阅读说明：本技术 一种磷脂酰糖苷的制备方法及其应用 (Preparation method and application of phosphatidyl glycoside ) 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,具体涉及一种磷脂酰糖苷的制备方法及其应用。本发明通过磷脂酶D水解磷脂酰胆碱,同时与糖类底物作用,生成磷脂酰糖苷。实现了酶法合成磷脂酰糖苷,并证实了所得到的磷脂酰糖苷具有预防减轻阿尔兹海默病症的能力,能改善行为认知障碍。(The invention belongs to the technical field of active substance screening, and particularly relates to a preparation method and application of phosphatidyl glycoside. The invention hydrolyzes phosphatidylcholine through phospholipase D, and reacts with carbohydrate substrate to generate phosphatidyl glycoside. Realizes the enzymatic synthesis of the phosphatidyl glycoside, and proves that the obtained phosphatidyl glycoside has the capacity of preventing and relieving Alzheimer disease and can improve behavior cognitive disorder.)

1. A preparation method of phosphatidyl glycoside is characterized in that: phosphatidylcholine is hydrolyzed by phospholipase D and reacts with a carbohydrate substrate to produce a phosphatidyl glycoside.

2. The method for producing a phosphatidylglycoside of claim 1, wherein: the phosphatidylcholine was thoroughly mixed with silica gel powder prior to reaction.

3. The method for producing a phosphatidylglycoside of claim 1, wherein: the phospholipase D and the carbohydrate substrate are mixed and dissolved in a phosphate buffer before the reaction.

4. The method for producing a phosphatidylglycoside according to any one of claims 1 to 3, wherein: mixing phosphatidylcholine with a solution of phospholipase D and a saccharide substrate, fully stirring at the temperature of 35-45 ℃ for 12h, and centrifuging to collect precipitate.

5. The method for producing a phosphatidylglycoside of claim 4, wherein: mixing petroleum ether and ethanol solution at a volume ratio of 4:1, and extracting the reactant for 3 times.

6. The method for producing a phosphatidylglycoside of claim 5, wherein: and combining the petroleum ether phases and removing the organic solvent to obtain the high-purity phosphatidyl glycoside.

7. The method for producing a phosphatidylglycoside of claim 1, wherein: the glycosyl substrate is derived from monosaccharides, oligosaccharides, polysaccharides, amino sugars and sugar alcohols with different configurations, specifically including glucose, trehalose, ribose, deoxyribose, erythrose, threose, arabinose, xylose, lyxose, mannose, galactose, UDP-glucose, rhamnose, fucose, ADP-glucose, CDP-glucose, GDP-glucose, TDP-glucose, sucrose, maltose, gentiobiose, lactose, cellobiose, raffinose, stachyose, sorbitol, xylitol, ribitol, mannitol, glucitol, glucosamine and galactosamine; the phosphatidylcholine substrate is derived from soybean, egg yolk, and phosphatidylcholine extracted from marine animals.

8. A phosphatidyl glycoside produced by the method of claim 1, wherein: the sn-1 and sn-2 positions are connected to form fatty acid, and the sn-3 position has a phospholipid compound with a polar head composed of a phosphate-linked glycosyl.

9. An application of phosphatidyl glycoside in the preparation for treating neurodegenerative diseases and the application in the preparation for preventing, improving and treating Alzheimer's disease are provided.

10. The use of claim 9, wherein: the product comprises phosphatidyl glycoside with a pharmacologically effective concentration.

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 phosphatidyl glycoside.

Background art:

alzheimer's Disease (AD) is a neurodegenerative disease, the most common and devastating dementia at present, and is recognized by World Health Organization (WHO) as one of the first problems of world public health, affecting nearly 5000 people 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). In addition, loss of synaptic and neuronal function, reactive gliosis, oxidative and inflammatory damage are also major morbidity features. 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.

Phosphatidylglucoside (ptglc) is the only phosphatidylglycoside compound discovered so far, and pure ptglc successfully isolated from the brain of fetal rodents has only one fatty acid combination: i.e., the sn-1 and sn-2 chains are stearic acid (C18: 0) and arachidic acid (C20: 0), respectively. PtdGlc is also present in human neutrophils with a fatty acid composition comprising C18:0/C18:0 and C18:0/C20: 0. Phosphatidylglucosides, glycosylated lipids, have been found to play a fundamental role in a variety of cellular processes. Phosphatidylglucosides have been shown to be involved in nerve conduction, particularly the axonal regulation associated with TrkA and TrkC. The metabolite of phosphatidylglucoside, lysophosphatidyl- β -d-glucoside (LysoPtdGlc), a hydrophilic glycerophospholipid, was also shown to play a role in activating GPR55 in morphologically specific rejection guidance of spinal sensory axons. Although the importance of phosphatidylglycosides in the brain is known, the effect of ingestion of phosphatidylglycosides on neurodegenerative diseases has not yet been investigated. Meanwhile, due to the limitation of the separation technique, it is extremely difficult to purify the phosphatidylglucoside to a high purity.

The invention content is as follows:

the technical problem to be solved by the invention is that the effect of taking phosphatidyl glycoside on neurodegenerative diseases is not researched; meanwhile, due to the limitation of the separation technique, it is extremely difficult to purify the phosphatidylglucoside to a high purity.

In order to solve the problems, the invention realizes the enzymatic synthesis of the phosphatidyl glycoside by using the phospholipase D, and proves that the obtained phosphatidyl glycoside has the capability of preventing and relieving Alzheimer's disease and can improve behavioral cognitive disorder.

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

a method for preparing phosphatidyl glycoside comprises hydrolyzing phosphatidyl choline with phospholipase D, and reacting with saccharide substrate to obtain phosphatidyl glycoside.

Furthermore, the phosphatidylcholine is fully mixed with the silica gel powder before reaction, so that the subsequent phosphatidylcholine substrate can be uniformly dispersed in a reaction system.

Further, the phospholipase D and the carbohydrate substrate are mixed and dissolved in a phosphate buffer solution before the reaction, so as to provide an aqueous environment required by the enzyme reaction.

Further, the phosphatidylcholine mixed with the silica gel powder is mixed with phospholipase D (10-40U/ml) and a solution of a saccharide substrate (phosphatidylcholine: saccharide substrate is 1:40-1:10mol/mol), then the mixture is fully stirred for 12 hours at the temperature of 35-45 ℃, and then the precipitate is collected by centrifugation, and during the period, the phospholipase D can catalyze and hydrolyze choline groups in the phosphatidylcholine and perform a lipid exchange reaction with the saccharide substrate to generate the required phosphatidyl glycoside. Changes in temperature and time or deficiencies in carbohydrate substrates can lead to the production of by-products such as phosphatidic acid in the substrate.

Further, after mixing petroleum ether and ethanol solution according to a ratio of 4:1(V/V), adding the reacted precipitate, stirring for ten minutes, standing for layering, taking away an upper layer petroleum ether phase, extracting the reacted precipitate for 3 times, adding petroleum ether with the same volume in each time in an equivalent manner, wherein the product phosphatidyl sugar is dissolved in the upper layer petroleum ether phase, the redundant choline and the substrate serine are flocculent precipitate, and the redundant substrate phosphatidyl choline is dissolved in an ethanol layer.

Further, the petroleum ether phases are combined and removed to obtain the high-purity phosphatidyl glycoside.

Further, the glycosyl substrate (constituting the polar head of the phosphatidyl glycoside) is derived from monosaccharides, oligosaccharides, polysaccharides, aminosugars, and sugar alcohols of different configurations, specifically including glucose, trehalose, ribose, deoxyribose, erythrose, threose, arabinose, xylose, lyxose, mannose, galactose, UDP-glucose, rhamnose, fucose, ADP-glucose, CDP-glucose, GDP-glucose, TDP-glucose, sucrose, maltose, gentiobiose, lactose, cellobiose, raffinose, stachyose, sorbitol, xylitol, ribitol, mannitol, glucitol, glucosamine, galactosamine; the phosphatidylcholine substrate is derived from soybean, egg yolk, and phosphatidylcholine extracted from marine animals (squid, sea cucumber, cod, salmon, mackerel, mussel, oyster, sea urchin, starfish, and conch).

The phosphatidyl glycoside prepared by the method is a phospholipid compound with sn-1 and sn-2 connected by fatty acid and sn-3 position having polar head composed of glycosyl connected by phosphate, and the purity of the phospholipid compound is more than 85%.

An application of phosphatidyl glycoside in the preparation for treating neurodegenerative diseases and the application in the preparation for preventing, improving and treating Alzheimer's disease are provided.

The product comprises phosphatidyl glycoside with a pharmacologically effective concentration.

The product is a medicine or a special medicine food, a health product and a biological preparation, and preferably the medicine or the special medicine food.

The invention has the beneficial effects that:

(1) the purity of the prepared phosphatidyl glycoside is more than 85%.

(2) The prepared phosphatidyl glycoside can obviously reduce the behavior cognitive function of mice with Alzheimer disease, improve the brain Abeta aggregation and Tau protein aggregation, can be used for preparing medicines for preventing and treating diseases such as Alzheimer disease and the like, and has wide market application prospect.

Drawings

FIG. 1 is a water maze result statistical chart showing the improvement effect of phosphatidyl glucoside and phosphatidyl trehaloside 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 phosphatidylglucoside and phosphatidyltrehaloside prepared according to the present invention on the expression level of A β 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 shows the improvement effect of phosphatidyl glucoside and phosphatidyl trehaloside prepared by the present invention on Tau protein expression level and Tau protein aggregation pathology of APP/PS1 Alzheimer's disease mice 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.