CN109125342A - The new application of ozone carburetion preventing/treating periodontosis - Google Patents
The new application of ozone carburetion preventing/treating periodontosis Download PDFInfo
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Abstract
The invention discloses the new applications of ozone carburetion preventing/treating periodontosis, and the beneficial effects of the invention are as follows ozone carburetion to have good effect for preventing/treating periodontosis.Ozone carburetion antibacterial activity height, natural environmental-protective, Small side effects can provide a kind of new selection for the prevention and treatment of periodontosis.
Description
Technical field
The invention belongs to pharmaceutical technology fields, are related to ozone carburetion in preparation for preventing and/or treating periodontal medicine
In application.
Background technique
Periodontitis is to invade chronic infectious disease caused by Periodontal Supporting Tissue as bacterial plaque microorganism, with periodontal connective group
Knit degradation, absorption of alveolar bone be main feature, be cause China adult lose tooth the main reason for.
Periodontitis is mainly related with the field planting of Grain-negative anaerobe, especially gum porphin beautiful jade monad, tool core shuttle
Bacillus assembles the main pathogenic bacteria that bacillus etc. is considered as periodontosis with unwrapping wire.So the main purpose of periodontitis treatment is to disappear
Except the biomembrane of pathogen.Mechanical debridement art is to treat traditional initial step of periodontitis on gum, under gum, to Doctors' skill requirement
High and operation is more difficult.In addition, being only difficult fully erased periodontal pathogen by mechanical debridement art.Therefore some local antimicrobials and
Locally or systemically antibiotic is used to increase mechanical removal effect.However, locally and systemically antibiotic treatment have it is several apparent
Disadvantage, it is such as antibacterial that there is selectivity, lead to bacterial drug resistance and host's adverse reaction.Most popular antibacterial in periodontal treatment
Agent Chlorhexidine (CHX) can lead to mucous membrane and fall off, and dental stain and the sense of taste change.In addition, even if the CHX of low concentration to gum at
Fibrocyte is also toxic, to reduce the generation of collagen and non-collagen, hinders periodontal healing.Common periodontal is locally anti-
There are also 3% hydrogen peroxide (H for microbial inoculum2O2), but a large amount of bubbles can be generated when hydrogen peroxide effect, it carries out under deeper oral pocket gum
It may result in pneumoderm when flushing.Therefore, selection antibacterial activity is high, safety is good, Small side effects complementary antimicrobials
Object has important value to the treatment of periodontosis.
Ozone (O3) it is a kind of light blue gas with fishy smell, it is oxygen (O2) allotrope, have extremely strong oxygen
The property changed.Ozone is unstable, can voluntarily be decomposed into oxygen, and room temperature half-life is 20-30min.Under normal temperature and pressure, ozone is in water
Solubility than 25 times of air, it is 13 times higher than oxygen.Early stage is applied primarily to remove the harmful in drinking water and sanitary sewage
Close object.It is now discovered that ozone all has stronger killing effect to bacterium, fungi, virus.Also there are many reports to point out in recent years smelly
Oxygen also has the effects that anti-inflammatory, anti-oxidant, promotion organization healing, hemostasis.For ozone application there are mainly three types of form, gaseous state is smelly at present
Oxygen, liquid ozone and ozone carburetion.Ozone carburetion is to be passed through ozone gas using oil as carrier and be wherein made, economic and environment-friendly,
Stability is good, is not easily decomposed, can long-term preservation, experiment and clinical application feasibility it is higher.Studies have reported that ozone carburetion
It applies and obtains preferable curative effect in the diseases such as angiocarpy, gynaecology, skin, liver, joint and stomach, ozone carburetion is to large intestine
The various bacterias such as bacillus, staphylococcus aureus, candida albicans, virus have significant killing effect.However about ozonisation
The research that oil is applied in periodontal disease is seldom, to main pathogenic bacteria (the gum porphin beautiful jade monad, tool core shuttle of three kinds of periodontosis
Bacillus assembles bacillus with unwrapping wire) antibacterial effect and minimum antibacterial, bacteriocidal concentration research have not been reported.The present invention passes through phase
Experimental study is closed, confirms ozone carburetion from many aspects to the antibacterial action of above-mentioned three kinds of periodontosis main pathogenic bacterias, for its conduct
The application offer for preventing and treating periodontal medicine may.
Summary of the invention
The purpose of the present invention is to provide the new application of ozone carburetion preventing/treating periodontosis, beneficial effects of the present invention
It is that ozone carburetion is used for preventing/treating periodontosis with good effect.Ozone carburetion antibacterial activity height, natural environmental-protective, pair are made
With small, a kind of new selection can be provided for the prevention and treatment of periodontosis.
Ozone carburetion preventing/treating periodontosis.
Further, fungistatic effect of the ozone carburetion to gum porphin beautiful jade monad, Fusobacterium nucleatum, companion unwrapping wire aggregation bacillus.
Further, the ozone carburetion of low concentration, which has, assembles bacillus to gum porphin beautiful jade monad, Fusobacterium nucleatum, with unwrapping wire
Antibacterial activity.
Further, ozone carburetion has to gum porphin beautiful jade monad, Fusobacterium nucleatum, with unwrapping wire aggregation bacillus biofilm formation
Inhibiting effect.
Further, biofilm of the ozone carburetion to gum porphin beautiful jade monad, Fusobacterium nucleatum, companion unwrapping wire aggregation bacillus
Removing it is effective, and the scavenging effect of gum porphin beautiful jade monad, Fusobacterium nucleatum biofilm is significantly stronger than with putting
Line assembles bacillus.
Detailed description of the invention
Fig. 1 is fungistatic effect figure of the ozone carburetion to gum porphin beautiful jade monad, Fusobacterium nucleatum, companion unwrapping wire aggregation bacillus.
Fig. 2 is the shadow that ozone carburetion assembles bacillus biofilm formation to gum porphin beautiful jade monad, Fusobacterium nucleatum, companion's unwrapping wire
Loud statistical chart；
Fig. 3 is the shadow that ozone carburetion assembles bacillus biofilm to gum porphin beautiful jade monad, Fusobacterium nucleatum, companion's unwrapping wire
Loud statistical chart.
Specific embodiment
The present invention is described in detail With reference to embodiment.
Experimental strain: gum porphin beautiful jade monad (Porphyromonas gingivalis, P.gingivalis, Pg)
ATCC33277, Fusobacterium nucleatum (Fusobacterium nucleatum, F.nucleatum, Fn) ATCC23726, companion's unwrapping wire
Assemble bacillus (Actinobacillus actinomycetemcomitans, A.actinomycetemcomitans, Aa)
ATCC29523 is provided by mouth disease research National Key Laboratory of Nanjing Medical University.
Recovery, culture and the preparation of bacteria suspension of bacterium: by the gum porphin beautiful jade monad ATCC33277 frozen, tool core shuttle
Bacillus ATCC23726, it is recovered in BHI culture medium respectively with unwrapping wire aggregation bacillus ATCC29523 international standard strain, is put into 37 DEG C
Constant-temperatureanaerobic anaerobic case (80%N2, 10%CO2, 10%H2) culture, wherein (gum porphin beautiful jade monad has core shuttle bar to obligate anaerobe
Bacterium) culture 72h, facultative anaerobic bacteria (with unwrapping wire assemble bacillus) culture 48h after, take its pure culture to be inoculated in Liquid Culture respectively
Culture increases bacterium 48h under same culture conditions in base, and using turbidimetry for Determination bacteria suspension concentration, phosphate buffer (PBS) is prepared
Concentration is 1 × 108CFU/mL and 2 × 106The bacterium bacteria suspension of CFU/mL is spare.
1 AGP test of embodiment tests (punch method) assessment ozone carburetion to gum porphin beautiful jade monad, Fusobacterium nucleatum, companion
The fungistatic effect of unwrapping wire aggregation bacillus.
Aseptically, 1 × 10 that 100ul has diluted is taken8The bacteria suspension of CFU/mL aseptic inoculation ring even spread
In on the culture dish (90mm) of agar medium containing BHI (25ml), stand several minutes.It is equal on culture dish with the punch of 6mm
It is even to make a call to 4 holes, and the distance between two holes should be greater than 24mm, hole should be greater than 15mm at a distance from culture dish edge.Add in every hole
Enter the corresponding reagent of 100ul, experimental group is ozone carburetion (90mg/ml), negative control group PBS, and positive controls are
0.12% Chlorhexidine and 1% jodine glycerin.It is put into 37 DEG C of constant-temperatureanaerobic anaerobic case (80%N2, 10%CO2, 10%H2) culture 48h
(gum porphin beautiful jade monad culture 72h), visually observes, at the same taken pictures with bacterium colony automatic counter for counting, to measure and record inhibition zone straight
Diameter is indicated with mm.Using the region for visually having no bacterium colony growth as inhibition zone edge, there is a small amount of indecipherable tiny bacterium in inhibition zone
It falls including disregarding.All experiments repeat at least three times.
The micro broth dilution method of embodiment 2 is probed into ozone carburetion and is gathered to gum porphin beautiful jade monad, Fusobacterium nucleatum, with unwrapping wire
Collect the antibacterial activity of bacillus.
Experiment is divided into blank group (culture medium without bacteria suspension), (culture medium containing bacteria suspension contains two groups of negative control group
The bacteria suspension culture medium of PBS), two groups of positive controls (bacteria suspension culture medium, the iodine containing various concentration of the Chlorhexidine containing various concentration
The bacteria suspension culture medium of glycerol), experimental group (the bacteria suspension culture medium of the carburetion of ozone containing various concentration).Two are pressed with BHI culture medium
Times dilution method respectively by 0.12% Chlorhexidine, 1% jodine glycerin and the ozone carburetion of 90mg/ml be diluted to 1:2,1:4,1:8,
Totally 11 concentration gradients of 1:16,1:32,1:64,1:128,1:256,1:512,1:1024,1:2048.To the every Kong Yi of 96 orifice plates
Secondary reagent (two groups of the negative control group BHI and 100ul for being separately added into 100ul that the good various concentration of the above-mentioned dilution of 100ul is added
PBS), then by 100ul configured 2 × 106The bacterial suspension inoculation of CFU/mL makes every hole final bacterial concentration 1 in each hole
×106CFU/mL.But bacteria suspension is not added in blank group, and every hole is only the sterile BHI culture medium of 200ul.3 multiple holes of every group of setting.
It is put into 37 DEG C of constant-temperatureanaerobic anaerobic case (80%N2, 10%CO2, 10%H2) in cultivate 48-72h, it is all that visually observe liquid in micropore limpid
Growing without muddy or bottom without precipitating is then minimal inhibitory concentration (Minimum Inhibitory Concentration, MIC).
10ul will be taken to be spread evenly across on BHI agar medium with aseptic inoculation ring after liquid blending in hole, is put into 37 DEG C of constant-temperatureanaerobic anaerobics
Case (80%N2, 10%CO2, 10%H2) in cultivate 48-72h, observation without bacterial growth is then minimum bactericidal concentration (Minimum
Bactericidal Concentration, MBC).All experiments repeat at least three times.
3 crystal violet staining assay of embodiment is assessed ozone carburetion and is assembled to gum porphin beautiful jade monad, Fusobacterium nucleatum, with unwrapping wire
The influence of bacillus biofilm formation.
The acquisition of saliva: the nonirritant static saliva of three normal adults is taken, is taken after 10000 × g centrifugation 20min
Clearly, it with the sterile disposable filter filtration sterilization in the aperture 0.22um, is stored for future use after packing in -80 DEG C.
Construct the external adhesive model of saliva coated: the saliva of 10ul is added in every hole in 96 orifice plates, under 37 DEG C of environment
1h is stood, extra saliva, naturally dry under aseptic condition are discarded.
It is 2 × 10 that the concentration that 100ul has diluted, which is added, to 96 orifice plates7The bacteria suspension of CFU/mL, then it is separately added into 100ul
1/4 × MIC, 1/2 × MIC, MIC, 2 × MIC, 4 × MIC, five kinds of concentration corresponding reagent, make every hole final bacterial concentration 1 ×
107CFU/mL.Only to add the hole of bacteria suspension and BHI culture medium as negative control hole, only to add the hole of sterile BHI culture medium to make
For blank well.3 multiple holes of every group of setting.It is put into 37 DEG C of constant-temperatureanaerobic anaerobic case (80%N2, 10%CO2, 10%H2) in culture for 24 hours after,
Original liquid is discarded, PBS is washed three times to remove suspended bacterial, and movement is light in the process, in order to avoid destroy established biomembrane.So
The fixed 15min of methanol of 200ul is added afterwards, 0.1% crystal violet that 200ul is added after drying is protected from light at room temperature is incubated for 20min,
With deionized water by excessive dyestuff wash clean, dry at room temperature.Every hole adds ethyl alcohol dissolution and the cell of 200ul 95%
In conjunction with dyestuff, be protected from light at room temperature be incubated for 20min.OD value is read at 595nm with microplate spectrophotometer.All experiments are extremely
Less in triplicate.Biofilm formation rate (%)=[(experimental group OD- blank group OD)/(control group OD- blank group OD)] ×
100%.
4 crystal violet staining assay of embodiment is assessed ozone carburetion and is assembled to gum porphin beautiful jade monad, Fusobacterium nucleatum, with unwrapping wire
The influence of bacillus biofilm.
The acquisition of saliva: the nonirritant static saliva of three normal adults is taken, is taken after 10000 × g centrifugation 20min
Clearly, it with the sterile disposable filter filtration sterilization in the aperture 0.22um, is stored for future use after packing in -80 DEG C.
Construct the external adhesive model of saliva coated: the saliva of 10ul is added in every hole in 96 orifice plates, under 37 DEG C of environment
1h is stood, extra saliva, naturally dry under aseptic condition are discarded.
It is 1 × 10 that the concentration that 200ul has diluted, which is added, to 96 orifice plates7The bacteria suspension of CFU/mL is put into 37 DEG C of constant-temperatureanaerobic anaerobics
Case (80%N2, 10%CO2, 10%H2) in after culture for 24 hours, discard original liquid, the sterile BHI culture medium of 100ul be added, then
Be separately added into 1/4 × MIC of 100ul, 1/2 × MIC, MIC, 2 × MIC, 4 × MIC, five kinds of concentration corresponding reagent, with containing only
There is the hole of bacteria suspension and BHI culture medium as negative control hole, to only have the hole of sterile BHI culture medium as blank well.Every group
3 multiple holes are set.It is put into 37 DEG C of constant-temperatureanaerobic anaerobic case (80%N2, 10%CO2, 10%H2) in culture for 24 hours after, discard original liquid,
PBS is washed three times to remove suspended bacterial, and movement is light in the process, in order to avoid destroy established biomembrane.Then it is added 200ul's
Methanol fixes 15min, and 0.1% crystal violet that 200ul is added after drying is protected from light at room temperature is incubated for 20min, will with deionized water
Excessive dyestuff wash clean, dries at room temperature.Every hole adds the dyestuff of the ethyl alcohol dissolution and cell combination of 200ul 95%, room
It is protected from light under temperature and is incubated for 20min.OD value is read at 595nm with microplate spectrophotometer.All experiments are at least in triplicate.It is raw
Object film total amount (%)=[(experimental group OD- blank group OD)/(control group OD- blank group OD)] × 100%.
Experimental result:
1. ozone carburetion is to the fungistatic effect of gum porphin beautiful jade monad, Fusobacterium nucleatum, companion unwrapping wire aggregation bacillus.
As a result as shown in Figure 1, ozone carburetion has apparent bacteriostasis to above-mentioned three kinds of periodontosis main pathogenic bacterias, especially
It is to be substantially better than 0.12% Chlorhexidine, 1% jodine glycerin to the fungistatic effect of gum porphin beautiful jade monad and Fusobacterium nucleatum.Wherein
A, B are fungistatic effect audio-visual picture of the ozone carburetion to gum porphin beautiful jade monad in figure；C is suppression of the ozone carburetion to Fusobacterium nucleatum
Bacterium effect audio-visual picture；D is ozone carburetion to the fungistatic effect audio-visual picture with unwrapping wire aggregation bacillus；E is ozone carburetion to above-mentioned three
The statistical chart of bacterium inhibition zone size is planted, antibacterial circle diameter is indicated with mean ± standard error, phase between experimental group and positive controls
Mutually relatively: P < 0.001 * P < 0.05, * * P < 0.01, * * *；Compared with negative control group: #P < 0.05, ##P < 0.01, ###P <
0.001。
2. ozone carburetion is to the antibacterial activity of gum porphin beautiful jade monad, Fusobacterium nucleatum, companion unwrapping wire aggregation bacillus.
The results are shown in Table 1, and ozone carburetion is 0.0439mg/ml, to tool to the MIC and MBC of gum porphin beautiful jade monad
The MIC and MBC of core Fusobacterium are 0.1758mg/ml, are 5.6250mg/ml to the MIC with unwrapping wire aggregation bacillus, MBC is
11.2500mg/ml.This is the result shows that the ozone carburetion of low concentration can play the role of anti-Periodontal Pathogens.
The micro broth dilution method of table 1. assesses ozone carburetion to the minimal inhibitory concentrations (MIC) of three kinds of Periodontal Pathogens and most
Small bacteriocidal concentration (MBC).
3. influence of the ozone carburetion to gum porphin beautiful jade monad, Fusobacterium nucleatum, companion's unwrapping wire aggregation bacillus biofilm formation.
As a result as shown in Fig. 2, being compared with untreated fish group: * P < 0.05, * * P < 0.01, * * * P < 0.001.MIC: most
Low Mlc；MBC: minimum bactericidal concentration.
Ozonisation oil concentration can largely inhibit gum porphin beautiful jade monad when being 1/4 × MIC, but 0.12% Chlorhexidine only exists
MIC, 1% jodine glycerin only just plays inhibiting effect to gum porphin beautiful jade monad biofilm formation in 1/2 × MIC concentration, and presses down
Rate processed is low compared with ozone carburetion；Suppression of the ozone carburetion in 1/4 × MIC, 1/2 × MIC concentration to Fusobacterium nucleatum biofilm formation
Rate processed is high compared with 0.12% Chlorhexidine, 1% jodine glycerin；Ozone carburetion assembles bacillus biomembrane to unwrapping wire in 1/4 × MIC concentration
The inhibiting rate of formation is higher than 0.12% Chlorhexidine, 1% jodine glycerin.The result shows that ozone carburetion is in low concentration to above-mentioned three kinds
The formation of Periodontal Pathogens biomembrane has good inhibiting effect.
4. influence of the ozone carburetion to gum porphin beautiful jade monad, Fusobacterium nucleatum, companion's unwrapping wire aggregation bacillus biofilm.
As a result as shown in figure 3, being compared with untreated fish group: * P < 0.05, * * P < 0.01, * * * P < 0.001.MIC: most
Low Mlc；MBC: minimum bactericidal concentration.
Ozone carburetion is effective to the removing of the biofilm of three kinds of Periodontal Pathogens, and to gum porphin beautiful jade unit cell
The scavenging effect of bacterium, Fusobacterium nucleatum biofilm, which is significantly stronger than, assembles bacillus with unwrapping wire.
It is right by comparing ozone carburetion and two kinds of clinically used auxiliary antibacterial agents (0.12% Chlorhexidine, 1% jodine glycerin)
The antibacterial effect of three kinds of periodontosis main pathogenic bacterias explores its potential application foreground in periodontosis prevention and treatment.Inventor passes through
Experimental results demonstrate ozone carburetion to gum porphin beautiful jade monad, Fusobacterium nucleatum, with the shape of unwrapping wire aggregation bacillus and its biomembrane
Inhibiting effect is played at development, and probes into its minimal inhibitory concentration (MIC), minimum bactericidal concentration to this three kinds of bacteriums
(MBC), minimum to inhibit biomembrane concentration (MBIC) and minimum elimination biomembrane concentration (MBEC), to play prevention and treatment periodontosis
Effect has potential new drug development value.The ozone carburetion of low concentration can play periodontosis main pathogenic bacteria relatively strong
Inhibiting effect, and it has the characteristics that natural, economical, environmental protection, Small side effects, is it as the auxiliary antibacterial for treating periodontosis
Agent provides potential application prospect.
The above is only not to make limit in any form to the present invention to better embodiment of the invention
System, any simple modification that embodiment of above is made according to the technical essence of the invention, equivalent variations and modification,
Belong in the range of technical solution of the present invention.
Claims (5)
1. ozone carburetion preventing/treating periodontosis.
2. according to ozone carburetion preventing/treating periodontosis described in claim 1, it is characterised in that: the ozone carburetion is to gum
Porphin beautiful jade monad, Fusobacterium nucleatum, companion's unwrapping wire assemble the fungistatic effect of bacillus.
3. according to ozone carburetion preventing/treating periodontosis described in claim 1, it is characterised in that: the ozonisation of the low concentration
Oil has to gum porphin beautiful jade monad, Fusobacterium nucleatum, with the antibacterial activity of unwrapping wire aggregation bacillus.
4. according to ozone carburetion preventing/treating periodontosis described in claim 1, it is characterised in that: the ozone carburetion is to gum
Porphin beautiful jade monad, Fusobacterium nucleatum have inhibiting effect with unwrapping wire aggregation bacillus biofilm formation.
5. according to ozone carburetion preventing/treating periodontosis described in claim 1, it is characterised in that: the ozone carburetion is to gum
The removing for the biofilm that porphin beautiful jade monad, Fusobacterium nucleatum, companion's unwrapping wire assemble bacillus is effective, and to gum porphin beautiful jade list
The scavenging effect of born of the same parents bacterium, Fusobacterium nucleatum biofilm, which is significantly stronger than, assembles bacillus with unwrapping wire.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20060074129A1 (en) * | 2002-04-08 | 2006-04-06 | Mirabal Jesus M | Method for obtaining ozonized oils and vegetable fats and use of said products for pharmaceutical and cosmetic purposes |
| CN102178632A (en) * | 2011-05-06 | 2011-09-14 | 石平安 | Application of ozonized oil to preparation of raw materials of medical health-care products |
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2018
- 2018-09-17 CN CN201811080356.5A patent/CN109125342A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20060074129A1 (en) * | 2002-04-08 | 2006-04-06 | Mirabal Jesus M | Method for obtaining ozonized oils and vegetable fats and use of said products for pharmaceutical and cosmetic purposes |
| CN102178632A (en) * | 2011-05-06 | 2011-09-14 | 石平安 | Application of ozonized oil to preparation of raw materials of medical health-care products |
Non-Patent Citations (4)
| Title |
|---|
| HUTH，等: "Effectiveness of ozone against periodontal pathogenic microorganisms", 《EUROPEAN JOURNAL OF ORAL SCIENCES》 * |
| PATEL，等: "Effect of subgingival application of topical ozonated olive oil in the treatment of chronic periodontitis: a randomized, controlled, double blind, clinical and microbiological study", 《MINERVA STOMATOLOGICA》 * |
| SHOUKHEBA，等: ""The effects of subgingival application of ozonated olive oil gel in patient with localized aggressive periodontitis. A clinical and bacteriological study"", 《TANTA DENTAL JOURNAL》 * |
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Application publication date: 20190104 |