Application of plasmalogen in assisting in gaining muscle and reducing fat in aerobic exercise and relieving skeletal muscle and kidney injury caused by exercise

Abstract

The invention discloses application of plasmalogen in assisting in gaining muscle and reducing fat in aerobic exercise and relieving skeletal muscle and kidney injury caused by exercise, and belongs to the technical field of biological medicine. According to the invention, a model of mouse skeletal muscle and kidney injury caused by exhaustion movement is established in vivo, and skeletal muscle and kidney injury is relieved by intervention of a sea squirt phospholipid extract and sea squirt plasmalogen respectively. Skeletal muscle injury and metabolic waste entering blood of a C57BL / 6J mouse caused by continuous exhaustion exercise for 7 days greatly increase the kidney burden. The sea squirt phospholipid extract and the sea squirt plasmalogen relieve muscle and kidney injury caused by exhaustive exercise and improve exercise performance.

Description

Technical Field

[0001] The present invention relates to the application of plasmalogen in assisting muscle growth and fat reduction during aerobic exercise and alleviating skeletal muscle and kidney damage caused by exercise, belonging to the technical field of biomedicine. Background Art

[0002] Ascidian sea squirts are the most common marine invertebrates, belonging to the phylum Chordata and subphylum Urochordata. Ascidian sea squirts are rich in phospholipids, particularly plasmalogens, which have previously been reported to alleviate Alzheimer's disease and neurodegenerative diseases. Ascidian sea squirts are commonly consumed as sashimi in countries like Japan and South Korea, and phospholipids extracted from ascidian sea squirts are more reliably safe for consumption. As major components of cell membranes, energy sources, and exogenous antioxidants, phospholipids may protect the human body through multiple pathways. Based on this, we explored the possible role of ascidian plasmalogens during exercise.

[0003] Exercise is an important way to maintain human health. Various aerobic exercises, such as yoga, long-distance running, and swimming, are considered effective means of weight loss. However, in addition to burning fat, exercise and weight loss can also damage skeletal muscle. Therefore, how to promote fat loss and minimize skeletal muscle loss after exercise is crucial for athletic performance.

[0004] In addition, with the rapid expansion of the sports population, cases of sports injuries have frequently occurred, causing social concern. For people's daily exercise, choosing the right exercise intensity, maintaining adequate rest time, and timely rehydration after exercise are important measures to alleviate sports injuries. However, for the military, sports practitioners, and people who want to lose weight and keep fit, they need to participate in high-intensity exercise to meet their exercise needs. Providing sports supplements is an effective strategy to combat sports injuries, and sugary sports drinks may aggravate oxidative stress after exercise and stimulate sports injuries. Therefore, exploring a non-sugar sports supplement is of great significance. There is an urgent need for a health food that can relieve the damage to skeletal muscle and kidney caused by long-term aerobic exercise, especially exhaustive exercise, to meet the needs of high-intensity exercise, while promoting fat consumption and reducing skeletal muscle loss after exercise. Summary of the Invention

[0005] In response to the current situation, the present invention aims to provide a plasmalogen extracted from sea squirts to enhance athletic performance and alleviate skeletal muscle and kidney damage caused by aerobic exercise, particularly exhaustive exercise (EX). Exhaustive exercise damages skeletal muscle and kidneys. The plasmalogen provided by the present invention can reduce skeletal muscle and kidney damage, reduce skeletal muscle mass loss, and improve athletic performance in mice, providing a protective effect on exercise.

[0006] The present invention provides an application of plasmalogen in preparing a product for assisting muscle gain and fat loss during aerobic exercise; the muscle gain and fat loss is to reduce muscle loss and increase fat consumption during aerobic exercise.

[0007] The product of the present invention can be used as a health product for weight loss through aerobic exercise. It can reduce muscle loss during aerobic exercise while increasing fat consumption and improving exercise capacity. It can be used as an effective weight loss product.

[0008] The present invention also provides a use of plasmalogen in preparing a product for preventing, improving and / or treating skeletal muscle damage and kidney damage caused by aerobic exercise.

[0009] In one embodiment, the aerobic exercise includes but is not limited to exhaustive exercise.

[0010] In one embodiment, the product includes, but is not limited to, one or more of a drug, a health food, a food for special medical purposes, a food, a functional food, a feed, a feed additive, or a pet food;

[0011] In one embodiment, the product is a sports food, a sports supplement, or a sports nutrition product.

[0012] In one embodiment, the food is a health drink.

[0013] In one embodiment, the preparation method of the plasmalogen is as follows:

[0014] (i) adding water to the inner sheath of the ascidian and mixing the mixture and then homogenizing the mixture to obtain a homogenate;

[0015] (ii) centrifuging the homogenate obtained in step (i) to obtain a precipitate, mixing the precipitate with methanol, dichloromethane, and water, stirring, and centrifuging to obtain a supernatant;

[0016] (iii) adding water and dichloromethane to the supernatant obtained in step (ii), allowing the mixture to stand, collecting the lower organic phase, collecting the precipitate by rotary evaporation, and washing the precipitate with hexane to obtain a scinothiolane extract;

[0017] (iv) dissolving the ascidian phospholipid extract obtained in step (iii) in dichloromethane, purifying the total lipids by silica gel column chromatography, eluting the neutral esters in the purified lipids by using dichloromethane and methanol as eluents, and collecting the eluate; subjecting the eluate to thin layer silica gel plate chromatography, and confirming the color development with an iodine reagent, and then rotary evaporating to remove the residual organic solvent to obtain a crude ascidian lipid extract;

[0018] (v) dissolving the crude ascidian lipid extract obtained in step (iv) in n-hexane, adding phospholipase A1 and sodium citrate buffer for incubation, extracting the phospholipids treated with phospholipase A1 with n-hexane and isopropanol, collecting the hexane layer, washing it with physiological saline, and rotary evaporating the n-hexane to obtain plasmalogen.

[0019] In one embodiment, the dosage form of the drug is a liquid preparation or a solid preparation;

[0020] In one embodiment, the dosage form of the drug includes but is not limited to granules, capsules, tablets, pills or oral solutions;

[0021] In one embodiment, the drug further comprises a pharmaceutically acceptable excipient;

[0022] In one embodiment, the pharmaceutical excipients include: any one or more of solubilizers, emulsifiers, colorants, binders, disintegrants, fillers, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrities, pH regulators, buffers, plasticizers, defoamers, thickeners, humectants, filter aids, and release retardants.

[0023] In one embodiment, in step (i), the mass volume ratio (g / ml) of the ascidian inner sheath to water is: (1-2): (1-2);

[0024] In one embodiment, in step (ii), the methanol is added in a ratio of (90-100) to the ascidian homogenate (ml / g): (70-80), the dichloromethane is added in a ratio of (40-50) to the ascidian homogenate (ml / g): (70-80), and the water is added in a ratio of (30-40) to the ascidian homogenate (ml / g): (70-80);

[0025] In one embodiment, in step (iii), the dichloromethane is added in a ratio (ml / g) of (40-50) to the ascidian homogenate: (70-80), and the water is added in a ratio (ml / g) of (30-40) to the ascidian homogenate: (70-80);

[0026] In one embodiment, in step (iv), the dichloromethane is added to the ascidian homogenate at a ratio (ml / g) of (8-10): (70-80); the eluent chloromethane and methanol are mixed at a volume ratio of (10-12):1;

[0027] In one embodiment, in step (v), the pH of the sodium citrate buffer is 4-5; the added enzyme activity of the phospholipase A1 is 400-450 U; and the incubation time is 2-5 h.

[0028] The present invention also provides a use of plasmalogen in preparing products for resisting fatigue and improving exercise performance.

[0029] In one embodiment, the product includes, but is not limited to, one or more of a drug, a health food, a food for special medical purposes, a food, a functional food, a feed, a feed additive, or a pet food;

[0030] In one embodiment, the preparation method of the plasmalogen is as follows:

[0031] (i) adding water to the inner sheath of the ascidian and mixing the mixture and then homogenizing the mixture to obtain a homogenate;

[0032] (ii) centrifuging the homogenate obtained in step (i) to obtain a precipitate, mixing the precipitate with methanol, dichloromethane, and water, stirring, and centrifuging to obtain a supernatant;

[0033] (iii) adding water and dichloromethane to the supernatant obtained in step (ii), allowing the mixture to stand, collecting the lower organic phase, collecting the precipitate by rotary evaporation, and washing the precipitate with hexane to obtain a scinothiolane extract;

[0034] (iv) dissolving the ascidian phospholipid extract obtained in step (iii) in dichloromethane, purifying the total lipids by silica gel column chromatography, eluting the neutral esters in the purified lipids by using dichloromethane and methanol as eluents, and collecting the eluate; subjecting the eluate to thin layer silica gel plate chromatography, and confirming the color development with an iodine reagent, and then rotary evaporating to remove the residual organic solvent to obtain a crude ascidian lipid extract;

[0035] (v) dissolving the crude ascidian lipid extract obtained in step (iv) in n-hexane, adding phospholipase A1 and sodium citrate buffer for incubation, extracting the phospholipids treated with phospholipase A1 with n-hexane and isopropanol, collecting the hexane layer, washing it with physiological saline, and rotary evaporating the n-hexane to obtain plasmalogen.

[0036] In one embodiment, the dosage form of the drug is a liquid preparation or a solid preparation;

[0037] Preferably, the dosage form of the drug includes but is not limited to granules, capsules, tablets, pills or oral liquids;

[0038] Preferably, the drug further comprises a pharmaceutically acceptable excipient;

[0039] Preferably, the pharmaceutical excipients include: any one or more of solubilizers, emulsifiers, colorants, adhesives, disintegrants, fillers, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrities, pH regulators, buffers, plasticizers, defoamers, thickeners, humectants, filter aids and release retardants.

[0040] The present invention also provides a drug for preventing, improving and / or treating skeletal muscle damage and kidney damage caused by aerobic exercise, wherein the drug contains plasmalogen, and the aerobic exercise includes but is not limited to exhaustive exercise.

[0041] The preparation method of the plasmalogen is as follows:

[0042] (i) adding water to the inner sheath of the ascidian and mixing the mixture and then homogenizing the mixture to obtain a homogenate;

[0043] (ii) centrifuging the homogenate obtained in step (i) to obtain a precipitate, mixing the precipitate with methanol, dichloromethane, and water, stirring, and centrifuging to obtain a supernatant;

[0044] (iii) adding water and dichloromethane to the supernatant obtained in step (ii), allowing the mixture to stand, collecting the lower organic phase, collecting the precipitate by rotary evaporation, and washing the precipitate with hexane to obtain a scinothiolane extract;

[0045] (iv) dissolving the ascidian phospholipid extract obtained in step (iii) in dichloromethane, purifying the total lipids by silica gel column chromatography, eluting the neutral esters in the purified lipids by using dichloromethane and methanol as eluents, and collecting the eluate; subjecting the eluate to thin layer silica gel plate chromatography, and confirming the color development with an iodine reagent, and then rotary evaporating to remove the residual organic solvent to obtain a crude ascidian lipid extract;

[0046] (v) dissolving the crude ascidian lipid extract obtained in step (iv) in n-hexane, adding phospholipase A1 and sodium citrate buffer for incubation, extracting the phospholipids treated with phospholipase A1 with n-hexane and isopropanol, collecting the hexane layer, washing it with physiological saline, and rotary evaporating the n-hexane to obtain plasmalogen.

[0047] In one embodiment, in step (i), the mass volume ratio (g / ml) of the ascidian inner sheath to water is: (1-2): (1-2);

[0048] In one embodiment, in step (ii), the methanol is added in a ratio of (90-100) to the ascidian homogenate (ml / g): (70-80), the dichloromethane is added in a ratio of (40-50) to the ascidian homogenate (ml / g): (70-80), and the water is added in a ratio of (30-40) to the ascidian homogenate (ml / g): (70-80);

[0049] In one embodiment, in step (iii), the dichloromethane is added in a ratio (ml / g) of (40-50) to the ascidian homogenate: (70-80), and the water is added in a ratio (ml / g) of (30-40) to the ascidian homogenate: (70-80);

[0050] In one embodiment, in step (iv), the dichloromethane is added to the ascidian homogenate at a ratio (ml / g) of (8-10): (70-80); the eluent chloromethane and methanol are mixed at a volume ratio of (10-12): 1;

[0051] In one embodiment, in step (v), the pH of the sodium citrate buffer is 4-5; the added enzyme activity of the phospholipase A1 is 400-450 U; and the incubation time is 2-5 h.

[0052] In one embodiment, the dosage form of the drug is a liquid preparation or a solid preparation. Optionally, the dosage form of the drug includes but is not limited to granules, capsules, tablets, pills or oral liquids; preferably, the drug further includes pharmaceutically acceptable excipients; preferably, the pharmaceutical excipients include: any one or more of solubilizers, emulsifiers, colorants, adhesives, disintegrants, fillers, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrins, pH regulators, buffers, plasticizers, defoamers, thickeners, humectants, filter aids and release retardants.

[0053] In one embodiment, in step (i), the mass volume ratio (g / ml) of the ascidian inner sheath to water is: (1-2): (1-2);

[0054] In one embodiment, in step (ii), the methanol is added in a ratio of (90-100) to the ascidian homogenate (ml / g): (70-80), the dichloromethane is added in a ratio of (40-50) to the ascidian homogenate (ml / g): (70-80), and the water is added in a ratio of (30-40) to the ascidian homogenate (ml / g): (70-80);

[0055] In one embodiment, in step (iii), the dichloromethane is added in a ratio (ml / g) of (40-50) to the ascidian homogenate: (70-80), and the water is added in a ratio (ml / g) of (30-40) to the ascidian homogenate: (70-80);

[0056] In one embodiment, in step (iv), the dichloromethane is added to the ascidian homogenate at a ratio (ml / g) of (8-10): (70-80); the eluent chloromethane and methanol are mixed at a volume ratio of (10-12): 1;

[0057] In one embodiment, in step (v), the pH of the sodium citrate buffer is 4-5; the added enzyme activity of the phospholipase A1 is 400-450 U; and the incubation time is 2-5 h.

[0058] Beneficial effects

[0059] (1) The ascidian plasmalogen prepared by the present invention reduces the mass loss of gastrocnemius, soleus and skeletal muscles of mice caused by aerobic exercise, especially exhaustive exercise, and increases the mass consumption of brown fat and subcutaneous fat.

[0060] (2) The present invention establishes a mouse skeletal muscle and kidney damage model caused by aerobic exercise (exhaustive exercise) in vivo, and uses sea squamous phospholipid extract and sea squamous acetal phospholipid intervention to alleviate skeletal muscle and kidney damage. After 7 consecutive days of exhaustive exercise, the skeletal muscle damage and metabolic waste entering the blood of C57BL / 6J mice greatly increased the burden on the kidneys. Sea squamous phospholipid extract (100 mg / kg) and sea squamous acetal phospholipid (100 mg / kg, 50 mg / kg) alleviated the muscle and kidney damage caused by exhaustive exercise and improved exercise performance. Sea squamous extract intervention also alleviated the occurrence of inflammatory oxidation and cell apoptosis in skeletal muscle. Molecular docking and qPCR results showed that sea squamous phospholipids also upregulated the PI3K / mTOR / Akt pathway, promoted muscle synthesis and protein expression, and thus alleviated muscle damage.

[0061] Therefore, the ascidian plasmalogen prepared by the present invention improves the exercise performance of mice and prevents skeletal muscle and kidney damage in mice caused by exhaustive exercise. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] To more clearly illustrate the technical solution of this embodiment, the following briefly introduces the drawings required for the description of this embodiment. Obviously, the drawings described below are some embodiments of the present application. For those skilled in the art, other drawings can be derived from these drawings without inventive effort.

[0063] Figure 1 : HPLC chromatogram of ascidian plasmalogen.

[0064] Figure 2 : Exercise time and distance of mice in different groups.

[0065] Figure 3 : Skeletal muscle injury indicators in serum and skeletal muscle and kidney pathological staining images of mice in different groups.

[0066] Figure 4 : Flowchart of animal experiments.

[0067] Figure 5 : Comparison of DPPH clearance rates of four phospholipids; PLS: ascidian plasmalogen; AE: ascidian phospholipid extract; SL: soybean lecithin; EL: egg lecithin; lowercase letters on the columns in the figure indicate significant differences among different groups (p<0.05), and the same letters indicate no significant differences among the groups (p>0.05).

[0068] Figure 6 :(a) Serum creatinine and urea nitrogen levels in mice; (b) KIM-1 and NGAL gene expression in mouse kidney; (c) HE staining results of mouse kidney; PLS: ascidian plasmalogen + EX; AE: ascidian phospholipid extract + EX; EX: exhaustive exercise; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, or not significant (ns).

[0069] Figure 7 (a) SOD and CAT gene expression in skeletal muscle of mice in different groups; (b) SOD, CAT, GPX, and MDA levels in skeletal muscle of mice in different groups. PLS: ascidian plasmalogen + EX; AE: ascidian phospholipid extract + EX; EX: exhaustive exercise; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, or not significant (ns).

[0070] Figure 8 :(a) TNF-α, IL-1β, TGF-β and IL-10 levels in mouse skeletal muscle; (b) TNF-α, IL-1β and IL-10 gene expression in mouse skeletal muscle; Note: PLS: ascidian plasmalogen + EX; AE: ascidian phospholipid extract + EX; EX: exhaustive exercise; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, or not significant (ns).

[0071] Figure 9: Bax and Bcl2 gene expression levels in mouse skeletal muscle; Note: PLS: ascidian plasmalogen + EX; AE: ascidian phospholipid extract + EX; EX: exhaustive exercise; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, or not significant (ns). DETAILED DESCRIPTION

[0072] To make the objectives, technical solutions and advantages of the present invention more clear, the embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.

[0073] The sources of raw materials involved in the following examples are:

[0074] Sea squirts were purchased from Green Fresh Food Store; methanol, chloroform, hexane, and isopropanol were purchased from Sinopharm Shanghai; phospholipase A1 was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; SPF-grade C57BL / 6J mice (20 ± 2 g, about 6 weeks old) were purchased from Changzhou Cavens Company. Animal ethics were approved by the Animal Experiment Center of Jiangnan University (Animal Ethics Number JN.No20240830c0401010

[421] ). Soybean lecithin (SL) and egg lecithin (EL) were purchased from Sichuan Huakang Raw Materials Co., Ltd.

[0075] The detection methods involved in the following embodiments are:

[0076] Determination of phospholipid content in scinowin extracts:

[0077] The phospholipid content of scidia phospholipid extracts and scidia plasmalogens was determined using the molybdenum blue colorimetric method. Weigh 0.2197 g of potassium dihydrogen phosphate into a 200 mL volumetric flask and add deionized water to mix thoroughly to prepare a 10 μg / mL potassium dihydrogen phosphate standard solution. Dissolve 2.5 g of ammonium molybdate in 100 mL of deionized water to prepare 2.5% ammonium molybdate. Dissolve 5 g of ascorbic acid in 45 mL of deionized water to prepare 10% ascorbic acid. In six 10 mL centrifuge tubes, add 0.5 mL of nitric acid, followed by 0 mL, 0.1 mL, 0.2 mL, 0.3 mL, 0.4 mL, and 0.5 mL of potassium dihydrogen phosphate standard solution. Add 3.5 mL, 3.4 mL, 3.3 mL, 3.2 mL, 3.1 mL, and 3 mL of ultrapure water, respectively, 0.5 mL of 2.5% ammonium molybdate, and 0.5 mL of 10% ascorbic acid. Mix thoroughly and compare color at 820 nm.

[0078] Example 1: Preparation of plasmalogen

[0079] The specific steps are as follows:

[0080] 1. Preparation of plasmalogen

[0081] (1) Take two fresh sea squirts, about 60 ± 5 g, remove the black unusable part of the outer skin, and take out the yellow inner sheath, weighing about 40 ± 2 g. Cut the inner sheath into small pieces, add purified water (1:1, g / ml), and crush using a wall crusher, 15 seconds each time, 5 seconds apart, repeat 3 times, and collect 80 g of sea squirt homogenate.

[0082] (2) Place the ascidian homogenate in a 50 mL centrifuge tube, pre-cool it at 4°C, centrifuge it at 10,000 rpm for 1 hour, and collect the ascidian meat precipitate.

[0083] (3) Transfer the tunicate precipitate to a 1L beaker, add 100mL of methanol, 50mL of dichloromethane, and 40mL of ultrapure water, and stir at room temperature for 1 hour to fully extract the lipids in the tunicate. At this time, the tunicate is mixed in the solution and appears yellow-brown.

[0084] The ascidian flesh was removed by filtration and the supernatant was collected;

[0085] (4) Add 50 mL of dichloromethane and 40 mL of water to the supernatant obtained in step (3), let stand at 4°C to separate the layers, and collect the lower yellow-brown organic phase using a separatory funnel. The organic phase is then rotary evaporated at 40°C to collect the precipitate, which is then washed three times with hexane to obtain an ascidians phospholipid extract (AE).

[0086] (5) The sea squirt phospholipid extract obtained in step (4) was dissolved in 10 mL of dichloromethane and the total lipids were purified by silica gel column chromatography. Dichloromethane: methanol (10:1, v / v) was used as an eluent to remove the neutral esters in the purified lipids, and the eluent was collected. The eluent was subjected to thin layer silica gel plate chromatography (TLC), and the eluent was chloroform with an Rf value of 0.2, and iodine reagent was used for color development. After confirmation by iodine reagent color development, a crude sea squirt lipid extract was obtained;

[0087] (6) The crude lipid extract of the ascidian collected in step (5) was dissolved in 10 ml of n-hexane, and 10 ml of phospholipase A1 (40 U / mL) and 20 mL of sodium citrate buffer (pH 4.5) were added. After incubation for 4 hours, the crude lipid extract of the ascidian treated with phospholipase A1 was extracted with n-hexane:isopropanol (4:1, v / v). The hexane layer was collected and washed with physiological saline. The n-hexane was rotary evaporated to dryness to obtain ascidian plasmalogen (PLS).

[0088] 2. Detection of plasmalogen

[0089] The collected scinic phospholipid extract and scinic plasmalogen were diluted with hexane to 100 μg / mL, ultrasonicated at room temperature for 15 minutes, fully dispersed, and filtered through a 0.45 μm organic phase membrane to prepare samples for liquid chromatography-mass spectrometry.

[0090] Chromatographic conditions:

[0091] Mobile phase A was 60% acetonitrile + 40% water; mobile phase B was 90% isopropanol + 10% acetonitrile. The flow rate was 0.260 mL / min, the detection wavelength was 254 nm, the column temperature was 30°C, and the working time was 21 minutes (gradient elution is shown in Table 1). Mass spectrometry conditions: Waters MALDI SYNAPT quadrupole time-of-flight mass spectrometer; positive ion m / z detection range 300-1000, spray voltage 4500 V, and nebulizer gas 0.8 Bar.

[0092] Table 1: Mobile phase elution gradient

[0093]

[0094] Liquid chromatography test results Figure 1 As shown in Table 2, the types and proportions of phospholipid compounds in acetal phospholipids are shown in Table 2.

[0095] Table 2: The ten most abundant plasmalogens in ascidian plasmalogens

[0096]

[0097] The results in Table 2 show the proportion and peak time of the 10 most abundant phospholipids in the sea scirrhosa phospholipid extract. The peak time of phospholipids is concentrated between 12.68min and 19.16min. It is proved that the proportion of phospholipids in the extracted sea scirrhosa phospholipids is 87.77% of the total phospholipids. Liquid chromatography-mass spectrometry analysis was performed and the ten most abundant phospholipids in the purified sea scirrhosa phospholipids were statistically analyzed. The proportion of phospholipids increased from 46.52% in the sea scirrhosa phospholipid extract to 87.77%, and PE (P-18:0_20:4) increased from 6.304% to 19.429%, indicating that column chromatography and phospholipase A1 enzymatic hydrolysis removed other non-phospholipid components and significantly improved the purity of sea scirrhosa phospholipids.

[0098] The most abundant plasmalogen in ascidian is PE (P-18:0_20:4), accounting for 19.429% of the total phospholipids.

[0099] Example 2: Comparison of antioxidant activity of sea squirt extract and two lecithins

[0100] Soybean lecithin and egg lecithin are the two most common lecithins. They also play the role of drug delivery, liver protection and anti-oxidation in medicine. Therefore, these two lecithins are selected for free radical scavenging ability. The sea scissor phospholipid extract AE, sea scissor plasmalogen PLS, soybean lecithin and egg lecithin prepared in Example 1 were prepared into a 10 mg / mL solution with ultrapure water, and ultrasonically shaken and mixed for use. The antioxidant activity of the four phospholipids was determined according to the instructions of the DPPH free radical scavenging kit. The results are as follows. Figure 5 shown.

[0101] The results show:

[0102] The DPPH scavenging rates of 10 mg / mL scinic acid phosphatidylcholine extract, scinic acid plasmalogen, soybean lecithin and egg lecithin were compared.

[0103] Example 3: Effects of plasmalogens on exercise capacity and renal damage

[0104] The phospholipid extract (AE) and plasmalogen (PLS) of sciatic acid were diluted to 10 mg / mL with normal saline and stored at 4°C until use. 30 C57BL / 6J mice (male, 6-8 weeks old, 20±2 g) were purchased from Changzhou Cavens Animal Ethics Number JN.No20240830c0401010

[421] . All mice were housed in a laboratory cage at 21±1°C and 60%±5% humidity with a 12-hour light / dark cycle and free access to food and water. Each group of mice was gavaged with drugs once a day ( Figure 4 ).

[0105] The animals were divided into five groups: 50 mg / kg PLS + EX group, 100 mg / kg PLS + EX group, AE + EX group, Con group, and EX group. Days 1 to 7 were the acclimation period;

[0106] On days 8-14, the 50 mg / kg PLS+EX group and the 100 mg / kg PLS+EX group were gavaged with an aqueous solution of plasmalogen (50 mg / kg / d and 100 mg / kg / d), respectively; the AE+EX group was gavaged with an oscillin extract (100 mg / kg / d); the Con and EX groups were gavaged with an equal volume of normal saline daily for 7 consecutive days. During the administration period, the 50 mg / kg PLS+EX group, the 100 mg / kg PLS+EX group, the AE+EX group, and the EX group performed adaptive exercise on a treadmill at 15 m / min for 30 minutes every day;

[0107] One hour after the last oral gavage, mice in the 50 mg / kg PLS + EX group, 100 mg / kg PLS + EX group, AE + EX group, and EX group underwent the first exhaustive exercise on a treadmill. The exhaustive exercise speed was shown in Table 3 , and then exhaustive exercise was performed for 7 days according to the speed in Table 3 ;

[0108] 24 hours after the last exhaustive exercise, the mice were killed and their blood, skeletal muscle and kidney tissues were collected for biochemical index detection and histopathological observation.

[0109] Table 3: Exhaustive exercise speed of mice

[0110]

[0111]

[0112] After each group of mice were sacrificed, blood was collected from the eyeballs and allowed to stand at room temperature for 1 hour. Serum was collected by centrifugation at 3000 rpm at 4°C for 15 minutes and stored at -80°C. The kidneys, soleus, gastrocnemius, and quadriceps muscles were collected and weighed. The left kidney was cut in half and placed in a 1.5 mL centrifuge tube with the left soleus and gastrocnemius muscles containing 4% paraformaldehyde. Half of the blood was placed in a 1.5 mL centrifuge tube containing 30% sucrose solution and allowed to stand at room temperature for 1 day. The remaining kidney and skeletal muscle tissue were placed in a clean 1.5 mL centrifuge tube and stored at -80°C.

[0113] 2. Experimental results:

[0114] (1) Changes in muscle mass (Table 4)

[0115] Table 4: Changes in body weight, soleus muscle, gastrocnemius muscle and quadriceps muscle mass of mice in different groups

[0116]

[0117] The results, as shown in Table 4, show that compared to the Con group, mice in the EX group experienced a significant decrease in body weight, and sciatic phospholipid treatment restored the mice's body weight. Changes in skeletal muscle mass were calculated for the soleus, gastrocnemius, and quadriceps femoris muscles. As shown in Table 4, after exhaustive exercise, soleus muscle mass was restored relative to that of unexercised mice. 100 mg / kg of sciatic plasmalogen and sciatic phospholipid extract restored soleus muscle mass. Gastrocnemius and quadriceps femoris muscle mass also decreased after exercise. Both brown fat and subcutaneous fat mass decreased in the EX group compared to the Con group, and sciatic plasmalogen treatment further increased the loss of fat mass. The decrease in body weight and fat mass after exhaustive exercise suggests that exhaustive exercise can be an effective means of weight loss. However, skeletal muscle mass also decreases after exhaustive exercise, which often negatively impacts athletic performance. Sciatic plasmalogen treatment mitigated skeletal muscle mass loss and increased fat loss, suggesting that sciatic plasmalogen supplementation is an effective strategy for muscle building in fitness enthusiasts and fat loss in weight loss enthusiasts.

[0118] (2) Improvement of athletic ability

[0119] The results are shown in Table 5 below:

[0120] Table 5: Exercise capacity of different groups

[0121]

[0122]

[0123] The results show:

[0124] Skeletal muscle status is directly related to exercise capacity. The total distance and daily exercise time of mice during exhaustive exercise for 7 days were counted. Figure 2 The results showed that sustained exhaustive exercise led to a decrease in mice's exercise time, muscle strength, and endurance. All mice showed a significant decrease in exhaustive exercise time on the second day compared to the first day. Over the following six days, exercise time fluctuated but showed no significant downward or upward trend. However, administration of different doses of scidin increased the mice's exercise time.

[0125] The total running distance of mice without sciatic phospholipid intervention was 28740±1187m, while intervention with 100mg / kg sciatic phospholipid (35784±1127m) and 100mg / kg sciatic phospholipid extract (33859±1737m) significantly increased the running distance of C57BL / 6J mice during 7 days of exhaustive exercise. In summary, sciatic phospholipid intervention improved the running ability of mice, supporting the use of sciatic phospholipid as a sports supplement to improve exercise performance. (3) Contents of creatine kinase, lactate, and lactate dehydrogenase

[0126] Exhaustive exercise can trigger a series of stress states in the body. Excessive muscle damage, such as rhabdomyolysis, can cause a large amount of metabolic waste to enter the bloodstream, increasing the burden on the kidneys and impairing renal function. Creatine kinase is a skeletal muscle function enzyme, primarily present in skeletal and cardiac muscle cells.

[0127] The results are shown in Table 6 below:

[0128] Table 6: Contents of creatine kinase, lactate, and lactate dehydrogenase in different groups

[0129] Mouse groups Creatine kinase (U / L) Lactic acid(mM) Lactate dehydrogenase (U / L) 50mg / kg PLS+EX group 1178±173.7 5.826±0.1294 616.8±45.44 100 mg / kg PLS+EX group 1062±82.78 5.860±0.1320 543.6±19.02 Con group 1093±98.29 5.237±0.3010 425.2±32.85 EX Group 1954±268.2 7.255±0.2916 768.6±67.69

[0130] The results show:

[0131] After muscle injury, serum creatine kinase increases rapidly. Figure 3 After exhaustive exercise, the creatine kinase level of mice in the EX group (1954±268.2U / L) was significantly higher than that in the normal group (1178±141.5U / L), indicating that the 7-day exhaustive exercise caused muscle damage. Both 100mg / kg and 50mg / kg of ascidian plasmalogen significantly reduced serum CK levels, dropping to 1062±82.78U / L and 1178±173.7U / L, respectively. The high-dose ascidian plasmalogen had lower creatine kinase levels. While 100mg / kg of ascidian phospholipid extract also downregulated creatine kinase levels, but without significant changes (1547±229.3U / L), this suggests that purified plasmalogen plays a key role in reducing creatine kinase production.

[0132] Further examination of the serum lactate and lactate dehydrogenase levels showed that the levels in the EX group increased significantly, further confirming the damage to skeletal muscle caused by exhaustive exercise. Scidin reversed the levels of these two metabolic wastes. Compared with the 50 mg / kg PLS+EX group (616.8±45.44 U / L), the 100 mg / kg PLS+EX group showed a lower serum concentration (543.6±19.02 U / L); the lactate dehydrogenase content of the 100 mg / kg AE+EX group was 621.9±59.94 U / L.

[0133] (4) Histopathological analysis

[0134] Serum indicators indicate skeletal muscle damage in mice after exhaustive exercise. To determine the specific extent of damage, further analysis of skeletal muscle pathology is required. The soleus muscle, which is constantly under tension during exercise and participates in energy metabolism, exhibits the most pronounced decrease in mass after exercise. Therefore, the soleus muscle was selected as the primary target for skeletal muscle damage and was analyzed using HE staining.

[0135] Histopathological analysis showed that the skeletal muscle in the Con group was neat and dense, with no obvious cell deformation or nucleus shedding. In the EX group, skeletal muscle cell melt damage and muscle fiber rupture occurred, which was alleviated after intervention with sciatic phospholipids. No obvious inflammatory infiltration or neutrophil accumulation occurred in the soleus muscles of mice undergoing exhaustive exercise training. Protein casts and tubular vacuolation appeared in the kidneys of mice in the EX group, indicating that exhaustive exercise still caused kidney damage. This may be due to the accumulation of excessive metabolic waste generated by muscle damage in the renal tubules and further destruction of the renal tubular structure. This phenomenon was alleviated after administration of different doses of sciatic phospholipids.

[0136] In summary, intervention with ascidian plasmalogen alleviated skeletal muscle and kidney damage caused by exhaustive exercise, providing a new sports protection solution for the military and sports professionals.

[0137] (5) Effect of sea squirt extract on alleviating kidney damage in mice caused by exhaustive exercise

[0138] Exercise-induced skeletal muscle damage often stimulates kidney damage. Detect serum creatinine and urea nitrogen levels. Figure 6 In (a), all groups of mice showed no significant changes (p>0.05), which may be because the kidney damage of mice after 7 days of exhaustive exercise did not reach the standard of acute kidney injury or the creatinine and urea nitrogen in the mice returned to normal levels after 24 hours;

[0139] KIM-1 and NGAL are rapidly expressed when renal function is impaired. To further verify the renal damage caused by exhaustive exercise, qPCR was used to detect the expression of KIM-1 and NGAL in mouse kidneys. Figure 6 As shown in (b): Both genes in the EX group increased significantly compared with the Con group, especially KIM-1, whose expression level in the exhaustion exercise group (8.724±0.260) was 8 times that of the normal group (1.007±0.078). Prevention with cytosphingolipids significantly alleviated this trend and was closer to the Con group.

[0140] In order to further determine whether 7 days of exhaustive exercise caused damage to the mouse kidneys, the mouse kidney sections were subjected to pathological examination and analysis, such as Figure 6 As shown in (c), the kidneys of mice in the EX group developed protein casts and tubular vacuolation, indicating that exhaustive exercise still caused kidney damage. This may be due to the accumulation of excessive metabolic waste products generated by muscle damage in the renal tubules, further damaging the tubular structure. Administration of various doses of scidin alleviated this phenomenon, and HE staining of renal tissue showed no obvious pathological changes.

[0141] In summary, although serum creatinine urea nitrogen (CUN) is sensitive and is clinically used as the main criterion for judging acute kidney injury, the kidneys of mice still suffered damage after 7 days of exhaustive exercise even though the blood CUN did not change significantly. This shows that it is necessary to use multiple indicators to detect kidney health and protect the kidneys during exercise.

[0142] (6) Effect of sea squirt extract on alleviating oxidative stress in skeletal muscle of mice induced by exhaustive exercise

[0143] Exhaustive exercise-induced acute kidney injury is associated with the production of metabolic waste in skeletal muscle. Scidin alleviates renal injury from exhaustive exercise, which may be related to the lower skeletal muscle damage after scidin intervention. Appropriate exercise can increase the expression of endogenous antioxidant enzymes and enhance the body's antioxidant system. However, the intense energy metabolism of skeletal muscle during excessive exercise places a huge burden on mitochondria, which in turn leads to lipid oxidation and oxidative stress.

[0144] The gene expressions of antioxidant enzymes SOD and CAT in skeletal muscle were detected. Figure 7 Results (a) in the figure show increased expression of both antioxidant enzymes in the EX group, demonstrating that 7 days of exhaustive exercise increased antioxidant gene expression in C57BL / 6J mice. After sphingolipid administration, antioxidant enzyme expression decreased compared to the EX group and was closer to that of the Con group. The plasmalogen in sphingolipid itself acts as an exogenous antioxidant enzyme, contributing to its antioxidant effects. Based on this, it is speculated that exogenous supplementation with plasmalogen inhibits oxidative damage in mice and reduces antioxidant enzyme activation.

[0145] In order to further explore the role of scidin in exercise-induced oxidative stress, the content of antioxidant enzymes in skeletal muscle was detected by Elisa method, such as Figure 7 (b) The activities of SOD, CAT, and GPX in the EX group were significantly lower than those in the Con group, indicating that even though exercise increased antioxidant enzyme expression, oxidative stress still occurred in skeletal muscle. MDA content in the EX group reached 16.61±1.02mmol / mg, significantly increased compared to the Con group (10.60±0.74mmol / mg) (p<0.001). However, scidinylphospholipids reversed this phenomenon and approached that of the normal control group, indicating that scidinylphospholipids still exerted antioxidant effects without promoting antioxidant enzyme expression.

[0146] (7) Effect of sea squirt extract on alleviating skeletal muscle inflammation in mice induced by exhaustive exercise

[0147] There was no obvious neutrophil accumulation in the HE staining results of soleus muscle. To explore whether 7 days of exhaustive exercise led to inflammatory response in mice, qPCR assay was performed on the genes of three inflammatory factors, such as Figure 8As shown in (a), exhaustive exercise significantly increased the expression of TNF-α and IL-1β genes in mouse skeletal muscle (p<0.001), and pretreatment with scidin-phosphatidylcholine alleviated this phenomenon. Compared with the normal group, the expression of the anti-inflammatory factor IL-10 decreased in the EX group. The three scidin-phosphatidylcholine compounds increased IL-10 expression, indicating that scidin-phosphatidylcholine inhibited the inflammatory response in skeletal muscle at the genetic level after exhaustive exercise.

[0148] Further detection of inflammatory factors in mouse skeletal muscle was performed. Figure 8 In (b), exhaustive exercise increased the accumulation of pro-inflammatory factors TNF-α, IL-1β, and TGF-β in the skeletal muscle of mice, proving that mice had an inflammatory response under exhaustive exercise. After intervention with 100 mg / kg of sciatic phospholipid extract, the increase in inflammatory factors caused by exhaustive exercise was alleviated. However, the content of pro-inflammatory factors was higher than that of the sciatic plasmalogen group (100 mg / kg, 50 mg / kg). Sciatic phospholipids increased the accumulation of the anti-inflammatory factor IL-10, and this protein was significantly decreased in the EX group (p < 0.05). This shows that sciatic phospholipids alleviated the skeletal muscle inflammatory response induced by exhaustive exercise, and the 100 mg / kg sciatic phospholipid extract had a lower effect on alleviating inflammation than sciatic plasmalogen.

[0149] (8) Effect of sea squirt extract on the apoptosis of skeletal muscle cells in mice induced by exhaustive exercise

[0150] Skeletal muscle cell death is an important prerequisite for the entry of cell contents into the blood and the resulting impairment of renal function. Exhaustive exercise, severe mechanical damage, and energy metabolism-induced oxidative stress and inflammatory responses can all lead to skeletal muscle cell apoptosis. The expression levels of skeletal muscle cell apoptosis factors Bax and Bcl2 genes were detected. Figure 9 As shown, the expression of Bax in the EX group (2.990±0.396) was nearly three times that of the Con group (1.002±0.041), and the expression of Bcl2 (0.488±0.025) was only 0.5 times that of the Con group (1.003±0.052). Pretreatment with scidin-phospholipids alleviated cell apoptosis, especially when 100 mg / kg of scidin-phospholipids was added, the expression of apoptotic genes was closest to normal levels.

[0151] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. The use of plasmalogen in preparing a product for assisting muscle growth and fat loss during aerobic exercise, characterized in that: The muscle-building and fat-reducing method is to reduce muscle loss and increase fat consumption during aerobic exercise.

2. The use of plasmalogens in the preparation of products for preventing, improving and / or treating skeletal muscle damage and kidney damage caused by aerobic exercise.

3. The use according to claim 1 or 2, characterized in that The product includes but is not limited to one or more of medicine, health food, food for special medical purposes, food, functional food, feed, feed additive or pet food; preferably, the product is sports food or sports supplement or sports nutrition product; Preferably, the preparation method of the plasmalogen is as follows: (i) adding water to the inner sheath of the ascidian and mixing the mixture and then homogenizing the mixture to obtain a homogenate; (ii) centrifuging the homogenate obtained in step (i) to obtain a precipitate, mixing the precipitate with methanol, dichloromethane, and water, stirring, and centrifuging to obtain a supernatant; (iii) adding water and dichloromethane to the supernatant obtained in step (ii), allowing the mixture to stand, collecting the lower organic phase, collecting the precipitate by rotary evaporation, and washing the precipitate with hexane to obtain a scinothiolane extract; (iv) dissolving the ascidian phospholipid extract obtained in step (iii) in dichloromethane, purifying the total lipids by silica gel column chromatography, eluting the neutral esters in the purified lipids by using dichloromethane and methanol as eluents, and collecting the eluate; subjecting the eluate to thin layer silica gel plate chromatography, and confirming the color development with an iodine reagent, and then rotary evaporating to remove the residual organic solvent to obtain a crude ascidian lipid extract; (v) dissolving the crude ascidian lipid extract obtained in step (iv) in n-hexane, adding phospholipase A1 and sodium citrate buffer for incubation, extracting the phospholipids treated with phospholipase A1 with n-hexane and isopropanol, collecting the hexane layer, washing it with physiological saline, and rotary evaporating the n-hexane to obtain plasmalogen.

4. The use according to any one of claims 1 to 3, characterized in that: The dosage form of the drug is a liquid preparation or a solid preparation; Preferably, the dosage form of the drug includes but is not limited to granules, capsules, tablets, pills or oral liquids; Preferably, the drug further comprises a pharmaceutically acceptable excipient; Preferably, the pharmaceutical excipients include: any one or more of solubilizers, emulsifiers, colorants, adhesives, disintegrants, fillers, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrities, pH regulators, buffers, plasticizers, defoamers, thickeners, humectants, filter aids and release retardants.

5. The use according to any one of claims 1 to 4, characterized in that: In step (i), the mass volume ratio of the ascidian inner sheath to water is: (1-2): (1-2); Preferably, in step (ii), the methanol is added in a ratio of (90-100): (70-80) to the ascidian homogenate, the dichloromethane is added in a ratio of (40-50): (70-80) to the ascidian homogenate, and the water is added in a ratio of (30-40): (70-80) to the ascidian homogenate; Preferably, in step (iii), the dichloromethane is added in a ratio of (40-50) to the ascidian homogenate: (70-80), and the water is added in a ratio of (30-40) to the ascidian homogenate: (70-80); Preferably, in step (iv), the dichloromethane is added to the ascidian homogenate in a ratio of (8-10): (70-80); the eluent chloromethane and methanol are mixed in a volume ratio of (10-12):1; Preferably, in step (v), the pH of the sodium citrate buffer is 4-5; the added enzyme activity of the phospholipase A1 is 400-450 U; and the incubation time is 2-5 h.

6. Application of plasmalogens in the preparation of products that resist fatigue and improve athletic performance.

7. The use according to claim 6, characterized in that The products include, but are not limited to, one or more of medicines, health foods, foods for special medical purposes, foods, functional foods, feeds, feed additives or pet foods; Preferably, the preparation method of the plasmalogen is as follows: (i) adding water to the inner sheath of the ascidian and mixing the mixture and then homogenizing the mixture to obtain a homogenate; (ii) centrifuging the homogenate obtained in step (i) to obtain a precipitate, mixing the precipitate with methanol, dichloromethane, and water, stirring, and centrifuging to obtain a supernatant; (iii) adding water and dichloromethane to the supernatant obtained in step (ii), allowing the mixture to stand, collecting the lower organic phase, collecting the precipitate by rotary evaporation, and washing the precipitate with hexane to obtain a scinothiolane extract; (iv) dissolving the ascidian phospholipid extract obtained in step (iii) in dichloromethane, purifying the total lipids by silica gel column chromatography, eluting the neutral esters in the purified lipids by using dichloromethane and methanol as eluents, and collecting the eluate; subjecting the eluate to thin layer silica gel plate chromatography, and confirming the color development with an iodine reagent, and then rotary evaporating to remove the residual organic solvent to obtain a crude ascidian lipid extract; (v) dissolving the crude ascidian lipid extract obtained in step (iv) in n-hexane, adding phospholipase A1 and sodium citrate buffer for incubation, extracting the phospholipids treated with phospholipase A1 with n-hexane and isopropanol, collecting the hexane layer, washing it with physiological saline, and rotary evaporating the n-hexane to obtain plasmalogen.

8. The use according to claim 6 or 7, characterized in that The dosage form of the drug is a liquid preparation or a solid preparation; Preferably, the dosage form of the drug includes but is not limited to granules, capsules, tablets, pills or oral liquids; Preferably, the drug further comprises a pharmaceutically acceptable excipient; Preferably, the pharmaceutical excipients include: any one or more of solubilizers, emulsifiers, colorants, adhesives, disintegrants, fillers, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrities, pH regulators, buffers, plasticizers, defoamers, thickeners, humectants, filter aids and release retardants.

9. A drug for preventing, improving and / or treating skeletal muscle damage and kidney damage caused by aerobic exercise, characterized in that: The medicine contains plasmalogen, and the preparation method of the plasmalogen is as follows: (i) adding water to the inner sheath of the ascidian and mixing the mixture and then homogenizing the mixture to obtain a homogenate; (ii) centrifuging the homogenate obtained in step (i) to obtain a precipitate, mixing the precipitate with methanol, dichloromethane, and water, stirring, and centrifuging to obtain a supernatant; (iii) adding water and dichloromethane to the supernatant obtained in step (ii), allowing the mixture to stand, collecting the lower organic phase, collecting the precipitate by rotary evaporation, and washing the precipitate with hexane to obtain a scinothiolane extract; (iv) dissolving the ascidian phospholipid extract obtained in step (iii) in dichloromethane, purifying the total lipids by silica gel column chromatography, eluting the neutral esters in the purified lipids by using dichloromethane and methanol as eluents, and collecting the eluate; subjecting the eluate to thin layer silica gel plate chromatography, and confirming the color development with an iodine reagent, and then rotary evaporating to remove the residual organic solvent to obtain a crude ascidian lipid extract; (v) dissolving the crude ascidian lipid extract obtained in step (iv) in n-hexane, adding phospholipase A1 and sodium citrate buffer for incubation, extracting the phospholipids treated with phospholipase A1 with n-hexane and isopropanol, collecting the hexane layer, washing it with physiological saline, and rotary evaporating the n-hexane to obtain plasmalogen.

10. The drug according to claim 9, characterized in that The dosage form of the drug is a liquid preparation or a solid preparation. Optionally, the dosage form of the drug includes but is not limited to granules, capsules, tablets, pills or oral liquids. Preferably, the drug further includes pharmaceutically acceptable excipients. Preferably, the pharmaceutical excipients include: any one or more of solubilizers, emulsifiers, colorants, adhesives, disintegrants, fillers, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesive agents, integrities, pH regulators, buffers, plasticizers, defoamers, thickeners, humectants, filter aids and release retardants. Preferably, in step (i), the mass volume ratio of the ascidian inner sheath to water is: (1-2): (1-2); Preferably, in step (ii), the methanol is added in a ratio of (90-100): (70-80) to the ascidian homogenate, the dichloromethane is added in a ratio of (40-50): (70-80) to the ascidian homogenate, and the water is added in a ratio of (30-40): (70-80) to the ascidian homogenate; Preferably, in step (iii), the dichloromethane is added in a ratio of (40-50) to the ascidian homogenate: (70-80), and the water is added in a ratio of (30-40) to the ascidian homogenate: (70-80); Preferably, in step (iv), the dichloromethane is added to the ascidian homogenate in a ratio of (8-10): (70-80); the eluent chloromethane and methanol are mixed in a volume ratio of (10-12): 1; Preferably, in step (v), the pH of the sodium citrate buffer is 4-5; the added enzyme activity of the phospholipase A1 is 400-450 U; and the incubation time is 2-5 h.

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