Artificial synthesis method and application of compound theanine diglucoside TFG

Abstract

The invention belongs to the technical field of chemistry, and particularly relates to an artificial synthesis method and application of a compound theanine diglucoside TFG. Theanine-fructosyl-glucose (TFG) is a Maillard reaction product, has obvious antioxidant activity, and is not systematically studied at present, so that the antioxidant activity of the theanine-fructosyl-glucose (TFG) can be obviously improved, and the antioxidant activity of the theanine-fructosyl-glucose (TFG) can be obviously improved. The invention provides an efficient artificial synthesis method of the compound TFG, the synthesis cost is reduced, a material basis is provided for subsequent research, and the method has the characteristics of economy, high efficiency, simplicity in operation, safety and the like, and is suitable for industrial production. Subsequently, activity screening of HT22 mouse hippocampal neuronal cells and HUVEC human vascular endothelial cells in vitro proves that the compound TFG can remarkably relieve cell oxidative damage induced by hydrogen peroxide (H2O2). Based on the synergistic protection effect of the TFG on neurons and vascular endothelial systems, the TFG is suitable for developing antioxidant drugs for treating Alzheimer's disease.

Description

Technical Field

[0001] This invention belongs to the field of chemical technology and specifically relates to a method for the artificial synthesis and use of a compound called theanine disaccharide TFG. Disclosed are the compound's synthesis method and its antagonistic effect on hydrogen peroxide (H2O2)-induced damage to mouse hippocampal neurons and human vascular endothelial cells (HUVECs). Background Art

[0002] Theanine (L-Theanine), a characteristic functional amino acid in tea, accounts for over 50% of the total free amino acids in tea. Its glycoside derivatives, produced through the Maillard reaction, are an important research area in the deep processing of tea. Yu Zaiyuan et al. reported a method for glycosylation of theanine with maltose in a methanol system at 65°C for 18 hours, producing a maltitol-based theanine mixture (theanine disodium) containing four isomers, two of which are represented by Formula (I) and Formula (II), respectively. However, this method has significant drawbacks: the reaction time is lengthy (18 hours) and the product is a complex mixture, making the isolation and purification of the target compound difficult. This has severely hampered the functional research and application development of theanine disodium derivatives.

[0003] In the field of Maillard reaction mechanism research, Yukio Suzuki et al. found that changes in the content of arginine and sugar substances during the processing of red ginseng significantly affect the degree of browning, confirming that the concentration of amino compounds and reducing sugars is a key factor in regulating the progress of the Maillard reaction. Zhao Jing et al. further studied the effect of heating time on the content of the Maillard reaction products AF (the product of the reaction of arginine and glucose) and AFG (the product of the reaction of arginine and maltose) in red ginseng. The results showed that the content of both products increased significantly with the extension of preheating time, revealing the dose-effect relationship between reaction time and product formation. These studies all indicate that the optimization of reaction solvent, temperature, and time parameters plays a decisive role in the formation of Maillard reaction products.

[0004] These deficiencies severely hinder the controlled synthesis and application of theanine disaccharide derivatives, necessitating the development of efficient and specific new preparation systems. Based on our laboratory's experience in solvent screening and reaction condition optimization in the study of ginseng Maillard reaction products, we innovatively proposed the use of glacial acetic acid as a reaction medium to construct a directed glycosylation reaction system for theanine and maltose. Compared to existing technologies, this invention addresses the problems of low reaction efficiency and complex products in traditional methods through solvent innovation, providing a new technical path for the controlled synthesis and functional development of theanine disaccharide. Summary of the Invention

[0005] TFG is present in low concentrations in tea leaves and is difficult and costly to separate. Our research team innovatively used acetic acid solution as the reaction medium to establish a directional Maillard reaction system for theanine and maltose, obtaining single-configuration TFG. This significantly shortened the reaction time to 1 / 9 of the traditional method (compared to 18 hours for the methanol system, this method was optimized to 2 hours), enabling artificial synthesis and large-scale preparation and reducing the difficulty of separating the mixture.

[0006] The present invention provides a TFG artificial synthesis method and an optimal process screening method with simple operation, easy control of reaction conditions and reaction temperature, readily available raw materials and high synthesis rate.

[0007] The compound of the present invention is named theanine disodium glycoside, and its molecular formula is: 19 H 34 N2O 13 , the structural formula is as follows: (I)

[0008] The present invention is achieved through the following technical solutions:

[0009] The artificial synthesis method of the compound TFG of the present invention consists of the following steps:

[0010] A certain proportion of theanine and maltose are placed in an acidic reaction medium, mixed evenly, and then synthesized under high temperature conditions to synthesize theanine disaccharide. The specific steps are as follows:

[0011] A. Accurately weigh theanine (L-Theanine) and D-(+)-maltose at a mass ratio of 1:1 to 1:5. Place them in a 20-fold volume of reaction medium (based on theanine mass, g / mL) with a concentration of 75% to 100%.

[0012] B. Place in a constant temperature shaker at 60-95°C for 60-140 minutes.

[0013] C. After the reaction, the product was concentrated to dryness using a rotary evaporator, then passed through a polyacrylamide (Bio-gel P-2) column and eluted with a 0.2% acetic acid aqueous solution at a flow rate of 0.3 mL / min. Fractions were collected and lyophilized to obtain the compound TFG.

[0014] As a preferred technical solution of the present invention, the reaction medium is acetic acid aqueous solution of different concentrations (v / v), namely 75%, 80%, 85%, 90%, 95% acetic acid aqueous solution and glacial acetic acid, and the preferred reaction medium is glacial acetic acid.

[0015] As a preferred technical solution of the present invention, the mass ratios of theanine to maltose are 1:1, 1:2, 1:3, 1:4, and 1:5, respectively, with the preferred mass ratio being 1:2.

[0016] As a preferred technical solution of the present invention, the selected reaction temperature range is 60, 70, 80, 85, 90, and 95°C, and the preferred reaction temperature is 85°C.

[0017] As a preferred technical solution of the present invention, the selected reaction time range is 60, 80, 100, 120, 140, and 160 min, and the preferred reaction time is 120 min.

[0018] The advantages of the present invention over the prior art are:

[0019] For the first time, the artificial synthesis and preparation of the single-configuration compound theanine disaccharide was achieved, and the existing related technologies were summarized and optimized. After comprehensive investigation, a method with simpler operation, low cost and high synthesis rate was obtained. After further purification and separation, the purity can reach more than 97%.

[0020] The present invention further provides the use of the compound TFG in the preparation of antioxidant drugs and / or health products, specifically, the use of the compound TFG in the preparation of products for preventing, treating or improving oxidative stress in HT22 cells induced by hydrogen peroxide (H2O2).

[0021] Alternatively, the application is based on the improvement effect of TFG on HUVEC cell damage induced by hydrogen peroxide (H2O2), which activates the intracellular antioxidant signaling pathway, inhibits the oxidative stress response, and protects the barrier function of vascular endothelial cells.

[0022] Alternatively, the application is specifically for preparing a drug for preventing or treating oxidative stress-related diseases, including but not limited to hydrogen peroxide-induced endothelial cell function damage and oxidative damage-related vascular lesions.

[0023] Based on the above-mentioned neuron-endothelial protection mechanism, this TFG application is particularly suitable for the development of antioxidant drugs for the treatment of Alzheimer's disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of the Maillard reaction route of TFG of the present invention

[0025] Figure 2 HPLC-ELSD spectrum of TFG of the present invention

[0026] Figure 3 The TFG of the present invention 13 C-NMR spectrum

[0027] Figure 4 The TFG of the present invention 1 H-NMR spectrum

[0028] Figure 5 HMBC spectrum of TFG of the present invention

[0029] Figure 6 HSQC spectrum of TFG of the present invention

[0030] Figure 7 HR-ESI-MS spectrum of TFG of the present invention DETAILED DESCRIPTION

[0031] The artificial synthesis method and antioxidant activity of the present invention will be further described below with reference to specific examples, but the scope of protection of the present invention is not limited thereto.

[0032] Theanine disodium glycoside TFG was isolated from tea leaves by our research group in the early stage and identified as follows:

[0033] TFG is generated by the Maillard reaction (e.g. Figure 1 As shown), detected by HPLC-ELSD ( Figure 2 ), with an absorption peak retention time of 23.83 min. Further one-dimensional nuclear magnetic resonance (1D NMR) analysis showed that the compound's hydrogen spectrum (1H NMR) displayed 25 hydrogen proton signals, mainly distributed in the chemical shift range of 3.0-4.0 ppm; carbon spectrum (13C NMR) confirmed that the molecule contained 19 carbon atoms, of which the sugar moiety showed 11 carbon signals. Characteristic peaks at chemical shifts of 100.57 and 95.41 ppm indicated the presence of two sugar units (such as Figure 3 and Figure 4 By heteronuclear multiple bond correlation spectroscopy (HMBC) analysis, long-range correlation signals between H-3′ and C-1′′ were observed, thereby determining the connection mode between sugar groups (such as Figure 5 and Figure 6 ). In addition, if Figure 7 High-resolution mass spectrometry (HR-ESI-MS) showed m / z 497.2027 [MH]⁻ (calculated value C 19 H 34 N2O 13 : 498.497). Based on the above NMR and mass spectrometry data, the molecular formula of the compound was confirmed to be C 19 H 34 N2O 13 As shown in formula (Ⅰ).

[0034] (I)

[0035] The structural formula of the isomers is as follows (II): (II)

[0036] Table 1 Compound TFG1 H-NMR and 13 C-NMR chemical shift

[0037] Detection of crude synthetic theanine disodium glycoside

[0038] 1.1 Detection method: High performance liquid chromatography-evaporative light scattering detection (HPLC-ELSD) was used to detect the content of the obtained crude synthetic product.

[0039] 1.2 Sample preparation: Each group accurately weighed 5.0 mg of the crude theanine disodium glycoside and diluted it to a 2 mL volumetric flask with 0.1 mol / L hydrochloric acid solution to obtain a liquid injection sample with a concentration of 2.5 mg / mL.

[0040] 1.3 Chromatographic Conditions: Yuexu Ultimate® Amino Acid Plus II column (4.6 mm × 300 mm), evaporative light scattering detector, drift tube temperature at 120°C, carrier gas flow rate at 3.2 L / min, mobile phase consisting of 0.3% heptafluorobutyric acid solution (A) and acetonitrile (B), gradient elution: 0–10 min, 100% A; 10–15 min, 100%–93% A; 15–40 min, 93%–50% A; 40–41 min, 50%–100% A; 41–60 min, 100% A; column temperature at 30°C, injection volume of 20 μL, flow rate at 1.0 mL / min. To prepare the 0.3% heptafluorobutyric acid mobile phase: 997 mL of water, add 3 mL of heptafluorobutyric acid, mix thoroughly, and degas by ultrasonication.

[0041] Example 1:

[0042] 10 g of theanine and 20 g of maltose were accurately weighed and placed in 200 mL of 75% acetic acid. The mixture was then placed in a shaker at 85°C for 120 min. The reaction was immediately concentrated using a rotary evaporator and then passed through a polyacrylamide (Bio-gel P-2) column. Elution was performed with 0.2% acetic acid at a flow rate of 0.3 mL / min. Fractions were collected and lyophilized to obtain the compound TFG. No obvious browning was observed during the reaction. The experiment was repeated three times. After purification, the product yield was 55.6% and the purity of TFG was 96.2%.

[0043] Example 2:

[0044] 10 g of theanine and 20 g of maltose were accurately weighed and placed in 200 mL of 80% acetic acid. The mixture was then placed in a shaker at 85°C for 120 min. The reaction was immediately concentrated using a rotary evaporator and then passed through a polyacrylamide (Bio-gel P-2) column. Elution was performed with 0.2% acetic acid at a flow rate of 0.3 mL / min. Fractions were collected and lyophilized to obtain the compound TFG. No obvious browning was observed during the reaction. The experiment was repeated three times. After purification, the product yield was 60.1% and the purity of TFG was 96.5%.

[0045] Example 3:

[0046] 10 g of theanine and 20 g of maltose were accurately weighed and placed in 200 mL of 85% acetic acid. The mixture was then placed in a shaker at 85°C for 120 minutes. The reaction was immediately concentrated using a rotary evaporator and passed through a polyacrylamide (Bio-gel P-2) column. Elution was performed with 0.2% acetic acid at a flow rate of 0.3 mL / min. Fractions were collected and lyophilized to obtain the compound TFG. The experiment was repeated three times. After purification, the product yield was 61.1% and the purity of TFG was 96.2%.

[0047] Example 4:

[0048] Accurately weigh 10 g of theanine and 20 g of maltose in 200 mL of 90% acetic acid. Mix thoroughly and place in a shaker at 85°C for 120 minutes. Immediately concentrate using a rotary evaporator and then pass through a polyacrylamide (Bio-gel P-2) column. Elution with 0.2% acetic acid at a flow rate of 0.3 mL / min is performed. Fractions are collected and lyophilized to obtain the compound TFG. The experiment was repeated three times. After purification, the yield of the product was 62.6%, and the purity of TFG was 95.8%.

[0049] Example 5:

[0050] Accurately weigh 10 g of theanine and 20 g of maltose in 200 mL of 95% acetic acid. Mix thoroughly and place in a shaker at 85°C for 120 min. Immediately concentrate using a rotary evaporator and then pass through a polyacrylamide (Bio-gel P-2) column. Elution with 0.2% acetic acid at a flow rate of 0.3 mL / min is performed. Fractions are collected and lyophilized to obtain the compound TFG. This experiment was repeated three times. After purification, the yield of the product was 64.1%, and the purity of TFG was 96.1%.

[0051] Example 6:

[0052] Accurately weigh 10 g of theanine and 20 g of maltose in 200 mL of glacial acetic acid. Mix thoroughly and place in a shaker at 85°C for 120 min. Immediately concentrate using a rotary evaporator and then pass through a polyacrylamide (Bio-gel P-2) column. Elution with 0.2% acetic acid at a flow rate of 0.3 mL / min is performed. Fractions are collected and lyophilized to obtain the compound TFG. The experiment was repeated three times. After purification, the yield of the product was 65.6%, and the purity of TFG was 97.2%.

[0053] Example 7:

[0054] Accurately weigh 10 g of theanine and 20 g of maltose in 200 mL of glacial acetic acid. Mix thoroughly and place in a 60°C water bath shaker for 120 min. Immediately concentrate using a rotary evaporator and then pass through a polyacrylamide (Bio-gel P-2) column. Elution with 0.2% acetic acid at a flow rate of 0.3 mL / min is performed. Fractions are collected and lyophilized to obtain the compound TFG. The experiment was repeated three times. After purification, the yield of the product was 59.7%, and the purity of TFG was 96.5%.

[0055] Example 8:

[0056] Accurately weigh 10 g of theanine and 20 g of maltose in 200 mL of glacial acetic acid. Mix thoroughly and place in a 70°C water bath shaker for 120 min. Immediately concentrate using a rotary evaporator and then pass through a polyacrylamide (Bio-gel P-2) column. Elution with 0.2% aqueous acetic acid at a flow rate of 0.3 mL / min is performed. Fractions are collected and lyophilized to obtain the compound TFG. The experiment was repeated three times. After purification, the yield of the product was 60.2%, and the purity of TFG was 96.1%.

[0057] Example 9:

[0058] Accurately weigh 10 g of theanine and 20 g of maltose in 200 mL of glacial acetic acid. Mix thoroughly and place in a shaker at 80°C for 120 min. Immediately concentrate using a rotary evaporator and then pass through a polyacrylamide (Bio-gel P-2) column. Elution with 0.2% acetic acid at a flow rate of 0.3 mL / min is performed. Fractions are collected and lyophilized to obtain the compound TFG. The experiment was repeated three times. After purification, the yield of the product was 63.7%, and the purity of TFG was 95.9%.

[0059] Example 10:

[0060] Accurately weigh 10 g of theanine and 20 g of maltose in 200 mL of glacial acetic acid. Mix thoroughly and place in a shaker at 85°C for 120 min. Immediately concentrate using a rotary evaporator and then pass through a polyacrylamide (Bio-gel P-2) column. Elution with 0.2% acetic acid at a flow rate of 0.3 mL / min is performed. Fractions are collected and lyophilized to obtain the compound TFG. The experiment was repeated three times. After purification, the yield of the product was 65.6%, and the purity of TFG was 97.2%.

[0061] Example 11:

[0062] Accurately weigh 10 g of theanine and 20 g of maltose in 200 mL of glacial acetic acid. Mix thoroughly and place in a 90°C water bath shaker for 120 min. Immediately concentrate using a rotary evaporator and then pass through a polyacrylamide (Bio-gel P-2) column. Elution with 0.2% acetic acid at a flow rate of 0.3 mL / min is performed. Fractions are collected and lyophilized to obtain the compound TFG. The experiment was repeated three times. After purification, the yield of the product was 64.3%, and the purity of TFG was 96.8%.

[0063] Example 12:

[0064] Accurately weigh 10 g of theanine and 20 g of maltose in 200 mL of glacial acetic acid. Mix thoroughly and place in a 95°C water bath shaker for 120 min. Immediately concentrate using a rotary evaporator and then pass through a polyacrylamide (Bio-gel P-2) column. Elution with 0.2% acetic acid at a flow rate of 0.3 mL / min is performed. Fractions are collected and lyophilized to obtain the compound TFG. The experiment was repeated three times. After purification, the yield of the product was 63.6%, and the purity of TFG was 96.2%.

[0065] Example 13:

[0066] Accurately weigh 10 g of theanine and 20 g of maltose in 200 mL of glacial acetic acid. Mix thoroughly and place in a shaker at 85°C for 60 min. Immediately concentrate using a rotary evaporator and pass through a polyacrylamide (Bio-gel P-2) column. Elution with 0.2% acetic acid at a flow rate of 0.3 mL / min is performed. Fractions are collected and lyophilized to obtain the compound TFG. The experiment was repeated three times. After purification, the yield of the product was 59.9%, and the purity of TFG was 95.4%.

[0067] Example 14:

[0068] Accurately weigh 10 g of theanine and 20 g of maltose in 200 mL of glacial acetic acid. Mix thoroughly and place in a shaker at 85°C for 80 min. Immediately concentrate using a rotary evaporator and then pass through a polyacrylamide (Bio-gel P-2) column. Elution with 0.2% acetic acid at a flow rate of 0.3 mL / min is performed. Fractions are collected and lyophilized to obtain the compound TFG. The experiment was repeated three times. After purification, the yield of the product was 62.2%, and the purity of TFG was 96.2%.

[0069] Example 15:

[0070] Accurately weigh 10 g of theanine and 20 g of maltose in 200 mL of glacial acetic acid. Mix thoroughly and place in a shaker at 85°C for 100 min. Immediately concentrate using a rotary evaporator and pass through a polyacrylamide (Bio-gel P-2) column. Elution with 0.2% acetic acid at a flow rate of 0.3 mL / min is performed. Fractions are collected and lyophilized to obtain the compound TFG. The experiment was repeated three times. After purification, the yield of the product was 64.6%, and the purity of TFG was 95.9%.

[0071] Example 16:

[0072] Accurately weigh 10 g of theanine and 20 g of maltose in 200 mL of glacial acetic acid. Mix thoroughly and place in a shaker at 85°C for 120 min. Immediately concentrate using a rotary evaporator and then pass through a polyacrylamide (Bio-gel P-2) column. Elution with 0.2% acetic acid at a flow rate of 0.3 mL / min is performed. Fractions are collected and lyophilized to obtain the compound TFG. The experiment was repeated three times. After purification, the yield of the product was 65.6%, and the purity of TFG was 97.2%.

[0073] Example 17:

[0074] 10 g of theanine and 20 g of maltose were accurately weighed and placed in 200 mL of glacial acetic acid. The mixture was then placed in a shaker at 85°C for 140 min. The reaction was immediately concentrated using a rotary evaporator and passed through a polyacrylamide (Bio-gel P-2) column. Elution was performed with 0.2% acetic acid at a flow rate of 0.3 mL / min. Fractions were collected and lyophilized to obtain the compound TFG. The experiment was repeated three times. After purification, the yield of the product was 64.9%, and the purity of TFG was 96.9%.

[0075] Example 18:

[0076] 10 g of theanine and 20 g of maltose were accurately weighed and placed in 200 mL of glacial acetic acid. The mixture was then placed in a shaker at 85°C for 160 min. The reaction was immediately concentrated using a rotary evaporator and passed through a polyacrylamide (Bio-gel P-2) column. Elution was performed with 0.2% acetic acid at a flow rate of 0.3 mL / min. Fractions were collected and lyophilized to obtain the compound TFG. The experiment was repeated three times. After purification, the yield of the product was 63.2%, and the purity of TFG was 96.5%.

[0077] Example 19:

[0078] Accurately weigh 10 g of theanine and 10 g of maltose, add them to 200 mL of glacial acetic acid, mix thoroughly, and place in a shaker at 85°C for 120 min. Immediately after reaction, the mixture was concentrated using a rotary evaporator and then passed through a polyacrylamide (Bio-gel P-2) column. Elution was performed with 0.2% aqueous acetic acid at a flow rate of 0.3 mL / min. Fractions were collected and lyophilized to obtain the compound TFG. The experiment was repeated three times. After purification, the yield of the product was 64.2%, and the purity of TFG was 96.8%.

[0079] Example 20:

[0080] Accurately weigh 10 g of theanine and 20 g of maltose in 200 mL of glacial acetic acid. Mix thoroughly and place in a shaker at 85°C for 120 min. Immediately concentrate using a rotary evaporator and then pass through a polyacrylamide (Bio-gel P-2) column. Elution with 0.2% acetic acid at a flow rate of 0.3 mL / min is performed. Fractions are collected and lyophilized to obtain the compound TFG. The experiment was repeated three times. After purification, the yield of the product was 65.6%, and the purity of TFG was 97.2%.

[0081] Example 21:

[0082] 10 g of theanine and 30 g of maltose were accurately weighed and placed in 200 mL of glacial acetic acid. The mixture was then placed in a shaker in a constant-temperature water bath at 85°C for 120 min. The reaction was immediately concentrated using a rotary evaporator and then passed through a polyacrylamide (Bio-gel P-2) column. Elution was performed with 0.2% aqueous acetic acid at a flow rate of 0.3 mL / min. Fractions were collected and lyophilized to obtain the compound TFG. The experiment was repeated three times. After purification, the yield of the product was 64.4%, and the purity of TFG was 96.5%.

[0083] Example 22:

[0084] Accurately weigh 10 g of theanine and 40 g of maltose in 200 mL of glacial acetic acid. Mix thoroughly and place in a shaker at 85°C for 120 min. Immediately concentrate using a rotary evaporator and then pass through a polyacrylamide (Bio-gel P-2) column. Elution with 0.2% acetic acid at a flow rate of 0.3 mL / min is performed. Fractions are collected and lyophilized to obtain the compound TFG. The experiment was repeated three times. After purification, the yield of the product was 63.5%, and the purity of TFG was 96.1%.

[0085] Example 23:

[0086] Accurately weigh 10 g of theanine and 50 g of maltose in 200 mL of glacial acetic acid. Mix thoroughly and place in a shaker at 85°C for 120 min. Immediately concentrate using a rotary evaporator and pass through a polyacrylamide (Bio-gel P-2) column. Elution with 0.2% acetic acid at a flow rate of 0.3 mL / min is performed. Fractions are collected and lyophilized to obtain the compound TFG. The experiment was repeated three times. After purification, the yield of the product was 60.2%, and the purity of TFG was 95.8%.

[0087] According to the above embodiment, the results can be seen from Table 2: by regulating the synthesis of TFG with different concentrations of acetic acid, the experimental results show that when the concentration of acetic acid is glacial acetic acid, the product yield and purity can reach an ideal state during the reaction.

[0088] Table 2. Statistical table of the effects of different concentrations of acetic acid on TFG synthesis

[0089] As shown in Table 2, the yield is the highest when the reaction medium is glacial acetic acid, and it is economical and applicable.

[0090] According to the above embodiment, the results can be seen from Table 3: TFG was synthesized by regulating time and temperature. The results showed that the yield of the synthesized product reached the maximum when the temperature was 85°C and the time was 120 min.

[0091] Table 3. Statistical table of the effects of different time and temperature on TFG synthesis

[0092] As shown in Table 3, by controlling the time and temperature respectively through the control variable method, the optimal synthesis temperature of TFG is 85℃ and the optimal time is 120 min.

[0093] According to the above examples, the results are shown in Table 4: by controlling the mass ratio of theanine to maltose during the reaction, it can be concluded that the mass ratio of theanine to maltose for synthesizing TFG is 1:2.

[0094] Table 4. Statistical table of the effect of the mass ratio of theanine to maltose on the synthesis of TFG during the reaction

[0095] Example 24

[0096] The compound TFG of the present invention has a protective effect on H2O2-induced mouse hippocampal neuronal cells HT22 cells.

[0097] 1. Drug preparation

[0098] TFG (structural formula shown in Formula I, previously referred to as TFG, hereinafter referred to as TFG) and TFG (structural formula shown in Formula II, reported in CN201180038302.2) were dissolved in DMEM high-glucose medium to prepare a 1 mM stock solution. The stock solution was then diluted to concentrations of 1.25, 2.5, 5, and 10 μM, respectively. H₂O₂ was diluted in DMEM high-glucose medium to a 100 mM solution. TFG is inherently unstable and should be prepared freshly before use. The entire process must be performed under sterile conditions. After dissolution, the solution must be filtered through a 0.22 μM filter membrane before use.

[0099] 2. Cell culture

[0100] HT22 cell lines (mouse hippocampal neurons) were purchased from the Shanghai Cell Resource Center, Chinese Academy of Sciences, and cultured in a thermostat at 37°C and 5% CO2 in a thermostat. The culture medium used was Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% FBS, 1% penicillin, and 1% streptomycin. For formal experiments, HT22 cells exhibited a normal karyotype as observed under a microscope, were adherent and in the logarithmic growth phase, and reached a density of 80–90%.

[0101] 3. MTT assay to test the effect of TFG on cell viability

[0102] HT22 cells were seeded in a 96-well culture plate and treated with H₂O₂ and various concentrations of TFG. Then, 20 μL of MTT solution (5 mg / mL) was added to each well. After incubation at 37°C for 3.5 hours, dark blue formazan crystals formed. The supernatant was discarded, and 150 μL of DMSO solution was added to each well to dissolve the formazan crystals. The plate was shaken and mixed for 5 minutes. The absorbance was then measured at 490 nm using a DR-200B microplate reader. The viability of the group treated with DMEM was normalized to 100%.

[0103] 4. Antioxidant activity (MDA, SOD, CAT, GSH) detection

[0104] HT22 cells were seeded in 6-well culture plates and treated with H2O2 and different concentrations of TFG. The cells were then mixed with RIPA lysis buffer (with protease inhibitors and phosphatase inhibitors added in proportion) for protein extraction. The MDA, SOD, CAT, and GSH detection kits were followed according to their instructions. The absorbance was measured using a DR-200B microplate reader, and the antioxidant activity was calculated based on the absorbance.

[0105] 5. Data processing

[0106] All data were expressed as mean ± standard deviation (mean ± SD) established in different experiments and analyzed by one-way analysis of variance (ANOVA) and Bonferroni post hoc test. Statistical graphs were generated by using GraphPad Prism 8.0.2 software (GraphPad Software, Inc, San Diego, USA). p <0.05, p <0.01 or p The difference was statistically significant when the value was <0.001.

[0107] 6. Results

[0108] The protective effects of TFG and TFG (II) on oxidative stress in mouse hippocampal neuronal cells HT22 induced by H2O2 are shown in Table 5. According to the MTT results, the MTT assay was used to determine the effect of TFG at different concentrations (1.25, 2.5, 5, and 10 μM) on the viability of HT22 cells with or without the addition of H2O2. The cell viability was significantly decreased after H2O2 treatment ( ** p <0.001), TFG showed better protective activity ( ### p < 0.001, # p < 0.05). TFG (Ⅱ) administered at the same dose showed lower protective activity ( # p < 0.05). Subsequently, the effects of different concentrations (1.25, 2.5, 5, and 10 μM) of TFG and TFG (II) on HT22 cells with or without H2O2 treatment were detected using MDA, SOD, CAT, and GSH assays. The results showed that H2O2 treatment significantly increased the MDA level in HT22 cells ( *** p < 0.001), SOD, CAT and GSH levels were significantly decreased ( *** p< 0.001); after TFG intervention, the abnormal changes of the above indicators were significantly improved. Among them, TFG (10 μM) showed the best protective effect and could significantly alleviate the oxidative stress damage of HT22 cells induced by H2O2 (compared with the H2O2 treatment group, ### p < 0.001; ## p < 0.01; # p < 0.05). The experimental data showed that TFG can effectively reduce the oxidative stress damage of HT22 cells induced by H2O2. It can be concluded that TFG has a better advantage in protecting HT22 cells from oxidative stress induced by H2O2. However, the biological activity of TFG (Ⅱ) (compared with the H2O2 treatment group, ## p < 0.01; # p < 0.05) was lower than that of TFG.

[0109] It can be concluded that the present reaction TFG has a better advantage in protecting HT22 cells from oxidative stress induced by H2O2.

[0110] Table 5 Effect of TFG on H2O2-induced HT22 cell viability

[0111] Table 6 Effects of TFG on basic oxidative indices (MDA / SOD / CAT / GSH) of HT22 cells induced by H2O2

[0112] Example 25

[0113] The compound TFG of the present invention has a protective effect on H2O2-induced human umbilical vein endothelial cells HUVEC cells.

[0114] 1. Drug preparation

[0115] TFG (structural formula shown in Formula I, previously referred to as TFG, hereinafter referred to as TFG) and TFG (structural formula shown in Formula II, reported in CN201180038302.2) were dissolved in DMEM high-glucose medium to prepare a 1 mM stock solution. The stock solution was then diluted to concentrations of 1.25, 2.5, 5, and 10 μM, respectively. H₂O₂ was diluted in DMEM high-glucose medium to a 100 mM solution. TFG is inherently unstable and should be prepared freshly before use. The entire process must be performed under sterile conditions. After dissolution, the solution must be filtered through a 0.22 μM filter membrane before use.

[0116] 2. Cell culture

[0117] HUVEC (human umbilical vein endothelial cell) cells were purchased from the Shanghai Cell Resource Center, Chinese Academy of Sciences, and cultured in a thermostat at 37°C and 5% CO2. The culture medium used was Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% FBS, 1% penicillin, and 1% streptomycin. Formal experiments were performed when HUVEC cells exhibited a normal karyotype, adhered to the culture medium and were in the logarithmic growth phase, reaching a cell density of 80–90%.

[0118] 3. MTT assay to test the effect of TFG on cell viability

[0119] HUVEC cells were seeded in 96-well culture plates and treated with H₂O₂ and varying concentrations of TFG. Then, 20 μL of MTT solution (5 mg / mL) was added to each well. After incubation at 37°C for 3.5 hours, dark blue formazan crystals formed. The supernatant was discarded, and 150 μL of DMSO solution was added to each well to dissolve the formazan crystals. The plate was shaken and mixed for 5 minutes. The absorbance was then measured at 490 nm using a DR-200B microplate reader. The viability of the group treated with DMEM was normalized to 100%.

[0120] 4. Antioxidant activity (MDA, SOD, CAT, GSH-Px) detection

[0121] HUVEC cells were seeded in 6-well culture plates and treated with H2O2 and different concentrations of TFG. The cells were then mixed with RIPA lysis buffer (with protease inhibitors and phosphatase inhibitors added in proportion) for protein extraction. The MDA, SOD, CAT, and GSH-Px detection kits were followed according to their instructions. The absorbance was measured using a DR-200B microplate reader, and the antioxidant activity was calculated based on the absorbance.

[0122] 5. Data processing

[0123] All data were expressed as mean ± standard deviation (mean ± SD) established in different experiments and analyzed by one-way analysis of variance (ANOVA) and Bonferroni post hoc test. Statistical graphs were generated by using GraphPad Prism 8.0.2 software (GraphPad Software, Inc, San Diego, USA). p <0.05, p <0.01 or p The difference was statistically significant when the value was <0.001.

[0124] 6. Results

[0125] The protective effect of TFG on H2O2-induced oxidative stress in human umbilical vein endothelial cells (HUVEC) is shown in Tables 7 and 8. According to the MTT results, the MTT assay was used to determine the effect of TFG at different concentrations (1.25, 2.5, 5, and 10 μM) on HUVEC cell viability with or without H2O2 addition. After H2O2 treatment, cell viability was significantly decreased ( *** p < 0.001), TFG showed better protective activity ( ### p < 0.001, # p < 0.05), while TFG (Ⅱ) ( # p < 0.05) was weaker than TFG. MDA, SOD, CAT and GSH-Px assays were used to test the effects of different concentrations (1.25, 2.5, 5 and 10 μM) of TFG and TFG (II) on HUVEC cells with or without H2O2 treatment. The results showed that H2O2 treatment significantly increased the MDA level in HUVEC cells ( *** p < 0.001), SOD, CAT and GSH-Px levels were significantly decreased ( *** p <0.001); after TFG intervention, the abnormal changes of the above indicators were significantly improved. Among them, TFG (10 μM) showed the best protective effect and could significantly alleviate the oxidative stress damage of HUVEC cells induced by H2O2 (compared with the H2O2 treatment group, ### p <0.001; ## p < 0.01; # p < 0.05). The experimental data showed that TFG can effectively reduce H2O2-induced oxidative stress damage in HUVEC cells. It can be concluded that TFG has a better advantage in protecting H2O2-induced oxidative stress in HUVEC cells. However, the biological activity of TFG (Ⅱ) (compared with the H2O2 treatment group, ## p < 0.01; # p < 0.05) was significantly lower than that of TFG.

[0126] Table 7 Effect of TFG on H2O2-induced HUVEC cell viability

[0127] Table 8 Effects of TFG on basic oxidative indices of HUVEC cells induced by H2O2 (MDA / SOD / CAT / GSH-Px)

[0128] The present invention has been described in detail above with reference to preferred embodiments and exemplary examples. However, it should be noted that these specific embodiments are merely illustrative explanations of the present invention and do not constitute any limitation on the scope of protection of the present invention. Various improvements, equivalent substitutions, or modifications may be made to the technical content of the present invention and its embodiments without departing from the spirit and scope of protection of the present invention, and all of these fall within the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the appended claims.

Claims

1. A method for synthesizing the compound theanine disodium glycoside TFG, characterized in that: include: A. Accurately weigh theanine (L-Theanine) and D-(+)-maltose at a mass ratio of 1:1 to 1:

5. Place them in 20 volumes (based on theanine mass, g / mL) of reaction medium with a concentration of 75% to 100%. B. Place in a constant temperature shaker at 60-95°C and incubate for 60-160 min. C. After the reaction, the product was immediately concentrated using a rotary evaporator and then passed through a polyacrylamide (Bio-gel P-2) column and eluted with a 0.2% acetic acid aqueous solution at a flow rate of 0.3 mL / min. The fractions were collected and lyophilized to obtain the compound TFG.

2. The artificial synthesis method according to claim 1, characterized in that The reaction medium is acetic acid aqueous solution with different concentrations (v / v), namely 75%, 80%, 85%, 90%, 95% acetic acid aqueous solution and glacial acetic acid.

3. The artificial synthesis method according to claim 1, characterized in that The mass ratio of theanine to maltose is 1:2, the theanine is measured as 10.0 g, the maltose is measured as 20.0 g, and the amount of glacial acetic acid used is 200 mL.

4. The artificial synthesis method according to claim 1, characterized in that The reaction temperature was 85°C.

5. The artificial synthesis method according to claim 1, characterized in that The reaction time was 120 min.

6. Use of the compound prepared according to the preparation method according to any one of claims 1 to 5 in the preparation of drugs and / or health products for preventing or treating oxidative stress-related diseases.

7. The use according to claim 6, characterized in that: The oxidative stress-related disease is a neurodegenerative disease, preferably Alzheimer's disease; wherein the TFG exerts its effect by inhibiting hydrogen peroxide (H2O2)-induced oxidative stress in hippocampal neurons and vascular endothelial cells.

8. A pharmaceutical composition for preventing or treating neurodegenerative diseases, characterized in that: The TFG comprises the pharmaceutical composition according to any one of claims 1 to 6 and medically acceptable excipients.

9. The drug according to claim 8, characterized in that The medicine is in the form of tablets, capsules, granules, ointments, suspensions, powders, injections, sprays or pills.

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