Bacteriostatic and anti-inflammatory composition containing 6-gingerol as well as preparation method and application thereof

Abstract

The invention provides an antibacterial and anti-inflammatory composition containing 6-gingerol as well as a preparation method and application of the antibacterial and anti-inflammatory composition, and belongs to the technical field of medicines. The preparation method comprises the following steps: extracting a gingerol crude extract from ginger, separating and purifying through a molecularly imprinted compound to obtain 6-gingerol, mixing the 6-gingerol with epigallocatechin gallate and resveratrol, and embedding the mixture in polyethylene glycol-poly epsilon-caprolactone-polylactic acid / vitamin E polyethylene glycol succinate micelles to prepare the 6-gingerol-containing antibacterial and anti-inflammatory composition. The bacteriostatic and anti-inflammatory composition containing 6-gingerol prepared by the invention has better water solubility and transdermal absorption performance, greatly improves the bioavailability, is high in drug loading capacity, has the characteristic of sustained and controlled release of drugs, has better antioxidant, anti-inflammatory and antibacterial effects, is low in preparation cost, is easy to realize industrial application, and has wide application prospects.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, in particular to a bacteriostatic and anti-inflammatory composition containing 6-gingerol and a preparation method and application thereof. BACKGROUND

[0002] Rheumatoid arthritis (RA) is a chronic systemic disease with unknown etiology, mainly inflammatory synovitis. Its onset may be related to genetics, infection, sex hormones, etc. The pathology of RA arthritis mainly includes synovial lining cell proliferation, interstitial inflammatory cell infiltration, microvascular neovascularization, vascular pannus formation, and destruction of cartilage and bone tissue, etc. Rheumatic arthritis is a common acute or chronic connective tissue inflammation. The rheumatic arthritis commonly referred to is one of the main manifestations of rheumatic fever, and is characterized by joint and muscle migratory soreness, redness, pain. It is related to infection with beta hemolytic streptococcus, and factors such as cold and humidity can induce the disease. The large joints of the lower limbs such as the knee and ankle are most commonly affected. The manifestations of these two diseases are chronic and difficult to cure, and pain is a common manifestation. Because of the long-term and intractable nature of the disease, an effective and side-effect-free symptomatic treatment (analgesic) drug is very important, and an appropriate administration route is equally important.

[0003] Gingerol is a series of phenolic compounds with long alkyl side chains found in ginger, which has very good anti-inflammatory and analgesic effects. Modern pharmacology shows that they are very strong cyclooxygenase inhibitors, thus exhibiting the activity of non-steroidal anti-inflammatory drugs. Gingerol capsules are oral preparations made from low-content ginger oil resin. Because of oral administration, long-term use has the common gastrointestinal irritation of non-steroidal anti-inflammatory drugs, and even serious complications such as bleeding or perforation. In addition, 6-gingerol faces many challenges in practical application: first, its poor water solubility leads to low bioavailability; second, it is chemically unstable and easily degrades under light and heat; third, it is rapidly metabolized in the body, with a short half-life, making it difficult to maintain an effective blood drug concentration. These limitations have severely restricted the clinical application value of 6-gingerol.

[0004] Therefore, it is necessary to develop a safe and effective 6-gingerol-containing preparation that can have a good effect on arthritis, improve bioavailability, and not cause other symptoms. SUMMARY

[0005] The present application aims to provide a bacteriostatic and anti-inflammatory composition containing 6-gingerol and a preparation method and application thereof, which has good water solubility and transdermal absorption performance, greatly improves its bioavailability, has a high drug loading capacity, and has the characteristics of controlled release of the drug, has good antioxidant, anti-inflammatory, and antibacterial effects, and has low preparation cost, easy industrial application, and broad application prospects.

[0006] The technical scheme of the present application is implemented as follows: The present application provides a preparation method of a 6-gingerol-containing bacteriostatic and anti-inflammatory composition. After gingerol crude extract is extracted from fresh ginger, 6-gingerol is obtained by separation and purification of a molecular imprinting compound, mixed with epigallocatechin gallate and resveratrol, embedded in polyethylene glycol-polyepsilon caprolactone-polylactic acid / vitamin E polyethylene glycol succinate micelles, and a 6-gingerol-containing bacteriostatic and anti-inflammatory composition is prepared.

[0007] As a further improvement of the present application, the following steps are included: S1. Extraction of gingerol: wash, dry and crush fresh ginger, add an ethanol solution, heat and reflux to extract, filter, recover ethanol from the filtrate, evaporate and concentrate to prepare gingerol crude extract; S2. Separation and purification of 6-gingerol: add gingerol crude extract to a deep eutectic solvent, add a molecular imprinting compound, stir and adsorb, filter, separate the molecular imprinting compound, elute 6-gingerol with an eluent, collect the eluate, concentrate and dry to prepare 6-gingerol; S3. Preparation of the composition: mix 6-gingerol, epigallocatechin gallate and resveratrol uniformly to prepare the composition; S4. Preparation of the 6-gingerol-containing bacteriostatic and anti-inflammatory composition: mix the composition, polyethylene glycol-polyepsilon caprolactone-polylactic acid and vitamin E polyethylene glycol succinate, add acetonitrile, stir and dissolve, drop into hot water, incubate and stir, remove the solvent under reduced pressure, wash, dry and prepare the 6-gingerol-containing bacteriostatic and anti-inflammatory composition.

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

[0009] As a further improvement of the present application, 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℃, then adding water, the water content is 20-50wt% of the total system, stirring and mixing for 2-4h; the mass ratio of the crude extract of gingerol, deep eutectic solvent, and molecular imprinting compound is 15-20:100-120:5-7, the eluent is 20-30wt% ethanol solution, and the preparation method of the molecular imprinting compound is as follows: gingerone is dissolved in acetonitrile, methyl methacrylate, glycidyl methacrylate and double bond ionic liquid are added, heated and stirred for reaction, then dimethyl acrylate, water and monoglyceride are added, stirred and mixed uniformly, then initiator and pore former are added, ultrasonic degassing, heating and polymerization reaction, filtration, washing with ethanol until no gingerone is detected in the eluent, drying to obtain the molecular imprinting compound.

[0010] As a further improvement of the present application, the mass ratio of gingerone, methyl methacrylate, glycidyl methacrylate, double bond ionic liquid, dimethyl acrylate, monoglyceride, initiator and pore former 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, the double bond ionic liquid is 1-vinyl-3-methylimidazole chloride salt, the pore former is diethyl ether, the initiator is azobisisobutyronitrile or azobisisovaleronitrile, the heating and stirring reaction temperature is 45-55℃, the time is 10-12h, and the heating and polymerization reaction temperature is 55-65℃, the time is 20-28h.

[0011] As a further improvement of the present application, 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 application, the mass ratio of the composition, polyethylene glycol-poly-epsilon-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 polyethylene glycol-poly-epsilon-caprolactone-polylactic acid is as follows: T1. Mix polyethylene glycol monomethyl ether and epsilon-caprolactone, add catalyst A, heat and stir under inert gas protection, add dichloromethane for dilution, precipitate the product with ice ethanol, filter, wash and dry to obtain polyethylene glycol-poly-epsilon-caprolactone-OH; T2. Add polyethylene glycol-poly-epsilon-caprolactone-OH and polylactic acid to toluene, add catalyst B, heat and stir under reflux, precipitate the product with ice ethanol, filter, wash and dry to obtain polyethylene glycol-poly-epsilon-caprolactone-polylactic acid.

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

[0014] The present application further protects a 6-gingerol-containing bacteriostatic and anti-inflammatory composition prepared by the above preparation method.

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

[0016] The present application has the following beneficial effects: The present application extracts gingerol from ginger and separates and purifies 6-gingerol by using a molecular imprinting compound, and the extraction has high purity, and the method is simple and convenient, and the molecular imprinting compound after separation can be reused, thereby reducing the production cost.

[0017] The prepared molecular imprinting compound takes gingerone as a template molecule, and the gingerone is a structural analogue of 6-gingerol, and compared with the 6-gingerol, the gingerone is more stable in chemical properties, is commercially available, and is low in price, and is more suitable as a template for large-scale polymerization. In addition, in the polymer monomer, the monomer glycidyl methacrylate with an epoxy group is used, and the ring opening reaction of the epoxy group and the hydroxyl group on the 6-gingerol molecule is used to fix the 6-gingerol molecule on the polymer in a highly ordered and stable manner, which greatly improves the specific recognition ability of the molecular imprinting polymer to 6-gingerol. In addition, an ionic liquid monomer is also added to the monomer, and the ionic bond energy of the ionic liquid monomer is much higher than that of the traditional covalent bond monomer, and the thermal decomposition temperature of the polymer skeleton formed after polymerization is generally improved, and the polymer is not easy to swell or degrade in strong acid, strong base or strong polar solvent, so that the service life of the polymer is longer. In addition, the cation skeleton of the ionic liquid monomer has a regular spatial structure, and no polar defect site easy to adsorb impurities, which can reduce the non-specific adsorption of the polymer to the interfering molecules (such as impurity phenols and fatty acids) in the system. The ionic liquid monomer has cation (such as imidazolium) and anion groups, and the hydroxyl group of the cation side chain of the ionic liquid monomer can form a double hydrogen bond with the phenolic hydroxyl group of 6-gingerol, and the hydrophobic group of the side chain can form a hydrophobic interaction with the alkyl chain of 6-gingerol, and the multiple synergistic effect is much stronger than the single hydrogen bond of the traditional monomer (such as methacrylic acid), which can more stably fix the template molecule and form an imprinting cavity with high matching of the spatial structure and the action site of the template, so that the specific binding of the polymer to the template molecule is obviously improved.

[0018] In addition, the prepared 6-gingerol is combined with epigallocatechin gallate and resveratrol, and the combination has obvious synergistic effect in antibacterial and anti-inflammatory effects. In the antibacterial and bacteriostatic mechanism, the epigallocatechin gallate mainly destroys the integrity of the bacterial cell membrane, the resveratrol inhibits the synthesis of bacterial nucleic acid, and the 6-gingerol interferes with the bacterial quorum sensing system, so that the three attack the pathogen from different ways, and multiple antibacterial mechanisms are generated. In the anti-inflammatory mechanism, the resveratrol has good antioxidant effect, the 6-gingerol and the epigallocatechin gallate selectively inhibit the release of inflammatory factors and block nucleation, and the combination of the three can significantly reduce the level of inflammatory factors.

[0019] The application encapsulates the active composition in a core-shell-crown structure micelle constructed by polyethylene glycol-poly epsilon caprolactone-polylactic acid (PEG-PCL-PLA) triblock polymer, to form a new type of nano drug delivery system. The micelle has performance significantly better than traditional polyethylene glycol-poly epsilon caprolactone (PEG-PCL) micelles: the inner core polylactic acid (PLA) domain enhances the affinity with 6-gingerol through hydrophobic interaction and hydrogen bonding, significantly improving the drug loading capacity; the middle poly epsilon caprolactone (PCL) layer can regulate the drug release behavior, realizing gradient drug release; the outer crown layer of polyethylene glycol (PEG) chain provides spatial stability and stealth effect, prolonging the in vivo circulation time. In addition, the micelle system significantly improves the water solubility and transdermal absorption performance of 6-gingerol, thereby greatly improving its bioavailability, providing a more efficient delivery strategy for the application of 6-gingerol in antibacterial and anti-inflammatory aspects.

[0020] The antibacterial and anti-inflammatory composition containing 6-gingerol prepared by the application has good water solubility and transdermal absorption performance, greatly improves the bioavailability, has high drug loading capacity, and has the characteristics of sustained and controlled release of drugs, has good antioxidant, anti-inflammatory and antibacterial effects, and has low preparation cost, easy to realize industrial application, and has broad application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiment or prior art description. Obviously, the drawings in the following description are only some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained without creative labor.

[0022] Figure 1 TEM image of the water dispersion of the antibacterial and anti-inflammatory composition containing 6-gingerol prepared in Example 1. DETAILED DESCRIPTION

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

[0024] Preparation Example 1: molecularly imprinted compound The preparation method is as follows: Preparation Example 1 Molecularly imprinted compound

[0025] Preparation Example 2 Molecularly imprinted compound The preparation method is as follows: Preparation Example 1 Molecularly imprinted compound

[0026] Preparation Example 3 Molecularly imprinted compound The preparation method is as follows: Preparation Example 1 Molecularly imprinted compound

[0027] Comparative Preparation Example 1 The difference compared with Preparation Example 3 is that no glycidyl methacrylate is 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 10 g of polyethylene glycol monomethyl ether and 7 g of ε-caprolactone, add 4 wt% of stannous octoate based on the mass of ε-caprolactone, heat to 135°C under nitrogen protection, stir for 28 h, dilute with 50 mL of dichloromethane, precipitate the product by adding 200 mL of ice ethanol for 1 h, filter, wash, and dry to obtain polyethylene glycol-poly-ε-caprolactone-OH; T2. Add 14 g of polyethylene glycol-poly-ε-caprolactone-OH and 4 g of polylactic acid to 200 mL of toluene, add 2 wt% of p-toluenesulfonic acid based on the mass of polylactic acid, heat to reflux and stir for 7 h, remove the solvent from the product under reduced pressure, precipitate by adding 100 mL of ice ethanol, 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 10 g of polyethylene glycol monomethyl ether and 7 g of ε-caprolactone, add 4 wt% of stannous octoate based on the mass of ε-caprolactone, heat to 135°C under nitrogen protection, stir for 28 h, dilute with 50 mL of dichloromethane, precipitate the product by adding 200 mL of ice ethanol for 1 h, filter, wash, and dry to obtain polyethylene glycol-poly-ε-caprolactone-OH; T2. Add 14 g of polyethylene glycol-poly-ε-caprolactone-OH and 4 g of polylactic acid to 200 mL of toluene, add 2 wt% of p-toluenesulfonic acid based on the mass of polylactic acid, heat to reflux and stir for 7 h, remove the solvent from the product under reduced pressure, precipitate by adding 100 mL of ice ethanol, filter, wash, and dry to obtain polyethylene glycol-poly-ε-caprolactone-polylactic acid. Example 1

[0034] The present embodiment provides a preparation method of a bacteriostatic and anti-inflammatory composition containing 6-gingerol, comprising the following steps: S1. Extraction of gingerol: wash, dry, and crush 10 g of fresh ginger, add 50 mL of 85 wt% ethanol solution, heat to reflux and extract for 2 h, filter, recover ethanol from the filtrate, and evaporate and concentrate to obtain crude gingerol extract; S2. Separation and purification of 6-gingerol: add 15 g of crude gingerol extract to 100 g of deep eutectic solvent, add 5 g of molecularly imprinted compound prepared in Preparation Example 1, stir for 30 min for adsorption, filter, separate the molecularly imprinted compound, elute 6-gingerol with 20 wt% ethanol solution, collect the eluate, and concentrate and dry to obtain 6-gingerol; The deep eutectic solvent is prepared by mixing choline chloride and glucose at a molar ratio of 1:1, heating to 100°C, adding water in an amount of 20 wt% of the total system, and stirring and mixing for 2 h; S3. Preparation of the composition: 7 mg of 6-gingerol, 2 mg of epigallocatechin gallate, and 1 mg of resveratrol were stirred and mixed for 10 min to prepare the composition; S4. Preparation of the bacteriostatic and anti-inflammatory composition containing 6-gingerol: 10 mg of the composition, 0.3 g of the polyethylene glycol-poly-epsilon-caprolactone-polylactic acid prepared in Preparation Example 4, and 0.3 g of vitamin E polyethylene glycol succinate were mixed, 1 mL of acetonitrile was added, stirred and dissolved, 10 mL of hot water at 45°C was added dropwise, and stirred for 20 min. The solvent was removed under reduced pressure, washed, and dried to prepare the bacteriostatic and anti-inflammatory composition containing 6-gingerol. The composition was dispersed in water to prepare a solution having a concentration of 1 mg / mL, and the morphological characteristics were observed using TEM after staining with phosphomolybdic acid, and the results are shown in FIG. 2. As shown in the figure, the particles of the preparation were uniform and spherical, and no significant aggregation of the particles was observed. Figure 1 Example 2

[0035] The present example provides a method for preparing a bacteriostatic and anti-inflammatory composition containing 6-gingerol, comprising the following steps: S1. Extraction of gingerol: 10 g of fresh ginger was washed, dried, and pulverized, 100 mL of 75 wt% ethanol solution was added, heated and refluxed for 4 h, filtered, the ethanol was recovered from the filtrate, and evaporated and concentrated to prepare a crude gingerol extract; S2. Isolation and purification of 6-gingerol: 20 g of the crude gingerol extract was added to 120 g of a deep eutectic solvent, 7 g of the molecularly imprinted compound prepared in Preparation Example 2 was added, stirred and adsorbed for 30 min, filtered, and the molecularly imprinted compound was separated. 6-gingerol was eluted using a 30 wt% ethanol solution, the eluate was collected, concentrated and dried to prepare 6-gingerol. The deep eutectic solvent was prepared by mixing choline chloride and glucose at a molar ratio of 1:3, heating to 95°C, adding water to a content of 50 wt% of the total system, and stirring and mixing for 4 h; 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 prepare the composition; S4. Preparation of the bacteriostatic and anti-inflammatory composition containing 6-gingerol: 15 mg of the composition, 0.5 g of the polyethylene glycol-poly-epsilon-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, stirred and dissolved, 10 mL of hot water at 45°C was added dropwise, and stirred for 20 min. The solvent was removed under reduced pressure, washed, and dried to prepare the bacteriostatic and anti-inflammatory composition containing 6-gingerol. Example 3

[0036] The present example provides a method for preparing a bacteriostatic and anti-inflammatory composition containing 6-gingerol, comprising the following steps: ​S1. Extraction of gingerol: 10 g of fresh ginger was washed, dried, and crushed, and then added to 75 mL of 80 wt% ethanol solution. The mixture was heated to reflux for 3 h, filtered, and the filtrate was concentrated by evaporation. The crude gingerol extract was obtained. S2. Isolation and purification of 6-gingerol: 17 g of the crude gingerol extract was added to 110 g of deep eutectic solvent, and 6 g of the molecularly imprinted compound prepared in Preparation Example 3 was added. The mixture was stirred for 30 min to adsorb the molecularly imprinted compound, filtered, and the molecularly imprinted compound was separated. The 6-gingerol was eluted with 25 wt% ethanol solution, and the eluate was collected, concentrated, and dried to obtain 6-gingerol. The deep eutectic solvent was prepared by mixing choline chloride and glucose at a molar ratio of 1:2, heating to 95°C, adding water to a content of 35 wt% of the total system, and stirring for 3 h. 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 the antibacterial and anti-inflammatory composition containing 6-gingerol: 12 mg of the composition, 0.4 g of the polyethylene glycol-polyε-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 and dissolved. The mixture was added dropwise to 10 mL of hot water at 45°C, and stirred for 20 min. The solvent was removed under reduced pressure, washed, and dried to obtain the 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 in Comparative Preparation Example 1.

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

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

[0040] The details are as follows: S3. Preparation of the composition: 2.5 mg of epigallocatechin gallate and 1 mg of 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] The details are 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 compared with Example 3 is that resveratrol is not added in step S3.

[0044] The details are as follows: S3. Preparation of the composition: 8.5 mg of 6-gingerol, 2.5 mg of epigallocatechin gallate, and 1 mg of resveratrol are stirred and mixed for 10 min to prepare the composition, which is the antibacterial and anti-inflammatory composition containing 6-gingerol.

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

[0046] The details are as follows: S1. Extraction of gingerols: 10 g of fresh ginger is washed, dried, and crushed, and then added to 75 mL of an 80 wt% ethanol solution, heated to reflux for 3 h, filtered, and the filtrate is recovered by evaporation and concentration to prepare a gingerol crude extract; S2. Isolation and purification of 6-gingerol: 17 g of the gingerol crude extract is added to 110 g of a deep eutectic solvent, and 56 g of the molecularly imprinted compound prepared in Preparation Example 3 is added, stirred and adsorbed for 30 min, filtered, and the molecularly imprinted compound is separated, and 6-gingerol is eluted using a 25 wt% ethanol solution, and the eluate is collected, concentrated, and dried to prepare 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 in an amount of 35 wt% of the total system, and stirring and mixing for 3 h; S3. Preparation of the composition: 8.5 mg of 6-gingerol, 2.5 mg of epigallocatechin gallate, and 1 mg of resveratrol are stirred and mixed for 10 min to prepare the composition, which is the antibacterial and anti-inflammatory composition containing 6-gingerol.

[0047] Comparative Example 7 The difference compared with Example 3 is that the polyethylene glycol-poly-ε-caprolactone-polylactic acid is replaced by an equal amount of polyethylene glycol-poly-ε-caprolactone.

[0048] Test Example 1: Encapsulation efficiency and drug loading test 100 μL of the antibacterial and anti-inflammatory composition containing 6-gingerol in Examples 1-3 and Comparative Example 7 (the solution after step S4 is added to 45°C hot water without removing the solvent under reduced pressure, washed, and dried) is taken in a 10 mL volumetric flask, and methanol is added to break the emulsion and make up to volume; a certain amount of the antibacterial and anti-inflammatory composition containing 6-gingerol is taken, filtered using a 0.22 μm microporous filter, and 100 μL of the filtrate is taken and made up to volume in the same way. Both solutions are filtered using a 0.22 μm microporous filter, and the concentration of 6-gingerol in the solution is analyzed by liquid chromatography. The calculation formulas for the encapsulation efficiency (EE) and drug loading (DL) are as follows: EE (%) = C2 / C1 x 100% DL (%) = (C2 x V) / W x 100% In the formula, C1 represents the concentration of 6-gingerol in the antibacterial and anti-inflammatory composition solution containing 6-gingerol after filtering through a microporous filter, C2 represents the total concentration of 6-gingerol measured after the preparation is directly demulsified, V represents the total volume of the preparation formula, and W represents the dosage of 6-gingerol in the preparation formula.

[0049] The results are shown in Table 1.

[0050] Table 1

[0051] As can be seen from the above table, the antibacterial and anti-inflammatory composition solution containing 6-gingerol prepared in Examples 1-3 has a high encapsulation rate of 6-gingerol and a high drug loading.

[0052] Test Example 2 Anti-inflammatory Test Mouse macrophage Raw264.7 cells were inoculated into a 96-well cell plate at 5 x 10 4 cells / well, and DMEM medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin was used; the antibacterial and anti-inflammatory composition containing 6-gingerol prepared in Examples 1-3 and Comparative Examples 1-7 was added to the medium (sample: medium mass ratio of 1:15), and pure water was used as a blank group instead of the sample, and then mixed and added to each cell well at 1 mL per well, 3 parallel groups per group, 1 μg / mL lipopolysaccharide was added for incubation for 48 h, and the supernatant was aspirated, and the concentrations of IL-6 and TNF-α were determined using a mouse IL-6 and TNF-α ELISA kit. The concentration reduction rate (%) of TNF-α and IL-6 was calculated using the following formula: Reduction rate (%) = (blank group concentration - test sample group concentration) / blank group concentration x 100%.

[0053] The results are shown in Table 2.

[0054] Table 2

[0055] As can be seen from the above table, the antibacterial and anti-inflammatory composition containing 6-gingerol prepared in Examples 1-3 has good in vitro anti-inflammatory effect.

[0056] Test Example 3 Antibacterial Test The antibacterial test was performed on the antibacterial and anti-inflammatory composition containing 6-gingerol prepared in Examples 1-3 and Comparative Examples 1-7 according to WS / T650-2019, and the bacteria selected were Staphylococcus aureus ATCC6538 and Escherichia coli ATCC25922, and the results are shown in Table 3.

[0057] The bacteriostatic rate was calculated according to the following formula: Bacteriostatic rate (%) = (amount of bacteria recovered in the control group - amount of bacteria recovered in the test group) / amount of bacteria recovered in the control group x 100% Unit: CFU / mL Table 3

[0058] As shown in the above table, the 6-gingerol-containing bacteriostatic and anti-inflammatory composition prepared in Examples 1-3 has good in-vitro bacteriostatic effect.

[0059] The above description is merely preferred embodiments of the present application but not to confine the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A process for the preparation of a bacteriostatic anti-inflammatory composition containing 6-gingerol, characterized in that, After extracting gingerol crude extract from fresh ginger, 6-gingerol is separated and purified by a molecular imprinting compound, mixed with epigallocatechin gallate and resveratrol, embedded in polyethylene glycol-polyepsilon caprolactone-polylactic acid / vitamin E polyethylene glycol succinate micelles to prepare an antibacterial and anti-inflammatory composition containing 6-gingerol.

2. The production method according to claim 1, characterized by, The method comprises the following steps: S1. Extraction of gingerol: wash, dry and crush fresh ginger, add ethanol solution, heat and reflux to extract, filter, recover ethanol from the filtrate, evaporate and concentrate to prepare gingerol crude extract; S2. Separation and purification of 6-gingerol: add gingerol crude extract to a deep eutectic solvent, add a molecular imprinting compound, stir and adsorb, filter, separate the molecular imprinting compound, elute 6-gingerol with an eluent, collect the eluate, concentrate and dry to prepare 6-gingerol; S3. Preparation of the composition: mix 6-gingerol, epigallocatechin gallate and resveratrol uniformly to prepare the composition; S4. Preparation of the antibacterial and anti-inflammatory composition containing 6-gingerol: mix the composition, polyethylene glycol-polyepsilon caprolactone-polylactic acid and vitamin E polyethylene glycol succinate, add acetonitrile, stir and dissolve, drop into hot water, keep warm and stir, remove the solvent under reduced pressure, wash and dry to prepare the antibacterial and anti-inflammatory composition containing 6-gingerol.

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

4. The preparation method according to claim 2, characterized in that, In step S2, the deep eutectic solvent is prepared by mixing choline chloride and glucose at a molar ratio of 1:1-3, heating to 95-100℃, adding water (water content is 20-50wt% of the total system), and stirring for 2-4h; the mass ratio of the gingerol crude extract, the deep eutectic solvent and the molecular imprinting compound is 15-20:100-120:5-7, the eluent is 20-30wt% ethanol solution, and the molecular imprinting compound is prepared as follows: gingerone is dissolved in acetonitrile, methyl methacrylate, glycidyl methacrylate and double-bond ionic liquid are added, heated and stirred for reaction, then dimethyl glycol acrylate, water and monoglyceride are added, stirred and mixed uniformly, ultrasonic degassing, heated and polymerized, filtered, washed with ethanol until no gingerone is detected in the eluate, and dried to prepare the molecular imprinting compound.

5. The preparation method according to claim 4, characterized in that, The mass ratio of the gingerone, methyl methacrylate, glycidyl methacrylate, double-bond ionic liquid, dimethyl glycol acrylate, monoglyceride, initiator and pore-forming agent 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, the double-bond ionic liquid is 1-vinyl-3-methylimidazole chloride salt, the pore-forming agent is diethyl ether, the initiator is azobisisobutyronitrile or azobisisovaleronitrile, the temperature for heat stirring reaction is 45-55℃, and the time is 10-12h, the temperature for heating polymerization is 55-65℃, and the time is 20-28h.

6. The preparation method according to claim 2, characterized in that, The mass ratio of the 6-gingerol, epigallocatechin gallate and resveratrol in step S3 is 7-10:2-3:1-2.

7. The preparation method according to claim 2, characterized in that, The mass ratio of the composition, polyethylene glycol-poly-epsilon-caprolactone-polylactic acid and vitamin E polyethylene glycol succinate in step S4 is 1-1.5:30-50:30-50, and the polyethylene glycol-poly-epsilon-caprolactone-polylactic acid is prepared as follows: T1. Polyethylene glycol monomethyl ether and epsilon-caprolactone are mixed, catalyst A is added, and the mixture is heated and stirred under the protection of inert gas, dichloromethane is added for dilution, the product is precipitated in ice ethyl ether, filtered, washed and dried to obtain polyethylene glycol-poly-epsilon-caprolactone-OH; T2. Polyethylene glycol-poly-epsilon-caprolactone-OH and polylactic acid are added to toluene, catalyst B is added, and the mixture is heated and stirred under reflux, the product is precipitated in ice ethyl ether, filtered, washed and dried to obtain polyethylene glycol-poly-epsilon-caprolactone-polylactic acid.

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

9. A bacteriostatic and anti-inflammatory composition containing 6-gingerol prepared by the preparation method of any one of claims 1-8.

10. Use of the bacteriostatic and anti-inflammatory composition containing 6-gingerol of claim 9 in the preparation of a drug for treating arthritis and an antibacterial drug.

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