Dihydromyricetin-based nitrogen-containing derivative, preparation method therefor, and use thereof

Abstract

A dihydromyricetin-based nitrogen-containing derivative, a preparation method therefor, and a use thereof. The dihydromyricetin-based nitrogen-containing derivative has a structure as shown in formula (I) below. The preparation method therefor comprises the following steps: adding dihydromyricetin to concentrated ammonia water at a solid-to-liquid ratio of 3:1-5:1 (g:L) and carrying out vortex treatment for 1-2 minutes at room temperature; adding 20-30% sulfuric acid while stirring until the pH of the reaction solution is adjusted to 7-8; after pre-freezing at -80ºC for 12 h, carrying out vacuum freeze-drying to obtain sample powder and storing the sample powder at -20ºC in a refrigerator; adding ethyl acetate to the sample powder for extraction, filtering an extraction solution by means of a 0.22 µm organic filter membrane to remove an ammonium sulfate salt, and then carrying out rotary evaporation in a water bath at 25ºC to remove ethyl acetate to obtain a concentrate; and carrying out vacuum freeze-drying on the concentrate to obtain a dihydromyricetin-based nitrogen-containing derivative powder having a purity exceeding 90%.

Description

A dihydromyricetin nitrogen-containing derivative and its preparation method and application Technical Field

[0001] The present invention relates to the technical field of organic synthesis, and in particular to a dihydromyricetin nitrogen-containing derivative and a preparation method and application thereof. Background Art

[0002] Dihydromyricetin (DMY) is a natural flavanol extracted from a woody vine (Vitis vinifera) of the genus Ampelopsis in the family Vitaceae. It has antioxidant, anti-inflammatory, organ protection (especially liver), neuroprotection, damage repair, anticancer, antibacterial, detoxification, cell death mediation, and lipid and glucose metabolism regulation activities.

[0003] At present, the changes of dihydromyricetin in cell culture medium are mostly found to be dimers, oxidized to quinone products and ring cleavage products.

[0004] There are few reports on nitrogen-containing derivatives of dihydromyricetin, and there are currently no studies on their high-purity preparation methods and activity. Technical Solutions

[0005] The purpose of the present invention is to propose a dihydromyricetin nitrogen-containing derivative and its preparation method and application, to solve the technical problem that most of the current processes require complex processes such as preparing liquid phases to separate and prepare the changed products of natural compounds, and to achieve the technical effect of large-scale and rapid preparation of dihydromyricetin nitrogen-containing derivatives.

[0006] In order to achieve the above technical objectives, the technical solution adopted by the present invention is:

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

[0008]

[0009] Formula (I)

[0010] The present invention also provides a method for preparing the above-mentioned dihydromyricetin nitrogen-containing derivative, comprising the following steps:

[0011] (1) Weigh the dihydromyricetin and add concentrated ammonia water at a solid-liquid ratio of 3:1−5:1 (g:L), and quickly vortex at room temperature for 1 min−2 min;

[0012] (2) Add sulfuric acid to the vortexed reaction solution while stirring until the pH of the reaction solution is 7-8;

[0013] (3) After the reaction solution is cooled, seal it and place it in a -80℃ freezer for 12 h, then perform vacuum freeze drying to obtain a preliminary powder containing a nitrogen-containing derivative of dihydromyricetin. After drying, seal the sample powder and store it in a -20℃ refrigerator for later use.

[0014] (4) Using excess ethyl acetate to extract the nitrogen-containing derivatives of dihydromyricetin in the preliminary powder by stirring on ice, then filtering the supernatant of the ethyl acetate extract using an organic filter membrane, concentrating the ethyl acetate extract using a rotary evaporator in a water bath at 25°C, and recovering the ethyl acetate;

[0015] Repeat the above extraction, filtration and rotary evaporation concentration process until the extract is almost colorless, combine the concentrates of all the extracts, and then rotary evaporate the extract in a water bath at 25°C to obtain a concentrate;

[0016] (5) The concentrate is freeze-dried again to obtain a powder of a nitrogen-containing derivative of dihydromyricetin with a purity exceeding 90%.

[0017] As a preferred embodiment, preferably, the purity of dihydromyricetin in step (1) is greater than 99%.

[0018] As a preferred embodiment, preferably, the mass concentration of sulfuric acid in step (2) is 20-30%.

[0019] As a preferred embodiment, preferably, in step (3), vacuum freeze drying is performed at a vacuum degree of 15-30 Pa, and the processing temperature is as follows:

[0020] -10℃(2 h)→-5℃(2 h)→0℃(2 h)→5℃(2 h)→10℃(2 h)→20℃(2 h).

[0021] As a preferred embodiment, preferably, the pore size of the organic filter membrane in step (4) is 0.22 µm.

[0022] In another aspect of the present invention, there is provided the use of nitrogen-containing derivatives of dihydromyricetin in inhibiting the activities of α-glucosidase and α-amylase.

[0023] In another aspect of the present invention, the use of nitrogen-containing dihydromyricetin derivatives in the preparation of hypoglycemic products is provided, including foods, medicines, and health products.

[0024] The use of the above-mentioned dihydromyricetin nitrogen-containing derivatives for reducing blood sugar in vitro can be applied in the fields of food, medicine and health products. Beneficial effects

[0025] The present invention is based on the principle that dihydromyricetin reacts with molecular oxygen to form DMYquinone, where ammonia undergoes nucleophilic addition to the carbonyl group of DMYquinone, followed by dehydration to form an imine intermediate, which is then converted into a nitrogen-containing dihydromyricetin derivative (as shown in Figure 1). A nitrogen-containing dihydromyricetin derivative is a compound formed by the rapid reaction of dihydromyricetin with concentrated ammonia, in which the 4′ hydroxyl group (OH) in the pyrogallol structure on the B ring of dihydromyricetin is replaced with an amino group (-NH2). This structural modification of dihydromyricetin yields a nitrogen-containing dihydromyricetin derivative with enhanced hypoglycemic activity, which can be used in the development of foods, pharmaceuticals, and health products with hypoglycemic activity.

[0026] By adopting the above technical solution, the present invention has the following beneficial effects compared with the prior art:

[0027] The dihydromyricetin nitrogen-containing derivative of the present invention is a compound containing 4'NH2 on the B ring generated by the rapid reaction of dihydromyricetin and concentrated ammonia water. The dihydromyricetin nitrogen-containing derivative can be quickly prepared in large quantities, which is conducive to studying the conversion process of dihydromyricetin in cell culture medium. Moreover, since the dihydromyricetin nitrogen-containing derivative has a better hypoglycemic effect than dihydromyricetin, it can be applied to the fields of food, medicine and health care products. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only 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.

[0029] FIG1 is a schematic diagram showing the reaction of dihydromyricetin and concentrated ammonia to form a nitrogen-containing derivative of dihydromyricetin;

[0030] FIG2 is a chromatogram of the nitrogen-containing derivatives of dihydromyricetin of the present invention;

[0031] FIG3 is a comparison diagram of the secondary mass spectrometry fragments of the nitrogen-containing derivatives of dihydromyricetin and dihydromyricetin of the present invention;

[0032] FIG4 is a hydrogen and carbon nuclear magnetic resonance spectrum of the nitrogen-containing derivative of dihydromyricetin of the present invention;

[0033] FIG5 is a comparative graph showing the inhibitory activities of the nitrogen-containing derivatives of dihydromyricetin and dihydromyricetin against α-glucosidase of the present invention;

[0034] FIG6 is a graph comparing the inhibitory activities of the nitrogen-containing derivatives of dihydromyricetin of the present invention and dihydromyricetin against α-amylase. Modes for Carrying Out the Invention

[0035] The present invention will be described in further detail below with reference to the accompanying drawings and examples. It is particularly noted that the following examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. Similarly, the following examples are only some embodiments of the present invention and are not intended to be exhaustive. All other embodiments obtained by those of ordinary skill in the art without creative effort are intended to fall within the scope of protection of the present invention.

[0036] In the following examples, the experimental methods are as follows:

[0037] 1. Ultra-high performance liquid chromatography-mass spectrometry method for the identification of nitrogen-containing derivatives of dihydromyricetin and detection of dihydromyricetin: A Waters ACQUITY UPLC H-Class system equipped with a quaternary solvent manager, sample manager, and PDA eλ detector was used. Analyses were performed on a Waters ACQUITY UPLC BEH C18 column (2.1 × 50 mm, 1.7 µm). The column temperature was set at 35°C, and the mobile phases consisted of 0.1% formic acid in water (phase A) and acetonitrile (phase B). The flow rate was 0.3 mL / min, and the injection volume was 1 µL. The linear gradient elution was: 15% B, 0-2 min; 15-45% B, 2-10 min; 45-15% B, 10-12 min; and 15% B, 12-15 min. The PDA detection wavelength range was 220-600 nm. MS / MS analysis was performed using a Waters XEVO G2-XS QTOF equipped with an electrospray ionization source (Waters, MA, USA). Mass spectral data were acquired in MS mode over a range of 10–1500 Da using sensitivity and negative electrospray ionization (ESI-) mode. The scan time was set to 0.5 s. Tuning parameters were 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 50 L / h, and desolvation gas flow 700 L / h.

[0038] 2. Identification of nitrogen-containing derivatives of dihydromyricetin by nuclear magnetic resonance: DMSO-d6 was used as solvent, tetramethylsilane (TMS) was used as internal standard, and the 400 MHz NMR spectrum was recorded on a Bruker AVANCE 400 M spectrometer (Bruker, Germany). 1 H and 100 MHz 13 C NMR spectroscopy.

[0039] 3. α-glucosidase inhibitory activity assay

[0040] The experiment was divided into blank, control, sample blank, and sample groups, with three replicates per group. Samples were loaded into a 96-well plate. 50 µL of inhibitor solution and 50 µL of α-glucosidase were added sequentially, mixed evenly, and incubated at 37°C for 10 minutes. After incubation, the plates were removed and 50 µL of substrate solution was added. Mix thoroughly and incubate at 37°C for 20 minutes. The reaction was terminated by adding 50 µL of anhydrous ethanol. The absorbance was measured at 405 nm, and the α-glucosidase inhibition rate of each sample was calculated according to the formula.

[0041] 4. α-amylase inhibitory activity assay

[0042] The experiment was divided into blank, control, sample blank, and sample groups, with three replicates per group. Samples were loaded into a 96-well plate. 50 µL of inhibitor solution and 50 µL of α-amylase were added, mixed thoroughly, and incubated at 37°C for 20 minutes. After the reaction, the plates were removed and 20 µL of substrate solution was added. Mix thoroughly and incubate at 37°C for 30 minutes. After the reaction, 100 µL of DNS colorimetric reagent was added, and the plates were incubated in a boiling water bath for 6 minutes. The absorbance was measured at 540 nm, and the α-amylase inhibition rate of each sample was calculated according to the formula.

[0043] Example 1

[0044] Weigh 200 mg of dihydromyricetin (greater than 99% purity) and add 50 mL of concentrated ammonia. Vortex at room temperature for 1 minute 30 seconds, then quickly pour into a large glass dish (200 mm diameter). Terminate the reaction by adding 25% sulfuric acid to the reaction solution in the glass dish while stirring until the pH reaches approximately 7-8. Cool the reaction solution in a fume hood, seal with plastic wrap, and prefreeze at -80°C overnight. Then, freeze-dry the solution in a large vacuum freeze dryer to obtain a preliminary powder. Specifically, vacuum freeze-drying was performed at a vacuum of 15-30 Pa. The drying temperature was as follows: -10°C (2 h) → -5°C (2 h) → 0°C (2 h) → 5°C (2 h) → 10°C (2 h) → 20°C (2 h). The target dihydromyricetin nitrogen-containing derivative in the preliminary powder was extracted using excess ethyl acetate with repeated stirring on ice until the extract was nearly colorless. All the extract concentrates were combined, and the ethyl acetate extract was filtered using a 0.22 µm organic filter membrane. The concentrate was rotary evaporated to obtain a concentrate. The concentrate was dissolved with a very small amount of methanol, and then a large amount of water was added. After pre-freezing at -80°C overnight, the dihydromyricetin nitrogen-containing derivative powder was freeze-dried.

[0045] The obtained powder of the nitrogen-containing derivative of dihydromyricetin 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 spectrum comparison with dihydromyricetin.

[0046] According to the detection spectrum of the prepared dihydromyricetin nitrogen-containing derivative detected by ultra-performance liquid chromatography-mass spectrometry (as shown in FIG2 ), the purity of the finally prepared dihydromyricetin nitrogen-containing derivative exceeded 95%.

[0047] According to the mass-to-charge ratio (m / z) of the nitrogen-containing derivative of dihydromyricetin in the negative ion mode of ultra-high performance liquid chromatography-mass spectrometry, which is 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. 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 (as shown in Figure 3), the chemical structure of the nitrogen-containing derivative of dihydromyricetin was deduced to be formula (1).

[0048]

[0049] Formula (1)

[0050] Table 1 Nitrogen-containing derivatives of dihydromyricetin and secondary ion fragments of dihydromyricetin

[0051] Compound retention time (min) Measured [MH]-(m / z) Theoretical [MH]-(m / z) Error (ppm) Fragment ion (m / z) Molecular formula Dihydromyricetin 1.53 19.0463 19.0453 193.0137, 215.0348, 233.0447, 257.0463, 301.0358 C15H12O8 Nitrogen-containing derivatives of dihydromyricetin 0.83 18.0623 18.0613 192.0295, 214.0504, 232.0611, 256.0612, 300.0511 C15H13O7N

[0052] In addition, according to the nitrogen-containing derivatives of dihydromyricetin in Table 2 1 H and 13 C spectrum data, the structure of the nitrogen-containing derivative of dihydromyricetin can be accurately identified as formula (1).

[0053] Table 2 NMR spectra of nitrogen-containing derivatives of dihydromyricetin 1 H and 13 C spectrum characteristics

[0054] Position C spectrum H spectrum 284.024.88 (d, J=10.7 Hz, 1H) 372.134.38 (d, J=10.7 Hz, 1H) 4198.04 5163.78 696.385.90 (d, J=1.8 Hz, 1H) 7167.53 895.445.85 (d, J=1.8 Hz, 1H) 9163.01 10100.83 1'124.85 2'6'106.966.37 (s, 2H) 3'5'144.64 4'124.77

[0055] Example 2

[0056] 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 Dulbecco's Modified Eagle Medium (DMEM) cell culture medium to dilute it to a 0.5 mM DMY in DMEM dilution. The solution was vortexed and incubated at 37°C for 15 hours. Every hour, 50 µL of the DMY in DMEM dilution was removed and added to 450 µL of ice-cold methanol. The solution was vortexed and centrifuged at 12,000 rpm for 10 minutes before analysis by ultra-performance liquid chromatography-mass spectrometry. The ion signal intensity changes of dihydromyricetin and its nitrogen-containing derivatives over a 15-hour period (Figure 4) indicate that dihydromyricetin is rapidly degraded in the cell culture medium, with some of the degradation occurring in the cell culture medium, with some of the degradation occurring in the cell culture medium in the form of nitrogen-containing dihydromyricetin derivatives. The nitrogen-containing derivatives of dihydromyricetin continued to increase within 0-4 h, and then continued to decrease. In addition, the nitrogen-containing derivatives of dihydromyricetin in the cell culture medium were identified by mass spectrometry. Their retention time and mass-to-charge ratio were the same as those in Table 1, proving that the nitrogen-containing derivatives of dihydromyricetin prepared by the present invention are the nitrogen-containing derivatives of dihydromyricetin in the cell culture medium.

[0057] Example 3

[0058] A series of dihydromyricetin nitrogen-containing derivatives and dihydromyricetin (1, 5, 10, 15, and 20 mM) were used to determine their hypoglycemic activity, including α-glucosidase and α-amylase inhibition assays. The activities of α-glucosidase and α-amylase are crucial for regulating blood glucose levels, and many natural products, as inhibitors of these two enzymes, can effectively lower blood glucose. Therefore, studying the inhibitory effect of dihydromyricetin nitrogen-containing derivatives on α-glucosidase is of great significance for the natural development and utilization of dihydromyricetin nitrogen-containing derivatives for their hypoglycemic efficacy.

[0059] As shown in Figure 5, the α-glucosidase inhibitory activity of the nitrogen-containing derivative of dihydromyricetin (N-DMY) continues to increase between 1 and 10 mM, and N-DMY has significantly better α-glucosidase inhibitory activity than dihydromyricetin (DMY) between 1 and 15 mM. As shown in Figure 6, the inhibitory ability of the nitrogen-containing derivative of dihydromyricetin (N-DMY) against α-amylase continues to increase between 1 and 20 mM, and is significantly better than that of dihydromyricetin (DMY). These results demonstrate that N-DMY has better in vitro hypoglycemic activity than DMY.

[0060] Example 4

[0061] A healthy tea drink with blood sugar lowering effect is made by compounding the nitrogen-containing derivative of dihydromyricetin with various other seasonings, milk or healthy ingredients.

[0062] The above descriptions are only some embodiments of the present invention and do not limit the scope of protection of the present invention. Any equivalent device or equivalent process transformation made by using the contents of the description and drawings of the present invention, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A dihydromyricetin nitrogen-containing derivative, characterized in that, The structural formula of the dihydromyricetin nitrogen-containing derivative is as shown in the following formula (I): Formula (I) 2. A method for preparing the nitrogen-containing derivative of dihydromyricetin as described in claim 1, characterized in that, comprises the following steps: (1) Weigh dihydromyricetin, add concentrated ammonia water according to a solid-liquid ratio of 3:1 - 5:1 (g:L), and quickly vortex at room temperature for 1 min - 2 min; (2) Add sulfuric acid to the vortexed reaction solution while stirring until the pH of the reaction solution is 7 - 8; (3) After the reaction solution cools, seal it and pre-freeze it at -80 °C for 12 h, then carry out vacuum freeze-drying treatment to obtain a preliminary powder containing the dihydromyricetin nitrogen-containing derivative; (4) Use an excessive amount of ethyl acetate to stir and extract the dihydromyricetin nitrogen-containing derivative in the preliminary powder on ice, then filter the supernatant of the ethyl acetate extract using an organic filter membrane, concentrate the ethyl acetate extract in a water bath at 25 °C using a rotary evaporator, and recover ethyl acetate; Repeat the above extraction, filtration, and rotary evaporation concentration processes until the extract is nearly colorless, combine the concentrated solutions of all extracts, and then rotary evaporate the extract in a water bath at 25 °C to obtain a concentrate; (5) Freeze-dry the concentrate again to obtain a dihydromyricetin nitrogen-containing derivative powder with a purity exceeding 90%.

3. The preparation method of the nitrogen-containing derivative of dihydromyricetin according to claim 2, wherein, In step (1), the purity of dihydromyricetin is greater than 99%.

4. The preparation method of the dihydromyricetin nitrogen-containing derivative according to claim 2, wherein, In step (2), the mass concentration of the sulfuric acid is 20 - 30%.

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

6. The preparation method of the dihydromyricetin nitrogen-containing derivative according to claim 2, characterized in that, In step (4), the pore size of the organic filter membrane is 0.22 µm.

7. Use of the dihydromyricetin nitrogen-containing derivative according to claim 1 in inhibiting the activities of α-glucosidase and α-amylase.

8. Use of the dihydromyricetin nitrogen-containing derivative according to claim 1 in the preparation of hypoglycemic products.

9. The application according to claim 8, characterized in that, The products include foods, beverages, drugs, and health products.

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