KR100445413B1 - The animal growth stimulating substance with korean traditional medicine and the preparing method of thereof - Google Patents

Abstract

The present invention relates to an animal growth promoter using a herbal medicine and a method for producing the same.

Animal growth promoting agent using the herbal medicine of the present invention is put into a hot water extractor at a ratio of 300 g of herbal medicine selected from the herbal medicines Kwakhyang, Eoseongcho, Pogongyoung, Clove, Gugija to 5,000 ml of distilled water 5,000 ml 3 hours at a temperature of 116 ~ 126 ℃ After extraction, the mixture was filtered to obtain an extract, and then refrigerated at a temperature of 3 to 5 ° C., and then, after feeding, the extract was diluted in water at a rate of 10% (v / v) to prepare an animal growth accelerator and then fed. .

Animal growth promoter using the herbal medicine of the present invention activates immune macrophages by significantly increasing iNOS (inducible Nitric Oxide Synthase) gene expression, insulin growth factor-1 (IGF-1) and interferon-gamma (IFN-γ) Increasing the serum concentration of the animal, increases the weight of the animal, and increases the viability when infected with the EMC virus.

Description

Growth promoter for animal using herbal medicine and its manufacturing method {THE ANIMAL GROWTH STIMULATING SUBSTANCE WITH KOREAN TRADITIONAL MEDICINE AND THE PREPARING METHOD OF THEREOF}

The present invention relates to an animal growth promoter using a herbal medicine and a method for producing the same.

In addition to the five nutrients of carbohydrates, fats, proteins, vitamins, and inorganic salts, growth-stimulating substances are provided for livestock animals in order to promote their growth, improve feed efficiency and improve the quality of their products. substances).

Growth promoters known to date include antibiotics, hormones, live bacteria, and ruminant fermentation promoters.

When antibiotics are used, they can inhibit pathogens to promote the growth of livestock animals and improve the productivity of livestock products such as meat, milk, and eggs, but antibiotics used in livestock remain in livestock products such as milk, meat, and eggs. There is a problem such as can cause antibiotic resistance.

Growth hormone, yeast (Yeast), Lactobacillus ( Lactobacillus ) and other methods to add the feed to feed, but there is a costly disadvantage.

Meanwhile, Korean Patent Application No. 10-1999-0021539 (Method for manufacturing chicken feed using Chinese herbal medicine) mixes crude protein, crude fat, crude fiber, and the like, and mixed herbs such as corn, soybean meal, limestone, etc. It is described that the mixed chicken feed, but the composition and manufacturing process has a complex disadvantage.

Korean Patent Application No. 10-1999-0015504 (Method of manufacturing pork feed containing fish paste) relates to a pig feed comprising 5-10 wt% of powdered fish vinegar in a pig feed consisting of corn, soybean paste, sage stick, etc. In addition, the composition and manufacturing method are complicated.

An object of the present invention is to provide a growth accelerator for animals that is cheap and excellent in effect while being manufactured in a simple manner using the herbal medicine.

1 is a process diagram of action in the macrophages of iNOS

Figure 2 is an analysis of iNOS gene expression enhancement in macrophages of mice following the administration of the growth promoter for animals of the present invention

Figure 3 is a measure of the concentration of insulin growth factor-1 (IGF-1) in the serum of rats after administration of the animal growth promoter of the present invention

Figure 4 is a measure of the concentration of interferon-gamma (IFN-γ) in the serum of rats after administration of the growth promoter for animals of the present invention

Figure 5 is a measure of weight change in rats after administration of the growth promoter for animals of the present invention

6 is a final weight change of the rat after administration of the growth promoter for animals of the present invention

Figure 7 is a survival curve measurement of the rats for EMC virus infection after administration of the growth promoter for animals of the present invention

The present invention relates to an animal growth promoter using a herbal medicine and a method for producing the same.

Animal growth promoting agent using the herbal medicine of the present invention is put into a hot water extractor at a ratio of 300 g of herbal medicine selected from the herbal medicines Kwakhyang, Eoseongcho, Pogongyoung, Clove, Gugija to 5,000 ml of distilled water 5,000 ml 3 hours at a temperature of 116 ~ 126 ℃ After extraction, the mixture was filtered to obtain an extract, and then refrigerated at a temperature of 3 to 5 ° C., and then, after feeding, the extract was diluted in water at a rate of 10% (v / v) to prepare an animal growth accelerator and then fed. .

Animal growth promoter using the herbal medicine of the present invention significantly increases the expression of iNOS (inducible Nitric Oxide Synthase, NOS-II) gene to activate macrophage (Macrophage) to enhance immune boosting.

In addition, the growth promoter for animals using the herbal medicine of the present invention increases the serum concentration of insulin growth factor-1 (IGF-1) and interferon-gamma (IFN-γ).

In addition, the growth promoter for animals using the herbal medicine of the present invention has the effect of increasing the weight of the feed animal.

In addition, the growth promoter for animals using the herbal medicine of the present invention has the effect of increasing the survival rate of animals infected with the EMC virus.

Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples, but these examples do not limit the scope of the present invention.

Example 1 Preparation of Animal Growth Promoter Using Herbal Medicine

300 g of each of the 13 herb listed in Table 1 was prepared.

5,000 ml of distilled water was added to each of the prepared 13 kinds of herbal medicines, and extracted by boiling water extractor (SAE IK Medical, Korea) at a temperature of 116 ~ 126 ℃ for 3 hours.

The extract was filtered to give an extract.

The extract was refrigerated at a temperature of 3 to 5 ° C.

Before feeding, the extract was diluted in water at a rate of 10% (V / V) to prepare an animal growth promoter of the present invention.

<Table 1> Herbal Medicine List

Chinese medicine Scientific name Kill Bambusae caulis in taeniam Eoseongcho Houttuyniae Herba Poongyoung Taraxaci Herba Nurro Echinopsis Radix Kwak Incense Agastachis Herba cinnamon Cinnamomi cortex Cloves Caryophylli Flos (Syzygium Aromaticum) White oyster Journey Room Ligusturum lucidum Two tooth Eucommiae cortex Fennel Foeniculi fructus Wolfberry Lycii Fructus Golden cherry Rosae Laevigatae Fructus

Experimental Example 1 Expression of iNOS (NOS-II) Gene in Mouse Macrophages

(1) Isolation and Culture of Macrophages in Mice

BALB / C mice were lethal to the cervical vertebra and injected with 6 ml of cold D-PBS in the abdominal cavity, lightly massage the abdomen for 1 minute, inhaled by suspension and separated macrophages into each well of a 24 well plate. After dispensing 10 ⁶ cells, starvation was performed for 1 hour.

Animal growth promoter prepared in Example 1 was treated in each well and co-cultured for 6 hours.

(2) reverse transcriptase-polymerase chain reaction (RT-PCR)

① RNA extraction

After the completion of the culture, the supernatant was removed, and the mouse immune cell membrane was popped using RNAzol ^B and RNA was extracted.

RNAzol ^B was added to CHCl 3 (chloroform) (40 μl / 400 μl RNAsol ^B ) in 1/10 amount, mixed with Vortex for 15 seconds, and left for 15 minutes on ice.

After centrifugation at 15,000 rpm for 15 minutes with a high speed centrifuge (4 ℃), take the supernatant, mix with an equal amount of iso-propanol and shake it slowly. After centrifugation for 5 minutes, remove the supernatant, add 1 ml of 80% EtOH / DEPC DW, vortex slightly, centrifuge at 15,000 rpm for 15 minutes, and remove the supernatant again. Dried.

Total RNA extracted from DEPC / D.W (0.05%) was dissolved in 20 μl of distilled water treated with diethyl pyrocarbonate (DEPC) and used for RT-PCR.

② Reverse Transcription-Polymerase Chain Reaction (RT-PCR)

The reverse transcription reaction denatures 3 μg of the prepared total RNA at 75 ° C. for 5 minutes, resulting in 2.5 μl 10 mM dNTPs mix, 1 μl random sequence hexanucleotides (25 pmole / 25 μl), and RNA inhibitor (inhibitor). 1 μl RNase inhibitor (20 U / μl), 1 μl 100 mM DTT, 4.5 μl 5 × RT buffer (250 mM Tris-HCl, pH 8.3, 375 mM KCl, 15 mM MgCl 2) 1 μl of M-MLV RT (200 U / μl) was added again and the final volume was 20 μl with DEPC treated distilled water.

The 20 μl reaction mixture was mixed well and then centrifuged at 2,000 rpm for 5 seconds to react for 60 minutes in a 37 ° C. constant temperature water bath to synthesize first-strand cDNA, and then left at 95 ° C. for 5 minutes to prepare M-mlV RT. After inactivation of the synthesized cDNA was used for PCR (polymerase chain reaction).

CDNA PCR

PCR was performed by reverse transcription-polymerase chain reaction using Primus 96 Legal PCR system (with high pressure lid, MWG in Germany).

The reaction uses 3 μl of the synthesized cDNA as a template, and primers for the template are 20 pmole / μl and antisense primer 20 to amplify β-actin and NOS-II. After mixing pmole / μl, 1 μl was added, again 3 μl 2.5 mM dNTPs, 3 μl 10 × PCR buffer (100 mM Tris-HCl, pH 8.3, 500 mM KCl, 15 mM MgCl ₂ ), 0.18 μl tack polymerase ( Taq polymerase) (5 U / μl) was added and sterile distilled water was added to a final volume of 30 μl, followed by predenaturation; 95 ° C, 5 minutes, denaturation; 95 ° C, annealing; 55 ° C, 1 minute, elongation; 72 ° C, 1 After 25 cycles of minutes, PCR was performed under postelongation conditions at 72 ° C. for 3 minutes.

Each PCR product was loaded into 1.2% agarose gel (20 μl) and analyzed by electrophoresis for 20 minutes at 120V.

The nucleotide sequence of the oligonucleotide is as follows;

Of NOS-II,

sence oligonucleotide 5'-ACCGATACAGTACAGTACAGA-3 '(SEQ ID NO: 1)

antisence oligonucleotide 5'-CCGATATTTAGATACGTTAAAC-3 '(SEQ ID NO: 2),

Of beta-actin,

sence oligonucleotide 5'-TGGAATCCTGATCCATGAAC-3 '(SEQ ID NO: 3)

antisence oligonucleotide 5'-TAAAACGCAGCTCAGTAGTCCG-3 '(SEQ ID NO: 4).

This experimental result is shown in FIG.

As shown in FIG. 2, when the LPS (lipopolysacchride) treatment of the mouse macrophages was used as a positive control, the NOS-II gene expression was significantly increased in the group treated with the animal growth promoter using pogongyoung, kwak, eosungcho, clove, and wolfberry. Thus, it has been shown to be effective in immune macrophage activation.

Experimental Example 2 Serum Concentration of Insulin Growth Factor-1 (IGF-1) after Administration of Animal Growth Accelerator

Serum concentrations of insulin growth factor-1 were measured after feeding 13 animal growth promoters prepared in Example 1 to experimental animals.

SD rats (4 weeks old, 80 ~ 100g) were purchased and used in the experiment after one week of adaptation in the animal laboratory of the College of Oriental Medicine, Daejeon University.

The feed was a solid feed for the rat (Samyang Yuji Feed Co., Ltd.), the negative water was first filtered through the water supply and sterilized using an ultraviolet sterilizer.

Breeding environment conditions of SPF-type animal room are temperature 23 ± 1 ℃, relative humidity 55 ± 5%, noise 60 phone or less, odor 20 ppm or less, lighting 150 ~ 300 Lux, lighting time 12 hours light cycle, ventilation frequency 13-15 It was reared in a time / hour.

Each group of rats were eight rats, and one group of control group, 13 groups of growth promoters prepared in Example 1, and one group of general herbal medicines were tested in a total of 15 groups.

20 ml / marily administered daily.

<Table 2> Placement of Rats

group Dosage Number of animals Remarks One fennel 8 Taking each herbal extract 2 Tofu 8 3 Eoseongcho 8 4 Kwak Hyang 8 5 Golden cherry 8 6 cloves 8 7 Nuro 8 8 White oyster 8 9 Wolfberry 8 10 Kill 8 11 Poongyoung 8 12 Journey Room 8 13 cinnamon 8 14 Chinese medicine 8 Taking herbal medicine on the market 15 Control 8 Dietary foods only

Serum concentrations of insulin growth factor-1 (IGF-1) were measured by selecting four rats reared for 4 weeks in each group.

The quantitative analysis of insulin growth factor-1 (IGF-1) was requested by the dual test center Co., Ltd. and the results are shown in FIG.

Serum concentration of insulin growth factor-1 (IGF-1) in rats was higher than that of control (approximately 1300 ng / ml), clove (1900 ng / ml), kwak (1800 ng / ml), and Echo (1900 ng / ml). Serum concentrations of insulin growth factor-1 (IGF-1) were 40-50% higher, pogongyoung (1700 ng / ml) and goji berry (2000 ng / ml).

Experimental Example 3 Measurement of Interferon-Gamma Concentration in Serum after Administration of Animal Growth Promoter

The concentration of interferon-gamma in serum was measured by IFN-γ ELISA kit (Pharmingen, USA) using the rats bred in Experimental Example 2.

Four rats were selected for each group, and 100 μl of rat serum was dispensed on a well plate.

After standing at room temperature for 1 hour, washed twice with washing buffer, and then treated with 100 μl of antibody Avidin-HRP conjugeted, left at room temperature for 1 hour, washed again, and 100 μl of TMB substrate was dispensed. After leaving for 30 minutes in the dark, 50 μl of stop solution was treated and the absorbance was measured at 450 nm of ELISA leader.

The results are shown in FIG.

As shown in FIG. 4, the serum concentration of interferon-gamma (IFN-γ) was measured as compared to the control group (30 pg / ml), goji berry (230 ng / ml), wagyang (260 ng / ml), and edible herb (170 pg / ml) showed a significant increase from 7 to 11 times, and clove and pogongyoung increased 2-3 times.

Experimental Example 4 Analysis on the Effect of Animal Growth Promoter on Weight Gain

Twelve kinds of animal growth promoters prepared in Example 1 were administered to experimental animals to test the effect of weight gain.

The mice were bred by the method of Experimental Example 2.

After administration of the growth promoter for animals, each individual was calibrated at 10 am at 2 am intervals for 4 weeks and then weighed accurately to 100 mg by balance.

The sample was measured by the amount of input at the time of input and the remaining sample at the time of weight measurement to measure the dietary amount by the difference from the input.

The results are shown in FIGS. 5 and 6.

In FIG. 5, a sharp change in body weight was observed for 4 weeks, and a significant difference in body weight was observed in comparison with the control group 2 weeks after the growth accelerator administration.

As shown in FIG. 6, the final body weight change after 4 weeks was as follows: clove (12.6%), eoseongcho (13.4%), pogongyoung (9.2%), kwagyang (10.7%), and wolfberry (15%) compared to the control (0%). There was a weight gain effect.

Experimental Example 5 Measurement of Survival Curve of Rats Against EMC Virus Infection after Administration of Animal Growth Promoter

Five rats of the SD series were used as one group for 72 hours by microvenous injection of EMC (Encephalomyocarditis) virus (× 10 ⁴ , × 10 ⁵ , × 10 ⁶ , × 10 ⁷ , × 10 ⁸ , × 10 ⁹ ) When observed, the LD ₅₀ value was measured by measuring the EMC virus concentration of 50% of the entire population.

Four rats of Experimental Example 2 were selected and microvenously injected with EMC virus (× 10 ⁷ ) at a LD ₅₀ concentration, and bred in an SPF breeding facility, and observed daily survival. The results are shown in FIG. 7.

In Gwakhyang, Eoseongcho, Pogongyoung, Clove and Gojija, life extension was observed 2 to 5 times compared to control.

In accordance with the present invention, iNOS gene expression increases macrophage activity, increases the concentration of insulin growth factor-1 and interferon-gamma in serum, increases body weight, and increases resistance to EMC virus. Provided are animal growth promoters and methods for preparing the same.

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Claims (2)

In the growth promoter for animals, The goji berries per 5,000 ml of distilled water were mixed at a rate of 300 g, Extracted by boiling water extractor at a temperature of 116 ~ 121 ℃ for 3 hours, After filtering to obtain goji berry extract, As a growth promoter for animals prepared by storing the gojija extract at a temperature of 3 ~ 5 ℃, dilution at a rate of 10% (V / V) in water at the time of feeding, Increasing iNOS gene expression promotes macrophage activity, increases the concentration of insulin growth factor-1 (IGF-1) and interferon-gamma (IFN-γ) in serum, increases body weight, EMC With features that increase resistance to viruses, Growth promoter for animals using wolfberry. In the production method of the growth promoter for animals, The goji berries per 5,000 ml of distilled water were mixed at a rate of 300 g, Extracted by boiling water extractor at a temperature of 116 ~ 121 ℃ for 3 hours, After filtering to obtain goji berry extract, Gojija extract is refrigerated at a temperature of 3 ~ 5 ℃, and diluted to 10% (V / V) in water at the time of feeding to prepare a growth accelerator, Method for producing animal growth promoter using wolfberry.