A refined component of deer blood peptide and a preparation method and application thereof

Abstract

The application relates to a refined component of deer blood peptide and a preparation method and application thereof, relates to the technical field of traditional Chinese medicines, and solves the problem of adverse reactions caused by long-term use of existing testosterone replacement therapy drugs. The component contains 23 polypeptides. After deer blood is stabilized in a 4 DEG C environment, supernatant is collected through centrifugation, freeze-drying is carried out, methanol is used for elution in a Bond Elut C18 reverse chromatographic column, eluate is collected, and nitrogen blowing drying is carried out to obtain the component. The application can be applied to the development of drugs for promoting testosterone secretion as active ingredients, and is expected to be further developed into food and health products with deer antler and the like as raw materials, and has excellent application prospect and commercial development value.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine technology, specifically to a refined component of deer antler blood peptides, its preparation method, and its application. Background Technology

[0002] Testosterone is the most abundant androgen secreted in men, with the most crucial physiological effects. It is primarily synthesized and secreted by interstitial cells in the testes, and its secretion process is precisely regulated by the hypothalamic-pituitary-testis axis (HPT axis). Simultaneously, testosterone secreted into the bloodstream can influence the release of gonadotropin-releasing hormone from the hypothalamus and luteinizing hormone from the pituitary gland through negative feedback mechanisms, forming a closed-loop regulatory network to maintain stable testosterone levels. After binding to androgen receptors, testosterone activates downstream signaling cascades, exerting multidimensional physiological regulatory functions in mammals. These include promoting osteoblast mineralization and maintaining bone metabolic homeostasis, driving skeletal muscle protein synthesis and metabolism, inducing accessory gland morphogenesis, and regulating spermatogenesis. It also plays a key regulatory role in hematopoietic function, glucose and lipid metabolism balance, and mental and cognitive states. Its physiological functions span the entire male development process from embryonic development to aging, making it an indispensable core hormone for maintaining normal physiological function. Senescence of testicular interstitial cells can directly lead to gonadal dysfunction and induce various age-related diseases, severely impacting men's quality of life.

[0003] As people age, the physiological functions of human tissues and organs progressively decline, and hypogonadism is one of the typical characteristics of aging. Unlike female reproductive aging, which is clearly marked by ovarian depletion and the cessation of menstruation, male testosterone levels decline slowly with age. Clinical data shows that the incidence of testicular dysfunction in men over 60 years of age reaches 20% to 30%, and rises to about 50% in those over 80 years of age. Low testosterone levels not only directly lead to male sexual dysfunction (such as erectile dysfunction), but also significantly increase the risk of anemia, muscle and bone loss, osteoporosis, glucose and lipid metabolism disorders, metabolic syndrome, and other diseases, seriously affecting the health and quality of life of middle-aged and elderly men.

[0004] Currently, the core clinical intervention for insufficient testosterone secretion is testosterone replacement therapy (TRT). While it can rapidly increase serum testosterone levels and improve related symptoms, long-term use has significant safety limitations. On the one hand, exogenous testosterone may interfere with the physiological feedback regulation mechanism of the HPT axis, leading to further suppression of endogenous testosterone secretion and causing endocrine axis dysfunction. Discontinuation of TRT can exacerbate the patient's own gonadal dysfunction. On the other hand, long-term TRT may increase the risk of benign prostatic hyperplasia and prostate cancer, and is closely related to adverse cardiovascular events such as heart attack, stroke, and venous thrombosis. Its safety and tolerability are insufficient to meet the long-term management needs of individuals with age-related testosterone decline. Furthermore, there are currently no safe and effective preventive measures or targeted interventions for testosterone reduction caused by natural aging, leaving a significant unmet clinical need.

[0005] Traditional Chinese medicine has a long history of application and natural advantages in the treatment of chronic diseases and the maintenance of reproductive function. Deer antler blood, a precious blood resource extracted from newly shaving deer antlers, has been recorded in important medical classics throughout history. The Compendium of Materia Medica clearly states that it "treats impotence, replenishes deficiency, benefits essence and blood, and stops lower back pain...those suffering from severe pain will be cured immediately upon drinking it." Its traditional efficacy of "tonifying kidney yang and benefiting essence and blood" is highly consistent with the clinical needs of testosterone secretion regulation and reproductive function improvement, providing important traditional medicine theoretical support and research direction for the development of safe and effective testosterone secretion promoters.

[0006] Deer antlers, the unossified young antlers of male sika deer or red deer, are a classic and precious medicinal material in Traditional Chinese Medicine for "tonifying kidney yang and replenishing essence and blood." As a rare example of a periodically regenerating organ among mammals, the regeneration process requires the complete reconstruction of skin, blood vessels, cartilage, and nerve tissue, and is closely related to the periodic regulation of the endocrine system. The antler-growing period (spring and summer) is the key stage for antler regeneration. During this time, the deer's HPT axis is active, hormone levels are in dynamic equilibrium, and the blood is rich in various bioactive components such as polypeptides and active factors. Previous studies have confirmed that deer plasma during the antler-growing stage can significantly promote skin regeneration in rats. These studies focus on skin tissue repair and regenerative medicine, only demonstrating the bioactivity of deer blood peptides in skin wound repair. They do not involve any research on reproductive endocrine regulation or testosterone secretion, nor do they analyze or verify the regulatory relationship between deer blood peptides and the hypothalamus-pituitary-testis axis (HPT axis). Furthermore, existing research on deer antler stem cells only confirms that their secreted active factors can indirectly participate in the hormone secretion regulation network related to some tissue regeneration, without involving research on testosterone synthesis and secretion in testicular interstitial cells, and has no direct technical connection with the field of testosterone secretion regulation.

[0007] Although deer blood has traditionally been recorded as having the effects of "tonifying kidney yang and replenishing essence and blood," and modern regenerative medicine research has also confirmed its bioactivity in tissue repair, current technology has not yet established a direct technical link between these two effects and testosterone secretion regulation. Furthermore, no systematic research has been conducted on the active components in deer antler blood that regulate testosterone secretion. Three core technological gaps remain in this field that have not yet been effectively addressed. First, the basis of the active substances is unclear. Current technology has not systematically isolated, purified, and structurally identified polypeptide components in deer antler blood that may be related to reproductive endocrine regulation. There is no public disclosure of the amino acid sequences, effective content ranges, and ratios of any peptides with testosterone-promoting activity. Those skilled in the art cannot determine the core substances in deer antler blood that promote testosterone secretion from existing technology, nor can they obtain guidance on the combination and ratio of related active peptides. Second, a targeted preparation process has not been established. Current technology has not disclosed any method for the targeted enrichment of polypeptides with testosterone-promoting activity from deer antler blood. Conventional methods used in the field for processing deer antler blood, such as enzymatic hydrolysis, water extraction and alcohol precipitation, and ordinary ultrafiltration, either damage the deer antler or... The spatial structure and bioactivity of natural endogenous peptides in blood can only yield crude extracts of total peptides from deer antler blood, which are complex in composition and unstable in activity. This makes it impossible to achieve targeted enrichment and precise purification of specific active peptides. Those skilled in the art cannot obtain process guidance for preparing target active components from existing technologies. Thirdly, direct bioactivity has not been verified. Existing technologies do not have any in vitro or in vivo experimental data that directly verify the promotion of testosterone synthesis and secretion by deer antler blood peptides using testicular interstitial cells as the core research model. Nor have they conducted relevant research on the repair activity of testicular interstitial cell function decline and testosterone secretion inhibition caused by oxidative damage. The actual effect of deer antler blood in the field of testosterone secretion regulation has not been confirmed by any direct experimental data in existing technologies.

[0008] Meanwhile, there are widespread technical biases and cognitive limitations in this field regarding testosterone secretion regulation and the development of deer antler blood resources, directly restricting research and development directions in this area. In the clinical field, it is generally believed that only exogenous testosterone replacement therapy can rapidly and effectively increase serum testosterone levels for insufficient testosterone secretion. Natural product components can only serve as an adjunct to this approach and cannot directly and effectively promote endogenous testosterone secretion from testicular interstitial cells, nor can they reverse the inhibition of testosterone secretion caused by oxidative damage. Therefore, natural deer antler blood components are not considered a core research direction for alternative testosterone replacement therapy. In the field of natural product research, it is generally believed that natural endogenous peptides in deer antler blood are easily degraded by enzymes in the body and have low bioavailability. Artificial enzymatic hydrolysis is necessary to obtain small molecule peptides to exert stable biological activity. Naturally occurring endogenous peptides do not possess practical medicinal or functional food development value. Therefore, conventional research in this field focuses on the enzymatic hydrolysis of deer antler blood peptides and does not pay attention to the isolation and activity studies of natural endogenous peptides. Furthermore, it is generally believed in the art that deer antler blood has a complex composition and contains many interfering substances. Conventional separation and purification processes can only yield crude extracts of total peptides with uncertain composition and large batch-to-batch variations, failing to obtain refined peptide compositions with clear components, fixed ratios, and stable activity. This makes it difficult to achieve precise regulation of efficacy and quality control. Therefore, there is no attempt to isolate and identify peptide compositions with specific sequences from deer antler blood for testosterone secretion regulation. These technical biases and cognitive limitations prevent those skilled in the art from actively attempting to isolate and enrich natural endogenous peptide compositions from deer antler blood for testosterone secretion regulation, based on existing technologies. Summary of the Invention

[0009] To address the problem of adverse reactions caused by long-term use of existing testosterone replacement therapy drugs, this invention proposes a refined deer antler blood peptide component, its preparation method, and its application.

[0010] The specific technical solution of the present invention is as follows: A refined deer antler blood peptide component comprises the following ingredients in parts by weight: SEQ ID NO.1: 1-10 parts of the following peptides: GLC (glycine-leucine-cysteine ​​tripeptide), 1-10 parts; RYG (arginine-tyrosine-glycine tripeptide), 1-15 parts; SEQ ID NO.2: 1-30 parts of the following peptides: NFT (asparagine-phenylalanine-threonine tripeptide), 1-20 parts; SRN (serine-arginine-asparagine tripeptide), 1-10 parts; PVD (proline-valine-aspartic acid tripeptide), 1-30 parts; SEQ ID NO.3: 1-30 parts of the following peptides: TYH (threonine-tyrosine-histidine tripeptide), 1-30 parts; SEQ ID NO.4: 1-60 parts of the following peptides: SEQ ID NO.5: 1-20 parts of the following peptides; SEQ ID NO.6: 2-55 parts of the following peptides; SEQ ID NO.7: 1-50 parts of the following peptides. The following peptides are listed: SEQ ID NO. 8 (1-55 parts); SEQ ID NO. 9 (1-45 parts); SEQ ID NO. 10 (2-80 parts); SEQ ID NO. 11 (5-150 parts); SEQ ID NO. 12 (2-80 parts); VQA (valine-glutamine-alanine tripeptide) (2-65 parts); ASM (alanine-serine-methionine tripeptide) (2-60 parts); SEQ ID NO. 13 (5-100 parts); SEQ ID NO. 14 (5-140 parts); SEQ ID NO. 15 (1-40 parts).

[0011] Preferably, the component comprises the following parts by weight: One sample of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO.1; one sample of GLC; two samples of RYG; three samples of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO.2; three samples of NFT; two samples of SRN; two samples of PVD; two samples of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO.3; two samples of TYH; three samples of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO.4; one sample of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO.5; five samples of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO.6; four samples of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO.7; four samples of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO.8; four samples of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO.9; five samples of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO.10; twelve samples of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO.11; seven samples of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO.12; six samples of VQA; six samples of ASM; nine samples of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO.13; SEQ ID 12 polypeptides consisting of the amino acid sequence shown in NO.14; 3 polypeptides consisting of the amino acid sequence shown in SEQ ID NO.15.

[0012] The present invention also provides a method for preparing the above-mentioned purified deer antler blood peptide components, comprising the following steps: Deer antler blood was collected, stabilized at 4°C, centrifuged, and the supernatant was collected and lyophilized to obtain lyophilized deer antler blood serum powder. The powder was eluted with methanol in a Bond Elut C18 reverse chromatography column, and the eluent was collected and dried under nitrogen to obtain the purified deer antler blood fraction.

[0013] Preferably, the antler blood is extracted from antlers during the newly antler-growing stage. Specifically, the newly antler-growing stage refers to the period after a male deer has its first antler harvested, when new antlers begin to grow.

[0014] Preferably, the stabilization time in the 4℃ environment is 30~60 min.

[0015] Preferably, the centrifugation speed is 3000~5000 r / min, and the centrifugation time is 20~60 min.

[0016] Preferably, the elution specifically includes the following steps: Activate Bond Elut C18 with methanol for 2-8 h, elute with 3-8 times the volume of ultrapure water at 0.5-1 mL / min, reconstitute the lyophilized deer antler serum powder with ultrapure water to prepare a solution of 1-3 mg / mL, and repeatedly load the sample 3-8 times at 0.5-1 mL / min. Then elute the Bond Elut C18 packing with 3-8 times the volume of 30-100% methanol at 0.5-1 mL / min and collect the eluent.

[0017] The present invention also provides an application of the above-mentioned refined deer antler blood peptide component, specifically as an active ingredient in the preparation of a drug that promotes testosterone secretion.

[0018] Compared with the prior art, the specific beneficial effects of the present invention are as follows: This invention clarifies for the first time the active substance basis of deer antler blood regulating testosterone secretion, systematically isolates, purifies, and identifies eight specific polypeptides with testosterone-promoting activity, and discloses their effective content range and ratio relationship, solving the technical problem in the field of being unable to determine the core substance of deer antler blood that promotes testosterone secretion. Furthermore, it establishes for the first time a targeted preparation process for testosterone-promoting active polypeptides from deer antler blood, achieving targeted enrichment and precise purification of specific active peptides, overcoming the industry pain point that conventional processes destroy the activity of natural polypeptides and only yield crude extracts with complex components.

[0019] This invention overcomes the clinical misconception that natural products can only be used as auxiliary conditioning methods and cannot directly and effectively promote endogenous testosterone secretion or reverse testosterone secretion inhibition caused by oxidative damage. It confirms that natural endogenous peptides from deer antler blood can serve as a core research direction for testosterone replacement therapy. It also overcomes the research misconception that natural endogenous peptides from deer antler blood are easily degraded, have no development value, and must undergo artificial enzymatic hydrolysis to obtain active small molecule peptides. This invention confirms that natural endogenous peptides possess stable biological activity and medicinal and functional food development value.

[0020] The refined components provided by this invention can promote the proliferation of testicular interstitial cells and significantly enhance the vitality of damaged TM3 testicular interstitial cells, showing significant effects within a concentration range of 100-400 μg / mL. Even at low concentrations of 25 μg / mL and 50 μg / mL, the natural refined components still exhibit significant proliferative effects. It can effectively improve testosterone secretion disorders in testicular interstitial cells, strongly promote testosterone synthesis and secretion, and significantly increase the testosterone content in cell supernatant. Furthermore, the natural refined components have a wider effective concentration range and more significant effects. This invention can be used as an active ingredient in drug development to promote testosterone secretion, and it also holds promise for further development into food and health products using deer antler and other raw materials, possessing excellent application prospects and commercial development value.

[0021] Compared to the core limitations of long-term clinical testosterone replacement therapy (TRT), the refined components of this invention work by promoting endogenous testosterone secretion. This does not interfere with the physiological feedback regulation of the hypothalamus-pituitary-testis axis, does not inhibit endogenous testosterone secretion, does not aggravate gonadal dysfunction, and has no related safety risks of increasing the risk of prostate hyperplasia / cancer or adverse cardiovascular events. It is perfectly suited to the long-term conditioning needs of people with age-related testosterone reduction, filling the gap in safe and effective intervention methods for testosterone reduction caused by natural aging. Attached Figure Description

[0022] Figure 1 HPLC analysis of the refined components in Example 1 ESI MS 2 Chromatogram; Figure 2 Information on compounds in the purified deer antler blood peptides identified in Example 1; Figure 3 HPLC analysis of the refined components in Example 2 ESI MS 2 Chromatogram; Figure 4 HPLC analysis of the refined components in Example 3 ESI MS 2 Chromatogram; Figure 5 Figure 1 shows the cell viability results of H2O2-induced TM3 testicular interstitial cell models in different treatment groups. Figure 6 Figure 1 shows the results of H2O2-induced oxidative damage detection of testicular interstitial cells in different treatment groups; Figure 7 The image shows the ELISA results of testosterone secretion in testicular interstitial cells of H2O2-induced oxidative damage TM3 in different treatment groups. Detailed Implementation

[0023] To make the technical solutions of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. It should be noted that the following embodiments are only used to better understand the technical solutions of the present invention and should not be construed as limiting the present invention.

[0024] Example 1. Step S1: Take 10 mL of deer antler blood, let it stand at 4℃ for 40 min, then centrifuge at 4000 r / min for 30 min. Take the supernatant, store it at -80℃ overnight, and then freeze-dry it to obtain lyophilized powder.

[0025] Step S2: Reconstitute the lyophilized powder with ultrapure water to prepare a 2 mg / mL solution. Load this solution five times sequentially onto a Bond Elut C18 chromatography column at a flow rate of 0.8 mL / min. Then, elute with 5 times the packing volume of ultrapure water at a flow rate of 0.8 mL / min and collect the eluent. Next, elute with 5 times the packing volume of 50% methanol at a flow rate of 0.8 mL / min and collect the eluent. Finally, dry under nitrogen to obtain the purified deer antler blood peptide fraction.

[0026] High-performance liquid chromatography-mass spectrometry (HPLC-MS) detection of the purified deer antler blood peptide fraction obtained in this embodiment: (1) Preparation of test solution: Accurately weigh 1 mg of purified component, add 1 mL of 50% methanol to dissolve, filter through a 0.22 μm filter membrane to obtain test solution.

[0027] (2) UPLC QE Orbitrap The chromatographic conditions for MS determination were as follows: the instrument was UPLC. QE Orbitrap MS ultra-high performance liquid chromatography-high resolution mass spectrometry; Column: XBridge™ Premier Peptide BEH C18 (130Å 2.5μm, 2.1×100mm); Flow rate: 0.3mL / min; Column temperature: 30℃; Injection volume: 10 μL; Mobile phase A: acetonitrile, Mobile phase B: 0.1% formic acid aqueous solution. Gradient elution: 0~5 min, 5% A; 10~20 min, 5%~15% A; 20~25 min, 15%~33% A; 25~35 min, 35% A; 35~50 min.

[0028] (3) Mass spectrometry conditions: Electrospray ionization source in positive (ESI+) ion mode, scanning range of m / z 66~1500. Mass spectrometry parameters were set as follows: sheath gas flow rate: 35 L / min; auxiliary gas flow rate: 15 L / min; scanning gas flow rate: 1 L / min; gas temperature: 350 ℃; capillary voltage: 3.5 kV. Full spectrum scan was performed at resolutions of 70000 (Full MS) and 17500 (dd-MS2), with collision energies of 30, 35, and 40 eV.

[0029] The total ion chromatogram of the purified deer antler blood peptide fraction in positive ion mode is shown below. Figure 1 As shown, based on HPLC ESI MS 2The composition of the refined deer antler blood peptide was analyzed, and preliminary identification information of the compounds in the refined deer antler blood peptide is available in [link to relevant documentation]. Figure 2 23 compounds were identified: SSPP (1 sample); GLC (1 sample); RYG (2 samples); EPGP (3 samples); NFT (3 samples); SRN (2 samples); PVD (2 samples); FQNAL (2 samples); TYH (2 samples); LDDLKGA (3 samples); RNPSP (1 sample); GFQNAL (5 samples); FLTPQ (4 samples); VDPENF (4 samples); GPKYW (4 samples); AEGGGVR (5 samples); DHSKETNSNELS (12 samples); EPVF (7 samples); VQA (6 samples); ASM (6 samples); ATPGEK (9 samples); EGGGVR (12 samples); ALEVAPGA (3 samples). Example 2. Step S1: Take 10 mL of deer antler blood, let it stand at 4℃ for 40 min, then centrifuge at 4000 r / min for 30 min. Take the supernatant, store it at -80℃ overnight, and then freeze-dry it to obtain lyophilized powder.

[0030] Step S2: Reconstitute the lyophilized powder with ultrapure water to prepare a 2 mg / mL solution. Load this solution five times sequentially onto a Bond Elut C18 chromatography column at a flow rate of 0.8 mL / min. Then, elute with 5 times the packing volume of ultrapure water at a flow rate of 0.8 mL / min and collect the eluent. Next, elute with 5 times the packing volume of 30% methanol at a flow rate of 0.8 mL / min and collect the eluent for each fraction. Finally, dry under nitrogen to obtain the purified deer antler blood peptide fraction.

[0031] High-performance liquid chromatography-mass spectrometry (HPLC-MS) detection of the purified deer antler blood peptide fraction obtained in this embodiment: (1) Preparation of test solution: Accurately weigh 1 mg of purified component, add 1 mL of 50% methanol to dissolve, filter through a 0.22 μm filter membrane to obtain test solution.

[0032] (2) UPLC QE Orbitrap The chromatographic conditions for MS determination were as follows: Instrument: UPLC QE Orbitrap MS ultra-high performance liquid chromatography-high resolution mass spectrometry; Column: XBridge™ Premier Peptide BEH C18 (130Å 2.5μm, 2.1×100mm); Flow rate: 0.3mL / min; Column temperature: 30℃; Injection volume: 10 μL; Mobile phase A: acetonitrile, Mobile phase B: 0.1% formic acid aqueous solution. Gradient elution: 0~5 min, 5% A; 10~20 min, 5%~15% A; 20~25 min, 15%~33% A; 25~35 min, 35% A; 35~50 min.

[0033] (3) Mass spectrometry conditions: Electrospray ionization source in positive (ESI+) ion mode, scanning range of m / z 66~1500. Mass spectrometry parameters were set as follows: sheath gas flow rate: 35 L / min; auxiliary gas flow rate: 15 L / min; scanning gas flow rate: 1 L / min; gas temperature: 350 ℃; capillary voltage: 3.5 kV. Full spectrum scanning was performed at resolutions of 70000 (Full MS) and 17500 (dd-MS2), with collision energies of 30, 35, and 40 eV. The total ion chromatogram of the purified deer antler blood peptide obtained in this embodiment in positive ion mode is shown in the figure. Figure 3 As shown. Based on HPLC-ESI-MS 2 The composition of the refined deer antler blood peptide was analyzed, and the compound components in the component were the same as those in Example 1.

[0034] Example 3. Step S1: Take 10 mL of deer antler blood, let it stand at 4℃ for 40 min, then centrifuge at 4000 r / min for 30 min. Take the supernatant, store it at -80℃ overnight, and then freeze-dry it to obtain lyophilized powder.

[0035] Step S2: Reconstitute the lyophilized powder with ultrapure water to prepare a 2 mg / mL solution. Load this solution five times sequentially onto a Bond Elut C18 chromatography column at a flow rate of 0.8 mL / min. Then, elute with 5 times the packing volume of ultrapure water at a flow rate of 0.8 mL / min and collect the eluent. Next, elute with 5 times the packing volume of 100% methanol at a flow rate of 0.8 mL / min and collect the eluent for each fraction. Finally, dry under nitrogen to obtain the purified deer antler blood peptide fraction.

[0036] High-performance liquid chromatography-mass spectrometry (HPLC-MS) detection of the purified deer antler blood peptide fraction obtained in this embodiment: (1) Preparation of test solution: Accurately weigh 1 mg of purified component, add 1 mL of 50% methanol to dissolve, filter through a 0.22 μm filter membrane to obtain test solution.

[0037] (2) UPLC QE Orbitrap The chromatographic conditions for MS determination were as follows: Instrument: UPLC QE Orbitrap MS ultra-high performance liquid chromatography-high resolution mass spectrometry; Column: XBridge™ Premier Peptide BEH C18 (130Å 2.5μm, 2.1×100mm); Flow rate: 0.3mL / min; Column temperature: 30℃; Injection volume: 10 μL; Mobile phase A: acetonitrile, Mobile phase B: 0.1% formic acid aqueous solution. Gradient elution: 0~5 min, 5% A; 10~20 min, 5%~15% A; 20~25 min, 15%~33% A; 25~35 min, 35% A; 35~50 min.

[0038] (3) Mass spectrometry conditions: Electrospray ionization source in positive (ESI+) ion mode, scanning range of m / z 66~1500. Mass spectrometry parameters were set as follows: sheath gas flow rate: 35 L / min; auxiliary gas flow rate: 15 L / min; scanning gas flow rate: 1 L / min; gas temperature: 350 ℃; capillary voltage: 3.5 kV. Full spectrum scanning was performed at resolutions of 70000 (Full MS) and 17500 (dd-MS2), with collision energies of 30, 35, and 40 eV. The total ion chromatogram of the purified deer antler blood peptide obtained in this embodiment in positive ion mode is shown in the figure. Figure 4 As shown. Based on HPLC-ESI-MS 2 The composition of the refined deer antler blood peptide was analyzed, and the compound components in the component were the same as those in Example 1.

[0039] The components of the refined component in the above three embodiments are as follows: SSPP, 1-10 parts; GLC, 1-10 parts; RYG, 1-15 parts; EPGP, 1-30 parts; NFT, 1-20 parts; SRN, 1-10 parts; PVD, 1-30 parts; FQNAL, 1-30 parts; TYH, 1-30 parts; LDDLKGA, 1-60 parts; RNPSP, 1-20 parts; GFQNAL, 2-55 parts; FLTPQ, 1-50 parts; VDPENF, 1-55 parts; GPKYW, 1-45 parts; AEGGGVR, 2-80 parts; DHSKETNSNELS, 5-150 parts; EPVF, 2-80 parts; VQA, 2-65 parts; ASM, 2-60 parts; ATPGEK, 5-100 parts; EGGGVR, 5-140 parts; ALEVAPGA, 1-40 parts.

[0040] Example of results. Effects of refined deer antler blood peptides on cell viability in a hydrogen peroxide-induced testicular interstitial cell injury model: (1) Experimental cells: Mouse testicular interstitial cells (TM3 cells) were cultured in DMEM containing 10% FBS. Cells were cultured at 37°C and 5% CO2, and passaged every two days. When the cells reached approximately 80% confluence, they were digested with trypsin and then passaged. Cells in the logarithmic growth phase were used for experimental studies.

[0041] (2) Establishment of the TM3 cell model induced by hydrogen peroxide (H2O2): Take TM3 cells in the logarithmic growth phase and use 5 × 10⁻⁶ cells. 3 TM3 cells were seeded at a density of 100 cells / well in 96-well plates and cultured for 24 h. The cells were then treated with H2O2 at concentrations of 25, 50, 100, 200, and 400 μmol / L. After 2 h of treatment, 10 μL of CCK-8 reagent was added to each well, and the cells were incubated for 30 min. The absorbance was then measured at 450 nm using a microplate reader. All experiments were repeated three times. Cell viability under different H2O2 concentrations was assessed using the CCK-8 assay. Cells with a viability of 50-60% or higher were selected for subsequent experiments.

[0042] (3) Experimental design, grouping, and dosing regimen: Take TM3 cells in the logarithmic growth phase and use 5 × 10⁻⁶ cells. 3Cells were seeded at a density of 100 cells / well in 96-well plates and cultured at 37°C in a 5% CO2 incubator for 24 h. After cell adhesion and growth, the cells were randomly divided into four groups: blank control group, model group, 50% ethanol-refined deer antler blood peptide group (LRXW), 30% ethanol-refined deer antler blood peptide group (LRXS), and 100% ethanol-refined deer antler blood peptide group (LRXB). First, the cells in each group were synchronized for 12 h using serum-free medium. Then, 100 μmol / L H2O2 was added to the model group, LRXW group, LRXS group, and LRXB group, and the cells were incubated for 2 h to establish the cell model. After modeling, the corresponding drug solutions were added to the LRXW group, LRXS group, and LRXB group, respectively, to achieve final concentrations of 25, 50, 100, 200, and 400 μg / mL for each concentration gradient. The blank control group and model group were added with an equal volume of serum-free medium. All groups were then cultured for another 24 h using standard methods.

[0043] (4) Testosterone content determination: After drug administration, the cells were incubated at 37 °C in a 5% CO2 incubator for 24 h. The cell culture medium from each well was then aspirated and transferred to centrifuge tubes. The cells were centrifuged at 3000 r / min at 4 °C for 20 min. The supernatant was collected after centrifugation and stored at -20 °C for later use. A mouse testosterone ELISA kit was used, and the procedure was strictly followed according to the kit instructions. The absorbance of each well was measured using a microplate reader, and the testosterone content in the cell supernatant was calculated.

[0044] (5) Experimental results: The effects of different concentrations of H2O2 on TM3 cell viability were investigated to screen for suitable concentrations for inducing oxidative damage. Results are shown in [Figure number missing]. Figure 5 .Depend on Figure 5 It is evident that the oxidative damage of H2O2 to TM3 cells is concentration-dependent; with increasing H2O2 concentration, the degree of oxidative damage to cells significantly increases, and cell viability decreases in a gradient manner. Specifically, the survival rate of TM3 cells treated with 100 μmol / L H2O2 was 56.67%, significantly lower than that of the control group. P The concentration is <0.05, which is within the standard range (50%~60%) for constructing oxidative damage models, indicating that this concentration is suitable for subsequent experiments.

[0045] After treating H2O2-induced damaged TM3 cells with different concentrations of LRXW, LRXS, and LRXB for 24 h, the cell proliferation results are as follows: Figure 6 As shown. Compared with the model group, both the LRXW group and the LRXS group significantly increased the proliferation level of TM3 cells in the concentration range of 100~400 μg / mL. P<0.05. Furthermore, the LRXW group also showed a significantly higher concentration at 50 μg / mL compared to the model group ( P <0.05, the LRXB group only showed a significant increase compared to the model group at 200 μg / mL ( P <0.05). The above results suggest that the LRXW group, LRXS group and LRXB group can all promote H2O2-induced TM3 cell proliferation at certain concentrations, and the LRXW group also showed a promoting effect at a lower concentration.

[0046] After treating H2O2-induced oxidative damage TM3 cells with LRXW, LRXS, and LRXB drugs for 24 h, cell supernatants were collected, and testosterone secretion levels were detected by ELISA. The experimental results are as follows: Figure 7 As shown. Compared with the model group, both the LRXW group and the LRXS group significantly promoted testosterone secretion from TM3 cells within the concentration range of 100~400 μg / mL, and the testosterone content in the supernatant was significantly increased ( P The concentration of testosterone secretion increased significantly with increasing drug concentration (<0.05), and both components showed a clear concentration-dependent effect on promoting testosterone secretion, meaning that testosterone secretion gradually increased with increasing drug concentration. Further analysis showed that the LRXW group, even at a low concentration of 50 μg / mL, could significantly increase testosterone secretion levels in TM3 cells. P <0.05), which also showed a concentration-dependent characteristic, while neither the LRXS group nor the LRXB group showed a significant promoting effect at low concentrations (50 μg / mL).

[0047] Notably, at a concentration of 400 μg / mL, the testosterone content in the cell supernatant of the LRXW group was significantly higher than that of the model group and almost equal to that of the blank group. This suggests that at this concentration, the LRXW group can completely reverse the H2O2-induced inhibition of testosterone secretion in TM3 cells, and even restore it to normal physiological secretion levels. In summary, the LRXW, LRXS, and LRXB groups can all effectively improve H2O2-induced testosterone secretion disorders in TM3 cells and promote cellular testosterone synthesis and secretion at certain concentrations. Among them, the LRXW group has a wider effective concentration range and a more significant effect in promoting testosterone secretion. These experimental results provide direct experimental evidence for clarifying the material basis of the deer antler blood refined components (LRXW, LRXS, and LRXB groups) in improving insufficient testosterone secretion in men caused by natural aging, and also lay the foundation for further in-depth research on its regulatory mechanism and the development of related functional products.

[0048] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for preparing a refined component of deer antler blood peptides, characterized in that, Includes the following steps: Deer antler blood was collected, stabilized at 4°C, centrifuged, and the supernatant was collected and lyophilized to obtain deer antler blood serum lyophilized powder. The powder was eluted with 30-100% methanol in a Bond Elut C18 reverse chromatography column, and the eluent was collected and dried under nitrogen to obtain the purified deer antler blood fraction. The refined deer antler blood peptide component comprises the following components in parts by weight: 1-10 parts of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO.1; 1-10 parts of GLC; 1-15 parts of RYG; 1-30 parts of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO.2; 1-20 parts of NFT; 1-10 parts of SRN; 1-30 parts of PVD; 1-30 parts of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO.3; 1-30 parts of TYH; 1-60 parts of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO.4; 1-20 parts of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO.5; 2-55 parts of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO.6; 1-50 parts of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO.7; 1-55 parts of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO.8; 1-45 parts of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO.9; 2-80 parts of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO.10; 5-150 parts of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO.11; SEQ SEQ ID NO.12: 2-80 parts of polypeptide; VQA: 2-65 parts; ASM: 2-60 parts; SEQ ID NO.13: 5-100 parts of polypeptide; SEQ ID NO.14: 5-140 parts of polypeptide; SEQ ID NO.15: 1-40 parts of polypeptide.

2. The method for preparing the refined deer antler blood peptide component according to claim 1, characterized in that, The deer antler blood is extracted from newly antler-growing deer antlers.

3. The method for preparing the refined deer antler blood peptide component according to claim 1, characterized in that, The stabilization time in the 4℃ environment is 30~60 min.

4. The method for preparing the refined deer antler blood peptide component according to claim 1, characterized in that, The centrifugation speed is 3000~5000 r / min, and the centrifugation time is 20~60 min.

5. The method for preparing the refined deer antler blood peptide component according to claim 1, characterized in that, The elution process specifically includes the following steps: Activate Bond Elut C18 with methanol for 2-8 h, elute with 3-8 times the volume of ultrapure water at 0.5-1 mL / min, reconstitute the lyophilized deer antler serum powder with ultrapure water to prepare a solution of 1-3 mg / mL, and repeatedly load the sample 3-8 times at 0.5-1 mL / min. Then elute the Bond Elut C18 packing with 3-8 times the volume of 30-100% methanol at 0.5-1 mL / min and collect the eluent.

6. The application of a refined deer antler blood peptide component prepared by the preparation method according to any one of claims 1 to 5, characterized in that, It is used as an active ingredient in the preparation of drugs that promote testosterone secretion.

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