Modified sea cucumber peptide, preparation method thereof and use thereof in improving sexual function

Abstract

The present invention relates to the technical field of sea cucumber peptides, and more specifically to modified sea cucumber peptides, their preparation methods, and uses for improving sexual function. The method comprises adding a synthesized sea cucumber peptide to a hydrochloric acid solution and heat-treating it, adjusting the pH to neutral, dialyzing it, and freeze-drying it to obtain a primary modified sample; preparing a primary modified sample liquid, adding sodium tripolyphosphate to the primary modified sample liquid, reacting it with magnetic stirring, dialyzing it, and freeze-drying it to obtain a modified sea cucumber peptide. The modified sea cucumber peptide has significantly improved foaming ability and foaming stability, and is mixed with yam powder to form an emulsion with significantly improved foaming ability and foaming stability. These modified sea cucumber peptides and emulsions have the significant effect of improving the sexual function of male mice.

Description

Technical Field

[0001] The present invention relates to the technical field of sea cucumber peptides, and in particular to a modified sea cucumber peptide, a preparation method thereof, and an application thereof in improving sexual function. Background Art

[0002] Sea cucumber peptides are made from fresh sea cucumbers. After protease hydrolysis and other processes, they are separated and purified to obtain small molecule peptides composed of 2 to 12 amino acids or polypeptides with larger molecular weights. Components with a molecular weight of less than 2000U account for more than 90%. The extracted sea cucumber peptides are active peptides with the advantages of good solubility, stability, and easy absorption and digestion. Yu Yihao et al. (Study on the anti-fatigue effect and mechanism of sea cucumber peptides, a master's thesis of Jiangnan University in 2021) selected and synthesized sea cucumber peptide G (HyP) LQADY based on the results of 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, reduced the phosphorylation level of NF-κB, and increased the phosphorylation level of STAT3. Zuo Aihua et al. (High-throughput HPLC-MS / MS analysis and identification of sea cucumber oligopeptides and activity screening [J]. Food Industry Science and Technology, 2020, 41(03):12-17)) used high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) to analyze and identify the peptides and source proteins in sea cucumber oligopeptides. The results showed that 88 small molecule peptides were identified in sea cucumber oligopeptides, including a large number of unreported small molecule active peptides, derived from 19 sea cucumber proteins, mainly collagen, α-Ⅰ type collagen, glycine-rich collagen, type II collagen fibers and extracellular matrix protein 3. After online database activity screening, a total of 9 potential antihypertensive active peptides were obtained. Further structure-activity relationship analysis revealed that the core peptide RPQYPQYPS has potential antihypertensive activity and is expected to be developed as an antihypertensive peptide. The discovery of small molecule sea cucumber peptides and their active functions through existing technologies provides a basis for the further development of these sea cucumber peptides. Summary of the Invention

[0003] In view of this, the present invention provides a modified sea cucumber peptide and its preparation method and use for improving sexual function

[0004] One of the purposes of the present invention is to provide a method for preparing a modified sea cucumber peptide, comprising the steps of:

[0005] (1) adding the synthesized sea cucumber peptide to a hydrochloric acid solution to a final concentration of 3 wt % and stirring;

[0006] (2) heat treating the reaction solution obtained in step (1) for 10 minutes;

[0007] (3) adjusting the pH value of the solution obtained in step (2) to neutral, dialyzing, and freeze-drying to obtain an initial modified sample;

[0008] (4) dissolving the initial modified sample in an ammonia-ammonium chloride buffer solution at pH 8.0 to prepare an initial modified sample solution, adding sodium tripolyphosphate to the initial modified sample solution, and performing a magnetic stirring reaction;

[0009] (5) The reaction solution obtained in step (4) is dialyzed and freeze-dried to obtain a modified sea cucumber peptide.

[0010] In step (1) of the preparation method, the concentration of the hydrochloric acid solution is 0.05 to 0.15 mol / L, the final concentration of the sea cucumber peptide is 2.5 to 3.5 wt%, and the stirring treatment time is 1 to 3 hours. 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 treatment time is 2 hours.

[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 10 minutes.

[0012] In step (2) of the preparation method, a wet heat treatment is performed at a temperature of 105-120°C. Preferably, the wet heat treatment is performed in a sealed chamber (eg, a wet heat sterilizer or a pressure cooker). Preferably, the wet 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 is 4-8 wt %, and the reaction time is 1-2 h. Preferably, the final concentration of sodium tripolyphosphate is 6 wt %, and the reaction time is 1.5 h.

[0015] In steps (3) and (5) of the preparation method, an SP131060 cellulose dialysis bag is used for dialysis.

[0016] One of the objectives of the present invention is to provide a modified sea cucumber peptide obtained by the preparation method.

[0017] One of the purposes of the present invention is to provide a method for preparing an emulsion, comprising the steps of: mixing the modified sea cucumber peptide and yam polysaccharide obtained by the preparation method in equal weights, dissolving the mixture in distilled water, adjusting the pH to 7.0 with a PBS solution having a pH of 7.4, stirring the mixture at a constant speed, and homogenizing the mixture at 28,000 r / min for 3 minutes.

[0018] One of the objectives of the present invention is to provide the use of the modified sea cucumber peptide prepared by the preparation method or the emulsion prepared by the preparation method in the preparation of products for improving sexual function.

[0019] Beneficial effects:

[0020] The present invention obtains two modified sea cucumber peptides with enhanced solubility by subjecting two special sea cucumber peptides to a special modification process. Furthermore, it was found that these two modified sea cucumber peptides significantly improve the emulsification ability and emulsion stability of the sea cucumber peptides. Compared with unmodified sea cucumber peptides, the modified sea cucumber peptides exhibit significantly enhanced foaming ability and foaming stability. The present invention also combines the two modified sea cucumber peptides with yam powder to produce an emulsion with significantly enhanced foaming ability and foaming stability.

[0021] In addition, through mouse experiments on these modified sea cucumber peptides and the emulsions prepared therefrom, it was found that the modified sea cucumber peptides and emulsions prepared by the present invention can increase the serum testosterone and NO levels and the corpus cavernosum cGMP levels of male mice, reduce the corpus cavernosum PDE5 levels, and shorten the mice's mounting latency, indicating that these modified sea cucumber peptides and emulsions have a significant effect on improving the sexual function of male mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 These are the solubility results of sea cucumber peptides (G(HyP)LQADY, RPQYPQYPS) and modified sea cucumber peptides (Group 1, Group 2, Group 3, Group 4) at different pH values.

[0023] Figure 2 These are the emulsification ability results of sea cucumber peptides (G(HyP)LQADY, RPQYPQYPS) and modified sea cucumber peptides (Group 1 (Group1), Group 2 (Group2), Group 3 (Group3), Group 4 (Group4)) at different pH values.

[0024] Figure 3 These are the emulsification stability results of sea cucumber peptides (G(HyP)LQADY, RPQYPQYPS) and modified sea cucumber peptides (Group 1, Group 2, Group 3, Group 4) at different pH values.

[0025] Figure 4 These are the foaming results of sea cucumber peptides (G(HyP)LQADY, RPQYPQYPS) and modified sea cucumber peptides (Group 1, Group 2, Group 3, and Group 4).

[0026] Figure 5These are the foaming stability results of sea cucumber peptides (G(HyP)LQADY, RPQYPQYPS) and modified sea cucumber peptides (Group 1, Group 2, Group 3, and Group 4).

[0027] Figure 6 The results of serum testosterone levels in different groups of mice are shown in the figure. In the figure, "ns" indicates no statistical difference; "*" indicates p < 0.05, indicating statistical difference; "**" indicates p < 0.001, indicating statistical difference.

[0028] Figure 7 The results of serum NO levels in different groups of mice are shown in the figure. In the figure, "ns" indicates no statistical difference; "*" indicates p < 0.05, which indicates a statistical difference; "**" indicates p < 0.001, which indicates a statistical difference.

[0029] Figure 8 The results of cGMP content in the corpus cavernosum of mice in different groups are shown in the figure. In the figure, "ns" indicates no statistical difference; "*" indicates p < 0.05, indicating statistical difference.

[0030] Figure 9 The results of PDE5 content in the corpus cavernosum of mice in different groups are shown in the figure. In the figure, "ns" indicates no statistical difference; "*" indicates p<0.05, indicating statistical difference.

[0031] Figure 10 Figure 2 shows the results of the mounting latency in different groups of male mice during the mating experiment. In the figure, "ns" indicates no statistical difference; "*" indicates a statistically significant difference at p < 0.05; and "**" indicates a statistically significant difference at p < 0.001. DETAILED DESCRIPTION

[0032] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to the following examples. It should be understood that the specific examples described herein are merely illustrative of the present invention and are not intended to limit the present invention. Reagents not described in detail herein are all conventional reagents and are commercially available; methods not specifically described in detail are all conventional experimental methods and are known in the art.

[0033] Example 1: Modification of sea cucumber peptide

[0034] 1. Preparation of modified sea cucumber peptide

[0035] Group 1: Chemically synthesized sea cucumber peptide G (HyP) LQADY was added to a 0.1 mol / L hydrochloric acid solution to a final concentration of 3 wt %. After stirring for 2 h, the reaction solution was heat-treated at 110°C for 10 min (autoclave), the pH was adjusted to neutral with 0.1 mol / L NaOH solution, and dialyzed for 24 h at 4°C using an SP131060 cellulose dialysis bag (100-500, 31 mm, 3.1 ML / CM), and freeze-dried to obtain a primary modified sample. The primary modified sample was prepared with a pH 8.0 ammonia-ammonium chloride buffer to a 0.02 g / mL primary modified sample solution, sodium tripolyphosphate was added to a final concentration of 6 wt %, magnetic stirring was applied, and the reaction was allowed to proceed for 1.5 h. The sample was dialyzed for 24 h using an SP131060 cellulose dialysis bag, and freeze-dried to obtain a modified sea cucumber peptide.

[0036] Group 2: The same modification steps were applied to the sea cucumber peptide RPQYPQYPS.

[0037] Group 3: Chemically synthesized sea cucumber peptide RPQYPQYPS was added to a 0.1 mol / L hydrochloric acid solution to a final concentration of 3 wt%. After stirring for 2 h, the reaction solution was wet-heat treated at 110°C for 10 min, the pH was adjusted to neutral with 0.1 mol / L NaOH solution, and dialyzed at 4°C using an SP131060 cellulose dialysis bag (100-500, 31 mm, 3.1 ML / CM) for 24 h. The sample was freeze-dried to obtain a modified sample.

[0038] Group 4: The sea cucumber peptide RPQYPQYPS was prepared into a 0.02 g / mL primary modified sample solution using ammonia-ammonium chloride buffer solution at pH 8.0, and sodium tripolyphosphate was added with a final concentration of 6 wt%. The solution was stirred magnetically and reacted for 1.5 h. The sample was dialyzed using an SP131060 cellulose dialysis bag for 24 h and freeze-dried to obtain the modified sea cucumber peptide.

[0039] 2. Determination of free ammonia concentration by deamidation with hydrochloric acid method

[0040] 50 mg of the initially modified sample was weighed and placed in a hydrolysis tube. Then, 5 mL of sulfuric acid (final concentration: 3 mol / L) was added. The tube was sealed with an alcohol burner and hydrolyzed at 110°C for 24 h. After hydrolysis, the hydrolyzate was neutralized and diluted to a certain concentration. The absorbance of the diluted hydrolyzate was measured at 625 nm. The total amount of glutamine residues in the sample was calculated based on the standard curve. The total amide content was determined according to Wagner and Gueguen (1995). Deamidation (%) = free ammonia content in the sample / total amide content in the sample × 100%. The deamidation degrees of the modified sea cucumber peptides obtained in Groups 1, 2, and 3 were 58.63%, 56.49%, 57.82%, and 0.65%, respectively, indicating that the sea cucumber peptides in Groups 1, 2, and 3 were all successfully deamidated.

[0041] 3. Phosphorylation determination of modified sea cucumber peptides

[0042] The degree of protein phosphorylation was determined using the molybdenum blue colorimetric method. The specific steps are as follows:

[0043] Potassium dihydrogen phosphate is used as the standard, and placed in a 25mL colorimetric tube. 2.0mL of 5% ammonium molybdate solution is added in sequence, shaken, and after standing for a few seconds, 1.0mL of 2% sodium sulfite solution and 1.0mL of 0.5% hydroquinone solution are added respectively, shaken, water is added to the scale, and after standing for 30 minutes, zeroed with a reference, and the absorbance is measured at a wavelength of 660nm. The mass concentration of the phosphate standard solution (in terms of P) is used as the abscissa and the corresponding absorbance is used as the ordinate to draw a standard curve, and the standard curve equation is obtained. Take 20mg of modified sea cucumber peptide in a beaker, add 1mL of concentrated sulfuric acid and concentrated nitric acid respectively, heat on an electric stove until smoking, then add 1mL of 30% hydrogen peroxide solution after cooling, and then slowly heat. Repeat the above steps until the smoke in the bottle stops and the solution is colorless, transparent or light yellow. After cooling, add 1mL of 6mol / L hydrochloric acid, heat on an electric stove to completely decompose the acid, and transfer to a 50mL volumetric flask to make up the volume. 5 mL was taken and the absorbance was measured according to the standard curve operation method. The phosphate content (measured as P, the same below) was calculated based on the regression curve. The results showed that the phosphate content (measured as P) of the modified sea cucumber peptides obtained in Groups 1, 2, 3, and 4 was 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 freeze-dried powder was dissolved in distilled water to prepare a 0.05 mg / mL solution. The pH was adjusted with hydrochloric acid and sodium hydroxide solutions. The solution was centrifuged at 20,000 g for 15 minutes. The protein content of the supernatant was determined using a Lowry kit. Solubility = protein content in supernatant / total protein content × 100%.

[0047] The results are as follows Figure 1 As shown in the figure, the solubility of the unmodified sea cucumber peptides is low, especially RPQYPQYPS. After the same modification steps, the solubility of the modified sea cucumber peptides obtained in Group 1 and Group 2 increased, indicating that the above modification steps can improve the solubility of sea cucumber peptides.

[0048] 2. Determination of emulsification and emulsion stability

[0049] The emulsification test method is as follows: weigh 1.0g of unmodified sea cucumber peptide or modified sea cucumber peptide freeze-dried powder and dissolve it in 20mL of water. Adjust the pH to different levels with hydrochloric acid and sodium hydroxide solution. Then add 20mL of salad oil. Set the internal cutting homogenizer to 28,000 rpm to mix the oil and water for 1 minute. Centrifuge at 1200 rpm for 5 minutes. The emulsion stability test method is as follows: heat the above samples in an 80℃ water bath for 30 minutes, cool them, and centrifuge them at 1200 rpm for 5 minutes.

[0050] The calculation formulas for emulsification and emulsion stability are as follows:

[0051] Emulsification capacity (%) = height of emulsified layer (mm) / total height of liquid in centrifuge tube (mm)

[0052] Emulsion stability (%) = height of liquid layer that still maintains emulsification state (mm) / original emulsion layer height (mm)

[0053] like Figure 2 、 3 As shown in the figure, it is shown that under acidic and alkaline conditions, the modified sea cucumber peptide can significantly improve the emulsification ability and emulsification stability of sea cucumber peptide. Generally, the emulsification ability is due to the introduction of phosphate groups, which changes the protein conformation, stretching the protein molecules, exposing a large number of hydrophobic groups, and connecting with non-polar oil molecules, while the hydrophilic groups connect with water molecules, reducing the surface tension when the two are in contact, increasing the binding capacity of oil and water, and facilitating the formation of more strong interfacial films, reducing the ability of oil droplets to aggregate.

[0054] 4. Evaluation of foaming properties and foam stability

[0055] The unmodified sea cucumber peptides G (HyP) LQADY and RPQYPQYPS mentioned above and the modified sea cucumber peptides provided in groups 1 to 4 were used.

[0056] Weigh 1.5g of unmodified or modified sea cucumber peptide and dissolve it in 25mL of distilled water. Adjust the pH to 7.0 with pH 7.4 PBS solution and stir at a constant speed for 10 minutes. Continue adding distilled water to 50mL as the test solution. Set the internal homogenizer to 28,000 rpm for 3 minutes to prepare a foaming emulsion. Record the volume of the foam (V0) (mL) that appears on the surface to represent the foaming property. After standing for 30 minutes, record the foam volume (V30) (mL). The foaming property and foam stability are calculated as follows: Foaming property (%) = 100% × V0 / 50; Foam stability (%) = 100% × V30 / V0.

[0057] In addition, in this experiment, the modified sea cucumber peptides provided by Groups 1 to 4 were mixed with an equal weight of yam powder (Henan Jiuding Junjian Biotechnology 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 with a pH of 7.4. The mixture was stirred at a constant speed for 10 minutes, and distilled water was continued to be added to 50 mL as the test solution. The internal cutting homogenizer was adjusted to 28,000 r / min and worked for 3 minutes to prepare a foaming emulsion, which was respectively used as Groups 5 to 8, and their foaming properties and foam stability were also tested.

[0058] Protein foaming property and foam stability are the two most commonly used indicators for evaluating protein foaming properties. Foaming property is a property that occurs at the liquid / air interface and is related to the interfacial tension between the two phases. Disordered proteins are easily concentrated at the liquid / air interface and have sufficient viscosity and strength, which is conducive to the formation of more stable foam. Figure 4 、 5 As shown in the results, compared with the unmodified sea cucumber peptide, the foaming ability of the modified sea cucumber peptide was significantly improved (P<0.05). It can be seen that the modification treatment enhances the disorder of the molecules, so that the liquid / gas interface after dissolving in water has sufficient viscosity and strength, and the modified sea cucumber peptide foam formed is more stable. Although groups 3 and 4 can also be modified, their foaming performance and foaming stability are not as good as those of group 2 (P<0.01), indicating that only the modification method of group 2 can obtain an emulsion with better foaming performance and foaming stability. In addition, there is no significant change in the foaming performance and foaming stability of groups 5 to 8 relative to groups 1 to 4, indicating that the addition of yam powder to the emulsion does not affect its emulsion performance.

[0059] Example 3: Biological performance study

[0060] 1. Materials and Methods

[0061] 1. Experimental Animals

[0062] Healthy clean-grade adult ICR mice, half male and half female, weighing 18-22 g, 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 care laboratory met national clean-grade standards, maintained a temperature range of (25 ± 1)°C, a relative humidity of 50% to 60%, and maintained a 12h / 12h light / dark cycle.

[0063] 2. Group experiment

[0064] Male mice were randomly divided into blank group (Blank), negative group (Neg), sea cucumber peptide 1 group (G(HyP)LQADY), sea cucumber peptide 2 group (RPQYPQYPS), group 1 (Group 1), group 2 (Group 2), group 3 (Group 2), group 4 (Group 2), group 5 (Group 5), group 6 (Group 6), group 7 (Group 7), and group 8 (Group 8) according to body weight. Blank mice were gavage-treated with distilled water. The negative group was gavage-treated with 0.5 g / kg body weight of yam polysaccharide per day. The sea cucumber peptide 1 group was gavage-treated with 0.5 g / kg body weight of sea cucumber peptide G(HyP)LQADY per day. The sea cucumber peptide 2 group was gavage-treated with 0.5 g / kg body weight of sea cucumber peptide RPQYPQYPS per day. Group 1 was gavage-treated with 0.5 g / kg body weight of modified sea cucumber peptide G(HyP)LQADY per day. Group 2 was gavage-treated with 0.5 g / kg body weight of modified sea cucumber peptide RPQYPQYPS per day. Group 3 was gavaged with 0.5 g / kg body weight per day of the modified sea cucumber peptide provided by Group 3. Group 4 was gavaged with 0.5 g / kg body weight per day of the modified sea cucumber peptide provided by Group 4. Group 5 was gavaged with 0.5 g / kg body weight per day of the emulsion provided by Group 5 (an emulsion of modified sea cucumber peptide G (HyP) LQADY and yam polysaccharide, the mixing mass ratio of which was 1:1, and the mixture was prepared into a homogenous emulsion according to the above-mentioned foaming test process). Group 6 was gavaged with 0.5 g / kg body weight per day of the emulsion provided by Group 6 (an emulsion of modified sea cucumber peptide RPQYPQYPS and yam polysaccharide, the mixing mass ratio of which was 1:1, and the mixture was prepared into a homogenous emulsion according to the above-mentioned foaming test process). Group 7 was gavaged with 0.5 g / kg body weight per day of the emulsion provided by Group 7 (an emulsion of modified sea cucumber peptide RPQYPQYPS and yam polysaccharide, the mixing mass ratio of which was 1:1, and the mixture was prepared into a homogenous emulsion according to the above-mentioned foaming test process). Group 8 was gavaged with 0.5 g / kg body weight of the emulsion provided by Group 8 (an emulsion of modified sea cucumber peptide RPQYPQYPS and yam polysaccharide, mixed at a mass ratio of 1:1, and the mixture was prepared into a homogenized emulsion according to the foaming test process described above). Each group of mice was gavaged once daily for 45 consecutive days.

[0065] 3. Mating experiment

[0066] On the 45th day of intervention, male mice in each group underwent mating experiments, and the mice's mounting latency was recorded. Each experiment was conducted 60 minutes after the last drug administration. Female mice were subcutaneously injected with 5 μg of estradiol benzoate 48 hours before the experiment, and 5 μg of progesterone was subcutaneously injected 5 hours before the experiment to induce estrus. During the mating experiment, a male mouse was placed in the mating box (30 cm × 15 cm × 15 cm). After acclimation for 5 minutes, a female mouse was immediately placed in the box. The time from the time the mouse was placed in the cage to the first mounting (i.e., the mounting latency) was recorded. The experiment was conducted in a low-light, quiet environment, and the observation time was from 20:00 to 23:00.

[0067] 4. Determination of biochemical indicators

[0068] Male mice in each group were weighed 30 minutes after the last administration. Blood was collected from the eyeballs and placed in a 4°C refrigerator for 3 hours. The supernatant was then centrifuged at 3500 rpm for 10 minutes, and the NO and testosterone levels were determined. After blood collection, the mice were sacrificed by cervical dislocation. The corpus cavernosum tissue on both sides of the penis was dissected in an ice bath, and the cGMP and PDE5 levels were determined. Serum NO levels were determined according to the instructions for the NO detection kit (Beyotime Biotechnology Research Institute). Serum testosterone levels were determined using the testosterone ELISA kit (No. PT872, Beyotime). Cyclic guanosine monophosphate (cGMP) and phosphodiesterase 5 (PDE5) assays were purchased from Beijing Andy Huatai Technology Co., Ltd.

[0069] 6. Statistical methods

[0070] The experimental data were expressed as mean ± standard deviation (x ± s), and statistical analysis was performed using SPSS 22.0 software.

[0071] 2. Results

[0072] like Figure 6 As shown, compared with the blank group, there was no statistical difference in the serum testosterone content of the negative group, sea cucumber peptide 1 group, sea cucumber peptide 2 group, group 3, and group 4 mice. However, group 1 was significantly higher than the blank group, and group 2 was significantly higher than the blank group, indicating that the modified sea cucumber peptides provided by groups 1 and 2, respectively, can increase the serum testosterone content of mice. However, groups 3 and 4 could not increase the serum testosterone content of mice. In addition, there were statistical differences between group 5 and group 1, group 6 and group 2, and group 8 and group 4, indicating that the combination of modified sea cucumber peptides and yam powder to form an emulsion is beneficial to provide the modified sea cucumber peptide to increase the serum testosterone content of mice.

[0073] like Figure 7As shown, compared with the blank group, there was no statistical difference in the serum NO content of the negative group, sea cucumber peptide group 1, sea cucumber peptide group 2, group 3, and group 4. However, group 1 was significantly higher than the blank group, and group 2 was significantly higher than the blank group, indicating that the modified sea cucumber peptides provided by groups 1 and 2, respectively, can increase the serum NO content of mice. However, groups 3 and 4 could not increase the serum NO content of mice. In addition, there were statistical differences between group 6 and group 2, group 7 and group 3, and group 8 and group 4, indicating that the combination of modified sea cucumber peptide and yam powder to make an emulsion is beneficial to provide the modified sea cucumber peptide to increase the NO content in the serum of mice.

[0074] like Figure 8 As shown, compared with the blank group, there was no statistical difference in the cGMP content of the mouse corpus cavernosum in the negative group, sea cucumber peptide 1 group, sea cucumber peptide 2 group, group 3, and group 4. Group 1 was significantly higher than the blank group, and group 2 was significantly higher than the blank group, indicating that the modified sea cucumber peptides provided by groups 1 and 2, respectively, can increase the cGMP content of the mouse corpus cavernosum. Groups 3 and 4 were unable to increase the cGMP content of the mouse corpus cavernosum. In addition, there were statistical differences between group 6 and group 2, group 7 and group 3, and group 8 and group 4, indicating that the combination of modified sea cucumber peptide and yam powder to make an emulsion is beneficial to providing the modified sea cucumber peptide to enhance the cGMP content of the mouse corpus cavernosum.

[0075] like Figure 9 As shown, compared with the blank group, there was no statistical difference in the PDE5 content of the mouse cavernous body in the negative group, sea cucumber peptide group 1, sea cucumber peptide group 2, group 3, and group 4. However, group 1 was significantly higher than the blank group, and group 2 was significantly higher than the blank group, indicating that the modified sea cucumber peptides provided by groups 1 and 2 can reduce the PDE5 content of the mouse cavernous body, while groups 3 and 4 cannot reduce the PDE5 content of the mouse cavernous body.

[0076] like Figure 10 As shown in the figure, compared with the blank group, there was no statistical difference in the mounting latency in the male mice mating experiment among the negative group, sea cucumber peptide 1 group, sea cucumber peptide 2 group, group 3, and group 4. However, group 1 was significantly higher than the blank group, and group 2 was significantly higher than the blank group, indicating that the modified sea cucumber peptides provided by groups 1 and 2, respectively, can reduce the mounting latency in the male mice mating experiment, while groups 3 and 4 cannot reduce the mounting latency in the male mice mating experiment.

[0077] This study showed that intervention with modified sea cucumber peptides and their combination with yam powder in an emulsion increased serum testosterone and NO levels and corpus cavernosum cGMP levels in male mice, decreased corpus cavernosum PDE5 levels, and shortened the mice's mounting latency. Unmodified sea cucumber peptides did not exhibit these biological activities.

[0078] The strength of male sexual function is the result of the combined effects of many factors inside and outside the body, such as testosterone secreted by interstitial cells of the testicles. Testosterone promotes the expression of nitric oxide synthase by regulating the expression of sexual function-related genes, thereby catalyzing the synthesis of NO from L-arginine. For example, NO inhibits the production of PDE5 and promotes the synthesis of guanylate cyclase to reduce the hydrolysis of cGMP in the body, allowing the penile arterioles and corpus cavernosum smooth muscle to relax, and blood is rapidly pumped into the cavernous sinus to cause erection. The above is a relatively well-studied pathway in the process of erection, and the determination of its key factor content can indirectly reflect the ability to have an erection. It can be seen that the modified sea cucumber peptides and emulsions prepared by the present invention can increase the serum testosterone and NO content and the corpus cavernosum cGMP level of male mice, reduce the corpus cavernosum PDE5 level, and shorten the mouse mounting latency, 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 specific 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 thought of by any technician familiar with this technical field within the technical scope disclosed by the present invention should be covered by the scope of protection of the present invention.

Claims

1. A method for modifying sea cucumber peptide RPQYPQYPS, characterized in that: Including steps: (1) Add the synthesized sea cucumber peptide RPQYPQYPS to a 0.1 mol / L hydrochloric acid solution to a final concentration of 3 wt % and stir for 2 h; (2) wet heat treatment of the reaction solution obtained in step (1) at 110°C for 10 min; (3) Adjust the pH value of the solution obtained in step (2) to neutral, dialyze using SP131060 cellulose dialysis bag at 4°C for 24 hours, and freeze-dry to obtain the initial modified sample; (4) Dissolving the initial modified sample in ammonia-ammonium chloride buffer at pH 8.0 to prepare a 0.02 g / mL initial modified sample solution, adding sodium tripolyphosphate with a final concentration of 6 wt% to the initial modified sample solution, and reacting under magnetic stirring for 1.5 h; (5) The reaction solution obtained in step (4) is dialyzed and freeze-dried to obtain modified sea cucumber peptide.

2. The modification method according to claim 1, characterized in that In step (3), the pH was adjusted to neutral using 0.1 mol / L NaOH solution.

3. A method for preparing an emulsion, characterized in that: Including steps: The modified sea cucumber peptide prepared by the modification method according to any one of claims 1 to 2 is mixed with yam polysaccharide by weight, dissolved in distilled water, adjusted to pH 7.0 with PBS solution of pH 7.4, stirred at a constant speed of 28000 r / min for 3 minutes, and homogenized.

4. Use of the modified sea cucumber peptide prepared by the method according to any one of claims 1 to 2 or the emulsion prepared by the preparation method according to claim 3, the use comprising: preparing a preparation for increasing serum testosterone and NO levels in male mice; Or a product for increasing the cGMP level in the corpus cavernosum of male mice is prepared.

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