CN104606085A - Novel efficient antimicrobial chlorhexidine acetate nanoemulsion gargle and preparation method thereof - Google Patents

Novel efficient antimicrobial chlorhexidine acetate nanoemulsion gargle and preparation method thereof Download PDF

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CN104606085A
CN104606085A CN201410273137.4A CN201410273137A CN104606085A CN 104606085 A CN104606085 A CN 104606085A CN 201410273137 A CN201410273137 A CN 201410273137A CN 104606085 A CN104606085 A CN 104606085A
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emulsion
chlorhexidine acetate
nano
collutory
parts
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CN104606085B (en
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孙红武
刘开云
李云飞
李世音
郭刚
邹全明
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Third Military Medical University TMMU
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Abstract

This invention provides an efficient antimicrobial chlorhexidine acetate nanoemulsion gargle and a preparation method thereof. The nanoemulsion gargle provided by the invention is prepared from the following raw and supplemental materials by weight: 0.2-1.5 parts of chlorhexidine acetate, 8-30 parts of a surfactant, 4-15 parts of a cosurfactant, 3-20 parts of an oil phase, 0.05-1 part of an oral cavity refreshing agent, and 34.45-69.0 parts of distilled water. The nanoemulsion gargle provided by the invention can obviously improve the solubility, has extremely stable quality, and especially has the function of obviously enhancing the in vitro and in vivo antimicrobial effect on oral pathogenetic bacteria represented streptococcus mutans and the like by cell membrane destruction. The product provided by the invention has the characteristics of convenient use, low cost, remarkable curative effect, no alcohol, safety and no toxicity, no stimulation, no side effect and no drug resistance, and is a novel oral cavity sterilization, disinfection and cleaning product. The gargle can be widely applied to periodontitis, gingivitis, oral ulcer, dental plaque, dental bleeding, halitosis and other oral diseases.

Description

A kind of new and effective antibacterial chlorhexidine acetate nano-emulsion collutory and preparation method thereof
Technical field
The invention belongs to field of medicaments, relate to a kind of nano-emulsion collutory efficiently can treating oral disease, particularly a kind of new and effective antibacterial chlorhexidine acetate nano-emulsion collutory and preparation method thereof.
Background technology
Because internal environment of oral cavity (humidity, temperature and nutriture) is particularly suitable for microbial growth, so oral cavity is that in human body, microbe number exists maximum positions.There are some researches show: there are 10,000,000,000 antibacterials in each gram of tartar (tooth rust) of normal healthy people, in oral cavity, antibacterial is more much more than the quantity of skin surface.Therefore, oral cavity cleaning not in time, can cause halitosis, various focusing depths represented, oral ulcer, decayed tooth, periodontal disease various diseases, thus the health of serious harm people.The clean of oral cavity becomes the first step of health of people health and the important component part of daily life.At present, usually clean to oral cavity, adopts though most and brushes teeth, to use between dental floss, toothpick, tooth the external force such as brush, oral rinsing device, oral cleansing lotion to reach cleaning action, for the antibacterial grown in oral cavity, particularly the people of serious, the periodontal inflammation of abnormal flavour, can not reach and effectively dispel.And brush teeth be confined to get up morning after and before sleep in evening, use also inconvenient, cleaning not in time, not thoroughly, have the drawbacks such as wearing and tearing to tooth, is difficult to the oral cavity cleaning requirement meeting people.Collutory is as a kind of novel oral cavity cleaning and nursing product, progress in the life of mediocrity, the impulsive force that it not only can utilize liquid to flow in mouth, to remove the food debris of delay, can also utilize the effective ingredient added in collutory to remove the malignant bacteria in oral cavity.It is packaged in portable Packaging Bottle, can use at any time, can thoroughly overcome mouth care not in time, toothpaste uses inconvenient, the clean drawback such as not thorough, thus cleaning teeth, tooth-whitening, removal dental plaque can be played, kill the effects such as oral cavity noxious bacteria, caries prevention, fresh breath.It especially plays certain positive role to the treatment of such as some oral diseases such as various focusing depths represented, has the effects such as cleaning oral cavity, caries prevention and periodontitis.Therefore, it is very popular and widely use in recent years.At present, on market, collutory kind is a lot, mainly contains the compound preparation of the chemicals such as fluoride, triclosan (the chloro-2-dihydroxy diphenyl ether of 2,4,4-tri-), hibitane (chlorhexidine), metronidazole, silver nitrate and Chinese herbal medicine.And most widely used fluoride and silver nitrate.At present, though most widely used fluoride can form fluor-apatite protective layer (CaF2) at enamel surface, strengthen tooth acid-resisting, it may cause fluorosis, osteofluorosis, dental fluorosis after using, the generation of a large amount of F resistant Strain can also be caused, lower curative effect; Nerve fiber cell in silver nitrate Neng Shi dentinal tubule solidifies and forms protective layer; reduce neural reflex; slow down the symptoms such as pain; but can be corrosive and zest to soft tissue; the simultaneously excessive meeting of Ag+ is harmful, and Ag+ and intraoral reducing substances react and generate Ag and cause tooth blackening sometimes.And other antibiotic directly suppress or kill oral cavity bacterium, but antibiotic abuse easily causes bacterial resistance widely, thus lessens the curative effect.Compound Chinese medicinal preparation conventional in existing collutory has Herba Senecionis Scandentis, Rhizoma Smilacis Glabrae, Cacumen Securinegae Suffruticosae, Folium pterocaryae stenopterae, hops etc., but due to Chinese herbal medicine extract that traditional water extraction method, solvent extraction method are obtained inevitably have that active matter content is low, content is difficult to determine, solvent composition is difficult to problems such as removing, gained composition is too complicated, be very easy to affect its use mouthfeel.If add more essence to improve the mouthfeel of product, too much use essence may have potential threat to the health of human body again.
Therefore, though have many various collutorys at present, chase after its final curative effect all not satisfactory, at most just play the effect postponing pain or cleaning teeth.Many dental patient are difficult to edible cold and hot sour-sweet hard grade food, particularly dental caries patient, usually hurt like hell, serious have to pull out tooth, also has some patients, because there is heredity and pathological factor, and can not get effective treatment and control, cause tooth to occur too early loosening or come off.From medical point, ideal is not had to treat amplification and medicine efficiently at present, the department of stomatology generally sodium-chloride water solution that adopts is gargled more, the problem of interim solution pain, but do not reach the object of healing, from health care angle, sell various collutory and medicated toothpaste in the market, time only plays general health-care effect, very micro-to general oral disease curative effect.And the collutory on market is mostly containing alcoholic content, alcohol content in some collutory is up to 26%, professor Luo Binximo of the restorative dentistry of University of Newcastle represents, ethanol itself can destroy the private film keeping oral cavity moistening, make xerostomia, and then cause halitosis, oral cavity also can be made to produce uncomfortable prickling sensation.
Domestic and international research shows, a kind of broad-spectrum antiseptic non-antibiotic class, is had therapeutical effect by the disinfecting drug chlorhexidine acetate being widely used in various apparatus and operation at present to oral disease.Now research shows, it is combined by the phosphate in lipopolysaccharide and the carboxyl in chlorhexidine acetate, thus the transhipment of interference cell film, make small-molecule substance overflow the outer mechanism of born of the same parents, thus cause bacterial death.But chlorhexidine acetate is low effect disinfectants, and due to its dissolubility in water low (0.2g/100g), just due effect must can be played with other preparations are composite.Only a small amount of after passing through dissolve with ethanol at present, and other Chinese medicines, chemicals (the most frequently used fluoride and benzalkonium bromide) compatibility improve its antibacterial activity.But compatibility conbined usage, as fluoride to the certain drug resistance greatly of antibacterial, can produce a large amount of F resistant Strain, yet can with the serious side effect such as fluorosis, osteofluorosis, dental fluorosis; Benzalkonium bromide is a kind of quaternaries cation surface disinfectant, is widely used in the external sterilizings such as operation, utensil and place at present.The smelly fragrance of its abnormal smells from the patient, taste is extremely bitter, aqueous solution react acid, volume foam is produced during jolting, can not oral disinfecting be used for, and allergic conjunctivitis, visual deterioration, contact dermatitis can be caused, cause nausea with after 3% solution coloclysis number minute, cold sweat is lethal eventually dies.Dead serious side effects also can be being caused as vaginadouche.Therefore, prepare and use non-antibiotic class, single chlorhexidine acetate composition, do not add ethanol, a kind of new and effective antibacterial nano-emulsion collutory that just can significantly improve antibacterial potentiation especially seems important and particularly urgent.
Summary of the invention
According to demand and the deficiency in above-mentioned field, the invention provides a kind of new and effective antibacterial nano-emulsion collutory.This nano-emulsion collutory, utilize nanotechnology to improve drug solubility, use non-antibiotic class, single chlorhexidine acetate composition, and do not add ethanol, described nano-emulsion collutory can significantly improve antibacterial potentiation, is particularly useful for dental caries and other suffering from oral diseases.
A kind of high-efficiency antimicrobial dental caries chlorhexidine acetate nano-emulsion collutory, it is characterized in that, be made up of the raw material of following weight portion: chlorhexidine acetate 0.2 ~ 1.5 part, 8 ~ 30 parts, surfactant, cosurfactant 4 ~ 15 parts, oil phase 3 ~ 20 parts, oral cleansing lotion 0.05 ~ 1 part, distilled water 34.45 ~ 69.0 parts.
Described medicine chlorhexidine acetate, described substrate concentration scope: 0.2 ~ 1.5 part.
Described surfactant is one or more in Polysorbate or polyoxyethylene fatty acid ester; Described Polysorbate is one or more in tween 80, tween 85, polysorbas20, and described polyoxyethylene fatty acid ester is one in polyoxyethylene hydrogenated Oleum Ricini 40, polyoxyethylene castor oil 40 or its combination; The effective dose of described component is respectively tween 80: 10 ~ 30 parts, tween 85: 10 ~ 30 parts, polysorbas20: 8 ~ 20 parts, polyoxyethylene hydrogenated Oleum Ricini 40 (RH40): 10 ~ 30 parts, polyoxyethylene castor oil 40 (EL40): 10 ~ 30 parts.
Described cosurfactant is: sorbitan fatty acid ester is or/and the monohydric alcohol of C2 ~ C4 or polyhydric alcohol; Described sorbitan fatty acid ester is one in sorbester p37, sorbester p17 and more than one combinations; The monohydric alcohol of described C2 ~ C4 or polyhydric alcohol are any one in n-butyl alcohol, glycerol, 1,3 butylene glycol, 1,3-PD; The effective dose of described cosurfactant is respectively Arlacel-85: 4 ~ 15 parts, sorbester p17: 5 ~ 15 parts; Glycerol: 5 ~ 10 parts; Propylene glycol: 4.8 ~ 15 parts; 1,3 butylene glycol: 5 ~ 10 parts, n-butyl alcohol: 5 ~ 10 parts.
Described oil phase is any one in IPM, GTCC, liquid paraffin, vegetable oil and Oleum Glycines, and the effective dose of wherein said oil phase is respectively IPM:3 ~ 20 part, liquid paraffin: 5 ~ 15 parts, GTCC:3 ~ 15 part, edible vegetable oil: 4 ~ 15% parts, edible soybean oil: 5 ~ 15%.
Described oral cleansing lotion is: any one or its combination in natural Herba Menthae, natural Broneolum Syntheticum, Oleum menthae, Mentholum; Its effective dose is respectively natural Herba Menthae: 0.05 ~ 1 part, natural Broneolum Syntheticum: 0.05 ~ 1 part, Oleum menthae: 0.05 ~ 1 part, Mentholum: 0.05 ~ 1 part.
After the infinite dilution of described nano-emulsion collutory water, its particle diameter is 1 ~ 100nm.
The preparation method of above-mentioned nano-emulsion collutory, specifically comprises step as follows:
(1) each material in composition of raw materials is taken by proportional quantity;
(2) surfactant, cosurfactant, chlorhexidine acetate are stirred and fully dissolve, obtain the solution 1 of clarification;
(3) in the solution 1 of clarification, add the oil phase of proportional quantity again, slowly add the aqueous phase of the proportional quantity of 70% under stirring, obtain the solution 2 clarified;
(4) add in the solution 2 of clarification after the oral cleansing lotion of proportional quantity being dissolved in solvent.
(5) solution 2 of clarification is slowly added to the aqueous phase of residue proportional quantity in 20 DEG C ~ 25 DEG C, fully stir, until form clear, nano-emulsion collutory that viscosity is little;
(6) then filtration sterilization, fill, is sealed to the chlorhexidine acetate collutory of different size.
When described oral cleansing lotion be natural Herba Menthae or natural Broneolum Syntheticum time, described solvent is water; When described oral cleansing lotion be Oleum menthae or Mentholum time, described solvent is propylene glycol.
Surfactant is that nano-emulsion forms necessary material, and its Main Function reduces interfacial tension to form interfacial film, impels nano-emulsion to be formed; Increase the dissolubility of principal agent, guarantee the stability of preparation.Surfactant is divided into the six large classes such as nonionic surfactant, cationic surfactant, anion surfactant, block copolymer, zwitterionic surfactant, fluorinated surfactant.Because nonionic surfactant has nontoxic, good biocompatibility, more stable in the solution, be not subject to the impact of strong electrolyte, inorganic salts, soda acid, and good with the compatibility of other surfactants, numerous advantages such as haemolysis is less and be subject to applying.Meanwhile, in the forming process of nano-emulsion, when hydrophile-lipophile balance (HLB) value of surfactant is equal with the HLB value needed for oil or close, easily form the highly stable nano-emulsion of character.HLB value can prepare w/o type nano-emulsion at the surfactant of 4 ~ 7, and the surfactant of HLB value in 8 ~ 18 can prepare O/W type nano-emulsion.Therefore, the present invention adopts surface active agent tween class and the polyoxyethylene fatty acid ester class of high hlb, and these surfactants are nontoxic non-stimulated, and good biocompatibility, has certain Nutrition, is the better adjuvant of the various nano-emulsion of preparation.Preferred: Tween 80, polysorbate85, polyoxyethylene castor oil (EL40), polyoxyethylene hydrogenated Oleum Ricini (RH-40).
The formation of nano-emulsion also requires of short duration negative surface tension.And cosurfactant can make surfactant have larger absorption at oil-water interface by the hydrophilic and oleophilic value (HLB) of adjustment form surface-active agent, assist surfactant to reduce interfacial tension, reduce the repulsive force between surfactant molecule; Increase interface motion, reduce interface crooking ability when nano-emulsion is formed, be inserted in interfacial film, promote the formation of the film that radius of curvature is very little, expand nano-emulsion forming region, be conducive to preparing stay-in-grade nano-emulsion.The present invention is its main cosurfactant with spans (85,80) the most frequently used at present, yet select the low chain alcohols solvent (glycerol, 1 with the effect strengthening cosurfactant simultaneously, 3-butanols, n-butyl alcohol, propylene glycol), can allow use in nano-emulsion keep permeating and contacting between oil phase and interfacial film upper surface active agent molecule, and be easy to form interfacial film with surfactant, thus be more conducive to the formation of nano-emulsion and stablize.
In nano-emulsion forming process, oil is that matching surface activating agent participates in forming stabilized nanoscale breast, and their molecular weight is little, effect and the cosurfactant of micromolecule oil phase are similar, easily be embedded in surfactant, jointly form interfacial film with it, thus impel the formation of nano-emulsion.When the HLB of the surfactant of the present invention needed for oil phase is close with surfactant, the principle of the emulsion stabilization formed, select liquid paraffin, edible vegetable oil, GTCC, edible soybean oil, isopropyl myristate etc., the equal edible of these oil phase materials, nontoxic non-stimulated, good biocompatibility, has certain Nutrition, is the major medical adjuvant preparing various nano-emulsion.
In the present invention, also specific aim adds oral cleansing lotion: natural Herba Menthae, Oleum menthae, Mentholum, natural Broneolum Syntheticum, make its mouthfeel better.
The chlorhexidine acetate that the present invention adopts is a kind of wide spectrum non-antibiotic class antimicrobial drug, and it can be combined with sialoglycoprotein, and facing adhesion protein is reduced, and interference bacterial plaque is formed; In addition, hibitane also can be combined with bacterial cell exo polysaccharides, antibacterial is not easily adsorbed onto and obtains on film, reach the object of prevention and minimizing periodontal disease and carious disease.Chlorhexidine acetate is adsorbed on the permeability barrier of bacterial cytoplasm film, changes the permeability of bacterial cell membrane, cellular content is spilt, thus suppresses and kill bacteria.Research report, it is extremely sensitive to some staphylococcus, Streptococcus mutans, streptococcus salivarius, Candida albicans, escherichia coli, anaerobism bacterium acidi propionici; The medium degree of bloodthirsty streptococcus is responsive.At present, due to chlorhexidine acetate dissolubility in water extremely low (0.2g/100g), it is only a small amount of by after dissolve with ethanol at present, and other Chinese medicines, limited in oral disease after chemicals compatibility.Collutory of the present invention be easy to promote, economic, practical, safe, efficient non-antibiotic medicament or preparation, can effective caries prevention.
Collutory of the present invention is especially by suppressing the key link of cariogenic formation: produce acid, slightly solubility extracellular polysaccharide, biomembranous formation makes it have dental caries well to treat and preventive effect, soft in collutory of the present invention, side effect can not be brought to oral cavity and the person.
The preparation method of chlorhexidine acetate nano-emulsion collutory of the present invention, specifically comprises step as follows:
(1) preparation of surfactant phase takes surfactant by formula proportion, and helps surface activity composite in proportion, calculates the HLB value of this system, stirs.The HLB value of common emulsifying agent can be found in some chemical industry handbooks, " the chemical products handbook " of such as chemical industry publishing house.Because hydrophile-lipophile balance (HLB) value of surfactant has additive properties, available quality averaging method obtains the HLB value of surfactant.Such as, after two kinds of surfactant A, B mix, the hydrophile-lipophile balance HLBAB value of its mixed surfactant is HLBAB=(WAHLBA+WBHLBB)/(WA+WB)
WA, WB in formula---the quality of blend surfactants A, B;
The HLB value of HLBA, HLBB---surfactant A, B.
(2) surfactant of described amount and cosurfactant, chlorhexidine acetate are stirred dissolve completely, obtain the solution clarified;
(3) preparation of oil phase is according to the HLB value of surfactant phase, selects oil, adjusts its ratio, make the HLB value needed for its emulsifying close with the HLB of surfactant phase.Add the oil phase of described amount, under stirring, slowly add the aqueous phase of recipe quantity 70%, obtain the solution clarified;
(4) natural for the oral cleansing lotion of described amount Herba Menthae or natural Broneolum Syntheticum are dissolved in water, or Oleum menthae and Mentholum be dissolved in propylene glycol after add in prescription.
(5) slowly add to recipe quantity distilled water at 20 DEG C ~ 25 DEG C fully to stir, until form clear, nano-emulsion collutory that viscosity is little.
(6), after then using high speed centrifugation, transmission and grain size analysis Detection job qualified, filtration sterilization, fill, is sealed to the chlorhexidine acetate collutory of different size.
A kind of high-efficiency antimicrobial chlorhexidine acetate nano-emulsion collutory outward appearance of the present invention is colourless transparent liquid, has good stability.Ageing stability refers to that nanometer emulsion oral liquid places 1 year in room temperature natural trend condition, and outward appearance, content, granularity, current potential and pH extend in time and significant change do not occur.This chlorhexidine acetate nano-emulsion collutory is transparent lastingly, does not find muddiness or precipitation, then illustrate that ageing stability is good.This collutory is placed in test tube, and sealing, there is not the wild effect such as obvious layering and medicine precipitation, flocculation, detects that particle diameter is in 1 ~ 100nm scope by Particle Size Analyzer and transmission electron microscope in centrifugal 30min clear under the rotating speed of 13000r/min.
MIC experiment shows, sees Fig. 6.The MIC of chlorhexidine acetate nano-emulsion collutory is 0.4ug/ml, and the MIC of chlorhexidine acetate aqueous solution is 0.8ug/ml, and the MIC of compound hibitane acetate solution is 1.0ug/ml.Chlorhexidine acetate nano-emulsion is 2 times of its aqueous solution to Streptococcus mutans drug effect, is 2.5 times of Chlorhexidine Acetate In Compound.The present invention obtains reasonable antibacterial effect and is that the present invention adopts suitable prescription and proportioning, adds suitable abluent, adopts nanotechnology and technique to prepare new and effective antimicrobial mouthwash.Perhaps, these effects are the common synergism adopting suitable surfactant, cosurfactant, oil phase, freshener.
Chlorhexidine acetate nano-emulsion collutory is to testing result Fig. 8 of the MIC of escherichia coli, candida albicans, Streptococcus mutans, lactobacillus.Result is found out, the MIC of nano-emulsion collutory of the present invention to escherichia coli, candida albicans, Streptococcus mutans and lactobacillus is respectively 0.8ug/ml, 3.2ug/ml, 0.4ug/ml, 3.2ug/ml.The microemulsion disinfectant of chlorhexidine acetate folk prescription and compound recipe to escherichia coli, candida albicans, Streptococcus mutans and lactobacillus be 3.2ug/ml and 1.6ug/ml, 6.4ug/ml and 4.8ug/ml, 1.6ug/ml and 0.8ug/ml, 6.4ug/ml and 3.2ug/ml respectively.Nano-emulsion collutory of the present invention is chlorhexidine acetate folk prescription and compound recipe microemulsion 4 times and 2 times to escherichia coli, candida albicans, Streptococcus mutans and lactobacillus antibacterial effect respectively, 2 times and 1.5 times, 4 times and 2 times, 2 times and 1 times.Patent of the present invention at the antibacterial activity of escherichia coli, candida albicans, Streptococcus mutans, lactobacillus apparently higher than chlorhexidine acetate folk prescription and compound recipe microemulsion.Patent of the present invention obtains desirable antibacterial effect and is that the present invention adopts suitable prescription and proportioning (only in prescription, it is large that chlorhexidine acetate folk prescription and compound recipe microemulsion have selected toxicity, volatile ethyl acetate, and the present invention have selected and do not have volatility, the oil phase of safer edible (medicinal)), add suitable abluent, adopt nanotechnology and technique to prepare new and effective antimicrobial mouthwash.Perhaps, these effects are adopt the common synergism of suitable surfactant, cosurfactant, oil phase, freshener to improve the antibacterial effect of new and effective collutory.
The present invention, compared with other prior art, has following beneficial effect:
1. solve chlorhexidine acetate slightly solubility problem, preparing chlorhexidine acetate nano-emulsion collutory can greatly improve chlorhexidine acetate dissolubility, has good solubilization.
2. the chlorhexidine acetate nano-emulsion collutory of preparation has that viscosity is low, good stability, dispersibility are strong, absorb the features such as rapid, medicine is wrapped in kernel oil phase, also there is slow release and targeting, and can improve medicine bioavailability, prolong drug half-life in vivo, heighten the effect of a treatment, reduce toxic and side effects.
3. the diameter of aspirin particle of chlorhexidine acetate nano-emulsion collutory of the present invention is between 1 ~ 100nm, and mean diameter is 63.13nm, is be dissolved in by chlorhexidine acetate in surfactant and cosurfactant, is titrated to even, transparent nano-emulsion with distilled water.
4. the formula that adopts of the present invention and method simple possible, with low cost, can so that large-scale industrial production.Product of the present invention is easy to use, evident in efficacy, soft, can not bring side effect, good palatability to oral cavity and the person.
5. the present invention its can thoroughly penetrate Dental Plaque nucleus, effective ingredient is directly acted on its kernel, kill to cause the pathogenic bacterium such as the anaerobe of various oral disease, Candida albicans, staphylococcus glucose coccus, pyococcus, Streptococcus mutans and control it and grow, minimizing nosocomial infection and postoperation of oral cavity infect, sterilization tooth protection, counteract oral malodour.Shown by sterilization kinetic results, it after 5 minutes, just can kill the antibacterial of 95% to Streptococcus mutans effect;
6. the present invention can kill the pathogenic bacterium causing periodontitis, gingivitis, oral ulcer, dental plaque, dental hemorrhage, halitosis fast, safety non-toxic, non-stimulated, have no side effect, do not develop immunity to drugs, be Novel mouth sterilization, sterilizing clean-keeping articles for use, also can be used for artificial tooth and soak and sterilizing.
Accompanying drawing illustrates:
Fig. 1 is chlorhexidine acetate nano-emulsion collutory prescription screening figure;
Fig. 2 is the drafting of chlorhexidine acetate nano-emulsion collutory pseudo-ternary phase diagram;
Fig. 3 is chlorhexidine acetate nano-emulsion collutory qualification result figure;
Fig. 4 is chlorhexidine acetate nano-emulsion collutory HPLC content detection figure;
Fig. 5 is chlorhexidine acetate nano-emulsion collutory stability test result;
Fig. 6 is that chlorhexidine acetate nano-emulsion collutory is to Streptococcus mutans MIC result figure;
Fig. 7 antibacterial and sterilization kinetic results figure that is chlorhexidine acetate nano-emulsion collutory to Streptococcus mutans;
Fig. 8 is that chlorhexidine acetate nano-emulsion collutory detects comparing result figure to different bacterium MIC;
Fig. 9 is that chlorhexidine acetate nano-emulsion collutory produces acid to Streptococcus mutans and glucosan affects result figure;
Figure 10 is that chlorhexidine acetate nano-emulsion collutory is on the impact of Streptococcus mutans biofilm formation;
Figure 11 is that chlorhexidine acetate nano-emulsion collutory is to Streptococcus mutans biofilm surface form atomic force microscope observation result figure;
Figure 12 is that chlorhexidine acetate nano-emulsion collutory is to pharmacodynamic evaluation in the body of Streptococcus mutans;
Figure 13 is the cell membrane disruption evaluation of chlorhexidine acetate nano-emulsion collutory to Streptococcus mutans.
Detailed description of the invention
For better the object, technical solutions and advantages of the present invention being described, by the description of detailed description of the invention, the invention will be further described, but this is not limitation of the present invention, those skilled in the art are according to basic thought of the present invention, various amendment or improvement can be made, but only otherwise depart from basic thought of the present invention, all within the scope of the present invention.
Embodiment:
Embodiment 1: chlorhexidine acetate nano-emulsion collutory (100g)
1) chlorhexidine acetate 0.5g, propylene glycol 4.8g, tween 80 19.6g, IPM6g, natural Herba Menthae 0.1g, distilled water 69g is taken.
2) surfactant of described amount, cosurfactant and chlorhexidine acetate are mixed, and be that 50 ~ 100r/m constant temperature blender with magnetic force stirs at rotating speed, allow chlorhexidine acetate dissolve completely;
3) add oil phase 25 DEG C of waters bath with thermostatic control of described amount again, and be that 100 ~ 200r/m constant temperature blender with magnetic force stirs at rotating speed, slowly add about 70% recipe quantity distilled water, obtain the solution clarified;
4) natural for the oral cleansing lotion of described amount Herba Menthae is dissolved in water, or Oleum menthae and Mentholum be dissolved in propylene glycol after add in prescription.
5) slowly add to recipe quantity distilled water at 20 DEG C ~ 25 DEG C fully to stir, until form clear, nano-emulsion collutory that viscosity is little.
6), after then using high speed centrifugation, transmission and grain size analysis Detection job qualified, filtration sterilization, fill, is sealed to the chlorhexidine acetate collutory of different size.
The nano-emulsion mouthwash formulation appearance colorless of chlorhexidine acetate of preparation, clear, particle diameter are between 1 ~ 100nm, not stratified after 13000rpm, 10min high speed centrifugation, drug loading is 0.5%, envelop rate is 90%, and after room temperature places 1 year, nano-emulsion is without significantly flocculation, layering and medicine are separated out.
Embodiment 2
Take chlorhexidine acetate 0.5g, propylene glycol 15g, tween 80 30g, medicinal IPM20g, natural Herba Menthae 0.05g, distilled water 34.45g.
Embodiment 3
Take chlorhexidine acetate 0.5g, glycerol 5g, tween 85 30g, Arlacel-80 4g, medicinal IPM10g, natural Broneolum Syntheticum 0.05g, distilled water 50.45g.Preparation method is with embodiment 1.
Embodiment 4
Take chlorhexidine acetate 1.0g, glycerol 5g, tween 80 20g, Arlacel-80 10g, medicinal GTCC15g, natural Herba Menthae 1.0g, distilled water 48g.Preparation method is with embodiment 1.
Embodiment 5
Take chlorhexidine acetate 1.0g, glycerol 10g, tween 85 8g, Tween 80 20g, medicinal IPM3g, Mentholum 1.0g, distilled water 57g.Preparation method is with embodiment 1.
Embodiment 6
Take chlorhexidine acetate 1.5g, propylene glycol 5g, tween 80 10g, tween 20 20g, medicinal liquid paraffin 15g, Oleum menthae 1.0g, distilled water 47.5g.Preparation method is with embodiment 1.
Embodiment 7
Take chlorhexidine acetate 1.0g, n-butyl alcohol 5g, RH-4030g, Arlacel-85 4g, medicinal paraffin 10g, Oleum menthae 0.05g, distilled water 49.95g.Preparation method is with embodiment 1.
Embodiment 8
Take chlorhexidine acetate 1.0g, 1,3 butanediol 15g, tween 85 20g, medicinal IPM10g, Mentholum 0.05g, distilled water 53.95g.Preparation method is with embodiment 1.
Embodiment 9
Take chlorhexidine acetate 0.2g, propylene glycol 10g, Tween 80 20g, tween 85 10g, medicinal IPM10g, natural Broneolum Syntheticum 1g, distilled water 48.8g.Preparation method is with embodiment 1.
Embodiment 10
Take chlorhexidine acetate 0.2g, propylene glycol 15g, EL-4030g, medicinal IPM15g, distilled water 39.8g.Preparation method is with embodiment 1.
Embodiment 11
Take chlorhexidine acetate 1g, EL-4010g, RH-4010g, Arlacel-85 10g, medicinal liquid paraffin 5g, Oleum menthae 0.5g, distilled water 63.5g.Preparation method is with embodiment 1.
Embodiment 12
Take chlorhexidine acetate 0.5g, EL-4020g, medicinal GTCC10g, Oleum menthae 1.0g, distilled water 68.5g.Preparation method is with embodiment 1.
Embodiment 13
Take chlorhexidine acetate 1.0g, glycerol 5g, EL-4030g, Arlacel-85 15g, Oleum Glycines 10g, natural Broneolum Syntheticum 1.0g, distilled water 38g.Preparation method is with embodiment 1.
Embodiment 14
Take chlorhexidine acetate 1.0g, glycerol 5g, RH-4030g, Arlacel-80 15g, vegetable oil 10g, Oleum menthae 0.1g, distilled water 38.9g.Preparation method is with embodiment 1.
Embodiment 15
Take chlorhexidine acetate 1.0g, RH-4015g, EL4015g, Arlacel-80 15g, vegetable oil 4g, Oleum menthae 1g, distilled water 49g.Preparation method is with embodiment 1.
Embodiment 16
Take chlorhexidine acetate 1.0g, RH-4015g, Tween 80 15g, glycerol-8015g, vegetable oil 15g, natural Herba Menthae 1.0g, distilled water 38g.Preparation method is with embodiment 1.
Embodiment 17
Take chlorhexidine acetate 1.0g, polysorbate85 20g, Tween 80 15g, 1,3 butanediol-8015g, GTCC4g, Mentholum-0.5g, distilled water 44.5g.Preparation method is with embodiment 1.
Embodiment 18
Take chlorhexidine acetate 1.0g, EL-4010g, RH-4010g, 1,3 butanediol 5g, Oleum Glycines 15g, natural Herba Menthae 1.0g, distilled water 57g.Preparation method is with embodiment 1.
Embodiment 19
Take chlorhexidine acetate 1.0g, polysorbas20 8g, tween 85 25g, n-butyl alcohol 5g, GTCC4g, natural Broneolum Syntheticum 1.0g, distilled water 56g.Preparation method is with embodiment 1.
Embodiment 20
Take chlorhexidine acetate 1.0g, tween 20 15g, tween 80 15g, n-butyl alcohol 10g, Oleum Glycines 3g, natural Broneolum Syntheticum 0.5g, distilled water 55.5g.Preparation method is with embodiment 1.
experimental example
Test material: chlorhexidine acetate (content 98.3%, the safe Pharmaceuticals Ltd in Jinzhou, Liaoning nine); Chlorhexidine acetate reference substance (Nat'l Pharmaceutical & Biological Products Control Institute); Polyoxyethylene hydrogenated Oleum Ricini (RH40), polyoxyethylene castor oil (EL40), BASF Corp. of Germany produces, and purchases to Beijing Feng Lijingqiu commerce and trade Co., Ltd; CRODA company of GTCC Britain produces, and purchases to Beijing Feng Lijingqiu commerce and trade Co., Ltd; Oleum Glycines, Oleum Arachidis hypogaeae semen are all purchased to Shandong Lu Hua company limited; Tween 80, polysorbate85, sorbester p17, sorbester p37, polysorbas20, glycerol, propylene glycol, liquid paraffin, 1,3 butanediols, n-butyl alcohol are purchased to Chinese Shanghai traditional Chinese medicines group.Brain-heart infusion medium (Brain heart infusion, BHI) (BD, New Jersey, USA); 24 porocyte culture plates (Costar3524, USA); Coverslips (Thermo, Rochester, USA).Strain: Streptococcus mutans UA159 (ATCC700610) (ATCC, Virginia, USA), escherichia coli (ATCC8099), candida albicans (ATCC10231) all ATCC are bought, and laboratory is preserved and provided; Lactobacillus is separated from southwestern hospital clinical, and laboratory is preserved and provided.SD rat, SPF, female, in 3 week age, purchase to Chongqing Institute of Chinese Medicine.
Instrument and equipment: transmission electron microscope (Dutch PHILIPS company); Current potential and Particle Size Analyzer (NanoZS90, Malvern company of Britain); HPLC (Waters-E2695, the U.S.); ND-1000 ultraviolet spectrophotometer (NanoDrop company of the U.S.); Microplate reader (Biorad6.0, Japan), micro-aerobic incubator (Thermo Scitinfic), bacterial colony counting instrument (Shineso Science & Technology Co., Ltd., Zhejiang Hangzhou); Scanning electron microscope (AMRAY1000B, USA); Atomic Mechanics microscope (existing instrument of National Immunization preparation technique Engineering Research Center such as (IPC-208B, University Of Chongqing).
1. the screening of the optimum prescription of the nano-emulsion collutory of chlorhexidine acetate
(1) screening of surfactant and cosurfactant:
With the method Surfactant Tween 80 of screening chlorhexidine acetate nano-emulsion oil phase, polysorbate85, polysorbas20, polyoxyethylene castor oil (EL40), polyoxyethylene hydrogenated Oleum Ricini (RH40) and cosurfactant (CoSF), namely the same method of sorbester p17, sorbester p37 and ethanol, glycerol, propylene glycol screens the saturated concentration of chlorhexidine acetate.
To the selection result of above-mentioned surfactant, surfactant, oil phase, see Fig. 1 a to Fig. 1 c.To the solubility test result of above-mentioned surfactant be: dissolubility size is Tween 80 (16.16 ± 0.82mg/g), polyoxyethylene hydrogenated Oleum Ricini (RH40) (13.23 ± 0.56mg/g), polyoxyethylene castor oil (EL40) (10.28 ± 0.17mg/g), polysorbate85 (8.45 ± 0.08mg/g), polysorbas20 (4.23 ± 0.06mg/g) successively, Fig. 1 a.The solubility test result of above-mentioned cosurfactant is from big to small as ethanol (9.60 ± 0.31mg/g), propylene glycol (7.73 ± 0.30mg/g), sorbester p37 (4.35 ± 0.11mg/g), sorbester p17 (3.95 ± 0.03mg/g), n-butyl alcohol (2.56 ± 0.09mg/g), 1,3 butanediols (2.09 ± 0.46mg/g), are shown in Fig. 1 b.
(2) screening of oil phase:
Select the oil of IPM (C14), GTCC (C18), liquid paraffin (C16 ~ C20), Oleum Glycines, Semen Allii Tuberosi wet goods 5 kinds of different carbon chain length, 20 DEG C of viscosity sizes are followed successively by liquid paraffin (110 ~ 230mPas), GTCC (25.0 ~ 33.0mPas), Oleum Glycines (8.5mPas), IPM (5 ~ 6mPas), vegetable oil (8.5mPas).Get above-mentioned 5 kinds of oil respectively appropriate, put respectively in tool plug conical flask, add excessive chlorhexidine acetate, 25 DEG C of water-baths, jolt 24h and arrive balance.The dissolubility of chlorhexidine acetate in various oil phase is measured by HPLC method.
Fig. 1 c result shows: IPM (C14), liquid paraffin, GTCC, Oleum Glycines, Oleum Brassicae campestris are respectively 3.08 ± 0.15mg/g, 2.27 ± 0.09mg/g, 1.53 ± 0.08mg/g, 0.89 ± 0.06mg/g, 0.52 ± 0.05mg/g, maximum at the dissolubility of IPM, be 3.08mg/g.This may be relevant with viscosity with the carbochain length of IPM, is conducive to the distribution of medicine in oil phase and dissolving.Therefore, select IPM as the oil phase of the optimum of chlorhexidine acetate nano-emulsion.All oil phases, surfactant and cosurfactant have larger dissolubility (being greater than 0.2mg/ml), meet and can prepare nano-emulsion requirement.But due to ethanol, there is volatility and have strong impulse to oral mucosa, select all oil phase, surfactant and the cosurfactant except he thinks, but according to the pseudo-ternary phase diagram region that it is maximum, finally determine its surfactant and cosurfactant interworking is more excellent is combined as: Tween 80/sorbester p17, polysorbate85/sorbester p37, RH40/ propylene glycol, EL40/ propylene glycol.
(3) drafting of pseudo-ternary phase diagram
With filtering out surfactant and cosurfactant, Comprehensive Designing experimental group, by 9:1,8:2,7:3,6:4,5:5,4:6,3:7,2:8,1:9 (mass ratio of SF and CoSF), takes SF-CoSF, add selected oil phase, vortex shakes, and drips water, observes the change of each group, with centrifugal (13,000rpm, 30min) stability and particle diameter be main evaluation criterion, thus determine SF and CoSF forming nano-emulsion.3 summits using surfactants/cosurfactants (mixed surfactant), oil phase and deionized water as equilateral triangle, draw pseudo-ternary phase diagram, point on phasor every bar limit represents the proportionate relationship between corresponding two components respectively, and phasor any point represents the mass percentage of each component in corresponding system.According to oil, water, mixed surfactant at the mass fraction of critical point, draw pseudo-ternary phase diagram, determine maximum nano-emulsion district.The evaluation criterion evaluation criterion of nano-emulsion is: the liquid of preparation is clear or translucent; There is blue-opalescent; After high speed centrifugation (13000r/m, 30min) is centrifugal, stablize not stratified; Tyndall phenomenon is had after directional light incidence; By transmission electron microscope and laser particle analyzer, detect that emulsion droplet is between 1 ~ 100nm.If the mixing material of preparation is creamy white, having scattering phenomenon after directional light incidence, is then Emulsion; If clear, stiff, then it is gel.Also liquid crystal can be produced in phase in version process simultaneously.Utilize liquid crystal to have refractive power to exist, in polarizing microscope detects, there will be light and shade change, so often distinguish liquid crystal and gel with it, is liquid crystal as shinny, not shinny gel.
The result of the drafting of pseudo-ternary phase diagram, is shown in Fig. 2 a to Fig. 2 d.As can be seen from Fig. 2 a to Fig. 2 d, when ratio (Km) value of surfactant and cosurfactant increases, the region of nano-emulsion first increases rear minimizing.When its ratio is 4:1, the region of its nano-emulsion is maximum, and when its ratio is 2:1, its nano-emulsion region is minimum.According to the result of pseudo-ternary phase diagram, thus determine that the ratio of preferred aspect is 4:1.
2. chlorhexidine acetate nano-emulsion gargle preparation
According to Sequential design thought, Yi Shui, oil, SF/CoSF are three-phase, adopt titrimetry to draw pseudo-ternary phase diagram.Select centrifugal (13,000rpm, 30min) to stablize, diameter is prescription candidate regions in the nano-emulsion district of 1 ~ 100nm.Accurately take the chlorhexidine acetate of recipe quantity, the surfactant of recipe quantity and cosurfactant (surfactant and cosurfactant are that the above-mentioned 4 kinds of more excellent interworkings screened combine), the oil phase of recipe quantity, first add aqueous solution or the buffer of about 70%, stir, finally progressively add water or buffer, obtain the nanoemulsions of clear.Can find out from Fig. 3 a, the faint yellow clear liquid of chlorhexidine acetate nano-emulsion outward appearance of preparation.The optimization formulation of the chlorhexidine acetate nano-emulsion prepared that finally shows according to drawing pseudo-ternary phase diagram is: Tween 80: propylene glycol: IPM (19.2%:4.8%:6%), chlorhexidine acetate concentration 0.5% and water 56.5%, Oleum menthae 5%.This optimized prescription that following Performance Detection all adopts as shown.
3. the form of acetic acid nano-emulsion collutory, granularity and potentiometric detection
A small amount of nano-emulsion is appropriate, after dilute with water 100 times, drop in and be covered with on the copper mesh of supporting film, blot with filter paper after static 10min, drip 2% phosphotungstic acid (pH is 7.4) solution negative staining 3min on copper mesh again, naturally volatilize, with transmission electron microscope observation and photographs, the results are shown in Figure 3b.Get chlorhexidine acetate nano-emulsion collutory appropriate, measure with laser granulometry with after appropriate distilled water diluting, the particle diameter of nano-emulsion and particle size distribution are shown in Fig. 3 c and Fig. 3 d.
Fig. 3 a result shows, all in spheroidal under Electronic Speculum, inside is oil phase, sees that Fig. 3 b is from Electronic Speculum, and random selecting is no less than 500 drops and measures particle diameter, and obtain nano-emulsion droplet size range at 1 ~ 100nm, mean diameter is 63.0nm.Fig. 3 b result shows, the chlorhexidine acetate nano-emulsion mean diameter 63.13nm of experiment preparation.The particle being less than 70nm accounts for 70%; The particle being less than 90nm accounts for 95%, and the particle size distribution range of visible obtained nano-emulsion collutory is narrow, and particle diameter is more even.Fig. 3 d result shows, the chlorhexidine acetate nano-emulsion average potential-67.13nm of experiment preparation, belongs to stabilising system scope.
4. the quality testing of chlorhexidine acetate nano-emulsion collutory and Detection of Stability
Chlorhexidine acetate HPLC detection method is set up: chromatographic condition: HPLC:Waters (E2695, USA); Chromatographic column: ZORABX SB-C18 (5um, 4.6mm × 250mm): sample size: 10ul; Mobile phase: acetonitrile: 0.02M-K2HPO4 (pH=2.0) (70/30, v/v); Determined wavelength: 275nm; Column temperature: 20 DEG C.Chlorhexidine acetate reference substance dissolve with methanol is become 0,25,50,75,100,200ng/mL.Under above-mentioned chromatographic condition, detect its peak area of variable concentrations, according to variable concentrations and peak area, the standard curve equation of drafting.Get chlorhexidine acetate nano-emulsion collutory 0.2mL, 13000r/m, 30min, postprecipitation adds 10ml ethanol, and 0.22um filtering with microporous membrane HPLC measures its amount of not wrapping up; Supernatant adds the dehydrated alcohol of two volumes, and after shaking up breakdown of emulsion, 13000r/m, 30min, get cleer and peaceful precipitation and add 10ml ethanol respectively, and 0.22um filtering with microporous membrane HPLC measures the amount of not wrapping up.With its envelop rate of formulae discovery and drug loading.
Drug loading=(precipitation capacity after the supernatant+breakdown of emulsion after breakdown of emulsion) × 100%/sample gross mass.
Envelop rate=(actual drug drug loading/theoretical drug drug loading) × 100%
The appearance time of the HPLC figure of chlorhexidine acetate reference substance, chlorhexidine acetate nano-emulsion collutory is basically identical, near 4.87min.The standard curve drawn, is shown in Fig. 4, and linear relationship is good, and standard curve equation is Y=14782X-17189 (R 2=0.9963, linear equation scope 25-1000ug/mL), have higher dependency and wider detection range, blank nano-emulsion does not produce obvious peak at this place, illustrates that its separating effect is better.Because of this detection method, this can be used for envelop rate and the drug loading that HPLC detects chlorhexidine acetate.According to standard curve, the drug loading recording chlorhexidine acetate nano-emulsion collutory is 5mg/ml, and its envelop rate is greater than 90%.
Get chlorhexidine acetate nano-emulsion collutory 3 batches, often criticize 3 parts, place 12 months under 25 DEG C of relative humidity 60 ± 5% conditions.Sampling in every 3 months once, respectively at 0,30,60,90,120,180,360 day, first observes cosmetic variation before and after high speed centrifugation (13,000rpm, 10min); And its content HPLC is detected, current potential and granularity Nano ZS90 current potential and Particle Size Analyzer detect, and survey its pH value with pH meter.
Stability experiment result shows, chlorhexidine acetate nano-emulsion collutory above-mentioned free in, all there is obvious change in the outward appearance before and after high speed centrifugation, also without producing, significantly flocculation, layering and medicine precipitation phenomenon occurs.Its content, granularity and current potential, pH are recorded and the results are shown in Figure 5.As can be seen from Figure 5, there is not obvious change in its content, granularity and current potential, pH.The above results fully confirms, its chlorhexidine acetate nano-emulsion quality prepared is highly stable.
Because the pathogenic bacterium in oral cavity are numerous, and the Streptococcus mutans pathogenic bacterium of generally acknowledging that to be oral cavity main, following research is just that representative is studied with Streptococcus mutans.
5. chlorhexidine acetate nano-emulsion is to the antibacterial potentiation of Streptococcus mutans
Experiment material and grouping: 0.2% chlorhexidine acetate aqueous solution group (writes a Chinese character in simplified form CHX, take 0.2g chlorhexidine acetate and join 100g distilled water, stirring and dissolving is complete), 0.5% chlorhexidine acetate nano-emulsion collutory group (writes a Chinese character in simplified form CNE, adopt preparation technology of the present invention, optimized prescription be: Tween 80: propylene glycol: IPM (19.2%:4.8%:6%, i.e. specific embodiment 1), chlorhexidine acetate concentration 0.5% and water 56.5%, Oleum menthae 5%).Blank group (is write a Chinese character in simplified form, MNE), Chlorhexidine Acetate In Compound collutory group (is write a Chinese character in simplified form, FCHX, with reference to Chinese patent 96115216.3 major pharmaceutical component: 0.2% sodium fluoride, 0.02% chlorhexidine acetate, 0.01% natural sweetener and 0.075% essence, preparation method for being each component of formula disclosed in the application for a patent for invention of 96115261.3 and consumption thereof according to application number, and according to dental caries treating agent prepared by method disclosed in it).
The mensuration of MIC: combine preliminary experiment in early stage, chlorhexidine acetate aqueous solution group, chlorhexidine acetate nano-emulsion collutory, Chlorhexidine Acetate In Compound collutory are diluted to respectively containing chlorhexidine acetate 1,2,4,6,8,10ug/ml.The bacterium liquid that UA159 (Streptococcus mutans ATCC700610) cultivates is adjusted to OD=1.0.Draw and dilute 1:10000 bacterium liquid 1.8ml doubly with BHI, by above-mentioned variable concentrations chlorhexidine acetate solution 0.2ml, put into 37 DEG C of micro-aerobic incubators, cultivate 24h, every hole is got except 200ul liquid, detects its absorption values by microplate reader at 595nm wavelength place.
MIC experiment shows, sees Fig. 6.The MIC of chlorhexidine acetate nano-emulsion collutory is 0.4ug/ml, and the MIC of chlorhexidine acetate aqueous solution is 0.8ug/ml, and the MIC of compound hibitane acetate solution is 1.0ug/ml.Chlorhexidine acetate nano-emulsion is 2 times of its aqueous solution to Streptococcus mutans drug effect, is 2.5 times of Chlorhexidine Acetate In Compound.
The mensuration of MBC: draw the bacterium liquid 5ul that above-mentioned MIC cultivates, instillation BHI solid culture plate, the coating of L rod, puts into 37 DEG C of micro-aerobic incubators, cultivates 24h, observe the growing state of antibacterial.
MBC result shows, the BHI flat board of the chlorhexidine acetate nano-emulsion collutory of 0.4ug/ml does not grow antibacterial; The BHI flat board of the chlorhexidine acetate aqueous solution of 0.8ug/ml does not grow antibacterial; The BHI flat board of the compound chlorhexidine gargle in vitro of 1ug/ml does not grow antibacterial.Therefore, the MBC of chlorhexidine acetate nano-emulsion is 0.4ug/ml, and the MBC of chlorhexidine acetate aqueous solution is 0.8ug/ml, and the MBC of compound hibitane acetate solution is 1.0ug/ml.The result of MIC and MBC confirms, chlorhexidine acetate nano-emulsion can significantly improve Streptococcus mutans active.
Sterilization kinetics: the bacterium liquid that ATCC700610 cultivates is adjusted to OD=0.6.Draw ATCC700610 bacterium liquid 1.8ml, by 2,4,8ug/ml chlorhexidine acetate aqueous solution group, chlorhexidine acetate nano-emulsion collutory group, put into 37 DEG C of micro-aerobic incubators, cultivate 0,5,10,15,30,60,120,240,480 and 720min.After hatching different time, with BHI dilution 0,10 1, 10 2, 10 3with 10 4doubly, get 5 μ L dilute solutions dull and stereotyped to BHI, be placed on 37 DEG C of 24h, count by Automatic Colony Counter.
Sterilization kinetics Fig. 7 a result shows, the nano-emulsion collutory that we observe has the bactericidal activity faster and more powerful than CHX.The display of sterilization kinetic results, antibacterial effect and chlorhexidine acetate have remarkable dose-dependent effect.Result of study shows, the concentration C NE of 0.8 μ g/mL than CHX (73.33%) have fast (5min) bactericidal effect to Streptococcus mutans (95.07% bacterial death).And when antibacterial all can kill by the chlorhexidine acetate nano-emulsion collutory of 0.8 μ g/mL in 480min.After chlorhexidine acetate nano-emulsion collutory effect 5min, time-killing kinetic curve obviously reduces, and chlorhexidine acetate aqueous solution just occurs obviously to change at 15min.Result confirms, chlorhexidine acetate nano-emulsion collutory significantly shortens it to Streptococcus mutans effect than its aqueous solution.
Antibacterial kinetics: chlorhexidine acetate aqueous solution group, chlorhexidine acetate nano-emulsion collutory, blank elements are not diluted to containing chlorhexidine acetate 2,4,6,8,10ug/ml.The bacterium liquid that ATCC700610 cultivates is adjusted to OD=1.Draw the bacterium liquid 1.8ml that ATCC700610 BHI fluid medium dilutes 1000 times, by above-mentioned variable concentrations chlorhexidine acetate solution 0.2ml, put into 37 DEG C of micro-aerobic incubators, cultivate 0,10,20,30,60,120,240,360,480min, every hole is got except 200ul liquid, detects its absorption values by microplate reader at 595nm wavelength place.
Antibacterial kinetic results, is shown in Fig. 7 b.From Fig. 7 b, along with the prolongation of time, its OD raises gradually, when cultivating 60min, there is not significant change in the OD of antibacterial, and at 240min, obvious change does not occur with 60min yet when chlorhexidine acetate nano-emulsion, and chlorhexidine acetate aqueous solution at 240min apparently higher than its nano-emulsion.These show, than its aqueous solution significant prolongation, it antibacterially does the time to chlorhexidine acetate nano-emulsion.
Its MIC of above-mentioned MIC, MBC, MBC, sterilization and antibacterial dynamic test result confirm, chlorhexidine acetate nano-emulsion has significant antibacterial activity to Streptococcus mutans, obvious shortening, to the sterilizing time of Streptococcus mutans, extends its Developing restraint to Streptococcus mutans.
6. chlorhexidine acetate nano-emulsion collutory is to the detection of the MIC of escherichia coli, candida albicans, Streptococcus mutans and lactobacillus
Experiment material and grouping: 0.5% chlorhexidine acetate nano-emulsion collutory group (writes a Chinese character in simplified form CNE, adopt preparation technology of the present invention, optimized prescription be: Tween 80: propylene glycol: IPM (19.2%:4.8%:6%, i.e. specific embodiment 1), chlorhexidine acetate concentration 0.5% and water 56.5%, Oleum menthae 5%).Compound recipe chlorhexidine acetate microemulsion disinfectant group (is write a Chinese character in simplified form, FCHME, with reference to Chinese patent CN103081940A embodiment 1, major pharmaceutical component: oneself is fixed for 0.5% acetic acid, 0.5% benzalkonium bromide, 25%RH40,8.3% propylene glycol, 2.7% ethyl acetate); Chlorhexidine acetate microemulsion disinfectant group (write a Chinese character in simplified form, CHME, with reference to Chinese patent CN103081940A embodiment 1, major pharmaceutical component: 0.5% acetic acid is own determines 25%RH40,8.3% propylene glycol, 2.7% ethyl acetate; Preparation method for being each component of formula disclosed in the application for a patent for invention of 96115261.3 and consumption thereof according to application number, and according to chlorhexidine acetate microemulsion disinfectant prepared by method disclosed in it)
Grope at combination preliminary experiment in early stage, chlorhexidine acetate nano-emulsion collutory, chlorhexidine acetate microemulsion disinfectant group, chlorhexidine acetate microemulsion disinfectant group are diluted to respectively containing chlorhexidine acetate 1,2,4,8,16,32,48,64ug/ml.The bacterium liquid that escherichia coli (ATCC8099), candida albicans (ATCC10231), Streptococcus mutans (ATCC700610), lactobacillus are cultivated is adjusted to OD=1.0.Draw and dilute 1:10000 bacterium liquid 1.8ml doubly with BHI, by above-mentioned variable concentrations chlorhexidine acetate solution 0.2ml, put into 37 DEG C of micro-aerobic incubators, cultivate 24h, every hole is got except 200ul liquid, detects its absorption values by microplate reader at 595nm wavelength place.
Chlorhexidine acetate nano-emulsion collutory is to testing result Fig. 8 of the MIC of escherichia coli, candida albicans, Streptococcus mutans, lactobacillus.Fig. 8 result is found out, the MIC of nano-emulsion collutory of the present invention to escherichia coli, candida albicans, Streptococcus mutans and lactobacillus is respectively 0.8ug/ml, 3.2ug/ml, 0.4ug/ml, 3.2ug/ml.The microemulsion disinfectant of chlorhexidine acetate folk prescription and compound recipe to escherichia coli, candida albicans, Streptococcus mutans and lactobacillus be 3.2ug/ml and 1.6ug/ml, 6.4ug/ml and 4.8ug/ml, 1.6ug/ml and 0.8ug/ml, 6.4ug/ml and 3.2ug/ml respectively.Nano-emulsion collutory of the present invention is chlorhexidine acetate folk prescription and compound recipe microemulsion 4 times and 2 times to escherichia coli, candida albicans, Streptococcus mutans and lactobacillus antibacterial effect respectively, 2 times and 1.5 times, 4 times and 2 times, 2 times and 1 times.
7. chlorhexidine acetate nano-emulsion produces acid impact to the suppression of Streptococcus mutans
Chlorhexidine acetate aqueous solution group, chlorhexidine acetate nano-emulsion collutory group are diluted to respectively containing chlorhexidine acetate 2,4,6,8ug/ml.The bacterium liquid that ATCC700610 cultivates is adjusted to OD=1.0.ATCC700610 bacterium liquid BHI fluid medium is diluted 1000 times afterwards draw 1.8ml put into 24 porocyte culture plates, by above-mentioned variable concentrations chlorhexidine acetate solution 0.2ml, put into 37 DEG C of micro-aerobic incubators, cultivate 24h, every hole is got except 1ml liquid, with precise digital display acidometer pH value determination.
Chlorhexidine acetate nano-emulsion, on the result of Streptococcus mutans acid production impact, is shown in Fig. 9 a.Can find out from Fig. 9 a, chlorhexidine acetate nano-emulsion and its aqueous solution are 0.2ug/ml, Streptococcus mutans are produced to the there was no significant difference of acid.When concentration is increased to 4ug/ml, the suppression acid production of chlorhexidine acetate nano-emulsion is obvious, and chlorhexidine acetate aqueous solution obviously could suppress Streptococcus mutans product acid just when concentration is increased to 8ug/ml, and the contrast of the nano-emulsion of blank, acid is produced without any remarkable inhibitory action to Streptococcus mutans.
8. chlorhexidine acetate nano-emulsion forms impact on Streptococcus mutans to slightly solubility extracellular polysaccharide
Chlorhexidine acetate aqueous solution group, chlorhexidine acetate nano-emulsion collutory group are diluted to respectively containing chlorhexidine acetate 2,4,6,8ug/ml.The bacterium liquid that ATCC700610 cultivates is adjusted to OD=1.0.ATCC700610 bacterium liquid BHI fluid medium is diluted 1000 times afterwards draw 1.8ml put into 24 porocyte culture plates, by above-mentioned variable concentrations chlorhexidine acetate solution 0.2ml, put into 37 DEG C of micro-aerobic incubators, cultivate 48h, collect supernatant, under 6000g condition, centrifugal 15min, 4 DEG C, abandon supernatant.Add 0.5mol/L NaOH1ml and repeat mixing, under 6000g condition, centrifugal 15min, washs 2 times, detects its content after the 0.1M NaOH dissolving of precipitation 2ml with Phenol sulfuric acid procedure.Each sample repeats 3 times.
Chlorhexidine acetate nano-emulsion, on the result that the slightly solubility extracellular polysaccharide of Streptococcus mutans affects, is shown in Fig. 9 b.Can find out from Fig. 9 b, chlorhexidine acetate nano-emulsion and its aqueous solution are 0.2ug/ml, Streptococcus mutans are produced to the there was no significant difference of sour slightly solubility extracellular polysaccharide.When concentration is increased to 4ug/ml, the inhibitory action of chlorhexidine acetate nano-emulsion is obvious, and chlorhexidine acetate aqueous solution works as the generation that concentration is increased to 8ug/ml ability obvious suppression Streptococcus mutans extracellular polysaccharide just, and the nano-emulsion contrast of blank, slightly solubility extracellular polysaccharide is produced without any remarkable inhibitory action to Streptococcus mutans.
By producing acid to it, slightly solubility extracellular polysaccharide observed result confirms, chlorhexidine acetate nanometer can suppress the generation of producing acid, extracellular polysaccharide.
9. chlorhexidine acetate nano-emulsion forms biofilm formation impact to Streptococcus mutans
(1) Streptococcus mutans biofilm formation amount
Chlorhexidine acetate aqueous solution group, chlorhexidine acetate nano-emulsion collutory group are diluted to respectively containing chlorhexidine acetate 500ug/ml.The bacterium liquid that ATCC700610 cultivates is adjusted to OD=1.0.ATCC700610 bacterium liquid BHI fluid medium is diluted 10000 times afterwards draw 2ml put into 24 porocyte culture plates, under putting into 80%N2,20%CO2,37 DEG C of conditions, micro-aerobic cultivation 48h takes out.Abandoning supernatant, add 0.2ml the nano-emulsion collutory and the aqueous solution effect 30min that contain 500ug/ml chlorhexidine acetate, be equipped with in PBS standard 96 orifice plate clean 2 ~ 3 times, drying at room temperature 30min, every hole adds the crystal violet 200 μ l of 0.1%, 15min dyes, and cleans 3 times, drying at room temperature 10min with PBS same method.Adding 95% ethanol 200 μ l/ hole in 96 orifice plates, micro-oscillator shakes 30min, be that its OD numerical value is detected at 570nm place by microplate reader at wavelength, every hole repeats 3 times, not contrast as radix containing the hole of bacteria suspension.
Chlorhexidine acetate nano-emulsion, affects result to the biofilm formation amount of Streptococcus mutans, sees Fig.10a.As can be seen from Figure 10, obviously reduce with same amount chlorhexidine acetate nano-emulsion and aqueous solution effect Streptococcus mutans biofilm biomass, and chlorhexidine acetate aqueous solution is obviously higher than the amount between blank group.And chlorhexidine acetate nano-emulsion, chlorhexidine acetate aqueous solution produce notable difference to the biofilm formation amount of blank group.
(2) its suppression biomembrane morphology of scanning electron microscopic observation
Chlorhexidine acetate aqueous solution group, chlorhexidine acetate nano-emulsion collutory group are diluted to respectively containing chlorhexidine acetate 2ug/ml.The bacterium liquid that ATCC700610 cultivates is adjusted to OD=1.0.ATCC700610 bacterium liquid BHI fluid medium is diluted 10000 times afterwards draw 1.8ml put into 24 porocyte culture plates, by above-mentioned variable concentrations chlorhexidine acetate solution 0.2ml.Micro-aerobic cultivation 24h and 48h under putting into 80%N2,20%CO2,37 DEG C of conditions.Its plastic sheet is carefully taken out, fix with 2.5% glutaraldehyde, respectively dewater 1 time with 50%, 70%, 80%, 90% ethanol successively, then 100% dehydration of alcohol 2 times are used, isoamyl acetate dewaters 2 times, is 5min at every turn, CO2 lyophilization 5h, carrier surface is gold-plated powder under vacuum, and SEM observes.
Chlorhexidine acetate nano-emulsion collutory, to the biofilm formation apperance result of Streptococcus mutans, is shown in Figure 10 b-Figure 10 e.Can find out from figure, with chlorhexidine acetate nano-emulsion collutory and the discovery after 2000,4000,8000 times of amplifications of aqueous solution effect Streptococcus mutans biofilm formation cosmetic variation of same amount chlorhexidine acetate, nano-emulsion collutory density is well below its aqueous solution, but nano-emulsion collutory and aqueous solution are to no significant difference between biomembranous form.
(3) atomic force microscope observation is to the morphology of biomembrane three dimensional structure
Chlorhexidine acetate aqueous solution group, chlorhexidine acetate nano-emulsion collutory group are diluted to respectively containing chlorhexidine acetate 2ug/ml.The bacterium liquid that ATCC700610 cultivates is adjusted to OD=1.0.ATCC700610 bacterium liquid BHI fluid medium is diluted 10000 times afterwards draw 1.8ml put into 24 porocyte culture plates, by above-mentioned variable concentrations chlorhexidine acetate solution 0.2ml.Micro-aerobic cultivation 48h under putting into 80%N2,20%CO2,37 DEG C of conditions.Its plastic sheet is carefully taken out, cleans 3 times gently with aseptic PBS, naturally dry, use Atomic Mechanics microscope direct observing.All images all by automatic smoothing process to eliminate the low frequency noise sweeping direction slowly.Calculate its arithmetic average root, mean square deviation, maximum height, degree of skewness, steepness.
Act on Streptococcus mutans biomembrane with high-resolution atomic force microscope Dichlorodiphenyl Acetate hibitane nano-emulsion and aqueous solution to observe and the results are shown in of three-dimensional reconstruction, Figure 11 a-Figure 11 e.From the X-Y scheme of Figure 10, can find out, the biofilm thickness of nano-emulsion collutory is starkly lower than aqueous solution.Figure 11 graphics is then found out, the biomembranous density that nano-emulsion collutory is formed is starkly lower than its aqueous solution.His arithmetic average root, mean square deviation, maximum height, nano-emulsion collutory is all lower than its aqueous solution, and degree of skewness and steepness are then contrary.
Its biomembranous formation volume, outward appearance and three dimensional structure are proved, the amount that chlorhexidine acetate nano-emulsion obviously suppresses Streptococcus mutans to be formed, the density simultaneously suppressing formation and thickness, but its biofilm formation Streptococcus mutans form is obviously changed without generation.
(10) chlorhexidine acetate nano-emulsion Streptococcus mutans is caused to rodents model body in pharmacodynamic study
Experiment material and grouping: experiment is divided into: model control group, blank nano-emulsion group, 2mg/ml chlorhexidine acetate nano-emulsion collutory group, 5mg/ml chlorhexidine acetate nano-emulsion collutory group, 2mg/ml chlorhexidine acetate aqueous solution group, 5mg/ml chlorhexidine acetate suspension group.60 Female sexual rats of SPF (Chongqing Institute of Chinese Medicine) in three week age are divided into 6 groups at random, often organize 10, raise and freely drink water under SPF environment, infect front chow diet, with Keys2000 dental caries feedstuff after infecting.Carry out according to Chinese patent (a kind of Streptococcus mutans infects and cariogenic method for building animal model, ZL201210558790.6), first remove miscellaneous bacteria with 1% chloromycetin, 1% ampicillin, 1% Carbenicillin for three days on end.After antibiotic is removed, after all rats are anaesthetized, 1 × 109CFU/ml700610 bacterium liquid of oral cavity instillation 0.2ml.Every day 2 times, for three days on end.Infect latter 5th day, except model group animal, all the other every treated animals are anaesthetized the different solutions of instillation 0.2ml, every day 2 times, continuous 28 days.The 14th day and 28 days upon administration, take rat oral cavity after anesthesia with aseptic cotton carrier, used physiological saline solution 100 times to dilute 5ul and instill light saliva culture medium, micro-aerobic condition of culture cultivates 3 days, counts with bacterial colony counting instrument.The 70th day after infection, all animals were condemned to death, and its tooth Keys (1958) marking system evaluates the dental caries of each treated animal.
14 days and 28 days after successive administration, rat oral cavity clump count the results are shown in Figure figure .12a.Continuous 14 days and administration in 28 days twice daily, the mouth clump count of 2mg/ml and 5mg/ml CNE is significantly lower than the CHX of same concentrations.Meanwhile, along with the prolongation of administration time, the clump count in oral cavity reduces.To the dental caries appraisal result of all animals, see Figure 12 b to Figure 12 d.As can be seen from Figure, the score of the enamel caries of all drug treating treated animals is significantly lower than blank nano-emulsion group and model control group.The mark of the enamel caries of 2mg/ml and 5mg/ml CNE, shallow dental caries and middle dental caries is detailed CHX lower than same concentrations.In addition, along with drug level increases, the enamel caries of its dental caries, shallow dental caries and middle dental caries mark obviously decline.The representative graph figure of its every treated animal dental caries can find out, green arrow presentation surface (enamel caries) pathological changes; Blue arrow represents slight dentin (shallow dental caries) pathological changes; Red arrow instruction moderate dentin (middle dental caries) pathological changes.As can be seen from Figure 12, CHX and the matched group of same concentrations is also starkly lower than by the dental caries degree of 2mg/ml and 5mg/ml CNE.Therefore, this result confirms, CNE can improve the antibacterial effect of this medicine and reduce dental caries degree.
(11) chlorhexidine acetate nano-emulsion is studied the destruction of Streptococcus mutans cell membrane
Chlorhexidine acetate aqueous solution group, chlorhexidine acetate nano-emulsion collutory group are diluted to respectively containing chlorhexidine acetate 2ug/ml.The bacterium liquid that ATCC700610 cultivates is adjusted to OD=1.0.ATCC700610 bacterium liquid BHI fluid medium is diluted 10000 times afterwards draw 1.8ml put into 24 porocyte culture plates, by above-mentioned variable concentrations chlorhexidine acetate solution 0.2ml.Micro-aerobic cultivation 48h under putting into 80%N2,20%CO2,37 DEG C of conditions.Centrifugal to culture fluid 6000g, 10min rear use 2.5% glutaraldehyde is fixed.After specimen is fixing with 2% (w/v) osmium 0.1M PBS at room temperature 1 hour, with identical buffer solution three times, then dewater at a series of alcoholic solution (that is, 50%, 70%, 80%, 90%, 95% and 100%).Each sample is embedded in epoxy resin and reduces and uses oere horse ultramicrotome, observes with use tecnai-10TEM with uranyl acetate stain and alkaline lead citrate.
Streptococcus mutans cell membrane damage, is shown by transmission electron microscope observation, at Figure 13 a to Figure 13 d.Amplification 3.9 ten thousand times, after more obvious leak carries out process and CNE, Streptococcus mutans is observed to (Figure 13 is a) than CHX (Figure 13 b).At 9.3 ten thousand times, CHX shows complete, clearly to pick out cell membrane equally distributed endochylema.But, along with CNE shows the cell changing and destroy cell membrane process, as depicted in fig. 13 a.The appearance of cytoplasma membrane becomes from cell membrane local detachment.Compare, the degree of observing this breakage with CHX is serious (Figure 13 a and Figure 13 c).Cell membrane interrupts being more serious in 0.2 μ g/mL CNE group and CHX.These results show, CNE shows the Streptococcus mutans of stronger destruction membrane structure than the ability with the CHX of concentration.
ATC700610 Bacteria is cultivated (1 × 10 6cFU/ milliliter) to cultivate at 24 well culture plate 4 hours and CNE and CHX0.2,0.4 and 0.8 μ g/ml chlorhexidine acetate content assesses the integrity of cell membrane.By the antibacterial centrifugal 10min of 6000rpm cultivated, precipitation adds the physiological saline solution of 200ul, gets the micro-ultraviolet spectrophotometer of its 5ul and measures its absorption values at 260nm and 280nm.
The integrity result of cell membrane is shown in Figure 13 e and Figure 13 f.Can find out from Figure 13 e, the 260nm extinction number of degrees of 0.4 and 0.8 μ g/mL CNE, be significantly higher than the CHX (P=000, P=0.001, P < 0.01) of same concentrations.Due to destruction and the damage of cell membrane, the nucleic acid amount that 0.4 and 0.8 μ g/mL CNE leaks from cell membrane is apparently higher than the CHX of same concentrations.Can find out from Figure 13 f, the 280nm extinction number of degrees of 0.4 and 0.8 μ g/mL CNE, be significantly higher than the CHX (P=000, P=0.001, P < 0.01) of same concentrations.Due to destruction and the damage of cell membrane, the protein content that 0.4 and 0.8 μ g/mL CNE leaks from cell membrane is apparently higher than the CHX of same concentrations.Due to destruction and the damage of cell membrane, the CNE of 0.4 and 0.8 μ g/mL is 1.75 times, 1.78 times and 6.25 times and 4.43 times of its aqueous solution in 260 and 280nm absorbance.Above-mentioned result of study shows, chlorhexidine acetate nano-emulsion collutory is by destroying membrane structure and biomembranous formation, thus improves the antibacterial activity to Streptococcus mutans.
The present invention by solubility experiment, what successfully filter out take Tween 80/propylene glycol/IPM as the chlorhexidine acetate nano-emulsion collutory of representative, confirm with transmission electron microscope, high speed centrifugation, current potential and Particle Size Analyzer, HPLC, successfully prepare particle diameter at 1-100nm, envelop rate is greater than 90%, and drug loading is the chlorhexidine acetate nano-emulsion collutory of 0.5%.Confirm content, granularity, current potential, pH and its outward appearance research that its room temperature is placed 1 year, nano-emulsion collutory quality is extremely stable simultaneously.By external MIC, MBC, antibacterial and sterilization dynamic test and body in dental caries scoring confirm, nano-emulsion collutory has the activity of remarkable in-vitro antibacterial to Streptococcus mutans.Produce acid, slightly solubility extracellular polysaccharide and the research of biofilm biomass, outward appearance and three dimensional structure to it to find, nano-emulsion collutory can significantly suppress it to produce acid, slightly solubility extracellular polysaccharide and biofilm formation amount, form biomembranous Streptococcus mutans density, quantity and thickness, destruction bacterial cell membrane.
The all lists of references mentioned in invention are quoted as a reference all in this application, just quoted separately as a reference as each section of document, without the need to elaborating further, believe content disclosed before employing, those skilled in the art can apply to greatest extent, and chlorhexidine acetate pharmaceutical preparation stability, drug delivery amount, envelop rate, preparation technology and the drug effect aspect of these equivalent form of values except nano-emulsion list of references all can not arrive the application's appended claims limited range simultaneously.In addition, preferred specific embodiments above should be understood to only illustrate, but not limits the scope of the invention by any way.The above is only the preferred embodiment of the present invention but not limiting the scope of the invention; should be understood that; for those skilled in the art; under the premise without departing from the principles of the invention; can modify to technical solution of the present invention or equivalent replacement, these are revised or are equal to replacement and also should be considered as protection scope of the present invention.

Claims (9)

1. a high-efficiency antimicrobial dental caries chlorhexidine acetate nano-emulsion collutory, it is characterized in that, be made up of the raw material of following weight portion: chlorhexidine acetate 0.2 ~ 1.5 part, 8 ~ 30 parts, surfactant, cosurfactant 4 ~ 15 parts, oil phase 3 ~ 20 parts, oral cleansing lotion 0.05 ~ 1 part, distilled water 34.45 ~ 69.0 parts.
2. nano-emulsion collutory according to claim 1, described medicine chlorhexidine acetate, described substrate concentration scope: 0.2 ~ 1.5 part.
3. nano-emulsion collutory according to claim 2, is characterized in that, described surfactant is one or more in Polysorbate or polyoxyethylene fatty acid ester; Described Polysorbate is one or more in tween 80, tween 85, polysorbas20, and described polyoxyethylene fatty acid ester is one in polyoxyethylene hydrogenated Oleum Ricini 40, polyoxyethylene castor oil 40 or its combination; The effective dose of described component is respectively tween 80: 10 ~ 30 parts, tween 85: 10 ~ 30 parts, polysorbas20: 8 ~ 20 parts, polyoxyethylene hydrogenated Oleum Ricini 40 (RH40): 10 ~ 30 parts, polyoxyethylene castor oil 40 (EL40): 10 ~ 30 parts.
4. nano-emulsion collutory according to claim 3, described cosurfactant is: sorbitan fatty acid ester is or/and the monohydric alcohol of C2 ~ C4 or polyhydric alcohol; Described sorbitan fatty acid ester is one in sorbester p37, sorbester p17 and more than one combinations; The monohydric alcohol of described C2 ~ C4 or polyhydric alcohol are any one in n-butyl alcohol, glycerol, 1,3 butylene glycol, 1,3-PD; The effective dose of described cosurfactant is respectively Arlacel-85: 4 ~ 15 parts, sorbester p17: 5 ~ 15 parts; Glycerol: 5 ~ 10 parts; Propylene glycol: 4.8 ~ 15 parts; 1,3 butylene glycol: 5 ~ 10 parts, n-butyl alcohol: 5 ~ 10 parts.
5. nano-emulsion collutory according to claim 4, described oil phase is any one in IPM, GTCC, liquid paraffin, vegetable oil and Oleum Glycines, and the effective dose of wherein said oil phase is respectively IPM:3 ~ 20 part, liquid paraffin: 5 ~ 15 parts, GTCC:3 ~ 15 part, edible vegetable oil: 4 ~ 15% parts, edible soybean oil: 5 ~ 15%.
6. according to the arbitrary described nano-emulsion collutory of Claims 1 to 5, described oral cleansing lotion is: any one or its combination in natural Herba Menthae, natural Broneolum Syntheticum, Oleum menthae, Mentholum; Its effective dose is respectively natural Herba Menthae: 0.05 ~ 1 part, natural Broneolum Syntheticum: 0.05 ~ 1 part, Oleum menthae: 0.05 ~ 1 part, Mentholum: 0.05 ~ 1 part.
7., according to the arbitrary described nano-emulsion collutory of claim 1 ~ 6, be 1 ~ 100nm with its particle diameter after water infinite dilution.
8. the preparation method of the nano-emulsion collutory described in claim 1 ~ 7, specifically comprises step as follows:
(1) each material in composition of raw materials is taken by proportional quantity;
(2) by surfactant, cosurfactant and chlorhexidine acetate, stir and fully dissolve, obtain the solution 1 of clarification;
(3) in the solution 1 of clarification, add the oil phase of proportional quantity again, slowly add the aqueous phase of the proportional quantity of 70% under stirring, obtain the solution 2 clarified;
(4) add in the solution 2 of clarification after the oral cleansing lotion of proportional quantity being dissolved in solvent.
(5) solution 2 of clarification is slowly added to the aqueous phase of residue proportional quantity in 20 DEG C ~ 25 DEG C, fully stir, until form the nano-emulsion collutory of clear;
(6) then filtration sterilization, fill, is sealed to the chlorhexidine acetate collutory of different size.
9. method according to claim 8, is characterized in that, when described oral cleansing lotion be natural Herba Menthae or natural Broneolum Syntheticum time, described solvent is water; When described oral cleansing lotion be Oleum menthae or Mentholum time, described solvent is propylene glycol.
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