Gallic acid derivative as well as preparation method and application thereof

Abstract

The invention relates to the technical field of food processing, in particular to a gallic acid derivative as well as a preparation method and application thereof. The preparation method of the propyl gallate comprises the following steps: mixing gallic acid, alcohol and a catalyst, heating to a reaction temperature under a condensation reflux condition, carrying out an esterification reaction, and then carrying out post-treatment to obtain a gallic acid derivative; the alcohol is n-propyl alcohol, and the catalyst is sulfonic acid resin. The gallic acid derivative obtained by the preparation method provided by the invention not only improves the stability of gallic acid, but also enhances the antioxidant activity of gallic acid, and the prepared gallic acid derivative has higher application value in the fields of food, medicine, cosmetic production and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of food processing, and in particular to a gallic acid derivative and a preparation method and application thereof. Background Art

[0002] Economic forests are a vital component of forest resources, offering significant ecological, economic, and social benefits. As woody oilseed production increases annually, the number of byproducts generated during production and processing also increases. Underutilized byproducts not only waste resources but also increasingly harm the ecological environment. Woody oilseed processing byproducts contain active substances such as polyphenols, proteins, and fatty acids, with relatively high levels of polyphenols. Gallic acid, a polyphenolic compound, is present in many woody oilseed processing byproducts, with relatively high levels found in walnut shells, walnut husks, and oil-tea cattails. Gallic acid exhibits excellent antioxidant, antibacterial, and anti-inflammatory properties, but its low lipid solubility results in poor bioavailability. It is also susceptible to degradation, oxidation, isomerization, and polymerization, lacking long-term stability. The unsaturated bonds in its molecular structure and its strong antioxidant capacity make it sensitive to light, heat, pH changes, enzyme activity, and the presence of oxygen, limiting its application in food, pharmaceutical, and cosmetic production. Summary of the Invention

[0003] In order to solve the above technical problems, improve the stability of gallic acid and effectively protect its biological activity from loss, modify gallic acid to obtain high-efficiency active substances, and further expand the application of woody oil processing by-products. The present invention provides a gallic acid derivative and a preparation method and application thereof. The gallic acid derivative obtained by the preparation method provided by the present invention not only improves the stability of gallic acid, but also enhances the antioxidant activity of gallic acid. The prepared gallic acid derivative has high application value in the fields of food, medicine and cosmetics production.

[0004] In a first aspect, the present invention provides a method for preparing a gallic acid derivative, comprising: mixing gallic acid, an alcohol, and a catalyst, then heating the mixture to a reaction temperature under condensation reflux conditions and conducting an esterification reaction, followed by post-processing to obtain the gallic acid derivative; the alcohol is n-propanol, the catalyst is a sulfonic acid resin, and the mass ratio of the gallic acid, alcohol, and catalyst is 5-15:20-50:1-1.5. The method for preparing the gallic acid derivative provided by the present invention, through optimization of the preparation process and raw materials, produces a gallic acid derivative that not only improves the stability of gallic acid but also enhances its antioxidant activity. The prepared gallic acid derivative has high application value in the fields of food, medicine, and cosmetics production.

[0005] Preferably, the mass ratio of gallic acid, n-propanol and sulfonic acid resin is 7-13:25-35:0.7-1.3.

[0006] The present invention can reduce the need for excessive feeding by optimizing the ratio of gallic acid to n-propanol, save raw materials, reduce costs, and help reduce the occurrence of side reactions and improve the yield of esters.

[0007] More preferably, the raw materials for preparing propyl gallate are: 10±2 parts of gallic acid, 30±2 parts of n-propanol, and 1±0.2 parts of sulfonic acid resin, calculated by mass.

[0008] More preferably, the reaction temperature is 105-125°C and the reaction time is 5.5-6.5 hours. By optimizing the reaction conditions, the reaction efficiency and product purity can be significantly improved, side reactions (such as degradation or polymerization) caused by excessively high or low temperatures can be avoided, the selectivity of the target product can be improved, and a more complete reaction can be achieved. At the appropriate temperature, the activity of the catalyst can be optimized, ensuring that the product has high stability and antioxidant activity.

[0009] Preferably, the preparation method of the gallic acid derivative comprises: 1) Gallic acid and alcohol are mixed, sulfonic acid resin is added and stirred, and the mixture is heated to the reaction temperature under condensation reflux conditions to carry out esterification reaction.

[0010] 2) Cooling and filtering the reaction product of step 1), collecting the filtrate and performing rotary evaporation to remove alcohol.

[0011] 3) The filtrate treated in step 2) is mixed with water, heated to dissolve and filtered, and then cooled until the product is precipitated and filtered through a sand core.

[0012] 4) Drying the product obtained in step 3) to obtain a gallic acid derivative.

[0013] By optimizing the above steps, the reaction process is made more efficient and the product is purer. The final gallic acid derivative is more suitable for subsequent structural characterization and further functional analysis.

[0014] Preferably, in step 1), the reaction is carried out in a reaction vessel equipped with a magnetic stirrer and a reflux condenser.

[0015] Preferably, in step 2), the mixture is cooled to 55-65° C. and then filtered.

[0016] Preferably, in step 2), the rotary evaporation temperature is 55-65°C.

[0017] More preferably, in step 2), excess n-propanol is removed by rotary evaporation at 60±2°C using a water pump.

[0018] Preferably, in step 3), the ratio of the amount of water added to the amount of gallic acid used is 8 mL:1 g.

[0019] More preferably, in step 3), the solution is heated to 68-72° C. to dissolve, filtered while hot, and then cooled to 8-12° C. to precipitate the product and then filtered through a sand core.

[0020] In the present invention, the above optimization steps can improve the efficiency of the esterification reaction and optimize the purity of the final product by controlling the temperature, time and solvent dosage. By optimizing these conditions, the reaction process is more efficient, the solvent and impurities are better removed, the purity and yield of the product are improved, and the stability and antioxidant activity of the product are further ensured. The prepared gallic acid derivative has better application effects and has higher market value in the fields of food, medicine and cosmetics.

[0021] Preferably, in step 4), the drying time is 24-30 hours and the temperature is 95-105°C.

[0022] The preparation method of the gallic acid derivatives provided by the present invention can improve the purity, stability and antioxidant activity of the product through optimization of reaction raw materials and proportions, reaction process, heating under condensation reflux conditions, rotary evaporation to remove alcohol, cooling precipitation and sand core filtration, thereby providing higher product performance and market value. The prepared gallic acid derivatives have broad application prospects in multiple fields such as food, medicine, and cosmetics.

[0023] In a second aspect, the present invention provides gallic acid derivatives prepared by the above-mentioned preparation method of gallic acid derivatives.

[0024] In a third aspect, the present invention provides the use of gallic acid derivatives produced by the above-mentioned preparation method in food, medicine, and cosmetics. The gallic acid derivatives provided by the present invention can significantly improve stability and antioxidant activity, and have higher application value in food preservation, drug preservation, anti-aging cosmetics, and other aspects.

[0025] The beneficial effect of the present invention is at least that: the gallic acid derivatives provided by the present invention not only improve the stability of gallic acid, but also enhance the antioxidant activity of gallic acid. The prepared gallic acid derivatives have high application value in the fields of food, medicine and cosmetics production. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions in the present invention or the prior art, a brief introduction is given below to the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0027] Figure 1 These are the test results of the DPPH free radical scavenging ability of propyl gallate generated at different feed ratios and reaction temperatures in the embodiments of the present invention.

[0028] Figure 2 It is the Fourier transform infrared spectrum of gallic acid and its derivatives in the embodiment of the present invention.

[0029] Figure 3 It is the nuclear magnetic resonance spectrum of gallic acid and its derivatives in the examples of the present invention.

[0030] Figure 4 This is the thermogravimetric analysis result of gallic acid and its derivatives in the examples of the present invention.

[0031] Figure 5 These are the test results of the DPPH free radical scavenging ability of gallic acid and its derivatives at different concentrations in the examples of the present invention.

[0032] Figure 6 These are the test results of the ABTS free radical scavenging ability of gallic acid and its derivatives at different concentrations in the examples of the present invention.

[0033] Figure 7 This is the establishment of the SH-SY5Y cell oxidative stress model caused by H2O2 in the examples of the present invention.

[0034] Figure 8 This is the effect of gallic acid and its derivatives on the activity of SH-SY5Y nerve cells in the examples of the present invention.

[0035] Figure 9 This is the protective effect of gallic acid and its derivatives in the examples of the present invention on H2O2-induced oxidative stress damage in SH-SY5Y neurons. DETAILED DESCRIPTION

[0036] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention are described clearly and completely below. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0037] The endpoints of the ranges and any values disclosed herein are not limited to the precise ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values can be combined with each other to form one or more new numerical ranges, and these numerical ranges should be considered to be specifically disclosed in the present invention.

[0038] Where specific techniques or conditions are not specified in the examples of the present invention, the techniques or conditions described in the literature in the field or in the product specifications were followed. All devices, instruments, reagents, etc. used, where the manufacturer is not specified, are conventional products available through regular channels. All experimental reagents and raw materials involved are commercially available, and all reagents are analytically pure.

[0039] In the examples of the present invention, gallic acid (Article No.: G823263) was purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0040] Example 1 This embodiment provides a propyl gallate and a preparation method thereof, and the preparation steps are as follows: Step 1: Pour 10 g of gallic acid and 30 g of n-propanol into a reaction flask equipped with a magnetic stirrer and a reflux condenser.

[0041] Step 2: Add 1 g of sulfonic acid resin, stir evenly, and heat to 110° C. under condensation reflux conditions for reaction for 6 h.

[0042] Step 3: Cool to 60°C and filter while hot to collect the filtrate.

[0043] Step 4: Remove excess n-propanol by rotary evaporation at 60°C.

[0044] Step 5: Add 100 mL of deionized water, heat to 70°C to dissolve, filter while hot, cool to about 10°C to precipitate the product, and filter with a sand core.

[0045] Step 6: The product obtained by the reaction was placed in a drying oven and dried at 100° C. for 27 h. Propyl gallate was weighed and had a purity of 96.07% (Table 1).

[0046] Example 2 The same method as Example 1 was adopted, except that "10 g gallic acid and 30 g n-propanol" in step 1 was changed to "10 g gallic acid and 20 g n-propanol".

[0047] Example 3 The same method as Example 1 was adopted, except that "10 g gallic acid and 30 g n-propanol" in step 1 was changed to "10 g gallic acid and 40 g n-propanol".

[0048] Example 4 The same method as in Example 1 is adopted, except that "raising the temperature to 110° C." in step 2 is changed to "raising the temperature to 100° C.".

[0049] Example 5 The same method as in Example 1 is adopted, except that "raising the temperature to 110° C." in step 2 is changed to "raising the temperature to 120° C.".

[0050] Table 1

[0051] Example 6 The propyl gallate prepared in Examples 1-5 was subjected to the following tests.

[0052] 1) Purity test: (1) Liquid chromatography conditions: a) Chromatographic column: C18 column, 250 mm long, 4.6 mm inner diameter, 5 μm particle size, or equivalent.

[0053] b) Mobile phase A: 0.5% formic acid in water, mobile phase B: methanol.

[0054] c) Elution gradient: 0-5 min: mobile phase (A) 50%; 5 min-15 min: mobile phase (A) decreased from 50% to 20%; 15 min-20 min: mobile phase (A) 20%; 20 min-25 min: mobile phase (A) decreased from 20% to 10%; 25 min-27 min: mobile phase (A) increased from 10% to 50%; 27 min-30 min: mobile phase (A) 50%.

[0055] d) Column temperature: 35°C.

[0056] e) Injection volume: 5 μL.

[0057] f) Detection wavelength: 280nm.

[0058] (2) Preparation of standard curve: Inject the prepared standard working solutions of a series of concentrations into the liquid chromatograph respectively, and draw a standard curve with the concentration of the standard working solution as the horizontal axis and the response value (such as peak area, peak height, absorbance, etc.) as the vertical axis.

[0059] (3) Determination of propyl gallate: The sample solution is injected into a high performance liquid chromatograph to obtain the response value of the corresponding chromatographic peak, and the purity of propyl gallate in the test solution is obtained according to the standard curve.

[0060] The results are shown in Table 1. Single-factor experiments were conducted to evaluate the effects of different conditions on product purity. The experiment examined two factors: feed ratio and temperature. The results showed that at a feed ratio of 1:3 and a temperature of 110°C, purity reached 96.07%.

[0061] 2) DPPH radical scavenging ability of propyl gallate generated at different feed ratios and reaction temperatures: The DPPH radical scavenging assay was performed according to the kit instructions. The steps are as follows: First, weigh an appropriate amount of propyl gallate and dissolve it in the extract solution (provided with the kit). Then, dilute the propyl gallate with the extract solution to different concentrations (25, 50, 100, 200, 400, and 800 μmol / L) using a two-fold dilution method. DPPH was dissolved in 4.05 mL of anhydrous ethanol and stored in the dark until use. 10 μL of sample solution and 190 μL of DPPH / ethanol solution (provided with the kit) were added to a 96-well plate, vortexed to mix, and allowed to stand at room temperature in the dark for 30 minutes. The absorbance was measured at 515 nm, with three replicates per well. The scavenging rate was calculated as follows: DPPH radical scavenging rate (%) = [[A blank - (A test - A control)] ÷ A blank] × 100%.

[0062] The results are as follows Figure 1 (a) Figure 1 As shown in (b): In the concentration range of 25-800 μmol / L, when the feed ratio is 1:3, i.e. "10g gallic acid and 30g n-propanol" and the reaction temperature is 110℃, the gallic acid propyl ester generated by the reaction has a stronger ability to scavenge DPPH free radicals.

[0063] 3) Test of ABTS scavenging ability of propyl gallate generated at different feed ratios and reaction temperatures: The ABTS free radical scavenging experiment was performed according to the kit instructions. The operating steps are as follows: First, weigh an appropriate amount of propyl gallate and dissolve it in the extract (provided by the kit). Then, dilute the propyl gallate with the extract to different concentrations (6.25, 12.5, 25, 50, 100, 200, 400 μmol / L) using the two-fold dilution method. Take 10 μL of sample solution, 20 μL of reagent IV working solution (provided by the kit), and 170 μL of ABTS working solution (provided by the kit), add them to a 96-well plate, mix thoroughly, and stand at room temperature in the dark for 6 minutes. Measure the absorbance at 405 nm, and set up three groups of repeated experiments for each well. The same concentration of Vc was selected as the positive control standard for antioxidant capacity. The calculation formula for the scavenging rate is: ABTS scavenging capacity (mM) = -1.6195 × Asample + 1.3378.

[0064] Figure 1 (c) Figure 1(d) In the concentration range of 6.25-400 μmol / L, the propyl gallate produced when the feed ratio was 1:3, i.e., "10 g gallic acid and 30 g n-propanol" and the reaction temperature was 110°C, had a stronger ability to scavenge ABTS free radicals.

[0065] Experimental Example 7 The propyl gallate prepared in Example 1 was subjected to the following tests.

[0066] 1) Fourier transform infrared spectroscopy analysis: Weigh 1 mg of gallic acid or its derivatives and grind them carefully with potassium bromide powder at a ratio of 1:100 to ensure that the gallic acid or its derivatives are completely dispersed in the potassium bromide. After mixing, press the mixture into a thin sheet and scan the sample on an infrared spectrometer at a wavelength range of 4000-400 cm -1 .

[0067] The results are as follows Figure 2 As shown in the infrared spectra of gallic acid and propyl gallate, 3497-3283 cm -1 The peak at 2970cm is the stretching vibration absorption peak of OH on gallic acid. -1 and 2882cm -1 The peak at 1663 cm is the stretching vibration absorption peak of CH on the saturated carbon, which is due to the alkane chain on propyl gallate. -1 The peak at 1694 cm is the stretching vibration absorption peak of the carboxyl C=O on gallic acid, and the peak at 1694 cm -1 The peak at 1618 cm is the stretching vibration absorption peak of C=O on the ester group of propyl gallate, indicating that propyl gallate was generated by the reaction. -1 and 1537cm -1 The absorption peak at 1427cm is the skeleton vibration absorption peak of the benzene ring in gallic acid. -1 and 1310cm -1 The absorption peak at 1465 cm is the in-plane bending vibration absorption peak of CH. -1 The absorption peak at 1310 cm is the bending vibration absorption peak of -CH2- on propyl gallate, which is caused by the methylene group in the alkane chain on propyl gallate. -1 , 1100 cm -1 and 1038cm -1 The absorption peak at 1100 cm is the in-plane bending vibration absorption peak of CH. -1 The alkyl chain of propyl gallate is responsible for the 1269-1213 cm -1The stretching vibration absorption peak of CO on gallic acid is at 1246-1197 cm -1 This is due to the structural change of gallic acid caused by propyl gallate. 867-640 cm -1 The out-of-plane bending vibration absorption peak of CH is at 989 cm -1 The out-of-plane bending vibration absorption peak of CH appears at , which is caused by the alkane chain on propyl gallate.

[0068] 2) Nuclear magnetic resonance spectroscopy hydrogen spectrum analysis: Weigh 5 mg of gallic acid and its derivatives, add 0.5 mL of deuterated DMSO and ultrasonically dissolve them, then add them to a special NMR test tube. At a constant temperature of 25°C, the gallic acid and its derivatives were analyzed by NMR system. 1 H NMR ( 1 H NMR) analysis, and finally analyzed using MestReNova software.

[0069] The results are as follows Figure 3 As shown, Figure 3 In (a), δ 12.22 (s, 1H), δ 9.18 (s, 2H), δ 8.83 (s, 1H), δ 6.93 (s, 2H); Figure 3 In (b), δ 9.24 (s, 2H), δ 8.92 (s, 1H), δ 6.93 (s, 2H), δ 4.10 (s, 2H), δ 1.65 (s, 2H), δ 0.93 (s, 3H). exist 1 In the H NMR spectrum, the chemical shift δ 10.5-13 ppm is the chemical signal of hydrogen on the carboxyl group. Comparison between gallic acid and its derivatives shows that the signal of the derivative at δ 12.48 ppm disappears, indicating that the hydrogen on the carboxyl group is replaced, showing a weak signal or no signal; δ 6.5-8 ppm is the electrochemical signal of the hydrogen of the phenolic hydroxyl group on the benzene ring; δ 8-9 ppm is the electrochemical signal of the hydrogen bond on the benzene ring, indicating that gallic acid has been successfully synthesized into propyl gallate.

[0070] 3) Thermal stability test analysis: Weigh (10 ± 1) mg of gallic acid and its derivatives and place them in the alumina crucible of the thermogravimetric analyzer. Open the gas line, set the nitrogen atmosphere and gas flow rate to 40 mL / min, set the heating rate to 15°C / min, and set the heating range from room temperature to 500°C.

[0071] The results are as follows Figure 4 Shown by: Figure 4As shown in (a), when the temperature rises from 50°C to 208.5°C, gallic acid experiences virtually no mass loss. The decomposition temperature, at which the mass loss of gallic acid reaches 10%, is 261.3°C. The decomposition rate reaches its maximum at 274.22°C, where the mass loss reaches 25%. The decomposition rate reaches its second peak at 346.2°C, where the mass loss reaches 61.2%. At 503.15°C, decomposition is essentially complete, with the weight loss complete and a residual content of approximately 23.16%.

[0072] Depend on Figure 4 As shown in (b), when the temperature rises from 50°C to 190.4°C, propyl gallate undergoes virtually no mass loss. The decomposition temperature, at which the mass loss of propyl gallate is 10%, is 257.7°C. The decomposition rate reaches its maximum at 303.6°C, and at this temperature, the mass loss of gallic acid reaches 50%. The decomposition rate reaches its second peak at 458.7°C, at which the mass loss of gallic acid reaches 81.2%. At 503.97°C, decomposition is essentially complete, with weight loss complete and a residual content of approximately 8.03%. This is consistent with the thermal stability of gallic acid. Before 292.1°C, the modified propyl gallate exhibits greater thermal stability than gallic acid, and above this temperature, gallic acid exhibits enhanced stability.

[0073] 4) Test on the DPPH free radical scavenging ability of gallic acid and its derivatives: The same method as 2) in Example 6 was used, except that the test object "propyl gallate" was changed to "gallic acid and its derivatives (propyl gallate)".

[0074] The results are as follows Figure 5 The results show that within the concentration range of 25-800 μmol / L, the scavenging effect of gallic acid and its derivatives on DPPH free radicals is directly proportional to their concentration, that is, as the concentration of gallic acid increases, its scavenging efficiency against DPPH free radicals also increases. The results show that gallic acid and its derivatives have scavenging activity against DPPH free radicals, and the derivatives have stronger scavenging ability against DPPH free radicals, that is, the derivatives have stronger antioxidant activity.

[0075] 5) ABTS scavenging ability test of gallic acid and its derivatives: The same method as 3) in Example 6 was used, except that the test object "propyl gallate" was changed to "gallic acid and its derivatives (propyl gallate)".

[0076] The results are as follows Figure 6The results show that within the concentration range of 6.25-400 μmol / L, the scavenging effect of gallic acid and its derivatives on ABTS free radicals is directly proportional to their concentration, i.e., as the concentration of gallic acid increases, its scavenging efficiency against ABTS free radicals also increases. The results indicate that gallic acid and its derivatives have scavenging activity against ABTS free radicals, and the derivatives have stronger scavenging ability against ABTS free radicals, indicating that the derivatives have stronger antioxidant activity.

[0077] 6) Establishment of H2O2 oxidative damage model in SH-SY5Y cells: (1) When the SH-SY5Y cells grow to about 85-95%, they can be plated. After accurate counting, the cells are plated at 5×10 5 The cells were evenly inoculated into a 96-well plate at a density of 100 μL per well, with 5 replicate wells per group. The plates were placed in a cell culture incubator (37°C, saturated humidity, 5% CO2) and cultured for 24 h.

[0078] (2) After the cells adhered to the wall, a blank control group and a H2O2 injury group (25, 50, 100, 200, 400 μmol / L) were set up. The blank group was cultured with DMEM, and the H2O2 injury group was cultured with DMEM containing H2O2 at the corresponding concentration. The cells were placed in an incubator (37°C, saturated humidity, 5% CO2) and cultured for 24 h.

[0079] (3) At the end of the incubation period, the plate was removed from the clean bench and 10 μL of CCK-8 working solution was added to each well. The plate was then placed back into the incubator (37°C, saturated humidity, 5% CO2) and incubated for 1.5 h. After the incubation period, the absorbance of each well was measured using a microplate reader at 450 nm.

[0080] The results are as follows Figure 7 As shown in the figure, cell viability was measured 24 hours after H2O2 treatment. Cell viability decreased with increasing H2O2 concentration, reaching approximately 50% at 200 μmol / L, meeting the modeling requirements. Therefore, a 200 μmol / L H2O2 concentration was used for subsequent modeling.

[0081] 7) Effects of Gallic Acid and Its Derivatives on SH-SY5Y Cell Viability: (1) When the SH-SY5Y cells grow to about 85-95%, they can be plated. After accurate counting, the cells are plated at 5×10 5 The cells were evenly inoculated into a 96-well plate at a density of 100 μL per well, with 5 replicate wells per group. The plates were placed in a cell culture incubator (37°C, saturated humidity, 5% CO2) and cultured for 24 h.

[0082] (2) After the cells adhered to the wall, a blank group, a gallic acid group, and a gallic acid derivative group (2.5, 5, 10, 20, 40, 80, 160, and 320 μmol / L) were set up. After treating the cells according to the different treatment groups, they were placed in the incubator (37°C, saturated humidity, 5% CO2) again and cultured for 24 h.

[0083] (3) At the end of the incubation period, the plate was removed from the clean bench and 10 μL of CCK-8 working solution was added to each well. The plate was then placed back into the incubator (37°C, saturated humidity, 5% CO2) and incubated for 1.5 h. After the incubation period, the absorbance of each well was measured using a microplate reader at 450 nm.

[0084] The results are as follows Figure 8 As shown in the results, compared with the blank control group, the cell viability of cells treated with gallic acid and gallic acid derivatives showed a trend of first increasing and then decreasing with increasing concentration. Within the concentration range of 2.5-20 μmol / L, the cell survival rate showed an increasing trend with increasing concentration of gallic acid and its derivatives. Cell viability was maximum at 20 μmol / L, and cell viability decreased significantly when the concentration continued to increase. This is because low concentrations of gallic acid and its derivatives are beneficial to cell growth and proliferation, but when reaching a certain concentration, they show a certain inhibitory effect or toxicity. Therefore, gallic acid and its derivatives were subsequently selected at concentrations of 10, 20, and 40 μmol / L to act together with H2O2.

[0085] 8) Protective effects of gallic acid and its derivatives on H2O2-induced oxidative stress damage in SH-SY5Y neurons: (1) When the SH-SY5Y cells grow to about 85-95%, they can be plated. After accurate counting, the cells are plated at 5×10 5 The cells were evenly inoculated into a 96-well plate at a density of 100 μL per well, with 5 replicate wells per group. The plates were placed in a cell culture incubator (37°C, saturated humidity, 5% CO2) and cultured for 24 h.

[0086] (2) After the cells adhered to the wall, a blank group, a H2O2 model group, and a gallic acid intervention group / gallic acid derivative intervention group (10, 20, and 40 μmol / L) were set up. The blank group and the H2O2 injury group were replaced with an equal volume of DMEM, and the gallic acid intervention group was replaced with DMEM containing the corresponding concentration of gallic acid. The cells were placed in an incubator (37°C, saturated humidity, 5% CO2) for 1 hour before H2O2 was added to establish the model. The H2O2 injury group and the gallic acid intervention group were treated with 200 μmol / L H2O2. The blank group was added with an equal volume of DMEM and placed in an incubator (37°C, saturated humidity, 5% CO2) for another 24 hours.

[0087] (3) At the end of the incubation period, the plate was removed from the clean bench and 10 μL of CCK-8 working solution was added to each well. The plate was then placed back into the incubator (37°C, saturated humidity, 5% CO2) and incubated for 1.5 h. After the incubation period, the absorbance of each well was measured using a microplate reader at 450 nm.

[0088] The results are as follows Figure 9 As shown in the results, compared with the control group, the cell viability of the H2O2 model group decreased significantly. However, gallic acid and its derivatives, at three concentrations of 10, 20, and 40 μmol / L, were able to improve the survival rate of the oxidatively damaged cell model. Gallic acid and its derivatives showed the strongest protective effect against oxidative stress-damaged cells at a concentration of 40 μmol / L, increasing cell viability from 52.66% to 94.55% and 128.89%, respectively. These results indicate that gallic acid derivatives have a more protective effect against H2O2-induced oxidative damage in cells, indicating that they possess superior antioxidant properties.

[0089] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A method for preparing a gallic acid derivative, characterized in that: include: Gallic acid, alcohol and a catalyst are mixed, and then the temperature is raised to a reaction temperature under condensation reflux conditions to carry out an esterification reaction, and then post-processed to obtain a gallic acid derivative; The alcohol is n-propanol, the catalyst is sulfonic acid resin; the mass ratio of the gallic acid, alcohol and catalyst is 5-15:20-50:1-1.

5.

2. The method for preparing a gallic acid derivative according to claim 1, wherein The mass ratio of the gallic acid, n-propanol and sulfonic acid resin is 7-13:25-35:0.7-1.

3.

3. The method for preparing a gallic acid derivative according to claim 1 or 2, wherein: The reaction temperature is 100-185°C, and the reaction time is 5-12 hours; Preferably, when the alcohol is n-propanol, the reaction temperature is 105-125° C., and the reaction time is 5.5-6.5 h.

4. The method for preparing a gallic acid derivative according to any one of claims 1 to 3, wherein: include: 1) Gallic acid and alcohol are mixed, sulfonic acid resin is added and stirred, and the mixture is heated to the reaction temperature under condensation reflux conditions to carry out esterification reaction; 2) cooling and filtering the reaction product of step 1), collecting the filtrate and performing rotary evaporation to remove alcohol; 3) mixing the filtrate treated in step 2) with water, heating to dissolve and filtering, then cooling until the product precipitates and filtering with sand core suction; 4) Drying the product obtained in step 3) to obtain a gallic acid derivative.

5. The method for preparing a gallic acid derivative according to claim 4, wherein: In step 1), the reaction is carried out in a reaction vessel equipped with a magnetic stirrer and a reflux condenser; and / or, in step 2), the mixture is cooled to 55-65° C. and then filtered.

6. The method for preparing a gallic acid derivative according to claim 4 or 5, characterized in that: In step 3), the ratio of the amount of water added to the amount of gallic acid is 8 mL:1 g.

7. The method for preparing a gallic acid derivative according to any one of claims 4 to 6, characterized in that: In step 3), the solution is heated to 68-72°C to dissolve the solution, filtered while hot, and then cooled to 8-12°C to precipitate the product and filter it through a sand core.

8. The method for preparing a gallic acid derivative according to any one of claims 4 to 7, characterized in that: In step 4), the drying time is 24 to 30 hours.

9. A gallic acid derivative obtained by the method for preparing a gallic acid derivative according to any one of claims 1 to 8.

10. Use of the gallic acid derivatives prepared by the method for preparing the gallic acid derivatives according to any one of claims 1 to 8 in food, medicine and cosmetics.

Images