KR20140140171A - Immunity-enhancing composition for the prevention and treatment of gastroenteritis of nursery pigs comprising extract of artemisia - Google Patents

Abstract

Disclosed is a composition for preventing and treating gastroenteritis of nursery pigs characterized by containing an extract of Artemisia. The composition shows an effect of preventing and treating gastroenteritis through an activity of enhancing immunity of nursery pigs. Provided is a composition of achieving effect of preventing and treating gastroenteritis through an immunity-enhancing effect, not showing an effect of treating gastroenteritis based on antibacterial activity. In the composition, the Artemisia extract is characterized by extracting Artemisia with isopropanol.

Description

FIELD OF THE INVENTION [0001] The present invention relates to an immunomodulating composition for preventing and treating enteritis of a piglet including an extract of Mugwort,

The present invention relates to a composition capable of preventing or treating enteritis of piglets using an extract of mugwort. More specifically, the present invention relates to a composition capable of preventing or treating enteritis of piglets when feeding an extract obtained by extracting mugwort with isopropanol to a feed to feed piglets.

In animal husbandry, the mortality rate of livestock and poultry is very high due to bacterial enteritis of animals. Bacterial enteritis is a disease in which the intestinal mucosa is damaged by a bacterial infection, and bacterial enterocolitis, which is common in livestock, especially in pigs, includes colitis, salmonellosis, proliferative ileitis, colitis,

In order to reduce mortality caused by these bacterial enteritis, antibiotics and antigens are added to feed almost all when raising livestock or poultry. The functions of these antibiotics are largely divided into promotion of growth, prevention of disease, and improvement of feed utilization rate. However, antibiotics used indiscriminately in the process of raising livestock have problems such as the appearance of resistant bacteria to antibiotics, the ingestion of human antibiotics through meat, and environmental pollution.

Therefore, there has been a demand for the development of a composition for the treatment and prevention of enteritis of piglets without using antibiotics. In addition, by improving the immunity of animals not merely treating enteritis by antibacterial action, There is a need for a composition that achieves the effect of prevention and increases immunity.

Korean Patent Laid-Open Publication No. 10-2013-0013712

Accordingly, the present invention provides a composition for treating and preventing enteritis of piglets, which does not show the therapeutic effect of enteritis on the basis of antibacterial action, but which achieves the preventive and therapeutic effect of enteritis through immunostimulating action .

As means for achieving the object to be solved, the present invention discloses the following means.

A composition for preventing or treating enteritis of a piglet characterized by comprising an extract of Mugwort is disclosed.

In the composition, the mugwort extract is a composition for preventing or treating enteritis of a piglet characterized by extracting mugwort with isopropanol.

Disclosed is a composition for preventing or treating enteritis of pigs, which is characterized in that the composition for preventing or treating enteritis of pigs is expressed through immunostimulating action.

When the composition of the present invention is mixed with feed and fed to piglets, productivity and disease-improving effect of piglets can be achieved, and immunity can be activated, thereby preventing and treating enteritis.

Brief Description of the Drawings Fig. 1 is a diagram showing the results of HPCL analysis of a standard sample of attitude ecidine.
Fig. 2 is a graph showing the results of HPLC analysis of a standard sample of yaffatiline. Fig.
FIG. 3 is a graph showing the results of HPLC analysis of Mugwort extract. FIG.
Figure 4 shows the total polyphenol content of mugwort extract and by-products.
Figure 5 shows the total flavonoid content of mugwort extract and by-products.
Figure 6 shows the antibacterial activity of isopropanol extract and hot water extract of mugwort. E: Extract of hot water (Salmonella), F: Extract of hot water (Clostridium), A: Extract of Isopropanol (Escherichia coli), B: Isopropanol extract (Salmonella) Dime))
FIG. 7 shows a photograph of the small intestine of the group inoculated with Salmonella (left photograph) and the group of Salmonella inoculation + mugwort extract (right photograph).
Fig. 8 shows photographs of a group inoculated with E. coli (left image) and a group to which Escherichia coli inoculation + Mugwort extract was administered (right image).
FIG. 9 shows small villus photographs of the control group (left photograph) and the group to which mugwort extract was administered (right photograph).
Fig. 10 shows the fecundity of piglets (left photograph) and non-piglets (right photograph) fed with mugwort extract after inoculation with Salmonella.
Fig. 11 shows the change of T cells in the blood on the second day of enteritis induction.
Fig. 12 shows the change of T cells in the blood on the 7th day after enteritis induction.
Figure 13 shows changes in IL-6 concentration in the blood after enteritis induction.
Fig. 14 shows changes in IL-1? Concentration in blood after enteritis induction.
FIG. 15 is a graph showing the growth of diarrhea-induced treatment during the period of inoculation of Salmonella.
Fig. 16 is a graph showing the growth of diarrhea during the period of treatment with E. coli inoculation. Fig.

Pigs can be exposed to many diseases as soon as they are born. The most threatening diseases are digestive diseases. Enteritis or diarrhea occurs in all ages, but the most common is piglets. Major bacteria causing enteritis or diarrhea in pigs include Escherichia coli, Clostridium, Salmonella, and Pseudomonas aeruginosa

This is caused by a pathogenic Escherichia coli infection, and the enterotoxin has a diathermal (LT) and heat-stable (ST) toxin. Escherichia coli, which secretes enterovirus, invades the small intestine and attaches to the barrier, causing disease. The most common attachment factors of E. coli are K88, K99, 987P and F41. Hemagglutination of pathogenic Escherichia coli is known to be various, such as O138, O139, O141, and O149. Hemolytic Escherichia coli acts as a major pathogen in pigs. Once E. coli attaches to the intestinal epithelium, E. coli rapidly proliferates to produce intestinal toxins, damaging the intestinal epithelial cells, causing loss of body fluids, and diarrhea by interfering with the absorption of nutrients.

On the other hand, as a method for treating enteritis or diarrhea of such piglets, natural or synthetic antibiotics having an antibiotic action have been used for bacteria causing enteritis. However, the use of antibiotics in livestock is not desirable.

On the other hand, mugwort is a wild herb that grows around us, and has been used in general folk medicine for a long time. It has been separated from yaffatilin and postosocidin, and the mugwort extract itself has been found to promote the biosynthesis of gastrointestinal prostaglandins, Has been used in such treatments.

The appearance of mugwort is perennial herb, height 45 ~ 120cm, stem is upright and circular. The vagina is rigid and basal cotton, covered with soft white fur, and the roots are branched. Also, mugwort is highly adaptable to the climate, and fertility of the soil increases, so it is one of the representative plants that are common even in our surroundings because it is strong enough to be grown in a vacant land in one corner because its adaptability increases. Mugwort contains essential oil, and the essential ingredient of essential oil is cineol, which is 25 ~ 30%. The praxis effect (cough stopping effect) contains telpinene 4 ol, a relatively good ingredient. In addition, it is known that beta -carriophorene, linalool, altemic alcohols, camphor, pourneol and the like are contained. The petroleum ether extract of mugwort contains stigmasterol,? -Sitosterol,? -Amyrin and acetic acid, acetic acid,? -Pinene and? -Pinene, ferenol.

However, to date, there has been no report that the mugwort extract can increase the immunity of piglets to prevent or treat enteritis.

In this situation, the present inventors have surprisingly found that although the extract of mugwort, specifically isopropanol extract of mugwort, does not exhibit antibiotic action or antibacterial action, it can prevent or treat enteritis of the piglet in a manner of increasing the immunity of piglets Leading to the present invention.

That is, the present invention discloses a composition for preventing or treating enteritis of a piglet including an extract of mugwort. The mugwort mugwort raw material used in the present invention is obtained by obtaining domestic mugwuga (Chinese wormwood) and Chinese mugwort, drying them completely and cutting them into a certain size. Isopropanol was separately added to the cut samples, and the immersion step for 24 hours was repeated twice to separate the extract and the byproducts. The extract was concentrated twice under reduced pressure to prepare a concentrate by evaporating the solvent. And the solvent was evaporated and recovered in the form of a dried product. The extract is concentrated twice at a reduced pressure, and the efficacy of the extract is about 8 times higher than that of the raw material. The extract of the mugwort includes the oil tillin and the oriental osteidine.

On the other hand, the isopropanol extract of mugwort did not exhibit antimicrobial activity in itself, and thus it was presumed that it was not effective for enteritis and diarrhea of piglets. However, according to the inventors' study, surprisingly, isopropanol extract of mugwort promoted the immunity activity of piglets I was able to cure enteritis or diarrhea.

In other words, according to the study of the present inventor, it was confirmed that the mugwort extract activates innate immunity by inducing IL-1β and IL-6 in the piglet, and the mugwort extract has an early, For example, the ratio of CD4 T cells that were elevated as opposed to the initial level after a certain period of time, for example, 7 days, was normalized to normal level after 2 days from infection, The effect of restoring to the same level was confirmed.

In other words, the mugwort extract was found to be effective for activating congenital immunity and inducing acquired immunity more strongly and effectively removing pathogens. Therefore, when the mugwort extract is mixed with the feed and fed to piglets, treatment and prevention effects of enteritis or diarrhea can be achieved.

Hereinafter, the present invention will be described by way of examples.

Example 1 - Preparation of Mugwort extract

Prepare 1kg of dried domestic and Chinese mugwort, cut to 0.5cm or less, and add 9kg of isopropanol to the mugweed raw material. After soaking for 24 hours, the extract and the by-product are separated, and isopropanol is added to the by-product again and re-extracted for additional 24 hours. The extract is concentrated twice under reduced pressure (60 ° C) to give a concentrate.

Example 2_ Identification of active ingredients

The contents of the active ingredients of the mugwort extract, that is, the tillin and the positional osteidine, were analyzed by HPLC. The HPLC analysis conditions are shown in Table 1. The detector of HPLC (Hitachi, Japan) was detected at 350 nm using a UV-Vis detector and analyzed using an Inertsil ODS II8 column (5 μm, 250 × 46 mm) using MeOH-0.4% H 3 PO 4 as an eluent and measuring 1 ml / Each active ingredient content was analyzed by injecting 10 μl of each sample liquid at a flow rate. The results are shown in Table 1 below.

Time (minutes) Sol. A Sol. B MeOH (%) 0.4% H ₃ PO ₄ 0 50 50 10 70 30 30 70 30

To establish the analysis of the active ingredient content, analysis was first carried out on the standard samples of the tiline and posacin, and the results are shown in FIG. 1 and FIG. 2, respectively. After analyzing the standard samples, we analyzed the mugwort extract and the reduced-pressure concentrate, and extracted 50g of Chinese mugwort and domestic mugwort extract, respectively, Are shown in [Table 2] and [Figure 3]. The content of active ingredients in the mugwort extract of Chinese mugwort was higher than that of mugwort extract in the same manufacturing process.

division Active ingredient content (mg / g) Yupatiline Posture osidine Chinese mugwort
(Lady) Extract (concentrated) 2.164 0.746 Domestic wormwood
(Fortified wormwood) Extract (concentrated) 1.312 0.483

Example 3 Evaluation of physiological activity of Mugwort extract

A variety of antioxidative activities were evaluated to evaluate the physiological activity of Mugwort extract. Antioxidant activity was evaluated by DPPH radical scavenging activity, ABTS radical scavenging activity evaluation, total polyphenol and total flavonoid content analysis. The mugwort extracts used in this study were mugwort Chinese mugworts and domestic mugworts. DPPH radical scavenging activity was determined by adding 0.8 ml of 2 × 10 -4 M DPPH (Sigma-Aldrich) solution (99.9%, dissolved in ethanol) to 0.2 ml of sample, shaking for 10 seconds with a vortex mixer, and using a spectrophotometer The electron donating ability was determined by absorbance measurement at 525 nm. ABTS radical scavenging activity was determined by incubating 7.4 mM potassium 2,2'-azino-bis- (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS, Sigma-Aldrich) and 2.6 mM potassium persulphate The solution was diluted with methanol using a molar extinction coefficient (ε = 3.6 × 104 M-1 cm -1) at 735 nm so that the absorbance value was 1.4 to 1.5, and 50 μl of the extract was added to 1 ml of the diluted ABTS + Was measured after 30 minutes.

The results of analysis of DPPH and ABTS radical scavenging ability of Mugwort extract are shown in Table 3.

Sample name IC50
(mg / g) ABTS
(mg trolox / g) Chinese mugwort
(Lady) extract 84.99 2.91 + 0.04 Domestic wormwood
(Fortified wormwood) extract 812.89 1.5 ± 0.13

Total phenolic compounds content of mugwort extract was measured by Folin-Denis method (AOAC, 1984). Mugwort extract (0.2 ml) is extracted three times with 75 ml of methanol (20 ml) and then filtered (Whatman No. 2). The extract is mixed and concentrated under reduced pressure at 40 ° C and then recovered in 20 ml of 75% methanol solution. 5 ml of distilled water and 2 ml of 10% Folin-Ciocalteau's phenol reagent were added to 1 ml of the prepared sample extraction solution, and the mixture was allowed to stand for 3 minutes. 2 ml of 7.5% Na2CO3 solution was added thereto, diluted with distilled water and adjusted to 20 ml, The absorbance at 760 nm was measured using a VIS spectrophotometer, and the concentration was expressed in terms of the total phenol content from a standard curve prepared by the same method using chloro genic acid as a reference material. The flavonoid contents were mixed with 1 ml of 2% aluminum chloride methanolic solution in 1 ml of extract prepared at 1 mg / ml, reacted at room temperature for 15 minutes, and then absorbance was measured using a microplate reader at 430 nm. Total flavonoid content (mg / g) was calculated by preparing a standard curve using quercetin (6.25-100 μg / ml).

The results of analysis of the total polyphenol and flavonoid contents of the mugwort extract are shown in [Fig. 4] and [Fig. 5].

Example 4_ Antimicrobial activity of Mugwort extract

Isopropanol extract (10-fold concentration) and hot-water extract concentrate (10-fold concentration) of mugwort extract were prepared and their antimicrobial activity was investigated. Escherichia coli , Salmonella typhimurium and Clostridium perfringens strains were used as the indicator strains for the antimicrobial activity. E. coli, Salmonella, and Clostridium colony were inoculated into LB (Difco, USA) and cultured at 37 ° C for 24 hours. The antimicrobial activity was evaluated using a paper disc (φ8 mm, Whatman). 1 ml of the inoculum was diluted with 0.1% peptone water to make about 10 ⁵ CFU / ml, and 0.1 ml of the inoculum was mixed with Muller-Hinton medium. After loading 20 μl each in sterilized paper discs, the cells were incubated at 37 ° C for 24 hours, and the size of the clear zone (㎜) around the disc was measured to evaluate the antimicrobial activity.

As a result of evaluation of the antimicrobial activity of the mugwort extract, there was no antimicrobial activity in the isopropanol extract (concentrate), and antimicrobial activity was confirmed in E. coli, Salmonella and Clostridium strains in the hot water extract (concentrate). This suggests that the main component of isopropanol extract is not related to the antimicrobial activity against strains causing gastrointestinal diseases, and that the treatment of enteritis and diarrhea of mugwort extract is not due to antimicrobial activity.

EXPERIMENTAL EXAMPLE 1 Effect of Mugwort Extract on Enteritis and Diarrhea

Cysteamine injection and intestinal infestation induced intestinal infestation by inducing pathogenic microorganisms. E. coli Fimbriae type F18ac, which is a harmful microorganism isolated from the feces of pigs in which diarrhea was detected in the test pigs, was isolated three days after the feeding of the test diets. , Enterotoxin type LT, ST) and Salmonella typhimurium were inoculated at 1 × ^{10 8} cfu / ml. For the feeding of piglets infected with harmful microorganisms causing enteritis, 0.2% of the mugwort extract prepared in Example 1 was added to the commercially available yeast extract . After ingestion of harmful microorganisms, symptoms such as feed intake, body weight and diarrhea were observed for 14 days. After completion of the test, autopsy was performed to obtain intestinal tissue samples and the lesion index of the tissues was observed. T cells and cytokines were also collected from the blood samples collected on the 2nd, 7th, and 14th days of experiment to observe the immunological indicators. the results are as follow.

① small intestinal tissue change

As an index of the effect of productivity improvement, it was possible to confirm the relationship between the occurrence of lesion of the tissue and the intestinal mucosal tissue photograph of the small intestine absorbing site of nutrients (Fig. 7 to Fig. 9) It was confirmed that the damage could be cured.

Also, the efficacy of Mugwort extract could be confirmed from the actual feces condition of piglets. (Fig. 10)

As a result, it was confirmed that the mugwort extract had a therapeutic effect on diarrhea caused by Escherichia coli and Salmonella.

② T cell changes in blood of enteritis-induced pigs

The blood samples taken at 2, 7, and 14 days of age were used to examine the effect of mugwort extract on the immune status of the weaning pigs in enteric diarrhea. The changes of CD4 and CD8 T cells in the blood were analyzed by flow cytometry on the second day after inoculation with diarrhea caused by inoculation with cysteamine (Cys), salmonella (Sal) and E. coli, which cause enteritis. Respectively. As shown in FIG. 11, CD4 T cells (CD4 + CD8-) were significantly decreased when treated with only E. coli. However, it was confirmed that the reduced CD4 T cells were recovered by treatment with mugwort extract (labeled as Artville in the figure). However, in the case of Salmonella (Sal), the result was opposite to that of E. coli. This is probably due to the fact that Salmonella does not stay in the intestines like Escherichia coli but penetrates into the cells and directly affects immune cells as well as epithelial cells.

In the CD8 T cells (CD8 + CD4-), it was increased about 2 times (~ 10% -> ~ 20%) in all enteritis inducing groups (Cys, Ecoli, Sal). When the extract was treated with mugwort extract, it was decreased to the control level in all enteritis - induced groups. CD4 + CD8 + T cells were increased by enteritis induction compared to the control group, and no change by the mugwort extract was observed. Overall, the mugwort extract on the second day of enteral induction seems to antagonize the changes of CD4 + T cells by E. coli and Salmonella.

On the other hand, the results on the 7th day of enteritis induced were similar to those of the 2nd day. CD4 + T cells showed a tendency to increase as a result of enteritis induction, but this phenomenon did not appear when the mugwort extract was added (Fig. 12). In the case of CD8 + T cells, it was increased by about 10% in Salmonella-induced enterocolitis induced by mugwort extract treatment. However, CD4 + CD8 + T cells did not show any change.

③ cytokine change in blood of inducing enteritis

Changes in blood IL-6 levels were observed on days 2, 7, and 14 after induction of enteritis. As a result, no significant change was observed on day 2, but very high IL-6 was measured in the mugwort extract-treated group on the 7th day (FIG. 13).

IL-1β in blood was higher at 2 and 7 days after administration of matsutake extract supplemented diet and in the quasi-group treated with sithiamine (FIG. 14).

These results suggest that the mugwort extract induces IL-1β and IL-6, thereby activating innate immunity. Mugwort extracts recovered normal levels of CD4 + T cells reduced by E. coli at the early stage (day 2), and recovered normal levels of CD4 T cells at day 7 as opposed to the early stage. In the case of salmonella which can survive inside the cell, the ratio of CD8 + T cells is increased at 7th day by addition of mugwort extract. In conclusion, the mugwort extract can activate the innate immune system and induce more aggressive immunity, which is effective in removing pathogens effectively.

EXPERIMENTAL EXAMPLE 2 Effect of Mugwort Extract on Productivity of Sugar Pepper

(CON), the feed group of the present invention (commercial feed containing 2% of the mugwort extract of Example 1, invented product), cysteamine injected group (E + invention), Salmonella inoculation group (SAL), Salmonella inoculation + Invention of cysteamine inoculation + Invention feed group (C + invention), E. coli in inoculation group, And feed group (S + invention). Escherichia coli and Salmonella were inoculated in the same manner as Experimental Example 1, and the effects on productivity after 14 days of inoculation with harmful microorganisms caused by enteritis were as follows. As can be seen from the table, there was no significant difference in the addition of the developed product compared to the control which did not cause gastritis or enteritis, but in all the treatments which caused the enteritis, The results were confirmed. These results were also found to affect feed efficiency as well as feed intake. It was once again confirmed that Salmonella diarrhea or Escherichia coli diarrhea caused by the infant stage of piglets may affect the growth of piglets and cause a decrease in finishing performance. If the diarrhea caused by the pathogenic fungus infection at the infant stage is prevented by the addition of the present invention product, the productivity can be obtained without significant difference from the normal healthy infant.

Items CON invention CYS C + Inventions E. coli E + Invention SAL S + Invention SEM P-value BW gain (kg) 8.12 ^ab 9.05 ^a 1.99 ^e 7.04 ^abc 4.92 ^cd 8.36 ^ab 2.97 ^de 6.42 ^bc 0.65 <0.0001 ADG
(kg / d) 0.579 ^ab 0.646 ^a 0.142 ^e 0.502 ^abc 0.351 ^cd 0.597 ^ab 0.212 ^de 0.459 ^bc 0.05 <0.0001 ADFI
(g / d) 627.5 ^a 622.6 ^c 492.3 ^h 615.4 ^f 625.4 ^b 619.8 ^e 621.8 ^d 614.9 ^g 0 <0.0001 F / G 1.10 ^b 1.00 ^b 3.89 ^a 1.26 ^b 1.93 ^b 1.07 ^b 3.55 ^a 1.37 ^b 0.38 <0.0001

Experimental Example 3

We examined the growth of diarrhea during the period by inoculation of pathogenic bacteria. As shown in FIGS. 15 and 16, in the case of inoculation with pathogenic bacteria isolated from piglets with diarrhea in order to induce enteritis, the growth of the seedlings not containing the feed of the present invention is improved. Especially, in case of inoculation of salmonella with severe diarrhea, it showed significant improvement compared to the treatment not added.

The mugwort extract of the present invention is mixed with the feed and fed to piglets, thereby preventing or treating enteritis or diarrhea.

Claims (4)

A composition for preventing or treating enteritis of piglets, which comprises the mugwort extract. The composition for preventing or treating enterocolitis of piglets according to claim 1, wherein the mugwort extract is obtained by extracting mugwort with isopropanol. The composition for preventing and treating enterocolitis of piglets according to claim 1, wherein the effect of preventing or treating enteritis of piglets is expressed through immunostimulating action. A composition for immunizing a piglet comprising an isopropanol extract of mugwort.

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