Solubilizing process and application of dihydromyricetin in ampelopsis grossedentata

Abstract

The invention discloses a solubilizing process and application of dihydromyricetin in ampelopsis grossedentata, and relates to the technical field of solubilizing of dihydromyricetin in ampelopsis grossedentata, and the solubilizing process comprises an extraction process of dihydromyricetin and solubilizing of dihydromyricetin. The extraction process of dihydromyricetin comprises the steps of content determination of dihydromyricetin in ampelopsis grossedentata, methodology investigation, comparison of extraction solvents, cyclodextrin type investigation, single factor experiment and response surface optimization experiment. Solubilization of dihydromyricetin comprises inclusion and analysis of dihydromyricetin by an air jet pulverization technology, a preparation method of the compound, characterization determination of the three compounds and physicochemical property analysis of the compounds. According to the solubilizing process of dihydromyricetin in ampelopsis grossedentata and the application of dihydromyricetin in ampelopsis grossedentata, it is found that an auxiliary material with good water solubility is used for embedding dihydromyricetin to prepare an inclusion compound preparation, the water solubility of a medicine can be improved, meanwhile, the medicine can be protected against the influence of the external environment, the chemical stability of the embedded medicine is enhanced, and then the original bioavailability is also improved.

Description

Technical Field

[0001] The invention relates to the technical field of solubilization of dihydromyricetin in rattan tea, and specifically to a solubilization process and application of dihydromyricetin in rattan tea. Background Art

[0002] Rattan tea is a woody vine plant of the genus Agave in the family Vitaceae. It is widely distributed in the area south of the Yangtze River in China. The surface of rattan tea is often covered with special white crystals. It tastes slightly bitter but then turns sweet. It is widely used and has a long history. Domestic ethnic minorities process and prepare its young leaves as tea, which is also called moldy tea, white tea, sweet tea vine, nectar tea, etc. It has been recorded in the "Compilation of Chinese Herbal Medicines", "Quality Standards for Chinese Medicinal Materials in Hubei Province", "Guangxi List of Medicinal Plants", etc.

[0003] Tengcha is cool in nature, slightly bitter and astringent, with a light sweet aftertaste; it has the effects of clearing away heat and detoxifying, calming the liver and purging fire, reducing swelling and dampness, promoting blood circulation, detoxifying alcohol and clearing turbidity; it is used to treat icteric hepatitis, sore throat and wind-heat cold, and also has anti-inflammatory and analgesic, antioxidant, anti-tumor, lipid-lowering and blood sugar-lowering, liver protection and immunity enhancement. At present, tengcha has been regarded as a kind of medicine and food, and has been widely concerned and recognized by the public.

[0004] Modern pharmacological research has found that rattan tea contains a variety of chemical components, such as flavonoids, polysaccharides, tea polyphenols, volatile oils and trace elements. The content of flavonoids is the most effective ingredient in rattan tea. So far, a total of 35 flavonoids have been isolated and identified from rattan tea, including dihydromyricetin, myricetin, rutin and other substances. Among them, dihydromyricetin has the highest content, accounting for about 80% of the total flavonoid content of rattan tea.

[0005] Dihydromyricetin, also known as ampelopsis, is a polyphenolic dihydroflavonol in the flavonoids. Dihydromyricetin appears as white needle-shaped crystals or powder. It is easily soluble in methanol, ethanol and acetone, slightly soluble in ethyl acetate, and difficult to dissolve in chloroform and petroleum ether. The solubility of dihydromyricetin is poor at low temperature and room temperature, and it is easily soluble in hot water (when the temperature is >100°C, it will lead to irreversible oxidation reaction).

[0006] Because the dihydromyricetin component in rattan tea has poor water solubility and is easily oxidized, its use in the fields of medicine and product development is seriously affected. Summary of the invention

[0007] In view of the deficiencies in the prior art, the present invention provides a solubilization process and application of dihydromyricetin in rattan tea, which solves the problems raised in the above-mentioned background technology.

[0008] To achieve the above objectives, the present invention is implemented by the following technical scheme: a solubilization process of dihydromyricetin in rattan tea, including a dihydromyricetin extraction process and a dihydromyricetin solubilization process;

[0009] The extraction process of dihydromyricetin includes the content determination of dihydromyricetin in rattan tea, methodological investigation, comparison of extraction solvents, investigation of cyclodextrin types, single factor experiment and response surface optimization experiment;

[0010] The solubilization of dihydromyricetin includes the inclusion and analysis of dihydromyricetin by air flow milling technology, the preparation method of the complex, the characterization and determination of three complexes, and the analysis of the physical and chemical properties of the complexes;

[0011] The inclusion and analysis of dihydromyricetin by airflow pulverization technology: based on the best extraction process of dihydromyricetin, dihydromyricetin: cyclodextrin (2:1, 1:1, 1:2, 1:4, 1:6) were weighed, preliminarily mixed with a mortar, and then crushed and included in an airflow pulverizer to obtain inclusion compound powder; the inclusion rate and drug loading rate of powders with different ratios were calculated by liquid phase determination, thereby completing the screening of inclusion ratio;

[0012] The preparation method of the composite is as follows:

[0013] (1) preparing a dihydromyricetin complex by freeze drying technology, weighing a certain amount of sample and cyclodextrin and ethanol solution, heating and stirring under a magnetic stirrer; after the reaction, taking out the solution, cooling it and filtering it; then recovering 30-45% ethanol by rotary evaporation, and freeze drying the solution for 48 hours to obtain a complex powder;

[0014] (2) mixing the prepared dihydromyricetin and cyclodextrin according to a certain proportion, and fully mixing them in a mortar for 5 minutes to prepare a grinding mixture of the two;

[0015] (3) using air flow milling technology to mix dihydromyricetin and cyclodextrin to obtain inclusion powder of the two;

[0016] The physical and chemical properties of the complex are analyzed as follows:

[0017] (1) Solubility test: 0.05 g of DMY and DMY / M-β-CD grinding mixture, air flow and freeze-dried inclusion complex were weighed and added to a test tube containing 5 mL of distilled water; the solution was completely dissolved at 25±1°C to make the solubility of the solution reach saturation; then, the solution in the test tube was filtered with a filter membrane, the filtrate was dissolved with purified water, shaken and fixed to volume, diluted to an appropriate multiple, and sampled and measured according to the dihydromyricetin content determination method, so as to obtain the solubility of DMY and the three DMY / M-β-CD complexes;

[0018] (2) Stability test: accurately weigh three equal amounts of DMY / M-β-CD complex and place them in weighing bottles to ensure uniform thickness distribution; place the sample in a 60°C thermostat for 10 days, take samples on the 5th and 10th days, dissolve them in purified water to prepare 1 mg / mL solutions, filter them through a filter membrane, collect the filtrate and sample it for determination, and examine its high temperature stability based on the changes in concentration and appearance;

[0019] The three complexes were placed in a constant temperature and humidity chamber (RH75%±5%) for 10 days in the same manner as above, and samples were taken on the 5th and 10th days, dissolved in purified water, and prepared into a 1 mg / mL solution, which was then filtered with a filter membrane, and the filtrate was collected and sampled for determination, and its high humidity stability was examined by concentration and appearance;

[0020] (3) In vitro antioxidant assay, including ABTs + Free radical scavenging ability study and DPPH free radical scavenging ability study.

[0021] Optionally, the specific steps of determining the content of dihydromyricetin are as follows:

[0022] (1) Chromatographic conditions and system suitability requirements are set, and the chromatographic column is selected as C 18 Fill, C 18设 250mm×4.6mm, particle size 5μm; mobile phase is acetonitrile-0.05% phosphoric acid, volume ratio 15:85, 0-12min; flow rate is 1mL / min; column temperature is 30℃; detection wavelength is 290nm; theoretical plate number is ≥5000; detection sensitivity: 1AUFS;

[0023] (2) Preparation of reference solution: Use a precision balance to weigh 12.5 mg of dihydromyricetin into a 25 mL volumetric flask, wait until all the methanol is dissolved, and dilute to the mark to obtain a 0.5 mg / mL reference solution;

[0024] (3) Preparation of test solution: 0.5±0.001 g of rattan tea powder was taken, the powder was passed through a 40-mesh sieve, poured into 20 mL of methanol, ultrasonically treated for 20 min, the extract was poured out, the operation was repeated once, the residue was washed with 4 mL of methanol, the extracts were combined and transferred to a 50 mL volumetric flask, the volume was fixed to the mark with methanol, shaken, filtered, 10 mL of the initial filtrate was discarded, and the solution was transferred to a 100 mL volumetric flask, the volume was fixed again after all the methanol was dissolved, and the test solution was obtained after filtration. The determination method was to accurately aspirate 5 μL of the two solutions and determine them using a chromatograph.

[0025] Optionally, the specific steps of the methodological investigation are as follows:

[0026] (1) Linear relationship investigation: 20 mg of dihydromyricetin was accurately weighed and transferred to a 10 mL volumetric flask for dilution with methanol, and the solution was completely dissolved with the aid of ultrasound. The solution was then fixed to volume and shaken to obtain a 2 mg / mL stock solution. The solution was diluted to volume with methanol to the mark using the binary method to prepare solutions of different concentrations of 2 mg / mL, 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL, and 0.125 mg / mL. The solution was injected according to the chromatographic conditions used in the determination of dihydromyricetin content, and the concentration was observed and recorded to examine the linear relationship between the concentration and the peak area.

[0027] (2) Precision experiment: Prepare the sample solution according to the method, and measure the same solution 6 times according to the chromatographic conditions in the determination of dihydromyricetin. Use the peak area value to directly reflect the precision, and calculate the mean and RSD value of the 6 results;

[0028] (3) Stability experiment: Based on the chromatographic conditions used in the determination of dihydromyricetin, the same sample solution was tested at 0 h, 2 h, 4 h, 8 h, 12 h, and 24 h after preparation. The stability was reflected by the peak area value, and the mean and RSD were calculated.

[0029] (4) Repeatability experiment: 6 test solutions were prepared in parallel and measured according to the chromatographic conditions in the above dihydromyricetin content determination to examine its repeatability and RSD value;

[0030] (5) Recovery experiment: 9 portions of 0.25 g rattan tea sample were accurately weighed, and 2 mL, 4 mL, and 6 mL of the reference solution with a content of 0.5 mg / mL were added to 10 mL volumetric flasks, respectively. The solution was diluted to the scale with methanol and shaken to prepare 0.1 mg / mL, 0.2 mg / mL, and 0.3 mg / mL test solutions. The filtrate was filtered through a filter membrane and the filtrate was measured according to the chromatographic and injection conditions in the above-mentioned dihydromyricetin content determination. The average recovery was calculated.

[0031] Optionally, the extraction solvents are compared, and methanol and ethanol are selected as solvents for extraction. At the same time, the effect of hot water extraction and the change in extraction rate after adding cyclodextrin are compared and investigated. The extraction conditions are proposed in the experiment: sample and cyclodextrin are mixed in a 1:1 ratio, ultrasonic extraction is performed for 20 minutes, and the solid-liquid ratio is 1:40. The effects of these three solvents on the extraction rate and the effect of adding cyclodextrin on the extraction rate are compared. Based on the results and comprehensive analysis, the final extraction solvent is determined.

[0032] Optionally, the cyclodextrin types are investigated, and the sample is mixed with cyclodextrin in a 1:1 ratio, the extraction time is 20 min, the solid-liquid ratio is 1:20, the ethanol concentration is 50%, and the temperature is 70°C, to study the effects of different cyclodextrin types on the extraction rate of dihydromyricetin, wherein the cyclodextrin types are α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, HP-β-cyclodextrin, M-β-cyclodextrin, and SBE-β-cyclodextrin; the auxiliary extraction effects of different cyclodextrins are compared to determine the selected cyclodextrin type.

[0033] Optionally, the specific steps of the single factor experiment are as follows:

[0034] (1) The effect of extraction time on the extraction rate of dihydromyricetin complex. When the solid-liquid ratio was 1:50, the extraction temperature was 60°C, and the alcohol concentration was 50%, the effects of the selection of extraction time of 20min, 30min, 40min, 50min, and 60min on the extraction rate of dihydromyricetin were analyzed;

[0035] (2) The effect of alcohol concentration on the extraction rate of dihydromyricetin complex. When the solid-liquid ratio was 1:50, the extraction time was 50 min, and the temperature was 60°C, the effects of different ethanol concentrations of 30%, 40%, 50%, 60%, and 70% on the extraction rate of dihydromyricetin were analyzed respectively;

[0036] (3) The effect of extraction temperature on the extraction rate of dihydromyricetin complex. When the solid-liquid ratio was 1:50, the extraction time was 50 min, and the alcohol concentration was 50%, the effect of the difference in extraction temperature of 30°C, 40°C, 50°C, 60°C, and 70°C on the extraction rate of dihydromyricetin was analyzed;

[0037] (4) The effect of the solid-liquid ratio on the extraction rate of dihydromyricetin complex. When the extraction time was 50 min, the alcohol concentration was 50%, and the temperature was 50°C, the effects of the differences in the solid-liquid ratios of 1:30, 1:40, 1:50, 1:60, and 1:70 on the extraction rate of dihydromyricetin were analyzed.

[0038] Optionally, the response surface optimization experiment is based on the effects of ethanol concentration, solid-liquid ratio, extraction temperature and time on the extraction rate and compared, and the extraction time has little effect on the extraction rate; then combined with the response surface optimization method and principle, three factors of solid-liquid ratio, extraction temperature and ethanol concentration are selected for experimental design; the response surface experiment is designed using Design-Expert.V10.0 software, and the solid-liquid ratio (A), extraction temperature (B), and ethanol concentration (C) are set as independent variables, and the response value is the dihydromyricetin extraction rate (Y), and a quadratic orthogonal design experiment with 3 factors, 3 levels and 17 test points is formulated; using dihydromyricetin as the response surface, the optimal extraction conditions are determined.

[0039] Optionally, the ABTs +The specific steps for free radical scavenging ability study are as follows:

[0040] A. Take 200 μL of diluted ABTs + The free radical solution was added to 50 μL of sample solution with different concentration gradients (7.81 μM, 15.63 μM, 31.25 μM, 62.5 μM, 125 μM), and the obtained solution was shaken rapidly for 30 seconds and placed in the dark for another 6 minutes;

[0041] B. Abs at 734 nm were measured by UV / visible spectroscopy, and vitamin C (Vc) was used as a positive control; the clearance rate of each sample was expressed as SA (%); the 50% effective concentration (IC 50 ) is inferred from the SA (%) and concentration of the sample;

[0042] C. Replace the ABTS aqueous solution with an equal volume of water and add it to 200 μL of sample solutions of different concentrations. After 6 minutes of full reaction, set the detection wavelength to 734 nm and measure the absorbance of the reaction solution.

[0043] D. Replace the sample solutions of different concentrations with 50 μL of purified water, then add 200 μL of ABTS aqueous solution into 50 μL of purified water, quickly shake and mix, react fully at room temperature for 6 minutes, and then measure the absorbance of the reaction solution at a wavelength of 734 nm;

[0044] All samples were tested three times in a row, and the scavenging rates of dihydromyricetin and the three complexes were calculated with reference to the equation of ABTS free radicals. The free radical scavenging rates of the three complexes were compared with the calculated results of dihydromyricetin to demonstrate whether the use of cyclodextrin inclusion can significantly increase the antioxidant capacity of dihydromyricetin itself.

[0045] The specific steps for studying the DPPH free radical scavenging ability are as follows:

[0046] A. Take 100 μL of DDPPH test solution and add it to 100 μL of dihydromyricetin with different concentrations and the three complex solutions. Make sure to shake the mixture thoroughly and allow it to react completely for 30 minutes.

[0047] B. Abs at 517 nm were measured by UV / visible spectroscopy, with Vc as a positive control; the clearance rate of each sample was expressed as SA (%);

[0048] C. Then, an equal volume of ethanol was used to replace the alcohol solution of DPPH, and the solution was added to 100 μL of sample solutions of different concentrations in turn; after 30 minutes of reaction time, the solution was placed at a detection wavelength of 517 nm to measure the absorbance of the reaction solution;

[0049] D. Replace the sample solutions of different concentrations with 100 μL of purified water, then add 100 μL of ethanol solution of DPH to 100 μL of purified water, mix quickly, and react completely at room temperature for 30 min. Measure the absorbance of the reaction solution at a wavelength of 517 nm.

[0050] All samples were tested three times in succession, and the scavenging rates of dihydromyricetin and the three complexes were calculated based on the DPPH free radical equation. The free radical scavenging rates of the three inclusion complexes were compared with the results calculated for dihydromyricetin to demonstrate whether the use of cyclodextrin inclusion complexes significantly improved the antioxidant capacity of dihydromyricetin itself.

[0051] An application of dihydromyricetin in rattan tea according to any one of the above claims, comprising preparing an inclusion compound based on the dihydromyricetin extracted above by freeze-drying, wherein the inclusion compound is specifically dihydromyricetin effervescent granules;

[0052] The preparation method of dihydromyricetin effervescent granules is as follows:

[0053] Step 1: select 50% ethanol with a solid-liquid ratio of 1:70, heat and stir at 60°C, filter and concentrate the extract, and set aside;

[0054] Step 2: Select the wet granulation method, take 1 mL of the concentrated solution of the dihydromyricetin complex and mix it with the acid source: 30% mixed acid mass fraction (15% each of citric acid and tartaric acid), 3% sweetener (sucrose), filler (10% mannitol) evenly divided into two parts, 20% sodium bicarbonate, mix them with the acid and alkali source respectively, and pass through an 80-mesh sieve;

[0055] Step 3: Heat the lubricant in a 70°C water bath until it melts. When it is in a molten state, quickly pour in the acid and alkali source and a wetting agent (2% sodium chloride), and stir until the polyethylene glycol cools down. The lubricant is 5% polyethylene glycol 4000.

[0056] Step 4: sieve the prepared granules, fully dry the wet granules, and then granulate them.

[0057] The present invention provides a solubilization process and application of dihydromyricetin in rattan tea, which has the following beneficial effects:

[0058] The solubilization process of dihydromyricetin in the rattan tea selects the process method of cyclodextrin-assisted extraction to achieve extraction and inclusion solubilization, saving raw materials and time costs, and combines the response surface optimization method to carry out the best process screening, and finally significantly improve the original extraction rate and stability of dihydromyricetin; the airflow crushing technology is used to determine the inclusion rate and drug loading rate of powders with different inclusion ratios, so as to achieve the purpose of crushing and inclusion, and then obtain the dihydromyricetin inclusion compound; through structural characterization and physical and chemical property research, it is judged whether the application of the inclusion process can play a role in solubilizing and improving the bioavailability of dihydromyricetin; on this basis, the preparation of dihydromyricetin effervescent granules is carried out, which provides new ideas and reference value for the development of dihydromyricetin related products, and has certain innovation;

[0059] Cyclodextrin has both strong hydrophilicity on the outer surface and hydrophobic cylindrical cavity structure, so it is often used to improve the water solubility of the included drug. Non-toxicity and high biodegradability are the advantages of cyclodextrin, so it can be widely used in the fields of food, cosmetics and medicine. Cyclodextrin-assisted inclusion extraction is a new green, environmentally friendly and low-cost process method that can achieve the extraction and inclusion of active ingredients in drugs, simplify the process flow, and be efficient and time-saving. When the drug forms an inclusion complex with cyclodextrin, it can maximize the retention of active ingredients, enhance the utilization and stability of the drug, reduce irritation, slow down the release rate, improve the bitterness of the drug, and transform the drug from liquid to powder.

[0060] The solubilization process of dihydromyricetin in the rattan tea uses HPLC to complete the DMY content determination method analysis, and the concentration is in the range of 0.1-2 mg / mL, and there is a linear relationship with the peak area; at the same time, the analysis method has good precision, stability, repeatability and recovery rate, and can be used as an effective method for DMY content determination;

[0061] The solubilization process of dihydromyricetin in the rattan tea, the freeze-drying method can achieve complete inclusion between DMY and M-β-CD, presenting an observable amorphous state; the inclusion powder was analyzed through physical and chemical property experiments, among which the solubility experiment showed that the solubility of the three complexes in water was significantly improved compared with dihydromyricetin; the stability experiment showed that after the dihydromyricetin formed the inclusion state, the stability was significantly improved compared with before; the characterization and determination method used in the experiment can not only identify and test substances, but also conduct in-depth exploration of the specific mechanism of action of the inclusion complex, with the significant advantages of accuracy, intuitiveness and efficiency, and can prove whether dihydromyricetin enters the cavity of cyclodextrin, and the mechanism of action of the two forming an inclusion complex. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 This is a schematic diagram of the process in Embodiment 1 of the present invention. DETAILED DESCRIPTION

[0063] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0064] Embodiment 1: Figure 1 As shown, the solubilization process of dihydromyricetin in rattan tea includes the extraction process of dihydromyricetin and the solubilization of dihydromyricetin;

[0065] The extraction process of dihydromyricetin includes the content determination of dihydromyricetin in rattan tea, methodological investigation, comparison of extraction solvents, investigation of cyclodextrin types, single factor experiment and response surface optimization experiment;

[0066] The specific steps for determining the content of dihydromyricetin are as follows:

[0067] (1) Chromatographic conditions and system suitability requirements are set, and the chromatographic column is selected as C 18 Fill, C 18设 250mm×4.6mm, particle size 5μm; mobile phase is acetonitrile-0.05% phosphoric acid, volume ratio 15:85, 0-12min; flow rate is 1mL / min; column temperature is 30℃; detection wavelength is 290nm; theoretical plate number is ≥5000; detection sensitivity: 1AUFS;

[0068] (2) Preparation of reference solution: Use a precision balance to weigh 12.5 mg of dihydromyricetin into a 25 mL volumetric flask, wait until all the methanol is dissolved, and dilute to the mark to obtain a 0.5 mg / mL reference solution;

[0069] (3) Preparation of test solution: 0.5±0.001 g of rattan tea powder was taken, the powder was passed through a 40-mesh sieve, poured into 20 mL of methanol, ultrasonically treated for 20 min, the extract was poured out, the operation was repeated once, the residue was washed with 4 mL of methanol, the extracts were combined and transferred to a 50 mL volumetric flask, the volume was fixed to the mark with methanol, shaken, filtered, 10 mL of the initial filtrate was discarded, and the solution was transferred to a 100 mL volumetric flask, the volume was fixed again after all the methanol was dissolved, and the test solution was obtained after filtration. The determination method was to accurately aspirate 5 μL of the two solutions and determine them using a chromatograph.

[0070] The specific steps of the methodological investigation are as follows:

[0071] (1) Linear relationship investigation: 20 mg of dihydromyricetin was accurately weighed and transferred to a 10 mL volumetric flask for dilution with methanol, and the solution was completely dissolved with the aid of ultrasound. The solution was then fixed to volume and shaken to obtain a 2 mg / mL stock solution. The solution was diluted to volume with methanol to the mark using the binary method to prepare solutions of different concentrations of 2 mg / mL, 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL, and 0.125 mg / mL. The solution was injected according to the chromatographic conditions used in the determination of dihydromyricetin content, and the concentration was observed and recorded to examine the linear relationship between the concentration and the peak area.

[0072] (2) Precision experiment: Prepare the sample solution according to the method, and measure the same solution 6 times according to the chromatographic conditions in the determination of dihydromyricetin. Use the peak area value to directly reflect the precision, and calculate the mean and RSD value of the 6 results;

[0073] (3) Stability experiment: Based on the chromatographic conditions used in the determination of dihydromyricetin, the same sample solution was tested at 0 h, 2 h, 4 h, 8 h, 12 h, and 24 h after preparation. The stability was reflected by the peak area value, and the mean and RSD were calculated.

[0074] (4) Repeatability experiment: 6 test solutions were prepared in parallel and measured according to the chromatographic conditions in the above dihydromyricetin content determination to examine its repeatability and RSD value;

[0075] (5) Recovery experiment: 9 portions of 0.25 g rattan tea sample were accurately weighed, and 2 mL, 4 mL, and 6 mL of the reference solution with a content of 0.5 mg / mL were added to 10 mL volumetric flasks, respectively. The solution was diluted to the scale with methanol and shaken to prepare 0.1 mg / mL, 0.2 mg / mL, and 0.3 mg / mL test solutions. The filtrate was filtered through a filter membrane and the filtrate was measured according to the chromatographic and injection conditions in the above-mentioned dihydromyricetin content determination. The average recovery was calculated.

[0076] For the comparison of extraction solvents, methanol and ethanol were selected as solvents for extraction. At the same time, the effect of hot water extraction and the change in extraction rate after adding cyclodextrin were compared. The experimental extraction conditions were as follows: sample and cyclodextrin were mixed in a ratio of 1:1, ultrasonic extraction was performed for 20 minutes, and the solid-liquid ratio was 1:40. The effects of these three solvents on the extraction rate were compared, as well as the effect of adding cyclodextrin on the extraction rate. Based on the results and comprehensive analysis, the final extraction solvent was determined.

[0077] The types of cyclodextrin were investigated, and the sample was mixed with cyclodextrin in a ratio of 1:1, the extraction time was 20 min, the solid-liquid ratio was 1:20, the ethanol concentration was 50%, and the temperature was 70 °C. The effects of different types of cyclodextrin on the extraction rate of dihydromyricetin were studied. The types of cyclodextrin were α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, HP-β-cyclodextrin, M-β-cyclodextrin, and SBE-β-cyclodextrin. The auxiliary extraction effects of different cyclodextrins were compared to determine the selected type of cyclodextrin.

[0078] The specific steps of the single factor experiment are as follows:

[0079] (1) The effect of extraction time on the extraction rate of dihydromyricetin complex. When the solid-liquid ratio was 1:50, the extraction temperature was 60°C, and the alcohol concentration was 50%, the effects of the selection of extraction time of 20min, 30min, 40min, 50min, and 60min on the extraction rate of dihydromyricetin were analyzed;

[0080] (2) The effect of alcohol concentration on the extraction rate of dihydromyricetin complex. When the solid-liquid ratio was 1:50, the extraction time was 50 min, and the temperature was 60°C, the effects of different ethanol concentrations of 30%, 40%, 50%, 60%, and 70% on the extraction rate of dihydromyricetin were analyzed respectively;

[0081] (3) The effect of extraction temperature on the extraction rate of dihydromyricetin complex. When the solid-liquid ratio was 1:50, the extraction time was 50 min, and the alcohol concentration was 50%, the effect of the difference in extraction temperature of 30°C, 40°C, 50°C, 60°C, and 70°C on the extraction rate of dihydromyricetin was analyzed;

[0082] (4) The effect of the solid-liquid ratio on the extraction rate of dihydromyricetin complex. When the extraction time was 50 min, the alcohol concentration was 50%, and the temperature was 50°C, the effects of the differences in the solid-liquid ratios of 1:30, 1:40, 1:50, 1:60, and 1:70 on the extraction rate of dihydromyricetin were analyzed.

[0083] In the response surface optimization experiment, the effects of ethanol concentration, solid-liquid ratio, extraction temperature and time on the extraction rate were compared and the extraction time had little effect on the extraction rate. Combining the response surface optimization method and principle, three factors, solid-liquid ratio, extraction temperature and ethanol concentration, were selected for experimental design. The response surface experiment was designed using Design-Expert.V10.0 software, with solid-liquid ratio (A), extraction temperature (B) and ethanol concentration (C) as independent variables, and the response value as dihydromyricetin extraction rate (Y). A quadratic orthogonal design experiment with 3 factors, 3 levels and 17 test points was developed. Taking dihydromyricetin as the response surface, the optimal extraction conditions were determined.

[0084] ABTs + The specific steps for free radical scavenging ability study are as follows:

[0085] A. Take 200 μL of diluted ABTs + The free radical solution was added to 50 μL of sample solution with different concentration gradients (7.81 μM, 15.63 μM, 31.25 μM, 62.5 μM, 125 μM), and the obtained solution was shaken rapidly for 30 seconds and placed in the dark for another 6 minutes;

[0086] B. Abs at 734 nm were measured by UV / visible spectroscopy, and vitamin C (Vc) was used as a positive control; the clearance rate of each sample was expressed as SA (%); the 50% effective concentration (IC 50 ) is inferred from the SA (%) and concentration of the sample;

[0087] C. Replace the ABTS aqueous solution with an equal volume of water and add it to 200 μL of sample solutions of different concentrations. After 6 minutes of full reaction, set the detection wavelength to 734 nm and measure the absorbance of the reaction solution.

[0088] D. Replace the sample solutions of different concentrations with 50 μL of purified water, then add 200 μL of ABTS aqueous solution into 50 μL of purified water, quickly shake and mix, react fully at room temperature for 6 minutes, and then measure the absorbance of the reaction solution at a wavelength of 734 nm;

[0089] All samples were tested three times in a row, and the scavenging rates of dihydromyricetin and the three complexes were calculated with reference to the equation of ABTS free radicals. The free radical scavenging rates of the three complexes were compared with the calculated results of dihydromyricetin to demonstrate whether the use of cyclodextrin inclusion can significantly increase the antioxidant capacity of dihydromyricetin itself.

[0090] The specific steps for studying the DPPH free radical scavenging ability are as follows:

[0091] A. Take 100 μL of DDPPH test solution and add it to 100 μL of dihydromyricetin with different concentrations and the three complex solutions. Make sure to shake the mixture thoroughly and allow it to react completely for 30 minutes.

[0092] B. Abs at 517 nm were measured by UV / visible spectroscopy, with Vc as a positive control; the clearance rate of each sample was expressed as SA (%);

[0093] C. Then, an equal volume of ethanol was used to replace the alcohol solution of DPPH, and the solution was added to 100 μL of sample solutions of different concentrations in turn; after 30 minutes of reaction time, the solution was placed at a detection wavelength of 517 nm to measure the absorbance of the reaction solution;

[0094] D. Replace the sample solutions of different concentrations with 100 μL of purified water, then add 100 μL of ethanol solution of DPH to 100 μL of purified water, mix quickly, and react completely at room temperature for 30 min. Measure the absorbance of the reaction solution at a wavelength of 517 nm.

[0095] All samples were tested three times in a row, and the scavenging rates of dihydromyricetin and the three complexes were calculated according to the DPPH free radical equation. The free radical scavenging rates of the three inclusion complexes were compared with the results calculated for dihydromyricetin to demonstrate whether the use of cyclodextrin inclusion complexes significantly improved the antioxidant capacity of dihydromyricetin itself.

[0096] The solubilization of dihydromyricetin includes the inclusion and analysis of dihydromyricetin by air flow milling technology, the preparation method of the complex, the characterization and determination of three complexes, and the analysis of the physical and chemical properties of the complexes;

[0097] The inclusion and analysis of dihydromyricetin by airflow pulverization technology: based on the best extraction process of dihydromyricetin, dihydromyricetin: cyclodextrin (2:1, 1:1, 1:2, 1:4, 1:6) were weighed, preliminarily mixed with a mortar, and then crushed and included in an airflow pulverizer to obtain inclusion compound powder; the inclusion rate and drug loading rate of powders with different ratios were calculated by liquid phase determination, thereby completing the screening of inclusion ratio;

[0098] The preparation method of the composite is as follows:

[0099] (1) preparing a dihydromyricetin complex by freeze drying technology, weighing a certain amount of sample and cyclodextrin and ethanol solution, heating and stirring under a magnetic stirrer; after the reaction, taking out the solution, cooling it and filtering it; then recovering 30-45% ethanol by rotary evaporation, and freeze drying the solution for 48 hours to obtain a complex powder;

[0100] (2) mixing the prepared dihydromyricetin and cyclodextrin according to a certain proportion, and fully mixing them in a mortar for 5 minutes to prepare a grinding mixture of the two;

[0101] (3) using air flow milling technology to mix dihydromyricetin and cyclodextrin to obtain inclusion powder of the two;

[0102] The physical and chemical properties of the complex are analyzed as follows:

[0103] (1) Solubility test: 0.05 g of DMY and DMY / M-β-CD grinding mixture, air flow and freeze-dried inclusion complex were weighed and added to a test tube containing 5 mL of distilled water; the solution was completely dissolved at 25±1°C to make the solubility of the solution reach saturation; then, the solution in the test tube was filtered with a filter membrane, the filtrate was dissolved with purified water, shaken and fixed to volume, diluted to an appropriate multiple, and sampled and measured according to the dihydromyricetin content determination method, so as to obtain the solubility of DMY and the three DMY / M-β-CD complexes;

[0104] (2) Stability test: accurately weigh three equal amounts of DMY / M-β-CD complex and place them in weighing bottles to ensure uniform thickness distribution; place the sample in a 60°C thermostat for 10 days, take samples on the 5th and 10th days, dissolve them in purified water to prepare 1 mg / mL solutions, filter them through a filter membrane, collect the filtrate and sample it for determination, and examine its high temperature stability based on the changes in concentration and appearance;

[0105] The three complexes were placed in a constant temperature and humidity chamber (RH75%±5%) for 10 days in the same manner as above, and samples were taken on the 5th and 10th days, dissolved in purified water, and prepared into a 1 mg / mL solution, which was then filtered with a filter membrane, and the filtrate was collected and sampled for determination, and its high humidity stability was examined by concentration and appearance;

[0106] (3) In vitro antioxidant assay, including ABTs + Free radical scavenging ability study and DPPH free radical scavenging ability study.

[0107] Example 2: Application of dihydromyricetin in rattan tea, specifically dihydromyricetin effervescent granules;

[0108] The preparation method of dihydromyricetin effervescent granules is as follows:

[0109] Step 1: select 50% ethanol with a solid-liquid ratio of 1:70, heat and stir at 60°C, filter and concentrate the extract, and set aside;

[0110] Step 2: Select the wet granulation method, take 1 mL of the concentrated solution of the dihydromyricetin complex and mix it with the acid source: 30% mixed acid mass fraction (15% each of citric acid and tartaric acid), 3% sweetener (sucrose), filler (10% mannitol) evenly divided into two parts, 20% sodium bicarbonate, mix them with the acid and alkali source respectively, and pass through an 80-mesh sieve;

[0111] Step 3: Heat the lubricant in a 70°C water bath until it melts. When it is in a molten state, quickly pour in the acid and alkali source and a wetting agent (2% sodium chloride), and stir until the polyethylene glycol cools down. The lubricant is 5% polyethylene glycol 4000.

[0112] Step 4: sieve the prepared granules, fully dry the wet granules, and then granulate them.

[0113] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A solubilization process for dihydromyricetin in rattan tea, characterized in that: Including the extraction process of dihydromyricetin and the solubilization of dihydromyricetin; The extraction process of dihydromyricetin includes the content determination of dihydromyricetin in rattan tea, methodological investigation, comparison of extraction solvents, investigation of cyclodextrin types, single factor experiment and response surface optimization experiment; The solubilization of dihydromyricetin includes the inclusion and analysis of dihydromyricetin by air flow milling technology, the preparation method of the complex, the characterization and determination of three complexes, and the analysis of the physical and chemical properties of the complexes; The inclusion and analysis of dihydromyricetin by airflow pulverization technology, based on the above-mentioned optimal extraction process of dihydromyricetin, dihydromyricetin: cyclodextrin (2:1, 1:1, 1:2, 1:4, 1:6) were weighed, preliminarily mixed with a mortar, and then crushed and included in an airflow pulverizer to obtain inclusion compound powder; the inclusion rate and drug loading rate of powders with different ratios were calculated by liquid phase determination, thereby completing the screening of inclusion ratio; The preparation method of the composite is as follows: (1) preparing a dihydromyricetin complex by freeze drying technology, weighing a certain amount of sample and cyclodextrin and ethanol solution, heating and stirring under a magnetic stirrer; after the reaction, taking out the solution, cooling it and filtering it; then recovering 30-45% ethanol by rotary evaporation, and freeze drying the solution for 48 hours to obtain a complex powder; (2) mixing the prepared dihydromyricetin and cyclodextrin according to a certain proportion, and fully mixing them in a mortar for 5 minutes to prepare a grinding mixture of the two; (3) using air flow milling technology to mix dihydromyricetin and cyclodextrin to obtain inclusion powder of the two; The physical and chemical properties of the complex are analyzed as follows: (1) Solubility test: 0.05 g of DMY and DMY / M-β-CD grinding mixture, airflow and freeze-dried inclusion complex were weighed and added into test tubes containing 5 mL of distilled water respectively; the solubility of the solution was saturated by dissolving it completely at 25 ± 1 °C; Subsequently, the solutions in the test tubes were filtered with filter membranes, the filtrate was dissolved with purified water, shaken and fixed to volume, diluted to an appropriate multiple, and sampled and measured according to the dihydromyricetin content determination method, thus obtaining the solubility of DMY and three DMY / M-β-CD complexes; (2) Stability test: accurately weigh three equal amounts of DMY / M-β-CD complex and place them in weighing bottles to ensure uniform thickness distribution; place the sample in a 60°C thermostat for 10 days, take samples on the 5th and 10th days, dissolve them in purified water to prepare 1 mg / mL solutions, filter them through a filter membrane, collect the filtrate and sample it for determination, and examine its high temperature stability based on the changes in concentration and appearance; The three complexes were placed in a constant temperature and humidity chamber (RH75%±5%) for 10 days in the same manner as above, and samples were taken on the 5th and 10th days, dissolved in purified water, and prepared into a 1 mg / mL solution, which was then filtered with a filter membrane, and the filtrate was collected and sampled for determination, and its high humidity stability was examined by concentration and appearance; (3) In vitro antioxidant assay, including ABTs + Free radical scavenging ability study and DPPH free radical scavenging ability study.

2. The solubilization process of dihydromyricetin in rattan tea according to claim 1, characterized in that: The specific steps of determining the content of dihydromyricetin are as follows: (1) Setting of chromatographic conditions and system suitability requirements, where the chromatographic column is selected as C 18 Fill, C 18设 250mm×4.6mm, particle size 5μm; mobile phase is acetonitrile-0.05% phosphoric acid, volume ratio 15:85, 0-12min; flow rate is 1mL / min; column temperature is 30℃; detection wavelength is 290nm; theoretical plate number is ≥5000; detection sensitivity: 1AUFS; (2) Preparation of reference solution: Use a precision balance to weigh 12.5 mg of dihydromyricetin into a 25 mL volumetric flask, wait until all the methanol is dissolved, and dilute to the mark to obtain a 0.5 mg / mL reference solution; (3) Preparation of the test solution: 0.5±0.001 g of rattan tea powder was taken, the powder was passed through a 40-mesh sieve, poured into 20 mL of methanol, and ultrasonically treated for 20 min. The extract was poured out and the operation was repeated once. The residue was washed with 4 mL of methanol, the extracts were combined and transferred to a 50 mL volumetric flask, and the volume was adjusted to the mark with methanol. The solution was shaken and filtered. 10 mL of the initial filtrate was discarded and the solution was transferred to a 100 mL volumetric flask. The methanol was completely dissolved and the volume was adjusted again. After filtering, the test solution was obtained. The determination method is to accurately pipette 5 μL of the two solutions and determine them using a chromatograph.

3. The solubilization process of dihydromyricetin in rattan tea according to claim 1, characterized in that: The specific steps of the methodological investigation are as follows: (1) Linear relationship investigation: 20 mg of dihydromyricetin was accurately weighed and transferred to a 10 mL volumetric flask for dilution with methanol, and the solution was completely dissolved with the aid of ultrasound. The solution was then fixed to volume and shaken to obtain a 2 mg / mL stock solution. The solution was diluted to volume with methanol to the mark using the binary method to prepare solutions of different concentrations of 2 mg / mL, 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL, and 0.125 mg / mL. The solution was injected according to the chromatographic conditions used in the determination of dihydromyricetin content, and the concentration was observed and recorded to examine the linear relationship between the concentration and the peak area. (2) Precision experiment: Prepare the sample solution according to the method, and measure the same solution 6 times according to the chromatographic conditions in the determination of dihydromyricetin. Use the peak area value to directly reflect the precision, and calculate the mean and RSD value of the 6 results; (3) Stability experiment: Based on the chromatographic conditions used in the determination of dihydromyricetin, the same sample solution was tested at 0 h, 2 h, 4 h, 8 h, 12 h, and 24 h after preparation. The stability was reflected by the peak area value, and the mean and RSD were calculated. (4) Repeatability experiment: 6 test solutions were prepared in parallel and measured according to the chromatographic conditions in the above dihydromyricetin content determination to examine its repeatability and RSD value; (5) Recovery experiment: 9 portions of 0.25 g rattan tea sample were accurately weighed, and 2 mL, 4 mL, and 6 mL of the reference solution with a content of 0.5 mg / mL were added to 10 mL volumetric flasks, respectively. The solution was diluted to the scale with methanol and shaken to prepare 0.1 mg / mL, 0.2 mg / mL, and 0.3 mg / mL test solutions. The filtrate was filtered through a filter membrane and the filtrate was measured according to the chromatographic and injection conditions in the above-mentioned dihydromyricetin content determination. The average recovery was calculated.

4. The solubilization process of dihydromyricetin in rattan tea according to claim 1, characterized in that: The extraction solvents were compared, methanol and ethanol were selected as solvents for extraction, and the effect of hot water extraction and the change in extraction rate after adding cyclodextrin were compared. The extraction conditions were as follows: sample and cyclodextrin were mixed in a 1:1 ratio, ultrasonic extraction was performed for 20 minutes, and the solid-liquid ratio was 1:

40. The effects of the three solvents on the extraction rate were compared, as well as the effect of adding cyclodextrin on the extraction rate. The final extraction solvent was determined based on the results and comprehensive analysis.

5. The solubilization process of dihydromyricetin in rattan tea according to claim 1, characterized in that: The cyclodextrin types were investigated, and a 1:1 mixture of sample and cyclodextrin, an extraction time of 20 min, a solid-liquid ratio of 1:20, an ethanol concentration of 50%, and a temperature of 70°C were prepared to study the effects of different cyclodextrin types on the extraction rate of dihydromyricetin. The cyclodextrin types were α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, HP-β-cyclodextrin, M-β-cyclodextrin, and SBE-β-cyclodextrin. The auxiliary extraction effects of different cyclodextrins were compared to determine the selected cyclodextrin type.

6. The solubilization process of dihydromyricetin in rattan tea according to claim 1, characterized in that: The specific steps of the single factor experiment are as follows: (1) The effect of extraction time on the extraction rate of dihydromyricetin complex. When the solid-liquid ratio was 1:50, the extraction temperature was 60°C, and the alcohol concentration was 50%, the effects of the selection of extraction time of 20min, 30min, 40min, 50min, and 60min on the extraction rate of dihydromyricetin were analyzed; (2) The effect of alcohol concentration on the extraction rate of dihydromyricetin complex. When the solid-liquid ratio was 1:50, the extraction time was 50 min, and the temperature was 60°C, the effects of different ethanol concentrations of 30%, 40%, 50%, 60%, and 70% on the extraction rate of dihydromyricetin were analyzed respectively; (3) The effect of extraction temperature on the extraction rate of dihydromyricetin complex. When the solid-liquid ratio was 1:50, the extraction time was 50 min, and the alcohol concentration was 50%, the effect of the difference in extraction temperature of 30°C, 40°C, 50°C, 60°C, and 70°C on the extraction rate of dihydromyricetin was analyzed; (4) The effect of the solid-liquid ratio on the extraction rate of dihydromyricetin complex. When the extraction time was 50 min, the alcohol concentration was 50%, and the temperature was 50°C, the effects of the differences in the solid-liquid ratios of 1:30, 1:40, 1:50, 1:60, and 1:70 on the extraction rate of dihydromyricetin were analyzed.

7. The solubilization process of dihydromyricetin in rattan tea according to claim 1, characterized in that: The response surface optimization experiment is based on the influence of ethanol concentration, solid-liquid ratio, extraction temperature and time factors on the extraction rate and comparison, and the extraction time has a small influence on the extraction rate; then combined with the response surface optimization method and principle, three factors of solid-liquid ratio, extraction temperature and ethanol concentration are selected for experimental design; the response surface experiment is designed using Design-Expert.V10.0 software, the solid-liquid ratio (A), extraction temperature (B) and ethanol concentration (C) are set as independent variables, the response value is the dihydromyricetin extraction rate (Y), and a quadratic orthogonal design experiment with 3 factors, 3 levels and 17 test points is formulated; and dihydromyricetin is used as the response surface to determine the optimal extraction conditions.

8. The solubilization process of dihydromyricetin in rattan tea according to claim 1, characterized in that: The ABTs + The specific steps for free radical scavenging ability study are as follows: A. Take 200 μL of diluted ABTs + The free radical solution was added to 50 μL of sample solution with different concentration gradients (7.81 μM, 15.63 μM, 31.25 μM, 62.5 μM, 125 μM), and the obtained solution was shaken rapidly for 30 seconds and placed in the dark for another 6 minutes; B. Abs at 734 nm were measured by UV / visible spectroscopy, and vitamin C (Vc) was used as a positive control; the clearance rate of each sample was expressed as SA (%); the 50% effective concentration (IC 50 ) is inferred from the SA (%) and concentration of the sample; C. Replace the ABTS aqueous solution with an equal volume of water and add it to 200 μL of sample solutions of different concentrations. After 6 minutes of full reaction, set the detection wavelength to 734 nm and measure the absorbance of the reaction solution. D. Replace the sample solutions of different concentrations with 50 μL of purified water, then add 200 μL of ABTS aqueous solution into 50 μL of purified water, quickly shake and mix, react fully at room temperature for 6 minutes, and then measure the absorbance of the reaction solution at a wavelength of 734 nm; All samples were tested three times in a row, and the scavenging rates of dihydromyricetin and the three complexes were calculated with reference to the equation of ABTS free radicals. The free radical scavenging rates of the three complexes were compared with the calculated results of dihydromyricetin to demonstrate whether the use of cyclodextrin inclusion can significantly increase the antioxidant capacity of dihydromyricetin itself. The specific steps for studying the DPPH free radical scavenging ability are as follows: A. Take 100 μL of DDPPH test solution and add it to 100 μL of dihydromyricetin with different concentrations and the three complex solutions. Make sure to shake the mixture thoroughly and allow it to react completely for 30 minutes. B. Abs at 517 nm were measured by UV / visible spectroscopy, with Vc as a positive control; the clearance rate of each sample was expressed as SA (%); C. Then, an equal volume of ethanol was used to replace the alcohol solution of DPPH, and the solution was added to 100 μL of sample solutions of different concentrations in turn; after 30 minutes of reaction time, the solution was placed at a detection wavelength of 517 nm to measure the absorbance of the reaction solution; D. Replace the sample solutions of different concentrations with 100 μL of purified water, then add 100 μL of ethanol solution of DPH to 100 μL of purified water, mix quickly, and react completely at room temperature for 30 min. Measure the absorbance of the reaction solution at a wavelength of 517 nm. All samples were tested three times in succession, and the scavenging rates of dihydromyricetin and the three complexes were calculated based on the DPPH free radical equation. The free radical scavenging rates of the three inclusion complexes were compared with the results calculated for dihydromyricetin to demonstrate whether the use of cyclodextrin inclusion complexes significantly improved the antioxidant capacity of dihydromyricetin itself.

9. An application of dihydromyricetin in rattan tea according to any one of claims 1 to 8, characterized in that: The invention comprises preparing an inclusion compound based on the dihydromyricetin extracted as above by freeze-drying, wherein the inclusion compound is specifically dihydromyricetin effervescent granules; The preparation method of dihydromyricetin effervescent granules is as follows: Step 1: select 50% ethanol with a solid-liquid ratio of 1:70, heat and stir at 60°C, filter and concentrate the extract, and set aside; Step 2: Select the wet granulation method, take 1 mL of the concentrated solution of the dihydromyricetin complex and mix it with the acid source: 30% mixed acid mass fraction (15% each of citric acid and tartaric acid), 3% sweetener (sucrose), filler (10% mannitol) evenly divided into two parts, 20% sodium bicarbonate, mix them with the acid and alkali source respectively, and pass through an 80-mesh sieve; Step 3: Heat the lubricant in a 70°C water bath until it melts. When it is in a molten state, quickly pour in the acid and alkali source and a wetting agent (2% sodium chloride), and stir until the polyethylene glycol cools down. The lubricant is 5% polyethylene glycol 4000. Step 4: sieve the prepared granules, fully dry the wet granules, and then granulate them.

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