A ha-adh-dhm conjugate for improving solubility and biological activity of dihydromyricetin and a preparation method thereof

Abstract

This invention discloses a HA-ADH-DHM conjugate for improving the solubility and bioactivity of dihydromyricetin and its preparation method, belonging to the pharmaceutical field. The preparation method of the HA-ADH-DHM conjugate of this invention includes the following steps: (1) performing an amino dehydration reaction on hyaluronic acid and adipic acid dihydrazide to obtain HA-ADH; (2) performing a dehydration condensation reaction on the HA-ADH and dihydromyricetin to prepare the HA-ADH-DHM conjugate. This invention significantly improves the solubility of dihydromyricetin (DHM) in water, enhancing the absorption and efficacy of DHM in vivo.

Description

Technical Field

[0001] This invention relates to the pharmaceutical field, specifically to a HA-ADH-DHM conjugate for improving the solubility and bioactivity of dihydromyricetin and its preparation method. Background Technology

[0002] Ampelopsis grossedentata leaves, also known as vine tea, are a traditional ethnic medicinal resource widely distributed in southwestern my country. They have long been used by the Tujia, Miao, and Yao ethnic groups in western Hunan and Guangxi to treat chronic pharyngitis, dermatitis, jaundice hepatitis, and damp-heat arthralgia, possessing traditional effects such as clearing heat and detoxifying, promoting diuresis and reducing swelling, and calming the liver and lowering blood pressure. In 2013, vine tea was approved by the National Health Commission as a new resource food raw material. Modern pharmacological studies have shown that vine tea possesses multiple biological activities, including antioxidant, lipid-lowering, anti-inflammatory, and liver-protective effects. Its main active ingredient is dihydromyricetin (DHM), which can reach 30-40% of the dry weight of vine tea, indicating high development potential.

[0003] DHM, a flavonoid natural compound, has been proven to possess various pharmacological activities, including antioxidant, anti-inflammatory, regulation of glucose and lipid metabolism, and hepatoprotective effects, with high safety profile, providing a solid foundation for its development as a natural drug or functional food. However, current research and practical applications have revealed significant limitations in DHM regarding solubility, water stability, and bioavailability. Its water solubility is extremely low (approximately 0.2 mg / mL), gastrointestinal absorption is slow, and the first-pass effect is significant, resulting in oral bioavailability of less than 5%. Furthermore, DHM exhibits uneven distribution in vivo and weak accumulation in target organs, further affecting its efficacy. These issues have become key bottlenecks restricting the in-depth development and industrial transformation of DHM. Summary of the Invention

[0004] This invention addresses the problems of poor solubility and low bioavailability of dihydromyricetin by providing an HA-ADH-DHM conjugate and its preparation method to improve the solubility and bioactivity of dihydromyricetin. This invention significantly improves the solubility of dihydromyricetin (DHM) in water and enhances its absorption and efficacy in vivo.

[0005] This invention first provides a method for preparing HA-ADH-DHM conjugates, comprising the following steps:

[0006] (1) Hyaluronic acid and adipic dihydrazide undergo an amino dehydration reaction to obtain HA-ADH;

[0007] (2) The HA-ADH and dihydromyricetin were subjected to a dehydration condensation reaction to prepare the HA-ADH-DHM conjugate.

[0008] In the above preparation method, the mass ratio of hyaluronic acid to adipic dihydrazide is 1:1.3 to 1.5, specifically 1:1.38;

[0009] The molar ratio of HA-ADH and dihydromyricetin is 1:1 to 1.5, specifically 1:1.48.

[0010] In the above preparation method, the temperature of the amino dehydration reaction is room temperature;

[0011] The time for the amino dehydration reaction is 8 to 12 hours, specifically 10 hours;

[0012] The amino dehydration reaction is carried out in an inert atmosphere;

[0013] The dehydration condensation reaction was carried out at room temperature.

[0014] The dehydration condensation reaction takes 5 to 12 hours, specifically 10 hours.

[0015] In the above preparation method, step (1) specifically includes the following: mixing hyaluronic acid, activator and water, reacting to activate the carboxyl group of hyaluronic acid; then adding adipic acid dihydrazide to the above-obtained system, adjusting the pH of the system to 4.0 to 5.0 (specifically 4.7), and carrying out an amino dehydration reaction in an inert atmosphere to obtain the HA-ADH.

[0016] More specifically, step (1) includes the following: mixing hyaluronic acid and water, then adding an activator at 0°C to react and activate the carboxyl group of hyaluronic acid; then adding adipic acid dihydrazide to the above-obtained system, adjusting the pH of the system to 4.0-5.0, and carrying out an amino dehydration reaction in an inert atmosphere to obtain the HA-ADH.

[0017] In the above preparation method, after the amino dehydration reaction, there are further steps of adjusting the pH of the system to neutral, dialyzing, and lyophilizing the supernatant.

[0018] Specifically, the pH of the system is neutral when using NaOH solution.

[0019] In the above preparation method, the activator is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride;

[0020] The mass ratio of hyaluronic acid to activator is 1:0.6 to 0.7, specifically 1:0.6;

[0021] The reaction of the hyaluronic acid, activator and water is carried out at room temperature; the reaction time is 0.5 to 2 hours; and the reaction is carried out in an inert atmosphere.

[0022] In the above preparation method, in step (1), the mass ratio of hyaluronic acid to water is 100 mg: 1-3 mL; specifically, it can be 100 mg: 1 mL.

[0023] In the above preparation method, step (2) specifically includes the following: mixing HA-ADH, dihydromyricetin and water, adjusting the pH of the system to 10.0 to 11.0, and carrying out a dehydration condensation reaction to obtain the HA-ADH-DHM conjugate.

[0024] In the above preparation method, the mass ratio of dihydromyricetin to water is 50-55 mg:1 mL, specifically 54.5 mg:1 mL;

[0025] Adjust the pH of the system using triethylamine;

[0026] The dehydration condensation reaction is followed by a freeze-drying step.

[0027] In the above preparation method, the room temperature is known to those skilled in the art and is 15–35°C.

[0028] This invention further provides the HA-ADH-DHM conjugate prepared by the above preparation method.

[0029] Finally, the present invention also provides the use of the above-mentioned HA-ADH-DHM conjugate in the preparation of any of the following drugs:

[0030] 1) Medications for the prevention and / or treatment of metabolic disorder-related diseases

[0031] The metabolic disorder-related diseases include: diabetes (such as type 2 diabetes) and its complications, fatty liver (such as non-alcoholic fatty liver disease), steatohepatitis (such as non-alcoholic steatohepatitis), and metabolic syndrome (such as those with obesity, insulin resistance, or hyperlipidemia).

[0032] 2) Medications that improve glucose and lipid metabolism, including those that lower blood sugar and regulate blood lipids (such as lowering triglycerides);

[0033] 3) Drugs with anti-inflammatory and cell-protective effects; including those that improve insulin resistance and reduce inflammation of the liver or adipose tissue;

[0034] 4) Medications with neuroprotective effects are used to improve diabetes-related cognitive impairment or sleep disorders.

[0035] Specifically, the metabolic syndrome is characterized by one or more of the following symptoms: hyperglycemia, obesity, hyperlipidemia, insulin resistance, inflammation, and non-alcoholic fatty liver disease.

[0036] This invention uses hyaluronic acid (HA) as a drug carrier and prepares HA-adipic acid dihydrazide (ADH) through an amino dehydration reaction. Then, HA-ADH is dehydrated and condensed with DHM to synthesize the HA-ADH-DHM conjugate. The solubility of this conjugate in water is more than 1000 times higher than that of DHM. In vitro cell experiments and in vivo animal experiments have shown that the HA-ADH-DHM conjugate of this invention has high safety, improves insulin resistance and glucose and lipid metabolism disorders, and its effect is significantly better than that of DHM.

[0037] This invention successfully overcomes the technical bottleneck that limits the clinical application of DHM due to its poor solubility and low absorption rate. This invention provides a novel, highly efficient, and safe formulation of DHM for the treatment of diabetes, inflammation, fatty liver disease, metabolic syndrome, and related diseases, laying a solid foundation for the industrial production and clinical translation of DHM. It also provides new ideas and methods for drug delivery technology of natural products. Attached Figure Description

[0038] Figure 1 The synthetic route for the HA-ADH-DHM conjugate is shown.

[0039] Figure 2 The results are Fourier transform infrared spectra.

[0040] Figure 3 The particle size and zeta potential of the HA-ADH-DHM conjugate are given.

[0041] Figure 4 The results are the 1H NMR spectra of HA, HA-ADH, and HA-ADH-DHM conjugates; among them, Figure 4 (a) shows the 1H NMR spectrum (D2O, 300MHz) of the HA-ADH-DHM conjugate of HA and HA-ADH, and (b) shows the integrated 1H NMR spectrum (300MHz) of the HA-ADH-DHM conjugate.

[0042] Figure 5 The morphological characteristics of the HA-ADH-DHM conjugate are shown.

[0043] Figure 6 The image shows the UV absorption spectrum of the HA-ADH-DHM conjugate.

[0044] Figure 7 The effects of DHM and HA-ADH-DHM conjugate treatment on cytotoxicity and cellular glucose uptake were investigated; among them, Figure 7 In the table, A represents the effect of DHM and HA-ADH-DHM on glucose uptake in AML-12 hepatocytes, and B represents the effect of DHM and HA-ADH-DHM on the survival rate of AML-12 hepatocytes.

[0045] Figure 8 The effects of DHM and HA-ADH-DHM conjugate treatment on oleic acid-induced lipid accumulation in hepatocytes; among which, Figure 8 In the table, A represents the effect of DHM and HA-ADH-DHM on lipid accumulation in AML-12 hepatocytes, and B represents the effect of DHM and HA-ADH-DHM on the survival rate of AML-12 hepatocytes.

[0046] Figure 9 The in vivo evaluation results of the HA-ADH-DHM conjugate improving glucose tolerance in insulin-resistant mice; among which, Figure 9 In the figure, A represents the acute glucose tolerance test, and B represents the area under the curve of graph A. Detailed Implementation

[0047] The present invention will be further described in detail below with reference to specific embodiments. The embodiments given are only for illustrating the present invention and are not intended to limit the scope of the present invention.

[0048] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0049] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are averaged.

[0050] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0051] Example 1: Preparation of HA-ADH-DHM conjugate

[0052] The synthetic route for the HA-ADH-DHM conjugate is shown below. Figure 1 .

[0053] 1. Synthesis of HA-ADH

[0054] (1) Accurately weigh 300.0 mg of hyaluronic acid (HA) into a flask, add 3 mL of distilled water as a solvent, and stir until the solution is clear;

[0055] (2) Add 181.7 mg of EDCI activator (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride) to the solution in (1) at 0℃ and react at room temperature for 2 h under argon protection to activate the carboxyl group of HA.

[0056] (3) Add 413.3 mg of adipic acid dihydrazide (ADH) to the system obtained in (2) until it is completely dissolved, then adjust the pH of the system to 4.7 with hydrochloric acid solution, and react at room temperature for 10 h under argon protection;

[0057] (4) After the reaction was completed, the pH was adjusted to neutral with 1 mol / L NaOH solution, and the mixture was dialyzed with distilled water for 24 h. The liquid in the dialysis bag (3,500 kDa) was collected and then freeze-dried to obtain the product HA-ADH.

[0058] 2. Synthesis of HA-ADH-DHM conjugates

[0059] (1) Dissolve 100 mg of HA-ADH (0.23 mmol) in 2 mL of distilled water, add 109 mg of DHM (0.34 mmol), and adjust the pH to 10 with triethylamine;

[0060] (2) After reacting at room temperature (20-25℃) for 10 h, the mixture was freeze-dried to obtain the HA-ADH-DHM conjugate.

[0061] Weigh a small amount of sample HA, HA-ADH, or HA-ADH-DHM conjugate and mix it with dry KBr. After grinding and pressing into a pellet, the sample is detected using a Fourier transform infrared spectroscopy (FT-IR) instrument. The results are shown in the figure. Figure 2 Structural analysis of HA, HA-ADH, and HA-ADH-DHM was performed using infrared absorption spectroscopy. FT-IR results showed that HA has a peak density at 1043 cm⁻¹. -1 1610cm -1 and 3420cm -1 An absorption peak appears at 1660 cm⁻¹, which can be attributed to the stretching vibration peak of the CO single bond, the stretching vibration peak of the carboxyl group (COOH), and the stretching vibration peak of the hydroxyl group (OH) in hyaluronic acid. Meanwhile, HA-ADH shows an absorption peak at 1660 cm⁻¹. -1 The presence of newly formed peaks at 2856 cm⁻¹ is attributed to the stretching vibration of the amide (NC=O), indicating the formation of new amide bonds between HA and ADH. The HA-ADH-DHM peak is observed at 2856 cm⁻¹. -1 The absorption peaks are attributed to the torsional and resonant vibrations of the aromatic C=N double bonds.

[0062] The hydration kinetics diameter and zeta potential of the prepared HA-ADH-DHM conjugate dispersion (dispersion solvent: distilled water, conjugate concentration: 10 mg / mL) were determined using a Malvern particle size analyzer. The results are as follows: Figure 3 As shown, the average particle size of the HA-ADH-DHM conjugate is 95.27±4.47 nm, and the Zeta potential is -22.83±0.03 mV.

[0063] Weigh an appropriate amount (5-10 mg) of the sample powder to be tested, use D2O as solvent, and determine the composition of HA, HA-ADH, and HA-ADH-DHM conjugates using a 300 MHz fully digital superconducting nuclear magnetic resonance hydrogen spectrometer. 11H-NMR. First, hyaluronic acid modified with adipic acid dihydrazide (HA-ADH) was synthesized via a carbodiimide coupling reaction. According to the 1H NMR spectrum, HA-ADH showed a new peak corresponding to the ADH proton, indicating successful synthesis of HA-ADH with a grafting rate of approximately 40%. Second, HA-ADH-DHM was synthesized via a Schiff base reaction. According to the 1H NMR spectrum, the HA-ADH-DHM conjugate showed a new peak corresponding to the DHM proton, indicating successful synthesis of HA-ADH-DHM with a grafting rate of approximately 61.25%. 1 The structure of the reaction product was confirmed by H-NMR, and the results are as follows: Figure 4 As shown.

[0064] The prepared HA-ADH-DHM dispersion (dispersion solvent: distilled water, conjugate concentration: 10 mg / mL) was dropped onto a 230-mesh copper grid, and the morphology of the particles was observed using a transmission electron microscope. The results showed that the HA-ADH-DHM conjugate was spherical and morphologically intact. Figure 5 ).

[0065] Take 1 mg / mL of DHM and HA-ADH-DHM (dissolve 10 mg of DHM and HA-ADH-DHM separately in methanol and distilled water, respectively, and dilute to volume in a 10 mL volumetric flask to obtain a 1 mg / mL solution (stock solution)) and dilute to a concentration of 12 μg / mL. Measure the absorbance of each group using a UV-Vis spectrophotometer. The results are shown in the table below. Figure 6 The HA-ADH-DHM conjugate exhibits the characteristic absorption peak of DHM, indicating that nanoparticles containing DHM were successfully obtained.

[0066] Example 2, Solubility Determination

[0067] Prepare a series of DHM or HA-ADH-DHM solutions of known concentrations (1, 5, 10, 20, 50 μg / mL; DHM dissolved in methanol; HA-ADH-DHM dissolved in distilled water). Measure the absorbance using UV-Vis spectrophotometry (λmax = 290 nm, DHM) and plot a standard curve.

[0068] Solubility determination: Preparation of saturated aqueous solutions of DHM or HA-ADH-DHM conjugates: 1) Dissolution process: Add 100 mg of excess DHM to 10 mL of water (far exceeding the saturation volume), and add 10 g of HA-ADH-DHM to 10 mL of water (far exceeding the saturation volume). 2) Heating and stirring: Magnetic stirring in a water bath at 40-50℃ for 2-4 hours (avoid high temperatures, as DHM may be unstable above 60℃); 3) Ultrasonic assistance: Ultrasonic treatment (40 kHz, 30 minutes) can accelerate dissolution; 4) Filtration and collection: Pass the hot solution through a 0.22 μm filter membrane to remove undissolved solids. Cool the filtrate to room temperature (25℃) and filter again. 5) Verification of saturation: Evaporate 1 mL of the filtrate to dryness, weigh, and calculate the concentration.

[0069] Take a saturated aqueous solution of DHM or HA-ADH-DHM conjugate, centrifuge and filter it, and measure the UV absorbance of the supernatant. Calculate the solubility (mg / mL) based on the above standard curve.

[0070] Calculate the increase in water solubility: Increase factor = HA - ADH - DHM solubility / DHM solubility

[0071] The results are shown in Table 1. The solubility of the HA-ADH-DHM conjugate in water is 755 mg / mL, which is 1060 times higher than the solubility of DHM (0.712 mg / mL). The solubility has changed from "very slightly soluble" to "easily soluble".

[0072] Table 1. Solubility of DHM and HA-DHM conjugates

[0073] solute solubility Solubility DHM 0.712 mg / mL Slightly dissolving HA-ADH-DHM 755mg / mL Easily soluble

[0074] Note: "Frequently soluble" means that 1g of solute can dissolve in 1 to less than 10mL of solvent; "Very slightly soluble" means that 1g of solute can dissolve in 1000 to less than 10000mL of solvent.

[0075] Example 3: In vitro evaluation of the safety and glucose uptake activity of the HA-ADH-DHM conjugate.

[0076] 1. MTT assay for cell safety of HA-ADH-DHM conjugate

[0077] (1) Collect normal liver AML-12 cells from mice in the logarithmic growth phase and adjust the cell suspension concentration to 1×10⁻⁶. 4 Cells per 100 μL were used to adjust the concentration of the cell suspension with DMEM / F12 complete medium (containing 10% FBS, 1% penicillin, 1% streptomycin (v / v)), 1% insulin-transferrin-selenium (v / v), and 0.004% (v / v) dexamethasone (stock solution 1 mg / mL).

[0078] (2) Inoculate 100 μL of the above cell suspension into each well of a 96-well plate and incubate at 37°C (5% CO2 concentration, v / v) for 24 h.

[0079] (3) Grouping according to experimental requirements: blank group (without cells), control group, and drug administration group (10 parallel wells).

[0080] (4) Drug administration group: Replace with 100 μL of DMEM / F12 medium containing DHM (200 μg / mL) or HA-ADH-DHM (12.5-200 μg / mL) and 2% (v / v) FBS, and continue incubation for 24 h. Prepare a 200 mg / mL stock solution of DHM or HA-ADH-DHM with DMSO. When using, dilute the stock solution with DMEM / F12 medium containing 2% FBS to the required concentration.

[0081] Control group: Replace with 100 μL of DMEM / F12 medium containing the same volume of DMSO and 2% (v / v) FBS, and continue incubation for 24 h.

[0082] Blank group: Replace with 100 μL of DMEM / F12 medium containing the same volume of DMSO and 2% (v / v) FBS, and continue incubation for 24 h.

[0083] (5) Terminate the culture, discard the culture medium, add 20 μL of 5.0 g / L tetramethylazobium bromide (MTT) solution to each well, and incubate for another 4 h in a cell culture incubator at 37 ℃.

[0084] (6) After 4 hours, remove the supernatant (be careful not to remove the purple crystals at the bottom of the well). Add 100 μL of DMSO to each well, shake at room temperature for 10 minutes to completely dissolve the blue formazan crystals, place in an ELISA reader, and then measure the absorbance at 570 nm.

[0085] Cell viability = (Drug-treated group - Blank group) / (Control group - Blank group)

[0086] Experimental results show that ( Figure 7 A) The HA-ADH-DHM conjugate did not produce cytotoxicity within the measured concentration range (12.5-200 μg / mL), and significantly increased cell survival at 100 and 200 μg / mL, indicating that the HA-ADH-DHM conjugate has high safety and no cytotoxicity was observed within the detection range.

[0087] 2. Cellular glucose uptake experiment

[0088] (1) Collect normal liver AML-12 cells from mice in the logarithmic growth phase and adjust the cell suspension concentration to 1×10⁻⁶. 4 Cells per 100 μL were used to adjust the concentration of the cell suspension with DMEM / F12 complete medium (containing 10% FBS, 1% penicillin and 1% streptomycin (v / v)), 1% insulin-transferrin-selenium (v / v), and 0.004% (v / v) dexamethasone (stock solution 1 mg / mL).

[0089] (2) Inoculate 100 μL of the above cell suspension into each well of a 96-well plate and incubate at 37°C (5% CO2 concentration, v / v) for 24 h.

[0090] (3) Grouping according to experimental requirements: blank group (without cells), control group, and drug administration group (10 parallel wells).

[0091] (4) Drug administration group: Replace the culture medium with 100 μL of DMEM / F12 medium containing DHM (200 μg / mL) or HA-ADH-DHM (12.5-200 μg / mL) and 2% (v / v) FBS, and continue incubation for 24 h (DHM or HA-ADH-DHM stock solution and final drug administration solution are prepared as above).

[0092] Control group: Replace with 100 μL of DMEM / F12 medium containing the same volume of DMSO and 2% (v / v) FBS, and continue incubation for 24 h.

[0093] Blank group: Replace with 100 μL of DMEM / F12 medium containing the same volume of DMSO and 2% (v / v) FBS, and continue incubation for 24 h.

[0094] (5) Terminate the culture, discard the culture medium, wash once with PBS, add 50 μL of 100 μmol / L fluorescently labeled 2-deoxyglucose (2-Deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose, 2-NBDG) solution to each well, and incubate for another 30 min in a cell culture incubator at 37℃.

[0095] (6) After 30 min, aspirate the supernatant (be careful not to aspirate the cells at the bottom of the well), and wash twice with PBS buffer. Add 100 μL of PBS buffer to each well, place the well in a microplate reader, and then detect the fluorescence absorption at an excitation wavelength of 488 nm and an emission wavelength of 520 nm. Calculate the cellular glucose uptake rate using the following formula:

[0096] Cellular glucose uptake rate = [OD(glucose uptake in the treatment group) ÷ OD(cell viability in the treatment group)] / [OD(glucose uptake in the control group) ÷ OD(cell viability in the control group)] × 100%

[0097] like Figure 7 As shown in Figure B, compared with 200 μg / mL DHM, the HA-ADH-DHM conjugate significantly improved insulin-stimulated cellular glucose uptake in the measured concentration range (12.5-200 μg / mL). This result indicates that the HA-ADH-DHM conjugate exhibits better insulin sensitivity enhancement than DHM, can more effectively promote cellular glucose uptake, and has a greater potential to reduce blood glucose concentration.

[0098] Example 4, Performance Evaluation

[0099] 1. In vitro evaluation of the inhibitory activity of HA-ADH-DHM conjugate on lipid accumulation in hepatocytes

[0100] (1) Mouse AML-12 cells 1×10 4 Each well was seeded with 100 μL of DMEM / F12 complete medium (containing 10% FBS, 1% penicillin, 1% streptomycin (v / v), and 1% insulin-transferrin-selenium (v / v)) and 0.004% (v / v) dexamethasone (stock solution 1 mg / mL). The plates were incubated at 37°C (5% CO2, v / v) for 24 h.

[0101] (2) Grouping according to experimental requirements: control group (without cells), model group and drug administration group (10 parallel wells).

[0102] (3) Drug treatment group: The DMEM / F12 complete medium was replaced with 100 μL of DMEM / F12 medium containing 1% (v / v) FBS. The drug treatment group was simultaneously given DHM (200 μg / mL) or HA-ADH-DHM (12.5-200 μg / mL) (DHM or HA-ADH-DHM was prepared as a 200 mg / mL stock solution with DMSO. When using, the stock solution was added to the DMEM / F12 medium containing 1% FBS according to the required final concentration and mixed thoroughly). After culturing for 24 h, the medium was replaced with 100 μL of 1% FBS DMEM / F12 medium containing 1 mmol / L oleic acid (OA) (containing 1% FBS (v / v, OA stock solution concentration of 10 mmol / L) for 24 h of stimulation.

[0103] Control group: The culture medium was replaced with 100 μL of DMEM / F12 medium containing the same volume of DMSO and 1% (v / v) FBS, and cultured for 24 h. Then it was replaced with 100 μL of DMEM / F12 medium containing 1% FBS (1% FBS (v / v)) and cultured for 24 h.

[0104] Model group: The culture medium was replaced with 100 μL of DMEM / F12 medium containing the same volume of DMSO and 1% (v / v) FBS. After culturing for 24 h, it was replaced with 100 μL of DMEM / F12 medium containing 1 mmol / L oleic acid (OA) (containing 1% FBS (v / v), and the OA stock solution concentration was 10 mmol / L) for 24 h of stimulation.

[0105] All culture conditions were incubated in an incubator at 37°C and a CO2 concentration of 5% (v / v).

[0106] (4) Terminate the culture, discard the OA modeling solution, wash once with PBS buffer, and add 100 μL of 4% paraformaldehyde to each well to fix the cells. Subsequently, remove the paraformaldehyde, wash once with PBS, add 0.5% Oil Red O (ORO) solution (ORO dissolved in PBS, w / v) for staining, discard excess Oil Red, and wash twice with PBS buffer. Add 100 μL of DMSO to each well to dissolve the Oil Red, and incubate at room temperature with shaking for 10 min until the Oil Red is completely dissolved. Measure the absorbance at 358 nm using a microplate reader (all results are converted to the OD value of the control group as a percentage).

[0107] The experimental results are shown in Figure 8 ,Depend on Figure 8 As shown in A, compared with the control group, the model group (MOD) cells showed significant lipid accumulation (P<0.05). In the DHM and 100, 50, 25, and 12.5 μg / L HA-ADH-DHM conjugate pretreatment groups, OA-induced cellular lipid accumulation was significantly alleviated (P<0.05), and the 12.5 μg / mL HA-ADH-DHM conjugate was more effective than DHM. Furthermore, due to… Figure 8 As shown in section B, compared with the model group, cells treated with the HA-ADH-DHM conjugate still maintained a higher survival rate, indicating that the HA-ADH-DHM conjugate did not produce cytotoxicity and had high safety. These experimental results demonstrate that the HA-ADH-DHM conjugate has high safety, and compared with DHM, this conjugate not only has improved solubility but also significantly enhanced activity in inhibiting lipid accumulation in hepatocytes.

[0108] 2. In vivo evaluation of the effect of HA-ADH-DHM conjugate on glucose tolerance in insulin-resistant mice

[0109] The experimental procedure was as follows: Sixty 5-week-old male C57BL / 6J mice were housed in a standard environment with a constant room temperature of (22±2)℃, relative humidity of (50±5)%, a 12h / 12h light / dark cycle, and free access to food and water. The housing location was the SPF-grade animal laboratory of the Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences. After one week of acclimatization, the mice were randomly divided into two groups. Ten mice were fed a standard diet (D12450J) and the remaining 50 mice were fed a high-fat diet (Research Diet, D12492) containing 60% fat. After four weeks of feeding, the mice's weight and fasting blood glucose were measured. Mice fed a high-fat diet with a fasting blood glucose greater than 6.1 mmol / L were considered to be in a hyperglycemic state. They were then divided into five groups of ten mice each: (1) HFD + saline group; (2) HFD + MET (25 mg / kg metformin positive drug group); (3) HFD + DHM group (200 mg / kg); (4) HFD + HA-ADH-DHM group (200 mg / kg); (5) HFD + HA-ADH group (200 mg / kg).

[0110] An acute glucose tolerance test (OGTT) was conducted to examine the effects of different drug administration groups on glucose tolerance in insulin-resistant mice. In the OGTT, mice were fasted for 12 hours but allowed free access to water. Fasting blood glucose was measured. Subsequently, the drug administration groups were administered the listed drugs (solvent being 0.5% sodium carboxymethyl cellulose) by gavage at a dose of 0.1 mL / 10 g. The control group and the HFD model group were administered the same volume of 0.5% sodium carboxymethyl cellulose by gavage daily. 30 minutes later, a glucose solution (2 g / kg) was administered by gavage at a dose of 0.1 mL / 10 g. Blood glucose was measured at 30, 60, and 90 minutes using a handheld glucometer from the tail vein. A line graph of blood glucose changes was plotted. The area under the curve (AUC) was calculated.

[0111] Experimental results: The OGTT results showed that the fasting blood glucose and blood glucose at all time points after glucose loading were significantly higher in the HFD group than in the control group, and the AUC-OGTT was significantly higher in the HFD group than in the control group, indicating that HFD induced glucose intolerance in mice. Figure 9 DHM intervention showed a certain reduction in blood glucose levels at 60 and 90 minutes after glucose loading, and a trend towards a decrease in the area under the curve (AUC-OGTT), but without significant difference. Compared with the HFD group, HA-ADH-DHM intervention significantly reduced blood glucose levels at 30, 60, and 90 minutes after glucose loading, and significantly reduced the area under the curve (AUC-OGTT), indicating that HA-ADH-DHM significantly improved glucose intolerance induced by HFD.

Claims

1. A method for preparing a HA-ADH-DHM conjugate, comprising the following steps: (1) Hyaluronic acid and adipic dihydrazide undergo an amino dehydration reaction to obtain HA-ADH; (2) The HA-ADH and dihydromyricetin were subjected to a dehydration condensation reaction to prepare the HA-ADH-DHM conjugate.

2. The preparation method according to claim 1, characterized in that: The mass ratio of hyaluronic acid to adipic acid dihydrazide is 1:1.3 to 1.5; The molar ratio of HA-ADH and dihydromyricetin is 1:1 to 1.

5.

3. The preparation method according to claim 1 or 2, characterized in that: The temperature for the amino dehydration reaction is room temperature; The amino dehydration reaction takes 8–12 hours; The dehydration condensation reaction was carried out at room temperature. The dehydration condensation reaction takes 5 to 12 hours.

4. The preparation method according to any one of claims 1-3, characterized in that: Step (1) specifically includes the following: hyaluronic acid, activator and water are mixed and reacted to activate the carboxyl group of hyaluronic acid; then adipic acid dihydrazide is added to the above-obtained system, the pH of the system is adjusted to 4.0 to 5.0, and an amino dehydration reaction is carried out in an inert atmosphere to obtain the HA-ADH.

5. The preparation method according to claim 4, characterized in that: The amino dehydration reaction is followed by steps of adjusting the pH of the system to neutral, dialysis, and lyophilizing the supernatant.

6. The preparation method according to claim 4 or 5, characterized in that: The activator is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride; The mass ratio of hyaluronic acid to activator is 1:0.6 to 0.7; The reaction of the hyaluronic acid, activator and water is carried out at room temperature; the reaction time is 0.5 to 2 hours; and the reaction is carried out in an inert atmosphere.

7. The preparation method according to any one of claims 1-6, characterized in that: Step (2) specifically includes the following: HA-ADH, dihydromyricetin and water are mixed, the pH of the system is adjusted to 10.0 to 11.0, and a dehydration condensation reaction is carried out to obtain the HA-ADH-DHM conjugate.

8. The preparation method according to claim 7, characterized in that: The mass ratio of the dihydromyricetin to the volume ratio of water is 50-55 mg: 1 mL; Adjust the pH of the system using triethylamine; The dehydration condensation reaction is followed by a freeze-drying step.

9. The HA-ADH-DHM conjugate prepared by the preparation method according to any one of claims 1-8.

10. The use of the HA-ADH-DHM conjugate of claim 9 in the preparation of any of the following pharmaceutical products: 1) Medications for the prevention and / or treatment of diabetes; 2) Medications for the prevention and / or treatment of fatty liver; 3) Medications that lower blood lipids; 4) Medications that lower blood sugar; 5) Medications for the prevention and / or treatment of metabolic syndrome; 6) Medications for the prevention and / or treatment of metabolic disorder-related diseases; 7) Drugs with anti-inflammatory and cell-protective effects; 8) Drugs with neuroprotective effects.

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