Composition comprising isoprenylated flavonoid glycosides and uses thereof

Abstract

The application discloses a composition containing isoprenyl flavonoid glycosides and application thereof. The composition contains isoprenyl flavonoid glycosides and excipients, wherein the isoprenyl flavonoid glycosides are icariin or a salt thereof, and the excipients contain glycyrrhizic acid salt. The composition is effective for preventing and / or treating oxidative stress damage, diabetes, hyperlipidemia, fatty liver and the like; in particular, the content of total cholesterol (TC), triglyceride (TG) and low-density lipoprotein cholesterol (LDL-C) in serum can be effectively reduced, the composition has good anti-hyperlipidemia activity and hypolipidemic effect, and is expected to be further developed into a medicine or a functional health product.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a composition containing isopentenyl flavonoid glycosides (icariin or its salt) and its uses. Background Technology

[0002] Icariin (CAS No.: 489-32-7) is a pale yellow needle-shaped crystal that is soluble in ethanol. It can increase blood flow to the heart and brain, promote hematopoietic function, immune function and bone metabolism, and also has the effects of tonifying the kidney and strengthening yang, and anti-aging.

[0003] Dipotassium glycyrrhizate (DG) 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.

[0004] Hyperlipidemia, also known as hyperlipidemia, refers to a group of diseases characterized by elevated levels of cholesterol and / or triglycerides in the blood. Hyperlipidemia is a major cause of cardiovascular and cerebrovascular diseases, with an incidence rate of approximately 7% in the Chinese population, primarily affecting middle-aged and elderly individuals. One of the main harms of hyperlipidemia is its role in causing atherosclerosis, which can lead to coronary heart disease, ischemic stroke, and other diseases. Furthermore, hyperlipidemia is also a significant risk factor for hypertension, impaired glucose tolerance, and diabetes.

[0005] Currently, the commonly used lipid-lowering drugs in clinical practice mainly fall into the following two categories: (1) Statins: Common ones include simvastatin, atorvastatin, rosuvastatin, etc. The main mechanism of action of this class of drugs is to lower cholesterol, and the effect is good; (2) Fibrates: Common ones include fenofibrate, bezafibrate, etc. The main function of this class of drugs is to lower triglycerides.

[0006] To date, no literature has been found reporting the use of icariin or its combination with dipotassium glycyrrhizate for the prevention and / or treatment of hyperlipidemia.

[0007] In view of this, the present invention is hereby proposed. Summary of the Invention

[0008] To address the problems and / or deficiencies of existing technologies, one objective of this invention is to provide a composition comprising isopentenyl flavonoid glycosides (icariin or its salts). This composition is effective in preventing and / or treating oxidative stress damage, diabetes, hyperlipidemia, fatty liver, and other conditions; in particular, it effectively reduces the levels of total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C) in serum, exhibiting good anti-hyperlipidemia activity / lipid-lowering effects, and holds promise for further development into a pharmaceutical product.

[0009] The present invention provides a composition comprising an isopentenyl flavonoid glycoside and an excipient (e.g., a pharmaceutically acceptable excipient), wherein the isopentenyl flavonoid glycoside is icariin or a salt thereof, and the excipient comprises glycyrrhizate.

[0010] In any of the above technical solutions (the compositions described), the mass ratio of the isopentenyl flavonoid glycoside compound to the glycyrrhizic acid is 1:2 to 50 (e.g., mass ratios of 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, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, etc.). Preferably, the mass ratio of the isopentenyl flavonoid glycoside compound to the glycyrrhizate is 1:5 to 25; More preferably, the mass ratio of the isopentenyl flavonoid glycoside compound to the glycyrrhizate is 1:9 to 17; for example, the mass ratio of the isopentenyl flavonoid glycoside compound to the glycyrrhizate is 1:15.

[0011] In any of the above technical solutions (the compositions described), the isopentenyl flavonoid glycoside compound is icariin; 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. Preferably, the glycyrrhizic acid salt is selected from dipotassium glycyrrhizate or disodium glycyrrhizate.

[0012] In any of the above technical solutions (the compositions described), the excipients coat the isopentenyl flavonoid glycosides; preferably, the excipients coat the isopentenyl flavonoid glycosides to form nanoparticles; more preferably, the average particle size of the nanoparticles is 0.5 to 300 nm (e.g., average particle sizes of 1 nm, 5 nm, 10 nm, 15 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 150 nm, 200 nm, 250 nm, 300 nm, etc.), and even more preferably 1 to 100 nm.

[0013] In any of the above technical solutions (the compositions described), the composition may be a drug encapsulated by a carrier such as microspheres, nanoparticles, or liposomes; preferably, it may be a drug encapsulated by nanoparticles.

[0014] In any of the above technical solutions (the compositions described), the composition may be carrier nanoparticles, carrier liposomes, etc.; preferably, carrier nanoparticles.

[0015] In any of the above technical solutions (the compositions described), the isopentenyl flavonoid glycosides exist in an amorphous form.

[0016] In any of the above technical solutions (the compositions described), the encapsulation efficiency of the isopentenyl flavonoid glycoside compound is at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, etc.); preferably, the encapsulation efficiency of the isopentenyl flavonoid glycoside compound is ≥90% or ≥95%.

[0017] In any of the above technical solutions (the compositions described), the composition is a liquid formulation, a semi-solid formulation, or a solid formulation; and / or, the isopentenyl flavonoid glycoside compound in the composition is in a therapeutically effective amount.

[0018] In any of the above technical solutions (the compositions described), the composition is a liquid formulation, and the solvent of the liquid formulation is selected from water, sodium carboxymethyl cellulose aqueous solution, or PBS buffer.

[0019] In any of the above technical solutions (the compositions described), the liquid formulation is a nanomicelle formulation.

[0020] In any of the above technical solutions (the compositions described), when the content of isopentenyl flavonoid glycosides in the liquid formulation is 1 mg / ml, the liquid formulation satisfies one or more of the following conditions ① to ③: ① The average micelle size of the liquid formulation is 0.5–300 nm (e.g., 1 nm, 5 nm, 10 nm, 15 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 150 nm, 200 nm, 250 nm, 300 nm, etc.), preferably 1–100 nm, more preferably 50–95 nm; ② The polydispersity index of the liquid formulation is ≤0.5, preferably ≤0.3; ③ The zeta potential of the liquid formulation is -20 to 0 mV (e.g., -20 mV, -19 mV, -18 mV, -17 mV, -16 mV, -15 mV, -14 mV, -13 mV, -12 mV, -11 mV, -10 mV, -9 mV, -8 mV, -7 mV, -6 mV, -5 mV, -2 mV, -1 mV, etc.), preferably -10 to 0 mV; Preferably, when the content of isopentenyl flavonoid glycosides in the liquid formulation is 1 mg / ml, the liquid formulation simultaneously satisfies conditions ① to ③.

[0021] In any of the above technical solutions (the compositions described), the composition is prepared by a method comprising the following steps: dissolving the isopentenyl flavonoid glycoside compound and the excipients in an organic solvent, and then removing the organic solvent, thereby obtaining the product; Preferably, the organic solvent is an alcohol solvent; and / or, the amount of the organic solvent used per milligram of the isopentenyl flavonoid glycoside is 0.1 to 20 mL (e.g., 0.1 ml, 0.2 ml, 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, 20 ml, etc.), preferably 2 to 10 mL; More preferably, the organic solvent is ethanol.

[0022] In any of the above technical solutions (the compositions described), the compositions are compositions for the prevention and / or treatment of oxidative stress damage, diabetes, hyperlipidemia and / or fatty liver. Alternatively, the composition may be used as an α-D-glucosidase inhibitor, a pancreatic lipase inhibitor, and / or a cholesterol esterase inhibitor.

[0023] The present invention also provides the use of the compositions described in any of the preceding claims in the preparation of medicaments for the prevention and / or treatment of oxidative stress damage, diabetes, hyperlipidemia and / or fatty liver; For example, Use of any of the preceding compositions in the preparation of a medicament for the prevention and / or treatment of oxidative stress damage; Alternatively, the use of any of the preceding compositions in the preparation of a medicament for the prevention and / or treatment of diabetes; Alternatively, the use of any of the preceding compositions in the preparation of a medicament for the prevention and / or treatment of hyperlipidemia; Alternatively, the use of any of the preceding compositions in the preparation of a medicament for the prevention and / or treatment of fatty liver.

[0024] Regarding the definition of terms used, unless otherwise stated, the initial definition provided for a term in this document applies to that term throughout the document; for terms not specifically defined herein, the meaning that a person skilled in the art would give them should be given based on the disclosure and / or context and common knowledge in the art.

[0025] The term "pharmaceutically acceptable" means that a drug is generally chemically or physically compatible with other components that make up a drug dosage form and physiologically compatible with receptors.

[0026] The term "excipients" refers to substances other than the active ingredient that are included in a dosage form.

[0027] 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.

[0028] The beneficial effects of this invention mainly include the following aspects: (1) This invention develops a new use for isopentenyl flavonoid glycosides (icariin or its salts): they can be used to prepare drugs for the prevention and / or treatment of oxidative stress damage, diabetes, hyperlipidemia and / or fatty liver; (2) In vitro and / or in vivo test results show that isopentenyl flavonoid glycosides (icariin or its salts) and their combinations have good efficacy in preventing and / or treating oxidative stress damage, diabetes, hyperlipidemia, fatty liver and other diseases; For example, the inhibitory activity of icariin-glycyrrhizic acid dipotassium (ICA-DG) on α-D-glucosidase is IC50. 50 The concentration was 0.056 ± 0.009 mg / ml (calculated as ICA), which has potential application value in the prevention, treatment or adjuvant treatment of diabetes. And / or, the inhibitory activity IC50 against lipase and cholesterol esterase 50 The concentrations were 1.117±0.164 mg / ml and 0.659±0.055 mg / ml (calculated as ICA), respectively, demonstrating good anti-hyperlipidemia activity; And / or, can effectively reduce the levels of total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C) in serum, and has a good lipid-lowering effect; And / or, can reduce and / or alleviate fatty infiltration of the liver, and has a liver-protective effect; (3) The test results also showed that the combination of isopentenyl flavonoid glycosides (icariin or its salt) and glycyrrhizic acid salt has high safety, good biocompatibility, low cytotoxicity, good stability, and is easy to dissolve, release and / or absorb, which improves the bioavailability of active ingredients. The two have a synergistic effect and achieve better therapeutic effects than any single component. Attached Figure Description

[0029] Figure 1 This is a diagram showing the appearance of the ICA-DG obtained in Example 1.

[0030] Figure 2 The images shown are SEM images of the microstructures of ICA, DG, DG&ICA, and ICA-DG in Example 1. Image B shows the microstructure of ICA, image C shows the microstructure of DG, image D shows the microstructure of DG&ICA, and image E shows the microstructure of ICA-DG.

[0031] Figure 3 The XRD comparison spectra of ICA, DG, DG&ICA, and ICA-DG in Example 1 are shown.

[0032] Figure 4 The FT-IR comparison spectra of ICA, DG, DG&ICA, and ICA-DG in Example 1 are shown.

[0033] Figure 5 The chart shows the encapsulation efficiency of ICA-DG obtained under different ICA and DG ratios in Example 2.

[0034] Figure 6 This is a comparison chart of the water solubility of ICA, DG&ICA, and ICA-DG in Example 2.

[0035] Figure 7 The solubility charts (4 h) of ICA, DG&ICA and ICA-DG in different solvents in Example 2 are shown.

[0036] Figure 8 The solubility charts (24 h) of ICA, DG&ICA and ICA-DG in different solvents in Example 2 are shown.

[0037] Figure 9 This is a graph showing the ICA retention rate after 12 weeks of storage in the ICA-DG solid state in Example 2.

[0038] Figure 10 The chart shows the ICA retention rate of the ICA-DG aqueous solution in Example 2 after 3 weeks of storage.

[0039] Figure 11 This is a comparison chart of the in vitro release curves of ICA and ICA-DG in Example 3.

[0040] Figure 12 This is a chart comparing the hemolysis rates of ICA, DG, DG&ICA, and ICA-DG in Example 4.

[0041] Figure 13 This is a comparison of blood vessels before and after contact between ICA, DG, DG&ICA, and ICA-DG and the chicken embryo allantoic membrane (CAM) in Example 4.

[0042] Figure 14 This is a comparison chart of trypan blue absorption after ICA, DG, DG&ICA, and ICA-DG came into contact with the chicken embryo allantoic membrane (CAM) in Example 4.

[0043] Figure 15 This is a comparison chart of the cytotoxicity test results of ICA, DG, DG&ICA, and ICA-DG in Example 4.

[0044] Figure 16 This is a comparative chart showing the effects of ICA, DG, DG&ICA, and ICA-DG in preventing and / or treating oxidative stress damage in Example 6.

[0045] Figure 17 This is a comparison chart of the effects of ICA, DG, DG&ICA, and ICA-DG on ROS levels in Example 6.

[0046] Figure 18 This is a comparative chart showing the effects of ICA, DG, DG&ICA, and ICA-DG on SOD levels in Example 6.

[0047] Figure 19 This is a comparative chart showing the effects of ICA, DG, DG&ICA, and ICA-DG on serum TC in Example 9.

[0048] Figure 20 This is a comparative chart showing the effects of ICA, DG, DG&ICA, and ICA-DG on serum TG in Example 9.

[0049] Figure 21 This is a comparative chart showing the effects of ICA, DG, DG&ICA, and ICA-DG on serum LDL-C in Example 9.

[0050] Figure 22 This is a comparative chart showing the effects of ICA, DG, DG&ICA, and ICA-DG on serum HDL-C in Example 9. Detailed Implementation

[0051] The present invention 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 invention, but not all embodiments, and are only used to illustrate the present invention, and should not be regarded as a limitation on the scope of protection of the present invention.

[0052] In the embodiments of the present invention, experiments without specific conditions are conducted according to conventional conditions in the art or conditions recommended by the manufacturer. The reagents and instruments used, unless otherwise specified, are all conventional products that can be purchased commercially.

[0053] Icariin (abbreviated as "ICA"): purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0054] Dipotassium glycyrrhizinate (DG): purity ≥98%, purchased from Shaanxi Fujie Pharmaceutical Co., Ltd.

[0055] Trypan Blue (abbreviated as "TB"): Purchased from Beijing Solarbio Technology Co., Ltd.

[0056] α-D-glucosidase, pancreatic lipase, cholesterol esterase, 4-nitrophenyl-α-D-glucopyranoside, and p-nitrophenyl butyrate were purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0057] Male Kunming mice (8 weeks old): purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd. All animals were healthy and had no clinically observable physical abnormalities.

[0058] In the embodiments of the present invention, the comparison between the two sets of data is analyzed using SPSS Statistics 25 software (Student's t-test). P <0.05 indicates statistical significance.

[0059] In the following examples, the percentages used are mass percentages.

[0060] Example 1: Preparation of ICA-DG Take 20 mg of icariin (ICA) and mix it with 180 mg, 220 mg, 260 mg, 300 mg, and 340 mg of dipotassium glycyrrhizate (DG) in weight ratios of 1:9, 1:11, 1:13, 1:15, and 1:17, respectively. Dissolve the mixture completely in 50 ml of ethanol. Then, evaporate the ethanol completely using a rotary evaporator at 35°C. Pass the resulting powder through an 80-mesh sieve; this is ICA-DG (white powder). The ICA-DG obtained with a weight ratio of ICA:DG = 1:15 has the following appearance: Figure 1 As shown.

[0061] 1.1 SEM Observation The microstructure of ICA-DG powder was observed using a scanning electron microscope (TESCAN MIRA LMS, Czechia). SEM images of ICA, DG, DG&ICA (a physical mixture of ICA and DG at a weight ratio of 1:15), and ICA-DG (the ICA-DG obtained from the aforementioned weight ratio of ICA:DG=1:15) are shown in the following figures. Figure 2 As shown in B, C, D, and E.

[0062] SEM results showed that the ICA contained aggregated rod-like structures. Figure 2 -B), DG is spherical ( Figure 2 -C), DG&ICA shows ICA separated from or attached to DG spheres ( Figure 2 -D), ICA-DG shows them as clusters of nanoparticles with diameters of approximately 1–100 nm ( Figure 2 -E).

[0063] 1.2 XRD Testing XRD patterns were obtained using an X-ray diffractometer (XRD, D / max-2400, Rigaku, Japan) with a scanning range of 5°–70°. XRD patterns for ICA, DG, DG&ICA (a physical mixture of ICA and DG at a weight ratio of 1:15), and ICA-DG (ICA-DG obtained from the aforementioned weight ratio of ICA:DG=1:15) are shown below. Figure 3 As shown.

[0064] The results showed that the XRD pattern of ICA had characteristic peaks at 2θ = 6.88°, 8.18°, 9.06° and 10.14°, while these characteristic peaks disappeared in the XRD pattern of ICA-DG, indicating that the ICA in ICA-DG is amorphous.

[0065] 1.3 Fourier Transmission Infrared Spectroscopy (FT-IR) Test Fourier transform infrared spectroscopy (FTIR, Nicolet iS10, Thermo Fisher, Madison, WI, USA) was used to study the intermolecular interactions between DG and ICA in ICA-DG. The FT-IR spectra of ICA, DG, DG&ICA (a physical mixture of ICA and DG at a weight ratio of 1:15), and ICA-DG (ICA-DG obtained from the aforementioned weight ratio of ICA:DG=1:15) are shown below. Figure 4 As shown.

[0066] The results showed that the ICA spectrum contained 3360.0 cm⁻¹. -1 (OH tensile vibration), 2930.5cm -1 (Stretching vibration of aromatic groups), 1650.5cm -1 (Aromatic ring C=C tensile vibration), 1600.0cm -1 (Stretching vibration of olefin C=C), 1260.0 cm -1 (CO tensile vibration) and 1060.0cm -1 The infrared absorption peaks of the ICA-DG were observed, but no new infrared absorption peaks appeared in the spectrum of the ICA-DG, indicating that no chemical reaction occurred during the preparation of the ICA-DG and that the ICA was successfully encapsulated in the DG.

[0067] Example 2: ICA-DG Performance Testing 2.1 Encapsulation ratio The ICA-DG obtained in Example 1 was dissolved in water (ICA concentration approximately 1 mg / ml), filtered through a 0.22 μm filter membrane, and the unencapsulated ICA was separated by filtration. The ICA-DG solutions before and after filtration were diluted with methanol to disrupt the micelles. The ICA concentration was determined by high-performance liquid chromatography (HPLC). The encapsulation rate is the ratio of the ICA concentration detected after filtration to the ICA concentration detected before filtration.

[0068] HPLC: Agilent ZORBAX SB-C18 column (250 mm × 4.6 mm, 5 μm), column temperature 26℃; acetonitrile-water = 70:30 (v / v) as mobile phase; flow rate 1.0 ml / min; detection wavelength 270 nm; G1367A injector (injection volume 20 μl); ICA retention time 4.3–4.6 min.

[0069] The encapsulation efficiency of ICA-DG obtained by different weight ratios of icariin (ICA) and dipotassium glycyrrhizate (DG) (1:9, 1:11, 1:13, 1:15, 1:17) is shown in the figure. Figure 5As shown; the results show that the encapsulation efficiency of ICA-DG obtained by a weight ratio of ICA:DG=1:15 is (99.07±0.56)%.

[0070] 2.2 Water solubility Solubility of ICA-DG: It dissolves in water, presenting as a pale yellow, transparent solution (ICA-DG obtained in Example 1 with a weight ratio of ICA:DG=1:15, and an ICA concentration of 1 mg / ml), with a pH of approximately 6. ICA and DG&ICA (a physical mixture of ICA and DG at a weight ratio of 1:15) form a suspension in water (ICA content of 1 mg / ml), such as... Figure 6 As shown.

[0071] The aforementioned pale yellow transparent solution (ICA-DG obtained in Example 1 with a weight ratio of ICA:DG=1:15 and an ICA concentration of 1 mg / ml) can be observed to contain spherical or near-spherical particles with a uniform size distribution under a transmission electron microscope (TEM).

[0072] 2.3 Solubility Excess ICA, DG & ICA (a physical mixture of ICA and DG in a weight ratio of 1:15), and the ICA-DG obtained in Example 1 (weight ratio ICA:DG=1:15) were added to 1 ml of the following liquids: water, phosphate-buffered saline (PBS) (pH=6.8), simulated gastric juice (SGF) (pH=1.2), and simulated intestinal juice (SIF) (pH=6.8). After incubation at 37°C and 100 rpm for 4 h or 24 h, the mixture was centrifuged, filtered through a 0.22 μm filter membrane, diluted with methanol, and the ICA concentration was determined using the aforementioned HPLC method.

[0073] The results of the solubility measurements at 4 h and 24 h are as follows: Figure 7 and Figure 8 As shown ("*" indicates: compared to ICA, P <0.05; "&" indicates: compared to DG&ICA, P <0.05). The results showed that, compared with ICA, the solubility of ICA in ICA-DG in water, PBS, SIF, and SGF was increased by more than 5200 times.

[0074] 2.4 Micelle size, polydispersity index, and zeta potential The ICA-DG obtained in Example 1 (weight ratio ICA : DG = 1:15) was prepared into an aqueous solution (ICA concentration of 1 mg / ml). The micelle size, zeta potential and polydispersity index (PDI) were determined at room temperature using a Zetasizer Nano ZS90 (dynamic light scattering, DLS).

[0075] The results showed that the average micelle size of the ICA-DG aqueous solution was 73.39±20.21 nm, with a narrow particle size distribution (PDI=0.217±0.021) and a Zeta potential of -(8.37±0.82) mV.

[0076] 2.5 Storage stability The ICA-DG obtained in Example 1 (weight ratio ICA:DG=1:15) was stored at 4℃ and 25℃ in the dark for 12 weeks, respectively. Samples were taken every three weeks, and the encapsulation efficiency was determined using the aforementioned HPLC method. The ICA retention rate was calculated using the following formula: ICA retention rate = encapsulation efficiency data at sampling / initial encapsulation efficiency data. The initial encapsulation efficiency was (99.07±0.56)%. The results are as follows: Figure 9 As shown.

[0077] The results showed that ICA-DG exhibited good stability in the solid state. After 12 weeks of storage at 4℃ and 25℃, the retention rate of ICA was still as high as (97.98±1.67)% and (97.54±1.58)%, respectively.

[0078] The ICA-DG obtained in Example 1 (weight ratio ICA:DG=1:15) was prepared into an aqueous solution (ICA concentration of 1 mg / ml). It was stored at 25°C in the dark for 3 weeks. Samples were taken on days 1, 11, and 21, and the encapsulation efficiency was determined using the aforementioned HPLC method, and the ICA retention rate was calculated. The results are as follows: Figure 10 As shown.

[0079] The results showed that after 3 weeks (21 days) of storage in aqueous solution, the retention rate of ICA was (93.71±2.00)%, indicating that ICA-DG also exhibited good storage stability in aqueous solution.

[0080] In addition, ICA-DG also has good pH stability and salt stability, meaning that ICA-DG aqueous solutions can maintain a high encapsulation efficiency over a wide range of pH values ​​or sodium chloride concentrations.

[0081] Example 3 In vitro release test 3.1 The in vitro release characteristics of ICA-DG were analyzed and evaluated using the dialysis method. Test solutions: (1) ICA-DG aqueous solution (ICA-DG obtained by weight ratio of ICA:DG=1:15 in Example 1, with an ICA concentration of 1 mg / ml); (2) ICA suspension: ICA dispersed in water, with an ICA content of 1 mg / ml.

[0082] 1 ml of the above test solution was placed in a dialysis bag (molecular weight cutoff [MWCO] = 3500 Da), immersed in 100 ml of phosphate buffer (pH = 6.8), and incubated at 37°C with a shaker at 100 rpm. At each set time point, 1 ml of dialysis solution was collected (with 1 ml of phosphate buffer added simultaneously), filtered through a 0.22 μm filter membrane, and detected using the aforementioned HPLC method. The in vitro release curve is shown below. Figure 11 As shown ("*" indicates: compared to ICA, P <0.05).

[0083] The results showed that the cumulative release of ICA at 1 h, 4 h and 24 h was only (1.30±0.52)%, (3.07±0.98)% and (4.71±1.15)%, respectively, while the cumulative release of ICA-DG at 1 h, 4 h and 24 h was (5.81±2.47)%, (23.34±5.61)% and (59.79±7.04)%, respectively; ICA-DG showed a faster in vitro release rate compared to ICA.

[0084] 3.2 In vitro digestion assay and bioavailability Simulated in vitro artificial digestive fluids at different stages were used to digest ICA and ICA-DG, respectively.

[0085] Oral stage: Dissolve 20 mg of test powder in 10 ml of phosphate buffer (pH=6.8) containing 2 mg of α-amylase for 5 minutes; Gastric stage: Adjust the pH of the test solution to 2 with hydrochloric acid, then add 1330 mg of pepsin and dissolve completely, incubate for 1 hour (SGF); Intestinal stage: Adjust the pH of the test solution to 7.0 with sodium hydroxide, then add 250 mg of lipase and 50 mg of bile salts, incubate for 2 hours (SIF). Collect the test solution from each stage, centrifuge at 10000 rpm for 30 minutes, and measure the ICA content in the supernatant.

[0086] Calculate bioavailability using the following formula: Bioavailability (%) = Msss / Mst × 100%.

[0087] Mss: represents the ICA content in the supernatant; Mst: represents the total ICA content.

[0088] The results showed that the bioavailability of both ICA and ICA-DG in SGF was less than 10%, while the bioavailability of ICA and ICA-DG upon transfer to SIF was 47.13±3.15% and 88.54±0.31%, respectively, indicating that the bioavailability of ICA-DG in SIF was significantly higher than that of ICA. p <0.05).

[0089] Example 4 Biocompatibility and safety test 4.1 Hemolysis test For ICA, DG, DG&ICA (a physical mixture of ICA and DG in a weight ratio of 1:15), and ICA-DG (the ICA-DG obtained in Example 1 with a weight ratio of ICA:DG=1:15), hemolysis tests were performed using physiological saline as a negative control and 0.1% Triton X-100 as a positive control (refer to: CN 115212221 A).

[0090] From an appearance standpoint, except for the positive control group where the supernatant was blood-red, indicating obvious hemolysis, the colors of the other groups were similar to those of the saline negative control group, with hemolysis rates below 5% (e.g., Figure 12 As shown in the figure, it meets the safety requirements, indicating that ICA-DG has good blood biocompatibility.

[0091] 4.2 Chicken embryo chorioallantoic membrane (HET-CAM) - trypan blue staining test The experimental chicken embryos were randomly divided into the following 7 groups, with 3 to 5 embryos in each group: ① Negative control: 0.9% NaCl solution (physiological saline); ② Positive control: 0.1M NaOH solution; ③ ICA group: aqueous suspension, 1 mg / ml; ④ DG group: aqueous solution, 15 mg / ml; ⑤ DG&ICA group (physical mixture of ICA and DG in a weight ratio of 1:15): aqueous suspension, ICA content 1 mg / ml; ⑥ ICA-DG group (ICA-DG obtained by weight ratio of ICA:DG=1:15 in Example 1): aqueous solution, ICA concentration is 1 mg / ml; ⑦ ICA-DG group (ICA-DG obtained by weight ratio of ICA:DG=1:15 in Example 1): aqueous solution, ICA concentration is 5 mg / ml.

[0092] After the aforementioned 7 groups of solutions or suspensions were contacted with the chicken embryo allantoic membrane (CAM) for 5 minutes, the vascular condition of the CAM was as follows: Figure 13As shown in the figure; then, trypan blue staining was performed and the trypan blue absorption was measured, and the results are as follows. Figure 14 As shown ("*" indicates: compared to the negative control group (0.9% NaCl solution), P <0.05).

[0093] The results showed that the positive control group (0.1M NaOH solution) exhibited symptoms such as congestion and thrombosis, as well as a high absorption of trypan blue, indicating that it was severely irritating; while the ICA-DG group was basically similar to the saline group and did not show obvious irritation, which, on the other hand, demonstrates that ICA-DG has good biocompatibility and safety.

[0094] 4.3 Cytotoxicity test The effects of DG, ICA, DG&ICA (a physical mixture of ICA and DG at a weight ratio of 1:15), and ICA-DG (the ICA-DG obtained in Example 1 with a weight ratio of ICA:DG=1:15) on HepG2 cells were detected using the MTT assay: higher cell viability indicated lower cytotoxicity; the results are as follows. Figure 15 As shown.

[0095] The results showed that at 24 h, ICA and ICA-DG did not exhibit cytotoxicity in the range of ICA concentration from 7.8 to 500 μg / ml (aqueous solution), while the physical mixture of DG and ICA showed some cytotoxicity at an ICA concentration of 500 μg / ml.

[0096] Example 5: In vitro antioxidant activity test The antioxidant activities of ICA, DG, DG&ICA (a physical mixture of ICA and DG in a weight ratio of 1:15) and ICA-DG (the ICA-DG obtained in Example 1 with a weight ratio of ICA:DG=1:15) were detected by 2,2'-hydrazine-bis(3-ethylbenzothiazoline-6-sulfonic acid) diamine salt (ABTS method) and 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH method), respectively, with vitamin C (Vc) as a positive control.

[0097] The results of both the ABTS and DPPH methods showed that the ICA-DG of the present invention exhibited stronger antioxidant activity compared with ICA; among them, the antioxidant activity values ​​of DG, ICA, DG&ICA, ICA-DG and vitamin C determined by the ABTS method at 250 μg / ml and 5 minutes were 0.39, 0.44, 0.54, 0.64 and 0.73 equivalents of Trolox (water-soluble vitamin E), respectively.

[0098] Example 6: Prevention and / or treatment of oxidative stress damage H2O2 was used as an oxidant to induce oxidative stress damage in HepG2 cells (H2O2 concentration 1 mM, 24 hours; MEM medium); the cells were divided into the following experimental groups: ① Positive control group: No drugs were added; ② Vc control group: 31.3 μg / ml; ③ ICA group: 31.3 μg / ml (safe dose); ④ DG group: 468.8 μg / ml; ⑤. DG&ICA group: ICA (31.3 μg / ml) & DG (468.8 μg / ml); ⑥ ICA-DG group (ICA-DG obtained by weight ratio of ICA:DG=1:15 in Example 1): ICA 31.3 μg / ml.

[0099] In multi-well cell culture plates, HepG2 cells were treated with the aqueous solutions of the aforementioned experimental groups for 24 hours. Then, H2O2 was added to each well to a final concentration of 1 mM, and the cells were cultured for another 24 hours. Cell viability was used to assess the effectiveness of each experimental group in preventing and / or treating oxidative stress damage. The results are as follows: Figure 16 As shown; and the levels of reactive oxygen species (ROS) and superoxide dismutase (SOD) in the cells were measured using a kit, and the results are as follows. Figure 17 and 18 As shown in the figure. A separate experimental group without any added drugs or H2O2 was set up as a normal control group.

[0100] Figure 16 (* indicates: compared with the normal control group, P <0.05; "#" indicates: compared with the positive control group, P <0.05; "&" indicates: compared to group Vc, P <0.05; "@" indicates: compared to the ICA group, P The results (<0.05) showed that ICA-DG can effectively prevent and / or treat oxidative stress damage, with a cell survival rate as high as (91.76±4.86)%, which is close to the level of the normal control group.

[0101] Figure 17 and 18 (* indicates: compared with the normal control group, P <0.05; "#" indicates: compared with the positive control group, P <0.05; "&" indicates: compared to group Vc, P <0.05; "@" indicates: compared to the ICA group, P <0.05; "%" indicates: compared to group DG, P<0.05; "^" indicates: compared to the DG&ICA group, P Results (<0.05) showed that ICA-DG could regulate changes in ROS and SOD caused by cellular oxidative stress, restoring them to normal levels.

[0102] Example 7 Prevention and / or treatment of diabetes Inhibition of α-D-glucosidase is an important method for the prevention and / or treatment of diabetes. The in vitro antidiabetic activity of ICA-DG (ICA-DG obtained in Example 1 with a weight ratio of ICA:DG=1:15) was evaluated from the perspective of α-D-glucosidase inhibitory activity: using 4-nitrophenyl α-D-glucopyranoside (PNPG) as a chromogenic substrate, a yellow solution was produced through the cleavage action of α-D-glucosidase.

[0103] For each test solution, the inhibitory activity is expressed as IC50. 50 (Calculated based on inhibition percentage data at different concentrations). Mix 50 μl of test solution with 50 μl of α-D-glucosidase (0.5 U / ml) in a microplate, incubate at 37°C for 10 minutes, then add 50 μl of PNPG (10 mM) and continue incubation for 15–20 minutes. Measure absorbance at 405 nm. Acarbose was used as a positive control, and phosphate buffer was used as a control solution.

[0104] Calculate the inhibition percentage (%) using the following formula: Inhibition percentage (%) = [1 - (A)] 测试溶液 -A 测试空白 ) / (A 对照溶液 -A 对照空白 )]×100%.

[0105] in, A 测试溶液 : Indicates the absorbance measured by adding the enzyme test solution; A 测试空白 : Indicates the absorbance measured without adding enzyme to the test solution; A 对照溶液 : This indicates the absorbance measured with enzyme added to the control solution (without a test solution); A 对照空白 : This indicates the absorbance measured in the control solution without enzyme (no test solution).

[0106] The test results for antidiabetic activity are shown in Table 1.

[0107] Table 1. α-D-glucosidase inhibitory activity (n = 3)

[0108] The results showed that the ICA-DG of the present invention exhibited inhibitory activity against α-D-glucosidase. 50 The concentration was 0.056 ± 0.009 mg / ml (based on ICA), lower than the ICA, DG, and DG & ICA. 50 The value indicates that ICA-DG has superior antidiabetic activity and has potential application value in the prevention, treatment or adjunctive treatment of diabetes.

[0109] Example 8: Prevention and / or treatment of hyperlipidemia The in vitro anti-hyperlipidemia activity of ICA-DG (the ICA-DG obtained in Example 1 with a weight ratio of ICA:DG=1:15) was evaluated from the perspective of the inhibitory activity of pancreatic lipase and cholesterol esterase: p-nitrobenzene oxide was generated by the cleavage of pancreatic lipase or cholesterol esterase using 4-nitrophenyl butyrate (PNPB) as a substrate.

[0110] For each test solution, the inhibitory activity is expressed as IC50. 50 (Calculated based on inhibition percentage data at different concentrations). Mix 20 μl of test solution with 150 μl of pancreatic lipase or cholesterol esterase in a microplate, incubate at 37°C for 10 minutes, then add 20 μl of PNPB (10 mM) and continue incubation for 15–20 minutes. Measure absorbance at 405 nm. Orlistat was used as a positive control for pancreatic lipase, simvastatin as a positive control for cholesterol esterase, and Tris-HCl as a control solution.

[0111] Calculate the inhibition percentage (%) using the following formula: Inhibition percentage (%) = [1 - (A)] 测试溶液 -A 测试空白 ) / (A 对照溶液 -A 对照空白 )]×100%.

[0112] in, A 测试溶液 : Indicates the absorbance measured by adding the enzyme test solution; A 测试空白 : Indicates the absorbance measured without adding enzyme to the test solution; A 对照溶液 : This indicates the absorbance measured with enzyme added to the control solution (without a test solution); A 对照空白 : This indicates the absorbance measured in the control solution without enzyme (no test solution).

[0113] The test results for anti-hyperlipidemia activity are shown in Table 2.

[0114] Table 2 Enzyme inhibitory activity (n = 3)

[0115] The results showed that the ICA-DG of this invention exhibits inhibitory activity against lipase and cholesterol esterase. 50 The values ​​were 1.117±0.164 mg / ml and 0.659±0.055 mg / ml (based on ICA), lower than the ICA, DG, and DG&ICA values. 50 Values, especially, ICA-DG showed better results than simvastatin (ICD). 50 =0.847±0.025 mg / ml) showed stronger inhibitory activity against cholesterol esterase; this indicates that ICA-DG has good anti-hyperlipidemia activity.

[0116] Example 9 Prevention and / or treatment of hyperlipidemia (in vivo experiments) Fifty Kunming mice were randomly divided into 10 groups and treated with the following drugs: ① Healthy control group: No medication was administered; ② Negative control group (NS group): administered normal saline; ③ Fenofibrate treatment group (FEN group): Dosage 60 mg / kg; ④ Simvastatin treatment group (SIM group): Dosage 10 mg / kg; ⑤ ICA treatment group: Dosage 60 mg / kg; ⑥ DG treatment group: dosage 900 mg / kg; ⑦ DG&ICA treatment group: Dosage (60 mg / kg ICA and 900 mg / kg DG); ⑧ Low-dose ICA-DG (L) (ICA-DG obtained by weight ratio of ICA:DG=1:15 in Example 1) treatment group: dosage 20 mg / kg (calculated as ICA); ⑨ Medium-dose ICA-DG (M) (ICA-DG obtained by weight ratio of ICA:DG=1:15 in Example 1) Treatment group: Dosage 40 mg / kg (calculated as ICA); ⑩ High-dose ICA-DG (H) (ICA-DG obtained by weight ratio of ICA:DG=1:15 in Example 1) treatment group: dosage 60 mg / kg (calculated as ICA).

[0117] Mice in each group were orally administered the drug once daily for 7 days. Except for the healthy control group, on the evening of day 7, mice in groups ②–⑩ were intraperitoneally injected with poloxamer 407 (P-407, 300 mg / kg) to induce hyperlipidemia. Mice were sacrificed 24 hours after P-407 injection, and blood was collected to prepare serum. Total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) in the serum were measured using a kit. Liver tissue was collected and fixed in 4% formalin for further histopathological examination.

[0118] The results of the determination of TC, TG, LDL-C and HDL-C are as follows: Figures 19-22 As shown ("*" indicates: compared with the healthy control group, P <0.05; "#" indicates: compared to group NS, P <0.05; "&" indicates: compared to the FEN group, P <0.05; "$" indicates: compared to the SIM group, P <0.05; "@" indicates: compared to the ICA group, P <0.05; "%" indicates: compared to group DG, P <0.05; "^" indicates: compared to the DG&ICA group, P <0.05; "+" indicates: compared with the low-dose ICA-DG group, P <0.05; "!" indicates: compared with the medium-dose ICA-DG group, P <0.05).

[0119] The results show: (1) Compared with the NS group (administered saline), DG&ICA and medium and high doses of ICA-DG can effectively reduce the levels of total cholesterol (TC), triglycerides (TG) and low-density lipoprotein cholesterol (LDL-C) in serum, and have a good lipid-lowering effect. (2) In particular, the effect of high-dose ICA-DG in reducing TC (2.87±1.11 mM), TG (1.59±0.61 mM), and LDL-C (0.50±0.34 mM) is even better than that of clinical drugs such as fenofibrate and simvastatin, which further indicates that ICA-DG has good anti-hyperlipidemia activity and has potential application value in the prevention, treatment or adjuvant treatment of hyperlipidemia. It is expected to be further studied and developed into a lipid-lowering drug and / or functional food. (3) The results of liver histopathological observation show that ICA-DG can reduce and / or alleviate fatty infiltration in the liver and promote the liver to recover to a healthy state; ICA-DG can also be used as a potential drug for fatty liver and / or as a functional food for liver protection.

[0120] Of course, the present invention can also have other various embodiments. Without departing from the spirit and essence of the present invention, those skilled in the art can make various corresponding changes and / or modifications according to the present invention, and these corresponding changes and / or modifications should all fall within the protection scope of the appended claims.

Claims

1. A composition comprising isopentenyl flavonoid glycosides and excipients, wherein, The isopentenyl flavonoid glycoside is icariin, and the excipient is glycyrrhizate. The mass ratio of the isopentenyl flavonoid glycoside compound to the glycyrrhizate is 1:9-17; The glycyrrhizic acid salt is selected from dipotassium glycyrrhizate.

2. The composition according to claim 1, characterized in that: The mass ratio of the isopentenyl flavonoid glycoside compound to the glycyrrhizate is 1:

15.

3. The composition according to claim 1 or 2, characterized in that: In the composition, the excipients coat the isopentenyl flavonoid glycosides.

4. The composition according to claim 3, characterized in that: The excipients coat the isopentenyl flavonoid glycosides to form nanoparticles.

5. The composition according to claim 4, characterized in that: The average particle size of the nanoparticles is 0.5–300 nm.

6. The composition according to claim 5, characterized in that: The average particle size of the nanoparticles is 1–100 nm.

7. The composition according to claim 1 or 2, characterized in that: The isopentenyl flavonoid glycosides exist in an amorphous form.

8. The composition according to claim 1 or 2, characterized in that: The encapsulation efficiency of the isopentenyl flavonoid glycoside compound is at least 80%.

9. The composition according to claim 8, characterized in that: The encapsulation efficiency of the isopentenyl flavonoid glycosides is ≥90% or ≥95%.

10. The composition according to claim 1 or 2, characterized in that: The composition is a liquid, semi-solid, or solid formulation; the isopentenyl flavonoid glycoside in the composition is in a therapeutically effective amount.

11. The composition according to claim 10, characterized in that: The composition is a liquid formulation, and the solvent for the liquid formulation is selected from water, sodium carboxymethyl cellulose aqueous solution, or PBS buffer.

12. The composition according to claim 11, characterized in that: The liquid formulation is a nanomicelle formulation.

13. The composition according to claim 11, characterized in that: When the content of the isopentenyl flavonoid glycoside in the liquid formulation is 1 mg / ml, the liquid formulation satisfies one or more of the following conditions ① to ③: ① The average micelle size of the liquid formulation is 0.5–300 nm; ② The polydispersity index of the liquid formulation is ≤0.5; ③ The zeta potential of the liquid formulation is -20 to 0 mV.

14. The composition according to claim 13, characterized in that: The average micelle size of the liquid formulation is 1–100 nm.

15. The composition according to claim 14, characterized in that: The average micelle size of the liquid formulation is 50–95 nm.

16. The composition according to claim 13, characterized in that: The polydispersity index of the liquid formulation is ≤0.

3.

17. The composition according to claim 13, characterized in that: The zeta potential of the liquid formulation is -10 to 0 mV.

18. The composition according to claim 13, characterized in that: When the content of the isopentenyl flavonoid glycoside compound in the liquid formulation is 1 mg / ml, the liquid formulation simultaneously satisfies conditions ① to ③.

19. The composition according to claim 1 or 2, characterized in that: The composition is prepared by a method comprising the following steps: dissolving the isopentenyl flavonoid glycoside compound and the excipient in an organic solvent, and then removing the organic solvent, thereby obtaining the composition.

20. The composition according to claim 19, characterized in that: The organic solvent is an alcohol solvent; and / or, the amount of the organic solvent used per milligram of the isopentenyl flavonoid glycoside is 0.1 to 20 mL.

21. The composition according to claim 20, characterized in that: The amount of organic solvent used per milligram of the isopentenyl flavonoid glycoside compound is 2 to 10 mL.

22. The composition according to claim 19, characterized in that: The organic solvent is ethanol.

23. The composition according to claim 1 or 2, characterized in that: The composition is for the prevention and / or treatment of oxidative stress damage, diabetes, hyperlipidemia and / or fatty liver.

24. Use of the composition according to any one of claims 1 to 23 in the preparation of a medicament for the prevention and / or treatment of oxidative stress damage.

25. Use of the composition according to any one of claims 1 to 23 in the preparation of a medicament for the prevention and / or treatment of diabetes.

26. Use of the composition according to any one of claims 1 to 23 in the preparation of a medicament for the prevention and / or treatment of hyperlipidemia.

27. Use of the composition according to any one of claims 1 to 23 in the preparation of a medicament for the prevention and / or treatment of fatty liver.

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