A nucleic acid composition of protamine and its preparation method and anti-tumor application

Abstract

The application belongs to the technical field of nucleic acid extraction, and particularly relates to a nucleic acid composition of fish sperm protein, a preparation method thereof and anti-tumor application. The preparation method of the nucleic acid composition of fish sperm protein comprises the following steps: selection, rinsing, extraction, maturation, sterilization, enzymolysis, concentration, drying, impurity removal, alcohol precipitation and desalination. The nucleic acid composition of fish sperm protein prepared by the method has good anti-tumor effect, saves resources and has high bioavailability.

Description

Technical Field

[0001] This invention belongs to the field of nucleic acid extraction technology, specifically relating to a fish spermatin nucleic acid composition, its preparation method, and its anti-tumor application. Background Technology

[0002] Fish testes are the testicular tissue of mature male fish. Due to their distinctive odor, they are unpalatable to people and have long been discarded without being fully utilized. Protamine, extracted from fish testes, has become an important pharmaceutical raw material. As a natural preservative, protamine has broad-spectrum antibacterial activity and is safe and non-toxic. Protamine can also be used as a preservative for meat products.

[0003] The main components of fish testes are basic proteins and deoxyribonucleic acid (DNA). The basic proteins are rich in arginine, lysine, and histidine, while the DNA contains nucleotides such as adenine deoxyribonucleic acid (dAMP), guanine deoxyribonucleic acid (dGMP), cytosine deoxyribonucleic acid (dCMP), and thymine deoxyribonucleic acid (dTMP). It can be seen that fish testes have comprehensive and rich nutritional value.

[0004] Current methods of extracting and utilizing fish spermatophores only collect protamine, discarding the deoxyribonucleic acid (DNA) contained within, significantly reducing the effective value of the spermatophores. Furthermore, due to the stringent preservation requirements for pharmaceutical raw materials, a large amount of fish spermatophore tissue generated in ordinary aquatic product markets is discarded. Therefore, seeking comprehensive and practical processing and production technologies for fish spermatophores to achieve high-value utilization and protect the environment is of great significance.

[0005] Relevant patent documents were retrieved:

[0006] Patent number CN118496283A, published on August 16, 2024, discloses a method for obtaining polydeoxyribonucleic acid (PDRN) from fish testes, comprising the following steps: 1) removing blood vessels and disrupting tissue in fresh fish testes; 2) adding the disrupted fish testes to a DNA extraction buffer and stirring evenly; 3) adding protease and SDS solution to the extraction buffer, heating in a water bath, and stirring; 4) centrifuging the extraction buffer at 4°C, discarding the precipitate and collecting the supernatant; 5) mixing the supernatant with a saturated sodium chloride aqueous solution, centrifuging at 4°C, and collecting the supernatant; 6) adding the centrifuged supernatant to pre-cooled anhydrous ethanol for precipitation, and collecting the precipitate. This invention has fewer steps and is simple to operate. The extraction method is relatively gentle and safe during PDRN acquisition, with high PDRN extraction efficiency, high purity, and bright white color.

[0007] Relevant non-patent literature was retrieved: The literature title is "Experimental Study on Protamine Inhibiting Angiogenesis and Inducing Tumor Cell Apoptosis," journal name is *Chinese Journal of Cancer*, volume number 2002, (02): 18-20. This study explored the therapeutic mechanism of protamine in treating malignant tumors. An animal tumor model was established, and protamine treatment was combined with immunohistochemistry and in situ DNA nick-end labeling to detect intratumoral blood vessel density and apoptosis index. The results showed that the blood vessel density in the experimental group was significantly lower than that in the control group (P < 0.01). Simultaneously, tumor cell apoptosis was observed, with an apoptosis index of (7.67 ± 1.70)%. Conclusion: Protamine can significantly reduce intratumoral blood vessel density and induce apoptosis; the therapeutic effect comes from inhibiting angiogenesis and inducing tumor cell apoptosis.

[0008] The prior art represented by the aforementioned documents has at least the following unresolved technical problems or defects: The above literature only extracts and utilizes protamine and deoxyribonucleic acid individually. Protamine is extracted from fish spermatophores and used as a natural preservative, or only deoxyribonucleic acid is extracted. The comprehensive utilization and other medicinal value of fish spermatophores have not been realized.

[0009] In solving the above problems or overcoming the above defects, the present invention encountered the following difficulties and obstacles: The technical challenge of this invention lies in the effective separation of deoxyribonucleic acid (DNA) and nucleoprotein (protamine precursor) during the extraction process and the subsequent re-dissolution during enzymatic hydrolysis. DNA remains in the supernatant, while nucleoprotein precipitates out. The difficulty lies in controlling the degree of nucleoprotein denaturation; it must denature and separate without completely losing its solubility, otherwise re-dissolution and enzymatic hydrolysis will be difficult. Experiments compared hydrochloric acid, sulfuric acid (listed in the pharmacopoeia), alkaline solution (sodium hydroxide), and anionic surfactants (SDS), ultimately selecting sodium hydroxide as the most effective denaturing agent. Summary of the Invention

[0010] The purpose of this invention is to provide: a fish spermatogonium nucleic acid composition, its preparation method and anti-tumor application, and related technologies, to solve technical problems such as low extraction efficiency and poor anti-tumor effect, or a combination thereof.

[0011] Terminology Explanation: Unless otherwise defined, all technical terms in this document have the same meanings as commonly understood by one of ordinary skill in the art to which the subject matter of the claims pertains. Unless otherwise stated, all patents, patent inventions, and publications cited in this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this document, the definitions in this chapter shall prevail.

[0012] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0013] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0014] In a first aspect, the present invention provides a method for preparing a fish testis protein nucleic acid composition, comprising the following steps: (1) Take fish spermatophores, add washing liquid, stir and soak, remove solvent, wash, crush to obtain fish spermatophore powder; (2) Add sodium chloride solution to dissolve and extract the fish spermatophore fragments obtained in step (1) to obtain an extract; mature the extract, keep it warm, and cool it; then add sodium hydroxide to adjust the pH, centrifuge, and collect the precipitate and the centrifuged clear liquid respectively; (3) Soak the precipitate obtained in step (2) in water, heat to dissolve, continue to heat and keep warm, then cool down, add protease to hydrolyze, inactivate, concentrate, sterilize, cool and dry to obtain protein powder; (4) Add the centrifuged clear liquid obtained in step (3) to SDS solution, stir and let stand, centrifuge to remove the precipitate, repeat the addition of SDS solution, stir and let stand, centrifuge to remove the precipitate, and obtain the centrifuged clear liquid after impurity removal. Precipitate the centrifuged clear liquid after impurity removal with alcohol to obtain filamentous material; desalt the filamentous material to obtain solid material; enzymatically hydrolyze the solid material to obtain enzymatic hydrolysate; concentrate the enzymatic hydrolysate, sterilize, and dry to obtain deoxynucleotide powder; (5) Mix the protein powder and deoxynucleotide powder according to the specified ratio to obtain the final product.

[0015] Preferably, the fish testes in step (1) are salmon or carp testes.

[0016] Preferably, the washing liquid in step (1) is composed of sodium citrate and sodium chloride, and the molar ratio of the two is 0.01-0.02:0.10-0.15, more preferably 0.01:0.14.

[0017] Preferably, the amount of washing liquid added in step (1) is 2-5 times the mass of the fish testes.

[0018] Preferably, the soaking time in step (1) is 10-20 minutes.

[0019] Preferably, the solvent removal method in step (1) is filtration, draining, or centrifugation.

[0020] Preferably, the washing in step (1) is performed 1-3 times until the washing waste liquid is no longer bloody.

[0021] Preferably, the mass concentration of sodium chloride in step (2) is 8%-12%, more preferably 10%.

[0022] Preferably, the amount of sodium chloride added in step (2) is 20-30 times the mass of the fish spermatophore fragments, more preferably 25 times.

[0023] Preferably, the dissolution time in step (2) is 24-48 hours.

[0024] Preferably, the heating temperature in step (2) is 80-85°C, the holding time is 10-15 min, and the temperature is cooled to below 40°C.

[0025] Preferably, the pH in step (2) is 9-11, more preferably 11.

[0026] Preferably, the amount of water added in step (3) is 30-50 times the mass of the precipitate, more preferably 32 times.

[0027] Preferably, the heating temperature for the aging process in step (3) is 75-85°C, more preferably 80°C.

[0028] Preferably, the heating and heat preservation step (3) involves heating to 90-100℃ and then holding for 45-65 minutes.

[0029] More preferably, the heating and heat preservation step (3) is to heat to 92°C and keep warm for 50 minutes.

[0030] Preferably, the temperature reduction in step (3) is reduced to 50-60°C, more preferably to 55°C.

[0031] Preferably, the protease in step (3) is trypsin and alkaline protease, wherein the amount of trypsin added is 0.2%-1% of the precipitate mass, and the amount of alkaline protease added is 0.2%-0.6% of the precipitate mass.

[0032] More preferably, the amount of trypsin added is 0.6% of the precipitate mass, and the amount of alkaline protease added is 0.4% of the precipitate mass.

[0033] Preferably, the enzymatic hydrolysis time in step (3) is 5-7 hours, more preferably 6 hours.

[0034] Preferably, the inactivation temperature in step (3) is 90-100℃ and the time is 10-15min.

[0035] More preferably, the inactivation temperature in step (3) is 92°C and the time is 10 min.

[0036] Preferably, the concentration temperature in step (3) is 65℃-70℃, and the sugar content is concentrated to 25%-35%.

[0037] More preferably, the concentration in step (3) is carried out at a temperature of 69°C until the sugar content is 28%.

[0038] Preferably, the sterilization temperature in step (3) is 90-100℃ and the time is 35-50min.

[0039] More preferably, the sterilization temperature in step (3) is 92°C and the time is 40 min.

[0040] Preferably, the sterilization in step (3) can also be performed by UHT sterilization at 138±1℃ for 4 seconds.

[0041] Preferably, the cooling temperature in step (3) is 60-65°C, more preferably 65°C.

[0042] Preferably, the drying in step (3) is spray drying.

[0043] Preferably, the amount of SDS solution added in step (4) is 8%-15% of the mass of the centrifuged clear liquid, more preferably 10%; the mass concentration of the SDS solution is 4%-6%, more preferably 5%.

[0044] Preferably, the stirring time in step (4) is 10-15 min, and the settling time is 30-70 min.

[0045] More preferably, the stirring time in step (4) is 10 min and the settling time is 50 min.

[0046] Preferably, the number of times this operation is repeated in step (4) is 1-3 times, more preferably 2 times.

[0047] Preferably, the specific operation of alcohol precipitation in step (4) is as follows: add 1-2 times the volume of the centrifuged clear liquid after impurity removal to edible ethanol with a volume concentration of 75%-95%, let stand for 10-15 minutes, and take out the filamentous material (the upper layer of coarse filamentous material has more impurities, and the lower layer of fine filamentous material has high purity; after rotating and winding, take it out).

[0048] More preferably, the specific operation of alcohol precipitation in step (4) is as follows: add 1.5 times the mass of the centrifuged clear liquid after impurity removal to edible ethanol with a volume concentration of 75%, let stand for 10-15 minutes, and then remove the filamentous material.

[0049] Preferably, the specific operation of desalination in step (4) is as follows: wash the filamentous material 2-4 times with edible ethanol with a volume concentration of 30%-60% at 8-12 times its mass, and then centrifuge.

[0050] More preferably, the specific operation of desalination in step (4) is as follows: wash the filamentous material three times with 50% edible ethanol at a volume concentration of 10 times its mass, and then centrifuge it.

[0051] Preferably, the specific operation of enzymatic hydrolysis in step (4) is as follows: dissolve the solid in 30-55 times the amount of water, heat to 82-90℃ and maintain for 10-30 min, cool down to 65-70℃, adjust the pH to 5.5-6.5 with dilute hydrochloric acid, add 2%-8% of the solid mass of nuclease to dissolve, maintain at 65-70℃ for 3-5 h, stir, and heat to 90-100℃ to inactivate the enzyme for 10-15 min.

[0052] More preferably, the specific operation of enzymatic hydrolysis in step (4) is as follows: dissolve the solid in 50 times the amount of water, heat to 85°C and keep for 20 minutes, cool down to 68°C, adjust the pH to 6 with dilute hydrochloric acid, add 5% of the solid mass of nuclease to dissolve, keep at 68°C for 4 hours, stir, and heat to 92°C to inactivate the enzyme for 10 minutes.

[0053] Preferably, the specific operation of concentration in step (4) is as follows: cool the enzymatic hydrolysate to a temperature of 60-68°C, filter it with a microporous resin column, and concentrate it to a liquid sugar content of 25%-35%.

[0054] More preferably, the specific operation of concentration in step (4) is as follows: cool the enzymatic hydrolysate to a temperature of 65°C, filter it with a microporous resin column, and concentrate it to a liquid sugar content of 30%.

[0055] Preferably, the sterilization temperature in step (4) is 90-100℃ and the time is 30-50min.

[0056] More preferably, the sterilization temperature in step (4) is 92°C and the time is 40 min.

[0057] Preferably, the drying in step (4) is spray drying.

[0058] Preferably, the mass ratio of protein powder to deoxynucleotide powder in step (5) is 80-93:7-20.

[0059] More preferably, the mass ratio of protein powder to deoxynucleotide powder in step (5) is 80-86:14-20.

[0060] More preferably, the mass ratio of protein powder to deoxynucleotide powder in step (5) is 80:20.

[0061] Secondly, the present invention provides a fish spermatogonium nucleic acid composition prepared by the above-described preparation method.

[0062] Thirdly, the present invention provides the application of the fish spermatogonium nucleic acid composition prepared by the above preparation method in the preparation of antitumor drugs.

[0063] Preferably, the drug further includes pharmaceutically acceptable excipients.

[0064] The present invention has the following beneficial effects: The fish spermatogonium nucleic acid composition prepared by this method contains basic proteins that are small molecule peptides that have been enzymatically hydrolyzed, and DNA that has been hydrolyzed into deoxynucleotide monomers. The human body's absorption and utilization efficiency is higher than that of conventionally extracted fish protamine or deoxyribonucleic acid. Experiments have shown that it can inhibit the growth of tumor cells and has anti-tumor effects. The fish testis protein nucleic acid composition prepared by this method contains high levels of basic amino acids (arginine, lysine, histidine) and deoxynucleotides, which can be added to various foods to improve their nutritional value. Deoxynucleotides are essential substances for the human body and are the building blocks of human DNA. They can participate in DNA synthesis, promote tissue repair and cell regeneration, and improve tissue dysfunction caused by damage or disease. This method has relaxed requirements for fish testes as raw materials, and can extract testes at stages III and IV of fish maturity. A large amount of fish testis tissue generated in ordinary aquatic product markets can be used for production. The proteins and deoxyribonucleic acid contained in fish testes are comprehensively extracted and utilized, which improves the use value of fish testis raw materials, while also reducing the waste of aquatic resources and mitigating environmental pollution. The proteases used in this method (0.2%-1% trypsin and 0.2%-0.6% alkaline protease) are incubated at 50-60℃ for 6 hours. The alkaline amino acid peptides obtained by the synergistic hydrolysis of protamine by the two enzymes have smaller molecular structures than those obtained by conventional single trypsin hydrolysis products, and are more easily digested and absorbed by the human body. This method involves first dissolving and extracting, then aging. The extract is heated to 75-85℃ and held for 10-15 minutes, then cooled to below 40℃. This process can unwind the double helix structure of DNA, which is more conducive to the complete dissolution and extraction of fish spermatogonium raw materials than the conventional method of aging and then dissolving. It can also promote the separation of protamine and deoxyribonucleic acid. In this method, the centrifuged liquid containing DNA is further treated with 8%-15% SDS solution at a mass concentration of 4-6% to remove protein impurities. SDS is an anionic surfactant that denatures and flocculates proteins. After standing, the flocs naturally agglomerate and float to the liquid surface, resulting in higher solid-liquid separation efficiency. This method simplifies centrifugation and filtration steps compared to conventional methods, thereby improving production efficiency. Attached Figure Description

[0065] Figure 1 This is a typical fluorescence intensity diagram of tumor cells after sample processing. The red fluorescent particles in the diagram are human non-small cell lung cancer (A549) cells.

[0066] Figure 2 The fluorescence intensity of tumor cells after sample treatment is compared with that of the model control group. p<0.05, p<0.001; compared with Comparative Example 1, # p<0.05, ## p<0.01, ### p<0.001; compared with Comparative Example 2, $ p<0.05, $$ p<0.01, $$$ p<0.001; compared with Comparative Example 3, & p<0.05, &&& p<0.001.

[0067] Figure 3 This is a typical map of the quasi-metastatic distance of tumor cells after sample processing. The red fluorescent particles in the map are human non-small cell lung cancer (A549) cells.

[0068] Figure 4 This represents the distance of tumor cell metastasis after sample treatment, compared with the model control group. p<0.05, p<0.01, p<0.001; compared with Comparative Example 1, # p<0.05, ### p<0.001; compared with Comparative Example 2, $ p<0.05, $$$ p<0.001; compared with Comparative Example 3, && p<0.01, &&& p<0.001. Detailed Implementation

[0069] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention. Those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and these should also fall within the scope of protection claimed by the present invention. The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all instruments, devices, equipment, reagents, products, etc., used in the embodiments of the present invention are obtained through conventional commercial means.

[0070] Example 1 (1) After selecting fresh salmon testes, add 3 times the weight of the salmon testes washing solution (composed of sodium citrate and sodium chloride, with a molar ratio of 0.01:0.14) and soak for 15 minutes, then drain. Add 2 times the weight of the salmon testes washing solution again and soak for 10 minutes, then drain. Crush into salmon testes fragments less than 10 mm. (2) The fish spermatozoa fragments obtained in step (1) were added to a 10% sodium chloride solution with a mass of 25 times their weight, dissolved for 48 hours, heated to 85°C and held for 12 minutes, then cooled to 38°C and added to a 28% NaOH solution to adjust the pH to 11. After standing for 30 minutes, the mixture was centrifuged and the precipitate and the supernatant were collected separately. (3) Soak the precipitate obtained in step (2) in 32 times its weight of water and heat it to 80°C to dissolve it. Then heat it to 92°C and keep it warm for 50 minutes. Then cool it down to 55°C and add 0.6% of the precipitate mass of trypsin and 0.4% of the precipitate mass of alkaline protease. Keep it warm for 6 hours. Then heat it to 92°C and keep it warm for 10 minutes. Then cool it down to 69°C and concentrate it until the sugar content is 28%. Then heat it to 92°C and keep it warm for 40 minutes. Then cool it down to 65°C and spray dry it at 200°C / 90°C. Collect the dry powder (protein powder). (4) Add the centrifuged liquid obtained in step (2) to 10% of its weight of SDS solution (mass concentration of 5%), stir for 10 min, let stand for 50 min, centrifuge to remove the precipitate, repeat twice; add the centrifuged liquid to 1.5 times the mass of the centrifuged liquid in ice-cold edible ethanol (volume concentration of 75%), let stand for 10 min, take out the filamentous material, wring it dry, soak it in 10 times the volume concentration of 50% edible ethanol for 30 min, take it out, repeat 3 times to obtain solid; put the solid into 50 times its mass of water and heat to 85℃ for 20 min, then cool down to 68℃, adjust the pH to 6 with dilute hydrochloric acid, add 5% of the solid mass of nuclease and keep warm for 4 h, then heat up to 95℃ to inactivate the enzyme for 10 min, then cool down to 65℃ and concentrate to 30% sugar content, continue to heat up to 92℃ and keep warm for 40 min, then cool down to 65℃, spray dry, temperature control 190℃ / 90℃, collect dry powder II (deoxynucleotide powder) (5) Mix dry powder one (protein powder) and dry powder two (deoxynucleotide powder) at a ratio of 80:20 until homogeneous.

[0071] Example 2 The difference from Example 1 is that the amount of trypsin added is 0.2% of the precipitate mass, and the amount of alkaline protease added is 0.6% of the precipitate mass.

[0072] Example 3 The difference from Example 1 is that the amount of trypsin added is 1% of the amount of precipitate, and the amount of alkaline protease added is 0.2% of the amount of precipitate.

[0073] Example 4 The difference from Example 1 is that the amount of SDS solution added is 8% of the mass of the centrifuged supernatant.

[0074] Example 5 The difference from Example 1 is that the amount of SDS solution added is 15% of the mass of the centrifuged supernatant.

[0075] Example 6 The difference from Example 1 is that the mass ratio of protein powder to deoxynucleotide powder is 86:14.

[0076] Example 7 The difference from Example 1 is that the mass ratio of protein powder to deoxynucleotide powder is 93:7.

[0077] Comparative Example 1 The difference from Example 1 is that the amount of trypsin added is 1% of the precipitate mass, and no alkaline protease is added; otherwise, it is the same as Example 1.

[0078] Comparative Example 2 The difference from Example 1 is that the amount of SDS solution added is 20% of the mass of the centrifuged clear liquid, and the rest is the same as in Example 1.

[0079] Comparative Example 3 The difference from Example 1 is that the specific operation of step (2) is as follows: the fish spermatophore fragments obtained in step (1) are heated to 85°C and then added to a 10% sodium chloride solution with a mass of 25 times that of the fish spermatophore fragments. The solution is dissolved for 48 hours, cooled to 38°C, and then a 28% NaOH solution is added to adjust the pH to 11. After standing for 30 minutes, the solution is centrifuged and the precipitate and the supernatant are collected respectively. The rest is the same as in Example 1.

[0080] Example 1: Antitumor Efficacy Detection Positive control: Paclitaxel, white powder, batch number J2126744, Shanghai Aladdin Biochemical Technology Co., Ltd., solvent is DMSO.

[0081] Wild-type AB strain zebrafish were bred by natural paired mating. Zebrafish at 2 days post-fertilization (dpf) were used for the anti-tumor efficacy analysis of the fish sperm protein nucleic acid composition. All zebrafish were raised in fish culture water at 28 °C (water quality: 200 mg of instant sea salt was added to every 1 L of reverse osmosis water, with a conductivity of 450 - 550 μS / cm; pH 6.5 - 8.5; hardness 50 - 100 mg / L CaCO3). The experimental animal use license number is: SYXK (Zhe) 2022 - 0004. The feeding management complied with the requirements of international AAALAC accreditation (accreditation number: 001458), and the IACUC ethical review number is: IACUC - 2026 - 202601090002 - 01.

[0082] Human non-small cell lung cancer (A549) cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% double antibody under the conditions of 37 °C and 5% CO2.

[0083] 1. Experimental instruments, consumables and reagents Dissecting microscope (SZX7, OLYMPUS, Japan); CCD camera (VertA1, Shanghai Tusen Vision Technology Co., Ltd., China); Precision electronic balance (CP214, OHAUS, USA); Ultrasonic cleaner (JP - 010T, Shenzhen Jiemeng Cleaning Equipment Co., Ltd., China); Electrically focused continuous zoom fluorescence microscope (AZ100, Nikon, Japan); Microinjector (IM - 300, Narishige, Japan); Pulling needle instrument (PC - 10, Narishige, Japan); 6-well plate (Zhejiang Beilamber Biotechnology Co., Ltd., China); CO2 incubator (3111, Thermo Fisher Scientific, USA); Double-sided single-person purification workbench (SW - CJ - 215KS, Shanghai Sujing Industrial Co., Ltd., China); Inverted microscope (CKX53, OLYMPUS, Japan); Tabletop large-capacity low-speed centrifuge (TD5A, Changsha Yingtai Instrument Co., Ltd., China).

[0084] Methylcellulose (lot number C2004046, Shanghai Aladdin Biochemical Technology Co., Ltd., China); DMEM medium (lot number 8122785, Gibco, China); fetal bovine serum (FBS, lot number N2603053p, Thermo Fisher Scientific, New Zealand); 0.25% Trypsin-EDTA trypsin (lot number 2186962, Thermo Fisher Scientific (China) Co., Ltd., USA); CM-DiI trypsin (lot number 2335589, Thermo Fisher Scientific (China) Co., Ltd., USA); penicillin-streptomycin antibiotics (lot number 2585616, Gibco, USA); PBS phosphate buffer (catalog number BL601A, Biosharp, China).

[0085] 2. Experimental Methods 2.1 Determination of Maximum Detectable Concentration (MTC) Wild-type AB strain zebrafish, 3 days post-fertilization (3 dpf), were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well (experimental group). The samples were administered in water (concentrations shown in Table 1), and a normal control group was also included. Each well contained 3 mL of the sample. After treatment at 28℃ for 48 h, the mean chromosomal conversion ratio (MTC) of the samples relative to normal zebrafish was determined.

[0086] 2.2 Evaluation of efficacy in inhibiting tumor growth Human non-small cell lung cancer (A549) cells were labeled with CM-DiI and transplanted into the yolk sac of 2 dpf wild-type AB strain zebrafish via microinjection, with approximately 200 cells transplanted per fish, to establish a zebrafish tumor transplantation model.

[0087] Model zebrafish were cultured at 35℃ to 3 days post-flop (dpf). At 3 dpf, zebrafish with good consistency of transplanted tumor cells were selected under a microscope and randomly assigned to 6-well plates, with 30 zebrafish treated in each well. Water-soluble samples (concentrations shown in Table 2) were administered, along with a positive control of paclitaxel at a concentration of 2.00 μg / mL. A model control group was also included, with a volume of 3 mL per well. After two more days of treatment at 35℃, 10 zebrafish from each experimental group were randomly selected and photographed under a fluorescence microscope. Data were acquired using NIS-Elements D3.20 advanced image processing software, and the fluorescence intensity of human non-small cell lung cancer (A549) cells was analyzed. The statistical analysis results of this index were used to evaluate the tumor-inhibiting efficacy of the samples. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS software, and p < 0.05 was considered statistically significant.

[0088] 2.3 Evaluation of efficacy in inhibiting tumor metastasis Human non-small cell lung cancer (A549) cells were labeled with CM-DiI and transplanted into the yolk sac of 2 dpf wild-type AB strain zebrafish via microinjection, with approximately 200 cells transplanted per fish, to establish a zebrafish tumor transplantation model.

[0089] Model zebrafish were cultured at 35℃ to 3 days post-flop (dpf). At 3 dpf, zebrafish with good consistency of transplanted tumor cells were selected under a microscope and randomly assigned to 6-well plates, with 30 zebrafish treated in each well. The samples (concentrations shown in Table 3) were administered in water, along with a positive control of paclitaxel at a concentration of 2.00 μg / mL. A model control group was also included, with a volume of 3 mL per well. After two more days of treatment at 35℃, 10 zebrafish from each experimental group were randomly selected and photographed under a fluorescence microscope. ImageJ advanced image processing software was used to analyze and collect data to analyze the metastatic distance of human non-small cell lung cancer (A549) cells. The statistical analysis results of this index were used to evaluate the tumor metastasis inhibition efficacy of the samples. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS software, and p < 0.05 was considered statistically significant.

[0090] 3. Experimental Results 3.1 MTC Table 1 Results of the concentration exploration experiment for antitumor efficacy of samples (n=30)

[0091] As shown in the table above, the antitumor efficacy of the fish spermatoprotein nucleic acid composition in Example 1 is 2000 μg / mL in zebrafish.

[0092] 3.2 Evaluation of efficacy in inhibiting tumor growth At the same concentration (1000 μg / mL), the antitumor efficacy of Examples 1-5 was superior to that of Comparative Examples 1-3; the antitumor efficacy of Example 6 was superior to that of Comparative Examples 1 and 3, but showed no statistical difference compared to Comparative Example 2; the antitumor efficacy of Example 7 was superior to that of Comparative Example 3, but showed no statistical difference compared to Comparative Examples 1 and 2. See Table 2 for details. Figure 1 and Figure 2 .

[0093] Table 2. Experimental results evaluating the tumor growth inhibition efficacy of the samples (n=10)

[0094] Note: Compared with the model control group, p<0.05, p<0.001; compared with Comparative Example 1, # p<0.05, ##p<0.01, ### p<0.001; compared with Comparative Example 2, $ p<0.05, $$ p<0.01, $$$ p<0.001; compared with Comparative Example 3, & p<0.05, &&& p<0.001.

[0095] 3.3 Evaluation of efficacy in inhibiting tumor metastasis At the same concentration (1000 μg / mL), the antitumor efficacy of Examples 1-6 was superior to that of Comparative Examples 1-3; Example 7 showed no statistically significant difference compared to Comparative Examples 1, 2, and 3. See Table 3 for details. Figure 3 and Figure 4 .

[0096] Table 3. Experimental results evaluating the efficacy of the samples in inhibiting tumor metastasis (n = 10)

[0097] Note: Compared with the model control group, p<0.01, p<0.001; compared with Comparative Example 1, # p<0.05, ### p<0.001; compared with Comparative Example 2, $ p<0.05, $$$ p<0.001; compared with Comparative Example 3, && p<0.01, &&& p<0.001.

[0098] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for preparing a fish testis protein nucleic acid composition, characterized in that, Includes the following steps: (1) Take fish spermatophores, add washing liquid, stir and soak, remove solvent, wash, crush to obtain fish spermatophore powder; (2) Add sodium chloride solution to dissolve and extract the fish spermatophore fragments obtained in step (1) to obtain an extract; The extract is then aged, kept at a constant temperature, and cooled. Then add sodium hydroxide to adjust the pH, centrifuge, and collect the precipitate and the supernatant separately; (3) Soak the precipitate obtained in step (2) in water, heat to dissolve, continue to heat and keep warm, then cool down, add protease to hydrolyze, inactivate, concentrate, sterilize, cool and dry to obtain protein powder; (4) Add the centrifuged clear liquid obtained in step (2) to SDS solution, stir and let stand, centrifuge to remove the precipitate, add SDS solution again, stir and let stand, centrifuge to remove the precipitate, and obtain the centrifuged clear liquid after removing impurities. Precipitate the centrifuged clear liquid after removing impurities with alcohol to obtain filamentous material; desalt the filamentous material to obtain solid material; enzymatically hydrolyze the solid material to obtain enzymatic hydrolysate; concentrate the enzymatic hydrolysate, sterilize, and dry to obtain deoxynucleotide powder; (5) Mix the protein powder and deoxynucleotide powder according to the specified ratio to obtain the final product; In step (3), the proteases are trypsin and alkaline protease. The amount of trypsin added is 0.2%-1% of the precipitate mass, and the amount of alkaline protease added is 0.2%-0.6% of the precipitate mass.

2. The preparation method according to claim 1, characterized in that, The fish testes mentioned in step (1) are salmon or carp testes; the washing solution is composed of sodium citrate and sodium chloride, with a molar ratio of 0.01-0.02:0.10-0.15; the amount of washing solution added is 2-5 times the mass of the fish testes.

3. The preparation method according to claim 1, characterized in that, The mass concentration of sodium chloride in step (2) is 8%-12%; the amount of sodium chloride added is 20-30 times the mass of fish spermatophore fragments.

4. The preparation method according to claim 1, characterized in that, The amount of water added in step (3) is 30-50 times the amount of precipitate, the heating temperature for ripening is 75-85℃, and the heat preservation after heating is 90-100℃ for 45-65 minutes.

5. The preparation method according to claim 1, characterized in that, The enzymatic hydrolysis time in step (3) is 5-7 hours; the concentration temperature is 65℃-70℃, and the sugar content is concentrated to 25%-35%.

6. The preparation method according to claim 1, characterized in that, The amount of SDS solution added in step (4) is 8%-15% of the mass of the centrifuged clear liquid; the mass concentration of the SDS solution is 4%-6%.

7. The preparation method according to claim 1, characterized in that, The specific operation of alcohol precipitation in step (4) is as follows: add 1-2 times the volume of edible ethanol with a volume concentration of 75%-95% to the centrifuged clear liquid after impurity removal, let it stand for 10-15 minutes, and then remove the filamentous material. The specific desalination operation is as follows: wash the filamentous material 2-4 times with 8-12 times its mass of edible ethanol with a volume concentration of 30-60%, and then centrifuge. The specific operation of the enzymatic hydrolysis is as follows: dissolve the solid in 30-55 times the amount of water, heat to 82-90℃ and maintain for 10-30 minutes, cool down to 65-70℃, adjust the pH to 5.5-6.5 with dilute hydrochloric acid, add 2-8% of the solid mass of nuclease to dissolve, maintain at 65-70℃ for 3-5 hours, stir, and heat to 90-100℃ to inactivate the enzyme for 10-15 minutes; The specific operation of concentration is as follows: cool the enzymatic hydrolysate to a temperature of 60-68℃, filter it with a microporous resin column, and concentrate it to a liquid sugar content of 25%-35%.

8. The preparation method according to claim 1, characterized in that, The mass ratio of protein powder to deoxynucleotide powder in step (5) is 80-93:7-20.

9. A fish spermatogonium nucleic acid composition prepared by the preparation method according to any one of claims 1-8.

10. The use of the fish spermatogonium nucleic acid composition prepared by the preparation method according to any one of claims 1-8 in the preparation of antitumor drugs.

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