Pharmaceutical composition containing pterostilbene and its preparation method and pharmaceutical use

Abstract

The application discloses a kind of pharmaceutical compositions, it includes stilben compound and pharmaceutically acceptable adjuvant;Wherein, the stilben compound is Pterostilbene or its pharmaceutically acceptable salt, the pharmaceutically acceptable adjuvant includes glucosyl tangeritin and / or glycyrrhizic acid salt.The in vitro and / or in vivo test results show that the pharmaceutical composition provided by the application has good curative effect for preventing and / or treating drug-induced liver injury;Meanwhile, it can also significantly reduce serum transaminase level, alleviate the symptoms such as liver and spleen edema, and also has good inhibitory effect on proinflammatory cytokines.The test results further show that stilben compound and pharmaceutically acceptable adjuvant (including glucosyl tangeritin and / or glycyrrhizic acid salt) combination, good safety, and easy to dissolve, release and / or absorption etc., improve the bioavailability of Pterostilbene (PTE), have synergistic effect, and have good application prospect.

Description

Technical Field

[0001] This application belongs to the field of chemical pharmaceuticals, specifically relating to a pharmaceutical composition containing pterostilbene and its preparation method and pharmaceutical use. Background Technology

[0002] Acetaminophen, also known as paracetamol, is one of the most common antipyretic analgesics. It is mainly used for fever caused by the common cold or influenza, and also for relieving mild to moderate pain. It has a very wide range of applications.

[0003] However, excessive dosage or prolonged use of medication can damage the liver, causing partial hepatocyte necrosis, manifested as abnormal liver function indicators such as serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT). Generally, liver injury caused by drugs and / or their metabolites is termed drug-induced liver injury.

[0004] In recent years, the incidence of drug-induced liver injury has increased significantly in clinical practice, especially in Europe and the United States, where it is showing an increasingly serious trend. Overuse and abuse of acetaminophen is one of the main causes of this type of disease. After onset, serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) rise, leading to hepatocyte necrosis, which in turn can cause cirrhosis and even death.

[0005] Currently, N-acetylcysteine ​​(NAC) is mainly used as a treatment for this type of drug-induced liver injury. Although it is the most effective drug at present, it also has certain limitations and drawbacks (see: CN 112535694 A). There are no other better and more effective treatment options available.

[0006] Pterostilbene (PTE), CAS No. 537-42-8, is an effective ingredient derived from plants such as sandalwood and blueberry. When combined with skin-softening lotion, it can whiten and reduce inflammation, delay aging, restore cell vitality, and maintain skin elasticity. In addition, it also has pharmacological activities such as anti-cancer, anti-inflammatory, antioxidant, and analgesic effects. However, due to the following disadvantages, Pterostilbene's development and utilization in skin care products, pharmaceutical preparations, and other fields are seriously affected: (1) poor water solubility; (2) the molecular structure contains phenolic hydroxyl groups, making the structure unstable, especially in aqueous solution; (3) poor permeability to biological membranes and low bioavailability (see: CN 108371636 A, CN 113350284 A, etc.).

[0007] Dipotassium glycyrrhizate (DG), CAS No.: 68797-35-3, is a white or off-white powder with anti-inflammatory, anti-allergic, and moisturizing effects. In the pharmaceutical industry, it is mainly used for cough suppression and expectoration, gastric ulcers, acute and chronic gastritis, eczema, pruritus, as well as for the treatment of cancer and the prevention and treatment of AIDS.

[0008] Alpha-glucosyl hesperidin (AGH), CAS No. 161713-86-6, is a polysaccharide mainly used in cosmetics and other fields.

[0009] To date, no literature has been found on the use of a combination of pterostilbene, dipotassium glycyrrhizate, and α-glucosylhesperidin for the prevention, treatment, and / or relief of liver injury (e.g., drug-induced liver injury). Summary of the Invention

[0010] In view of the problems and / or deficiencies of the prior art, one of the objectives of this invention is to provide a pharmaceutical composition comprising a stilbene compound and pharmaceutically acceptable excipients. This pharmaceutical composition can be used to prepare drugs for the prevention and / or treatment of drug-induced liver injury, or to lower serum transaminase levels, or as an inhibitor of pro-inflammatory cytokines, exhibiting relatively good therapeutic effects.

[0011] One or more embodiments of this application provide a pharmaceutical composition comprising a stilbene compound and a pharmaceutically acceptable excipient; wherein the stilbene compound is pterostilbene or a pharmaceutically acceptable salt thereof, and the pharmaceutically acceptable excipient comprises glucosyl hesperidin and / or glycyrrhizate.

[0012] In one or more embodiments, the weight ratio of the stilbene compound to the pharmaceutically acceptable excipient is 1:5 to 50 (e.g., 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:40 or 1:50).

[0013] In one or more embodiments, the weight ratio of the stilbene compound to the pharmaceutically acceptable excipient is 1:10 to 20.

[0014] In one or more embodiments, the stilbene compound is pterostilbene; and / or, the glucosyl hesperidin is α-glucosyl hesperidin; and / or, the glycyrrhizic acid salt is selected from one or more of sodium glycyrrhizate, disodium glycyrrhizate, potassium glycyrrhizate, dipotassium glycyrrhizate, ammonium glycyrrhizate, and diammonium glycyrrhizate.

[0015] In one or more embodiments, the glycyrrhizic acid salt is dipotassium glycyrrhizate or disodium glycyrrhizate.

[0016] In one or more embodiments, the pharmaceutically acceptable excipients comprise glucosyl hesperidin and glycyrrhizate.

[0017] In one or more embodiments, the weight ratio of glucosyl hesperidin to glycyrrhizate is 1:2 to 20 (e.g., 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19 or 1:20).

[0018] In one or more embodiments, the weight ratio of glucosyl hesperidin to glycyrrhizate is 1:5 to 12.

[0019] In one or more embodiments, the encapsulation efficiency of the stilbene compound is at least 80%.

[0020] In one or more embodiments, the encapsulation efficiency of the stilbene compound is ≥90% or ≥95%.

[0021] In one or more embodiments, the pharmaceutical composition is a solid or liquid formulation; and / or, the stilbene compound in the pharmaceutical composition is in a therapeutically effective amount.

[0022] In one or more embodiments, the pharmaceutical composition is a liquid formulation, wherein the solvent for the liquid formulation is selected from pharmaceutically acceptable water or PBS buffer.

[0023] In one or more embodiments, when the concentration of stilbene compounds in the liquid formulation is 1 mg / mL, the liquid formulation satisfies the following conditions ① and / or ②:

[0024] ① The average diameter of the micelles in the liquid formulation is 1-50 nm; preferably 2-15 nm;

[0025] ② The zeta potential of the liquid formulation is -20 to 0 mV; preferably -5 to -0.1 mV.

[0026] In one or more embodiments, when the concentration of stilbene compounds in the liquid formulation is 1 mg / mL, the liquid formulation simultaneously satisfies conditions ① and ②.

[0027] In one or more embodiments, the pharmaceutical composition is prepared by a method comprising the following steps: dispersing or dissolving a stilbene compound and a pharmaceutically acceptable excipient together in an organic solvent, mixing them thoroughly, and then removing the organic solvent by rotary evaporation at 35–45°C.

[0028] In one or more embodiments, the organic solvent is an alcohol solvent; and / or, the amount of the organic solvent used per milligram of the stilbene compound is 0.5 to 20 mL (e.g., 0.5 mL, 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, 11 mL, 12 mL, 13 mL, 14 mL, 15 mL, 16 mL, 17 mL, 18 mL, 19 mL, or 20 mL).

[0029] In one or more embodiments, the alcohol solvent is methanol and / or ethanol; and / or, the amount of the organic solvent used per milligram of the stilbene compound is 1.2 to 2.5 mL.

[0030] In one or more embodiments, the pharmaceutical composition is a pharmaceutical composition for the prevention and / or treatment of drug-induced liver injury.

[0031] In one or more embodiments, the drug-induced liver injury is drug-induced liver injury caused by acetaminophen.

[0032] In one or more embodiments, the pharmaceutical composition is a pharmaceutical composition for reducing serum transaminase levels.

[0033] In one or more embodiments, the transaminase is alanine aminotransferase (ALT) and / or aspartate aminotransferase (AST).

[0034] In one or more embodiments, the pharmaceutical composition is a pro-inflammatory cytokine inhibitor.

[0035] In one or more embodiments, the pro-inflammatory cytokine inhibitor is an HMGB1 inhibitor, an IL-1β inhibitor, an IL-6 inhibitor, an NF-κB inhibitor, or a TNF-α inhibitor.

[0036] One or more embodiments of this application provide a method for preparing the pharmaceutical composition of this application, comprising the following steps: dispersing or dissolving a stilbene compound and a pharmaceutically acceptable excipient together in an organic solvent, mixing them, and then removing the organic solvent by rotary evaporation at 35-45°C, thereby obtaining the pharmaceutical composition.

[0037] In one or more embodiments, the organic solvent is an alcohol solvent; and / or, the amount of the organic solvent used per milligram of the stilbene compound is 0.5 to 20 mL.

[0038] In one or more embodiments, the alcohol solvent is methanol and / or ethanol; and / or, the amount of the organic solvent used per milligram of the stilbene compound is 1.2 to 2.5 mL.

[0039] One or more embodiments of this application provide the use of the pharmaceutical composition of this application in the preparation of a medicament for the prevention and / or treatment of drug-induced liver injury.

[0040] One or more embodiments of this application provide the use of the pharmaceutical composition of this application in the preparation of a medicament for reducing serum transaminase levels.

[0041] One or more embodiments of this application provide the use of the pharmaceutical compositions described herein in the preparation of inhibitors of pro-inflammatory cytokines;

[0042] In one or more embodiments, the drug-induced liver injury is drug-induced liver injury caused by acetaminophen.

[0043] In one or more embodiments, the transaminase is alanine aminotransferase (ALT) and / or aspartate aminotransferase (AST).

[0044] In one or more embodiments, the pro-inflammatory cytokine inhibitor is an HMGB1 inhibitor, an IL-1β inhibitor, an IL-6 inhibitor, an NF-κB inhibitor, or a TNF-α inhibitor.

[0045] Regarding the definitions of terms used in this invention, unless otherwise stated, the initial definitions provided herein apply to the term throughout the text; for terms not specifically defined herein, the meanings that a person skilled in the art should give them should be based on the disclosure and / or context and common knowledge in the field.

[0046] The term "pharmaceutical acceptable" generally refers to a drug that is chemically or physically compatible with other components that make up a drug dosage form and physiologically compatible with receptors.

[0047] The term "excipients" refers to substances other than the active ingredient that are included in a dosage form.

[0048] The term "therapeutic effective dose" refers to the amount of a drug compound administered to a patient that is sufficient to effectively treat the disease. Therapeutic effective doses vary depending on the drug compound, the type of disease, the severity of the disease, the patient's age, and other factors, and may be routinely adjusted as appropriate by those skilled in the art.

[0049] There are no particular restrictions on the administration method of the drug of the present invention. Representative administration methods include, but are not limited to, oral, parenteral (intravenous, intramuscular or subcutaneous), and local administration.

[0050] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), or with the following components: (a) filler or compatibilizer; (b) thickener, such as carboxymethyl cellulose and its salts; (c) humectant; (d) disintegrant; (e) slowing agent; (f) absorption accelerator; (g) wetting agent; (h) adsorbent; and (i) lubricant. In capsules, tablets, and pills, the dosage form may also contain a buffer.

[0051] Solid dosage forms such as tablets, sugar pills, capsules, pellets, and granules can be prepared using coatings and shell materials, such as casings and other materials known in the art. They may contain opaque agents, and the release of the active compound in such dosage forms can be delayed in a portion of the digestive tract. If necessary, the active compound may also be formed into microcapsules with one or more of the excipients described above.

[0052] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, or tinctures. In addition to the active compound, liquid dosage forms may contain inert diluents conventionally used in the art, such as water or other solvents, as well as solubilizers and emulsifiers. Besides these inert diluents, the drug may also contain adjuvants such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, and fragrances. Suspensions may contain suspending agents in addition to the active compound.

[0053] Drugs intended for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions, or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents, or excipients are also included.

[0054] Pharmaceutical dosage forms for topical administration include ointments, powders, patches, sprays, and inhalers. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be necessary.

[0055] The beneficial effects of this application mainly include the following aspects:

[0056] (1) In vitro and / or in vivo test results show that the pharmaceutical composition provided by the present invention (containing stilbene compounds and pharmaceutically acceptable excipients) has good efficacy in preventing and / or treating drug-induced liver injury, can play a good role in protecting the liver, can effectively reduce the mortality rate caused by the disease and prolong its survival; at the same time, it can also significantly reduce serum transaminase levels, relieve symptoms such as edema of the liver and spleen, and also has a good inhibitory effect on pro-inflammatory cytokines;

[0057] (2) The experimental results further show that the combination of stilbene compounds and pharmaceutically acceptable excipients (including glucosyl hesperidin and / or glycyrrhizate) has good safety and is easy to dissolve, release and / or absorb, which improves the bioavailability of pterostilbene (PTE) and has a synergistic effect, achieving a better therapeutic effect than any single component. Attached Figure Description

[0058] Figure 1 The image shows the appearance of the PTE-DG-AGH composition of this application obtained in Example 1.

[0059] Figure 2 The image shows an FT-IR comparison of the PTE-DG-AGH composition obtained in Example 1 of this application.

[0060] Figure 3 This is a DSC comparison chart of the PTE-DG-AGH composition of this application obtained in Example 1.

[0061] Figure 4 The image shows an XRD comparison of the PTE-DG-AGH composition obtained in Example 1 of this application.

[0062] Figure 5 This is a comparison chart showing the solubility of the PTE-DG-AGH composition of this application obtained in Example 1 in PBS.

[0063] Figure 6 The micelle size changes of the PTE-DG-AGH composition of this application obtained in Example 1 after storage at 4°C and 25°C for 2 to 6 weeks.

[0064] Figure 7 The encapsulation efficiency change of the PTE-DG-AGH composition of this application obtained in Example 1 after storage at 4°C and 25°C for 2 to 6 weeks.

[0065] Figure 8 This is a comparison chart of the antioxidant activity of the PTE-DG-AGH composition of this application obtained in Example 1 (ABTS method: different incubation times at a fixed concentration).

[0066] Figure 9 This is a comparison chart of the antioxidant activity of the PTE-DG-AGH composition of this application obtained in Example 1 (ABTS method: different concentrations under fixed culture time).

[0067] Figure 10 This is a comparison chart of the antioxidant activity of the PTE-DG-AGH composition of this application obtained in Example 1 (FRAP method: different incubation times at a fixed concentration).

[0068] Figure 11This is a comparison chart of the antioxidant activity of the PTE-DG-AGH composition of this application obtained in Example 1 (FRAP method: different concentrations under fixed culture time).

[0069] Figure 12 This is a comparison chart of the in vitro release curves of the PTE-DG-AGH composition of this application obtained in Example 1 in PBS.

[0070] Figure 13 This is a comparison chart of the in vitro release curves of the PTE-DG-AGH composition of this application obtained in Example 1 in SGF / SIF.

[0071] Figure 14 The image shows a comparison of blood vessels before and after contact with the chicken embryo allantoic membrane (CAM) of the PTE-DG-AGH composition of this application obtained in Example 1.

[0072] Figure 15 This is a comparison chart of trypan blue absorption when the PTE-DG-AGH composition of this application obtained in Example 1 contacts the allantoic membrane (CAM) of chicken embryos.

[0073] Figure 16 The in vivo pharmacokinetic study of the PTE-DG-AGH composition of this application obtained in Example 1: the change of PET content in plasma over time.

[0074] Figure 17 In vivo tissue distribution study of the PTE-DG-AGH composition of this application obtained in Example 1: PTE content in organs such as heart and liver 4 hours after administration.

[0075] Figure 18 In vivo tissue distribution study of the PTE-DG-AGH composition of this application obtained in Example 1: PTE content in gastrointestinal tissues such as stomach and duodenum 4 hours after administration.

[0076] Figure 19 In vivo tissue distribution study of the PTE-DG-AGH composition of this application obtained in Example 1: PTE content in organs such as heart and liver 8 hours after administration.

[0077] Figure 20 In vivo tissue distribution study of the PTE-DG-AGH composition of this application obtained in Example 1: PTE content in gastrointestinal tissues such as stomach and duodenum 8 hours after administration.

[0078] Figure 21 This is a comparison of the liver weight to body weight ratios of mice in different groups after injection of acetaminophen.

[0079] Figure 22 This is a comparison of the ratio of spleen weight to body weight in mice of different groups after injection of acetaminophen.

[0080] Figure 23 This is a comparison of the serum aspartate aminotransferase (AST) levels in mice from different groups after acetaminophen injection.

[0081] Figure 24 This is a comparison of serum alanine aminotransferase (ALT) levels in mice from different groups after acetaminophen injection.

[0082] Figure 25 Images show the appearance of liver tissue in mice after injection of acetaminophen.

[0083] Figure 26 Comparative images of the histopathological observations of the livers of mice in different groups after injection of acetaminophen, stained with hematoxylin and eosin, under a microscope.

[0084] Figure 27 This is a comparison chart showing the percentage of hepatocyte necrosis area in each group of mice after injection of acetaminophen.

[0085] Figure 28 This is a comparison of superoxide dismutase (SOD) levels in the liver tissues of mice in different groups after injection of acetaminophen.

[0086] Figure 29 This is a comparison of malondialdehyde (MDA) levels in the liver tissues of mice in different groups after injection of acetaminophen.

[0087] Figure 30 This is a comparison of HMGB1 levels in the liver tissues of mice in different groups after injection of acetaminophen.

[0088] Figure 31 This is a comparison of IL-1β levels in the liver tissues of mice in different groups after injection of acetaminophen.

[0089] Figure 32 This is a comparison of IL-6 levels in the liver tissues of mice in different groups after injection of acetaminophen.

[0090] Figure 33 This is a comparison of NF-κB levels in the liver tissues of mice in different groups after injection of acetaminophen.

[0091] Figure 34 This is a comparison of TNF-α levels in the liver tissues of mice in different groups after injection of acetaminophen. Detailed Implementation

[0092] The present application will be clearly and completely described below with reference to specific embodiments. Those skilled in the art will understand that the embodiments described below are some embodiments of the present application, but not all embodiments, and are only used to illustrate the present application, and should not be regarded as a limitation on the scope of protection of the present application.

[0093] In this application, unless otherwise specified, the conditions shall be in accordance with the standard conditions or the conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they shall be conventional products that can be purchased commercially.

[0094] For example:

[0095] Pterostilbene (abbreviated as PTE): Purchased from Aladdin Ltd. (Shanghai, China).

[0096] Dipotassium glycyrrhizinate (DG): purity ≥98%, purchased from Shaanxi Fujie Pharmaceutical Co., Ltd.

[0097] α-glucosyl hesperidin (AGH): Purchased from Cosfa International Trading Co., Ltd. (Shanghai, China).

[0098] Trypan Blue (TB) and Acetaminophen (APAP): Purchased from Beijing Solarbio Technology Co., Ltd.

[0099] Male SD rats (approximately 220–250 g) and male C57BL / 6 mice (8 weeks old) were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd. All animals were healthy and showed no clinically observable physical abnormalities.

[0100] In this application, SPSS Statistics 24 software was used for data analysis, and P<0.05 indicates significance.

[0101] Example 1

[0102] Solvent evaporation method: 40 mg of pterostilbene (PTE) and 600 mg of excipients (containing 61 mg α-glucosylhesperidin (AGH) and 539 mg dipotassium glycyrrhizate (DG)) were completely dissolved in ethanol (the amount of ethanol can be selected from 60 ml to 70 ml, and 70 ml is used in this example). Then, the ethanol was completely removed by vacuum evaporation at 40 °C using a rotary evaporator to obtain the PTE-DG-AGH composition of this application, which is a white powder solid with the appearance as shown. Figure 1 As shown.

[0103] The FT-IR, DSC, and XRD spectra of the aforementioned PTE-DG-AGH composition of this application are shown in the figures below. Figure 2 , Figure 3 and Figure 4PTE, DG & AGH physical mixture (ratio: 61 mg α-glucosyl hesperidin and 539 mg dipotassium glycyrrhizate) and PTE & DG & AGH physical mixture (ratio: 40 mg pterostilbene, 61 mg α-glucosyl hesperidin and 539 mg dipotassium glycyrrhizate) served as controls.

[0104] Example 2

[0105] 1. Solubility

[0106] Excess PTE and the PTE-DG-AGH composition of this application (obtained in Example 1) were added to 1 ml of test solution: water, phosphate buffer (commercially available PBS powder from Wuhan Saiwei Biotechnology Co., Ltd., dissolved in distilled water, concentration 0.01M, pH 7.2-7.4 at 25℃), PBS (pH = 7.4), stirred at 37℃ for 24 h, filtered through a 0.22 μm filter membrane, diluted with methanol, and the PTE concentration was determined by high performance liquid chromatography (HPLC) to calculate the solubility.

[0107] The concentration of PTE in the sample was determined by HPLC:

[0108] Chromatographic column: Agilent ZORBAX SB-C18 (250mm×4.60mm, 5μm), column temperature 25℃; mobile phase: methanol-water = 70:30 (volume ratio), flow rate 1.0ml / min; detection wavelength 312nm; injection volume 20μl.

[0109] The results show:

[0110] (1) The PTE-DG-AGH composition of this application is readily soluble in water or PBS, and its appearance is shown in the figure. Figure 5 PTE, DG & AGH physical mixture (ratio: 61 mg α-glucosyl hesperidin and 539 mg dipotassium glycyrrhizate) and PTE & DG & AGH physical mixture (ratio: 40 mg pterostilbene, 61 mg α-glucosyl hesperidin and 539 mg dipotassium glycyrrhizate) were used as controls.

[0111] (2) The solubility of PTE in water and PBS is 5.11±0.45μg / ml and 4.99±1.36μg / ml, respectively, while the solubility of PTE in the PTE-DG-AGH composition of this application in water and PBS is 31395.81±8380.53μg / ml and 42791.61±11697.12μg / ml, respectively.

[0112] 2. Micellar size and zeta potential

[0113] The PTE-DG-AGH composition of this application obtained in Example 1 was prepared into an aqueous solution (PTE concentration 1.0 mg / mL), and the micelle size and Zeta potential were determined at 25 °C using a Zetasizer Nano ZS90 (dynamic light scattering method, DLS).

[0114] The results showed that the average micelle size of the aqueous solution of the PTE-DG-AGH composition of this application was 8.85±1.14 nm, and the average Zeta potential was -0.90±0.51 mV.

[0115] 3. Encapsulation rate

[0116] The PTE-DG-AGH composition obtained in Example 1 was prepared into an aqueous solution (PTE concentration 1.0 mg / mL), filtered through a 0.22 μm filter membrane, and the unencapsulated PTE was separated by filtration. The solutions of the PTE-DG-AGH composition before and after filtration were diluted with methanol to disrupt the micelles. The PTE concentration in the solution was determined by the aforementioned HPLC method; the encapsulation efficiency was the ratio (%) of the PTE concentration detected after filtration to the PTE concentration detected before filtration.

[0117] The results showed that the initial (on the day of preparation) encapsulation efficiency was 99.90% ± 0.02%.

[0118] 4. Storage stability evaluation

[0119] The PTE-DG-AGH composition obtained in Example 1 was sealed in a glass bottle and stored in the dark at 4°C and 25°C for 6 weeks, respectively. Every two weeks (weeks 2, 4, and 6), the PTE-DG-AGH composition was prepared into an aqueous solution (PTE concentration 1.0 mg / mL). The micelle size and encapsulation efficiency were tested according to the aforementioned method. The results are shown in the figure below. Figure 6 and Figure 7 .

[0120] The results showed that the PTE-DG-AGH composition of this application exhibited good stability after being stored at 4°C or 25°C for 6 weeks, with little change in average micelle size and encapsulation efficiency.

[0121] Example 3

[0122] In vitro antioxidant activity

[0123] The antioxidant activities of PTE, the physical mixture of DG & AGH (ratio: 61 mg α-glucosyl hesperidin and 539 mg dipotassium glycyrrhizate), and the PTE-DG-AGH composition of this application (obtained in Example 1) were detected by 2,2'-hydrazine-bis(3-ethylbenzothiazoline-6-sulfonic acid) diamine salt (ABTS method) and ferric reduction / antioxidant capacity method (FRAP), respectively. The results are shown in the figures below. Figure 8 (ABTS method: different incubation times at a fixed concentration) Figure 9 (ABTS method: different concentrations under fixed culture time) Figure 10 (FRAP method: different incubation times at a fixed concentration) and Figure 11 (FRAP method: different concentrations under fixed culture time).

[0124] The results of ABTS and FRAP tests showed that the PTE-DG-AGH composition of this application exhibited stronger antioxidant activity compared with PTE.

[0125] Example 4

[0126] In vitro drug release assay

[0127] (1) The aqueous solution (PTE concentration 1.0 mg / mL) and 1 mL of PTE suspension (PTE concentration 1.0 mg / mL) of the PTE-DG-AGH composition (obtained in Example 1) of this application were placed in dialysis bags (molecular weight cutoff = 3500 Da), immersed in 100 mL of phosphate-buffered saline (PBS) (pH = 7.4), and cultured at 37°C with a shaker at 100 rpm. At different time points, 1 mL of culture medium was taken (with 1 mL of phosphate-buffered saline added simultaneously), and the PTE concentration in the 1 mL of culture medium was detected by the aforementioned HPLC method. The in vitro release curve in PBS is shown in [Figure number missing]. Figure 12 .

[0128] (2) The aqueous solution (PTE concentration 1.0 mg / mL) and 1 mL of PTE suspension (PTE concentration 1.0 mg / mL) of the PTE-DG-AGH composition (obtained in Example 1) of this application were placed in dialysis bags (molecular weight cutoff = 3500 Da). For the first 2 hours, they were incubated in 100 mL of simulated gastric fluid SGF (pH = 1.2) at 37°C with a shaker at 100 rpm. Then, they were transferred to 100 mL of simulated intestinal fluid SIF (pH = 6.8) and incubated again at 37°C with a shaker at 100 rpm. At different time points, 1 mL of culture medium (SGF or SIF) samples were collected (with an equal volume of SGF or SIF added simultaneously). The PTE concentration in the 1 mL of culture medium was detected by the aforementioned HPLC method. The in vitro release curve in SGF / SIF is shown in [Figure number missing]. Figure 13 .

[0129] The results show:

[0130] (1) After 24 hours, the cumulative release of PTE in PBS was 12.84% ± 1.35%, while the cumulative release of PTE in PBS in the PTE-DG-AGH composition of this application was 69.73% ± 2.47%.

[0131] (2) In the first 2 hours, the cumulative release of PTE in SGF was 4.20% ± 0.57%, and the cumulative release of PTE in SGF in the PTE-DG-AGH composition of this application was 5.15% ± 0.22%; after 24 hours, the cumulative release of PTE in SIF was 10.23% ± 1.63%, and the cumulative release of PTE in SIF in the PTE-DG-AGH composition of this application was 50.29% ± 0.92%.

[0132] Example 5

[0133] 1. Hemolysis test

[0134] Physiological saline was used as a negative control (0% hemolysis), and 0.1% Triton X-100 was used as a positive control (100% hemolysis) for the hemolysis test.

[0135] The results showed that the hemolysis rate of the PTE-DG-AGH composition (obtained in Example 1) in aqueous solution was less than 0.1% when the concentration of PTE in the aqueous solution was 0.1 mg / ml, and the hemolysis rate of the PTE in the aqueous solution was less than 1% when the concentration of PTE in the aqueous solution was 0.2 to 2 mg / ml, which is lower than the safety value of 5%, indicating that the PTE-DG-AGH composition of this application has good blood biocompatibility.

[0136] 2. Chicken embryo chorioallantoic membrane (HET-CAM) - trypan blue staining test

[0137] The experimental chicken embryos were randomly divided into the following 6 groups, with 3 embryos in each group:

[0138] ① Negative control: 0.9% NaCl solution (physiological saline);

[0139] ② Positive control: 0.1M NaOH solution;

[0140] ③ DG & AGH physical mixing: aqueous solution (ratio: 61 mg α-glucosyl hesperidin and 539 mg dipotassium glycyrrhizate);

[0141] ④PTE: 8 mg / ml suspension;

[0142] ⑤PTE&DG&AGH physical mixture: suspension, PTE content is 8mg / ml (ratio: 40mg pterostilbene, 61mg α-glucosylhesperidin and 539mg dipotassium glycyrrhizate);

[0143] ⑥ The PTE-DG-AGH composition of this application (obtained in Example 1): aqueous solution with a PTE concentration of 8 mg / ml.

[0144] After the aforementioned sample solution was in contact with the chicken embryo allantoic membrane (CAM) for 5 minutes, the vascular condition of the CAM was as follows: Figure 14 As shown in the figure; then, trypan blue staining was performed and the trypan blue absorption was measured, and the results are as follows. Figure 15 As shown.

[0145] The results showed that the positive control (0.1M NaOH) group exhibited symptoms such as congestion and thrombosis, as well as a high amount of trypan blue absorption, indicating that it had severe irritation. In contrast, the PTE-DG-AGH composition group of this application was basically similar to the saline (normal) group and did not show obvious irritation, which, on the other hand, demonstrates the safety of the PTE-DG-AGH composition of this application.

[0146] Example 6

[0147] 1. Pharmacokinetic studies in rats

[0148] Twelve male SD rats were randomly divided into two groups. Before the experiment, the rats were fasted for 24 hours but allowed free access to water. One group was administered PTE suspension (0.5 wt% sodium carboxymethyl cellulose aqueous solution as the dispersion solvent, PTE concentration 10 mg / mL; dosage 100 mg / kg), while the other group was administered the PTE-DG-AGH composition solution (obtained in Example 1) of this application (0.5 wt% sodium carboxymethyl cellulose aqueous solution as the solvent, PTE concentration 10 mg / mL; dosage 100 mg / kg). At 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, and 12 hours after administration, 0.5 mL blood samples were drawn from the tail vein, collected in anticoagulant tubes, centrifuged, stored at -20°C, and then analyzed by HPLC.

[0149] To determine plasma drug concentrations, 100 μL of plasma was mixed with 200 μL of an acetonitrile solution of pinosylvine (2 μg / mL, internal standard), centrifuged at 10,000 rpm for 10 min, and the supernatant was filtered through a 0.22 μm filter and injected into an HPLC system (retention time of pinosylvine internal standard: 5.0–5.3 min; retention time of PTE: 10.1–10.7 min). Data were analyzed using the pharmacokinetic program Drug and Statistics 2.0. The results are shown in Table 1 and [Table data would be inserted here]. Figure 16 .

[0150] Table 1. Pharmacokinetic parameters of PTE after administration of PTE and the PTE-DG-AGH composition of this application (n=6)

[0151] parameter PTE The PTE-DG-AGH composition of this application <![CDATA[C max (μg / mL)]]> 2.175 4.940 <![CDATA[T max (min)]]> 240 60 <![CDATA[T 1 / 2 (h)]]> 1.76 1.65 <![CDATA[AUC 0-t (ng / mL h)]]> 13115.72 34832.25

[0152] in

[0153] Peak drug concentration (C) maxPeak concentration: The highest plasma drug concentration that can be achieved after administration. This is the maximum plasma drug concentration on the time-time curve.

[0154] Peak time (T) max Peak time to drug action: The time required for the drug to reach its highest concentration (peak concentration) on the human plasma concentration curve after administration.

[0155] Elimination half-life (T) 1 / 2 Half-life of a drug refers to the time required for the drug's highest concentration in plasma to decrease by half.

[0156] Area under the drug-time curve (AUC): The area enclosed by the blood drug concentration curve on the time axis; this parameter represents the bioavailability of the drug (the degree to which the drug is absorbed and utilized in the human body). A larger AUC indicates higher bioavailability, and vice versa.

[0157] The results showed that PTE reached its peak concentration T at 240 min. max =2.175 μg / ml, its area under the curve (AUC) is 2.175 μg / ml. 0-t =13115.72 ng / ml·h, while the PTE in the PTE-DG-AGH composition of this application reaches a peak concentration T of 13115.72 ng / ml·h at 60 min. max =4.940 μg / ml, its area under the curve (AUC) is 4.940 μg / ml. 0-t = 34832.25 ng / ml·h; These figures indicate that, compared with PTE, the PTE in the PTE-DG-AGH composition of this application has better bioavailability.

[0158] 2. Evaluation of in vivo tissue distribution

[0159] Twenty-four SD rats were randomly divided into two groups to assess the distribution of PTE and the PTE-DG-AGH composition of this application in major organs and gastrointestinal tissues. The administration procedure and dosage were the same as described in the previous "Pharmacokinetic Study in Rats". At 4 h and 8 h after administration, 12 rats from each group were randomly sacrificed, and their organs (including heart, liver, spleen, lung, kidney and brain) and the entire gastrointestinal tract (further divided into stomach, duodenum, jejunum, ileum, cecum, colon and rectum) were removed, weighed, and stored at -20°C. Then, HPLC analysis was performed (as mentioned before, pinosylvine was used as an internal standard).

[0160] Four hours after administration, the PTE levels in organs such as the heart, liver, spleen, lungs, kidneys, and brain were as follows: Figure 17 The PTE content in gastrointestinal tissues such as the stomach, duodenum, jejunum, ileum, cecum, colon, and rectum is shown in the table below. Figure 18 Eight hours after administration, the PTE levels in organs such as the heart, liver, spleen, lungs, kidneys, and brain were as follows: Figure 19 The PTE content in gastrointestinal tissues such as the stomach, duodenum, jejunum, ileum, cecum, colon, and rectum is shown in the table below. Figure 20 (*: indicates that P < 0.05 compared to the PTE group).

[0161] The results show:

[0162] (1) The PTE content in organs such as the heart, liver, spleen, lungs, kidneys, and brain of the PTE-DG-AGH composition of this application at 4h and 8h after administration was higher than the PTE content in organs such as the heart, liver, spleen, lungs, kidneys, and brain at 4h and 8h after administration of PTE (e.g., ...). Figure 17 and Figure 19 As shown in the figure, this indicates that compared with PTE alone, the PTE in the PTE-DG-AGH composition of this application can more easily enter organs such as the heart, liver, spleen, lungs, kidneys, and brain, so as to better exert the pharmacological effects of PTE, especially the protection and / or treatment of the liver.

[0163] (2) The PTE content in the gastrointestinal tissues such as the stomach, duodenum, jejunum, ileum, cecum, colon, and rectum at 4 h and 8 h after administration of the PTE-DG-AGH composition of this application was higher than the PTE content in the same gastrointestinal tissues at 4 h and 8 h after administration of PTE (e.g., ...). Figure 18 and Figure 20 As shown in the figure, this indicates that, compared to PTE alone, the PTE in the PTE-DG-AGH composition of this application is also more easily absorbed and utilized by gastrointestinal tissues.

[0164] Example 7

[0165] Healthy male C57BL / 6 mice were randomly divided into the following 9 groups, with 16 mice in each group:

[0166] ① Healthy control group;

[0167] ②PBS treatment group;

[0168] ③ Positive (NAC: N-acetylcysteine) treatment group: Dosage, 150 mg / kg;

[0169] ④DG&AGH physical therapy group (ratio: 61mg α-glucosyl hesperidin and 539mg dipotassium glycyrrhizate): dosage, dipotassium glycyrrhizate 539mg / kg and α-glucosyl hesperidin 61mg / kg;

[0170] ⑤PTE suspension treatment group: Dosage, 40 mg / kg;

[0171] ⑥PTE&DG&AGH physical therapy combination group (ratio: 40mg pterostilbene, 61mg α-glucosylhesperidin and 539mg dipotassium glycyrrhizinate): dosage, pterostilbene 40mg / kg, dipotassium glycyrrhizinate 539mg / kg and α-glucosylhesperidin 61mg / kg;

[0172] ⑦ The low-dose treatment group (10 mg / kg PTE) of the PTE-DG-AGH composition of this application (obtained in Example 1);

[0173] ⑧ The medium-dose treatment group (20 mg / kg PTE) of the PTE-DG-AGH composition of this application (obtained in Example 1);

[0174] ⑨ High-dose treatment group (40 mg / kg PTE) of the PTE-DG-AGH composition of this application (obtained in Example 1).

[0175] The mice were administered the drug for 7 consecutive days (the healthy control mice were fed the same amount of PBS). The mice were fasted for 12 hours before administration on day 7 and injected with acetaminophen (400 mg / kg) 1 hour after administration on day 7. The healthy control mice were injected with the same amount of PBS.

[0176] 1. Liver to body weight ratio & Spleen to body weight ratio

[0177] Three hours after injection of APAP (acetaminophen) or PBS, mice in each group were sacrificed, and serum, liver, and spleen samples were collected for analysis. The samples were weighed, and the liver-to-body weight ratio and spleen-to-body weight ratio were calculated to evaluate the degree of liver and spleen edema. The results are shown in the table below. Figure 21 and Figure 22 (Where, * indicates P < 0.05 compared to the healthy control group; # indicates P < 0.05 compared to the PBS group; & indicates P < 0.05 compared to the NAC group; $ indicates P < 0.05 compared to the DG&AGH group; @ indicates P < 0.05 compared to the PTE group; % indicates P < 0.05 compared to the PTE&DG&AGH group; ^ indicates P < 0.05 compared to the low-dose group of the PTE-DG-AGH composition of this application; + indicates P < 0.05 compared to the medium-dose group of the PTE-DG-AGH composition of this application).

[0178] The results showed that the PTE-DG-AGH composition of this application can effectively relieve and / or prevent symptoms such as hepatosplenic edema caused by excessive APAP (acetaminophen).

[0179] 2. Serum Aspartate Aminotransferase (AST) and Alanine Aminotransferase (ALT) Tests

[0180] The levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in mouse serum were measured using a commercially available kit (Nanjing Jiancheng Bioengineering Institute). (AST and ALT are two important indicators reflecting the severity of liver damage.) The results are shown in the table below. Figure 23 and Figure 24 (Where, * indicates P < 0.05 compared to the healthy control group; # indicates P < 0.05 compared to the PBS group; & indicates P < 0.05 compared to the NAC group; $ indicates P < 0.05 compared to the DG&AGH group; @ indicates P < 0.05 compared to the PTE group; % indicates P < 0.05 compared to the PTE&DG&AGH group; ^ indicates P < 0.05 compared to the low-dose group of the PTE-DG-AGH composition of this application).

[0181] The results showed that the PTE-DG-AGH composition of this application can effectively inhibit or avoid the sudden increase in serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) caused by excessive APAP (acetaminophen), and has good liver protection and hepatoprotective effects.

[0182] 3. Histological examination

[0183] Formalin-fixed liver (see) Figure 25 Embedded in paraffin, sectioned, stained with hematoxylin and eosin, and evaluated histopathologically under a microscope (see [link to article]). Figure 26 The ratio of hepatocyte necrosis area to total area was used for semi-quantitative analysis, and the results are shown in [Figure number missing]. Figure 27 (Where, * indicates P < 0.05 compared to the healthy control group; # indicates P < 0.05 compared to the PBS group; & indicates P < 0.05 compared to the NAC group; $ indicates P < 0.05 compared to the DG&AGH group; @ indicates P < 0.05 compared to the PTE group; % indicates P < 0.05 compared to the PTE&DG&AGH group; ^ indicates P < 0.05 compared to the low-dose group of the PTE-DG-AGH composition of this application; + indicates P < 0.05 compared to the medium-dose group of the PTE-DG-AGH composition of this application).

[0184] The results showed that the proportion of hepatocyte necrosis area in the PBS group mice was as high as 67.65±5.66%, while the hepatocyte necrosis area in the high-dose treatment group (40mg / kg PTE) and the NAC treatment group of the present application was 0%. The PTE-DG-AGH composition of the present application can effectively inhibit or avoid the symptoms such as hepatocyte necrosis, liver edema, and hemorrhage caused by excessive APAP (acetaminophen), and has good liver protection and hepatoprotective effects.

[0185] 4. Detection of superoxide dismutase (SOD) and malondialdehyde (MDA)

[0186] Liver tissue was homogenized with physiological saline. The supernatant after centrifugation was used to detect SOD and MDA levels using a commercially available kit (Beyotime Biotechnology, Shanghai, China). Results are expressed as U / mg protein. (See attached table.) Figure 28 and Figure 29 (Where, * indicates P < 0.05 compared to the healthy control group; # indicates P < 0.05 compared to the PBS group; & indicates P < 0.05 compared to the NAC group; $ indicates P < 0.05 compared to the DG&AGH group; @ indicates P < 0.05 compared to the PTE group; % indicates P < 0.05 compared to the PTE&DG&AGH group; ^ indicates P < 0.05 compared to the low-dose group of the PTE-DG-AGH composition of this application; + indicates P < 0.05 compared to the medium-dose group of the PTE-DG-AGH composition of this application).

[0187] The results showed that the PTE-DG-AGH composition of this application can effectively inhibit or avoid the significant decrease in superoxide dismutase (SOD) level in liver tissue caused by excessive APAP (acetaminophen), and can also effectively inhibit or avoid the significant increase in malondialdehyde (MDA) level in liver tissue caused by excessive APAP (acetaminophen), thus exhibiting good liver protection and hepatoprotective effects.

[0188] 5. Enzyme-linked immunosorbent assay (ELISA)

[0189] The levels of different cytokines (e.g., pro-inflammatory cytokines) in mouse liver were detected using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (Shanghai Enzyme-Link Biotechnology Co., Ltd.).

[0190] HMGB1 (referring to high-mobility group box 1, an important late-stage pro-inflammatory factor; studies have shown that HMGB1 is an important late-stage inflammatory mediator of endotoxin lethality and plays an important role in the pathogenesis of various diseases such as sepsis, tumors, and arthritis).

[0191] IL-1β (an important pro-inflammatory cytokine with strong pro-inflammatory activity, capable of inducing various pro-inflammatory mediators);

[0192] IL-6 (an important inflammatory cytokine, mainly expressed in inflammatory responses, etc.);

[0193] NF-κB (an important transcription activator involved in various pathological processes such as inflammation and acute response, cell proliferation, apoptosis, and viral infection);

[0194] TNF-α (a pro-inflammatory cytokine whose dysfunction is believed to be associated with many diseases; TNF-α inhibitors have been reported to be the first-line treatment for autoimmune diseases such as rheumatoid arthritis, ankylosing spondylitis, and psoriatic arthritis).

[0195] See results Figures 30-34 (Where, * indicates P < 0.05 compared to the healthy control group; # indicates P < 0.05 compared to the PBS group; & indicates P < 0.05 compared to the NAC group; $ indicates P < 0.05 compared to the DG&AGH group; @ indicates P < 0.05 compared to the PTE group; % indicates P < 0.05 compared to the PTE&DG&AGH group; ^ indicates P < 0.05 compared to the low-dose group of the PTE-DG-AGH composition of this application; + indicates P < 0.05 compared to the medium-dose group of the PTE-DG-AGH composition of this application).

[0196] The results showed that the PTE-DG-AGH composition of this application can effectively inhibit the expression of cytokines such as HMGB1, IL-1β, IL-6, NF-κB, and TNF-α, and has good preventive and / or therapeutic effects on various inflammations caused by these factors. It can be used as a potential inhibitor of HMGB1, IL-1β, IL-6, NF-κB, or TNF-α, providing more options for clinical drug use.

[0197] Of course, this application may have other various implementations. Without departing from the spirit and essence of this application, those skilled in the art can make various corresponding changes and / or modifications based on this application. All such corresponding changes and / or modifications should fall within the protection scope of the appended claims.

Claims

1. A pharmaceutical composition comprising a stilbene compound and a pharmaceutically acceptable excipient; wherein, The stilbene compound is pterostilbene or a pharmaceutically acceptable salt thereof, and the pharmaceutically acceptable excipients include glucosyl hesperidin and glycyrrhizate; the glucosyl hesperidin is α-glucosyl hesperidin; the glycyrrhizate is dipotassium glycyrrhizate or disodium glycyrrhizate; the weight ratio of the stilbene compound to the pharmaceutically acceptable excipient is 1:5 to 50; the weight ratio of glucosyl hesperidin to glycyrrhizate is 1:2 to 20. The pharmaceutical composition is a liquid formulation; The average diameter of the micelles in the liquid formulation is 2–15 nm; The zeta potential of the liquid formulation is -5 to -0.1 mV.

2. The pharmaceutical composition according to claim 1, characterized in that, The weight ratio of the stilbene compound to the pharmaceutically acceptable excipient is 1:10 to 20.

3. The pharmaceutical composition according to claim 1, characterized in that, The weight ratio of glucosyl hesperidin to glycyrrhizate is 1:5 to 12.

4. The pharmaceutical composition according to any one of claims 1 to 3, characterized in that, The encapsulation efficiency of the stilbene compound is at least 80%.

5. The pharmaceutical composition according to claim 4, characterized in that, The encapsulation efficiency of the stilbene compounds is ≥90% or ≥95%.

6. The pharmaceutical composition according to any one of claims 1 to 3, characterized in that, The stilbene compounds in the pharmaceutical composition are in a therapeutically effective amount.

7. The pharmaceutical composition according to claim 6, characterized in that, The pharmaceutical composition is a liquid formulation, and the solvent for the liquid formulation is selected from pharmaceutically acceptable water or PBS buffer.

8. The pharmaceutical composition according to any one of claims 1 to 3, characterized in that, The pharmaceutical composition is prepared by a method comprising the following steps: dispersing or dissolving a stilbene compound and a pharmaceutically acceptable excipient together in an organic solvent, mixing them thoroughly, and then removing the organic solvent by rotary evaporation at 35–45°C.

9. The pharmaceutical composition according to claim 8, characterized in that, The organic solvent is an alcohol solvent; and / or, the amount of the organic solvent used per milligram of the stilbene compound is 0.5 to 20 mL.

10. The pharmaceutical composition according to claim 9, characterized in that, The alcohol solvent is methanol and / or ethanol; and / or, the amount of the organic solvent used per milligram of the stilbene compound is 1.2 to 2.5 mL.

11. The pharmaceutical composition according to any one of claims 1 to 3, characterized in that, The pharmaceutical composition described herein is a pharmaceutical composition for the prevention and / or treatment of drug-induced liver injury.

12. The pharmaceutical composition according to claim 11, characterized in that, The drug-induced liver injury mentioned refers to drug-induced liver injury caused by acetaminophen.

13. A method for preparing the pharmaceutical composition according to any one of claims 1 to 12, characterized in that, Includes the following steps: The stilbene compound and pharmaceutically acceptable excipients are dispersed or dissolved together in an organic solvent, mixed well, and then the organic solvent is removed by rotary evaporation at 35–45 °C to obtain the product.

14. The preparation method according to claim 13, characterized in that, The organic solvent is an alcohol solvent; and / or, the amount of the organic solvent used per milligram of the stilbene compound is 0.5 to 20 mL.

15. The preparation method according to claim 14, characterized in that, The alcohol solvent is methanol and / or ethanol; and / or, the amount of the organic solvent used per milligram of the stilbene compound is 1.2 to 2.5 mL.

16. Use of the pharmaceutical composition according to any one of claims 1 to 12 in the preparation of a medicament for the prevention and / or treatment of drug-induced liver injury, wherein the drug-induced liver injury is drug-induced liver injury caused by acetaminophen.

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