Composition for resisting oxidation and inhibiting porphyromonas gingivalis and application thereof

Abstract

The invention relates to a composition for resisting oxidation and inhibiting porphyromonas gingivalis and application of the composition, and belongs to the technical field of oral care. The invention provides a composition for resisting oxidation and inhibiting porphyromonas gingivalis. The composition is prepared from a mangnolia officinalis bark extract and hinokiol. The mangnolia officinalis bark extract and hinokiol are screened from numerous existing components, it is found that the composition formed by combining and compounding the mangnolia officinalis bark extract and hinokiol has a synergistic interaction effect, the antibacterial effect of the composition can be improved, especially the antibacterial effect on porphyromonas gingivalis can be improved, and the inhibition effect is higher.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of oral care, in particular to a composition for inhibiting Porphyromonas gingivalis and its application. BACKGROUND

[0002] Gum health is an important gateway to the overall health of the human body, and Porphyromonas gingivalis (P. gingivalis) as the main pathogenic bacteria of periodontal disease has become a pathogen of focus in the field of oral health. Porphyromonas gingivalis, P. gingivalis Currently, the methods for inhibiting P. gingivalis include mechanical debridement, chemical bacteriostatic agents, antibiotics, etc., but these methods have certain limitations.

[0003] Mechanical debridement can remove part of the plaque biofilm, but it is limited by the complexity of oral anatomy, and the biofilm removal effect in hidden areas such as the root bifurcation and alveolar bone depression is poor, and it cannot prevent the bacteria from resisting mechanical force through the biofilm matrix, and the gingival protease secreted by the bacteria can still continuously damage the periodontal tissue; simple mechanical operation cannot completely remove the pathogenic bacteria. Chemical bacteriostatic agents have significant side effects: long-term use of components represented by chlorhexidine gluconate and cetylpyridinium chloride can disrupt the balance of oral microecology, induce excessive proliferation of Candida albicans or tooth pigmentation, and high-concentration preparations can also cause damage to oral mucosa. Antibiotics can produce drug resistance, and although metronidazole and tetracycline can inhibit planktonic bacteria in the short term, their penetration into the biofilm is limited, and the recurrence rate is high after drug withdrawal, and the detection rate of drug-resistant strains is increasing year by year, and long-term systemic use of drugs can also cause gastrointestinal discomfort, aggravate liver and kidney function, and other systemic risks.

[0004] In addition, the mechanism of free radicals in damaging periodontal tissue is manifested in multiple aspects. First, they directly induce lipid peroxidation of cell membranes and interfere with normal cell metabolism; they can also cause DNA strand breaks or base modifications, inducing apoptosis or even necrosis. Secondly, free radicals are important regulators of inflammation. More importantly, free radicals can activate the matrix metalloproteinase family, which can efficiently degrade collagen fibers in periodontal ligaments and gingival connective tissue, directly leading to alveolar bone resorption and attachment loss. In terms of bone metabolism, free radicals promote the differentiation and activation of osteoclasts while inhibiting the function of osteoblasts, disrupting the balance between bone formation and resorption, and accelerating the destruction of periodontal support tissues.

[0005] Therefore, it is urgent to develop a complex system based on natural active ingredients, which can significantly enhance the inhibitory effect on P. gingivalis and antioxidant efficacy through synergistic effect, and reduce the dosage of single component; the complex system also needs to be adapted to mouthwash, toothpaste, oral spray and other oral care products, to provide a safer and milder solution for the prevention and adjuvant therapy of periodontal disease, and meet the urgent needs of the current oral care market for "green bacteriostatic" technology. SUMMARY

[0006] The present application aims to overcome the deficiencies of the prior art and provide an antioxidant and Porphyromonas gingivalis inhibiting composition and application thereof.

[0007] To achieve the above-mentioned object, the technical scheme adopted by the present application is as follows: In a first aspect, the present application provides an antioxidant and Porphyromonas gingivalis inhibiting composition, which consists of Magnolia officinalis bark extract and hinokitiol.

[0008] Magnolia officinalis bark extract is a natural active ingredient extracted from the bark of Magnolia officinalis in the Magnoliaceae family, mainly containing Honokiol and Magnolol, etc. Magnolia officinalis bark extract has multiple biological activities, and it can effectively inhibit the excessive secretion of sebaceous glands and has obvious inhibitory effect on Malassezia furfur and Propionibacterium acnes.

[0009] Hinokitiol (also known as hinokitiol) is a natural monoterpene compound derived from the plant "Chamaecyparis". Its chemical structure is a unique seven-membered ring phenolic ketone, and it belongs to an environmentally friendly bioactive ingredient. The most prominent feature of this ingredient is its high safety. As a plant-derived natural molecule, it has good biocompatibility, is mild and non-irritating to the skin and mucous membranes, and is not easy to induce drug resistance with long-term use, and is widely used in daily chemical, pharmaceutical and food industries. In terms of application, hinokitiol has the functions of dandruff removal, itching relief, scalp microecological regulation, and oil secretion control, and is commonly used in hair care products. In addition, it can also be used as a food preservative, fruit and vegetable preservative, and wood mildew inhibitor, which reflects its safety advantage in multiple fields of application.

[0010] The present application selects Magnolia officinalis bark extract and hinokitiol from a large number of existing components, and finds that the combination of the two has a synergistic effect, which can improve the bacteriostatic and antioxidant effects of the composition, especially the bacteriostatic effect on Porphyromonas gingivalis.

[0011] As a preferred embodiment of the first aspect, the mass ratio of the Magnolia officinalis bark extract and the hinokitiol is 1: (2-10).

[0012] As a preferred embodiment of the first aspect, the mass ratio of the Magnolia officinalis bark extract and the hinokitiol is 1: (4-10).

[0013] As a preferred embodiment of the first aspect, the mass ratio of the Magnolia officinalis bark extract and the hinokitiol is 1:4.

[0014] The present application studies the ratio of Magnolia officinalis bark extract and hinokitiol in the composition by Chou-Talalay model, when CI <1, indicating that there is a synergistic effect; CI =1, indicating that there is an additive effect; CI >1, indicating that there is an antagonistic effect. It is found that when the mass ratio of Magnolia officinalis bark extract and hinokitiol is in the range of 1: (2-10), CI the value <1, indicating that the two have a synergistic effect in this range. When the mass ratio of Magnolia officinalis bark extract and hinokitiol is in the range of 1: (4-10), CI the value is in the range of 0.638-0.699, indicating that the synergistic effect of the two can be improved in this ratio range. When the mass ratio of Magnolia officinalis bark extract and hinokitiol is 1:4, CI the value is 0.638, indicating that the synergistic effect of the two is the strongest when the mass ratio is this value.

[0015] In the second aspect, the present application provides the use of the composition of the first aspect in the preparation of an oral care product.

[0016] Porphyromonas gingivalis causes a series of oral diseases through its special pathogenic mechanism, and the composition of the present application can effectively inhibit Porphyromonas gingivalis. Therefore, the composition of the present application is used in an oral care product, which can effectively prevent and improve oral diseases caused by Porphyromonas gingivalis.

[0017] As a preferred embodiment of the second aspect, the content of the composition for inhibiting Porphyromonas gingivalis in the oral care product is 0.02-0.2% by mass percentage.

[0018] As a preferred embodiment of the second aspect, the product includes toothpaste and mouthwash.

[0019] In the third aspect, the present application provides the use of the composition of the first aspect in the preparation of a product for preventing or improving oral diseases.

[0020] Since the composition of the present application can effectively inhibit Porphyromonas gingivalis, the composition of the present application is used in a product for preventing or improving oral diseases, which can prevent or improve oral diseases.

[0021] As a preferred embodiment of the third aspect, the oral disease is any one of halitosis, acute tooth, dental caries, periodontal disease, gingivitis, periodontitis, pulpitis, stomatitis, oral mucositis, oral mucosal ulcer, and oral mucosal ulcer.

[0022] Halitosis, acute dental caries, periodontitis, gingivitis, pulpitis, stomatitis, oral mucositis, oral mucosal ulcers, and thrush are all oral diseases caused by oral pathogens. Inhibiting these oral pathogens can treat these oral diseases. In the oral environment, *Porphyromonas gingivalis* is a major pathogen causing oral lesions; therefore, the composition of this invention has the effect of preventing or improving oral diseases.

[0023] Fourthly, the present invention provides the use of the composition described in the first aspect in antibacterial products.

[0024] As a preferred embodiment of the fourth aspect, the bacteria is Porphyromonas gingivalis.

[0025] The composition of the present invention has a certain antibacterial effect, especially against Porphyromonas gingivalis, with a MIC of 30 μg / mL, while the MICs of Magnolia officinalis bark extract or physalisol alone are 160 μg / mL and 40 μg / mL, respectively. Obviously, the composition of the present invention has a stronger antibacterial ability, so it can be used as an antibacterial active ingredient in the preparation of antibacterial products.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention, through systematic screening of the compound ratio of Magnolia officinalis bark extract and physalisol, found that when the mass ratio of Magnolia officinalis bark extract to physalisol is 1:(2-10), the CI < 1, indicating a synergistic antibacterial effect against Porphyromonas gingivalis. Specifically, when the mass ratio of Magnolia officinalis bark extract to physalisol is in the range of 1:(4-10), the CI ≈ 0.64–0.70, showing a stronger synergistic effect. This compound system not only reduces the dosage of single components and improves safety, but also provides a scientific basis for the development of oral care products such as mouthwash, toothpaste, and oral sprays. Attached Figure Description

[0027] Figure 1 This is a schematic diagram of the antioxidant results in Example 1; Figure 2 This is a schematic diagram of the antioxidant results in Example 2; Figure 3 This is a schematic diagram of the antioxidant results in Example 3; Figure 4 This is a schematic diagram of the antioxidant results in Example 4; Figure 5 This is a schematic diagram of the antioxidant results in Example 5; Figure 6 This is a schematic diagram of the antioxidant results for Comparative Example 1; Figure 7 This is a schematic diagram of the antibacterial ring containing the mouthwash from Example 4. Detailed Implementation

[0028] In order to better illustrate the purposes, technical solutions and advantages of the present application, the present application will be further described below in combination with specific examples.

[0029] The Magnolia officinalis bark extract used in the present application is from Nanning Houxin Biotechnology Co., Ltd., and the product code is HM98. It is a white to off-white powder with a unique odor of Magnolia officinalis and is soluble in organic solvents such as ethanol. The production process of the extract uses supercritical extraction and molecular distillation technology, and there is no organic solvent residue throughout the process. The microorganisms and heavy metals meet the requirements of the “Cosmetic Safety Technical Specifications” (2015 edition). The drying loss is less than or equal to 2%. The total phenol content (honokiol and magnolol) of Magnolia officinalis is greater than or equal to 98%.

[0030] The hinokitiol used in the present application is from Guangzhou Hengguang Composite Material Co., Ltd. Its quality specification is: white to light yellow crystalline powder in appearance, active matter content not less than 98%, drying loss not more than 2%, and burning residue less than 1%.

[0031] The Melaleuca alternifolia oil used in the present application is from Southern Cross Botanicals Pty Ltd (affiliated to IFF | Lucas Meyer Cosmetics Company) and its trade name is Melafresh™ T96. Its quality specification is: colorless to yellow clear flowing liquid with characteristic odor, and the content of the main component 4-terpineol is not less than 96%, and its chiral ratio ((+ / -) 4-TERPINEOL) is controlled in the range of 1.8-2.4:1.

[0032] The preparation method of the tea tree leaf extract is as follows: after the tea tree leaves are crushed, a water / ethanol / acetone mixed solution (the volume ratio of water, ethanol and acetone is 1:1:1) is added at a solid-liquid ratio of 1g:15mL, and ultrasonic is performed at 40-50℃ for 1.5 hours. Then, the tea tree leaf extract powder is obtained by filtering through a 0.22μm filter membrane, removing the water / ethanol / acetone mixed solution, and dissolving the tea tree leaf extract powder in ethyl acetate at a concentration of 2% by mass, removing the precipitate, and retaining the filtrate.

[0033] The components and their proportions of the compositions of Examples 1-5 and Comparative Examples 1-3 are shown in Table 1: Table 1 Components and their mass ratios of the compositions of Examples 1-5 and Comparative Examples 1-3 Comparative Example 4 The difference between Comparative Example 4 and Example 4 is that the tea tree leaf extract is used instead of the Magnolia officinalis bark extract.

[0034] Comparative Example 5 The difference between Comparative Example 5 and Example 4 is that Melaleuca alternifolia oil was used instead of cypressin.

[0035] Test Example 1: Minimum Inhibitory Concentration (MIC) Test 1. Experimental materials Porphyromonas gingivalis ( Porphyromonas gingivalis ,Pg,ATCC 33277), purchased from Guangdong Provincial Microbial Culture Collection Center GDMCC.

[0036] Blood agar plates (TSA + 5% defibrinated sheep blood), brain and heart extract broth (BHI), vitamin K1 (0.1 mg / mL), heme chloride (0.5 mg / mL), Beijing Luqiao Technology Co., Ltd.; dimethyl sulfoxide, Sinopharm Group Pharmaceutical Co., Ltd.; Magnolia officinalis bark extract, Nanning Houxin Biotechnology Co., Ltd.; cypressin, Guangzhou Hengguang Composite Materials Co., Ltd.

[0037] 2. Experimental apparatus Biosafety cabinet AC2-4S1, Singapore ESCO Technology Co., Ltd.; Biochemical incubator BD240, Binder Environmental Testing Equipment (Shanghai) Co., Ltd.; 0.01% electronic analytical balance PX84ZH, Ohaus Instruments (Shanghai) Co., Ltd.; Autoclave SX-700, Tomy Digital Biology, Japan; 20-200µL pipettes, 100-1000µL pipettes, Eppendorf, Germany.

[0038] 3. Experimental Methods 3.1 Preparation of culture medium Preparation of brain and heart extract broth culture medium: Weigh 24.5g of brain and heart extract broth culture medium into 1L of distilled water, heat to boiling until completely dissolved, autoclave at 121℃ for 15min, and set aside. Remove oxygen from the broth in an anaerobic environment 24 hours before use. When culturing microorganisms, add 100μL each of vitamin K1 and heme chloride to every 10mL of broth culture medium.

[0039] 3.2 Activation of bacterial strains and preparation of bacterial suspension Dissolve the lyophilized bacterial culture in pre-deoxygenated sterile water, inoculate onto blood agar plates, and anaerobically incubate at 37°C for 7-10 days, then subculture 1-2 times. When the colonies on the blood agar plates turn from white to black, pick colonies in good growth condition and transfer them to brain and heart extract broth medium. Grind the culture finely and incubate anaerobically at 37°C for 48 hours. Centrifuge 10 mL of the bacterial suspension in a centrifuge tube for 2 minutes, remove the supernatant, collect the lower layer of cells, and then inoculate 10 μL into brain and heart extract broth medium. After incubating as described above, adjust the bacterial suspension concentration to 1.0 × 10⁻⁶.9 CFU / mL, diluted 50-fold in medium, ready for use.

[0040] 3.3 Preparation of reagents Preparation of Magnolia officinalis bark extract stock solution: Using brain heart infusion broth BHI medium as diluent, 2% (v / v) ethanol was added to prepare the stock solution of corresponding concentration, and then ultrasonic assisted dissolution was performed. The concentration of Magnolia officinalis bark extract stock solution was 6400 μg / mL.

[0041] Preparation of hinokitiol stock solution: Using brain heart infusion broth BHI medium as diluent, 2% (v / v) ethanol was added to prepare the stock solution of corresponding concentration, and then ultrasonic assisted dissolution was performed. The concentration of hinokitiol stock solution was 3200 μg / mL.

[0042] Preparation of composition stock solution: According to the ratio of Magnolia officinalis bark extract and hinokitiol in Table 1, using brain heart infusion broth BHI medium as diluent, 6% dimethyl sulfoxide was added to prepare the stock solution of corresponding concentration. The concentration of composition stock solution was 1000 μg / mL.

[0043] 3.4 Measurement of MIC value of sample by 96-well plate method 3.4.1 Preparation of working solution and sample addition According to the paper "Study on the antibacterial function of quercetin extracted from Osmanthus fragrans in vitro against Porphyromonas gingivalis and Fusobacterium nucleatum" published in the journal "Occupation and Health" in December 2024, the double dilution method was used: The MIC of Magnolia officinalis bark extract was determined by the double dilution method. The Magnolia officinalis bark extract stock solution was diluted to 320 μg / mL with brain heart infusion broth BHI medium, and 100 μL was added to the first well of the 96-well plate. The BHI medium was double diluted to the following concentrations: 320, 160, 80, 40, 20, 10, 5, 2.5, 1.25, 0.63 μg / mL. In the 96-well plate, 100 μL of the corresponding concentration of working solution was added to wells 1 to 10; wells 11 and 12 were positive and negative control wells, and 100 and 200 μL of brain heart infusion broth BHI medium was added, respectively.

[0044] The MIC of hinokitiol was determined by the double dilution method. The hinokitiol stock solution was diluted to 160 μg / mL with brain heart infusion broth BHI medium, and 100 μL was added to the first well of the 96-well plate. The BHI medium was double diluted to the following concentrations: 160, 80, 40, 20, 10, 5, 2.5, 1.25, 0.63, 0.31 μg / mL; wells 11 and 12 were positive and negative control wells, and 100 and 200 μL of brain heart infusion broth BHI medium was added, respectively.

[0045] Example 1 to Example 5, Comparative Example 3, Comparative Example 4 MIC determination of the composition: the composition mother liquor was diluted with brain heart infusion broth BHI medium into the following concentrations: 160, 140, 120, 100, 80, 60, 40, 20, 10, 5 μg / mL. In a 96-well plate, 100 μL of the working solution of the corresponding concentration was added to wells 1 to 10 respectively; wells 11, 12 were positive and negative control wells, 100, 200 μL of brain heart infusion broth BHI medium was added respectively.

[0046] MIC determination of the composition of Comparative Example 1: the composition mother liquor was diluted with brain heart infusion broth BHI medium into the following concentrations: 300, 280, 260, 240, 220, 200, 180, 160, 140, 120 μg / mL. In a 96-well plate, 100 μL of the working solution of the corresponding concentration was added to wells 1 to 10 respectively; wells 11, 12 were positive and negative control wells, 100, 200 μL of brain heart infusion broth BHI medium was added respectively.

[0047] MIC determination of the composition of Comparative Example 2: the composition mother liquor was diluted with brain heart infusion broth BHI medium into the following concentrations: 280, 260, 240, 220, 200, 180, 160, 140, 120, 100 μg / mL. In a 96-well plate, 100 μL of the working solution of the corresponding concentration was added to wells 1 to 10 respectively; wells 11, 12 were positive and negative control wells, 100, 200 μL of brain heart infusion broth BHI medium was added respectively.

[0048] MIC determination of the composition of Comparative Example 5: the composition mother liquor was diluted with brain heart infusion broth BHI medium into the following concentrations: 520, 480, 440, 400, 360, 320, 280, 240, 200, 160 μg / mL. In a 96-well plate, 100 μL of the working solution of the corresponding concentration was added to wells 1 to 10 respectively; wells 11, 12 were positive and negative control wells, 100, 200 μL of brain heart infusion broth BHI medium was added respectively.

[0049] 3.4.2 Bacterial solution addition The diluted bacterial suspension was about 2.0 x 10 7 CFU / mL. 100 μL of the diluted bacterial solution was added to wells 1 to 11 in a 96-well plate, at this time the final content of the bacterial solution concentration in each well should be 1.0 x 10 7 CFU / mL.

[0050] 3.4.3 Incubation and result interpretation The 96-well plate was taken out after 48 h of anaerobic culture at 37°C and observed under dark background. No clear bright turbidity appeared in the well, indicating that no bacteria grew in the well. At this time, the minimum concentration of the sample solution was the MIC of P. gingivalis.

[0051] 3.5 Synergy index calculation for inhibiting P. gingivalis The "Chou-Talalay" model is an effective method for quantifying synergistic effects, and the combination index (CI) is used to evaluate the combined effect of each composition. Therefore, the synergistic effect of each composition is evaluated by the Chou-Talalay model, and the calculation formula is as follows: CI C a , C b are the MICs of components A and B, respectively; C A , C B represent the concentrations of each component when the composition reaches the MIC, and the results of C A or C B = composition MIC experimental data x the proportion of component A or B in the composition. CI <1 indicates that there is a synergistic effect, CI the lower the value, the better the synergistic effect; CI = 1 indicates that there is an additive effect; CI > 1 indicates that there is an antagonistic effect.

[0052] 4. Experimental results 4.1 Single drug antibacterial results The MIC test results of Magnolia officinalis bark extract and hinokitiol on P. gingivalis are shown in Table 2. Magnolia officinalis bark extract has broad-spectrum antibacterial activity, and the main active ingredients include phenolic compounds such as honokiol. It can destroy the structure of bacterial cell membranes, inhibit enzyme activity, and interfere with biofilm formation, thereby effectively inhibiting the growth of P. gingivalis. The MIC of Magnolia officinalis bark extract alone is 160 μg / mL. Hinokitiol is a natural phenolic compound with strong antibacterial and antioxidant activity. It can penetrate the biofilm and inhibit key enzymes in bacterial metabolism, and is particularly effective against anaerobic bacteria. The MIC of hinokitiol alone is 40 μg / mL. Both Magnolia officinalis bark extract and hinokitiol have good antibacterial effects on P. gingivalis, but the required concentration is high when used alone, which may have problems such as cytotoxicity, limiting its application in actual products. Therefore, the synergistic effect of Magnolia officinalis bark extract and hinokitiol on inhibiting P. gingivalis was further explored.

[0053] Table 2 Single drug antibacterial results ​4.2 Composition Bacteriostatic Test Results Tables 3-10 are the MIC test results of the composition of Magnolia officinalis bark extract and hinokitiol against Porphyromonas gingivalis. In Examples 1-5, i.e., when the mass ratio of Magnolia officinalis bark extract to hinokitiol is in the range of (1:10) to (1:2), CI <1, the bacteriostatic effect against Porphyromonas gingivalis is synergistic; wherein, when the mass ratio of Magnolia officinalis bark extract to hinokitiol is in the range of (1:10) to (1:4) CI ≈0.64-0.70, the synergistic effect is strong; the MIC of the composition of Example 4 is reduced to 30 μg / mL, which is only 18.8% of the MIC of Magnolia officinalis bark extract alone and 75% of the MIC of hinokitiol alone, significantly reducing the amount of each component. In Comparative Examples 1-3, i.e., when the mass ratio of Magnolia officinalis bark extract to hinokitiol is in the range of (1:1) to (4:1), CI >1, the inhibition against Porphyromonas gingivalis is antagonistic. Therefore, from Tables 3-10, when the mass ratio of Magnolia officinalis bark extract to hinokitiol is 1:4 (Example 4), the synergistic effect is best. When tea tree leaf extract is used to replace Magnolia officinalis bark extract in Example 4, CI >1, the inhibition against Porphyromonas gingivalis is antagonistic; when melaleuca alternifolia oil is used to replace hinokitiol in Example 4, CI >1, the inhibition against Porphyromonas gingivalis is antagonistic, as shown in Tables 11 and 12.

[0054] Table 3 Synergistic Test Results of Example 1 (μg / mL) Table 4 Synergistic Test Results of Example 2 (μg / mL) Table 5 Synergistic Test Results of Example 3 (μg / mL) Table 6 Synergistic Test Results of Example 4 (μg / mL) Table 7 Synergistic Test Results of Example 5 (μg / mL) Table 8 Synergistic Test Results of Comparative Example 1 (μg / mL) Table 9 Synergistic Test Results of Comparative Example 2 (μg / mL) Table 10 Synergistic Test Results of Comparative Example 3 (μg / mL) Table 11 Synergy test results of comparative example 4 (μg / mL) Table 12 Synergy test results of comparative example 5 (μg / mL) Antioxidant test of test example 2 composition 1. Experimental method 1.1 Reagents and instruments Reagents: 2,2-diphenyl-1-picrylhydrazyl, Shanghai Maikelin Biochemical Technology Co., Ltd.; 95% ethanol, National Pharmaceutical Group Chemical Reagent Co., Ltd.; Magnolia officinalis bark extract, Nanning Houxin Biotechnology Co., Ltd.; cembrene, Guangzhou Hengguang Composite Material Co., Ltd.

[0055] Equipment: SYNERGY H1 fluorescence microplate reader, BioTek, USA; 20-200 μL pipette, 100-1000 μL pipette, Eppendorf, Germany.

[0056] 3.2 Preparation of sample solution Preparation method of sample solution of example 1-5, comparative example 1 and Magnolia officinalis bark: weigh the sample into a 50 ml volumetric flask, and then add 95% ethanol to the mark. After fully dissolving, 2500 μg / ml is obtained. Take 25 ml solution into a 50 ml volumetric flask, and then add 95% ethanol to the mark. After dilution, 1250 μg / ml is obtained. Similarly, 2500 μg / ml, 1250 μg / ml, 625 μg / ml, 321.5 μg / ml and 156.25 μg / ml sample solutions with different mass concentrations are obtained.

[0057] Preparation method of cembrene sample solution: weigh 500 mg of cembrene into a 50 ml volumetric flask, and then add 95% ethanol to the mark. After fully dissolving, 10000 μg / ml is obtained. Take 25 ml solution into a 50 ml volumetric flask, and then add 95% ethanol to the mark. After dilution, 5000 μg / ml is obtained. Similarly, 2500 μg / ml, 1250 μg / ml and 625 μg / ml sample solutions with different mass concentrations are obtained.

[0058] 1.2 Determination of DPPH free radical scavenging rate DPPH solution was prepared with 95% ethanol at 0.2 mmol / L, and the prepared DPPH should be stored in the dark. 2 mL of DPPH solution and 2 mL of sample solution with different mass concentrations were mixed, and the absorbance (A1) at 517 nm was measured after reaction at room temperature for 30 min in the dark. Then, 2 mL of 95% ethanol was used to replace the DPPH solution (i.e., 2 mL of 95% ethanol and 2 mL of sample solution with different mass concentrations were mixed), and the absorbance (A2) at 517 nm was measured. Finally, 2 mL of 95% ethanol was used to replace the sample solution (i.e., 2 mL of 95% ethanol and 2 mL of DPPH solution), and the absorbance (A0) at 517 nm was measured.

[0059] 200 μL of the solution was taken in a 96-well plate using a pipette, and the absorbance at 517 nm was measured using an enzyme marker.

[0060] The calculation formula of DPPH radical scavenging rate (R1, %) is as follows: 1.3 Plotting and IC50 (Half maximal inhibitory concentration) calculation The logarithm of the concentration of each sample to be tested (Log Drug Concentration) was taken as the abscissa, and the DPPH radical scavenging rate of each sample to be tested (DPPH radical scavenging activity / %) was taken as the ordinate, and a curve was plotted to obtain the IC 50 .

[0061] 1.3 Calculation of synergistic index of DPPH radical scavenging According to the reference "Study on Inhibition of P. acnes and Scavenging of Free Radicals by Three Plant Components and Their Combinations" published in "China Detergent Industry", the synergistic effect of each combination was evaluated using the Chou-Talalay model, and the calculation formula is as follows: Wherein, Ca-b is the IC 50 of component A and B when acting alone, C A-B represents the concentration of each component when reaching IC 50 , and the result of C A-B = IC 50 of the combination × proportion of A-B components in the combination. CI <1 indicates synergistic effect; CI =1 indicates additive effect; CI >1 indicates antagonistic effect.

[0062] 2, Test results DPPH radical scavenging experiment is a classic method for evaluating antioxidant capacity, and the lower the IC50 (half-inhibitory concentration), the stronger the antioxidant activity. The results of the DPPH radical scavenging experiment are shown in Table 13. The results show that all the test combinations (Examples 1-5) exhibit significant synergistic antioxidant effects, with the synergistic index (CI) being much less than 1 (range 0.076-0.094), indicating that there is a strong synergistic effect between the Magnolia officinalis bark extract and hinokitiol. Among them, Example 5 (Magnolia officinalis bark extract: hinokitiol = 1:2) has the lowest IC50 value (167.3 μg / mL) and the strongest antioxidant activity, which is significantly improved compared with Magnolia officinalis bark extract (IC50 = 771.9 μg / mL) and hinokitiol (IC50 = 27248 μg / mL) alone. The IC50 in Comparative Example 1 is 779.1 μg / mL, although the CI is close to 1, but its IC50 is greater than Magnolia officinalis bark extract (IC50 = 771.9 μg / mL) alone.

[0063] Table 13 Synergistic antioxidant experiment results In summary, from Table 1, Tables 3-8, and Table 13, it can be seen that the antibacterial effect of the Magnolia officinalis bark extract and hinokitiol in Example 4 is the best, and the antioxidant effect is only second to that in Example 5, while the antioxidant effect of the Magnolia officinalis bark extract and hinokitiol in Example 5 is the best, but the antibacterial effect is not as good as that in Examples 1-4. Therefore, Example 4 is selected as the best scheme of the present application, and the composition prepared under this ratio has the best antibacterial effect and strong antioxidant property.

[0064] Application Example 4 The composition prepared in Example 4 is used in mouthwash 1. Mouthwash formula and preparation method 1.1 The mouthwash formula is shown in Table 14: Table 14 Mouthwash formula containing the composition of Example 4 Note: The water in phase D in Table 14 is premixed sodium citrate with a fixed amount of 1%.

[0065] 1.2 The preparation method of the mouthwash includes the following steps: (1) Add the water in phase A to a stirring pot, then add disodium EDTA and dipotassium glycyrrhizinate, start stirring, and heat to 80°C while stirring, and keep for 30 minutes; (2) Start cooling to 55°C, and add the Stevia rebaudiana extract and sodium benzoate in phase B to the stirring pot; (3) Premix the raw materials in phase C, heat appropriately, stir to dissolve, and reserve; (4) When the mixing pot cools to 45°C, add the premixed C phase into the mixing pot; (5) If necessary, use phase D to adjust the pH to 5.5-6.0; (6) Once the test is passed, the material can be discharged.

[0066] 2. Mouthwash antibacterial ring test 2.1 Experimental Methods The above-mentioned mouthwash was used to test the antibacterial zone. The test method was as follows: Experimental materials: Porphyromonas gingivalis ( Porphyromonas gingivalis Pg, ATCC 33277), blood agar plate (TSA + 5% defibrinated sheep blood).

[0067] Experimental method: Place the bacterial plates containing the added samples in anaerobic refrigeration at 4℃ for 4 hours or overnight for pre-diffusion, then take them out and place them upright in anaerobic culture at 36℃±1℃ for 36-72 hours. Measure the diameter of the inhibition zone formed by the test sample and the blank control group with vernier calipers.

[0068] 2.2 Experimental Results The test results are shown in Table 15 and Figure 1 As shown, the inhibition zone size of the above-mentioned mouthwash against Porphyromonas gingivalis was 19.92 mm. According to the NCCLS standard, it showed high sensitivity to Porphyromonas gingivalis, indicating that the mouthwash containing Example 4 has a strong inhibitory ability against Porphyromonas gingivalis.

[0069] Table 15 shows the antibacterial zone test results for the mouthwash from Example 4. 3. Sensory testing and detection of pH value, harmful substances and microorganisms. 3.1 Test Method In accordance with the requirements of the national standard GB / T 43543-2023 "Mouthwash", the mouthwash containing Example 4 was subjected to sensory, pH, harmful substance and microbial tests. The sensory tests included appearance, color and odor. Harmful substances included lead, arsenic, mercury, cadmium and methanol. Microbial tests included total bacterial count, total mold and yeast count, thermotolerant coliforms, Staphylococcus aureus and Pseudomonas aeruginosa.

[0070] The appearance and color test method is: at room temperature and natural light conditions, 25 ml sample is poured into a colorimetric tube, and observed visually at a distance of 30 cm. The test method of odor is directly performed by manual smell. The pH value is tested according to GB / T 43543-2023. The methanol content is tested according to QB / T 5703. Lead, arsenic, mercury, cadmium, total bacterial count, total mold and yeast count, heat-resistant coliform group, Staphylococcus aureus and Pseudomonas aeruginosa are tested according to the “Cosmetic Safety Technology Standard” (2015 edition). If these test items meet the requirements of GB / T 43543-2023 “mouthwash”, it is determined to be qualified; if it does not meet the requirements of GB / T 43543-2023 “mouthwash”, it is determined to be unqualified.

[0071] 3.2 Test results The specific test results are shown in Table 16: Table 16 Sensory test and pH value, harmful substance and microbial detection results As can be seen from Table 16, the appearance, odor, pH value, lead, arsenic, mercury, cadmium, methanol, total bacterial count, total mold and yeast count, heat-resistant coliform group, Staphylococcus aureus and Pseudomonas aeruginosa test items of the mouthwash containing the composition of Example 4 meet the requirements of the national standard GB / T 43543-2023 “mouthwash”.

[0072] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application and not to limit the scope of protection of the present application. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present application.

Claims

1. An antioxidant and Porphyromonas gingivalis inhibiting composition, characterized by comprising, The composition consists of Magnolia officinalis bark extract and hinokitiol.

2. The composition of claim 1, wherein The mass ratio of the Magnolia officinalis bark extract and the hinokitiol is 1: (2-10).

3. The composition of claim 2, wherein The mass ratio of the Magnolia officinalis bark extract and the hinokitiol is 1: (4-10).

4. Use of the composition according to any one of claims 1-3 in the manufacture of an oral care product.

5. The use according to claim 4, wherein the compound is ###0002### The content of the composition for inhibiting Porphyromonas gingivalis in the oral care product is 0.02-0.2% by mass.

6. The use according to claim 4, wherein the compound is ###0002### The product includes toothpaste and mouthwash.

7. Use of the composition according to any one of claims 1-3 in the manufacture of a product for preventing or improving oral diseases.

8. Use according to claim 7, wherein the compound is ###0002### The oral disease is any one of halitosis, acute tooth, dental caries, periodontal disease, gingivitis, periodontitis, pulpitis, stomatitis, oral mucositis, oral mucosal ulcer, and aphthous ulcer.

9. Use of the composition according to any one of claims 1-3 in a bacteriostatic product.

10. Use according to claim 9, wherein The bacterium is Porphyromonas gingivalis.

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