Perinereis aibuhitensis extract, preparation method thereof and application of perinereis aibuhitensis extract in delaying senescence

Abstract

The invention discloses a perinereis aibuhitensis extract as well as a preparation method and application of the perinereis aibuhitensis extract in delaying senescence, and belongs to the technical field of biological medicines. Comprising the following steps: homogenizing perinereis aibuhitensis, adding alkaline protease for enzymolysis, centrifuging, taking supernate, passing through an ultrafiltration membrane, and collecting a perinereis aibuhitensis ultrafiltration component which is less than 5kDa, namely the perinereis aibuhitensis extract. The perinereis aibuhitensis extract provided by the invention has significant application value in preparation of functional food, health care products, cosmetics or medicines for delaying senescence, resisting oxidation, relieving heat stress injury, improving reproductive capacity and prolonging life, and provides theoretical basis and key technical support for development of marine-derived anti-aging products.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a perinereis aibuhitensis extract, a preparation method thereof and application thereof in delaying aging. BACKGROUND

[0002] Aging is a complex biological process characterized by a gradual decline in physiological function, leading to an increased susceptibility to disease and ultimately death. With the intensification of global aging, delaying aging has become a hot issue of global concern. The accumulation of various cellular and molecular damage during the aging process is the core mechanism of aging, and the excessive production of reactive oxygen species is one of the key factors. Therefore, antioxidant is the key to delaying aging. Current measures for antioxidant mainly include antioxidant supplements and lifestyle interventions. Antioxidant active substances from natural products have been found to have the effect of delaying aging in improving the body's oxidative stress state, such as cod collagen peptide, abalone peptide, manila moon snail peptide, etc. Bioactive substances derived from the ocean have unique structures and high biological activity due to the high pressure and high salt environment, thereby having high-value utilization value in developing anti-aging functional foods, cosmetics, etc.

[0003] Perinereis aibuhitensis Perinereis aibuhitensis Perinereis aibuhitensis is rich in nutrients such as amino acids and proteins, and is known as the "sea Cordyceps". The total amino acid content of Perinereis aibuhitensis cultivated in Zhanjiang is 11.73%, and the contents of methionine, histidine, lysine, arginine and tyrosine, which have strong total antioxidant capacity, account for 24.81% of the total amino acids. Perinereis aibuhitensis is rich in crude protein, and the crude protein content of both natural and cultivated Perinereis aibuhitensis reaches more than 60%. Zhu Guoping found that the enzymatic product of Perinereis aibuhitensis with a molecular weight less than 5kDa has good DPPH free radical scavenging effect. Huang Lin et al. observed the antioxidant activity of Perinereis aibuhitensis extract by DPPH method, and confirmed that Perinereis aibuhitensis has strong reducing power and DPPH free radical scavenging capacity. Liu et al. compared the physicochemical properties of Perinereis aibuhitensis hydrolysate freeze-dried and spray-dried, and found that both freeze-dried and spray-dried powders have strong antioxidant activity. In summary, Perinereis aibuhitensis is rich in protein, has rich amino acid content, and has good antioxidant activity, which is an ideal raw material for preparing anti-aging active ingredients.

[0004] Caenorhabditis elegans Caenorhabditis elegansCaenorhabditis elegans is a classical invertebrate model, which has the characteristics of short life span, easy observation, short experimental period and complete genome sequence. Because of its short life cycle and genetic similarity to humans, it is widely used as a model organism in aging research. At the same time, the signal pathway of nematode is highly conserved in humans, and the human homologous genes account for 60-80%, which indicates that it is highly feasible and valuable to use Caenorhabditis elegans to study the aging mechanism related to humans. It is an ideal animal model for studying aging phenotype and signal pathway mechanism. At present, there are a large number of studies on the application of nematode model to antioxidant, anti-aging, lipid metabolism regulation and prevention and treatment of neurodegenerative diseases at home and abroad, so it is highly reliable to use nematode to evaluate the nutritional and functional properties of active ingredients of natural products. SUMMARY

[0005] The purpose of the present application is to provide a Perinereis aibuhitensis extract and its preparation method and application in delaying aging, so as to solve the problems existing in the prior art. The present application takes Perinereis aibuhitensis as raw material and Caenorhabditis elegans as model organism to determine the in vitro and in vivo antioxidant activities of the ultrafiltration components of the Perinereis aibuhitensis enzymatic products, explore the effects of the intervention of the ultrafiltration components of the Perinereis aibuhitensis enzymatic products on the life span, movement ability, reproductive capacity, lipofuscin accumulation degree and expression amount of aging genes of Caenorhabditis elegans, and preliminarily discuss the mechanism of the ultrafiltration components of the Perinereis aibuhitensis enzymatic products in delaying aging. The experimental results can provide a theoretical basis for developing Perinereis aibuhitensis products with the effect of delaying aging.

[0006] To achieve the above-mentioned purpose, the present application provides the following solutions: One of the technical solutions of the present application is a preparation method of Perinereis aibuhitensis extract, which comprises the following steps: The Perinereis aibuhitensis is homogenized, and alkaline protease is added for enzymatic hydrolysis. The supernatant is obtained by centrifugation, and the Perinereis aibuhitensis ultrafiltration components less than 5 kDa are collected by passing through an ultrafiltration membrane, which are the Perinereis aibuhitensis extract.

[0007] The second technical solution of the present application is the Perinereis aibuhitensis extract prepared by the preparation method.

[0008] The third technical solution of the present application is the application of the Perinereis aibuhitensis extract in preparing products for improving reproductive capacity.

[0009] The fourth technical solution of the present application is the application of the Perinereis aibuhitensis extract in preparing products for delaying aging.

[0010] The fifth technical solution of the present application is the application of the Perinereis aibuhitensis extract in preparing antioxidant products.

[0011] The sixth technical solution of the present application is the application of the Perinereis aibuhitensis extract in preparing products for relieving heat stress damage.

[0012] The application provides the application of the Perinereis aibuhitensis extract in the preparation of a product for prolonging life.

[0013] Based on the technical scheme, the application has the following technical effects: The alkaline protease enzymolysis combined with ultrafiltration separation technology is adopted to efficiently obtain the active component of the Perinereis aibuhitensis with a molecular weight less than 5 kDa under mild conditions, and the active component is easy to be produced in large scale, has good stability and low cost. The obtained extract is rich in protein (60.55%) and has small molecular weight and is easy to be absorbed and utilized by human bodies; the Perinereis aibuhitensis is used as the raw material, is widely available and rich in nutrition, and is an ideal material for developing the marine source anti-aging functional factor. The extract has multiple anti-aging effects, for example, the average life of the Caenorhabditis elegans is prolonged by 13.9% and the reproduction capacity is improved by 20.65% when the concentration of the extract is 4 mg / mL, the aging movement disorder is significantly improved, and the accumulation of the lipofuscin, an aging marker, is significantly reduced. The extract can significantly enhance the resistance of the Caenorhabditis elegans to acute oxidative stress and heat stress, and the oxidative stress survival rate is improved by 22.9% and the heat stress survival rate is improved by 23.33% when the concentration of the extract is 4 mg / mL, and the extract has excellent antioxidant and cell protection activities. The Perinereis aibuhitensis extract has significant application value in the preparation of functional food, health products, cosmetics or medicines for delaying aging, resisting oxidation, relieving heat stress damage, improving reproduction capacity and prolonging life, and provides a theoretical basis and key technical support for developing the marine source anti-aging product. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 The figure is the hydroxyl radical scavenging capacity of EPU-1, EPU-2 and EPU-3. A. The hydroxyl radical scavenging rate curves of the three components; B. The IC50 values of the hydroxyl radical scavenging rate of the three components. 50 **** represents significant difference P <0.0001, same below.

[0015] Figure 2 The figure is the superoxide anion radical scavenging capacity of EPU-1, EPU-2 and EPU-3. A. The superoxide anion scavenging rate curves of the three components; B. The IC50 values of the superoxide anion scavenging rate of the three components.

[0016] Figure 3 The figure is the influence of the Perinereis aibuhitensis ultrafiltration component on the life of the Caenorhabditis elegans under acute oxidative stress. A. The 1 mg / mL treatment group of the three components; B. The 4 mg / mL treatment group of the three components; C. The 8 mg / mL treatment group of the three components.

[0017] Figure 4Effects of the three kinds of Nereis super-filtered components on the life span of nematodes. Among them, A. different dose concentrations of EPU-1 treatment group; B. different dose concentrations of EPU-2 treatment group; C. different dose concentrations of EPU-3 treatment group.

[0018] Figure 5 Effects of the three kinds of Nereis super-filtered components on the life span of nematodes. Among them, A. different dose concentrations of EPU-1 treatment group; B. different dose concentrations of EPU-2 treatment group; C. different dose concentrations of EPU-3 treatment group.

[0019] Figure 6 Effects of the three kinds of Nereis super-filtered components on the movement ability of nematodes. Among them, A. head and tail swing times on the 3rd day; B. head and tail swing times on the 6th day; C. head and tail swing times on the 9th day; "*" indicates significant difference P<0.05; "**" indicates significant difference P<0.01; "***" indicates significant difference P<0.01; "****" indicates significant difference P<0.0001.

[0020] Figure 7 Effects of the three kinds of Nereis super-filtered components on the reproduction ability of nematodes. Among them, "*" indicates significant difference P<0.05 .

[0021] Figure 8 Effects of the three kinds of Nereis super-filtered components on the fluorescence intensity of lipofuscin in nematodes.

[0022] Figure 9 Effects of the three kinds of Nereis super-filtered components on the relative content of lipofuscin in nematodes. Among them, "****" indicates significant difference P< 0.0001.

[0023] Figure 10 Effects of EPU-1 on the expression level of IIIS pathway related genes of nematodes. Among them, A. daf-2 gene level expression; B. age-1 gene level expression; C. daf-16 gene level expression; D. skn-1 gene level expression; "*" indicates significant difference P<0.05; "**" indicates significant difference P<0.01. DETAILED DESCRIPTION

[0024] The technical solutions described in the present application are conventional solutions in the art if not specifically stated, and the reagents or raw materials used are purchased from commercial channels or are already disclosed if not specifically stated.

[0025] The present application provides a preparation method of Perinereis aibuhitensis extract, comprising the following steps: The Perinereis aibuhitensis is homogenized, alkaline protease is added for enzymolysis, the supernatant is taken after centrifugation, and the Nereis super-filtered components less than 5 kDa are collected by passing through a ultrafiltration membrane, that is, the Perinereis aibuhitensis extract.

[0026] In some specific embodiments, the homogenization condition is homogenization with water at a ratio of 1:5 (g:mL) of material to water; the amount of alkaline protease is 3000 u / g; the enzymolysis condition is pH=8.5, enzymolysis temperature is 50℃, and enzymolysis time is 7 h.

[0027] In some specific embodiments, the centrifugation condition is centrifugation at 8000 r / min at 4℃ for 20 min.

[0028] The present application also provides a Perinereis aibuhitensis extract prepared by the preparation method.

[0029] The present application also provides application of the Perinereis aibuhitensis extract in preparation of a product for improving reproductive capacity.

[0030] The present application also provides application of the Perinereis aibuhitensis extract in preparation of a product for delaying aging.

[0031] The present application also provides application of the Perinereis aibuhitensis extract in preparation of an antioxidant product.

[0032] The present application also provides application of the Perinereis aibuhitensis extract in preparation of a product for relieving heat stress damage.

[0033] The present application also provides application of the Perinereis aibuhitensis extract in preparation of a product for prolonging life span.

[0034] In some specific embodiments, the Perinereis aibuhitensis extract prolongs the life span of the nematode by down-regulating genes daf-2 and age-1 and up-regulating genes daf-16 and skn-1, thereby interfering with the growth and development, energy metabolism and stress resistance of the nematode.

[0035] Example 1 1. Materials and methods 1.1. Materials and instruments Perinereis aibuhitensis: Suixi Tengfei Biotechnology Co., Ltd.; N2 wild-type Caenorhabditis elegans and OP50 Escherichia coli: Fujian Shangyuan Biological Science and Technology Co., Ltd.; Alkaline protease (200 U / mg): Shanghai Yuanye Biotechnology Co., Ltd.; Phenanthroline, epigallocatechin gallate (EGTA), tris(hydroxymethyl) aminomethane: National Pharmaceutical Group Chemical Reagent Co., Ltd.; Ferrous sulfate: Guangzhou Chemical Reagent Factory; NGM nematode culture medium: Shijiazhuang Simo Technology Co., Ltd.; LB agar: Beijing Land Bridge Technology Co., Ltd. LB broth, agar powder: Beijing Solabio Technology Co., Ltd. Tetramisole hydrochloride, 5-fluorodeoxyuridine (FUdR): Shanghai Maikelin Biotechnology Co., Ltd. Sodium hypochlorite: Shanghai Yianhua Technology Co., Ltd. Sodium hydroxide: Xilong Scientific Co., Ltd.

[0036] DF-101T heat collecting constant temperature heating magnetic stirrer: Shanghai Kesheng Instrument Co., Ltd. Thermo Lynx 6000 high-speed floor centrifuge: Thermo Fisher Scientific, USA Varioskan LUX full-automatic enzyme marker: Thermo, USA Masterflex L / S ultrafiltration equipment: Shanghai Merscy Scientific Instrument Co., Ltd. EYELA N-1300 rotary evaporator: Shanghai Ailang Instrument Co., Ltd. LGJ-12 freeze dryer: Beijing Songyuan Huaxing Technology Development Co., Ltd. FOSS automatic Kjeldahl nitrogen determination instrument Kjeltec 8200: Shanghai Ruixin International Trade Co., Ltd. SOX606 full-automatic fat determination instrument: Haikongshi Technology Co., Ltd. B60F upright fluorescence microscope: Guangzhou Daoyi Science and Technology Co., Ltd. SW-CJ-2F double-person single-face vertical air supply purification workbench: Suzhou Bolier Purification Equipment Co., Ltd. SRL7045 body microscope: Beijing Tiannuo Xiang Science and Technology Instrument Co., Ltd. FQD-96C fluorescent quantitative PCR instrument: Hangzhou Borys Technology Co., Ltd. SMA4000 micro nucleic acid quantification instrument: Meilin Hengtong (Beijing) Instrument Co., Ltd. 9600 gene amplification instrument: Zhuhai Black Horse Biotechnology Co., Ltd.

[0037] 1.2 Experimental method 1.2.1 Preparation of perinacean ultrafiltration components Fresh Perinereis exuviae were homogenized and water was added at a ratio of 1 : 5 (g : mL). Alkaline protease was added at a ratio of 3000 u / g of raw material, and the pH was adjusted to 8.5. The mixture was stirred in a constant-temperature water bath at 50°C for 7 h, and then the enzyme was inactivated in a boiling water bath for 10 min. The mixture was centrifuged at 8000 r / min for 20 min at 4°C, and the supernatant was collected. The supernatant was sequentially filtered through 8 kDa and 5 kDa ultrafiltration membranes to obtain Perinereis exuviae ultrafiltration components with molecular weights greater than 8 kDa (EPU-3), 5-8 kDa (EPU-2), and less than 5 kDa (EPU-1). The components were freeze-dried for later use.

[0038] 1.2.2 Evaluation of the in vitro antioxidant activity of Perinereis exuviae ultrafiltration components 1.2.2.1 Determination of hydroxyl radical scavenging capacity The experimental method of Zhu et al. was used with appropriate modifications. The following were added to a test tube in order: 1.2 mL 1, 10-phenanthroline (5 mmol / L), 0.8 mL phosphate buffer (0.2 mol / L, pH=7.4), 2 mL sample solution, 1.2 mL EDTA-2Na (15 mmol / mL), 1.2 mL FeSO4 (5 mmol / mL), 1.6 mL H2O2 (0.1%), and water to a final volume of 10 mL. The mixture was mixed and incubated in a water bath at 37°C for 90 min. After centrifugation, the absorbance was measured at a wavelength of 536 nm using a UV spectrophotometer. The damage group: instead of the sample, ultrapure water was used, and the other steps were the same as for the sample tube. The non-damage group: instead of H2O2, ultrapure water was used, and the other steps were the same as for the damage tube. Three replicates were performed for each concentration, and the results were expressed as the hydroxyl radical scavenging rate: ; In the formula, A0 represents the absorbance value of the sample group, A1 represents the absorbance value of the damage group, and A2 represents the absorbance value of the non-damage group.

[0039] 1.2.2.2 Determination of superoxide anion scavenging capacity The experimental method of Alashi et al. was used with appropriate modifications. The following were added to a test tube: 1 mL sample, 4.5 mL Tris-HCL buffer (0.01 mol / L, pH=8.2), and the mixture was incubated at 25°C for 20 min. Then, 4.2 mL ultrapure water and 0.3 mL preheated pyrogallol solution at 25°C were added, and the mixture was quickly mixed and poured into a cuvette. The absorbance A1 was measured at a wavelength of 325 nm every 0.5 min for a total of 5 min. The absorbance change rate ΔA1 of the control solution was calculated, and the same volume of water was used as a blank control to measure the absorbance change rate ΔA0. The final results were expressed as the superoxide anion scavenging rate: ; Wherein: ΔA0 represents the rate of change of absorbance over time; ΔA1 represents the rate of change of absorbance over time for the control solution.

[0040] 1.2.3 Culture and synchronization of C. elegans Refer to the method of Wei Runxia with slight modification: Preparation of nematode growth medium (NGM): After autoclaving the medium at 121 ℃, pour it into a 60 mm sterile culture dish and cool it to solidify. Add 50 μL of OP50 bacterial solution to the culture plate, dry it, and culture the bacterial plaque in a 20 ℃ incubator to obtain a nematode culture plate. Preparation of nematode lysis solution: Mix 1 mol / L NaOH solution, 10% sodium hypochlorite, and sterile water in a volume ratio of 1:1:8, and use immediately after preparation.

[0041] Synchronization of nematodes: Wash the nematodes to be oviposited into a 2 mL centrifuge tube with M9 buffer, and let it stand until the nematodes are precipitated. Remove the supernatant after repeating the washing 3 times, add 1 mL of lysis solution, and shake for 1-2 min to completely lyse the nematodes. Centrifuge for 30 s, remove the supernatant to obtain the egg precipitate, and repeat the washing with M9 buffer for 5-6 times until the pungent smell disappears. Transfer the washed eggs to NGM medium containing OP50, and incubate in a 20 ℃ incubator for 48 h to obtain synchronized nematodes.

[0042] 1.2.4 Effect of E. brevis ultrafiltration components on nematodes under acute oxidative stress injury Collect L4 synchronized nematodes, and treat them with EPU-1, EPU-2, and EPU-3 at concentrations of 1, 4, and 8 mg / mL, respectively. Set up a control group, with 3 replicates in each group, and 30 nematodes in each replicate. After 3 days of culture, transfer the nematodes to a culture plate containing 10 μL of 30% hydrogen peroxide per 10 mL of medium. Observe every 1 h, count the number of surviving, disappearing, and dead nematodes, and draw a survival curve until all nematodes die.

[0043] 1.2.5 Effect of E. brevis ultrafiltration components on nematodes under heat stress injury Collect L4 synchronized nematodes, and treat them with EPU-1, EPU-2, and EPU-3 at concentrations of 1, 4, and 8 mg / mL, respectively. Set up a control group, with 3 replicates in each group, and 30 nematodes in each replicate. After 3 days of culture, transfer the nematodes to a culture plate without food, and incubate in a 35 ℃ incubator for 8 h. Then transfer them to a 20 ℃ incubator for recovery for 12 h, count the number of surviving nematodes, and calculate the survival rate.

[0044] 1.2.6 Effect of Nereis Ultrafiltration Components on the Lifespan of Nematodes Collect L4 synchronized nematodes, and randomly transfer the nematodes to NGM plates containing OP50 E. coli and 150 mmol / L FUdR, and treat with 50 μL of EPU-1, EPU-2, and EPU-3 at different concentrations (1, 4, and 8 mg / mL), and use ultrapure water instead of the samples in the control group, 3 parallels in each group, 30 worms in each parallel, and culture in a 20°C incubator. The day of transfer is recorded as day 0, and every 24 h, replace the NGM plates and record the number of surviving nematodes, the number of disappeared nematodes, and the number of dead nematodes (slightly touch with a needle without any reaction and in a rigid state, which is considered dead), until all nematodes die, and draw a survival curve.

[0045] 1.2.7 Effect of Nereis Ultrafiltration Components on the Movement Ability of Nematodes Collect L4 synchronized nematodes, and treat the nematodes according to the method of 1.2.6, set up a control group, 3 parallels in each group, 5 nematodes in each parallel, and observe and record the number of movements of the nematodes on days 3, 6, and 9 during the culture period. The method for measuring the movement of nematodes is as follows: pick one nematode and place it in M9 buffer, and after 30 s of adaptation, observe and record the number of movements of the nematode within 10 s (one back-and-forth swing of the head and tail of the nematode is counted as one movement).

[0046] 1.2.8 Effect of Nereis Ultrafiltration Components on the Reproduction Ability of Nematodes Collect L4 synchronized nematodes, and treat the nematodes according to the method of 1.2.6 (without FUdR), set up a control group, 6 parallels in each group, 1 nematode in each parallel, and record day 1 as the day when the nematodes start laying eggs, and start counting the number of eggs laid at the same time, replace the culture plates and count every 24 h, until the end of egg laying, and count the total number of eggs laid.

[0047] 1.2.9 Effect of Nereis Ultrafiltration Components on the Accumulation of Lipofuscin in Nematodes Collect L4 synchronized nematodes, and treat the nematodes according to the method of 1.2.6, set up a control group, 3 parallels in each group, 15 nematodes in each parallel, after 5 d of culture, anesthetize the nematodes with 0.1% tetramisole hydrochloride solution, transfer to 2% agarose slides, observe under a fluorescence microscope and collect pictures, and analyze the average fluorescence intensity of the nematodes using Image J software.

[0048] 1.2.10 Determination of the expression level of aging-related genes Collect L4 synchronized nematodes, treat with EPU-1 (4 mg / mL) for 72 h, set up a control group, 3 parallels in each group, rinse with M9 buffer solution into 2 mL EP tubes, about 1000 worms per tube, -80°C quick freezing. Total RNA is extracted using a kit and cDNA is synthesized by reverse transcription. Real-time quantitative PCR is performed using SYBR dye method. PCR amplification conditions: 95°C pre-denaturation for 2 min, 95°C denaturation for 10 s, 60°C annealing for 30 s, a total of 45 cycles. The mRNA expression levels of genes daf-2, daf-16, age-1, skn-1 are evaluated by RT-qPCR, actin is the internal reference gene, and the gene expression is calculated by 2 -ΔΔCt -ΔΔCt method, and the primer design is shown in Table 1.

[0049] Table 1 Primer sequences used in quantitative polymerase chain reaction

[0050] 1.3 Data processing The experiments were repeated 3 times, and the experimental data were processed, analyzed and plotted using GraphPad Prism 9 and Image J software, and significant analysis was performed using IBM SPSS Statistics 27, and the experimental results were expressed as mean ± standard deviation.

[0051] 2 Results and analysis 2.1 Evaluation of the in vitro antioxidant activity of Perinereis nuntia ultrafiltration components 2.1.1 Hydroxyl radical scavenging capacity The hydroxyl radical scavenging rates of different mass concentrations of Perinereis nuntia ultrafiltration components are shown in Figure 1 Table 1. All three Perinereis nuntia ultrafiltration components have certain hydroxyl radical scavenging capacity, and there is a dose relationship with the mass concentration. With the increase of concentration, the hydroxyl radical scavenging rates of the three ultrafiltration components show an upward trend. The IC 50 values of EPU-1, EPU-2 and EPU-3 are 5.64 ± 0.11, 6.21 ± 0.01 and 7.02 ± 0.01 mg / mL, respectively, and the hydroxyl radical scavenging capacity of EPU-1 is significantly higher than that of EPU-2 and EPU-3 P<0.0001 .

[0052] 2.1.2 Superoxide anion scavenging capacity As shown in Figure 2 , the superoxide anion radical scavenging capacity of the three Perinereis nuntia ultrafiltration components increases with the increase of mass concentration, and there is a dose relationship, and EPU-1 has the highest scavenging rate and EPU-3 has the lowest scavenging rate. The IC 50The values ​​were 1.45±0.05, 2.30±0.11, and 3.14±0.09 mg / mL, respectively. EPU-1's superoxide anion scavenging capacity was significantly higher than that of EPU-2 and EPU-3. P<0.0001 ).

[0053] 2.2 Effects of ultrafiltration components from *Nematodes nervosa* on the lifespan of nematodes damaged by acute oxidative stress Hydrogen peroxide, a common reactive oxygen species in cells, can cause oxidative damage to the structure and molecular function of cells when excessive amounts are induced. This invention determined the effects of different concentrations of ultrafiltration components from *Nematodes nereis* on the lifespan of nematodes under hydrogen peroxide-induced acute oxidative stress (results are shown in Table 3). Figure 3 ).like Figure 3 As shown in Figure A, at a concentration of 1 mg / mL, the survival rates of the EPU-1, EPU-2, and EPU-3 groups increased by 6.6%, 3.7%, and 2.9%, respectively, with no significant difference compared to the control group. P> 0.05 );like Figure 3 As shown in Figure B, at a concentration of 4 mg / mL, the survival rate of nematodes in the EPU-1 treatment group was significantly increased by 22.9% ( P< 0.05 The EPU-2 and EPU-3 groups prolonged the average lifespan of nematodes to some extent, but without significant change. P>0.05 );like Figure 3 In C, when the concentration was increased to 8 mg / mL, the survival rate of the EPU-1 group significantly increased by 13.4% ( P<0.05 However, there were no significant changes in the EPU-2 and EPU-3 groups. P>0.05 In summary, EPU-1 has a good free radical scavenging ability. By eliminating free radicals in the body, it can reduce the damage caused by free radicals, improve the antioxidant stress resistance of nematodes, and thus delay aging.

[0054] Table 2. Effects of ultrafiltration components on lifespan of Nematodes under acute oxidative stress.

[0055] 2.3 Effects of ultrafiltration components from *Nematodes nervosa* on heat stress damage in nematodes High temperatures of 35°C cause metabolic disorders in nematodes, leading to the accumulation of reactive oxygen species and resulting in cell and tissue damage, thereby shortening their lifespan. For example... Figure 4 As shown in Figure A, after heat stress treatment, the survival rate of the control group was 55.83%, while the survival rates of nematodes in the EPU-1, EPU-2, and EPU-3 treatment groups (1 mg / mL) were 66.67%, 60.83%, and 56.67%, respectively. Compared with the control group, the EPU-1 group showed a significant increase of 10.84%. P<0.01The EPU-2 and EPU-3 groups showed increases of 5.00% and 0.84% ​​respectively, but the differences were not statistically significant. P>0.05 );like Figure 4 As shown in Figure B, the survival rate of nematodes in the EPU-1 and EPU-2 treatment groups at a concentration of 4 mg / mL was significantly increased by 23.33% ( P< 0.0001 ) and 13.33% P<0.01 ), while the EPU-3 group improved by 5.83% ( P>0.05 The EPU-1 group showed the best results; Figure 4 As shown in Figure C, the survival rate of nematodes in the EPU-1 and EPU-2 treatment groups at a concentration of 8 mg / mL was significantly increased by 15.00%. P< 0.001 ) and 9.17% P<0.05 The EPU-3 group improved by 3.33% ( P>0.05 The results showed that EPU-1, EPU-2, and EPU-3 could improve the survival rate of nematodes after heat stress to a certain extent, with EPU-1 having a stronger effect. In conclusion, the ultrafiltration components of *Nematodes nereis* have a protective effect against heat stress damage in nematodes and can improve their heat stress resistance.

[0056] 2.4 Effects of ultrafiltration components on nematode lifespan Lifespan is the most important indicator for measuring the aging of nematodes. For example... Figure 5 As shown in Table 4, compared with the control group, all three polychaete ultrafiltration components at different concentrations prolonged the average lifespan of nematodes, shifting the survival curves to the right. Under normal culture conditions, the average lifespan of nematodes was 11.49 ± 0.56 days. After treatment with different concentrations (1, 4, 8 mg / mL) of EPU-1 component, compared with the control group, the average lifespan of nematodes was prolonged by 8.4%, 13.9%, and 7.3%, respectively, with the effect being most significant at a concentration of 4 mg / mL. P<0.05 This extended the maximum lifespan of nematodes from 18 days to 22 days. While both EPU-2 and EPU-3 groups showed some degree of extension in nematode lifespan, the difference was not significant. P>0.05 In summary, the ultrafiltration components of *Nematodes nervosa* can prolong the lifespan of nematodes, with EPU-1 showing the best effect at a mass concentration of 4 mg / mL.

[0057] Table 3. Effects of ultrafiltration components on the lifespan of *C. elegans*

[0058] 2.5 Effects of ultrafiltration components on the motility of Nematodes The motility of nematodes can directly reflect the degree and state of aging of their nervous system. This invention uses the number of head-tail swings per 10 seconds in M9 buffer as a physiological indicator to explore the effect of polychaete ultrafiltration components on the motility of nematodes. Experimental results show that on days 3, 6, and 9, the number of swings in the control group nematodes was 14.82, 7.87, and 2.33, respectively. Over time, the number of head-tail swings significantly decreased. P<0.05 ).like Figure 6 As shown in Figure A, after 3 days of treatment with the ultrafiltration fraction of *Nematodes*, the motility of the nematodes was similar to that of the control group, with no significant difference. P>0.05 );like Figure 6 As shown in Figures B and C, with increasing time, the number of movements in the control group decreased on days 6 and 9. In contrast, the nematodes treated with all three polychaete ultrafiltration fractions showed increased head and tail movements. At a concentration of 4 mg / mL, the EPU-1 group exhibited 11.47 and 8.20 movements on days 6 and 9, respectively, representing increases of 3.60 and 5.87 movements compared to the control group, significantly increasing the number of nematode movements. P<0.001 In summary, the three polychaete ultrafiltration components can delay the aging of the nematode's nervous system, thereby improving the motility of aging nematodes.

[0059] 2.6 Effects of ultrafiltration components on nematode reproduction capacity Reproductive capacity is a key indicator of aging in many organisms. Aging leads to a shortened oviposition period and a significant decrease in egg production in nematodes. To investigate the effect of ultrafiltration components in *Nematodes* on extending nematode lifespan and thus their reproductive capacity, the total number of eggs produced by nematodes was statistically analyzed. Figure 7 As shown, the total number of eggs laid in the control group was 147.67. Compared with the control group, the number of eggs laid by nematodes increased at sample concentrations of 1 and 8 mg / mL, but the difference was not statistically significant. P>0.05 However, at a sample concentration of 4 mg / mL, the number of eggs laid by nematodes in the EPU-1 group was 178.17, which was significantly increased by 20.65% compared to the control group. P<0.05 In summary, the ultrafiltration components of *Nematodes nervosa* extended the lifespan of nematodes without reducing their reproductive capacity, and EPU-1 significantly promoted nematode reproduction at a sample concentration of 4 mg / mL.

[0060] 2.7 Effects of ultrafiltration fractions from *Nematocystis jirovecii* on lipofuscin accumulation in nematodes Lipofuscin, also known as "age plaques," is a marker of aging in nematodes. This invention analyzes the accumulation of lipofuscin in nematodes to explore the anti-aging effect of polychaete ultrafiltration components on nematodes. Figure 8 , Figure 9 As shown, compared with the control group, different concentrations of EPU-1, EPU-2, and EPU-3 all significantly reduced the accumulation of lipofuscin in nematodes.P<0.0001 ); with EPU-1 group, the average fluorescence intensity of EPU-1 nematodes at low and high dose concentrations was significantly less than that of EPU-3 group (P < 0.05) P<0.0001 ), and there was no significant difference with EPU-2 group (P > 0.05) P>0.05 ), and the relative average fluorescence intensity of EPU-1, EPU-2 and EPU-3 nematodes was 18.67 ± 6.48%, 44.85 ± 10.20% and 83.43 ± 6.04% respectively at a dose concentration of 4 mg / mL, and the effect of EPU-1 on reducing the accumulation of lipofuscin in nematodes was significantly better than that of EPU-2 and EPU-3 (P < 0.05) P<0.0001 ). In summary, all three kinds of super-filtered components of Arenicola can reduce the accumulation of lipofuscin in nematodes, and the effect of EPU-1 is the most significant.

[0061] 2.8 Effect of EPU-1 on the expression level of nematode aging-related genes The insulin / insulin-like growth factor (IGF) signaling (IIS) pathway is one of the typical signaling pathways that affect nematode lifespan, with high conservation, regulating many processes and traits of animals, including growth and development, energy metabolism, reproduction and stress resistance, etc. Down-regulating the insulin / insulin-like growth factor (IGF) signaling (IIS) pathway is a known intervention measure to prolong lifespan. The longevity of C. elegans is related to the increased expression level of daf-16 and the decreased expression level of daf-2. The missense mutation of daf-2 makes the lifespan of C. elegans double, which is one of the most important pioneering findings in the field of aging research. daf-2 is a growth factor receptor of the IIS pathway, which plays a major role in nematode growth and metabolism. daf-2 can down-regulate the metabolic activity of nematodes, reduce the expression of daf-2, and reduce the activity of the downstream kinase cascade, while activating the transcription factor daf-16, thereby prolonging the lifespan. The daf-16 gene of C. elegans is a FOXO homolog, which is a key transcription target of the IIS pathway, and plays an active role in regulating biological processes such as lifespan, stress and reproduction, and increasing its expression can prolong the lifespan of nematodes. age-1 is one of the classic longevity genes in nematodes, which encodes the catalytic subunit of phosphatidylinositol 3-kinase (PI3K), and PI3K is a key component of the insulin / insulin-like growth factor (IIS) signaling pathway, which plays an important regulatory role in metabolism, growth and lifespan; skn-1 is a key transcription factor in C. elegans, which is a functional homolog of Nrf2 protein in mammals, and plays an important regulatory role in stress response and metabolic homeostasis.

[0062] In order to preliminarily understand the mechanism of the action of the Nereis enzymatic product on prolonging the life of the nematode, the present application further explores the EPU-1 in three ultrafiltration components which has a significant effect on delaying the life and improving the stress resistance of the nematode, and determines the expression of the aging genes of the nematode after the intervention of the EPU-1. The experimental results are shown in Table 1 Figure 10 As shown in Table 1, after the intervention of the EPU-1, compared with the control group, the mRNA expression levels of daf-2 and age-1 are significantly reduced (A, B), which are reduced by 21% (A) and 18% (B) respectively, and the mRNA expression levels of daf-16 and skn-1 are significantly improved (C, D), which are increased by 28% (C) and 23% (D) respectively. Figure 10 P<0.01 P< Figure 10 P<0.01 P<0.05 Therefore, the EPU-1 can intervene in the growth and development, energy metabolism, stress resistance and the like of the nematode by down-regulating the genes daf-2 and age-1 and up-regulating the genes daf-16 and skn-1, so as to prolong the life of the nematode.

[0063] Obviously, the above embodiments of the present application are only examples for clearly illustrating the present application, and are not intended to limit the embodiments of the present application. Based on the above description, other different forms of changes or variations can be made by the ordinary skilled in the art. It is not necessary or possible to exhaust all the embodiments. Any modification, equivalent replacement and improvement made within the spirit and principle of the present application should be included in the protection scope of the claims of the present application.​​​​​

Claims

1. A method for preparing an extract of Perinereis borbonica, characterized in that, The method comprises the following steps: The Perinereis aibuhitensis is homogenized and added with alkaline protease for enzymolysis, and the supernatant is obtained by centrifugation, and the Perinereis aibuhitensis ultrafiltration component less than 5kDa is collected by passing through an ultrafiltration membrane, so as to obtain the Perinereis aibuhitensis extract.

2. The production method according to claim 1, characterized by, The homogenization is performed by adding water in a ratio of 1:5 (g:mL), the amount of the alkaline protease is 3000 u / g, the enzymolysis is performed at pH=8.5 and a temperature of 50℃ for 7 h.

3. The production method according to claim 1, characterized by, The centrifugation is performed at 8000 r / min at 4℃ for 20 min.

4. The Perinereis aibuhitensis extract prepared by the method of any one of claims 1-3.

5. The Perinereis aibuhitensis extract of claim 4 is used for preparing a product for improving reproductive capacity.

6. The Perinereis aibuhitensis extract of claim 4 is used for preparing a product for delaying aging.

7. The Perinereis aibuhitensis extract of claim 4 is used for preparing an antioxidant product.

8. The Perinereis aibuhitensis extract of claim 4 is used for preparing a product for relieving heat stress damage.

9. The Perinereis aibuhitensis extract of claim 4 is used for preparing a product for prolonging life.

10. Use according to claim 9, characterized in that, The Perinereis aibuhitensis extract prolongs the life of the nematode by down-regulating genes daf-2 and age-1 and up-regulating genes daf-16 and skn-1, and interfering with the growth and development, energy metabolism and stress resistance of the nematode.

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