阅读说明：本技术 一种epa/ara型缩醛磷脂的制备方法及其应用 (Preparation method and application of EPA/ARA type plasmalogen ) 是由 薛长湖 刘炎峻 徐杰 姜晓明 李兆杰 丛培旭 王玉明 王静凤 于 2020-05-18 设计创作，主要内容包括：本发明属于活性物质筛选技术领域,一种EPA/ARA型缩醛磷脂的制备方法与应用。用PLA1酶连续水解磷脂复合物,然后利用超临界CO<Sub>2</Sub>流体萃取技术从酶解磷脂中提取得到EPA/ARA型缩醛磷脂。本发明所制备的EPA/ARA型缩醛磷脂,纯度在80％以上,与EPA型缩醛磷脂或者ARA型缩醛磷脂相比,更有显著降低阿尔兹海默病症小鼠的行为认知功能,改善大脑Aβ聚集,Tau蛋白聚集,可用于制备预防和治疗阿尔兹海默病症等疾病的药物,具有广阔的市场应用前景。(The invention belongs to the technical field of active substance screening, and relates to a preparation method and application of EPA/ARA type plasmalogen. Continuous hydrolysis of phospholipid complexes with PLA1 enzyme followed by supercritical CO 2 The EPA/ARA type plasmalogen prepared by the invention has the purity of more than 80 percent, and compared with the EPA type plasmalogen or the ARA type plasmalogen, the EPA/ARA type plasmalogen has the advantages of remarkably reducing the behavior cognitive function of mice with Alzheimer disease, improving the aggregation of brain A β and the aggregation of Tau protein, can be used for preparing medicines for preventing and treating diseases such as Alzheimer disease and the like, and has wide market application prospect.)

1. A preparation method of EPA/ARA type plasmalogen is characterized in that: continuous hydrolysis of phospholipid complexes with PLA1 enzyme followed by supercritical CO₂The EPA/ARA type plasmalogen is extracted from the enzymolysis phospholipid by a fluid extraction technology.

2. The method for producing an EPA/ARA type plasmalogen according to claim 1, wherein: the preparation steps of the phospholipid complex are as follows: freezing and vacuum drying marine animals, and crushing to obtain powder with the fat content of 5-30%; and performing subcritical extraction on the powder to obtain the phospholipid complex.

3. The method for producing an EPA/ARA type plasmalogen according to claim 2, wherein: the marine animal is one or more of cod, salmon, Spanish mackerel, mussel, Concha Ostreae, sea urchin, starfish, conch, sea cucumber, and Loligo chinensis Gray.

4. The method for producing an EPA/ARA type plasmalogen according to claim 2, wherein: the subcritical extraction condition is that the solvent-to-material ratio is 1:1-1.5: 1; the working pressure of the extraction tank is as follows: 0.3-0.8 MPa; the extraction temperature is as follows: 30-50 ℃; extraction time: 30-60 minutes; temperature of the separation tank: 50-70 ℃.

5. The method for producing an EPA/ARA type plasmalogen according to claim 1, wherein: the enzymolysis steps are as follows: controlling the substrate concentration to be 2-10g/mL, adding 2-10% of enzyme (phospholipase A1, Lectiase Ultra) for enzymolysis, reacting for 5-20h, adding 10-20% of water, reacting at 40-50 ℃ and 200 r/min.

6. The method for producing an EPA/ARA type plasmalogen according to claim 1, wherein: supercritical CO₂The fluid extraction steps are as follows: passing the enzymolysis phospholipid product through supercritical CO₂Extracting with ethanol mixed fluid to obtain high-purity plasmalogen; wherein the extraction conditions comprise extraction temperature of 40-50 ℃, extraction pressure of 35MPa and extraction time of 5 h.

7. An EPA/ARA type plasmalogen prepared by the process of claim 1, characterized in that: the plasmalogen is a plasmalogen mixture in which sn-1 position fatty acid is connected with a glycerol skeleton by an alkenyl ether bond, and sn-2 position fatty acid is EPA (eicosapentaenoic acid) and ARA (arachidonic acid).

8. The EPA/ARA-type plasmalogen according to claim 7, wherein: the fatty acid composition of the plasmalogen comprises EPA and ARA, the ratio of EPA to ARA fatty acid is 2: 1-1: 1, and the total amount of EPA and ARA accounts for more than 50% of the total fatty acid composition.

9. An EPA/ARA type plasmalogen according to claim 7 for use in the prevention and treatment of amelioration of Alzheimer's disease.

10. An article of manufacture for treating alzheimer's disease, comprising: the preparation comprises 1% to 100% of the EPA/ARA-type phospholipid complex or EPA/ARA-type plasmalogen according to claim 1.

The technical field is as follows:

the invention belongs to the technical field of active substance screening, and particularly relates to a preparation method and application of EPA/ARA type plasmalogen.

Background art:

alzheimer's Disease (AD) is a neurodegenerative disease, the most common and devastating dementia at present, and is recognized by the World Health Organization (WHO) as one of the first problems of public health worldwide. Atrophy of hippocampus and cortex of patients with alzheimer's disease occurs, and the most important neuropathological markers are hyperphosphorylation of Tau protein and excessive accumulation of neurofibrillary tangles and amyloid plaque deposition by amyloid β (Α β). More importantly, alzheimer's disease has a higher lethality rate. Unfortunately, current drug therapy for AD is still in its infancy, providing symptomatic relief without slowing down the disease progression, and due to the high cost of society and healthcare, high morbidity and mortality, and lack of an effective treatment modality, alzheimer's disease has become a pressing issue.

Plasmalogens (plasmalogens) are a unique class of membrane glycerophospholipids, in which the sn-1 position of the glycerol backbone is a long-chain enol with an olefinic ether linkage, and the sn-2 position is mostly a polyunsaturated fatty acid. Plasmalogen is widely present in animal tissue, constitutes 15-20% of cell membrane phospholipid component, and has high content (more than or equal to 50%) in brain, heart, kidney, etc. The levels of ethanolamine plasmalogens (PlsEtn) in brain tissue and serum of patients with Alzheimer's Disease (AD) were reduced, while the plasmalogen content was significantly reduced by 40 mol% in the early AD stage and by 10 mol% in the gray matter at the early stage. At the same time, more and more studies have shown that plasmalogen deficiency and systemic peroxisome dysfunction may also be specific markers for AD pathology. Exogenous plasmalogens have also been shown to possess a variety of biological activities. Plasmalogen has strong susceptibility to chemical hypoxia of macrophages in vitro and ROS generators, and can also inhibit Neuro2a apoptosis. Dietary intake of plasmalogens is therefore one of the possible treatments for alzheimer's disease, but studies of improvement of senile dementia by EPA/ARA type plasmalogens have not been reported.

Essential fatty acids play a key role in the brain and mediate many of the processes altered by Alzheimer's Disease (AD), where essential fatty acids can be subdivided into two omega-3 and omega-6. The major omega-3 fatty acids are eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), etc., while the major omega-6 fatty acids are Arachidonic Acid (AA) and Linoleic Acid (LA). In the brain, AA, EPA and DHA are the most important three fatty acids, constituting the majority of free fatty acids. Arachidonic acid and its products have been shown to be involved in a variety of functions of the brain, including signaling, neurotransmitter release, neuronal gene expression, sleep cycle regulation, and the like. AA uptake has been shown to promote cell survival and enhance neuronal growth. Abnormalities in AA metabolism are also associated with a number of cerebral and psychological disorders, such as epilepsy, stroke, alzheimer's disease, parkinson's disease, schizophrenia and mood disorders. The research on the function of EPA is more proved at present, the effect of EPA on the inhibition of inflammation and neuroprotection is also proved to be reduced, and the EPA level in the brain of a patient with neurodegenerative disease is also proved to be reduced.

The existing extraction method of plasmalogen comprises extraction and purification of an organic reagent and extraction of the organic reagent after enzymatic conversion, and the adoption of the method has several problems: first, free fatty acids cannot be completely removed; secondly, complete separation of lysophospholipids from plasmalogens is difficult to achieve, and solvent residues are a disadvantage, which limits the scope of applications.

The invention content is as follows:

the technical problem to be solved by the invention is that the existing extraction method of plasmalogen comprises organic reagent extraction and purification and organic reagent extraction after enzymatic conversion, and the method has several problems: first, free fatty acids cannot be completely removed; secondly, complete separation of lysophospholipids from plasmalogens is difficult to achieve, and solvent residues are a disadvantage, which limits the scope of applications.

In order to solve the problems, the invention adopts the principle of PLA1 hydrolysis and acyl migration for the first time to continuously hydrolyze sn-1 and sn-2 fatty acids of impurity phospholipid to obtain α -glycerophospholipid with different polarities, and then uses supercritical CO to perform hydrolysis₂The plasmalogen is extracted from the enzymolysis phosphatide by the fluid extraction technology, and the industrialization of the plasmalogen is realized.

In order to achieve the purpose, the invention is realized by the following technical scheme:

a method for preparing EPA/ARA type plasmalogen comprises continuously hydrolyzing phospholipid complex with PLA1 enzyme, and performing supercritical CO extraction₂The EPA/ARA type plasmalogen is extracted from the enzymolysis phospholipid by a fluid extraction technology.

Further, the preparation steps of the phospholipid complex are as follows: freezing and vacuum drying marine animals, and crushing to obtain powder with the fat content of 5-30%; and performing subcritical extraction on the powder to obtain the phospholipid complex.

Further, the marine animal is one or more of cod, salmon, Spanish mackerel, mussel, Concha Ostreae, sea urchin, starfish, conch, sea cucumber, and Loligo chinensis Gray. The marine animals are rich in two important polyunsaturated fatty acids of EPA and ARA, and are suitable for preparing EPA/ARA type plasmalogen.

Further, the subcritical extraction condition is that the solvent ratio is 1:1-1.5: 1; the working pressure of the extraction tank is as follows: 0.3-0.8 MPa; the extraction temperature is as follows: 30-50 ℃; extraction time: 30-60 minutes; temperature of the separation tank: at 50-70 deg.C to fully extract total phospholipids and reduce impurities (such as neutral lipid).

Further, the enzymolysis step is as follows: controlling the concentration of the substrate to be 2-10g/mL, adding 2-10% of enzyme (phospholipase A1, Lectiaseultra) for enzymolysis, reacting for 5-20h, adding 10-20% of water, reacting at 40-50 ℃ and 200 r/min. Specifically hydrolyzing fatty acid of sn-1 by PLA1, and simultaneously migrating the sn-2 fatty acid to the sn-1 position due to steric hindrance and continuously hydrolyzing the sn-1 fatty acid and the sn-2 fatty acid of impurity phospholipid to obtain a mixture of alpha-glycerophospholipid, free fatty acid and plasmalogen, wherein the condition can fully hydrolyze fatty acid of impurity phospholipid, and the parameter setting outside the condition can lead to the retention of hemolytic phospholipid and reduce the subsequent product purity.

Further, supercritical CO₂The fluid extraction steps are as follows: passing the enzymolysis phospholipid product through supercritical CO₂And (3) carrying out an ethanol mixed fluid extraction process to obtain high-purity plasmalogen, wherein the extraction conditions comprise extraction temperature of 40-50 ℃, extraction pressure of 35MPa and extraction time of 5h, and the parameters are set to fully remove free fatty acid and α -glycerophospholipid in the product.

The EPA/ARA type plasmalogen prepared by the method is a plasmalogen mixture of fatty acid at sn-1 position and glycerin skeleton which are connected by vinyl ether bond, and fatty acid connected at sn-2 position is EPA (eicosapentaenoic acid) and ARA (arachidonic acid).

Further, the fatty acid composition of the plasmalogen comprises EPA and ARA, the ratio of the EPA to the ARA fatty acid is 2: 1-1: 1, and the total amount of the EPA and the ARA accounts for more than 50% of the total fatty acid composition.

The EPA/ARA type plasmalogen of the present invention is one or a mixture of plasmalogen choline (pPC), plasmalogen ethanolamine (pPE), plasmalogen serine (pPS), plasmalogen inositol (pPI), plasmalogen acid (pPA).

An application of the EPA/ARA type plasmalogen in preventing, treating and improving Alzheimer's disease is provided. The EPA/ARA type plasmalogen has the capability of preventing and relieving Alzheimer disease, and can improve behavior cognitive disorder.

Furthermore, the EPA/ARA type plasmalogen product is preferably special medical food, health care product, medicine or biological preparation, and the dosage is 0.5mg-50mg plasmalogen per day for an adult.

Further, when the EPA/ARA type plasmalogen is used for preparing a product for preventing and treating Alzheimer disease, the EPA/ARA fatty acid ratio is 2: 1-1: 1, and the total amount of EPA and ARA accounts for more than 50% of the fatty acid composition.

Further, the preparation comprises the EPA/ARA type phospholipid complex or the EPA/ARA type plasmalogen according to claim 1 in a pharmacologically effective concentration (1% to 100%).

The invention has the beneficial effects that:

(1) the EPA/ARA type plasmalogen prepared by the invention has the purity of more than 80 percent.

(2) Compared with EPA type plasmalogen or ARA type plasmalogen, the EPA/ARA type plasmalogen prepared by the invention has the advantages that the behavior cognitive function of mice with Alzheimer diseases is remarkably reduced, the brain Abeta aggregation and Tau protein aggregation are improved, the EPA/ARA type plasmalogen can be used for preparing medicines for preventing and treating diseases such as Alzheimer diseases, and the market application prospect is wide.

Drawings

FIG. 1 is a water maze result statistical chart showing the improvement effect of EPA/ARA type plasmalogens prepared by the invention on the behavior cognition level of APP/PS1 Alzheimer disease mice in the embodiment 2.

FIG. 2 is a graph showing the improvement effect of EPA/ARA type plasmalogen prepared by the present invention on the expression level of A beta 1-42 in APP/PS1 Alzheimer's disease mice in example 2. (black and white picture on top and color picture on bottom)

FIG. 3 is a graph showing the improvement effect of EPA/ARA type plasmalogen prepared by the present invention on the Tau protein expression level and Tau protein aggregation pathology of APP/PS1 Alzheimer's disease mouse in example 2. (black and white picture on top and color picture on bottom)

The specific implementation mode is as follows:

in order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are a part of the embodiments of the present invention, but not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.