Dihydromyricetin nitrogen-containing derivative, and preparation method and application thereof

Abstract

This invention proposes a nitrogen-containing dihydromyricetin derivative, its preparation method, and its application. The nitrogen-containing dihydromyricetin derivative has the structure shown in formula (1). The preparation method includes the following steps: adding concentrated ammonia water to dihydromyricetin at a solid-liquid ratio of 3:1-5:1 (g:L) and vortexing at room temperature for 1-2 minutes; adding 20-30% sulfuric acid while stirring until the pH of the reaction solution is adjusted to 7-8; pre-freezing at -80℃ for 12 hours and then freeze-drying under vacuum to obtain sample powder, which is stored in a -20℃ refrigerator; extracting the sample powder with ethyl acetate, filtering the extract through a 0.22μm organic filter membrane to remove ammonium sulfate, and then removing ethyl acetate by rotary evaporation in a 25℃ water bath to obtain a concentrate; and freeze-drying the concentrate under vacuum to obtain a nitrogen-containing dihydromyricetin derivative powder with a purity of over 90%.

Description

Technical Field

[0001] This invention relates to the field of organic synthesis technology, and in particular to a dihydromyricetin nitrogen-containing derivative, its preparation method, and its application. Background Technology

[0002] Dihydromyricetin (DMY) is a natural flavanol, mostly extracted from a woody vine (vine tea) of the genus *Vitis* in the Vitaceae family. It has antioxidant, anti-inflammatory, organ-protective (especially liver), neuroprotective, damage-repairing, anti-cancer, antibacterial, detoxifying, cell death-mediating, and lipid and glucose metabolism-regulating activities.

[0003] Currently, the products of dihydromyricetin in cell culture media are mostly found to be dimers, quinone products, and cycloclast products.

[0004] There are few reports on nitrogen-containing derivatives of dihydromyricetin, and there are currently no research reports on their high-purity preparation methods and activities. Summary of the Invention

[0005] The purpose of this invention is to propose a nitrogen-containing dihydromyricetin derivative, its preparation method, and its application, thereby solving the technical problem that most current methods require complex processes, such as liquid phase preparation, to separate and prepare natural compound metabolites, and achieving the technical effect of large-scale and rapid preparation of nitrogen-containing dihydromyricetin derivatives.

[0006] To achieve the above-mentioned technical objectives, the technical solution adopted by this invention is as follows:

[0007] A nitrogen-containing derivative of dihydromyricetin, characterized in that the structural formula of the nitrogen-containing derivative of dihydromyricetin is shown in the following formula (I):

[0008]

[0009] The present invention also provides a method for preparing the above-mentioned dihydromyricetin nitrogen-containing derivative, comprising the following steps:

[0010] (1) Weigh out the dihydromyricetin and add it to concentrated ammonia water at a solid-liquid ratio of 3:1-5:1 (g:L), and vortex it quickly at room temperature for 1-2 minutes.

[0011] (2) Add sulfuric acid to the reaction solution after vortexing, stirring while adding until the pH of the reaction solution is 7-8;

[0012] (3) After the reaction solution is cooled, the container is sealed and pre-frozen at -80℃ for 12 hours. Then, vacuum freeze-drying is performed to obtain a preliminary powder containing dihydromyricetin nitrogen derivative. After drying, the sample powder is sealed and stored in a refrigerator at -20℃ for later use.

[0013] (4) Use excess ethyl acetate to stir on ice to extract the nitrogen-containing derivative of dihydromyricetin from the preliminary powder. Then filter the supernatant of the ethyl acetate extract using an organic filter membrane. Concentrate the ethyl acetate extract using a rotary evaporator in a water bath at 25°C and recover the ethyl acetate.

[0014] Repeat the above extraction, filtration and rotary evaporation concentration process until the extract is nearly colorless. Combine all the concentrated extracts and then rotary evaporate the extracts in a 25°C water bath to obtain the concentrate.

[0015] (5) The concentrate was freeze-dried again to obtain a nitrogen-containing derivative powder of dihydromyricetin with a purity of over 90%.

[0016] As a preferred embodiment, preferably, the purity of dihydromyricetin in step (1) is greater than 99%.

[0017] As a preferred embodiment, the mass concentration of sulfuric acid in step (2) is preferably 20-30%.

[0018] As a preferred embodiment, step (3) is preferably performed under vacuum of 15-30 Pa, with the following processing temperatures:

[0019] -10℃(2h)→-5℃(2h)→0℃(2h)→5℃(2h)→10℃(2h)→20℃(2h).

[0020] As a preferred embodiment, the pore size of the organic filter membrane in step (4) is preferably 0.22 μm.

[0021] In another aspect of the invention, the use of dihydromyricetin nitrogen derivatives in inhibiting α-glucosidase and α-amylase activity is provided.

[0022] In another aspect of the invention, the use of dihydromyricetin nitrogen-containing derivatives in the preparation of hypoglycemic products is provided. These products include food, pharmaceuticals, and health supplements.

[0023] The above-mentioned nitrogenous derivatives of dihydromyricetin can be used in vitro to lower blood sugar in the fields of food, pharmaceuticals and health products.

[0024] The principle upon which this invention is based is as follows: dihydromyricetin reacts with molecular oxygen to generate DMYquinone, where an ammonia molecule undergoes nucleophilic addition to the carbonyl group of DMYquinone, followed by dehydration to generate an imine intermediate, which is finally converted into a nitrogen-containing derivative of dihydromyricetin (as shown in the appendix). Figure 1(As shown). Dihydromyricetin nitrogen-containing derivatives are compounds formed by the rapid reaction of dihydromyricetin and concentrated ammonia, in which the 4′ hydroxyl group (OH) in the pyrogallol structure on the B ring of dihydromyricetin is replaced by an amino group (-NH2). This modification of the original structure of dihydromyricetin yields dihydromyricetin nitrogen-containing derivatives with higher hypoglycemic activity, which can be used to develop foods, pharmaceuticals, and health products with hypoglycemic activity.

[0025] By adopting the above technical solution, the present invention has the following beneficial effects compared with the prior art:

[0026] This invention relates to a nitrogen-containing derivative of dihydromyricetin, which rapidly reacts with concentrated ammonia to generate a compound containing 4′NH2 on the B ring. This enables the rapid and large-scale preparation of the nitrogen-containing derivative of dihydromyricetin, which is beneficial for studying the transformation process of dihydromyricetin in cell culture media. Furthermore, since the nitrogen-containing derivative of dihydromyricetin has a better hypoglycemic effect than dihydromyricetin, it can be applied in the fields of food, pharmaceuticals, and health products. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 A schematic diagram illustrating the principle of the reaction between dihydromyricetin and concentrated ammonia to produce a nitrogen-containing derivative of dihydromyricetin.

[0029] Figure 2 The chromatogram of the nitrogen-containing derivative of dihydromyricetin of this invention is shown below.

[0030] Figure 3 This is a comparison image of secondary mass spectrometry fragments of the nitrogen-containing derivative of dihydromyricetin and dihydromyricetin of the present invention.

[0031] Figure 4 The proton and carbon NMR spectra of the nitrogen-containing derivative of dihydromyricetin of this invention are shown below.

[0032] Figure 5 This is a comparison diagram of the inhibitory activities of the nitrogen-containing derivative of dihydromyricetin and dihydromyricetin relative to α-glucosidase in this invention.

[0033] Figure 6 This is a comparison diagram of the inhibitory activities of the nitrogen-containing derivative of dihydromyricetin and dihydromyricetin relative to α-amylase. Detailed Implementation

[0034] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be particularly noted that the following embodiments are for illustrative purposes only and do not limit the scope of the invention. Similarly, the following embodiments are only some, not all, embodiments of the present invention, and all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0035] In the following embodiments, the experimental methods are as follows:

[0036] 1. Identification of Nitrogen-Containing Derivatives of Dihydromyricetin and Detection Method of Dihydromyricetin using Ultra-High Performance Liquid Chromatography-Mass Spectrometry: A Waters ACQUITY UPLC H-Class system equipped with a quaternary solvent manager, sample manager, and PDAeλ detector was used. A C18 column (2.1×50mm, 1.7μm) was used on a Waters ACQUITY UPLC BEH, with the column temperature set at 35℃. The mobile phase was 0.1% formic acid in water (phase A) and acetonitrile (phase B), the flow rate was 0.3mL / min, and the injection volume was 1μL. Linear gradient elution was used: 15% B, 0-2min; 15-45% B, 2-10min; 45-15% B, 10-12min; 15% B, 12-15min. The PDA detection wavelength was in the range of 220-600nm. MS / MS analysis was performed using a Waters XEVO G2-XS QTOF equipped with an electrospray ionization source (Waters, MA, USA). Mass spectrometry data were acquired in MSE mode within the range of 10–1500 Da in both sensitivity and ESI-mode. The scan time was set to 0.5 s. Tuning parameters were set as follows: capillary voltage 2 kV, sampling cone voltage 40 V, source bias 80 V, source temperature 120 °C, desolvation temperature 450 °C, cone gas flow rate 50 L / h, and desolvation gas flow rate 700 L / h.

[0037] 2. Detection method for nitrogen-containing derivatives of dihydromyricetin by nuclear magnetic resonance: DMSO-d6 is used as the solvent, tetramethylsilane (TMS) is used as the internal standard, and the concentration is recorded at 400 MHz on a Bruker AVANCE 400M spectrometer (Bruker, Germany). 1 H and 100MHz 13 C NMR spectrum.

[0038] 3. Assay for α-glucosidase inhibitory activity

[0039] The experiment consisted of a blank group, a control group, a sample blank group, and a sample group, with three replicates in each group. Samples were added to 96-well plates, with 50 μL of inhibitor solution and 50 μL of α-glucosidase added sequentially. After thorough mixing, the plates were incubated at 37°C for 10 min. Afterward, 50 μL of substrate solution was added, and the mixture was thoroughly mixed. The plates were then incubated at 37°C for 20 min. Finally, 50 μL of anhydrous ethanol was added to terminate the reaction. The absorbance was measured at 405 nm, and the inhibition rate of α-glucosidase in each sample was calculated using the formula.

[0040] 4. Assay for α-amylase inhibitory activity

[0041] The experiment consisted of a blank group, a control group, a sample blank group, and a sample group, with three replicates in each group. Samples were added to 96-well plates, with 50 μL of inhibitor solution and 50 μL of α-amylase added sequentially. After thorough mixing, the plates were incubated at 37°C for 20 min. Afterward, 20 μL of substrate solution was added, and the mixture was thoroughly mixed and incubated at 37°C for 30 min. Finally, 100 μL of DNS chromogenic reagent was added, and the plates were incubated in a boiling water bath for 6 min. The absorbance was measured at 540 nm, and the inhibition rate of α-amylase in each sample was calculated using the formula.

[0042] Example 1

[0043] Weigh 200 mg of dihydromyricetin with a purity greater than 99%, add 50 mL of concentrated ammonia, vortex at room temperature for 1 min 30 s, and then quickly pour into a large glass dish (200 mm in diameter). Add 25% sulfuric acid to the reaction solution in the glass dish to terminate the reaction, stirring constantly until the final pH of the reaction solution in the dish reaches approximately 7-8. Cool the reaction solution in a fume hood, seal with plastic wrap, and pre-freeze at -80℃ overnight. Then, freeze-dry using a large vacuum freeze dryer to obtain a preliminary powder. Specifically, vacuum freeze-drying is performed at a vacuum degree of 15-30 Pa, with the following processing temperatures: -10℃ (2 h) → -5℃ (2 h) → 0℃ (2 h) → 5℃ (2 h) → 10℃ (2 h) → 20℃ (2 h). The target dihydromyricetin nitrogen derivative in the preliminary powder was extracted by repeated stirring on ice with excess ethyl acetate until the extract was nearly colorless. All extract concentrates were combined and filtered through a 0.22 μm organic filter membrane. The extract was then rotary evaporated to obtain a concentrate. The concentrate was dissolved in a very small amount of methanol, and then a large amount of water was added. The mixture was then pre-frozen at -80°C overnight and freeze-dried to obtain the dihydromyricetin nitrogen derivative powder.

[0044] The obtained dihydromyricetin nitrogen-containing derivative powder was dissolved in mass spectrometry-grade methanol, and the structure of the substance was identified by ultra-high performance liquid chromatography-mass spectrometry and nuclear magnetic resonance, combined with the spectrum comparison with that of dihydromyricetin.

[0045] The detection chromatograms of the prepared dihydromyricetin nitrogen-containing derivatives were analyzed using ultra-high performance liquid chromatography-mass spectrometry (as shown in the attached figure). Figure 2 As shown in the figure, the final dihydromyricetin nitrogen-containing derivative obtained has a purity of over 95%.

[0046] Based on the mass-to-charge ratio (m / z) of the nitrogen-containing derivative of dihydromyricetin obtained by ultra-high performance liquid chromatography-mass spectrometry in negative ion mode being 318.0621, its chemical formula is deduced to be C. 15 H 13 O7N, in addition, the secondary ion fragments of the nitrogen-containing derivatives of dihydromyricetin are m / z 192.0295, m / z 214.0504, m / z 256.0612, m / z 232.0611 and m / z 300.0511. Compare this to the chemical formula of dihydromyricetin (C). 15 H 12 O8 and secondary ion fragments m / z 193.0137, m / z 215.0348, m / z 233.0447, m / z 257.0463 and m / z 301.0358 (see attached) Figure 3 As shown in the figure, the chemical structure of the nitrogen-containing derivative of dihydromyricetin is derived as formula (1).

[0047]

[0048] Table 1. Nitrogen-containing derivatives of dihydromyricetin and secondary ion fragments of dihydromyricetin by mass spectrometry

[0049]

[0050] In addition, according to the dihydromyricetin nitrogen-containing derivatives in Table 2 1 H and 13 C-spectral data can accurately identify the structure of the nitrogen-containing derivative of dihydromyricetin as formula (1).

[0051] Table 2. Nuclear magnetic resonance (NMR) of nitrogen-containing derivatives of dihydromyricetin 1 H and 13 C-spectral characteristics

[0052]

[0053] Example 2

[0054] Dihydromyricetin with a purity greater than 99% was dissolved in dimethyl sulfoxide to prepare a 5 mM dihydromyricetin stock solution. 100 μL of the 5 mM dihydromyricetin stock solution was added to 900 μL of Duchenne Modified Eagle Medium (DMEM) to dilute it to 0.5 mM DMEM dilution of DMY. After vortexing and mixing thoroughly, the solution was incubated at 37°C for 15 h. Every 1 h, 50 μL of the incubated DMEM dilution of DMY was added to 450 μL of ice-cold methanol, vortexed, centrifuged at 12000 rpm for 10 min, and then analyzed using ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS / MS). The changes in ion signal intensity of dihydromyricetin and its nitrogenous derivative over 15 h are shown in the attached graph. Figure 4 As shown in the figure, dihydromyricetin rapidly degrades in cell culture medium, with some of it being converted into nitrogen-containing dihydromyricetin derivatives. The nitrogen-containing dihydromyricetin derivatives continuously increase within 0-4 hours, then decrease. Furthermore, mass spectrometry analysis of the nitrogen-containing dihydromyricetin derivatives in the cell culture medium showed that their retention time and mass-to-charge ratio were the same as those in Table 1, proving that the nitrogen-containing dihydromyricetin derivatives prepared in this invention are indeed the nitrogen-containing dihydromyricetin derivatives found in the cell culture medium.

[0055] Example 3

[0056] A series of nitrogenous derivatives of dihydromyricetin and dihydromyricetin (1, 5, 10, 15, and 20 mM) at the same concentrations were used to determine their hypoglycemic activity, including inhibition experiments on α-glucosidase and α-amylase. The activities of α-glucosidase and α-amylase play a crucial role in regulating blood glucose levels, and many natural products, acting as inhibitors of these two enzymes, can effectively lower blood glucose. Therefore, studying the inhibitory effect of nitrogenous derivatives of dihydromyricetin on α-glucosidase is of great significance for the natural development and utilization of the hypoglycemic efficacy of nitrogenous derivatives of dihydromyricetin.

[0057] From the appendix Figure 5 It can be seen that the α-glucosidase inhibitory activity of the nitrogenous derivative of dihydromyricetin (N-DMY) continuously increases between 1-10 mM, and between 1-15 mM, N-DMY exhibits significantly superior α-glucosidase inhibitory activity compared to dihydromyricetin (DMY). Figure 6 It can be seen that within the range of 1-20 mM, the inhibitory effect of the nitrogenous derivative of dihydromyricetin (N-DMY) on α-amylase continuously increases and is significantly better than that of dihydromyricetin (DMY). These results reflect that N-DMY has better in vitro hypoglycemic activity than DMY.

[0058] Example 4

[0059] A healthy tea drink with blood sugar-lowering effects is made by combining dihydromyricetin nitrogen derivatives with various other flavorings, milk, or health ingredients.

[0060] The above description is only a part of the embodiments of the present invention and does not limit the scope of protection of the present invention. Any equivalent device or equivalent process transformation made based on the content of the present invention specification and drawings, or direct or indirect application in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A method for preparing a nitrogen-containing derivative of dihydromyricetin, characterized by, The structural formula of the dihydromyricetin nitrogen-containing derivative is shown in the following formula (I): Formula (I) The preparation method of the dihydromyricetin nitrogen-containing derivative comprises the following steps: (1) Dihydromyricetin was weighed, and the solid-liquid ratio was 3:1 5:1 (g:L) concentrated ammonia water was added, and quickly vortexed at room temperature for 1 min 2 min; (2) After vortexing, sulfuric acid is added to the reaction solution while stirring until the pH of the reaction solution is 7-8; (3) After the reaction solution is cooled, it is sealed and pre-frozen at-80℃ for 12 h, and then vacuum freeze-drying treatment is performed to obtain a preliminary powder containing the dihydromyricetin nitrogen-containing derivative; (4) An excess of ethyl acetate is used to extract the dihydromyricetin nitrogen-containing derivative in the preliminary powder on ice, and then the supernatant of the ethyl acetate extract is filtered using an organic filter membrane, the ethyl acetate extract is concentrated using a rotary evaporator under a 25℃ water bath, and ethyl acetate is recovered; The above extraction, filtration and rotary evaporation concentration processes are repeated until the extract is nearly colorless, the concentrated solutions of all the extracts are combined, and the extract is rotary evaporated under a 25℃ water bath to obtain a concentrate; (5) The concentrate is freeze-dried again to obtain a dihydromyricetin nitrogen-containing derivative powder with a purity of more than 90%.

2. The method for preparing dihydromyricetin nitrogen-containing derivatives according to claim 1, characterized in that, The purity of the dihydromyricetin in step (1) is greater than 99%.

3. The method of preparing a nitrogen-containing derivative of dihydromyricetin according to claim 1, characterized in that, The mass concentration of the sulfuric acid in step (2) is 20-30%.

4. The method of preparing a nitrogen-containing derivative of dihydromyricetin according to claim 1, characterized in that, In step (3), the vacuum freeze-drying treatment is performed under a vacuum degree of 15-30 Pa, and the treatment temperature is as follows: -10℃, 2 h →-5℃, 2 h →0℃, 2 h →5℃, 2 h →10℃, 2 h →20℃, 2 h.

5. The method of preparing a nitrogen-containing derivative of dihydromyricetin according to claim 1, characterized in that, The pore size of the organic filter membrane in step (4) is 0.22 µm.

6. The dihydromyricetin nitrogen-containing derivative prepared by the preparation method of any one of claims 1-5 is used for inhibiting the activities of α-glucosidase and α-amylase for non-therapeutic purposes.

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