KR100785375B1 - Feed for prevention of heavy metal poisoning - Google Patents

Abstract

Feed for the prevention of heavy metal poisoning is provided to prevent a disease-causing factor of livestock or a pet and supply higher value-added functional feed. Feed for the prevention of heavy metal poisoning comprises 2ppm of organic selenium and 2~4ppm of the powder of dregs obtained from the distillation of medicinal herbs. The powder of dregs obtained from the distillation of medicinal herbs includes licorice and polyphenol. The licorice contains liquiritigenin capable of preventing cells from being damaged by heavy metals, and most of the liquiritigenin is included in the powder of dregs obtained from the distillation of medicinal herbs. In addition, polyphenol has an antioxidant effect and so promote the health of animals. The organic selenium such as selenomethionine, selenocysteine, or methylselenocysteine has anti-oxidant, anti-cancer, and anti-inflammatory effects.

Description

Feed for the prevention of heavy metal poisoning. {Feed for prevention of heavy metal poisoning}

1 is a schematic diagram of Liposome,

2 is a graph showing the weight change of the SD during the test period,

3 is a graph showing the oxidation index during the test period,

4 is a graph showing the oxidation index of serum during the test period,

5 is a graph showing the levels of sGOT and sGPT in blood.

The assessment of the degree of deposition of heavy metals in biological samples ranges from food and feed stability assessments to clinical trials for diagnosis and prescription. It has been the subject of continuous research up to the latest research for prescriptions. In general, a technique for testing the degree of deposition of heavy metals to take a part of the body, such as blood, urine, hair, etc. in order to determine the health of the living body and its impact on a specific environment, but the blood is commonly used In the case of the urine and urine specimens, there is a problem of the cumbersome sampling process and the variation of the diversity of the growing environment, and the temporal and spatial limitations on the tracking of various types of medical history of the subject, and the degree of symptoms of intoxication from the beginning of the onset. Not enough to track it. On the other hand, hair is easy to collect samples and has the advantage of being able to grasp the external and internal influences on the growth environment for the last 3-4 months. The method of investigation is expected to be a very useful diagnostic.

Attempts to cure heavy metal poisoning symptoms identified through diagnosis have been addressed as a major concern regardless of western medicine and oriental medicine. To date, studies have shown that various polyphenols contained in plants suppress heavy metal poisoning symptoms. Heavy metal detoxification methods known to date include methods using the sorption capacity of indigestible adhesive polymer sugars such as cellulose, pectin, chitosan, alginic acid, and lignin, and diuretic accelerators such as beer, yeast, microbial metabolites, and similar hormones. Methods for increasing cellular immunity using a release method, vitamins, antioxidants, and the like are known, and research on liquirigenin as a substance having direct detoxification ability is also underway.

On the other hand, the development of functional feed has also been steadily progressed, and in the Korean Patent Application Publication No. 0181679 Selenium-containing feed composition and the method of producing livestock products containing selenium, selenium is highly accumulated by feeding a feed composition containing 0.05-5 ppm of selenium methionine. The present invention provides a method for producing livestock products, and in the combination feed containing selenium as an active ingredient and a method for producing functional livestock and fruit vegetables using the same, 0.1-2.0 organic and inorganic selenium The company has provided a method for producing livestock products containing a large amount of selenium by feeding the compound feed with the addition of ppm. However, the above methods are intended to produce livestock products containing selenium, which is far from the purpose of preventing and treating heavy metal poisoning in livestock or pets.

In addition, in the Republic of Korea Patent Publication No. 10-0463805, dog food and manufacturing method: 5.5% cheongung, 4.2% sanjoin, 4.2% raw paper, Fuxin 4.2%, 5.5% turmeric, 5.5% half ginseng, 5.5% ginseng, 2.8% killed 2.8%, Golden 4.2%, Licorice 2.7%, Peony 5.5%, Insect Repellent 4.2%, Creation 5.5%, Thick 4.2%, Dermis 4.2%, Cod 4.2%, Hyunsam 2.8%, Hyangbu 5.5%, Gwakyang 2.8%, Jujube 5.5% Provided dog feed consisting of, ginger 2.7%, vigor 4.2%, in Korean Patent Publication No. 10-0495605, the herbal feed additive for broiler chicken and its manufacturing method 10% by weight of gold, 15% by weight of Seokchangpo, 10% by weight of edible vinegar, safflower An herbal feed additive containing 30% by weight, wolfberry 10% by weight, airborne 10% by weight, and 15% by weight glucose was provided. The feed additives, or feeds contain the components of the herbal medicine, but the purpose of adding the herbal medicine components has nothing to do with the prevention and treatment of heavy metal poisoning symptoms.

An object of the present invention is to provide a feed for preventing and treating heavy metal poisoning symptoms, to prevent disease factors of livestock and pets and to maintain health.

Feed for the prevention of heavy metal poisoning of the present invention contains herbal medicine and organic selenium.

Herbal medicine is the residue left after distilling the herbal medicine contains the ingredients of various herbs, among which licorice plays a role in harmonizing various herbs, so the main medicine included in most herbal medicine is included in the herbal medicine. Licorice contains liquiritigenin to prevent cell damage due to heavy metals.Liquirigenin is not soluble in water, so it is not extracted during the manufacture of Chinese medicine. . In addition, the polyphenol component contained in the herbal medicine has an antioxidant effect, which helps to improve the health of the animal, and can greatly reduce the cost by using the herbal medicine instead of expensive herbal medicine.

Selenium is an antioxidant, anti-cancer, anti-inflammatory action, and is a substance that shows the effect of activating the thyroid function and immune function. Since inorganic selenium is highly toxic, organic selenium such as selenomethionine, selenocysteine, and methyl selenocysteine is mainly used for ingestion.

In the present invention, it is used after drying, powdering and receiving the herbal medicine distillation residue of medicinal herb from Yoonsun Giper Co., Ltd. in consideration of economic value as a salary source of liquiriginine in licorice, which is a substance for preventing and treating heavy metal poisoning symptoms. Organic selenium was supplied from Hannew Farm Co., Ltd. and used. In order to evaluate the capability of the herbal medicine and the organic selenium raw material, the antioxidant capacity known as the representative function of the material was tested by preparing Liposome. In addition, as a result of analyzing the hair according to the breeding of the dog through ICP (Inductivity Coupled Plasma) -AES (Ultima-2C), the degree of heavy metal deposition of the dogs with the history of inflammation was high. The anti-inflammatory test was also conducted.

Experimental Example 1 Antioxidant and Anti-inflammatory Capacity Test Using Liposome as Substrate

Liposomes are prepared by suspending phospholipids in aqueous solutions. Liposomes are closed vesicles of lipid bilayers, which are liquid phases, and have the most similar form to biofilms. The phenomenon of liposomes originates from the nature of phospholipid molecules. That is, since it is an amphiphilic compound having an intramolecular polar group and a nonpolar group, the polar group is directed toward the aqueous solution side, and the nonpolar group tends to be collected by hydrophobic interaction. In the present invention, Liposome was used as a biofilm model for antioxidant activity evaluation.

Experimental Example 1-1 Preparation of Liposome

Put 50mg of EYPC (Egg yolk phosphatidylcholine) into the test tube, remove the organic solvent with Nitrogen gas, and fractionate in 1ml by using RP column (Lichroprep Lobar column, RP-8, 40 ~ 63mm, 240 × 10mm, Merck) After (CHCl ₃ -MeOH-H ₂ O (1: 10: 0.5, v / v), 1.8ml / min), each fraction unit was developed using HPTLC to confirm the elution fraction (elution range of about 35-55ml). , HPTLC plate (RP-8 F254s, Merck), CHCl ₃ -MeOH-H ₂ O (1: 10: 0.5, v / v), Iodine vapor (chromo-phosphorus)). The elution fractions were combined to remove the solvent, dissolved in CHCl ₃ -MeOH (95: 5, v / v), 5ml was placed in a test tube, applied to form a thin film on the outer wall of the tube, the solvent was removed with nitrogen gas and vacuum desiccated. Drying in the furnace for 30 minutes further removed the solvent completely. 0.7 ml of Tris-HCl buffer was added and stirred for 1 minute with Vortex to peel off the film layer, dispersed for 30 seconds on a sonicator to make 200-5,000 nm, and then polycarbonate membrane (Pore size) in LipoFast (Avestin Co., Ottawa, Canada). 100 nm) was used to prepare a large unilamella vesicle (LUV) having a size of 100 nm.

Experimental Example 1-2 FOX Assay (Ferrous Oxidation in Xylenol orange assay)

The oxidation of lipids can be explained by the production of hydroperoxide and the production of malonaldehyde. In other words, the antioxidant effect simply refers to a mechanism for inhibiting and reducing the production of the two substances described above. Hydroperoxide is a peroxide that occurs during the rancidity of lipids and is a source of oxidative stress in the living body. Therefore, the antioxidant effect of the herbal medicine and organic selenium can be confirmed as an inhibitory effect on the substance, for which FOX assay was performed. As shown in Table 1, AAPH (2,2'-azobis-2-amidinopropane-dihydrochloride), which is an oxidizing agent, was added to Liposome at a concentration of 60 mg / kg, and the functional substances to be measured were adjusted to meet the concentrations shown in Table 2, respectively. Added.

[Experimental Method 1-2] FOX Assay

Xylenol orange 38mg, 440mg is dissolved in 450ml HPLC grade methanol, 49mg Ammonium ferrous sulfate is dissolved in 50ml 250mM sulfuric acid and added to the above solvent. Final solvent was prepared by dissolving Xylenol Orange 100μmol / l, 4mM BHT, 25mM sulfuric acid and Ammonium ferrous sulfate in 250% mol / l in 90% methanol. Measure and check the numerical change (keep 3.92 × 10 ⁴ / Mcm or more). The solution is measured by preparing a calibration curve with a standard material and reacting with 300 μl of Liposome in the dark at a reaction time of 37 ° C. for 10 minutes.

Experimental Example 1-3 TBArs assay (Thiobarbituric acid reactive substance assay)

As a representative measure of antioxidant capacity, the amount of Malonaldehyde continuously increased by lipid oxidation was measured by TBArs assay. Oxidation-inducing agent AAPH (2,2'-azobis-2-amidinopropane-dihydrochloride) was added to Liposome at a concentration of 60 mg / kg, and the functional material to be measured was added to meet the concentrations listed in Table 3.

[Test Method 1-3] TBArs assay

300 μl of Liposome and the functional substance to be measured are placed in a test tube, SDS (200 μl, 8.1% in water, v / v), acetic acid (1.5ml, 20%, w / v, 15% NaOH (w / v) PH 4.0), 2-thiobarbituric acid (1.5 ml, 0.8% in water, w / v), BHT (0.5 ml, 0.08% in MeOH, w / v) is added. This was reacted at 95 ° C. for 60 minutes in a water bath and allowed to cool at room temperature, and then measured for absorbance at 532 nm using 1,1,3,3-tetramethoxypropane as a standard.

Experimental Example 1-4 Hyaluronidase inhibition assay

Hyaluronidase is an enzyme that hydrolyzes mucopolysaccharides. In addition to its ability to decompose hyaluronic acid, hyaluronidase is known as a prophylactic agent that is involved in capillary permeability and causes acute edema. Hyaluronidase is most active at pH 3-4 and is generally inactive in the body. Although a certain concentration is maintained in tissues, the amount is increased due to allergies, vascular permeable inflammatory reactions, and tissue damage. The functional material to be measured was added according to the concentrations shown in [Table 4].

[Test Method 1-4] Hyaluronidase inhibition assay

Dissolve Hyaluronidase in Acetate buffer (7,900 Units / ml), and add 20 µl of the sample containing the substance to be measured. In this case, MeOH is used as a control. 100 μl of 12.5 mM CaCl ₂ was added thereto and reacted in a 37 ° C. water bath for 20 minutes. Hyaluronic acid is added to the above reaction material (12mg / 5ml, acetate buffer), which is then reacted at 37 ° C. for 40 minutes, and 100 μl of 0.4N NaOH and 0.4M Potassium tetraborate are added. Again, the reaction in 100 ℃ / 3 min in a water bath and allowed to cool to room temperature. Dimethylaminobezaldehyde sol. 3 ml is added and reacted for 20 minutes in a water bath of 37 ° C., then the absorbance is measured at 585 nm to evaluate its ability based on the following formula.

Hyaluronidase inhibitor (%) = [(ODcontrol-ODsample) / ODcontrol] × 100

[Experimental Results] Statistical Processing

The measured data were analyzed using the SAS Statistical Package Program, and the results were calculated using the mean and standard deviation. The mean value of each group was analyzed by Duncan's multiple range test after variance analysis. ( p <0.05)

[Experiment 1] Antioxidant and Anti-inflammatory Capacity Test Using Liposome as Substrate

Antioxidant and anti-inflammatory properties of Medicinal Herbs and organo selenium were confirmed by FOX assay and TBArs test. The concentrations used in the test were applied up to 5 ~ 100ppm, respectively. For comparison, Ascorbate, a natural antioxidant, and BHT, a synthetic antioxidant, were tested in the same way. In order to induce artificial oxidative stress due to heavy metals, AAPH was co-treated with LUV, and each capacity was evaluated by quantifying the amount of peroxide increased by AAPH.

TABLE 1

TABLE 2

TABLE 3

TABLE 4

As shown in Table 1, the concentration of Hydroperoxide and Malonaldehyde was high as AAPH was treated. As shown in Table 2 and Table 3, as a result of treating the measurement target with antioxidant function with AAPH, the antioxidant effect was improved as the concentration of the measurement target functional material increased in all the test groups. In particular, in the group treated with Medicinal Herb Park and organic selenium, a strong antioxidant effect appeared compared to the separate treatment group, confirming the synergistic effect due to the simultaneous treatment.

The anti-inflammatory test was carried out by focusing on the correlation between dog hair and etiology, and the inhibitory effect of hyaluronidase as an anti-inflammatory effect by the use of the functional substance to be measured was examined. As can be seen in Table 4, the herbal medicine had a significant level of inhibitory effect of Hyaluronidase, whereas organic selenium and Ascorbate did not have a significant effect. However, when the organic selenium and the herbal medicine was treated simultaneously, the inhibitory effect was improved compared to the treatment with the herbal medicine alone, showing a synergistic effect.

According to Experimental Example 1, the organic selenium and the herbal medicinal herb showed better antioxidative and anti-inflammatory effects than the mixture, and the higher the concentration, the better the effect. However, current feed management legislation requires that selenium in feed should not exceed 2 ppm. In addition, as a result of feeding the feed by adding medicinal herb to the dog, when the medicinal herb exceeds 4ppm, the feed intake of the dog was drastically lowered, which means that the aroma and taste of the medicinal herb added to the dog do not fit the taste of the dog. It was judged that. Therefore, it was determined that the concentrations of selenium and herbal medicine added to the actual feed should be 2 ppm or less of organic selenium and 4 ppm or less of herbal medicine.

Experimental Example 2 Animal Test

The test animals used in this test are SD (Sprague-Dawley male rats, weighing approximately 180g and 6weeks old), and each test group is as described in Table 5 below. The test animals were placed in a stainless steel cage with 6 weights per group uniformly for each treatment group by the complete randomization method, and then adapted to the surrounding environment for 1 week. The total test period was 6 weeks. At the beginning of the initial test, the heavy metal treatment group was intraperitoneally administered the heavy metal standard prepared to the limit of food control law, and the AAPH (2, 2'-azobis-2-amidinopropane-dihydrochloride) treatment group. About 24 hours before slaughter, AAPH preparations were intraperitoneally administered. The functional substance to be measured was administered orally once a day for each group, and diet was fed without restriction on general feed and water. Hair was collected three times at the same back area each time, immediately before the start of the test, three weeks after the start of the test, and six weeks after the start of the test.

The body weight was measured every week during the test period. After 6 weeks, the whole body was slaughtered to determine the detoxification ability, and the liver and blood were collected to perform heavy metal deposition and oxidation index evaluation, sGOT, and sGPT tests. It was.

TABLE 5

Experimental Example 2-1 Evaluation of Oxidation Index of Serum and Liver

Liver tissues that are detoxified in vivo are quite sensitive to antioxidant effects. Since liver tissue exists in the form of a complexed Lipo-protein of a series of proteins and lipids, peroxide production is rapidly increased by oxidative stress, and TBArs experiments were conducted to verify oxidative stress in liver tissue (Ohkawa et al , 1979), hepatic tissue also examined the trend of heavy metal changes in tissues.

Experimental Example 2-2 Enzymatic assay

sGOT and sGPT are enzymes that are sensitive to oxidative stress, such as AAPH, and are rapidly secreted into the blood by the liver when oxidative stress is induced. Therefore, since the blood titer appears to be high due to oxidative stress, it was used as a measurement index to distinguish oxidative stress caused by AAPH and heavy metal induction. All tests were analyzed using an analysis kit supplied by Eiken.

Experimental Example 2-3 Heavy Metal Deposition Test

Hair was collected three times from the same site each time, divided into initial, middle, and slaughtered, and the collected hair was tested for heavy metal deposition using ICP-AES immediately after collection. Liver and blood were also tested in the same manner immediately after collection.

[Experimental Results] Statistical Processing

The measured data were analyzed using the SAS Statistical Package Program, and the results were calculated using the mean and standard deviation. The mean value of each group was analyzed by Duncan's multiple range test after variance analysis. ( p <0.05)

[Test Result 2] Animal Test

The change in body weight during the test period is shown in FIG. 2, and according to FIG. 2, no significant difference was found between the test groups.

[Experiment 2-1] Oxidation Index Evaluation Test

Oxidation index evaluation of the serum and liver samples extracted from the test animal carcass was tested by the TBArs test, the results are shown in Figures 3 and 4.

According to the results shown in FIGS. 3 and 4, there is no difference in the tendency between the serum and the liver. The malonaldehyde level was high in the heavy metal treatment group, thereby confirming the production of free radicals by the heavy metal. In addition, it was confirmed that the antioxidant effect of the mixture of herbal medicine and organic selenium. However, since the effect of oxidative stress inhibition was small compared to the concentration ratio of the herbal medicine used in the test, it was judged that the concentration of the herbal medicine used for the inhibition was close to saturation.

[Experiment 2-2] Blood sGOT and sGPT titer test

Blood sGOT and sGPT titer test results are shown in FIG. 5.

According to FIG. 5, blood titers of sGOT and sGPT secreted into the blood by external stress also showed almost the same tendency as the oxidative index evaluation test. That is, in the case of AAPH and heavy metal treatment group, the sGOT and sGPT titers were rapidly increased, but it was confirmed that the trend was stabilized again by using organic selenium and herbal medicine. However, as in the oxidation index evaluation test, it was judged that the concentration of the herbal medicine was close to saturation, considering that there was no significant difference between treatment groups having different concentrations of the herbal medicine.

[Experimental Result 2-3] Evaluation Test for Inhibitory Effect of Heavy Metal Deposition in Hair, Liver and Blood

The distribution of heavy metals in collected animal hair and liver and blood samples collected at the end of the study was determined to correlate the final goal of this study with hair analysis and liver and blood heavy metal analysis. The measurement results of the hair samples are shown in Table 6, the liver sample measurement results in Table 7, and the blood sample measurement results in Table 8, in ppm units.

TABLE 6

TABLE 7

TABLE 8

As a result of the hair analysis of Table 6, the amount of heavy metals deposited in the hair gradually decreased as the test period progressed. However, the reduction rate was relatively low in the heavy metal treatment group, and the reduction rate was significantly higher in the organic selenium and herbal medicine mixture treatment group. In particular, the highest improvement was found in the 2ppm organic selenium and 4ppm medicinal herbs, and accordingly, the dietary supplementation could be expected to have anti-oxidant effect and liver function protection by sGOT and sGPT titer improvement. The distribution of heavy metals in the liver and blood of Table 7 and Table 8 also showed similar trends with the hair analysis, and it was possible to derive a correlation between the hair analysis and the tissue analysis classified into liver and blood. However, the difference according to the concentration of herbal medicine could not be confirmed in the liver and blood, which is considered to be due to the detoxification and buffering capacity, which are typical functions of the liver.

Taken together, herbal medicines and organic selenium mixtures lower the concentration of heavy metals deposited in the body and have antioxidant and anti-inflammatory effects. Therefore, when added to feeds, it prevents diseases caused by heavy metal poisoning in livestock or pets. And be curable.

Organic selenium and Chinese herbal medicine powder are effective in antioxidant, anti-inflammatory and heavy metal removal when added to feed. However, when a large amount of organic selenium is added, it is not preferable not only because it exceeds the allowable value of the feed management method, but also because the cost increases, and the herbal medicine powder does not have a large difference in effect in the range of 2-4 ppm. The difference is expected to be small, the feed containing a large amount of herbal medicine has a problem that does not fit the pet's taste. Therefore, it is considered that it is preferable to include 2 ppm of organic selenium and 2-4 ppm of herbal powder in the feed.

Feed according to the present invention has the effect of preventing and treating poisoning symptoms due to heavy metals accumulated in the body of livestock or pets by containing herbal medicine and organic selenium, and also has an antioxidant and anti-inflammatory effect. Therefore, it helps to prevent disease factors of livestock and pets and maintains health, and can also contribute to revitalizing stagnant domestic feed industry by supplying high value-added functional raw materials.

Claims (2)

Organic selenium 2ppm, Chinese medicine powder containing 2-4ppm heavy metal poisoning disease prevention feed. The pet food for preventing heavy metal poisoning diseases of claim 1.

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