A targeted anti-inflammatory and antibacterial oral care compound and its preparation process

Abstract

The present invention relates to the technical field of oral care products, and specifically to a targeted anti-inflammatory and sterilizing oral care complex and a process for preparing the same. The liposome complex is a liposome co-loaded with a curcumin-luteolin metal zinc complex wrapped by a betaine surfactant, which can be used for the preparation of oral care products such as mouthwash, toothpaste, gel, etc., and the luteolin-zinc metal complex and curcumin are respectively encapsulated in the hydrophilic center and the hydrophobic bilayer of the liposome, and then the liposome surface is modified by a betaine surfactant, and the liposome surface is positively charged, which can be targeted to the gingival inflammation site. This technical solution solves the technical problem that the oral care products used to alleviate and treat gingivitis in the prior art are not targeted and have limited effects, and can penetrate deeply into the inflammation site, release curcumin and luteolin metal zinc complex, synergistically fight bacteria, remove free radicals, and achieve rapid anti-inflammatory effects.

Description

Technical Field

[0001] The invention relates to the technical field of oral care products, and in particular to an oral care compound with targeted anti-inflammatory and sterilization properties and a preparation method thereof. Background Art

[0002] Gingivitis is the early manifestation of oral inflammation. It is mainly caused by the accumulation of dental biofilm and is an inflammation in specific areas. It is concentrated in the edge of the gingival tissue or the gingival papilla, and does not involve the underlying bone. When suffering from gingivitis, the local gingival tissue edema causes the gingival margin to thicken and no longer adhere to the tooth surface. The gingival papilla becomes rounded and enlarged, accompanied by symptoms of bleeding when brushing teeth. Gingivitis is a persistent chronic disease that poses a continuous risk of periodontitis. Although not all patients with gingivitis will develop periodontitis, the management of gingivitis is considered to be both a primary prevention strategy for periodontitis control and a secondary prevention strategy for recurrent periodontitis. Therefore, effective prevention and treatment of gingivitis has a positive significance for maintaining the health of gingival tissue.

[0003] Prevention and treatment of gingivitis, patients with strong oral care awareness will go to the hospital regularly for professional staff to remove deposited tartar and plaque (commonly known as cleaning teeth), and then use antibacterial agents. But the frequency of preventive cleaning teeth is recommended to be once a year or once every six months, which is too frequent and will cause damage to teeth and gums. In daily prevention, oral care products (such as mouthwash, toothpaste) containing antibacterial agents have a certain effect on the prevention of gingivitis, but because gingivitis only occurs locally, long-term and large-scale use of oral care products containing antibacterial agents not only kills oral harmful bacteria, but also kills indiscriminately the beneficial bacteria in the oral cavity, which is very likely to cause dysbacteriosis and lead to other oral problems. Therefore, targeted gingivitis medication is one of the best choices. How to prepare a kind of oral care products with strong targeting for the prevention and treatment of gingivitis, and give full play to the anti-gingivitis effect of the functional ingredients, is a problem to be solved in the art. Summary of the invention

[0004] The purpose of the present invention is to provide a targeted anti-inflammatory and antibacterial oral care compound to solve the technical problems in the prior art of oral care products for relieving and treating gingivitis that the targeting is not strong and the effect is limited.

[0005] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0006] A liposome complex for oral care with targeted anti-inflammatory and sterilization properties, wherein the raw materials thereof include, by weight, 0.1-1 part of curcumin, 0.2-1 part of luteolin-zinc metal complex, 2-10 parts of liposomes, and 0.15-0.25 parts of betaine-type surfactant.

[0007] Furthermore, the luteolin-zinc metal complex is obtained by thermal reflux reaction of luteolin and zinc salt in a molar ratio of 2:1;

[0008] Preferably, the conditions for the thermal reflux reaction are: at a pH value of 4 and a reaction temperature of 60° C., the thermal reflux reaction is carried out for 4 hours, and the reaction solvent is anhydrous ethanol.

[0009] Furthermore, the raw materials of the liposome are cholesterol, soybean phosphatidylcholine and DMG-PEG 2000 in a molar ratio of 40:12:1.5;

[0010] Preferably, the raw materials of liposomes are dispersed in chloroform, and the chloroform is evaporated to form a film, and the film is hydrated and freeze-dried to obtain liposomes.

[0011] Furthermore, the betaine-type surfactant includes lauryl amide propyl betaine and / or coconut oil hydroxysulfobetaine.

[0012] The technical solution also provides a method for preparing a liposome complex for oral care with targeted anti-inflammatory and sterilization properties, which is characterized by comprising the following steps performed in sequence:

[0013] S1: preparing a solution A containing 0.2% to 1% by mass of luteolin-zinc metal complex, and a solution B containing 0.1% to 1% by mass of curcumin and 2% to 10% by mass of liposome;

[0014] S2: In an environment of 50-60°C, solution B is added dropwise at a rate of 30-60 drops / min to solution A which is stirred at 200-500 r / min. After the addition is completed, stirring is continued for 1-3 hours to obtain a mixed emulsion;

[0015] S3: adding a betaine-type surfactant to the mixed emulsion, stirring and filtering; and then freeze-drying to obtain a freeze-dried liposome complex.

[0016] The technical solution also provides an application of a targeted anti-inflammatory and antibacterial oral care liposome complex in the preparation of oral care products, wherein the oral care products include mouthwash, toothpaste and gel.

[0017] Furthermore, in parts by weight, the formula of the mouthwash is: 1-5 parts of freeze-dried liposome complex, 0.2-1.5 parts of sweetener, 4-20 parts of moisturizer, 0.1-0.8 parts of fragrance, 0.1-0.8 parts of preservative, and the balance is water; the formula of the gel or toothpaste is: 4-11 parts of freeze-dried liposome complex, 5-65 parts of moisturizer, 10-40 parts of abrasive, 0.3-2 parts of thickener, 0.1-2 parts of sweetener, 0.1-1.8 parts of preservative, 0.5-1.5 parts of fragrance, and the balance is water.

[0018] Further, the sweetener includes at least one or more of saccharin sodium, xylitol, sucralose, steviol glycosides, and aspartame;

[0019] The moisturizing agent includes at least one of sorbitol, glycerol, and polyethylene glycol;

[0020] The friction agent includes at least one of silicon dioxide and calcium hydrogen phosphate;

[0021] The thickener comprises at least one of sodium carboxymethyl cellulose, hydroxyethyl cellulose, guar gum, xanthan gum, carrageenan and carbomer;

[0022] The preservative includes at least one of parabens, benzyl alcohol and phenoxyethanol.

[0023] Furthermore, the toothpaste is prepared by the following method: the freeze-dried liposome complex is re-dissolved in water and mixed with polyethylene glycol in the moisturizer, and then mixed with the flavoring agent, the remaining moisturizers in the formula, and the remaining water in the formula to obtain a mixture A; an abrasive, a preservative, and a surfactant are added to the mixture A, and stirred evenly to obtain a mixture B; a fragrance is added to the mixture B, and the mixture is homogenized and stirred again, and vacuum degassing is performed to obtain the toothpaste.

[0024] The technical scheme also provides an oral care product of a liposome complex for oral care with targeted anti-inflammatory and antibacterial properties, wherein the oral care product contains a moisturizer, and the moisturizer includes polyethylene glycol; the liposome complex is mixed with polyethylene glycol after redissolution, and then mixed with the remaining raw materials of the oral care product to prepare the oral care product.

[0025] The technical principle and beneficial effects of this technical solution are:

[0026] This technical solution adopts the method of adding betaine-type surfactants, which can not only modify the surface charge of liposomes, but also serve as freeze-drying protective agents when the liposome complex is freeze-dried, so that after the freeze-dried liposomes are rehydrated, their particle size and ζ potential are stable, and the drug retention rate is more than 80%. The double-layer drug-loaded liposomes wrapped with betaine surfactants obtained after freeze-drying and rehydration have luteolin zinc complexes in the hydrophilic layer and curcumin adsorbed in the hydrophobic layer. Under the acidic conditions (pH=4.5-6) ​​of the inflammatory site where oral biofilms are gathered, the carboxylate radicals in the betaine-type surfactants can be protonated, increasing the positive charge on the surface of the liposomes, improving the adsorption of the liposomes and the bacterial biofilms (negatively charged on the bacterial surface) at the inflammatory site, and realizing the active targeting effect of the complex.

[0027] Curcumin and Clerodendrum oxyphylla extract are both widely present natural flavonoid compounds. Curcumin is the most medically valuable part of turmeric extract, with strong anti-inflammatory and antioxidant abilities. However, curcumin has low water solubility, poor chemical stability, and low oral bioavailability (animal experiments found that the oral absorption rate was only 1%), which greatly limits the utilization of curcumin. Although there are literature reports that preparing curcumin into an emulsion can improve its stability, in a system with a pH lower than 7, curcumin can only stably exist at 85% after one month, while in a system with a pH greater than 7, the content of curcumin decreases rapidly, and the color changes, and crystals precipitate. The pH values ​​of most oral care products currently on the market (such as toothpaste, mouthwash, etc.) are neutral to alkaline, and the pH of calcium carbonate-based toothpaste is even mostly above 8. Curcumin is directly added to oral care products as an active ingredient, and a large amount of attenuation cannot guarantee the efficacy, and even precipitation affects the stability of the product. Luteolin has received extensive attention and research for its strong antioxidant activity (Luteolin has the strongest antioxidant activity compared with quercetin and capsaicin under equimolar concentration conditions). However, the water solubility of luteolin is poor at room temperature, which limits its clinical application. Some literature has studied the formation of luteolin metal complexes with metal zinc (II) or copper (II), which not only increases the solubility and stability of luteolin, but also its metal complex has much better antibacterial activity against a variety of bacteria than the parent (luteolin). However, like the use of a large number of antibacterial agents, there are still problems such as low specificity and low efficacy. This technical solution combines luteolin and curcumin in the form of positively charged liposomes to enhance the therapeutic effects of the two active ingredients. Luteolin and curcumin can not only scavenge inflammatory free radicals at the site of inflammation, but also synergistically fight bacteria and kill bacteria. The curcumin and luteolin zinc complex liposomes wrapped with betaine-type surfactants can actively target the biofilm in the oral inflammation site and achieve highly effective anti-inflammatory and antibacterial effects. The freeze-dried liposome complex is added to mouthwash, toothpaste, and gel as an anti-inflammatory and antibacterial ingredient to obtain an oral care product with high efficiency in anti-inflammatory and antibacterial properties.

[0028] In summary, luteolin-zinc metal complex and curcumin are entrapped in the hydrophilic center and hydrophobic bilayer of liposomes, respectively, and then the surface of liposomes is modified by betaine-type surfactants to form liposomes co-loaded with positively charged curcumin-luteolin metal zinc complex, which can target the site of gingival inflammation, penetrate deeply into the inflammatory site, and release curcumin and luteolin metal zinc complex. Curcumin and luteolin metal zinc complex can synergistically enhance antibacterial effects, scavenge free radicals, and achieve rapid anti-inflammatory effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a typical microscopic image of the liposome complex of Example 2.

[0030] Figure 2 This is an evaluation of the effects of the combination of curcumin and luteolin-zinc monomers of Example 3.

[0031] Figure 3 These are the test results of the free radical scavenging effects of different liposome complexes in Example 3.

[0032] Figure 4 This is a photo of the typical appearance of the toothpaste containing the liposome complex of Example 4 after the accelerated test. DETAILED DESCRIPTION

[0033] The present invention is further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following examples and experimental examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used can be obtained from commercial sources.

[0034] The overall situation of this technical solution is as follows:

[0035] The ingredients and dosage of the oral care complex (liposome complex) for targeted anti-inflammatory and sterilization are as follows, by weight: 0.1-1 part of curcumin (CAS: 458-37-7), 2-10 parts of liposomes, 0.2-1 part of luteolin-zinc metal complex, and 0.15-0.25 part of betaine-type surfactant.

[0036] Among them, liposomes are cholesterol, soybean phosphatidylcholine (CAS: 97281-47-5) and DMG-PEG 2000 (1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000, CAS: 160743-62-4) prepared by thin film dispersion method, with a molar ratio of 40:12:1.5, that is, all raw materials (a total of 53.5mmol of three substances) are weighed and dissolved in 30mL of chloroform, and slowly blown dry with nitrogen to form a layer of uniformly dispersed film at the bottom of the bottle. After rehydration, the liposomes are hydrated. The rehydrated liposomes were subjected to conventional freeze-drying treatment to obtain dry powder liposomes. The liposomes in the above liposome complex formula are measured in the state of freeze-dried powder.

[0037] The luteolin-zinc metal complex was prepared by the following method: 2 mmol of luteolin (CAS: 491-70-3) was weighed into a round-bottom flask filled with 100 mL of anhydrous ethanol, 1 mmol of zinc salt (zinc chloride was used in this experiment) was added, the pH was adjusted to 4 with hydrochloric acid, and the mixture was refluxed and stirred at about 60°C for 4 hours. The reflux reaction product was purified and evaporated to dryness by conventional methods to obtain a solid luteolin-zinc metal complex.

[0038] Betaine-type surfactants are composed of a quaternary ammonium salt type cation part and a carboxylate type anion part. Lauramide propyl betaine (CAS: 4292-10-8), cocoamidopropyl hydroxysulfonyl betaine (CAS: 68139-30-0) and the like can be selected.

[0039] Process steps:

[0040] (1) adding luteolin-zinc metal complex into deionized water to prepare solution A, wherein the mass percentage of the complex is 0.2%-1%;

[0041] (2) Adding curcumin and liposomes into an anhydrous ethanol solution to prepare solution B, wherein the mass percentage of curcumin is 0.1%-1%, and the mass percentage of liposomes is 2%-10%.

[0042] (3) Under 50-60°C water bath conditions (preferably 60°C), magnetically stir solution A at 200-500 r / min (preferably 300 r / min), and slowly drip solution B into solution A at a rate of 30-60 drops per minute (preferably 50 drops per minute). Solution A and solution B are dripped and mixed in equal volumes. After dripping, the temperature is maintained and stirring is continued for 1-3 h (preferably 2 h) to allow the two solutions to fully blend.

[0043] (4) Under room temperature, add the formulated amount of betaine surfactant to the above mixed solution, stir thoroughly, filter with a 0.22 μm microporous filter membrane, and freeze-dry in a conventional manner. The optional freeze-drying procedure is: use a freeze dryer for conventional freeze drying for 24 hours (constant temperature freeze drying, cold trap temperature -45°C).

[0044] The freeze-dried liposome complex is added into mouthwash, toothpaste and gel as an anti-inflammatory and antibacterial ingredient to obtain an oral care product with high efficiency of anti-inflammatory and antibacterial properties.

[0045] Among them, for mouthwash products, the raw materials include, by weight: 1-5 parts of freeze-dried liposome complex, 0.2-1.5 parts of sweetener, 4-20 parts of moisturizer, 0.1-0.8 parts of fragrance, 0.1-0.8 parts of preservative, and the balance is water.

[0046] For gel or toothpaste products, the raw materials include, by weight: 4-11 parts of freeze-dried liposome complex, 5-65 parts of moisturizer, 10-40 parts of abrasive, 0.3-2 parts of thickener, 0.1-2 parts of sweetener, 0.1-1.8 parts of preservative, 0.5-1.5 parts of fragrance, and the balance is water.

[0047] The sweetener is selected from a combination of one or more of saccharin sodium, xylitol, sucralose, stevioside, and aspartame. In each embodiment and comparative example of the present invention, saccharin sodium is selected as the sweetener, and other sweeteners or combinations thereof have substantially the same effect as saccharin sodium.

[0048] The moisturizer is selected from a combination of one or more of sorbitol, glycerol, and polyethylene glycol. In each embodiment and comparative example of the present invention, the moisturizer is a combination of sorbitol and polyethylene glycol, and the addition mass ratio is 25:3. Other moisturizers or combinations thereof have substantially the same effect as the combination.

[0049] The friction agent is selected from one of silicon dioxide and calcium hydrogen phosphate or a combination of the two. In each embodiment and comparative example of the present invention, the friction agent is selected from silicon dioxide, calcium hydrogen phosphate or a combination of the two (silicon dioxide + calcium hydrogen phosphate), and the effect is basically the same as that of silicon dioxide.

[0050] The thickener is selected from one or more combinations of sodium carboxymethyl cellulose, hydroxyethyl cellulose, guar gum, xanthan gum, carrageenan, and carbomer. In each embodiment and comparative example of the present invention, a combination of sodium carboxymethyl cellulose and carrageenan is selected, and the added mass ratio of sodium carboxymethyl cellulose and carrageenan is 4:1. The effects of other combinations are basically the same as those of the combination.

[0051] Fragrances are fragrances that adjust the aroma of products.

[0052] The preservative is selected from a combination of one or more of parabens, benzyl alcohol, and phenoxyethanol. In each embodiment and comparative example of the present invention, benzyl alcohol is selected as the preservative, and the effect of selecting other preservatives or combinations is substantially the same as that of benzyl alcohol.

[0053] Example 1: Graded Inhibitory Concentration (FIC) of Luteolin-Zinc and Curcumin in Liposome Complexes

[0054] The agar dilution method was used to dissolve luteolin-zinc and curcumin (pre-dissolved with a small amount of 1% sodium hydroxide) in agar medium, and then the test bacteria were inoculated to determine the minimum inhibitory concentration (MIC) of the antibacterial (inhibitory) substances to inhibit the growth of the test bacteria.

[0055] The FIC index (MIC A药联用 / MIC A药单用 +MIC B药联用 / MIC B药单用The FIC index (Fractional Inhibitory Concentration Index) is an indicator used to evaluate the synergistic, additive, irrelevant or antagonistic effects of two or more antimicrobial drugs when used in combination. A药联用 MIC A药单用 MIC B药联用 MIC B药单用 All of them are measured by conventional means in the prior art, and will not be described in detail here. A药联用 It refers to the minimum inhibitory concentration of drug A against a specific microorganism when used in combination with drug B. Similarly, MIC B药联用 , which can be measured by conventional chessboard dilution method or time-kill curve method. When the FIC index is ≤0.75, it indicates that the two have a synergistic effect; when 0.75 < FIC index ≤1, it indicates that the two have an additive effect; when 1 < FIC index ≤2, it indicates that the two are unrelated; when the FIC index is >2, the two have an antagonistic effect. The MIC values ​​of luteolin-zinc and curcumin and the FIC index after the combination of the two are shown in Table 1. According to the test results, the FIC values ​​of luteolin-zinc and curcumin against Streptococcus mutans, Porphyromonas gingivalis, Candida albicans and the combination of the two were obtained, and referring to the judgment criteria of the FIC index, the FIC index of each group was less than 0.75, indicating that luteolin-zinc and curcumin have a synergistic effect on Streptococcus mutans, Porphyromonas gingivalis and Candida albicans. In summary, the experimental results show that the combination of luteolin-zinc and curcumin has a synergistic effect on Streptococcus mutans, Porphyromonas gingivalis and Candida albicans.

[0056] Table 1: Antimicrobial susceptibility test of luteolin-zinc and curcumin against Streptococcus mutans

[0057]

[0058] Example 2: Stability study of liposome complexes

[0059] Tests 1 to 5 are representative examples of the preparation of curcumin-luteolin metal zinc complex co-loaded liposomes encapsulated by lauramidopropyl betaine for targeted anti-inflammatory and antibacterial effects of the present invention. The composition and content information are shown in Table 2 (the preparation process uses the aforementioned optimal process). The liposomes obtained have a light yellow translucent colloidal solution with obvious Tyndall phenomenon; the microscopic morphology is spherical (see the attached typical image). Figure 1 ), the particle size is uniform, about 100nm, and the zeta potential is about +35mV. The stability results of the liposomes listed in the above tests 1 to 5 are shown in Table 2. After the liposomes are freeze-dried and rehydrated, their zeta potential, liposome particle size, drug loading and encapsulation efficiency are all stable. If the addition ratio exceeds the limit, unstable phenomena such as stratification, discoloration and precipitation will occur.

[0060] Table 2: Liposomes with different composition ratios in Tests 1 to 5 and their property parameters (betaine-type surfactant specifically uses lauroyl amide propyl betaine)

[0061]

[0062]

[0063] Example 3: In vitro free radical scavenging test of liposome complexes

[0064] In terms of anti-free radical effect, the combined use of luteolin-zinc and curcumin can produce a synergistic effect. The synergistic effect of the mixed monomers in scavenging free radicals was evaluated by the equivalent method (the results are shown in Figure 2 ), the half scavenging rates (IC50) of the two monomer samples of luteolin-zinc and curcumin on free radicals are X and Y values ​​respectively, and a straight line is drawn to connect the IC50 values ​​of luteolin-zinc and curcumin when used alone. This straight line is the isobolite line, and then when luteolin-zinc and curcumin are used in combination at different proportions to scavenging 50% of free radicals, different points are plotted with the concentrations of the two monomers as the horizontal and vertical coordinates. If this point is below the isobolite line, it is a synergistic effect, if it falls on the line, it is an additive effect, and if it is above the isobolite line, it is an antagonistic effect. Figure 2 The results showed that the points of scavenging three free radicals by luteolin-zinc and curcumin in a mixed monomer ratio of 1:8 to 8:1 all fell below the equivalent curve, proving that luteolin-zinc and curcumin showed a synergistic effect in scavenging free radicals. It can be seen that when the mass ratio of luteolin-zinc and curcumin is in the range of 1:8 to 8:1, the two substances have a synergistic effect in antioxidant effect. The above synergistic effect not only appears when luteolin-zinc and curcumin are used separately, but also in the liposome complex containing the two substances. The following is an antioxidant experimental study on liposome complexes.

[0065] In order to verify the anti-inflammatory effect (e.g., anti-free radical effect) of the liposome complex of the present invention, liposome complexes without adding luteolin-zinc (comparative test 1 in Table 3), without adding curcumin (comparative test 2 in Table 3), and without adding betaine-type surfactant (comparative test 3 in Table 3) were prepared. The preparation process used the aforementioned optimal process, except that the missing component was not added in the process step or the process step was omitted.

[0066] Table 3: Liposome complexes with different component ratios in comparison tests 1 to 3 (betaine-type surfactant specifically uses lauroyl amide propyl betaine)

[0067]

[0068] The preparation method of the above-mentioned liposome complex is carried out with reference to the preferred process steps described above. In the scheme without adding luteolin-zinc, the dropping step is omitted, and the anhydrous ethanol solution of curcumin and liposomes is directly stirred to ensure emulsification, and the other steps refer to the preferred process steps above. In the scheme without adding curcumin, the anhydrous ethanol solution of liposomes is directly prepared for complex preparation (without adding curcumin), and the other steps refer to the preferred process steps above. In the scheme without adding betaine-type surfactant, it is only necessary to omit the step of adding the substance, and the other steps refer to the preferred process steps above.

[0069] Comparative Tests 1 to 3 and Test 3 in Example 2 were selected to conduct in vitro free radical scavenging experiments.

[0070] Reactive oxygen species (ROS) free radicals (such as H2O2, ·OH, O2· - ) is physiologically produced during cell metabolism. When oral inflammation occurs, their balance is disrupted and ROS is produced, resulting in oxidative stress. Oxidative stress is an important process in the pathogenesis of oral inflammation. Inflammation leads to the production of a large amount of ROS, which further aggravates tissue damage at the site of inflammation and develops into chronic inflammation. Removing excess ROS at the site of inflammation and reducing oxidative stress have a positive and beneficial effect on quickly eliminating inflammation.

[0071] Hydrogen peroxide (H2O2) removal: A hydrogen peroxide kit (Nanjing Jiancheng Bioengineering Institute) was used to detect the performance of the liposome complexes in test 3 and comparative test 1-3 in removing H2O2. Specific method: The freeze-dried liposomes were rehydrated into a 4% solution, incubated with 2mM H2O2 at 37°C for 2 hours, and then molybdic acid was added and the absorbance at 405nm was detected using an ELISA reader to obtain the hydrogen peroxide removal rate.

[0072] Hydroxyl radical scavenging test: The freeze-dried liposomes of test 3 and comparative tests 1-3 were rehydrated into a 4% solution, incubated with 2 mM H2O2, 250 μM 3,3',5,5'-tetramethylbenzidine (TMB), and 1 mM ferrous sulfate in acetic acid-sodium acetate buffer at room temperature in the dark for 5 minutes, and the absorbance at 652 nm was detected using a UV-visible spectrometer.

[0073] Remove superoxide anions (O2 - ) Performance test: Superoxide anion determination kit (Nanjing Jiancheng Bioengineering Institute) was used to detect the ability of freeze-dried liposome reconstituted water solutions of test 3 and comparative tests 1-3 to remove O2· -Specific method: rehydrate the freeze-dried liposomes into a 4% solution, add it to the working solution prepared according to the instructions, keep it in a constant temperature water bath at 37°C for 40 minutes, add the color developer solution, let it stand for 10 minutes, and then use an enzyme marker to detect the absorbance value at 550nm.

[0074] Results Figure 3 (mean ± standard deviation, n = 3), Figure 3 The in vitro antioxidant performance test results of the liposomes prepared by Test 3 of Table 2 and Comparative Tests 1-3 of Table 3 are statistically shown, which show that: Test 3, i.e., liposomes containing luteolin-zinc and curcumin and protected by betaine, showed better antioxidant properties against three different types of reactive oxygen species, and had a strong scavenging ability against different types of reactive oxygen species.

[0075] Regarding the scavenging effect of liposome complexes on H2O2, test 3 uses a liposome complex containing a combination of luteolin-zinc (0.6 parts) and curcumin (0.5 parts), and the clearance rate (average value, n = 3) is 82.7%; comparative test 1 uses only a liposome complex containing curcumin (0.5 parts), and the clearance rate (average value, n = 3) is 34.33%; comparative test 2 uses only a liposome complex containing luteolin-zinc (0.6 parts), and the clearance rate (average value, n = 3) is 44.67%. Test 3, comparative test 1 and comparative test 2 differ only in the use or non-use of luteolin-zinc and / or curcumin, and the other raw material compositions and preparation methods are consistent. From the experimental data, it can be seen that the clearance rate of the combined use is greater than the sum of the clearance rates of the individual uses (i.e.: 82.7%>34.33%+44.67%), which shows that the combined use of luteolin-zinc and curcumin can synergistically enhance the antioxidant effect of the composition. In addition, the liposome complex prepared by the formula of this scheme "0.1-1 part of curcumin, 2-10 parts of liposomes, 0.2-1 part of luteolin-zinc metal complex, 0.15-0.25 part of betaine-type surfactant" also has the phenomenon of synergistically increasing the antioxidant effect of the complex by luteolin-zinc and curcumin, which is not limited to the formula composition shown in the specific experiment.

[0076] Similarly, for the ·OH scavenging effect, Test 3 used a liposome complex containing a combination of luteolin-zinc and curcumin, with a clearance rate of 93%; Comparative Test 1 used only a liposome complex containing curcumin, with a clearance rate of 52%; Comparative Test 2 used only a liposome complex containing luteolin-zinc, with a clearance rate of 39.7%. -In terms of scavenging effect, test 3 used a liposome complex containing luteolin-zinc and curcumin, with a scavenging rate of 93.3%; in comparison test 1, a liposome complex containing only curcumin was used, with a scavenging rate of 38%; in comparison test 2, a liposome complex containing only luteolin-zinc was used, with a scavenging rate of 48%. From the experimental data, it can be seen that for other free radicals (·OH and O2· - ), the synergistic effect of luteolin-zinc and curcumin in the liposome complex also exists (i.e., for ·OH: 93%>52%+39.7%; for O2· - :93.3%>38%+48%).

[0077] In addition, when comparing Test 3 and Comparative Test 3, the difference is whether betaine-type surfactants are used. The original purpose of using betaine-type surfactants in the technical solution is to increase the targeting of liposomes (positively charged). In the actual research process, other effects of betaine-type surfactants were found, namely, anti-free radical effects. The anti-free radical effect of the composition of Test 3 is improved relative to that of Comparative Test 3, which illustrates the dual role of betaine-type surfactants in clearing free radicals and increasing targeting.

[0078] Example 4: Application of liposome complex in the preparation of toothpaste

[0079] The liposome complex of this solution can be used in the preparation of oral care products, and this embodiment is described by taking toothpaste as an example. The following is the general formula (see Table 4) and preparation method of toothpaste in Tests 6-8 and Comparative Test 4.

[0080] A toothpaste containing anti-inflammatory and antibacterial liposomes, comprising anti-inflammatory and antibacterial liposomes, abrasives, thickeners, moisturizers, flavoring agents, surfactants, essences and water. In parts by weight, the components are: 4-12 parts of freeze-dried liposome complexes (the raw material composition of the liposomes is: 0.6 parts of luteolin-zinc metal complex, 0.6 parts of curcumin, 8 parts of liposomes, 0.2 parts of betaine surfactant, prepared by referring to the above-mentioned preferred preparation process), 5-65 parts of moisturizers, 10-40 parts of abrasives, 0.3-2 parts of thickeners, 0.1-2 parts of sweeteners, 0.1-1.8 parts of preservatives, 0.5-1.5 parts of aromatics, and the balance is water.

[0081] Table 4: Proportions of liposome-containing toothpaste ingredients in Tests 6-8 and Comparative Test 4

[0082]

[0083] The preparation process of toothpaste is as follows (tests 6-8):

[0084] (1) First, the freeze-dried liposomes are fully hydrated and dispersed in a small amount of water to obtain a rehydrated liposome, which is added to polyethylene glycol-8 and stirred evenly, and then the remaining humectants (sorbitol), flavoring agents and the remaining water are added in sequence and stirred evenly to obtain a mixture A;

[0085] (2) adding abrasive, preservative and surfactant to the mixture A and stirring to obtain a mixture B;

[0086] (3) homogenizing and stirring the mixture B for 5 to 10 minutes;

[0087] (4) Add fragrance to mixture B, stir homogenously again, and perform vacuum degassing to obtain toothpaste.

[0088] The preparation process of the toothpaste in comparative test 4 is somewhat different from the above method, specifically:

[0089] After the liposomes were rehydrated, they were not first dispersed in polyethylene glycol-8, but directly mixed with the humectant (polyethylene glycol + sorbitol), flavoring agent and the remaining water to obtain mixture A. The remaining steps were the same as above.

[0090] According to the above process, 4 groups of toothpastes were prepared, namely Test 6-8 and Comparative Test 4. According to the "Technical Guidelines for Cosmetic Stability Assessment", the four groups of toothpastes were placed at 45℃±2℃, 75%RH±5%RH for 3 months, and key indicators such as viscosity and consistency were tested. The experimental results show that when the toothpaste was prepared according to the above process, in Test 6-8, the structure of the liposomes was stable and the overall stability of the toothpaste was good; Comparative Example 4 and Example 8 have the same components, but the preparation process does not fully mix the liposome rehydrate with polyethylene glycol in advance, but directly mixes the liposome rehydrate with sorbitol, flavoring, polyethylene glycol-8, water, etc., which may lead to a large amount of drug leakage in the liposome rehydrate in the toothpaste, abnormal increase in the viscosity of the toothpaste, and the surface of the paste is not smooth, with obvious yellowing, water separation, and shelling (see Figure 4 , left is test 8, right is comparison test 4), the toothpaste is unstable.

[0091] It can be seen that one of the key technical points in preparing oral care products containing liposome complexes is that polyethylene glycol is needed as a part of the moisturizer in oral care products (toothpaste, mouthwash, gel), and the polyethylene glycol is first fully mixed with the reconstituted liposome complex to obtain the liposome complex-polyethylene glycol, which is used as a whole and then mixed with other raw materials to prepare oral care products to ensure the stability of the liposome complex and its compatibility with other ingredients of the oral care products.

[0092] Example 5: Animal Experiment

[0093] The toothpaste containing liposomes prepared in Test 6 of Example 4 was used to test the therapeutic effect of animal periodontitis.

[0094] Animal modeling: The rat periodontitis model was established by compressing the gums with ligatures. The specific operation is as follows: the rats were anesthetized by intraperitoneal injection of 10% chloral hydrate at a dose of 5 mL / kg, and then fixed. The mandibular posterior molars of the rats were cleaned, and the subgingival of the selected teeth was explored with a periodontal probe. Then the ligature was placed on the mesiobuccal side of the teeth. By compressing between the mesiobuccal and distal sides of the teeth, the tightness of the ligature was moderate to simulate the local compression of gingivitis, and the end of the ligature was fixed. Successful modeling criteria: symptoms of redness, swelling and bleeding of the gums, average attachment loss of all teeth ≥ 0.6 mm, and average attachment loss of adjacent teeth < 3 mm (Wang Dan, Liu Yudi, Shao Liping, et al. Effects of astragaloside I on the remodeling of periodontal tissues in orthodontic rats with periodontitis [J]. Journal of Practical Stomatology, 2023, 39(6): 722-729.).

[0095] The rats with successful modeling were divided into a control group and an experimental group using a random number table method, with 30 animals in each group. The rats in the experimental group used a cotton swab dipped in the toothpaste slurry prepared in Test 6 (the Test 6 toothpaste was prepared into toothpaste slurry at a ratio of 1:4) to repeatedly smear the gingivitis area for 3 minutes at a fixed time every day, while the control group used a cotton ball with clean water to wipe the area for the same time, twice a day, with an interval of 8-12 hours between the two times, for one week.

[0096] Index observation: The probing depth (PD), plaque index (PLI), gingival bleeding index (SBI) of the two groups of rats were investigated respectively, and the expression levels of TNF-α, IL-6, and IL-12 in serum were detected. The detection steps refer to the instructions of the kit.

[0097] Probing depth (PD) is an important indicator for evaluating the severity of periodontitis and the effect of treatment. The deeper the probing depth, the more severe the periodontitis. The plaque index (PLI) indicates the presence of plaque bands, with a score of 0-4 points. The lower the score, the milder the inflammatory response. The sulcus bleeding index (SBI) indicates the degree of gingival bleeding, with a score of 0-5 points. The higher the score, the more severe the bleeding (Xu Wang, Zhao Qin, Deng Tingchao, et al., Effects of Porphyromonas gingivalis infection on periodontal indicators, NLRP-3 inflammasome pathway and Th17 / Treg cell balance in patients with chronic periodontitis [J]. Advances in Modern Biomedicine, 2022, 22(23): 4555-4559.). SPSS20.0 was used to establish a database and perform analysis. The quantitative data were compared using two independent sample t-tests (inter-group comparisons) or paired design data t-tests (intra-group comparisons). p < 0.05 indicated that the difference was statistically significant. The results are shown in Tables 5, 6, and 7.

[0098] Table 5: Comparison of periodontal conditions of the two groups of rats before and after intervention

[0099]

[0100] Table 6: Changes in serum TNF-α, IL-6, and IL-12 levels in the two groups of rats before and after intervention (unit: pg / mg)

[0101]

[0102] Table 7: Comparison of therapeutic effects between two groups of rats [n (%)]

[0103] Group Significant effect efficient invalid Total Effectiveness Control group (n=30) 12(40) 14(46.7) 4(12.13) 26(87.87) Experimental group (n=30) 17(56.7) 11(36.6) 2(6.7) 28(93.3) p \ ＜0.05 \ ＜0.05

[0104] As can be seen from Table 5, the PD, PLI and SBI indexes of rats increased significantly after periodontitis modeling. After intervention, the PD, PLI and SBI indexes of rats in the experimental group were significantly lower than those in the control group, indicating that toothpaste containing liposomes can significantly reduce the depth of periodontal pockets, improve dental plaque index and reduce gingival bleeding.

[0105] As can be seen from Table 6, the levels of TNF-α, IL-6 and IL-12 in rats increased significantly after periodontitis modeling. After intervention, the levels of TNF-α, IL-6 and IL-12 in the experimental group were significantly lower than those in the control group. The treatment effect of the research group was significantly better than that of the control group, indicating that toothpaste containing liposomes can reduce the production of inflammatory factors, inhibit the inflammatory response of periodontal tissues, and maintain the stability of the immune system.

[0106] It can be seen from Table 7 and the above results that toothpaste containing liposomes can effectively reduce inflammatory damage to periodontal tissues in periodontitis model rats, significantly improve the symptoms of periodontitis, and show a good therapeutic effect.

[0107] The above is only an embodiment of the present invention, and the common knowledge such as the known specific structure and characteristics in the scheme is not described in detail here. It should be pointed out that for those skilled in the art, several deformations and improvements can be made without departing from the structure of the present invention, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. A liposome complex for oral care with targeted anti-inflammatory and antibacterial properties, characterized in that: The raw materials include, by weight: 0.1-1 part of curcumin, 0.2-1 part of luteolin-zinc metal complex, 2-10 parts of liposomes, and 0.15-0.25 parts of betaine type surfactant.

2. The oral care liposome complex for targeted anti-inflammatory and sterilization according to claim 1, characterized in that: The luteolin-zinc metal complex is obtained by thermal reflux reaction of luteolin and zinc salt in a molar ratio of 2:1; Preferably, the conditions for the thermal reflux reaction are: at a pH value of 4 and a reaction temperature of 60° C., the thermal reflux reaction is carried out for 4 hours, and the reaction solvent is anhydrous ethanol.

3. The oral care liposome complex for targeted anti-inflammatory and sterilization according to claim 1, characterized in that: The raw materials of the liposome are composed of cholesterol, soybean phosphatidylcholine and DMG-PEG 2000 in a molar ratio of 40:12:1.5; Preferably, the raw materials of liposomes are dispersed in chloroform, and the chloroform is evaporated to form a film, and the film is hydrated and freeze-dried to obtain liposomes.

4. The liposome complex for oral care with targeted anti-inflammatory and sterilization according to claim 1, characterized in that: The betaine-type surfactant includes lauryl amide propyl betaine and / or cocoyl hydroxysulfobetaine.

5. A method for preparing a targeted anti-inflammatory and antibacterial liposome complex for oral care according to any one of claims 1 to 4, characterized in that: The method includes the following steps in sequence: S1: preparing a solution A containing 0.2% to 1% by mass of luteolin-zinc metal complex, and a solution B containing 0.1% to 1% by mass of curcumin and 2% to 10% by mass of liposome; S2: In an environment of 50-60°C, solution B is added dropwise at a rate of 30-60 drops / min to solution A which is stirred at 200-500 r / min. After the addition is completed, stirring is continued for 1-3 hours to obtain a mixed emulsion; S3: adding a betaine-type surfactant to the mixed emulsion, stirring and filtering; and then freeze-drying to obtain a freeze-dried liposome complex.

6. Use of a targeted anti-inflammatory and antibacterial oral care liposome complex according to any one of claims 1 to 4 in the preparation of oral care products, characterized in that: The oral care products include mouthwash, toothpaste and gel.

7. Use of a targeted anti-inflammatory and antibacterial oral care liposome complex according to claim 6 in the preparation of oral care products, characterized in that: In parts by weight, the formula of the mouthwash is: 1-5 parts of freeze-dried liposome complex, 0.2-1.5 parts of sweetener, 4-20 parts of moisturizer, 0.1-0.8 parts of fragrance, 0.1-0.8 parts of preservative, and the balance is water; the formula of the gel or toothpaste is: 4-11 parts of freeze-dried liposome complex, 5-65 parts of moisturizer, 10-40 parts of abrasive, 0.3-2 parts of thickener, 0.1-2 parts of sweetener, 0.1-1.8 parts of preservative, 0.5-1.5 parts of fragrance, and the balance is water.

8. Use of a targeted anti-inflammatory and antibacterial oral care liposome complex according to claim 7 in the preparation of oral care products, characterized in that: The sweetener includes at least one or more of saccharin sodium, xylitol, sucralose, steviol glycosides, and aspartame; The moisturizing agent includes at least one of sorbitol, glycerol, and polyethylene glycol; The friction agent includes at least one of silicon dioxide and calcium hydrogen phosphate; The thickener comprises at least one of sodium carboxymethyl cellulose, hydroxyethyl cellulose, guar gum, xanthan gum, carrageenan and carbomer; The preservative includes at least one of parabens, benzyl alcohol and phenoxyethanol.

9. Use of a targeted anti-inflammatory and antibacterial oral care liposome complex according to claim 8 in the preparation of oral care products, characterized in that: The toothpaste is prepared by the following method: re-dissolving the freeze-dried liposome complex with water, mixing it with polyethylene glycol in the moisturizer, and then mixing it with the flavoring agent, the remaining moisturizers in the formula, and the remaining water in the formula to obtain a mixture A; adding an abrasive, a preservative, and a surfactant to the mixture A, stirring evenly to obtain a mixture B; adding a fragrance to the mixture B, stirring homogenously again, and vacuum degassing to obtain the toothpaste.

10. An oral care product comprising a targeted anti-inflammatory and antibacterial oral care liposome complex according to any one of claims 1 to 4, characterized in that: The oral care product contains a moisturizer, which includes polyethylene glycol; the liposome complex is mixed with the polyethylene glycol after redissolution, and then mixed with the remaining raw materials of the oral care product to prepare the oral care product.

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