Multi-target anti-senile dementia activity holothuria scabra-derived lipid complex as well as separation component and application thereof

Abstract

The invention discloses a multi-target anti-senile dementia activity holothuria scabra-derived lipid compound as well as a separation component and application thereof, and belongs to the field of medicines and functional foods. The preparation method of the holothuria scabra lipid compound comprises the following steps: extracting holothuria scabra total lipid by using a mixed solvent of chloroform and methanol, deoiling by using cold acetone, and then separating and collecting precipitate to obtain the holothuria scabra lipid compound. The holothuria scabra separation component is obtained by carrying out silica gel column chromatography on the holothuria scabra lipid compound, and eluting by taking a mixed solution of dichloromethane, methanol and / or water as an eluent, so as to obtain a primary component with activity after elution; respectively carrying out normal-phase column chromatography on the two components with the best activity, and eluting by using a mixed solvent of ethyl acetate and normal hexane and / or a mixed solvent of dichloromethane and methanol to obtain a secondary component; the holothuria scabra lipid complex and the separated component thereof have remarkable Abeta neurotoxicity resistance, neuroinflammation resistance, intracellular oxidative stress resistance, free radical scavenging and acetylcholin esterase inhibition activity.

Description

Technical Field

[0001] The invention relates to the fields of medicine and functional food, and in particular to a sea cucumber-derived lipid complex with multi-targeted anti-Alzheimer's activity and its separated components and applications. Background Art

[0002] Alzheimer's disease (AD), also known as senile dementia, has become the "fourth killer" that endangers the health of the elderly, bringing a heavy burden to families and society. However, the efficacy of traditional clinical drugs such as donepezil and memantine for the prevention and treatment of AD is limited, and the development of new drugs has been stagnant. Existing pathological studies have shown that the occurrence of AD is related to multiple complex factors and their interactions, such as the deposition of amyloid beta peptide (Aβ) between brain neurons, neurofibrillary tangles caused by hyperphosphorylation of Tau protein in neurons, neuroinflammation, oxidative stress, cholinergic deficiency, etc. Therefore, the development of drugs or nutrients targeting multiple targets is of great significance for the intervention of AD.

[0003] Lipids are important components of the brain. Modern biomedical research shows that the active lipid components in sea cucumbers that protect nerves and improve memory are of great value in preventing and treating AD. For example, cerebrosides extracted from Acaudinamolpadioides from cold temperate waters and phospholipids extracted from North Atlantic sea cucumbers / Cucumaria frondosa have been reported to have neuroprotective or anti-neuroinflammatory effects.

[0004] Holothuria scabra, scientific name Holothuria scabra (Jaeger, 1833), commonly known as Mingyu ginseng, Shashen, Mingbalu ginseng, white ginseng, is widely distributed in tropical waters of the Indian Ocean and the Western Pacific Ocean. In my country, it is mainly distributed in Guangxi, Guangdong, Hainan and other places. It is a high-quality tropical edible sea cucumber with high nutritional value, economic value and health care function. The ethanol-soluble or insoluble components of crude phospholipids of Holothuria scabra have been reported to inhibit LPS-induced BV2 cell neuroinflammatory response and Aβ, MPP + The effect of causing SH-SY5Y neuronal cell damage (Zhang Yongping et al. Chinese invention patent CN110327358B; Song Cai et al. Chinese invention patent CN110478374B).

[0005] However, the pathogenesis of Alzheimer's disease involves a wider range of targets. Although cerebrosides, phospholipids and other lipids from sea cucumbers and other sea cucumbers have been reported to have different anti-Alzheimer's disease-related effects, there are no reports of sea cucumber active lipids that simultaneously have anti-Aβ neurotoxicity, anti-neuroinflammation, anti-intracellular oxidative stress, direct free radical scavenging and acetylcholinesterase inhibition effects. This sea cucumber lipid that can simultaneously target multiple Alzheimer's disease pathogenesis is expected to have better medicinal and food application value for preventing and treating Alzheimer's disease and improving memory. Summary of the invention

[0006] The purpose of the present invention is to provide a rough sea cucumber-derived lipid complex with multi-targeted anti-Alzheimer's activity and its separated components and applications, so as to solve the problems existing in the above-mentioned prior art. By optimizing the extraction method of sea cucumber lipids and based on a variety of Alzheimer's activity evaluation systems, active lipids with multi-targeted anti-Alzheimer's effects and their separated components are tracked and prepared. These rough sea cucumber lipids and their separated components can be used to prepare drugs and / or functional foods that assist in improving memory for different targets, laying a theoretical foundation for the research and development of new drugs for the treatment of Alzheimer's disease.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The invention provides a rough sea cucumber lipid extract. The rough sea cucumber lipid extract comprises a rough sea cucumber lipid complex and separated components thereof. The preparation method of the rough sea cucumber lipid complex comprises extracting the rough sea cucumber total lipid by using a mixed solvent of chloroform and methanol, deoiling by cold acetone, and then separating and collecting precipitates to obtain the rough sea cucumber lipid complex; the rough sea cucumber separated components are obtained by subjecting the rough sea cucumber lipid complex to silica gel column chromatography (the filler of the vacuum column used is 200-300 mesh silica gel), eluting with a mixed solution of dichloromethane, methanol and / or water as an eluent, and after elution, obtaining an active primary component by multi-target anti-Alzheimer's activity screening; the primary component is subjected to multi-target anti-Alzheimer's activity screening, and two components with the best activity are respectively subjected to normal phase column chromatography (the filler of the silica gel column used is 300-400 mesh silica gel), and eluting with a mixed solvent of ethyl acetate and n-hexane and / or a mixed solvent of dichloromethane and methanol to obtain a secondary component.

[0009] Preferably, the method for preparing the sea cucumber lipid complex comprises the following steps:

[0010] After the rough sea cucumber is crushed into powder, a mixed solvent of chloroform and methanol is added at a mass volume ratio of 1: (3-10), ultrasonic extraction is performed for 20-30 minutes, and then suction filtration is performed to collect the filtrate, and chloroform is added to the filter residue at a mass volume ratio of 1: (1-3), and the extraction is repeated 1-3 times, and the filtrate is combined, and then 1-3 times the volume of potassium chloride solution is added and mixed, and after standing and stratification, the lower layer liquid is collected and concentrated to obtain the total lipid of the rough sea cucumber;

[0011] Add 10-40 times volume of cold acetone to the total lipid of the rough sea cucumber, extract, centrifuge, collect the precipitate, and obtain the rough sea cucumber lipid complex.

[0012] Preferably, the volume ratio of chloroform to methanol is 1:2; the concentration of potassium chloride is 0.5%-1.5%, and 0.5-1.5% KCl solution can be replaced by 0.5-1.5% NaCl solution or water; the Rf value of the sea cucumber lipid complex on a silica gel plate with a mixed solution of chloroform, methanol and water in a volume ratio of 65:22:4 as a developing agent is 0.95, 0.8-0.9, 0.57-0.65, 0.45-0.5, 0.35-0.4 and 0.2.

[0013] The above-mentioned static stratification method can be replaced by the centrifugal stratification method, but the separation effect and economy are best in the chloroform / methanol solvent system.

[0014] Preferably, the method for preparing the isolated components of sea cucumber comprises the following steps:

[0015] The mixed solution of dichloromethane, methanol and / or water includes: a mixed solvent of dichloromethane and methanol, a mixed solvent solution of dichloromethane, methanol and water, and a mixed solvent of dichloromethane and methanol; more preferably, in the mixed solvent of dichloromethane and methanol, the volume ratio of dichloromethane to methanol is 1:0 and 24:1; in the mixed solution of dichloromethane, methanol and water, the volume ratio of dichloromethane, methanol and water is 95:8:1; and in the mixed solvent of dichloromethane and methanol, the volume ratio of dichloromethane to methanol is 6:1, 1:1 and 0:1.

[0016] Preferably, after multi-target anti-Alzheimer's activity screening, the highly active primary components Fr.B, Fr.E and Fr.G are obtained.

[0017] Among them, on a silica gel plate with a mixed solution of chloroform, methanol and water in a volume ratio of 65:22:4 as the developing solvent, the Rf value of Fr.B is 0.95 and 0.85-0.9, the Rf value of Fr.E is 0.6-0.7, and the Rf value of Fr.G is 0.4-0.7, 0.2-0.4 and 0-0.2.

[0018] Preferably, the two most active primary components Fr.B and Fr.E are subjected to normal phase column chromatography respectively, wherein the Fr.B is eluted with the following mixed solvents in sequence: a mixed solvent of ethyl acetate and n-hexane, and a mixed solvent of dichloromethane and methanol; more preferably, in the mixed solvent of ethyl acetate and n-hexane, the volume ratio of ethyl acetate to n-hexane is 1:20, 1:10, 1:5 and 1:1, respectively; and in the mixed solvent of dichloromethane and methanol, the volume ratio of dichloromethane to methanol is 1:0, 40:1, 24:1, 10:1, 5:1, 1:1 and 0:1, respectively; the Fr.E is eluted with a mixed solvent of dichloromethane and methanol, more preferably, in the mixed solvent of dichloromethane and methanol, the volume ratio of dichloromethane to methanol is 20:1, 10:1, 6:1, 3:1, 1:1 and 0:1, respectively.

[0019] Preferably, after normal phase column chromatography, the Fr.B obtains a component Fr.Bd with good anti-multi-target and anti-Alzheimer's activity, and the Rf value of the Fr.Bd on a silica gel plate with a mixed solution of chloroform, methanol and water in a volume ratio of 65:22:4 as the developing solvent is 0.95; after normal phase column chromatography, the Fr.E obtains components Fr.Eb and Fr.Ed with good anti-multi-target and anti-Alzheimer's activity, wherein on a silica gel plate with a mixed solution of chloroform, methanol and water in a volume ratio of 65:22:4 as the developing solvent, the Rf values ​​of the Fr.Eb and the Fr.Ed are 0.6-0.64 and 0.61-0.67, respectively.

[0020] Preferably, the rough sea cucumber lipid complex, Fr.B, Fr.Bd, Fr.E, Fr.Eb, Fr.Ed and Fr.G are analyzed by liquid chromatography-mass spectrometry, and the liquid chromatography conditions are: the chromatographic column model is Waters ACQUITY UPLC BEHRP18; the mobile phase is a mobile phase A and a mobile phase B with a volume ratio of 4:6, the mobile phase A is a methanol solution containing 0.1% formic acid, and the mobile phase B is an acetonitrile solution containing 0.1% formic acid; the gradient elution conditions are: 0-8min, 40% mobile phase A; the flow rate is 0.2mL / min;

[0021] Among them, the rough sea cucumber lipid complex, Fr.B, Fr.Bd, Fr.E, Fr.Eb, Fr.Ed and Fr.G have retention times of 0.5-0.6min, 0.75-0.9min, 0.9-1.0min, 1.0-1.1min, 1.2-1.3min, 1.35-1.48min, 1.48-1.58min, 1.75-1.89min, 2.1-2.25min, 2.65-2.8min, 3.0-3.3min, 3.3-3.48min, 4.4-4.6min, and 6.0-6.3 in the positive ion mode. min, and their total contents were 90.66%, 86.32%, 90.35%, 82.23%, 91.85, 73.16% and 88.64% respectively; the corresponding negative ion mode contained a group of chromatographic peaks with retention times of 0.52-0.67min, 1.1-1.2min, 1.2-1.32min, 1.4-1.6min, 1.7-1.87min, 2.4-2.6min and 6.0-6.3min, and their total contents were 69.64%, 81.95%, 79.08%, 70.54%, 77.54%, 66.50% and 75.01% respectively.

[0022] The present invention also provides the use of the sea cucumber lipid extract in any of the following items:

[0023] Application in the preparation of drugs for resisting neuronal cell damage;

[0024] Use in the preparation of drugs with anti-neuritis activity;

[0025] Application in the preparation of drugs for removing active oxygen in neuronal cells;

[0026] Application in the preparation of drugs for scavenging DPPH free radical activity;

[0027] Application in the preparation of drugs for inhibiting acetylcholinesterase activity;

[0028] Application in the preparation of food that assists in improving memory function;

[0029] The sea cucumber lipid extract includes any one of the sea cucumber lipid complex, Fr.B, Fr.Bd, Fr.E, Fr.Eb, Fr.Ed and Fr.G.

[0030] The present invention also provides a medicine having anti-neuronal cell damage and / or anti-neuritis activity and / or scavenging active oxygen in neuronal cells and / or scavenging DPPH free radical activity and / or inhibiting acetylcholinesterase activity and / or a functional food for assisting in improving memory, comprising any one of the rough sea cucumber lipid complex, Fr.B, Fr.Bd, Fr.E, Fr.Eb, Fr.Ed and Fr.G.

[0031] The present invention discloses the following technical effects:

[0032] The present invention obtains a group of rough sea cucumber lipid complexes and their separated components by extracting rough sea cucumbers. Experiments have confirmed that the separated rough sea cucumber lipid complexes and their separated components have the ability to significantly inhibit nerve cell damage caused by Aβ, inhibit NO production induced by lipopolysaccharide in brain-derived BV-2 microglial cells, remove intracellular reactive oxygen species caused by Aβ, directly remove DPPH free radical activity and inhibit acetylcholinesterase activity; and have no toxic side effects on normal cells within the scope of application, and can be used as a drug with high activity, high yield, non-toxicity and natural source for inhibiting acetylcholinesterase activity;

[0033] In addition, the preparation method of the rough sea cucumber lipid complex and its separated components has high yield. The rough sea cucumber lipid complex and its separated components use the rough sea cucumber, an edible tropical economic sea cucumber species that is naturally caught or cultured, as raw materials. The material source is natural and extensive. The obtained rough sea cucumber lipid complex and its separated components are non-toxic and will not cause adverse reactions. The preparation method is simple and the cost is low. Therefore, the rough sea cucumber lipid complex and its separated components provided by the present invention have multi-target anti-Alzheimer's activity, can be used to prepare drugs that resist neuronal cell damage, anti-neuritis activity, scavenging reactive oxygen in neuronal cells, scavenging DPPH free radical activity and inhibiting acetylcholinesterase activity, and have great potential for further research and development into new drugs for the treatment of Alzheimer's disease and / or functional foods that assist in improving memory. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0035] Figure 1 It is a thin layer chromatography image of the sea cucumber lipid complex with multi-target anti-Alzheimer's activity and its separated components of the present invention;

[0036] Figure 2It is a chromatogram of the main components of the rough sea cucumber lipid complex and its separated components with multi-target anti-Alzheimer's activity of the present invention analyzed by liquid chromatography-mass spectrometry; wherein A: positive ion mode base peak chromatogram of the rough sea cucumber lipid complex and its separated components; B: positive ion mode total ion flow diagram of the rough sea cucumber lipid complex and its separated components; C: negative ion mode base peak chromatogram of the rough sea cucumber lipid complex and its separated components; D: negative ion mode total ion flow diagram of the rough sea cucumber lipid complex and its separated components;

[0037] Figure 3 This is a graph showing the results of the test of inhibiting acetylcholinesterase activity of the sea cucumber lipid complex with multi-targeted anti-Alzheimer's activity and its separated components of the present invention;

[0038] Figure 4 This is a graph showing the test results of the scavenging activity of the sea cucumber lipid complex with multi-target anti-Alzheimer's disease activity and its separated components on DPPH free radicals; A: bar chart, B: DPPH free radical staining silica gel plate development;

[0039] Figure 5 This is a graph showing the toxicity test results of the sea cucumber lipid complex with multi-target anti-Alzheimer's activity and its separated components on BV-2 cells;

[0040] Figure 6 This is a graph showing the results of the activity test of the sea cucumber lipid complex and its separated components with multi-target anti-Alzheimer's activity in inhibiting NO production in BV-2 cells;

[0041] Figure 7 This is a test result diagram of the cytotoxicity of the sea cucumber lipid complex and its separated components with multi-target anti-Alzheimer's activity on HT-22 cells;

[0042] Figure 8 The present invention is a rough sea cucumber lipid complex with multi-target anti-Alzheimer's activity and its separated components against Aβ 25-35 Figure 1. Test results of protective activity of HT-22 cell damage induced by toxic peptides;

[0043] Fig. 9 The present invention is a rough sea cucumber lipid complex with multi-target anti-Alzheimer's activity and its separated components against Aβ 25-35 Results of the scavenging activity test of the toxin peptide-induced reactive oxygen species (ROS) produced in HT-22 cells; A: fluorescence image, B: fluorescence intensity statistics. DETAILED DESCRIPTION

[0044] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0045] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0046] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0047] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0048] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0049] Example 1 Preparation of Sea Cucumber Lipid Complex and Its Separated Components

[0050] 1. Experimental methods

[0051] 1.1 The preparation method of the sea cucumber lipid complex and its separated components comprises the following steps:

[0052] S1. After washing the surface of the light-dried rough sea cucumber with pure water, soak it in pure water in a clean oil-free container for two days, dry the surface water stains, crush it with a grinder, freeze the crushed sea cucumber particles at -20℃ overnight, and freeze-dry it with a freeze dryer. The freeze-dried sea cucumber particle sample is crushed again into fine powder. Add 6 times (volume: weight ratio (g:mL), the same below) of chloroform-methanol mixed solvent (volume ratio 1:2) to this powder, ultrasonically extract for 20 minutes, filter, collect the filtrate, add 2 times the volume of chloroform to the filter residue, ultrasonically extract for 30 minutes, filter to obtain the filtrate, and repeat the extraction twice. Combine the filtrates extracted three times, add 2 times the volume of 0.88% KCl solution to it and mix well, let it stand for 3 hours and then separate, collect the lower layer liquid, and concentrate it by rotary evaporation to obtain the total lipid of rough sea cucumber. Add 10 to 40 times the volume of cold acetone (precooled at 4°C for more than 4 hours) to the total lipids, shake and fully extract, then pass through high-speed refrigerated centrifugation (4°C, 10,000r / min, 15min), take the precipitate obtained by centrifugation to obtain the rough sea cucumber lipid complex, which contains iodine-colored spots with Rf values ​​of 0.95, 0.8-0.9, 0.57-0.65, 0.45-0.5, 0.35-0.4, and 0.2 on a Merck 60F254 silica gel plate with chloroform: methanol: water = 65:22:4 (volume ratio) as the developing solvent.

[0053] S2. The rough sea cucumber lipid complex obtained in step S1 was subjected to silica gel column chromatography (the filler of the silica gel column was 200-300 mesh silica gel), and was eluted and separated with the following reagents: dichloromethane / methanol (volume ratio = 1:0, 24:1), dichloromethane / methanol / water (volume ratio = 95:8:1), dichloromethane / methanol (volume ratio = 6:1, 1:1, 0:1), and the components obtained after elution were spotted by TLC and iodine color development was used as a reference. After combining the components, 7 primary components (Fr.A~Fr.G) were obtained; through multi-target anti-Alzheimer's activity screening, components Fr.B, Fr.E and Fr.G were strongly active components, and Fr.B was eluted in Merck HPLC with chloroform: methanol: water = 65:22:4 as the developing solvent. The 60F254 silica gel plate contains iodine color spots with Rf values ​​of 0.95 and 0.85-0.9. Under this condition, Fr.E contains iodine color spots with Rf values ​​of 0.6-0.7. Under this condition, Fr.G contains iodine color spots with Rf values ​​of 0.4-0.7, 0.2-0.4, and 0-0.2. Figure 1 .

[0054] S3. According to the results of multi-target anti-Alzheimer's activity screening, the highly active primary components Fr.B and Fr.E obtained in step S2 were further subjected to normal phase column chromatography using 300-400 mesh silica gel, wherein:

[0055] 1) The primary component Fr.B was continuously eluted and collected with ethyl acetate / n-hexane (volume ratios of 1:20, 1:10, 1:5, 1:1), and dichloromethane / methanol (volume ratios of 1:0, 40:1, 24:1, 10:1, 5:1, 1:1, 0:1) and TLC spot plate iodine color development, and then combined to obtain 7 secondary components: Fr.Ba~Fr.Bg. Through multi-target anti-Alzheimer's activity screening, the secondary component Fr.Bd was a strong active component, which showed an iodine color spot with an Rf value of 0.95 on a Merck 60F254 silica gel plate with chloroform: methanol: water = 65:22:4 as the developing solvent, as shown in FIG. Figure 1 ;

[0056] 2) The primary component Fr.E was continuously eluted and collected with dichloromethane / methanol (volume ratio = 20:1, 10:1, 6:1, 3:1, 1:1, 0:1) and iodine-stained by TLC, and then combined to obtain 7 secondary components: Fr.Ea~Fr.Eg. Through multi-target anti-Alzheimer's activity screening, the secondary components Fr.Eb and Fr.Ed were strong active components, which showed iodine-stained spots with Rf values ​​of 0.6-0.64 and 0.61-0.67 on Merck 60F254 silica gel plate with chloroform: methanol: water = 65:22:4 as the developing solvent, as shown in Figure 2. Figure 1 .

[0057] The multi-target anti-Alzheimer's activity screening in the above preparation process includes: DPPH free radical scavenging activity test, acetylcholinesterase (AChE) inhibition activity test, cytotoxicity and anti-inflammatory activity test on BV-2 cells, toxicity to HT-22 neuronal cells and protective activity against Aβ-induced HT-22 neuronal damage, and scavenging activity against Aβ-induced intracellular reactive oxygen species (ROS) in HT-22 cells. For details, see Application Examples 1 to 7.

[0058] 1.2 Liquid chromatography-mass spectrometry analysis

[0059] The main components of the rough sea cucumber lipid complex and its separated components obtained above were analyzed by liquid chromatography-mass spectrometry. The sample was dissolved in mass spectrometry pure methanol: acetonitrile (volume ratio 4:6) (50 μg / mL), and the injection volume was 1 μL. Methanol containing 0.1% formic acid was used as mobile phase A, and acetonitrile containing 0.1% formic acid was used as mobile phase B. The gradient elution conditions of the sample were as follows: 40% A (0-8min), flow rate 0.2mL / min. The scanning range of the mass spectrometer setting was m / z 100-1700, electrospray ion source, positive ion + negative ion mode. The parameters of the mass spectrometer setting were as follows: ion source temperature: 120°C; capillary voltage: 1.6KV; sampling cone voltage: 40V; source offset voltage: 80V; collision energy: 25-55eV; desolvation temperature: 450°C; cone gas flow rate: 50L / hr; desolvation gas flow rate: 800L / hr. LC-MS / MS data were collected by a Waters Acquity UHPLCDAD Xevo G2-XS Q-Tof liquid chromatography-mass spectrometer, and the chromatographic column used was Waters ACQUITY UPLC BEH RP18 (2.1×50mm, 1.7μm). The lipidome analysis function lipidomics of MS-DIAL metabolomics analysis software was used to annotate the main characteristic components.

[0060] 2. Experimental results

[0061] The isolated and prepared rough sea cucumber lipid complex (Crude) with strong multi-targeted anti-Alzheimer's activity and its separated components (Fr.B, Fr.E, Fr.G, Fr.Bd, Fr.Eb and Fr.Ed) were subjected to liquid chromatography-tandem mass spectrometry analysis in positive and negative ion modes. The results are as follows Figure 2As shown in Tables 1 and 2, it can be seen that the rough sea cucumber lipid complex and its separated components mainly contain several chromatographic peaks with retention times in the following ranges under the positive ion mass spectrometry detection mode: 0.5-0.6min, 0.75-0.9min, 0.9-1.0min, 1.0-1.1min, 1.2-1.3min, 1.35-1.48min, 1.48-1.58min, 1.75-1.89min, 2.1-2.25min, 2.65-2.8min, 3.0-3.3min, 3.3-3.48min, 4.4-4.6min, 6.0-6.3min. Under the negative ion mass spectrometry detection mode, it mainly contains several chromatographic peaks with retention times in the following ranges: 0.52-0.67min, 1.1-1.2min, 1.2-1.32min, 1.4-1.6min, 1.7-1.87min, 2.4-2.6min and 6.0-6.3min. The main characteristic parent ions in these chromatographic peaks and their annotated molecular formula information are shown in Tables 1 and 2.

[0062] Table 1

[0063]

[0064]

[0065]

[0066] Table 2

[0067]

[0068]

[0069] The multi-target anti-Alzheimer's activity screening experiments for the rough sea cucumber lipid complex (Crude) and its separated components (Fr.B, Fr.E, Fr.G, Fr.Bd, Fr.Eb and Fr.Ed) prepared in Example 1 are as follows: Application Example 1 to Application Example 7.

[0070] Application Example 1 Test on the inhibition of acetylcholinesterase activity by sea cucumber lipid complex and its isolated components

[0071] Acetylcholinesterase is a classic target enzyme of Alzheimer's disease. Inhibition of acetylcholinesterase can alleviate the degradation of acetylcholine, a neurotransmitter in the synaptic cleft, thereby improving memory. The sea cucumber lipid complex prepared in Example 1 of the present invention and its separated components were used as samples, and anti-acetylcholinesterase drugs were used to screen the commonly used electric eel acetylcholinesterase (AChE). The in vitro anti-acetylcholinesterase activity experiment of the sea cucumber lipid complex and its separated components was carried out. The specific experimental method and experimental results are as follows:

[0072] 1. Experimental methods

[0073] Dissolve the sample to be tested in DMSO solution to a concentration of 1 mg / mL. Perform the experiment according to the following reagent ratios, with at least 3 parallels set up for each group.

[0074] Sample group (D sample ): 5 μL sample solution with different gradients + 45 μL PBS + 10 μL AChE + 20 μL DTNB;

[0075] The control group (D control ): 5 μL sample solution with different gradients + 45 μL PBS + 10 μL BSA + 20 μL DTNB;

[0076] Blank group (D blank ): 5μL DMSO+45μL PBS+10μL AChE+20μL DTNB;

[0077] Blank control group (D sample blank ): 5μL DMSO+45μL PBS+10μL BSA+20μL DTNB.

[0078] After the sample loading was completed, the 96-well plate was placed in a 37°C constant temperature incubator for 10 min, 20 μL of iodinated thioacetylcholine (ATCI) was added to each well, and then incubated at 37°C for 20 min. The optical density value D at 405 nm was measured using a microplate reader. 405 nm Donepezil Hydrochloride was used as the positive control (final concentration 50 μM). The calculation formula of AChE inhibition rate is as follows:

[0079]

[0080] 2. Experimental results

[0081] The results of the test on the inhibition of acetylcholinesterase activity of the sea cucumber lipid complex and its separated components of the present invention are as follows: Figure 3As shown, it can be seen that when the concentration of each sample is 50 μg / mL, the inhibition rate of the rough sea cucumber lipid complex (Crude) on acetylcholinesterase activity reaches 74.52%, the inhibition rate of the separated component Fr.B reaches 23.09%, the inhibition rate of the component Fr.E reaches 52.87%, the inhibition rate of the component Fr.G reaches 53.69%, the inhibition rate of the component Fr.Bd reaches 41.07%, the inhibition rate of the component Fr.Eb reaches 34.42%, and the inhibition rate of the component Fr.Ed reaches 26.3%, indicating that the rough sea cucumber lipid complex prepared by the present invention and its separated components have the activity of inhibiting acetylcholinesterase.

[0082] Application Example 2 Free Radical Scavenging Activity Test of Sea Cucumber Lipid Complex and Its Separated Components

[0083] Alzheimer's disease and various neurodegenerative diseases are accompanied by excessively high free radical levels in the nerve center and oxidative damage to the nervous system, so substances that can scavenge free radicals play an important role in the development of anti-Alzheimer's drugs and health products. The sea cucumber lipid complex prepared in Example 1 of the present invention and its separated components were used as samples, and antioxidants were used to screen the commonly used DPPH free radicals. The in vitro free radical scavenging activity experiment of the sea cucumber lipid complex and its separated components was carried out. The specific experimental methods and experimental results are as follows:

[0084] 1. Experimental method 1 (96-well plate solution system test method)

[0085] Dissolve the sample to be tested in DMSO solution to a concentration of 0.5 mg / mL. Perform the experiment according to the following reagent ratios, with at least 3 parallels per group.

[0086] Sample group (A sample ): 50 μL sample solution with different gradients + 50 μL DPPH (0.16 mM methanol solution);

[0087] The control group (A control ): 50 μL sample solution with different gradients + 50 μL methanol;

[0088] Blank group (A blank ): 50 μL DMSO + 50 μL methanol;

[0089] Blank control group (A sample blank ): 50 μL DMSO + 50 μL DPPH (0.16 mM methanol solution).

[0090] After the sample addition is completed, the 96-well plate is placed in the dark to react for 30 minutes, and the 96-well plate is taken out and the absorbance at 517nm is measured with an ELISA reader, with vitamin C (VC) as the positive control (final concentration is 250μg / mL). The calculation formula for DPPH clearance is as follows:

[0091]

[0092] 2. Experimental method 2 (silicone plate test method)

[0093] Dissolve the samples to be tested in methanol to a concentration of 2 mg / mL. Use a capillary to spot 5 μL of each sample on a GF254 thin layer chromatography silica gel plate. Dry the organic solvent and evenly spray 1.6 mM DPPH methanol solution on the silica gel plate in an atomized manner. Place the silica gel plate in a dark place for 15 minutes. The empty spots on the purple background are observed to be samples with free radical scavenging activity.

[0094] 3. Experimental results

[0095] The antioxidant properties test results of the sea cucumber lipid complex and its isolated components of the present invention are as follows Figure 4 As shown. Figure 4 As can be seen from A, when the concentration of each sample is 250 μg / mL, the scavenging rate of DPPH free radicals by Crude Sea Cucumber Lipid Complex (Crude) reaches 61.39%, the scavenging rate of separated component Fr.B reaches 7.12%, the scavenging rate of component Fr.E reaches 47.3%, the scavenging rate of component Fr.G reaches 62.88%, the scavenging rate of component Fr.Bd reaches 26.53%, the scavenging rate of component Fr.Eb reaches 24.39%, and the scavenging rate of component Fr.Ed reaches 24.72%. Figure 4 As can be seen in B, on the purple background of the silica gel plate stained with DPPH free radicals, the Crude Sea Cucumber Lipid Complex (Crude), component Fr.B, component Fr.Bd and component Fr.G have more obvious spots at the spots, showing strong free radical scavenging activity, among which the activity of component Fr.B and component Fr.Bd in the silica gel plate test method is stronger than that in the 96-well plate liquid system test method. The above results show that the Crude Sea Cucumber Lipid Complex and its separated components prepared by the present invention have antioxidant activity.

[0096] Application Example 3 Toxicity test of sea cucumber lipid complex and its isolated components on BV-2 cells

[0097] Cytotoxicity is a simple cell killing event caused by cells or chemical substances, which is independent of the cell death mechanism of apoptosis or necrosis. In drug screening, it is necessary to detect the cytotoxicity of specific substances. Therefore, cytotoxicity tests can screen the toxicity of compounds. Cytotoxicity tests are also required in anti-inflammatory experiments to exclude false positives. The rough sea cucumber lipid complex prepared in Example 1 of the present invention and its separated components were used as samples, and their potential cytotoxicity to microglial cells BV-2 was evaluated by CCK-8 method.

[0098] 1. Experimental methods

[0099] A. Experimental Design

[0100] Blank group: BV-2 cells + DMEM medium;

[0101] Experimental group: BV-2 cells + DMEM culture medium + different concentrations of sea cucumber lipid complexes and their separated component samples.

[0102] B. BV-2 cell plating

[0103] When the cell density in the culture flask reaches about 80%, digest the cells with 0.25% trypsin, add 3mL of fresh complete culture medium, blow repeatedly to evenly mix, and absorb most of the cell suspension into a 5mL sterilized and dried EP tube for temporary storage. Keep a small amount of cell suspension, add 5mL of complete culture medium, and mix well. Take 10μL of cell suspension in the EP tube and mix with an equal volume of trypan blue for staining, and count the cells with a cell counter to obtain the cell concentration. According to 1×10 4 Cells / well, 100 μL / well, were inoculated on a 96-well plate, marked, and placed in a cell constant temperature incubator at 37° C. (containing 5% carbon dioxide) for 24 h.

[0104] C. Sample Processing

[0105] Fresh DMEM (serum-free) was used to dilute the samples of the sea cucumber lipid complex and its separated components (8 mg / mL, dissolved in anhydrous ethanol). According to the preliminary experimental results, the final dilution concentration gradient of the sea cucumber lipid complex was 0.1, 1, 3, 5, 10, 20 μg / mL; the final dilution concentration gradient of the first-level components Fr.B and Fr.E was 2, 3, 4, 5, 6, 7 μg / mL; the final dilution concentration gradient of the first-level component Fr.G was 0.05, 0.1, 0.2, 0.3, 0.4, 0.5 μg / mL; the final dilution concentration gradient of the second-level component Fr.Bd was 0.1, 1, 2, 3, 4, 5 μg / mL; the final dilution concentration gradient of the second-level component Fr.Eb was 0.1, 1, 2, 3, 4, 5 μg / mL; the final dilution concentration gradient of Fr.Ed was 0.1, 0.4, 0.8, 1.2, 1.6, 2 μg / mL.

[0106] After dilution, the complete medium in the wells was discarded, and different concentration gradients of the rough sea cucumber lipid complex or its separated components were added to the corresponding wells, and cultured in a cell constant temperature incubator at 37°C (containing 5% carbon dioxide) for 24 hours, with 4 parallels for each group.

[0107] D. Detection

[0108] After BV-2 was cultured for 24 hours, the 96-well plate was taken out, 10 μL of CCK-8 was added to each well in the dark, and the cells were incubated in an incubator for 1 hour. The absorbance was measured at a wavelength of 450 nm using an enzyme marker to calculate the cell viability.

[0109] 2. Experimental results

[0110] The results of the toxicity test on cells of the sea cucumber lipid complex and its separated components of the present invention are as follows: Figure 5 As shown, it can be seen that within the tested range, the separated components of the sea cucumber lipid complex are non-toxic to BV-2 cells, and the sea cucumber lipid complex is non-toxic to BV-2 cells at a concentration not higher than 5 ug / mL.

[0111] Application Example 4 Anti-neuroinflammatory activity test of sea cucumber lipid complex and its isolated components

[0112] Adding LPS to induce inflammation in microglial BV-2 cells can cause them to generate and release NO. When anti-inflammatory drugs are added, the amount of NO generated and released will decrease. Therefore, the amount of NO in cells can be tested as a screening model for anti-neuroinflammatory drugs. The Bio-Sky Nitric Oxide Kit was used to quantitatively detect the NO level of BV-2 cells induced by lipopolysaccharide (LPS) and not induced by LPS in the samples of rough sea cucumber lipid complexes and their separated components.

[0113] A. Experimental Design

[0114] Blank group: BV-2 cells + DMEM medium;

[0115] Control group: BV-2 cells + DMEM medium + LPS (1 μg / mL);

[0116] Experimental group: BV-2 cells + DMEM culture medium + LPS (1 μg / mL) + different concentration gradients of rough sea cucumber lipid complexes and their separated component samples.

[0117] B. Cell plating

[0118] BV-2 Press 2×10 4 Cells / well, 100 μL / well, were seeded in 96-well plates.

[0119] C. Samples of sea cucumber lipid complexes and their separated components at different concentration gradients and LPS induction

[0120] The corresponding samples in Example 4 were diluted with gradient dilution at different concentrations, where the final concentration of the sea cucumber lipid complex ranged from 1 to 6 μg / mL). Samples were added, with 4 parallels per group. The 96-well plate was placed in an incubator for 1 hour, and then LPS-induced cells (final concentration: 1 μg / mL) were added and cultured for 24 hours.

[0121] D. Detection

[0122] According to the instructions of the Bio-Tech NO kit, a NO standard curve was prepared. 1M nitrite standard was diluted with DMEM to different concentrations of 0, 1, 2.5, 5, 10, 20, 40, 50, 80, and 100 μM. 30 μL of standard samples of different concentrations were taken, 30 μL of sulfonamide solution was added, and the samples were placed in a biosafety cabinet away from light for 5 minutes. Then, 30 μL of hydrochloric acid-naphthylamine solution was added, and the samples were placed away from light for 5 minutes. The absorbance was measured at 540 nm using an enzyme reader. Finally, the NO standard curve was drawn after calculation using Microsoft Excel. The regression equation was: y = 0.0074x + 0.0523 (R 2 =0.9996).

[0123] After calculating different concentrations of the sea cucumber lipid complex and its separated component samples according to the above method, the corresponding NO values ​​can be obtained by substituting them into the regression equation.

[0124] 2. Experimental results

[0125] The results of the test on the activity of the sea cucumber lipid complex and its separated components in inhibiting the production of NO in cells are as follows: Figure 6 As shown, it can be seen that the NO concentration in the blank group is very low, basically between 0.5 and 2 μmol / L, while the NO concentration of BV-2 increased significantly after LPS induction. The NO concentration of cells treated with different concentrations of Crude and its separated components Fr.B, Fr.E, Fr.G, Fr.Bd, Fr.Eb, and Fr.Ed decreased significantly, showing a significant anti-inflammatory effect. The optimal anti-inflammatory concentrations were 6 μg / mL, 5 μg / mL, 5 μg / mL, 0.3 μg / mL, 4 μg / mL, 10 μg / mL, and 2 μg / mL, respectively. This shows that Crude and its separated components can significantly inhibit the production of NO in BV-2 cells and have anti-neuroinflammatory activity.

[0126] Application Example 5 Toxicity test of sea cucumber lipid complex and its isolated components on HT-22 cells

[0127] In order to detect the cytotoxicity of the sea cucumber lipid complex and its isolated fractions on neurons, the potential cytotoxicity of the sea cucumber lipid complex and its isolated fractions on HT-22 cells was evaluated using a colorimetric assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT).

[0128] A. Experimental Design

[0129] Blank group: HT-22 cells + DMEM medium;

[0130] Experimental group: HT-22 cells + DMEM medium + sea cucumber lipid complex and its separated component samples.

[0131] B. HT-22 cell plating

[0132] HT-22 cells were cultured at 3×10 4 The cells / well, 100 μL / well, were inoculated on a 96-well plate, marked, and placed in a cell constant temperature incubator at 37° C. (containing 5% carbon dioxide) for 24 h.

[0133] C. Sample processing of sea cucumber lipid complex and its isolated components

[0134] Fresh DMEM (serum-free) was used to dilute the rough sea cucumber lipid complex and its separated components Fr.B, Fr.E and Fr.G (8 mg / mL, dissolved in anhydrous ethanol), and the final dilution concentration gradient was 5, 10, 20, 30, 40, 50, 60, 80, 100, 120 μg / mL, while the final dilution concentration gradient of the separated component Fr.Bd was 1, 10, 20, 30, 40, 50 μg / mL, and the final dilution concentration gradient of Fr.Eb and Fr.Ed was 1, 10, 20, 30, 40, 50 μg / mL.

[0135] After dilution, the complete medium in the wells of the cell culture plate was discarded, and different concentration gradients of the rough sea cucumber lipid complex or its separated components were added to the corresponding wells, and cultured in a cell constant temperature incubator at 37°C (containing 5% carbon dioxide) for 48 hours, with 4 parallels for each group.

[0136] D. Detection

[0137] After 48 hours of culture, dilute MTT with fresh DMEM in the dark, discard the sample-containing medium in the well, add 100 μL MTT (final concentration: 1 μg / mL) to each well, and incubate in the incubator. After 4 hours, take out the 96-well plate, slowly aspirate the upper solution in the well in the dark, and add 100 μL DMSO to each well, shake on a shaker for 10 minutes to fully dissolve the purple formazan. Finally, use an enzyme marker to detect the absorbance at a wavelength of 570 nm to measure the cell viability.

[0138] 2. Experimental results

[0139] The toxicity test results of the sea cucumber lipid complex and its separated components on HT-22 neuronal cells are as follows: Figure 7 As shown, it can be seen that within the tested range, except for the isolated component Fr.G, the rough sea cucumber lipid complex and other isolated components are not toxic to HT-22 cells, and the isolated component Fr.G is also not toxic to HT-22 cells at a concentration not higher than 10 μg / mL.

[0140] Application Example 6 Testing of the neuroprotective effect of sea cucumber lipid complex and its isolated components on HT-22 cells

[0141] Neuronal damage is the root cause of neurodegenerative diseases such as Alzheimer's disease. The present invention uses the MTT method to evaluate the 25-35 The cell viability of HT-22 neuronal cells induced by injury was investigated, and the preventive effect of the sea cucumber lipid complex and its isolated components against neuronal injury was evaluated.

[0142] 1. Experimental methods

[0143] A. Experimental Design

[0144] Blank group: HT-22 cells + DMEM medium;

[0145] Control group: HT-22 cells + DMEM medium + Aβ 25-35 (5 μmol / L) + anhydrous ethanol (same as the highest concentration in the experimental group);

[0146] Experimental group: HT-22 cells + DMEM medium + Aβ 25-35 (5μmol / L)+Sea cucumber lipid complex and its separated component samples.

[0147] B. HT-22 cell plating

[0148] The method is the same as that in Application Example 5.

[0149] C. Sample processing and manipulation of sea cucumber lipid complexes and their isolated components

[0150] The HT-22 cells cultured for 24 h were taken out, the culture medium was discarded, and different concentration gradients of sea cucumber lipid complexes or their separated components diluted with DMEM were added to different wells, and the cells were cultured in a cell constant temperature incubator at 37°C (containing 5% carbon dioxide) for 2 h, then taken out and Aβ was added. 25-35 , so that the volume of each well is 100 μL, Aβ 25-35The final concentration was 5 μmol / L, and the final concentrations of the rough sea cucumber lipid complex and the primary components Fr.B, Fr.E, and Fr.G were 10, 20, 30, 40, 50, and 60 μg / mL, while the final concentrations of the secondary separation components Fr.Bd, Fr.Eb, and Fr.Ed were the same as in Application Example 5. They were placed in a constant temperature incubator and cultured for 48 hours, with 4 parallels in each group.

[0151] D. Detection

[0152] The method is the same as that in Application Example 5.

[0153] 2. Experimental results

[0154] According to the cell viability Figure 8 As shown, it can be seen that HT-22 cells 25-35 The cell viability was significantly reduced under induction, and the cell samples treated with different concentrations of Crude and its separated components Fr.B, Fr.E, Fr.G, Fr.Bd, Fr.Eb, and Fr.Ed had a preventive effect on this neuronal damage, and their respective optimal protective concentrations were 40μg / mL, 40μg / mL, 40μg / mL, 60μg / mL, 0.4μg / mL, 1μg / mL, and 20μg / mL, respectively. This shows that the Crude lipid complex and its separated components of the present invention have the activity of preventing neuronal damage.

[0155] Application Example 7 Test of the intracellular reactive oxygen scavenging activity of sea cucumber lipid complex and its isolated components on HT-22 cells

[0156] The neurotoxic mechanism of Aβ includes inducing an increase in intracellular reactive oxygen species. Substances that can remove intracellular reactive oxygen species have a protective effect on nerves. The molecular probe DCFH-DA itself has no fluorescence and can freely pass through the cell membrane. After entering the cell, it can be hydrolyzed by the intracellular esterase to generate non-fluorescent DCFH. The intracellular ROS can oxidize it into fluorescent DCF. The fluorescence of DCF is detected to measure the level of intracellular ROS. The present invention uses the Biyuntian ROS reactive oxygen species detection kit to evaluate the intracellular reactive oxygen levels of HT-22 cells, as well as the scavenging effect of the rough sea cucumber lipid complex and its separated components.

[0157] 1. Experimental methods

[0158] A. Experimental Design

[0159] Blank group: HT-2 cells + DMEM medium;

[0160] Control group: HT-22 cells + DMEM medium + Aβ (5 μmol / L) + anhydrous ethanol (the same as the highest concentration of the experimental group);

[0161] Experimental group: HT-22 cells + DMEM culture medium + Aβ (5 μmol / L) + sea cucumber lipid complex and its separated component samples.

[0162] B. Cell plating

[0163] HT-22 cells were collected at 1.9×10 5 The cells were plated at 500 μL / well on a 24-well plate and placed in a cell incubator at 37°C (containing 5% carbon dioxide) for 24 hours after labeling.

[0164] C. Effect of sea cucumber lipid complex and its separated components on Aβ 25-35 Induced ROS scavenging

[0165] The 24-well plate was removed, the culture medium was discarded, and the plate was rinsed twice with PBS. 400 μL of different concentrations of sea cucumber lipid complexes or their separated components were added to the corresponding wells. After incubation in the incubator for 2 h, Aβ was added to the wells. 25-35 (Final concentration: 5 μmol / L), and the final concentration of the sample was 40 μg / mL of the rough sea cucumber lipid complex, 40 μg / mL of the first-level components Fr.B, Fr.E and Fr.G, 1 μg / mL of the second-level components Fr.Bd, and 40 μg / mL of the second-level components Fr.Eb and Fr.Ed. Continue to culture for 48 hours, and set up 3 parallels for each group.

[0166] D. Loading of DCFH-DA fluorescent probe

[0167] Dilute 10 mmol DCFH-DA 1000 times with DMEM to prepare DCFH-DA probe working solution. Take the above cultured 24-well plate, discard the culture medium, rinse the cells 3 times with PBS, add 200 μL DCFH-DA probe working solution, and rinse the cells at least 3 times with PBS to remove the residual probe in the cells and background to avoid affecting the photography.

[0168] E. Detection

[0169] The 24-well plate was placed on a fluorescent inverted microscope to observe the fluorescence of the cells and take photos. The fluorescence intensity was processed and the data were sorted using Image J software.

[0170] 2. Experimental results

[0171] The present invention is a method for treating Aβ with the sea cucumber lipid complex and its separated components 25-35 The results of the inhibitory and scavenging effects of ROS in HT-22 cells induced by Fig. 9 As shown, it can be seen that there is no Aβ 25-35The intracellular ROS content (intuitively reflected by fluorescence intensity) of the control group (Blank) was very low; while the addition of Aβ 25-35 The fluorescence intensity caused by intracellular ROS in the model group (Control) was significantly enhanced; while when the rough sea cucumber lipid complex (Crude) and its separated components Fr.B, Fr.E, Fr.G, Fr.Bd, Fr.Eb, and Fr.Ed were added, the fluorescence intensity caused by intracellular ROS was significantly reduced. This shows that the rough sea cucumber lipid complex and its separated components can significantly inhibit the intracellular ROS level and have intracellular antioxidant activity.

[0172] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A rough sea cucumber lipid extract, characterized in that The rough sea cucumber lipid extract comprises a rough sea cucumber lipid complex and its separated components, wherein the preparation method of the rough sea cucumber lipid complex comprises extracting the total lipid of the rough sea cucumber by using a mixed solvent of chloroform and methanol, deoiling by cold acetone, and then separating and collecting the precipitate to obtain the rough sea cucumber lipid complex; the rough sea cucumber separated component is obtained by subjecting the rough sea cucumber lipid complex to silica gel column chromatography, eluting with a mixed solution of dichloromethane, methanol and / or water as an eluent, and after elution, obtaining an active primary component through multi-target anti-Alzheimer's activity screening; the primary component is subjected to multi-target anti-Alzheimer's activity screening, and the two components with the best activity are subjected to normal phase column chromatography respectively, and eluted with a mixed solvent of ethyl acetate and n-hexane and / or a mixed solvent of dichloromethane and methanol to obtain a secondary component.

2. The sea cucumber lipid extract according to claim 1, characterized in that The preparation method of the sea cucumber lipid complex comprises the following steps: After the rough sea cucumber is crushed into powder, a mixed solvent of chloroform and methanol is added at a mass volume ratio of 1 g: (3-10) mL, ultrasonic extraction is performed for 20-30 min, and then suction filtration is performed to collect the filtrate, and chloroform is added to the filter residue at a mass volume ratio of 1 g: (1-3) mL, and the extraction is repeated 1-3 times, the filtrate is combined, and then 1-3 times the volume of potassium chloride solution is added and mixed, and after standing and stratification, the lower layer liquid is collected and concentrated to obtain the total lipid of the rough sea cucumber; Add 10-40 times volume of cold acetone to the total lipid of the rough sea cucumber, extract, centrifuge, collect the precipitate, and obtain the rough sea cucumber lipid complex.

3. The sea cucumber lipid extract according to claim 2, characterized in that The volume ratio of chloroform to methanol is 1:2; the concentration of potassium chloride is 0.5%-1.5%; the Rf values ​​of the sea cucumber lipid complex on a silica gel plate using a mixed solution of chloroform, methanol and water in a volume ratio of 65:22:4 as a developing agent are 0.95, 0.8-0.9, 0.57-0.65, 0.45-0.5, 0.35-0.4 and 0.

2.

4. The sea cucumber lipid extract according to claim 1, characterized in that The method for preparing the isolated components of sea cucumber comprises the following steps: The mixed solution of dichloromethane, methanol and / or water includes: a mixed solvent of dichloromethane and methanol, a mixed solution of dichloromethane, methanol and water, and a mixed solvent of dichloromethane and methanol.

5. The sea cucumber lipid extract according to claim 1, characterized in that After multi-target anti-Alzheimer's activity screening, highly active primary components Fr.B, Fr.E and Fr.G were obtained. Among them, on a silica gel plate with a mixed solution of chloroform, methanol and water in a volume ratio of 65:22:4 as the developing agent, the Fr.BRf value was 0.95 and 0.85-0.9, the Fr.E had an Rf value of 0.6-0.7, and the Fr.G had an Rf value of 0.4-0.7, 0.2-0.4 and 0-0.

2.

6. The sea cucumber lipid extract according to claim 5, characterized in that The two most active primary components Fr.B and Fr.E were subjected to normal phase column chromatography respectively, wherein Fr.B was eluted with the following mixed solvents in sequence: a mixed solvent of ethyl acetate and n-hexane, and a mixed solvent of dichloromethane and methanol; and Fr.E was eluted with a mixed solvent of dichloromethane and methanol.

7. The sea cucumber lipid extract according to claim 6, characterized in that After normal phase column chromatography, the Fr.B is subjected to normal phase column chromatography to obtain a component Fr.Bd with good anti-multi-target and anti-Alzheimer's activity. The Rf value of the Fr.Bd on a silica gel plate using a mixed solution of chloroform, methanol and water in a volume ratio of 65:22:4 as a developing agent is 0.95; after normal phase column chromatography, the Fr.E is subjected to normal phase column chromatography to obtain components Fr.Eb and Fr.Ed with good anti-multi-target and anti-Alzheimer's activity. The Rf values ​​of the Fr.Eb and Fr.Ed on a silica gel plate using a mixed solution of chloroform, methanol and water in a volume ratio of 65:22:4 as a developing agent are 0.6-0.64 and 0.61-0.67, respectively.

8. The sea cucumber lipid extract according to claim 7, characterized in that The sea cucumber lipid complex, Fr.B, Fr.Bd, Fr.E, Fr.Eb, Fr.Ed and Fr.G were analyzed by liquid chromatography-mass spectrometry, and the liquid chromatography conditions were as follows: the chromatographic column model was Waters ACQUITY UPLC BEH RP18; the mobile phase was a mobile phase A and a mobile phase B with a volume ratio of 4:6, the mobile phase A was a methanol solution containing 0.1% formic acid, and the mobile phase B was an acetonitrile solution containing 0.1% formic acid; the gradient elution conditions were: 0-8min, 40% mobile phase A; the flow rate was 0.2mL / min; Among them, the rough sea cucumber lipid complex, Fr.B, Fr.Bd, Fr.E, Fr.Eb, Fr.Ed and Fr.G have retention times of 0.5-0.6min, 0.75-0.9min, 0.9-1.0min, 1.0-1.1min, 1.2-1.3min, 1.35-1.48min, 1.48-1.58min, 1.75-1.89min, 2.1-2.25min, 2.65-2.8min, 3.0-3.3min, 3.3-3.48min, 4.4-4.6min, and 6.0-6.3 in the positive ion mode. min, and their total contents were 90.66%, 86.32%, 90.35%, 82.23%, 91.85, 73.16% and 88.64% respectively; the corresponding negative ion mode contained a group of chromatographic peaks with retention times of 0.52-0.67min, 1.1-1.2min, 1.2-1.32min, 1.4-1.6min, 1.7-1.87min, 2.4-2.6min and 6.0-6.3min, and their total contents were 69.64%, 81.95%, 79.08%, 70.54%, 77.54%, 66.50% and 75.01% respectively.

9. Use of the lipid extract of sea cucumber in any of the following: Application in the preparation of drugs for resisting neuronal cell damage; Use in the preparation of drugs with anti-neuritis activity; Application in the preparation of drugs for removing active oxygen in neuronal cells; Application in the preparation of drugs for scavenging DPPH free radical activity; Application in the preparation of drugs for inhibiting acetylcholinesterase activity; Application in the preparation of food that assists in improving memory function; The sea cucumber lipid extract comprises any one of the sea cucumber lipid complex, Fr.B, Fr.Bd, Fr.E, Fr.Eb, Fr.Ed and Fr.G described in claim 8.

10. A drug having anti-neuronal cell damage and / or anti-neuritis activity and / or scavenging active oxygen in neuronal cells and / or scavenging DPPH free radical activity and / or inhibiting acetylcholinesterase activity and / or a functional food for assisting in improving memory, characterized in that: It includes any one of the rough sea cucumber lipid complex described in claim 8, Fr.B, Fr.Bd, Fr.E, Fr.Eb, Fr.Ed and Fr.G.

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