Method for improving bioavailability of flavonoid compounds and application thereof

Abstract

The invention belongs to the technical field of health care product development, and particularly relates to a method for improving the bioavailability of flavonoid compounds and application of the flavonoid compounds. The method comprises the following steps: (1) mixing a flavonoid compound, beta-cyclodextrin and water to obtain a mixed solution; (2) carrying out ultrasonic treatment on the mixed solution for 8-12 minutes to obtain an inclusion compound; (3) dialyzing the inclusion compound for 20-28 hours, wherein the liquid retained in a dialysis bag is a composite solution; the ultrasonic power in the step (2) is 55-65 W, the ultrasonic treatment lasts for 3-5 s each time, and the ultrasonic treatment is stopped for 0.8-1.2 s. The embedding rate of the composite solution prepared by the method for embedding the flavonoid compound is greater than or equal to 58.19%, and the bioavailability of the composite solution after gavage of mice is greater than or equal to 1.28%, which is higher than the bioavailability of the flavonoid compound after single gavage. And a basis is provided for development of health-care foods or medicines for preventing and / or treating Alzheimer's disease.

Description

Technical Field

[0001] The present invention belongs to the technical field of health product development, and particularly relates to a method for improving the bioavailability of flavonoids and its application. Background Art

[0002] Flavonoids have various functions and also have certain effects in treating neurodegenerative diseases such as Alzheimer's disease (AD). Through a series of in vivo and in vitro experiments in the early stage, it was explored that flavonoids have significant therapeutic and improving effects on AD. However, their bioavailability is low.

[0003] The prior art lacks a method for improving the bioavailability of flavonoids. The products prepared by this method are dedicated to the development of health foods or drugs for treating AD, providing favorable support for the prevention and treatment of AD.

[0004] Based on this, the present invention is proposed. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a composite solution with a high embedding rate and high bioavailability of flavonoids, and particularly relates to a method for improving the bioavailability of flavonoids and its application.

[0006] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0007] The present invention provides a method for improving the bioavailability of flavonoids, comprising the following steps:

[0008] (1) Mix flavonoids, β-cyclodextrin and water to obtain a mixed solution;

[0009] (2) Ultrasonic the mixed solution for 8 - 12 min to obtain an inclusion complex;

[0010] (3) Dialyze the inclusion complex for 20 - 28 h, and the liquid retained in the dialysis bag is the composite solution;

[0011] The power of ultrasonic in step (2) is 55 - 65 W, and each ultrasonic lasts for 3 - 5 s with a pause of 0.8 - 1.2 s.

[0012] Preferably, the flavonoids are epigallocatechin gallate or epigallocatechin gallate.

[0013] Preferably, the mass ratio of the flavonoids to β-cyclodextrin in the mixture is 1:2 - 4;

[0014] The mass-volume ratio of the flavonoids to water in the mixture is 6 - 10 mg:mL.

[0015] Preferably, the molecular weight cut-off of the dialysis bag in step (3) is 900-1100 Da.

[0016] The present invention also provides a composite solution obtained by the method described above. The encapsulation rate of flavonoids encapsulated in the composite solution is ≥58.19%; the bioavailability of flavonoids in the composite solution is ≥1.28%.

[0017] The present invention also provides the application of the composite solution obtained by the method described above in the preparation of foods or drugs for improving the bioavailability of flavonoids.

[0018] The present invention also provides the application of the composite solution obtained by the method described above in the preparation of foods or drugs for treating Alzheimer's disease.

[0019] The present invention also provides the application of the composite solution described above in the preparation of foods or drugs for improving the bioavailability of flavonoids.

[0020] The present invention also provides the application of the composite solution described above in the preparation of foods or drugs for treating Alzheimer's disease.

[0021] The present invention provides a method for improving the bioavailability of flavonoids and its application. The method of the present invention comprises the following steps: (1) mixing flavonoids, β-cyclodextrin and water to obtain a mixed solution; (2) ultrasonicating the mixed solution for 8-12 min to obtain an inclusion complex; (3) dialyzing the inclusion complex for 20-28 h, and the liquid retained in the dialysis bag is the composite solution; the power of the ultrasonic wave in step (2) is 55-65 W, each ultrasonic wave lasts for 3-5 s, and the pause is 0.8-1.2 s. The encapsulation rate of flavonoids encapsulated in the composite solution prepared according to the method of the present invention is ≥58.19%, and the bioavailability of flavonoids after intragastric administration of the composite solution to mice is ≥1.28%, which is higher than the bioavailability of intragastric administration of flavonoids alone. It provides a basis for the development of health foods or drugs for preventing and / or treating Alzheimer's disease. Description of the Drawings

[0022] Figure 1 Standard curves plotted for reference substances at different concentrations;

[0023] Figure 2 Standard curves plotted for standard solutions at different concentrations;

[0024] Figure 3 Peak intensity of ECG detected in plasma after intragastric administration of ECG;

[0025] Figure 4 Peak intensity of ECG detected in plasma after intragastric administration of ECG;

[0026] Figure 5 It is the peak intensity of ECG detected in plasma after intragastric administration of ECG.

[0027] Figure 6 It is the peak intensity of ECG detected in plasma after intragastric administration of the composite solution.

[0028] Figure 7 It is the peak intensity of ECG detected in plasma after intragastric administration of the composite solution.

[0029] Figure 8 It is the peak intensity of ECG detected in plasma after intragastric administration of the composite solution.

[0030] Figure 9 It is the peak intensity of ECG in the plasma of the control group mice.

[0031] Figure 10 It is the peak intensity of ECG in the plasma of the control group mice.

[0032] Figure 11 It is a comparison graph of the bioavailability of flavonoids (the vertical axis is bioavailability). Specific implementation mode

[0033] The present invention provides a method for improving the bioavailability of flavonoids, comprising the following steps:

[0034] (1) Mix flavonoids, β-cyclodextrin and water to obtain a mixed solution;

[0035] (2) Ultrasonicate the mixed solution for 8 - 12 min to obtain an inclusion complex;

[0036] (3) Dialyze the inclusion complex for 20 - 28 h, and the liquid retained in the dialysis bag is the composite solution;

[0037] The power of the ultrasonication in step (2) is 55 - 65 W, preferably 60 W; each ultrasonication lasts for 3 - 5 s, preferably 4 s; the pause is 0.8 - 1.2 s, preferably 1.0 s; the ultrasonication time is preferably 10 min, and this ultrasonication time only refers to the ultrasonication time and does not include the pause time.

[0038] In the present invention, the flavonoids are epicatechin gallate (ECG) or epigallocatechin gallate (EGCG), preferably epicatechin gallate.

[0039] In the present invention, the mass ratio of the flavonoids to β-cyclodextrin in the mixture is 1:2 - 4, preferably 1:3;

[0040] The mass-volume ratio of the flavonoids to water in the mixture is 6 - 10 mg:1 mL, preferably 8 mg:1 mL.

[0041] In the present invention, β-cyclodextrin is a cyclic oligosaccharide composed of 7 glucose molecules linked by β-1,4-glycosidic bonds, with a slightly conical hollow cylindrical three-dimensional structure, having a hydrophilic outer surface and a hydrophobic inner cavity. Flavonoids, especially the ECG molecule, have a certain hydrophobic part, and its size and shape are suitable for entering the hydrophobic cavity of β-cyclodextrin, and a stable inclusion complex is formed through non-covalent interactions such as van der Waals forces, hydrogen bonds, and hydrophobic interactions between molecules.

[0042] In the present invention, the molecular weight cut-off of the dialysis bag in step (3) is 900-1100 Da, preferably 1000 Da;

[0043] In the present invention, the molecular weight of β-cyclodextrin after ultrasound is 1100 Da, and the molecular weight of flavonoids, especially the ECG molecule, is more than 400 Da. Therefore, a dialysis bag with a molecular weight cut-off of 1000 Da is selected to precipitate the unentrapped flavonoids.

[0044] In the present invention, during dialysis, the dialysis bag containing the inclusion complex is placed in pure water.

[0045] The present invention also provides a composite solution obtained by the described method. The encapsulation rate of the flavonoids encapsulated in the composite solution is ≥57.4%; the bioavailability of the flavonoids in the composite solution

[0046] ≥1.28%.

[0047] The present invention also provides the application of the composite solution obtained by the described method in the preparation of foods or drugs for improving the bioavailability of flavonoids.

[0048] The present invention also provides the application of the composite solution obtained by the described method in the preparation of foods or drugs for treating Alzheimer's disease.

[0049] The present invention also provides the application of the described composite solution in the preparation of foods or drugs for improving the bioavailability of flavonoids.

[0050] The present invention also provides the application of the described composite solution in the preparation of foods or drugs for treating Alzheimer's disease.

[0051] The following combines examples to detail the technical solutions provided by the present invention, but they cannot be understood as limiting the protection scope of the present invention.

[0052] In the examples of the present invention, the flavonoid is epicatechin gallate (ECG), purchased from Shanghai Macklin Biochemical Co., Ltd.

[0053] Example 1

[0054] Take 150 mg of β-cyclodextrin and 50 mg of ECG and add them to a centrifuge tube. Add 5 mL of water and stir well to dissolve them to form a mixed solution. Ultrasonically treat the mixed solution with an ultrasonic power of 60 W. The ultrasonic time lasts for 4 s each time, with a pause of 1 s in between, and the total ultrasonic time is 10 min (excluding the pause time) to promote the entry of ECG molecules into the hydrophobic cavity of β-cyclodextrin to form an inclusion complex. The molecular mass of β-cyclodextrin in the inclusion complex is 1100 Da, and the molecular mass of ECG is 400 Da. Therefore, a 1000 Da dialysis bag is selected. Place the inclusion complex in a 1000 Da dialysis bag and dialyze it in clear water for 24 h to precipitate the unembedded ECG. After dialysis, the solution retained in the dialysis bag is the composite solution.

[0055] Experimental Example 1

[0056] Determine the embedding situation of ECG embedded in β-cyclodextrin in the composite solution

[0057] (1) Preparation of reference solution

[0058] Accurately weigh an appropriate amount of ECG powder, dissolve it with deionized water to prepare working solutions of 0, 2.5, 5, 10, and 20 mg / mL, and filter them with a 0.22 μm water-based microporous filter membrane to obtain reference solutions of different concentrations.

[0059] (2) Preparation of mobile phase buffer

[0060] Accurately weigh 2.72 g of potassium dihydrogen phosphate and place it in a 1 L volumetric flask. After dissolving it with deionized water, adjust the pH to 2.5 with phosphoric acid, make up the volume, and filter it with a 0.45 μm water-based microporous filter membrane to obtain a 20 mM KH2PO4 buffer mobile phase for use.

[0061] (3) Liquid chromatography

[0062] The gradient elution program is as follows: from 0 to 15 min, the acetonitrile in pump B is maintained at 13%; from 15 to 25 min, the acetonitrile in pump B linearly increases from 13% to 20%, remains for 5 min, and then decreases to 13% B within 2 min; detection wavelength: 278 nm; flow rate: 1.0 mL / min; column temperature: 30 °C; injection volume: 10 μL; chromatographic column: Agilent chromatographic column SB-C18. Detect the peak areas of the reference solutions of different concentrations and the composite solution of Example 1 at 278 nm in sequence.

[0063] Take the concentration values of the reference solutions of different concentrations as the abscissa and the peak areas as the ordinate to plot a standard curve. The results are shown in Table 1 and Figure 1 as follows.

[0064] Table 1 Peak areas of different reference solutions

[0065] ECG concentration (mg / mL) 0 2.5 5 10 20 Peak area 57437 6656376 12180671 22608661 42624227

[0066] The standard curve is: Y = 2100641*X + 1070670.

[0067] Substitute the peak area of the composite solution, 13294325, into the standard curve to calculate the concentration of ECG in the composite solution. The concentration of ECG in the composite solution obtained is 5.819 mg / mL.

[0068] Calculate the embedding situation of the composite solution embedding ECG according to the formula.

[0069] Embedding rate % = Concentration of ECG in the composite solution / Initial concentration of ECG.

[0070] Finally, it is obtained that the embedding rate of the composite solution = 5.819 / 10 = 58.19%.

[0071] Experimental Example 2

[0072] Determination of bioavailability

[0073] Preparation of the ECG standard curve:

[0074] Weigh a certain mass of ECG powder respectively to prepare working solutions with concentrations of 0, 25, 50, 100, and 150 μg / mL. Pass the working solutions through a 0.22 μm aqueous microfiltration membrane to obtain standard solutions with different concentrations.

[0075] Collection of mouse orbital blood:

[0076] Randomly divide 8 mice into 2 experimental groups and 1 control group, with 3 mice in each experimental group and 2 mice in the control group. The control group mice were gavaged with 0.3 mL / head of PBS, the experimental group 1 mice were gavaged with an ECG solution at a dose of 10 mg / kg (0.3 mL per head), and the experimental group 2 mice were gavaged with the composite solution of Example 1 at a dose of 10 mg / kg (0.3 mL per head). After 15 minutes, the 3 groups of mice were treated respectively; blood was collected from the orbital venous plexus and placed in a centrifuge tube, centrifuged at 306×g for 10 minutes to separate the plasma. All samples were stored at -80°C for future measurement.

[0077] Sample pretreatment:

[0078] Take 100 μL of plasma, dilute it to 1.0 mL with 10% formic acid, and process it with a solid-phase extraction column according to the following steps: The HLB column is pre-washed with 1.0 mL of acetone-formic acid mixture (volume ratio of acetone to formic acid is 9:1, the same below) and 1.0 mL of formic acid aqueous solution (volume ratio of formic acid to water is 1:9). Take 1.0 mL of the sample supernatant and load it into the filtration column at a flow rate of 1.0 mL / min. Add 1.0 mL of acetone-formic acid mixture to the filtration column for elution at a flow rate of 1.0 mL / min. Place the eluate in a fume hood to completely evaporate the solvent. Re-dissolve the remaining precipitate with 100 μL of formic acid aqueous solution.

[0079] Standard Curve and Determination of ECG Content in Plasma

[0080] Determination was carried out by HPLC method. Mobile phase A was acetonitrile, mobile phase B was formic acid with a volume fraction of 10%, and the detection wavelength was 280 nm. An elite C18 chromatographic column (250 mm × 4.6 mm, 5 μm) was selected for analysis, the column temperature was 30 °C, the flow rate was 1.0 mL / min, the injection volume was 20 μL, and each sample was analyzed for 10 min. The mobile phase gradient elution conditions were: 0 - 15 min, 5% - 15% A; 15 - 21 min, 15% - 28% A; 21 - 22 min, 28% - 40% A; 22 - 24 min, 40% - 60% A; 24 - 27 min, 60% - 5% A; 27 - 30 min, 5% A.

[0081] Using the peak area of the standard solution at different concentrations as the ordinate and the concentration as the abscissa, a standard curve was plotted. The results are shown in Table 2 and Figure 2 as follows.

[0082] Table 2 Peak Areas of Standard Solutions at Different Concentrations

[0083] ECG concentration (μg / ml) 0 25 50 100 150 Peak area 0 53392 101751 195442 259430

[0084] The obtained standard curve was: Y = 1740X + 8903, R 2 = 0.9914.

[0085] The peak intensity of ECG detected in the plasma after intragastric administration of ECG in experimental group 1 is as Figures 3 - 5 shown.

[0086] After intragastric administration of the compound solution in experimental group 2, the peak intensity of ECG detected in the plasma is as Figures 6 - 8 shown.

[0087] The peak intensity of ECG detected in the plasma of the control group mice is as Figures 9 - 10 shown.

[0088] The peak intensity was converted into peak area and substituted into the standard curve, and the content of ECG obtained is shown in Table 3. The bioavailability calculated according to the formula is shown in Table 3. The bioavailabilities of experimental group 1 and experimental group 2 were analyzed, and the results are as Figure 11 shown. Figure 11 Among them, β-cyclodextrin-ECG represents the group of experimental group 2 intragastrically administered with the composite solution, and ECG represents experimental group 1 intragastrically administered with ECG alone.

[0089] Bioavailability = content of ECG in mouse blood / total amount of intragastrically administered ECG × 100%.

[0090] Table 3 Bioavailability of flavonoids in different groups

[0091]

[0092] Table 3 and Figure 11 It can be seen that intragastrically administering ECG embedded with β-cyclodextrin (experimental group 2) to mice can significantly improve the bioavailability of ECG.

[0093] The present invention provides a method for improving the bioavailability of flavonoids and its application. The method of the present invention comprises the following steps: (1) mixing a flavonoid, β-cyclodextrin and water to obtain a mixed solution; (2) ultrasonicating the mixed solution for 8 - 12 min to obtain an inclusion complex; (3) dialyzing the inclusion complex for 20 - 28 h, and the liquid retained in the dialysis bag is the composite solution; the power of the ultrasonicating in step (2) is 55 - 65 W, each ultrasonicating lasts for 3 - 5 s, and pauses for 0.8 - 1.2 s. The embedding rate of the flavonoid embedded in the composite solution prepared according to the method of the present invention is ≥58.19%, and the bioavailability after intragastrically administering the composite solution to mice is ≥1.28%, which is higher than the bioavailability of intragastrically administering the flavonoid alone. It provides a basis for the development of health foods or drugs for preventing and / or treating Alzheimer's disease.

[0094] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for improving the bioavailability of flavonoids, characterized in that: The steps include: (1) mixing flavonoids, β-cyclodextrin and water to obtain a mixed solution; (2) ultrasonically treating the mixed solution for 8 to 12 minutes to obtain an inclusion compound; (3) dialyzing the inclusion compound for 20 to 28 hours, wherein the liquid retained in the dialysis bag is the complex solution; The power of the ultrasound in step (2) is 55-65W, and each ultrasound lasts for 3-5s, with a pause of 0.8-1.2s.

2. The method according to claim 1, characterized in that The flavonoid compound is epicatechin gallate or epigallocatechin gallate.

3. The method according to claim 2, characterized in that The mass ratio of the flavonoid compound to β-cyclodextrin is 1:2-4; The mass volume ratio of the flavonoid compound mixed with water is 6-10 mg:1 mL.

4. The method according to claim 3, characterized in that The molecular weight cutoff of the dialysis bag in step (3) is 900 to 1100 Da.

5. The composite solution obtained by the method according to any one of claims 1 to 4, characterized in that The embedding rate of the flavonoid compound in the composite solution is ≥58.19%; and the bioavailability of the flavonoid compound in the composite solution is ≥1.28%.

6. Use of the composite solution obtained by the method according to any one of claims 1 to 4 in the preparation of food or medicine for improving the bioavailability of flavonoids.

7. Use of the composite solution obtained by the method according to any one of claims 1 to 4 in preparing food or medicine for treating Alzheimer's disease.

8. Use of the composite solution according to claim 5 in preparing food or medicine for improving the bioavailability of flavonoids.

9. Use of the composite solution according to claim 8 in preparing food or medicine for treating Alzheimer's disease.

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