A method for improving bioavailability of puerarin

Abstract

The application provides a method for improving the bioavailability of puerarin, and belongs to the field of biological preparation, wherein puerarin and berberine are prepared into a puerarin-berberine compound, preferably hydrochloric acid berberine, and the molar ratio of the puerarin-berberine compound is 1:1, the method can improve the bioavailability of puerarin by tens of times, and further significantly improves the effect of puerarin on treating metabolic diseases caused by high-fat diet, improves physiological functions from multiple aspects, and has an excellent industrial application prospect.

Description

Technical Field

[0001] This invention provides a method for improving the bioavailability of puerarin, belonging to the field of biomaterial preparation technology. Background Technology

[0002] Puerarin, also known as puerarin flavonoid, is an isoflavone derivative with coronary vasodilatory effects isolated from the traditional Chinese medicine kudzu root. It is found in the roots of the legumes *Pueraria lobata* (Willd) Ohwi and *Pueraria thunbergiana* Benth. It has antipyretic, sedative, and coronary blood flow-increasing effects, and protects against acute myocardial hemorrhage induced by posterior pituitary extract. Clinically, it is used for angina pectoris and hypertension. Appearance: White to slightly yellow crystalline powder. Solubility: Soluble in methanol, slightly soluble in ethanol, sparingly soluble in water, insoluble in chloroform or ether. While commercially available puerarin is a stable compound, clinical use has revealed low bioavailability. Furthermore, it is difficult to prepare salts, as salts are unstable. Even when prepared as salts, bioavailability cannot be significantly improved. This problem remains unresolved in the research field, and whether improving bioavailability can significantly enhance the efficacy of related drugs is also an unknown question in this area. Summary of the Invention

[0003] In order to solve the above-mentioned technical problems, the applicant intends to provide a method for improving the bioavailability of puerarin, the method involving the preparation of puerarin-berberine complex from puerarin and berberine.

[0004] Furthermore, in the above method, the berberine is berberine hydrochloride.

[0005] Furthermore, in the above method, the molar ratio of puerarin to berberine in the puerarin-berberine complex is 1:0.1-10.

[0006] Further, in the above method, the preparation method of the puerarin-berberine complex is as follows: puerarin is dissolved in an alkaline solution, and berberine is prepared as an aqueous solution; or puerarin and berberine are separately prepared as aqueous solutions, an alkaline solution is added, the mixture is stirred and precipitated, and the precipitate is obtained by filtration. Further, in the above method, the weight ratio of puerarin to berberine is 1:0.1-10.

[0007] Furthermore, in the above method, the molar ratio of puerarin to berberine is 1:0.5-2.

[0008] Furthermore, in the above method, the molar ratio of puerarin to berberine is 1:0.8-1.5.

[0009] Furthermore, in the above method, the alkaline solution is selected from KOH or NaOH solution.

[0010] Furthermore, the present invention provides a puerarin-berberine complex obtained by the above method.

[0011] Furthermore, the present invention provides the application of puerarin-berberine complex in the preparation of a medicament for treating metabolic diseases caused by a high-fat diet.

[0012] In this invention, the puerarin-berberine complex is mainly puerarin-berberine salt, so they can be generally considered to be the same name.

[0013] This invention provides a method for improving the bioavailability of puerarin. The method innovatively synthesizes puerarin with berberine, revealing that the two components interact to form a novel complex. This complex exhibits excellent puerarin bioavailability, differing from previous teachings that berberine promotes the absorption of puerarin bioavailability. Previous methods for improving bioavailability have not achieved such a significant leap. This method is simple, involving dissolving puerarin and berberine separately, then adding an alkaline solution to drive the reaction, precipitating the new complex. This is a novel preparation method. Evaluation indicators from various high-fat diet models show that this method significantly improves the bioavailability of puerarin, while the dissolution and bioavailability of berberine remain unchanged. This demonstrates that the significant improvement in efficacy of the prepared salts stems from the increased bioavailability of puerarin, while the effect of berberine remains almost unchanged, indirectly reflecting the significance of this method in improving puerarin bioavailability. Attached Figure Description

[0014] Figure 1 This is the NMR spectrum of berberine puerarin salt;

[0015] Figure 2 This is the NMR spectrum of a physical mixture of berberine and puerarin;

[0016] Figure 3 This is the infrared spectrum of berberine puerarin salt;

[0017] Figure 4 This is the infrared spectrum of a physical mixture of berberine and puerarin;

[0018] Figure 5 This is a DSC diagram of berberine puerarin salt;

[0019] Figure 6 This is a DSC diagram of a physical mixture of berberine and puerarin;

[0020] Figure 7This is a single-crystal diffraction pattern of berberine puerarin salt;

[0021] Figure 8 This is a cell packing diagram of berberine puerarin salt;

[0022] Figure 9 This is the cumulative dissolution curve of puerarin in the sample in water;

[0023] Figure 10 This is a cumulative dissolution curve of berberine in water;

[0024] Figure 11 The plasma concentration-time curve of puerarin in mice was obtained by gavage administration of various samples to C57 mice.

[0025] Figure 12 The plasma concentration-time curves of berberine in mice were obtained by gavage administration of various samples to C57 mice.

[0026] Figure 13 The study compared liver triglyceride levels in different drug-treated experimental animals in a high-fat diet-induced metabolic disease model of C57 mice.

[0027] Figure 14 The comparison of plasma cholesterol levels in experimental animals from different drug administration groups was conducted in a high-fat diet-induced metabolic disease model in C57 mice.

[0028] Figure 15 The study compared the low-density lipoprotein cholesterol levels in the plasma of experimental animals in different drug administration groups in a high-fat diet-induced metabolic disease model of C57 mice.

[0029] Figure 16 The study compared the triglyceride levels in the plasma of experimental animals in different drug administration groups in a high-fat diet-induced metabolic disease model of C57 mice.

[0030] Figure 17 The comparison of blood glucose levels in experimental animals of different drug administration groups in a high-fat diet-induced metabolic disease model of C57 mice;

[0031] Figure 18 The study compared the plasma alanine aminotransferase (ALT) levels in experimental animals from different drug administration groups in a high-fat diet-induced metabolic disease model in C57 mice.

[0032] Figure 19 The study compared the levels of aspartate aminotransferase (AST) in the plasma of experimental animals in different drug administration groups in a high-fat diet-induced metabolic disease model of C57 mice.

[0033] Figure 20 This is the result of Oil Red staining of liver tissue sections from experimental animals in different drug administration groups in a high-fat diet-induced metabolic disease model of C57 mice. Detailed Implementation

[0034] The following examples are used to further illustrate, but are not limited to, the present invention. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. The rats and mice described below were provided by Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0035] The beneficial effects of the traditional Chinese medicine composition described in this invention will be further illustrated through the following experiments.

[0036] Example 1: Determination of the cumulative dissolution curves of puerarin and berberine in water

[0037] Preparation of Berberine-Puerariae Salt: 1. Add 5g of berberine chloride (molecular weight: 371.86) to 300mL of water and heat to 80℃ to dissolve. Separately, add 5.6g of puerariae (molecular weight: 416.36) to 100mL of water, and add 0.49g of potassium hydroxide until the system becomes colorless and clear. While stirring at room temperature, slowly add the above berberine aqueous solution. After the addition is complete, stir at room temperature and let stand; a solid precipitates. Filter to obtain the solid, dry at 50℃ for 5h to obtain 9.5g of berberine-puerariae salt.

[0038] (Molar ratio approximately 1:1)

[0039] Preparation of the berberine-puerarin mixture: 7.5 g of berberine chloride and 8.4 g of puerarin were placed in a mortar and repeatedly ground and mixed to obtain 15.8 g of the berberine-puerarin mixture (molar ratio 1:1).

[0040] berberine baicalin salt NMR 1 H NMR determination

[0041] The prepared berberine-puerarin salt and berberine-puerarin mixture were analyzed by 1H NMR spectroscopy.

[0042] Preparation of Berberine-Puerariae Salt: 10 g of berberine chloride was added to 300 mL of water and heated to 80 °C to dissolve. Separately, 5.6 g of puerariae was added to 100 mL of water, followed by 0.98 g of potassium hydroxide, until the system became colorless and clear. The above berberine aqueous solution was slowly added dropwise with stirring at room temperature. After the addition was complete, the mixture was stirred at room temperature and allowed to stand, resulting in the precipitation of a solid. The solid was filtered and dried at 50 °C for 5 h to obtain 9.5 g of berberine-puerariae salt. (Molar ratio of puerariae to berberine: 1:2)

[0043] Preparation of Berberine-Puerariae Salt: 3. Add 5g of berberine chloride to 300mL of water and heat to 80℃ to dissolve. Separately, add 11.2g of puerariae to 100mL of water, and add 0.49g of potassium hydroxide until the system becomes colorless and clear. While stirring at room temperature, slowly add the above berberine aqueous solution. After the addition is complete, stir at room temperature and let stand; a solid precipitates. Filter to obtain the solid, dry at 50℃ for 5h to obtain 9.5g of berberine-puerariae salt. (Molar ratio of puerariae to berberine: 1:0.5)

[0044] In the above preparation method, the applicant tried to mix and dissolve berberine and puerarin in water, or dissolve them separately in water and then mix them, and then add the alkali solid. It was found that it was almost impossible to obtain the relevant complex solid, which also proves the randomness and specificity of the preparation method.

[0045] Figure 1 This is the 1H NMR spectrum of berberine-puerarin salt.

[0046] 1H NMR(500MHz,DMSO-d6)δ9.88(s,1H),8.92(s,1H),8.19(d,J=9.1Hz,1H),7.98(d,J=9.0Hz,1H),7.94(s,1H), 7.79(s,1H),7.53(d,J=9.0Hz,1H),7.34(d,J=8.4Hz,2H),7.08(s,1H),6.75(d,J=8.3Hz,2H),6.32(d,J=9.0 Hz,1H),6.17(s,2H),4.92(d,J=7.9Hz,2H),4.83(s,2H),4.59(d,J=9.9Hz,1H),4.09(s,3H),4.06(s,3H),4. 03(t,J=9.3Hz,1H),3.66(d,J=11.5Hz,1H),3.42(d,J=5.1Hz,3H),3.23(t,J=8.4Hz,2H),3.20-3.13(m,4H).

[0047] Figure 2 The 1H NMR spectrum of a physical mixture of berberine and puerarin.

[0048] 1H NMR (600MHz, DMSO-d6) δ9.89(s,1H),9.60(s,1H),8.93(s,1H),8.32(s,1H),8.18(d,J=9.1Hz,1H),7.99(d,J=9.0Hz, 1H),7.90(d,J=8.8Hz,1H),7.78(s,1H),7.44–7.32(m,2H),7.07(s,1H),7.04(d,J=8.8Hz,1H),6.85–6.75(m,2H),6.1 7(s,2H),5.04(d,J=16.1Hz,2H),4.93(t,J=6.4Hz,2H),4.89–4.76(m,2H),4.56(s,1H),4.10(s,3H),4.06(s,3H),4. 03(d,J=10.2Hz,1H),3.78–3.68(m,1H),3.46(s,1H),3.29(t,J=8.6Hz,1H),3.27–3.22(m,2H),3.20(t,J=6.4Hz,2H).

[0049] Depend on Figure 1 , 2 The comparison revealed that berberine-puerarin salt is a completely new substance and no longer belongs to a mixture.

[0050] Figure 3 , Figure 4 Infrared spectra of berberine-puerarin salt and physical mixtures of berberine and puerarin. Appropriate amounts of each sample were mixed with KBr and then compressed into tablets. The scanning range was 400-4000 cm⁻¹. -1 As shown in the figure, the infrared spectrum of berberine-puerarin salt is at 3549 cm⁻¹. -1 The characteristic absorption peak of berberine disappears at 2901 cm⁻¹. -1 The characteristic absorption peak of puerarin disappeared at 441.5 cm⁻¹. -1 503.3cm -1 536.1cm -1 New characteristic absorption peaks appeared in all of them.

[0051] Figure 5 , Figure 6This is a DSC spectrum of berberine-puerarin salt and a physical mixture of berberine and puerarin. For each sample, 2-5 mg was accurately weighed and placed in an aluminum crucible, with an empty crucible of the same type used as a reference. The protective atmosphere was 99% nitrogen, the flow rate was 60 mL / min, the sample scan rate was 10 °C / min, and the scan range was 25-280 °C. As shown in the figure, berberine-puerarin salt exhibits an endothermic peak at 97.3 cel, while the physical mixture of berberine and puerarin shows endothermic peaks at 96.7 cel, 150.4 cel, and 197.2 cel.

[0052] Further structural analysis was performed on the prepared berberine puerarin salt crystals. The crystal analysis parameters are shown below, and the crystal structure diagram (ignoring the solvent part) is as follows. Figure 7 As shown, the unit cell packing diagram is as follows: Figure 8 As shown.

[0053] Berberine puerarin salt crystal data and structure:

[0054]

[0055] Chromatographic conditions for puerarin determination: Column: Agilent ZORBAX SB-C 18 Column (4.6 × 250 mm, 5 μm); Mobile phase: methanol-water (25:75); Column temperature: 30℃; Detection wavelength: 250 nm; Flow rate: 1 mL·min -1 Injection volume: 10 μL.

[0056] Chromatographic conditions for berberine determination: Column: Kromasil C 18 Column: 4.6 × 150 mm, 5 μm; Mobile phase: acetonitrile-0.05 mol / L sodium dihydrogen phosphate (adjusted to pH 3 with phosphoric acid) (28:72); Column temperature: room temperature; Detection wavelength: 345 nm; Flow rate: 1 mL / min -1 Injection volume: 10 μL.

[0057] Samples were taken according to the paddle method specified in the appendix of the 2015 edition of the Chinese Pharmacopoeia, with a rotation speed of 100±1 r / min, a water bath temperature of 37℃, and water as the dissolution medium. The dosage was equivalent to 16 mg of berberine and 18 mg of puerarin. 2 mL samples were taken at 0.25, 0.5, 1, 2, 3, 4, 6, 8, 10, 12, and 24 h, respectively, and filtered through a 0.45 μm filter membrane. Simultaneously, 2 mL of dissolution medium was added at the same temperature. 1 mL of the filtrate was diluted with the mobile phase, and the cumulative dissolution amount was determined using the above HPLC method. Figure 9-10 The cumulative dissolution curves of berberine and puerarin in each sample are shown. Figure 9-10It is evident that only 2.9% of puerarin dissolves from the physical mixture after 24 hours. After the berberine-puerarin salt is formed, the solubility of puerarin significantly increases, with a cumulative dissolution of 30% over 4 hours.

[0058] Berberine water-puerarin salt slowed down the dissolution rate of berberine.

[0059] Example 2: Bioavailability of puerarin in mice

[0060] Chromatographic conditions for puerarin: Column: Agilent ZORBAX SB-C 18 Column (4.6 × 250 mm, 5 μm); Mobile phase: methanol - 10 mmol / L -1 Ammonium acetate buffer-acetonitrile (70:20:10); column temperature: 30℃; detection wavelength: 250nm; flow rate: 0.2mL·min -1 Injection volume: 5 μL; Internal standard (IS): hesperidin.

[0061] Mass spectrometry conditions: Ion source: electrospray ionization (ESI); voltage: 5500V; temperature: 350℃; multiple reaction ion detection (MRM) mode for positive ion detection; monitored ion pairs: puerarin m / z 417.2 / 267.2, IS m / z 611.2 / 303.2.

[0062] Chromatographic conditions for berberine determination: Column: Shim-pack XR-ODSⅡ column (3mm×75mm, 2.3μm); Internal standard (IS): berberine hydrochloride; Column temperature: 30℃; Injection volume: 10μL; Mobile phase: A is 0.5% formic acid aqueous solution, B is acetonitrile, gradient elution (0-4min, 85% A + 15% B; 5-6min, 20% A + 80% B); Flow rate: 0.5mL / min.

[0063] Mass spectrometry conditions: Ion source: electrospray ionization (ESI); IS: 5500V; Temperature: 550℃; CUR: 20V; CE: 35V; DP: 50V; CAD: mediam; Detection mode: positive ion mode; Monitored ion pairs: BBR m / z 398.2 / 308.2, IS m / z 392.1 / 312.1.

[0064] One hundred and sixty-five C57 mice were randomly divided into three groups and administered puerarin, a physical mixture of berberine and puerarin, and berberine-puerarin salt by gavage, respectively. Five mice from each group were sacrificed at 0.25, 0.5, 1, 2, 4, 6, 8, 12, 16, 20, and 24 hours after administration. 500 μL of blood was collected and placed in an EP tube containing 1% heparin sodium. The tube was centrifuged at 3000 rpm for 10 min at 4°C. 300 μL of plasma was collected in a 1.5 mL EP tube, and 300 μL of 10 mmol / L ammonium acetate buffer and 20 μL of internal standard solution were added. After mixing, the mixture was loaded onto a solid-phase extraction column, washed with 1.0 mL of double-distilled water, and eluted with 0.5 mL of methanol. The eluent was collected, dried under nitrogen at 45°C, reconstituted with 150 μL of mobile phase, and injected in 5 μL for analysis. Figure 11-12 The drug-time curves for puerarin and berberine in each sample are shown. Figure 11-12 It is evident that the bioavailability of puerarin in the berberine-puerarin physical mixture is the same as that of puerarin monotherapy, while the bioavailability of puerarin in the berberine-puerarin salt is significantly higher than that of puerarin monotherapy and the berberine-puerarin physical mixture. The bioavailability of berberine in the berberine-puerarin salt is the same as that of berberine monotherapy and the berberine-puerarin physical mixture.

[0065] Table 1

[0066]

[0067] Table 2

[0068]

[0069] Example 3

[0070] Application of berberine-puerarin salt

[0071] Its characteristic is its use in the preparation of drugs for treating metabolic-related diseases. This invention establishes models of hyperlipidemia, hyperglycemia, obesity, hypercoagulability, and fatty liver disease in C57 mice by feeding them a high-sugar, high-fat diet. Experimental Design

[0072] Eight-week-old C57 mice were randomly divided into five groups: control group (normal diet), model group (high-sugar and high-fat diet), puerarin group (high-fat diet + puerarin monotherapy), berberine and puerarin physical mixture group (high-fat diet + berberine and puerarin physical mixture), and berberine-puerarin salt group (high-fat diet + berberine-puerarin salt), with six mice in each group.

[0073] The animals in each group were administered the drugs by gavage. The control group and model group mice were given 10 mL / kg / d of distilled water. In the puerarin, berberine-puerarin physical mixture group and berberine-puerarin salt group, the berberine dose was 100 kg / d and the puerarin dose was 120 mg / kg / d, administered once daily for 8 consecutive weeks.

[0074] Animals were weighed weekly. After administration, they were euthanized by intraperitoneal injection of 1 mL of 20% chloral hydrate, blood was collected from the eyes, and the liver was harvested. Blood biochemical parameters were measured using an automated biochemical analyzer. The results showed that berberine-puerarin salt reduced triglycerides in the liver, lowered blood lipids and blood sugar, and reduced weight gain. These effects were superior to those of a physical mixture of berberine and puerarin, and even more superior to puerarin alone. Therefore, berberine and puerarin salt can effectively prevent and treat metabolic diseases such as hyperlipidemia, hyperglycemia, obesity, platelet aggregation, and fatty liver disease.

[0075] 1. Effect of berberine-puerarin salt on triglyceride content in the liver of experimental animals

[0076] The results of liver triglyceride content determination are shown in Figure 13 And Table 3.

[0077] Table 3 Results of liver triglyceride content determination

[0078]

[0079] In animals raised on a high-fat diet, triglyceride levels in the liver were significantly higher than in the control group. Berberine-puerarin salt effectively reduced the increase in liver triglyceride levels induced by a high-fat diet, and the reduction was more significant than that of the physical mixture of berberine and puerarin. Puerarin itself did not significantly reduce liver triglycerides.

[0080] 2. Effects of berberine-puerarin salt on blood lipids and blood glucose in experimental animals

[0081] Blood lipid testing mainly targets the levels of cholesterol, low-density lipoprotein, and triglycerides in blood plasma.

[0082] The results of plasma cholesterol levels are shown in Figure 14 And in Table 4.

[0083] Table 4 Total Cholesterol Content in Plasma

[0084]

[0085] The results of plasma low-density lipoprotein levels were shown in Figure 15 And in Table 5.

[0086] Table 5. Plasma Low-Density Lipoprotein-Cholesterol Content

[0087]

[0088] The results of plasma triglyceride levels are shown in Figure 16 And in Table 6.

[0089] Table 6 Plasma Triglyceride Content

[0090]

[0091] The results of blood glucose (Glucose) levels are shown in Figure 17 and Table 7

[0092] Table 7 Blood Glucose Content

[0093]

[0094]

[0095] After a high-fat diet model was established, blood glucose, cholesterol, low-density lipoprotein, and triglycerides in animal plasma increased significantly. The physical mixture of berberine and puerarin showed a blood glucose and lipid-lowering effect. Berberine-puerarin salt effectively reduced the lipid elevation induced by a high-fat diet, and the reduction was more significant than that of the berberine-puerarin physical mixture. Puerarin alone did not have a significant blood glucose or lipid-lowering effect.

[0096] 3. Effects of berberine-puerarin salt on liver function in experimental animals

[0097] Figure 18 Table 8 shows the comparison of alanine aminotransferase (ALT) levels in mice in each group after the intervention.

[0098] Table 8. Alanine aminotransferase (ALT) levels

[0099]

[0100] As shown in the figure, ALT levels in the model group were significantly elevated compared to the blank control group. Compared to the model group, berberine-puerarin salt significantly reduced ALT levels induced by a high-fat diet, and the reduction was more significant than that of the berberine-puerarin physical mixture. Puerarin alone did not significantly reduce ALT levels.

[0101] Figure 19 Table 9 shows the comparison of aspartate aminotransferase (AST) levels in mice in each group after the intervention.

[0102] Table 9 Aspartate aminotransferase (AST) levels

[0103]

[0104] As shown in the figure, there was no significant difference in AST levels between the model group and the blank control group. There were also no significant differences in puerarin, berberine-puerarin salt, and berberine-puerarin physical mixtures compared to the model group.

[0105] Figure 20 The results of oil red staining of liver tissue sections from experimental animals.

[0106] As shown in the figure, compared with the blank control group, the high-fat diet group showed a significant increase in lipid deposition in the liver tissue; the berberine-puerarin salt group showed a significant reduction in oil red staining of the liver, indicating a decrease in liver lipid deposition. These results suggest that drug intervention can inhibit the deposition of triglycerides and other neutral fats in the liver, improving hepatic steatosis, and the degree of improvement in liver tissue degeneration is significantly stronger than that in the berberine-puerarin physical mixture group. Puerarin itself does not significantly inhibit the deposition of triglycerides and other neutral fats in the liver. Therefore, increasing the bioavailability of puerarin can simultaneously inhibit the opportunistic effects of hepatic steatosis. This is a new function brought about by the complex after improving the bioavailability of puerarin through relevant methods.

Claims

1. A method for preparing berberine puerarin salt, characterized in that, 5g of berberine chloride was added to 300mL of water and heated to 80℃ to dissolve. Separately, 5.6g of puerarin was added to 100mL of water, and 0.49g of potassium hydroxide was added until the system became colorless and clear. The above berberine aqueous solution was slowly added dropwise while stirring at room temperature. After the addition was complete, the mixture was stirred at room temperature and allowed to stand. A solid precipitated out. The solid was filtered and dried at 50℃ for 5 hours to obtain 9.5g of berberine puerarin salt.

2. A berberine puerarin salt, characterized in that, It is prepared by the method described in claim 1.

3. The use of the berberine puerarin salt according to claim 2 in the preparation of drugs that improve the bioavailability of puerarin.

4. The use of berberine puerarin salt according to claim 2 in the preparation of a medicament for treating metabolic diseases caused by a high-fat diet.

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