Method for extracting high purity polydeoxyribonucleotides from salmon testes

Abstract

The present disclosure relates to a method of extracting high purity polydeoxyribonucleotides (PDRN) from salmon testes, the method comprising: 1) isolating semen and immature testis region from the testes of salmon, 2) gently grinding the immature testis region and then diluting with artificial seminal plasma, 3) treating the dilution with a predetermined concentration of human chorionic gonadotropin (hCG) to induce artificial maturation of testis cells into sperm, 4) after a predetermined time of hCG treatment, centrifuging the testis dilution and collecting potentially motile sperm, 5) extracting PDRN from the collected sperm. The method enables extraction of 100 to 200 times higher purity PDRN raw material from 20 mL (max 50 mL) of sperm (semen) collected from salmon at one time. Furthermore, the method enables extraction of PDRN at a high yield in a cost-effective and economically viable manner.

Description

Technical Field

[0001] This invention relates to a method for extracting high-purity polydeoxyribonucleic acid (PDRN) from salmon testes, and more specifically to a method for extracting high-purity PDRN by treating immature testes with hormones, which is currently used in other applications. Background Technology

[0002] Polydeoxyribonucleotides (PDRNs) are active substances present in living organisms that promote tissue regeneration (evolution). PDRNs are mixtures of chain-like deoxyribonucleotide polymers consisting of 50 to 2000 base pairs. PDRNs are cell growth activators and play a special role in the regeneration of tissues (such as ligaments, tendons, and skin) and the relief of inflammation in the human body.

[0003] PDRN is present in very small amounts only in the human placenta. PDRN raises ethical concerns and presents difficulties in pharmaceutical production. For these reasons, PDRN from fish has been proposed as an alternative to human PDRN. Trout and salmon semen and testes are rich in nucleic acids, but more importantly, they offer a qualitative advantage. DNA is composed of nucleotides, each containing a pentose sugar, a phosphate group, and a nitrogenous base such as adenine, guanine, thymine, or cytosine. The balance of this combination in trout and salmon is 96.5% identical to that in humans. This similarity is significant because all nucleic acids are usable and not discarded.

[0004] PDRN is approved for wound treatment and cosmetic purposes in European and other countries. PDRN preparation primarily involves collecting semen from salmon or trout, isolating the DNA from the semen, and fragmenting the DNA. DNA isolation techniques used for PDRN preparation have been technologically developed and applied to mammals and other species. For example, phenol and chloroform are used to isolate DNA from fish tissues.

[0005] Polydeoxyribonucleotides (PDRNs), as DNA fragments, are prepared from salmon semen containing sperm and are used as therapeutic agents and cosmetic materials with excellent pharmacological activity.

[0006] PDRN is used as a high-purity DNA fragment for pharmacological purposes. In this case, sperm fluid (or semen) is centrifuged, washed with buffer, and extracted with ethanol or a mixture of sodium acetate and isopropanol to obtain PDRN. This extraction process is based on the principle used by many patents and manufacturers.

[0007] Semen from the testes contains a large number of spermatids, spermatocytes, and spermatogonia, all of which differentiate into sperm during gametogenesis. In particular, semen contains more trophoblasts than these reproductive cells; trophoblasts are responsible for providing nutrients to the reproductive cells.

[0008] like Figure 1 As shown, the testes of fish mature when attached to the dorsal wall of the body cavity. During sexual maturity, the growth of germ cells leads to an increase in the gonadal cell index (GSI). The GSI increases by 20% to 50% in females and by 1% to 5% in males. That is, the number of testes obtained from males is not significant.

[0009] On the other hand, PDRN extraction necessarily involves centrifugation of semen, enrichment of sperm (or spermatocytes), and separation of sperm using organic solvents. Sperm consists of DNA (i.e., PDRN) as the genome, mitochondria containing ATP that enables tail movement, and the acrosome that allows sperm to penetrate the egg membrane. PDRN is the target substance of this invention.

[0010] The PDRN content in products extracted from semen using organic solvents differs fundamentally from that extracted from testes. Extraction from semen yields PDRN of higher purity; conversely, when extracting PDRN from testes containing trophoblast cells using organic solvents, various substances soluble in the organic solvents are inevitably extracted.

[0011] In CN107287186A (“Patent Document 1”), PDRN is obtained from semen, which is referred to as semen. KR10-2018-0066331 (“Patent Document 2”), which was ultimately rejected in Korea, describes freeze-drying the testes and extracting with an organic solvent to obtain PDRN. The Korean patent disclosure acknowledges that the purity of PDRN obtained from semen differs from that obtained from testes and reveals that high-purity PDRN can be extracted from sperm (semen) with the highest efficiency.

[0012] Therefore, washing and organic solvent extraction have been established as fundamental techniques for PDRN extraction. Consequently, numerous patents have recently been issued, and papers have been published regarding the use of PDRN based on its pharmacological activity or cosmetic effects rather than on the methods of PDRN extraction.

[0013] Once sperm is produced in the salmon's testes, the semen is collected. The abdomen is then dissected to collect the immature testes, followed by further processing such as freeze-drying. The inventors surprisingly discovered that diluting the salmon's testes with seminal plasma, depending on their maturity, and then treating them with hormones such as human chorionic gonadotropin (hCG), results in the production of large quantities of sperm (or semen). For example, 100-200 times the amount of semen can be obtained from a single salmonid.

[0014] The composition of seminal plasma (NaCl, 40 mM KCl, 1 mM CaCl2, 20 mM Tris-HCl) was determined based on the seminal mineral concentration information described in "Rosengrave P, Taylor H, Montgomerie R, Metcalf V, McBride K and Gemmell NJ, 2008, Chemical composition of seminal and ovarian fluids of chinook salmon (Oncorhynchustshawytscha) and their effects on sperm motility traits. CBP A 152, 123-129" and the information on artificial semen described in "Bartlett MJ, Steeves TE, Gemmell NJ and Rosengrave PC, 2017, Sperm competition risk drives rapid ejaculate adjustments mediated by seminal fluid. eLIFE 6:e28811".

[0015] The semen mentioned in Patent Document 1 as a raw material for PDRN is salmon semen, not an extract of the testes.

[0016] EP 0226254B1 (“Patent Document 3”) establishes a method for extracting PDRN from the placenta. However, this patent only mentions that the process is also applicable to other tissues in other animals, without describing any specific examples. In contrast, the present invention proposes a method for extracting PDRN from salmon testes, starting with collecting testes from live or fresh salmon and washing the testes to remove blood. This method is fundamentally different from methods for preparing PDRN from motile sperm produced from the testes, although the same PDRN source (i.e., testes) is used in both methods.

[0017] The inventors focused on extracting PDRN from immature testes, rather than from semen or mature testes, which are widely used in the art. They discovered that treating immature testes with suitable hormones, particularly human chorionic gonadotropin (hCG), could produce mature sperm from which high-purity PDRN could be extracted. This invention was based on this discovery.

[0018] Existing technical documents

[0019] Patent documents

[0020] (Patent Document 1) CN107287186A is titled "Method for separating polydeoxyribonucleic acid from fish semen, polydeoxyribonucleic acid obtained by said method and its application".

[0021] (Patent Document 2) KR10-2018-0066331 is titled "Method for extracting PDRN from fish semen and testes".

[0022] (Patent Document 3) EP 0226254B1 is entitled "Method for obtaining biologically active non-informative pure polydeoxyribonucleotides and corresponding products".

[0023] Non-patent documents

[0024] (Non-Patent Literature 1)(Journal Article 1) Hyo Won KIM, Jeong Hyeon KIM, Dae Geun KIM, MinHwan JEONG, Seung Chul JI, Sang Keun SANG, Cheol Min AHN, Jeong In MYEONG, DaeJung KIM. 2018. Effects of hCG dosage and injection interval on sexual maturity of cultured male eels. Journal of the Korean Society for Fisheries and Marine Science Education, 30, 1578-1586.

[0025] (Non-Patent Literature 2)(Journal Article 2) OHTA H. and Tanaka H., 1997. Relationship between serum levels of human chorionic gonadotropin (hCG) and 11-ketotestosterone and induced maturation in male Japanese eels after a single injection. Aquaculture 153, 123-134.

[0026] (Non-Patent Literature 3)(Project Report 1) DAL Young Kim, Seong Gon Kim, Tong Soo Kim. 2016. Adaptability Study of Korean Mandarin Fish to Compound Feed - Test of Growth Rate of 1-Year-Old Individuals Adapted to Compound Feed. 2016 Practical Research Project Report. Gyeonggi Provincial Ocean and Fisheries Research Institute, Korea. 84-91.

[0027] (Non-Patent Document 4)(Project Report 2) Suncheonhyang University, South Korea, 2011. Manual for Artificial Breeding of Endangered Freshwater Fish. Ministry of Environment, North Korea, pp. 93. Summary of the Invention

[0028] One object of the present invention is to provide a method for artificially producing sperm by treating the testes with hCG in order to extract a large amount of PDRN from the sperm.

[0029] According to the present invention, a method for extracting high-purity polydeoxyribonucleic acid (PDRN) from salmon testes includes: 1) separating semen and immature testicular regions from salmon testes; 2) gently grinding the immature testicular regions and then diluting them with artificial seminal plasma; 3) treating the diluted solution with a predetermined concentration of human chorionic gonadotropin (hCG) to induce artificial sexual maturation of testicular cells into sperm; 4) centrifuging the testicular diluted solution and collecting potentially motile sperm after a predetermined time of hCG treatment; and 5) extracting PDRN from the collected sperm.

[0030] The present invention will now be described in detail.

[0031] In the method of the present invention, the salmon is preferably a species belonging to the subfamily Salmoninae, Gynosteminae and Salmoninae, and is preferably a river salmon caught in an area extending 10 kilometers upstream from the estuary.

[0032] In the method of the present invention, in step 1), the immature testicular regions have varying degrees of maturity, exhibiting a relatively high maturity characterized by sperm motility of at least 0.7%, and in step 3), 25 to 200 IU hCG is used relative to each gram of testes.

[0033] In the method of the present invention, in step 4), the predetermined time is preferably 10 minutes to 1 hour, and the immature testicular region preferably includes spermatogonia or spermatocytes. Preferably, the method of the present invention further includes, before step 5), suspending the sperm and adding fresh water to the suspension to allow the sperm to consume ATP, thereby obtaining high-purity PDRN.

[0034] hCG is an abbreviation for human chorionic gonadotropin, which is a hormone that can be detected in the urine of pregnant women.

[0035] When hCG is injected into carp, crucian carp, or goldfish raised at 12°C or lower year-round, fertilized eggs can be obtained at any time. Specifically, injecting hCG at a dose of 500 IU / kg or 500 IU / egg induces gonadal maturation in both females and males, resulting in natural fertilization of eggs within 12 to 18 hours after injection (from dawn to the following morning). Injecting salmon pituitary extract into female eels leads to sexual maturation of gametes (eggs), while weekly injections of hCG at a concentration of 500 IU / kg can mature sperm in 3 months. Mature female eggs and mature sperm meet to form fertilized eggs.

[0036] In one embodiment of the invention, semen and immature testicular regions with different degrees of maturity are separated from the testes of salmon. The immature testicular regions with high maturity are collected. The collected immature testicular regions are gently ground and diluted with artificial seminal plasma. The diluted solution is treated with hCG of a predetermined concentration. The testicular diluted solution is centrifuged 10-30 minutes after hCG treatment to collect potentially motile sperm and use it as raw material for PDRN.

[0037] In other words, this invention relates to the preparation of high-purity PDRN from artificially produced motile sperm from the testes of fresh salmon. The maturity of fresh testes varies by region, but the closer to the genital pore, the more mature the spermatogonia. Due to the maturation of hCG, a spermatogonia undergoes meiosis to produce spermatids and sperm (up to four), resulting in a higher yield than the total wet weight of the testes used, and ultimately leading to an increase in PDRN production. In the examples below, hCG treatment increased the dry weight (excluding precipitate) of sperm derived from spermatids by 1.6 times, and the amount of PDRN with a final purity of at least 82% was 2.3 to 3.9 times that of the untreated group. Furthermore, high-purity PDRN was obtained on average at a higher yield of 40.1 times from a single collection of semen from a male, and PDRN was prepared with even higher purity when mature sperm were exposed to ambient water to deplete their ATP.

[0038] About 30-50 mL of semen can be obtained from a male salmon, but currently only a limited amount (about 10-20 mL) can be obtained because most of the semen in South Korea is used for fertilization.

[0039] The method of this invention can produce PDRN raw material with a purity up to 100-200 times higher from 20 mL (maximum 50 mL) of sperm (sperm) collected naturally from salmon in a single batch. In other words, PDRN prepared from salmon testes using the method of this invention is more concentrated and purer than PDRN obtained from expensive live semen. According to the method of this invention, PDRN can be prepared in high yield from the semen and testes of trout or salmon. Furthermore, compared to conventional methods used by other companies, the method of this invention enables the extraction of PDRN in a more cost-effective and economically feasible manner. Attached Figure Description

[0040] These and / or other aspects and advantages of the invention will become apparent and more readily understood from the following description of embodiments taken in conjunction with the accompanying drawings, in which:

[0041] Figure 1 Transverse and longitudinal cross-sectional views showing the spatial location of immature gonads (common in both males and females) in the body cavity of common fish;

[0042] Figure 2This is an enlarged longitudinal sectional view of the immature gonads (used in both male and female fish) in the body cavity of a common fish;

[0043] Figure 3 A cross-sectional view showing the structure of sperm cells in a typical fish (left) and a photomicrograph showing sperm in the semen of a typical fish (right); and

[0044] Figure 4 This is a diagram showing the location of different areas of the testes in a mature male salmon. Detailed Implementation

[0045] The various steps of the method according to the present invention will be described.

[0046] Step 1: Separate the semen from the immature testicular region

[0047] Semen and immature testicular regions were separated from the salmon's testes. Figure 4 The semen leaking from region D is collected and used as raw material. After the semen is completely removed from region D, region C is separated from region D by cutting at a predetermined location.

[0048] refer to Figure 4 The testes were divided into a region D where semen leaked out and immature testicular regions A, B, and C. Specifically, most sperm from testicular regions A and B were inactive before hCG treatment, and approximately 1% of sperm from testicular region C were motile. Sperm motility in mature semen from testicular region D was 100%.

[0049] Step 2: Grinding and diluting the immature testicular region

[0050] The immature testicular regions A, B, and C were ground and diluted with artificial seminal plasma. The grinding was performed in the following order: First, the testes were cut into small pieces with scissors to peel off the membranes surrounding the sperm cells. Then, the exposed testicular regions A, B, and C were gently squeezed with gloved hands.

[0051] The seminal plasma consisted of 80 mM NaCl, 40 mM KCl, 1 mM CaCl2, and 20 mM Tris-HCl, particularly 159.26 ± 8.84 mM sodium (Na), 33.72 ± 2.01 mM potassium (K), 133.04 ± 5.96 mM chloride (Cl), 1.68 ± 0.2 mM calcium (Ca), and 0.988 ± 0.13 mM magnesium (Mg) (see Hatef A, Et. Al., 2007. Aquaculture Research 38, 1175-1181). The seminal plasma also contained total protein as an organic component (0.75 ± 0.14 mg / 100 mL). -1 ), cholesterol (2.86±0.58 mg·L) -1) and glucose (3.81±1.04 mM·L) -1 ).

[0052] Step 3: hCG processing

[0053] hCG is an abbreviation for human chorionic gonadotropin. Diluted hCG at concentrations of 25-200 IU / g is used to induce artificial sexual maturation of testicular cells into sperm. Specifically, the diluted hCG is treated with a predetermined amount while slowly agitated using a low-speed impeller agitator. It should be noted that high-speed impeller agitation can cause cell rupture, increasing the likelihood of impurities.

[0054] hCG treatment increased sperm motility from testicular region A to approximately 3%, while increasing it to 35% and 89% from regions B and C, respectively. In summary, using testicular region C, which comprises 45% of the total testicular weight, to produce mature sperm resulted in high-purity PDRN that was virtually free of impurities (see [link to treatment]). Figure 4 (and Table 1).

[0055] Step 4: Centrifuge

[0056] Centrifuge the testis dilution at 1000-10000 RCF (relative centrifugal force) for at least 20 minutes and collect the precipitate of potentially motile sperm.

[0057] Step 5: PDRN extraction

[0058] Polydeoxyribonucleic acid (PDRN) is extracted from collected sperm. Various methods known in the art are applicable to PDRN extraction, and therefore their description is omitted here. The present invention is characterized by producing artificial semen and extracting high-purity PDRN from it, as described above, rather than by PDRN extraction.

[0059] For example, PDRN can be extracted through the following process. First, sperm is treated with lysis buffer, frozen, and powdered. DNA is separated by centrifugation and proteins are removed for purification. Subsequently, the DNA is precipitated by centrifugation and purified by washing. Finally, the DNA is fragmented by one of the following processes: (1) restriction digestion, (2) high-frequency acoustic energy transfer, (3) atomization, (4) sonication, and (5) needle shearing.

[0060] The invention will be explained in more detail with reference to the following embodiments. However, these embodiments are given to provide those skilled in the art with a thorough understanding of the invention, and several other modifications may be made, and are not intended to limit the invention.

[0061] Example 1: Sperm motility (%) in different regions of salmon testes before and after hCG treatment.

[0062] Immature areas (excluding area D where semen leaks out) are collected by cutting open the testes of male salmon. See Figure 4 Before hCG treatment, sperm motility from testicular regions A and B was almost zero, sperm motility from testicular region C was as low as 1%, and sperm motility in mature semen was 100%. After hCG treatment, sperm motility from testicular region A increased to approximately 3%, while sperm motility from testicular regions B and C increased to 35% and 89%, respectively. These results conclude that using testicular region C, which accounts for 45% of the total weight of the testes, to produce mature sperm results in high-purity PDRN that is virtually free of impurities (see [link to hCG treatment]). Figure 4 (and Table 1).

[0063] Table 1. Sperm motility and weight percentage of different regions of salmon testes before and after treatment with 100 IU / 2g hCG (%)

[0064]

[0065] Testicular regions of male salmon caught in a river in October were treated with three different types of hormones: hCG, luteinizing hormone-releasing hormone (LhRH), and DHP. When treated with LhRH and DHP, sperm motility was as low as 5% and 4%, respectively. In contrast, the experimental group treated with hCG had sperm motility as high as 80%, producing semen with very few impurities (Table 2).

[0066] Table 2. Viability (%) of sperm in region C of salmon testis when treated with different types of hormones.

[0067]

[0068] Even after hCG treatment, sperm motility in salmon caught at sea did not increase (Table 3). Treatment of testicular region C of salmon returning to rivers from September to December with hCG showed no substantial difference in sperm motility since the river salmon were already mature (Table 4). Sperm motility was compared based on distance from the estuary bar. Sperm motility in male salmon caught at the estuary bar (distance = 0 m) and 1 km upstream of the estuary bar was 65% and 85%, respectively. In contrast, sperm motility in male salmon caught 2 km and 3 km upstream of the estuary bar was as high as 97% and 98%, respectively (Table 5).

[0069] Table 3. Viability (%) of C sperm in the testicular region of river salmon (2 km upstream of the estuary) and sea salmon after hCG treatment.

[0070]

[0071] Table 4. Viability (%) of sperm in testicular region C of river salmon (2 km upstream of the estuary) caught in different months after hCG treatment.

[0072]

[0073] Table 5. Viability (%) of sperm in testicular region C of river salmon captured at different distances from the estuary after hCG treatment.

[0074]

[0075] The distance from the estuary refers to the distance from the estuary to the point where the salmon travel upstream before being caught. hCG has almost no effect on sea salmon, but it works on salmon returning to the river because they have already begun to reach sexual maturity. The results in Table 5 show that hCG is more effective on salmon caught upstream than on salmon caught closer to the sea.

[0076] Example 2: Sperm motility (%) in testicular region C of salmon under different hCG treatments.

[0077] Testicular region C of male river salmon was diluted with a buffer solution and treated with different concentrations of hCG. When region C was untreated, sperm motility was very low (2%). As the hCG concentration increased to 50 IU / g, sperm motility steadily increased, and when treated with hCG at concentrations of 50-200 IU / g, sperm motility was ≥82% (Table 6).

[0078] Table 6. Sperm motility (%) in testicular region C of river salmon caught 2 km upstream of the estuary in October when treated with different concentrations of hCG (IU / g testes).

[0079]

[0080] *Measured 10 minutes after hCG treatment

[0081] Sperm motility changes over time were compared after treatment with hCG concentrations shown in Table 6. There was no difference in sperm motility in the untreated control group over time. Sperm motility in the 25 IU / g testis treatment group increased to a maximum of 73% at 1 hour post-treatment and reached as high as 85% after 10 minutes. Sperm motility in the 50 IU / g testis group was ≥94% after 20–30 minutes post-treatment. However, sperm motility decreased to 83% at 60 minutes post-treatment and then dropped sharply to 32%. Sperm motility in the 100 IU / g and 200 IU / g testis groups began to decline after 30 and 20 minutes, respectively. That is, sperm motility decreased in the 50–200 IU / g testis treatment groups when treatment time exceeded 20 minutes (Table 7).

[0082] Table 7. Viability (%) of C sperm in testicular regions of river salmon caught 2 km upstream of the estuary in October after treatment with different concentrations of hCG (IU / g testes) and for a period of time after treatment.

[0083]

[0084] Example 3: PDRN extraction rate (%) of testicular region C treated with hCG under different conditions.

[0085] In this embodiment, testicular region C of 10g male river salmon was used. For comparison, 30mL of semen, typically extracted from male river salmon, was used (Table 8). The testicular region was treated with hCG at concentrations of 50 and 100 IU / g of testis. After hCG treatment, ≥80% of the top layer was collected and defined as the supernatant.

[0086] The dry weight of the untreated group was 5.10 g, less than that of the hCG-treated group but greater than that of the semen (2.89 g). The greater dry weight of the untreated group compared to the semen is attributed to the presence of heavier trophoblasts. The greater dry weight of the hCG-treated group compared to the untreated group is attributed to hCG treatment stimulating spermatogonia to meiosis into spermatids, resulting in an increase in cell number. The lower dry weight of the supernatant after hCG treatment is attributed to certain components (approximately 20%), such as trophoblasts, that did not differentiate into spermatids.

[0087] The extraction rates of PDRN from the untreated group and the hCG-treated testicular region were 10.2% and 14.7%, respectively. The extraction rates from the supernatant and semen were 7.0-7.2% and 6.1%, respectively. PDRN with a content (or purity) of 10% and 45-52% was obtained from the untreated group and 10g of hCG-treated testicular region. Conversely, PDRN with a purity ≥92% was obtained from the supernatant and semen.

[0088] Based on these results, the amount of PDRN extracted from a male salmon was calculated. The results showed that 0.26 g of PDRN was obtained from the semen of a male salmon, and 0.16 g of PDRN was obtained from the untreated testis region of a male salmon. In contrast, 5.77 g and 7.63 g of PDRN were obtained from the testis region treated with hCG at concentrations of 50 and 100 IU / g, respectively. High-purity PDRN (≥10.51 g) was obtained from the supernatant regardless of the hCG concentration. The PDRN yield in the untreated group and the testis region treated with 50 IU hCG was 0.6 times and 21.8 times higher than that in the semen, respectively. In contrast, the PDRN yield in the supernatant after treating the testis with hCG at concentrations of 50 and 100 IU / g was 39.7 times and 40.1 times higher than that in the semen, respectively.

[0089] Since the supernatant is a suspension containing a high proportion of sperm, when exposed to ambient water (freshwater), the sperm become motile, consuming ATP in their mitochondria. This effect is thought to increase the purity of PDRN. Because immotile sperm are precipitated and removed in this process, efficiency needs to be considered. However, due to the high PDRN content extracted from sperm (≥92%), this consideration did not lead to a significant increase in efficiency.

[0090] Table 8. PDRN levels in the testes of river salmon caught 2 km upstream of the estuary in October after treatment with hCG at concentrations of 0, 50, and 100 IU / g for 20 minutes.

[0091]

[0092] 1) The preparation method involves thoroughly mixing the testicular region immediately after hCG treatment, allowing the mixture to stand for 10 minutes, and then collecting ≥80% of the top layer.

[0093] 2) The higher dry weight after hCG treatment is thought to be due to the increase in cell number caused by meiosis into sperm (chromosomes).

[0094] Although preferred embodiments of the invention have been explained in detail, the scope of the invention is not limited thereto. Those skilled in the art will understand that various modifications can be made without departing from the spirit and scope of the invention.

Claims

1. A method for extracting high-purity polydeoxyribonucleotides (PDRN) from salmon testes, the method comprising: 1) Semen and immature testicular regions were separated from the testes of salmon; 2) The immature testicular regions were ground and then diluted with artificial semen; 3) The diluted solution was treated with 25 to 200 IU of human chorionic gonadotropin (hCG) per gram of testes to induce artificial sexual maturation of testicular cells into sperm; 4) The testicular diluted solution was centrifuged 10 minutes to 1 hour after hCG treatment and the potentially motile sperm were collected; 5) PDRN was extracted from the collected sperm. The salmon are river salmon caught in an area extending 1-10 kilometers upstream from the estuary; in step 1), the immature testes are separated by distinguishing parts of different maturity, so that the immature testicular area has a higher maturity and the sperm motility of the immature testicular area is at least 0.7%.

2. The method according to claim 1, wherein, The salmon in question belongs to a subfamily consisting of the subfamily Salmoninae, Gynosteminae, and Salmoninae.

3. The method according to claim 1, wherein, The immature testicular region includes spermatogonia or spermatocytes.

4. The method according to claim 1, before step 5), further includes suspending the sperm and adding fresh water to the suspension to cause the sperm to consume ATP, thereby obtaining high-purity PDRN.

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