A method for the artificial synthesis and uses of the compound theanine disaccharide TFG

Abstract

This invention belongs to the field of chemical technology, specifically relating to a method for the artificial synthesis of theanine-fructosyl-glucose (TFG) and its applications. Theanine-fructosyl-glucose (TFG) is a Maillard reaction product with significant antioxidant activity, but it has not yet been systematically studied. This study provides an efficient method for the artificial synthesis of TFG, reducing synthesis costs and providing a material basis for subsequent research. This method is economical, efficient, simple to operate, and safe, making it suitable for industrial production. Subsequently, in vitro activity screening of HT22 mouse hippocampal neurons and HUVEC human vascular endothelial cells confirmed that TFG can significantly alleviate hydrogen peroxide (H2O2)-induced cellular oxidative damage. Based on its synergistic protective effect on neurons and the vascular endothelial system, TFG is suitable for developing antioxidant drugs for the treatment of Alzheimer's disease.

Description

Technical Field

[0001] This invention belongs to the field of chemical technology, specifically relating to a method for the artificial synthesis of theanine disaccharide TFG and its uses. The synthesis method of this compound and its antagonistic effect on hydrogen peroxide (H2O2)-induced damage to mouse hippocampal neurons and human vascular endothelial cells (HUVECs) are disclosed. Background Technology

[0002] L-Theanine, a characteristic functional amino acid of tea, accounts for more than 50% of the total free amino acids in tea. Its glycoside derivatives generated through the Maillard reaction are an important research direction in the field of deep tea processing. In existing technologies, Yu Zaiyuan et al. reported a method involving reflux at 65°C for 18 hours in a methanol system to induce glycosylation between L-theanine and maltose, preparing a mixture of maltitol-theanine isomers (theanine disaccharides), two of which are shown in formulas (I) and (II), respectively. However, this method has significant drawbacks: the reaction time is lengthy (18 hours) and the product is a complex mixture, making the separation and purification of the target compound difficult, severely restricting the functional research and application development of theanine disaccharide derivatives.

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

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

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

[0006] This invention provides a simple method for the artificial synthesis of TFG, which is easy to operate, allows for easy control of reaction conditions and temperature, has readily available raw materials, and achieves a high synthesis rate, as well as the optimal process screening.

[0007] The compound described in this invention is named theanine disaccharide, and its molecular formula is: C 19 H 34 N2O 13 The structural formula is as follows:

[0008] (I)

[0009] This invention is achieved through the following technical solution:

[0010] The method for artificially synthesizing the compound TFG described in this invention comprises the following steps:

[0011] Theanine and maltose in a certain proportion are placed in an acidic reaction medium, mixed evenly, and then synthesized into theanine disaccharide under high temperature conditions. The specific steps are as follows:

[0012] A. Accurately weigh L-Theanine and D-(+)-maltose in a mass ratio of 1:1 to 1:5, and place them in a reaction medium with a volume of 20 times (g / mL based on the mass of theanine), and the concentration of the reaction medium should be 75% to 100%.

[0013] B. Place in a constant temperature shaking incubator and react at 60~95℃ for 60-140 min;

[0014] C. After the reaction was completed, the mixture was concentrated to dryness using a rotary evaporator, and then passed through a polyacrylamide (Bio-gel P-2) column, eluted with 0.2% acetic acid aqueous solution at a flow rate of 0.3 mL / min. The fraction was collected, lyophilized, and the compound TFG was obtained.

[0015] As a preferred embodiment of the present invention, the reaction medium is an aqueous solution of acetic acid of different concentrations (v / v), namely 75%, 80%, 85%, 90%, 95% aqueous solution of acetic acid and glacial acetic acid, with glacial acetic acid being the preferred reaction medium.

[0016] As a preferred embodiment of the present invention, the mass ratio of theanine to maltose is 1:1, 1:2, 1:3, 1:4, or 1:5, with a preferred mass ratio of 1:2.

[0017] As a preferred embodiment of the present invention, the selected reaction temperature range is 60, 70, 80, 85, 90, or 95°C, with a preferred reaction temperature of 85°C.

[0018] As a preferred technical solution of the present invention, the selected reaction time range is 60, 80, 100, 120, 140, or 160 min, with a preferred reaction time of 120 min.

[0019] The advantages of this invention over the prior art are:

[0020] For the first time, the artificial synthesis of theanine disaccharide with a single configuration was achieved. Existing related technologies were summarized and optimized. After comprehensive evaluation, a simpler, lower-cost, and higher-yield method was obtained. After further purification and separation, the purity can reach over 97%.

[0021] The present invention further provides the application of the compound TFG in the preparation of antioxidant drugs and / or health products, specifically, in the preparation of drugs for the prevention, treatment or improvement of oxidative stress induced by hydrogen peroxide (H2O2) in HT22 cells.

[0022] Alternatively, the application is based on the ameliorative effect of TFG on hydrogen peroxide (H2O2)-induced HUVEC cell damage, by activating intracellular antioxidant signaling pathways, inhibiting oxidative stress response, and protecting the vascular endothelial cell barrier function.

[0023] Alternatively, the application may specifically be the preparation of drugs for the prevention or treatment of oxidative stress-related diseases, including but not limited to hydrogen peroxide-induced endothelial cell dysfunction and oxidative damage-related vascular lesions.

[0024] Based on the aforementioned neuronal-vascular endothelial protective mechanism, this TFG application is particularly suitable for developing antioxidant drugs to treat Alzheimer's disease. Attached Figure Description

[0025] Figure 1 This is a schematic diagram of the Maillard reaction pathway of TFG in this invention.

[0026] Figure 2 This is the HPLC-ELSD spectrum of TFG in this invention.

[0027] Figure 3 For the TFG of this invention 13 C-NMR spectrum

[0028] Figure 4 For the TFG of this invention 1 H-NMR spectrum

[0029] Figure 5 The HMBC spectrum of the TFG of this invention.

[0030] Figure 6 The HSQC spectrum of the TFG of this invention.

[0031] Figure 7 The HR-ESI-MS spectrum of TFG in this invention. Detailed Implementation

[0032] The artificial synthesis method and antioxidant activity of the present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0033] The theanine disaccharide TFG was previously isolated from tea by our research group and identified as follows:

[0034] TFG is generated by the Maillard reaction (e.g. Figure 1 As shown), detected by HPLC-ELSD ( Figure 2 The absorption peak retention time was 23.83 min. Further one-dimensional nuclear magnetic resonance (1D NMR) analysis showed that the compound's 1H NMR spectrum exhibited 25 hydrogen proton signals, mainly distributed in the 3.0–4.0 ppm chemical shift range; 13C NMR confirmed the presence of 19 carbon atoms in the molecule, with the glycosyl group showing 11 carbon signals. The characteristic peaks at chemical shifts of 100.57 and 95.41 ppm indicate the presence of two glycosyl units (e.g., 2H NMR). Figure 3 and Figure 4 Through heteronuclear multi-bond correlation spectroscopy (HMBC) analysis, long-range correlation signals between H-3′ and C-1′′ were observed, thereby determining the linkage mode between glycosyl groups (e.g., Figure 5 and Figure 6 In addition, such as Figure 7 High-resolution mass spectrometry (HR-ESI-MS) showed an m / z of 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 this compound is confirmed to be C. 19 H 34 N2O 13 As shown in equation (Ⅰ).

[0035] (I)

[0036] The structural formula of the isomer is shown in formula (II):

[0037] (II)

[0038] Table 1. Compound TFG 1 H-NMR and 13 C-NMR chemical shift

[0039]

[0040] Detection of crude theanine disaccharide compounds

[0041] 1.1 Detection method: The content of the crude compound was determined by high performance liquid chromatography-evaporative light scattering detection (HPLC-ELSD).

[0042] 1.2 Sample preparation: 5.0 mg of crude theanine disaccharide was accurately weighed for each group and diluted to 2 mL in a volumetric flask with 0.1 mol / L hydrochloric acid solution to obtain the liquid phase injection sample with a concentration of 2.5 mg / mL.

[0043] 1.3 Chromatographic conditions: Ultimate® Amino Acid Plus second-generation amino acid-specific column (4.6 mm × 300 mm), evaporative light scattering detector, drift tube temperature 120℃, carrier gas flow rate 3.2 L / min, using 0.3% heptafluorobutyric acid solution (A) and acetonitrile (B) as the mobile phase, 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 30℃, injection volume 20 μL, flow rate 1.0 mL / min. Preparation of the 0.3% heptafluorobutyric acid mobile phase: Take 997 mL of water, add 3 mL of heptafluorobutyric acid, mix well, and degas by sonication.

[0044] Example 1:

[0045] Accurately weigh 10 g of theanine and 20 g of maltose, place them in 200 mL of 75% acetic acid, mix well, and then place the mixture on a constant temperature water bath shaker at 85℃ for 120 min. Immediately after the reaction, concentrate the mixture using a rotary evaporator, then pass it through a polyacrylamide (Bio-gel P-2) column, eluting with 0.2% acetic acid aqueous solution at a flow rate of 0.3 mL / min. Collect the fraction, freeze-dry it, and obtain the compound TFG. The reaction showed no obvious browning. The experiment was repeated three times. After purification, the yield of the product was 55.6%, and the purity of TFG was 96.2%.

[0046] Example 2:

[0047] Accurately weigh 10 g of theanine and 20 g of maltose, place them in 200 mL of 80% acetic acid, mix well, and then place the mixture on a constant temperature water bath shaker at 85℃ for 120 min. Immediately after the reaction, concentrate the mixture using a rotary evaporator, then pass it through a polyacrylamide (Bio-gel P-2) column, eluting with 0.2% acetic acid aqueous solution at a flow rate of 0.3 mL / min. Collect the fraction, freeze-dry it, and obtain the compound TFG. The reaction showed no obvious browning. The experiment was repeated three times. After purification, the yield of the product was 60.1%, and the purity of TFG was 96.5%.

[0048] Example 3:

[0049] Accurately weigh 10 g of theanine and 20 g of maltose, place them in 200 mL of 85% acetic acid, mix well, and then place the mixture on a constant temperature water bath shaker at 85℃ for 120 min. Immediately after the reaction, concentrate the mixture using a rotary evaporator, then pass it through a polyacrylamide (Bio-gel P-2) column, eluting with 0.2% acetic acid aqueous solution at a flow rate of 0.3 mL / min. Collect the fraction, freeze-dry it, and obtain the compound TFG. The experiment was repeated three times, and after purification, the yield of the product was 61.1%, and the purity of TFG was 96.2%.

[0050] Example 4:

[0051] Accurately weigh 10 g of theanine and 20 g of maltose, place them in 200 mL of 90% acetic acid, mix well, and then place the mixture on a constant temperature water bath shaker at 85℃ for 120 min. Immediately after the reaction, concentrate the mixture using a rotary evaporator, then pass it through a polyacrylamide (Bio-gel P-2) column, eluting with 0.2% acetic acid aqueous solution at a flow rate of 0.3 mL / min. Collect the fraction, freeze-dry it, and 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%.

[0052] Example 5:

[0053] Accurately weigh 10 g of theanine and 20 g of maltose, place them in 200 mL of 95% acetic acid, mix well, and then place the mixture on a constant temperature water bath shaker at 85℃ for 120 min. Immediately after the reaction, concentrate the mixture using a rotary evaporator, then pass it through a polyacrylamide (Bio-gel P-2) column, eluting with 0.2% acetic acid aqueous solution at a flow rate of 0.3 mL / min. Collect the fraction, freeze-dry it, and obtain the compound TFG. The experiment was repeated three times. After purification, the yield of the product was 64.1%, and the purity of TFG was 96.1%.

[0054] Example 6:

[0055] Accurately weigh 10 g of theanine and 20 g of maltose, place them in 200 mL of glacial acetic acid, mix well, and then place the mixture on a constant temperature water bath shaker at 85 °C for 120 min. Immediately after the reaction, concentrate the mixture using a rotary evaporator, then pass it through a polyacrylamide (Bio-gel P-2) column, eluting with 0.2% acetic acid aqueous solution at a flow rate of 0.3 mL / min. Collect the fraction, freeze-dry it, and obtain the compound TFG. The experiment was repeated three times, and after purification, the yield of the product was 65.6%, and the purity of TFG was 97.2%.

[0056] Example 7:

[0057] Accurately weigh 10 g of theanine and 20 g of maltose, place them in 200 mL of glacial acetic acid, mix well, and then place the mixture on a shaker in a 60℃ water bath for 120 min. Immediately after the reaction, concentrate the mixture using a rotary evaporator, then pass it through a polyacrylamide (Bio-gel P-2) column, eluting with 0.2% acetic acid aqueous solution at a flow rate of 0.3 mL / min. Collect the fraction, freeze-dry it, and 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%.

[0058] Example 8:

[0059] Accurately weigh 10 g of theanine and 20 g of maltose, place them in 200 mL of glacial acetic acid, mix well, and then place the mixture on a shaker in a 70℃ water bath for 120 min. Immediately after the reaction, concentrate the mixture using a rotary evaporator, then pass it through a polyacrylamide (Bio-gel P-2) column, eluting with 0.2% acetic acid aqueous solution at a flow rate of 0.3 mL / min. Collect the fraction, freeze-dry it, and obtain the compound TFG. The experiment was repeated three times, and after purification, the yield of the product was 60.2%, and the purity of TFG was 96.1%.

[0060] Example 9:

[0061] Accurately weigh 10 g of theanine and 20 g of maltose, place them in 200 mL of glacial acetic acid, mix well, and then place the mixture on a constant temperature water bath shaker at 80 °C for 120 min. Immediately after the reaction, concentrate the mixture using a rotary evaporator, then pass it through a polyacrylamide (Bio-gel P-2) column, eluting with 0.2% acetic acid aqueous solution at a flow rate of 0.3 mL / min. Collect the fraction, freeze-dry it, and obtain the compound TFG. The experiment was repeated three times, and after purification, the yield of the product was 63.7%, and the purity of TFG was 95.9%.

[0062] Example 10:

[0063] Accurately weigh 10 g of theanine and 20 g of maltose, place them in 200 mL of glacial acetic acid, mix well, and then place the mixture on a constant temperature water bath shaker at 85 °C for 120 min. Immediately after the reaction, concentrate the mixture using a rotary evaporator, then pass it through a polyacrylamide (Bio-gel P-2) column, eluting with 0.2% acetic acid aqueous solution at a flow rate of 0.3 mL / min. Collect the fraction, freeze-dry it, and obtain the compound TFG. The experiment was repeated three times, and after purification, the yield of the product was 65.6%, and the purity of TFG was 97.2%.

[0064] Example 11:

[0065] Accurately weigh 10 g of theanine and 20 g of maltose, place them in 200 mL of glacial acetic acid, mix well, and then place the mixture on a shaker in a 90℃ water bath for 120 min. Immediately after the reaction, concentrate the mixture using a rotary evaporator, then pass it through a polyacrylamide (Bio-gel P-2) column, eluting with 0.2% acetic acid aqueous solution at a flow rate of 0.3 mL / min. Collect the fraction, freeze-dry it, and 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%.

[0066] Example 12:

[0067] Accurately weigh 10 g of theanine and 20 g of maltose, place them in 200 mL of glacial acetic acid, mix well, and then place the mixture on a shaker in a 95°C water bath for 120 min. Immediately after the reaction, concentrate the mixture using a rotary evaporator, then pass it through a polyacrylamide (Bio-gel P-2) column, eluting with 0.2% acetic acid aqueous solution at a flow rate of 0.3 mL / min. Collect the fraction, freeze-dry it, and 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%.

[0068] Example 13:

[0069] Accurately weigh 10 g of theanine and 20 g of maltose, place them in 200 mL of glacial acetic acid, mix well, and then place the mixture on a constant temperature water bath shaker at 85 °C for 60 min. Immediately after the reaction, concentrate the mixture using a rotary evaporator, then pass it through a polyacrylamide (Bio-gel P-2) column, eluting with 0.2% acetic acid aqueous solution at a flow rate of 0.3 mL / min. Collect the fraction, freeze-dry it, and 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%.

[0070] Example 14:

[0071] Accurately weigh 10 g of theanine and 20 g of maltose, place them in 200 mL of glacial acetic acid, mix well, and then place the mixture on a constant temperature water bath shaker at 85℃ for 80 min. Immediately after the reaction, concentrate the mixture using a rotary evaporator, then pass it through a polyacrylamide (Bio-gel P-2) column, eluting with 0.2% acetic acid aqueous solution at a flow rate of 0.3 mL / min. Collect the fraction, freeze-dry it, and 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%.

[0072] Example 15:

[0073] Accurately weigh 10 g of theanine and 20 g of maltose, place them in 200 mL of glacial acetic acid, mix well, and then place the mixture on a constant temperature water bath shaker at 85 °C for 100 min. Immediately after the reaction, concentrate the mixture using a rotary evaporator, then pass it through a polyacrylamide (Bio-gel P-2) column, eluting with 0.2% acetic acid aqueous solution at a flow rate of 0.3 mL / min. Collect the fraction, freeze-dry it, and 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%.

[0074] Example 16:

[0075] Accurately weigh 10 g of theanine and 20 g of maltose, place them in 200 mL of glacial acetic acid, mix well, and then place the mixture on a constant temperature water bath shaker at 85 °C for 120 min. Immediately after the reaction, concentrate the mixture using a rotary evaporator, then pass it through a polyacrylamide (Bio-gel P-2) column, eluting with 0.2% acetic acid aqueous solution at a flow rate of 0.3 mL / min. Collect the fraction, freeze-dry it, and obtain the compound TFG. The experiment was repeated three times, and after purification, the yield of the product was 65.6%, and the purity of TFG was 97.2%.

[0076] Example 17:

[0077] Accurately weigh 10 g of theanine and 20 g of maltose, place them in 200 mL of glacial acetic acid, mix well, and then place the mixture on a constant temperature water bath shaker at 85 °C for 140 min. Immediately after the reaction, concentrate the mixture using a rotary evaporator, then pass it through a polyacrylamide (Bio-gel P-2) column, eluting with 0.2% acetic acid aqueous solution at a flow rate of 0.3 mL / min. Collect the fraction, freeze-dry it, and 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%.

[0078] Example 18:

[0079] Accurately weigh 10 g of theanine and 20 g of maltose, place them in 200 mL of glacial acetic acid, mix well, and then place the mixture on a constant temperature water bath shaker at 85℃ for 160 min. Immediately after the reaction, concentrate the mixture using a rotary evaporator, then pass it through a polyacrylamide (Bio-gel P-2) column, eluting with 0.2% acetic acid aqueous solution at a flow rate of 0.3 mL / min. Collect the fraction, freeze-dry it, and 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%.

[0080] Example 19:

[0081] Accurately weigh 10 g of theanine and 10 g of maltose, place them in 200 mL of glacial acetic acid, mix well, and then place the mixture on a constant temperature water bath shaker at 85℃ for 120 min. Immediately after the reaction, concentrate the mixture using a rotary evaporator, then pass it through a polyacrylamide (Bio-gel P-2) column, eluting with 0.2% acetic acid aqueous solution at a flow rate of 0.3 mL / min. Collect the fraction, freeze-dry it, and 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%.

[0082] Example 20:

[0083] Accurately weigh 10 g of theanine and 20 g of maltose, place them in 200 mL of glacial acetic acid, mix well, and then place the mixture on a constant temperature water bath shaker at 85 °C for 120 min. Immediately after the reaction, concentrate the mixture using a rotary evaporator, then pass it through a polyacrylamide (Bio-gel P-2) column, eluting with 0.2% acetic acid aqueous solution at a flow rate of 0.3 mL / min. Collect the fraction, freeze-dry it, and obtain the compound TFG. The experiment was repeated three times, and after purification, the yield of the product was 65.6%, and the purity of TFG was 97.2%.

[0084] Example 21:

[0085] Accurately weigh 10 g of theanine and 30 g of maltose, place them in 200 mL of glacial acetic acid, mix well, and then place the mixture on a constant temperature water bath shaker at 85℃ for 120 min. Immediately after the reaction, concentrate the mixture using a rotary evaporator, then pass it through a polyacrylamide (Bio-gel P-2) column, eluting with 0.2% acetic acid aqueous solution at a flow rate of 0.3 mL / min. Collect the fraction, freeze-dry it, and 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%.

[0086] Example 22:

[0087] Accurately weigh 10 g of theanine and 40 g of maltose, place them in 200 mL of glacial acetic acid, mix well, and then place the mixture on a constant temperature water bath shaker at 85℃ for 120 min. Immediately after the reaction, concentrate the mixture using a rotary evaporator, then pass it through a polyacrylamide (Bio-gel P-2) column, eluting with 0.2% acetic acid aqueous solution at a flow rate of 0.3 mL / min. Collect the fraction, freeze-dry it, and 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%.

[0088] Example 23:

[0089] Accurately weigh 10 g of theanine and 50 g of maltose, place them in 200 mL of glacial acetic acid, mix well, and then place the mixture on a constant temperature water bath shaker at 85℃ for 120 min. Immediately after the reaction, concentrate the mixture using a rotary evaporator, then pass it through a polyacrylamide (Bio-gel P-2) column, eluting with 0.2% acetic acid aqueous solution at a flow rate of 0.3 mL / min. Collect the fraction, freeze-dry it, and obtain the compound TFG. The experiment was repeated three times, and after purification, the yield of the product was 60.2%, and the purity of TFG was 95.8%.

[0090] According to the above embodiments, the results shown in Table 2 are as follows: by controlling 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 the ideal state during the reaction.

[0091] Table 2. Statistical table of the effect of different concentrations of acetic acid on TFG synthesis

[0092]

[0093] As shown in Table 2, the yield is highest when glacial acetic acid is used as the reaction medium, and it is also economical.

[0094] According to the above embodiments, the results shown in Table 3 are as follows: by controlling the time and temperature to synthesize TFG, the results show that the yield of the synthesized product reaches the maximum when the temperature is 85℃ and the time is 120 min.

[0095] Table 3. Statistical table of the effects of different times and temperatures on TFG synthesis

[0096]

[0097] As shown in Table 3, by controlling the time and temperature respectively using the controlled variable method, the optimal synthesis temperature of TFG was found to be 85℃ and the optimal time was 120 min.

[0098] According to the above embodiments, the results shown in Table 4 indicate that by controlling the mass ratio of theanine to maltose during the reaction, the mass ratio of theanine to maltose in the synthesis of TFG is 1:2.

[0099] Table 4. Statistical table of the effect of the mass ratio of theanine to maltose on the synthesis of TFG during the reaction.

[0100]

[0101] Example 24

[0102] The present invention relates to the protective effect of compound TFG against H2O2-induced HT22 cells in mouse hippocampal neurons.

[0103] 1. Drug preparation

[0104] TFG (structural formula as shown in Formula I, i.e., the aforementioned TFG, hereinafter referred to as TFG) and TFG (structural formula as shown in Formula II, reported in CN201180038302.2) were dissolved in DMEM high-glucose medium to prepare a stock solution with a concentration of 1 mM, which was then diluted to concentrations of 1.25, 2.5, 5, and 10 μM, respectively. H2O2 was diluted with DMEM high-glucose medium to make a 100 mM solution. TFG is inherently unstable and must be prepared fresh for each use. The entire process must be carried out under aseptic conditions. After the drug is dissolved, it must be filtered through a 0.22 μM filter membrane before use.

[0105] 2. Cell Culture

[0106] HT22 cell line (mouse hippocampal neurons) was purchased from the Shanghai Cell Resource Center, Chinese Academy of Sciences, and cultured in a Thermo incubator at 37°C and 5% CO2. The culture medium used was DMEM containing 10% FBS, 1% penicillin, and 1% streptomycin. The conditions for the formal experiments were: under a microscope, HT22 cells showed normal karyotype, were adherent, and were in the logarithmic growth phase, with a cell density of 80-90%.

[0107] 3. MTT assay for the effect of TFG on cell viability

[0108] HT22 cells were seeded in 96-well plates and treated with H2O2 and different concentrations of TFG. Then, 20 μL of MTT solution (5 mg / mL) was added to each well, and the plates were incubated at 37°C for 3.5 h. After the formation of deep blue formazan crystals, the supernatant was discarded. 150 μL of DMSO solution was added to each well to dissolve the formazan crystals, and the plates were shaken to mix for 5 min. The absorbance was then measured at 490 nm using a DR-200B microplate reader. Groups treated with DMEM medium were normalized to 100% viability.

[0109] 4. Antioxidant activity (MDA, SOD, CAT, GSH) detection

[0110] HT22 cells were seeded in 6-well culture plates and treated with H2O2 and different concentrations of TFG. Then, they were 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 tested according to the instructions, and the absorbance values ​​were measured using a DR-200B microplate reader. The antioxidant activity was calculated based on the absorbance values.

[0111] 5. Data Processing

[0112] All data are expressed as mean ± standard deviation (Mean ± SD) established in different experiments, and analyzed using one-way ANOVA and Bonferroni post-hoc tests. Statistical graphs were generated using GraphPad Prism 8.0.2 software (GraphPad Software, Inc., San Diego, USA). p <0.05、 p <0.01 or p <0.001 indicates a statistically significant difference.

[0113] 6. Results

[0114] The protective effects of TFG and TFG(II) against H2O2-induced oxidative stress in mouse hippocampal neurons HT22 cells are shown in Table 5. According to the MTT assay, the effect of different concentrations of TFG (1.25, 2.5, 5, and 10 μM) on HT22 cell viability with or without H2O2 treatment was determined. H2O2 treatment significantly decreased cell viability. ** p <0.001%, TFG showed good protective activity ( ### p < 0.001, # p < 0.05). Administration of the same dose of TFG(II) 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 and without H2O2 treatment were detected using MDA, SOD, CAT, and GSH kits. The results showed that H2O2 treatment significantly increased the MDA level in HT22 cells ( *** p< 0.001), SOD, CAT and GSH levels were significantly reduced ( *** p < 0.001); and 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 H2O2-induced oxidative stress damage in HT22 cells (compared with the H2O2 treatment group, ### p < 0.001; ## p < 0.01; # p < 0.05). These experimental data indicate that TFG can effectively alleviate H2O2-induced oxidative stress damage in HT22 cells. Therefore, TFG has a significant advantage in reducing H2O2-induced oxidative stress in HT22 cells. However, the biological activity exhibited by TFG(II) (compared to the H2O2-treated group) is... ## p < 0.01; # p < 0.05) is lower than TFG.

[0115] Therefore, it can be concluded that the TFG reaction has a good advantage in stimulating H2O2-induced oxidative stress in HT22 cells.

[0116] Table 5. Effects of TFG on H2O2-induced viability of HT22 cells

[0117]

[0118] Table 6. Effects of TFG on H2O2-induced basic oxidative indicators (MDA / SOD / CAT / GSH) in HT22 cells

[0119]

[0120] Example 25

[0121] The present invention describes the protective effect of compound TFG against H2O2-induced human umbilical vein endothelial cells (HUVECs).

[0122] 1. Drug preparation

[0123] TFG (structural formula as shown in Formula I, i.e., the aforementioned TFG, hereinafter referred to as TFG) and TFG (structural formula as shown in Formula II, reported in CN201180038302.2) were dissolved in DMEM high-glucose medium to prepare a stock solution with a concentration of 1 mM, which was then diluted to concentrations of 1.25, 2.5, 5, and 10 μM, respectively. H2O2 was diluted with DMEM high-glucose medium to make a 100 mM solution. TFG is inherently unstable and must be prepared fresh for each use. The entire process must be carried out under aseptic conditions. After the drug is dissolved, it must be filtered through a 0.22 μM filter membrane before use.

[0124] 2. Cell Culture

[0125] HUVEC cell line (human umbilical vein endothelial cells) was purchased from the Shanghai Cell Resource Center, Chinese Academy of Sciences, and cultured in a Thermo incubator at 37°C and 5% CO2. The culture medium used was DMEM containing 10% FBS, 1% penicillin, and 1% streptomycin. The conditions for formal experiments were: under a microscope, the HUVEC cells showed normal karyotype, were adherent, and were in the logarithmic growth phase, with a cell density of 80-90%.

[0126] 3. MTT assay for the effect of TFG on cell viability

[0127] HUVEC cells were seeded in 96-well plates and treated with H2O2 and different concentrations of TFG. Then, 20 μL of MTT solution (5 mg / mL) was added to each well, and the plates were incubated at 37°C for 3.5 h. After the formation of deep blue formazan crystals, the supernatant was discarded. 150 μL of DMSO solution was added to each well to dissolve the formazan crystals, and the plates were shaken to mix for 5 min. The absorbance was then measured at 490 nm using a DR-200B microplate reader. Groups treated with DMEM medium were normalized to 100% viability.

[0128] 4. Antioxidant activity (MDA, SOD, CAT, GSH-Px) detection

[0129] HUVEC cells were seeded in 6-well culture plates and treated with H2O2 and different concentrations of TFG. Then, they were 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 tested according to the instructions, and the absorbance values ​​were measured using a DR-200B microplate reader. The antioxidant activity was calculated based on the absorbance values.

[0130] 5. Data Processing

[0131] All data are expressed as mean ± standard deviation (Mean ± SD) established in different experiments, and analyzed using one-way ANOVA and Bonferroni post-hoc tests. Statistical graphs were generated using GraphPad Prism 8.0.2 software (GraphPad Software, Inc., San Diego, USA). p <0.05, p <0.01 or p <0.001 indicates a statistically significant difference.

[0132] 6. Results

[0133] The protective effect of TFG against H2O2-induced oxidative stress in human umbilical vein endothelial cells (HUVECs) is shown in Tables 7 and 8. According to the MTT assay, the effect of different concentrations of TFG (1.25, 2.5, 5, and 10 μM) on HUVEC cell viability with or without H2O2 treatment was determined. H2O2 treatment significantly decreased cell viability. *** p < 0.001), TFG showed good protective activity ( ### p < 0.001, # p < 0.05), while TFG(Ⅱ) ( # p < 0.05) compared to TFG, the activity was weaker. The effects of different concentrations (1.25, 2.5, 5, and 10 μM) of TFG and TFG(II) on HUVEC cells with and without H2O2 treatment were detected using an MDA, SOD, CAT, and GSH-Px kit. The results showed that H2O2 treatment significantly increased the MDA level in HUVEC cells ( < 0.05). *** p < 0.001), SOD, CAT and GSH-Px levels were significantly reduced ( *** p <0.001); and 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 H2O2-induced oxidative stress damage in HUVEC cells (compared with the H2O2 treatment group, <0.001). ### p <0.001; ## p < 0.01; # p< 0.05). These experimental data indicate that TFG can effectively alleviate H2O2-induced oxidative stress damage in HUVEC cells. Therefore, TFG has a significant advantage in reducing H2O2-induced oxidative stress in HUVEC cells. However, the biological activity exhibited by TFG(II) (compared to the H2O2-treated group) is... ## p < 0.01; # p < 0.05) significantly lower than TFG.

[0134] Table 7 Effects of TFG on H2O2-induced HUVEC cell viability

[0135]

[0136] Table 8 Effects of TFG on H2O2-induced basic oxidation parameters (MDA / SOD / CAT / GSH-Px) in HUVEC cells

[0137]

[0138] 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 invention and do not constitute any limitation on the scope of protection of the invention. Various improvements, equivalent substitutions, or modifications can be made to the technical content and embodiments of the present invention without departing from the spirit and scope of protection of the invention, and all such modifications fall within the scope of protection of the present invention. The scope of protection of the present invention is defined by the appended claims.

Claims

1. The application of the compound theanine disaccharide TFG in the preparation of drugs for the prevention or treatment of oxidative stress-related diseases. The structure of TFG is: 。 2. The application according to claim 1, characterized in that: The oxidative stress-related diseases mentioned are neurodegenerative diseases.

3. The application according to claim 2, characterized in that: Neurodegenerative diseases are selected from Alzheimer's disease; wherein, the TFG exerts its effect by inhibiting hydrogen peroxide-induced oxidative stress in hippocampal neurons and vascular endothelial cells.

4. A pharmaceutical composition for the prevention or treatment of neurodegenerative diseases, characterized in that, The pharmaceutical composition comprises the compound theanine disaccharide TFG and a medically acceptable excipient, wherein the structure of TFG is: 。 5. The pharmaceutical composition according to claim 4, characterized in that, The pharmaceutical composition is in the form of tablets, capsules, granules, ointments, suspensions, powders, injections, sprays, or pills.

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