阅读说明：本技术 一种阿魏酸丁酯的制备方法及其在治疗或者预防阿兹海默症中的功能应用 (Preparation method of ferulic acid butyl ester and functional application of ferulic acid butyl ester in treating or preventing Alzheimer's disease ) 是由 石玉刚 吴煜 朱陈敏 于 2019-08-27 设计创作，主要内容包括：本发明公开了一种阿魏酸丁酯的绿色制备方法及其应用,实验结果表明阿魏酸丁酯具有较好的预防和治疗阿兹海默症的效果。阿魏酸丁酯可以有效抑制β-淀粉样蛋白单体的聚集,同时,它能通过清除神经细胞内氧自由基和激活细胞内抗氧化应激系统的方式达到治疗效果。该制备方法首先以天然产物阿魏酸作为原料制备了一种生物相容性较好的功能性离子液体,再通过构建稳定离子液滴微反应器,将含有酶的离子液体与SiO<Sub>2</Sub>制备成Pickering乳液,并以此构建宏观连续流动柱式反应系统,突破了反应平衡、催化剂分离与传统间歇反应器的限制。操作简单、转化率高,且酶活耐久性高,重复利用率高,节约成本。(The invention discloses a green preparation method of ferulic acid butyl ester and application thereof, experimental results show that ferulic acid butyl ester has better effect of preventing and treating Alzheimer's disease, ferulic acid butyl ester can effectively inhibit β -amyloid monomer aggregation, and simultaneously, ferulic acid butyl ester can achieve treatment effect by removing oxygen free radicals in nerve cells and activating an intracellular antioxidant stress system 2 Preparing Pickering emulsion, constructing a macroscopic continuous flow column type reaction system by using the Pickering emulsion, and breaking through reaction balance, catalyst separation and traditional batch reactionThe limitation of the device. The method has the advantages of simple operation, high conversion rate, high enzyme activity durability, high repeated utilization rate and cost saving.)

1. The application of the ferulic acid butyl ester in the preparation of the functional additive is characterized in that the functional additive is used for preventing and treating the Alzheimer disease.

2. The use of ferulic acid butyl ester of claim 1 in the preparation of a functional additive, wherein the functional additive is used to inhibit β amyloid protein from precipitating as a more neurotoxic oligomer.

3. The use of ferulic acid butyl ester according to claim 1, for the preparation of a functional additive for scavenging reactive oxygen species, ROS, accumulated in nerve cells.

4. The use of ferulic acid butyl ester according to claim 1 in the preparation of a functional additive, wherein the functional additive is used to activate antioxidant stress systems in nerve cells.

5. The preparation method of the ferulic acid butyl ester is characterized by comprising the following steps:

(1) drying the silicon dioxide nanoparticles at 100-120 ℃ for 4-10 hours, and uniformly dispersing the silicon dioxide nanoparticles in toluene under the assistance of ultrasound to obtain a suspension;

(2) to the above suspensionAdding (CH)₃)₂SiCl₂And CH₃(CH₂)₅NH₂Stirring for 4-10 h at the constant temperature of 50-60 ℃ under the protection of nitrogen, centrifuging to obtain solid particles, and flushing the solid particles with toluene and methanol to obtain dimethyl modified silicon dioxide particles;

(3) dissolving dodecyl betaine in methanol, adding equal molar amount of ferulic acid into the solution, stirring at 25 deg.C for 1 hr, removing methanol by rotary evaporation, vacuum drying the solid at 25 deg.C for 24 hr to obtain ionic liquid [ C₁₂Bet][Fal]；

(4) Uniformly dispersing the dimethyl modified silicon dioxide particles into n-octane, and adding ionic liquid [ C ] into the solution₁₂Bet][Fal]Mixing with candida antarctica enzyme, and homogenizing in a homogenizer to obtain pickering emulsion;

(5) adding the solution into a packed column with the bottom having an aperture of 4.5-9 μm and an inner diameter of 2cm to obtain an ion droplet microreactor packed column;

(6) simultaneously injecting a 0.4M n-butanol-n-octane solution and a 0.1M n-octane solution of ferulic acid into the packed column through a pump, controlling the flow rates of the two solutions to be 2mL/h, keeping the packed column at a constant temperature of 45 ℃, collecting an effluent, then taking silica gel as a stationary phase and petroleum ether/ethyl acetate as a mobile phase, and separating and purifying a target product of ferulic acid butyl ester according to gradient elution.

Technical Field

The invention relates to the fields of functional food additives, medicines and the like, and relates to preparation of a ferulic acid ester compound and functional application of the ferulic acid ester compound in preventing and treating Alzheimer's disease.

Background

According to the research of World Health Organization (WHO), about forty million patients with Alzheimer's disease are existed all over the world, however, no drug can effectively treat the disease, with the deep research, it is found that β -deposition of amyloid protein outside Neuron cells and Neurofibrillary Tangles (NTFs) formed by abnormal phosphorylation of Tau protein lead to nerve cell damage (Acta Neurophophathol.1991, 82, 239-.

At present, the pathogenesis related to AD is mainly the neurotoxic effect of A β precipitate, oxygen pressure and the formation of Reactive Oxygen Species (ROS), wherein the abnormal precipitation of A β in the brain is one of the important causes of nerve injury, excessive A β leads to the formation of increased oxygen pressure related to A β, which causes the accumulation of intracellular ROS, which is also the pathogenesis of several diseases in human, such as cancer and neurological diseases, therefore, the development of antioxidant strategies, the capture of intracellular ROS by antioxidants, and the inhibition or reduction of oxygen pressure, is also an effective method for preventing and treating AD (Pharmaceutical Research,2011,28(11), 2771-shaped 2779).

Experiments of the subject find that the hydrophilic lipophilic property of phenolic acid in a food system and a cell system can be effectively changed by grafting an alkyl chain on a hydroxyl group of the phenolic acid, so as to enhance the antibacterial effect (Journal of agricultural and food Chemistry,2018, 66(45),12088-12101.) and the antioxidant capacity (Pharmaceutical Research,2013,30, 1979-1989.) at present, application Research of alkyl ferulate is mainly focused on the antioxidant performance of the alkyl ferulate in the food system, Research on neuroprotection and prevention or treatment of Alzheimer's disease is limited, particularly related mechanism Research is less, it is reported that ferulic acid in animal experiments has the effect of protecting nerve damage caused by amyloid β (prepared by Toxosan, H.S.., 2004,27, 120. Ethyl ferulate has the effect of inhibiting the nerve damage caused by amyloid protein β (European stress-induced by European stress-induced protein kinase, European stress-induced cell repair, European stress-induced amyloid protein (European stress-induced amyloid protein) and the effect of inhibiting the antioxidant activity of amyloid protein (European stress-induced by European stress-induced amyloid polypeptide kinase, European stress-induced amyloid polypeptide, European stress-induced cell repair, European stress-induced protein, European stress-induced cell repair, European stress-induced injury, induced injury.

Disclosure of Invention

The invention aims to provide a new application of ferulic acid butyl ester, and experimental results show that ferulic acid butyl ester has a neuroprotective effect and has great potential for preventing and treating Alzheimer's disease.

The technical scheme of the invention is as follows:

the application of ferulic acid butyl ester in the preparation of functional additives can be used for preventing and treating Alzheimer's disease.

Experimental results show that the ferulic acid butyl ester can inhibit β -amyloid protein deposition, reduce the accumulation of intracellular ROS, activate an antioxidant stress system, protect nerve cells and achieve the effect of preventing and treating the Alzheimer's disease.

The invention also provides a green and efficient preparation method of the ferulic acid butyl ester, which comprises the following steps:

preferably, the silica nanoparticles are modified by the Stoer method, 1g of the modified silica nanoparticles are dried at 120 ℃ for 4h and uniformly dispersed in 12mL of toluene with the aid of ultrasound.

To the above suspension was added 3mmol (CH)₃)₂SiCl₂And 6mmol CH₃(CH₂)₅NH₂And stirring for 4 hours at the constant temperature of 60 ℃ under the protection of nitrogen. Centrifuging to obtain solid particlesThe solid particles were washed with toluene and methanol to give dimethyl-modified silica particles.

0.01mol of dodecyl betaine was dissolved in 20mL of methanol. Adding equal molar amount of ferulic acid into the solution, stirring at 25 deg.C for 1 hr, removing methanol by rotary evaporation, and vacuum drying the solid at 25 deg.C for 24 hr to obtain [ C ]₁₂Bet][Fal]。

0.195g of the above dimethyl-modified silica particles was uniformly dispersed in 2.4mL of n-octane, and 6.5g of an ionic liquid [ C ] was added to the solution₁₂Bet][Fal]And 80. mu.L of Candida antarctica enzyme (lipase CALB) at a concentration of 4mg/mL pH 8.0, followed by homogenization in a homogenizer (8000rpm, 2min) to give a Pickering emulsion.

And adding the solution into a packed column with the bottom provided with a pore diameter of 4.5-9 mu m and the inner diameter of 2cm to obtain the ion droplet microreactor packed column.

Simultaneously injecting 0.4M n-butanol-n-octane solution and 0.1M n-octane solution of ferulic acid into the packed column by pump, controlling flow rate of both at 2mL/h, and keeping the packed column at constant temperature of 45 deg.C. Collecting the effluent, then using silica gel as a stationary phase and petroleum ether/ethyl acetate as a mobile phase, and carrying out gradient elution to separate and purify a target product, namely ferulic acid butyl ester (FAC 4).

Experimental results show that compared with ferulic acid in the prior art, the ferulic acid butyl ester can more effectively inhibit β -amyloid from precipitating, has a better inhibition effect on intracellular ROS (reactive oxygen species) triggered by 6-ODHA (deoxyribose nucleic acid) in a PC12 neuroma cell, and has a stronger inhibition effect on intracellular ROS triggered by β -amyloid in a PC12 neuroma cell, so that the ferulic acid butyl ester can be used as a functional additive with higher potential for preventing and treating Alzheimer's disease.

Drawings

FIG. 1 shows the results of experiments on the inhibition of β -amyloid protein deposition by ferulic acid and ferulic acid ester derivatives thereof according to the invention.

FIG. 2 is the experimental results of the inhibition of intracellular ROS induced by 6-ODHA by ferulic acid butyl ester and ferulic acid of the invention in PC12 neuroma cells;

FIG. 3 is a graph showing the experimental results of the inhibition of intracellular ROS induced by β -amyloid in PC12 neuroma cells by ferulic acid butyl ester and ferulic acid of the present invention;

FIG. 4 is an image of a hyperendocrinological imaging system of butyl ferulate and ferulic acid of the invention on intracellular ROS induced by β -amyloid in PC12 neuroma cells;

FIG. 5 is a Western blot of the butyl ferulate of the invention in PC12 neuroma cells against activation of the oxidative stress system by β -amyloid.

Detailed Description

The invention is further described with reference to specific examples.