A bacteriostatic anti-inflammatory composition containing 6-gingerol and its preparation method and application

Abstract

The application provides a 6-gingerol-containing bacteriostatic anti-inflammatory composition and a preparation method and application thereof, and belongs to the technical field of medicines. After gingerol crude extract is extracted from ginger, the 6-gingerol is separated and purified through a molecular imprinting compound, mixed with epigallocatechin gallate and resveratrol, embedded in polyethylene glycol-poly epsilon-caprolactone-polylactic acid / vitamin E polyethylene glycol succinate micelles, and a 6-gingerol-containing bacteriostatic anti-inflammatory composition is prepared. The 6-gingerol-containing bacteriostatic anti-inflammatory composition prepared by the application has good water solubility and transdermal absorption performance, greatly improves the bioavailability, has high drug loading, has the characteristics of slow-release and controlled-release of the drug, has good antioxidant, anti-inflammatory and antibacterial effects, has low preparation cost, is easy to realize industrial application, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to an antibacterial and anti-inflammatory composition containing 6-gingerol, its preparation method, and its application. Background Technology

[0002] Rheumatoid arthritis (RA) is a chronic, systemic disease of unknown etiology, primarily characterized by inflammatory synovitis. Its pathogenesis may be related to genetics, infection, and sex hormones. The pathology of RA mainly involves synovial lining cell proliferation, extensive inflammatory cell infiltration in the interstitium, microvascular angiogenesis, pannus formation, and destruction of cartilage and bone tissue. Rheumatic arthritis is a common acute or chronic inflammatory disease of connective tissue. Commonly referred to as rheumatic arthritis is one of the main manifestations of rheumatic fever, clinically characterized by migratory aches, redness, swelling, and pain in the joints and muscles. It is associated with group A streptococcal infection, and cold and dampness can trigger the disease. Large joints of the lower extremities, such as the knee and ankle, are most commonly affected. Both diseases are chronic and difficult to cure, with pain being a common symptom. Due to the long-term and persistent nature of the disease, a highly effective symptomatic treatment (analgesic) with few side effects is crucial, and the appropriate route of administration is equally important.

[0003] Gingerol is a series of phenolic compounds with long alkyl side chains discovered in ginger. It possesses excellent anti-inflammatory and analgesic effects. Modern pharmacology shows that they are very potent cyclooxygenase inhibitors, thus exhibiting the activity of nonsteroidal anti-inflammatory drugs (NSAIDs). Gingerol capsules are oral preparations made from a low content of ginger oleoresin. Because of oral administration, long-term use can lead to gastrointestinal irritation, a common symptom of NSAIDs, and even serious complications such as bleeding or perforation. Furthermore, 6-gingerol faces several challenges in practical applications: firstly, its poor water solubility results in low bioavailability; secondly, it is chemically unstable and easily degrades under light and heat; and thirdly, it is rapidly metabolized in vivo with a short half-life, making it difficult to maintain effective blood drug concentrations. These limitations severely restrict the clinical application value of 6-gingerol.

[0004] Therefore, there is a need to develop a safe and effective formulation containing 6-gingerol that can have a good effect on arthritis, improve bioavailability, and not cause other symptoms. Summary of the Invention

[0005] The purpose of this invention is to propose an antibacterial and anti-inflammatory composition containing 6-gingerol, its preparation method and application. It has good water solubility and transdermal absorption performance, which greatly improves its bioavailability. It has a high drug loading capacity and the characteristics of sustained-release drug release. It has good antioxidant, anti-inflammatory and antibacterial effects, and the preparation cost is low. It is easy to realize industrial application and has broad application prospects.

[0006] The technical solution of this invention is implemented as follows: This invention provides a method for preparing an antibacterial and anti-inflammatory composition containing 6-gingerol. After extracting crude gingerol from ginger, 6-gingerol is obtained by molecular imprinting compound separation and purification. It is then mixed with epigallocatechin gallate and resveratrol and embedded in polyethylene glycol-poly(ε-caprolactone-polylactic acid / vitamin E polyethylene glycol succinate micelles to obtain an antibacterial and anti-inflammatory composition containing 6-gingerol.

[0007] As a further improvement to the present invention, the following steps are included: S1. Extraction of gingerol: Wash, dry and crush fresh ginger, add it to ethanol solution, heat and reflux to extract, filter, recover ethanol from filtrate, evaporate and concentrate to obtain crude gingerol extract; S2. Separation and purification of 6-gingerol: Crude gingerol extract was added to a deep eutectic solvent, a molecularly imprinted compound was added, the mixture was stirred and adsorbed, filtered, the molecularly imprinted compound was separated, 6-gingerol was eluted with an eluent, the eluent was collected, concentrated and dried to obtain 6-gingerol. S3. Preparation of the composition: 6-gingerol, epigallocatechin gallate, and resveratrol were mixed evenly to obtain the composition; S4. Preparation of an antibacterial and anti-inflammatory composition containing 6-gingerol: The composition, polyethylene glycol-poly(ε-caprolactone-polylactic acid), and vitamin E polyethylene glycol succinate were mixed, acetonitrile was added, the mixture was stirred and dissolved, and then added dropwise to hot water. The mixture was kept warm and stirred, the solvent was removed under reduced pressure, and the mixture was washed and dried to obtain an antibacterial and anti-inflammatory composition containing 6-gingerol.

[0008] As a further improvement of the present invention, the concentration of the ethanol solution in step S1 is 75-85 wt%, the solid-liquid ratio of ginger to ethanol solution is 1:5-10 g / mL, and the heating and reflux extraction time is 2-4 h.

[0009] As a further improvement of the present invention, the deep eutectic solvent in step S2 is obtained by mixing choline chloride and glucose in a molar ratio of 1:1-3, heating to 95-100°C, adding water (20-50 wt% of the total system), and stirring for 2-4 hours; the mass ratio of the crude gingerol extract, deep eutectic solvent, and molecularly imprinted compound is 15-20:100-120:5-7, the eluent is a 20-30 wt% ethanol solution, and the preparation method of the molecularly imprinted compound is as follows: gingerone is dissolved in acetonitrile, methyl methacrylate, glycidyl methacrylate, and double-bonded ionic liquid are added, the mixture is heated and stirred, then ethylene glycol dimethacrylate, water, and monoglyceride are added, the mixture is stirred and mixed evenly, an initiator and a porogen are added, the mixture is degassed by ultrasound, heated and polymerized, filtered, washed with ethanol until gingerone is no longer detectable in the eluent, and dried to obtain the molecularly imprinted compound.

[0010] As a further improvement of the present invention, the mass ratio of gingerone, methyl methacrylate, glycidyl methacrylate, double-bonded ionic liquid, ethylene glycol dimethacrylate, monoglyceride, initiator, and porogen is 0.2-0.3:0.3-0.4:0.2-0.5:0.5-1:3-4:0.02-0.03:0.01-0.03:3-5, wherein the double-bonded ionic liquid is 1-vinyl-3-methylimidazolium chloride; the porogen is diethyl ether; the initiator is azobisisobutyronitrile or azobisisovalerate; the heating and stirring reaction temperature is 45-55℃ for 10-12 h; and the heating polymerization reaction temperature is 55-65℃ for 20-28 h.

[0011] As a further improvement of the present invention, the mass ratio of 6-gingerol, epigallocatechin gallate, and resveratrol in step S3 is 7-10:2-3:1-2.

[0012] As a further improvement of the present invention, the mass ratio of the composition, polyethylene glycol-poly(ε-caprolactone-polylactic acid), and vitamin E polyethylene glycol succinate in step S4 is 1-1.5:30-50:30-50, and the preparation method of the polyethylene glycol-poly(ε-caprolactone-polylactic acid) is as follows: T1. Polyethylene glycol monomethyl ether and ε-caprolactone were mixed, catalyst A was added, and the mixture was heated and stirred under inert gas protection. Dichloromethane was added for dilution, and the product was precipitated with ice-cold diethyl ether. The mixture was filtered, washed, and dried to obtain polyethylene glycol-polyε-caprolactone-OH. T2. Polyethylene glycol-poly(ε-caprolactone-OH) and polylactic acid were added to toluene, catalyst B was added, and the mixture was heated under reflux with stirring. The product was precipitated by adding ice-cold diethyl ether, filtered, washed, and dried to obtain polyethylene glycol-poly(ε-caprolactone-polylactic acid).

[0013] As a further improvement of the present invention, in step T1, the mass ratio of polyethylene glycol monomethyl ether to ε-caprolactone is 8-10:4-7, the catalyst A is stannous octoate, and the amount added is 2-4 wt% of ε-caprolactone; the temperature of the heating and stirring reaction is 125-135°C, and the time is 20-28 h; in step T2, the mass ratio of polyethylene glycol-polyε-caprolactone-OH to polylactic acid is 10-14:3-4, the catalyst B is p-toluenesulfonic acid, and the amount added is 1-2 wt% of polylactic acid; the time of the heating, reflux, and stirring reaction is 5-7 h.

[0014] The present invention further protects an antibacterial and anti-inflammatory composition containing 6-gingerol prepared by the above-mentioned preparation method.

[0015] The present invention further protects the use of the above-mentioned antibacterial and anti-inflammatory composition containing 6-gingerol in the preparation of medicaments for treating arthritis and in the preparation of antibacterial drugs.

[0016] The present invention has the following beneficial effects: This invention extracts gingerol from ginger and uses molecularly imprinted compounds to separate and purify 6-gingerol. The extraction purity is high, and the method is simple and convenient. The separated molecularly imprinted compounds can be reused, thereby reducing production costs.

[0017] The molecularly imprinted compound prepared in this invention uses gingerone as a template molecule. Gingerone is a structural analog of 6-gingerol and, compared to 6-gingerol, it is more chemically stable, commercially available, and inexpensive, making it more suitable as a template for large-scale polymerization. Furthermore, glycidyl methacrylate, a monomer with an epoxy group, is used in the polymer monomer. The epoxy group reacts with the hydroxyl groups on the 6-gingerol molecule in a ring-opening reaction, immobilizing the 6-gingerol molecule in a highly ordered and stable manner on the polymer, greatly enhancing the specific recognition ability of the molecularly imprinted polymer for 6-gingerol. In addition, an ionic liquid monomer is added to the monomer composition. The ionic bond energy of the ionic liquid monomer is much higher than that of traditional covalent monomers, resulting in a generally higher thermal decomposition temperature of the polymer backbone after polymerization. It is also less prone to swelling or degradation in strong acids, strong bases, or highly polar solvents, thus extending the polymer's lifespan. Moreover, the cationic backbone of the ionic liquid monomer has a regular spatial structure and lacks polar defect sites that easily adsorb impurities, reducing the non-specific adsorption of interfering molecules (such as impurity phenols and fatty acids) in the system. Ionic liquid monomers possess both cationic (e.g., imidazolium) and anionic groups. The hydroxyl groups on the cationic side chains of ionic liquid monomers can form double hydrogen bonds with the phenolic hydroxyl groups of 6-gingerol, while the hydrophobic groups on the side chains can form hydrophobic interactions with the alkyl chains of 6-gingerol. This multi-synergistic effect is far stronger than the single hydrogen bond effect of traditional monomers (e.g., methacrylic acid), which can more stably fix the template molecule and form an imprinted cavity that is highly matched with the spatial structure and interaction site of the template, thus significantly improving the specific binding of the polymer to the template molecule.

[0018] Furthermore, this invention combines the prepared 6-gingerol with epigallocatechin gallate and resveratrol, exhibiting a significant synergistic effect in both antibacterial and anti-inflammatory activities. In the antibacterial mechanism, epigallocatechin gallate primarily disrupts bacterial cell membrane integrity, resveratrol inhibits bacterial nucleic acid synthesis, and 6-gingerol interferes with the bacterial quorum sensing system. These three agents simultaneously attack pathogens through different pathways, resulting in a multi-pronged antibacterial mechanism. In the anti-inflammatory mechanism, resveratrol possesses good antioxidant activity, while 6-gingerol and epigallocatechin gallate selectively inhibit the release of inflammatory factors and block nucleation. The combined use of these three agents significantly reduces the levels of inflammatory factors.

[0019] This invention encapsulates an active composition within a core-shell-crown structure micelle constructed from a polyethylene glycol-poly(ε-caprolactone)-polylactic acid (PEG-PCL-PLA) triblock polymer, forming a novel nano-drug delivery system. This micelle exhibits significantly superior performance compared to traditional PEG-PCL micelles: its core PLA domains enhance affinity for 6-gingerol through hydrophobic and hydrogen bonding interactions, significantly increasing drug loading; the intermediate PCL layer modulates drug release behavior, enabling gradient drug release; and the outer PEG chains provide spatial stability and stealth effects, prolonging in vivo circulation time. Furthermore, this micelle system significantly improves the water solubility and transdermal absorption of 6-gingerol, thereby greatly enhancing its bioavailability and providing a more efficient delivery strategy for the antibacterial and anti-inflammatory applications of 6-gingerol.

[0020] The antibacterial and anti-inflammatory composition containing 6-gingerol prepared by this invention has good water solubility and transdermal absorption performance, which greatly improves its bioavailability. It has a high drug loading capacity and features sustained-release drug release. It has good antioxidant, anti-inflammatory and antibacterial effects, and has low preparation cost, making it easy to realize industrial application and showing broad application prospects. Attached Figure Description

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

[0022] Figure 1 This is a TEM image of the aqueous dispersion of the antibacterial and anti-inflammatory composition containing 6-gingerol prepared in Example 1. Detailed Implementation

[0023] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] Preparation Example 1: Molecularly Imprinted Compounds The preparation method is as follows: 0.2 g gingerone was dissolved in 30 mL acetonitrile, and 0.3 g methyl methacrylate, 0.2 g glycidyl methacrylate, and 0.5 g 1-vinyl-3-methylimidazolium chloride were added. The mixture was heated to 45 °C and stirred for 10 h. Then, 3 g ethylene glycol dimethacrylate, 15 g water, and 0.02 g monoglyceride were added and stirred for 30 min. Finally, 0.01 g azobisisobutyronitrile and 3 g diethyl ether were added. The mixture was degassed by ultrasonication at 500 W for 20 min, heated to 55 °C, and polymerized for 20 h. The mixture was filtered, washed with ethanol until gingerone was undetectable in the eluent, and dried to obtain the molecularly imprinted compound.

[0025] Preparation Example 2: Molecularly Imprinted Compounds The preparation method is as follows: 0.3 g gingerone was dissolved in 30 mL acetonitrile, and 0.4 g methyl methacrylate, 0.5 g glycidyl methacrylate, and 1 g 1-vinyl-3-methylimidazolium chloride were added. The mixture was heated to 55 °C and stirred for 12 h. Then, 4 g ethylene glycol dimethacrylate, 15 g water, and 0.03 g monoglyceride were added and stirred for 30 min. Finally, 0.03 g azobisisovalerate and 5 g diethyl ether were added. The mixture was degassed by ultrasonication at 500 W for 20 min, heated to 65 °C, and polymerized for 28 h. The mixture was filtered, washed with ethanol until gingerone was undetectable in the eluent, and dried to obtain the molecularly imprinted compound.

[0026] Preparation Example 3: Molecularly Imprinted Compounds The preparation method is as follows: 0.25 g gingerone was dissolved in 30 mL acetonitrile, and 0.35 g methyl methacrylate, 0.35 g glycidyl methacrylate, and 0.7 g 1-vinyl-3-methylimidazolium chloride were added. The mixture was heated to 50 °C and stirred for 11 h. Then, 3.5 g ethylene glycol dimethacrylate, 15 g water, and 0.025 g monoglyceride were added and stirred for 30 min. Finally, 0.02 g azobisisobutyronitrile and 4 g diethyl ether were added. The mixture was degassed by ultrasonication at 500 W for 20 min, heated to 60 °C, and polymerized for 24 h. The mixture was filtered, washed with ethanol until gingerone was undetectable in the eluent, and dried to obtain the molecularly imprinted compound.

[0027] Comparative Preparation Example 1 The difference compared to Preparation Example 3 is that glycidyl methacrylate was not added.

[0028] The preparation method is as follows: 0.25 g of gingerone was dissolved in 30 mL of acetonitrile, and 0.35 g of methyl methacrylate and 1.05 g of 1-vinyl-3-methylimidazolium chloride were added. The mixture was heated to 50 °C and stirred for 11 h. Then, 3.5 g of ethylene glycol dimethacrylate, 15 g of water and 0.025 g of monoglyceride were added and stirred for 30 min. Then, 0.02 g of azobisisobutyronitrile and 4 g of diethyl ether were added. The mixture was degassed by ultrasonication at 500 W for 20 min, heated to 60 °C and polymerized for 24 h. The mixture was filtered, washed with ethanol until gingerone was undetectable in the eluent, and dried to obtain the molecularly imprinted compound.

[0029] Comparative Preparation Example 2 The difference from Preparation Example 3 is that 1-vinyl-3-methylimidazolium chloride was not added.

[0030] The preparation method is as follows: 0.25 g gingerone was dissolved in 30 mL acetonitrile, and 0.35 g methyl methacrylate and 1.05 g glycidyl methacrylate were added. The mixture was heated to 50 °C and stirred for 11 h. Then, 3.5 g ethylene glycol dimethacrylate, 15 g water and 0.025 g monoglyceride were added and stirred for 30 min. Then, 0.02 g azobisisobutyronitrile and 4 g diethyl ether were added. The mixture was degassed by ultrasonication at 500 W for 20 min, heated to 60 °C and polymerized for 24 h. The mixture was filtered, washed with ethanol until gingerone was undetectable in the eluent, and dried to obtain the molecularly imprinted compound.

[0031] Preparation Example 4: Polyethylene glycol-poly(ε-caprolactone-polylactic acid) The preparation method is as follows: T1. Mix 8g of polyethylene glycol monomethyl ether and 4g of ε-caprolactone, add 2wt% of stannous octoate (based on the mass of ε-caprolactone), heat to 125℃ under nitrogen protection, stir and react for 20h, add 50mL of dichloromethane to dilute, add 200mL of ice-cold diethyl ether to precipitate the product for 1h, filter, wash, and dry to obtain polyethylene glycol-polyε-caprolactone-OH; T2. Add 10g of polyethylene glycol-poly(ε-caprolactone-OH) and 3g of polylactic acid to 200mL of toluene, add 1wt% p-toluenesulfonic acid (based on the mass of polylactic acid), heat under reflux and stir for 5h, remove the solvent from the product under reduced pressure, add 100mL of ice-cold diethyl ether to precipitate, filter, wash, and dry to obtain polyethylene glycol-poly(ε-caprolactone-polylactic acid).

[0032] Preparation Example 5: Polyethylene glycol-poly(ε-caprolactone-polylactic acid) The preparation method is as follows: T1. Mix 10g polyethylene glycol monomethyl ether and 7g ε-caprolactone, add 4wt% stannous octoate (based on the mass of ε-caprolactone), heat to 135℃ under nitrogen protection, stir and react for 28h, add 50mL dichloromethane to dilute, add 200mL ice-cold diethyl ether to precipitate the product for 1h, filter, wash, and dry to obtain polyethylene glycol-polyε-caprolactone-OH; T2. Add 14g of polyethylene glycol-poly(ε-caprolactone-OH) and 4g of polylactic acid to 200mL of toluene, add 2wt% p-toluenesulfonic acid (based on the mass of polylactic acid), heat under reflux and stir for 7h, remove the solvent from the product under reduced pressure, add 100mL of ice-cold diethyl ether to precipitate, filter, wash, and dry to obtain polyethylene glycol-poly(ε-caprolactone-polylactic acid).

[0033] Preparation Example 6: Polyethylene glycol-poly(ε-caprolactone-polylactic acid) The preparation method is as follows: T1. Mix 9g of polyethylene glycol monomethyl ether and 5.5g of ε-caprolactone, add 3wt% of stannous octoate (based on the mass of ε-caprolactone), heat to 130℃ under nitrogen protection, stir and react for 24h, add 50mL of dichloromethane to dilute, add 200mL of ice-cold diethyl ether to precipitate the product for 1h, filter, wash, and dry to obtain polyethylene glycol-polyε-caprolactone-OH; T2. Add 12g of polyethylene glycol-poly(ε-caprolactone-OH) and 3.5g of polylactic acid to 200mL of toluene, add 1.5wt% p-toluenesulfonic acid (based on the mass of polylactic acid), heat under reflux and stir for 6h, remove the solvent from the product under reduced pressure, add 100mL of ice-cold diethyl ether to precipitate, filter, wash, and dry to obtain polyethylene glycol-poly(ε-caprolactone-polylactic acid). Example 1

[0034] This embodiment provides a method for preparing an antibacterial and anti-inflammatory composition containing 6-gingerol, comprising the following steps: S1. Extraction of gingerol: Wash, dry and crush 10g of fresh ginger, add 50mL of 85wt% ethanol solution, heat and reflux for 2h, filter, recover ethanol from filtrate, evaporate and concentrate to obtain crude gingerol extract. S2. Separation and purification of 6-gingerol: 15g of crude gingerol extract was added to 100g of deep eutectic solvent, and 5g of the molecularly imprinted compound prepared in Preparation Example 1 was added. The mixture was stirred and adsorbed for 30min. After filtration, the molecularly imprinted compound was separated. 6-gingerol was eluted with 20wt% ethanol solution. The eluent was collected, concentrated and dried to obtain 6-gingerol. The deep eutectic solvent is prepared by mixing choline chloride and glucose in a molar ratio of 1:1, heating to 100°C, adding water (20 wt% of the total system), and stirring for 2 hours. S3. Preparation of the composition: 7 mg 6-gingerol, 2 mg epigallocatechin gallate and 1 mg resveratrol were stirred and mixed for 10 min to obtain the composition; S4. Preparation of an antibacterial and anti-inflammatory composition containing 6-gingerol: 10 mg of the composition, 0.3 g of polyethylene glycol-poly(ε-caprolactone-polylactic acid) prepared in Example 4, and 0.3 g of vitamin E polyethylene glycol succinate were mixed. 1 mL of acetonitrile was added, and the mixture was stirred to dissolve. The solution was then added dropwise to 10 mL of 45°C hot water, and the mixture was kept warm and stirred for 20 min. The solvent was removed under reduced pressure, and the mixture was washed and dried to obtain the antibacterial and anti-inflammatory composition containing 6-gingerol. The composition was dispersed in water to a concentration of 1 mg / mL. Morphological characteristics were observed using TEM after phosphomolybdic acid staining. The results are as follows: Figure 1 As shown in the figure, the formulation particles are uniformly spherical, and no obvious particle aggregation was observed. Example 2

[0035] This embodiment provides a method for preparing an antibacterial and anti-inflammatory composition containing 6-gingerol, comprising the following steps: S1. Extraction of gingerol: Wash, dry and crush 10g of fresh ginger, add 100mL of 75wt% ethanol solution, heat and reflux for 4h, filter, recover ethanol from filtrate, evaporate and concentrate to obtain crude gingerol extract. S2. Separation and purification of 6-gingerol: 20g of crude gingerol extract was added to 120g of deep eutectic solvent, and 7g of the molecularly imprinted compound prepared in Preparation Example 2 was added. The mixture was stirred and adsorbed for 30min. After filtration, the molecularly imprinted compound was separated. 6-gingerol was eluted with 30wt% ethanol solution. The eluent was collected, concentrated and dried to obtain 6-gingerol. The deep eutectic solvent is prepared by mixing choline chloride and glucose in a molar ratio of 1:3, heating to 95°C, adding water (50 wt% of the total system), and stirring for 4 hours. S3. Preparation of the composition: 10 mg of 6-gingerol, 3 mg of epigallocatechin gallate, and 2 mg of resveratrol were stirred and mixed for 10 min to obtain the composition; S4. Preparation of an antibacterial and anti-inflammatory composition containing 6-gingerol: 15 mg of the composition, 0.5 g of polyethylene glycol-poly(ε-caprolactone-polylactic acid) prepared in Preparation Example 5, and 0.5 g of vitamin E polyethylene glycol succinate were mixed, 1 mL of acetonitrile was added, and the mixture was stirred to dissolve. The mixture was then added dropwise to 10 mL of hot water at 45 °C, kept warm and stirred for 20 min, the solvent was removed under reduced pressure, and the mixture was washed and dried to obtain an antibacterial and anti-inflammatory composition containing 6-gingerol. Example 3

[0036] This embodiment provides a method for preparing an antibacterial and anti-inflammatory composition containing 6-gingerol, comprising the following steps: S1. Extraction of gingerol: Wash, dry and crush 10g of fresh ginger, add 75mL of 80wt% ethanol solution, heat and reflux for 3h, filter, recover ethanol from filtrate, evaporate and concentrate to obtain crude gingerol extract. S2. Separation and purification of 6-gingerol: 17g of crude gingerol extract was added to 110g of deep eutectic solvent, and 6g of the molecularly imprinted compound prepared in Preparation Example 3 was added. The mixture was stirred and adsorbed for 30min. After filtration, the molecularly imprinted compound was separated. 6-gingerol was eluted with 25wt% ethanol solution. The eluent was collected, concentrated and dried to obtain 6-gingerol. The deep eutectic solvent is prepared by mixing choline chloride and glucose in a molar ratio of 1:2, heating to 95°C, adding water (35 wt% of the total system), and stirring for 3 hours. S3. Preparation of the composition: 8.5 mg of 6-gingerol, 2.5 mg of epigallocatechin gallate and 1 mg of resveratrol were stirred and mixed for 10 min to obtain the composition; S4. Preparation of an antibacterial and anti-inflammatory composition containing 6-gingerol: 12 mg of the composition, 0.4 g of polyethylene glycol-poly(ε-caprolactone-polylactic acid) prepared in Preparation Example 6, and 0.4 g of vitamin E polyethylene glycol succinate were mixed, 1 mL of acetonitrile was added, and the mixture was stirred to dissolve. The mixture was then added dropwise to 10 mL of hot water at 45 °C, kept warm and stirred for 20 min, the solvent was removed under reduced pressure, and the mixture was washed and dried to obtain an antibacterial and anti-inflammatory composition containing 6-gingerol.

[0037] Comparative Example 1 The difference from Example 3 is that the molecularly imprinted compound was prepared by Comparative Preparation Example 1.

[0038] Comparative Example 2 The difference from Example 3 is that the molecularly imprinted compound was prepared by Comparative Preparation Example 2.

[0039] Comparative Example 3 The difference from Example 3 is that 6-gingerol was not added in step S3.

[0040] Specifically as follows: S3. Preparation of the composition: 2.5 mg epigallocatechin gallate and 1 mg resveratrol were stirred and mixed for 10 min to obtain the composition.

[0041] Comparative Example 4 The difference from Example 3 is that epigallocatechin gallate was not added in step S3.

[0042] Specifically as follows: S3. Preparation of the composition: 8.5 mg of 6-gingerol and 1 mg of resveratrol were stirred and mixed for 10 min to obtain the composition.

[0043] Comparative Example 5 The difference from Example 3 is that resveratrol was not added in step S3.

[0044] Specifically as follows: S3. Preparation of the composition: 8.5 mg of 6-gingerol and 2.5 mg of epigallocatechin gallate were stirred and mixed for 10 min to obtain the composition.

[0045] Comparative Example 6 The difference from Example 3 is that step S4 was not performed.

[0046] Specifically as follows: S1. Extraction of gingerol: Wash, dry and crush 10g of fresh ginger, add 75mL of 80wt% ethanol solution, heat and reflux for 3h, filter, recover ethanol from filtrate, evaporate and concentrate to obtain crude gingerol extract. S2. Separation and purification of 6-gingerol: 17g of crude gingerol extract was added to 110g of deep eutectic solvent, and 56g of the molecularly imprinted compound obtained in Preparation Example 3 was added. The mixture was stirred and adsorbed for 30min. After filtration, the molecularly imprinted compound was separated. 6-gingerol was eluted with 25wt% ethanol solution. The eluent was collected, concentrated and dried to obtain 6-gingerol. The deep eutectic solvent is prepared by mixing choline chloride and glucose in a molar ratio of 1:2, heating to 95°C, adding water (35 wt% of the total system), and stirring for 3 hours. S3. Preparation of the composition: 8.5 mg of 6-gingerol, 2.5 mg of epigallocatechin gallate and 1 mg of resveratrol were stirred and mixed for 10 min to obtain the composition, which is the antibacterial and anti-inflammatory composition containing 6-gingerol.

[0047] Comparative Example 7 The difference from Example 3 is that polyethylene glycol-poly(ε-caprolactone)-polylactic acid is replaced by an equal mass of polyethylene glycol-poly(ε-caprolactone).

[0048] Test Example 1: Anti-inflammatory Test Mouse macrophages Raw264.7 were seeded into 96-well cell plates at 5 × 10⁶ cells / well. 4Cells / well were cultured in DMEM medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin. The antibacterial and anti-inflammatory compositions containing 6-gingerol prepared in Examples 1-3 and Comparative Examples 1-7 were added to the medium (sample:medium mass ratio 1:15). The blank group was replaced with purified water. After mixing, 1 mL was added to each cell well, with three replicates per group. 1 μg / mL lipopolysaccharide was added, and incubation continued for 48 h. The supernatant was collected, and the concentrations of IL-6 and TNF-α were measured using a mouse IL-6 and TNF-α ELISA kit. The reduction rates (%) of TNF-α and IL-6 concentrations were calculated using the following formulas: Reduction rate (%) = (Concentration of blank group - Concentration of test sample group) / Concentration of blank group × 100%.

[0049] The results are shown in Table 1.

[0050] Table 1

[0051] As can be seen from the table above, the antibacterial and anti-inflammatory compositions containing 6-gingerol prepared in Examples 1-3 of the present invention have good in vitro anti-inflammatory effects.

[0052] Test Example 2: Antibacterial Test According to WS / T650-2019, the antibacterial and anti-inflammatory compositions containing 6-gingerol prepared in Examples 1-3 and Comparative Examples 1-7 were tested for antibacterial properties. The selected bacterial strains were Staphylococcus aureus ATCC6538 and Escherichia coli ATCC25922. The results are shown in Table 2.

[0053] The antibacterial rate is calculated using the following formula: Inhibition rate (%) = (Recovered bacteria in control group - Recovered bacteria in experimental group) / Recovered bacteria in control group × 100% Unit: CFU / mL Table 2

[0054] As can be seen from the table above, the antibacterial and anti-inflammatory compositions containing 6-gingerol prepared in Examples 1-3 of the present invention have good in vitro antibacterial effects.

[0055] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing an antibacterial and anti-inflammatory composition containing 6-gingerol, characterized in that, Includes the following steps: S1. Extraction of gingerol: Wash, dry and crush fresh ginger, add it to ethanol solution, heat and reflux to extract, filter, recover ethanol from filtrate, evaporate and concentrate to obtain crude gingerol extract; S2. Separation and purification of 6-gingerol: Crude gingerol extract was added to a deep eutectic solvent, a molecularly imprinted compound was added, the mixture was stirred and adsorbed, filtered, the molecularly imprinted compound was separated, 6-gingerol was eluted with an eluent, the eluent was collected, concentrated and dried to obtain 6-gingerol; the mass ratio of crude gingerol extract, deep eutectic solvent, and molecularly imprinted compound was 15-20:100-120:5-7; The molecularly imprinted compound is prepared as follows: gingerone is dissolved in acetonitrile, methyl methacrylate, glycidyl methacrylate and 1-vinyl-3-methylimidazolium chloride are added, and the mixture is heated and stirred. Then, ethylene glycol dimethacrylate, water and monoglyceride are added, and the mixture is stirred and mixed evenly. Initiator and porogen are added, the mixture is degassed by ultrasound, and the polymerization reaction is carried out by heating. The mixture is filtered, washed with ethanol until gingerone is no longer detectable in the eluent, and dried to obtain the molecularly imprinted compound. The mass ratio of gingerone, methyl methacrylate, glycidyl methacrylate, double bond ionic liquid, ethylene glycol dimethacrylate, monoglyceride, initiator and porogen is 0.2-0.3:0.3-0.4:0.2-0.5:0.5-1:3-4:0.02-0.03:0.01-0.03:3-5. S3. Preparation of the composition: 6-gingerol, epigallocatechin gallate, and resveratrol are mixed evenly to obtain the composition; the mass ratio of 6-gingerol, epigallocatechin gallate, and resveratrol is 7-10:2-3:1-2. S4. Preparation of an antibacterial and anti-inflammatory composition containing 6-gingerol: The composition, polyethylene glycol-poly(ε-caprolactone-polylactic acid), and vitamin E polyethylene glycol succinate are mixed, acetonitrile is added, the mixture is stirred and dissolved, and then added dropwise to hot water. The mixture is kept warm and stirred, the solvent is removed under reduced pressure, and the mixture is washed and dried to obtain an antibacterial and anti-inflammatory composition containing 6-gingerol; the mass ratio of the composition, polyethylene glycol-poly(ε-caprolactone-polylactic acid), and vitamin E polyethylene glycol succinate is 1-1.5:30-50:30-50.

2. The preparation method according to claim 1, characterized in that, The concentration of the ethanol solution in step S1 is 75-85 wt%, the solid-liquid ratio of ginger to ethanol solution is 1:5-10 g / mL, and the heating and reflux extraction time is 2-4 h.

3. The preparation method according to claim 1, characterized in that, The deep eutectic solvent in step S2 is prepared by mixing choline chloride and glucose in a molar ratio of 1:1-3, heating to 95-100°C, adding water (20-50 wt% of the total system), and stirring for 2-4 hours; the eluent is a 20-30 wt% ethanol solution.

4. The preparation method according to claim 1, characterized in that, The porogen is diethyl ether; the initiator is azobisisobutyronitrile or azobisisovalerate; the heating and stirring reaction temperature is 45-55℃ for 10-12 hours; and the heating polymerization reaction temperature is 55-65℃ for 20-28 hours.

5. The preparation method according to claim 1, characterized in that, The preparation method of polyethylene glycol-poly(ε-caprolactone)-polylactic acid in step S4 is as follows: T1. Polyethylene glycol monomethyl ether and ε-caprolactone were mixed, catalyst A was added, and the mixture was heated and stirred under inert gas protection. Dichloromethane was added for dilution, and the product was precipitated with ice-cold diethyl ether. The mixture was filtered, washed, and dried to obtain polyethylene glycol-polyε-caprolactone-OH. T2. Polyethylene glycol-poly(ε-caprolactone-OH) and polylactic acid were added to toluene, catalyst B was added, and the mixture was heated under reflux with stirring. The product was precipitated by adding ice-cold diethyl ether, filtered, washed, and dried to obtain polyethylene glycol-poly(ε-caprolactone-polylactic acid).

6. The preparation method according to claim 5, characterized in that, In step T1, the mass ratio of polyethylene glycol monomethyl ether to ε-caprolactone is 8-10:4-7, the catalyst A is stannous octoate, and the amount added is 2-4 wt% of ε-caprolactone. The heating and stirring reaction temperature is 125-135℃, and the time is 20-28 h. In step T2, the mass ratio of polyethylene glycol-polyε-caprolactone-OH to polylactic acid is 10-14:3-4, the catalyst B is p-toluenesulfonic acid, and the amount added is 1-2 wt% of polylactic acid. The heating, reflux, and stirring reaction time is 5-7 h.

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