KR101053640B1 - Nandrolone Production Method - Google Patents

Abstract

The present invention relates to a method for producing nandrolone, and specifically, a method for producing nandrolone using testis of boar or lydig cells of boar testis and bovine testis It relates to a method for separating interstitial cells. Nandrolone is mainly manufactured by chemical methods, but the production cost is remarkably high by the conventional chemical production method, and the produced nandrolone involves various side effects such as decreased male reproductive function and cardiovascular disease when used. In addition, the present invention not only obtains nandrolone in a simple and easy manner by using bovine testes or epilepsy cells of the testis, but also is derived from natural substances, and thus, the risk of side effects is lower than that produced by chemical production methods. The industrial effect will be very large.

Nandrolone, pig, testis, stromal cells

Description

Nandrolone production method {PREPARATION METHOD OF NANDROLONE}

The present invention relates to a method of preparing nandrolone.

Anabolic steroids generally refer to chemically synthesized male hormones or hormones similar in chemical structure to male hormones, which can play a role in promoting muscle growth. These anabolic steroids have been purified by male hormones first found in animal testicles and their chemical structure has been revealed, and many pharmaceutical companies have begun to produce hormones that are more potent than male hormones in nature. It is derived from referring to the male hormone derivative synthesized by changing the hormone.

Sex hormones (steroid hormones) play an important role in preparing the body to induce skeletal and muscle growth and genital development, producing the next generation. In particular, male hormones function to promote muscle growth and male genital development.

In this respect, anabolic steroids are known to have a more potent effect than male hormones that exist in nature with respect to muscle production. However, excessive use of it has been reported to have side effects such as decreased male reproductive function and cause cardiovascular disease. For this reason, the Olympic Committee banned athletes from taking anabolic steroids in 1964. Among the anabolic steroids, nandrolone is rarely made or produced in very small amounts in most animals, including humans, while the muscle growth promoting effect is much stronger than general male hormones produced in the human body. Being a representative prohibited drug.

On the other hand, anabolic steroids, and more specifically, nandrolone, restore the weakened muscles of patients suffering from burns, surgery, radiation therapy and AIDS, and prevent muscle wasting disease from negative side effects such as those mentioned above. It is used to treat. In addition, it is used as anemia treatment or osteoporosis treatment. And, in the case of nandrolone, it is also used as a component of contraceptives. Anabolic steroids, such as testosterone or androstenedione, which are used as treatments for men with sexual dysfunction, have been reported to be converted to dihydrotestosterone by the enzyme 5α-reductase to cause prostate cancer. have. On the other hand, in the case of nandrolone, the degree of conversion to dihydrotestosterone by an enzyme called 5α-reductase is very low, thereby reducing this problem.

In the case of the nandrolone, it is currently produced mainly by chemical methods. However, the chemical production method of nandrolone is significantly higher in manufacturing cost, the manufacturing process is complicated, and the production of nandrolone produced by the chemical production method, compared with the natural origin, that is, the reduction of male reproductive function, It is known to be accompanied by various side effects such as cardiovascular disease. Therefore, the nandrolone isolated from the natural substance can reduce the side effects of the artificially produced steroids, and is excellent in the treatment or improvement effect not only in the treatment of human diseases, but also in the reproductive function of men, and natural supplement foods. It is expected to be very high.

On the other hand, most of the domestic pig farming farms are using the method of castration and breeding the boar of the boar immediately after birth in order to produce the high quality pork by removing the odor generated from the male pork. However, this castration removes other effective male steroids along with the substance that generates boar's swell, which slows down the growth of the boar and puts stress on pigs in childhood, resulting in longer breeding periods. It is causing economic damage.

To solve this problem, castration when a pig is old requires a lot of effort compared to castration at birth after 2 weeks of age, and does not actually boil between two weeks after birth. If this is not the case, farmers usually give up castration and raise pigs. In the case of such non-taxeous pigs, they are graded as low-grade pigs compared to the caste during the shipping process, which causes the economic loss of the farm.

In addition, non-castrated pigs that do not use these castrations are extracted from the slaughterhouse during the slaughter process, and the extracted testes are currently of low industrial value and are provided to restaurants for roasting only a small portion, and most of them are slaughter by-products. It was disposed of or partly recycled as animal feed.

The present invention is to provide a method for producing nandrolone in order to improve the problems of the prior art as described above.

Moreover, an object of this invention is to provide the composition for nandrolone manufacture.

In order to achieve the above object, the present invention provides a method for producing nandrolone. Specifically, the production method may be a method of using lydig cells isolated from testis of boar and a method of extracting bovine testis.

In addition, the present invention provides a composition for preparing nandrolone in order to achieve the above object.

The present inventors have a large amount of nandrolone, which is generally not well-produced in other animals, is contained in the boar's testis, and in particular, the stromal cells of the boar's testis are produced with various steroid hormones, including nandrolone, and are cultured by separating the stromal cells. In this case, an in vitro method can produce nandrolone, and in the case of 19-nortestoterone produced by the bovine stromal stromal cells, less conversion to dihydrotestosterone by 5α-reductase is possible. It was confirmed that the present invention was completed.

Hereinafter, the present invention will be described in more detail.

The present invention relates to a method of producing nandrolone. The present invention also relates to a composition for producing nandrolone.

The method for producing nandrolone of the present invention may be at least one selected from the group consisting of a method of using interstitial cells of pig testis and a method of using a testis extract of swine.

Specifically, the method using the interstitial cells of the swine testis comprises the steps of culturing the interstitial cells of the swine testes in a culture medium; And separating nandrolone from the cell culture medium in which the stromal cells are cultured.

The culture may be a conventional animal cell culture, for example, may be DMEM, DMEM, DMEM / F12 or DMEM containing FBS. The cell culture medium in which the stromal cells are cultured may or may not contain stromal cells.

According to an embodiment of the present invention, the production method

a) separating stromal cells from porcine testes;

b) culturing the isolated stromal cells in the culture medium; And

c) separating the nandrolone from the cell culture medium in which the stromal cells are cultured.

The a) step may be performed by a conventional method for separating animal cells, for example, may be a percoll gradient method using a percoll, but preferably may be performed by a subculture method that is a method of separating interstitial cells without using a percoll. .

According to a recent report by Pharmacia, the producer of percoll, percoll, a silica particle coated with PVP, has a slightly toxic PVP component and, when used in a salted culture, the combination of PVP and silica This weakens and finally the silica gel (silica gel) is exposed in the culture, there is a problem that can affect the DNA structure of the cells to be separated.

Therefore, the production method of the present invention for the final mass production of nandrolone is preferably carried out by a method that can replace the percoll gradient method using a percoll.

The interstitial cell separation method of the present invention was completed by confirming that the interstitial cells of boars are superior in adhesion ability to other cells in the bovine testes. Provides a way to separate. The cell separation method of the present invention is a method capable of separating a large number of cells at low cost in a short time, safe from the risk of cell damage, which is a problem in the percoll gradient method using percoll.

The stromal cell separation method may be a subculture, and specifically, the stromal cell separation method may include: cutting a pig testis; Enzymatically treating the sliced pig testis; Culturing the enzyme treated pig testis in a culture solution; Separating the cells from the culture medium in which the pig testis has been cultured; And it may be a method comprising the step of subcultured to the cultured cells.

The subculture is a step of culturing the separated cells in a culture solution contained in a culture dish (dish); And separating the cells which are not attached to the culture vessel from the culture medium in which the cells are cultured. The passaging method was completed by confirming that the interstitial cells of the swine testis have excellent adhesion ability compared with the cells of other swine testis, and more specifically, the isolated cells are cultured in a culture solution in a dish. The process may be performed for 30 minutes to 20 hours, preferably 1 hour to 10 hours, more preferably 1 hour 30 minutes to 8 hours after starting the culture of the separated cells in the culture solution contained in the culture vessel.

Incubation time of the isolated cells may be affected by the age of the pigs from which the cells are separated. For example, in the case of pigs 1 week to 4 weeks old, the culture time of the cells may be 1 hour to 3 hours, preferably 1 hour 30 minutes to 2 hours, and 5 months to 9 months, preferably 6 months after birth. For pigs that are 8 months old, the incubation time of the cells may be 4 hours to 10 hours, preferably 5 hours to 8 hours.

The culture solution may be a culture solution of a conventional animal cell, and the culture medium in which the pig testis or cells are cultured may or may not include cells.

The process of enzymatically processing the sliced pig testis may be performed by mixing the sliced pig testis with an enzyme solution, mixing the mixture solution, and recovering the supernatant, wherein the enzyme solution is an animal cell containing a proteolytic enzyme. It may be an enzyme solution commonly used in the separation method, for example, may include trypsin and EDTA.

Step b) may be carried out by conventional animal cell culture conditions and culture methods. In addition, the cell culture step may be performed by further treating the hormone in the culture. The hormone is preferably androstenedione (androstenedione), progesterone (progesterone) and testosterone (testosterone), more preferably androstenedione may be.

Step c) may use a conventional method for separating steroid hormones from cell culture. For example, the method may use high performance liquid chromatography or EIA kit, depending on the required purity. The conditions of the high performance liquid chromatography may be carried out under conventional conditions for separating steroid hormones, preferably nandrolone.

In addition, the present invention relates to a composition for preparing nandrolone, including interstitial cells of porcine testis.

The present inventors have completed the present invention by confirming that extremely little or no androlone, which is present in most animals, is produced in interstitial cells of swine testis.

Since the interstitial cells of the swine testis can continuously produce nandrolone through a relatively several passages, when using a composition for preparing a nadrrone containing the interstitial cells, mass production of nandrolone is possible.

The composition for preparing nandrolone may further include a culture solution for culturing the interstitial cells in addition to the interstitial cells and a substance for promoting nandrolone production.

The culture may be a conventional animal cell culture used for culturing animal cells, for example, may be DMEM or DMEM containing FBS. The culture solution may be one that does not contain interstitial cells.

In addition, the nandrolone production-promoting substance may mean a substance that can be added to the culture medium of stromal cells to act on the stromal cells, thereby promoting the production of androlone production of stromal cells, for example, hormones or steroid hormones. . The hormone is preferably at least one selected from the group consisting of androstenedione (androstenedione), progesterone (testgeone) and testosterone (testosterone), more preferably androstenedione.

The present invention also relates to a method for producing nandrolone using a pig testis extract. In contrast to other animals, the inventors of the present invention contain a large amount of nandrolone in the case of swine testis, and when the optimal extraction solvent and extraction method for extracting nandrolone are selected and applied, high purity nandrolone is easily The present invention was completed by confirming that it could be produced.

The testis is an organ that makes sperm, which is a male germ cell, and is also called gohan in mammals. In the testes, mainly male hormones such as sperm and androgen are produced. On the other hand, boar testis, that is, the extracted boar testis, is only provided to restaurants for roasting only a portion, most of which is disposed of as slaughter by-products or partially recycled as animal feed. The value of use is very low.

The testis may be preferably to remove all the indefinite. The testis may be cut after removing all the indefinite, for the production of testis extract. The cutting process can be performed using a tissue homogenizer.

The extract may preferably be an animal extract. Animal extract means a substance extracted from the body of an animal, that is, a substance obtained by dissolving and separating a specific substance in a solid or liquid to be extracted using a solvent.

The extraction solvent of the present invention is water, 50% to 100% methanol, alcohol having 1 to 4 carbon atoms such as ethanol, polar solvent such as ethyl acetate and organic solvents such as non-polar solvent of chloroform, ether, hexane or dichloromethane, these It may be a mixed solvent of, preferably chloroform or a mixture of alcohol having 1 to 4 carbon atoms and chloroform, such as 50% to 100% methanol, ethanol, more preferably 50% to 100% ethanol or methanol It may be a chloroform mixture. In addition, the alcohol having 1 to 4 carbon atoms and chloroform mixture is 5: 1 to 1: 5 (chloroform: ethanol), preferably 3: 1 to 1: 3, most preferably 2: 1 to 1 based on the volume ratio It may be a mixture of 2 :. In addition, the extraction solvent may be used at least one type.

Extract of the present invention may be prepared according to the production method of a conventional animal extract, specifically may be cold extraction, hot extraction or heat extraction, etc., may be used a conventional extraction device, ultrasonic grinding extractor or fractionator. In addition, the extraction process may be performed at 4 ℃ to 120 ℃, but is not limited thereto.

Specifically, the nandrolone production method of the present invention may include extracting by adding an extraction solvent to the testes of pigs.

The prepared extract may be further performed at least one selected from the group consisting of filtration, concentration and drying. Specifically, the filtration may be performed using a filter paper or sodium sulfate and a filter paper or a reduced pressure filter, the concentration may be concentrated under reduced pressure using a vacuum condenser, for example, a rotary evaporator, the drying may be carried out by lyophilization. have.

According to one embodiment of the invention the nandrolone production method of the present invention can be carried out by the method described in FIG. More specifically, the process of removing all the indefinite to obtain a testis; It may include the step of preparing and homogenizing the mixed solution by adding an extraction solvent to the obtained testes and after leaving the mixed solution to stand, removing the supernatant. Removing the supernatant may be performed using a separatory funnel. The removing of the supernatant may further include any one selected from the group consisting of removing the supernatant and concentrating the obtained extract, filtration and drying to remove the solvent. It may be carried out using a vacuum concentrator, the filtration step may be carried out using a filter paper or filter paper and sodium sulfate, the drying step may be carried out using a rotary pressure reducer or a water bath method.

The inventor of the present invention completed the present invention by confirming that the fatty acid accumulated in the extract can be removed by adding NaOH to the mixed solution. Therefore, the present invention may further comprise the step of adding NaOH to the pig testis extract.

The nandrolone production method may further include adding an aqueous solvent having 1 to 4 carbon atoms in addition to extracting the extract by adding an extraction solvent to the testes of pigs to prepare a mixed solution; Adding NaOH to the mixed solution and applying heat; And after adding ether, separating the ether layer.

The C 1-4 alcohol solution is preferably a mixture of 1 to 4 carbon atoms and water in a volume ratio of 95: 5 to 70:30 (alcohol: water), more preferably 90:10 to 80:20 It may be. The applying of the heat may be performed at 50 ° C. to 95 ° C., preferably 70 ° C. to 90 ° C., for 10 minutes to 120 minutes, preferably for 30 minutes to 60 minutes. have.

The nandrolone production method may further include a step of concentrating the separated ether layer, and may further include a step of obtaining nandrolone by adding chloroform to the concentrate obtained through the concentration step.

In addition, the method for producing nandrolone of the present invention may further perform a method commonly used to separate steroids from the mixture with respect to the obtained ether layer or chloroform-added concentrate. For example, The method for separation can be high performance liquid chromatography.

Most of the methods of production of nandrolone of the present invention are discarded as slaughter by-products or partly recycled to livestock feed, such as testis of boar, which is of very low industrial value, or epilepsy isolated from it. A method for producing nandrolone that can be used for the treatment of various diseases by using a Leydig cell is not only simple and inexpensive, but also obtained by the chemical method. Alternatively, since it is extracted from the natural products that are being taken for food, the industrial effect is very large in that the risk of side effects is extremely low.

Hereinafter, preferred embodiments of the present invention will be described. However, the following examples are only preferred embodiments of the present invention, and the present invention is not limited to the following examples.

Example 1 Isolation, Culture and Production of Nandrolone Using Porcine Testicular Stromal Cells

[Experimental Materials and Equipment]

1) DMEM / F12: (Dulbecco's Modified Egle's Medium / Nutrient Mixture F-12 Ham, Sigma D8900)

2) Amphoteripcin B: Gibco Manufactory in UK (Invitrogen co., USA)

3) 25% Trpsin-EDTA: Gibco 25200 (Invitrogen co., USA)

4) Penicillin-Streptomycine: Gibco 15140-122 (Invitrogen co., USA)

5) Fetal Bovine Serum (FBS): Hyclone

6) Centrifuge 5810R: Eppendorf

7) 10 ml Pipette: Falcon

8) Tissue Culture Dish 100 x 22 mm: Falcon

9) Iso tomp 215 water bath: Fisher Scientific

10) Testosterone (TES, Wako pure pure chemical industries.208-08341.Japan)

11) Androstenedione (And, 19-Nor-4 androstene-3.17 dione, Wako pure chemical industries. 143-06061)

12) Progesterone (Pro, progesterone,> = 99%) Sigma chemical co.P8783 (St. Louis, USA)

Example 1-1 Primary culture of porcine testicular stromal cells (initial culture)

Pig testis leydig cells used in this study were collected from farm and slaughterhouses of two-week-old and seven-month-old Landrace pigs, respectively, at 75 µg / ml penicillin G (Sigma, USA) and 50 µg / ml. After washing with sterile physiological saline (0.9% NaCl) added streptomycin sulfate (Sigma, USA), it was stored in a bottle containing sterile physiological saline and transported to the laboratory.

Pig testes carried to the laboratory were washed again three times with fresh sterile saline solution and alcohol, respectively, and then disclosed in the experiment. The washed testes were harvested by cutting each sample to about 10 × 10 mm, and the collected testes were finely chopped and added to 0.25% trypsin-EDTA (Gibco, USA), followed by vortexing for 30 minutes. After the vortexing, the solution was immersed for 10 minutes, and the supernatant of the mixed solution was recovered and centrifuged with DMEM / F-12 (Sigma, USA) culture medium to which 10% FBS was added to remove trysin-EDTA. After removing the trysin-EDTA, cells were separated by DMEM / F-12 culture medium to which 10% FBS was added.

The isolated cells were dispensed with 100% 20 mm dish (Falcon, USA) of DMEM / F-12 with 10% FBS and 5% CO _2. And 37 ° C., the cells isolated from the testes of 2 weeks old pigs were cultured for 2 hours, and the cells isolated from the testes of pigs 7 months old for 6 hours. After each hour of incubation, non-bottom cells were removed and cultured for 12 days with fresh DMEM / F-12 medium replaced every 48 hours. When the incubation time was excessively longer than the above time, it was observed that cells other than the interstitial cells adhered and grew. In this case, when the percoll gradient method was performed, the yield of the interstitial cells was very low.

After incubation for each of the incubation time, after subculture by removing the non-bottom cells with the culture medium and adding a new culture medium, 0.25% trypsin-EDTA when the donor cells grew more than 90% in the dish After the treatment was suspended and divided by about 1/3 was carried out incubation. In order to confirm that the isolated cells are interstitial cells, the cell culture solution was analyzed by centrifugation using a percoll gradient. As a result, it was confirmed that 90% of all cells were interstitial cells, which was significantly or more compared with the conventional report. It was confirmed that it is an excellent result.

In addition, the separation method of the interstitial cells separated by subculture using the cell adhesion ability showed a small morphological change compared to the case of using the percoll gradient method, and even if the cells were passaged for a long period of time. It was confirmed that the state of was good. From the above results, it was recognized that the passage method of the present invention has an advantageous effect of reducing damage to cells than the separation culture by percoll.

Example 1-2 Hormonal Treatment of Porcine Testicular Stromal Cells

Hormonal treatment was performed for 24 hours by adding androstenedione, nortestosterone, testosterone, and progesterone to DMEM / F12 and 10% FBS medium at 100 nM concentration, respectively. After 24 hours of treatment, the medium was harvested and stored at −20 ° C. until the EIA assay was performed on the harvested medium.

Example 1-3 Analysis of Nandrolone Content Using EIA KIT

The content of nandrolone was performed using EIA KIT (19-Nortestosterone-EIA (Euro-Diagnostica B. V. 5082NOR1p, Netherlands)), and experimental materials and equipment related thereto are as follows.

[Experimental Materials and Equipment]

1) Microtitre plate

2) Substrate solution (in dark, at room temperature before use, storage at 4 ℃)

3) Conjugate solution (in dark, stored at room temperature before use, stored at 4 ℃, long-term use at 20 ℃)

4) Store at room temperature and store at 4 ℃ before use. Dissolve with 4 ml of dilution buffer in power state.)

5) Antibody solution (Storage should be stored at 4 ℃ and dissolved in 4 ml of power dilution buffer)

6) The medium to be used for hormonal analysis was dissolved in a constant temperature water bath at 37 ℃ and sterilized in 3rd

Diluted 10 times, 20 times in distilled water, EIA analysis

The plate for the analysis of nandrolone content using the EIA KIT is shown in FIG. 2. 100 μl of zero standard was added to A1 well of FIG. 2, and 50 μl of zero standard was added to B1 well, and standard 1 to 6 were added to each well of B, C, D, E, F, G and H, respectively. Was added. 50 μl of sample to be analyzed was added to the remaining wells, and 25 μl of Enzyme conjugate solution and 25 μl of Antibody solution were added to the remaining wells (standard and sample wells) except for the A1 well.

The process was carried out and the plate was blocked by using silver foil incubated at 4 ℃ for 1 hour and washed three times with rinsing buffer. 100 μl of Substrate solution was added to the plate after the washing, incubated for 30 minutes, 100 μl of Stop solution was added thereto, and an optical density (OD) value was measured at 450 nm. Table 3 and shown in Figures 3 and 4.

TABLE 1

TABLE 2

[Table 3]

As shown in Tables 1 to 3 and FIGS. 3 and 4, three kinds of hormones of testosterone, androstenedione, and progesterone were treated to stromal cells, and media were collected after 24 hours, and Nandrolone was treated with EIA kit. As a result, the amount of nandrolone was the lowest in the control, and the amount of nandrolone appeared in the order of androstenedione, progesterone, and testosterone. It was confirmed that it can produce. Thus, the androstenedione was found to be the best nandrolone production promoter.

In addition, from the above results, the composition comprising porcine testes stromal cells and the culture medium of the cells can be used for screening (ie, screening) the screen for the production of nandrolone or an inhibitor, and the stromal cells as described above. There is also provided a method capable of searching for nandrolone production or inhibitor by measuring the amount of change in the production of nandrolone after adding a candidate for promoting nandrolone production or a candidate for promoting inhibition of nandrolone to the culture medium.

Example 2: Nandrolone Production Using Pork Testis Extract

[Experimental reagents and instruments]

1) chloroform: HPLC grade, SK Chemical Co. (Seoul, Korea)

2) methanol: HPLC grade, Merck Co. (Darmstadt, Germany)

3) 1000 mL measuring cylinder: Hanil Chemical (Seoul, Korea)

4) 1000 mL beaker: Hanil Chemical (Seoul, Korea)

5) Separation funnel: Hanil Chemical (Seoul, Korea)

6) Tissue homogenizer: Ultra-Turrax T25, IKA Co. (USA

7) Rotary vacuum dryer: 1200 type, Eyela Co. (Tokyo, Japan)

Example 2-1 Preparation of Porcine Testis Extract

Preparation of Low Concentrate Extract

The testes for fat extraction removed all the negatives and separated the pure testes. The isolated testes were pre-cut into 10-30 g size for grinding into a tissue homogenizer and stored frozen at -20 ° C freezer. As a solvent for extracting the testis fat, a mixed solution in which chloroform and ethanol were mixed at a volume ratio of 1: 1 was used. A test piece between 25 and 50 g was placed in a 1000 mL beaker, followed by addition of 8 times the chloroform / ethanol mixed solution, followed by homogenization for 3 minutes. After homogenization, remove all residues using Whatman No.2 filter paper, and put all the filtrates in 250 mL separatory funnel to make products containing fat, and add a small amount of 0.9% saline solution. Shake it lightly and let stand for about 10 minutes. The separated lower layer was placed in a recovery bottle, and the supernatant was once again shaken with physiological saline, and left for about 20 minutes. At this time, the separated lower layer was combined with the recovery bottle. This process is repeated one more time, for a total of three times, and the lower floors are all placed in the same collection bottle. The lower layer recovered in the recovery bottle was still standing for 24 hours because it still contained some water layer, and the upper layer separated by standing was discarded using pasteur pipette. After disposing of the upper layer, the remaining organic solvent was added to the Whatman No. 2 filter paper with 10 g of sodium sulphate in a flask of a round-type vacuum condenser and filtered. The mixture was concentrated to 50 ° C. in a rotary vacuum concentrator until all solvents were removed and the residue was recovered and further processed.

Preparation of Highly Concentrated Extract

Preparation of high concentration extract was carried out in the following manner.

First, a solution in which a large amount of fat and other steroidal substances were first extracted from the testis using the chloroform / ethanol mixed solution was used. The organic solvent was volatilized using a rotary pressure reducer, and about 3 times of the ethanol / distilled water mixed solution (= 85/15, v / v) of the remaining solution was mixed well and the contents were mixed. 25 mL of a 50% NaOH solution was added thereto, and the mixture was cooled to room temperature after boiling at 80 ° C. for 45 minutes. The pH was adjusted to neutral with 10% sulfuric acid solution. The contents were divided in a separatory funnel, ether was added by half of the contents, shaken well, mixed well, and allowed to stand still for 10 minutes to separate layers. When the layers were separated cleanly, the lower layer was put in a new separatory funnel and separated once more to extract as much as possible. After that, the lower layer was discarded, and all the ethers of the upper layer were collected, washed with distilled water, and purified. The ether layer was placed in a rotary vacuum concentrator, and completely dried and concentrated. To recover the concentrate, a small amount of chloroform was added to dissolve and then stored in a 3 mL sample bottle for freezing. It was also used for encapsulation.

Example 2-2 Content Analysis of Nandrolone

Nandrolone content analysis was performed in the same manner using the same EIA KIT as in Example 1-3.

More specifically, after adding 100 μl of zero standard to A1 well, 50 μl of zero standard was added to B1 well, and standard 1 to 6 were added to B, C, D, E, F, and G H 1 wells, respectively. 50 μl of sample to be analyzed was added to the remaining wells, and 25 μl of Enzyme conjugate solution and 25 μl of Antibody solution were added to the remaining wells (standard and sample wells) except for A1 wells. After the above process, the plate was blocked with light paper and placed at 4 ° C for 1 hour, and then washed 3 times with rinsing buffer. After the plate was washed, 100 μl of Substrate solution was added and left for 30 minutes. Then, 100 μl of Stop solution was added thereto, and the OD value (optical density) was measured at 450 nm. The results are shown in Tables 4, 5, and 6. Indicated.

[Table 4]

TABLE 5

As shown in Table 4 and Table 5, it was confirmed that the testicular extract contained an average of 61.13 ng of nandrolone per 1 ml and 825.22 ng of nandrolone per 1 g of testes.

From the above results, it has been confirmed that the extract of swine testis contains an industrially available degree of nandrolone, and thus, when the production of nandrolone using the swine testis extract is carried out in a cheap and simple process, It was confirmed that it is possible to produce nandrolone with fewer side effects than the nandrolone produced.

1 is a flowchart showing a method for producing nandrolone using a pig testis extract according to an embodiment of the present invention.

Figure 2 shows a plate for analyzing the Nandrolone content of the interstitial cell culture of pig testis according to an embodiment of the present invention using EIA KIT.

3 is a graph showing a standard curve of nandrolone according to an embodiment of the present invention.

Figure 4 is a graph showing the concentration of nandrolone in the stromal cell culture medium according to the hormone treatment according to an embodiment of the present invention. In FIG. 4, C means control, T means testosterone, A means androstenedione, and P means progesterone.

5 shows a plate for analyzing the Nandrolone content of the pig testis extract according to an embodiment of the present invention using EIA KIT.

6 is a graph showing a standard curve of nandrolone obtained from a pig extract according to an embodiment of the present invention.

Claims (11)

a) culturing the stromal cells of porcine testis in a culture medium; And b) a method for producing nandrolone using interstitial cells of swine testis, comprising the step of separating the nandrolone from the cell culture medium in which the stromal cells are cultured. delete The method of claim 1, The step a) is an androstenedione (androstenedione), progesterone (progesterone) and testosterone (testosterone) by adding an additional one or more selected from the group consisting of nandrolone production method for culturing interstitial cells. 4. The method of claim 1 or 3, wherein the interstitial cells of the swine testis are obtained by subculture. Nandrolone production composition comprising interstitial cells of porcine testis. The method of claim 5, Wherein the composition further comprises a DMEM culture medium containing DMEM (Dulbecco's modified Eagle's medium) or FBS (fetal bovine serum). The method of claim 5, The composition is a composition for producing nandrolone, further comprising one or more nandrolone production promoting material selected from the group consisting of androstenedione (androstenedione), progesterone (progesterone) and testosterone (testosterone). Nandrolone production method comprising the step of separating the nandrolone from the pig testis extract. The method of claim 8, The nandrolone production method comprises the steps of obtaining a pig testis extract by adding an extraction solvent to one or more selected from the group consisting of water, alcohol having 1 to 4 carbon atoms, ethyl acetate, chloroform, ether, hexane and dichloromethane in pig testis. Nandrolone production method comprising a. 10. The method of claim 9, The nandrolone production method further comprises the step of additionally adding NaOH to the pig testis extract. The method of claim 10, The nandrolone production method further comprises the step of additionally adding ether to the pig testis extract and obtaining only an ether layer.

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