Princess Sea Anemone Peptides and Their Applications

Abstract

This invention provides princess sea anemone polypeptides and their applications. Using princess sea anemones as material, the invention extracts total protein from the anemones by lysing the cells with cell lysis buffer, followed by enzymatic hydrolysis using alkaline protease and centrifugation to obtain small-molecule princess sea anemone polypeptides. The princess sea anemone polypeptides prepared by this invention possess anti-activation, anti-hypoxia, and antioxidant activities, and can be applied in the preparation of anti-hypoxia agents, anti-heat stress agents, and antioxidants. In particular, they can be used in the preparation of anti-hypoxia drugs and anti-heat stress drugs, further contributing to the industrial development of princess sea anemone resources.

Description

Technical Field

[0001] This invention relates to the field of sea anemone technology, and particularly to Princess Sea Anemone polypeptide and its applications. Background Technology

[0002] Sea anemones, also known as sea chrysanthemums, are skeletal animals belonging to the phylum Coelenterata, class Anthozoa, and order Actiniaria. Over 1100 species of Actiniaria have been recorded worldwide, belonging to approximately 400 genera and 50 families. China currently accounts for about one-tenth of the global sea anemone species, distributed in all sea areas, and showing a clear trend of decreasing distribution from tropical to temperate zones. Common sea anemones in my country include: *Anemone simonii*, *Anemone japonicus*, *Anemone chinensis*, and *Anemone chinensis*. Most sea anemones have a cylindrical body shape, with tentacles arranged radially in several whorls around the mouth, covered with stinging cells for defense and predation.

[0003] Sea anemones possess various medicinal properties, including nourishing yin and yang, astringing and consolidating, and eliminating dampness and killing parasites. Traditional Chinese medicine has historically used them to treat conditions such as anal prolapse, tinea corporis, pinworm infection, and hemorrhoids. As early as ancient times, sea anemones were used to treat certain diseases in my country, as recorded in the *Chinese Materia Medica* and *Chinese Medicinal Animal Records*. Research on the chemical composition of sea anemones began in the 1970s. They contain abundant cytotoxic and neurotoxic substances, as well as protein and polypeptide compounds with cytolytic effects. Furthermore, with ongoing research, various small molecule compounds, such as glycerides, sterols, alkaloids, sesquiterpenes, and pyrimidines, have been isolated from sea anemones. In addition, the chemical components of sea anemones exhibit a wide range of physiological activities, including insecticidal, hypotensive, cardiotonic, antibacterial, and analgesic effects, making them highly valuable for research.

[0004] In recent years, the efficacy of marine bioactive peptides in the pharmaceutical field has attracted increasing attention. Marine organisms contain a rich variety of protein resources; however, due to our lack of understanding of the ocean, these resources have not yet been well developed. With the advancement of technology, the development and utilization of marine natural products in the pharmaceutical field has become a hot topic. Sea anemones possess a variety of chemical components with diverse activities, thus exhibiting extremely high medicinal value and a very broad application prospect. It is known that Zhang Junshun et al. studied the structure and function of sea anemone polypeptide neurotoxins; Zheng Shuzhen et al. isolated two polysaccharide substances from sea anemones collected from the South China Sea's *Schefflera* group that can effectively enhance the contractility of isolated animal myocardium, and the results showed that both had significant antihypertensive activity and good medicinal research value; Yuan Lin et al. used alkaline protease to enzymatically hydrolyze small molecule polypeptides from mantle anemones and verified their significant insecticidal activity. It is evident that certain achievements have been made in the research of the chemical components and physiological activities of sea anemones, but further research on the mechanisms of action and the activity of small molecule chemical substances should be intensified. Sea anemones are diverse, and research on many species is lacking. This invention aims to explore sea anemones with high medicinal value and further enhance their development value. Summary of the Invention

[0005] In view of this, the present invention proposes Princess Sea Anemone polypeptide and its application, prepares Princess Sea Anemone polypeptide, and finds that it has good activity in anti-stress, anti-hypoxia and anti-oxidation.

[0006] The technical solution of this invention is implemented as follows:

[0007] This invention provides the application of Princess Sea Anemone polypeptide in the preparation of anti-hypoxia agents, anti-heat stress agents, and antioxidants, particularly in the preparation of anti-hypoxia drugs and anti-heat stress drugs. The Princess Sea Anemone polypeptide is a small molecule Princess Sea Anemone polypeptide, a polypeptide complex with a molecular weight of less than 1000 Da obtained from Princess Sea Anemone as a raw material.

[0008] Furthermore, the Princess Sea Anemone polypeptide is used in the preparation of anti-hypoxia drugs, and the drug concentration of the Princess Sea Anemone polypeptide is 4-16 g / kg.

[0009] Furthermore, the Princess Sea Anemone polypeptide is used in the preparation of anti-heat stress drugs, wherein the drug concentration of the Princess Sea Anemone polypeptide is 0.5-2.0 mg / ml.

[0010] This invention also provides a method for preparing Princess Sea Anemone polypeptide, comprising the following steps:

[0011] (1) Degreasing: Rinse the Princess sea anemone with pure water, break it, soak the broken sea anemone in isopropyl alcohol, change the isopropyl alcohol every 3.5-4.5 hours, soak 5-6 times, then wash off the isopropyl alcohol with pure water, drain the water, and you will get the degreased Princess sea anemone.

[0012] (2) Preparation of Princess Anemone Protein Powder: Take the defatted Princess Anemone from step (1), add pure water, then add the mixed RIPA lysis buffer and PMSF protease inhibitor, sonicate for 8-12 min, let stand at room temperature for 25-35 min, centrifuge at low temperature and high speed at 2-5℃ and 9000-11000r / min for 15-25 min, and discard the precipitate; freeze the collected supernatant and break it into crushed ice, freeze dry to obtain Princess Anemone Protein Powder;

[0013] The ratio of the above-mentioned Princess Sea Anemone to pure water, RIPA lysis buffer and PMSF protease inhibitor is 5g: 40-60mL: 400-600 μL: 4-6μL;

[0014] (3) Proteolytic hydrolysis of Princess Anemone: Take the Princess Anemone protein powder from step (2) and add pure water. The mass-to-volume ratio of the material to the liquid is 1:4-6 g / L. Add alkaline protease and perform enzymatic hydrolysis in a water bath at pH 8-9, enzyme dosage of 3500-4500 U / g, and hydrolysis temperature of 55-65℃. After hydrolysis, the enzyme is inactivated. Then, centrifuge at 10000-12000 r / min, filter the supernatant and collect it. Adjust the pH of the supernatant to 6.5-7.5 to obtain the hydrolyzed peptides. Freeze-dry to obtain Princess Anemone peptides.

[0015] Compared with the prior art, the beneficial effects of the present invention are:

[0016] (1) The princess sea anemone polypeptide prepared by the present invention has anti-activation, anti-hypoxia activity and antioxidant activity, and can be used in the preparation of anti-hypoxia agents, anti-heat stress agents and antioxidants. In particular, it can be used in the preparation of anti-hypoxia drugs and anti-heat stress drugs, which can further help the industrial development of sea anemone resources.

[0017] (2) The present invention uses Princess Sea Anemone as raw material to produce a product with multiple drug activities, which is conducive to further developing the medicinal value of sea anemone.

[0018] (3) In this invention, the Princess Sea Anemone is used as material. The total protein of the Princess Sea Anemone is extracted by adding cell lysis buffer to lyse the sea anemone cells. Then, the small molecule Princess Sea Anemone polypeptide is obtained by enzymatic hydrolysis through alkaline protease hydrolysis and centrifugation. Attached Figure Description

[0019] Figure 1 Princess Anemone.

[0020] Figure 2 SDS-PAGE electrophoresis image of Princess Anemone protein; Note: M is a low molecular weight protein marker; lanes 1, 2, 3, 4, and 5 are water-extracted proteins from Princess Anemone lysate; lanes 6 and 7 are Princess Anemone protein polypeptides after enzymatic hydrolysis.

[0021] Figure 3 Scavenging rates of DPPH free radicals by undigested sea anemone protein and enzymatically hydrolyzed sea anemone polypeptide; Note: The control group was undigested Princess sea anemone protein; * indicates statistical difference with respect to the control group, and *** indicates extremely significant statistical difference with respect to ...

[0022] Figure 4 Effects of enzymatic hydrolysis of *Princess anemone* polypeptides on survival time in mice under normobaric hypoxia; Note: Negative control group was 0.9% physiological saline; positive control group was 3 × 10⁻⁶. -4 g / kg flunarizine hydrochloride; compared with the negative control group, ** indicates P < 0.01, indicating a significant difference.

[0023] Figure 5 Effects of enzymatic hydrolysis of peptides from *Princess anemone* on the lifespan of nematodes under heat stress. Detailed Implementation

[0024] To better understand the technical content of this invention, specific embodiments are provided below to further illustrate the invention.

[0025] Unless otherwise specified, the experimental methods used in the embodiments of this invention are all conventional methods.

[0026] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available.

[0027] Example

[0028] 1. Materials and Methods

[0029] 1.1 Experimental Materials

[0030] Princess sea anemone, collected from the South China Sea, washed with deionized water, and stored frozen at -20°C in a laboratory for later use. See details for specific morphology. Figure 1 .

[0031] 1.2 Main Reagents

[0032] The reagents and materials required for the experiment are listed in Table 1.

[0033] Table 1. Main Experimental Drugs and Reagents

[0034]

[0035] 1.3 Experimental Equipment

[0036] The equipment required for the experiment is listed in Table 2.

[0037] Table 2 Main Experimental Equipment

[0038]

[0039] 1.4 Extraction of total protein from Princess Sea Anemone

[0040] Rinse the Princess Sea Anemone multiple times with pure water, then crush it into small tissues using a crusher. Soak the crushed Princess Sea Anemone in isopropyl alcohol for 4 hours each time, 5-6 times, until the isopropyl alcohol solution does not change color. Then rinse off the isopropyl alcohol with pure water, drain the water in a fume hood, and store at -20℃ for later use.

[0041] Take 5g of defatted Princess anemone, add 50mL of pure water, then add 500μL of RIPA lysis buffer and 5μL of LPMSF protease inhibitor, and sonicate for 10min. Almost no large tissue fragments were observed. After standing at room temperature for 30min, centrifuge at 10000r / min for 20min at 4℃, and discard the precipitate. Freeze the collected supernatant and break it into ice fragments. Freeze-dry the supernatant using a freeze dryer to obtain Princess anemone protein powder, and store it at -20℃ for later use.

[0042] 1.5 Determination of total protein and polypeptides in Princess Sea Anemone

[0043] According to the instructions for the BCA test kit, first prepare the BCA working solution, then prepare the BSA standard, and then use the microplate method to add an appropriate volume of the test sample into the microplate and mix it with the working solution. After mixing, the sample is reacted at 37°C for 30 minutes. The absorbance value is recorded at 562nm and a standard curve is plotted.

[0044] 1.6 SDS-PAGE analysis of total protein and peptides in Princess Sea Anemone

[0045] Prepare the gel according to the instructions. Mix 16 μL of sample aqueous solution with 4 μL of 5× Loading Buffer. Add 20 μL of protein marker to a centrifuge tube. Boil all centrifuge tubes at 100℃ for 5 min, centrifuge at 1000 rpm for 5 min at room temperature, and then pipette 10 μL of the supernatant and place it close to the sample loading tank. Load the sample slowly and electrophoresis for about 3.5 h. Then stain with Coomassie Brilliant Blue solution on a shaker for at least 8 h. After complete staining, repeatedly destain with the prepared destaining solution until the blue color of the base gel disappears. At this point, obvious anemone protein bands can be observed. Observe and record the electrophoresis results using a fully automated gel imaging system.

[0046] 1.7 Enzymatic hydrolysis of total protein in Princess Sea Anemone

[0047] Take 2g of Princess Sea Anemone protein powder and add 10mL of pure water. Add alkaline protease and perform enzymatic hydrolysis in a water bath at pH 8, enzyme dosage of 4000U / g, and 60℃. After complete hydrolysis, boil at high temperature to inactivate the enzyme. Then centrifuge at 11000r / min for 10min, repeating the centrifugation process multiple times. Collect the supernatant after filtering through a 0.45μm microporous membrane. Adjust the pH of the supernatant to 7 using a pH adjuster to obtain the enzymatically hydrolyzed peptide, namely the Princess Sea Anemone peptide, which is a peptide complex with a molecular weight of less than 1000Da. Analyze the enzymatic hydrolysis effect by electrophoresis and calculate the hydrolysis yield. Freeze-dry the remaining product and store it at -20℃ for later use.

[0048]

[0049] 1.8 Free radical scavenging experiment of enzymatically hydrolyzed peptides

[0050] The working solution was prepared by adding anhydrous ethanol to powder A from the experimental kit according to the instructions. For the control tube, 400 μL of sample was mixed with 600 μL of 80% methanol. For the assay tube, 400 μL of sample was mixed with 600 μL of working solution. For the blank tube, 400 μL of 80% methanol was mixed with 600 μL of working solution. The mixture was reacted in the dark for 30 min wrapped in aluminum foil, then centrifuged at 4000 rpm for 5 min. An appropriate amount of supernatant was placed in a cuvette for absorbance measurement. The instrument was zeroed with anhydrous ethanol, and the wavelength was set to 517 nm. The absorbance values ​​at this wavelength were measured and recorded as A (blank), A (control), and A (assay). The sample clearance rate was calculated using the formula.

[0051]

[0052] 1.9 Experiment on the anti-hypoxia activity of enzymatically hydrolyzed peptides

[0053] Thirty healthy male mice weighing 30±2g were randomly divided into five groups: a flunarizine hydrochloride positive control group (flunarizine hydrochloride capsules dissolved at the human dosage), a negative control group (equal volume of 0.9% physiological saline), and high, medium, and low dose groups of sea anemone enzymatically hydrolyzed peptides (16g / kg, 8g / kg, and 4g / kg, respectively). Mice were administered the drug via gavage once daily for 10 days, during which they had free access to food and water. On the tenth day, 3 hours after gavage, the mice were placed in a 250mL wide-mouth Erlenmeyer flask, the flask opening was sealed with a rubber stopper, and then further sealed with plastic wrap to create a hypoxic, pressure-controlled environment. The time from sealing the flask opening to the mice ceasing respiration was observed and recorded.

[0054] 1.10 Experiment on the improvement of stress resistance in nematodes by enzymatic hydrolysis of peptides

[0055] 1.10.1 Preparation of Escherichia coli culture medium

[0056] Place 2g of peptone and 2.4g of NaCl in a 1000mL Erlenmeyer flask. Add 20mL of 1M phosphate buffer (108.3g KH₂PO₄ and 35.6g K₂HPO₄ dissolved in 1000mL deionized water, adjusted to pH 6). Add deionized water to a final volume of 800mL (initial culture medium). Autoclave at 121℃ for 15min. Then, add 0.8mL each of 5mg / mL cholesterol solution dissolved in ethanol (unsterilized), 1M MgSO₄ (246.47g MgSO₄·7H₂O, autoclaved, to a final volume of 1000mL), and 1MCaCl (111g CaCl₂, to a final volume of 1000mL). Shake well to obtain the complete culture medium. Inoculate with *E. coli* and incubate at 37℃ in a shaker. The culture medium can be temporarily stored at 4℃. When needed, incubate at 37℃ in a shaker for one day before reuse.

[0057] 1.10.2 Preparation of nematode growth medium

[0058] Take 2g of peptone, 16g of agar, and 2.4g of NaCl and place them in a 1000mL Erlenmeyer flask. Add 20mL of 1M phosphate buffer (108.3g KH2PO4 and 35.6g K2HPO4 dissolved in 1000mL distilled water, adjusted to pH=6) and 12.5mg / L 5-fluorouracil (to inhibit nematode reproduction). Add distilled water to a final volume of 800mL. Autoclave at 121℃ for 15min. Then add 0.8mL each of 5mg / L cholesterol solution dissolved in ethanol (unsterilized), 1M CaCl2 (sterilized), and 1M MgSO4 (sterilized) sequentially. Shake well to obtain complete culture medium. Spread the E. coli culture medium, which has been placed on a 37℃ shaker one day in advance, onto the solid culture medium. Incubate at 37℃ for one day until the solid culture medium is fully colonized with E. coli for subsequent use.

[0059] 1.10.3 Synchronization of Nematodes

[0060] The experiment used *Caenorhabditis elegans*. A culture medium containing a large number of oviparous hermaphroditic nematodes was selected in the prior stages. M9 buffer (3g KH₂PO₄, 5g NaCl, 6g Na₂HPO₄ dissolved in 1L of ultrapure water, autoclaved, and then 1mL of 1mol / L HCl was added) was then added. The culture medium was repeatedly washed with MgSO4 to remove nematodes and eggs attached to the bacterial growth. The washing solution containing nematodes was collected in a sterile 2.0 mL centrifuge tube. The nematodes were washed with 1 mL of M9 buffer and centrifuged at 1000 rpm for 1 min. The supernatant was removed as much as possible with a pipette. This process was repeated 3 times. Then, 1 mL of cell lysis buffer was added to lyse the nematodes. The nematodes were observed under a microscope until most of them were lysed. The nematodes were centrifuged at 6000 rpm for 1 min and the supernatant was discarded as much as possible. The nematodes were washed with an appropriate amount of M9 buffer and centrifuged several times. The washed eggs were transferred to a pre-prepared nematode growth medium and cultured at 20°C for two days. After two days, the nematodes basically developed into L4 stage larvae, thus completing the nematode synchronization.

[0061] 1.10.4 Effects of sea anemone peptides on the lifespan of nematodes under heat stress

[0062] After synchronization treatment, nematodes were randomly divided into 5 groups, with 3 replicates per group and 30 nematodes per plate. These included a control group (0 mg / mL) and experimental groups containing different concentrations of enzymatically hydrolyzed peptides from *Princess Anemone* (0.5, 1.0, 2.0, and 2.5 mg / mL). After 2 days of incubation at 20°C, the synchronized nematodes were transferred to a 37°C incubator. The number of surviving and dead nematodes was recorded every 0.5 h (nematodes were considered dead if they did not respond to gentle touch with a fine platinum wire) until all nematodes died. Survival curves under heat stress were plotted based on the nematode survival time.

[0063] 2 Results and Discussion

[0064] 2.1 Extraction and determination of total protein content from sea anemones

[0065] From SDS-PAGE electrophoresis results ( Figure 2 As can be seen, the Princess Sea Anemone extracted using the lysis buffer method has relatively abundant low molecular weight proteins, mainly distributed between 25kDa and 17kDa, with a small amount distributed around 30kDa. Therefore, this study provides convenient conditions for subsequent enzymatic hydrolysis of peptides. BCA assay results showed that the total protein content of Princess Sea Anemone extracted from 100g of dried powder was 0.98g, with a concentration of 1.82mg / mL.

[0066] 2.2 DPPH· Free Radical Scavenging Experiment

[0067] Because free radicals in living organisms are extremely unstable and exist for a very short time, while DPPH· is a relatively stable free radical, DPPH· is used to generate free radicals to replace those in living organisms, thus evaluating the free radical scavenging ability of antioxidants. According to Figure 3 It was found that the free radical scavenging rate of sea anemone peptides was significantly improved after enzymatic hydrolysis compared with that before enzymatic hydrolysis (P<0.05). Furthermore, the free radical scavenging ability increased with increasing sea anemone peptide concentration, exhibiting a clear dose-response relationship. This indicates that enzymatically hydrolyzed sea anemone peptides can exert good antioxidant activity.

[0068] 2.3 Anti-hypoxia experiment of enzymatically hydrolyzed peptides

[0069] Mice were placed in wide-mouthed Erlenmeyer flasks, and the flasks were sealed tightly with rubber stoppers to ensure a closed environment. Initially, the mice appeared relatively calm, but after a few minutes, they began to struggle and attempt to escape. This behavior continued for several minutes before the mice began to curl up, collapse, and convulse. Their breathing changed from rapid to slow until it finally stopped, and their limbs turned grayish-white. Specific experimental data are shown in Table 3. Compared with the negative control group, the survival time of the positive control group and the low, medium, and high-dose experimental groups was prolonged to varying degrees. In particular, the survival times of the positive control group and the high-dose group reached 35.05±4.57 min and 39.04±9.03 min, respectively, representing increases of 23.38% (P<0.01) and 37.42% (P<0.01) compared with the negative control group. Figure 4 Observations revealed that, within the selected drug concentrations, the survival time of mice in each dose group of sea anemone polypeptide increased with the increase of sea anemone enzymatic hydrolysis polypeptide concentration, exhibiting a significant dose-response relationship. This indicates that sea anemone enzymatic hydrolysis polypeptide can prolong the survival time of mice under hypoxic conditions in a closed environment at normal pressure and exert anti-hypoxia activity.

[0070] Table 3. Effects of Princess Sea Anemone Enzymatic Hydrolysate Peptides on Survival Time of Mice Under Normative Pressure Closed Hypoxic Environment

[0071]

[0072] Note: The negative control group was 0.9% saline; the positive control group was 3×10⁻⁶ saline solution. -4 g / kg flunarizine hydrochloride; compared with the negative control group, **P<0.01 indicates a statistically significant difference.

[0073] 2.4 Effects of enzymatic hydrolysis of peptides on the lifespan of nematodes under heat stress

[0074] Numerous studies have confirmed that the extended lifespan of nematodes is closely related to their enhanced stress resistance. This enhanced stress resistance is one of the reasonable explanations for the extended lifespan of nematodes. Table 4 shows the effect of enzymatically hydrolyzed peptides from *Princess anemone* on the lifespan of nematodes under heat stress. The average lifespan of the control group was 3.60 ± 0.52 h, with the longest survival time being 4.5 h. At 37℃, the analysis of the effect of different concentrations of enzymatically hydrolyzed peptides from *Princess anemone* on the heat stress resistance of nematodes showed that, among the four set doses, the average lifespan of the 0.5 mg / mL group reached 4.13 ± 0.27 h, which was 14.5% longer than the control group, with the longest survival time reaching 6.5 h. The average lifespan of nematodes in the 1 mg / mL group reached 3.87 ± 0.24 h, which was 7.4% longer than that of the control group. However, the average lifespan of nematodes in the 2 mg / mL group was only 3.0% longer than that of the control group. This indicates that enzymatically hydrolyzed peptides from *Princess anemone* can enhance the heat stress resistance of nematodes within a certain concentration range; however, concentrations exceeding this range do not enhance their heat stress resistance. Figure 5 The lifespan curve of nematodes with a concentration of 0.5 mg / mL shifted significantly to the right compared to the control group, which to some extent indicates that sea anemone peptides within a certain concentration range can enhance the heat stress resistance of nematodes.

[0075] Table 4. Effects of enzymatically hydrolyzed polypeptides from *Princess Sea Anemone* on the lifespan of nematodes under heat stress.

[0076]

[0077]

[0078] Note: Compared with the control group, * indicates P<0.05, which is statistically significant;

[0079] 3. Conclusion

[0080] This experiment used a water extraction method with lysis buffer to extract relatively abundant low molecular weight Princess sea anemone protein, and then used alkaline protease to enzymatically hydrolyze the sea anemone protein to obtain enzymatically hydrolyzed peptides, with a degree of hydrolysis reaching 60.2%.

[0081] (I) In this DPPH free radical scavenging experiment, it was also found that the free radical scavenging ability of the enzymatically hydrolyzed sea anemone peptides was better than that of the unhydrolyzed ones. Furthermore, as the concentration of the enzymatically hydrolyzed peptides increased, the corresponding scavenging rate also increased, showing a clear dose-response relationship between the two.

[0082] (II) The mouse anti-hypoxia experiment proved that the enzymatic hydrolysis of sea anemone peptides can exert a certain anti-hypoxia activity, which significantly prolongs the survival time of mice in a normal pressure hypoxic environment.

[0083] (III) Nematodes can live on laboratory agar culture media, have a short lifespan, and are easy to observe and count, making them excellent model organisms. The academic community generally recognizes that nematodes have irreplaceable advantages in the fields of anti-oxidation and anti-aging research. Anti-stress experiments using *C. elegans* have demonstrated that enzymatic hydrolysis of peptides in *Anemone przewalskii* within a certain concentration range can prolong the survival time of nematodes under heat stress conditions, thus enhancing their heat stress resistance.

[0084] The princess sea anemone polypeptide prepared by this invention has anti-activation, anti-hypoxia, and antioxidant activities, and can be used in the preparation of anti-hypoxia agents, anti-heat stress agents, and antioxidants. In particular, it can be used in the preparation of anti-hypoxia drugs and anti-heat stress drugs, which can further help the industrial development of sea anemone resources.

[0085] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of Princess Sea Anemone Polypeptide, characterized in that, The Princess Sea Anemone polypeptide is used in the preparation of anti-hypoxia agents and / or anti-heat stress agents and / or antioxidants; The preparation method of the Princess Sea Anemone polypeptide includes the following steps: take Princess Sea Anemone protein powder, add water, add alkaline protease, and enzymatically hydrolyze in a water bath. After enzymatic hydrolysis, the protein is inactivated. Then, centrifuge at 10,000-12,000 r / min, filter the supernatant and collect it, adjust the pH of the supernatant to 6.5-7.5 to obtain the enzymatically hydrolyzed polypeptide, freeze-dry it, and the Princess Sea Anemone polypeptide is obtained. The mass-to-volume ratio of the Princess Sea Anemone Protein Powder to water is 1:4-6 (g / L); the conditions for water bath enzymatic hydrolysis are: pH 8-9, enzyme dosage of 3500-4500 U / g, and hydrolysis temperature of 55-65℃. The Princess Anemone Protein Powder is prepared by the following steps: taking defatted Princess Anemone, adding water, then adding the mixed RIPA lysis buffer and PMSF protease inhibitor, sonicating for 8-12 minutes, letting stand at room temperature, centrifuging at low temperature and high speed for 15-25 minutes, and discarding the precipitate; freezing the collected supernatant and breaking it into crushed ice, freeze-drying it to obtain Princess Anemone Protein Powder. The ratio of Princess sea anemone to water, RIPA lysis buffer, and PMSF protease inhibitor is 5g:40-60 mL:400-600 μL:4-6 μL; the centrifugation temperature for the low-temperature high-speed centrifugation is 2-5°C, and the centrifugation speed is 9000-11000 r / min; the standing time at room temperature is 25-35 min; the specific degreasing operation is as follows: rinse the Princess sea anemone with water, crush it, soak the crushed sea anemone in isopropanol, change the isopropanol every 3.5-4.5 hours, soak 5-6 times, then wash off the isopropanol with water, drain the water, and the product is obtained.

2. The application of the Princess Sea Anemone Polypeptide according to claim 1, characterized in that, The Princess Anemone polypeptide is a polypeptide complex with a molecular weight of less than 1000 Da obtained from Princess Anemone as a raw material.

3. Princess Sea Anemone Polypeptide, characterized in that, The preparation method of the Princess Sea Anemone Polypeptide includes the following steps: (1) Rinse the Princess sea anemone with pure water, crush it, soak the crushed sea anemone in isopropyl alcohol, change the isopropyl alcohol every 3.5-4.5 hours, soak 5-6 times, then wash off the isopropyl alcohol with pure water, drain the water, and obtain the degreased Princess sea anemone. (2) Take the defatted Princess anemone from step (1), add pure water, then add the mixed RIPA lysis buffer and PMSF protease inhibitor, sonicate for 8-12 min, let stand at room temperature for 25-35 min, centrifuge at low temperature and high speed at 2-5°C and 9000-11000r / min for 15-25 min, and discard the precipitate; freeze the collected supernatant and break it into crushed ice, freeze dry to obtain Princess anemone protein powder; The ratio of the above-mentioned Princess Sea Anemone to pure water, RIPA lysis buffer and PMSF protease inhibitor is 5g: 40-60 mL: 400-600 μL: 4-6 μL; (3) Take the Princess Anemone Protein Powder from step (2) and add pure water. The mass-to-volume ratio of the material to the liquid is 1:4-6 g / L. Add alkaline protease and perform enzymatic hydrolysis in a water bath at pH 8-9, enzyme dosage of 3500-4500 U / g, and hydrolysis temperature of 55-65℃. Inactivate the enzyme after hydrolysis. Then centrifuge at 10000-12000 r / min, filter the supernatant and collect it. Adjust the pH of the supernatant to 6.5-7.5 to obtain the enzymatically hydrolyzed polypeptide. Freeze-dry to obtain the Princess Anemone polypeptide.

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