Abdominal distention hippocampal peptide and application thereof in preparation of anti-fatigue products

Abstract

The invention relates to the field of preparation and application of biological peptides, in particular to a swelling hippocampal peptide and application of the swelling hippocampal peptide in preparation of anti-fatigue products. Five active peptide sequences with high biological activity are obtained through research, animal behavioral experiments prove that the belly distention hippocampal peptide has a remarkable anti-fatigue effect and can prolong the swimming exhaustion time and enhance the muscle strength, the anti-fatigue effect of the belly distention hippocampal peptide is verified, and a new way is provided for development of anti-fatigue hippocampal products.

Description

Technical Field

[0001] The present invention relates to the field of preparation and application of bioactive peptides, and particularly to a puffy seahorse peptide and its application in the preparation of anti-fatigue products. Background Art

[0002] Exercise-induced fatigue can be roughly divided into central fatigue and peripheral fatigue, which are a common and complex multi-dimensional symptom. Exercise-induced fatigue involves a series of complex physiological changes, such as oxidative damage, energy consumption, mitochondrial dysfunction, and reduced muscle contractility. When severe, it can induce various diseases, including depression, Parkinson's disease, Alzheimer's disease, obesity, and anxiety. Therefore, food intake, as the safest and most effective method for preventing and relieving fatigue, has become a current research hotspot.

[0003] The seahorse is an important high-value marine fish in China's aquaculture economy. There are various bioactive substances in seahorses, and they are known as the "Southern Ginseng". Currently, the research on the 5 highly medicinal seahorses included in the Chinese Pharmacopoeia 2020 Edition is mainly on the three-spot seahorse. However, the three-spot seahorse is difficult to breed and it is difficult to meet the market demand. Chinese Patent CN115057921A discloses an antioxidant and anti-fatigue active collagen peptide of the grey seahorse and its large-scale preparation method. However, there are numerous proteins in seahorses, and with the passage of time and environmental changes, the types and contents of these proteins also continuously change. For example, there are significant differences in the types and contents of proteins in large seahorses and small seahorses, pregnant seahorses and non-pregnant seahorses. This leads to completely different pharmacological activities of seahorses at different times and physiological environments.

[0004] Due to its strong disease resistance, high survival rate, and fast growth rate, the puffy seahorse has become the preferred choice for aquaculture in recent years. However, there is currently little research on the biological activity of the puffy seahorse, especially in terms of anti-fatigue, and the specific functional protein components have not been clarified. Therefore, it remains to be studied whether short peptides of the puffy seahorse can be obtained by enzymatic hydrolysis to obtain proteins with a determined sequence, and further improve the anti-fatigue efficacy of puffy seahorse products. Summary of the Invention

[0005] The technical problem to be solved by the present invention is that there is currently little research on the biological activity of the puffy seahorse, especially in terms of anti-fatigue, and the specific functional protein components have not been clarified, which is a technical and market gap that needs to be filled.

[0006] To solve the above problems, the present invention provides a pygmy seahorse peptide and its application in the preparation of anti-fatigue products. Five active peptide sequences with high biological activity have been obtained through research. Animal behavior experiments have demonstrated that the pygmy seahorse peptide has a significant anti-fatigue effect, can extend the swimming exhaustion time, enhance muscle strength, verify the role of the pygmy seahorse peptide in anti-fatigue, and provide a new approach for the development of seahorse anti-fatigue products.

[0007] To achieve the above object, the present invention is realized by the following technical means: An application of a pygmy seahorse peptide in the preparation of anti-fatigue products; the pygmy seahorse peptide includes polypeptides with the amino acid sequences shown in SEQ ID NO.1 - 5: SEQ ID NO.1: GGPQMPGPM.

[0008] SEQ ID NO.2: DDDIFPM.

[0009] SEQ ID NO.3: ASGPMG.

[0010] SEQ ID NO.4: GGGFDFIS.

[0011] SEQ ID NO.5: DHFLFD.

[0012] The preparation method of the above-mentioned pygmy seahorse peptide includes the following steps: Take pygmy seahorses, dissolve them with Tris-HCl buffer containing 0.1 mM EDTA, add 5% papain, vortex for 30 s, then place in a 37 °C water bath and stir magnetically; after the reaction is completed, immediately add 10% TCA to a final concentration of 5% v / v, mix well, ice bath, and then centrifuge. The supernatant collected is the crude peptide sample; Load the supernatant into a dialysis bag with a molecular weight cut-off of 3 kDa and dialyze with ultrapure water for 4 hours to remove small molecule impurities; freeze-dry the dialyzed peptide solution to obtain a white powdery peptide sample and store it at -20 °C to obtain the said pygmy seahorse peptide.

[0013] Further, add buffer according to a material-liquid ratio of 1:5.

[0014] Further, the parameters of magnetic stirring are 150 rpm and stir for 4 hours.

[0015] Further, the ice bath time is 10 minutes. To inactivate the enzyme and precipitate undigested proteins.

[0016] Furthermore, the centrifugation parameters are centrifugation at 12,000 rpm for 10 minutes at 4°C. The higher rotation speed enables the peptide components to be released into the extraction solution faster, shortening the extraction time. Meanwhile, it is suitable for large-scale production: the high-efficiency extraction process can increase the raw material processing capacity per unit time, is suitable for industrial production, and reduces costs.

[0017] The above amino acid sequence is the Hippocampus abdominalis peptide shown in SEQ ID NO.1-5.

[0018] Furthermore, the Hippocampus abdominalis peptide with the amino acid sequence shown in SEQ ID NO.1-5 is artificially synthesized.

[0019] The hippocampus peptide mainly enhances the body's endurance by regulating oxidative stress. The active amino acid residues rich in the hippocampus peptide can activate the Nrf2 antioxidant pathway, up-regulate the activities of antioxidant enzymes such as superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), scavenge free radicals, reduce oxidative damage caused by exercise, and relieve muscle soreness.

[0020] The beneficial effects of the present invention are as follows: (1) Animal experiments prove that the Hippocampus abdominalis peptide can significantly relieve physical fatigue and can significantly prolong the exhaustion time in the aerobic swimming exhaustion experiments under aerobic and anaerobic respiration; at the same time, through the pulling force experiment and the pole climbing experiment, it is found that it can significantly enhance muscle strength, providing a new direction for the product development of the Hippocampus abdominalis peptide.

[0021] (2) It gives reasonable guidance for the application of the Hippocampus abdominalis peptide in anti-fatigue, and has a broad market prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is a graph of the duration of weight-bearing swimming of mice ingesting 9% of the hippocampus mixed peptide; Figure 2 It is a graph of the duration of weight-bearing swimming of mice ingesting 4% of the hippocampus mixed peptide; Figure 3 It is a graph of the duration of pole climbing of mice ingesting the hippocampus peptide; Figure 4 It is a graph of the muscle pulling force of mice ingesting the hippocampus peptide; Figure 5 It is a graph of the duration of weight-bearing swimming of mice ingesting 9% of the single hippocampus peptide; Figure 6 It is a graph of the duration of weight-bearing swimming of mice ingesting 4% of the single hippocampus peptide. DETAILED DESCRIPTION OF THE INVENTION

[0023] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0024] In addition, all materials used in the embodiments of the present invention are obtained by purchasing from the market unless otherwise specified.

[0025] Example 1: A pachypharynx hippocampus peptide is prepared by the following method: Take 2 g of pachypharynx hippocampus samples (purchased from Weihai Yinze Biotechnology Co., Ltd.), dissolve them in 10 ml of Tris-HCl buffer (containing 0.1 mM EDTA), add 0.1 g of papain, vortex for 30 s, then place in a water bath at 37 °C and react with magnetic stirring (150 rpm) for 4 hours. After the reaction, immediately add 10% TCA to a final concentration of 5% (v / v), mix well, ice-bath for 10 minutes to inactivate the enzyme and precipitate undigested proteins, and then centrifuge (12,000 rpm, 10 minutes, 4 °C) to collect the supernatant, which is the crude peptide sample. Load the supernatant into a dialysis bag (cut-off molecular weight 3 kDa) and dialyze with ultrapure water for 4 hours (change water 3 - 4 times) to remove small molecule impurities. Freeze-dry the dialyzed peptide solution to obtain a white powdery peptide sample and store it at -20 °C for later use. Papain (800,000 U / g, G8430-25) is purchased from Solarbio Science & Technology Co., Ltd.

[0026] 1. Research on the anti-fatigue effect of pachypharynx hippocampus peptide on mice: 1.1 Experimental design: ICR male mice are divided into 2 groups: the model group and the hippocampus peptide group. The model group is given drinking water and ordinary rodent feed; the hippocampus peptide group is given hippocampus peptide solution (100 mg / kg body weight) by gavage, ordinary rodent feed, and normal drinking water. After a single intake of hippocampus peptide, 9% load-bearing (mainly aerobic respiration) swimming exhaustion experiments are carried out at 2 h, 4 h, and 8 h respectively; after a full rest for 3 days, 4% load-bearing (mainly anaerobic respiration) swimming exhaustion experiments and pole climbing experiments are carried out respectively 4 h after a single intake; 14 days after the intake of hippocampus peptide, the muscle strength of the mice is tested using a tensiometer.

[0027] 1.2 Detection conditions: The sequences of the eluted peptides were analyzed using an UltiMate 3000 UHPLC system (Thermo Fisher Scientiiffc), an ACQUITY UPLC Peptide CSH C18 column (2.1×100 mm, 1.7 μm), and a TripleTOF 5600+ system. The parameters were as follows: positive ion mode, spray voltage 3600 eV, capillary temperature 320 °C, and signal intensity threshold 1.6 e5.

[0028] Amino acid composition of Hippocampus abdominalis peptide: Total amino acid composition analysis was performed using an automatic amino acid analyzer (Biochrom 30 Ltd). 0.1 g of the sample was weighed into an ampoule, 5 mL of 6 mol / mL hydrochloric acid solution was added, and acid hydrolysis was carried out at 110 °C for 24 h. After cooling, the volume was made up to 25 mL with ultrapure water. Then, 1 mL of the solution was deacidified to dryness and then 1 mL of sample buffer was added. The solution was filtered through a 0.22 μm filter membrane and loaded into a liquid phase vial for on-machine analysis. The content of amino acids was expressed in mg / g.

[0029] 1.3 Experimental results: The top 5 amino acids with the highest contents in the amino acid composition of Hippocampus abdominalis peptide were, in sequence: glutamic acid (15.82%), glycine (10.51%), alanine (9.30%), arginine (8.27%), and proline (7.50%). The content of essential amino acids accounted for 27.2%, which was close to the ideal amino acid pattern.

[0030] The experimental results showed that after a single intake of Hippocampus abdominalis peptide for 2 h, 4 h, and 8 h, the swimming exhaustion times of mice with 9% body weight load were as shown in Figure 1. There were significant differences at 2 h, 4 h, and 8 h. In addition, the swimming exhaustion times of mice with 4% body weight load after a single intake of Hippocampus abdominalis peptide were as Figure 2 shown. Hippocampus abdominalis peptide could also significantly increase the swimming exhaustion duration of mice. The above results indicated that Hippocampus abdominalis peptide had a significant and rapid effect on swimming exhaustion dominated by aerobic and anaerobic metabolism.

[0031] Next, a pole climbing experiment was carried out after a single intake of Hippocampus abdominalis peptide for 2 h, 4 h, and 8 h. There were significant differences at 2 h, 4 h, and 8 h, and the effect was most obvious at 8 h. The above experimental results indicated that Hippocampus abdominalis peptide had a significant enhancing effect on muscle endurance. In addition, according to the muscle tensile test results, the muscle tensile force of mice increased significantly after the intake of Hippocampus abdominalis peptide compared with the control group, indicating that long-term intake of hippocampus peptide could enhance the muscle strength of mice.

[0032] Example 2: Based on the results of Example 1, the peptide sequences obtained by de novo sequencing were retrieved in the NCBI database for hippocampal protein peptide sequences, and the results showed that the peptide sequences were not publicly available. The search parameters were set as follows: potential dynamic modification: oxidation (M, P, K); enzyme specificity: non-specific; fragment mass tolerance: 0.02 Da; precursor mass tolerance: 10 ppm; false discovery rate (FDR) of polypeptide identification results ≤ 1%. Peptide Ranker was used to predict the possibility of the identified peptide sequences having biological activity, and the test results are as follows.

[0033] Table 1: Scoring table for the biological activity of the identified peptide sequences: 。

[0034] 2. Study on the anti-fatigue effect of Hippocampus abdominalis peptides on mice: 1.1 Experimental design: The same as Example 1.

[0035] 1.2 Sample pretreatment: The predicted bioactive peptides of Hippocampus abdominalis were biosynthesized separately, and synthesized by Shanghai Sangon Biotech Co., Ltd. through Fmoc-solid phase peptide synthesis method. They are GGPQMPGPM (①), DDDIFPM (②), ASGPMG (③), GGGFDFIS (④), DHFLFD (⑤). After HPLC and MS sequence analysis, the purity of the ten peptide segments was greater than 98.89%. Chemically synthesized polypeptides have the characteristics of high purity and good clinical application safety due to the controllability of raw materials and processes. However, the biological activity of the synthesized polypeptides needs to be further verified.

[0036] 1.3 Experimental results: The experimental results showed that 4 hours after a single intake of the five predicted active Hippocampus abdominalis single peptides, the swimming exhaustion time of the mice with 9% load was shown in Figure 5. Compared with the control group, all 5 Hippocampus abdominalis single peptides had significant anti-fatigue effects; in addition, the swimming exhaustion time of the mice with 4% load after a single intake of Hippocampus abdominalis peptides was as Figure 6 shown, and the Hippocampus abdominalis single peptides could also significantly increase the swimming exhaustion duration of the mice. The above results indicate that the synthesized Hippocampus abdominalis single peptides have a significant and rapid effect on swimming exhaustion mainly aerobic and mainly anaerobic.

[0037] The present invention discloses the sequences of active peptides from Hippocampus abdominalis and their application in anti-fatigue products. The molecular weights of the Hippocampus abdominalis peptides are mainly short peptides with a molecular weight of < 1000 Da. The present invention discloses five active peptide sequences with high biological activity: GGPQMPGPM (①), DDDIFPM (②), ASGPMG (③), GGGFDFIS (④), and DHFLFD (⑤); through swimming exhaustion, pole climbing experiments, and muscle tensile strength tests, the anti-fatigue effects of the hippocampal active peptides were explored; highly pure peptides were obtained by chemical synthesis, and the biological activities of the peptides were verified using the above experiments; the understanding of Hippocampus abdominalis active peptides as an anti-fatigue product was improved.

[0038] Finally, it should be noted that although the above embodiments describe the specific implementation manners of the present invention, they do not limit the present invention; those skilled in the art should understand that these are only examples, and the protection scope of the present invention is defined by the appended claims. All modifications or equivalent replacements should be included within the protection scope of the present invention.

Claims

1. Application of a ventral-bulging seahorse peptide in the preparation of an anti-fatigue product, characterized in that: The belly-swelling seahorse peptide includes polypeptides with amino acid sequences shown in SEQ ID NO.1-5.

2. The preparation method of the seahorse abdominal peptide according to claim 1, characterized in that It includes the following steps: Take belly-swelling seahorses, dissolve them in Tris-HCl buffer containing 0.1 mM EDTA, add 5% papain, vortex for 30 s, then place in a 37 °C water bath and stir magnetically for reaction; after the reaction ends, immediately add 10% TCA to a final concentration of 5% v / v, mix well, place in an ice bath, and then centrifuge to collect the supernatant, which is the crude peptide sample; Load the supernatant into a dialysis bag with a molecular weight cut-off of 3 kDa, dialyze with ultrapure water for 4 hours to remove small molecule impurities; freeze-dry the dialyzed peptide solution to obtain a white powdery peptide sample, store it at -20 °C, and thus obtain the belly-swelling seahorse peptide.

3. The preparation method according to claim 2, characterized in that: Add buffer in a ratio of 1:5 of material to liquid.

4. The preparation method according to claim 2, wherein: The parameters for magnetic stirring are 150 rpm and stir for 4 hours.

5. The preparation method according to claim 2, characterized in that: The ice bath time is 10 minutes.

6. The preparation method according to claim 2, characterized in that: The parameters for centrifugation are centrifuge at 12,000 rpm for 10 minutes at 4 °C.

7. The belly-swelling seahorse peptide prepared by any of the methods according to claims 2-6.

8. The belly - swelling hippocampal peptide according to claim 1, characterized in that: The amino acid sequences are shown in SEQ ID NO.1-5.

9. The venter distension seahorse peptide according to claim 8, wherein: The belly-swelling seahorse peptide is artificially synthesized.

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