composed of polysaccharides and injected into a gel or oil-based oil backing agent used as a backing agent for an inactivated vaccine. An adjuvant therapeutic agent for enhancing the therapeutic efficacy of an animal comprising the composition of claim 7 or 8 as an active ingredient, the composition consisting of a polysaccharide, lyophilized and diluted with sterile distilled water upon use. An animal feed additive comprising the composition of claim 7 or 8 as an active ingredient, wherein the composition is composed of polysaccharides and is made in powder form to be added to the animal feed. 13. The animal feed additive according to claim 12, wherein the animal feed is selected from the group consisting of animal powder feed, calf substitute oil or granulated feed of piglets. The animal feed additive according to claim 13, wherein the content of the powdered active ingredient is in the range of 1-2g per kg of feed.