Sipunculus nudus peptide as well as extraction method and application thereof

Abstract

The invention relates to the technical field of peptide extraction, in particular to sipunculus nudus peptide and an extraction method and application thereof. According to the specific technical scheme, the method comprises the following steps: 1) crushing a sipunculus nudus raw material, adding water, adding sodium hydrogen sulfite and ascorbic acid, and stirring and soaking for 10-15 minutes; 2) grinding the soaked raw materials, and adjusting the pH value of the slurry to be alkaline; (3) adding alkali enzyme into the slurry, carrying out microwave enzymolysis for 45-50 minutes, adding papaya peptidase B, continuously carrying out microwave enzymolysis for 45-50 minutes, heating, inactivating, cooling, carrying out centrifugal filtration, and collecting filtrate; and 4) sequentially carrying out microfiltration, ultrafiltration and sodium filtration on the obtained filtrate to obtain the product. Microwave radiation is adopted for assisting enzymolysis, energy can be provided for an enzymolysis system, the temperature needed by enzymolysis can be maintained, the enzymolysis efficiency is greatly improved, the prepared sipunculus nudus peptide can stabilize iron ions, a safe and efficient iron supplementing preparation can be developed, meanwhile, reproductive function decline caused by obesity can be comprehensively repaired, the advantage of multi-dimensional conditioning is achieved, and the sipunculus nudus peptide has a good application prospect. And the method is suitable for metabolism-related reproductive disorder treatment.

Description

Technical Field

[0001] This invention relates to the field of peptide extraction technology, specifically to a prickly ash peptide, its extraction method, and its application. Background Technology

[0002] The naked Sipunculus nudus, commonly known as the sand worm, sea ginseng, or sand sausage, is a dioecious species widely distributed in tropical, subtropical, and temperate seas worldwide. In China, it is mainly found in Hainan, Taiwan, Guangdong, Guangxi, Fujian, and Shandong, with the Beibu Gulf (Guangxi, Guangdong, and Hainan) having the richest natural resources. It belongs to the family Sipunculidae and the genus Sipunculus. This warm-water, dioecious species primarily inhabits sandy or muddy coastal areas, feeding on benthic diatoms and organic detritus. It is distributed throughout the intertidal zone, with higher yields near the subtidal zone. Adult Sipunculus nudus are typically 10-25 cm long, with a burrow depth of 50-80 cm. The burrow is circular, sloping at the front and almost vertical at the back, a feature fishermen call "flower eye." The optimal salinity for the naked Sipunculus nudus is 20‰-35‰.

[0003] Naked Sipunculus nudus is a green and healthy seafood product rich in various nutrients, with crisp and tender meat and a fresh and fragrant taste. Historical records indicate that Sipunculus nudus has the effects of promoting lactation, nourishing yin and reducing internal heat, and clearing the lungs and resolving phlegm, and it was called "sea medicine" in ancient my country. Fishermen along the southeastern coast of my country call Sipunculus nudus "marine cordyceps," while people in southern Fujian call it "animal ginseng." Current research on the medicinal components of Sipunculus nudus shows that its polysaccharide extract can promote the proliferation of mouse lymphocytes, help monitor the immune system and clear diseased cells, and play an important role in the body's immune system. Liu Yongqiang et al. found through analysis of the nutritional components of Sipunculus nudus that it contains abundant amino acids and vitamin B2, which can enhance the exercise endurance and anti-fatigue ability of mice. Zheng Zhihong's research on the wound healing mechanism in mice shows that Sipunculus nudus contains abundant arginine and proline, which have the effect of promoting wound healing. Cao Yuping found through analysis of the nutritional protein of Sipunculus nudus that it can enhance phagocytosis and immune function. In addition, Sipunculus nudus also has the effects of delaying aging, anti-radiation, and antioxidant activity. Therefore, *Sipunculus nudus* possesses various bodily regulatory functions and has the potential to be developed into health foods or medicines that regulate human functions. However, current research on the extraction and processing of peptides from naked *Sipunculus nudus* is limited. Exploring the various beneficial peptide bioactive substances in *Sipunculus nudus*, extracting and applying these peptides to benefit human health, is one direction for the deep processing and utilization of *Sipunculus nudus*. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a method for extracting and applying Sipunculus spp. peptides. The peptides are extracted from dried / fresh Sipunculus spp. using microwave enzymatic hydrolysis technology and can be widely used for the treatment and health maintenance of sub-healthy individuals.

[0005] To achieve the above objectives, the present invention provides the following technical solution: This invention discloses a method for extracting peptides from *Sipunculus nudus*, comprising the following steps: 1) After crushing the naked checkered chafer raw material, add water, sodium bisulfite and ascorbic acid, stir and soak for 10-15 minutes; 2) Grind the soaked raw materials, adjust the pH of the slurry to alkaline, and keep the temperature at 10-50℃; 3) Add alkaline enzyme to the slurry and stir and microwave for 45-50 min at a power of 2-10 kW. Add papain B and continue stirring and microwave for 45-50 min. Heat to inactivate, cool to 45-50℃, centrifuge and filter, and collect the filtrate. 4) The obtained filtrate was subjected to microfiltration, two ultrafiltrations, and sodium filtration in sequence to obtain the square star insect peptide.

[0006] Preferably, in step 1), the naked Sipunculus raw material is crushed to 50-100 mesh, the amount of water added is 10-40 times the weight of the naked Sipunculus raw material, and the temperature of the water is 25-30℃.

[0007] Preferably, in step 1), the amount of sodium bisulfite added is 3-4‰ of the mass of the naked Sipunculus nudus raw material; the amount of ascorbic acid added is 0.5-1‰ of the mass of the naked Sipunculus nudus raw material.

[0008] Preferably, in step 3), the temperature for stirring and microwave enzymatic hydrolysis is 50-55℃; the temperature for continued stirring and microwave enzymatic hydrolysis is 55-60℃; and the temperature for inactivation by heating is 85-90℃.

[0009] Preferably, in step 3), the alkaline enzyme is Carsberg protease or Novo protease, and the amount added is 1%-3% of the weight of the naked Sipunculus nudus raw material; the amount added of papainase B is 2%-4% of the weight of the naked Sipunculus nudus raw material.

[0010] Preferably, in step 4), the microfiltration is performed using a 30,000 Da membrane capable of intercepting molecules up to 30,000 Da; the two ultrafiltration operations are as follows: the first ultrafiltration uses a 10,000 Da membrane, and the second ultrafiltration uses a 5,000 Da membrane; the sodium filtration is performed using a 300 Da membrane.

[0011] Correspondingly, the extraction method described above yields a type of stellaria peptide.

[0012] Correspondingly, a type of Sipunculus spp. peptide powder is obtained by freeze-drying the Sipunculus spp. peptide extracted by the extraction method.

[0013] Accordingly, the application of the square-shaped sipunculus peptide extracted by the extraction method or the square-shaped sipunculus peptide powder in the preparation of iron chelating agents or in the preparation of drugs for treating low TM3 cell viability and low TM4 cell viability caused by palmitic acid.

[0014] Accordingly, the application of the square-patterned cinnabar peptide extracted by the extraction method or the square-patterned cinnabar peptide powder in the preparation of drugs for treating hyperglycemia, reduced sperm count, sperm abnormalities, low sperm motility, low testicular index, and low epididymal index caused by obesity.

[0015] The present invention has the following beneficial effects: 1. The extraction method of this invention is simple, easy to control, low in cost, and easily scalable for industrial production. Using microwave radiation-assisted enzymatic hydrolysis not only provides energy to the hydrolysis system and maintains the required temperature, but also significantly improves hydrolysis efficiency, reduces hydrolysis time, and ensures thoroughness and accuracy of hydrolysis, providing a new approach for the industrial production of active ingredients from *Sipunculus nudus*. No harmful substances are generated, meeting clean production standards; the structure of small molecule peptides is not damaged, maintaining peptide activity and increasing the extraction rate of *Sipunculus nudus* peptides, thus enhancing the edible value and physiological functions of *Sipunculus nudus*; the use of multiple filtration techniques for separation and purification results in a simple production process, high extraction rate, and significant economic benefits.

[0016] 2. The iron-binding activity of the *Sipunculus nudus* peptide prepared in this invention is 15.3% of that of dammar spleen ferritin. It exhibits high natural biocompatibility, no side effects, and can stabilize iron ions, making it suitable for iron-deficient individuals. It can be used to develop safe and efficient iron supplements while reducing gastrointestinal irritation. Simultaneously, it can significantly enhance the viability of TM3 and TM4 cells damaged by pyogenic encephalopathy (PA), upregulate the expression of testosterone synthase protein and reproductive-related genes, repairing cell function at the molecular level, and providing a natural raw material for reproductive cell damage protection drugs.

[0017] 3. The *Sipunculus nudus* peptide prepared in this invention exhibits superior weight-loss effects at medium to high doses compared to orlistat, rapidly reducing body weight, body fat, and Lee's index in obese mice, reversing the obesity phenotype associated with a high-fat diet, and is natural, gentle, and free of the potential risks of chemical drugs, making it suitable for obesity treatment. Furthermore, it can rapidly reduce persistent hyperglycemia in obese mice, enhance glucose metabolism, and improve insulin resistance, providing a natural and comprehensive conditioning solution for metabolic syndrome characterized by obesity and hyperglycemia, and reducing the risk of diabetes.

[0018] 4. The square-grid sipunculus peptide prepared in this invention can simultaneously increase the testicular and epididymal indices, increase sperm count and motility, reduce the malformation rate, comprehensively repair the decline in reproductive function caused by obesity, and has multi-dimensional conditioning advantages, making it suitable for the treatment of metabolic-related reproductive disorders. Attached Figure Description

[0019] Figure 1 The ability of Sipunculus peptide to enrich iron; Figure 2 The ABTS cationic free radical scavenging ability of the square-grid sipunculid peptide; Figure 3 The DPPH free radical scavenging ability of the square-grid sipunculid peptide; Figure 4 The effect of sipunculid peptide on PA-induced cell viability decline; Figure 5 The effect of Sipunculus peptide on the expression of key cellular proteins; Figure 6 The effect of key gene expression in Sipuncula lataniae peptide cells; Figure 7 Weight changes in obese mice over 12 weeks of modeling obesity. Figure 8 The effect of square-grid sipunculi peptide on Lee's index in obese mice; Figure 9 The effect of Sipunculus peptide on body weight in obese mice; Figure 10 The effect of Sipunculus peptide on blood glucose concentration in obese mice; Figure 11 The effect of square-grid sipunculi peptide on the epididymal index of obese mice; Figure 12 The effect of square-grid sipunculi peptide on the testicular index in obese mice; Figure 13 The effect of sipunculid peptide on sperm motility in obese mice; Figure 14 The effect of sipunculid peptide on sperm abnormality rate in obese mice; Figure 15 The effect of sipunculid peptide on the total sperm count in obese mice; Figure 16 The effect of Sipunculus peptide on epididymal tissue in obese mice; Figure 17 The effect of Sipunculus peptide on testicular tissue in obese mice; Figure 18 The effect of sipunculid peptide on adipose tissue in obese mice. Detailed Implementation

[0020] 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 embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] Unless otherwise specified, the technical means used in the implementation examples are conventional means well known to those skilled in the art.

[0022] This invention discloses a method for extracting peptides from *Sipunculus nudus*, comprising the following steps: 1) After pulverizing the raw material of *Sipunculus nudus* to 50-100 mesh, add water at 25-30℃, the amount of water being 10-40 times the weight of the raw material. Add sodium bisulfite at 3-4‰ of the raw material's weight and ascorbic acid at 0.5-1‰ of the raw material's weight, stir and soak for 10-15 minutes to obtain a mixture. This invention uses sodium bisulfite and ascorbic acid as protective agents to prevent oxidation and deterioration of the raw material.

[0023] 2) Grind the soaked raw materials (i.e., the mixture of step 1) in a colloid mill for 30 minutes, adjust the pH of the slurry to alkaline, preferably pH=8.5, and the temperature is 10-50℃.

[0024] 3) Place the slurry in a microwave-safe container with a power of 2-10 kW, add alkaline enzyme, stir and microwave for 45-50 min at a temperature of 50-55℃ to hydrolyze serine peptide bonds and amide bonds; after the first step of enzymatic hydrolysis is completed, continue to add papainase B at 2%-4% of the weight of the naked Sipunculus nudus raw material in step 1), stir and microwave for 45-50 min at a temperature of 55-60℃ to hydrolyze carboxyl amino acid or aromatic amino acid peptide bonds; after completion, raise the temperature to 85-90℃ for inactivation, cool to 45-50℃, centrifuge and filter, and collect the filtrate; wherein, the alkaline enzyme is Carsberg protease or Novo protease, and the amount added is 1%-3% of the weight of the naked Sipunculus nudus raw material in step 1).

[0025] 4) The obtained filtrate was subjected to microfiltration, two ultrafiltrations, and sodium filtration in sequence to obtain the square-patterned star insect peptide. The microfiltration was performed using a 30,000 Da membrane capable of intercepting peptides with a molecular weight of 30,000 Da; the two ultrafiltrations were performed as follows: the first ultrafiltration used a 10,000 Da membrane, and the second ultrafiltration used a 5,000 Da membrane, to intercept peptides with molecular weights of 10,000 Da and 5,000 Da, respectively; the sodium filtration was performed using a 300 Da membrane.

[0026] Furthermore, in step 4), the filtrate is concentrated using an RO membrane after sodium filtration.

[0027] This invention discloses a Sipunculus spp. peptide powder, which is obtained by freeze-drying the extracted Sipunculus spp. peptide.

[0028] This invention discloses the application of the square-patterned sipunculus peptide obtained by the extraction method or the square-patterned sipunculus peptide powder in the preparation of iron chelating agents or in the preparation of drugs for treating low TM3 cell viability and low TM4 cell viability caused by palmitic acid.

[0029] The invention also discloses the application of the square-patterned sipunculus peptide extracted by the extraction method or the square-patterned sipunculus peptide powder in the preparation of a drug for treating hyperglycemia, reduced sperm count, sperm abnormalities, low sperm motility, low testicular index, and low epididymal index caused by obesity.

[0030] The present invention will be further described below with reference to specific embodiments.

[0031] Example 1 A method for extracting peptides from *Sipunculus nudus* using microwave-assisted enzymatic hydrolysis includes the following steps: 1) After crushing the fresh raw material of naked square Sipunculus worms to a particle size of 60 mesh, add 20 times the weight of naked square Sipunculus worms in water at 25-30℃, and add 3‰ sodium bisulfite and 1‰ ascorbic acid of naked square Sipunculus worms by weight. Stir and soak for 15 minutes. 2) Grind the soaked raw materials in a colloid mill, adjust the pH of the slurry to 8.5, and set the temperature to 30℃; 3) Place the slurry in a microwave jar with a power of 2-10kw, adjust the microwave power to 2kw, add Carsberg protease or Novo protease, the amount added is 1%-1.5% of the weight of naked Sipunculus nudus raw material, stir and microwave enzymatic hydrolysis at 55℃ for 45min, add papain B, the amount added is 2%-2.5% of the weight of naked Sipunculus nudus raw material, continue stirring and microwave enzymatic hydrolysis at 55℃ for 45min, raise the temperature to 85℃ to inactivate, cool to 45℃, centrifuge and filter with a 400-mesh filter cloth three-legged centrifuge, and collect the filtrate; 4) The obtained filtrate is subjected to microfiltration, ultrafiltration, and sodium filtration with a 30,000 membrane that can intercept molecular weight of 30,000 Da in sequence, followed by ultrafiltration with a 10,000 membrane, ultrafiltration with a 5,000 membrane, and sodium filtration with a 300 Da membrane. The filtrate is then concentrated with an RO membrane.

[0032] The square-shaped sipunculus peptides extracted by the above method were prepared into square-shaped sipunculus peptide powder by freeze drying, and the amino acid and protein contents were detected. The detection items, results and methods are shown in Table 1 below.

[0033] Table 1. Detection results of the top 15 protein contents in Sipuncula pectin powder.

[0034] Example 2 A method for extracting peptides from *Sipunculus nudus* using microwave-assisted enzymatic hydrolysis includes the following steps: 1) After crushing the dried naked square sipunculus raw material to a particle size of 50 mesh, add 20 times the weight of the dried naked square sipunculus in water at 25°C, and add 4‰ sodium bisulfite and 1‰ ascorbic acid by weight of naked square sipunculus. Stir and soak for 10 minutes. 2) Grind the soaked raw materials in a colloid mill, adjust the pH of the slurry to 8.5, and keep the temperature at 10-50℃; 3) Place the slurry in a microwave jar with a power of 2-10kw, adjust the microwave power to 2kw, add Carsberg protease or Novo protease, the amount added is 1.5%-2% of the weight of naked Sipunculus nudus raw material, stir and microwave enzymatically hydrolyze at 50℃ for 45min, add papain B, the amount added is 2.5%-3% of the weight of naked Sipunculus nudus raw material, continue stirring and microwave enzymatically hydrolyze at 55℃ for 45min, raise the temperature to 85℃ to inactivate, cool to 45℃, centrifuge and filter with a 400-mesh filter cloth three-legged centrifuge, and collect the filtrate; 4) The obtained filtrate is subjected to microfiltration, ultrafiltration, and sodium filtration with a 30,000 Da membrane that can intercept molecular weight of 30,000 Da in sequence.

[0035] Example 3 A method for extracting peptides from *Sipunculus nudus* using microwave-assisted enzymatic hydrolysis includes the following steps: 1) After crushing the dried naked square sipunculus raw material to a particle size of 100 mesh, add 20 times the weight of the dried naked square sipunculus in water at 30°C, and add 3‰ sodium bisulfite and 0.5‰ ascorbic acid by weight of naked square sipunculus. Stir and soak for 15 minutes. 2) Grind the soaked raw materials in a colloid mill, adjust the pH of the slurry to 8.5, and set the temperature to 50℃; 3) Place the slurry in a microwave jar with a power of 2-10kw, adjust the microwave power to 2kw, add Carsberg protease or Novo protease, the amount added is 2.5%-3% of the weight of naked Sipunculus nudus raw material, stir and microwave enzymatic hydrolysis at 55℃ for 50min, add papain B, the amount added is 3%-4% of the weight of naked Sipunculus nudus raw material, continue stirring and microwave enzymatic hydrolysis at 60℃ for 50min, raise the temperature to 90℃ to inactivate, cool to 50℃, centrifuge and filter with a 400-mesh filter cloth three-legged centrifuge, and collect the filtrate; 4) The obtained filtrate is subjected to microfiltration, ultrafiltration, and sodium filtration with a 30,000 membrane that can intercept molecular weight of 30,000 Da in sequence, followed by ultrafiltration with a 10,000 membrane, ultrafiltration with a 5,000 membrane, and sodium filtration with a 300 Da membrane. The filtrate is then concentrated with an RO membrane.

[0036] In vitro activity test of the square-patterned sipuncula peptide obtained in Example 1: 1. The total iron content of *Sipunculus nudus* peptide was determined using the total iron-binding capacity kit developed in Nanjing. Reagents were prepared according to the kit instructions. 0.1 g of *Sipunculus nudus* peptide was dissolved in 1 mL of phosphate buffer to prepare the sample solution. This was repeated in 3 tubes. The sample and the kit working solution were mixed and allowed to stand for 10 minutes. Iron adsorbent was added, and the mixture was allowed to stand for another 5 minutes. The supernatant was collected by centrifugation. The supernatant was reacted with a chromogenic reagent, and the absorbance was measured at 520 nm using a microplate reader. Horse spleen ferritin was used as a control group. The results are as follows: Figure 1 As shown, the results indicated that the average total iron content of horse spleen ferritin (HSF) was 11940 μmol / L, while the average total iron content of sipunculid peptide (SN) was 1825 μmol / L. This suggests that sipunculid peptide has a strong iron-binding capacity, with an iron-binding activity of approximately 15.3% that of horse spleen ferritin. Therefore, sipunculid peptide is a good iron chelating agent and may be used in the future development of oral iron supplements.

[0037] 2. The in vitro antioxidant activity of *Sipunculus nudus* peptide was determined using the ABTS kit from Suzhou Greens. 0.05 g of *Sipunculus nudus* peptide and vitamin C (as a control) were weighed and diluted with enzyme-free water to different concentrations: *Sipunculus nudus* peptide at concentrations of 1000, 2000, 5000, and 10000 μg / mL, and vitamin C at concentrations of 2, 5, 10, and 50 μg / mL. After mixing with the working solution in the kit, the solution was applied to wells, with three replicates for each concentration. After standing at room temperature in the dark for 6 minutes, 200 μL was taken and the absorbance was measured at 734 nm using a microplate reader. The results are as follows: Figure 2 As shown, the results indicate that the ABTS of fusiforme peptide and vitamin C are similar. ＋ The free radical scavenging ability of both increased with increasing concentration showed a dose-dependent increase, and the antioxidant activity also increased accordingly; however, the free radical scavenging ability of vitamin C was much higher than that of fusiforme peptide, making it impossible to compare them at the same concentration.

[0038] 3. The in vitro antioxidant activity of *Sipunculus nudus* peptide was determined using the Nanjing Jiancheng DPPH free radical scavenging kit. 0.05 g of *Sipunculus nudus* peptide and vitamin C (as a reference standard) were weighed and dissolved separately in 200 μL of enzyme-free water. Then, 800 μL of 80% methanol was added to dilute the solution to the final volume. Subsequently, using 80% methanol as the diluent, the peptide solution was serially diluted to 100, 500, 1000, and 2000 μg / mL, and the vitamin C solution was serially diluted to 2, 5, 8, and 10 μg / mL to prepare the test sample solutions. The supernatant of each concentration was taken and thoroughly mixed with 80% methanol and the working solution of the kit. The mixture was incubated at room temperature in the dark for 30 minutes. Three replicates were set for each concentration. After centrifugation, 200 μL of the supernatant was taken and the absorbance was measured at 517 nm using a microplate reader. The results are as follows: Figure 3As shown, the test results indicate that the DPPH free radical scavenging capacity of both psyllidin and vitamin C increases in a dose-dependent manner with increasing concentration, and their antioxidant activity also increases accordingly; however, the free radical scavenging capacity of vitamin C is much higher than that of psyllidin, making it impossible to compare them at the same concentration.

[0039] In vitro cell activity of the square-patterned sipunculus peptide obtained in Example 1: 1. Mouse testicular interstitial TM3 cell line was cultured in DMEM / F-12 medium containing 5% bovine serum peptide for 24 hours. Then, 0.4 mM palmitic acid (PA, Obe cell model), 10 μg / mL Sipunculus pentaphyllum peptide, 50 μg / mL Sipunculus pentaphyllum peptide, 100 μg / mL Sipunculus pentaphyllum peptide, and 500 μg / mL Sipunculus pentaphyllum peptide were added respectively. Cell viability was analyzed after 24 hours. Results are as follows: Figure 4 As shown in Figure A, the results indicate that, compared with the PA group, the cell viability of the Ctrl blank control group, the 10 μg / mL, 50 μg / mL, 100 μg / mL, and 500 μg / mL groups was significantly different, indicating that the pteropus peptide can resist the decrease in TM3 cell viability caused by PA.

[0040] Mouse testicular Sertoli cell line TM4 was cultured in DMEM / F-12 medium containing 5% bovine serum peptide for 24 hours. Then, 0.4 mM palmitic acid (PA), 40 μg / mL Sipunculus pentaphyllum peptide, 80 μg / mL Sipunculus pentaphyllum peptide, 100 μg / mL Sipunculus pentaphyllum peptide, 120 μg / mL Sipunculus pentaphyllum peptide, and 240 μg / mL Sipunculus pentaphyllum peptide were added, respectively. Cell viability was analyzed after 24 hours. Results are as follows: Figure 4 As shown in B, the results indicated that, compared with the PA group, the cell viability of the Ctrl blank control group, the 40 μg / mL, 80 μg / mL, 100 μg / mL, 120 μg / mL, and 240 μg / mL groups was significantly different, with the 240 μg / mL group showing the best effect. This suggests that the peptone can resist the decrease in TM4 cell viability caused by PA.

[0041] like Figure 5 Western blot results showed that the expression levels of testosterone synthase proteins such as CYP17A1, CYP11A1, StAR, PCNA, Vimentin, and GATA4 in the mouse testicular interstitial TM3 cell line decreased with PA stress. However, fusiforme peptides could alleviate this effect of PA, indicating that fusiforme peptides can promote the secretion of testosterone synthase and improve the fertility of animals.

[0042] like Figure 6Real-time quantitative PCR results showed that the expression levels of genes such as PCNA, Vimentin, GATA4, and WT1 in mouse testicular support cell line TM4 decreased in PA-induced Obe cells, while fusiform peptide could alleviate the effects of PA on Obe.

[0043] Example 1: Obtaining the in vivo mouse activity of the square-patterned sipunculus peptide: 1. Sixty 3-week-old male C57BL / 6 mice were divided into 6 groups of 10 each and housed in an SPF (Special Feeding Free) environment. After one week of temporary rearing, 50 mice were fed a 60% high-fat diet (HFD), while the other 10 were fed a maintenance diet for 12 weeks (until the mice were 16 weeks old). Weight was measured every 2 weeks. Figure 7 The results show that there was a significant difference in body weight between the control group and the HFD-fed group after week 4, indicating that the obese mouse model was successfully established.

[0044] After successful modeling, 50 obese mice were divided into 5 groups of 10 each. The control group (Ctrl group) and HFD group were administered physiological saline by gavage daily. The HFD+SN-L group, HFD+SN-M group, and HFD+SN-H group were administered 50 mg / kg / d, 100 mg / kg / d, and 150 mg / kg / d, respectively. The orlistat positive control group (HFD+Ot group) was administered 60 mg / kg / d by gavage.

[0045] like Figure 8 As shown, after 8 weeks of gavage administration (24 weeks old mice), compared with the control group, the Lee's index of mice in the HFD+SN-L group, HFD+SN-M group, HFD+SN-H group and HFD+Ot group was significantly reduced, indicating that the square sipunculus peptide can promote weight loss in mice, and the weight loss effect of the HFD+SN-M group or the HFD+SN-H group is better than that of the positive control group HFD+Ot group.

[0046] like Figure 9 As shown, compared with the HFD group, the body weight of mice in the HFD+SN-L group, HFD+SN-M group, HFD+SN-H group and HFD+Ot group decreased significantly during the treatment process and approached that of the Ctrl blank control group. Among them, the body weight of mice in the HFD+SN-M group at week 20 was even lower than that of the Ctrl blank control group and the HFD+Ot group, indicating that the pterocaryon peptide significantly promoted weight loss in mice.

[0047] 2. Oral glucose tolerance test (OGTT): Mice were administered glucose (2.0 g / kg) by gavage after a 12-hour fast. Blood glucose levels were measured using a Roche glucometer at 15, 30, 60, 90, and 120 minutes post-administration. Glucose tolerance was assessed by calculating the area under the curve (AUC). Figure 10As can be seen, the blood glucose level in the HFD group remained high, while the blood glucose levels in the Ctrl blank control group, HFD+SN-L group, HFD+SN-M group, HFD+SN-H group and HFD+Ot group decreased rapidly. Among them, the HFD+SN-L group showed the fastest decrease, indicating that the pterosa peptide can promote the glucose metabolism of obese mice.

[0048] 3. Epididymal index (the ratio of epididymal weight to body weight) results are as follows: Figure 11 As shown, the epididymal index of the HFD model group was at a low level, indicating that the reproductive capacity of obese mice caused by high-fat diet was reduced. However, at the end of the experiment in week 20, the epididymal index of the HFD+SN-L group, HFD+SN-M group, HFD+SN-H group and HFD+Ot group gradually recovered to the level of the Ctrl blank control group. Furthermore, there was no significant difference in the treatment effect among the three groups of HFD+SN-M group, HFD+SN-H group and HFD+Ot group, indicating that the pterocaryon peptide can promote the recovery of reproductive performance in obese mice.

[0049] The epididymis was stained with HE, and the results were as follows: Figure 16 As shown, obese mice had narrow epididymal ducts (C, D) and low sperm count. Supplementation with different concentrations of Sipunculus tetragoni peptides led to re-expansion of the epididymal ducts and a significant increase in sperm density (EJ).

[0050] The testicular index (the ratio of testicular weight to body weight) results are as follows: Figure 12 As shown, the testicular index of the HFD group was at a low level, indicating that the reproductive capacity of obese mice caused by high-fat diet was reduced. However, at the end of the experiment in week 20, the testicular index of the HFD+SN-L group, HFD+SN-M group, HFD+SN-H group and HFD+Ot group gradually recovered to the level of the Ctrl blank control group. Moreover, there was no significant difference in the treatment effect among the three groups of HFD+SN-M group, HFD+SN-H group and HFD+Ot group, indicating that the pterosa peptide can promote the recovery of reproductive performance in obese mice.

[0051] HE staining of the testes yielded the following results: Figure 17 As shown, the seminiferous epithelium of the testes of obese mice is loose and even sloughed off, and the spermatogenic cells are arranged in a disordered manner (C, D); supplementation with different concentrations of Sipunculus nudus peptide can effectively improve this phenomenon (EJ).

[0052] The fat was stained with HE, and the results are as follows: Figure 18 As shown, compared with the Ctrl group (A), the HFD group (B) exhibited enlarged adipocytes with varying cell morphologies. Supplementation with *Sipunculus nudus* peptide significantly improved lipid metabolism abnormalities and lipid deposition in obese mice (C, D, E). The positive control Ot group showed a similar trend (F). Figure 18C is the HFD+SN-L group, D is the HFD+SN-M group, and E is the HFD+SN-H group.

[0053] Sperm motility results as follows Figure 13 As shown, the sperm motility of the HFD group was at a low level, indicating that the reproductive capacity of obese mice caused by the high-fat diet was reduced. However, at the end of the experiment in week 20, the sperm motility of the HFD+SN-L group, HFD+SN-M group, HFD+SN-H group and HFD+Ot group gradually recovered to the level of the Ctrl blank control group. Furthermore, there was no significant difference in the treatment effect among the three groups of HFD+SN-M group, HFD+SN-H group and HFD+Ot group, indicating that the pterosa peptide can promote the recovery of reproductive performance in obese mice.

[0054] Sperm deformity rate results as follows Figure 14 As shown, the sperm abnormality rate in the HFD group was about 43%, indicating that the sperm abnormality rate in obese mice caused by a high-fat diet was increased. However, at the end of the experiment in week 20, the sperm abnormality rates in the HFD+SN-L, HFD+SN-M, HFD+SN-H, and HFD+Ot groups gradually returned to the level of the Ctrl blank control group. Furthermore, there was no significant difference in treatment effect among the three groups of HFD+SN-M, HFD+SN-H, and HFD+Ot, indicating that the pteris vittata peptide can promote the recovery of normal sperm morphology in obese mice.

[0055] Sperm count results as follows Figure 15 As shown, compared to the Ctrl blank control group, the sperm count per male mouse was 2.3 × 10⁻⁶. 6 In the HFD model group, the sperm count per male mouse was 1.5 × 10⁶. 6 In the HFD+SN-L, HFD+SN-M, HFD+SN-H, and HFD+Ot groups, sperm count gradually recovered to the level of the Ctrl blank control group, indicating that stellaria peptide can promote the recovery of sperm count in obese mice.

[0056] The activity data of the square-patterned sipunculus peptides prepared in Examples 2 and 3 were not significantly different from those in Example 1, and therefore the activity data of the square-patterned sipunculus peptides prepared in Examples 2 and 3 will not be supplemented here.

[0057] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for extracting peptides from *Sipunculus nudus*, characterized in that: Includes the following steps: 1) After crushing the naked checkered chafer raw material, add water, sodium bisulfite and ascorbic acid, stir and soak for 10-15 minutes; 2) Grind the soaked raw materials, adjust the pH of the slurry to alkaline, and keep the temperature at 10-50℃; 3) Add alkaline enzyme to the slurry and stir and microwave for 45-50 min at a power of 2-10 kW. Add papain B and continue stirring and microwave for 45-50 min. Heat to inactivate, cool to 45-50℃, centrifuge and filter, and collect the filtrate. 4) The obtained filtrate was subjected to microfiltration, two ultrafiltrations, and sodium filtration in sequence to obtain the square star insect peptide.

2. The extraction method according to claim 1, characterized in that: In step 1), the naked Sipunculus nudus raw material is crushed to 50-100 mesh, and the amount of water added is 10-40 times the weight of the naked Sipunculus nudus raw material, and the temperature of the water is 25-30℃.

3. The extraction method according to claim 1, characterized in that: In step 1), the amount of sodium bisulfite added is 3-4‰ of the mass of the naked Sipunculus nudus raw material; the amount of ascorbic acid added is 0.5-1‰ of the mass of the naked Sipunculus nudus raw material.

4. The extraction method according to claim 1, characterized in that: In step 3), the temperature for microwave enzymatic hydrolysis with stirring is 50-55℃; the temperature for continued microwave enzymatic hydrolysis with stirring is 55-60℃; and the temperature for inactivation by heating is 85-90℃.

5. The extraction method according to claim 1, characterized in that: In step 3), the alkaline enzyme is Carsberg protease or Novo protease, and the amount added is 1%-3% of the weight of the naked Sipunculus nudus raw material; the amount added is 2%-4% of the weight of the naked Sipunculus nudus raw material.

6. The extraction method according to claim 1, characterized in that: In step 4), the microfiltration is performed using a 30,000 Da membrane capable of intercepting molecules up to 30,000 Da; the two ultrafiltration operations are as follows: the first ultrafiltration uses a 10,000 Da membrane and the second ultrafiltration uses a 5,000 Da membrane; the sodium filtration is performed using a 300 Da membrane.

7. A type of Sipunculus peptide extracted by the extraction method according to any one of claims 1-6.

8. A kind of Sipunculus nipponense peptide powder, characterized in that: The square-shaped sipunculus peptide extracted by the extraction method according to any one of claims 1-6 is obtained by freeze-drying.

9. The use of the square-patterned sipunculus peptide extracted by the extraction method according to any one of claims 1-6 or the square-patterned sipunculus peptide powder according to claim 8 in the preparation of iron chelating agents or in the preparation of drugs for treating low TM3 cell viability and low TM4 cell viability caused by palmitic acid.

10. The use of the square-patterned cinnabar peptide extracted by the extraction method according to any one of claims 1-6 or the square-patterned cinnabar peptide powder according to claim 8 in the preparation of a drug for treating hyperglycemia, decreased sperm count, sperm abnormalities, low sperm motility, low testicular index, and low epididymal index caused by obesity.

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