CN117017834A - Flos Caryophylli and cortex Cinnamomi essential oil composition, and its preparation method and application - Google Patents
Flos Caryophylli and cortex Cinnamomi essential oil composition, and its preparation method and application Download PDFInfo
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- CN117017834A CN117017834A CN202310916535.2A CN202310916535A CN117017834A CN 117017834 A CN117017834 A CN 117017834A CN 202310916535 A CN202310916535 A CN 202310916535A CN 117017834 A CN117017834 A CN 117017834A
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- China
- Prior art keywords
- essential oil
- streptococcus mutans
- composition
- flos caryophylli
- oil composition
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/92—Oils, fats or waxes; Derivatives thereof, e.g. hydrogenation products thereof
- A61K8/922—Oils, fats or waxes; Derivatives thereof, e.g. hydrogenation products thereof of vegetable origin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/54—Lauraceae (Laurel family), e.g. cinnamon or sassafras
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/61—Myrtaceae (Myrtle family), e.g. teatree or eucalyptus
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q11/00—Preparations for care of the teeth, of the oral cavity or of dentures; Dentifrices, e.g. toothpastes; Mouth rinses
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Chemical & Material Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Medicinal Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Epidemiology (AREA)
- Biotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Organic Chemistry (AREA)
- Botany (AREA)
- Medical Informatics (AREA)
- Alternative & Traditional Medicine (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Birds (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Oral & Maxillofacial Surgery (AREA)
- Communicable Diseases (AREA)
- Oncology (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention belongs to the technical field of oral care, and particularly relates to a flos caryophylli and cortex cinnamomi essential oil composition, and a preparation method and application thereof. The composition is prepared by mixing flos caryophylli essential oil and cinnamon bark essential oil according to the volume ratio of 1:1-4, and the two produce synergistic effects, can be used as an oral antibacterial agent and an anticaries agent, and has inhibition effects on growth, acid production, biofilm formation and adhesion of streptococcus mutans, and the essential oil composition has potential for preventing dental caries caused by the streptococcus mutans.
Description
Technical Field
The invention belongs to the technical field of oral care, and particularly relates to a flos caryophylli and cortex cinnamomi essential oil composition, and a preparation method and application thereof.
Background
Caries is a disease in which hard tissues of the tooth body are chronically destroyed under the influence of a plurality of factors mainly including bacteria, and is mainly represented by tooth body demineralization, staining, formation of caries and the like.
Caries is mainly associated with the expression of virulence factors of streptococcus mutans and its acidogenesis and biofilm formation, streptococcus mutans (Streptococcus mutans, s.mutans) is a major cariogenic bacteria in the oral cavity, also known as plaque, a variety of biological films formed on hard surfaces of teeth. Streptococcus mutans can create advantageous living space for other acidogenic and acidic species by forming rich Extracellular Polysaccharide (EPS) and a low pH environment.
Streptococcus mutans (S.mutans) initially adheres to the tooth surface, creating an insoluble glucan layer. Dextran is synthesized by glycosyltransferase (Gtfs) and helps to form plaque matrix polysaccharides, thereby accelerating the maturation of plaque. Streptococcus mutans also has the ability to metabolize carbohydrates in food and release organic acids, such as lactic acid, as a byproduct. The released organic acid can lower the pH value of dental plaque and dissolve dental enamel. Caries development is mainly related to carbohydrate content in the diet and frequency of consumption. Sucrose is the most cariogenic carbohydrate, as it is the major metabolite of streptococcus mutans. Streptococcus mutans utilizes carbohydrate-producing acidic metabolites, resulting in acidic destruction and demineralization, and removal of mineral material, thereby leading to caries. This sucrose-dependent mechanism is also based on Gtfs, involving several cariogenic-related virulence factors such as glucan-binding proteins (Gbps). gbpB, which encodes a surface-associated glucan-binding protein (Gbp), is also an essential factor for bacterial adhesion, as this protein mediates interactions between the cell surface and glucan. In addition, streptococcus mutans expresses the spaP gene, which contributes to the adhesion of Streptococcus mutans and thus, reduces the number of cariogenic bacteria, reduces acid synthesis, and prevents plaque biofilm formation, is also the most effective strategy for preventing caries.
Currently, methods for prevention and treatment of dental caries generally include laser caries prevention, immune caries prevention, pit and fissure occlusion treatment, drug control, and daily care control. Among them, the most commonly used technology of daily care combined with drug control is to add a compound/composition with caries prevention to an oral care agent, and fluorine-containing toothpaste is one of the commonly used methods of maintaining oral hygiene, which has high clinical efficacy, but the use of fluoride-containing chemicals causes changes in intestinal and oral flora, even staining of teeth, vomiting and oral cancer. Triclosan (TCS) is a commonly used antibacterial/antifungal agent found in certain toothpastes and mouthwashes. Although an effective antimicrobial, the widespread use of triclosan in everyday products will result in antibiotic resistance. Ethanol is one of the major active ingredients of most mouthwashes on the market, with some products having ethanol concentrations even as high as 27%. The use of such high concentrations of ethanol in these products stimulates the oral cavity and dries. Thus, there is a need to develop a new antibacterial caries-preventing oral care composition.
Chinese patent application 01129070.6 discloses a composition for treating toothache caused by dental caries, which is prepared from herba asari, fructus Zanthoxyli, rhizoma Ligustici Chuanxiong, herba Menthae, plumbum Preparatium and Camphora as raw materials, wherein the composition contains Plumbum Preparatium and can cause lead poisoning after long-term use. The Chinese patent application 201010173234.8 discloses a multifunctional mouthwash which is prepared from disinfectants, caries-preventing agents, cleaning agents, scents, sweeteners, stabilizers and deionized water, and has the effects of inhibiting and removing oral bacteria, effectively preventing tooth decay and removing halitosis, wherein the caries-preventing agents are one or more of potassium fluoride, sodium fluoride and sodium monofluorophosphate, the substances contain fluorine, and the side effects such as fluorine poisoning are caused after long-term use, and the former two substances are corrosive, so that the mouthwash still has a certain safety risk.
Disclosure of Invention
Aiming at the defects existing in the prior art, the technical problem to be actually solved by the invention is to provide the flos caryophylli and cortex cinnamomi essential oil composition, and the preparation method and the application thereof.
The plant essential oil has great advantages in bacteriostasis and anti-biofilm aspects, and has the characteristics of safety and no side effect, and the plant essential oil just meets the requirements of serving as an oral care product. The essential oil has effects of resisting inflammation, relieving pain, resisting bacteria, stopping bleeding, and resisting pathogenic microorganism, and has good effect in protecting health of teeth and oral cavity.
The invention provides a composite essential oil composition, which comprises clove flower essential oil and cinnamon bark essential oil.
Preferably, the volume ratio of the flos caryophylli essential oil to the cortex cinnamomi essential oil is 1:0.8-10.
Further preferably, the volume ratio of the lilac flower essential oil to the cinnamon bark essential oil is 1:1:1-4.
Preferably, the effective components of the eugenol essential oil comprise 72-73% of eugenol and 16-18% of caryophyllene, and further preferably 72.88% of eugenol and 16.98% of caryophyllene in percentage by mass.
Preferably, the effective components of the cinnamon bark essential oil comprise, by mass, 55% -65% of (E) -cinnamaldehyde and 9% -11% of cinnamyl acetate, and further preferably 60.98% of (E) -cinnamaldehyde and 10.95% of cinnamyl acetate.
Preferably, the lilac flower essential oil and the cinnamon bark essential oil are recovered by water distillation.
The invention also relates to a preparation method of the composition, and the lilac flower essential oil and the cinnamon bark essential oil are mixed to obtain the composition.
The invention also relates to the application of the composition in preparing medicines or oral care products for inhibiting oral bacteria
Preferably, the oral bacteria include Streptococcus mutans and the composition is capable of treating dental caries.
Preferably, the medicament or product is capable of inhibiting the growth, acid production, biofilm formation and adhesion capacity of Streptococcus mutans.
Compared with the prior art, the invention has the beneficial effects that:
(1) The flos caryophylli essential oil and the cortex cinnamomi essential oil have synergistic effect after being mixed;
(2) The composition in the technical proposal can be added into oral products (such as toothpaste, mouthwash, dentifrice and the like), has obvious inhibition effect on streptococcus mutans (Streptococcus mutans, S.mutans) of oral pathogenic bacteria and inhibition effect on growth, acidogenesis, biofilm formation and adhesion of the streptococcus mutans, and the essential oil composition has potential for preventing decayed teeth caused by the streptococcus mutans;
(3) The essential oil is a mixture of various volatile aromatic components, has small molecular weight, high fat solubility and high volatility compared with other extracts, can quickly penetrate or destroy bacterial phospholipid bilayer membranes, enter the inside of cells to play a role, and takes effect quickly; the compound essential oil provided by the technical scheme has obvious synergistic effect compared with single essential oil.
Drawings
FIG. 1 is a graph showing the effect of essential oils of examples 1-3 and comparative examples 1-4 on zone of inhibition diameter of Streptococcus mutans;
FIG. 2 is a time-sterilization graph of example 1 essential oil versus Streptococcus mutans;
FIG. 3 is a graph showing the effect of essential oils of example 1 on Streptococcus mutans biofilm;
FIG. 4 is a graph showing the effect of essential oils of example 1 on the expression of Streptococcus mutans virulence factors vicR and relA;
FIG. 5 is a graph showing the effect of essential oils of example 1 on the expression of Streptococcus mutans virulence factors gtfB and gbpB;
FIG. 6 is a graph showing the effect of essential oils of example 1 on the expression of Streptococcus mutans virulence factors gtfD and gtfC;
FIG. 7 is a graph showing the effect of essential oils of example 1 on the expression of Streptococcus mutans virulence factors spaP and brpA.
Detailed Description
The technical solutions of the embodiments of the present invention are further clearly described, and the described embodiments are only a part of the present invention, which are used to explain the present invention, but not to limit the present invention, so that other embodiments obtained by other persons skilled in the art without creative efforts fall within the protection scope of the present invention.
Flos Caryophylli (Syzygium aromaticum) essential oil, purchased from Shanghai of China, limited liability company, cat No.: PEC1601;
cinnamon (Cinnamomum zeylanicum) essential oil, purchased from Shanghai, limited liability company, cat No.: PEC2201;
streptococcus mutans Streptococcus mutans, available from the China academy of sciences center for culture Collection of microorganisms, accession number: ATCC700610;
brain heart extract Broth (BHI) medium, BR, purchased from OXOID;
nutrient agar, BR, purchased from beijing oborgasm biotechnology limited.
Example 1
The essential oil composition comprises the following components in parts by volume: 1 part of flos caryophylli essential oil and 4 parts of cinnamon essential oil.
Example 2
The essential oil composition comprises the following components in parts by volume: :2 parts of flos caryophylli essential oil and 3 parts of cinnamon essential oil.
Example 3
The essential oil composition comprises the following components in parts by volume: 1 part of flos caryophylli essential oil and 1 part of cinnamon essential oil.
Comparative example 1
The essential oil composition comprises the following components in parts by volume: 3 parts of flos caryophylli essential oil and 2 parts of cortex cinnamomi essential oil.
Comparative example 2
The essential oil composition comprises the following components in parts by volume: 4 parts of flos caryophylli essential oil and 1 part of cinnamon essential oil.
Comparative example 3
Single component: flos Caryophylli essential oil.
Comparative example 4
Single component: cinnamon essential oil.
Test example 1 bacteriostatic Activity test
1. Preparation of culture medium
Preparation of liquid culture medium: weighing 37g of brain-heart leaching liquid broth (Brain Heart Infusion Broth, BHI) for culture, adding distilled water into a beaker for dissolution, fixing the volume to 1L, and finally packaging into proper conical flasks, and sealing by using a sealing film; sterilizing at 120deg.C for 20 min.
Preparation of solid medium: weighing 37g of brain-heart leaching liquid broth (Brain Heart Infusion Broth, BHI) culture medium and 15g of agar in a beaker, adding a proper amount of distilled water, heating for dissolving, stirring uniformly, adjusting pH value, sub-packaging in conical flasks with proper size, and sealing with sealing film; sterilizing at 120deg.C for 20min, cooling to below 50deg.C, packaging into sterile petri dishes, and cooling on a clean bench.
2. Preparation of bacterial suspension
The bacterial liquid is selected and cultivated for 24 hours in nutrient agar streak, then single colony with good growth vigor is selected and inoculated into a liquid culture medium, and cultivated for 16 hours under the anaerobic condition at 37 ℃. Then after stepwise dilution, the plate is coated and adopted to count bacterial colony, calculate bacterial concentration and make 10 6 -10 7 CFU/mL suspension.
3. Measurement of zone of inhibition diameter
The diameter of the inhibition zone is measured by a filter paper sheet method. 200. Mu.L of the prepared bacterial suspension (2X 10) 6 CFU/mL) was uniformly spread on BHI agar medium, then, three sterile filter paper sheets having a diameter of 6mm were placed in each dish, 3 μl of essential oil was dropped in the center of the filter paper sheets, and incubated at 37 ℃ for 24 hours with an equal volume of sterile water as a blank control, and the diameter of the zone of inhibition was measured, and the measurement was repeated three times for each essential oil to average. The judgment standard of the inhibition zone experiment is the inhibition index calculated by positive control chlorhexidine.
4. Statistical treatment
All experiments were performed in triplicate. Experimental data were analyzed using SPSS19.0 software and the overall mean of each group was compared using one-way analysis of variance (ANOVA). Mapping was performed using Origin 8.5.1.
5. Antibacterial Activity test on Streptococcus mutans
The essential oils of examples 1-3 and comparative examples 1-4 were tested for their bacteriostatic activity using a filter paper sheet method, and the results of measurements on all zone diameters are shown in FIG. 1. The results show that the lilac flower essential oil and the cinnamon bark essential oil have synergistic antibacterial effect after being compounded according to the volume ratio of 1:4, 2:3 and 1:1. The compound essential oil has the antibacterial circle diameter larger than that of single essential oil, and the compound essential oil with the volume ratio of the lilac flower essential oil to the cinnamon bark essential oil of 4:1 and 3:2, and shows good synergistic effect, and specific antibacterial effect data are shown in table 1.
TABLE 1 antibacterial Activity test results
Test example 2 determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC)
MIC of compound essential oil in example 1 on Streptococcus mutans was determined using a micro broth dilution method. Since the essential oil is insoluble in water, it is mixed with 1:1 (v/v) DMSO: tween20 and BHI broth (as an emulsifier for the essential oil, facilitating contact with bacteria). Adding an essential oil solution of known concentration to 1×10 6 In CFU/mL bacterial suspension, shake culture is carried out for 24 hours at 37 ℃. The positive control group was 1% chlorhexidine, with 1:1 (v/v) DMSO: tween20 and BHI broth. The negative control group was DMSO/Tween 20 and BHI liquid medium containing 0.1% and 1:1 (v/v). The essential oil concentration corresponding to the fact that the growth of bacteria in the culture medium can be inhibited by naked eyes is the lowest inhibitory concentration, and the growth of the bacteria is expressed as turbidity.
After MIC determination, 100. Mu.L of each of the bacterial suspension with the lowest inhibitory concentration and the bacterial suspension with the concentration above the lowest inhibitory concentration was plated on BHI agar dishes, and anaerobic cultured at 37℃for 24 hours. The concentration of essential oil corresponding to bacterial liquid in which bacterial colony is not grown in the solid culture medium is MBC value. The test results are shown in Table 2.
TABLE 2 Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of essential oils
Test example 3 time-kill test on Streptococcus mutans
The minimum bactericidal concentration of the essential oil of lilac flower and cinnamomum cassia bark was determined to be 0.625 mul/mL by an agar dilution method, and the time-bactericidal curve of the essential oil composition in example 1 was measured to reflect the effect of MBC concentration on killing bacteria at different times. Streptococcus mutans (1X 10) was cultured in BHI medium 6 CFU/mL), and the mixed bacterial liquid is placed in a shaking table at 37 ℃ and 180rpm for shake culture under the MBC concentration. The bacterial suspension was spread on solid BHI agar medium every 1 day, cultured for 24 hours, and the change in the number of single colonies in the dish was recorded. Experiments were repeated 3 times and averaged. And drawing a sterilization curve by taking the action time as an abscissa and taking the natural logarithmic value of the viable count as an ordinate.
Bacterial suspension without essential oil composition is used as a negative control group, bacterial suspension with 1% chlorhexidine is used as a positive control group, and flos caryophylli and cortex cinnamomi essential oil composition with the ratio of 1:4 in the embodiment 1 of the invention is used as an experimental group; the time-sterilization curve of the essential oil composition at a concentration of 0.625. Mu.L/mL against Streptococcus mutans within 10 hours is shown in FIG. 2. The streptococcus mutans bacteria number of the blank control group gradually increases in the whole culture period. The compound essential oil of flos Caryophylli and cortex Cinnamomi in the ratio of 1:4 in the example 1 has sustained killing effect on Streptococcus mutans, and the more bacteria killed with time, the slightly enhanced sterilizing effect with time.
Test example 4 test of bacteriostatic mechanism against Streptococcus mutans
1. Influence on the acid producing ability of Streptococcus mutans
Testing the effects of the compositions of example 1 on the acid generating ability of Streptococcus mutans at MIC, 1/2MIC, 1/4MIC and 1/8MIC concentrations, adding essential oils of different concentrations to 2mL of a composition containing 1% glucose at a concentration of 1X 10 7 CFU/mL deformed sprocketIn the bacterial suspension. The initial pH1 was measured by a pH meter, and after anaerobic incubation in an incubator at 37℃for 24 hours, pH2 was again measured, and the difference DeltapH between the two pH values was the effect on acid production, and three repeated experiments were performed for each group, and the results are shown in Table 3. There were 3 parallel controls for each concentration.
TABLE 3 influence of Compound essential oils of different concentrations on acid generating Capacity of Streptococcus mutans
| Experimental group | Concentration of the composition | pH1 | pH2 | △pH |
| 1 | MIC | 7.04±0.00 | 6.99±0.02 | 0.05±0.02 |
| 2 | 1/2MIC | 7.04±0.00 | 7.01±0.03 | 0.03±0.03 |
| 3 | 1/4MIC | 7.04±0.00 | 7.01±0.01 | 0.03±0.01 |
| 4 | 1/8MIC | 7.04±0.00 | 5.39±0.06 | 1.65±0.06 |
| Control group | 0μL/mL | 7.04±0.00 | 4.75±0.03 | 2.29±0.03 |
The pH value change is measured to determine whether the compound essential oil with different concentrations inhibits the acidity production of the streptococcus mutans at the concentration of 1% of sucrose. After 24h incubation, the pH of the blank group without compound essential oil was reduced from 7.04 by 4.75. The lilac flower essential oil and cinnamon bark essential oil composition with the concentrations of MIC, 1/2MIC, 1/4MIC and 1/8MIC has obvious inhibition effect on the reduction of the pH value.
2. Effects on Streptococcus mutans biofilm
(1) Safranin staining method for observing morphology of biological film
Biofilm formation was measured by safranin staining and was observed with the naked eye and photographed. In a 35mm polystyrene culture dish, the compound essential oil of flos Caryophylli and cortex Cinnamomi at a ratio of 1:4 of example 1 is used as a study object, and essential oils with different concentrations are added into BHI broth containing 1% sucrose. With Streptococcus mutans seed cultures (2.0X10) 7 CFU/mL) was inoculated with the broth and cultured for 24h. After incubation, the supernatant was removed and the dish was rinsed with distilled water.
(2) Observation of the Effect of the amount of biofilm formation by Crystal Violet staining
The bacterial suspension and prepared liquid medicine of different concentrations are mixed and inoculated into 100 mu L of 96-well plates according to the proportion of 1:1 (v/v), and each concentration is 5 compound wells. After being mixed evenly by light blowing and sucking with a sterile straw, the 96-well plate is placed in a constant temperature bacteria incubator for anaerobic culture at 37 ℃. Bacterial-only BHI broth without essential oil was inoculated as a control group. After 24h incubation, the 96-well plates were removed, the mixture was aspirated, rinsed twice with sterile water, and stained with 0.4% crystal violet for 15min at 0.1 mL. The cells were rinsed three times with sterile water, 100. Mu.L of 95% ethanol solution was placed in each well, and the OD was measured at 540nm of the UV spectrophotometer with shaking for decolorization. The inhibition was calculated according to the formula and the results are shown in table 4.
Inhibition = (1-OD experimental group/OD control group) ×100%
The effect of 1:4 ratio of syringa and cinnamomum cassia compound essential oil at MIC, 1/2MIC, 1/4MIC and 1/8MIC concentration on Streptococcus mutans biological membrane was determined. The biofilm formation by Streptococcus mutans on the surface of the dishes was observed by safranin staining and crystal violet staining as shown in FIG. 3. The results show that the biological membrane structure is destroyed at MIC, surface bacteria die and fall off, the activity of the S.mutans biological membrane is reduced, and the concentration dependence exists.
TABLE 4 Effect of Compound essential oils at different concentrations on Streptococcus mutans biofilm
| Experimental group | Concentration of composition (μL/mL) | OD value | Inhibition ratio (%) |
| 1 | MIC | 0.26 | 86.41 |
| 2 | 1/2MIC | 0.45 | 76.54 |
| 3 | 1/4MIC | 0.56 | 70.48 |
| 4 | 1/8MIC | 0.82 | 56.76 |
| Control group | 0μL/mL | 1.90 | - |
3. Effect on Streptococcus mutans virulence factors
3.1RNA extraction method
(1) Collecting Streptococcus mutans grown to logarithmic phase, and preparing concentration of 10 5 CFU/mL of Streptococcus mutans suspension;
(2) Three different concentrations of essential oils, 1/8MIC, 1/4MIC and 1/2MIC, of the compound essential oil of example 1 were added thereto;
(3) Culturing at 37deg.C for 24h, centrifuging at 5000r/min for 5min;
(4) Washing with PBS and transferring the bacteria into an RNase-free centrifuge tube, centrifuging at 5000r/min for 5min, and then carefully removing the supernatant;
(5) Grinding and mashing a sample in liquid nitrogen, adding 1mL of Transzol, blowing by a gun head uniformly to completely crush cells, standing for 5min, adding chloroform to remove protein, standing for 5-10min,10000g, centrifuging for 15min at 4 ℃, then taking the supernatant in a new RNase-free centrifuge tube, adding an equal amount of ethanol, adding into an RNase-free column, passing through the column, centrifuging for 30 seconds at 12000g and 4 ℃, and discarding the supernatant;
(6) 500. Mu.L of CB9 solution is added, 12000g is centrifuged for 1min and repeated 1 time;
(7) 500. Mu.L WB9 solution was added and the mixture was centrifuged 1min at 12000g and repeated 1 time;
(8) Spin 2min,12000g;
(9) The RNase-free column was placed in a new RNase-free centrifuge tube, dried at room temperature for 10min, and then added with 20. Mu.L of RNase-free water to stand for 2min,12000g, and centrifuged for 1min.
3.2 reverse transcription
Table 5 shows the reverse transcription reaction system.
TABLE 5 reverse transcription reaction system
After the addition of the reagents was completed, the tube was placed in a PCR apparatus at 37℃for 5 minutes.
Synthesis of cDNA: after adding the reagents according to Table 6, the centrifuge tube was placed into a PCR instrument for reaction at 37℃for 15min; reacting for 5min at 50 ℃; the reaction was carried out at 98℃for 5min.
TABLE 6 reverse transcription reaction system
3.3 real-time fluorescence quantification
Primer synthesis and design: the full-length sequence of mRNA of each target gene was obtained from GenBank, and primers were designed using software Primer 5.0. The primer sequence of the target gene and the primer sequence of the 16S rRNA of the internal reference gene are shown as 7.
Table 7 primer sequence for target gene and primer sequence for 16S rRNA of internal reference gene
RT-PCR: according to Table 8, the various substances in the table were added to a 200. Mu.L centrifuge tube, mixed well, and placed in a PCR apparatus, and set: pre-denaturation at 95℃for 2min; deforming at 95 ℃ for 30s; annealing at 57 ℃ for 30s; extending at 72 ℃ for 30s; x 38 cycles; the reaction was carried out under reaction conditions of 72℃for 2 min.
TABLE 8 fluorescent quantitative PCR reaction System
Note that: the fluorescent dye includes a 10 x buffer,green 1, taq enzyme, dNTPs.
The compound essential oil of the example 1 is taken as a study object, the streptococcus mutans is treated by an essential oil composition with a subminimum inhibitory concentration (1/8-1/2 MIC) and a negative control group (chlorhexidine 1%) for culturing for 24 hours, and the influence of the compound essential oil on the expression of virulence factors of the streptococcus mutans is detected by adopting a real-time fluorescence quantitative PCR method. As shown in fig. 4-7, mRNA expression levels of brpA, gbpB, gtfB, gtfC, gtfD, vicR, spaP and relA genes were significantly reduced after treatment with the compound essential oils.
The results show that the compound essential oil concentration of example 1 is 1/2MIC, the number of bacteria is reduced, the colony effect among bacteria is weakened, and the expression of virulence factors is reduced. Inhibition of biofilm formation by inhibition of gtfB, gtfC, gtfD and vicR, inhibition of acid production by inhibition of brpA and relA expression. The spaP, gbpB plays a key role in the adhesion of Streptococcus mutans to tooth surfaces.
The invention provides a flos caryophylli essential oil and cortex cinnamomi essential oil composite essential oil. The ratio of the finally optimized flos caryophylli essential oil to the cortex cinnamomi essential oil is 1:1-4, wherein the specific implementation proportion is 1:4, 2:3 and 1:1 respectively, and the optimal proportion is 1:4. The active ingredients of the flos caryophylli essential oil are 72.88 percent of eugenol and 16.98 percent of caryophyllene. The effective components of the cinnamon essential oil comprise (E) -cinnamaldehyde 60.98% and cinnamyl acetate 10.95%. The flos Caryophylli essential oil and cortex Cinnamomi essential oil are recovered by water distillation.
The flos caryophylli essential oil and cortex cinnamomi essential oil composition has good antibacterial effect on streptococcus mutans, and has obvious synergistic effect; the essential oil composition has obvious inhibition effect on the growth, acid production capacity, biological film formation capacity and adhesion capacity of the streptococcus mutans, so that the essential oil composition has potential for preventing dental caries caused by the streptococcus mutans.
The foregoing detailed description is directed to one of the possible embodiments of the present invention, which is not intended to limit the scope of the invention, but is to be accorded the full scope of all such equivalents and modifications so as not to depart from the scope of the invention.
Claims (10)
1. The composite essential oil composition is characterized in that the raw materials of the composition comprise flos caryophylli essential oil and cortex cinnamomi essential oil.
2. The essential oil composition according to claim 1, wherein the volume ratio of the lilac flower essential oil to the cinnamon bark essential oil is 1:0.8-10.
3. The essential oil composition according to claim 1, wherein the volume ratio of the lilac flower essential oil to the cinnamon bark essential oil is 1:1-4.
4. The essential oil composition according to claim 1, wherein the effective components of the lilac flower essential oil comprise, in mass percent, 72% -73% of eugenol and 16% -18% of caryophyllene.
5. The essential oil composition according to claim 1, wherein the effective components of the cinnamon bark essential oil comprise (E) -cinnamaldehyde 55% -65% and cinnamyl acetate 9% -11% by mass.
6. The essential oil composition of claim 1, wherein the lilac flower essential oil and the cinnamon bark essential oil are both recovered by water distillation.
7. A method for preparing the composition according to any one of claims 1 to 6, wherein the lilac flower essential oil and the cinnamon bark essential oil are mixed.
8. Use of a composition according to any one of claims 1 to 6 or a composition obtainable by a process according to claim 7 for the preparation of a medicament or oral care product for inhibiting oral bacteria.
9. The use according to claim 8, wherein the oral bacteria comprise streptococcus mutans and the composition is capable of treating dental caries.
10. The use according to claim 8, wherein the medicament or care product is capable of inhibiting the growth, acid production, biofilm formation and/or adhesion capacity of streptococcus mutans.
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