Hydrogel containing motherwort powder or alkaloid thereof as well as preparation and application of hydrogel

Abstract

The invention belongs to the field of traditional Chinese medicine preparations, and particularly relates to a hydrogel containing motherwort powder or alkaloid thereof as well as preparation and application of the hydrogel. According to the invention, novel carrier-free hydrogel of motherwort powder or alkaloid of motherwort and polyphenol molecules is combined to generate supramolecular hydrogel with a new structure, so that a supramolecular drug synthesized by self-assembly enhances the effect of '1 + 1 > 2' compared with a monomer component, and the hydrogel preparation can be prepared without adding special auxiliary materials; pharmacodynamic tests show that the compound also has good safety, simplicity and convenience, and has potential application value in the aspects of anti-inflammation, anti-oxidation, angiogenesis promotion and wound repair promotion.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine technology, specifically relating to a hydrogel containing Leonurus japonicus powder or its alkaloids, and its preparation and application. Background Technology

[0002] Normal wound healing generally progresses through four phases: coagulation, inflammation, repair, and maturation. Diabetic wounds, however, take 12-13 months longer to heal than normal wounds and often fail to follow the normal healing process. While the exact mechanisms underlying poor wound healing in diabetes are not fully understood, unbalanced inflammatory responses, oxidative stress, high blood sugar levels, lack of angiogenesis, and a significantly higher risk of bacterial infection are considered major contributing factors. Therefore, effective repair of diabetic wounds remains a challenging problem worldwide and a pressing issue for healthcare systems.

[0003] Currently, clinical treatment for diabetic foot primarily focuses on targeted therapy based on the symptoms. Diabetic wounds can be classified according to the severity of infection, including simple cellulitis, deep soft tissue infection, necrotizing fasciitis, and chronic osteomyelitis. Depending on the location and type of infection, mild infections require antibiotics targeting Staphylococcus aureus and Streptococcus pneumoniae; while infections progressing to deep soft tissue or more severe conditions necessitate thorough debridement of necrotic tissue and broad-spectrum antibiotic treatment. Although numerous antibiotic-based treatment strategies exist, long-term antibiotic use has led to the emergence of drug-resistant bacteria such as MRSA. Dressings are crucial materials in the treatment of chronic wounds. Ideal wound dressings should possess the following characteristics: (1) good biocompatibility, without causing allergic or immune reactions; (2) aiding in wound insulation, gas exchange, and debris removal; (3) effectively eliminating harmful microorganisms at the wound site and preventing wound infection; (4) maintaining a moist environment at the wound site, promoting related cell proliferation and migration, enhancing collagen synthesis, and reducing scar formation; and (5) being easy to change or remove without causing secondary damage to the wound. Clinically used novel dressings include hydrocolloids, films, hydrogels, sponge (foam) dressings, and alginate dressings. In recent years, researchers have designed hydrogel dressings with multiple functions to promote wound healing, specifically addressing the complex microenvironment of chronic inflammation and susceptibility to infection in diabetic foot wounds.

[0004] Leonurine is an alkaloid derived from the traditional Chinese medicine Leonurus japonicus, with the molecular formula C. 14 H 21 N3O5 possesses excellent antioxidant, anti-inflammatory, and free radical scavenging pharmacological effects with minimal toxicity. Therefore, it exhibits high activity in anti-inflammatory, angiogenesis-promoting, and oxidative stress-improving activities. However, the poor water solubility and low bioavailability of leonurine limit its clinical application.

[0005] Chlorogenic acid is a polyphenolic compound derived from the traditional Chinese medicine honeysuckle, with the molecular formula C6H2O. 16 H 18 O9, with its molecular structure containing ester bonds, polyphenols, and ortho- and tho-dihydroxyl groups, possesses a variety of pharmacological effects, including antibacterial, antioxidant, antitumor, and immunomodulatory properties. Epidemiological surveys, intervention studies, and animal experiments have shown that chlorogenic acid plays an important role in regulating glucose and lipid metabolism and improving insulin resistance.

[0006] Based on existing technologies, this invention aims to develop a novel carrier-free hydrogel formulation of Leonurus japonicus powder or its alkaloids and polyphenol molecules. The inventors have discovered that Leonurus japonicus powder or its main active components, leonurine and stachydrine, along with polyphenols such as chlorogenic acid and m-gallic acid, can form hydrogels without the need for polymeric carrier dressings. This allows for the development of new application forms such as oral and topical hydrogels, and possesses superior anti-inflammatory and blood-activating effects. This invention is of great significance for the discovery and development of structurally defined natural hydrogel drugs from traditional Chinese medicine.

[0007] However, most previously reported hydrogels are based on high molecular weight polymers. Currently, hydrogels used clinically are mainly antibacterial hydrogel dressings, primarily in ointment form, with few transdermal patches. From a materials science perspective, large-scale production of stable hydrogel dressings is a necessary step towards practical application. However, monomers remaining during the preparation process are easily encapsulated within the hydrogel and difficult to remove. For example, acrylamide monomers can easily remain in polyacrylamide gels, posing a significant challenge to the material's stability and biosafety.

[0008] Furthermore, from the perspective of the biofunctionality of gels, if the gel is loaded with drugs or other bioactive molecules, such as VEGF growth factor, it is necessary to consider how to store the dressing to ensure that the internal drugs or other bioactive molecules do not deteriorate. Different patients have different degrees of wound damage, immune systems, and self-healing abilities, so it is impossible to determine whether a drug-loaded hydrogel dressing, with a fixed drug content and release efficiency, will have a universal therapeutic effect on all patients. Summary of the Invention

[0009] To address the aforementioned shortcomings in the prior art, this invention provides a traditional Chinese medicine hydrogel for wound healing, as well as its preparation and application. This hydrogel is safe and highly effective, and can effectively solve the problems existing in the prior art.

[0010] One objective of this invention is to provide a hydrogel containing motherwort powder or motherwort alkaloids, along with polyphenols and / or flavonoids or other components. Furthermore, this invention provides a method for preparing a hydrogel compound formed by the active ingredients of motherwort and polyphenol molecules.

[0011] Furthermore, the leonurus alkaloids described in this invention are any one or a mixture of leonurine and stachydrine, the polyphenols are any one or a mixture of chlorogenic acid and gallic acid, and the flavonoids are puerarin.

[0012] Furthermore, the hydrogel contains leonurine, chlorogenic acid and a pH adjuster; leonurine, puerarin and a pH adjuster; or any combination of leonurine, gallic acid and a pH adjuster.

[0013] More preferably, the hydrogel contains leonurine and puerarin; stachydrine and puerarin; leonurine and chlorogenic acid; stachydrine and chlorogenic acid; leonurine and gallic acid; stachydrine and gallic acid; leonurine and gallic acid and chlorogenic acid; leonurine and stachydrine and chlorogenic acid; a combination of leonurine, stachydrine and gallic acid; any combination of leonurine, stachydrine, gallic acid and chlorogenic acid; preferably, the hydrogel contains leonurine and puerarin; stachydrine and puerarin; leonurine and chlorogenic acid; stachydrine and chlorogenic acid; any combination of leonurine and gallic acid or stachydrine and gallic acid; even more preferably, the hydrogel contains leonurine and chlorogenic acid; any combination of leonurine and puerarin or leonurine and gallic acid.

[0014] A second objective of this invention is to provide the application of the hydrogel in the preparation of a treatment to promote wound healing, wherein the hydrogel is selected from oral hydrogels or topical hydrogels.

[0015] Furthermore, the molar ratio of leonurine to polyphenol molecules is 1:0.1-20; more preferably, it is any combination of 3:1, 1:0.5, 1:3, 1:10, and 1:20; even more preferably, it is 3:1; the molar ratio of leonurine to flavonoid molecules is 1:0.1-20; more preferably, it is any combination of 3:1, 1:0.5, 1:3, 1:10, and 1:20; even more preferably, it is 3:1.

[0016] Further, the molar ratio of leonurine to chlorogenic acid is 1:0.1-20; more preferably, it is any combination of 3:1, 1:0.5, 1:3, 1:10, and 1:20; even more preferably, it is 3:1; the molar ratio of leonurine to puerarin is 1:0.1-20; more preferably, it is any combination of 3:1, 1:0.5, 1:3, 1:10, and 1:20; even more preferably, it is 3:1; the molar ratio of leonurine to gallic acid is 1:0.1-20; more preferably, it is any combination of 3:1, 1:0.5, 1:3, 1:10, and 1:20; even more preferably, it is 3:1.

[0017] This invention provides a method for preparing a hydrogel agent by any of the following methods:

[0018] Preparation method 1:

[0019] (1) Weigh out the active alkaloids or powder of Leonurus japonicus and the polyphenol molecules or flavonoid molecules separately for later use;

[0020] (2) Add water and pH adjuster to the motherwort active alkaloids or powder in step (1) to form an aqueous solution, and obtain solution A;

[0021] (3) Polyphenol molecules or flavonoid molecules are dissolved in water by heating or sonication to obtain a clear and transparent solution B;

[0022] (4) Mix and stir the solution A prepared in step (2) with the solution B prepared in step (3), place it in an ultrasonic crusher for ultrasonic crushing or heating treatment, centrifuge to obtain precipitate or colloid, wash with water and centrifuge, repeat three times to obtain the product;

[0023] The pH adjuster is selected from any one of 2-formylphenylboronic acid, citric acid, fumaric acid, and adipic acid; more preferably, the pH adjuster is selected from 2-formylphenylboronic acid; the concentration of the pH adjuster is 0.01wt% to 10wt%; the ultrasonic disruption power is 20-70W, the ultrasonic disruption time is more than 3min; and the centrifugation speed during centrifugation purification is 3500-13000rpm.

[0024] This invention provides a second method for preparing hydrogel agents:

[0025] (1) Weigh out the active alkaloids or powder of Leonurus japonicus and the polyphenol molecules or flavonoid molecules separately for later use;

[0026] (2) Mix the pH adjuster with water to form a pH adjusting solution;

[0027] (3) Mix the active alkaloids or powder of Leonurus japonicus from step (1) with polyphenol molecules or flavonoid molecules evenly, add them to the pH adjustment solution prepared in step (2), and vortex to mix evenly.

[0028] (4) Place the solution system from step (3) into an ultrasonic crusher for ultrasonic crushing or heat treatment.

[0029] Centrifugation yields a precipitate or colloid, which is then washed with water and centrifuged again. This process is repeated three times to obtain the final product.

[0030] Furthermore, the pH adjuster is selected from 2-formylphenylboronic acid, acetic acid, hydrochloric acid, sulfuric acid, citric acid, lactic acid, fumaric acid, and adipic acid; the concentration of the pH adjuster is 0.01wt% to 10wt%; the ultrasonic disruption power is 20-70W, the ultrasonic disruption time is more than 3min; and the centrifugation speed during centrifugation purification is 3500-13000rpm.

[0031] Furthermore, the preparation method described herein is carried out by any of the following methods:

[0032] Preparation method 1:

[0033] (1) Weigh out the active alkaloids or powder of Leonurus japonicus and the polyphenol molecules or flavonoid molecules separately for later use;

[0034] (2) Add water and 2-formylphenylboronic acid to the motherwort active alkaloids or powder in step (1) to form an aqueous solution, and obtain solution A, wherein the concentration of 2-formylphenylboronic acid is 1 wt%.

[0035] (3) Polyphenol molecules or flavonoid molecules are dissolved in water by heating or sonication to obtain a clear and transparent solution B;

[0036] (4) Mix and stir the solution A prepared in step (2) with the solution B prepared in step (3), place it in an ultrasonic crusher for ultrasonic crushing or heating treatment, centrifuge to obtain precipitate or colloid, wash with water and centrifuge, repeat three times to obtain the solution. The ultrasonic crushing power is 70W and the ultrasonic crushing time is more than 3 minutes. The centrifugation speed during centrifugation purification is 10000 rpm.

[0037] Or preparation method two:

[0038] (1) Weigh out the active alkaloids or powder of Leonurus japonicus and the polyphenol molecules or flavonoid molecules separately for later use;

[0039] (2) Mix 2-formylphenylboronic acid with water to form a 2-formylphenylboronic acid solution, wherein the concentration of 2-formylphenylboronic acid is 1 wt%.

[0040] (3) Mix the active alkaloids or powder of Leonurus japonicus from step (1) with polyphenol molecules or flavonoid molecules evenly, add them to the 2-formylphenylboronic acid solution prepared in step (2), and vortex to mix evenly.

[0041] (4) Place the solution system from step (3) in an ultrasonic crusher for ultrasonic crushing or heating treatment, centrifuge to obtain a precipitate or colloid, wash with water and centrifuge again, repeat three times to obtain the final product, wherein the ultrasonic crushing power is [value missing].

[0042] 70W, ultrasonic disruption time is more than 3 minutes; centrifugation speed during purification is 10000 rpm.

[0043] The third objective of this invention is to provide applications of the obtained hydrogel compounds, specifically the in vitro anti-inflammatory, ROS-scavenging, cell proliferation-promoting, angiogenesis-promoting, and wound healing-promoting applications of four carrier-free supramolecular hydrogel compounds. The hydrogels have the effects of promoting cell migration, promoting angiogenesis, scavenging ROS, and regulating macrophage polarization in vitro, and can be used to prevent or treat traumatic kidney injury, traumatic brain injury, colitis, and chronic wound repair.

[0044] Compared with the prior art, the advantages of the hydrogel of the present invention are as follows:

[0045] By investigating different molar ratios of feed, pH values, temperatures, ultrasonic crushing power, ultrasonic time, ultrasonic program, centrifugation speed, and other factors, the preparation process for forming supramolecular hydrogel compounds from Leonurus japonicus active alkaloids (leonurine and stachydrine) and polyphenolic components was determined. The structural information was determined using XRD, infrared spectroscopy, NMR, and mass spectrometry.

[0046] This invention discloses a novel carrier-free hydrogel of Leonurus japonicus powder or its alkaloids and polyphenol molecules. The hydrogel is efficiently formed through multiple mechanisms including electrostatic cross-linking, dynamic covalent bonds, and hydrogen bonding, combined with chlorogenic acid. Its microstructure is honeycomb-like. This not only solves the problem of poor solubility of Leonurus japonicus alkaloids preventing effective skin penetration, but also allows for the slow and continuous release of active drug molecules, prolonging bioavailability. This enables Leonurus japonicus powder or its alkaloids and polyphenol molecules to synergistically exert pharmacological effects at the lesion site.

[0047] This invention discloses a novel carrier-free hydrogel of Leonurus japonicus powder or its alkaloids and polyphenol molecules, which simultaneously achieves in-situ administration of multiple small molecules of traditional Chinese medicine in the form of a hydrogel, and can exert a synergistic effect, and has good clinical translational value.

[0048] This invention discloses a novel carrier-free hydrogel of Leonurus japonicus powder or its alkaloids and polyphenol molecules. The preparation method is simple, green, and easy to mass-produce. It is relatively easy to register as a medical device dressing and has the potential to be used in tissue engineering, showing broad prospects in biomedical applications.

[0049] In summary, compared with existing technologies, this invention has the following advantages: In this invention, a novel carrier-free hydrogel is formed by combining Leonurus japonicus powder or its alkaloids with polyphenol molecules through multiple mechanisms including electrostatic crosslinking, dynamic covalent bonds, and hydrogen bonds, efficiently driven by chlorogenic acid, without the need for excipient carriers. The Leonurus japonicus alkaloids and stachydrine possess anti-inflammatory and angiogenic capabilities, while the polyphenol molecules chlorogenic acid and gallic acid possess antioxidant activity. When these two types of components combine in pairs to form a new supramolecular hydrogel, not only does the self-assembled supramolecular drug enhance the "1+1>2" effect compared to the monomeric components—an enhancement difficult to predict without experimental verification—but it also brings about morphological changes after assembly, providing possibilities for other applications. Compared with existing technologies that combine traditional Chinese medicine monomeric components with polymeric carrier materials to prepare gels, this invention can produce hydrogel formulations without adding excipients, reducing raw material usage. While improving efficacy, it also has excellent safety and convenience, and has potential application value in anti-inflammatory, antioxidant, angiogenic, and wound healing applications. Attached Figure Description

[0050] Figure 1 The hydrogel agent contains leonurine and chlorogenic acid (3:1). The hydrogel of the present invention (abbreviated as CFL hydrogel) exhibits a honeycomb-like structure and presents a porous network structure.

[0051] Figure 2 The hydrogel agent contains leonurine and puerarin (1:3). The hydrogel of the present invention (abbreviated as CFL hydrogel) exhibits a honeycomb-like structure and presents a porous network structure.

[0052] Figure 3 The hydrogel agent contains leonurine and gallic acid (1:3). The hydrogel of the present invention (abbreviated as CFL hydrogel) exhibits a honeycomb-like structure and presents a porous network structure.

[0053] Figure 4 The hydrogel contains leonurine and chlorogenic acid (3:1), and under certain amplitude and frequency, its elastic modulus and viscous modulus remain within a certain range and increase to a certain extent.

[0054] Figure 5 The hydrogel agent contains leonurine and chlorogenic acid (3:1). The fluorescence intensity of the oxidative stress model gradually weakens after hydrogel treatment, and this weakening is concentration-dependent.

[0055] Figure 6 The hydrogel agent contains leonurine and chlorogenic acid (3:1). After the same scratch treatment, the CFL hydrogel group showed a significant healing effect after 48 hours. With the increase of concentration, the scratch closure rate increased significantly.

[0056] Figure 7 The hydrogel contains leonurine and chlorogenic acid (3:1). As shown in the in vitro tube formation experiment, the hydrogel group showed better cell vascular connection compared with free chlorogenic acid, leonurine and the combination of the two.

[0057] Figure 8 The hydrogel contains leonurine and chlorogenic acid (3:1), and compared with the free drug, the capillary length and branching points of the hydrogel group are significantly increased.

[0058] Figure 9 :right Figure 8 Further evidence, through quantitative analysis of the results, shows that the gel group has the largest relative tube length.

[0059] Figure 10 The hydrogel agent described contains leonurine and chlorogenic acid (3:1) in immunofluorescence images. After in vitro inflammatory stimulation, the morphology of RAW264.7 cells changed from round to spindle-shaped, and the fluorescence intensity increased after LPS stimulation. However, the fluorescence of the CD86 pro-inflammatory factor in the gel-treated group decreased in a concentration-dependent manner. The expression level of CD86 decreased after hydrogel treatment, demonstrating that CFL hydrogel can inhibit inflammatory responses in chronic wounds.

[0060] Figure 11 The hydrogel agent described contains leonurine and chlorogenic acid (3:1) in immunofluorescence images. After in vitro inflammatory stimulation, the morphology of RAW264.7 cells changed from round to spindle-shaped, and the fluorescence intensity increased after LPS stimulation. The enhancement of CD206 anti-inflammatory factor in the gel-treated group was concentration-dependent. The expression level of CD206 significantly increased after hydrogel treatment, demonstrating that CFL hydrogel can inhibit inflammatory responses in chronic wounds. Detailed Implementation

[0061] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings, but the scope of protection of the present invention is not limited to the following description.

[0062] The terms defined in this invention are as follows:

[0063] LPS*: Lipopolysaccharide.

[0064] CA 80: Free chlorogenic acid concentration 80 μg / ml.

[0065] Leo160: The concentration of free leonurine was 160 μg / ml.

[0066] Gel 80: Gel extract concentration 80 μg / ml.

[0067] Gel 160: Gel extract concentration 160 μg / ml.

[0068] Gel 320: Gel extract concentration 320 μg / m.

[0069] DAPI: A blue fluorescent dye used to label cell nuclei (DNA), thereby revealing the location and number of all cells.

[0070] CD86: A surface marker of M1 macrophages (pro-inflammatory type), representing the immune activation state of the cell.

[0071] Merge: Fluorescence images from different channels (such as DAPI blue, CD86 green, and CD206 red) are superimposed to observe colocalization relationships.

[0072] CD206: A characteristic surface marker of M2 macrophages (anti-inflammatory / repair type).

[0073] RAW264.7: A commonly used mouse-derived macrophage line widely used in in vitro immunology and inflammation research.

[0074] Example 1: Hydrogels prepared by reacting leonurine with chlorogenic acid and gallic acid respectively

[0075]

[0076] The preparation process is as follows:

[0077] (1) Weigh out the above amounts of Leonurus alkaloid and chlorogenic acid or gallic acid, and set them aside separately;

[0078] (2) Add water and 2-formylphenylboronic acid to the active alkaloids of Leonurus japonicus in step (1) to form an aqueous solution to obtain solution A, wherein the concentration of 2-formylphenylboronic acid is 1 wt%.

[0079] (3) Dissolve chlorogenic acid or gallic acid in an appropriate amount of water by heating or sonication to obtain a clear and transparent solution B.

[0080] (4) Mix and stir the solution A prepared in step (2) with the solution B in step (3), place it in an ultrasonic crusher for ultrasonic crushing or heating treatment, centrifuge to obtain precipitate or colloid, wash with water and centrifuge, repeat three times to obtain the precipitate. The ultrasonic crushing power is 70W and the ultrasonic crushing time is more than 3min. The centrifugation speed during centrifugation purification is 10000rpm.

[0081] Example 2: Hydrogel prepared from leonurine and puerarin

[0082]

[0083]

[0084] The preparation process is the same as described in Example 1.

[0085] Example 3: Hydrogel prepared from stachydrine and puerarin

[0086]

[0087] The preparation process is the same as described in Example 1.

[0088] Example 4: Hydrogel prepared from stachydrine, chlorogenic acid, and gallic acid

[0089]

[0090] The preparation process is the same as described in Example 1.

[0091] Example 5: Hydrogel prepared from Leonurus japonicus powder and chlorogenic acid

[0092]

[0093] The preparation process is the same as described in Example 1.

[0094] Electron microscopy was performed on the hydrogels prepared in Examples 1-5 above. The results showed that the best hydrogel was formed when the ratio was 3:1. Ratios of 1:0.1, 1:3, 1:15, and 1:20 could also form good hydrogels. However, the hydrogels formed at ratios of 1:0.05 and 1:30 could not achieve the same technical effect as other ratios, or even fail to form hydrogels.

[0095] Example 6: Hydrogels formed by different alkaline Chinese medicinal herbs and flavonoid molecules

[0096]

[0097] The preparation process is the same as described in Example 1.

[0098] Technical effect: The dosage is within the scope of this invention, but none of the above 6 combinations can form a stable honeycomb gel under an electron microscope. In other words, although the above products can be added arbitrarily under the guidance of the dosage technology of this invention, the final technical effect of this invention cannot be achieved.

[0099] Example 7: Leonurus alkaloids and chlorogenic acid respectively

[0100] component molar ratio Leonurine 3

[0101] Chlorogenic acid 1

[0102] The preparation process is as follows:

[0103] (1) Weigh out the above amounts of leonurine and chlorogenic acid and set them aside separately;

[0104] (2) Add water and 2-formylphenylboronic acid to the active alkaloids of Leonurus japonicus in step (1) to form an aqueous solution to obtain solution A, wherein the concentration of 2-formylphenylboronic acid is 0.1 wt%.

[0105] (3) Dissolve chlorogenic acid or gallic acid in an appropriate amount of water by heating or sonication to obtain a clear and transparent solution B.

[0106] (4) Mix and stir the solution A prepared in step (2) with the solution B in step (3), place it in an ultrasonic crusher for ultrasonic crushing or heating treatment, centrifuge to obtain precipitate or colloid, wash with water and centrifuge, repeat three times to obtain the precipitate. The ultrasonic crushing power is 30W and the ultrasonic crushing time is more than 3min. The centrifugation speed during centrifugation purification is 3500rpm.

[0107] Example 8: Leonurus alkaloids and chlorogenic acid respectively

[0108] component molar ratio Leonurine 3

[0109] Chlorogenic acid 1

[0110] The preparation process is as follows:

[0111] (1) Weigh out the above amounts of leonurine and chlorogenic acid and set them aside separately;

[0112] (2) Add water and 2-formylphenylboronic acid to the active alkaloids of Leonurus japonicus in step (1) to form an aqueous solution to obtain solution A, wherein the concentration of 2-formylphenylboronic acid is 10wt%.

[0113] (3) Dissolve chlorogenic acid or gallic acid in an appropriate amount of water by heating or sonication to obtain a clear and transparent solution B.

[0114] (4) Mix and stir the solution A prepared in step (2) with the solution B in step (3), place it in an ultrasonic crusher for ultrasonic crushing or heating treatment, centrifuge to obtain precipitate or colloid, wash with water and centrifuge, repeat three times to obtain the precipitate. The ultrasonic crushing power is 50W and the ultrasonic crushing time is more than 3 minutes. The centrifugation speed during centrifugation purification is 13000 rpm.

[0115] Example 9: Leonurus alkaloids and chlorogenic acid respectively

[0116] component molar ratio Leonurine 3

[0117] Chlorogenic acid 1

[0118] The preparation process is as follows:

[0119] (1) Weigh out leonurine and chlorogenic acid separately and set aside;

[0120] (2) Mix 2-formylphenylboronic acid with water to form a 2-formylphenylboronic acid solution, wherein the concentration of 2-formylphenylboronic acid is 1 wt%.

[0121] (3) Mix the active alkaloids or powder of Leonurus japonicus from step (1) with polyphenol molecules or flavonoid molecules evenly, add them to the 2-formylphenylboronic acid solution prepared in step (2), and vortex to mix evenly.

[0122] (4) Place the solution system from step (3) into an ultrasonic crusher for ultrasonic crushing or heating treatment, centrifuge to obtain precipitate or colloid, wash with water and centrifuge again, repeat three times to obtain the solution. The ultrasonic crushing power is 70W and the ultrasonic crushing time is more than 3 minutes. The centrifugation speed during centrifugation purification is 10000 rpm.

[0123] Example 10: Verification and Improvement of the Preparation Process

[0124] The inventors discovered that, in order to achieve good physical effects during the preparation process, other acidic solutions were used. 2-Formylphenylboronic acid, citric acid, fumaric acid, and adipic acid were screened and all achieved the same or similar effects. However, during the preparation process, acetic acid, hydrochloric acid, sulfuric acid, and lactic acid could not form honeycomb colloids, and no active substances were detected in sulfuric acid and hydrochloric acid.

[0125] Example 11: Preparation of gelling agent using conventional process, as detailed below:

[0126] 1) Sodium hyaluronate and CMC-Na are added to 50g of purified water, stirred and dispersed, and then allowed to stand for 24 hours to fully swell and form a uniform colloid.

[0127] 2) Add 3g of Leonurus japonicus alkaloid to 10g of purified water and stir until completely dissolved; add 1g of 50% ethanol solution to dissolve chlorogenic acid (using ethanol to enhance solubility and reduce chlorogenic acid oxidation), then mix the two together and add citrate-sodium citrate buffer to adjust the pH to 5.0-6.0.

[0128] 3) Slowly add the active ingredient mixture to the carrier colloid while stirring (300 r / min, 10 min), then add glycerol and the remaining purified water, and continue stirring until the system is homogeneous.

[0129] 4) Add ethylparaben, stir in a 60°C water bath for 30 minutes to dissolve and preliminarily sterilize. After cooling to room temperature, dispense into sterile containers to obtain a pale yellow transparent hydrogel.

[0130] Example 12: Optimal hydrogel and its preparation process

[0131] Group A: Leonurus alkaloid: Chlorogenic acid = 3:1 (calculated by molar ratio, of which leonurus alkaloid 37.36mg and chlorogenic acid 14.16mg);

[0132] Group B: Leonurus alkaloid: Gallic acid = 1:3 (calculated by molar ratio, of which leonurus alkaloid 37.36mg and gallic acid 14.16mg);

[0133] Group C: Leonurus alkaloid: Puerarin = 1:3 (calculated in molar ratio, of which leonurus alkaloid 37.36mg and puerarin 14.16mg);

[0134] Group D: Stachydrine: Puerarin = 1:3 (calculated by molar ratio, of which stachydrine 37.36mg and puerarin 14.16mg);

[0135] Group E: Stachydrine: Gallic acid = 1:3 (calculated by molar ratio, of which stachydrine 37.36mg and gallic acid 14.16mg);

[0136] Group F: Stachyine: Chlorogenic acid = 3:1 (calculated by molar ratio, of which 37.36 mg of stachydrine and 14.16 mg of chlorogenic acid);

[0137] Group G: Motherwort powder: chlorogenic acid = 3:1 (calculated by molar ratio, of which motherwort powder is 37.36mg and chlorogenic acid is 14.16mg);

[0138] Group H: Motherwort powder 186.8mg;

[0139] Group Q: Chlorogenic acid 70.8mg.

[0140] The preparation process is the same as described in Example 1.

[0141] II. Verification of the physicochemical properties:

[0142] 1) The hydrogel prepared in the above embodiments was further scanned by electron microscopy.

[0143] 1.1 Microscopic observation of hydrogel compounds using scanning electron microscopy (SEM)

[0144] 1.2 Rheological studies of hydrogel compounds

[0145] The specific method involves using a rheometer to perform dynamic time-scan tests to detect the elastic modulus (G) and viscous modulus (G), with an amplitude of 0.1% and a frequency of 10 rad / s.

[0146] 1.3 Reactive oxygen species experiments using hydrogel compounds

[0147] The procedure includes the following steps: seeding HUVEC cells into a plate, incubating them for 12 hours after complete dilution of H2O2 and the drug for another 12 hours, followed by incubation with DCFH-DA probe at 10 μM at 37°C in the dark for 20 minutes, washing them three times with PBS and observing the fluorescence intensity under a microscope.

[0148] 1.4L-929 cells were used for cell scratch assay.

[0149] The procedure included the following steps: First, L-929 cells were seeded into 6-well plates. The next day, after each well was 90% confluent, a crisscross pattern was created, and different groups of drugs were used for incubation. The plates were then incubated at 37°C for 24 hours and 48 hours, and images were taken at these time points. ImageJ software was used for analysis to calculate cell migration rate.

[0150] II) Chemical effects of the hydrogel in this invention

[0151] 2.1 Assay of angiogenesis using HUVEC cells

[0152] The main objective was to evaluate the pro-angiogenic effect of the obtained multi-molecular hydrogel compounds. The procedure included the following steps: First, the matrix gel stored at -20°C was incubated at 4°C overnight. A 100 μL pipette tip and a 24-well plate were pre-cooled to -80°C for 1 hour. The pre-cooled 24-well plate was placed on an ice pad, and the matrix gel on another ice pad. 20 μL of matrix gel was aspirated into each well, using a "pipette 1, dispense 2" technique to avoid air bubbles, and the gel was spread evenly in a circular motion with the pipette tip, avoiding touching the well edges. The plate was then incubated overnight at 4°C, without tilting. The next day, the plate was removed from the ice pad and placed in a clean bench. HUVEC cells were digested and counted. Different drug groups were prepared and diluted, and 500 μL of cell suspension was added to each well onto the matrix gel. The 24-well plate was then placed in a 37°C incubator. After 4-6 hours, the plate was removed and angiogenesis was observed under a microscope and photographed. The results were then used for quantitative analysis of capillary length and branching points.

[0153] 2.2 Immunofluorescence assay was performed on RAW264.7 cells to assess the expression levels of CD86 and CD206.

[0154] The procedure included the following steps: First, RAW264.7 cells were seeded at 150,000 cells per well for 24 hours. An inflammation model was constructed using LPS (lipopolysaccharide) (100 ng / ml). After 12 hours, different drugs were administered. The specific groups were: negative control (complete culture), positive control (12 hours of LPS + DMEM culture for 24 hours), and experimental groups (12 hours of LPS + different drug intervention groups for 24 hours). For immunofluorescence experiments, the treated wells were first washed three times with PBS, and cells were fixed with 4% paraformaldehyde at 4°C for 15 minutes. After 15 minutes, the paraformaldehyde was removed, and the cells were washed three times with PB for 5 minutes each time. Then, the cells were blocked with blocking solution at room temperature for 1 hour, washed three times with PBS, and incubated overnight with CD86 primary antibody at 4°C. The next day, the cells were warmed and the primary antibody was recovered. The wells were then washed three times with PBS for 5 minutes each time. Secondary antibody was added and incubated at room temperature in the dark for 2 hours. After 2 hours, the secondary antibody was recovered and the cells were washed three times. After adding DAPI and incubating at room temperature in the dark for 8 minutes, the old solution was discarded and the plate was rinsed three times with PBS before photographing the wells. The results were then analyzed in PS.

[0155] III) Analysis of Physicochemical Results

[0156] 3.1 Electron microscopy results are as follows Figure 1-3

[0157] in, Figure 1 The hydrogel agent contains leonurine and chlorogenic acid (3:1). The hydrogel of the present invention (abbreviated as CFL hydrogel) exhibits a honeycomb-like structure and presents a porous network structure.

[0158] Figure 2 The hydrogel agent contains leonurine and puerarin (1:3). The hydrogel of the present invention (abbreviated as CFL hydrogel) exhibits a honeycomb-like structure and presents a porous network structure.

[0159] Figure 3 The hydrogel agent contains leonurine and gallic acid (1:3). The hydrogel of the present invention (abbreviated as CFL hydrogel) exhibits a honeycomb-like structure and presents a porous network structure.

[0160] 3.2 Dynamic time-scan results of CFL hydrogel

[0161] Figure 4 The hydrogel contains leonurine and chlorogenic acid (3:1). Under certain amplitude and frequency, its elastic modulus and viscous modulus remain within a certain range and increase to a certain extent, indicating that CFL hydrogel has good extensibility and thixotropy and can be used as a hydrogel for external dressing.

[0162] 3.3 Results of ROS (Reactive Oxygen Species) Detection of Hydrogel

[0163] like Figure 5As shown, the fluorescence intensity of the constructed oxidative stress model gradually decreased after hydrogel treatment, and this decrease was concentration-dependent, indicating that the CFL hydrogel has the ability to scavenge reactive oxygen species.

[0164] 3.4 HUVEC cell scratch assay to evaluate CFL hydrogel migration images and quantification results

[0165] like Figure 6 As shown, after the same scratch treatment, the CFL hydrogel group exhibited a significant healing effect after 48 hours, and the scratch closure rate increased significantly with increasing concentration. This indicates that CFL hydrogel has the ability to promote cell proliferation and migration, and also has the potential to promote wound healing.

[0166] Figure 3.5 shows the results of angiogenesis experiments using HUVEC cells.

[0167] The reconstruction of the vascular network is a crucial process during wound healing. For example... Figure 7 As shown in the in vitro tube formation experiment, the hydrogel group exhibited better cell-vascular connectivity compared to free chlorogenic acid, leonurine, and the combination of the two. Figure 8 Quantitative analysis of the results showed that, compared with the free drug, the hydrogel group had significantly increased capillary length and branching points, with the hydrogel group exhibiting the largest relative capillary length. These results demonstrate that hydrogels can accelerate wound healing in early-stage diabetes by promoting angiogenesis. Figure 9 :right Figure 8 Further evidence, through quantitative analysis of the results, shows that the gel group has the largest relative tube length.

[0168] 3.6 Immunofluorescence images.

[0169] Figure 10 The hydrogel agent described contains leonurine and chlorogenic acid (3:1) in immunofluorescence images. After in vitro inflammatory stimulation, the morphology of RAW264.7 cells changed from round to spindle-shaped, and the fluorescence intensity increased after LPS stimulation. However, the fluorescence of the CD86 pro-inflammatory factor in the gel-treated group decreased in a concentration-dependent manner. The expression level of CD86 decreased after hydrogel treatment, demonstrating that CFL hydrogel can inhibit inflammatory responses in chronic wounds.

[0170] Figure 11 The hydrogel agent described contains leonurine and chlorogenic acid (3:1) in immunofluorescence images. After in vitro inflammatory stimulation, the morphology of RAW264.7 cells changed from round to spindle-shaped, and the fluorescence intensity increased after LPS stimulation. The enhancement of CD206 anti-inflammatory factor in the gel-treated group was concentration-dependent. The expression level of CD206 significantly increased after hydrogel treatment, demonstrating that CFL hydrogel can inhibit inflammatory responses in chronic wounds.

[0171] III. Experimental Study on the Healing Effect of the Hydrogel of the Present Invention on Skin Wounds in Rats

[0172] 3.1. Selection of rats and establishment of a diabetic rat model

[0173] Materials: Seventy-two male SD rats were selected for modeling, and the experiment was conducted at the Medical School of Nanhua University.

[0174] The model was established by fasting rats at 10 PM, measuring blood glucose and weighing them at 10 AM the next day to calculate the dosage. Subsequently, an intraperitoneal injection was administered (STZ dissolved in sodium citrate buffer to prepare a solution of 10 mg / ml, 60 mg / kg), for three consecutive days, with fasting for two hours after each injection at 10 AM. On the third day after the three-day injection period, a random blood glucose level greater than 17.6 mmol / L and a fasting blood glucose level greater than 11.1 mmol / L were considered successful model establishment. Tribromoethanol was then administered intraperitoneally for mild anesthesia. The rats' backs were shaved to expose the skin, covered with sterile drapes, and placed in a prone position. The backs were disinfected with 75% alcohol, and a 10 mm wound was made using multi-purpose punch forceps, marking the location 0.5 cm to one side of the spine on the rat's back. The marked area and surrounding skin were disinfected. After hemostasis, a rat skin wound model was established. The rats were returned to their cages, with free access to water and food, and housed individually.

[0175] Grouping: The rats after modeling were randomly divided into 12 groups of 6 rats each, namely the model control group, group A, group B, group C, group D, group E, group F, group G, group H, group Q, and group 11 of Example, as well as the positive control group (Shengji Yupi Ointment, National Drug Approval Number B20021045).

[0176] The nine rat groups in Example 12 and the rat groups in Example 11 applied their respective combined gels to the wounds twice daily, with a thickness of 1-2 mm; the positive control group strictly followed the instructions.

[0177] The rats in the model control group had their wounds treated with petroleum jelly twice a day for 14 consecutive days.

[0178] 2. Indicator Testing

[0179] The wound was photographed on days 0, 7, and 14 after modeling. A ruler was placed under the wound during photography to ensure consistent angle and distance for each shot. Imageproplus 6.0 image analysis software was used to measure the wound area and calculate the wound healing rate.

[0180] Healing rate = (Original wound area - Unhealed wound area) / Original wound area × 100%.

[0181] 3. Statistical Methods

[0182] Data processing was performed using GrapHpadPrism 9.0 software. Measurement data are expressed as mean ± standard deviation. This indicates that one-way ANOVA was used for comparisons among multiple groups, and pairwise comparisons between groups were performed using SNK-q, with P < 0.05 considered statistically significant.

[0183] 4. Experimental Results

[0184] Effect on skin wound healing rate

[0185] Table 1 Comparison of skin wound healing rates among different groups

[0186]

[0187] Note: Compared with the model control group, *P<0.05, **P<0.01; compared with the positive control group, #P<0.05, ##P<0.01; compared with group H, ￥P<0.05, ￥￥P<0.01; compared with group Q, @P<0.05, @@P<0.01; compared with group 9 of Example, &P<0.05, &&P<0.01;

[0188] The results are shown in Table 1. In this study, the hydrogel agent of the present invention can significantly improve the healing of skin wounds in rats. At 7 days and 14 days post-traumatic injury, the wound healing rate of the AG group was significantly higher than that of the model control group. In particular, at 14 days post-traumatic injury, the wound healing rate of the AG group was significantly higher than that of the positive control group and the combination of other embodiments (P < 0.05 or P < 0.01).

[0189] While the present disclosure has been specifically described above in conjunction with the accompanying drawings and examples, it is to be understood that the foregoing description does not limit the present disclosure in any way. Those skilled in the art can make modifications and variations to the present disclosure as needed without departing from its essential spirit and scope, and all such modifications and variations shall fall within the scope of the present disclosure.

Claims

1. A hydrogel agent, characterized in that, The hydrogel contains motherwort powder or motherwort alkaloids, along with flavonoids and / or polyphenols.

2. The hydrogel agent as described in claim 1, characterized in that, The leonurus alkaloids are any one or a mixture of leonurine and stachydrine, the polyphenols are any one or a mixture of chlorogenic acid and gallic acid, and the flavonoids are puerarin.

3. The hydrogel agent as described in claim 1, characterized in that, The hydrogel contains leonurine and puerarin; stachydrine and puerarin; leonurine and chlorogenic acid; stachydrine and chlorogenic acid; leonurine and gallic acid; stachydrine and gallic acid; leonurine and gallic acid and chlorogenic acid; leonurine and stachydrine and chlorogenic acid; a combination of leonurine, stachydrine and gallic acid; any combination of leonurine, stachydrine, gallic acid and chlorogenic acid; preferably, the hydrogel contains leonurine and chlorogenic acid; leonurine and puerarin; stachydrine and puerarin; stachydrine and chlorogenic acid; any combination of leonurine and gallic acid or stachydrine and gallic acid; furthermore, the hydrogel contains leonurine and chlorogenic acid; any combination of leonurine and puerarin or leonurine and gallic acid.

4. The application of the hydrogel agent according to any one of claims 1-3 in the preparation of a treatment to promote wound healing, wherein the hydrogel agent is selected from oral hydrogel agents or topical hydrogel agents.

5. The hydrogel agent according to any one of claims 1-3, characterized in that, The hydrogel contains leonurine, chlorogenic acid and a pH adjuster; leonurine, puerarin and a pH adjuster; or any combination of leonurine, gallic acid and a pH adjuster.

6. The hydrogel agent according to any one of claims 1-3, characterized in that, The molar ratio of leonurine to polyphenol molecules is 1:0.1-20; more preferably, it is any combination of 3:1, 1:0.5, 1:3, 1:10, and 1:20; even more preferably, it is 3:1; the molar ratio of leonurine to flavonoid molecules is 1:0.1-20; more preferably, it is any combination of 3:1, 1:0.5, 1:3, 1:10, and 1:20; even more preferably, it is 3:

1.

7. The hydrogel agent according to any one of claims 1-3, characterized in that, The molar ratio of leonurine to chlorogenic acid is 1:0.1-20; more preferably, it is any combination of 3:1, 1:0.5, 1:3, 1:10, and 1:20; even more preferably, it is 3:1; the molar ratio of leonurine to puerarin is 1:0.1-20; more preferably, it is any combination of 3:1, 1:0.5, 1:3, 1:10, and 1:20; even more preferably, it is 3:1; the molar ratio of leonurine to gallic acid is 1:0.1-20; more preferably, it is any combination of 3:1, 1:0.5, 1:3, 1:10, and 1:20; even more preferably, it is 3:

1.

8. The hydrogel agent according to any one of claims 1-3, characterized in that, The preparation method described herein is performed by any of the following methods: Preparation method 1: (1) Weigh out the active alkaloids or powder of Leonurus japonicus and the polyphenol molecules or flavonoid molecules separately for later use; (2) Add water and pH adjuster to the motherwort active alkaloids or powder in step (1) to form an aqueous solution, and obtain solution A; (3) Polyphenol molecules or flavonoid molecules are dissolved in water by heating or sonication to obtain a clear and transparent solution B; (4) Mix and stir the solution A prepared in step (2) with the solution B prepared in step (3), place it in an ultrasonic crusher for ultrasonic crushing or heating treatment, centrifuge to obtain precipitate or colloid, wash with water and centrifuge, repeat three times to obtain the product; Or preparation method two: (1) Weigh out the active alkaloids or powder of Leonurus japonicus and the polyphenol molecules or flavonoid molecules separately for later use; (2) Mix the pH adjuster with water to form a pH adjusting solution; (3) Mix the active alkaloids or powder of Leonurus japonicus from step (1) with polyphenol molecules or flavonoid molecules evenly, add them to the pH adjustment solution prepared in step (2), and vortex to mix evenly. (4) Place the solution system from step (3) into an ultrasonic crusher for ultrasonic crushing or heating treatment, centrifuge to obtain precipitate or colloid, wash with water and centrifuge again, repeat three times to obtain the final product.

9. The hydrogel agent as described in claim 8, characterized in that, The pH adjuster is selected from any one of 2-formylphenylboronic acid, citric acid, fumaric acid, and adipic acid; more preferably, the pH adjuster is selected from 2-formylphenylboronic acid; the concentration of the pH adjuster is 0.01wt% to 10wt%; the ultrasonic disruption power is 20-70W, the ultrasonic disruption time is more than 3min; and the centrifugation speed during centrifugation purification is 3500-13000rpm.

10. The hydrogel agent according to claim 8, characterized in that, The preparation method described herein is prepared by any of the following methods: Preparation method 1: (1) Weigh out the active alkaloids or powder of Leonurus japonicus and the polyphenol molecules or flavonoid molecules separately for later use; (2) Add water and 2-formylphenylboronic acid to the motherwort active alkaloids or powder in step (1) to form an aqueous solution, and obtain solution A, wherein the concentration of 2-formylphenylboronic acid is 1 wt%. (3) Polyphenol molecules or flavonoid molecules are dissolved in water by heating or sonication to obtain a clear and transparent solution B; (4) Mix and stir the solution A prepared in step (2) with the solution B prepared in step (3), place it in an ultrasonic crusher for ultrasonic crushing or heating treatment, centrifuge to obtain precipitate or colloid, wash with water and centrifuge, repeat three times to obtain the solution. The ultrasonic crushing power is 70W and the ultrasonic crushing time is more than 3 minutes. The centrifugation speed during centrifugation purification is 10000 rpm. Or preparation method two: (1) Weigh out the active alkaloids or powder of Leonurus japonicus and the polyphenol molecules or flavonoid molecules separately for later use; (2) Mix 2-formylphenylboronic acid with water to form a 2-formylphenylboronic acid solution, wherein the concentration of 2-formylphenylboronic acid is 1 wt%. (3) Mix the active alkaloids or powder of Leonurus japonicus from step (1) with polyphenol molecules or flavonoid molecules evenly, add them to the 2-formylphenylboronic acid solution prepared in step (2), and vortex to mix evenly. (4) Place the solution system from step (3) into an ultrasonic crusher for ultrasonic crushing or heating treatment, centrifuge to obtain precipitate or colloid, wash with water and centrifuge again, repeat three times to obtain the solution. The ultrasonic crushing power is 70W and the ultrasonic crushing time is more than 3 minutes. The centrifugation speed during centrifugation purification is 10000 rpm.

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