KR101297574B1 - Compositions for promoting degradation of androstenone comprising Pueraria lobata extracts - Google Patents

Abstract

The present invention relates to a composition for promoting androstenone degradation, and more particularly, to a composition for promoting androstenone degradation by 3β-HSD (3β-hydroxysteroid dehydrogenase) in hepatocytes containing the root extract as an active ingredient.
According to the present invention, it is possible to efficiently decompose androstenone by enhancing the expression of 3β-HSD, a degrading enzyme of androstenone, using the extract of the root, which is a natural extract. Therefore, the composition of the present invention can be usefully used as a safe and economical new non-cauterine boar deodorant technology, unlike the existing deodorant technology through physical and chemical castration methods.

Description

Composition for promoting degradation of androstenone containing brown root extract {Compositions for promoting degradation of androstenone comprising Pueraria lobata extracts}

The present invention relates to a composition for promoting androstenone degradation, and more particularly, to a composition for promoting androstenone degradation by 3β-HSD (3β-hydroxysteroid dehydrogenase) in hepatocytes containing the root extract as an active ingredient.

Boar taint is known to be caused by androstenone and skatole, a type of steroid hormone secreted during the maturation of boars, and it causes fatal flavors in meat. In order to remove the arch, castration, which is a method of removing the testicles through surgical procedures, has generally been performed before the maturation of the boar. This surgical castration method is relatively simple and inexpensive, but can effectively remove the houng, the case is mostly made for hogs.

However, these surgical castrations present serious problems in various aspects as follows. First of all, from an economic point of view, there is a loss due to decreased productivity and disease resistance caused by surgical procedures. One of the studies on the effect of reduced productivity due to castrates in boars prevents the natural assimilation of androgens, which increase red meat production.As a result, castrated pigs lose about 12% of feed efficiency compared to non-rated pigs, and It has been reported to be 6.5% lower (Jensen, MT et al. 1995. Animal Science 61, 293-304). In addition, excessive stress in childhood may cause a decrease in immunity in the body and consequently weaken disease resistance, which may actually provide a cause of mortality. These findings can be linked to the findings of the use of non-surgical castrations to improve the profitability of pork production by 30% (McGlone, J. J. et al. 1993. Journal of Animal Science 71, 1441-1446). Pig farming recognizes this as the most serious problem at the moment, so it is urgent to develop a technique to replace surgical castration.

Generally, boars have better growth-related abilities such as carcass weight and red meat production capacity than sows, but there is a problem in that meat quality and texture are degraded due to dung. In recent years, meat quality and texture of pork have been recognized as an important quality judgment measure of consumers, so that castration is almost entirely carried out in the case of fattening pigs, while bearing a decline in growth-related ability. Therefore, in view of industrially utilizing the boar's excellent red meat production capacity and overcoming the problem of deterioration of texture due to dung, it is urgent to develop a new dung removal method that can maintain the boar's male ability.

Furthermore, apart from hog-fining, the improvement of the number of boars in the boar due to surgical castration is severe. This is because if they are sent to slaughterhouses without screening after testing, they will lose their fertilization ability by juvenile as a child in order to reduce economic losses due to a drop in carcass grades and differences in the price of mercury. Indeed, the difference in meridian prices between castrated and non-taxrated pigs is an average unit price difference of 1,100 won / kg this year. As a result of the limitation of the number of capacity test, the selection strength of the sows is lowered, so that the genetic improvement is relatively reduced, and it is difficult to select high-quality sows. This happened.

Apart from the economic and industrial aspects, animal welfare related to animal castration (animal welfare) is increasing in importance mainly in developed countries, and Korea is also increasingly aware of the benefits, and new laws are being established. Korea is currently performing castration to remove testicles as a surgical procedure for millions of animals every year without any anesthesia for almost all fattening boars. However, Norway and Switzerland have already banned castration in consideration of animal welfare, and many developed countries around the world are considering similar methods. In Korea, too, time is not an exception for the advancement of livestock raising. Therefore, considering the various aspects of economic, industrial and social aspects, the development of a new non-caused boar manure removal technique must be made.

In order to overcome the problems of conventional castration, foreign countries have been studying for a long time, and Pfizer, Australia, has developed a breakthrough product called Improvac. Instead of removing the testicles through surgical procedures, the vaccine uses the pig's immune system to reduce the function of the testicles and produces castration effects, which have already been tested and commercialized in many countries. It is well known. The so-called castration vaccine (immuno castration) not only removes the stings through the effect of castration, but also has the advantage of increasing the growth capacity such as daily gain, feed intake, and back fat layer thickness compared to general castration pigs. (Dunshea, FR et al. 2001. Journal of Animal Science 79, 2524-2535).

However, this vaccine has several problems with its "efficacy." The principle of the vaccine is to induce castration by forming antibodies against GnRH secretion factor, which in turn neutralizes GnRH, leading to secondary production of gonadotropin (FSH), progesterone (LH) and testosterone. Reduce the growth and function of the testicles. This results in the same effect as castration, resulting in the removal of the odor, but consequently inhibits the release of male hormones, inhibiting the male-specific superior growth capacity, and impairing other hormone systems in the lower GnRH stage. Can also give Moreover, being a “hormone-like inhibitor” administered to living organisms can cause serious problems in safety as pork food when left in the blood. In addition, Pfizer's request for two ratings from the rating agency twice resulted in only 63% and 51% castration, respectively. S. et al. 2009. Theriogenology 71, 302-310). In addition, the stress-induced problems of vaccination can not be ignored. Apart from finishing pigs, there is a problem in that the selection strength is lowered because a large number of candidate pigs do not receive the ability test because the males lose their ability.

Improvac vaccines also have limited application in terms of cost. According to the domestic test, the improbacco vaccinated pigs have gained about 22,000 won per head due to the increase in pig meridion price due to the determination of castrated pigs. Nearly the cost of vaccines, combined from the producer's point of view, combined with the above mentioned castration rates, allows us to consider application once again. In addition, considering the expansion of the domestic pig industry, the payment of royalties for the use of products by foreign pharmaceutical companies and the relative waste of foreign currency will be burdensome along with the cost of feed imports.

In order to overcome the problems of the existing surgical castration method and the commercialized vaccine, research and development are needed to find out the direct factors that cause the sting and to effectively remove them. Technology development and preoccupation of technology are urgent. The present inventors have been found to be able to effectively remove the odor by decomposing androstenone by enhancing the expression of androstenone degrading enzyme 3β-HSD by treating the extract of the root extract, which is a natural product extract, to complete the present invention .

Accordingly, the main object of the present invention is to provide a composition for promoting androsterone degradation by 3β-HSD in hepatocytes containing the root extract as an active ingredient, which can effectively remove the androstenone which is the bovine scavenger substance of boar. have.

Another object of the present invention is to provide a feed additive for removing odor by androstenone in pigs, which can effectively remove odor in boars.

According to one aspect of the invention, the present invention provides a composition for promoting androstenone degradation by 3β-HSD (3β-hydroxysteroid dehydrogenase) in hepatocytes containing the root extract as an active ingredient.

The present invention is for the effective removal of androstenone (androstenone) that occurs in the testicles of secondary maturity as a major scavenging agent of boar meat existing in the back of the pig. The 3β-HSD (3β-hydroxysteroid dehydrogenase) is an enzyme involved in the metabolic process of androstenone synthesis and degradation in pigs, particularly in the production of androstenone in the testis, and in the liver, involved in the degradation of androstenone in the backfat. It is known to affect the accumulation of androstenone. In fact, it is known to correlate with the expression level of 3β-HSD in the liver and the amount of androstenone accumulated in the back fat. Therefore, by promoting the proliferation of hepatocytes and increasing the expression level of 3β-HSD in hepatocytes, androstenone degrading activity can be increased, and eventually the amount of androstenone accumulated in the back fat of pigs can be reduced.

In the composition for promoting androstenone degradation of the present invention, the androstenone degradation promotion is characterized by promoting hepatocyte proliferation and increasing 3β-HSD expression in hepatocytes. In the present invention, it was confirmed that various plant extracts can promote the hepatocyte proliferation and increase the expression of 3β-HSD in hepatocytes. As a result, black root, Schisandra chinensis, Cheongung and Astragalus extracts were selected as natural products that promote hepatocyte proliferation and increase the expression of 3β-HSD in hepatocytes (see FIGS. 1 to 3). (See FIG. 5). Therefore, the present inventors finally screened the root root as a natural product most suitable for decomposing androstenone, which is a cause of odor.

The root root ( Pueraria lobata ) used as an active ingredient in the composition of the present invention is the root of the root. Sesame is a legume vine plant with deciduous broad-leaved plants. It grows mainly in all sunny areas of the foothills below 100-1,200 m, and sometimes in fields or hills. Fruits are peduncles, 4-9 cm long, broad row, coarse hairs, ripen in September-October. In oriental medicine, the root is used as a medicine called euneun (발 根), sweating, fever is effective. The starch of the root is called brown powder, and it is edible. The bark of the stem is used as a raw material of galpo, and the boiled water is used as a drink.

The root extract of the present invention may be prepared according to a conventional method known in the art, that is, using a conventional solvent under conditions of conventional temperature and pressure.

As an extraction solvent for preparing the root extract of the composition of the present invention, one selected from the group consisting of water, ethanol, methanol, propylene glycol, butylene glycol, hydrous ethanol, hydrous propylene glycol, hydrous butylene glycol, glycerin The above solvent can be used.

According to a specific embodiment of the present invention, the root of the roots in distilled water and extracted by heating for 2-20 hours at 50-100 ℃ using an extractor equipped with a cooling condenser, or extracted for 1-15 days at 5-37 ℃, filtration, The root extract may be prepared through aging and concentration.

According to another aspect of the present invention, the present invention provides a feed additive for removing manure by androstenone in pigs containing the root extract as an active ingredient. Since the root extract of the present invention promotes hepatocyte proliferation and increases the activity of 3β-HSD expression in hepatocytes, when the root extract is added to the pig feed, it is possible to obtain a deodorizing effect by decomposing androstenone. Therefore, the root extract of the present invention can be usefully used as a feed additive for removing the hoof of pigs.

Deodorant feed additive of the present invention may be added to the feed by selecting an appropriate amount so long as it does not affect the diet of the feed to the pig due to the bitter taste of the root, preferably 0.1 to 10 relative to the total weight of the feed for pigs Swine feed can be prepared by adding% by weight. Pigs fed the mixed feed containing the feed additives to the pigs for a predetermined breeding period may have a characteristic in that the sting is removed compared to the pigs fed the general feed for the same breeding period.

As described above, according to the present invention, by using the root extract, which is a natural product extract, the expression of androstenone degrading enzyme 3β-HSD can be enhanced to efficiently decompose androstenone. Therefore, the composition of the present invention can be usefully used as a safe and economical new non-cauterine boar deodorant technology, unlike the existing deodorant technology through physical and chemical castration methods.

Figure 1 shows the results of screening substances promoting the division of hepatocytes using various plant extracts.
Figure 2 is a RT-PCR result of measuring the amount of RNA transcription of 3β-HSD using a variety of plant extracts.
Figure 3 is a Western blotting result of measuring the RNA expression of 3β-HSD using a variety of plant extracts.
Figure 4 shows the measurement results of androstenone in cell culture medium using GC mass.
Figure 5 is a table showing the results of in vitro efficacy evaluation whether the root extract promotes the degradation of androstenone.

Hereinafter, the present invention will be described in more detail with reference to Examples. Since these examples are only for illustrating the present invention, the scope of the present invention is not to be construed as being limited by these examples.

Preparation Example 1 Preparation of Plant Extract

The plant extract used in the experiment was extracted in the following manner. Dried licorice, sweet ginseng, rich man (Pangpo), brown root, turmeric, samchil, shiho, love leaf, Schisandra chinensis, Indong, Earl of Peony, Ogapi, Yin Yang Guk, Red Peony, Red Sewage, Cheonnja, Gugija, Cheongung, Tobokyeong, Astragalus 500 g Each was put in 5 kg of distilled water and extracted by heating at 70 ° C. for 3 hours using an extractor equipped with a cooling condenser, filtered through a 400 mesh filter cloth, cooled to room temperature, left to mature at 5-15 ° C. for 7 days, and filtered with a 0.45 filter. It was. The filtrate was completely removed from the solvent in a vacuum rotary evaporator to obtain plant extracts and used as a sample of the following experiment.

Example 1 Measurement of Promoting Division of Hepatocytes by Plant Extracts

Androstenone is a steroid hormone that is produced in the testes and is broken down by an enzyme called 3β-HSD (3β-hydroxysteroid dehydrogenase) in hepatocytes. Therefore, the present inventors first selected the natural product extract that affects the activity of hepatocytes.

First, various plant extracts were treated with HepG2, which is a hepatocyte, and a substance capable of promoting the division of hepatocytes was selected by MTT assay during cell culture of hepatocytes. The HepG2 cells used at this time were 5 × 10 ³ cells per well, and were cultured in 200 μl of RPMI medium (welGENE, Korea) containing 0.5% FBS (Fetal Bovine Serum) in 96 well plates. At this time, 1 μl (1: 200) of each natural product extract was added and incubated in a CO ₂ incubator for 3 days, and then treated with MTT at the last 4 hours to measure relative proliferation rate. The control used was 0.5% DMSO (dimethyl sulfoxide). At this time, the MTT assay process was performed according to the supplier's protocol using the MTS assay kit (promega). 100 μl of the culture solution was left on the 96well microplate and 20 μl of the MTS solution was added and mixed. After blocking the light with aluminum foil, the cells were incubated for 2 hours in a cell incubator and OD 490 was obtained from the microplate reader to measure the number of cells.

As a result of measuring the proliferation rate of hepatocytes as described above, natural roots, brown roots, samchil, Schisandra chinensis, Indong, Baekjak, Eumyangkwa, Red Peony, Dung Shou, Cheonnyeonja, Gugija, Cheongung, Tobokyeong, and Astragalus extracts, natural products affecting the activity of hepatocytes. Selected as extract (FIG. 1).

Example 2 Measurement of Increase of mRNA Transcription of 3β-HSD by Plant Extracts

Brown root, samchil, Schisandra chinensis, Schisandra chinensis, Indong, Earl of Peony, Yin Yang Guk, Red Peony, Red Sewage, Angelica, Gugija, Cheongung, Tobokyeong, Astragalus extracts, 3β-HSD expressing MLTC-1 cells (ATCC No. CRL) -2065) and 3β-HSD expression was measured by RT-PCR.

First, various extracts were treated to 3β-HSD expressing MLTC-1 cells, and then, MTT assay was used to select hepatocytes to promote hepatocyte division. The MLTC-1 cells used at this time were used 5 × 10 ⁴ cells per well, and cultured in 500 μl RPMI medium containing 10% FBS in a 24-well plate. At this time, 100 μg / ml of each natural product extract was added, incubated in a CO ₂ incubator for 5 days, and RNA was isolated and semiquantitative RT-PCR was performed. The control used at this time was used 0.5% DMSO.

In order to separate RNA, 200 μl of chloroform (Sigma) was added per 1 ml of Trizol (Invitrogen), and total RNA was isolated. Reverse transcription-polymerization chain polymerization was performed using 0.5 μg, 1 μg, and 2 μg of extracted RNA as a template. After cDNA was cloned by using the cDNA as a template, the primers were prepared as follows according to similar gene sequences obtained from the gene database, and 3β-HSD genes up to 577 bp were obtained through chain polymerization. The reverse transcriptase used was Superscript III (Invitrogen), and the polymerase used was Taq polymerase (Gene craft).

Forward primer: 5'-CCAACTCGTACAAGAAGATCGTCC-3 '

Reverse primer: 5'-GACAGTGTGATCAAGTGGCGGTTA-3 '

As described above, the 3β-HSD gene obtained by the chain polymerization reaction was loaded on an agarose gel. As a result, brown root, Schizandra chinensis, Cheongung, Astragalus extract increased RNA level of 3β-HSD than control (0.5% DMSO) (FIG. 2).

Example 3 Measurement of Increased Protein Expression of 3β-HSD by Plant Extracts

In order to verify the RT-PCR results, proteins were isolated from natural product treated cells and subjected to Western blot. Cells were first trypsinized and centrifuged, then the cell pellets were washed with PBS (phosphate buffered saline) and dissolved in cytosol (50 mM Tris / HCl, pH 7.2, 150 mM NaCl, 1% Triton X-100, 1 mg /). ml leupeptin, 1 mg / ml pepstatin, 2 mg / ml aprotinin, 200 mg / ml PMSF). Lysates were quantified by Braford assay and analyzed by SDS gel electrophoresis. It was then transferred to an Immobilon P membrane (Millipore corporation, Billerica, Mass., USA) and the ECL system with anti-3β-HSD antibody (rabbit IgG, Santa Cruz) and secondary antibody anti-rabbit IgG antibody (Santa Cruz). Western blot was performed using (GE healthcare). As a result, the roots increased the protein amount of 3β-HSD the highest than the control (0.5% DMSO) (Fig. 3).

Example 4 Determination of Effective Degradation of Androstenone of 3β-HSD by Root Extract

In vitro efficacy evaluation was performed to determine whether the root extract, which was found to promote division of hepatocytes and expression of 3β-HSD, promoted androstenone degradation as a result of natural screening.

First, as an analytical method using GC mass, an experiment was performed to measure androstenone in cell culture medium. For effective separation and assay, GC mass of Agilent 7890 (Young In Science) was analyzed. DB-1MS (30m × 0.25m × 0.25㎛) column was used as analytical conditions, and the gas flow rate was 1.0 ml / min. Temperature conditions were detector 280 ℃, inlet temperature 250 ℃, and the initial temperature of the oven was delayed for 2 minutes at 120 ℃ and then increased to 250 ℃ at 15 ℃ / min and then up to 300 ℃ at 5 ℃ / min. After rising, the temperature was maintained for 5 minutes. In order to measure androstenone, 999 μl of medium and 1 μl of 1000 ppm of androstenone were added as a control group, and only 1 ml of medium was used as a control and analyzed by GC mass. As a result, the peak of the androsethenone was analyzed in the experimental group (Fig. 4A), but was not detected in the control (Fig. 4B).

Then, to carry out an in vitro efficacy evaluation on whether the root extract promotes the degradation of androstenone, the experiment was conducted with the following design. All treatments contained DMSO (dissolved natural product in DMSO). The blank treatment group, the root treated MLTC-1 cells were composed of the experimental group, and the untreated root treated MLTC-1 cells as a control, the androstenone content was measured and compared. The MLTC-1 cells used at this time were 1 × 10 ⁵ cells per well, and were cultured in 1 ml of RPMI medium containing 10% FBS in a 24-well plate. At this time, 100 μg / ml of the root extract was added to a 24-well plate containing cells and incubated for 72 hours in a CO ₂ incubator.

Then, as a result of measuring the androstenone by the analysis method using the GC mass, it was analyzed that the androstenone level showed a significant decrease in the experimental medium with the added root compared to the medium without the added root, the control. Therefore, the level of androstenone was shown to be reduced by the root extract treatment (Fig. 5).

Claims (5)

A feed additive for removing odor by androstenone in pigs containing the root extract as an active ingredient.
The method of claim 1, wherein the removal of the odor by the androstenone is characterized in that the removal of the odor, characterized by the acceleration of hepatocyte proliferation and degradation of androstenone due to the increase of 3β- hydroxysteroid dehydrogenase (3β-HSD) expression in hepatocytes additive.
According to claim 1, wherein the root extract is extracted with at least one solvent selected from the group consisting of water, ethanol, methanol, propylene glycol, butylene glycol, hydrous ethanol, hydrous propylene glycol, hydrous butylene glycol, glycerin Deodorant feed additive.
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