NL2036639B1

NL2036639B1 - Preparation method for selenide dihydromyricetin

Info

Publication number: NL2036639B1
Application number: NL2036639A
Authority: NL
Inventors: Zhang Chi; Liu Xinping; Tan Mingchun; Shang Longchen
Original assignee: Zhang Chi
Priority date: 2023-12-22
Filing date: 2023-12-22
Publication date: 2024-08-13
Also published as: NL2036639A

Abstract

The present invention provides a preparation method for selenide dihydromyricetin (DMY), falling within the technical field of flavonoids development and utilization. DMY and sodium. selenite are included and used as raw materials to optimize the preparation process through single factor experiments and orthogonal experiments, and the structural characterization, stability, antibacterial and antioxidant activities experiments are performed on selenide DMY by atomic fluorescence, ultraviolet, infrared, TG and, XRD. Water‘ is used, as a solvent, effectively avoiding the problems of high cost and environmental pollution in the use of organic solvents, which is environmentally friendly and low—cost, and has great popularization and application value.

Description

P1958 /NLpd

PREPARATION METHOD FOR SELENIDE DIHYDROMYRICETIN

Technical field

The present invention relates to the technical field of fla- vonoids development and utilization, in particularly to a prepara- tion method for selenide dihydromyricetin (DMY).

Background

DMY is mostly extracted from a woody liana belonging to Ampe- lopsis, and is also extracted from Hovenia acerba. The main active component of DMY is a flavonoid compound. Studies have confirmed that DMY has many biological activities such as anti-tumor, anti- bacteria, anti-aging, anti-sensitivity, anti-fatigue, scavenging free radicals, anti-oxidation, anti-thrombosis, anti-tumor, anti- inflammatory and lowering blood glucose. DMY can be made into tab- lets, capsules and other products, and added to animal feed to in- crease meat quality and shorten a growth cycle. DMY is a special flavonoid compound, not only having the general characteristics of the flavonoid compound, but having the functions of relieving al- coholism, preventing alcoholic hepatitis and fatty liver, inhibit- ing the deterioration of liver cells and reducing the incidence of liver cancer, which is a good product for protecting liver and re- lieving hangover.

At present, selenium can't be synthesized in human body, which can only be supplemented by exogenous selenium. Organic selenides in selenium supplements can effectively avoid poisoning caused by inorganic selenium, and there are some problems related to poor water solubility (the solubility being only 263.54 mg/L at 25°C), poor stability, poor antioxidant activity, low intestinal mucosal permeability, short half-life in vivo, low pharmacological activity and easy discoloration. These problems have become the main bottleneck factors limiting the wide application of selenium, and the use of organic solvents is high-cost and pollutes the en- vironment.

Summary

Technical problem solved

Aiming at the shortcomings of the prior art, the present in- vention provides a preparation method for selenide DMY, which solves the problems of poor water sclubility, poor stability, poor antioxidant activity, low intestinal mucosal permeability, short half-life in vivo, low pharmacological activity, easy discolora- tion and high cost and environmental pollution caused by the use of organic solvents.

Technical solution

In order to achieve the above objectives, the present inven- tion is realized by the following technical solutions. A prepara- tion method for selenide DMY includes DMY and sodium selenite. DMY and sodium selenite are used as raw materials to optimize the preparation process through single factor experiments and orthogo- nal experiments, and the structural characterization, stability, antibacterial and antioxidant activities experiments are performed on selenide DMY by atomic fluorescence, ultraviolet, infrared, TG and XRD.

Preferably, the material ratio in the S3 may also be 1:0.25, 1:0.5, 1:1 or 1:2, the reaction time may also be 40, 60, 80 or 100 min, and the reaction temperature may also be 15, 35, 55 or 75°C; and in the reaction step of the S4, HCl is utilized to adjust the pH to 8, 7 or 2.

Preferably, a reaction condition of the S3 is that the reac- tion is carried out in an inert atmosphere (such as a nitrogen at- mosphere) to prevent oxygen from interfering with the reaction.

Preferably, a preparation method for selenide DMY includes the following steps:

Sl: material preparation preparing enough DMY and sodium selenite in proportion;

S2: solution selection selecting a saturated Na,CO; solution to cause reactants to be fully dissolved and reacted;

S3: reaction conditions setting a material ratio of DMY to sodium selenite as 1:3,

with a reaction time of 120 min and a reaction temperature of 95°C; and

S4: reaction steps a: dissolving a portion of DMY in the saturated Na;CO; solu- tion to obtain a DMY solution, b: gradually adding three portions of sodium selenite into the DMY solution and stirring a reaction mixture, c: reacting the mixture for 120 min at a reaction temperature of 95°C, and heating and refluxing the same, and d: adjusting a pH with HCl after the reacting, centrifuging the mixture, sequentially eluting the same with boiling distilled water and iced distilled water, and performing vacuum drying on the same to obtain powdery solid.

Advantageous effects

The present invention provides a preparation method for sele- nide DMY. The present invention has the following advantageous ef- fects. 1. According to the present invention, the preparation of selenide DMY by using water as the solvent has the advantages of environmental protection and low cost, and the stability and bio- logical activity of selenide DMY obtained by structural modifica- tion of DMY with selenium are improved. The preparation technology and related parameters of selenide DMY have a simple technological process, safe operations and low cost in production, which is suitable for batch processing, and can be widely used in experi- ments or industrial production. Water, selected as the solvent, effectively avoiding the problems of high cost and environmental pollution caused by the use of organic solvents, is environmental- ly friendly and low-cost, and has great popularization and appli- cation value. 2. According to the present invention, the active substance

DMY extracted and purified from natural product Vine tea is used as a substrate, and Na,CO; is used as a selenizing agent, and the structure of DMY is modified by selenization to prepare the organ- ic selenide DMY. The thermal stability of the obtained selenide

DMY in a range of 250°C-370°C is better than that of DMY, and the stability in an aqueous solution is good, and the antioxidant ac-

tivity is better than that of DMY, which is positively related to a sample amount. Therefore, the organic selenide DMY has good thermal stability, aqueous solution stability, antibacterial ac- tivity and strong antioxidant activity.

Brief description of the drawings

FIG. 1 is an ultraviolet spectrogram of the influence of a heating temperature on the stability of selenide DMY according to the present invention;

FIG. 2 is an ultraviolet spectrogram of the influence of a pH on the stability of selenide DMY according to the present inven- tion;

FIG. 3 is an ultraviolet spectrogram of the influence of a heating time on the stability of selenide DMY according to the present invention; and

FIG. 4 is a graph showing scavenging rates of DMY and sele- nide DMY to «OH radicals according to the present invention.

Detailed description

Technical solutions in the examples of the present invention will be described clearly and completely in the following with reference to the attached drawings in the examples of the present invention. Obviously, all the described examples are only some, rather than all examples of the present invention. Based on the examples in the present invention, all other examples obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

Example 1

As shown in FIGS. 1-4, an example of the present invention provides a preparation method for selenide DMY, including DMY and sodium selenite. DMY and sodium selenite are used as raw materials to optimize the preparation process through single factor experi- ments and orthogonal experiments, and the structural characteriza- tion, stability, antibacterial and antioxidant activities experi- ments are performed on selenide DMY by atomic fluorescence, ultra- violet, infrared, TG and XRD.

The material ratio in the 33 may also be 1:0.25, 1:0.5, 1:1 or 1:2, the reaction time may also be 40, 60, 80 or 100 min, and the reaction temperature may also be 15, 35, 55 or 75°C.

In the reaction step of the S4, HCl is utilized to adjust the

PH to 8, 7 or 2.

A reaction condition of the S3 is that the reaction is car- ried out in an inert atmosphere (such as a nitrogen atmosphere) to 5 prevent oxygen from interfering with the reaction.

Example 2

As shown in FIGS. 1-4, an example of the present invention provides a preparation method for selenide DMY, including the fol- lowing steps.

At S1: material preparation enough DMY and sodium selenite were prepared in proportion.

At S2: solution selection a saturated Na:CO: solution was selected to cause reactants to be fully dissolved and reacted.

At S3: reaction conditions a material ratio of DMY was set to sodium selenite as 1:3, with a reaction time of 120 min and a reaction temperature of 95°C.

At S4: reaction steps a: a portion of DMY was dissolved in the saturated Na,CO; so- lution to obtain a DMY solution, b: three portions of sodium selenite were gradually added in- to the DMY solution and a reaction mixture was stirred, c: the mixture was reacted for 120 min at a reaction tempera- ture of 95°C, heated and refluxed, and d: a pH was adjusted with HCl after the reacting, centri- fuged, sequentially eluted with boiling distilled water and iced distilled water, and vacuum dried to obtain powdery solid.

Example 3

As shown in FIGS. 1-4, an example of the present invention provides a preparation method, experiments and optimization for selenide DMY, including the following steps. (1) Single factor experiments: DMY and sodium selenite were weighed in proportion, an appropriate amount of distilled water was taken as a solvent, a pH of a reaction system was adjusted with a saturated Na:CO: solution, the reaction system was heated and refluxed, the pH was adjusted with HCl after the reaction, and the mixture was centrifuged, sequentially eluted with boiling dis-

tilled water and iced distilled water, and vacuum dried to obtain yellow-brown powdery solid. The single factor experiments were performed with material ratios of 1:0.25, 1:0.5, 1:1, 1:2 and 1:3 for reaction times of 40, 60, 80, 100 and 120 min at reaction tem- peratures of 15, 35, 55, 75 and 95°C, and pHs of the reaction sys- tem were 8, 7 and 2. When the material ratios were 1:0.25, 1:0.5, 1:1, 1:2 and 1:3, selenium source utilization ratios of selenide

DMY were 0.08%, 0.21%, 0.23%, 1.12% and 1.50%; when the reaction times were 40, 60, 80, 100 and 120 min, selenium source utiliza- tion rates of selenide DMY were 2.73%, 0.89%, 0.54%, 0.17% and 1.58%; and when the reaction temperatures were 15, 35, 55, 75 and 95°C, selenium source utilization rates of selenide DMY were 0.003%, 0.006%, 0.51%, 27.44% and 29.38%; when the pHs of the re- action system were 8, 7 and 2, selenium source utilization rates of selenide DMY were 0.54%, 1.43% and 0.07%; and finally, the se- lenium source utilization rate of selenide DMY was 29.38% when the ratio was 1:3, the reaction time was 120 min and the reaction tem- perature was 95°C. {2) Orthogonal experiments: according to the results of the single factor experiments, the material ratios were 1:0.25, 1:0.5, 1:1 and 1:2, the reaction times were 60, 80, 100 and 120 min, the reaction temperatures were 35, 55, 75 and 95°C, and four-factor and four-level orthogonal experiments were designed for the reaction system at pHs of 2, 7, 8 and 9. The utilization rates of selenium source were taken as performance indexes, the optimized process parameters for preparing selenide DMY were determined by SPSS23.0 analysis. The test results were shown in Table 1. (3) An optimized process for preparing selenide DMY: DMY and sodium selenite were weighted according to a material ratio of 1:0.25, an appropriate amount of distilled water was taken as a solvent, a pH of a reaction system was adjusted to 8, the reaction system was heated and refluxed at 95°C for 120 min, the pH was ad- justed with HCl after the reaction, and the mixture was centri- fuged, sequentially eluted with boiling distilled water and iced distilled water, and vacuum dried to obtain yellow-brown powdery solid. Under the optimized process conditions, a selenium source utilization rate of the prepared selenide DMY was 80.73%. A ultra- violet spectrum showed that there were structural characteristic peaks of flavonoids (208.5 nm) and flavonols (358.5 nm) in sele- nide DMY. An infrared spectrum showed that a benzene ring struc- ture still existed in selenide DMY, and C=0, -OH and Se* of DMY were selenized to form new rings, and a new characteristic absorp- tion peak of Se-0O appeared. TG showed that the thermal stability of selenide DMY was higher than that of DMY in a range of 250°C- 370°C, and XRD showed that crystal diffraction peaks of selenide

DMY were 8.96°, 11.92°, 14.58°, 23.46°, 26.88°, 29.62°, 36.14°, 43.54° and 45.26°. Selenide DMY is formed by chemical reactions, not simple physical mixing. (4) Stability experiments: 15 mL of sample solution was taken in a small test tube, heating temperatures, pHs and heating times were changed sequentially, and an ultraviolet spectrum was meas- ured in a range of 250-500 nm, where the heating temperatures were 10, 35, 55, 75, 95 and 100°C, pH values were 2, 4, 6, 7, 8, 10 and 12, and the heating timed were 10, 20, 30, 40, 50 and 60 min. The results of stability experiments were shown in FIGS. 1-3. From

FIG. 1, it could be seen that when the temperature was 10°C-100°C, positions of characteristic absorption peaks of selenide DMY did not change obviously, indicating that selenide DMY existed stably without obvious decomposition. From FIG. 2, it could be seen that characteristic absorption peaks of a selenide DMY aqueous solution with a PH value of 6 were consistent with that with a pH adjusted to be acidic, but different from that with pHs adjusted to be neu- tral, weakly alkaline and alkaline, indicating that selenide DMY was stable under acidic conditions and unstable under neutral and slightly alkaline conditions. From FIG. 3, it could be seen that positions of the characteristic absorption peaks of selenide DMY were basically consistent when the heating time was 10 min-60 min, indicating that it existed stably without decomposition. The sta- bility experiments showed that the selenide DMY aqueous solution had good stability. (5) Antioxidant activity experiments: 2 mL of sample solu- tions with different concentration gradients, 2 mL of 6 mmol/L

FesS0, solution, 2 mL of 6 mmol/L salicylic acid-ethanol solution and 2 ml of 6 mmol/L LH:0: solution were added to an appropriate amount of small test tubes, then an absorbance value was measured at 510 nm after holding in water bath at 37°C for 30 min, and an «OH radical scavenging rate (%) was calculated. The experimental results were shown in FIG. 4. In experimental ranges, when the sample concentration was 300 pg/mL, the scavenging rates of ¢CH radicals of DMY and selenide DMY were the highest, which were 31.40% and 56.40% respectively. The scavenging rate of selenide

DMY on sOH radicals was better than that of DMY, and the antioxi- dant experiments showed that selenide DMY had good antioxidant ac- tivity.

Table 1 Orthogonal experiment results

Serial A material B reaction Dreac temperature number ratio time (min) (°C) tion pH 1 2 3 1 1{1:0.25) 1{60) 1{35) 1{2} 12.63% 12.33% 12.55% 2 1 2{80) 2(55) 2(7) 0.54% 0.49% 0.51% 3 1 3{100} 3{75) 3{8) 50.59% 52.38% 50.89% 4 1 4{120) 4(95) 4(9) 71.88% 73.64% 72.76% 5 2{1:0.5) 1 2 3 0.19% 0.17% 0.15% 6 2 2 1 4 1.42% 2.08% 2.29% 7 2 3 4 1 3.23% 3.20% 3.22% 8 2 4 3 2 54.12% 54.42% 52.20% 9 3(1:1) 1 3 4 20.32% 20.44% 20.48% 10 3 2 4 3 21.02% 20.73% 20.87% 11 3 3 1 2 0.07% 0.07% 0.08% 12 3 4 2 1 1.13% 1.10% 0.72% 13 4(1:2) 1 4 2 7.31% 7.45% 7.39% 14 4 2 3 1 0.31% 0.29% 0.24% 4 3 2 4 1.75% 1.72% 1.73% 16 4 4 1 3 0.28% 0.29% 0.30% "While examples of the present invention have been shown and _ 15 described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations may be made herein without departing from the principles and spirit of the present invention, the scope of which is defined by the ap- pended claims and equivalents thereof.

Claims

CONCLUSIONS

1. A method for preparing selenide dihydromyricetin (DMY) comprising DMY and sodium selenite, wherein DMY and sodium selenite are used as raw materials to optimize the preparation process by single-factor experiments and orthogonal experiments, and wherein the structural characterization, stability, antibacterial and antioxidant activity experiments are carried out on selenide DMY by atomic fluorescence, ultraviolet, infrared, TG and XRD.

2. A method for preparing selenide DMY according to claim 1, comprising the steps of: S1: preparing material preparing sufficient DMY and sodium selenite in proportion; S2: solution selection choosing a saturated Na:CO; solution to completely dissolve and react the reactants; S3: reaction conditions setting a material ratio of DMY to sodium selenite of 1:3, with a reaction time of 120 min and a reaction temperature of 95°C; and S4: reaction steps a: dissolving one part of DMY in the saturated Na:CO: solution to obtain a DMY solution, b: gradually adding three parts of sodium selenite to the DMY solution and stirring a reaction mixture, c: reacting the mixture for 120 minutes at a reaction temperature of 95 °C, and heating and refluxing it, and d: adjusting the pH with HCl after the reaction, centrifuging the mixture, eluting with boiling distilled water and ice-cold distilled water successively, and vacuum-drying the mixture to obtain a powdery solid.

3. A method for preparing selenide DMY according to claim 1, wherein the material ratio in step S3 is also 1:0.25, 1:0.5,

can be 1:1 or 1:2, the reaction time can also be 40, 60, 80 or 100 minutes, and the reaction temperature can also be 15, 35, 55 or 75 °C; and in the reaction step of S4, HCl is used to adjust the pH to 8, 7 or 2.

4. The process for preparing selenide DMY according to claim 1, wherein a reaction condition of step S3 is that the reaction is carried out in an inert atmosphere, for example, a nitrogen atmosphere, to prevent oxygen from interfering with the reaction.