A ternary liquid supramolecular self-assembly system of NHDC / cyclodextrin / pyruvic acid and its preparation method

Abstract

The present invention discloses a ternary liquid supramolecular self-assembly system of NHDC / cyclodextrin / pyruvic acid and a preparation method thereof, belonging to the technical field of supramolecular. By selecting molecules compatible with neohesperidin dihydrochalcone, the types and addition amounts of the compatible molecules, the present invention provides a ternary liquid supramolecular self-assembly system of neohesperidin dihydrochalcone / cyclodextrin / pyruvic acid and a preparation method thereof. The neohesperidin dihydrochalcone / cyclodextrin / pyruvic acid system obtained by supramolecular self-assembly is in a stable liquid state, can be miscible with water in a specific ratio without precipitation, and effectively improves the water solubility, stability, antioxidant property and antibacterial property of neohesperidin dihydrochalcone.

Description

Technical Field

[0001] The invention relates to a ternary liquid supramolecular self-assembly system of NHDC / cyclodextrin / pyruvic acid and a preparation method thereof, belonging to the technical field of supramolecular technology. Background Art

[0002] Neomethyl hesperidin dihydrochalcone (NHDC) is a flavonoid derivative extracted from citrus plants and hydrogenated. It has the effects of inhibiting gastric acid secretion, increasing plasma high-density lipoprotein and having strong antioxidant effects, and can be applied to functional foods, cosmetics and medicines. However, due to its poor water solubility, it affects its application in water-containing products, and in medicine, it has fast metabolism and short retention time in the body. At present, the means to improve its solubility include physical methods, chemical methods and enzymatic methods, among which physical methods are favored because of their mildness and wide applicability. However, these methods are usually more complicated and may use organic solvents. Although they improve solubility, they have poor stability and cannot be used directly as raw materials. At the same time, most of the prior art focuses on the improvement of solubility, ignoring the importance of its antioxidant, antibacterial and sustained-release effects.

[0003] In drug preparation, it is very important to achieve long-term efficacy through sustained-release technology. Sustained-release technology can ensure the continuous release of drugs in the body, maintain drug concentrations at effective therapeutic levels, thereby reducing the frequency of administration, improving patient compliance, and minimizing the side effects caused by peak drug concentrations. At present, the existing technology is not stable enough for the sustained-release control of neomethyl hesperidin dihydrochalcone, and the release rate is greatly affected by the internal environment, resulting in fluctuations in drug concentration; secondly, the bioavailability is low, especially the absorption effect of the oral route is poor; thirdly, the preparation is easily affected by environmental factors during production, storage and transportation, resulting in drug degradation or failure of the sustained-release mechanism.

[0004] Patent CN 109730294 A discloses a method of encapsulating neomethyl hesperidin dihydrochalcone by coupling and grafting with glucan and soy protein isolate to improve its water solubility. However, the process requires the use of a special reaction device, and the process is relatively complicated, and the final solubility is 4g / L. Patent CN 109730295 A introduces another neomethyl hesperidin dihydrochalcone encapsulation technology. Although the water solubility is improved, the preparation process requires the use of special granulation equipment, resulting in an increase in processing costs, and the solubility is also 4g / L. Patent CN 109836513 A proposes a method of encapsulating neomethyl hesperidin dihydrochalcone with octenyl succinic acid α-cyclodextrin ester to improve its water solubility. However, the preparation process not only requires a large amount of organic solvents, but also increases the overall cost due to the high production cost of octenyl succinic acid α-cyclodextrin ester, and its solubility is only 2.52g / L. Patent CN 110156852 A uses enzyme-catalyzed synthesis of derivatives to achieve glycosylated new methyl hesperidin dihydrochalcone. This method significantly improves the solubility to 562.7 g / L, but still involves a relatively complex process flow.

[0005] Therefore, there is still a need for a simpler and greener processing process to more effectively solve the problems of poor water solubility and instability of neomethyl hesperidin dihydrochalcone. On this basis, the antioxidant and antibacterial properties of neomethyl hesperidin dihydrochalcone can be further improved and the sustained-release effect can be improved, which has extremely high practical and economic value. Summary of the invention

[0006] In view of the defects of the prior art, the present invention provides a ternary liquid supramolecular self-assembly system of neo-methyl hesperidin dihydrochalcone / cyclodextrin / pyruvic acid and a preparation method thereof by selecting molecules compatible with neo-methyl hesperidin dihydrochalcone, the types of compatible molecules and the addition amounts, aiming to solve the problems of poor water solubility of neo-methyl hesperidin dihydrochalcone in the prior art.

[0007] The first object of the present invention is to provide a method for preparing a new methyl hesperidin dihydrochalcone / cyclodextrin / pyruvic acid ternary liquid supramolecular self-assembly system, the method comprising:

[0008] The neomethyl hesperidin dihydrochalcone and pyruvic acid are mixed to prepare a neomethyl hesperidin dihydrochalcone / pyruvic acid mixture; the neomethyl hesperidin dihydrochalcone / pyruvic acid mixture is mixed with cyclodextrin to prepare a neomethyl hesperidin dihydrochalcone / cyclodextrin / pyruvic acid ternary liquid supramolecular self-assembly system.

[0009] As an optional embodiment of the present invention, the molar ratio of pyruvic acid, neomethyl hesperidin dihydrochalcone and cyclodextrin is 0.1-1:0.01:0.001-0.02;

[0010] Preferably, the molar ratio of pyruvic acid, neomethyl hesperidin dihydrochalcone and cyclodextrin is 0.1-0.6:0.01:0.001-0.01;

[0011] Preferably, the cyclodextrin includes one or more of β-cyclodextrin, γ-cyclodextrin, α-cyclodextrin, and hydroxypropyl-β-cyclodextrin.

[0012] As an optional method of the present invention, the neomethyl hesperidin dihydrochalcone and pyruvic acid are mixed by stirring the neomethyl hesperidin dihydrochalcone and pyruvic acid in air or inert gas at 20 to 120° C. for more than 2 hours at a stirring speed of 300 to 500 rpm.

[0013] As an optional method of the present invention, the mixture of neomethyl hesperidin dihydrochalcone / pyruvic acid and cyclodextrin is mixed by stirring the mixture of neomethyl hesperidin dihydrochalcone / pyruvic acid and cyclodextrin in air or inert gas at 20 to 120° C. for more than 2 hours;

[0014] Preferably, the mixture is stirred at 50-80°C for more than 2 hours.

[0015] As an optional embodiment of the present invention, the pH of the ternary liquid supramolecular self-assembly system of neomethyl hesperidin dihydrochalcone / cyclodextrin / pyruvic acid is 2.5-4.5.

[0016] As an optional embodiment of the present invention, the pH of the ternary liquid supramolecular self-assembly system may be 2.72, 3.67 or 4.36.

[0017] As an optional mode of the present invention, the mass fraction of the neomethyl hesperidin dihydrochalcone in the ternary liquid supramolecular self-assembly system can reach 25%.

[0018] As an optional mode of the present invention, the mass fraction of neomethyl hesperidin dihydrochalcone in the ternary liquid supramolecular self-assembly system can be 10%, 16% or 23%.

[0019] As an optional mode of the present invention, the ternary liquid supramolecular combination can be dissolved in water of a specific proportion, and can keep the mass fraction of neomethyl hesperidin dihydrochalcone in the aqueous solution at 0.1% to 18% without precipitation.

[0020] As an optional embodiment of the present invention, the mass fraction of neomethyl hesperidin dihydrochalcone in the aqueous solution can be 0.1%, 5% or 17.2%.

[0021] As an optional method of the present invention, the mixture is heated while stirring at a heating rate of 5°C / min. During the heating process, the mixture becomes liquid to form a liquid supramolecular self-assembly system. After cooling to room temperature, a uniform and stable ternary supramolecular self-assembly system is obtained at a cooling rate of 5°C / min; room temperature refers to 25°C.

[0022] As an optional mode of the present invention, the mass fraction of neomethyl hesperidin dihydrochalcone in the neomethyl hesperidin dihydrochalcone / cyclodextrin / pyruvic acid ternary liquid supramolecular self-assembly system is 10% to 20%.

[0023] As an optional method of the present invention, the neomethyl hesperidin dihydrochalcone / cyclodextrin / pyruvic acid ternary liquid supramolecular self-assembly system is mixed with water to prepare a neomethyl hesperidin dihydrochalcone / cyclodextrin / / pyruvic acid ternary liquid supramolecular self-assembly system aqueous solution, wherein the mass fraction of the neomethyl hesperidin dihydrochalcone in the aqueous solution is 0.1% to 18%.

[0024] As an optional mode of the present invention, when the mass fraction of the neomethyl hesperidin dihydrochalcone in the neomethyl hesperidin dihydrochalcone / cyclodextrin / pyruvic acid ternary liquid supramolecular self-assembly system in the aqueous solution is 0.1% to 18%, the system has good stability and will not precipitate.

[0025] As an optional embodiment of the present invention, the molar ratio of raw materials n (neomethyl hesperidin dihydrochalcone): n (pyruvic acid): n (cyclodextrin) is preferably 0.01: 0.1-1: 0.001-0.02; more preferably 0.01: 0.2-0.8: 0.002-0.01; most preferably 0.01: 0.2-0.5: 0.002-0.008.

[0026] As an optional embodiment of the present invention, the molar ratio of raw materials n (neomethyl hesperidin dihydrochalcone): n (pyruvic acid): n (cyclodextrin) is 0.01:0.2:0.002, 0.01:0.3:0.005 or 0.01:0.5:0.008.

[0027] As an optional mode of the present invention, heating is performed under an inert atmosphere or natural air conditions, preferably helium, nitrogen, argon, carbon dioxide or natural air.

[0028] The second object of the present invention is to provide a new methyl hesperidin dihydrochalcone / cyclodextrin / pyruvic acid ternary liquid supramolecular self-assembly system prepared by any of the above methods.

[0029] As an optional method of the present invention, in the neomethyl hesperidin dihydrochalcone / cyclodextrin / pyruvic acid ternary liquid supramolecular self-assembly system, pyruvic acid can effectively adjust the pH value of the system, and at the same time play a synergistic role with neomethyl hesperidin dihydrochalcone, so that the system has good antibacterial, moisturizing, antioxidant, soothing, repairing and other multiple effects.

[0030] As an optional method of the present invention, in the neomethyl hesperidin dihydrochalcone / cyclodextrin / pyruvic acid ternary liquid supramolecular self-assembly system, the water solubility of neomethyl hesperidin dihydrochalcone is changed by forming intermolecular hydrogen bonds. The process flow is green and simple, there is no environmental pollution problem, and it is suitable for large-scale preparation.

[0031] As an optional method of the present invention, the new methyl hesperidin dihydrochalcone / cyclodextrin / pyruvic acid ternary liquid supramolecular self-assembly system can be directly applied to the fields of cosmetics, medicine, food, etc. without the need for additional post-processing such as separation and purification.

[0032] As an optional method of the present invention, the new methyl hesperidin dihydrochalcone / cyclodextrin / pyruvic acid ternary liquid supramolecular self-assembly system prepared by the present invention can be directly diluted with water, and the maximum dilution up to 50 times can still maintain the stability of the system without solid precipitation, which is conducive to formulation application.

[0033] The third object of the present invention is to provide the application of any of the above methods or the above new methyl hesperidin dihydrochalcone / cyclodextrin / pyruvic acid ternary liquid supramolecular self-assembly system in the fields including but not limited to cosmetics, medicine or food.

[0034] As an optional mode of the present invention, the application in the field of food includes preparing common foods and special foods (health products, functional foods, dietary supplements) such as beverages, baked foods, food additives, fermented foods, etc.;

[0035] Applications in the pharmaceutical field include, but are not limited to, the preparation of liver-protecting drugs, hypoglycemic drugs, hypolipidemic drugs, anti-inflammatory drugs, antioxidant drugs, and intestinal flora regulating drugs;

[0036] Applications in the cosmetics field include, but are not limited to, the preparation of non-functional cosmetics, functional (whitening, soothing, moisturizing and anti-oxidation, anti-aging, anti-inflammatory, anti-free radical, etc.) cosmetics, and fragrances.

[0037] The fourth object of the present invention is to provide a product, which contains the above-mentioned new methyl hesperidin dihydrochalcone / cyclodextrin / pyruvic acid ternary liquid supramolecular self-assembly system;

[0038] Preferably, the product includes but is not limited to cosmetics, medicines or foods.

[0039] The fifth object of the present invention is to provide a method for simultaneously improving the solubility, antioxidant, antibacterial and sustained-release effects of neomethyl hesperidin dihydrochalcone, the method comprising: mixing neomethyl hesperidin dihydrochalcone and pyruvic acid to prepare a neomethyl hesperidin dihydrochalcone / pyruvic acid mixture; and mixing the neomethyl hesperidin dihydrochalcone pyruvic acid mixture and cyclodextrin to prepare a neomethyl hesperidin dihydrochalcone / cyclodextrin / pyruvic acid ternary liquid supramolecular self-assembly system.

[0040] As an optional embodiment of the present invention, the molar ratio of pyruvic acid, neomethyl hesperidin dihydrochalcone and cyclodextrin is 0.1-1:0.01:0.001-0.02;

[0041] Preferably, the molar ratio of pyruvic acid, neomethyl hesperidin dihydrochalcone and cyclodextrin is 0.1-0.6:0.01:0.001-0.01;

[0042] Preferably, the cyclodextrin includes: one or more of β-cyclodextrin, γ-cyclodextrin, α-cyclodextrin, and hydroxypropyl-β-cyclodextrin;

[0043] Preferably, the mixing of neomethyl hesperidin dihydrochalcone and pyruvic acid is performed by stirring the neomethyl hesperidin dihydrochalcone and pyruvic acid in air or inert gas at 20 to 120° C. for more than 2 hours;

[0044] Preferably, the mixing of the neomethyl hesperidin dihydrochalcone / pyruvic acid mixture and cyclodextrin is performed by stirring the neomethyl hesperidin dihydrochalcone / pyruvic acid mixture and cyclodextrin in air or inert gas at 20 to 120° C. for more than 2 hours;

[0045] Preferably, the mixture is stirred at 50-80°C for more than 2 hours.

[0046] Beneficial effects of the present invention:

[0047] The new methyl hesperidin dihydrochalcone / cyclodextrin / pyruvic acid system obtained by supramolecular self-assembly of the present invention is in a stable liquid state, can be miscible with water in a specific proportion without precipitation, and effectively improves the water solubility and stability of the new methyl hesperidin dihydrochalcone.

[0048] Specifically:

[0049] The present invention utilizes supramolecular self-assembly technology to successfully prepare a uniform, transparent and stable ternary liquid system of neomethyl hesperidin dihydrochalcone / cyclodextrin / pyruvic acid; in the system, the content of neomethyl hesperidin dihydrochalcone can reach 20%, which is significantly higher than the current conventional concentration, and when miscible with water, its mass fraction can reach 18% without solid precipitation;

[0050] In addition, the system is transparent, low-viscosity, and has good stability. It can be used directly as a raw material, showing excellent formulation application and product efficacy. At the same time, pyruvic acid can adjust the pH in the system and synergize with cyclodextrin to further enhance product performance. The preparation process of the system is simple and environmentally friendly, without the need for complex separation and purification. It is suitable for cosmetics, food, biomedicine and other fields, and has a wide range of application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 This is the infrared spectrum of the ternary liquid supramolecular self-assembly system prepared in Example 1;

[0052] Figure 2 is the DPPH scavenging rate of the aqueous solution of the ternary liquid supramolecular self-assembly system prepared in Example 1;

[0053] Figure 3 is the ABTS removal rate of the aqueous solution of the ternary liquid supramolecular self-assembly system prepared in Example 1;

[0054] Figure 4 The sustained release rate of the ternary liquid supramolecular self-assembly system prepared in Example 1 at different temperatures;

[0055] Figure 5 The sustained release rate of the ternary liquid supramolecular self-assembly system prepared in Comparative Example 2 at different temperatures;

[0056] Figure 6 This is a graph showing the properties of the system products of Examples 1 to 2 and Comparative Examples 1 to 7 after being left for 24 hours. DETAILED DESCRIPTION

[0057] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0058] In order to further illustrate the present invention, a novel methyl hesperidin dihydrochalcone / cyclodextrin / pyruvic acid ternary liquid supramolecular self-assembly system and a preparation method thereof provided by the present invention are described in detail below in conjunction with examples.

[0059] raw material:

[0060] Hydroxypropyl-β-cyclodextrin was purchased from Shanghai Haohong Biopharmaceutical Technology Co., Ltd.; product number: AL13A00113965; molecular weight: 1541.54;

[0061] α-Cyclodextrin was purchased from Anhui Zesheng Technology Co., Ltd.; product number: A01E080250; molecular weight: 972.85;

[0062] β-Cyclodextrin was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; product number: C804562; molecular weight: 1134.98;

[0063] γ-cyclodextrin was purchased from Anhui Zesheng Technology Co., Ltd.; product number: A01E0805155000; molecular weight: 1297.13;

[0064] Detection method:

[0065] The Fourier transform infrared spectroscopy test parameters are: Machine specifications: Nicolet iS5, Machine manufacturer: Thermo Fisher Scientific, USA, Experimental parameters: Scanning range is 450~4000cm -1 , resolution 4cm -1 .

[0066] High and low temperature cycle test: The ternary liquid supramolecular self-assembly system of neomethyl hesperidin dihydrochalcone / cyclodextrin / pyruvic acid was frozen at -5°C and then dissolved at 40°C. This was one cycle and then repeated. It was observed that the state after each dissolution could maintain a stable and uniform liquid state without solid precipitation. The example is the data of 15 cycles.

[0067] Example 1

[0068] 1. A method for preparing a new methyl hesperidin dihydrochalcone / cyclodextrin / pyruvic acid ternary liquid supramolecular self-assembly system, the steps are as follows:

[0069] 0.01 mol of neomethyl hesperidin dihydrochalcone (6.12 g) and 0.2 mol of pyruvic acid (17.6 g) were stirred in a sealed container and stirred at 400 rpm for 2 h at room temperature, then 0.002 mol of hydroxypropyl-β-cyclodextrin (3.08 g) was added, and the container was slowly heated in an air atmosphere, and after heating to 50° C., it was continuously heated at 400 rpm and stirred for 2 h, and the container was cooled to room temperature (25° C.) to obtain a ternary liquid supramolecular self-assembly system, in which the mass fraction of neomethyl hesperidin dihydrochalcone in the system was 23%, and the pH of the system was 4.36 when tested with a pH meter;

[0070] The ternary liquid supramolecular self-assembly system is miscible with water in a specific proportion so that the mass fraction of neomethyl hesperidin dihydrochalcone in the aqueous solution is 17.2%, that is, the solubility is 207.7 mg / g, and the system can maintain a stable and uniform liquid state, thereby obtaining an aqueous solution of the ternary liquid supramolecular self-assembly system.

[0071] The ternary liquid supramolecular self-assembly system and the ternary liquid supramolecular self-assembly system aqueous solution were subjected to high and low temperature cycle tests (cycled and stored at -5°C and 40°C for 15 times), and finally both systems were able to maintain a stable and uniform liquid state without solid precipitation.

[0072] The prepared uniform, transparent and stable ternary liquid supramolecular self-assembly system was placed at room temperature for 24 hours and then sampled and characterized by infrared spectrometer.

[0073] The infrared spectrum of the ternary liquid supramolecular self-assembly system is shown in Figure 1 As shown in the results, the ternary liquid supramolecular self-assembly system prepared in Example 1 has a wavelength of 3500-3200 cm -1 The absorption peak in the range corresponds to the absorption peak of the hydroxyl group (hydrogen bond). Compared with pyruvic acid, the ternary liquid supramolecular self-assembly system prepared in Example 1 has an obvious peak. Compared with neomethyl hesperidin dihydrochalcone and hydroxypropyl-β-cyclodextrin, the peak position is obviously moved to a low wave number, which is due to the interaction of a large number of hydrogen bonds generated in the system. In addition, relative to neomethyl hesperidin dihydrochalcone and hydroxypropyl-β-cyclodextrin, the peak position of the carbonyl group moves to a high wave number direction, which is due to the change in the carbonyl electron cloud density caused by the generation of a large number of hydrogen bonds within the system. The above results all confirm that pyruvic acid, hydroxypropyl-β-cyclodextrin and neomethyl hesperidin dihydrochalcone form a supramolecular self-assembly system through intermolecular hydrogen bonds.

[0074] 2. Performance testing

[0075] Triple distilled water was added to the ternary liquid supramolecular self-assembly system obtained in Example 1 to prepare aqueous solutions of the ternary liquid supramolecular self-assembly system with neomethylhesperidin dihydrochalcone concentrations of 0.1 mg / mL, 0.2 mg / mL, 0.4 mg / mL, 0.6 mg / mL, 0.8 mg / mL, and 1 mg / mL for antioxidant testing, with equal concentrations of neomethylhesperidin dihydrochalcone standard solutions and equal concentrations of pyruvic acid solutions as controls.

[0076] (1) DPPH free radical scavenging method was used to determine the antioxidant activity.

[0077] DPPH free radical has a strong absorption peak in the visible light with a wavelength of 517nm, and appears purple. When DPPH free radical combines with the electrons provided by the antioxidant sample, the purple color disappears and becomes colorless or light yellow. The absorbance before and after the reaction is measured with a UV-visible spectrophotometer to reflect the color change.

[0078] Weigh 5.0 mg of DPPH, dissolve it in an appropriate amount of anhydrous ethanol, protect it from light and sonicate it to make it fully dissolved, then make up the volume to 100 mL with anhydrous ethanol to prepare a DPPH solution with a concentration of 50 μg / mL.

[0079] Prepare the solution in a 96-well plate according to Table 1:

[0080] Table 1

[0081] Solution name A B C D DPPH solution 100μL - 100μL - Ternary liquid supramolecular self-assembly system aqueous solution / standard solution / pyruvic acid solution - - 100μL 100μL Anhydrous ethanol 100μL 200μL - 100μL

[0082] After the sample solution is mixed evenly, react at 25°C in the dark for 30 minutes and measure the absorbance value at a wavelength of 517 nm.

[0083] The free radical scavenging rate was calculated according to the following formula:

[0084]

[0085] P is the clearance rate, A a , A b , A c , A d , corresponding to the absorbance value of each well in the table.

[0086] The results are shown in Table 2 and Figure 2 As shown, the results indicate that the DPPH scavenging rate of the aqueous solution of the ternary liquid supramolecular self-assembly system is better than that of the single components neohesperidin dihydrochalcone and pyruvic acid.

[0087] Table 2 DPPH test results

[0088]

[0089] (2) Antioxidant activity was determined by ABTS free radical scavenging method.

[0090] After ABTS is oxidized, it generates a relatively stable ABTS free radical, which is blue-green and has a maximum absorption peak at 734nm wavelength of visible light. The sample reacts with the ABTS free radical to make it fade. The absorbance value after the reaction can be measured to compare the free radical scavenging rate. By comparing the scavenging ability with that of neomethyl hesperidin dihydrochalcone, the relative half-scavenging amount is used to judge the antioxidant capacity of the sample. 0 value.

[0091] Prepare 7mmol / L ABTS aqueous solution and 2.45mmol / L potassium persulfate aqueous solution, mix the above solutions in equal volumes, place in a dark environment for 12h, and dilute 30 times with deionized water to an absorbance value of A 734 nm =0.70±0.02, used as the measurement solution (ABTS solution).

[0092] Prepare the solution in a 96-well plate according to Table 3:

[0093] Table 3

[0094] Solution name A B C D ABTS solution 100μL - 100μL - Ternary liquid supramolecular self-assembly system aqueous solution / standard solution / pyruvic acid solution - - 100μL 100μL Deionized water 100μL 200μL - 100μL

[0095] After the sample solution is mixed evenly, react at 25°C in the dark for 5 minutes, and measure the absorbance value at a wavelength of 734 nm.

[0096] The free radical scavenging rate was calculated according to the following formula:

[0097]

[0098] P is the clearance rate, A a , A b , A c , A d , corresponding to the absorbance value of each well in the table.

[0099] The results are shown in Table 4 and Figure 3 As shown, the results indicate that the ABTS removal rate of the ternary liquid supramolecular self-assembly system in aqueous solution is superior to that of the single components neohesperidin dihydrochalcone and pyruvic acid.

[0100] Table 4 ABTS test results

[0101]

[0102] The above results show that the aqueous solution of the ternary liquid supramolecular self-assembly system exhibits excellent antioxidant capacity. Compared with the neohesperidin dihydrochalcone standard and pyruvic acid, the antioxidant capacity of the aqueous solution of the ternary liquid supramolecular self-assembly system is significantly improved, and it has a supramolecular synergistic effect.

[0103] (3) Antibacterial properties

[0104] Triple distilled water was added to the ternary liquid supramolecular self-assembly system obtained in Example 1 to prepare aqueous solutions of ternary liquid supramolecular self-assembly systems with different concentrations of neomethyl hesperidin dihydrochalcone. Antibacterial activity tests were performed, with triple distilled water as a control.

[0105] The overnight resuscitation culture of streptococci was inoculated into 5 mL of THB liquid medium at a volume of 1% v / v and incubated at 37°C and 5% CO 2 Incubate the cells in a humidified incubator for 24 hours. When the bacteria reach the mid-logarithmic growth stage and the bacterial count reaches 10 9 CFU / mL, take it out for later use.

[0106] Under aseptic operation in a biosafety cabinet, aqueous solutions of the ternary liquid supramolecular self-assembly system of different concentrations were added to a sterile 96-well culture plate, with the drug solution added to wells 1 to 9, 100 μL per well, and 100 μL of triple-distilled water was added to well 10 as a positive control.

[0107] The culture inoculum was diluted tenfold with the culture medium, and then added to the corresponding 96-well culture plate at 100 μL / well. The plate was sealed and placed at 37°C and 5% CO. 2 The solution was incubated in an incubator for 24 h to observe the antibacterial effect of the aqueous solution of the ternary liquid supramolecular self-assembly system.

[0108] After the drug and bacteria were co-incubated for 24 h, 100 μL was taken from each well and coated on THB plates at 37°C and 5% CO 2 The samples were incubated in an incubator for 24 hours and the lowest drug concentration that resulted in complete sterile growth was defined as the MIC. The test was meaningful only when bacteria in the positive control well (i.e., without drug) grew significantly.

[0109] The minimum complete inhibitory concentration plate experiment confirmed that the ternary liquid supramolecular self-assembly system had a significant inhibitory effect on Streptococcus mutans, Streptococcus suis serotype 2, and group A Streptococcus. The results are shown in Table 5. The minimum inhibitory concentration of the ternary liquid supramolecular self-assembly system was at the mg / mL level. That is, the ternary liquid supramolecular self-assembly system has good efficacy in antibacterial performance.

[0110] Table 5 Antibacterial effect

[0111]

[0112] (4) Sustained release effect

[0113] 15 mg of the ternary liquid supramolecular self-assembly system prepared in Example 1 was taken and placed in a dialysis bag, and the dialysis bag was suspended in 30 mL of a phosphate buffer solution with a pH of 7.4 and magnetically stirred at a constant temperature of 25° C., 33° C., 37° C. and 42° C. for in vitro drug release. The release was carried out for 10 h, and 3 mL of the solution was taken out from the solution every 30 min. The amount of released neomethyl hesperidin dihydrochalcone and pyruvic acid was determined by an ultraviolet spectrophotometer, and 3 mL of fresh phosphate buffer solution was supplemented at the same time.

[0114] The concentration of neomethyl hesperidin dihydrochalcone was measured at 282 nm using an ultraviolet spectrophotometer (the concentration of pyruvic acid was measured at 220 nm using an ultraviolet spectrophotometer). The cumulative percentage of drug release at different times was calculated based on the measured amounts of neomethyl hesperidin dihydrochalcone and pyruvic acid. The results are shown in FIG. Figure 4 shown.

[0115] The results show that the ternary liquid supramolecular self-assembly system has good sustained release effect at different temperatures. The release rate is less than 50% at 150 min and less than 70% at 600 min at 25-37°C; at 42°C, the release rate is 60% at 150 min and less than 90% at 600 min.

[0116] Example 2

[0117] A method for preparing a new methyl hesperidin dihydrochalcone / cyclodextrin / pyruvic acid ternary liquid supramolecular self-assembly system, the steps are as follows:

[0118] 0.01 mol of neomethyl hesperidin dihydrochalcone (6.12 g) and 0.3 mol of pyruvic acid (26.4 g) were placed in a sealed container and stirred at room temperature at 400 rpm for 4 h, and then 0.005 mol of α-cyclodextrin (4.86 g) was added, and the container was slowly heated in a natural air atmosphere, and after the temperature was raised to 60° C., the heating and stirring were continued for 4 h, and the container was cooled to room temperature to obtain a ternary liquid supramolecular self-assembly system, in which the mass fraction of neomethyl hesperidin dihydrochalcone in the system was 16%, and the pH of the system was 3.67 when tested with a pH meter;

[0119] The ternary liquid supramolecular combination is miscible with water in a specific proportion so that the mass fraction of neomethyl hesperidin dihydrochalcone in the aqueous solution is 5%, that is, the solubility is 52.6 mg / g, and the system can maintain a stable and uniform liquid state, thereby obtaining an aqueous solution of the ternary liquid supramolecular self-assembly system.

[0120] The prepared ternary liquid supramolecular self-assembly system and the aqueous solution of the ternary liquid supramolecular self-assembly system were subjected to high and low temperature cycle tests (cycled and stored at -5°C and 40°C for 15 times), and finally both systems were able to maintain a stable and uniform liquid state without solid precipitation.

[0121] Example 3

[0122] A method for preparing a new methyl hesperidin dihydrochalcone / cyclodextrin / pyruvic acid ternary liquid supramolecular self-assembly system, the steps are as follows:

[0123] 0.01 mol of neomethyl hesperidin dihydrochalcone (6.12 g) and 0.5 mol of pyruvic acid (44 g) were placed in a sealed container and stirred at room temperature at 400 rpm for 6 h, and then 0.008 mol of γ-cyclodextrin (10.38 g) was added, and the container was slowly heated in a natural air atmosphere, and after the temperature was raised to 80° C., the heating and stirring were continued for 6 h, and the container was cooled to room temperature to obtain a ternary liquid supramolecular self-assembly system, in which the mass fraction of neomethyl hesperidin dihydrochalcone in the system was 10%, and the pH of the system was 2.72 when tested by a pH meter;

[0124] The ternary liquid supramolecular combination is miscible with water in a specific proportion so that the mass fraction of neomethyl hesperidin dihydrochalcone in the aqueous solution is 0.1%, that is, the solubility is 1 mg / g, and the system can maintain a stable and uniform liquid state, thereby obtaining an aqueous solution of the ternary liquid supramolecular self-assembly system.

[0125] The ternary liquid supramolecular self-assembly system and the obtained ternary liquid supramolecular self-assembly system aqueous solution were subjected to high and low temperature cycle tests (cycled and stored at -5°C and 40°C for 15 times), and finally both systems were able to maintain a stable and uniform liquid state without solid precipitation.

[0126] Example 4

[0127] A method for preparing a new methyl hesperidin dihydrochalcone / cyclodextrin / pyruvic acid ternary liquid supramolecular self-assembly system, the steps are as follows:

[0128] 0.01 mol of neomethyl hesperidin dihydrochalcone (6.12 g) and 0.5 mol of pyruvic acid (44 g) were placed in a sealed container and stirred at room temperature at 400 rpm for 24 h, and then 0.008 mol of β-cyclodextrin (9.08 g) was added, and the container was slowly heated in a natural air atmosphere, and after heating to 80° C., the heating and stirring were continued for 6 h, and the container was cooled to room temperature to obtain a ternary liquid supramolecular self-assembly system, in which the mass fraction of neomethyl hesperidin dihydrochalcone in the system was 10%, and the pH of the system was 2.72 when tested with a pH meter;

[0129] The ternary liquid supramolecular combination is miscible with water in a specific proportion so that the mass fraction of neomethyl hesperidin dihydrochalcone in the aqueous solution is 0.1%, that is, the solubility is 1 mg / g, and the system can maintain a stable and uniform liquid state, thereby obtaining an aqueous solution of the ternary liquid supramolecular self-assembly system.

[0130] The ternary liquid supramolecular self-assembly system and the obtained ternary liquid supramolecular self-assembly system aqueous solution were subjected to high and low temperature cycle tests (cycled and stored at -5°C and 40°C for 15 times), and finally both systems were able to maintain a stable and uniform liquid state without solid precipitation.

[0131] Comparative Example 1

[0132] 0.01 mol of neomethyl hesperidin dihydrochalcone and 0.002 mol of hydroxypropyl-β-cyclodextrin were placed in a sealed container, and the container was slowly heated in a natural air atmosphere. After the temperature reached 50°C, heating and stirring were continued for 2 hours. As time went by, the system remained in a solid powder state.

[0133] Compared with Example 1, when only hydroxypropyl-β-cyclodextrin was selected as a single compatible molecule for the supramolecular self-assembly attempt, a uniform and transparent liquid state could not be obtained, but a solid powder state was obtained.

[0134] The solid powder supramolecular combination was miscible with water in a specific proportion, so that the mass fraction of neomethyl hesperidin dihydrochalcone in the aqueous solution was 17.2%, that is, the solubility was 207.7 mg / g. The system could not maintain a stable and uniform liquid state, and solid insoluble matter existed.

[0135] Comparative Example 2

[0136] 0.01 mol of neomethyl hesperidin dihydrochalcone and 0.2 mol of pyruvic acid were placed in a sealed container, and the container was slowly heated under natural air atmosphere. After the temperature reached 50°C, heating and stirring were continued for 2 hours. As time went by, the system became a uniform and transparent liquid state.

[0137] Compared with Example 1, only pyruvic acid was selected as a single compatible molecule for supramolecular self-assembly attempt, and a uniform and transparent liquid state was obtained. The ternary liquid supramolecular self-assembly system was miscible with a specific proportion of water, so that the mass fraction of neomethyl hesperidin dihydrochalcone in the aqueous solution was 17.2%, that is, the solubility was 207.7 mg / g, and the system could maintain a stable and uniform liquid state.

[0138] The in vitro sustained release effect of the ternary liquid supramolecular self-assembly system prepared in Comparative Example 2 was tested:

[0139] 30 mg of the ternary liquid supramolecular self-assembly system prepared in Comparative Example 2 was taken and placed in a dialysis bag, and the dialysis bag was suspended in 50 mL of a phosphate buffer solution with a pH of 7.4 and magnetically stirred at 25°C, 33°C, 37°C and 42°C for in vitro drug release. The release was performed for 10 hours, and 3 mL of the solution was taken out from the solution every 30 minutes to determine the amount of the released neomethyl hesperidin dihydrochalcone, and 3 mL of fresh phosphate buffer solution was added at the same time. The concentration of neomethyl hesperidin dihydrochalcone was determined at 282 nm using an ultraviolet spectrophotometer, and the cumulative percentage of drug release at different times was calculated based on the measured amount of neomethyl hesperidin dihydrochalcone. The results are shown in FIG. Figure 5 shown.

[0140] The results show that the ternary liquid supramolecular self-assembly system of Comparative Example 2 has no sustained release effect at different temperatures, the release amount reaches a stable state at 150 minutes, and the release amount does not change after 150 minutes.

[0141] Comparative Example 3

[0142] 0.01 mol of neomethyl hesperidin dihydrochalcone and 0.2 mol of pyruvic acid were placed in a sealed container and stirred for 2 hours, and then 0.002 mol of hydroxypropyl-β-cyclodextrin was added. The temperature of the container was slowly increased in a natural air atmosphere. After the temperature reached 100°C, the container was continuously heated and stirred for 2 hours. As time went on, the system became a uniform and transparent liquid state.

[0143] Compared with Example 1, only the heating temperature was increased, and the results were as follows Figure 6 As shown, the color of the system deepened, suggesting that a chemical reaction may have occurred during the reaction process.

[0144] Comparative Example 4

[0145] 0.01 mol of neomethyl hesperidin dihydrochalcone and 0.2 mol of pyruvic acid were placed in a sealed container and stirred for 2 hours, and then 0.002 mol of hydroxypropyl-β-cyclodextrin was added and stirred for 2 hours at room temperature. As time went by, the system became a solid-liquid mixture.

[0146] Compared with Example 1, only by lowering the heating temperature, a uniform and stable transparent liquid system cannot be obtained, and the system maintains a solid-liquid mixed state. The system is mixed with water in a specific proportion so that the mass fraction of neomethyl hesperidin dihydrochalcone is 17.2%, that is, the solubility is 207.7 mg / g. The system still cannot maintain a stable and uniform liquid state, and solid insoluble matter exists.

[0147] Comparative Example 5

[0148] 0.01 mol of neomethyl hesperidin dihydrochalcone and 0.2 mol of pyruvic acid were placed in a sealed container and stirred for 2 hours, and then 0.002 mol of hydroxypropyl-β-cyclodextrin was added. The temperature of the container was slowly increased in a natural air atmosphere. After the temperature reached 50°C, heating and stirring were continued for 1 hour. As time went by, the system became a solid-liquid mixture.

[0149] Compared with Example 1, only by shortening the heating time, a uniform and stable transparent liquid system cannot be obtained, and the system maintains a solid-liquid mixed state. The system is mixed with water in a specific proportion so that the mass fraction of neomethyl hesperidin dihydrochalcone is 17.2%, that is, the solubility is 207.7 mg / g. The system still cannot maintain a stable and uniform liquid state, and solid insoluble matter exists.

[0150] Comparative Example 6

[0151] 0.02 mol of neomethyl hesperidin dihydrochalcone and 0.2 mol of pyruvic acid were placed in a sealed container and stirred for 2 hours, and then 0.002 mol of hydroxypropyl-β-cyclodextrin was added. The temperature of the container was slowly increased in a natural air atmosphere. After the temperature reached 50°C, heating and stirring were continued for 2 hours. As time went by, the system became a solid-liquid mixture.

[0152] Compared with Example 1, only by increasing the feeding amount of neomethyl hesperidin dihydrochalcone, a uniform and stable transparent liquid system cannot be obtained, and the system maintains a solid-liquid mixed state. The system is mixed with water in a specific proportion so that the mass fraction of neomethyl hesperidin dihydrochalcone is 17.2%, that is, the solubility is 207.7 mg / g. The system still cannot maintain a stable and uniform liquid state, and solid insoluble matter exists.

[0153] Comparative Example 7

[0154] 0.01 mol of neomethyl hesperidin dihydrochalcone and 0.2 mol of deionized water were placed in a sealed container and stirred for 2 hours, and then 0.002 mol of hydroxypropyl-β-cyclodextrin was added. The temperature of the container was slowly increased in a natural air atmosphere. After the temperature reached 50°C, heating and stirring were continued for 2 hours. As time went by, the system became a solid-liquid mixture.

[0155] Compared with Example 1, only one of the system components was changed, and deionized water was used to try to replace pyruvic acid. A uniform and transparent liquid system could not be obtained. The system was a solid powder. The system was mixed with water in a specific proportion so that the mass fraction of neomethyl hesperidin dihydrochalcone was 17.2%, that is, the solubility was 207.7 mg / g. The system still could not maintain a stable and uniform liquid state, and solid insoluble matter existed.

[0156] Comparative Example 8

[0157] 0.01 mol of neomethyl hesperidin dihydrochalcone and 0.2 mol of mPEG2000-PLLA2000 were placed in a sealed container and stirred for 2 hours, and then 0.002 mol of hydroxypropyl-β-cyclodextrin was added. The temperature of the container was slowly increased under natural air atmosphere. After the temperature was increased to 50°C, heating and stirring were continued for 2 hours. As time went by, the system became a solid powder.

[0158] Compared with Example 1, only one of the system components was changed, and mPEG2000-PLLA2000 was selected to replace pyruvic acid. A uniform and transparent liquid system could not be obtained. The system was a solid powder. The system was mixed with water in a specific proportion so that the mass fraction of neomethyl hesperidin dihydrochalcone was 17.2%, that is, the solubility was 207.7 mg / g. The system still could not maintain a stable and uniform liquid state, and solid insoluble matter was present.

[0159] Comparative Example 9

[0160] 0.002 mol of hydroxypropyl-β-cyclodextrin and 0.2 mol of pyruvic acid were placed in a sealed container, and the temperature of the container was slowly increased in a natural air atmosphere. After the temperature reached 50°C, the container was continuously heated and stirred for 2 hours, and then cooled to room temperature. 0.01 mol of neomethyl hesperidin dihydrochalcone was added and stirred for 2 hours. As time went by, the system became a solid-liquid mixture.

[0161] Compared with Example 1, only the mixing order of the system was changed. Neomethyl hesperidin dihydrochalcone and pyruvic acid were first mixed and stirred, and then hydroxypropyl-β-cyclodextrin was added. The system was a solid-liquid mixture. The system was mixed with water in a specific proportion so that the mass fraction of neomethyl hesperidin dihydrochalcone was 17.2%, that is, the solubility was 207.7 mg / g. The system still could not maintain a stable and uniform liquid state, and solid insoluble matter existed.

[0162] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for preparing a ternary liquid supramolecular self-assembly system of neomethyl hesperidin dihydrochalcone / cyclodextrin / pyruvic acid, characterized in that: The method comprises: The neo-methyl hesperidin dihydrochalcone and pyruvic acid are mixed to prepare a neo-methyl hesperidin dihydrochalcone / pyruvic acid mixture; the neo-methyl hesperidin dihydrochalcone / pyruvic acid mixture is mixed with cyclodextrin to prepare a neo-methyl hesperidin dihydrochalcone / cyclodextrin / pyruvic acid ternary liquid supramolecular self-assembly system; The molar ratio of pyruvic acid, neomethyl hesperidin dihydrochalcone and cyclodextrin is 0.2-0.6:0.01:0.001-0.01; The step of mixing the neomethyl hesperidin dihydrochalcone / pyruvic acid mixture and cyclodextrin is to stir the neomethyl hesperidin dihydrochalcone / pyruvic acid mixture and cyclodextrin in air or inert gas at 50-80° C. for more than 2 hours.

2. The method according to claim 1, characterized in that Cyclodextrins include: One or more of β-cyclodextrin, γ-cyclodextrin, α-cyclodextrin, and hydroxypropyl-β-cyclodextrin.

3. The method according to claim 1, characterized in that: The step of mixing neomethyl hesperidin dihydrochalcone and pyruvic acid is to stir neomethyl hesperidin dihydrochalcone and pyruvic acid in air or inert gas at 20-120° C. for more than 2 hours.

4. The new methyl hesperidin dihydrochalcone / cyclodextrin / pyruvic acid ternary liquid supramolecular self-assembly system prepared by the method described in any one of claims 1 to 3.

5. Use of the method according to any one of claims 1 to 3 or the new methyl hesperidin dihydrochalcone / cyclodextrin / pyruvic acid ternary liquid supramolecular self-assembly system according to claim 4 in the preparation of cosmetics, medicines or foods.

6. The use according to claim 5, characterized in that: Applications in food preparation include preparation of beverages, baked goods, food additives, fermented foods; The application in the preparation of medicines includes the preparation of liver protection drugs, blood sugar lowering drugs, blood lipid lowering drugs, anti-inflammatory drugs, antioxidant drugs, and intestinal flora regulating drugs; Application in the preparation of cosmetics includes the preparation of fragrances.

7. A product, characterized in that The product contains the new methyl hesperidin dihydrochalcone / cyclodextrin / pyruvic acid ternary liquid supramolecular self-assembly system according to claim 4; The product includes a cosmetic, a medicine or a food.

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