Use of berberine and quercetin in combination for preparing a medicament for treating gastric ulcer and indigestion

Abstract

This invention discloses the application of berberine and quercetin in the preparation of drugs for treating gastric ulcers and indigestion, belonging to the field of self-assembled nanoparticle drug development technology. This invention prepares berberine and quercetin into an ethanol solution, then mixes them under a magnetic stirrer to allow them to self-assemble into nanoparticles. When applied to gastric ulcers, compared with single agents, it significantly improves the inhibition rate of gastric ulcers, and can also prevent constipation with few side effects.

Description

Technical Field

[0001] This invention relates to the field of self-assembled nanoparticle drug development technology, and more specifically to the application of berberine and quercetin in combination in the preparation of drugs for treating gastric ulcers and indigestion. Background Technology

[0002] Gastric ulcer is the most common type of peptic ulcer. It mainly refers to tissue damage beyond the muscularis mucosa caused by the digestive juices of the stomach, and is an inflammatory and necrotic lesion occurring between the cardia and pylorus. Typical gastric ulcer pain is characterized by its long duration, periodicity, and rhythmicity. With the accelerated pace of life, unhealthy lifestyle habits, and increased social, work, family, and psychological burdens, the incidence of gastric ulcers has been increasing year by year. Most commonly used anti-gastric ulcer drugs are Western medicines, which have the disadvantage of causing constipation, further burdening the gastrointestinal motility function on the basis of gastric ulcers. Therefore, finding an anti-gastric ulcer drug with significant efficacy and the ability to alleviate constipation is an urgent problem to be solved.

[0003] Berberine hydrochloride has protective effects on the gastric mucosa and anti-gastric ulcer properties, but long-term use can cause constipation as a side effect. Clinically, it is often combined with Amomum villosum to alleviate the side effects caused by berberine. Quercetin is the main component of Amomum villosum and is also an effective ingredient for relieving constipation. Animal experiments have shown that quercetin can effectively inhibit gastric acid secretion and has a protective effect against gastric ulcers.

[0004] Therefore, how to combine berberine and quercetin in the preparation of drugs for treating gastric ulcers is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention combines berberine (BBH) with quercetin (QR) to self-assemble nanoparticles, namely berberine-quercetin self-assembled nanoparticles (BBH-QRNPs), which can effectively synergistically reduce inflammation and repair gastric mucosa, while reducing the side effects caused by berberine administration, and treat gastrointestinal dysfunction in gastric ulcer disease.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] The application of berberine and quercetin in the preparation of drugs for treating gastric ulcers and indigestion.

[0008] The present invention also claims protection for a composition for treating gastric ulcers and functional dyspepsia, comprising berberine and quercetin in a mass ratio of 1:1.

[0009] This invention also claims protection for a method for preparing a self-assembled nanoparticle drug, prepared from the aforementioned composition for treating gastric ulcers and functional dyspepsia, comprising the following steps:

[0010] 1) Weigh the berberine and quercetin separately and dissolve them in anhydrous ethanol to prepare berberine-ethanol solution and quercetin-ethanol solution respectively;

[0011] 2) Stir the quercetin-ethanol solution with a magnetic stirrer. At the same time, add the berberine-ethanol solution dropwise to the quercetin-ethanol solution. After the addition is complete, stir to obtain the berberine-quercetin-ethanol solution.

[0012] 3) Berberine-quercetin-ethanol solution was added dropwise to purified water, stirred, frozen, and lyophilized to obtain berberine-quercetin self-assembled nanoparticle drugs.

[0013] The present invention also claims protection for a method for preparing a self-assembled nanoparticle drug, wherein in step 1), the concentrations of berberine-ethanol solution and quercetin-ethanol solution are both 2 mg / mL.

[0014] Preferably, in step 2), the volume ratio of berberine-ethanol solution to quercetin-ethanol solution is 1:1.

[0015] Preferably, in step 2), the rotation speed of the magnetic stirrer is 600 rpm.

[0016] Preferably, in step 2), a peristaltic pump is used to add the berberine-ethanol solution dropwise to the quercetin-ethanol solution at 3 rpm.

[0017] Preferably, in step 3), the volume of purified water is twice the volume of the berberine-quercetin-ethanol solution.

[0018] Preferably, in step 3), stirring is performed for 2 hours; freezing is performed at -80°C for 2 hours; and freeze-drying is performed in a freeze dryer for 48 hours.

[0019] Preferably, in step 3), the molar ratio of berberine to quercetin in the obtained drug is 1:1.26.

[0020] As can be seen from the above technical solution, compared with the prior art, the present invention is the first to use berberine and quercetin in combination to treat gastric ulcers. The gastric ulcer inhibition rate can reach 52.38%, and the carbon powder propulsion rate is as high as 70.01% ± 8.03%, which can effectively relieve constipation. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0022] Figure 1 The attached figure shows the particle size, DI, and Zate potential of BBH-QRNPs;

[0023] Figure 2 The attached figure shows the infrared absorption spectrum of BBH-QRNPs;

[0024] Figure 3 The attached figure shows the results of the BBH-QRNPs self-assembly model;

[0025] Figure 4 The attached diagram shows the interaction forces between berberine and quercetin.

[0026] Figure 5 The attached figure shows the number of hydrogen bonds involved in the interaction of BBH-QRNPs;

[0027] Figure 6 The attached figure shows the characterization of the gastric ulcer index in mice under different treatment groups;

[0028] Figure 7 The attached diagram illustrates the mechanism pathway of BBH-QRNPs nanoparticles in combating gastric ulcers.

[0029] Figure 8 The attached diagram shows the hysteresis effect of BBH-QRNPs;

[0030] Figure 9 The attached diagram shows the pathway of BBH-QRNPs' conduction mechanism. Detailed Implementation

[0031] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0032] The evaluation method used in the examples: Evaluation of the efficacy of BBH-QRNPs, the method is as follows:

[0033] (1) Evaluation of the efficacy of anti-gastric ulcer drugs

[0034] One hundred C57BL / 6J mice were randomly divided into 5 groups of 20 mice each. The model group was administered 300 mg / kg of aspirin aqueous solution daily for four days; on the fourth day, they were fasted but allowed free access to water for 12 hours. On the fifth day, they were given a 200 mg / kg aspirin ethanol solution prepared with 40% ethanol, and were dissected 4 hours later. The sucralfate group (300 mg / kg), berberine group (BBH, 130 mg / kg), and BBH-QRNPs group (380 mg / kg) received the same medication by gavage for the first four days in addition to the initial modeling treatment, and were given the same medication 2 hours before the aspirin ethanol solution on the fifth day. The control group was given an equal volume of physiological saline by gavage. After dissection, the gastric tissue was observed, and relevant gastric tissue parameters were measured.

[0035] (2) Evaluation of the efficacy of drugs for relieving constipation

[0036] One hundred C57BL / 6J mice were randomly divided into 5 groups of 20 mice each. The model group was given 300 mg / kg of aspirin aqueous solution daily for four consecutive days. On the fourth day, the mice were fasted but allowed free water for 12 hours. On the fifth day, 3 hours after receiving aspirin ethanol solution, the mice were given carbon powder solution (50 mg / mL, 1% sodium carboxymethyl cellulose solution) for 20 minutes before dissection. The sucralfate group (300 mg / kg), berberine group (BBH, 130 mg / kg), and BBH-QRNPs group (380 mg / kg) received the same treatment by gavage for the first four days in addition to the modeling treatment. On the fifth day, the mice were given aspirin ethanol solution 1 hour after the initial treatment. Two hours after the initial treatment, the mice were given carbon powder by gavage for 20 minutes before dissection. The length of the carbon powder was measured, and the gastric tissue was collected to detect the content of relevant indicators.

[0037] Example 1: Construction of a gastric ulcer model

[0038] The modeling method is as follows:

[0039] (1) The model group was given 300 mg / kg of aspirin aqueous solution every day for four consecutive days.

[0040] (2) On the fourth day, fasting was allowed but water was permitted for 12 hours. On the fifth day, an aspirin ethanol solution with a concentration of 200 mg / kg prepared with 40% ethanol was administered, and the body was dissected 4 hours later.

[0041] The successful establishment of the gastric ulcer model was confirmed by observing the degree of gastric damage and calculating the ulcer index in mice in the control and model groups after dissection. Combining two common ulcer models, compared to the severe gastric ulcer model induced by high-concentration ethanol, resulted in a relatively milder ulcer, which is more in line with the application scope of BBH-QRNPs and has greater clinical significance. Compared to the ulcer model induced by aspirin, the ulcer characteristics were more obvious, which is more conducive to analyzing the drug's efficacy and mechanism.

[0042] Example 2: Preparation of BBH-QRNPs

[0043] (1) Berberine and quercetin were dissolved in anhydrous ethanol to prepare berberine-ethanol solution and quercetin-ethanol solution with a concentration of 2 mg / mL, respectively.

[0044] (2) Stir the 2 mg / mL quercetin-ethanol solution at 600 rpm using a magnetic stirrer. At the same time, use a peristaltic pump to add the 2 mg / mL berberine solution dropwise to the quercetin solution at 3 rpm, with a volume ratio of 1:1.

[0045] (3) After the berberine has been completely added, continue stirring the mixed solution at 600 rpm for 2 hours. After two hours, the mixed solution is obtained.

[0046] (4) Prepare a pure aqueous solution with a volume twice that of the mixed solution. Add the mixed solution dropwise to the aqueous solution using the same method and conditions. Then, continue stirring the final mixed solution for 2 hours and freeze it at -80°C for 12 hours. Finally, freeze it in a freeze dryer for 48 hours to obtain yellow nanoparticle freeze-dried powder.

[0047] The prepared BBH-QRNPs lyophilized powder was diluted 20 times with pure water and placed in a particle size analyzer. The particle size, PDI, and Zeta potential of the nanoparticles were measured using a Malvern particle size analyzer. Figure 1 As shown in Table 1.

[0048] Table 1

[0049]

[0050]

[0051] Example 3: Structural identification of BBH-QRNPs by infrared characterization

[0052] Infrared characterization:

[0053] The infrared spectral wavenumber range is 500-4000 cm⁻¹ -1 The spectrometer has a resolution of 4cm. -1 The signal-to-noise ratio is 50000:1, and 32 scans were performed. Infrared characterization results show that... Figure 2 As shown, QR is at 3406.20cm. -1 The broad and strong absorption peak of the hydroxyl stretching vibration indicates a large number of hydroxyl groups in its structure; 1319.23 cm⁻¹ -1 The absorption peak at this location is the antisymmetric and symmetric stretching vibration of C=CH in BBH, which is 1599.18 cm⁻¹. -1 and 1508.06cm-1 The BBH-QRNPs lyophilized powders all retained the characteristic peaks of quercetin and berberine, with the same trend and no new absorption peaks, indicating that the chemical structure of quercetin and berberine in the nanoparticles has not changed, and the freeze-drying process has not affected the drugs.

[0054] Example 4 simulated the self-assembly process of berberine and quercetin in BBH-QRNPs using molecular simulation.

[0055] Molecular simulation

[0056] Partial charges of quercetin, berberine, and ethanol molecules were calculated using Gaussian16 with 6-31+g(d, p) basis functions. All atoms were parameterized using the OPLSS-AA force field and MKTOP, including bond parameters, angular parameters, and dihedral angles. The interaction between berberine and quercetin was studied using molecular dynamics (MD) simulations. The water molecule model was TIP3P. MD simulations were performed using GROMACS2021 software. Before the MD calculations, the steepest descent method was used to minimize the initial energy of each system, with a force tolerance of 1 kJ / (mol). -1 .nm -1 The maximum step size was 0.002 ps. Periodic boundary conditions were applied in all three directions. The leapfrog algorithm was used to integrate Newton's equations of motion. MD simulations were performed in the NPT ensemble for 20 ns. In the NPT simulations, a Berendsen isotropic pressure was maintained at 1 bar. The temperature was maintained at 298.15 K by a V-recalibrated thermostat. The LINCS algorithm was used to constrain the bond lengths of hydrogen atoms. Electrostatic interactions were calculated using the Particle-Mesh-Ewald (PME) method with fourth-order interpolation, with a grid spacing of [missing information]. Short-range van der Waals interactions employ The cutoff value was determined. After a 20 ns MD simulation, the number of hydrogen bonds and intermolecular interactions between berberine and quercetin were analyzed. The model results are as follows: Figure 3 As shown. The interaction force between berberine and quercetin is as follows. Figure 4 As shown, the number of hydrogen bonds is as follows Figure 5 As shown.

[0057] Example 5 evaluated the efficacy of BBH-QR NPs by calculating the ulcer index and ulcer inhibition rate.

[0058] One hundred and forty C57BL / 6J mice were randomly divided into seven groups of twenty mice each. The model group received 300 mg / kg of aspirin aqueous solution daily for four consecutive days; on the fourth day, they were fasted but allowed free access to water for 12 hours. On the fifth day, they received a 200 mg / kg aspirin ethanol solution prepared with 40% ethanol, and were dissected four hours later. The sucralfate (Urba, 300 mg / kg), berberine (BBH, 130 mg / kg), BBH-QRNPs (380 mg / kg), and BBH and QR physical mixture (Mix, 380 mg / kg) groups received the same medication by gavage for the first four days after modeling, and were administered the same medication 2 hours before the aspirin ethanol solution on the fifth day. The control group received an equal volume of physiological saline by gavage. After dissection, the gastric tissue was observed, and the degree of ulceration was assessed. Figure 6 As shown, the Gastric Ulcer Index (GUI) is a classic index that assesses the degree of gastric mucosal damage. It is calculated as follows: 0 = no damage, 1 = small hemorrhagic lesion, 2 = lesion < 2 mm (length of ulcer or erosion), 3 = lesion from 2 to 3 mm, 4 = lesion from 3 to 4 mm, and 5 = lesion > 4 mm. When the erosion width is greater than 1 mm, the score is doubled. Ulcer inhibition rate (%) = (Ulcer index of control group - Ulcer index of treatment group / Ulcer index of control group) × 100%. The ulcer index results for each group are shown in Table 2.

[0059] Table 2

[0060] blank 0.00 Model 10.50±1.05 Urba <![CDATA[5.17±0.98 * ]]> 50.79％ BBH <![CDATA[6.00±0.89 * ]]> 42.86％ QR <![CDATA[7.25±0.95 * ]]> 35.71％ Mix <![CDATA[6.75±1.71 * ]]> 52.38％ BBH-QRNPs <![CDATA[5.00±0.89 * ]]> 52.38％

[0061] Example 6 uses the animal model from Example 5 to investigate the mechanism of BBH-QRNPs in preventing gastric ulcers.

[0062] After dissecting the mice, stomach tissue was collected, cleaned, and frozen. The levels of endothelin (ET), gastrin (GAS), and serotonin (5-HT) in the stomach tissue were detected using an ELISA kit according to the manufacturer's instructions. Samples were diluted, reacted and washed at 37°C, then added to a microplate reader for further reaction and washing. Developing solutions A and B were added, followed by reaction. Finally, stop solution was added, and the OD values ​​were read and calculated at 450 nm using a microplate reader after 15 minutes. The results are shown below. Figure 7As shown. ET has a vasoconstrictive effect, and its elevated levels hinder the repair of gastric mucosa after injury; GAS also reduces the hexose content in the gastric mucosa, thus affecting the repair of damaged gastric mucosa; elevated 5-HT can cause vasoconstriction, reducing blood flow to the gastric mucosa, decreasing the gastric mucosa's defense function, and leading to gastric ulcers. Elevated levels of all three can lead to gastric mucosal damage and gastric ulcer formation. Compared with the control group, the model group showed significantly elevated levels of ET, GAS, and 5-HT, indicating a successful gastric ulcer model. The levels of berberine and BBH-QRNPs were both downregulated, and the downregulation effect of BBH-QRNPs was significantly different from that in the model group, making these indicators more similar to those in the control group.

[0063] Example 7: Evaluation of the hysteresis effect of BBH-QRNPs by carbon powder propulsion experiments.

[0064] One hundred and twenty C57BL / 6J mice were randomly divided into six groups of twenty each. The model group received 300 mg / kg of aspirin aqueous solution daily for four consecutive days; on the fourth day, they were fasted but allowed free water for 12 hours. On the fifth day, three hours after receiving aspirin ethanol solution, they were given carbon powder solution (50 mg / mL, 1% carboxymethyl cellulose sodium solution) for 20 minutes before dissection. The sucralfate group (300 mg / kg), berberine group (BBH, 130 mg / kg), berberine-quercetin physical mixture group (380 mg / kg), and BBH / QR-NP group (380 mg / kg) received the same treatment daily by gavage for the first four days after model establishment. On the fifth day, one hour after receiving aspirin ethanol solution, they were given carbon powder by gavage for 20 minutes, followed by dissection. The length of the carbon powder extrusion was measured and calculated. The results are shown in Table 3. Figure 8 As shown.

[0065] Carbon powder propulsion rate = Carbon powder propulsion length / Total small intestine length × 100%

[0066] Table 3

[0067] Blank group 68.46％±7.28％ Model group <![CDATA[49.54%±1.29% # ]]> Sucralfate group 46.68％±3.44％ Berberine group 51.96％±3.34％ Berberine and quercetin physical mixture group 58.88％±1.76％ Berberine-quercetin nanoparticles 70.01％±8.03％*

[0068] Example 8 uses the animal model from Example 7 to investigate the mechanism of BBH-QR NPs' conduction.

[0069] After dissecting the mice, stomach tissue was collected, cleaned, and frozen. The levels of motilin (MTL) and ghrelin in the stomach tissue were detected using an ELISA kit according to the manufacturer's instructions. The samples were diluted, reacted at 37°C, washed, and then reacted again using a microplate reader. Developing solutions A and B were added, followed by reaction. Finally, stop solution was added, and the OD values ​​were read and calculated at 450 nm using a microplate reader after 15 minutes. The results are shown below. Figure 9 MTL and ghrelin are important indicators of gastric motility. Compared with the control group, the levels of both MTL and ghrelin in the model group decreased. After administration of BBH-QRNPs, the levels of both increased, indicating that BBH-QRNPs enhance gastric motility by upregulating MTL and ghrelin levels, thereby relieving constipation caused by gastric ulcers.

[0070] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0071] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing a self-assembled nanoparticle drug for treating gastric ulcer, characterized in that, The process includes the following: 1) Weigh berberine and quercetin in a mass ratio of 1:1, then dissolve berberine and quercetin in anhydrous ethanol to prepare berberine-ethanol solution and quercetin-ethanol solution respectively. 2) Stir the quercetin-ethanol solution with a magnetic stirrer. At the same time, add the berberine-ethanol solution dropwise to the quercetin-ethanol solution. After the addition is complete, stir to obtain the berberine-quercetin-ethanol solution. 3) Berberine-quercetin-ethanol solution was added dropwise to purified water, stirred, frozen, and lyophilized to obtain berberine-quercetin self-assembled nanoparticle drugs; In step 1), the concentrations of both the berberine-ethanol solution and the quercetin-ethanol solution are 2 mg / mL; In step 2), the volume ratio of berberine-ethanol solution and quercetin-ethanol solution is 1:1; In step 2), the magnetic stirrer rotates at 600 rpm; In step 2), a peristaltic pump is used to add the berberine-ethanol solution dropwise to the quercetin-ethanol solution at 3 rpm; In step 3), the volume of purified water is twice the volume of the berberine-quercetin-ethanol solution; In step 3), stirring is for 2 hours; freezing is for 2 hours at -80°C; and freeze-drying is for 48 hours in a freeze dryer.

2. The drug prepared by the method for preparing self-assembled nanoparticle drugs for treating gastric ulcers as described in claim 1.

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