KR20080106789A - Glycyrrhiza extracts increasing immunity of animals and method for preparing thereof - Google Patents

Abstract

A Glycyrrhiza hot water extract, a method for preparing the extract, and an immune enhance containing the extract are provided to improve the immune increasing activity of animals. A Glycyrrhiza hot water extract is prepared by adding 1,000 mL of distilled water per 100 g of the dried Glycyrrhiza to soak Glycyrrhiza for 20-30 min and boiling it down for 150 min to obtain an extract; adding 800 mL of distilled water per 100 g of the dried Glycyrrhiza to the Glycyrrhiza remaining after the extraction and boiling it down for 150 min to obtain an extract; adding 700 mL of distilled water per 100 g of the dried Glycyrrhiza to the Glycyrrhiza remaining after the extraction and boiling it down for 150 min to obtain an extract; collecting the obtained extract and concentrating it; and dry-freezing the concentrated one.

Description

Licorice extract and method for preparing the same to enhance the immune function of the animal {Glycyrrhiza extracts increasing immunity of animals and method for preparing carrot}

The present invention relates to a licorice extract that enhances the immune ability of the animal.

In animals, the primary resistance to the invasion of various pathogenic microorganisms is neutrophils, macrophages, and lymphocytes, which are the main immune cells in the body, and pathogenic microorganisms when their activity is abnormal. This infection, proliferation, and onset. Immunopotentiating substances enhance the resistance to disease infection by enhancing the function of various immune cells that play a major role in defense against such diseases. These immune enhancing substances are known to exist in various organisms such as various animals, plants, and microorganisms, and many substances extracted therefrom have been developed and used as immune enhancers for humans and animals.

Licorice (Glycyrrhiza) is a medicinal plant of the Rosacea legume, whose roots are reddish brown and deep in the ground, and the stalks grow straight by 1 to 1.5m. The leaves are alternated with the double leaves, and in summer, butterfly-shaped indigo purple flowers bloom. The reddish brown root is sweet and is used as a sweetener or medicinal herb. Herbal medicine is evenly used in all medicines. Mongolia, the northeastern part of China, is native to the world and is grown as a herb. Licorice is known as Glycyrrhizin as an active ingredient, and other ingredients are recognized to inhibit gastric secretion and ulcerative action. Licorice extract has also been found to have estrogen-positive, antitussive, anti-inflammatory, anti-allergic, detoxification, and hyperlipidemic effects.

Immune enhancers are extracted from various plants such as ginseng and mushrooms, but the immune enhancers are extracted from licorice, which is a herbal medicine, to enhance immune activity and pathogens in neutrophils, macrophages, and lymphocytes (splenocytes). There have been no reports on the development of technologies related to nonspecific defense enhancement.

Therefore, the present inventors extracted the new useful substance from the licorice, which is a herbal medicine, and tested the effect on the activity of various immune cells of the extract to confirm the excellent immune enhancing activity of the extract and came to complete the present invention.

Therefore, an object of the present invention is to provide a novel substance that can improve the immune ability of the animal extracted from licorice, and using the licorice extract excellent in the animal's immune enhancing activity, animal enhancers, vaccine adjuvant, adjuvant therapy, etc. It can be used to develop

In order to achieve the above object, the present invention provides a licorice hot water extract with immuno-enhancing efficacy and a method for producing the licorice hot water extract with immuno-enhancing efficacy.

Hereinafter, the present invention will be described in detail.

Licorice hot water extract provided in the present invention

(Iii) adding 1000 ml of distilled water per 100 g of dried licorice, soaking licorice for 20-30 minutes, and then decocting the medicine for 150 minutes to collect the extract;

(Ii) adding 800 ml of distilled water per 100 g of dried licorice to the licorice after decoction of (iii), decocting the medicinal herbs for 150 minutes, and collecting the extract;

(Iii) adding 700 ml of distilled water per 100 g of dried licorice to the licorice after decoction of (ii), decocting medicinal herbs for 150 minutes, and collecting the extract;

(Iii) collecting and concentrating the extract extracted in steps (iii), (ii) and (iii); And

(Iii) freeze drying the licorice extract concentrated in step (iii);

It is manufactured through the process of.

When the hot water extract is prepared from 100 g of dry licorice, the extract extracted three times in the step (iii) is concentrated to 100 ml.

The licorice hot water extract may be used as an adjuvant, an adjuvants used as an additive to improve the efficacy of the vaccine, an adjuvant to enhance the therapeutic efficacy, and a feed additive.

In the case of an immunopotentiator, the licorice hot water extract provided by the present invention is produced in a lyophilized form, suspended in a diluent when used, and used as an injection. It is adsorbed on aluminum gel). In the case of adjuvant therapy is the same as in the case of an immune enhancer, when used as a feed additive is to use the licorice hot water extract provided by the present invention to make a powder form added to the feed.

The amount of licorice hot water extract used to show the immunopotentiation effect varies depending on the type of immune cells and animals, but it is 10 ㎍ / based on the volume of the formulation when used in the formulation of an immune enhancer, vaccine supplement, adjuvant or feed additive. It is preferable that the concentration is between 1 and 1 mg / ml, and when the immune cell test is regarded as a process for selecting an adjuvant, the actual applied concentration should be based on the effective concentration in the experimental animal. It is more preferable that it is μg / ml.

Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples, but the present invention is not limited only to these examples.

Reference Example 1 Preparation of Licorice Boiled Extract

The extract was prepared by boiling water extraction as follows. 100 g of licorice was washed in water to remove dust. At this time, since most of the drugs are water-soluble, the active ingredients of the drug were spilled over a long period of time, so that they were lightly washed. Washed licorice was put in a white bag for boiling water and put 1000ml of medicinal herbs and distilled water, soaked for about 20-30 minutes, soaked the medicine and then decocted the medicine for 150 minutes and collected the extract. Then, 800 ml of distilled water was added and the extracts were extracted for 150 minutes, the extracts were collected. Then, 700 ml of distilled water was added, the extracts were collected for 150 minutes, and the extracts were collected. The extracts were collected three times and concentrated to 100 ml. At this time, be careful not to burn the concentrate, and if burned, discarded and extracted again. The extract, concentrated to 100 ml, was lyophilized and the weight of the dried extract was measured. Licorice extract content extracted by the above extraction method was as follows. In other words,

Sample concentration; 100 ml

Dryness; 3.08g / 20ml

Extraction efficiency; 15.4 g / 100 g (%)

It was.

[Experimental Example 1] Random Migration Enhancement Effect of Neutrophil in Goat Blood of Licorice Boiling Extract

1.Isolation of neutrophils in goat blood and mixed treatment with secretion extract

Blood is drawn from the goat's jugular vein and mixed with anticoagulant ACD (ACD, sodium citrate, citric acid, dextrose) and centrifuged at 2,500 × g for 20 minutes to remove the buffy coat and then hemolyzed red blood cells. The neutrophils were collected by centrifugation for 5 minutes, and then suspended in AlMPM medium (RPMI1640) so that the number of neutrophils was 1 × 10 7 / ml. In order to mix neutrophils with licorice hot water extract, the dried extract in cold storage was suspended at 1mg / ml with AlMPI medium, diluted 10 times (100㎍ / ml), 100 times (10㎍ / ml) and the same amount. (30 μl) of neutrophil suspension (1 × 107 / ml) was mixed and treated at 37 ° C. for 1 hour in a CO ₂ incubator.

2. Effect of random migration

Jujugi ability, the power of the immune cells running there to detect bacteria or viruses, the greater Jujugi ability means that the activity of the immune cells is better.

10 μl of neutrophils mixed with licorice hot water extract were placed in a hole made 3 mm in diameter in an agarose plate, and fixed in 8% gurualdehyde solution after application at 37 ° C. for 18 hours in a carbon dioxide incubator. The gel was removed, washed and dried. The dried plate was stained with start stain (Stat stain, Volu-Sol, Inc.) to measure the distance of neutrophil drift under a microscope (MC-50T, MEIJI / Japan) Calculated together.

Sector area = πr ² -π (hole radius) ²

The effects of random neutrophils on goat blood neutrophils treated by dilution multiples with licorice hot water extract are shown in Table 1.

Effects of Randomized Efficacy on Neutrophils in Goat Blood Treated with Licorice Extract division Treatment concentration (/ ml) Sector area (mm ² ) Licorice Boil Extract 1mg 15.45 ± 0.11 ** 100 µg 17.51 ± 4.61 * 10 µg 21.28 ± 1.49 ** Control - 13.66 ± 3.28

The area of neutrophils treated with licorice hot water extract at concentrations of 1 mg / ml, 100 µg / ml and 10 µg / ml was 15.45, 17.51 and 21.28, respectively, showing a significant (p <0.01 ~ 0.05) difference compared to 13.66 of the control group. It was. When the licorice boiled water extract was treated, the immune activity of neutrophils was confirmed to be better than that of the control group.

Experimental Example 2 Investigation of Enhancement of Directional Migration of Neutrophil in Goat Blood of Licorice Boiling Extract

The middle hole of three holes in which 10 μl of neutrophils mixed with licorice boiling water extract were mixed in agarose plate with a diameter of 3 mm in agarose plate in the same manner as in the investigation of the random potency enhancement effect of Experimental Example 1 Into the other two holes, put 10 ml of phosphate buffered saline solution (PBS, pH 7.2) and chlorine complement which was opsonized with Zymosan A (Zymosan A, Sigma Z-4250), and then in a carbon dioxide incubator. After application at 37 ° C. for 3 to 5 hours, the mixture was fixed with 8% gurualaldehyde solution, the gel was removed, washed and dried. The dried plate was stained with start stain (Stat stain, Volu-Sol, Inc.), and the distance of neutrophils to phosphate buffered saline and opsonine treated chlorine complement was measured under a microscope (MC-50T, MEIJI / Japan), respectively. Measured and the difference in the drift distance of the neutrophils was calculated as in Equation 2 below.

Difference in the distance of neutrophils

= Distance resident with opsonized chlorine complement-Distance phosphate buffered saline

The effects on the directed chemotactic ability of neutrophils in goat blood treated with licorice hot water extract by dilution multiples are shown in Table 2 below.

Effect of neutrophils on directed direct chemotactic activity in goat blood treated with licorice water extract division Treatment concentration (/ ml) Joist distance (mm) Licorice Boil Extract 1mg 1.27 ± 0.42 100 µg 1.33 ± 0.31 10 µg 1.75 ± 0.06 ** Control - 1.24 ± 0.50

The directional drift distance of neutrophils treated with licorice boiled extract (10㎍ / ml) was 1.75mm, which was remarkably (p <0.01) difference compared to 1.24mm of control vortex, and it was not statistically significant but 100㎍ of licorice boiled extract Also treated with 1.33mm was larger than the control drift distance. When the licorice boiled extract was treated, the immune activity of neutrophils was confirmed to be better than that of the control group.

[Example 3] The antimicrobial peroxide goat blood neutrophils (neutrophil) of hot water extract of licorice (superoxide, O ₂ -) acid saengneung

Peroxides have biological defenses that fight pathogens such as bacteria and viruses and detoxify toxic substances. In general, when bacteria or viruses penetrate from outside, they make antibodies to fight or directly attack pathogens to protect the body. At this time, one of the cells that attacks directly is neutrophils, and in the process of attacking the pathogens, a significant amount of peroxide is produced. Kill the enemy.

50 μl of neutrophils (2 × 10 ⁶ / ml) mixed with licorice hot water extract, Earls balanced salt solution (EBSS), and chlorine complement 25 µl of each sonicated Zymosan suspension was placed in a hole of a co-culture microplate (Costar, 96 well) and operated at 37 ° C for 1 hour in a carbon dioxide incubator, followed by 100 µl of NBT (nitroblue tetrazolium) in the hole. (4mg / ml) was added and reacted for 3 hours in a carbon dioxide gas incubator. After dissolving formazan (formazan) in 100 μl of DMSO, absorbance was absorbed at an absorbing wavelength of 560 nm. ) And the amount of peroxide produced was measured. Peroxide production of neutrophils in goat blood treated with licorice hot water extract is shown in Table 3.

Peroxide Acid Production of Neutrophils in Goat Blood Treated with Licorice Boil Extract division Treatment concentration (/ ml) Optical Density (O.D) Licorice Boil Extract 1mg 0.3757 ± 0.072 100 µg 0.3805 ± 0.024 10 µg 0.4447 ± 0.111 * Control - 0.3690 ± 0.038

The peroxide generation reaction of neutrophils treated with 10 μg concentration of licorice hot water extract was significantly higher (p <0.05) compared to the control group, and it was found that the activity of neutrophils was enhanced when licorice hot water extract was treated.

Experimental Example 4 Production of Mouse Peritoneal Macrophages Nitric Oxide (NO)

1. Separation of mouse abdominal macrophage

Treatment of 25g of Icyal (ICR, ♂) mice with cervical distal bone method and intraperitoneal injection of 5ml phosphate buffered saline (PBS, pH 7.2) to collect intraperitoneal macrophages and washing twice with phosphate buffered saline It was suspended in AlMPM medium to 2 × 10 ⁶ / ml, and then 200 μl of the cultured microplate was incubated at 37 ° C. for 2 hours in a carbon dioxide gas 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 investigation

When macrophages devour microorganisms, they produce powerful enzymes and reactive oxygen radicals (ROR) or nitric oxide (NO) to process the phagocytosis, and thus the nitric oxide activity can be measured to determine the activity of macrophages.

A hot water extract of licorice rpm child medium in each hole microplate with macrophages 1mg / ml, 100㎍ / ml and 10㎍ / ml diluted so that the frequency division by the respective 50㎕ holes at different concentrations and the carbon dioxide gas incubator (CO ₂ incubator Incubated at 37 ° C. for 48 hours. A standard curve of sodium nitrite used as a standard solution was obtained by mixing 100 μl of the supernatant of each hole and 50 μl of Greases 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 production of mouse peritoneal macrophages treated with licorice hot water extracts by dilution multiples are shown in Table 4.

division Treatment concentration (ml) Optical Density (O.D) Nitric Oxide Concentration (micro-M) Licorice Boil Extract 1mg 0.1473 0.39 100 µg 0.1530 0.78 10 µg 0.1397 0.39 Control - 0.1273 <0.19 Standard nitric oxide - 0.1593 0.1347 0.1290 0.78 0.39 0.19

Nitric oxide was detected in all mouse intraperitoneal macrophage cultures treated with licorice water extract 1mg / ml, 100µg / ml and 10µg / ml, with 0.78 micromole being the most. It was confirmed that the activity of macrophages was enhanced when the licorice hot water extract was treated with an increased acid production of nitric oxide.

Experimental Example 5 Mouse Spleen Cell Proliferation of Licorice Boiling Extract

1. Isolation of Splenocytes

Igial (ICR, ♂) mouse of 25g was treated by cervical dislocation method, the abdominal wall was incised to separate the spleen, and the spleen cells were collected by the method of agitation. Red blood cells mixed in splenocytes were removed by hemolysis, washed twice with phosphate buffered saline, and suspended in AlfM medium so that the cell number was 2 × 10 ⁶ / ml, and 100 μl was dispensed into each hole of the microplate for coculture.

2. Proliferation investigation

The proliferation of splenocytes is directly related to the increase in the number of antibody-producing cells, confirming the increase of the number of splenocytes, it can be confirmed that the main function of the immune system has been enhanced.

In each hole containing mouse splenocytes, licorice hot water extract and ConA (15 µg / ml) diluted to 1 mg / ml, 100 µg / ml and 10 µg / ml with RMP medium were added to each hole. Μl was added thereto and incubated for 48 hours at 37 ° C. in a carbon dioxide gas incubator. 50 μl of empty reagent (MTT, 4mg / ml) was added to each hole of the plate, and the supernatant was removed after 4 hours of reaction at 37 ° C. in a carbon dioxide incubator. 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 absorption wavelength of 560 nm with an ELISA reader (Titertec multiscan) to measure the germination capacity.

The effects on the growth capacity of mouse splenocytes treated by diluting folds of licorice hot water extract are shown in Table 5.

Effect on the Growth Capacity of Mouse Spleen Cells Treated with Licorice Extract division Treatment concentration (/ ml) Optical Density (O.D) Licorice Boil Extract 1mg 0.2867 ± 0.02 ** 100 µg 0.2380 ± 0.009 ** 10 µg 0.2383 ± 0.02 * Control - 0.2157 ± 0.011

Splenocyte proliferation and optical density are proportional. In Table 5, it can be seen that the optical density was significantly higher than that of the control group when treated with licorice hot water extracts of 1 mg / ml, 100 µg / ml, and 10 µg / ml (p <0.01 ~ 0.05). The potentiation ability can be seen to be significantly higher than the control. This resulted in the treatment of licorice boiled water extract was confirmed that the function of the immune system is better than the control.

Experimental Example 6 Effect of Licorice Boiling Extract on the Production of Cytokines (Interleukin-2 and Gamma Interferon) in Mouse Blood

1. Mouse Inoculation and Blood Collection of Licorice Boiling Extract

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

2. Investigation of Interleukin-2 and Gamma Interferon Production

Cytokines are active substances that cause activated cells to act on other cells by reacting with an antigen. Interleukin is an immunomodulatory substance secreted from macrophages or lymphocytes. Among them, interleukin-2 is a T lymphocyte proliferation and differentiation cell. It is associated with the activity of toxic lymphocytes and macrophages. There are three known types of interferon, alpha, beta, and gamma, depending on the cells produced. In other words, what is produced in white blood cells is called interferon alpha, interferon beta, T lymphocytes, and macrophages are called interferon gamma. The three main functions of interferon are antiviral action, antiproliferative action and immunomodulatory action.

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 hole of the cytokine kit for each kind, and reacted for 3 hours at room temperature, and then washed 6 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 measured at an absorbing wavelength of 450 nm (ELISA reader, Titertec multiscan). ) And the production amount of interleukin-2 and gamma interferon using the standard curve of the standard.

Cytokine acid production in mice inoculated with licorice water extract is shown in Table 6 below.

Effects of Cytokine Acid Production on Licorice Bovine Extract Inoculated Mice Substance Inoculation concentration Interleukin-2 (IL-2) Gamma Interferon (IFN-gamma) Optical density (O.D) Production amount (pg / ml) Optical density (O.D) Production amount (pg / ml) Licorice extract 100 µg / ml 0.0865 <7.81 0.1110 15.6 Control 0.0685 <7.81 0.0790 <7.81

Gamma interferon was detected at 15.6 picograms (pg / ml) when inoculated with licorice extract, but the amount of interleukin-2 was less than 7.81 picograms. As a result, it was confirmed that the cytokine acid production in blood was improved compared to the control group, and in particular, the acid production of gamma interferon was increased.

Experimental Example 7 Protective Activity against Licorice Boiling Extract of Pathogenic Staphylococcus aureus

Pathogenic staphylococci were inoculated into mice and tested for nonspecific protective ability.

Licorice hot water 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 with Staphylococcus aureus (strain 289). Ten times (10MLD / 0.3ml) of the minimum lethal dose was inoculated into the abdominal cavity and the protective effect was investigated at 5 days survival rate. The results are shown in Table 7 below.

Protective Effect of Licorice Boiled Extracts Against Invasive Pathogenic Streptococcus Substance Inoculation (0.2ml, abdominal cavity) Attack % Defense Licorice extract 100 µg / ml 0.3 ml (10 mld) abdominal cavity 0/4 (100) Control - 0.3 ml (10 mld) abdominal cavity 4/4 (0)

The mice inoculated with licorice boiled extract survived all pathogenic staphylococcus challenge and exhibited a 100% protection rate, and all of the control mice died and confirmed excellent non-specific defense ability of the licorice boiled extract.

By using the licorice hot water extract provided by the present invention, it can be used as an adjuvant that can effectively treat and prevent animal diseases, as an adjuvants that can improve the efficacy of vaccines, and as an adjuvant therapy that can enhance the therapeutic efficacy. The effect is expected to be high, which will greatly reduce the economic damage caused by livestock diseases in livestock farmers and strengthen competitiveness with imported veterinary drugs.

Claims (6)

(Iii) adding 1000 ml of distilled water per 100 g of dried licorice, soaking licorice for 20-30 minutes, and then decocting the medicine for 150 minutes to collect the extract; (Ii) adding 800 ml of distilled water per 100 g of dried licorice to the licorice after decoction of (iii), decocting the medicinal herbs for 150 minutes, and collecting the extract; (Iii) adding 700 ml of distilled water per 100 g of dried licorice to the licorice after decoction of (ii), decocting medicinal herbs for 150 minutes, and collecting the extract; (Iii) collecting and concentrating the extract extracted in steps (iii), (ii) and (iii); And (Iii) freeze drying the licorice extract concentrated in step (iii); Licorice (Glycyrrhiza) boiling water extract having an immune enhancing effect produced through a process consisting of. (Iii) adding 1000 ml of distilled water per 100 g of dried licorice, soaking licorice for 20-30 minutes, and then decocting the medicine for 150 minutes to collect the extract; (Ii) adding 800 ml of distilled water per 100 g of dried licorice to the licorice after decoction of (iii), decocting the medicinal herbs for 150 minutes, and collecting the extract; (Iii) adding 700 ml of distilled water per 100 g of dried licorice to the licorice after decoction of (ii), decocting medicinal herbs for 150 minutes, and collecting the extract; (Iii) collecting and concentrating the extract extracted in steps (iii), (ii) and (iii); And (Iii) freeze drying the licorice extract concentrated in step (iii); Method of producing a licorice hot water extract having an immune enhancing effect through the process consisting of. An immunopotentiator comprising the licorice hot water extract of claim 1. A vaccine supplement containing the licorice boil extract of claim 1. An auxiliary treatment comprising the licorice hot water extract of claim 1. Feed additive containing the licorice boiled water extract of claim 1.