Application of pearl polypeptide chitosan liposome in intervention of caenorhabditis elegans aging

Abstract

The invention provides an application of a pearl polypeptide chitosan liposome in intervention of caenorhabditis elegans aging, further provides an application of the pearl polypeptide chitosan liposome in preparation of anti-aging food, drugs or cosmetics, and provides a preparation method of the pearl polypeptide chitosan liposome. The entrapment efficiency of the pearl polypeptide liposome is increased, the stability of the pearl polypeptide liposome is improved, the drug release speed is controlled, the pearl polypeptide is not prone to leakage, and the biological activity of the pearl polypeptide is better exerted. According to the pearl polypeptide chitosan liposome, polypeptide leakage is reduced, and the antioxidant activity of pearl polypeptide is improved. The invention provides a reference theoretical basis for further development, utilization and popularization of pearl polypeptide, and has a wide market application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and specifically relates to the application of pearl polypeptide chitosan liposomes in intervening in the aging of Caenorhabditis elegans. Background Art

[0002] Pearl polypeptide is a small molecule protein extracted from pearls, which contains a variety of active ingredients, including amino acids, minerals and various trace elements. The combination of these ingredients makes pearl polypeptide popular in skin care, anti-aging, immune enhancement and other aspects. For example, some studies have shown that it can effectively promote skin cell regeneration and slow down aging by penetrating deep into the skin to promote skin cell vitality, enhance skin elasticity and improve skin quality. Pearl polypeptide has both nutritional and functional properties, but its stability is poor and it is easily affected by temperature, moisture, etc. in the normal environment; it is easily degraded by proteases in the digestive tract after oral administration, and it will encounter various physiological barriers during the absorption process in the body, greatly reducing the bioavailability of active peptides in the body. When used in cosmetics, the transdermal absorption rate of pearl polypeptide is not high and it is easy to precipitate in the product. Therefore, a suitable delivery system needs to be found to improve the stability of pearl polypeptide and better exert its biological activity.

[0003] Selecting liposomes to encapsulate and deliver active peptides shows good prospects in improving the bioavailability of active peptides. Liposomes are spherical closed vesicles composed of phospholipid bilayers, which can effectively encapsulate hydrophilic, lipophilic and amphiphilic substances, protect active substances from external damage, and play a role in nutritional enhancement. Liposomes have the advantages of low toxicity, good biocompatibility, strong sustained-release properties, etc., so they are widely used in the fields of food, pharmacy, chemical industry and agriculture.

[0004] Polymers commonly used to modify liposomes include chitosan, pectin, sodium alginate, etc. Among them, chitosan has good biocompatibility, and the research on modifying liposomes with chitosan is relatively mature and has attracted much attention from researchers. It has been found that chitosan-modified liposomes can improve their physical stability and storage stability, and generally have a particle size less than 200 nm and the ability of complete transport and absorption. The thin film dispersion method is used to coat pearl polypeptide with soybean lecithin and cholesterol to prepare pearl polypeptide liposomes, and adding them to cosmetics solves the problem of insolubility or poor solubility of β-carotene. However, this liposome is sensitive to acids, heat, etc., and is prone to aggregation, fusion and flocculation during storage, resulting in leakage of the core material, too fast drug release during use, easy to produce burst release and difficult to control. Therefore, it is modified with the macromolecular material chitosan, and the sensitivity of liposomes to acids, heat, etc. can be improved, the stability can be enhanced, and the drug release rate can be controlled by the layer-by-layer coating technology, so that pearl polypeptide is not easy to leak.

[0005] To verify the anti-aging effect of pearl polypeptide chitosan liposomes, Caenorhabditis elegans was selected as a model animal in this study. Caenorhabditis elegans has become an ideal model for studying aging and related diseases, and also provides valuable experience for understanding the aging mechanism and developing anti-aging therapies. Summary of the Invention

[0006] The purpose of the present invention is to provide the application of pearl polypeptide chitosan liposomes in intervening in the aging of Caenorhabditis elegans, so as to provide a reference theoretical basis for the further development, utilization and popularization of pearl polypeptides.

[0007] The Caenorhabditis elegans described in the present invention has 60 - 80% of human homologous genes and at least 42% of human disease-related genes, and is an ideal model for exploring the pathogenesis and treatment methods of neurodegenerative diseases. Through the Caenorhabditis elegans lifespan experiment, it was found that pearl polypeptide chitosan liposomes can intervene in the aging of Caenorhabditis elegans, providing a powerful R & D model for the biological functions and molecular mechanisms of other natural source polysaccharide polypeptides and the further development and utilization of other anti-aging traditional Chinese medicine resources in the field of drugs for late-onset neurological diseases.

[0008] After the Caenorhabditis elegans is revived, it is cultured routinely, and specific experiments are carried out after homogenization.

[0009] The present invention provides the application of pearl polypeptide chitosan liposomes in intervening in the aging of Caenorhabditis elegans.

[0010] Specifically, the application includes the application of pearl polypeptide chitosan liposomes in prolonging the lifespan of Caenorhabditis elegans.

[0011] The specific technical scheme for the pearl polypeptide chitosan liposomes to prolong the lifespan of nematodes is as follows:

[0012] The experiment was divided into a blank group, a low-dose sample group, a medium-dose sample group, and a high-dose sample group, with 50 nematodes in each group. The N2 nematode strain was homogenized to collect eggs and cultured in S-medium. They were divided into groups on the first day of adulthood (d1). Every other day, the nematodes were transferred to a new medium to exclude the internally hatched nematodes, and the number of surviving nematodes was counted. The experiment continued until the last nematode died.

[0013] The application also includes the application of pearl polypeptide chitosan liposomes in enhancing the reproductive ability of Caenorhabditis elegans.

[0014] The specific technical scheme for the pearl polypeptide chitosan liposomes to enhance reproductive ability is as follows:

[0015] N2 was homogenized and cultured in S-medium. At the L4 stage, the nematodes were transferred to NGM plates coated with E. coli OP50, one nematode per plate, and divided into a drug administration group and a control group. On the first day of adulthood (d1), the nematodes were transferred to new culture plates, and the number of eggs on the previous culture plate was counted. The counting was carried out continuously for 5 days, and the average number of eggs laid per day in the drug administration group and the control group was compared.

[0016] The said application also includes the application of pearl polypeptide chitosan liposomes in accelerating the pharyngeal pumping rate of Caenorhabditis elegans;

[0017] The specific technical solution for the pearl polypeptide chitosan liposomes to accelerate the pharyngeal pumping rate is as follows:

[0018] Eggs of N2 were homogenized and collected and cultured in S-medium. On the first day of adulthood (d1), they were transferred to NGM plates coated with E. coli OP50. The drug was added to the E. coli OP50 plates in the drug administration group. Every day, the nematodes were transferred to new culture plates. On the 5th, 8th, and 10th days after adulthood, the number of pharyngeal pump movements of the nematodes was observed under a stereomicroscope for 1 minute for each nematode, and at least 10 nematodes were observed in each group. The pharyngeal pumping rate was calculated.

[0019] The said application also includes the application of pearl polypeptide chitosan liposomes in inhibiting the aggregation of polyglutamic acid in Caenorhabditis elegans.

[0020] The specific technical solution for the pearl polypeptide chitosan liposomes to inhibit the aggregation of polyglutamic acid in nematodes is as follows:

[0021] Eggs of AM141 were homogenized and collected and cultured in S-medium. At the L1 stage, they were divided into three groups and treated with the drug for 48 h. The nematodes were transferred to a glass slide, anesthetized with 10 mM sodium azide, and observed and photographed under a fluorescence microscope to quantitatively analyze the fluorescence accumulation of ployQ40::YFP.

[0022] The preparation of the said solid growth medium for Caenorhabditis elegans (NGM) is as follows: Weigh 3 g of NaCl, 2.5 g of peptone, and 17 g of agar respectively, add 1000 mL of distilled water, mix well, sterilize at 120 °C for 20 min, and quickly transfer to a 55 °C water bath to cool down. Then add the following sterilized solutions: 1 mol / L CaCl 2 (1 mL), 1 mol / L MgSO 4 (1 mL), 5 mg / L cholesterol solution (1 mL), 1 mol / L phosphate buffer (25 mL).

[0023] The said application also includes the application of pearl polypeptide chitosan liposomes in inducing oxidative stress in Caenorhabditis elegans.

[0024] The specific technical solution for the pearl polypeptide chitosan liposomes to cause oxidative stress in nematodes is as follows:

[0025] The animals were divided into 5 groups: blank group, model group (juglone), low, medium, and high dose groups of the test substance, with 50 nematodes in each group. After homogenously collecting the eggs of N2, they were cultured at 16 °C. On the 6th day of adulthood (d6), the nematodes were transferred to 96-well plates for culture, and different doses of the test substance and juglone (250 μmol / L) were added. Taking the addition of juglone as the 0 h, the survival status of Caenorhabditis elegans was counted every 24 h until all of them died. During the counting process, the Caenorhabditis elegans that were stiff and motionless and unresponsive to light and slight vibration were recorded as dead.

[0026] The application also includes the application of pearl polypeptide chitosan liposomes in improving the heat stress tolerance of Caenorhabditis elegans.

[0027] The specific technical solution of the pearl polypeptide chitosan liposomes for the heat stress of nematodes is as follows:

[0028] The animals were divided into 4 groups: blank group, low, medium, and high dose groups of the test substance, with 50 nematodes in each group. After homogenously collecting the eggs of N2, they were cultured at 20 °C. On the 6th day of adulthood (d6), the nematodes were transferred to 35 °C for culture, and the number of dead and surviving nematodes was counted every hour. The survival time of the nematodes under heat stress conditions at 35 °C was calculated, and the experiment was repeated 2 times.

[0029] The present invention also provides the application of pearl polypeptide chitosan liposomes in antioxidant activity. The application in antioxidant activity includes the application of pearl polypeptide chitosan liposomes in DPPH free radical scavenging and hydroxyl free radical scavenging.

[0030] The present invention also provides the application of pearl polypeptide chitosan liposomes in the preparation of anti-aging foods, drugs, or cosmetics.

[0031] When using the liposomes to prepare anti-aging products, according to actual needs, an effective amount of pearl polypeptide chitosan liposomes and a pharmaceutically acceptable carrier can be prepared into various dosage forms, including tablets, capsules, oral liquids, injections, powders, etc.

[0032] The preparation method of the pearl polypeptide chitosan liposomes obtained in the present invention includes the following steps:

[0033] 1) Coarsely crush high-quality pearls into submicron pearl powder using a hammer mill. Using lactic acid aqueous solution as a solvent, dissolve the pearl powder, adjust it to be slightly acidic, then add an appropriate amount of bioactive enzyme to catalytically decompose its protein. After ultrafiltration separation, nanofiltration concentration, and freeze-drying, pearl polypeptide is obtained;

[0034] 2) Preparation of liposomes by thin film dispersion method: Take a certain amount of soybean lecithin and cholesterol, dissolve them in chloroform, and then rotate and evaporate at 55 °C for 30 min to form a film. Place the rotary evaporation flask with the adhered film in a fume hood and dry it until there is no chloroform. Then add PBS buffer solution with Tween-80 at pH 7.4 and oscillate and mix evenly at the phase transition temperature (60 °C) for 1 h for hydration to obtain a crude liposome suspension. Ultrasonically treat the crude liposome suspension at 4 °C to obtain nano-liposomes;

[0035] 3) Preparation of chitosan-modified liposomes: Take a certain amount of chitosan and dissolve it in acetic acid solution, and stir magnetically for 3 h to obtain a chitosan acetic acid solution; Slowly drop the chitosan acetic acid solution into an equal amount of liposome solution, stir while dropping, and then stir magnetically for 1 h. To ensure that chitosan is fully coated on the surface of the liposome membrane, leave the above liposome suspension at 4 °C overnight to obtain chitosan-liposomes;

[0036] 4) Preparation of liposomes loaded with pearl polypeptides: Take a certain amount of pearl polypeptides and dissolve them in PBS at pH 7.4. The remaining steps are the same as the above method, and the obtained sample is freeze-dried to obtain pearl polypeptide chitosan liposomes.

[0037] Furthermore, when preparing liposomes, the ratio of pearl polypeptides to lecithin is 4:9 (w / w), the ratio of cholesterol to lecithin is 1:9 (w / w), the ratio of Tween-80 to lecithin is 2:9 (w / w), the chitosan concentration is 0.4% (w / w), the ultrasonic time is 6 min, and the ultrasonic power is 500 W.

[0038] Liposomes were prepared with the optimized parameters. The encapsulation efficiency of the pearl polypeptide liposomes was measured to be 83.13%. After modification with 0.4% chitosan, the encapsulation efficiency increased to 86.24%. This may be because the addition of chitosan slowed down the flow of liposomes and reduced the leakage of polypeptides; it may also be that the free polypeptides were embedded in the chitosan coating, increasing the encapsulation efficiency.

[0039] The particle size, PDI and potential of the liposomes were measured by a Malvern particle size analyzer, and the results were as follows: The particle size of the pearl polypeptide liposomes was 134.57 nm, and the PDI value was 0.641; after adding chitosan, the particle size increased to 146.22 nm, and the PDI value was 0.718. The zeta potential of the pearl polypeptide liposomes was -2.73 mV. After chitosan modification, the potential changed from negative to positive and increased to +0.86 mV.

[0040] The beneficial effects of the present invention are as follows:

[0041] The present invention uses Caenorhabditis elegans as a model and discovers through experiments that pearl polypeptide chitosan liposomes have a significant effect on delaying the aging of Caenorhabditis elegans, and their extension of the nematode lifespan shows a concentration gradient dependence. Through the analysis of its mechanism of action, it is found that the anti-aging effect of pearl polypeptide chitosan liposomes on Caenorhabditis elegans is achieved by reducing polyglutamate aggregation and activating the transcription factor DAF-16.

[0042] Meanwhile, the present invention uses chitosan modification and selects appropriate process parameters to increase the encapsulation rate of pearl polypeptide liposomes, improve the stability of pearl polypeptide liposomes, control the drug release rate, prevent the easy leakage of pearl polypeptides, and better exert their biological activities.

[0043] The present invention also discovers that the DPPH free radical scavenging rate of pearl polypeptide chitosan liposomes is higher than that of the raw material pearl polypeptides, and the hydroxyl free radical scavenging rate is significantly increased compared to the raw material pearl polypeptides. The pearl polypeptide chitosan liposomes of the present invention reduce polypeptide leakage and improve the antioxidant activity of pearl polypeptides.

[0044] The present invention can be used as an anti-aging product in foods, drugs, or cosmetics, providing a reference theoretical basis for the further development, utilization, and promotion of pearl polypeptides, and having broad market application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 Shows the effect of the ratio of polypeptide to lecithin on liposome preparation.

[0046] Figure 2 Shows the effect of the ratio of cholesterol to lecithin on liposome preparation.

[0047] Figure 3 Shows the effect of the ratio of Tween-80 to lecithin on liposome preparation.

[0048] Figure 4 Shows the effect of chitosan concentration on liposome preparation.

[0049] Figure 5 Shows the effect of ultrasonic power on liposome preparation.

[0050] Figure 6 Shows the effect of ultrasonic time on liposome preparation.

[0051] Figure 7 Shows the analysis of the Zeta potential and particle size of the samples, where Figures A and B are the potential and particle size distributions of pearl polypeptide liposomes respectively, and Figures C and D are the potential and particle size distributions of pearl polypeptide chitosan liposomes respectively.

[0052] Figure 8 Shows the DPPH free radical scavenging activity of the samples.

[0053] Figure 9 The hydroxyl radical scavenging activity of the sample.

[0054] Figure 10 The effect of the sample on the lifespan of C. elegans.

[0055] Figure 11 The effect of the sample on the reproductive ability of C. elegans.

[0056] Figure 12 The effect of the sample on the pharyngeal pumping rate of C. elegans.

[0057] Figure 13 The effect of the sample on oxidative stress in C. elegans.

[0058] Figure 14 The effect of the sample on heat stress in C. elegans.

[0059] Figure 15 The effect of the sample on polyglutamic acid in C. elegans. Figure A is the fluorescence photograph of polyglutamic acid in C. elegans treated with the sample, and Figure B is the quantitative graph of polyglutamic acid in C. elegans treated with the sample.

[0060] Figure 16 The effect of the sample on the subcellular localization of DAF-16 in C. elegans. Figure A is the fluorescence photograph of the subcellular localization of DAF-16 in C. elegans treated with the sample, and Figure B is the quantitative graph of the subcellular localization of DAF-16 in C. elegans treated with the sample. Detailed implementation mode

[0061] The following examples can enable those skilled in the art of this specialty to understand the present invention more comprehensively, but do not limit the present invention to the scope of the described examples.

[0062] Example 1 Preparation of pearl polypeptide chitosan liposomes

[0063] Take a certain amount of soybean lecithin and cholesterol, dissolve them in chloroform, and then rotate and evaporate at 55 °C for 30 min to form a film. Place the rotary evaporation flask with the adhered film in a fume hood and dry it until there is no chloroform. Then add PBS buffer solution with Tween-80 at pH 7.4 and oscillate and mix evenly at the phase transition temperature (60 °C) for 1 h to obtain a crude liposome suspension. Ultrasonically treat the crude liposome suspension at 4 °C to obtain nano-liposomes. Take a certain amount of chitosan and dissolve it in acetic acid solution, and stir magnetically for 3 h to obtain a chitosan acetic acid solution. Slowly drip the chitosan acetic acid solution into an equal amount of liposome solution, stir while dripping, and then stir magnetically for 1 h. The suspension is left overnight at 4 °C to obtain chitosan-liposomes. Take a certain amount of pearl polypeptide and dissolve it in PBS at pH 7.4, and the remaining steps are the same as the above method. The obtained sample is freeze-dried to obtain pearl polypeptide chitosan liposomes (PC-P). The pearl polypeptide liposomes not modified with chitosan are denoted as P-P.

[0064] Keeping other conditions unchanged, with the lecithin fixed at 0.10 g and the mass ratios of pearl polypeptide to lecithin being 1:2, 1:4, 1:6, 1:8, and 1:10, the effect of the ratio of lecithin to pearl polypeptide on the encapsulation efficiency of chitosan-modified polypeptide liposomes was investigated. The results showed that as the ratio of pearl polypeptide to lecithin increased, both the encapsulation efficiency and the particle size first increased and then decreased. When the drug-lipid ratio was 1:8, the encapsulation efficiency reached the maximum of 85.23%, and the particle size was 145 nm at this time. Considering comprehensively the effect of the ratio of pearl polypeptide to lecithin on chitosan-modified liposomes, the drug-lipid ratio of 1:8 was selected.

[0065] Keeping other conditions unchanged, the effect of the ratio of lecithin to cholesterol (w / w) (2:1, 3:1, 4:1, 5:1, 6:1) on the encapsulation efficiency of chitosan-modified pearl polypeptide liposomes was investigated. Cholesterol is a natural hydrophobic substance. Embedded between phospholipid molecules, it can enhance the flexibility of liposomes and play a role in stabilizing liposomes. When the ratio of lecithin to cholesterol is too large, cholesterol will cause a disturbing effect on the liposome membrane, destroying the bilayer structure of the liposome and making the liposome tend to an unstable state; when the ratio of lecithin to cholesterol is too small, there is less cholesterol and it cannot play a good role in fixing the bilayer structure of the liposome. The results showed that as the ratio of lecithin to cholesterol increased, the encapsulation efficiency first increased and then decreased. When the ratio of lecithin to cholesterol reached 5:1, the encapsulation efficiency reached the highest value of 85.65%, and the particle size was 143 nm at this time. Considering comprehensively, the ratio of lecithin to cholesterol of 5:1 was selected.

[0066] Keeping other conditions unchanged, the effect of the ratio of Tween-80 to lecithin (w / w) (1:10, 1:8, 1:6, 1:4, 1:2) on the encapsulation efficiency of chitosan-modified pearl polypeptide liposomes was investigated. Tween-80 is an oil-in-water emulsifier with amphiphilicity. In the liposome solution, the hydrophobic end of Tween-80 acts with the hydrophobic end of the phospholipid bilayer, and the hydrophilic end acts with the polar group phosphatidylcholine, which can maintain the microenvironment of the hydrophilic core of the liposome and reduce the leakage of polypeptides. When the addition amount of Tween-80 is too much, the particle size of the liposome may be smaller, and the probability of hydrophilic polypeptides in the hydrophilic core of the liposome becomes smaller. As shown by the results, within a certain range, the more the amount of Tween-80, the higher the encapsulation efficiency. When the ratio of Tween-80 to lecithin exceeds 1:6, the encapsulation efficiency gradually decreases from 86.11%. Tween-80 binds to phospholipids through hydrophobic interactions and forms a hydrophilic thin layer on the surface of the liposome. When the ratio of Tween-80 to lecithin is lower than 1:6, the emulsification degree is too high, the liposome leaks, and emulsion micelles are formed. Considering comprehensively, the ratio of Tween-80 to lecithin (w / w) of 1:6 was selected.

[0067] Keeping other conditions constant, the effects of chitosan concentration (w / w) (0.2%, 0.4%, 0.6%, 0.8%, 1.0%) on the encapsulation efficiency of chitosan-modified pearl polypeptide liposomes were investigated. When the chitosan concentration was 0.8%, the highest encapsulation efficiency of liposomes was 86.53%, and the particle size was 146 nm at this time. When encapsulating different substances, the required chitosan concentration of liposomes was also different. In summary, the appropriate chitosan concentration was 0.8% (w / w), and at this time, the particle size distribution of the liposome solution was uniform and relatively stable.

[0068] Keeping other conditions constant, the effects of ultrasonic power (200 W, 300 W, 400 W, 500 W, 600 W) on the encapsulation efficiency of chitosan-modified pearl polypeptide liposomes were investigated. When the ultrasonic power was 400 W, the highest encapsulation efficiency was 85.27%, and the particle size was 144 nm at this time. Considering comprehensively, the ultrasonic power of 500 W was selected.

[0069] Keeping other conditions constant, the effects of ultrasonic time (2 min, 4 min, 6 min, 8 min, 10 min) on the encapsulation efficiency of chitosan-modified pearl polypeptide liposomes were investigated. When the ultrasonic time was short, there were more large vesicle liposomes, which were easy to aggregate, the particle size distribution was uneven, and the stability was poor; when the ultrasonic time was too long, it would lead to polypeptide leakage. When the ultrasonic time was 8 min, the maximum encapsulation efficiency was 86.21%, and the particle size was 145 nm at this time. Therefore, the ultrasonic time of 8 min was selected.

[0070] In the above single-factor experiments, taking the encapsulation efficiency as the index and considering the particle size comprehensively, the optimal process for preparing PC-P was determined. The drug-lipid ratio was 1:8 (w / w), the ratio of cholesterol to lecithin was 1:5 (w / w), the ratio of Tween-80 to lecithin was 1:6 (w / w), the chitosan concentration was 0.8% (w / w), the ultrasonic power was 400 W, and the ultrasonic time was 8 min.

[0071] Example 2. Study on the physicochemical properties of pearl polypeptide chitosan liposomes PC-P

[0072] 1. Determination of encapsulation efficiency

[0073] The Folin - phenol method was used to determine the content of pearl polypeptides. The Folin - phenol reagent was diluted 10 times for use. Accurately weigh 0.0024 g of standard bovine serum albumin and prepare a standard solution with a concentration of 0.24 mg / mL. Respectively take 0, 0.1, 0.3, 0.5, 0.7, 0.9 mL of the standard solution, make up to 1 mL with distilled water. After adding 1 mL of alkaline copper reagent and mixing evenly, quickly add 4 mL of Folin - phenol reagent, mix evenly, and place in a water bath at 55 °C for 5 min. After cooling in a cold water bath for 5 min, measure the absorbance value at 650 nm. Take 1 mL of the sample to replace the above - mentioned standard solution, and the remaining steps are the same as those for making the standard curve. Take 3 mL of the sample and place it in a 30 kDa ultrafiltration centrifugal tube, centrifuge at 4000 r / min for 30 min, and record the filtrate volume V1. The filtrate is the sample containing free peptides. The operation of liposomes without encapsulated pearl polypeptides is the same as the above steps, and the filtrate (V0) is used as a blank. Record the polypeptide concentrations as C1 and C0 respectively. Take 2 mL of 0.06% (w / w) Triton 100 and add it to 1 mL of the sample, vortex and mix evenly. Take 1 mL of this mixed solution to replace the standard solution, and the subsequent steps are the same as those for the standard curve. Record the total peptide concentration as C2. The calculation method of the encapsulation efficiency EE is as follows: EE(%) = [1 - (C 1 V 1 -C 0 V 0 ) / 3C 2 ) * 100%

[0074] The encapsulation efficiency is an important indicator for evaluating the drug - loading property of liposomes. The results show that the encapsulation efficiency of P - P after optimization is 81.13%, and after chitosan modification, the encapsulation efficiency of the liposome is 86.24%. This may be because after adding chitosan, chitosan and phospholipids are adsorbed on the surface of the liposome through electrostatic interaction, slowing down the migration of polypeptides, reducing the fluidity of phospholipid molecules, and enabling more polypeptides to be encapsulated in the liposome. In addition, chitosan has a positive potential and the polypeptide surface shows a negative potential. It may be that chitosan binds to free polypeptides, and the polypeptides are embedded in the chitosan coating, increasing its encapsulation efficiency.

[0075] 2. Zeta potential and particle size

[0076] The zeta potential and particle size of the sample were measured using dynamic light scattering technology. The sample was diluted 50 times with 0.01 mmol / L, pH 7.0 PBS as the sample solution for standby. Set the temperature of the Malvern particle size analyzer to 25 ± 0.15 °C, select a polystyrene sample cell, and the other parameters are default. Absorb the sample solution to not exceed the scale of the sample cell, and pay attention to injecting slowly without bubbles. Test three parallels, and the results are averaged.

[0077] Particle size is an important indicator for measuring the stability, bioavailability, and solubility of bioactive compounds in liposomes. The polydispersity index (PDI) is an important indicator for measuring the uniformity of particle size distribution. A PDI value less than 0.4 indicates a narrow particle size distribution and a uniform particle size distribution. The particle size of liposomes was measured by a Malvern particle size analyzer, and the results showed that the particle size of P-P was 134.57 nm, and after modification with chitosan, the particle size increased to 146.22 nm. This is because the pKa of chitosan-NH 2 is 6.5, and it is easy to have electrostatic interaction with negatively charged groups under acidic conditions. Chitosan coats on the surface of negatively charged liposomes, increasing the particle size of chitosan. After adding chitosan, the PDI value increased from 0.641 to 0.718, indicating a uniform particle size distribution and a stable liposome solution.

[0078] The absolute value of the Zeta potential is closely related to the stability of the suspension. Generally, the larger the absolute value, the higher the stability of the system. The potential results showed that after adding chitosan, the zeta potential increased from -2.73 mV to +0.86 mV. The chitosan coating can delay the release of polypeptides and increase the stability of liposomes. In addition, negatively charged liposomes are more easily digested and absorbed. Therefore, the change of potential from negative to positive is beneficial to delaying the release of polypeptides in liposomes and improving their digestive stability.

[0079] 3. DPPH Free Radical Scavenging Activity

[0080] Take 19 test tubes. Set 1 tube as the control group, and divide the remaining 18 tubes into 3 groups. Add liposomes at a ratio of 1:1, chitosan-liposomes, and chitosan-acetic acid at a ratio of 1:1 to them. Then, add 0.4 mL, 0.8 mL, 1.2 mL, 1.6 mL, and 2 mL of the sample solution to the 18 test tubes in turn, make up to 2.00 mL with 50% methanol solution, and then add 3.00 mL of DPPH solution to each; add 2 mL of 50% methanol solution and 3 mL of DPPH to the other 1 tube as a control. After placing all the test tubes in the dark for 20 min, measure the absorbance at a wavelength of 517 nm. Calculate the scavenging rate, and make a standard curve with the volume as the abscissa and the scavenging rate as the ordinate.

[0081] The results showed that the DPPH free radical scavenging rates of pearl polypeptide liposomes and pearl polypeptide chitosan liposomes were both higher than that of the raw material pearl polypeptide. This may be because the DPPH free radical is a hydrophobic compound, and the addition of hydrophobic compounds or antioxidant substances (such as phospholipids, cholesterol, chitosan) in the system will increase the antioxidant activity of pearl polypeptide liposomes. At room temperature, the fluidity and permeability of the phospholipid membrane of liposomes are relatively high, and phospholipids are easily oxidized and degraded, resulting in polypeptide leakage and reduced activity. After adding chitosan, the interaction between chitosan and phospholipids slows down the fluidity of liposomes and reduces polypeptide leakage.

[0082] 4. Hydroxyl radical scavenging activity

[0083] To 27 test tubes, with 2 tubes as the control group and the remaining 25 test tubes evenly divided into five groups. Respectively add liposomes, chitosan - liposomes at a ratio of 1:1, and chitosan - acetic acid at a ratio of 1:1. Add 0.20, 0.30, 0.40, 0.50, 0.60 mL of the sample solution. Add distilled water to each test tube to make up 1 mL. Then, in sequence, add 1 mL of FeSO 4 , 1 mL of salicylic acid - absolute ethanol, and 1 mL of 3.0% hydrogen peroxide, and shake well. For the other two groups, add 1 mL of distilled water, 1 mL of FeSO 4 , 1 mL of salicylic acid - absolute ethanol, and 1 mL of 3.0% hydrogen peroxide. Then, after heating in a water bath at 37 °C for 30 min, measure the absorbance at a wavelength of 520 nm. Record each absorbance and calculate the scavenging rate. Use the sample volume as the abscissa and the scavenging rate as the ordinate to make the scavenging activity curve.

[0084] The results show that after loading pearl polypeptides into liposomes and chitosan - liposomes, under the same conditions, the hydroxyl radical scavenging rate is significantly increased compared with the raw material pearl polypeptides. This may be due to the substances in the wall material that can scavenge hydroxyl radicals. After modification with chitosan, the hydroxyl radical scavenging rate fluctuates. The activity is higher than that of pearl polypeptide liposomes at low and high doses, and lower than that of pearl polypeptide liposomes in the medium - dose range. It shows that the addition of chitosan reduces the leakage of polypeptides and has a better effect on maintaining the antioxidant activity of pearl polypeptides.

[0085] Example 3. Anti - aging activity of pearl polypeptide chitosan liposomes

[0086] 1. Recovery and conventional culture of nematodes

[0087] Take out the nematode cryopreservation tube from the - 80 °C ultra - low humidity refrigerator and thaw it naturally at room temperature. After complete thawing, centrifuge at 1500 rpm for 3 min, discard the supernatant, and add the remaining nematode suspension to the NGM plate coated with 4 times the amount of E. coli OP50 using a pipette tip. Rotate the plate to make it evenly distributed, and culture it in an incubator at 20 °C with a humidity of 40 - 60%.

[0088] 2. Nematode homogenization process

[0089] To ensure the accuracy of the experiment, before each batch of experiments, the nematodes need to be homogenized to ensure the consistency of their growth stages. When the nematodes enter the L4 stage (oviposition stage), all the nematodes are washed down into a 10 mL glass centrifuge tube with 4.5 mL of M9 solution. Then, 250 μL of sodium hypochlorite and 250 μL of sodium hydroxide (5 mol / L) are added. After mixing evenly, centrifuge at 1500 rpm for 3 min, discard the supernatant, add 5 mL of M9 solution, shake well to wash the worm body (the above steps are required to be completed within 10 min to avoid damaging the eggs), centrifuge at 1500 rpm for 3 min, discard the supernatant, aspirate the egg suspension and spot it onto an empty NGM dish, and incubate in an incubator at 20 °C. The hatched larvae are transferred to an NGM plate coated with OP50 for cultivation after 24 h.

[0090] 3. Nematode lifespan experiment

[0091] The experiment is divided into a blank group, a low-dose sample group (100 μg / L), a medium-dose sample group (200 μg / L), and a high-dose sample group (400 μg / L), with 50 nematodes in each group. The N2 nematode strain is homogenized, and the eggs are collected and cultured in S-medium. They are divided into groups on the first day of adulthood (d1). Every other day, the nematodes are transferred to a new medium to exclude the internally hatched nematodes, and the number of surviving nematodes is counted. The experiment continues until the last nematode dies. The experimental results show that compared with the control group, the high-dose groups of the sample PC-P can significantly extend the lifespan of N2 Caenorhabditis elegans, p < 0.05.

[0092] 4. Nematode reproductive ability experiment

[0093] The N2 is homogenized and cultured in S-medium. At the L4 stage, the nematodes are transferred to an NGM plate coated with E. coli OP50, one nematode per dish, and divided into a drug administration group and a control group. On the first day of adulthood (d1), the nematodes are transferred to a new culture dish, and the number of eggs on the previous culture dish is counted. The counting is continued for 5 days, and the average number of eggs laid per day in the drug administration group and the control group is compared. The experimental results show that the number of eggs laid by the nematodes in the high-dose groups of PC-P is increased compared with the blank group, but there is no significant difference, indicating that the extension of lifespan by PC-P does not come at the cost of reduced fertility.

[0094] 5. Nematode pharyngeal pumping movement rate experiment

[0095] Collect N2 homogenized eggs and culture them in S-medium. Transfer them to NGM plates coated with E. coli OP50 on the first day of adulthood (d1). Add the drug to the E. coli OP50 plates for the drug-treated group. Transfer the nematodes to new culture plates every day. Observe the number of pharyngeal pumping movements of the nematodes under a stereomicroscope on the 5th, 8th, and 10th days after adulthood for 1 minute for each nematode, and observe at least 10 nematodes in each group. Calculate the pharyngeal pumping rate. The results show that the pharyngeal pumping rates of the sample at medium dose on the 5th and 8th days of adulthood are significantly higher than those of the control group. The pharyngeal pumping rates of the high-dose group on the 5th, 8th, and 10th days of adulthood are significantly higher than those of the control group.

[0096] 6. Nematode Oxidative Stress Experiment

[0097] The nematodes were divided into 5 groups: blank group, model group (juglone), low, medium, and high-dose groups of the sample, with 50 nematodes in each group. After collecting N2 homogenized eggs, culture them at 16 °C. Transfer the nematodes to 96-well plates for culture on the 6th day of adulthood. Add different doses of the test substance and juglone (250 μmol / L). Starting from adding juglone as the 0 h, count the survival status of Caenorhabditis elegans every 24 h until all of them die. During the counting process, mark the Caenorhabditis elegans that are stiff and motionless and unresponsive to light and slight vibration as dead. The research shows that the redox reaction of juglone in nematodes will induce intracellular oxidative stress. The experimental data show that liposome PC-P has a protective effect on nematodes damaged by juglone oxidation and can extend the average survival time of nematodes to a certain extent. Among them, the average survival times of the nematodes in the medium and high-dose groups are significantly increased, indicating that a certain dose of pearl polypeptide chitosan liposome can increase the tolerance of nematodes to juglone oxidative damage.

[0098] 7. Heat Stress Experiment

[0099] The nematodes were divided into 4 groups: blank group, low, medium, and high-dose groups of the sample, with 50 nematodes in each group. After collecting N2 homogenized eggs, culture them at 20 °C. Transfer the nematodes to 35 °C for culture on the 6th day of adulthood. Count the number of dead and surviving nematodes every hour and calculate the survival time of the nematodes under heat stress conditions at 35 °C. Repeat 2 times. The research shows that while the lifespan of nematodes is extended, their tolerance to heat stress will also be improved to a certain extent. The experimental results show that the average survival times of the nematodes in the low, medium, and high-dose groups in the 35 °C high-temperature environment are extended by 2.3 h, 6.5 h, and 11.4 h respectively, indicating that pearl polypeptide chitosan liposome may extend the survival time of nematodes by improving their heat stress tolerance.

[0100] 8. Polyglutamic Acid Experiment in Nematodes

[0101] AM141 eggs were homogenously collected and cultured in S-medium. At the L1 stage, they were divided into three groups, and the samples were applied for 48 h. The nematodes were transferred onto a glass slide, anesthetized with 10 mM sodium azide, observed and photographed under a fluorescence microscope, and the accumulation of polyQ40::YFP fluorescence was quantified. The experimental results showed that, compared with the control group, the high-dose group of the sample could significantly inhibit the aggregation of polyglutamate in AM141 nematodes. After 48 h of treatment with the high-dose group of the sample, the number of polyQ40::YFP aggregation points in the body wall muscle layer of AM141 Caenorhabditis elegans individuals decreased from 85.5 in the control group to 65.2. This indicated that the sample had the effect of inhibiting aggregation and maintaining protein dynamic balance in polyQ Caenorhabditis elegans.

[0102] 9. Subcellular localization experiment of nematode DAF-16

[0103] The nematodes were divided into 4 groups: low, medium, and high-dose groups of the sample, with 10 nematodes in each group. TJ356 eggs were homogenously collected and cultured in S-medium. At the L1 stage, they were grouped, and the drug was applied for 24 h. The nematodes were transferred onto a glass slide, anesthetized with 10 mM sodium azide, observed and photographed under a fluorescence microscope. The experiment was repeated three times. In TJ356 transgenic nematodes, the GFP green fluorescent protein reporter gene was linked to the daf-16 gene, so the daf-16 could be localized by observing the GFP reporter gene under a fluorescence microscope. There were three localization situations of DAF-16 in the cell: cytoplasm, nuclear interstitium, and nucleus. The experimental results showed that the high-dose group of the sample could reduce the cytoplasmic part of DAF-16::GFP nematodes from 51.6% to 38.4%, while the nuclear part increased from 18.2% to 59.7%, significantly promoting the nuclear localization of DAF-16.

[0104] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention are all included in the protection scope of the present invention.

Claims

1. Application of pearl polypeptide chitosan liposomes in intervention of aging in Caenorhabditis elegans.

2. The use according to claim 1, characterized in that The application includes the application of pearl polypeptide chitosan liposome in extending the life span of Caenorhabditis elegans.

3. The use according to claim 1, characterized in that The application includes the application of pearl polypeptide chitosan liposome in enhancing the reproductive ability of Caenorhabditis elegans.

4. The use according to claim 1, characterized in that The application includes the application of pearl polypeptide chitosan liposome in accelerating the pharyngeal pump movement rate of Caenorhabditis elegans.

5. The use according to claim 1, characterized in that The application includes the application of pearl polypeptide chitosan liposome in inhibiting the aggregation of polyglutamic acid in Caenorhabditis elegans.

6. The use according to claim 1, characterized in that The application of pearl polypeptide chitosan liposomes in antioxidant activity includes the application of pearl polypeptide chitosan liposomes in scavenging DPPH free radicals and scavenging hydroxyl free radicals.

7. The use according to claim 1, characterized in that Application of pearl polypeptide chitosan liposome in preparing anti-aging food, medicine or cosmetics.

8. The use according to claim 7, characterized in that An effective amount of pearl polypeptide chitosan liposome and a pharmaceutically acceptable carrier can be prepared into various dosage forms, including tablets, capsules, oral liquids, injections, and powders.

9. The use according to any one of claims 1 to 8, characterized in that The preparation method of the pearl polypeptide chitosan liposome comprises the following steps: 1) Using a hammer mill to coarsely grind high-quality pearls into submicron pearl powder, using lactic acid aqueous solution as solvent to dissolve the pearl powder and adjust it to be slightly acidic, then adding an appropriate amount of bioactive enzyme to catalyze the decomposition of its protein, ultrafiltration separation and nanofiltration concentration, and freeze-drying to obtain pearl polypeptide; 2) Preparation of liposomes by thin film dispersion method: a certain amount of soybean lecithin and cholesterol were dissolved in chloroform, and then rotary evaporated at 55°C for 30 min to form a film, and the rotary evaporator with the film was placed in a fume hood to dry until there was no chloroform, and then PBS buffer with pH 7.4 containing Tween-80 was added and oscillated and mixed at the phase transition temperature (60°C) for 1 h to obtain a crude liposome suspension, and the crude liposome suspension was ultrasonically treated at 4°C to obtain nanoliposomes; 3) Preparation of chitosan modified liposomes: a certain amount of chitosan was dissolved in an acetic acid solution and magnetically stirred for 3 hours to obtain a chitosan acetic acid solution; the chitosan acetic acid solution was slowly added dropwise to an equal amount of the liposome solution while stirring, and then magnetically stirred for 1 hour. In order to allow the chitosan to be fully coated on the surface of the liposome membrane, the above liposome suspension was kept at 4°C overnight to obtain chitosan-liposomes; 4) Preparation of liposomes encapsulating pearl polypeptides: a certain amount of pearl polypeptides is dissolved in PBS at pH 7.4, and the remaining steps are the same as the above method. The obtained sample is freeze-dried to obtain pearl polypeptide chitosan liposomes.

10. The use according to claim 9, characterized in that The ratio of pearl polypeptide to lecithin is 4:9 (w / w), the ratio of cholesterol to lecithin is 1:9 (w / w), the ratio of Tween-80 to lecithin is 2:9 (w / w), the concentration of chitosan is 0.4% (w / w), the ultrasonic time is 6 min, and the ultrasonic power is 500W.

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