Preparation method of glycyrrhetinic acid berberine salt as well as product prepared by preparation method and application of glycyrrhetinic acid berberine salt

Abstract

The invention belongs to the technical field of biological medicines, and relates to a preparation method of glycyrrhetinic acid berberine salt, a product prepared by the preparation method and application of the product. The preparation method of the glycyrrhetinic acid berberine salt comprises the following specific steps: S1, dissolving berberine hydroxide into a solvent A to obtain a berberine hydroxide solution; and S2, adding glycyrrhetinic acid into the berberine hydroxide solution, fully reacting, and collecting a reaction product after the reaction is finished, thereby obtaining the glycyrrhetinic acid berberine salt. The glycyrrhetinic acid berberine salt prepared by the preparation method disclosed by the invention is high in yield (more than 90%) and high in purity (more than 90%), and can be prepared into nano particles through a nano precipitation method for treating liver cancer.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a preparation method of berberine glycyrrhetinic acid salt and a product prepared therefrom and application thereof. Background Art

[0002] Berberine (BB), also known as berberine, is a quaternary ammonium alkaloid isolated from the traditional Chinese medicine Coptis chinensis. It is the main active ingredient of Coptis chinensis for antibacterial effect. It is yellow needle-shaped crystals and bitter. It is widely distributed in the plant kingdom, and berberine is found in about 4 families and 10 genera. Existing studies have shown that berberine produces anti-tumor effects by inhibiting proliferation, promoting apoptosis, blocking cycles, inhibiting tumor migration and invasion, inducing autophagy, anti-inflammatory and anti-oxidative effects, and immunomodulation. There are more than 80 clinical trials worldwide studying the medicinal value of BB in various diseases such as anti-tumor. Its low water solubility and poor oral bioavailability limit its clinical application. Structural modification of BB is an effective way to solve this dilemma. Using anion substitution strategy, the anion part of BB is replaced with organic acid, which can not only keep the parent structure of BB unchanged, but also change the physical and chemical properties of BB to synergistically increase its pharmacological activity. Studies have shown that BB can enhance its anti-type 2 diabetes activity after anion substitution with organic acids such as citric acid, malic acid, succinic acid and fumaric acid.

[0003] Glycyrrhizic acid is the main active ingredient of the commonly used Chinese medicine licorice. Glycyrrhetinic acid (GA) is the active metabolite of glycyrrhizic acid in vivo. Glycyrrhetinic acid not only has anti-inflammatory, anti-ulcer, anti-allergic, anti-viral, and lipid-lowering effects, but also has certain anti-cancer effects. Chinese patent CN 101935334A discloses a berberine glycyrrhetinic acid enantiomeric salt and its preparation method application. The patent records that berberine glycyrrhetinic acid enantiomeric salt can be used to prepare anti-tumor drugs and drugs for treating diabetes. However, the yield of berberine glycyrrhetinic acid enantiomeric salt in the preparation method disclosed in the patent is about 50%, which is low in yield and purity. Summary of the invention

[0004] In view of the problems and shortcomings in the prior art, the present invention aims to provide a method for preparing berberine glycyrrhetinic acid salt and the products prepared therefrom and their applications.

[0005] To achieve the purpose of the invention, the technical solution adopted by the present invention is as follows:

[0006] The present invention first provides a method for preparing glycyrrhetinic acid berberine salt, the method comprising the following steps:

[0007] S1: dissolving berberine hydroxide in solvent A to obtain a berberine hydroxide solution;

[0008] S2: adding glycyrrhetinic acid to the berberine hydroxide solution to react fully, and collecting the reaction product after the reaction is completed to obtain glycyrrhetinic acid berberine salt.

[0009] According to the above-mentioned preparation method of glycyrrhetinic acid berberine salt, preferably, in step S2, the molar ratio of berberine hydroxide to glycyrrhetinic acid is (0.5-2):1.

[0010] According to the preparation method of the above-mentioned glycyrrhetinic acid berberine salt, preferably, when the solvent A is an organic solvent, the specific operation of step S2 is: add glycyrrhetinic acid to the berberine hydroxide solution, the molar ratio of berberine hydroxide to glycyrrhetinic acid is (0.5-2):1, stir and react at 40-60°C for 12-48h, after the reaction, remove the solvent A and dry the reaction solution to obtain the glycyrrhetinic acid berberine salt. More preferably, the molar ratio of berberine hydroxide to glycyrrhetinic acid is 1:1; the reaction temperature is 50°C, the reaction time is 12h; the solvent A is methanol, and the method of removing the solvent A is vacuum rotary evaporation.

[0011] According to the preparation method of the glycyrrhetinic acid berberine salt, preferably, when the solvent A is water, the specific operation of step S2 is: add glycyrrhetinic acid to the berberine hydroxide solution, the molar ratio of berberine hydroxide to glycyrrhetinic acid is (0.5-2):1, stir while adding, after all the glycyrrhetinic acid is added, react at 40-60°C for 6-24h, filter after the reaction, collect the precipitate, wash and dry the precipitate to obtain glycyrrhetinic acid berberine salt. More preferably, the molar ratio of berberine hydroxide to glycyrrhetinic acid is 1:1; the reaction temperature is 50°C, and the reaction time is 24h.

[0012] According to the preparation method of the above-mentioned berberine glycyrrhetinic acid salt, preferably, in step S1, the preparation method of the berberine hydroxide is: berberine hydrochloride is dissolved in solvent B to obtain a berberine hydrochloride solution; alkaline ion exchange resin is added to the berberine hydrochloride solution, mixed, reacted, filtered after the reaction is completed, the filtrate is collected, the solvent B in the filtrate is removed, and vacuum drying is performed to obtain berberine hydroxide.

[0013] According to the preparation method of the above-mentioned berberine glycyrrhetinic acid salt, preferably, the exchange ratio of berberine hydrochloride to the basic ion exchange resin is 0.15 mmol / g to 1.5 mmol / g. More preferably, the exchange ratio of berberine hydrochloride to the basic ion exchange resin is 0.15 mmol / g.

[0014] According to the preparation method of the above-mentioned berberine salt of glycyrrhetinic acid, preferably, the alkaline ion exchange resin needs to be activated before use, and the specific operation of the activation treatment is: adding the alkaline ion exchange resin into an alkaline solution for soaking treatment, and washing after soaking; adding the washed alkaline ion exchange resin into solvent C for soaking treatment, and drying after soaking to obtain an activated alkaline ion exchange resin.

[0015] According to the preparation method of the glycyrrhetinic acid berberine salt, preferably, the alkaline ion exchange resin is AMBERLYST TM A26OH, the alkali solution is a sodium hydroxide solution. More preferably, the concentration of the sodium hydroxide solution is 5%.

[0016] According to the above-mentioned preparation method of berberine glycyrrhetinic acid salt, preferably, the solvent B and solvent C are both organic solvents; more preferably, the organic solvent is at least one of methanol and ethanol; most preferably, the organic solvent is methanol.

[0017] According to the preparation method of berberine glycyrrhetinic acid salt, preferably, the specific operation of removing solvent B from the filtrate is: subjecting the filtrate to vacuum rotary evaporation at 40-70° C. More preferably, the specific operation of removing solvent B from the filtrate is: subjecting the filtrate to vacuum rotary evaporation at 50° C.

[0018] According to the preparation method of the glycyrrhetinic acid berberine salt, preferably, in step S1, the preparation method of berberine hydroxide is:

[0019] Dissolve berberine hydrochloride in solvent D to obtain a berberine hydrochloride solution; add silver oxide to the berberine hydrochloride solution for reaction, filter after the reaction to obtain a berberine hydroxide solution, remove the solvent in the berberine hydroxide solution to obtain berberine hydroxide.

[0020] According to the preparation method of the above-mentioned berberine glycyrrhetinic acid salt, preferably, the molar ratio of berberine hydrochloride to silver oxide is 1:(1-20); the reaction temperature of the berberine hydrochloride and silver oxide is 40-60°C, and the reaction time is 6-24h. More preferably, the molar ratio of berberine hydrochloride to silver oxide is 1:5; the reaction temperature of the berberine hydrochloride and silver oxide is 50°C, and the reaction time is 12h.

[0021] According to the above-mentioned preparation method of berberine glycyrrhetinic acid salt, preferably, the solvent D is water.

[0022] The second aspect of the present invention provides a glycyrrhetinic acid berberine salt product prepared by the preparation method described in the first aspect.

[0023] The third aspect of the present invention provides the use of the glycyrrhizic acid berberine salt product described in the second aspect in the preparation of a drug for preventing, treating or / and alleviating liver cancer.

[0024] The fourth aspect of the present invention provides a drug for preventing, alleviating and / or treating liver cancer. The drug includes glycyrrhetinic acid berberine salt nanoparticles. The glycyrrhetinic acid berberine salt nanoparticles are prepared by nanoprecipitation method using the glycyrrhetinic acid berberine salt product described in the second aspect as a raw material.

[0025] According to the above-mentioned medicine, the steps of the preparation method of glycyrrhizic acid berberine salt nanoparticles are as follows:

[0026] A. dissolving glycyrrhetinic acid berberine salt in dimethyl sulfoxide to obtain a glycyrrhetinic acid berberine salt solution;

[0027] B. Under ultrasonic conditions, the berberine glycyrrhetinic acid salt solution is added dropwise to a Tween-80 solution to obtain a mixed solution, the mixed solution is centrifuged, and the supernatant is removed to obtain nanoparticles; the nanoparticles are freeze-dried to obtain berberine glycyrrhetinic acid salt nanoparticles (BB-GANDs).

[0028] According to the above-mentioned medicine, preferably, the concentration of berberine glycyrrhetinic acid salt in the berberine glycyrrhetinic acid salt solution is 0.01 g / mL.

[0029] According to the above-mentioned medicine, preferably, the concentration of the Tween-80 solution is 0.002 g / mL.

[0030] According to the above-mentioned medicine, preferably, the power of the ultrasound is 240W.

[0031] According to the above-mentioned medicine, preferably, the medicine is in the form of an injection.

[0032] The principle of preparing glycyrrhetinic acid berberine salt by reacting berberine hydroxide with glycyrrhetinic acid in the present invention is that berberine hydroxide reacts with the carboxylate in glycyrrhetinic acid and dehydrates to obtain glycyrrhetinic acid berberine salt.

[0033] Compared with the prior art, the present invention has the following positive and beneficial effects:

[0034] (1) The present invention first obtains berberine hydroxide by ion exchange between berberine hydrochloride and ion exchange resin or reacts berberine hydrochloride with silver oxide to obtain berberine hydroxide, and then reacts berberine hydroxide with glycyrrhetinic acid to generate glycyrrhetinic acid berberine salt; compared with the existing preparation method, the preparation method of glycyrrhetinic acid berberine salt of the present invention has the advantages of high yield (>90%) and high purity (>90%) of glycyrrhetinic acid berberine salt, among which the highest yield of glycyrrhetinic acid berberine salt reaches 96.78%, and the highest purity reaches 97.82%, which are much higher than the existing preparation method.

[0035] (2) The glycyrrhetinic acid berberine salt prepared by the present invention can be prepared into nanoparticles (BB-GANDs) by nanoprecipitation method. The particle size of the nanoparticles is 307.8 nm and the Zeta potential is -13.11±2.11 mV, which is conducive to the in vivo circulation of BB-GA NDs. At the same time, BB-GANDs can be prepared into injectable drugs. The present invention further verifies that BB-GANDs have the effect of preventing, alleviating or / and treating liver cancer through in vitro cell experiments and in vivo mouse experiments. Since glycyrrhetinic acid has the characteristics of targeting and direct transmembrane delivery, BB-GANDs have liver cancer cell targeting through the synergistic effect of BB and GA, which greatly improves the targeting and therapeutic effect of BB-GANDs on liver cancer treatment. In addition, BB-GANDs have long-acting sustained release performance in vivo, without the need for additional carriers, and effectively improves the biosafety of berberine hydrochloride injected through the tail vein. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the embodiments or solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0037] Figure 1 The NMR images of BB, GA and BB-GA; BBGA represents berberine glycyrrhetinic acid salt (BB-GA);

[0038] Figure 2 : XRD patterns of GA, BB and BB-GA; wherein BBGA represents berberine glycyrrhetinic acid salt (BB-GA);

[0039] Figure 3 is the HPLC chart of BB-GA;

[0040] Figure 4 is the scanning electron microscopy image of BB-GANDs;

[0041] Figure 5 The hydrodynamic diameter and Zeta potential test results of BB-GANDs;

[0042] Figure 6 This is the result of hemolysis rate test of BB-GANDs;

[0043] Figure 7 Figure 1 is a graph showing the results of cell viability detection after BB-GA was co-incubated with liver cancer cells and normal liver cells; a is a graph showing the results of cell viability detection after BB-GA was co-incubated with liver cancer cells Huh7 and normal liver cells L02 for 48 hours; b is a graph showing the results of cell viability detection after liver cancer cells Huh7 were co-incubated with BB, GA, GA+BB, and BB-GA for 48 hours;

[0044] Figure 8 The figure shows the survival rate test results of Kunming mice under different treatment groups; Control represents the Control group, BB represents the BB group, GA represents the GA group, and BB-GA represents the BB-GA NDs group;

[0045] Fig. 9 The figures are tumor tissue pictures, tumor volume change curves and tumor inhibition rate detection results under different treatment groups; a is the tumor tissue picture, b is the tumor volume change curve, and c is the statistical result of tumor inhibition rate; data are expressed as mean ± SEM; compared with the control group: *p<0.05, **p<0.01. DETAILED DESCRIPTION

[0046] The following examples are only for further explanation of the present invention. It should be noted that all technical and scientific terms used in the present invention have the same meanings as in the technical field to which the present invention belongs unless otherwise specified. The experimental methods in the following examples without specifying specific conditions are all based on conventional techniques in the technical field, or according to the conditions recommended by the manufacturer; the reagents or instruments used without specifying the manufacturer are all conventional products that can be purchased commercially.

[0047] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0048] (I) Berberine hydrochloride reacts with ion exchange resin to prepare berberine hydroxide, and berberine hydroxide reacts with glycyrrhetinic acid to prepare glycyrrhetinic acid berberine salt

[0049] Example 1: Discussion on the exchange ratio of berberine hydrochloride and basic ion exchange resin

[0050] In order to explore the effect of the exchange ratio of berberine hydrochloride to alkaline ion exchange resin on the preparation of glycyrrhetinic acid berberine salt, the present invention carried out Examples 1-1 to 1-3. The specific contents of Examples 1-1 to 1-3 are as follows:

[0051] Example 1-1:

[0052] A method for preparing glycyrrhetinic acid berberine salt, comprising the following specific steps:

[0053] (1) Activation of basic ion exchange resin: Activate basic ion exchange resin AMBERLYST TM A26OH was placed in a chromatography column, and a peristaltic pump was used to circulate and soak the resin in a 5% sodium hydroxide solution at 0.7 mL / min for 12 hours, and then the resin was rinsed with purified water until neutral. The alkaline ion exchange resin was then added to methanol and soaked for 12 hours for activation. After the soaking, the methanol was removed and the resin was dried at 80-120°C to constant weight to obtain an activated alkaline ion exchange resin.

[0054] (2) Weigh 0.3021 g (0.81 mmol) of berberine hydrochloride and dissolve it in 35 ml of methanol to obtain a berberine hydrochloride solution; add activated alkaline ion exchange resin to the berberine hydrochloride solution, the exchange ratio of berberine hydrochloride to activated alkaline ion exchange resin (5.38 g) is 0.15 mmol / g, stir and react for 24 hours, filter after the reaction is completed, collect the filtrate, remove the solvent by reduced pressure rotary evaporation, and then vacuum dry to obtain berberine hydroxide.

[0055] (3) dissolving all the berberine hydroxide obtained in step (2) in 30 ml of methanol to obtain a berberine hydroxide solution, adding glycyrrhetinic acid to the berberine hydroxide solution, the molar ratio of berberine hydroxide to glycyrrhetinic acid being 1:1, and stirring the mixture at 50° C. for 12 h; after the reaction, removing the solvent from the reaction solution by vacuum rotary evaporation at 50° C., and then vacuum drying to obtain glycyrrhetinic acid berberine salt (BB-GA).

[0056] Embodiment 1-2:

[0057] The content of Example 1-2 is substantially the same as that of Example 1-1, except that in step (2), the exchange ratio of berberine hydrochloride to activated alkaline ion exchange resin is 0.75 mmol / g.

[0058] Embodiment 1-3:

[0059] The content of Example 1-3 is substantially the same as that of Example 1-1, except that in step (2), the exchange ratio of berberine hydrochloride to activated alkaline ion exchange resin is 1.5 mmol / g.

[0060] The yield and purity of the glycyrrhetinic acid berberine salt prepared in Examples 1-1 to 1-3 were tested, and the results are shown in Table 1.

[0061] Table 1 shows the effect of the exchange ratio of berberine hydrochloride to activated alkaline ion exchange resin on the preparation of glycyrrhetinic acid berberine salt

[0062]

[0063] As shown in Table 1, a lower exchange ratio of berberine hydrochloride to activated alkaline ion exchange resin is more conducive to the synthesis of BB-GA. Therefore, the exchange ratio of berberine hydrochloride to alkaline ion exchange resin is preferably 0.15 mmol / g.

[0064] Example 2: Discussion on the molar ratio of berberine hydroxide to glycyrrhetinic acid

[0065] In order to explore the effect of the molar ratio of berberine hydroxide to glycyrrhetinic acid on the preparation of glycyrrhetinic acid berberine salt, the present invention carried out Examples 2-1 to 2-3. The specific contents of Examples 2-1 to 2-3 are as follows:

[0066] Example 2-1:

[0067] The content of Example 2-1 is basically the same as that of Example 1-1, except that in step (3), the molar ratio of berberine hydroxide to glycyrrhetinic acid is 0.5:1.

[0068] Example 2-2:

[0069] The content of Example 2-2 is basically the same as that of Example 1-1, except that in step (3), the molar ratio of berberine hydroxide to glycyrrhetinic acid is 1.5:1.

[0070] Embodiment 2-3:

[0071] The content of Example 2-3 is basically the same as that of Example 1-1, except that in step (3), the molar ratio of berberine hydroxide to glycyrrhetinic acid is 2:1.

[0072] The yield and purity of the glycyrrhetinic acid berberine salt prepared in Examples 2-1 to 2-3 were tested, and the results are shown in Table 2.

[0073] Table 2 shows the effect of the molar ratio of berberine hydroxide to glycyrrhetinic acid on the preparation of glycyrrhetinic acid berberine salt

[0074]

[0075] As can be seen from Table 2, when the molar ratio of berberine hydroxide to glycyrrhetinic acid is 1:1, the yield and purity of glycyrrhetinic acid berberine salt are the highest. Therefore, the molar ratio of berberine hydroxide to glycyrrhetinic acid is preferably 1:1.

[0076] Embodiment 3:

[0077] The content of Example 3 is the same as that of Example 1-1, except that in step (3), the molar ratio of berberine hydroxide to glycyrrhetinic acid is 1.5:1, and the reaction is stirred at 40° C. for 48 h.

[0078] Embodiment 4:

[0079] The content of Example 4 is the same as that of Example 1-1, except that in step (3), the molar ratio of berberine hydroxide to glycyrrhetinic acid is 2:1, and the reaction is stirred at 60° C. for 24 h.

[0080] Embodiment 5:

[0081] The content of Example 5 is the same as that of Example 1-1, except that in step (3), the molar ratio of berberine hydroxide to glycyrrhetinic acid is 1:1, and the reaction is stirred at 50° C. for 24 h.

[0082] Embodiment 6:

[0083] The content of Example 6 is the same as that of Example 1-1, except that: in step (2), the exchange ratio of berberine hydrochloride to activated alkaline ion exchange resin is 0.75 mmol / g, and in step (3), the molar ratio of berberine hydroxide to glycyrrhetinic acid is 1:1, and the reaction is stirred at 60°C for 12h.

[0084] (ii) Berberine hydrochloride reacts with silver oxide to prepare berberine hydroxide, and berberine hydroxide reacts with glycyrrhetinic acid to prepare glycyrrhetinic acid berberine salt

[0085] Example 7: Molar Ratio of Berberine Hydroxide to Glycyrrhetinic Acid

[0086] In order to explore the effect of the molar ratio of berberine hydroxide to glycyrrhetinic acid on the preparation of glycyrrhetinic acid berberine salt, the present invention carried out Examples 7-1 to 7-3. The specific contents of Examples 7-1 to 7-3 are as follows:

[0087] Example 7-1:

[0088] A method for preparing glycyrrhetinic acid berberine salt, the specific steps are as follows:

[0089] (1) adding 0.2266 g (0.0006 mol) of berberine hydrochloride to 50 ml of purified water, heating and stirring at 50° C. until the berberine hydrochloride is completely dissolved, to obtain a berberine hydrochloride solution; adding silver oxide (the molar ratio of berberine hydrochloride to silver oxide is 1:5) to the berberine hydrochloride solution, reacting at 50° C. for 12 h, filtering after the reaction, to obtain a berberine hydroxide solution, wherein the pH of the berberine hydroxide solution is 10.45;

[0090] (2) adding glycyrrhetinic acid to the berberine hydroxide solution obtained in step (1), wherein the molar ratio of berberine hydroxide to glycyrrhetinic acid is 0.5:1, and stirring is performed while adding. During the glycyrrhetinic acid addition process, the pH of the reaction solution is monitored and controlled to be above 7.4. After all the glycyrrhetinic acid is added, reacting at 50° C. for 24 hours. After the reaction is completed, filtering and collecting the precipitate, washing and drying the precipitate to obtain glycyrrhetinic acid berberine salt.

[0091] Example 7-2:

[0092] The content of Example 7-2 is basically the same as that of Example 7-1, except that in step (2), the molar ratio of berberine hydroxide to glycyrrhetinic acid is 1:1.

[0093] Example 7-3:

[0094] The content of Example 7-3 is basically the same as that of Example 7-1, except that in step (2), the molar ratio of berberine hydroxide to glycyrrhetinic acid is 2:1.

[0095] The yield and purity of the glycyrrhetinic acid berberine salt prepared in Examples 7-1 to 7-3 were tested, and the results are shown in Table 3.

[0096] Table 3 shows the effect of the molar ratio of berberine hydroxide to glycyrrhetinic acid on the preparation of glycyrrhetinic acid berberine salt

[0097]

[0098] As can be seen from Table 3, when the molar ratio of berberine hydroxide to glycyrrhetinic acid is 1:1, the yield of glycyrrhetinic acid berberine salt is the best, so the molar ratio of berberine hydroxide to glycyrrhetinic acid is preferably 1:1.

[0099] Embodiment 8:

[0100] The content of Example 8 is the same as that of Example 7-1, except that: in step (1), the molar ratio of berberine hydrochloride to silver oxide is 1:1, the reaction temperature is 40°C, and the reaction time is 24h; in step (2), the molar ratio of berberine hydroxide to glycyrrhetinic acid is 1.5:1.

[0101] Embodiment 9:

[0102] The content of Example 9 is the same as that of Example 7-1, except that: in step (1), the molar ratio of berberine hydrochloride to silver oxide is 1:10, the reaction temperature is 60°C, and the reaction time is 6h; in step (2), the molar ratio of berberine hydroxide to glycyrrhetinic acid is 1:1.

[0103] Embodiment 10:

[0104] The content of Example 10 is the same as that of Example 7-1, except that: in step (1), the molar ratio of berberine hydrochloride to silver oxide is 1:20, the reaction temperature is 50°C, and the reaction time is 24h; in step (2), the molar ratio of berberine hydroxide to glycyrrhetinic acid is 1:1, the reaction temperature is 40°C, and the reaction time is 24h.

[0105] Embodiment 11:

[0106] The content of Example 11 is the same as that of Example 7-1, except that in step (2), the molar ratio of berberine hydroxide to glycyrrhetinic acid is 1:1, the reaction temperature is 60° C., and the reaction time is 6 h.

[0107] (III) Preparation of berberine glycyrrhizinate nanoparticles (BB-GA NDs)

[0108] Embodiment 12:

[0109] (1) Weigh 0.0805 g of BB-GA obtained in Example 7-2 and dissolve it in 8 mL of dimethyl sulfoxide solution.

[0110] (2) Weigh 0.2004 g of Tween-80 into a 200 mL conical flask, add 100 mL of purified water to prepare a 0.2% Tween-80 solution for later use.

[0111] (3) 1 mL of BB-GA dimethyl sulfoxide solution was added dropwise to the 0.2% Tween-80 solution using a pipette under the condition of an ultrasonic disruptor (240 W, 2 s on, 2 s off).

[0112] (4) The mixed solution was centrifuged, the supernatant was removed, and the mixture was washed three times with purified water and freeze-dried for 24 h to obtain berberine glycyrrhetinic acid nanoparticles (BB-GA NDs).

[0113] (IV) Characterization of glycyrrhetinic acid berberine salt

[0114] 1. MRI test

[0115] Berberine (BB), glycyrrhetinic acid (GA) and glycyrrhetinic acid berberine salt (BB-GA) prepared in Example 7-2 were subjected to nuclear magnetic resonance testing. The test results are as follows: Figure 1 shown.

[0116] Depend on Figure 1 It can be seen that the characteristic peaks of BB and GA are both present in BB-GA, and the hydrogen on the 43-position carboxyl group of GA disappears, proving that the hydrogen on the carboxyl group has been replaced, that is, BB-GA is successfully synthesized.

[0117] 2. X-ray diffraction test

[0118] The glycyrrhetinic acid berberine salt prepared in Example 7-2 was subjected to an X-ray diffraction test, and the results were as follows: Figure 2 shown.

[0119] Depend on Figure 2 It can be seen that by comparing the XPS spectra of BB and GA alone with the spectrum of BB-GA, it can be observed that the characteristic peaks of BB and GA have changed in BB-GA, indicating that they have formed new compounds through chemical bonding. At the same time, no characteristic peaks from impurities or unreacted raw materials are observed in the XPS spectrum, which indicates that the synthesis process is clean and the product purity is high.

[0120] 3. Purity test

[0121] The purity of the glycyrrhetinic acid berberine salt prepared in Example 7-2 was tested by high performance liquid chromatography.

[0122] The HPLC detection conditions are as follows: using a C18 reverse phase column (inner diameter 150 mm×4.6 mm, particle size 5 μm), the mobile phase is aqueous solution (A) and methanol (B), the flow rate is 1.0 mL / min, the methanol content is 10% in the first 5 minutes, the methanol content is 10% to 100% in the next 20 minutes, and the methanol content is 100% in the last 15 minutes. 5 μL of 2 mg / mL BB-GA is injected, and the detection wavelength is 267 nm.

[0123] The results are as follows Figure 3 shown.

[0124] Depend on Figure 3 It can be seen that the purity of the prepared glycyrrhetinic acid berberine salt is close to 100%.

[0125] (V) Characterization of berberine glycyrrhizic acid nanoparticles (BB-GA NDs)

[0126] The BB-GA NDs prepared in Example 12 were tested by scanning electron microscopy, and the test results were as follows: Figure 4 shown.

[0127] Depend on Figure 4 It can be seen that the prepared BB-GA NDs have uniform particle sizes and are all regular circles, which proves that the preparation of BB-GA NDs is successful.

[0128] The hydrodynamic diameter and Zeta potential of the BB-GA NDs prepared in Example 12 were tested. The test results are as follows: Figure 5 shown.

[0129] Depend on Figure 5It can be seen that the hydrodynamic diameter of BB-GA NDs is 307.8nm, and the Zeta potential is -13.11±2.11mV. The surface of BB-GA NDs carries negative charge, which is beneficial to the circulation of BB-GA NDs in vivo.

[0130] (VI) Hemolysis rate detection of BB-GA NDs

[0131] The BB-GA NDs prepared in Example 12 were prepared into 20 μg / mL, 50 μg / mL, 80 μg / mL, 100 μg / mL, and 200 μg / mL, respectively, and placed in a physiological saline suspension containing 2% red blood cells. After incubation at 37°C for 3 h, the absorbance at 545 nm was detected by an ultraviolet spectrophotometer, and the hemolysis rate was calculated according to the formula; 0.9% NaCl was used as a negative control and purified water was used as a positive control.

[0132] The hemolysis rate is calculated as follows: Hemolysis rate = (A i -A0) / (A1-A0)×100%, A i , absorbance value at a certain concentration; A0, absorbance value of negative group; A1, absorbance value of positive group. The hemolysis rate test results are as follows Figure 6 shown.

[0133] Depend on Figure 6 It can be seen that even if the concentration of BB-GA NDs reaches 200 μg / mL, the hemolysis rate is only 0.60%, which is far less than the standard of 5%. This shows that BB-GA NDs have good blood compatibility, are not easy to cause hemolysis, and can initially meet the requirements of intravenous administration.

[0134] (VII) In vitro targeting and efficacy verification of BB-GA

[0135] The CCK8 method was used to investigate the time- and concentration-dependent cytotoxicity of BB, GA, GA+BB, and BB-GA prepared in Example 7-2.

[0136] The experimental cells are: human liver cancer cell line Huh7 and human normal liver cell L02.

[0137] Experimental groups: There were five groups in the experiment, namely BB group, GA group, GA+BB group, and BB-GA group.

[0138] The specific experimental steps are as follows: 2500 liver cancer cells Huh7 in logarithmic growth phase and in good condition and 4500 normal human liver cells L02 in a well were placed in a 37°C, 5% CO2 incubator for overnight culture, and then BB, GA, BB-GA and a mixture of BB and GA were added at concentrations of 0, 1, 5, 10, 20, 40, 80, 160, 200 μM, respectively, and cultured for 48 hours. The drug-containing culture medium was removed and 100 μL of fresh complete culture medium was added. 10 μL of CCK-8 solution was added in the dark and incubated in the incubator for 1 hour. The absorbance was measured at a wavelength of 450 nm using an enzyme reader to determine the cell survival rate.

[0139] Data analysis: The cell proliferation inhibition rate and the half maximal inhibitory concentration (IC50) of the drug were calculated based on the absorbance values. The experiment was repeated 3 times. Inhibition rate (%) = (average OD value of the negative control group - average OD value of the drug administration group) / average OD value of the negative control group × 100%.

[0140] The experimental results are as follows Figure 7 shown.

[0141] Depend on Figure 7 It can be seen that the killing effect of BB-GA on tumor cells is better than the toxicity of monomer BB, GA, and a mixture of BB and GA (GA+BB); among them, the toxicity of BB-GA after co-incubation for 48 hours is 1.6 times, 3.9 times, and 1.5 times that of BB, GA, and GA+BB, respectively, and 3.71 times that of normal liver cells L02. This proves that the BB-GA prepared by the present invention integrates the synergistic anti-liver cancer effect of BB and GA.

[0142] (VIII) Intravenous injection acute toxicity test of BB-GA NDs

[0143] The acute toxicity of intravenous injection of BB-GA NDs prepared in Example 12 into mice was investigated by continuously observing the conditions of four groups of mice for 6 hours after a single intravenous administration.

[0144] The specific experimental steps are as follows:

[0145] Appropriate water and feed were provided to adapt the Kunming mice. At the end of the adaptation period, the Kunming mice were randomly divided into 4 groups (8 mice in each group), namely BB group, GA group, BB-GA NDs group and Control group; the Kunming mice in the BB group were intravenously injected with BB solution, and the injection dose of BB solution was 10 mg / kg body weight; the Kunming mice in the GA group were intravenously injected with GA solution, and the injection dose of GA solution was 12 mg / kg body weight; the Kunming mice in the BB-GA NDs group were intravenously injected with BB-GA NDs solution, and the injection dose of BB-GA NDs solution was 20 mg / kg body weight; the Kunming mice in the Control group were intravenously injected with PBS solution, and the injection dose was 10 mg / kg body weight.

[0146] The four groups of mice were observed continuously for 6 hours after a single intravenous administration (acute toxic reactions tend to occur within 6 hours after administration). Then, the BB group, GA group, BB-GA NDs group, and Control group were observed every day for 14 consecutive days. The survival and diet of the mice during the experiment were recorded. A survival curve was drawn based on the survival of each group of mice within 14 days. The results are shown in the figure. Figure 8 shown.

[0147] Depend on Figure 8 It can be seen that after intravenous injection of BB (10 mg / kg), severe toxic reactions occurred, and all died on the second day of administration. After intravenous injection of BB-GA NDs (20 mg / kg) and GA (12 mg / kg), no toxic reactions occurred. It is confirmed that the prepared BB-GANDs can reduce the toxic side effects of intravenous injection of BB.

[0148] (VIII) In vivo experimental verification of BB-GA NDs in the treatment of liver cancer

[0149] The efficacy of BB-GA NDs prepared in Example 12 in treating liver cancer was verified by in vivo experiments.

[0150] The specific experimental steps are as follows:

[0151] Twenty male Kunming mice, 5-7 weeks old, weighing 16-18 g, were purchased from the Experimental Animal Center of Guangxi Medical University, with animal production license number SYXK(Gui)2020-0003. The mice were raised in the SPF-grade mouse breeding room of the Experimental Animal Center of Guangxi Medical University. The experiment was carried out after the mice were adaptively raised for one week.

[0152] Three days before modeling, the right lower armpit of the mouse was depilated and iodine was applied to prevent infection. H22 cells in the logarithmic growth phase were taken, washed with D-PBS, centrifuged at 1000 rpm for 3 minutes, and the supernatant was discarded. Resuspended with pre-cooled D-PBS, and the cells were counted. The cell density was adjusted to 5×10 6 / mL and stored in an ice box to keep the cells in a lower metabolic state.

[0153] Before inoculation, use a 1000 μL pipette to disperse the cell suspension evenly, and use a 1 mL sterile insulin syringe to draw 100 μL for later use, that is, each 1 mL sterile insulin syringe contains 5x10 5 Hold the mouse with your left hand to expose the skin of the right lower armpit, insert the needle parallel to the mouse body with the bevel of the needle facing up, and after the cells are injected, a clear bulge is visible. Slowly rotate the needle out to complete the injection.

[0154] When the tumor volume reaches 50 mm 3 The mice were randomly divided into 4 groups: model (Control) group, BB-GA NDs low-dose group, BB-GA NDs high-dose group, and cisplatin (CDDP) group, with 5 mice in each group. The Kunming mice were weighed every two days, and the maximum diameter (length) and minimum diameter (width) of the mouse transplanted tumor were measured. Tumor volume = 0.5 × length × width 2 The transplanted tumor volume was calculated using the formula.

[0155] The dosage and route of administration are as follows:

[0156] ① Model (Control) group: 0.9% saline, 0.2 mL / mouse, injected into the tail vein, once every three days;

[0157] ②BB-GA NDs low-dose group: 10 mg / kg, 0.2 mL / mouse, tail vein injection, once every three days;

[0158] ③BB-GA NDs high-dose group: 15 mg / kg, 0.2 mL / mouse, tail vein injection, once every three days;

[0159] ④Cisplatin (CDDP) group: 2 mg / kg, 0.2 mL / mouse, injected into the tail vein, once every three days.

[0160] Animal sampling: The average tumor volume of any group grew to 1500mm 3 Around 2.5 s, samples were collected, tumor volume was measured, tumor volume changes were statistically analyzed, and tumor inhibition rate was calculated. Fig. 9 shown.

[0161] Depend on Fig. 9 It can be seen that after the treatment, compared with the Control group, BB-GA NDs showed a dose-dependent tumor inhibition effect, and the tumor inhibition rates were 57.08%±10.93% and 64.64%±7.71%, respectively ( Fig. 9 c) The experimental results show that the BB-GA NDs prepared by the present invention exhibit excellent anti-tumor effects.

[0162] The above embodiments are specific implementation methods of the present invention, but the implementation methods of the present invention are not limited to the above embodiments. Any other combination, change, modification, substitution and simplification that does not exceed the design concept of the present invention shall fall within the protection scope of the present invention.

Claims

1. A method for preparing glycyrrhetinic acid berberine salt, It is characterized in that The steps include: S1: dissolving berberine hydroxide in solvent A to obtain a berberine hydroxide solution; S2: adding glycyrrhetinic acid to the berberine hydroxide solution to react fully, and collecting the reaction product after the reaction is completed to obtain glycyrrhetinic acid berberine salt.

2. The method for preparing glycyrrhetinic acid berberine salt according to claim 1, It is characterized in that In step S2, the molar ratio of berberine hydroxide to glycyrrhetinic acid is (0.5-2):

1.

3. The method for preparing the glycyrrhetinic acid berberine salt according to claim 2, It is characterized in that When the solvent A is an organic solvent, the specific operation of step S2 is: adding glycyrrhetinic acid to the berberine hydroxide solution, stirring and reacting at 40-60° C. for 12-48 hours, and after the reaction is completed, removing the solvent A from the reaction solution and drying to obtain glycyrrhetinic acid berberine salt; When the solvent A is water, the specific operation of step S2 is: adding glycyrrhetinic acid to the berberine hydroxide solution while stirring, after all the glycyrrhetinic acid is added, reacting at 40-60° C. for 6-24 h, filtering after the reaction is completed, collecting the precipitate, washing and drying the precipitate to obtain glycyrrhetinic acid berberine salt.

4. The method for preparing the glycyrrhetinic acid berberine salt according to any one of claims 1 to 3, It is characterized in that In step S1, the preparation method of berberine hydroxide is: dissolving berberine hydrochloride in solvent B to obtain a berberine hydrochloride solution; adding alkaline ion exchange resin to the berberine hydrochloride solution, mixing, reacting, filtering after the reaction is completed, collecting the filtrate, removing the solvent B in the filtrate, and vacuum drying to obtain berberine hydroxide.

5. The method for preparing the glycyrrhetinic acid berberine salt according to claim 4, It is characterized in that The exchange ratio of the berberine hydrochloride to the basic ion exchange resin is 0.15 mmol / g to 1.5 mmol / g.

6. The method for preparing the glycyrrhetinic acid berberine salt according to any one of claims 1 to 3, It is characterized in that In step S1, the preparation method of berberine hydroxide is: Dissolve berberine hydrochloride in solvent D to obtain a berberine hydrochloride solution; add silver oxide to the berberine hydrochloride solution for reaction, filter after the reaction to obtain a berberine hydroxide solution, remove the solvent in the berberine hydroxide solution to obtain berberine hydroxide.

7. The method for preparing the glycyrrhetinic acid berberine salt according to claim 6, It is characterized in that The molar ratio of berberine hydrochloride to silver oxide is 1:(1-20); the reaction temperature of the berberine hydrochloride and silver oxide is 40-60° C., and the reaction time is 6-24 h; the solvent D is water.

8. A glycyrrhetinic acid berberine salt product prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the glycyrrhizic acid berberine salt product of claim 8 in the preparation of drugs for preventing, treating or / and alleviating liver cancer.

10. A drug, It is characterized in that The drug is used for preventing, alleviating and / or treating liver cancer, and comprises glycyrrhetinic acid berberine salt nanoparticles. The glycyrrhetinic acid berberine salt nanoparticles are prepared by nanoprecipitation method using the glycyrrhetinic acid berberine salt product of claim 8 as raw material.

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