KR101061257B1 - Natural antibiotics using bee venom and herbal extracts as an active ingredient and preparation method thereof - Google Patents

Abstract

The present invention relates to a natural antibiotic and a method for producing the same, and more particularly, to a natural antibiotic having an extract extracted from a specific hydrothermal condition with excellent antioxidant effect and bee venom with excellent antibacterial effect as an active ingredient. The natural antibiotics of the present invention have a high bactericidal effect on diarrhea-inducing bacteria in pigs, which can prevent and treat diseases of piglets, thereby improving productivity of piglets and providing pork without antibiotics remaining. In addition, since the bactericidal power against cow mastitis-causing bacteria is also high, the number of somatic cells in cow's milk can be significantly lowered, and it can replace the existing antibiotics. Therefore, it is possible to provide dairy products such as high-quality milk and cheese that have solved the problem of antibiotic residue in milk. Can be.

Bee venom, indongcho, eoseongcho, injin mugwort, green tea, antibiotic

Description

Natural antibiotics comprising bee-venoms and crude drug-extracts and Preparing method according to bee venom and herbal extracts

The present invention relates to a natural antibiotic extract and a method of preparing the same as an active ingredient extracted from a specific hot water conditions with excellent antioxidant effect and bee venom with excellent antibacterial effect, can replace the existing antibiotics applied to livestock, in particular pigs It is characterized by having a very good bactericidal power against diarrhea-inducing bacteria and cow mastitis-causing bacteria.

Bee venom is composed of a variety of components, dual peptides are anti-inflammatory, antibacterial, strong analgesic, immune enhancement. Bee venom has antimicrobial activity against both gram negative and positive bacteria, but is known to be stronger in gram positive bacteria. Most pharmacological action on bee venom is due to the activity of the entire poison component, a single major component group. The components and efficacy of bee venom are known as follows.

① Treatment of diseases of the immune system: Bee venom is effective in the treatment of most immune diseases, which enables the bee venom to stimulate the mammal's immune system to fight disease successfully. The two main immune actions of bee venom are to stimulate the organism's biological system, which in turn increases the body's defenses.

② anti-inflammatory action: The main components of bee venom are very effective in inhibiting inflammation. In the case of arthritis, infusion of melittin and apamin significantly suppressed edema. Protease inhibitors and adolamine treatment also inhibited 15-40% of edema.

③ neurotoxic effect: stinging bees cause pain and inflammation, and the substances causing them are effectively used in the development of a rare skin reaction or painkillers in mammals. In particular, it works by the action of apamin in bee venom, which is effective in various neuralgia, arthritis and rheumatoid arthritis, gout, myalgia, and strong therapeutic effect.

④ hemolysis: One of the most excellent effects of the bee sting is hemolysis. By melittin, phospholipase, etc., it absorbs and excretes blood, such as bruises and internal bleeding, and supplies new blood, oxygen, and nutrients to tissues for treatment and hemolysis.

⑤ vasodilation: the action of histamine substance is effective in expanding capillaries, arteries, predetermined veins, especially visceral vessels. Therefore, to treat diseases such as colds, frostbite, myalgia.

⑥ blood pressure lowering action: by the action of histamine substances to reduce the blood pressure is a powerful action. In particular, the concentration of 250 million 1/1 has a blood pressure lowering effect, and is excellent in the treatment of essential hypertension and the like.

⑦ autonomic nerve control action: When the human body is constantly stressed sympathetic nerve and parasympathetic nerve of the sympathetic nerve comes to this cause a lot of illness. The catecholamines and acylcholine, which are necessary to normalize the autonomic nerve, are contained in bee venom and it is effective in the treatment of psychosomatic menopausal disorder stress because it is a brain cell transporter.

⑧ antibacterial effect: antibacterial and antifungal effect is very good and effective in viral tumors. In particular, it has a high therapeutic effect on periodontitis, tonsillitis, stye, purulent diseases.

Lanicera japonica belonging to the evergreen tree ( Lanicera japonica ) is known to have physiological activities such as immune revival, anti-inflammatory, analgesic and antioxidant activity, and Artemisia capillaris has long been anti-inflammatory, antipyretic, edema, diuresis and It is widely used for the purpose of improving epilepsy, and is known to have pharmacological effects such as antioxidant activity. Houttuynia cordata is known to have diuretic, swelling, detoxification, edema, and antioxidant effects. The pharmacological effects of green tea ( Camellia sineis ) are widely known, and are known to be particularly excellent in antioxidant and anti-tumor efficiency. In addition, it is known that the daily weight gain and diarrhea frequency are greatly reduced when the mixture of Injin mugwort and Eoseongcho is added to the mixed feed and fed to weaning piglets.

Antibiotics have been widely used in pig feed since the 1950s, with the addition of antibiotics in pig feed to improve growth rates and improve feed efficiency, as well as to prevent and treat diseases (Hays, 1977; Zimmerman, 1986). . However, as public health interest in antibiotic-resistant bacteria increases along with antibiotic residues in meat products (Braude, 1978; Kunin, 1993; Cassell, 1995), research on the development of antibiotic substitutes in the swine industry is being actively conducted. Above all, livestock farms are planning to have a ban on most existing antibiotics in use since 2012, and there is an urgent need to develop substances to replace these antibiotics in natural materials.

Cow mastitis is a disease in which bacteria or fungi enter the cow's breast and cause inflammation, which is the most common disease in cows worldwide. In Korea, research on the crude oil sanitation system and the solution to this problem are being conducted in various ways. The pathogenesis and developmental factors of mastitis are very complicated and more than 80 kinds of causative organisms act, so there are many difficulties in treatment with certain antibiotics. In addition, the resistance rate is increased due to the use of antibiotics, and the treatment rate decreases.As a problem of antibiotics remaining in milk, it is necessary to set a period of absence for each antibiotic so that it is not necessary to develop hygienically safe and effective cow mastitis treatment. to be. Therefore, researches using natural materials as active agents for treating mastitis that can replace antibiotics causing these problems are being actively conducted.

Recently, there are reports that bee needle therapy is effective for promoting growth of animals, increasing immune function, and treating various diseases, and is actually used for preventing and treating diseases of animals and improving productivity in farms. Bee venom, which is a pure natural substance and has strong antibacterial and anti-inflammatory effects, has little risk of side effects and residues, and has been used for many years for diseases such as arthritis and gout as a bee sting therapy. It is becoming.

Therefore, the present inventors endeavored to improve the productivity of dairy products and livestock products, and as a result, they focus on the excellent antibacterial activity of bee venom and the excellent antioxidant power of herbal medicines such as Indongcho, Eoseongcho, Injin mugwort, and green tea leaves. New natural antibiotics have been completed. That is, an object of the present invention is to provide a natural antibiotic that can replace the existing antibiotic remaining in the livestock products.

Such natural antibiotics of the present invention is characterized by including bee venom; and herbal extract extracted from at least one mixture selected from Indongcho, Eoseongcho, Injin mugwort and green tea leaves; as an active ingredient.

In addition, the present invention relates to a method for producing the natural antibiotics, dry ground pulverized powder of one or two or more mixtures selected from Indongcho, Eoseongcho, Injin mugwort and green tea leaves at 100 ~ 120 ℃ and 1.0 ~ 1.2 atm hydrothermal conditions Cooling to reflux to obtain the extract; Concentrating and extracting the extract under reduced pressure to obtain a herbal extract; And after mixing the herbal extract and bee venom, the step of sterilization; characterized in that it comprises a.

As described above, the natural antibiotics of the present invention have a high bactericidal effect on diarrhea-inducing bacteria of pigs, thereby preventing and treating piglets' diseases, thereby improving productivity of piglets and providing pork with no antibiotics remaining. In addition, since the bactericidal power against cow mastitis-causing bacteria is also high, the number of somatic cells in cow's milk can be significantly lowered, and the existing antibiotics can be replaced, thereby providing a high-quality dairy product in which antibiotic residue problem in crude milk is solved.

The present invention relates to a natural antibiotic comprising bee venom and herbal extract as an active ingredient, bee venom; and herbal extract extracted from at least one mixture selected from indongcho, eoseongcho, jinjin mugwort and green tea leaves; There is this.

When explaining in detail the natural antibiotic of the present invention, the bee venom contains 0.01 to 5% by weight, 5 to 20% by weight of the herbal extract and 75 to 94.99% by weight of the solvent.

The type and amount of the solvent are not particularly limited, and water, distilled water, saline, etc., which can be well mixed with bee venom and the herbal extract, may be used, and most preferably, saline solution is used.

Hereinafter, the components of the natural antibiotics will be described in more detail.

Bee venom

The natural antibiotics of the present invention include bee venom, which is known to have an anti-inflammatory action, neurotoxin action, hemolytic action, vasodilation action, blood pressure lowering action, autonomic nerve control action, etc. and is effective in treating immune system diseases as an active ingredient. . Such bee venom includes 0.01 to 5% of the total weight of natural antibiotics in the present invention, where bee venom is less than 0.01% may cause a significant decrease in the antibacterial effect, and when used in excess of 5% It is preferable to use within the above range because there is a problem that may cause.

Herbal Extract

Lanicera japonica belonging to the evergreen tree ( Lanicera japonica ) is known to have physiological activities such as immune revival, anti-inflammatory, analgesic and antioxidant activity, and Artemisia capillaris has long been anti-inflammatory, antipyretic, edema, diuresis and It is widely used for the purpose of improving epilepsy, and is known to have pharmacological effects such as antioxidant activity. Houttuynia cordata is known to have diuretic, swelling, detoxification, edema and antioxidant effects. The pharmacological effects of green tea ( Camellia sineis ) are well known, and are known to be particularly excellent in antioxidant and anti-tumor efficiency. In addition, it is known that the daily weight gain and diarrhea frequency are greatly reduced when the mixture of Injin mugwort and Eoseongcho is added to the mixed feed and fed to weaning piglets.

The herbal extract of the present invention is an extract of hot water from a single species or a mixture of two or more selected from Indongcho, Echoseong, Injin mugwort and green tea leaves, and the herbal extract is 5 to 20% by weight based on the total weight of the natural antibiotic of the present invention. It is included. At this time, if the herbal extract is less than 5% by weight may cause a problem that the antioxidant efficiency is reduced, if the excess of 20% by weight may cause a problem and properties change to show cytotoxicity, the herbal extract within the above range It is preferable to use. In addition, the natural antibiotic of the present invention is made of the solvent in addition to 0.01 ~ 5% by weight of the bee venom and 5 ~ 20% by weight of the herbal extract.

Although the present invention is not limited thereto, the herbal extract is obtained by refluxing a single species or a mixture of two or more selected from Indongcho, Eungseongcho, Injin mugwort and green tea leaves under the hydrothermal conditions of 100 ~ 120 ℃ and 1.0 ~ 1.2 atm, The extract can be obtained by concentration under reduced pressure and filtration. Here, if the temperature is less than 100 ℃ may cause a problem that the water-soluble active ingredients are not sufficiently extracted, when the temperature is more than 120 ℃ may cause a problem to extract components other than the active ingredient, in a condition outside the 1.0 ~ 1.2 atm range Since reflux may cause a change in the components, it is preferable to obtain the extract under hydrothermal conditions within the above range. And reflux time should be enough for 2 hours.

When obtaining the herbal extract from the mixture of Indongcho, Eoseongcho, Injin mugwort and green tea leaves, hot water extraction from a mixture containing Indongcho, Eoseongcho, Injin mugwort and green tea leaves in a weight ratio of 1: 1.5 to 2.0: 1.0 to 2.0: 2.0 to 2.5 It is good to obtain by making it use, and it is preferable to use the mixture mixed by the said mixture weight ratio which this invention shows, from the point which raises antioxidant efficiency to the maximum.

Referring to the method for producing the natural antibiotic of the present invention will be described.

The natural antibiotics are refluxed for 1 hour 30 minutes to 2 hours 30 minutes in a dry grinding product of a single or two or more mixtures selected from Indongcho, Eoseongcho, Injin mugwort and green tea leaves at 100-120 ° C and 1.0-1.2 atm hydrothermal conditions Obtaining an extract; Concentrating and extracting the extract under reduced pressure to obtain a herbal extract; And after mixing the herbal extract and bee venom, it can be obtained through the step of sterilization.

The natural antibiotics of the present invention can be used in the form of feed additives of livestock, as well as mixed with pharmaceutically acceptable carriers, excipients (forming agents), diluents (powders, granules, capsules or injections) It can be prepared and applied in the form of.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the scope of the present invention is not limited by the following examples.

Example 1 Preparation of Natural Antibiotic

100 g of the mixture of dried Indongcho, Eoseongcho, Injin mugwort and green tea leaves in a weight ratio of 1: 2.0: 2.0: 2.5 was refluxed for 2 hours at 100 ° C and 1.2 atm conditions with 1L of distilled water to obtain an extract, and then concentrated under reduced pressure. ml of concentrated extract were obtained. The concentrated extract was filtered through a membrane (membrane filtration) of 0.45㎛ to obtain a herbal extract. Bee venom was isolated by using bee venom collecting device (Chungjin Tech, Korea) and impurities were removed to obtain pure bee venom. The isolated bee venom was obtained by liquid chromatography (AKTA explorer, Amersham Pharmacia Biotech USA) It was confirmed in comparison with phosphorus sigma bee venom, melittin, apamin, phospholipase.

0.1 g of the bee venom and 10 g of the herbal extract were placed in a beaker, filled with physiological saline to 100 ml gridline of the beaker, stirred and mixed, sterilized with a 0.2 μm membrane, and 0.1% by weight of bee venom extract 10 100 ml of natural antibiotic with weight percent was prepared.

Examples 2 to 3

To carry out the same as in Example 1, to prepare a natural antibiotic having the composition shown in Table 1.

Examples 4-8

Example 4 was carried out in the same manner as in Example 1, Example 4 from Indongcho, Example 5 from Eoseongcho, Example 6 from Injin mugwort and Example 7 using the herbal extract obtained from green tea leaves. In addition, Example 8 was prepared by mixing Indongcho, Eoseongcho and Injin mugwort in the same weight ratio to produce a herbal extract, using this to prepare a natural antibiotic having the composition shown in Table 1.

Comparative Example 1

In the same manner as in Example 1, without using the herbal extract, using only bee venom obtained in Example 1 and physiological saline to prepare a natural antibiotic having the composition shown in Table 1.

Comparative Example 2

In the same manner as in Example 1, without using bee venom, using only the herbal extract and the physiological saline obtained in Example 1 to prepare a natural antibiotic having the composition shown in Table 1.

Experimental Example 1: DPPH radical scavenging ability

DPPH (2,2-dihenyl-1-picrylhydrazyl) radical scavenging experiments of the natural antibiotics prepared in Examples 1 to 8 and Comparative Examples 1 and 2 were performed, and the results are shown in FIG. 1. DPPH antioxidant experiment was carried out at a concentration of 10% natural antibiotics prepared as in Example, and vitamin C was used as a positive control. And antioxidant capacity was calculated | required by following formula (1).

 Antioxidant Capacity = 100-(A / B × 100)

 Here, A is the absorbance of the sample at 517 nm, B is the absorbance of Blank at 517 nm.

Sterilization Test

Experimental Example 2-1: Sterilization test for wild strain isolated from pigs with diarrhea

The duration (PAE, postantibiotic effect) of the natural antibiotics prepared in Example 1 using wild strain isolated from pigs with diarrhea was measured by the method of Pankuch and Appelbaum (2006). After treatment with the culture at the same concentration as in the example, bee venom was removed by centrifugation and media dilution. Thereafter, the cells were replaced with fresh medium, incubated in a 37 ° C. incubator, and the bacterial counts were measured at 1 hour intervals. The results are shown in Table 2 below. The results can be said that the greater the PAE value, the better the antibacterial effect, the equation for obtaining the PAE is shown in Equation 2 below.

PAE = T-C

Here, T is the time taken to grow up to 1log ₁₀ in the test zone treated with natural antibiotics, and C is the time taken to grow up to 1log ₁₀ in the untreated zone.

* Number of inoculated bacteria: 2.5 ± 10 ⁵ CFU / ㎖

Experimental Example 2-2: Sterilization test for wild strain isolated from crude milk infected with cow mastitis

Sterilization duration of the natural antibiotics prepared in Example 1 using wild strain isolated from the milk infected with cow mastitis was measured by the same method as Experimental Example 2-1, the results are shown in Table 3 below.

* Number of inoculated bacteria: 2.5 ± 10 ⁵ CFU / ㎖

Experimental Example 3: Daily weight gain experiment of piglets

120 pigs (Landrace × Yorkshire) × Duroc) born from 8-term sows and 10 litters were published and placed 10 per pig. The control group, which did not receive bee venom, provided only basic feed. The administration of the natural antibiotics of Examples 1 to 8 and Comparative Examples 1 and 2 were auto-injected by diluting 1 ml on day 1 and 2 ml of weaning in physiological saline, and the specification management of conventional management methods such as vaccination and iron injection. Food and water were allowed to feed freely.

Body weight and feed intake were measured during birth, mammalian, and growth periods. Body weight and daily gain rate are shown in FIG. 2, survival rate is shown in FIG. 3, and feed efficiency and feed rate from birth to 60 days of age are shown in Table 4 below. Blood collection was performed by using a K3-EDTA Vacuum tube (Vacutainer, BD, USA) from the jugular vein at 20 days of age and analyzing blood cells using an automated blood analyzer (Hamat 8, SEAC, Italy). Shown in At this time, serum biochemical test was performed using a serum vacuum tube (Vacutainer, BD, USA), blood was collected, and serum was separated by centrifugation at 2,000 × g for 30 minutes at 4 ° C. Serum biochemical tests were performed using an automated biochemical analyzer (ADVIA1650, Byer, USA; Cobas Inetra, Roche, Switzerland), and all data were analyzed by variance analysis using Duncan's t-tests from SAS enterprise guide 3.0 (SAS). Was carried out.

division Non-treatment Example 1 Example 2 Example 3 Example 4 Example 5 Example 6 Example 7 Example 8 Comparative Example 1 Comparative Example 2 Feed rate 2.08 1.72 1.85 1.74 1.93 1.98 1.86 1.93 1.88 1.93 1.92 Feed efficiency 0.51 0.55 0.54 0.56 0.53 0.53 0.54 0.53 0.54 0.52 0.52

Experimental Example 4 Experiment for Measuring Somatic Cell Count in Crude Oil

In order to measure the effect on the reduction of somatic cell counts for cow mastitis, 15 natural cows and 15 minute cows in Yangpyeong, Gyeonggi-do, where the somatic cell count of crude milk was more than 999 million per ml, were compared with those of Examples 1 to 8 and Comparative Examples 1 to 2. Antibiotics were injected into the affected area of the cow once every day for 12 days at 12 ml for 5 days, and somatic cell counts were examined by collecting crude milk at 3 and 6 days after the initial injection. At this time, the control group was not treated with bee venom, but provided only basic feed. Somatic cell count in crude oil was aseptically collected in a sterile test tube and refrigerated, and then measured by using a Somacount 150 (Bentiey, USA) instrument to measure the somatic cell counts are shown in Table 6 below.

As described above, the natural antibiotics of the present invention are excellent in effect, and can replace the existing antibiotics, and can be used as feed additives of livestock, as well as pharmaceutically acceptable carriers and excipients (forming) agent), diluent (dilute) and the like can be prepared in the form of powder, granules, capsules or injections, etc. can be used for various applications. Through this, it is expected to provide non-antibiotic dairy products and livestock products to promote public health.

1 is a DPPH radical scavenging ability test results of Experimental Example 1.

2 is an experimental result of the weight gain and the daily gain rate of piglets in Experimental Example 3.

3 is an experimental result of the survival rate of piglets of Experimental Example 3.

Claims (5)

Bee Venom; and herbal extract extracted from at least one mixture selected from Indongcho, Eungseongcho, Injin mugwort and green tea leaves; Natural antibiotics as an active ingredient. The method of claim 1, 0.01 to 5% by weight of the bee venom, 5 to 20% by weight of the herbal extract and 75 to 94.99% by weight of the natural antibiotics. According to claim 1 or 2, wherein the herbal extract is Indongcho, Eoseongcho, Injin mugwort and green tea leaves 1: 1.5 to 2.0: 1.0 to 2.0: natural antibiotics, characterized in that it contains 2.0 to 2.5 by weight. Obtaining a extract by refluxing the dry ground product of at least one mixture selected from Indongcho, Eungseongcho, Injin mugwort and green tea leaves under hydrothermal conditions at 100-120 ° C. and 1-1.2 atm; Concentrating and extracting the extract under reduced pressure to obtain a herbal extract; And Mixing the herbal extract with bee venom and then sterilizing; Natural antibiotics manufacturing method comprising a. A feed additive, comprising the natural antibiotic of claim 1 or 2.

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