Sodium alginate nano-liposome embedded with squid-derived polypeptide

Abstract

The invention provides a sodium alginate nano-liposome embedded with squid-derived polypeptide, the sodium alginate nano-liposome comprises a core material and a wall material, the core material is squid-derived glycyl-prolyl-skeleton structure peptide, the wall material is sodium alginate, and the sodium alginate nano-liposome is prepared through high-pressure homogenization and molecular self-assembly. The sodium alginate-nano liposome prepared by the preparation method disclosed by the invention is relatively high in encapsulation efficiency which can reach 68.05%, the average particle size is 250.5 nm, the PDI value is 0.204, and the zeta potential is-58.4 mV. SA-LIP is small in particle size, good in monodispersity and high in stability. SA-LIP has a slow release property, and can continuously release squid-source glycyl-prolyl-skeleton structure peptide within 45-50 hours, so that the squid-source glycyl-prolyl-skeleton structure peptide can continuously play an active role in vivo. SA-LIP can protect the squid-source glycyl-prolyl-skeleton structure peptide from being damaged in the gastric digestion stage, the release rate is only 10-20%, and targeted release is achieved at the small intestine part.

Description

Technical Field

[0001] The invention belongs to the technical field of active biological products and oral delivery thereof, and particularly relates to a sodium alginate nanoliposome encapsulating a squid-derived polypeptide. Background Art

[0002] A glycyl-prolyl backbone peptide extracted from squid cartilage tissue has the typical amino acid composition of type II collagen and exhibits significant anti-inflammatory activity, effectively alleviating the symptoms of knee osteoarthritis in rats. The effects of oral administration of this squid-derived glycyl-prolyl backbone peptide on three signaling pathways, namely TLRs, NFκB, and Jak / Stat, were investigated at the mRNA and protein levels, revealing a pathway of action, namely the TLR4 / MyD88-NFκB-Jak / Stat signaling pathway. However, bioactive peptides are easily degraded after oral administration in the digestive tract, resulting in reduced bioactivity and limiting their practical application as nutritional or functional food ingredients. In vitro simulated digestion experiments revealed that squid-derived glycyl-prolyl backbone peptides are degraded by proteases after gastrointestinal digestion, disrupting their structure.

[0003] To address the poor gastrointestinal stability of bioactive peptides after oral administration, researchers have developed a variety of delivery systems to protect bioactive peptides, including liposomes, polymer nanoparticles, nanofibers, micelles, and inorganic nanoparticles, tailored to specific needs. Liposomes offer excellent biocompatibility, non-immunogenicity, good scalability, and high drug loading capacity, making them widely used for encapsulating both hydrophilic and hydrophobic bioactive peptides. Polymer nanoparticles offer excellent biocompatibility and are easily tailored to specific needs, enhancing their ability to protect and deliver bioactive peptides. Natural polymers, particularly adhesive polysaccharides such as chitosan, alginate, dextran, gum arabic, hyaluronic acid, and pectin, are particularly widely used. Inorganic nanoparticles exhibit high stability under both enzymatic and acidic conditions, possess a large surface area, and enhance membrane permeability. Currently, a growing body of research is focused on developing non-toxic and highly compatible carrier materials for encapsulating bioactive peptides using food ingredients such as polysaccharides, proteins, and lipids. Squid-derived glycyl-prolyl-backbone structure peptides are usually added as active ingredients to functional foods and nutritionally fortified foods. Bioactive peptides extracted from marine organisms usually have fishy and bitter tastes. These unpleasant odors will seriously affect the flavor of liquid and powder brewing products added with bioactive peptides, thereby reducing consumer acceptance.

[0004] Encapsulating squid-derived glycyl-prolyl-backbone peptides within liposome vesicles improves gastrointestinal digestibility while masking unpleasant odors and enhancing the sensory acceptance of liquid products. Soy lecithin, a natural and excellent emulsifier widely used in the food industry, is commonly used as the liposome wall material. However, conventional nanoliposomes suffer from poor thermal and acid-base stability, which to some extent limits their practical application. Summary of the Invention

[0005] The present invention aims to provide a sodium alginate-nanoliposome encapsulating a squid-derived glycyl-prolyl-backbone peptide (SSP), comprising a core material and a wall material, wherein the core material is the squid-derived glycyl-prolyl-backbone peptide and the wall material is sodium alginate, and the liposome is prepared by high-pressure homogenization and molecular self-assembly.

[0006] The present invention first provides a polypeptide with anti-inflammatory activity, wherein the polypeptide is a squid-derived glycyl-prolyl-skeleton structure peptide; The specific amino acid sequences of the polypeptides are GPAGPL (SEQ ID NO: 1), GPAGPN (SEQ ID NO: 2), GPEGPL (SEQ ID NO: 3), GPEGPLGAD (SEQ ID NO: 4), and GPEGPLGLP (SEQ ID NO: 5).

[0007] The polypeptide provided by the present invention can be used to prepare a product for anti-osteoarthritis. In an embodiment of the present invention, the product is sodium alginate-nanoliposome.

[0008] The present invention further provides a sodium alginate-nano liposome, the core material of which comprises the aforementioned polypeptide having anti-inflammatory activity. The sodium alginate-nano liposome is prepared by: 1) Dissolve a squid-derived glycyl-prolyl-backbone peptide with anti-inflammatory activity in water at 20-25°C using a solid-liquid shear method to prepare a 3-30 mg / mL solution. Adjust the pH to 6.0-6.5, filter through a 0.22 μm microporous membrane, and use it as a core material. 2) Dissolve soy lecithin powder in ethanol at 20-25°C to prepare a 35-45 mg / mL solution; 3) Slowly add the polypeptide solution to the soybean lecithin solution at a volume ratio of 10-20:1, and stir at 20-25°C for 20-30 minutes to fully mix. This is the core solution. The mass ratio of SSP to soy lecithin is 3:2-2:3, preferably 1:1; 4) Ultrasonicate the core solution at a temperature of 20-30°C, a power of 250-300 W, and a frequency of 30-40 kHz. The ultrasonication time is 18-22 minutes, preferably 20 minutes. 5) subjecting the core solution to high-pressure microfluidization homogenization at 20-25°C for three times at a homogenization pressure of 90-110 MPa, preferably 100 MPa; 6) After high-pressure homogenization, the core microcapsule-nanoliposome emulsion is obtained. 7) Prepare the shell material solution by dissolving sodium alginate in water at a concentration of 1-2 mg / mL at 20-25°C and stirring for 3-4 hours to fully dissolve. 8) Add the nanoliposome emulsion to the sodium alginate solution used as the shell wall material. Add the nanoliposome emulsion dropwise to 10 mL of the sodium alginate solution at 20-25°C while stirring at a stirring speed of 600 rpm and a dropwise addition rate of 1 mL / min. The mass ratio of soy lecithin to sodium alginate should be 3:2-5:2, preferably 2:1. After stirring for 60 minutes, the sodium alginate-nanoliposomes encapsulating the squid-derived glycyl-prolyl-backbone peptide are obtained.

[0009] The sodium alginate-nanoliposomes prepared by the present invention have a high encapsulation efficiency of up to 68.05%, an average particle size of 250.5 nm, a PDI value of 0.204, and a zeta potential of -58.4 mV. SA-LIP has a small particle size, good monodispersity, and strong stability.

[0010] SA-LIP has sustained-release properties and can continuously release squid-derived glycyl-prolyl-skeleton structure peptides within 45-50 hours, allowing it to continue to exert its active effects in the body.

[0011] SA-LIP can protect the squid-derived glycyl-prolyl-backbone structure peptide from being destroyed during the gastric digestion stage, with a release rate of only 10-20%, while achieving targeted release in the small intestine. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 This is the optimization diagram of the mass ratio of SSP and soybean lecithin.

[0013] Figure 2 This is a diagram for ultrasound time optimization.

[0014] Figure 3 This is the homogenization pressure optimization diagram.

[0015] Figure 4 This is the optimization diagram of the mass ratio of soybean lecithin and sodium alginate.

[0016] Figure 5Transmission electron microscopy image of SA-LIP.

[0017] Figure 6 This is the Fourier transform infrared spectrum of SA-LIP.

[0018] Figure 7 is the X-ray diffraction pattern of SA-LIP.

[0019] Figure 8 This is the digestion stability diagram of SA-LIP.

[0020] Figure 9 This is the in vitro cumulative release rate of SA-LIP.

[0021] Figure 10 This is the storage stability diagram of SA-LIP. DETAILED DESCRIPTION

[0022] The present invention uses sodium alginate to modify the surface of nanoliposomes to embed glycyl-prolyl-backbone structure peptides, which is beneficial for protecting the backbone structure peptides from the adverse environment in the stomach after oral administration and achieving targeted release in the small intestine, providing a theoretical basis for the application of glycyl-prolyl-backbone structure peptides.

[0023] In the following examples, the particle size, PDI value and zeta potential of SA-LIP were determined as follows: After the sample was diluted 10 times, its average particle size, zeta potential and PDI were measured using a Malvern Zetasizer Nano (ZS 90) nanoparticle size analyzer.

[0024] In the following examples, the encapsulation efficiency of SA-LIP was determined as follows: After centrifugation, the prepared SA-LIP was ultrafiltered through a 10 kDa molecular weight cutoff ultrafiltration tube. Unencapsulated SSP was filtered into the lower layer, and the lower filtrate was collected. The protein concentration of the collected lower filtrate and the SSP solution used to prepare the liposomes was determined using a BCA kit according to the manufacturer's instructions. The encapsulation efficiency was calculated using the following formula: Encapsulation efficiency = (m1-m2) / m2×100% Wherein, m1 is the total SSP content in LIP or SA-LIP; m2 is the unentrapped SSP content in LIP or SA-LIP.

[0025] In the following examples, the analysis method of transmission electron microscopy is as follows: After the SA-LIP was diluted to an appropriate multiple, it was dropped onto the copper grid and then counterstained with phosphotungstic acid. After the sample was dried, its microscopic morphology was observed using a transmission electron microscope and photographed.

[0026] In the following examples, the determination method of Fourier transform infrared spectroscopy is as follows: Before measurement, spectrally pure potassium bromide was dried at 110°C for at least 4 hours. The potassium bromide was thoroughly ground into a white powder in an agate mortar. An appropriate amount of the powder was placed in a mold and pressed into a tablet. The tablet was then placed in a Fourier transform infrared spectrometer for background scanning. Then, an appropriate amount of sample and potassium bromide were taken at a mass ratio of 1:100, thoroughly ground and mixed, and then pressed into a tablet. The background was subtracted before the sample was measured. The specific parameters were set as follows: scanning range 4000-400 cm -1 , 64 scans, resolution 1.0 cm -1 .

[0027] In the following examples, the X-ray diffraction analysis method is as follows: The crystal structure of the samples was determined by X-ray diffraction (XRD) with a scanning diffraction angle 2θ ranging from 4° to 60°, a scanning speed of 6° / min, and an accelerating voltage of 40 kV.

[0028] In the following examples, the digestion stability analysis method of SA-LIP is as follows: The pH of the sample solution was adjusted to 2.0, and pepsin was added to a final concentration of 2000 U / mL. The mixture was mixed thoroughly and allowed to react in a 37°C water bath with shaking for 2 h. The pH was then adjusted to neutral to inactivate the pepsin. Next, pancreatin was added to a final concentration of 100 U / mL, and the reaction was continued in a 37°C water bath with shaking for 4 h.

[0029] Samples were collected at 1, 2, 3, 4, 5, and 6 hours after digestion and divided equally into two parts. One part was immediately measured for particle size and zeta potential, and the other part was centrifuged and the supernatant was subjected to ultrafiltration centrifugation (molecular weight cutoff of 10 kDa). The filtrate in the lower layer of the ultrafiltration tube was collected and used to determine the release rate of SSP (expressed as hydroxyproline content) after inactivating the enzyme in a boiling water bath.

[0030] In the following examples, the method for determining the hydroxyproline content is as follows: (1) Reagent preparation: Buffer solution (pH 6.0): 15 g sodium hydroxide, 30 g citric acid, 90 g sodium acetate, 290 mL glycerol, add water to make 1 L of solution.

[0031] Chloramine T solution: Weigh 1.41 g of chloramine T, dissolve it in 100 mL of buffer, and mix thoroughly.

[0032] Color developer: Weigh 10 g of p-dimethylaminobenzaldehyde and dissolve it in 35 mL of 60% perchloric acid. Then, stir slowly and add 65 mL of isopropanol. Prepare the solution for immediate use.

[0033] Hydroxyproline (Hyp) standard solution: Accurately weigh a certain amount of hydroxyproline standard and dissolve it in ultrapure water to prepare a solution with a concentration of 1 mg / mL. Then dilute it to a 10 μg / mL Hyp standard solution.

[0034] (2) Drawing of the standard curve of hydroxyproline (Hyp): Prepare six stoppered test tubes, numbered 1, 2, 3, 4, 5, and 6. Add 2.0, 1.6, 1.2, 0.8, 0.4, and 0 mL of distilled water to each test tube, followed by 0, 0.4, 0.8, 1.2, 1.6, and 2.0 mL of Hyp standard solution. Add 1 mL of chloramine T solution and 1 mL of color developer to each test tube. After thorough mixing, heat the test tubes in a 60°C water bath for 15 minutes. Immediately rinse with cold water and cool for at least 3 minutes until the tubes return to room temperature. Finally, measure the absorbance of the samples at 560 nm to generate a standard curve.

[0035] (3) Sample determination: First, dilute the sample to the appropriate concentration. Place 2 mL of the diluted sample into an ampoule. Then, add 1 mL of ultrapure water and 1 mL of concentrated hydrochloric acid, sequentially. After filling with nitrogen, seal the ampoule and incubate at 130°C for 4 hours. Then, cool the solution to room temperature and accurately transfer it to a 100 mL volumetric flask. Add 2-3 drops of methyl red indicator to the flask, adjust the color of the solution to light yellow with 2 M sodium hydroxide solution, and finally bring it to volume with ultrapure water. Aspirate 2 mL of the diluted solution and perform the sample measurement according to the standard curve determination procedure.

[0036] In the following examples, the in vitro cumulative release rate analysis method of SA-LIP is as follows: The in vitro release rate of SSP from LIP, SA-LIP, CNP, and CANP was determined by dialysis. Ultrapure water adjusted to pH 2.0 was used as SGF, and PBS buffer was used as SIF. A 3 mL sample was placed in a dialysis bag (molecular weight cutoff, 10 kDa) in a centrifuge tube containing 30 mL of SGF and incubated in a shaking water bath at 37°C for 2 h. At 1 and 2 h, 1 mL of SGF was removed from the centrifuge tube and replaced with an equal amount of fresh SGF. After 2 h, the dialysis bag was transferred to a centrifuge tube containing 30 mL of SIF and incubated in a shaking water bath at 37°C for a further 46 h. At 3, 4, 6, 10, 16, 24, and 48 h of the total reaction time, 1 mL of SIF was removed and replaced with an equal amount of fresh SIF. The absorbance of the sample at 220 nm was measured using a UV spectrophotometer, and the cumulative release rate was calculated according to the following formula: Cumulative release rate = (SSP cumulative release amount) / (initial SSP content) × 100% In the following examples, the storage stability analysis method of SA-LIP is as follows: The prepared SA-LIP was sealed and stored in the dark at 4 ℃ and 25 ℃, and the particle size, PDI, zeta potential and encapsulation efficiency of the samples were measured on the 0th, 5th, 10th, 15th, 20th, 25th and 30th day of storage.

[0037] The present invention is described in detail below with reference to specific embodiments and accompanying drawings.

[0038] Example 1: Preparation of nanoliposomes encapsulating squid-derived glycyl-prolyl-backbone peptides To prepare squid-derived glycyl-prolyl backbone peptides, the squid necks were cleaned of meat and fat, retaining the cartilage. After cleaning, the mixture was wet-ground to an appropriate size. A 0.2 M NaOH solution was then added at a material-to-liquid ratio of 1:10. The mixture was then immersed under magnetic stirring for 18 hours. The cartilage precipitate was filtered and repeatedly washed until the pH was neutral. Distilled water was then added at a material-to-liquid ratio of 1:10 to adjust the pH. Alkaline protease and flavor enzyme were added at a mass ratio of 5:1, representing 2% of the substrate weight. The mixture was stirred thoroughly and then placed in a constant temperature shaking water bath for enzymatic hydrolysis at 50°C and 120 rpm for 4 hours. The hydrolyzate was inactivated by boiling in water, centrifuged, and the supernatant concentrated, dialyzed, and freeze-dried to obtain squid-derived glycyl-prolyl backbone peptides (SSPs).

[0039] Animal experiments have demonstrated that the prepared SSP peptides have anti-inflammatory effects. The specific amino acid sequences of some of these peptides are GPAGPL (SEQ ID NO: 1), GPAGPN (SEQ ID NO: 2), GPEGPL (SEQ ID NO: 3), GPEGPLGAD (SEQ ID NO: 4), and GPEGPLGLP (SEQ ID NO: 5). Artificial synthesis of these peptides also demonstrated that the five peptides with these specific amino acid sequences also have anti-inflammatory effects.

[0040] Soy lecithin (SL) was fully dissolved in anhydrous ethanol at a concentration of 40 mg / mL, SSP was dissolved in ultrapure water, and sodium alginate (SA) was fully dissolved in ultrapure water at a concentration of 2 mg / mL under magnetic stirring. The SSP solution was then added dropwise to the soybean lecithin emulsion at a specific ratio under magnetic stirring. After thorough stirring, the mixture was ultrasonically treated for a specific period of time and then subjected to high-pressure homogenization at a specific pressure to obtain SSP-encapsulated nanoliposomes (LIPs). The SSP solution was replaced with an equal amount of ultrapure water and the same steps were followed to prepare empty nanoliposomes (B-LIPs). The prepared LIPs were then added dropwise to the sodium alginate solution at a specific ratio under magnetic stirring. Stirring was continued for 1 h to obtain SSP-encapsulated sodium alginate nanoliposomes (SA-LIPs). B-LIPs were prepared under the same conditions under magnetic stirring to obtain empty sodium alginate nanoliposomes (B-SA-LIPs).

[0041] Effect of the mass ratio of SSP to soybean lecithin on the average particle size, PDI, zeta potential and encapsulation efficiency of SA-LIP Figure 1 As shown. The average particle size of the prepared SA-LIP is around 250 nm, and there is no significant difference. The PDI values ​​are all less than 0.3, indicating that they all have good monodispersity. The absolute values ​​of the zeta potential values ​​are all greater than 60 mV, indicating high stability. Taking the encapsulation efficiency as the main screening index, the mass ratio of SSP to SL is 1:1, which is the optimal condition. The SA-LIP prepared under this condition has the highest encapsulation efficiency, reaching 70.82%. At this time, its average particle size is 250.5 nm, PDI is 0.265, and zeta potential is -66.2 mV.

[0042] Effects of ultrasound time on the average particle size, PDI, zeta potential and encapsulation efficiency of SA-LIP Figure 2 As shown in the figure, with the extension of ultrasonic treatment time, the average particle size of the prepared SA-LIP first decreased and then increased, while the PDI value first increased and then decreased. The absolute values ​​of the zeta potential values ​​were all greater than 60 mV, indicating that the SA-LIP prepared with different ultrasonic treatment times had high stability. Using encapsulation efficiency as the main screening indicator, the optimal condition for ultrasonic treatment was 20 min. The SA-LIP prepared under this condition had the highest encapsulation efficiency, reaching 70.33%. At this time, its average particle size was 242.1 nm, PDI was 0.291, and zeta potential was -65.9 mV.

[0043] Homogenization pressure is an important factor affecting the particle size, stability, membrane structure and fluidity of liposomes. The effects of different homogenization pressures on the average particle size, PDI, zeta potential and encapsulation efficiency of SA-LIP are shown in Figure 2. Figure 3As shown in the figure. Samples prepared under higher homogenization pressures had smaller particle sizes, and their PDI values ​​first increased and then decreased with increasing homogenization pressure, with all PDI values ​​less than 0.310, indicating that the SA-LIPs prepared under different homogenization pressures all had good monodispersity. The absolute values ​​of the zeta potential values ​​were all greater than 60 mV, indicating that the prepared SA-LIPs had high stability. Using encapsulation efficiency as the primary screening criterion, a homogenization pressure of 100 MPa was the optimal condition. Under these conditions, the SA-LIP prepared had the highest encapsulation efficiency, reaching 68.04%. At this time, its average particle size was 228.9 nm, its PDI was 0.292, and its zeta potential was -61.4 mV.

[0044] Effect of the mass ratio of soybean lecithin (SL) and sodium alginate (SA) on the average particle size, PDI, zeta potential and encapsulation efficiency of SA-LIP Figure 4 As shown in the figure, the average particle size and PDI values ​​decreased with decreasing sodium alginate content. The absolute values ​​of the zeta potential of SA-LIP prepared at different SL:SA mass ratios were all around 60 mV, indicating high stability. The encapsulation efficiency of SA-LIP increased with increasing SL:SA mass ratios. Using encapsulation efficiency as the primary screening criterion, an SL:SA mass ratio of 2:1 was found to be optimal. Under these conditions, the SA-LIP prepared had the highest encapsulation efficiency, reaching 68.05%. At this point, its average particle size was 250.5 nm, its PDI was 0.204, and its zeta potential was -58.4 mV.

[0045] Example 2: Physicochemical properties of sodium alginate-nanoliposomes encapsulating squid-derived glycyl-prolyl-backbone peptides The SA-LIP prepared in Example 1 had an average particle size of 250.5 nm, a PDI of 0.204, a zeta potential of -58.4 mV, and an encapsulation efficiency of 68.05%. Figure 5 This is a transmission electron microscope image of SA-LIP at a magnification of 10,000 times. Its microstructure is a circular or elliptical structure with clear edges and good dispersion. This may be related to the high absolute value of SA-LIP's zeta potential, which can prevent particle aggregation through electrostatic repulsion, thereby improving its stability.

[0046] The chemical structures of the core and wall materials as well as the SSP-embedded nanoliposomes (LIPs) and SSP-embedded sodium alginate-nanoliposomes (SA-LIPs) can be determined by Fourier transform infrared spectroscopy (FTIR), e.g. Figure 6 The interface of the liposomal phospholipid bilayer can be characterized by two polar groups, C=O and P=O. In the spectrum of SL, 1737.67 cm −1The absorption peak at 1231.11 cm represents the stretching vibration of C=O. −1 The absorption peak at 2924.26 cm represents the symmetrical vibration of P=O. −1 and 2853.69 cm −1 The absorption peak at 2924.50 cm is mainly related to the symmetric and asymmetric stretching vibration of -CH2. Comparing the spectra of SL, B-LIP and LIP, the symmetric and asymmetric stretching vibration of -CH2 remains basically unchanged, and in B-LIP it is 2924.50 cm −1 and 2853.98 cm −1 , 2924.19 cm in LIP −1 and 2854.26 cm −1 Compared with the spectrum of LIP and SL, the absorption peak representing the P=O symmetric vibration shifted to 1237.12 cm −1 , which indicates that a new hydrogen bond is formed between SSP and SL. The 1059.88 cm −1 The absorption peak at 1064.69 cm-1 shifted to 1064.69 cm-1 in the LIP spectrum. −1 , indicating PO2 - The group participates in the formation of hydrogen bonds. SA at 1616.04 cm −1 There is a characteristic absorption peak at 1062.14 cm-1. Compared with the spectrum of LIP, the absorption peak intensity of SA-LIP at this location increases and the absorption peak becomes broader. −1 The absorption peak intensity at also increased, indicating that SA was successfully coated on the LIP surface.

[0047] Crystalline materials usually exhibit narrow and clear peak characteristics, while amorphous materials exhibit broad and fuzzy peaks. By analyzing the changes in the XRD patterns of single materials and after forming a composite system, the bonding mode between the components can be inferred. The XRD patterns of SSP, soybean lecithin (SL), sodium alginate (SA), LIP and SA-LIP are as follows: Figure 7 As shown in Figure 3 . SSP has a broad peak in the 18°-28° region, and the position of the LIP characteristic peak is similar to that of the SL, indicating that SSP is successfully encapsulated in the liposomes. Furthermore, the intensity of the LIP characteristic peak is slightly lower than that of the SL characteristic peak, likely due to a decrease in the proportion of crystalline structure after the addition of SSP. The position and intensity of the characteristic peaks in the XRD pattern of SA-LIP after sodium alginate coating the outer layer of LIP change, indicating that sodium alginate may interact with the liposomes.

[0048] Example 3: Digestive Stability of Sodium Alginate-Nanoliposomes Encapsulating Squid-Derived Glycyl-Prolyl-Backbone Peptide In a simulated gastrointestinal digestion environment, changes in particle size and zeta potential are important indicators for evaluating its stability during digestion. Changes in particle size directly reflect its aggregation or dispersion behavior in the digestive fluid, while changes in zeta potential reveal the dynamic changes in the particle surface charge. Figure 8 As shown in the figure. During the gastric digestion phase, the particle size of SA-LIP first increased and then decreased, with average particle sizes of 244.9, 291.9, and 268.2 nm at 0, 1, and 2 h, respectively. During the intestinal digestion phase, the particle size of SA-LIP showed a decreasing trend, decreasing significantly after 3 h, reaching an average particle size of 246.2 nm. After entering the simulated gastric fluid, the strongly acidic environment caused a significant change in the surface charge of SA-LIP. After 1 h, its zeta potential changed from -58.0 mV to -28.0 mV. However, after 2 h, its absolute value increased significantly, approaching the pre-digestion level. After entering the intestinal digestion phase, its absolute value of zeta potential decreased significantly, but stabilized after 2 h.

[0049] Using hydroxyproline (Hyp) in SSP as a detection marker, the release of SSP from SA-LIP after simulated gastrointestinal digestion was investigated. The release rate was 13.53% after 2 hours of gastric digestion and reached 63.95% after 6 hours of continuous gastrointestinal digestion. This indicates that the prepared SA-LIP effectively protects SSP from the adverse gastric environment and facilitates targeted release in the small intestine.

[0050] The peptides were embedded in sodium alginate-nanoliposomes, and their release at each stage of digestion was investigated by simulated digestion in vitro. Figure 8 As shown in (D), the release rate was 18.62% after 2 h of gastric digestion, and the release rate of the hexapeptide reached 81.40% after 6 h of gastrointestinal digestion, indicating that it can better protect the characteristic peptide segments of the backbone structure peptide during the gastric digestion stage and can achieve targeted release in the small intestine.

[0051] Example 4: In vitro release of sodium alginate-nanoliposomes encapsulating squid-derived glycyl-prolyl-backbone peptide The in vitro drug release of LIP and SA-LIP was studied by dialysis method with unencapsulated SSP as control. Both showed a sustained release effect on SSP. The results are shown in Figure 2. Figure 9As shown in the figure. Within the first 2 hours of acidic conditions, 40.77% of the free SSP was released, while the release rates of SSP from LIP and SA-LIP were 20.02% and 14.51%, respectively. Sodium alginate coating the outer layer of the nanoliposomes provides better protection for SSP in acidic environments. After 48 hours of continuous experimentation, the cumulative release rate of free SSP reached 83.63%, while the release rates of SSP from LIP and SA-LIP were 43.47% and 34.11%, respectively, demonstrating that the prepared LIP and SA-LIP possess excellent sustained-release properties.

[0052] Example 5: Storage stability of sodium alginate-nanoliposomes encapsulating squid-derived glycyl-prolyl-backbone peptide The storage stability of nanoparticles is one of the key factors in their practical application. It reflects the changes in the physical and chemical properties of nanoparticles during storage and transportation, which in turn affects their effectiveness in practical applications. The changes in particle size, zeta potential, PDI and encapsulation efficiency of SA-LIP during storage in the dark at 4 ℃ and 25 ℃ for 30 days are shown in the figure. Figure 10 The particle size of SA-LIP remained unchanged for 25 days at 4°C, but increased to 216.0 nm after 30 days (compared to 188.0 nm on day 0). At 25°C, the particle size increased with storage time, reaching a maximum average size of 366.1 nm on day 25 (compared to 187.9 nm on day 0). The PDI value of SA-LIP remained unchanged for 25 days at 4°C, but increased significantly to 0.403 on day 30 (compared to 0.285 on day 0). Within 5 days of storage at 25°C, the PDI value increased significantly from 0.280 to 0.489. The PDI value remained unchanged from days 5 to 25, but increased significantly to 0.547 on day 30. The absolute value of the zeta potential of SA-LIP decreased during storage at 4°C, changing from -60.9 mV to -30.3 mV after 30 days. At 25°C, the zeta potential of SA-LIP changed significantly within 5 days, from -60.9 mV to -15.7 mV. It then reached -28.0 mV on day 10 and remained unchanged for 20 days. During storage at 4°C, the encapsulation efficiency of SA-LIP was not significantly different from that measured on day 0. However, at 25°C, the encapsulation efficiency increased significantly from 63.36% to 78.54% within 5 days, decreased significantly to 73.14% on day 15, and remained unchanged until day 30.

[0053] In summary, the prepared SA-LIP has high storage stability and better storage stability at 4 ℃.

Claims

1. A polypeptide having anti-inflammatory activity, characterized in that The polypeptide is a squid-derived glycyl-prolyl-skeleton structure peptide.

2. The polypeptide according to claim 1, wherein The amino acid sequence of the polypeptide is any one or more of SEQ ID NOs: 1-5.

3. Use of the polypeptide according to claim 1 or 2 in the preparation of an anti-osteoarthritis product.

4. The use according to claim 3, characterized in that The product is a nanoliposome.

5. A nanoliposome, characterized in that: The core material of the nanoliposome comprises the polypeptide according to claim 1 or 2.

6. The nanoliposome according to claim 5, wherein Sodium alginate is used as the wall material of the nanoliposome.

7. The nanoliposome according to claim 5, wherein The preparation method of the nanoliposome comprises the following steps: 1) Dissolve a squid-derived glycyl-prolyl-backbone peptide with anti-inflammatory activity in water at 20-25°C using a solid-liquid shear method to prepare a 3-30 mg / mL solution. Adjust the pH to 6.0-6.5, filter through a 0.22 μm microporous membrane, and use it as a core material. 2) Dissolve soy lecithin powder in ethanol at 20-25°C to prepare a 35-45 mg / mL solution; 3) Slowly add the polypeptide solution to the soybean lecithin solution at a volume ratio of 10-20:1, and stir at 20-25°C for 20-30 minutes to fully mix. This is the core solution. 4) Ultrasonic treatment of the core solution at a temperature of 20-30°C, a power of 250-300 W, and a frequency of 30-40 kHz for 18-22 minutes; 5) subjecting the core solution to high-pressure microfluidization homogenization at 20-25°C for three times at a homogenization pressure of 90-110 MPa, preferably 100 MPa; 6) After high-pressure homogenization, the core microcapsule-nanoliposome emulsion is obtained. 7) Prepare the shell material solution by dissolving sodium alginate in water at a concentration of 1-2 mg / mL at 20-25°C and stirring for 3-4 hours to fully dissolve. 8) Add the nanoliposome emulsion to the sodium alginate solution of the shell wall material. Drop the nanoliposome emulsion into 10 mL of the sodium alginate solution at 20-25°C while stirring at a stirring speed of 600 r / min and a dropping rate of 1 mL / min.

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