WO2013051743A1 - Composition for use in liquid feed or as a feed additive - Google Patents

Abstract

The present invention relates to a composition for use in liquid feed or as a feed additive, and more particularly, to a composition for use in liquid feed or as a feed additive which contains an herbal extract extracted from an herbal medicine including cnidii rhizome, saposhnikoviae radix, angeicae gigantis radix, paeoniae radix, forsythiae fructus, menthae herba leaf, ephedrae herba, glaber salt, rhubarb, gypsum, playcodi radix, scutellaria radix, atractylodis rhizoma alba, fardeniae fructus, schizonepetae spica, ginger, talc, glycyrrhizae radix, epimedii herba, lonicerae flos, polygalae radix, and elecampane by using water or an organic solvent. Since the composition of the present invention may significantly improve the absolute value of body weight gain and the growth rate of livestock, as well as significantly increase vital sign scores, the composition may be effectively used for the efficient feeding and increasing of body weight of livestock, especially fragile weaning pigs.

Description

Drinking water or feed composition

The present invention relates to a composition for drinking water or feed addition, more specifically Porcine Reproductive and Respiratory Syndrome Virus (hereinafter referred to as 'PRRSV') and / or Porcine Circovirus type 2 (Porcine Circovirus Type 2, hereinafter referred to as 'PCV-2', the present invention relates to a composition for drinking water or for adding food which can improve various clinical symptoms caused by infection.

Feed is a substance that sustains livestock and supplies organic or inorganic nutrients necessary to produce milk, meat, eggs, fur, etc. A good feed is rich in nutrients, no harmful ingredients to livestock, and high in yield. It should always be readily available and always fresh and digestible. Grain is currently used in many parts of the feed for raising livestock, and the recent rapid increase in the population has increased the demand for grain for food, making it difficult to use grain as a feed source. It is limiting the development of livestock raising. Therefore, the feed industry has made a lot of efforts to maximize the efficiency of the feed and the development of feed that can replace the grain, various feeds that can perform additional functions such as improving the digestion and absorption rate of animals The development of additive solvents has become an important part of the feed industry.

Feed additives are a kind of supplementary feed, which is a feed that increases productivity by completely supplying essential nutrients or adding a specific effect to the feed, and is specifically an edible additive used for supplementation of deficiency, enhancement of feed efficiency, and growth growth. . Feed additives include salts, bone meal, vitamins, antibiotics, antibacterial agents, antioxidants, antifungal agents, enzymes, probiotics, amino acids and trace minerals. In general terms, a substance used to increase the effectiveness of the feed by adding or supplementing a small amount to a general feed mainly for energy and protein supply is called a feed additive, and is distinguished from an animal medicine for the purpose of preventing and treating diseases. The development of feed additive composition promotes the fattening and digestion of livestock, thereby maximizing feed efficiency and reducing feed consumption, and enabling high quality livestock production due to rapid growth and fattening. It is very important in that it can contribute.

On the other hand, the weak piglets in weaning pigs are generally 50% caused by porcine Circovirus Associated Disease (hereinafter referred to as 'PCVAD') caused by the combined infection of PRRSV and PCV-2. It is known.

The PRRSV is a causative virus of swine genital respiratory syndrome, which is a combination of reproductive disorders and respiratory infections. When the virus is infected, the disease causes a wide variety of diseases. From no economic loss to severe, up to 20% loss on farm production.

Reproductive disorders due to PRRSV infection are mainly characterized by late gestational or premature births, development of stillbirths and mummy piglets, concentrations of preterm weaning mortality, increased post-weaning mortality, and increased estrus These symptoms are concentrated in infected sows and piglets born from the sows. Most of the reproductive disorders caused by PRRSV infection return to the pre-infection state after 6 months from the beginning of infection.

Respiratory illnesses caused by PRRS infection can appear at all ages, especially in piglets and weaners. In this case, rapid double breathing, bleeding, conjunctivitis, sneezing, diarrhea, etc. are observed, and there may be a short period of temperature increase, and rarely neurological symptoms may be present. Respiratory diseases are characterized by more bacterial and viral pathogen secondary infections and multiple infections than PRRSV infection alone, in which case the pathogenicity is much more amplified.

The above clinical symptoms can be clearly observed in acute and severe progression, and in the case of subclinical infection or chronic progression, few distinct and characteristic clinical symptoms can be observed. Chronic infections occur mainly in weaning piglets and rearing stages. Bacterial and viral infections of the respiratory system under PRRSV attack cause rhinitis and pneumonia. As a result, average daily gains are lowered, feed demand is increased, and mortality after weaning is twice as high as the average mortality rate before PRRSV infection.

In addition, PCV-2 is a causative virus of post-weaning multisystemic wasting syndrome (PMWS), which is one of the most economically damaging diseases in Korea. Since weaning systemic wasting syndrome continues to reduce productivity by 20-40% in farms for at least six months to up to three years, farms with weaning systemic wasting syndrome fail to improve further productivity and worsen profitability. Diseases caused by PCV-2 infection include porcine dermatitis and nephropathy syndrome (PDNS) in addition to the systemic wasting syndrome described above. Recently, PCV-2 is a porcine respiratory disease complex. ), Abortions, stillbirths such as stillbirth, exudative epidermal inflammation, etc. are known to be involved.

The present inventors studied negative or feed additives that can ameliorate various clinical symptoms caused by the PCVAD. As a result, raw materials including yin and yang, gold and silver, raw paper, and nectar are included as a raw material of Bangpungtongsan. The present invention was completed by confirming that the herbal extract prepared by mixing and improving the weight gain and vital signs of the weaning pig (weak piglet) group affected by the PCVAD.

It is an object of the present invention to provide a composition for drinking water or feed comprising a herbal extract prepared by extracting a herbal mixture comprising yin and yang gwak, gold and silver, raw paper and Mok Hyang to the raw material of wind-breakable acid.

In order to achieve the above object, the present invention is a raw material of windproof Tongseong, cheongung, windproof, donkey, peony, pontoon, peppermint, ephedra, forget-me-not, rhubarb, gypsum, Gilgyeong, golden, baekchul, gardenia, mold, ginger, talc, licorice It provides a composition for drinking water or feed addition containing the herbal extract extracted by mixing the herbal medicine including yin and yang, gold and silver, paper and neck.

In order to prepare the herbal extract, first, the raw materials of windproof Tongseong, cheongung, windproof, Angelica, Peony, Yeongyo, Peppermint, ephedra, forget-me-not, rhubarb, gypsum, Gilgyeong, golden, baekrye, sanchija, hyungge, ginger, talc, licorice Extracting 1 ~ 10 hours, preferably 3 to 4 hours at 70 to 125 ℃ after adding 2 to 10 times the amount of water or an organic solvent, preferably water to the herbal medicine including Eum Yangkwa, gold and silver, paper and neck desirable. However, in addition to the hot water extraction as described above, extraction methods such as cold sediment extraction, reflux cooling extraction or ultrasonic extraction may be used. In the preparation of the herbal extract, the herbal medicine is 1 weight part in the yinyanggok cheongung, windproof, Angelica, Peony, Yeongyo, peppermint, ephedra, forget-me-not, rhubarb, gypsum, Gilgyeong, golden, baekchul, gardenia, mold, ginger, talc, licorice It can be used after mixing the gold, silver, paper and wood in a mixing ratio of 0.5 to 5 parts by weight, respectively, but the mixing ratio may be outside these ranges as long as a desired anti-viral effect is obtained. In addition, in addition to the herbal ingredients, in general, any medicines that can be used for the purpose of preventing or treating a cold or the flu may be included without limitation. In the present invention, the organic solvent may be used without the aldehyde and the aqueous solution of the alcohol, aldehyde and ketone of a ketone of the C ₁ -C ₄ alcohols, C ₁ -C _4, C ₁ -C ₄ limits, but limited to, It doesn't happen.

In preparing a composition for drinking or feed according to the present invention, the herbal extract is diluted in water and used as a drinking water, or formulated directly, or diluents such as wheat flour, starch, and textrin and cereals, rice hulls and skim rice bran. It can be formulated with feedstocks such as bran, oil and fat rich seed cakes.

In addition, the drinking water or feed addition composition of the present invention may be in the form of a dry or liquid preparation, and may further include one or more enzyme preparations. Enzyme preparations can be added in either dry or liquid form, lipolytic enzymes such as lipases, phytases that break down phytic acid to form phosphates and inositol phosphates, starches and glycogen Amylase, an enzyme that hydrolyzes an α-1,4-glycoside bond, and phosphatase, cellulose, an enzyme that hydrolyzes an organophosphate ester carboxymethylcellulase to break down cellulose, xylanase to break down xylose, maltase to hydrolyze maltose into two molecules of glucose, and It may be selected and used from the group consisting of sugar producing enzymes such as invertase which hydrolyzes saccharose to form a glucose-fructose mixture.

In addition, the compositions of the present invention may further comprise other non-pathogenic microorganisms. Microorganisms that can be added include Slyter, LLJ Animal Sci. 910-926) and yeasts such as Saccharomyces cerevisiae (Jhonson, DE et al. J. Anim. Sci., 1983, 56, 735-739; Williams, PEV et al, 1990, 211. ), Microorganisms of the genus Lactobacillus sp. That have physiological activity and organic matter degradability under anaerobic conditions such as cattle, but are not limited thereto.

In addition, feedstocks such as peanuts, peas, sugar beets, pulp, grain by-products, animal viscera flour and fishmeal flour, including various grains and soy protein may be appropriately used. The processing process is a process in which the feed material is compressed to a constant outlet under pressure in a state in which the feed material is filled, and in the case of protein, it is preferable to use an extrusion process in which the availability is increased. Extrusion has the advantages of denaturing proteins through heat treatment, destroying antienzyme factors, and increasing the activity of protease by enzymatic exposure to new sites by molecular structural changes. In addition, in the case of soy protein, the extrusion process improves the digestibility of the protein and inactivates antinutrients such as trypsin inhibitor, one of the inhibitors of protease present in soybean. Increasing digestion can increase the nutritional value of soy protein.

Representative examples of animals in which the compositions of the present invention can be used include livestock such as pigs, piglets, edible cattle, dairy cows, calves, sheep, goats, horses, rabbits, dogs, cats, and the like; Poultry such as chicks, chickens, domestic chickens, roosters, ducks, geese, turkeys, quails, birds, etc., but are not limited thereto. In addition, since the dosage is changed depending on the type of animal to be administered, the age, and the type of other feed components, it is difficult to define a constant, but in the case of the feed additive composition of the present invention, it is generally from 0.01 to 100 parts by weight of the blended feed. It is preferable to mix in an amount of 10 parts by weight and use it as a supplementary feed.

The compositions of the present invention can be usefully used to ameliorate the clinical symptoms caused by infection of swine genital respiratory syndrome virus and / or swine circovirus type 2 in particular. The clinical symptoms include weight loss, swine genital respiratory syndrome, weaning piglet systemic wasting syndrome, swine dermatitis renal failure syndrome, complex swine respiratory disease, miscarriage, stillbirth and exudative epidermal infection, but are not necessarily limited thereto.

Since the herbal extract of the present invention is a natural substance, there is no toxicity and can be continuously used in a large amount. As a result of conducting a toxicity test for mice, a safe substance having a 50% lethal dose (LD ₅₀ ) by at least 2 g / kg oral toxicity test Turned out to be. In addition, according to one embodiment of the present invention, when the composition of the present invention is administered, the absolute value of weight gain and weight gain is remarkable compared to the negative control group, which is the weak control pig group not fed the medicine, and the positive control group, which is the healthy pig group not fed the medicine. (See FIGS. 1 and 2).

In addition, according to another embodiment of the present invention, even when the composition of the present invention is administered in the average body weight, the weight gain is markedly different from that of the negative control group, which is a weak pig group not fed the medicine (see FIG. 3).

In addition, according to another embodiment of the present invention, when administering the composition of the present invention, although the vitality of the experimental group did not significantly increase until the week 0, week 1, the vital signs score compared to the negative control group after 2 weeks It shows a significant increase (see FIGS. 6 to 9).

The composition for drinking water or feed of the present invention can significantly improve the absolute value of the weight gain and weight gain of the livestock, and can also significantly increase the vital signs, so that the feed efficiency and weight gain of the livestock, especially the weak weaning piglets It can be usefully used.

1 is a graph showing the relative weight increase rate according to the RATIO parking, A (Week 0-1), B (Week 1-2), C (Week 2-3) and D (Week 3-4) Shows the rate of increase during

2 is a graph showing the absolute value of the weight gain according to the DIFF parking, A (week 0-1), B (week 1-2), C (week 2-3) and D (week 3-4) Weight gain).

Figure 3 is a graph showing the increase in the average body weight for 4 weeks, black is the positive control group, blue is the negative control group, red is the experimental group.

4 is an electrophoretic photograph showing that PRRSV (A) and CPV-2 (B) were detected in dead piglets, respectively.

FIG. 5 is a micrograph showing the symptoms of interstitial pneumomia (A) and pleuritis (B) in dead piglets, respectively (× 100).

Figure 6 is a graph showing the weekly vital signs scores of the negative control, positive control and experimental group.

7 to 9 are photographs showing the breeding state and the vitality state immediately after group separation (A) and 4 weeks (B) of the experimental group, the negative control group and the positive control group, respectively.

10 to 12 are graphs showing the number of cells having chromosomal abnormalities after 24 hours of treatment without S9 mix, 6 hours of treatment without S9 mix, and 6 hours of treatment with S9 mix.

Figures 13 to 16 respectively show the response curve of the base-substituted strain in the direct method, the administration response curve of the base-substituted strain in the metabolic activity method, the response curve of the frameshift-type strain in the direct method and the frameshift strain in the metabolic activity method. Is a graph showing the dose response curve.

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

However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited by the following examples.

Example 1 Preparation of Drinking Water or Feed Additives

All herbal medicines were purchased domestically and used as samples. 15 L of water was added to about 2,456.5 g of the selected herbal medicines having the composition shown in Table 1, and then left at room temperature for 1 hour. Thereafter, hot water extract was performed at 115 ° C. for 3 hours to obtain an herbal extract having a volume of about 11 L, and the herbal extract was lyophilized to obtain about 9.6 g of lyophilized product. The herbal extract was used in the present invention, and the test substance was mixed with water by 1% and used in experiments in the form of negative water.

Table 1

Raw material name	Weight (g)
Cheongung	84.5
Windproof	84.5
Donkey	84.5
Peony	84.5
Fellowship	84.5
Peppermint Leaf	84.5
Ephedra	84.5
sulphate of soda	84.5
rhubarb	84.5
gypsum	131
Street view	131
Gold	131
Whiteness	65.5
Gardener	65.5
Brother-in-law	65.5
ginger	225
talc	318.5
licorice	225
Gold coins	84.5
Yin Yang	84.5
Paper	84.5
elecampane	84.5

Example 2 Evaluation of Weight Gain

<2-1> RATIO parking and DIFF parking

Respiratory symptoms are present, the virus is diagnosed by serum tests, and a combination infection of PRRSV and PCV-2 viruses is confirmed, and the clinical signs of 20-day-old weaning piglets are about 60 to 70% of normal piglets with no vital symptoms. The experiment was conducted after adapting to the changed environment for one day. The experimental animals were housed in three cages provided with sufficient space at a temperature of 23 ± 3 ° C. and a humidity of 50 ± 5%. The test substance prepared in Example 1 was used for the experiment, and was carried out according to 'Formulation and preparation of test substance (NITR / SOP / TSB / 002_02)' in the standard work procedure of the Korea Food and Drug Administration. Weekly weight gain was calculated by weight measurement every week, and weight gain was measured for 4 weeks until weaning pigs were 49 days old. In order to evaluate the statistical significance of the experimental results, the number of individuals per each experimental group was 10, and the same experiment was repeated three times to analyze the data. The change value and error of test drug and control group were verified by t-test, and statistical program was used for SAS. The following two methods were applied for the increase rate.

(1) RATIO parking = (weight of the parking before weight of parking) / weight of parking before

(2) DIFF parking = the weight of the parking before the weight of the parking

The RATIO parking is a relative weight gain rate for all the parking, and represents the weight gain rate in a precise sense, and the DIFF parking is the absolute amount of the weight increased as the difference between the weight of the previous parking and the parking.

As a result, in the case of RATIO parking, the increase rate during the period from week 0 to week 1 was significantly different in both the negative control group, the weak control pig group not fed the composition of the present invention, the positive control group, the healthy control group not fed the composition of the present invention, and the drug administration group. None (p <0.01), it was confirmed that the gain ratio of the drug-administered group was significantly improved compared to both the negative control group and the positive control group after the 3rd to 4th week (p <0.01) (FIG. 1).

In addition, it was confirmed that the absolute value of the weight gain of the experimental group was significantly different from that of the negative control group, which was not fed the composition of the present invention, as the 3rd to 4th week in the DIFF parking (p <0.01) (Fig. 2).

<2-2> Mode of Increasing Average Weight

Compared with the negative control group of the weak control pig group not fed the composition of the present invention, the weight gain of the experimental group fed the composition of the present invention was significantly different (Fig. 3).

<2-3> Isolation of Causal Virus from Dead Weak Piglets

In the experimental procedure, two deaths (one week at two weeks and one week at three weeks) were observed in the negative control group, and the serum analysis of the two dead deaths was performed. Specifically, viral RNA and viral DNA were extracted from the two dead sera (PRRSV is RNA virus, PCV-2 is DNA virus), and then primers specific for PRRSV and PCV-2 were used (ORF7 of PRRSV). The gene region was amplified by ORF2) PCR of PCV-2. In this case, primers having a nucleotide sequence shown in SEQ ID NO: 1 and SEQ ID NO: 2 was used as a primer for PRRSV (Res Vet Sci. 2008 Aug; 85 (1): 184-93. Epub 2007 Dec 3. Simultaneous detection and genotyping of porcine reproductive and respiratory syndrome virus (PRRSV) by real-time RT-PCR and amplicon melting curve analysis using SYBR Green.MartE, Riera P, SitjM, Fang Y, Oliveira S, Maldonado J.), on PCV-2 As primers, primers having the nucleotide sequences set forth in SEQ ID NO: 3 and SEQ ID NO: 4 were used (J Vet Diagn Invest. 2003 Jul; 15 (4): 369-73. Detection of porcine circovirus type 2 in feces of pigs with or without enteric disease by polymerase chain reaction.Yang JS, Song DS, Kim SY, Lyoo KS, Park BK.). Thereafter, electrophoresis was performed to confirm that PRRSV and CPV-2 were positive (PRRSV: 300 bp, PCV-2: 300 bp) (FIG. 4).

In addition, gross morbidity was observed immediately after mortality, and the tissues were fixed with 10% buffered formalin, paraffin blocks were prepared, and analyzed by H & E staining. 5) This is a typical finding in respiratory symptoms caused by PRRSV and PCV-2 infection.

Example 3. Vital Sign Evaluation

The scoring of vital signs is described in Halina M. Zaleski, Roger R. Hacker, Variables related to the progress of parturition and probability of stillbirth in swine, The Canadian Veterinary Journal, 1993 February; 34 (2): 109-113. The method was changed and scored from 0 to 12 by combining muscle tone, skin condition, the ratio of duration of movement to lying for 30 minutes, the presence of clinical symptoms, feed intake, and negative intake. The vital signs scoring was performed at 3, 7, 10, 14, 17, 21, 24, 28 days of age.

As a result, although the vitality of the experimental group did not significantly increase until week 0 and week 1, the vital signs score was significantly increased compared to the negative control group after week 2 (FIGS. 6 to 9).

Example 4. Acute Toxicity Experiments

Acute toxicity test was performed using 7-week-old specific pathogen-free (SPF) SD (Sprague-Dawley) rats supplied by Dooyeol Biotech as follows. After 7 days of acquiring the experimental animals, 5 animals per group were administered to the control group and the herbal extracts prepared in Example 1 at doses of 500, 1,000 and 2,000 mg / kg, respectively. The experiment was performed by setting the experimental group. Animal mortality, clinical symptoms and body weight change (prior to administration, 1, 3, 7 and 14 days) were observed after one oral administration, and autopsy was performed to visually check for abnormalities of the thoracic cavity and abdominal organs.

As a result, no deaths or unusual general symptoms were observed in the experimental animals of all experimental groups, and the weight measurement showed normal weight gain in all experimental animals. At the end of the experiment, autopsy of all living animals revealed no unusual visual findings. As a result, the herbal extract did not show a toxic change even up to 2 g / kg in female and male rats, respectively, and the minimum lethal dose (LD ₅₀ ) was confirmed to be a safe substance of more than 2 g / kg.

Example 5. Chromosome Aberration Experiment

In order to evaluate the genotoxicity of the herbal extract prepared in Example 1 metabolic activation method (+ S9 mix) and applying metabolic enzyme system (S9) using ovarian embryonic cells (CHO-K1 cells) derived from Chinese hamster Chromosomal aberration experiments were performed in a direct method (-S9 mix). At this time, the composition of the S9 mix is as follows: 0.3 ml of S9 in 1 ml of S9 mix, 5 µmol of MgCl ₂ , 33 µmol of KCl, 5 µmol of G-6-P, 4 µmol of NADP, 4 µl of HEPES buffer. Mol included. Test substance was prepared by dissolving in sterile distilled water and diluting. In order to determine the treatment concentration of the test substance, cell proliferation inhibition experiments were performed, and the test substance was performed by applying eight concentration steps (39.06, 78.13 156.25, 312.5, 625, 1,250, 2,500, 5,000 ㎍ / ml). The concentration for the experiment was determined. Based on the results of the cell proliferation inhibition experiment, the highest treatment concentration of the test substance of this experiment was determined, and was determined as the following three concentration groups of azeotrope 2.

Direct method (-S9 mix, 24 hours continuous treatment group): 312.5, 625, 1,250 ㎍ / mL

Direct method (-S9 mix, 18 hours recovery group for 6 hours treatment): 312.5, 625, 1250 μg / ml

Metabolic activity method (+ S9 mix, 18 hours recovery group for 6 hours treatment): 625, 1,250, 2,500 ㎍ / ml

As a result of this experiment, the frequency of aberrant metaphases was not significantly increased at all treatment concentrations in the 24 hours continuous treatment group without direct metabolism activation (FIG. 10). In the 6-hour recovery 18-hour recovery group of the direct method, the significant increase in the frequency of abnormal midphase was not observed at all treatment concentrations compared to the negative control group (FIG. 11). The frequency of polyploidy (PP) and endoreduplication (ER) in both the 24-hour and 6-hour recovery groups did not show a statistically significant increase compared to the negative control group (Table 2).

TABLE 2

* Significant difference from negative control ( p <0.05)

a) processing time-recovery time

MMC: Mitomycin C (0.04 μg / ml), PP: ploidy, ER: intranuclear excretion

Gap: The undyed part is above the longitudinal axis of the chromosome, indicating that the width is as clear as the thickness of the chromosome.

In addition, when treated with the test substance for 6 hours using the metabolic activity method, the frequency of abnormal mesophase in all treatment groups was not statistically significant compared to the negative control group (FIG. 12). PP) and the frequency of intranuclear embryonic digestion (ER) also did not show a statistically significant increase compared to the negative control (Table 3).

TABLE 3

* Significant difference from negative control ( p <0.05)

a) processing time-recovery time

CPA: CyclophosphamideH ₂ O (10 μg / ml)

From the above results, it was confirmed that the herbal extract does not cause chromosomal aberration for CHO-K1 cells under the present experimental conditions.

Example 6 Microbial Return Mutation Experiment

In order to obtain basic data for judging the presence or absence of carcinogenicity of the herbal extract prepared in Example 1, microbial return mutation experiments were performed during genotoxicity experiments. In the experiment, histidine-required strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537) and WP2uvrA, a tryptophan-required strain of Escherichia coli, were used. The test substance was suspended in sterile distilled water and used for metabolic activity and for non-metabolism. At this time, the composition of the S9 mix used as a metabolite was as follows: 1 ml of S9 in 10 ml of S9 mix, 0.2 ml of 0.4 M MgCl ₂ /1.65 M KCl, 0.05 ml of 1.0 M Glucose-6-Phosphate, 0.1 M NADPH 0.4 ml, 0.1 M NADH 0.4 ml, 0.2 M Na-PBS 5 ml, distilled water 2.95 ml.

In order to determine the application concentration in this experiment, the concentration determination experiment was carried out in the 5 concentration group of azeotrope 2 with 5,000 ㎍ / plate as the highest concentration, and growth inhibition was suppressed in all strains regardless of the application of metabolic activity system. It was not observed (Table 4).

[Revision under Rule 91 03.02.2012]
Table 4

In the above, NaN ₃ is sodium azide, 9-AA is 9-aminoacridine hydrochloride hydrate, AF-2 is 2- (2-furyl) -3- (5-nitro-2-furyl) acrylamide) , 2-AA represents 2-aminoanthracene.

Based on the results of concentration determination experiments, the increase in the number of returned colonies was observed in the non-metabolized and non-metabolized groups compared with the negative control group in the treated group, but positively judged. No increase was observed (Table 5 and FIGS. 13-16).

[Revision under Rule 91 03.02.2012]
Table 5

From the above results, it was confirmed that the herbal extract does not cause a return mutation under the present experimental conditions.

Example 7 Micronucleus Experiments Using Mouse Bone Marrow Cells

Micronucleus experiments using bone marrow cells of male mice were performed to evaluate the genotoxicity of the herbal extract prepared in Example 1. In the micronucleus experiment, 7-week-old male mice were purified for 7 days in the preliminary experiment and the present experiment, and the test materials were administered orally by concentration. The test material was prepared by suspending in sterile saline solution, and in the preliminary experiments and the present experiment were treated in step concentration of 1,250, 2,500, 5,000 mg / kg.

As a result of preliminary experiments, no general symptoms were observed in all test substance-treated groups compared to the solvent control group. Observed according to the dose concentration (1,250, 2,500, 5,000 mg / kg) and the time of slaughter after dosing (24 hours, 48 hours), the number of polychromatic erythrocytes in total erythrocytes was significantly inhibited in proliferation of myeloid cells compared to the solvent control group. Did not appear.

Based on the results of the preliminary experiments, the main experiment was performed at 5,000 mg / kg as the highest dose, and autopsy and specimen preparation were performed 24 hours after administration. As a result, the number of polyinflammatory red blood cells in the total erythrocytes in the 5,000 mg / kg administration group showed a statistically significant difference compared to the solvent control (p <0.05). The frequency of micronucleus induction observed in about 2,000 polyinflammatory erythrocytes per subject did not show statistically significant results in all test substance administration groups (1,250, 2,500, 5,000 mg / kg) compared to the solvent control group. On the other hand, mitomycin C used as a positive control was statistically significant and significantly increased in the frequency of micronucleus induction compared to the solvent control (p <0.01). No statistically significant body weight change was observed in all dose groups after administration of the test substance compared to the solvent control group (Table 6).

Table 6

* Significant difference from negative control ( p <0.05) (one-way ANOVA)

** Significant difference from negative control ( p <0.01) (one-way ANOVA)

MNPCE: PCE with one or more micronuclei, PCE: polyinflammatory red blood cells, NCE: normal red blood cells, MMC: mitomycin C

From the above results, it was confirmed that the herbal extract does not affect the micronucleus formation of mouse bone marrow cells under the present experimental conditions.

Preparation example. Preparation of a feed composition comprising a herbal extract

The present inventors prepared the composition for feed addition comprising the herbal extract prepared in Example 1 to the composition of Table 7 below.

TABLE 7 Composition ratio of feed composition (unit; weight%)

	Herbal extract	Enzyme preparation	microbe	amino acid	Other
Preparation Example 1	100
Preparation Example 2	90	10
Preparation Example 3	80	10	10
Preparation Example 4	70	10	10	10
Preparation Example 5	60	15	15	8	2
Preparation Example 6	55	20	15	8	2

In Table 7, the enzyme preparation used a combination of phytase, cellulase, xylanase, maltase, and converting enzyme, and Aspergillus orissae was used as a non-pathogenic microorganism.

Claims (11)

1. Water or herbal medicines that include celestial, windproof, donkey, peony, duct, peppermint, ephedra, forget-me-not, rhubarb, gypsum, gilsheng, golden, white leach, gardenia, mold, ginger, talc, licorice, yin and yang, gold and silver A composition for drinking water or feed containing a herbal extract extracted with an organic solvent.
2. The method according to claim 1,

   1 part by weight of the yin and yang, cheongung, windproof, donkey, peony, yeonkyo, peppermint, ephedra, mango, rhubarb, gypsum, gilyeong, golden, white peach, wild gardenia, hedgehog, ginger, talc, licorice, gold and silver, paper and wood A composition extracted by mixing at a mixing ratio of 5 to 5 parts by weight.
3. The method according to claim 1,

   The organic solvent is selected from the group consisting of C ₁ -C ₄ alcohols, C ₁ -C ₄ ketones, C ₁ -C ₄ aldehydes and aqueous solutions of the alcohols, ketones and aldehydes.
4. The method according to claim 1,

   A composition further comprising one or more enzyme preparations.
5. The method according to claim 4,

   Said enzyme preparation is selected from the group consisting of lipase, phytase, amylase, phosphatase, carboxymethylcellulose, xylanase, maltase and convertase.
6. The method according to claim 1,

   The composition further comprises other non-pathogenic microorganisms.
7. The method according to claim 6,

   The microorganism is selected from the group consisting of microorganisms of the genus Lactobacillus, Aspergillus Orizae, Saccharomyces cerevisiae.
8. The method according to claim 1,

   Further a composition containing at least one amino acid.
9. The method according to any one of claims 1 to 8,

   A composition that ameliorates clinical symptoms caused by infection of the swine genital respiratory syndrome virus or swine circovirus type 2.
10. The method according to claim 9,

    The clinical symptoms are selected from the group consisting of weight loss, swine genital respiratory syndrome, weaning piglet systemic wasting syndrome, swine dermatitis renal failure syndrome, complex swine respiratory disease, miscarriage, stillbirth and exudative epidermal infection.
11. Feed containing the composition for drinking water or feed of claim 1.

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