Enzymatic preparation of diglycerides with improved efficacy in alzheimer's disease and use thereof

Abstract

The application belongs to the technical field of functional oil processing, and particularly relates to an enzymatic preparation method and application of a diglyceride with the effect of improving Alzheimer's disease. The method comprises the following steps: mixing low erucic acid rapeseed oil, glycerol and water, heating to obtain a pre-reaction mixture; adding a composite enzyme to react, then adding Candida antarctica lipase B and Pseudomonas fluorescens lipase to react; after the reaction is completed, performing molecular distillation to collect a light phase product, performing adsorption and filtration to collect a filtrate, and obtaining a diglyceride oil. The composite enzyme is composed of Mucor circinelloides lipase, Candida rugosa lipase and Candida antarctica lipase B with a weight ratio of 2-4:2:1. The method effectively improves the content of the diglyceride, and the prepared composition has the effect of preventing and treating Alzheimer's disease, which is manifested in the improvement of reaction ability and cognitive function, the inhibition of brain apoptosis and acetylcholinesterase, the enhancement of SOD and CAT activity, and the improvement of related gene expression.

Description

Technical Field

[0001] This invention belongs to the field of functional oil processing technology, and has an enzymatic preparation method for diglycerides that has the effect of improving Alzheimer's disease, as well as its application. Background Technology

[0002] Alzheimer's disease (AD) is a degenerative disease of the central nervous system, characterized by progressive cognitive and behavioral impairments. Early symptoms may include mild memory loss; as the disease progresses, symptoms such as language impairment, disorientation, impaired judgment, and mood and behavioral abnormalities emerge, severely impacting patients' daily living abilities and quality of life. Its pathological features include β-amyloid (Aβ) plaque deposition and tau protein tangles. The pathogenesis is complex, involving multiple factors such as genetics, metabolism, and inflammation. Among existing treatments, cholinesterase inhibitors can increase acetylcholine levels but are difficult to control disease progression and have gastrointestinal side effects; NMDA receptor antagonists can regulate glutamate activity and are used for moderate to severe patients, but cannot prevent deterioration and also cause adverse reactions such as dizziness. While research-stage drugs have theoretical basis, issues such as unclear efficacy and immunogenicity remain.

[0003] Diacylglycerol (DAG) is a functional lipid formed by the esterification of glycerol with two fatty acids. Due to its unique metabolic characteristics and physiological functions, it has attracted considerable attention in the food and pharmaceutical fields. Nutritionally, it provides energy to the body and accumulates little fat during metabolism, which is beneficial for maintaining body weight. Physiologically, it participates in cell signaling and regulates cell growth and differentiation. Regarding lipid regulation, some studies have shown that it can lower blood triglyceride levels and prevent cardiovascular disease. Currently, the industrial production of DAG mainly relies on chemical and enzymatic methods, which differ significantly in efficiency, safety, and product characteristics.

[0004] The chemical method uses a strong base (such as NaOH) as a catalyst to generate DAG through transesterification at high temperatures. While this method is fast and low-cost, the harsh reaction conditions lead to an increase in byproducts, including monoglycerides (MAG), trans fatty acids, and triglyceride residues. Studies have shown that the chemically produced products contain only 55%-65% 1,3-DAG and a high content of free fatty acids (FFA) of 10%-20%, requiring complex purification steps such as molecular distillation to meet food-grade standards. Furthermore, residual metal catalysts may cause cytotoxicity, making it difficult to meet the requirements for pharmaceutical applications.

[0005] Enzymatic methods utilize lipases to catalyze the esterification reaction of glycerol and fatty acids under mild conditions, offering advantages such as environmental friendliness and high selectivity. Patent CN118813729B discloses a method for preparing diglycerides and its application, comprising first partially hydrolyzing oils with a solvent under the action of a first lipase to obtain hydrolysate; then mixing the hydrolysate with glycerol and performing an esterification reaction using a second lipase; wherein the first lipase is a combination of lipase Lipozyme RM and Candida antarcticis lipase B. This patent can specifically synthesize 1,3-DAG, which is beneficial to the cardiovascular system, with high yield and promising application prospects. In their research on "Preparation of Diglycerides from Immobilized Aspergillus niger Enzymes with High Activity," Wang Taosuo et al. used rapeseed oil and glycerol as raw materials, employing a self-made immobilized Aspergillus niger lipase ANL@PMOS in a solvent-free system to catalyze the glycerolysis of rapeseed oil and glycerol to prepare diglycerides. Through single-factor experiments, they optimized the process conditions for the immobilized enzyme-catalyzed synthesis of diglycerides from rapeseed oil and glycerol, providing a new approach for the green preparation of diglycerides.

[0006] DAG may influence Alzheimer's disease by regulating energy metabolism, inflammatory responses, and lipid metabolism. However, current research is still in the exploratory stage, and the specific mechanisms and effects require further investigation. Therefore, analyzing the bottlenecks in DAG preparation technology, proposing a novel enzymatic hydrolysis process for DAG preparation, and verifying its intervention efficacy in AD pathological models can provide new insights for the intervention and treatment of AD. Summary of the Invention

[0007] To address the aforementioned shortcomings, this invention provides an enzymatic preparation method and application of diglycerides with Alzheimer's disease-improving effects. The method involves mixing low-erucic acid rapeseed oil, glycerol, and water, heating to obtain a pre-reaction mixture; adding a complex enzyme and reacting, followed by the addition of *Candida antarcticus* lipase B and *Pseudomonas fluorescens* lipase; after the reaction is complete, molecular distillation is performed, the light phase product is collected, adsorbed and filtered, and the filtrate is collected to obtain diglyceride oil. The complex enzyme consists of *Rhizopus oryzae* lipase, *Candida pumilum* lipase, and *Candida antarcticus* lipase B in a weight ratio of 2-4:2:1. This method effectively increases the diglyceride content, and the resulting composition has anti-Alzheimer's disease effects, manifested in improved responsiveness and cognitive function, inhibition of brain apoptosis and acetylcholinesterase, enhanced SOD and CAT activity, and improved expression of related genes.

[0008] The technical solution of this invention is as follows:

[0009] In a first aspect, the present invention provides a method for the enzymatic hydrolysis preparation of diglycerides with efficacy in improving Alzheimer's disease, the method comprising the following steps:

[0010] S1. Mix low-erucic acid rapeseed oil, glycerin and water, and heat to obtain a pre-reaction mixture;

[0011] S2. Add the complex enzyme to the pre-reaction mixture of step S1, react, and obtain the first reaction solution;

[0012] S3. Add Candida antarcticis lipase B and Pseudomonas fluorescens lipase to the first reaction solution of step S2, react, and obtain the second reaction solution.

[0013] S4. After the second reaction liquid is distilled, the light phase product is collected, adsorbed and filtered, and the filtrate is collected to obtain diglyceride oil.

[0014] Specifically, in step S1, the low-erucic acid rapeseed oil, glycerin, and water are in the following proportions: 25-40 parts by weight of low-erucic acid rapeseed oil, 5-12 parts by weight of glycerin, and 1-2 parts by weight of water.

[0015] Preferably, in step S1, the low-erucic acid rapeseed oil, glycerin, and water are in the following proportions: 30 parts low-erucic acid rapeseed oil, 10 parts glycerin, and 2 parts water.

[0016] Specifically, the heating described in step S1 is preheating to 40-45°C.

[0017] Preferably, the heating in step S1 is preheating to 45°C.

[0018] Specifically, the complex enzyme described in step S2 consists of Rhizopus miltiorrhiza lipase, Candida albicans lipase, and Candida antarcticis lipase B.

[0019] Preferably, the weight ratio of *Rhizopus oryzae* lipase, *Candida pumilum* lipase, and *Candida antarcticis* lipase is 2-4:2:1.

[0020] More preferably, the weight ratio of the *Rhizopus oryzae* lipase, *Candida pumilum* lipase, and *Candida antarcticis* lipase is 3:2:1.

[0021] Specifically, the reaction conditions described in step S2 include 200-300 rpm and 40-45°C for 4-6 hours.

[0022] Preferably, the reaction conditions in step S2 include 250 rpm and 45°C for 5 hours.

[0023] Preferably, the weight ratio of Candida antarcticis lipase B to Pseudomonas fluorescens lipase in step S3 is 3:1.

[0024] Specifically, the amount of Candida antarcticis lipase B added in step S3 is 1.5-3% w / v.

[0025] Preferably, the amount of Candida antarcticis lipase B added in step S3 is 1.5% w / v.

[0026] Specifically, the amount of fluorescent Pseudomonas lipase added in step S3 is 0.5-1% w / v.

[0027] Preferably, the amount of fluorescent Pseudomonas lipase added in step S3 is 0.5% w / v.

[0028] Specifically, the reaction conditions described in step S3 include: 200-300 rpm, 50-55℃ for 2-3 hours.

[0029] Preferably, the reaction conditions in step S3 include: 250 rpm, 555°C for 3 hours.

[0030] Specifically, the conditions for molecular distillation in step S4 are a temperature of 200-220°C, a vacuum of 150-250 Pa, and a scraping speed of 150-250 rpm.

[0031] Preferably, the conditions for molecular distillation in step S4 are a temperature of 210°C, a vacuum of 200 Pa, and a scraping speed of 200 rpm.

[0032] Specifically, the adsorption filtration described in step S4 includes: adding activated clay, stirring, and then filtering.

[0033] Preferably, the amount of activated clay added in step S4 is 1%-3%.

[0034] Preferably, the activated clay in step S4 has a pH value of 6.5-7.5.

[0035] Preferably, the stirring in step S4 is performed at 40-60 rpm and 70-80°C for 20-30 minutes.

[0036] More preferably, the stirring in step S4 is performed at 50 rpm and 80°C for 30 minutes.

[0037] Secondly, the present invention provides a diglyceride oil prepared by the above-described method.

[0038] The third invention provides a diglyceride composition comprising diglyceride oil, corn oil, peanut oil, and vitamin E.

[0039] Preferably, the diglyceride oil is a diglyceride oil prepared by the above method.

[0040] Specifically, the diglyceride composition comprises, by weight, 50-70 parts diglyceride oil, 15-30 parts corn oil, 5-15 parts peanut oil, and 0.03 parts vitamins.

[0041] Preferably, the diglyceride composition comprises, by weight, 60 parts diglyceride oil, 25 parts corn oil, 10 parts peanut oil and 0.03 parts vitamins.

[0042] Fourthly, the present invention provides the use of the diglyceride oil prepared by the above method, the above-mentioned diglyceride oil, or the above-mentioned diglyceride composition in the preparation of drugs for the prevention, treatment, and adjunctive treatment of Alzheimer's disease.

[0043] Preferably, the Alzheimer's disease drug improves responsiveness and cognitive function, inhibits brain apoptosis and acetylcholinesterase, enhances SOD and CAT activity, and improves the expression of related genes.

[0044] Fifthly, the present invention provides a medicament for the prevention, treatment, and adjunctive treatment of Alzheimer's disease, the medicament comprising diglyceride oil prepared by the above method, the above-mentioned diglyceride oil, or the above-mentioned diglyceride composition.

[0045] Specifically, the drug also includes pharmaceutically acceptable excipients.

[0046] Preferably, the pharmaceutically acceptable excipients include solvents, diluents, disintegrants, precipitation inhibitors, surfactants, flow aids, binders, lubricants, dispersants, suspending agents, isotonic agents, thickeners, emulsifiers, preservatives, stabilizers, hydrating agents, emulsification accelerators, buffers, absorbents, colorants, flavorings, sweeteners, ion exchangers, release agents, coating agents, flavoring agents, or antioxidants.

[0047] Preferably, the dosage form of the drug includes tablets, pills, powders, suspensions, gels, emulsions, creams, granules, capsules, suppositories, injections, sprays, or injections.

[0048] The beneficial effects of this invention are as follows:

[0049] (1) The present invention provides an enzymatic method for preparing diglycerides that have the effect of improving Alzheimer’s disease. The method effectively increases the content of diglycerides in diglyceride oil, providing a good basis for preparing diglyceride compositions for the prevention and treatment of Alzheimer’s disease.

[0050] (2) The present invention provides a diglyceride composition that has the effect of preventing and treating Alzheimer's disease, manifested in improving reaction ability and cognitive function, inhibiting brain apoptosis and acetylcholinesterase, enhancing SOD and CAT activity, and improving the expression of related genes. Attached Figure Description

[0051] Figure 1 This is a typical image of the zebrafish's movement trajectory after sample treatment; the blue dashed box in the image represents the blue area of ​​the cross maze.

[0052] Figure 2 The percentage of blue in zebrafish after sample treatment; ** in the figure represents p < 0.01 compared with the model group; *** represents p < 0.001 compared with the model group.

[0053] Figure 3 The value represents the difference in light and dark speed per minute between zebrafish after sample treatment; *** in the figure represents p<0.001 compared with the model group.

[0054] Figure 4 The values ​​represent the fluorescence values ​​of acetylcholinesterase (AChE) in zebrafish after sample treatment; * in the figure indicates p < 0.05 compared with the model group; *** indicates p < 0.001 compared with the model group.

[0055] Figure 5 This is a typical image of apoptotic cells in the brain of zebrafish after sample treatment; the yellow dashed box in the image represents the analysis area.

[0056] Figure 6 The fluorescence intensity of apoptotic cells in the zebrafish brain after sample treatment is shown; * in the figure represents p<0.05 compared with the model group; *** represents p<0.001 compared with the model group.

[0057] Figure 7 The figure shows the SOD activity of zebrafish after sample treatment; * indicates p < 0.05 compared with the model group; ** indicates p < 0.01 compared with the model group; *** indicates p < 0.001 compared with the model group.

[0058] Figure 8 The figure shows the CAT activity after sample treatment; * indicates p < 0.05 compared with the model group; *** indicates p < 0.001 compared with the model group.

[0059] Figure 9 for egr1 Relative gene expression levels; ** in the figure represent p < 0.01 compared to the model group.

[0060] Figure 10 for caspase3 Relative gene expression levels; * in the figure represents p < 0.05 compared with the model group; *** represents p < 0.001 compared with the model group.

[0061] Figure 11 for nrf2a Relative gene expression levels; ** in the figure represents p < 0.01 compared with the model group; *** represents p < 0.001 compared with the model group.

[0062] Figure 12 for bdnf Relative gene expression levels; *** in the figure represents p<0.001 compared with the model group. Detailed Implementation

[0063] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0064] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention.

[0065] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0066] The low-erucic acid rapeseed oil used in this invention was purchased from Chengdu Xinxing Grain and Oil Co., Ltd., with an erucic acid content of ≤3%; the corn oil was purchased from Shandong Yuhuang Grain and Oil Food Co., Ltd., with product number PY240916107; and the peanut oil was purchased from Shandong Jinsheng Grain and Oil Food Co., Ltd., with an oleic acid content of >75%.

[0067] The *Rhizopus oryzae* lipase used in this invention was purchased from Novozymes, with a transesterification activity of 250 IUN / g; *Candida pleuropsii* lipase was purchased from Hangzhou Chuangke Biotechnology Co., Ltd., with an enzyme activity of 30,000 U / g; *Candida antarcticis* lipase B was purchased from Hangzhou Chuangke Biotechnology Co., Ltd., with an enzyme activity of 50,000 U / g; and *Pseudomonas fluorescens* lipase was purchased from Hangzhou Chuangke Biotechnology Co., Ltd., with an enzyme activity of 20,000 U / g. All other raw materials were commercially available products, and their sources were not specifically limited.

[0068] Example 1

[0069] S1. Take 30 parts of low erucic acid rapeseed oil, add 10 parts of glycerol and 2 parts of water, mix, and preheat to 45°C to obtain a pre-reaction mixture.

[0070] S2. Add the compound enzyme to the pre-reaction mixture to a final concentration of 5% w / v, react at 250 rpm and 45°C for 5 h to obtain the first reaction solution; the compound enzyme is composed of Rhizopus oryzae lipase, Candida pleurisy lipase and Candida antarcticis lipase B, with a weight ratio of Rhizopus oryzae lipase, Candida pleurisy lipase and Candida antarcticis lipase of 3:2:1.

[0071] S3. Add Candida antarctica lipase B (final concentration 1.5% w / v) and Pseudomonas fluorescens lipase (final concentration 0.5% w / v) to the first reaction solution, react at 250 rpm and 55℃ for 3 h to obtain the second reaction solution.

[0072] S4. The second reaction solution is subjected to molecular distillation (temperature 210℃, vacuum degree 200Pa, scraping speed 200rpm) to collect the light phase product.

[0073] S5. The light phase product is heated to 65℃ to reduce viscosity, and 3% activated clay (pH 6.5-7.5) is added. The mixture is stirred at 50 rpm and 80℃ for 30 minutes and then filtered. The filtrate is diglyceride oil.

[0074] Example 2

[0075] S1. Take 40 parts of low erucic acid rapeseed oil, add 12 parts of glycerol and 2 parts of water, mix, and preheat to 42°C to obtain a pre-reaction mixture.

[0076] S2. Add the compound enzyme to the pre-reaction mixture to a final concentration of 6.5% w / v, react at 250 rpm and 42°C for 4 hours to obtain the first reaction solution; the compound enzyme is composed of Rhizopus oryzae lipase, Candida pleurisy lipase and Candida antarcticis lipase B, with a weight ratio of Rhizopus oryzae lipase, Candida pleurisy lipase and Candida antarcticis lipase of 2:2:1.

[0077] S3. Add Candida antarctica lipase B (final concentration 3% w / v) and Pseudomonas fluorescens lipase (final concentration 1% w / v) to the first reaction solution, react at 250 rpm and 55℃ for 3 h to obtain the second reaction solution.

[0078] S4. The second reaction solution is subjected to molecular distillation (temperature 210℃, vacuum degree 200Pa, scraping speed 200rpm) to collect the light phase product.

[0079] S5. The light phase product is heated to 65℃ to reduce viscosity, and 3% activated clay (pH 6.5-7.5) is added. The mixture is stirred at 50 rpm and 80℃ for 30 minutes and then filtered. The filtrate is diglyceride oil.

[0080] Example 3

[0081] S1. Take 25 parts of low erucic acid rapeseed oil, add 5 parts of glycerol and 1 part of water, mix, and preheat to 40°C to obtain a pre-reaction mixture.

[0082] S2. Add the compound enzyme to the pre-reaction mixture to a final concentration of 8% w / v, and react at 250 rpm and 40°C for 6 h to obtain the first reaction solution; the compound enzyme is composed of Rhizopus oryzae lipase, Candida pleurisy lipase and Candida antarcticis lipase B, and the weight ratio of Rhizopus oryzae lipase, Candida pleurisy lipase and Candida antarcticis lipase is 4:2:1.

[0083] S3. Add Candida antarctica lipase B (final concentration 1.5% w / v) and Pseudomonas fluorescens lipase (final concentration 0.5% w / v) to the first reaction solution, react at 250 rpm and 55℃ for 3 h to obtain the second reaction solution.

[0084] S4. The second reaction solution is subjected to molecular distillation (temperature 210℃, vacuum degree 200Pa, scraping speed 200rpm) to collect the light phase product.

[0085] S5. The light phase product is heated to 65℃ to reduce viscosity, and 3% activated clay (pH 6.5-7.5) is added. The mixture is stirred at 50 rpm and 80℃ for 30 minutes and then filtered. The filtrate is diglyceride oil.

[0086] Comparative Example 1

[0087] Comparative Example 1 differs from Example 1 only in steps S2 and S3. The specific operation methods for steps S2 and S3 of Comparative Example 1 are as follows:

[0088] S2. Add the compound enzyme to the pre-reaction mixture to a final concentration of 5% w / v, and react at 250 rpm and 45°C for 5 h to obtain the first reaction solution; the compound enzyme is composed of Rhizopus oryzae lipase, Candida pleurisy lipase and Candida antarcticis lipase B, and the weight ratio of Rhizopus oryzae lipase, Candida pleurisy lipase and Candida antarcticis lipase is 1:1:1.

[0089] S3. Add Candida antarctica lipase B (final concentration 0.5% w / v) and Pseudomonas fluorescens lipase (final concentration 1.5% w / v) to the first reaction solution, react at 250 rpm and 55℃ for 3 h to obtain the second reaction solution.

[0090] Comparative Example 2

[0091] The only difference between Comparative Example 2 and Example 1 is step S2. The specific operation method of step S2 in Comparative Example 2 is as follows:

[0092] S2. Add the compound enzyme to the pre-reaction mixture to a final concentration of 5% w / v, and react at 250 rpm and 45°C for 5 h to obtain the first reaction solution; the compound enzyme is composed of Rhizopus oryzae lipase, Candida pleurisy lipase and Candida antarcticis lipase B, and the weight ratio of Rhizopus oryzae lipase, Candida pleurisy lipase and Candida antarcticis lipase is 1:1:1.

[0093] Comparative Example 3

[0094] The only difference between Comparative Example 3 and Example 1 is step S2. The specific operation method of step S2 in Comparative Example 3 is as follows:

[0095] S2. Add the compound enzyme to the pre-reaction mixture to a final concentration of 5% w / v, and react at 250 rpm and 45°C for 5 h to obtain the first reaction solution; the compound enzyme consists of Rhizopus oryzae lipase and Candida antarcticis lipase B, with a weight ratio of Rhizopus oryzae lipase to Candida antarcticis lipase of 3:3.

[0096] Comparative Example 4

[0097] The only difference between Comparative Example 4 and Example 1 is step S3. The specific operation method of step S3 in Comparative Example 4 is as follows:

[0098] S3. Add Candida antarctica lipase B (final concentration 0.5% w / v) and Pseudomonas fluorescens lipase (final concentration 1.5% w / v) to the first reaction solution, react at 250 rpm and 55℃ for 3 h to obtain the second reaction solution.

[0099] Comparative Example 5

[0100] Comparative Example 5 differs from Example 1 only in step S3. The specific operation method of step S3 in Comparative Example 5 is as follows:

[0101] S3. Add Candida antarctica lipase B (final concentration 2.0% w / v) to the first reaction solution, react at 250 rpm and 55℃ for 3 h to obtain the second reaction solution.

[0102] Experimental Example 1: Determination of Diglyceride Content

[0103] The diglyceride oils prepared by the methods in Examples 1-3 and Comparative Examples 1-5 were analyzed for diglyceride content using liquid chromatography (LC), a technique provided by the prior art. The results are shown in Table 1.

[0104] Table 1. Percentage of diglycerides by mass

[0105]

[0106] The test results showed that the diglyceride oils prepared by the methods provided in Examples 1-3 of this invention had the highest diglyceride content, all exceeding 90%. Comparative Example 2 changed the weight ratio of *Rhizopus oryzae* lipase, *Candida foetida* lipase, and *Candida antarcticis* lipase, resulting in a 11.7% decrease in the diglyceride content of the oil. Comparative Example 4 changed the ratio between *Candida antarcticis* lipase B and *Pseudomonas fluorescens* lipase, resulting in a 14.1% decrease in the diglyceride content of the oil. Comparative Example 1 changed the weight ratio of *Rhizopus oryzae* lipase, *Candida foetida* lipase, and *Candida antarcticis* lipase, as well as the ratio between *Candida antarcticis* lipase B and *Pseudomonas fluorescens* lipase, resulting in a 17.6% decrease in the diglyceride content of the oil. It is evident that the method of the present invention can only achieve the highest diglyceride content under specific weight ratios of Rhizopus miltiorrhiza lipase, Candida albicans lipase, and Candida antarcticis lipase B, and specific weight ratios of Candida antarcticis lipase B to Pseudomonas fluorescens lipase.

[0107] Comparative Example 3 lacked Candida rhombiforme lipase in its complex enzyme, resulting in a diglyceride oil with a diglyceride content of only 74.9% by mass. Comparative Example 5 did not include Pseudomonas fluorescens lipase, leading to a diglyceride oil with a diglyceride content of only 79.7% by mass. These results indicate that the present invention can only achieve the highest diglyceride content under the conditions of the presence of a specific complex enzyme, Candida antarctice lipase B, and Pseudomonas fluorescens lipase.

[0108] Application Example 1: A diglyceride composition

[0109] By weight, 60 parts of diglyceride oil, 25 parts of corn oil, 10 parts of peanut oil and 0.03 parts of vitamin were mixed to obtain a diglyceride composition; the diglyceride oil used was the diglyceride oil prepared in Example 1.

[0110] Application Example 2: Application of diglyceride composition in improving Alzheimer's disease

[0111] 1. Preparation of the test sample

[0112] The diglyceride composition prepared in Example 1 was used as a solvent to prepare a 50% w / v diglyceride composition.

[0113] Positive control: donepezil hydrochloride, white powder, batch number 7169, solvent is DMSO.

[0114] 2. Laboratory animals

[0115] Zebrafish were raised in aquarium water at 28℃ (water quality: 200mg of instant sea salt added per 1L of reverse osmosis water, conductivity 450-550μS / cm; pH 6.5-8.5; hardness 50-100mg / L CaCO3). The laboratory animal use license number is: SYXK (Zhejiang) 2022-0004. The husbandry and management met the requirements of international AAALAC certification (certification number: 001458). The IACUC ethics review number is: IACUC-2024-10709-01.

[0116] 3. Experimental Methods

[0117] 3.1 Evaluation of the efficacy of cognitive function improvement

[0118] Wild-type AB strain zebrafish, 4 days post-fertilization (4 dpf), were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well. Samples (50% w / v diglyceride composition, concentrations shown in Table 2) were administered in water, along with a positive control of donepezil hydrochloride at a concentration of 3.33 μg / mL. Normal and model control groups were also included, with a volume of 3 mL per well. Except for the normal control group, all experimental groups were given aluminum chloride hexahydrate in water to establish a zebrafish dementia model. After treatment at 28℃ for 24 h, 5 zebrafish from each experimental group were randomly selected and placed in a cross-shaped module. The module was divided into four regions: yellow, blue, red, and green. Six modules were placed in each group. Data were collected using a behavioral analyzer, and the percentage of distance the zebrafish traveled within the blue region relative to the total distance traveled within the entire region within 10 minutes was analyzed. The statistical analysis results of this indicator were used to evaluate the efficacy of the sample in improving cognitive function. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS 26.0 software, and p < 0.05 indicated that the difference was statistically significant.

[0119] The measurement results are shown in Table 2. Figures 1-2 As shown, the 50% w / v diglyceride composition has the effect of improving cognitive function.

[0120] Table 2. Results of the experiment evaluating the efficacy of improving cognitive function (n=6)

[0121]

[0122] Note: In the table, ** indicates p < 0.01 compared with the model group; *** indicates p < 0.001 compared with the model group.

[0123] 3.2 Evaluation of the efficacy of improving responsiveness

[0124] Wild-type AB strain zebrafish (4 dpf) were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well (experimental group). Samples (50% w / v diglyceride composition, concentrations shown in Table 3) were administered in water, along with a positive control of donepezil hydrochloride at a concentration of 3.33 μg / mL. A normal control group and a model control group were also included, with a volume of 3 mL per well. Except for the normal control group, the remaining experimental groups were treated with aluminum chloride hexahydrate to establish a zebrafish dementia model. After treatment at 28℃ for 24 h, 10 zebrafish were randomly selected from each experimental group. The difference in light and dark stimuli per minute was measured using a behavioral analyzer. The statistical analysis results of this index were used to evaluate the efficacy of improving sample responsiveness. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS 26.0 software; p < 0.05 indicated statistical significance.

[0125] The measurement results are shown in Table 3 and Figure 3 As shown, the 50% w / v diglyceride composition has the effect of improving reactivity.

[0126] Table 3. Experimental results on the efficacy of corresponding ability improvement (n=10)

[0127]

[0128] Note: *** in the table represents p<0.001 compared with the model group.

[0129] 3.3 Evaluation of the inhibitory efficacy of acetylcholinesterase (AChE)

[0130] Wild-type AB strain zebrafish (4 dpf) were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well (experimental group). Samples (50% w / v diglyceride composition, concentrations shown in Table 4) were administered in aqueous solution to each experimental group. Donepezil hydrochloride (3.33 μg / mL) was used as a positive control. Normal and model control groups were also included. Each well contained 3 mL of solution. Except for the normal control group, all experimental groups were treated with aluminum chloride hexahydrate to establish a zebrafish dementia model. After treatment at 28℃ for 48 h, data were collected using an acetylcholinesterase assay kit and a multi-functional microplate reader. The fluorescence value of acetylcholinesterase in zebrafish was analyzed, and the statistical analysis results were used to evaluate the inhibitory efficacy of the samples on acetylcholinesterase. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS 26.0 software. p < 0.05 was considered statistically significant.

[0131] The measurement results are shown in Table 4 and Figure 4 As shown, the results indicate that the 50% w / v diglyceride composition has acetylcholinesterase inhibitory effects.

[0132] Table 4. Results of the experiment evaluating the efficacy of inhibiting acetylcholinesterase (n=10)

[0133]

[0134] Note: * in the table represents p < 0.05 compared with the model group; *** represents p < 0.001 compared with the model group.

[0135] 3.4 Evaluation of the efficacy of brain apoptosis inhibition

[0136] Wild-type AB strain zebrafish (4 dpf) were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well (experimental group). Samples (50% w / v diglyceride composition, concentrations shown in Table 5) were administered in aqueous solution, along with a positive control of donepezil hydrochloride at a concentration of 3.33 μg / mL. Normal and model control groups were also included, with a volume of 3 mL per well. Except for the normal control group, the remaining experimental groups were treated with aluminum chloride hexahydrate to establish a zebrafish dementia model. After treatment at 28℃ for 24 h, acridine orange (AO) staining was performed. After staining, 10 zebrafish from each experimental group were randomly selected and photographed under a fluorescence microscope. Data were analyzed and collected using NIS-ElementsD 3.20 advanced image processing software. The fluorescence intensity of apoptotic cells in the zebrafish brain was analyzed, and the statistical analysis results of this index were used to evaluate the inhibitory effect of the sample on brain apoptosis. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS 26.0 software; p < 0.05 indicated statistical significance.

[0137] The measurement results are shown in Table 5 and Figures 5-6 As shown, the results indicate that the 50% w / v diglyceride composition has the effect of inhibiting brain apoptosis.

[0138] Table 5. Experimental results evaluating the efficacy of inhibiting brain apoptosis (n=10)

[0139]

[0140] Note: * in the table represents p < 0.05 compared with the model group; *** represents p < 0.001 compared with the model group.

[0141] 3.5 Effects on superoxide dismutase (SOD)

[0142] Wild-type AB strain zebrafish (4 dpf) were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well (experimental group). Samples (50% w / v diglyceride composition, concentrations shown in Table 6) were administered in aqueous solution to each well. Donepezil hydrochloride (3.33 μg / mL) was used as a positive control. Normal control and model control groups were also included. Each well contained 3 mL, and three biological replicates were established. Except for the normal control group, the other experimental groups were treated with aluminum chloride hexahydrate to establish a zebrafish dementia model. After treatment at 28℃ for 48 h, SOD activity in zebrafish was analyzed using a multi-functional microplate reader (WST-1 method). The statistical analysis results were used to evaluate the effect of the samples on superoxide dismutase (SOD). Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS 26.0 software; p < 0.05 was considered statistically significant.

[0143] The measurement results are shown in Table 6 and Figure 7 As shown, the results indicate that the 50% w / v diglyceride composition has the effect of increasing SOD activity.

[0144] Table 6. Experimental results evaluating the effect on superoxide dismutase (SOD) (n=3)

[0145]

[0146] Note: In the table, * indicates p < 0.05 compared with the model group; ** indicates p < 0.01 compared with the model group; *** indicates p < 0.001 compared with the model group.

[0147] 3.6 Effects on catalase (CAT)

[0148] Wild-type AB strain zebrafish (4 dpf) were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well (experimental group). Samples (50% w / v diglyceride composition, concentrations shown in Table 7) were administered in aqueous solution to each well. Donepezil hydrochloride (3.33 μg / mL) was used as a positive control. Normal control and model control groups were also included. Each well contained 3 mL, and three biological replicates were established. Except for the normal control group, the remaining experimental groups were treated with aluminum chloride hexahydrate to establish a zebrafish dementia model. After treatment at 28℃ for 48 h, CAT activity in zebrafish was analyzed using a CAT assay kit (ammonium molybdate method) and a multi-functional microplate reader. The statistical analysis results were used to evaluate the effect of the samples on catalase (CAT). Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS 26.0 software; p < 0.05 was considered statistically significant.

[0149] The measurement results are shown in Table 7 and Figure 8As shown, the results indicate that the 50% w / v diglyceride composition has the effect of increasing CAT activity.

[0150] Table 7. Experimental results evaluating the effect on catalase (CAT) (n=3)

[0151]

[0152] Note: * in the table represents p < 0.05 compared with the model group; *** represents p < 0.001 compared with the model group.

[0153] 3.7 Effects on related genes

[0154] Wild-type AB strain zebrafish (4 dpf) were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well (experimental group). Samples (50% w / v diglyceride composition, concentrations shown in Table 8) were administered in water, along with a positive control of donepezil hydrochloride at a concentration of 3.33 μg / mL. Normal and model control groups were also included. Each well contained 3 mL, and three biological replicates were established. Except for the normal control group, all experimental groups were treated with aluminum chloride hexahydrate in water to establish a zebrafish dementia model. After treatment at 28℃ for 24 h, total RNA was extracted from each group of zebrafish using an automated nucleic acid extractor, and the total RNA concentration was measured using a UV-Vis spectrophotometer. 2.00 μg of total RNA from the zebrafish sample was used to synthesize 20.0 μL of cDNA according to the cDNA first-strand synthesis kit instructions, and the results were detected by q-PCR. β-actin , egr1 , caspase3 , nrf2a and bdnf Gene expression. (Using...) β-actin As an internal reference for gene expression, calculation egr1 , caspase3 , nrf2a and bdnf Relative RNA expression levels of genes. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS 26.0 software; p < 0.05 was considered statistically significant.

[0155] After sample processing, total RNA was extracted from zebrafish, and the RNA concentration and A260 / A280 ratio were determined using a UV-Vis spectrophotometer (Table 8). The A260 / A280 ratios were all between 1.8 and 2.2, indicating that the extracted total RNA from zebrafish was of good quality and could be used for subsequent q-PCR experiments. Primer sequences are shown in Table 9.

[0156] Table 8. Total RNA concentration and A260 / A280 ratio (n=3)

[0157]

[0158] Table 9 Primer sequence information

[0159]

[0160] Note: In the table, F represents the forward primer; R represents the reverse primer.

[0161] The measurement results are shown in Table 10 and Figures 9-12 As shown, the results indicate that the 50% w / v diglyceride composition can upregulate... egr1 , nrf2a , bdnf Genes and downregulation caspase3 Gene expression.

[0162] Table 10 Effects on the expression of related genes (n=3)

[0163]

[0164] Note: In the table, * indicates p < 0.05 compared with the model group; ** indicates p < 0.01 compared with the model group; *** indicates p < 0.001 compared with the model group.

[0165] The above detailed description is a specific illustration of one feasible embodiment of the present invention, and this embodiment is not intended to limit the patent scope of the present invention. It should be noted that all equivalent implementations or modifications that do not depart from the present invention should be included within the scope of the technical solution of the present invention. Therefore, the protection scope of the present invention should be determined by the appended claims.

Claims

1. A method for the enzymatic production of diglycerides having improved efficacy in Alzheimer's disease, characterized in that, The method comprises the following steps: S1, mixing low erucic acid rapeseed oil, glycerol and water, heating to obtain a pre-reaction mixture; S2, adding a complex enzyme to the pre-reaction mixture of step S1, reacting to obtain a first reaction liquid; S3, adding Candida antarctica lipase B and Pseudomonas fluorescens lipase to the first reaction liquid of step S2, reacting to obtain a second reaction liquid; S4, after molecular distillation of the second reaction liquid, collecting the light phase product, adsorbing and filtering, collecting the filtrate to obtain a diglyceride oil; The complex enzyme in step S2 is composed of Rhizomucor miehei lipase, Candida rugosa lipase and Candida antarctica lipase B; the weight ratio of the Rhizomucor miehei lipase, Candida rugosa lipase and Candida antarctica lipase B is 2-4:2:1; the addition amount of the complex enzyme is 5-8% w / v; the reaction conditions in step S2 include 200-300 rpm, 40-45℃ for 4-6 h; The weight ratio of the Candida antarctica lipase B and Pseudomonas fluorescens lipase in step S3 is 3:1; the addition amount of the Candida antarctica lipase B is 1.5-3% w / v; the addition amount of the Pseudomonas fluorescens lipase is 0.5-1% w / v; the reaction conditions in step S3 include 200-300 rpm, 50-55℃ for 2-3 h.

2. The method of claim 1, wherein, The low erucic acid rapeseed oil, glycerol and water in step S1 are 25-40 parts of low erucic acid rapeseed oil, 5-12 parts of glycerol and 1-2 parts of water by weight.

3. The diglyceride oil prepared by the method of any one of claims 1-2.

4. A diglyceride composition characterized in that, The diglyceride composition is composed of a diglyceride oil, corn oil, peanut oil and vitamin E; the diglyceride oil is the diglyceride oil prepared by the method of any one of claims 1-2 or the diglyceride oil of claim 3.

5. The diglyceride composition according to claim 4, characterized in that, The diglyceride composition comprises 50-70 parts of diglyceride oil, 15-30 parts of corn oil, 5-15 parts of peanut oil and 0.03 parts of vitamin E by weight.

6. The use of the diglyceride oil prepared by the preparation method of any one of claims 1-2, the diglyceride oil of claim 3 or the diglyceride composition of any one of claims 4-5 in the preparation of a drug for treating or adjuvant treating Alzheimer's disease.

7. A medicament for treating or adjuvant treatment of Alzheimer's disease, characterized in that it comprises a compound of the formula (I) as defined in claim 1. The drug comprises the diglyceride oil prepared by the preparation method of any one of claims 1-2, the diglyceride oil of claim 3 or the diglyceride composition of any one of claims 4-5.

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