Haematococcus pluvialis peptide and application thereof in preparation of products for improving male reproductive disorders

Abstract

The present application relates to the technical field of biology, and particularly relates to a Haematococcus pluvialis peptide and application thereof in preparation of products for improving male reproductive disorders. The present application extracts a highly active Haematococcus pluvialis peptide from Haematococcus pluvialis, and clearly defines the protection and improvement effect of the Haematococcus pluvialis peptide on chemical induction of mouse male reproductive disorders, thereby providing a new direction for resource utilization of Haematococcus pluvialis and treatment of male reproductive damage.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more particularly to a Haematococcus pluvialis peptide and its application in the preparation of products for improving male reproductive disorders. Background Technology

[0002] Haematococcus pluvialis, a single-celled green algae and a novel food resource, has attracted much attention due to its ability to efficiently accumulate natural astaxanthin. Related research and development are deepening, and the large-scale cultivation and deep processing industry is maturing. The industrial extraction of astaxanthin generates a large amount of algal residue as a byproduct. This residue contains up to 30% crude protein and a complete range of essential amino acids, making it a natural raw material for developing plant proteins and preparing bioactive peptides, possessing considerable processing and development potential. However, due to limitations in current processing technologies, the vast majority of algal residue has not yet been utilized as a resource, leading to environmental hazards such as waste pollution.

[0003] Chinese patent CN116217663A (South China Agricultural University, "An antioxidant polypeptide KFTPAP derived from Haematococcus pluvialis") discloses the method of obtaining hexapeptide KFTPAP from algal residue through alkaline protease hydrolysis, ultrafiltration, and HPLC purification, and verifying its antioxidant activity in nematodes. However, this patent only discloses a single peptide segment and lacks research on its efficacy in mammals and its protective effect on male reproduction.

[0004] Chinese patent CN112876548A (Dongguan University of Technology, "An antioxidant peptide from Chlorella and its preparation method") discloses the preparation of Chlorella antioxidant peptides by enzymatic hydrolysis and ultrasound-assisted extraction of Chlorella protein. However, this patent only discloses a single peptide segment, does not involve research on male reproductive protection, and cannot realize the resource utilization of algal residue.

[0005] Male reproductive dysfunction is a key cause of male infertility, affecting approximately 15% of couples of reproductive age worldwide. This issue is gradually evolving into a prominent public health and social challenge. From the perspective of pathogenic factors, male reproductive dysfunction can be mainly divided into two categories: on the one hand, external stimuli such as exogenous environmental pollutants and chemical drugs can directly damage reproductive tissues; on the other hand, endogenous physiological degenerative changes caused by natural aging can also continuously impair reproductive function. Given this situation, in-depth research into the pathogenesis of male reproductive disorders and the development of effective intervention methods are urgently needed. However, at present, the development and related research on Haematococcus pluvialis peptides are still relatively limited, and there are no studies that can confirm their ability to improve reproductive disorders. Summary of the Invention

[0006] The technical problem this invention aims to solve is that male reproductive disorders are gradually evolving into a prominent public health and social challenge, while the development and related research on Haematococcus pluvialis peptides are still relatively limited at present, and there is no research that can prove that they can improve reproductive disorders.

[0007] To address the aforementioned problems, this invention provides a Haematococcus pluvialis peptide and its application in the preparation of products for improving male reproductive disorders. A highly active Haematococcus pluvialis peptide was extracted from Haematococcus pluvialis residue, and the protective and improving effects of this Haematococcus pluvialis peptide on chemically induced male reproductive disorders in mice were clarified, providing a new direction for the resource utilization of Haematococcus pluvialis residue and the treatment of male reproductive damage.

[0008] To achieve the above objectives, the present invention is implemented through the following technical means: a Haematococcus pluvialis peptide, the amino acid sequence of which is the polypeptide shown in SEQ ID NO.1.

[0009] SEQ ID NO.1: FDSGGY.

[0010] The above-mentioned Haematococcus pluvialis peptides are used in the preparation of products for improving male reproductive disorders. These products include pharmaceuticals, health supplements, and functional foods.

[0011] This invention demonstrates through experiments that the Haematococcus pluvialis peptide FDSGGY has a significant ameliorative effect on male reproductive disorders induced by triptolide (TP), specifically manifested in improvements in sperm quality (TP significantly reduced sperm motility and count and increased abnormality rate in mice (p<0.01), which significantly recovered after FDSGGY intervention), testicular structure (TP caused seminiferous tubule vacuolation and disordered arrangement of spermatogenic cells, which reduced vacuolation and improved orderly arrangement after FDSGGY treatment), and reproductive hormones (FDSGGY significantly increased the levels of testosterone, luteinizing hormone, and follicle-stimulating hormone in model mice (p<0.01), alleviating damage to the reproductive axis). This may be because, on the one hand, FDSGGY can upregulate the secretion of hormones related to the reproductive axis (hypothalamus-pituitary-testis axis), alleviate the inhibition of the reproductive endocrine axis by TP, and promote endogenous testosterone synthesis and the initiation of spermatogenesis; on the other hand, it can repair the structure of seminiferous tubules, improve the arrangement of spermatogenic cells and the testicular spermatogenic microenvironment, thereby supporting spermatogenesis, increasing sperm count and motility, and reducing the rate of sperm abnormalities. The above mechanisms indicate that the protective effect of the peptides of this invention on the male reproductive system is mainly manifested in the regulation of reproductive endocrine function and spermatogenic tissue structure.

[0012] A method for preparing the above-mentioned Haematococcus pluvialis peptide, which is synthesized artificially.

[0013] The beneficial effects of this invention are as follows: The novel peptide FDSGGY obtained by this invention has a molecular weight of 644.24 Da, is non-toxic and non-allergenic, and can be used as a therapeutic drug to improve male reproductive disorders, which is beneficial to the high-value utilization of Haematococcus pluvialis residue. Attached Figure Description

[0014] Figure 1 This is the secondary structure diagram of FDSGGY.

[0015] Figure 2 This shows the effect of FDSGGY on sperm in a TP-induced mouse model. Figure A shows microscopic observation of sperm morphology, Figure B shows mouse sperm motility, Figure C shows mouse sperm count, and Figure D shows mouse sperm abnormality rate. # indicates a highly significant difference compared to the NC group (p<0.01), # indicates a significant difference compared to the model group (p<0.05), and ## indicates a highly significant difference compared to the model group (p<0.01).

[0016] Figure 3 This study investigated the effects of FDSGGY on the pathological morphology of testicular tissue in a mouse model of male reproductive disorders.

[0017] Figure 4 This shows the effects of FDSGGY on reproductive hormones in TP-induced male mice. Figure A shows serum testosterone levels, Figure B shows serum luteinizing hormone levels, and Figure C shows serum follicle-stimulating hormone levels. # indicates a highly significant difference compared to the NC group (p<0.01), # indicates a significant difference compared to the model group (p<0.05), and ## indicates a highly significant difference compared to the model group (p<0.01). Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0019] In addition, all materials used in the embodiments of the present invention, unless otherwise specified, were purchased from the market.

[0020] Example 1: The inventors previously extracted multiple peptides with molecular weights (Mw) < 3 kDa and lengths of 6-18 amino acids from Haematococcus pluvialis, exhibiting high thermal stability, acid-base stability, and gastrointestinal digestibility. The steps are as follows: 1. Preparation of Haematococcus pluvialis complex peptides: Accurately weigh an appropriate amount of defatted Haematococcus pluvialis and add it to distilled water at a ratio of 1:20 (w / v). After pretreatment with ultrasound at 220 W for 20 min, adjust the pH to 8 with 1 mol / L NaOH, add 3.2% (w / w) neutral protease, and hydrolyze in a 45℃ water bath for 3 h. After enzymatic hydrolysis, heat at 100 °C for 15 min to inactivate the enzyme, cool to room temperature, and centrifuge at 10000 r / min for 15 min at 4℃. Collect the supernatant to obtain the Haematococcus pluvialis enzymatic hydrolysate.

[0021] 2. Ultrafiltration separation of Haematococcus pluvialis complex peptides: The above enzymatic hydrolysate was filtered through a microporous membrane (0.22 μm pore size). Then, it was fractionated using an ultrafiltration system with an Mw cutoff of 3 kDa to obtain fractions with Mw < 3 kDa, which were then used for peptide sequence identification.

[0022] 3. LC-MS / MS identification of Haematococcus pluvialis complex peptide sequence: After desalting, the peptide sample was centrifuged and dried, then redissolved in Nano-LC mobile phase A (0.1% formic acid / water) and bottled for online LC-MS / MS analysis. The dissolved sample was loaded onto a nanoViper C18 pre-column (3 μm, 100 Å) at an appropriate volume, followed by a 20 μL wash for desalting. After desalting and retention on the pre-column, the sample was separated using a C18 reversed-phase column (Acclaim PepMap RSLC, 75 μm × 25 cm C18 - 2 μm 100 Å). The gradient used was a 60-minute increase in mobile phase B (80% acetonitrile, 0.1% formic acid) from 5% to 38%. Mass spectrometry was performed using a ThermoFisher Q Exactive plus system (ThermoFisher, USA) combined with a nano-spray Nano Flex ion source (ThermoFisher, USA), with a spray voltage of 1.9 kV and an ion transfer tube heating temperature of 320 °C.

[0023] Subsequently, the activity of bioactive peptides was predicted using the PeptideRanker website. Peptides with scores greater than 0.5 were selected, and further non-toxic and non-allergenic bioactive peptides were screened using online analysis tools such as ExPASy, ProtParam, Pepdraw, ToxinPred, and AllerTOP v2.0. The information for one of these peptides is as follows: Table 1: Peptide Information: .

[0024] Example 2: I. Experimental Methods: 1. Artificially synthesized polypeptides: The selected peptides were synthesized using artificial solid-phase synthesis at Sangon Biotech (Shanghai) Co., Ltd., with a peptide purity > 95%.

[0025] 2. The protective effect of Haematococcus pluvialis peptides on the improvement of triptolide (TP)-induced male reproductive disorders.

[0026] Animal experiment: Thirty-two SPF-grade male ICR mice aged 6-8 weeks were selected and, after one week of acclimatization, were randomly divided into four groups (n=8) for 21 days. The specific grouping and drug dosage are as follows.

[0027] Table 2: Grouping and Dosage: .

[0028] Specifically, a male mouse model of reproductive dysfunction was established by intraperitoneal injection of TP for 21 consecutive days. Sperm parameter measurement: (1) Sample processing: The experimental mice were fasted for 12 hours but allowed free access to water. After anesthetizing the mice, blood was drawn from the orbital rim and the mice were euthanized. The left and right epididymis of the mice were accurately removed and immediately placed in centrifuge tubes / culture dishes containing 5 mL of physiological saline, which had been preheated in a 37°C water bath. The epididymis was then cut into small pieces with clean ophthalmic scissors and incubated at 37°C for 5 minutes to ensure that the sperm could fully swim out of the epididymis. The corresponding sperm parameters were then evaluated. When collecting the sperm suspension, care should be taken to accurately aspirate it with a pipette. The sperm suspensions from different mice should be aspirated from the same location as much as possible to avoid errors.

[0029] (2) Sperm count determination: A hemocytometer containing 30 μL of sperm suspension was placed under a regular optical microscope for observation, and the sperm count was determined according to the red blood cell counting method. The number of sperm in the four medium-sized squares within the large central square was recorded under the microscope, following the principle of recording only one side (top, bottom, left, right) in an "S" shape. To avoid excessive error, the difference between the maximum and minimum sperm counts recorded in the selected squares had to exceed 20. The sperm counts from the four medium-sized squares were then summed.

[0030] Sperm count per milliliter of semen (10) 6 / mL) = Total sperm count in 4 medium squares / 4 × N × 10 × 10 3 ; Note: N is the dilution factor. The dilution factor for this experiment was 5-fold.

[0031] (3) Measurement of sperm motility: A hemocytometer containing 30 μL of sperm suspension was placed under a regular optical microscope for observation. The number of sperm that moved rapidly forward in a straight line was randomly evaluated among 200 sperm.

[0032] Sperm motility (%) = Number of fast-moving sperm / 200 × 100%.

[0033] (4) Determination of sperm abnormality rate: 30 μL of sperm suspension was dropped onto a clean glass slide, dried, and then fixed with methanol for 10 min. Then, an appropriate amount of eosin staining solution was added, and after drying and fixing for 1 h, it was rinsed with water. The sperm morphology was observed under a 100× ordinary optical microscope, and the sperm with intact structures were counted. The number of abnormal sperm in 200 sperm cells was assessed in each mouse. Sperm abnormalities were mainly manifested in the head, followed by the tail. The main types of abnormal sperm in the head included: banana-shaped head, double head, fat head, amorphous head, and forward-curved head; the main types of abnormal sperm in the tail included: folded tail and double tail.

[0034] Sperm deformity rate (%) = number of deformed sperm / 200 × 100%.

[0035] Pathological morphology of testicular tissue in a male reproductive disorder model mouse: Fresh testicular tissue was fixed with 4% paraformaldehyde. The fixed testicular tissue was rinsed thoroughly, washed with a series of ethanol solutions, embedded in paraffin, and sectioned (4 μm). After washing with xylene and ethanol, it was stained with hematoxylin and eosin, mounted with neutral resin, and observed under a light microscope.

[0036] TP induces male mouse reproductive hormones: Follow the operating procedures of the ELISA test kit (Jiangsu Jingmei Biotechnology Co., Ltd.).

[0037] Result verification: 1. Effects of FDSGGY on sperm in a TP-induced mouse model: Figure 2 As shown in the figure, compared with the normal group, TP significantly reduced sperm motility and sperm count in mice (p<0.01), increased sperm morphological abnormalities, increased number of abnormal sperm (&: represents abnormal sperm), and significantly increased sperm abnormality rate (p<0.01). FDSGGY intervention significantly restored sperm count and sperm motility, and reduced sperm abnormality rate (p<0.01). These results indicate that FDSGGY has a certain ameliorative effect on TP-induced spermatogenic dysfunction in male mice.

[0038] 2. Effects of FDSGGY on the pathological morphology of testicular tissue in a mouse model of male reproductive disorders, such as... Figure 3As shown in the figure, the testicular tissue structure in the NC group was normal, with spermatogenic cells at different developmental stages visible, and the spermatogenic cells within the seminiferous tubules were clearly layered and neatly arranged. The M group showed severe tissue vacuolation with irregular shapes, reduced spermatogenic cell layers, disordered arrangement, and enlarged gaps; only a small number of Sertoli cells, spermatogonia, and primary spermatocytes were present. After FDSGGY treatment, compared with the M group, the vacuolation of the testicular tissue was significantly improved, and the arrangement of spermatogenic cells was more orderly. These results indicate that FDSGGY can alleviate testicular tissue damage caused by TP.

[0039] 3. Results of the effects of FDSGGY on TP-induced reproductive hormones in male mice are as follows: Figure 4 As shown, the results indicate that FDSGGY significantly increased the level of reproductive hormones in mice in the TP model (p<0.01), maintained normal reproductive hormone levels to some extent, and alleviated damage to the reproductive axis in male mice.

[0040] Finally, it should be noted that although the above embodiments describe specific implementations of the present invention, they are not intended to limit the invention. Those skilled in the art should understand that these are merely illustrative examples, and the scope of protection of the present invention is defined by the appended claims. All modifications or equivalent substitutions should be included within the scope of protection of the present invention.

Claims

1. A Haematococcus pluvialis peptide, characterized in that: The amino acid sequence is that of the polypeptide shown in SEQ ID NO.

1.

2. The method for preparing Haematococcus pluvialis peptide according to claim 1, characterized in that: It is artificially synthesized.

3. The use of the Haematococcus pluvialis peptide according to claim 1 in the preparation of products for improving male reproductive disorders.

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