Sea cucumber peptide, preparation method and application of sea cucumber peptide in tonifying Yang and improving male sexual function
By modifying sea cucumber peptides and mixing them with yam polysaccharides to form an emulsion, the problems of insufficient solubility and emulsification ability of sea cucumber peptides were solved, significantly improving their biological activity, especially enhancing the sexual function of male mice.
Patent Information
- Application Number
- CN202511055357.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-12-12
- Publication Date
- 2025-10-28
AI Technical Summary
Existing sea cucumber peptides have shortcomings in terms of solubility, emulsification ability and emulsification stability, and their bioactivity is not significant in improving male sexual function.
Sea cucumber peptides were modified through a series of steps including hydrochloric acid treatment, heat treatment, dialysis, and reaction with sodium tripolyphosphate. Subsequently, they were mixed with yam polysaccharide to form an emulsion, which improved their solubility, emulsifying ability, and emulsion stability. Their bioactivity was then verified in mouse experiments.
Modified sea cucumber peptides significantly improved solubility, emulsifying ability and emulsification stability, and significantly improved male sexual function by increasing serum testosterone and NO levels in male mice, reducing PDE5 levels in the corpus cavernosum, and shortening the straddle latency.
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Figure CN120842315A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of sea cucumber peptide technology, specifically to sea cucumber peptides, their preparation methods, and their uses in enhancing male sexual function and improving male sexual function. Background Technology
[0002] Sea cucumber peptides are obtained from fresh sea cucumbers through processes such as enzymatic hydrolysis and separation and purification, yielding small molecule peptides or larger polypeptides composed of 2-12 amino acids. More than 90% of these peptides have a molecular weight below 2000U. The extracted sea cucumber peptides are active peptides with advantages such as good solubility, stability, and easy absorption and digestion. Yu Yihao et al. (Study on the Anti-fatigue Effect and Mechanism of Sea Cucumber Peptides, Master's Thesis, Jiangnan University, 2021) synthesized sea cucumber peptide G(HyP)LQADY based on the functional analysis of 134 sea cucumber peptides. At a concentration of 50 μg / mL, it significantly improved cell viability and antioxidant capacity, significantly reduced the content and expression of IL-6 and TNF-α, decreased the phosphorylation level of NF-κB, and increased the phosphorylation level of STAT3. Zuo Aihua et al. (High-throughput HPLC-MS / MS analysis and activity screening of sea cucumber oligopeptides [J]. Food Industry Technology, 2020, 41(03):12-17)) used high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) to analyze and identify peptides and their source proteins in sea cucumber oligopeptides. The results showed that 88 small molecule peptides were identified in the sea cucumber oligopeptides, containing a large number of previously unreported small molecule active peptides derived from 19 kinds of sea cucumber proteins, mainly collagen, α-I type collagen, glycine-rich collagen, type II collagen fibers, and extracellular matrix protein 3. Nine potential antihypertensive active peptides were obtained through online database activity screening. Further structure-activity relationship analysis revealed that the core peptide RPQYPQYPS has potential antihypertensive activity and is expected to be developed into an antihypertensive peptide. The discovery of small molecule sea cucumber peptides and their active functions through existing technologies provides a foundation for the further development of these sea cucumber peptides. Summary of the Invention
[0003] In view of this, the present invention provides modified sea cucumber peptides, their preparation method, and their use in improving sexual function.
[0004] One objective of this invention is to provide a method for preparing modified sea cucumber peptides, comprising the following steps:
[0005] (1) Add the synthesized sea cucumber peptide to the hydrochloric acid solution. The final concentration of the sea cucumber peptide added is 3wt%. Stir the solution.
[0006] (2) Heat treatment of the reaction solution obtained in step (1) for 10 min;
[0007] (3) Adjust the pH of the solution obtained in step (2) to neutral, dialyze, freeze dry, and obtain the initial modified sample;
[0008] (4) Dissolve the initial modified sample in an ammonia-ammonium chloride buffer solution with pH 8.0 to prepare an initial modified sample solution, add sodium tripolyphosphate to the initial modified sample solution, and stir magnetically to react;
[0009] (5) Dialyze the reaction solution obtained in step (4) and freeze-dry it to obtain modified sea cucumber peptide.
[0010] In step (1) of the preparation method, the concentration of the hydrochloric acid solution is 0.05–0.15 mol / L, the final concentration of the sea cucumber peptide is 2.5–3.5 wt%, and the stirring time is 1–3 h. Preferably, the concentration of the hydrochloric acid solution is 0.1 mol / L, the final concentration of the sea cucumber peptide is 3 wt%, and the stirring time is 2 h.
[0011] In step (2) of the preparation method, the reaction solution obtained in step (1) is subjected to wet heat treatment for 8 to 15 minutes. Preferably, the treatment time is 10 minutes.
[0012] In step (2) of the preparation method, a moist heat treatment at a temperature of 105–120°C is used. Preferably, a closed chamber is used for the moist heat treatment (e.g., a moist heat sterilizer or pressure cooker). Preferably, the moist heat treatment temperature is 110°C.
[0013] In step (3) of the preparation method, the pH is adjusted to neutral using 0.1 mol / L NaOH solution.
[0014] In step (4) of the preparation method, the final concentration of sodium tripolyphosphate added is 4-8 wt%, and the reaction time is 1-2 h. Preferably, the final concentration of sodium tripolyphosphate added is 6 wt%, and the reaction time is 1.5 h.
[0015] In steps (3) and (5) of the preparation method, SP131060 cellulose dialysis bags are used for dialysis.
[0016] One of the objectives of this invention is to provide modified sea cucumber peptides prepared by the aforementioned method.
[0017] One objective of this invention is to provide a method for preparing an emulsion, comprising the following steps: the method prepares modified sea cucumber peptide and yam polysaccharide in equal weight, mixes them, dissolves them in distilled water, adjusts the pH to 7.0 with PBS solution at pH 7.4, stirs at a uniform speed, and homogenizes at 28000 r / min for 3 min.
[0018] One of the objectives of this invention is to provide the use of the modified sea cucumber peptide prepared by the aforementioned preparation method or the emulsion prepared by the aforementioned preparation method in the preparation of products that enhance sexual function.
[0019] Beneficial effects:
[0020] This invention involves a specific modification process on two special sea cucumber peptides to obtain two modified sea cucumber peptides with improved solubility. Furthermore, it was found that these two modified sea cucumber peptides significantly improve the emulsifying and emulsion stabilizing abilities of sea cucumber peptides. Compared to unmodified sea cucumber peptides, the modified sea cucumber peptides exhibit significantly enhanced foaming ability and foaming stability. This invention also combines the two modified sea cucumber peptides with yam powder to create an emulsion with significantly improved foaming ability and foaming stability.
[0021] Furthermore, mouse experiments conducted on these modified sea cucumber peptides and their prepared emulsions revealed that the modified sea cucumber peptides and emulsions prepared in this invention can increase serum testosterone and NO levels and corpus cavernosum cGMP levels in male mice, reduce corpus cavernosum PDE5 levels, and shorten the mounting latency in mice, indicating that these modified sea cucumber peptides and emulsions have a significant effect on improving the sexual function of male mice. Attached Figure Description
[0022] Figure 1 The results show the solubility of sea cucumber peptides (G(HyP)LQADY, RPQYPQYPS) and modified sea cucumber peptides (Group 1, Group 2, Group 3, and Group 4) at different pH values.
[0023] Figure 2 The results show the emulsifying ability of sea cucumber peptides (G(HyP)LQADY, RPQYPQYPS) and modified sea cucumber peptides (Group 1, Group 2, Group 3, and Group 4) at different pH values.
[0024] Figure 3 The results show the emulsification stability of sea cucumber peptides (G(HyP)LQADY, RPQYPQYPS) and modified sea cucumber peptides (Group 1, Group 2, Group 3, and Group 4) at different pH values.
[0025] Figure 4 The foaming results are for sea cucumber peptides (G(HyP)LQADY, RPQYPQYPS) and modified sea cucumber peptides (Group 1, Group 2, Group 3, Group 4).
[0026] Figure 5The foaming stability results are for sea cucumber peptides (G(HyP)LQADY, RPQYPQYPS) and modified sea cucumber peptides (Group 1, Group 2, Group 3, Group 4).
[0027] Figure 6 The graph shows the serum testosterone levels in different groups of mice. In the graph, "ns" indicates no statistical difference; "*" indicates p<0.05, indicating a statistical difference; and "**" indicates p<0.001, indicating a statistical difference.
[0028] Figure 7 The graph shows the serum NO levels in different groups of mice. In the graph, "ns" indicates no statistical difference; "*" indicates p<0.05, indicating a statistical difference; and "**" indicates p<0.001, indicating a statistical difference.
[0029] Figure 8 The graph shows the cGMP content results in the corpus cavernosum of different groups of mice. In the graph, "ns" indicates no statistical difference; "*" indicates p<0.05, which is statistically significant.
[0030] Figure 9 The graph shows the results of PDE5 content in the corpus cavernosum of different groups of mice. In the graph, "ns" indicates no statistical difference; "*" indicates p<0.05, which is statistically significant.
[0031] Figure 10 The figure shows the mounting latency results in mating experiments of different groups of male mice. In the figure, "ns" indicates no statistical difference; "*" indicates p<0.05, indicating a statistical difference; and "**" indicates p<0.001, indicating a statistical difference. Detailed Implementation
[0032] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. Reagents not specifically described in detail in this invention are all conventional reagents and are commercially available; methods not specifically described in detail are all conventional experimental methods and can be obtained from the prior art.
[0033] Example 1: Modification of sea cucumber peptides
[0034] 1. Preparation of modified sea cucumber peptides
[0035] Group 1: Chemically synthesized sea cucumber peptide G(HyP)LQADY was added to a 0.1 mol / L hydrochloric acid solution, with a final concentration of 3 wt%. After stirring for 2 h, the reaction solution was subjected to moist heat treatment at 110℃ for 10 min (sterilization). The pH was adjusted to neutral with 0.1 mol / L NaOH solution, and dialyzed at 4℃ using an SP131060 cellulose dialysis bag (100-500, 31 mm, 3.1 mL / cm²) for 24 h. The solution was then freeze-dried to obtain the initial modified sample. The initial modified sample was prepared into a 0.02 g / mL initial modified sample solution using an ammonia-ammonium chloride buffer solution at pH 8.0. Sodium tripolyphosphate was added to a final concentration of 6 wt%, and the mixture was magnetically stirred for 1.5 h. The sample was then dialyzed using an SP131060 cellulose dialysis bag for 24 h and freeze-dried to obtain the modified sea cucumber peptide.
[0036] Group 2: The same modification steps were used for sea cucumber peptide RPQYPQYPS.
[0037] Group 3: Chemically synthesized sea cucumber peptide RPQYPQYPS was added to a 0.1 mol / L hydrochloric acid solution, with a final concentration of 3 wt%. After stirring for 2 h, the reaction solution was subjected to moist heat treatment at 110 °C for 10 min. The pH was adjusted to neutral with 0.1 mol / L NaOH solution, and dialyzed at 4 °C for 24 h using an SP131060 cellulose dialysis bag (100-500, 31 mm, 3.1 mL / cm). The solution was then freeze-dried to obtain the modified sample.
[0038] Group 4: The sea cucumber peptide RPQYPQYPS was prepared into a 0.02 g / mL initial modified sample solution using an ammonia-ammonium chloride buffer solution at pH 8.0. Sodium tripolyphosphate was added to a final concentration of 6 wt%, and the mixture was magnetically stirred and reacted for 1.5 h. The sample was dialyzed for 24 h using an SP131060 cellulose dialysis bag and then freeze-dried to obtain the modified sea cucumber peptide.
[0039] 2. Hydrochloric acid method for determining the concentration of free ammonia after deamidation
[0040] Weigh 50 mg of the initially modified sample and place it in a hydrolysis tube. Then add 5 mL of sulfuric acid with a final concentration of 3 mol / L. Seal the hydrolysis tube with an alcohol burner and hydrolyze at 110℃ for 24 h. After hydrolysis, neutralize the hydrolysate to neutral and dilute it by a certain factor. Measure the absorbance of the diluted hydrolysate at a wavelength of 625 nm. Calculate the total glutamine residues in the sample according to the standard curve. The method for determining the total amide content is as described by Wagner and Gueguen (1995). Deamidation degree % = Free ammonia content in the sample / Total amide content in the sample × 100%. The results showed that the deamidation degrees of the modified sea cucumber peptides obtained from groups 1, 2, and 3 were 58.63%, 56.49%, 57.82%, and 0.65%, respectively, indicating that the sea cucumber peptides from groups 1, 2, and 3 were successfully deamidated.
[0041] 3. Determination of phosphorylation of modified sea cucumber peptides
[0042] The degree of protein phosphorylation was determined using the molybdenum blue colorimetric method. The specific steps were as follows:
[0043] Using potassium dihydrogen phosphate as a standard, place it in a 25 mL colorimetric tube, add 2.0 mL of 5% ammonium molybdate solution, shake well, let stand for a few seconds, then add 1.0 mL of 2% sodium sulfite solution and 1.0 mL of 0.5% hydroquinone solution, shake well, add water to the mark, let stand for 30 min, zero with reference, and measure absorbance at 660 nm. Plot a standard curve with the mass concentration of phosphate standard solution (calculated as P) on the x-axis and the corresponding absorbance on the y-axis to obtain the standard curve equation. Take 20 mg of modified sea cucumber peptide in a beaker, add 1 mL of concentrated sulfuric acid and concentrated nitric acid respectively, heat on an electric furnace until fumes appear, then cool and add 1 mL of 30% hydrogen peroxide solution, then heat slowly again, repeating the above steps until no more fumes appear in the bottle and the solution is colorless and transparent or pale yellow. After cooling, add 1 mL of 6 mol / L hydrochloric acid, heat on an electric furnace to completely decompose the acid, transfer to a 50 mL volumetric flask and dilute to volume. Take 5 mL of the sample and measure the absorbance according to the standard curve procedure. Calculate the phosphate content (as P) based on the regression curve. The results showed that the phosphate content (as P) of the modified sea cucumber peptides obtained in groups 1, 2, 3, and 4 were 10.52%, 10.36%, 0.05%, and 3.65%, respectively, indicating that the sea cucumber peptides in groups 1, 2, and 4 were all phosphorylated.
[0044] Example 2: Study on the functional properties of sea cucumber peptides and modified sea cucumber peptides
[0045] 1. Solubility determination
[0046] Unmodified or modified sea cucumber peptide lyophilized powder was dissolved in distilled water to prepare a 0.05 mg / mL solution. The pH was adjusted using hydrochloric acid and sodium hydroxide solution. The solution was centrifuged at 20,000 g for 15 min, and the protein content in the supernatant was determined using a Lowry reagent kit. Solubility = (protein content in supernatant / total protein content) × 100%.
[0047] The results are as follows Figure 1 As shown, the unmodified sea cucumber peptides all exhibited low solubility, especially RPQYPQYPS. However, after undergoing the same modification steps, the solubility of the modified sea cucumber peptides obtained in both groups 1 and 2 increased, indicating that the above modification steps can improve the solubility of sea cucumber peptides.
[0048] 2. Determination of emulsifying properties and emulsion stability
[0049] The emulsifying property was determined as follows: 1.0 g of unmodified sea cucumber peptide or lyophilized sea cucumber peptide powder was dissolved in 20 mL of water. The pH was adjusted using hydrochloric acid and sodium hydroxide solution. Then, 20 mL of salad oil was added. The oil and water were mixed for 1 min at 28000 r / min using an internal homogenizer, and then centrifuged for 5 min at 1200 r / min. The emulsifying stability was determined as follows: The above sample was heated in an 80℃ water bath for 30 min, cooled, and then centrifuged for 5 min at 1200 r / min.
[0050] The formulas for calculating emulsifying properties and emulsion stability are as follows:
[0051] Emulsifying capacity (%) = Height of emulsified layer (mm) / Total liquid height in centrifuge tube (mm)
[0052] Emulsion stability (%) = Height of liquid layer still maintaining emulsion state (mm) / Original emulsion layer height (mm)
[0053] like Figure 2 , 3 As shown, modified sea cucumber peptides can significantly improve their emulsifying and emulsion stabilizing abilities under acidic and alkaline conditions. Emulsifying ability is typically due to the introduction of phosphate groups, which alters the protein conformation, causing the protein molecules to stretch and expose numerous hydrophobic groups that connect with nonpolar oil molecules, while the hydrophilic groups connect with water molecules. This reduces the surface tension at contact between the two, increasing the oil-water binding capacity, facilitating the formation of more and stronger interfacial films, and reducing oil droplet aggregation.
[0054] 4. Evaluation of foaming properties and foam stability
[0055] The modified sea cucumber peptides provided in groups 1 to 4 were obtained by using the unmodified sea cucumber peptides G(HyP)LQADY and RPQYPQYPS.
[0056] Weigh 1.5g of unmodified or modified sea cucumber peptides and dissolve them in 25mL of distilled water. Adjust the pH to 7.0 with pH 7.4 PBS solution and stir at a constant speed for 10min. Continue to add distilled water to 50mL as the test solution. Set the internal homogenizer to 28000r / min and run it for 3min to prepare a foaming emulsion. Record the volume of foam V0 (mL) that appears on the top, which represents the foaming property. After standing for 30min, record the foam volume V30 (mL) again. The formulas for calculating foaming property and foam stability are as follows: Foaming property (%) = 100% × V0 / 50; Foam stability (%) = 100% × V30 / V0.
[0057] In addition, the modified sea cucumber peptides provided in groups 1-4 were mixed with an equal weight of yam powder (Henan Jiuding Junjian Biomedical Technology Co., Ltd., white solid powder, the main component of which is yam polysaccharide (content is 50%)), dissolved in 25 mL of distilled water, and the pH was adjusted to 7.0 with 0.1 M PBS solution at pH 7.4. The mixture was stirred at a constant speed for 10 min, and then distilled water was added to 50 mL as the test solution. The internal homogenizer was set to 28000 r / min and worked for 3 min to prepare foaming emulsions, which were used as groups 5-8. Their foaming properties and foam stability were tested in the same way.
[0058] Foaming ability and foam stability are two of the most commonly used indicators for evaluating the foaming properties of proteins. Foaming property is a property that occurs at the liquid / gas interface and is related to the interfacial tension between the two phases. Disordered proteins tend to concentrate at the liquid / gas interface, possessing sufficient viscosity and strength, which is conducive to the formation of more stable foams. Figure 4 , 5 As shown, compared with unmodified sea cucumber peptides, modified sea cucumber peptides exhibited significantly improved foaming ability (P<0.05). This indicates that the modification treatment enhances the disorder of molecules, resulting in sufficient viscosity and strength at the liquid / gas interface after dissolving in water, leading to more stable modified sea cucumber peptide foam. While groups 3 and 4 can also be modified, their foaming performance and foaming stability are inferior to group 2 (P<0.01), indicating that only the modification method in group 2 can yield emulsions with better foaming performance and foaming stability. Furthermore, groups 5–8 showed no significant changes in foaming performance and foaming stability compared to groups 1–4, suggesting that adding yam powder to the emulsion does not affect its performance.
[0059] Example 3: Biological Performance Study
[0060] I. Materials and Methods
[0061] 1. Laboratory animals
[0062] Healthy, clean-grade adult ICR mice, half male and half female, weighing 18–22g, were provided by Jinan Pengyue Laboratory Animal Breeding Co., Ltd. The animals were housed in separate cages with free access to food and water. The animal housing laboratory met national clean-grade standards, with a temperature range of (25±1)℃, relative humidity of 50%–60%, and indoor lighting controlled according to a 12h / 12h light-dark cycle.
[0063] 2. Group Experiment
[0064] Male mice were randomly divided into four groups according to body weight: Blank, Negative, Sea Cucumber Peptide 1 (G(HyP)LQADY), Sea Cucumber Peptide 2 (RPQYPQYPS), Group 1, Group 2, Group 3, Group 4, Group 5, Group 6, Group 7, and Group 8. Blank mice were administered distilled water by gavage. Negative mice were administered 0.5 g / kg body weight of yam polysaccharide daily by gavage. Sea Cucumber Peptide 1 was administered 0.5 g / kg body weight of sea cucumber peptide G(HyP)LQADY daily by gavage. Sea Cucumber Peptide 2 was administered 0.5 g / kg body weight of sea cucumber peptide RPQYPQYPS daily by gavage. Group 1 was administered 0.5 g / kg body weight of modified sea cucumber peptide G(HyP)LQADY daily by gavage. Group 2 was administered 0.5 g / kg body weight of modified sea cucumber peptide RPQYPQYPS daily by gavage. Group 3: Administered via gavage 0.5 g / kg body weight daily of the modified sea cucumber peptide provided in Group 3. Group 4: Administered via gavage 0.5 g / kg body weight daily of the modified sea cucumber peptide provided in Group 4. Group 5: Administered via gavage 0.5 g / kg body weight daily of the emulsion provided in Group 5 (an emulsion of modified sea cucumber peptide G(HyP)LQADY and yam polysaccharide, mixed at a mass ratio of 1:1, the mixture prepared into a homogenized emulsion according to the foaming test procedure described above). Group 6: Administered via gavage 0.5 g / kg body weight daily of the emulsion provided in Group 6 (an emulsion of modified sea cucumber peptide RPQYPQYPS and yam polysaccharide, mixed at a mass ratio of 1:1, the mixture prepared into a homogenized emulsion according to the foaming test procedure described above). Group 7: Administered via gavage 0.5 g / kg body weight daily of the emulsion provided in Group 7 (an emulsion of modified sea cucumber peptide RPQYPQYPS and yam polysaccharide, mixed at a mass ratio of 1:1, the mixture prepared into a homogenized emulsion according to the foaming test procedure described above). Group 8 mice were administered the emulsion provided in Group 8 (a mixture of modified sea cucumber peptide RPQYPQYPS and yam polysaccharide in a 1:1 mass ratio, prepared as a homogenate according to the foaming test procedure described above) by gavage at a rate of 0.5 g / kg body weight daily. Each group of mice was gavage once daily for 45 consecutive days.
[0065] 3. Mating experiment
[0066] Male mice from each group were subjected to mating experiments on day 45 of the intervention, and the mounting latency was recorded. Each experiment was conducted 60 minutes after the last administration. Female mice were subcutaneously injected with 5 μg of estradiol benzoate 48 hours before the experiment, and subcutaneously injected with 5 μg of progesterone 5 hours before the experiment to induce estrus. During the mating experiment, one male mouse was placed in the mating box (30cm×15cm×15cm), and after 5 minutes of acclimatization, one female mouse was immediately placed in. The time from the time the mice were introduced into the box to the first mounting (i.e., the mounting latency) was recorded. The experiment was conducted in a low-light, quiet environment, and the observation period was from 20:00 to 23:00.
[0067] 4. Biochemical index determination
[0068] Thirty minutes after the last administration, the body weight of male mice in each group was measured. Blood was collected by removing the eyeballs and placed in a 4°C refrigerator for 3 hours, then centrifuged at 3500 rpm for 10 minutes. The supernatant was collected to determine the NO and testosterone levels. After blood collection, the mice were euthanized by cervical dislocation. The corpus cavernosum tissue from both sides of the penis was dissected in an ice bath, and the cGMP and PDE5 levels were measured. Serum NO levels were determined according to the NO detection kit instructions (Beyotime Biotechnology Research Institute). Serum testosterone content determination testosterone ELISA kit instructions (No.: PT872, Beyotime). Cycloguanosine monophosphate (cGMP) kit and phosphodiesterase 5 (PDE5) kit were purchased from Beijing Andy Huatai Technology Co., Ltd.
[0069] 6. Statistical methods
[0070] Experimental data are expressed as mean ± standard deviation (x ± s), and statistical analysis was performed using SPSS 22.0 software.
[0071] II. Results
[0072] like Figure 6 As shown, compared with the control group, there were no statistically significant differences in serum testosterone levels in the negative control group, sea cucumber peptide 1 group, sea cucumber peptide 2 group, group 3, and group 4. Group 1 and group 2 were significantly higher than the control group, indicating that the modified sea cucumber peptides provided by groups 1 and 2 could increase serum testosterone levels in mice. Groups 3 and 4, however, did not increase serum testosterone levels in mice. Furthermore, group 5 showed statistically significant differences compared to group 1, group 6 compared to group 2, and group 8 compared to group 4, suggesting that combining modified sea cucumber peptides with yam powder to form an emulsion is beneficial in enhancing the effect of modified sea cucumber peptides on increasing serum testosterone levels in mice.
[0073] like Figure 7As shown, compared with the control group, there were no statistically significant differences in serum NO levels in the negative control group, sea cucumber peptide 1 group, sea cucumber peptide 2 group, group 3, and group 4. Group 1 and group 2 were significantly higher than the control group, indicating that the modified sea cucumber peptides provided by groups 1 and 2 could increase serum NO levels in mice. Groups 3 and 4 did not increase serum NO levels in mice. Furthermore, group 6 showed statistically significant differences compared to group 2, group 7 compared to group 3, and group 8 compared to group 4, indicating that combining modified sea cucumber peptides with yam powder to form an emulsion is beneficial in enhancing the effect of modified sea cucumber peptides on increasing serum NO levels in mice.
[0074] like Figure 8 As shown, compared with the control group, there were no statistically significant differences in cGMP content in the corpora cavernosa of mice in the negative control group, sea cucumber peptide 1 group, sea cucumber peptide 2 group, group 3, and group 4. Group 1 and group 2 were significantly higher than the control group, indicating that the modified sea cucumber peptides provided by groups 1 and 2 could increase the cGMP content in the corpora cavernosa of mice. Groups 3 and 4, however, did not increase the cGMP content in the corpora cavernosa of mice. Furthermore, there were statistically significant differences between group 6 and group 2, group 7 and group 3, and group 8 and group 4, indicating that combining modified sea cucumber peptides with yam powder to form an emulsion is beneficial in enhancing the cGMP content in the corpora cavernosa of mice.
[0075] like Figure 9 As shown, compared with the control group, there were no statistically significant differences in PDE5 content in the corpus cavernosum of mice in the negative control group, sea cucumber peptide 1 group, sea cucumber peptide 2 group, group 3, and group 4. However, group 1 was significantly higher than the control group, and group 2 was significantly higher than the control group, indicating that the modified sea cucumber peptides provided by groups 1 and 2 can reduce the PDE5 content in the corpus cavernosum of mice, while groups 3 and 4 cannot reduce the PDE5 content in the corpus cavernosum of mice.
[0076] like Figure 10 As shown, compared with the control group, there was no statistically significant difference in the mounting latency in the mating experiment of male-dominated mice in the negative control group, sea cucumber peptide 1 group, sea cucumber peptide 2 group, group 3, and group 4. However, group 1 was significantly higher than the control group, and group 2 was significantly higher than the control group, indicating that the modified sea cucumber peptides provided by groups 1 and 2 can reduce the mounting latency in the mating experiment of male-dominated mice, while groups 3 and 4 cannot reduce the mounting latency in the mating experiment of male-dominated mice.
[0077] This study shows that intervention with modified sea cucumber peptides and their emulsion with yam powder increased serum testosterone and NO levels, decreased corpus cavernosum cGMP levels, and shortened corpus cavernosum PDE5 levels in male mice, while also shortening the straddle latency. Unmodified sea cucumber peptides did not exhibit these biological activities.
[0078] Male sexual function is the result of the combined effects of numerous internal and external factors. These include testosterone secreted by testicular interstitial cells, which regulates the expression of sex-related genes, promotes nitric oxide synthase expression, and catalyzes the synthesis of NO from L-arginine. NO, for example, inhibits PDE5 production and promotes guanylate cyclase synthesis, thus reducing cGMP hydrolysis in the body. This leads to relaxation of penile arterioles and corpus cavernosum smooth muscle, allowing blood to be rapidly pumped into the cavernous sinus, resulting in erection. These are relatively well-understood pathways in the erection process, and the content of key factors can indirectly reflect erectile function. Therefore, the modified sea cucumber peptides and emulsion prepared in this invention can increase serum testosterone and NO levels and corpus cavernosum cGMP levels in male mice, reduce corpus cavernosum PDE5 levels, and shorten the mounting latency in mice, indicating that these modified sea cucumber peptides and emulsions have a significant effect on improving the sexual function of male mice.
[0079] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.
Claims
1. A method for modifying sea cucumber peptides RPQYPQYPS or G(HyP)LQADY, characterized in that, Including the following steps: (1) Add the synthesized sea cucumber peptide RPQYPQYPS or G(HyP)LQADY to a hydrochloric acid solution with a concentration of 0.05~0.15mol / L. The final concentration of the added sea cucumber peptide is 2.5~3.5wt%, and stir for 2h. (2) The reaction solution obtained in step (1) was subjected to wet heat treatment at 110°C for 10 min; (3) Adjust the pH of the solution obtained in step (2) to neutral, dialyze at 4℃ using an SP131060 cellulose dialysis bag for 24h, freeze dry, and obtain the initial modified sample; (4) Dissolve the initial modified sample in an ammonia-ammonium chloride buffer solution at pH 8.0 to prepare an initial modified sample solution of 0.02 g / mL, add sodium tripolyphosphate to the initial modified sample solution to a final concentration of 6wt%, and stir magnetically for 1.5 h. (5) Dialyze the reaction solution obtained in step (4) and freeze-dry it to obtain modified sea cucumber peptide.
2. The modification method according to claim 1, characterized in that, In step (3), the pH is adjusted to neutral using 0.1 mol / L NaOH solution.
3. The modified sea cucumber peptide prepared by the preparation method according to claim 1 or 2.
4. A method for preparing an emulsion, characterized in that, Including the following steps: The modified sea cucumber peptide as described in claim 3 is mixed with yam polysaccharide in equal weight and dissolved in distilled water.
5. Use of the modified sea cucumber peptide according to claim 3 or the emulsion prepared by the method according to claim 4, wherein the use includes: Prepare products that enhance sexual function in male mice.
6. The use according to claim 5, wherein the improvement of male mouse sexual function is achieved by reducing the PDE5 level in the corpus cavernosum and shortening the mounting latency in mice.