CN1718197A - Pharmaceutical composition contg .bifidobacterium, prepn. method and medical use thereof - Google Patents

Abstract

A composite medicine for treating vaginitis is prepared from the bifidobacterium No. 47757732, the mixture of acid and alkali for making its pH=3.75-5.3, and the pharmacologically acceptable carrier. Its preparing process is also disclosed.

Description

A kind of pharmaceutical composition, preparation method and application in medical science that contains bacillus bifidus

Technical field

The present invention relates to a kind of pharmaceutical composition, be specifically related to a kind of be used for the treatment of intravaginal dysbacteriosis and colpitic pharmaceutical composition, preparation method and the application in medical science that contains bacillus bifidus.

Background technology

The equilibrated destruction of microecology in vaginas is the inducement of various gynecological inflammations, also is the result of various gynecological diseases.The treatment of colpitis mainly comprises antibacterial, microbial ecological agent two aspects.The treatment high specificity of antibacterial, effect is remarkable, but has a broad antifungal spectrum, selectivity is not strong, so side effect is more, and the relapse rate height.Existing single strain microbial ecological agent all with bacterial vaginitis as the treatment target spot, scope of application limitation.Simultaneously, the different section normal floras of vagina are distributed with certain rules, and promptly the deep is based on bacillus bifidus, and the stage casing is based on lactobacillus, and the inharmonious change of various like this normal floras can cause the vaginitis of different sections.

Aspect preparation, improve the viable bacteria survival rate as far as possible is the target that microbial ecological agent is pursued always, and the influence factor of its survival rate is mainly reflected in temperature, disperses aspects such as protectant use, oxygen content, acid-base value.The disclosed patent No. of the present inventor is 01128659.8, denomination of invention is inquired into for " a kind of bacillus bifidus, lactobacillus mixed live drop and preparation method " carried out some to this, and this invention focuses on the isolation and purification method that proposes a kind of vagina bacillus bifidus, lactobacillus.But also have following deficiency: 1, the antiinflammatory described in the description, infection, antibacterial, antitumor and immunoregulation effect are to infer according to the in vitro tests bibliographical information of bacillus bifidus, do not have concrete experimental data to support.Because Bifidobacterium and Lactobacillus contain strains up to a hundred respectively, each bacterial strain physicochemical property is not quite similar, and the therapeutic effect of generation is different certainly.As: be proved, Lactobacillus gasseri and Lactobacillus crispatus are absolutely void to escherichia coli and gonococcus, not only do not have antitumor and immunoregulation effect, and can also cause opportunistic infection to human body.Therefore, this deduction is not objective, has suitable uncertainty.2, aspect the indication of treatment, " antiinflammatory, infection, good antimicrobial effect " only mentioned in this invention, do not have specific adaptation therapeutic domain, also do not have concrete experimental result to support, and only is the deduction on the existing document basis.3, the acid-base value of not considering medicine simultaneously to the influence of the viable bacteria survival of lactobacillus and bifidus bacillus and growth conditions, to the influence of the condition of growth that suppresses pathogenic bacterium, to the influence of suitable condition mutually of local (vagina) medication environment acidity.4, in use and since be external with the balance liquid mixed dissolution, be difficult to keep away the pollution of assorted bacterium, aerobic environment can reduce the viable count of medicine simultaneously.And this invention does not relate to the course of treatment for the treatment of, so curative effect is difficult to judge with certain standard.5, added glycerol excipient can influence viable count in the preparation process.

Summary of the invention

One of purpose of the present invention is that to propose a kind of identification code that both had been suitable for be that 47757732 bacillus bifidus and/or identification code are the viable bacteria survival and the growth conditions of 44346222 lactobacillus, can suppress the growth of pathogenic bacterium again, again can with suitable pharmaceutical composition of local (vagina) medication environment acidity and preparation method thereof.

It is that 47757732 bacillus bifidus and/or identification code are the purposes in 44346222 the lactobacillus purposes in preparation treatment colpitis medicine, particularly medicines such as preparation treatment bacterial vaginitis, colpitis mycotica, gonococcal vaginitis that another object of the present invention provides identification code.

It is that 47757732 bacillus bifidus and identification code are 44346222 the purposes of lactobacillus in preparation treatment intravaginal dysbacteriosis medicine that another purpose of the present invention provides identification code.

It is that 47757732 bacillus bifidus and/or identification code are that 44346222 lactobacillus is removed the purposes in the medicines such as colon bacillus, staphylococcus aureus, gonococcus, Candida albicans in preparation that another purpose of the present invention provides identification code.

Realize the technical scheme of above-mentioned purpose:

A kind of pharmaceutical composition, contain the identification code for the treatment of effective dose and be 47757732 bacillus bifidus (hereinafter to be referred as bacillus bifidus) and/or identification code and be 44346222 lactobacillus (hereinafter to be referred as lactobacillus), an amount of acid, alkali and pharmacological-acceptable carrier, it is 3.75-5.3 that described an amount of acid, alkali make its mixed pharmaceutical composition pH value, pH value preferred 4～5.

Described acid is citric acid, tartaric acid, fumaric acid or adipic acid, and described alkali is sodium carbonate or sodium bicarbonate.

A kind of preparation of drug combination method comprises the steps:

(1) prepare respectively identification code be 47757732 bacillus bifidus and/identification code is the lyophilizing mycopowder of the lactobacillus of 44346222 (whether also having 44346022);

(2) with pharmacological-acceptable carrier drying, pulverizing back and an amount of acid, alkali mix homogeneously, the pH value that makes its mixture is 3.75-5.3;

(3) mycopowder that step (1) is made mixes the back press forming with the mixture that step (2) makes.

Further, described pharmacological-acceptable carrier comprises filler, dry adhesive and binding agent, and step (2) further comprises the steps:

(4) with filler, dry adhesive and binding agent drying, grinding and sieving;

(5) acid, filler, alkali, dry adhesive are mixed;

(6) in step (5), add binding agent, make the mixture soft material;

(7) with mixture soft material granulation and oven dry, make the mixture described in the step (2).

Further, described step (3) is that the mycopowder that earlier step (1) made mixes with a filler and mixes press forming afterwards again with mixture after grinding well dispersion.

Further, described pharmacological-acceptable carrier comprises filler lactose, starch, dry adhesive, filler Celluloasun Microcrystallisatum, disintegrating agent, dry adhesive low-substituted hydroxypropyl cellulose, binding agent 5%PVPK ₃₀Alcoholic solution (95%), described acid is tartaric acid, and described alkali is sodium bicarbonate, and step (2) further comprises the steps:

(4) the tartaric acid adding starch with weighing mixes, and sodium bicarbonate and the lactose with weighing adds and mix homogeneously again;

(5) in step (4), add again behind Celluloasun Microcrystallisatum and the low-substituted hydroxypropyl cellulose mix homogeneously with 5%PVPK ₃₀Alcoholic solution (95%) is made binding agent, the system soft material;

(6) soft material is used granulation, baking in granulator, make blank granule;

Step (3) further comprises the steps:

(7) mycopowder of weighing is ground well dispersion with an amount of lactose respectively;

(8) again with the blank granule equivalent mix homogeneously that progressively increases,

(9) add the lubricant mixing, compacting in flakes.

The invention provides identification code and be 47757732 bacillus bifidus and/or identification code and be the purposes in 44346222 the lactobacillus purposes in preparation treatment colpitis medicine, particularly medicines such as preparation treatment bacterial vaginitis, colpitis mycotica, gonococcal vaginitis.

The invention provides identification code and be 47757732 bacillus bifidus and identification code and be 44346222 the purposes of lactobacillus in preparation treatment intravaginal dysbacteriosis medicine.

The invention provides identification code and be 47757732 bacillus bifidus and/or identification code and be 44346222 lactobacillus and remove purposes in the medicines such as colon bacillus, staphylococcus aureus, gonococcus, Candida albicans in preparation.

The invention provides the purposes in the aforementioned pharmaceutical compositions purposes in preparation treatment colpitis medicine, particularly medicines such as preparation treatment bacterial vaginitis, colpitis mycotica, gonococcal vaginitis.

The invention provides above-mentioned identification code and be 47757732 bacillus bifidus and identification code and be 44346222 the purposes of lactobacillus in preparation treatment intravaginal dysbacteriosis medicine.

The invention provides the purposes of aforementioned pharmaceutical compositions in medicines such as preparation removing colon bacillus, staphylococcus aureus, gonococcus, Candida albicans.

Adopt technique scheme, in conjunction with the following embodiment that will describe in detail, the technique effect that the present invention gives prominence to is: 1, by being that 47757732 bacillus bifidus and/or identification code are to add bronsted lowry acids and bases bronsted lowry in the pharmaceutical composition of 44346222 lactobacillus containing identification code, and the pH value of control pharmaceutical composition is 3.75-5.3 (pH value preferred 4～5), simulated the PH environment of vagina, consistent with normal physiological conditions, thereby survival and the growth conditions of viable bacteria both be fit to, can suppress the growth of pathogenic bacterium again, again can be suitable mutually with local (vagina) medication environment acidity, can not cause stimulation to human body.In addition, PH is in 4～5 scopes, and the tablet compressibility is good, is convenient to make tablet.Confirm by pharmacodynamics test that 2, single vagina lactobacillus and bacillus bifidus and two kinds of bacterium thereof be mixed liquor in varing proportions, and the vaginitis pathogenic bacterium are all had the bactericidal effect of pressing down.And find, after single vagina lactobacillus or bacillus bifidus and two kinds of mixed in varing proportions concentration of bacterium thereof reach certain value (1 * 10 ⁷The CFU/ gram), the suppression ratio of pathogenic bacterium has been reached near 100%.And the ratio of Lactobacillus Jensenii and youth bacillus bifidus with press down to kill effect and do not have dependency relation.3, by interior therapeutic bacterial vaginitis experiment confirm: bigeminy viable bacteria pudendal lotion not only has the sure curative effect of treatment bacterial vaginitis, and when reaching therapeutic effect, has the effect of keeping or rebuilding intravaginal normal flora microecological environment.4, confirm by interior therapeutic colpitis mycotica and gonococcal vaginitis experimentation: a, bigeminy viable bacteria pudendal lotion are on the basis that produces effective therapeutical effect, tool is significantly rebuild the intravaginal microecological environment, replenish normal flora and correct the outstanding advantage of dysbacteriosis, to prophylactic recurrence or infect again and can produce positive effect.The intravaginal normal flora that b, bigeminy active bacteria formulation are rebuild can continue to bring into play therapeutical effect.Do not find in c, the clinical practice that it has apparent side effect, application is not restricted to breast-feeding female, and these are the advantage place of bigeminy viable bacteria pudendal lotion just.

The specific embodiment

One, the preparation of medicine:

1, the preparation of mycopowder: according to the patent No. is that 01128659.8 denomination of invention is the method described in " a kind of bacillus bifidus, lactobacillus mixed live drop and preparation method ", make through the API-20A identification code and be 44346022/44346222 lactobacillus strain and be encoded to 47757732 bifidobacterium species, be inoculated in strain in the fermentation tank respectively, be cultured to exponential phase, make lactobacillus bacterium mud and bacillus bifidus bacterium mud after centrifugal, bacterium mud concentration is not less than 1.0 * 10 ⁹The CFU/ gram adds and disperses protective agent, after about-70 ℃ of lyophilizing, adopts freeze-drying to make lactobacillus mycopowder and bacillus bifidus mycopowder.

The dispersion protective agent that adds can be skim milk, lactose, sodium glutamate, cysteine and starch etc., and wherein, the addition of cysteine is controlled between 0.01%～0.1%.The effect that adds cysteine is an oxygen reduction, makes oxygen content decline in the mycopowder, and the protection antibacterial is not destroyed in freeze-drying process simultaneously.When cysteine content is lower than 0.01% the effect not obvious, but since cysteine itself can the inhibition antibacterial growth, when cysteine content is higher than 0.1%, can kill viable bacteria on the contrary.

2, the preparation of dosage form:

(1) medicine is formed: comprise lactobacillus, bifidus bacillus, acid, alkali and other pharmaceutically acceptable carrier, the pH value of regulating the control preparation by acid, alkali is between the 3.75-5.3, and preferred pH value is between 4～5.

Because the variation of culture fluid pH value is bigger to the yield effect of lactobacillus and bifidus bacillus bacterium mud, experiment shows that the wet bacterium mud yield of cultivation 24h was the highest when PH was adjusted to 6.3-6.5.Therefore, by acid-alkali accommodation, the bacterium mud that makes made directly combine that to be used for human body be not too suitable behind the mycopowder with pharmaceutically acceptable carrier.

By in medicine, adding acid, alkali adjusting, the pH value of medicine is controlled between the 3.75-5.3, can guarantee the viable bacteria survival and the growth of lactobacillus and bifidus bacillus; Suppress the growth of pathogenic bacterium simultaneously, again can be suitable mutually with local (vagina) medication environment acidity, suitable human body is avoided being upset.

Alternative acid source has citric acid, tartaric acid, fumaric acid, adipic acid etc., and alkali source has sodium carbonate, sodium bicarbonate etc.

The tartaric acid that present embodiment selects stable in properties, be difficult for moisture absorption is acid source, and sodium bicarbonate is an alkali source.In order to obtain to suit mutually, help with local application environment acidity the optimum acidity (pH4～5) of viable bacteria survival, we have investigated acid, the alkali different proportion influence to acidity.Get each 5 of the effervescent tablets (is filler with conventional adjuvant) that different proportion acid, alkali makes and be dissolved in respectively in the 50ml distilled water, measure the pH value after the effervescent tablet dissolving.See Table 1:

Table 1

Acid/alkali (mass ratio)	pH
		0.7∶1 0.8∶1	5.64 5.28

0.9∶1 1.2∶1 1.4∶1 1.6∶1

5.03 4.02 3.75 3.59

As seen from the above table, the ratio of tartaric acid and sodium bicarbonate is chosen as 0.8: 1～and 1.4: 1, preferred 0.9: 1～1.2: 1.

Medicine is made tablet (being called for short clean cloudy effervescent tablet of bigeminy viable bacteria or effervescent tablet), pharmaceutically acceptable carrier comprise various filleies, binding agent, dry adhesive, etc., further, as required, also comprise disintegrating agent, lubricant, wetting agent and solvent etc.

Select for use lactose commonly used and starch as filler of the present invention.Select for use Celluloasun Microcrystallisatum as intergranular dry adhesive and filler.Select for use low-substituted hydroxypropyl cellulose as disintegrating agent and dry adhesive, add disintegrating agent and can make the rapid disintegrate of effervescent tablet behind the vagina medicinal, medicine is dispersed in mucomembranous surface in a big way, improves local blood drug level rapidly and plays a role, and do not have the greasy of suppository.Select for use magnesium stearate as lubricant.

The selection of binding agent: make tablet, certain rigidity must be arranged.Adopt suitable binding agent, help the tablet molding and increase tablet hardness.In addition, owing to contain certain acid source, alkali source respectively in the effervescent tablet, tablet easily moisture absorption and generated reactive gas and make sheet expand, break in put procedure, the increase of hardness can reduce inter-granular porosity in the tablet, and more effectively resists the intrusion of humid air in storage process.We select three kinds of different types of binding agents commonly used, with the Different concentrations of alcohol is solvent, be made into certain density solution respectively, granulate by the same prescription of drafting, tabletting then is molding particles, tablet hardness and disintegration to be the bonding effect that index is investigated the variable concentrations binding agent.Result such as table 2:

The bonding effect of table 2 variable concentrations binding agent

Binding agent	Shaping particles	Tablet hardness	Disintegration of tablet
				5％PVPK 30(95% ethanol) 6%PEG (95% ethanol) 5%HPMC (70% ethanol)	Good granule is looser good	8～10kg 8～10kg 10～14kg	1’～2’ 2’～3’ 3’～5’

Experimental result shows, adopts the 5%PVP alcoholic solution as the prepared granule of binding agent mouldability preferably to be arranged, and the tablet of being pressed has suitable hardness, and disintegration rate is very fast, and PVP can be dissolved in the high concentration ethanol, is applicable to that non-water granulates.HPMC (5%) binding agent is also more satisfactory, but needs the ethanol preparation of certain water content, can be suitable for different preparation technologies.

Table 3 has provided three experimental examples:

Screening of table 3 experimental example and result (unit of weight: gram)

Supplementary material	Experimental example 1	Experimental example 2	Experimental example 3
				(concentration is not less than 1.0 to mycopowder A	10	10	10

×10 9The CFU/ gram) (concentration is not less than 1.0 * 10 to mycopowder B 9The CFU/ gram) tartaric acid sodium acid carbonate lactose starch sweet mellow wine Celluloasun Microcrystallisatum low-substituted hydroxypropyl cellulose adhesive (5%PVPK30Ethanolic solution (95%)) dolomol tablet mouldability acidity hardness (Kg) gas release disintegration time limited (min)

10 140 150-200 50 100 40 qs, 10 general 5.00 5～6 8～11ml 3～4

10 165 150 180 100-120 40 qs, 10 good 4.32 9～10 8～11ml 2～3

10 150 150 100 180-120-qs, 10 general 4.73 6～7 8～12ml 4～5

As can be seen, the tablet that adopts experimental example 2 to prepare has mouldability preferably from above result, and acidity is in 4～5 scopes, and the tablet compressibility is good, and meet the requirements disintegration, is excellent than other two experimental examples.The tablet that the back effect experiment adopts experimental example 2 to make carries out.

(2) preparation technology:

Blank preparation of granules: under the environment of strictness [RT18 ± 1 ℃, relative humidity (45 ± 1) %, sterilizing room], all adjuvants are all dry in advance, pulverize and cross 100 mesh sieves.The tartaric acid of weighing is placed blender, add starch and mix, take by weighing sodium bicarbonate and lactose and add and mix homogeneously.Add again behind Celluloasun Microcrystallisatum and the low-substituted hydroxypropyl cellulose mix homogeneously with 5%PVPK ₃₀Alcoholic solution (95%) is made binding agent, and the system soft material is granulated with 18 mesh sieves in granulator, 50～60 ℃ of bakings, and in the control moisture content 2%, 30 mesh sieve granulate are removed 50 order fine powders and are got the blank granule of 30～50 orders.

Sample preparation: A, the B mycopowder of weighing are ground well dispersion with same amount of lactose respectively,, add tabletting behind the lubricant mixing, promptly get effervescent tablet, pack after the semi-manufactured goods quality passed examination again with the blank granule equivalent mix homogeneously that progressively increases.

This product is a microbial ecological agent, and effective ingredient is lactobacillus and bifidus bacillus.Because dosage little (account for adjuvant total amount 1%), so the design of the prescription of tablet is based on adjuvant.Because principal agent dosage is little and heat, the moist lability of biological product, must consider heat in blended uniformity of principal agent and adjuvant and the preparation processing process, wet influence to product quality.Therefore adopt the non-water direct hybrid technique of granulating, the blank granule of preparation increases progressively with low dose of principal agent equivalent and obtains the homodisperse granule of principal agent earlier, prepares effervescent tablet at the certain pressure lower sheeting again.Avoid heat, factor such as wet to the influence of active component and guarantee the uniformity of principal agent in the preparation.

According to experimental example 2 described prescriptions, adopt above-mentioned preparation technology, make 1000 in tablet (following called after effervescent tablet) altogether, after testing, contain lactobacillus and the bacillus bifidus sum is no less than 2.0 * 10 ⁵The CFU/ sheet, acidity average out to 4.26.

Two, the external pharmacodynamics test of the clean cloudy effervescent tablet of bigeminy viable bacteria

2.1 material and method

2.1.1 experiment material

(1) vagina Lactobacillus Jensenii LY2034, vagina bifidobacterium adolescentis BH0203 viable bacteria mycopowder.

(2) pathogenic species: colon bacillus ATCC2592 type strain and clinical strain, staphylococcus aureus ATCC25923 type strain and clinical strain, gonococcus clinical strain, Candida albicans clinical strain are provided by clinical laboratory of Xiangye No. 2 Hospital of Central South University and Xiangya Medical College, Zhongnan Univ microorganism and immunology teaching and research room.Serotype of strain morphology, biochemical character, pathogenic bacterium and cultural character are identified the standard of perfection that all meets this antibacterial ^[1]Gonococcus is identified by clinical laboratory of Xiangye No. 2 Hospital of Central South University by morphology and API-NH biochemical identification bar.Candida albicans is identified by clinical laboratory of Xiangye No. 2 Hospital of Central South University by morphology and API-C AUX biochemical identification bar.

(3) culture medium: MRS prescription culture medium (being used for lactobacillus, bacillus bifidus, Candida albicans cultivation); Common Nutrient medium (being used for bacillus coli, staphylococcus aureus cultivation), GC medium is available from the happy refreshed company in Guangzhou commodity.

2.1.2 experimental technique

(1) antibacterial nephelometer counting method ^[3]: add 1% sulphuric acid 9.95ml with 1.175% barium sulfate 0.05ml, with whole its turbidity of 530nm wavelength ratio tone, making absorption value is 0.1 behind the mixing, and this turbidity is 0.5 Maxwell unit, and bacterial population is equivalent to 5 * 10 ⁸/ ml.

(2) preparation of test organisms liquid and contrast: get four kinds of pathogenic bacterium with inoculating loop, streak inoculation is in corresponding culture dish, cultivate 24h for 37 ℃, Lactobacillus Jensenii, bifidobacterium adolescentis are inoculated in respectively in the liquid MRS culture medium, and packing 5ml fluid medium is used for contrast, places 37 ℃ of anaerobism to cultivate 24h jointly.Aseptic cotton carrier is collected pathogenic bacterium, and eluting and is adjusted each bacterium liquid phase with 0.9% normal saline and answered concentration 0.5 Maxwell unit in the test tube that 0.9% normal saline is housed.

2.1.3 extracorporeal disinfecting test: respectively get pathogenic bacterium bacterium liquid 0.1ml, coat the appropriate media plate with spreading rod, every kind of bacterium is repeated 5 plates, punch with the agar plate card punch, the punching diameter is 6mm, 6 of each plate punchings, compile to 1-6 number, add MRS fluid medium (blank group) successively, the vagina Lactobacillus Jensenii, the vagina bifidobacterium adolescentis, 1: 1 mixed liquor of Lactobacillus Jensenii and bifidobacterium adolescentis, 3: 1 mixed liquors of Zhan Shi lactobacillus and bifidobacterium adolescentis, 1: 3 each 200 μ l of mixed liquor of Zhan Shi lactobacillus and bifidobacterium adolescentis, 37 ℃ cultivate 24h after, be that 1/10 vernier caliper measurement presses down the diameter that sterilization encircles with degree of accuracy.The commercial product of gonococcus culture plate system, area is less; Lactobacillus: the ratio group of bacillus bifidus=1: 3 is not done.

2.1.4 external liquid minimal inhibitory concentration: respectively get the saturated type strain escherichia coli of 50 μ l and join in the 50ml cholate lactose medium, the type strain staphylococcus aureus that 50 μ l are saturated joins in the NaCl broth bouillon of 50ml 7.5%, each packing 5 pipe behind the mixing, every pipe 9ml, every pipe add the variable concentrations (1 * 10 with the blank culture medium dilution of MRS respectively ⁹CFU/ml, 1 * 10 ⁷CFU/ml, 1 * 10 ⁶CFU/ml, 1 * 10 ⁵CFU/ml, 1 * 10 ⁴CFU/ml, 1 * 10 ²CFU/ml, blank) Lactobacillus Jensenii/bifidobacterium adolescentis, put 37 ℃ cultivate 6-12h after, count escherichia coli, staphylococcus aureus quantity with Plating, and compare with the blank group, draw minimum Mlc.And mix with the Lactobacillus Jensenii different proportion by bifidobacterium adolescentis, concentration is 1 * 10 ⁴CFU/ml, the best of seeking different proportion presses down bactericidal effect.

2.2 experimental result

2.2.1 minimal inhibitory concentration: the effective dose according to the oral class active bacteria formulation of selling on market at present is chosen in 1 * 10 ²CFU/ml, 1 * 10 ⁴--1 * 10 ⁷CFU/ml, 1 * 10 ⁹The Lactobacillus Jensenii of CFU/ml/youth bacillus bifidus (general designation: probiotic bacteria) test by concentration.The result shows that Lactobacillus Jensenii, bifidobacterium adolescentis bacterium liquid have tangible pressing down to kill effect to vaginitis common bacteria escherichia coli and staphylococcus aureus.In as following table, as can be seen, do not add that pathogenic bacterium concentration has reached 2.0 * 10 in the test tube of probiotic bacteria ⁹CFU/ml adds 1 * 10 ²The CFU/ml probiotic bacteria all can produce certain inhibitory or killing effect, 1 * 10 to two kinds of pathogenic bacterium ⁴The bacterium liquid of concentration kills effect to pressing down of two kinds of pathogenic bacterium and obviously rises, and has reached more than 98%, but has increased probiotic bacteria to 1 * 10 again ⁶CFU/ml does not change its inhibitory or killing effect, concentration 1 * 10 ⁷The bacterium liquid of concentration has reached near 100% to the suppression ratio of pathogenic bacterium.And find, under the situation of valid density, the ratio of Lactobacillus Jensenii and bifidobacterium adolescentis with press down extremely effect and do not have dependency relation.See Table 4,5,6.

The youth bacillus bifidus of table 4 variable concentrations is to the inhibition effect of pathogenic bacterium

Youth bacillus bifidus concentration (CFU/ml)	Coliform count (CFU/ml)	Staphylococcus aureus number (CFU/ml)
			1×10 9 1×10 7 1×10 6 1×10 5 1×10 4 1×10 2Blank culture medium	0 0 1.0×10 5 3.8×10 5 2.4×10 5 6.4×10 8 1.5×10 9	0 0 1.2×10 5 1.8×10 5 2.6×10 5 5.8×10 8 0.8×10 9

According to table 4, can calculate the youth bacillus bifidus pressing down of escherichia coli and staphylococcus aureus killed effect: in concentration is 1 * 10 ²During CFU/ml to the suppression ratio of escherichia coli and staphylococcus aureus at 27%-57%, and be 1 * 10 in concentration ⁴Inhibitory action to two kinds of pathogenic bacterium during CFU/ml has all reached 99%, increases concentration again and suppresses the effect variation not quite, to 1 * 10 ⁷Suppression ratio has reached 100% during CFU/ml.

The Lactobacillus Jensenii of table 5 variable concentrations is to the inhibition effect of pathogenic bacterium

Lactobacillus Jensenii concentration (CFU/ml)	Coliform count (CFU/ml)	Staphylococcus aureus number (CFU/ml)
			1×10 9 1×10 7 1×10 6 1×10 5 1×10 4 1×10 2Blank culture medium	0 0 0.6×10 5 2.0×10 5 2.6×10 5 6.0×10 8 1.5×10 9	0 0 1.8×10 5 3.2×10 5 2.8×10 5 3.6×10 8 0.8×10 9

According to table 5, can calculate Lactobacillus Jensenii pressing down of escherichia coli and staphylococcus aureus killed effect: in concentration is 1 * 10 ²During CFU/ml to the suppression ratio of escherichia coli and staphylococcus aureus at 60%-65%, and be 1 * 10 in concentration ⁴Inhibitory action to two kinds of pathogenic bacterium during CFU/ml has all reached 99%, increases concentration again and suppresses the effect variation not quite, to 1 * 10 ⁷Suppression ratio has reached 100% during CFU/ml.

The bigeminy viable bacteria of table 6 different proportion is to the inhibition effect of pathogenic bacterium

Lactic acid: bifid ratio	Coliform count (CFU/ml)	Staphylococcus aureus number (CFU/ml)
			1: 41: 31: 21: 12: 13: 14: 1 blank culture mediums	2.0×10 5 1.0×10 5 2.8×10 5 0.8×10 5 1.8×10 5 2.3×10 5 1.7×10 5 1.5×10 9	3.2×10 5 2.6×10 5 1.0×10 5 1.2×10 5 2.4×10 5 0.8×10 5 2.0×10 5 0.8×10 9

Table 6 provides the inhibition effect of the bigeminy viable bacteria of different proportion to pathogenic bacterium.According to table 6, under the situation of valid density, blended Lactobacillus Jensenii of different proportion and bifidobacterium adolescentis kill effect to pressing down of escherichia coli and staphylococcus aureus, there was no significant difference between each group as can be seen.

2.2.2 press down bactericidal effect in the solid medium

Table 7 provides the bactericidal effect of bigeminy viable bacteria to four kinds of pathogenic bacterium, and according to shown in the table 7, single vagina lactobacillus and bacillus bifidus and two kinds of bacterium thereof be mixed liquor in varing proportions, and the bactericidal effect of pressing down is all arranged.Experiment shows, can form and press down the sterilization ring clearly, and blank culture medium is (for amplification is tested with lactobacillus and bacillus bifidus cofabrication, and place increase the collarium border) form to press down the sterilization ring, it is maximum that as seen range estimation presses down the sterilization ring with bacillus coli, staphylococcus aureus clinical strain, and the bigeminy viable bacteria is better than single bacillus bifidus.Then very approaching to gonococcus, Candida albicans and staphylococcus aureus.In the variant proportioning concentration, the equal proportion mixed liquor presses down the sterilization ring and is a bit larger tham other each groups and can obtains better to press down bactericidal effect to staphylococcus aureus, and its ring diameter is 30.4 ± 0.5.Total result is analyzed, find mixed vaccine press down that the sterilization ring all is a bit larger tham single bacterium press down sterilization ring (lactobacillus to bacillus coli except).Although as if but 1: 1 mixed bacterium can be obtained preferably bactericidal effect not statistically significant as can be seen from experimental data.

Table 7 bigeminy viable bacteria to the bactericidal effects of four kinds of pathogenic bacterium (sterilization ring diameter mm, X ± SD, n=5)

	Escherichia coli	Gonococcus	Candida albicans	Staphylococcus aureus
					Culture medium lactobacillus bacillus bifidus 1: 13: 11: 3	6 30.0±0.9 24.4±0.5 28.4±0.5 27.4±0.5 28.5±0.5	6 22.5±0.5 22.4±0.6 23.3±0.5 21.4±0.4	6 20.5±0.6 20.4±0.5 22.5±0.5 21.4±0.5 21.5±0.5	6 25.5±0.5 25.5±0.5 30.4±0.5 28.2±0.5 29.3±0.5

Statistics is used the one-wayANOVA statistical method of mean, to the effect of gonococcus and Candida albicans near (p＞0.05), the bactericidal action of single viable bacteria and mixed live is approaching, and Lactobacillus Jensenii and bifidobacterium adolescentis ratio are mixed bactericidal action in 1: 1,3: 1,1: 3 close (p＞0.05)

2.3 interpretation of result and discussion

For determine LY2034 strain of vagina Lactobacillus Jensenii and vagina hebephrenictype bacillus bifidus BH0203 strain external to the killing or inhibitory action of vaginitis pathogenic bacterium, and grope minimum Mlc, for follow-up in vivo test and preparation prescription provide test basis.Four kinds of vaginitis common pathogens of this test and Selection colon bacillus ATCC25922 and clinical strain, staphylococcus aureus ATCC25923 and clinical strain, gonococcus clinical strain, Candida albicans clinical strain are as being tried bacterium.Find that by solid plate punch method and liquid culture method Lactobacillus Jensenii and youth bacillus bifidus all have tangible inhibitory or killing effect to the vaginitis common pathogen.This inhibitory or killing effect possesses a kind of condition for the treatment of colpitis medicine of exploitation.From the angle analysis of amount, find that further this inhibitory or killing effect has certain concentration dependent.1 * 10 ⁴The Lactobacillus Jensenii of CFU/ml or youth bacillus bifidus have tangible inhibitory or killing effect to these two kinds of pathogenic bacterium, have reached 99.8% but kill effect, and along with increasing progressively of concentration, but it kill, and effect does not also have change, a plateau occurs, so, 1 * 10 ⁴CFU/ml be minimum press down sterilization dose (minimal inhibitory concentration, MIC).According to the rule of clinical application, reach maximum effect, 1 * 10 with minimum amount ⁴CFU/ml is the single therapy consumption.Make behind the tablet to guaranteeing curative effect, preparation prescription selects 1 * 10 ⁵The CFU/ sheet is good.Solid punch method and liquid culture method all find, under the valid density situation, the ratio that changes both is killed effect for pressing down of pathogenic bacterium and be there is no obvious change, and promptly this pressing down kills effect and do not have stack and antagonism.Consider the anatomical structure and the flora characteristic distributions of female genital tract, be that the vagina stage casing is based on lactobacillus, the vagina deep then is dominant with bacillus bifidus, therefore, this pudendal lotion is by the regularity of distribution under women's normal flora physiological status, select for use LY2034 strain of vagina Lactobacillus Jensenii and vagina hebephrenictype bacillus bifidus LY2034 strain bigeminy viable bacteria as the treatment strain, replenish the flora in stage casing and deep simultaneously, mixed proportion is 1: 1.

2.4 conclusion

1, Lactobacillus Jensenii and youth bacillus bifidus all have good inhibitory or killing effect external to common vaginitis pathogenic bacterium.

2, according to the principle of high-efficiency low-toxicity, follow-up preparation prescription selects 1 * 10 ⁵The CFU/ sheet is good.

3, according to the dissection and the flora segmentation characteristics of female genital tract, select the mix preparation of two kinds of bacterium for use, ratio is 1: 1, replenishes the flora of different sections simultaneously.

Three, the clean cloudy effervescent lamellar body giving drugs into nose of bigeminy viable bacteria is imitated and is learned experiment ()---the experiment of treatment bacterial vaginitis

3.1 purpose

With three kinds of clinical common pathogens that cause bacterial vaginitis: escherichia coli, staphylococcus aureus and gonococcus are injected the animal subject vagina and set up the vaginitis animal model, observe the clean cloudy effervescent tablet of bigeminy viable bacteria to colpitic therapeutical effect, the drug effect of quantitative assessment bigeminy active bacteria formulation.

3.2 material

5 number sword-shaped needles, 10ml syringe, aseptic paraffin oil, 0.2% ofloxacin injection (specification is 100ml for big pharmaceutical factory, Cologne, Sichuan, batch number A020216).60 of the female White Rabbits of New Zealand are provided by Xiangye No. 2 Hospital of Central South University's Animal Lab..The gonococcus special culture media is available from the happy refreshed company in Guangzhou; Manitol salt agar (selecting to cultivate golden Portugal bacterium); The red blue agar of Yi Mei (selecting to cultivate escherichia coli) is available from China Import And Export Commodity Inspection Technology Institute; MRS culture medium (cultivating lactobacillus and bacillus bifidus).Colon bacillus clinical strain bacterium liquid is mixed with 1.35 * 10 ⁸The concentration of CFU/ml; Staphylococcus aureus clinical strain bacterium liquid is made into 1.35 * 10 ⁸The concentration of CFU/ml, gonococcus clinical strain bacterium liquid is mixed with 6.3 * 10 ⁶The concentration of CFU/ml ^[1], these antibacterials provide by clinical laboratory of Xiangye No. 2 Hospital of Central South University and identify, and above-mentioned three kinds of pathogenic bacterium equal-volumes are mixed into the modeling bacterium.Bigeminy viable bacteria mycopowder is made by previous experiments example 2, and adjuvant is provided by pharmaceutical college of Central South University.

3.3 method

3.3.1. bacterial vaginitis modelling ^[2]: 60 female new zealand white rabbits, body weight 1.95 ± 0.30kg is coated with aseptic paraffin oil with 5 number sword-shaped needle silica gel tubes, slowly insert the White Rabbit vagina, the about 5-8cm of insertion depth, pumpback is determined to annotate a little air subsequently and guarantee that medicinal liquid all injects intracavity at intravaginal injection modeling bacterium liquid 0.75ml.12 White Rabbits are put to death in modeling the 6th day at random.Get the vaginal swab smear, Gram ' s dyeing microscopic examination, the clean degree classification of vagina; Three kinds of pathogenic bacterium selective mediums are cultivated and are done bacteriology's inspection; Get vagina tissue, fixing, the row histopathological examination.

3.3.2. zoopery: 48 vaginitis model White Rabbits are divided into 6 groups, 8 every group, are respectively low dose therapy group (7 * 10 ³CFU/ml), middle dosage treatment group (7 * 10 ⁴CFU/ml), the high-dose therapy group (7 * 10 ⁵CFU/ml), ofloxacin group (10mg/kg), adjuvant group, blank group ^[1]Every vaginitis animal pattern intravaginal liquid medicine injection 1ml, be 5 days the course of treatment.Respectively before treatment, treatment back measures body weight, anus temperature, observes the clean situation of growth of animal, active situation and pudendum, aseptic little cotton swab is adopted the vaginal swab smear, Gram ' s dyeing microscopic examination is checked the clean degree of vagina; Three kinds of pathogenic bacterium selective mediums and MRS culture medium culturing are done bacteriology's inspection; Put to death White Rabbit respectively, get vagina tissue 10% neutral formalin and fix, pathological examination is done in paraffin enclosed mass, section, HE dyeing.

3.3.3 the clean degree classification of vagina: with reference to " obstetrics and gynecology " the 5th edition system piece of writing teaching material

Under the I level mirror based on bacterium vaginae, and visible a large amount of epithelial cell.

Part bacterium vaginae and a small amount of pus cell and assorted bacterium are arranged under the II level mirror, and epithelial cell also as seen.

Visible down a small amount of bacterium vaginae of III level mirror and epithelial cell are with a large amount of pus cells and other assorted bacterium.

Absence of vagina bacillus under the IV level mirror except that a small amount of epithelial cell is arranged, mainly is pus cell and a large amount of assorted bacterium.

3.3.4 modeling success criterion ^[2]

1. white rabbit pudendum outward appearance: red and swollen, suppurate, fester for+, the pudendum cleaning is-.

2. vaginal swab smear, the clean degree classification of vagina: the III-IV level is+, the I-II level is-.

3. selective medium is cultivated: three kinds of antibacterials all are grown to+, no pathogenic bacterium are grown to-.

4. get vagina tissue, the row histopathological examination: have hyperemia, edema, necrosis and inflammatory cell infiltration for+, do not have congested, edema and inflammatory cell infiltration to be-.

4 criterions have 2 positives to think that promptly modeling is successful.

3.3.5 vaginitis is effective and criterion of cure: with reference to " obstetrics and gynecology " the 5th edition system piece of writing teaching material

1. the vagina part does not have hyperemia, edema, abnormal secretion thing

2. the clean degree classification of vagina III-IV level reverts to the II level for effective after the treatment, and the I level is defined as healing

3. the pathogenic bacterium selective medium is cultivated asepsis growth for curing

4. pathologic finding does not have edema, hemorrhage and inflammatory cell infiltration.

Criterion of cure: the vagina outward appearance is normal, clean degree I level, and the growth of no pathogenic bacterium, pathologic finding is normal, does not have changes such as hyperemia, edema, hemorrhage and inflammatory cell infiltration.

The improvement standard: the vagina outward appearance is by congestion and edema or suppurate and change into normally, and clean degree changes II level or I level into by the III-IV level, does not have or every plate minority bacterium colony that causes a disease, and pathologic finding is roughly normal.

3.3.6 cure rate and efficient calculating:

The number of animals of cure rate (%)=recovery from illness/respectively organize total number of animals * 100%

Effective percentage (%)=(number of animals of improvement)/respectively organize total number of animals * 100%

3.4 result

3.4.1 doe vaginitis modeling result:

3.4.1.1 the modeling of doe vaginitis detects: modeling the 6th day, observe animal pudendum outward appearance, put to death 12 animals at random and comprise the classification of vagina cleanliness factor, bacteriological detection.The vagina tissue pathological examination, whether successful to pass judgment on modeling.Found that the whole modelings successes of 12 does of execution, showing as the vagina outward appearance has in various degree redness, and secretions is unusual, clean degree III-IV level; Bacteriology checking is found pathogenic bacterium; Pathological examination is found vaginal mucosa hyperemia, edema, inflammatory cell infiltration.Be modeled as power 100%.See Table 8

12 made White Rabbit vaginitis model testing results of table 8

	2	15	21	35	26	30	33	39	40	41	43	54
													The clean degree classification of pudendum outward appearance vagina bacteriological detection vagina tissue pathologic finding	+ + + +	+ + + +	+ + + +	+ + + +	+ + + +	+ + + +	+ + + +	+ + + +	+ + + +	+ + + +	+ + + +	+ + + +

"+,-" symbol is according to the criterion gained in the table.Numeral is a number of animals.

3.4.1.2 being used for the treatment of 48 animal vaginal secretionies of observation detects: the vaginal secretions Gram is checked.The result shows that the vagina cleanliness factor of animal is III level and IV level before the treatment, 28 animals of III level wherein, and 20 animals of IV level meet colpitic diagnosis.Proof is not put to death 48 animals and has been formed vaginitis yet, photo under the vaginal secretions smear mirror of part animal subject, visible a large amount of pyocytes..

3.4.2 clean degree of vagina and curative effect before and after the treatment:

By the vaginal swab smear, the Gram result sees a large amount of pyocytes and pathogenic bacterium in the treatment provagina juice, and the male bacillus of no Gram ' s is treated under the 6th clear water surface and sees, and middle and high dosage smear is not seen pus bacterium and pathogenic bacterium, visible bacterium vaginae.Low dose group, pyocyte obviously reduces, and epithelial cell produces, and pathogenic bacterium reduce; And the ofloxacin arrangement of mirrors is relatively cleaner down, and no pyocyte and pathogenic bacterium do not have the male bacillus of Gram ' s yet.

According to clinical diagnosis bacterial vaginitis detection method commonly used, the vaginal secretion Gram is checked.The result shows as shown in table 9, and the vagina cleanliness factor of animal is III level and IV level before the treatment, meets the diagnosis of vagina.Bigeminy active bacteria formulation with low dosage is also obtained certain therapeutic effect, cure rate reaches 50%, the clean degree of III level and IV level transfers I-II level cleanliness factor to, effective percentage 100%, the bigeminy active bacteria formulation of middle and high dosage is close with the ofloxacin curative effect, can reach the effective percentage of 88% cure rate and 100%.Adjuvant group and blank group are invalid, only have 12.5% animal to take a turn for the better naturally, do not have the animal of recovery from illness.The efficacy result explanation, bigeminy viable bacteria system curative effect is comparatively sure, the angle analysis of amount, middle dosage is enough to reach therapeutic effect.

Table 9: the vagina cleanliness factor changes and curative effect before and after the treatment

Clean degree (model) (only) before the treatment

Treatment back clean degree (only)

Cure rate

Effective percentage

Grouping	n	I	II	III	IV	I	II	III	IV	(％)	(％)
												Dosage group high dose group ofloxacin group adjuvant group blank group in the low dose group	8 8 8 8 8 8	0 0 0 0 0 0	0 0 0 0 0 0	5 5 4 4 5 5	3 3 4 4 3 3	4 7 7 7 0 0	4 1 1 1 1 1	0 0 0 0 7 7	0 0 0 0 0 0	50％ 88％ 88％ 88％ 0 0	100％ 100％ 100％ 100％ 12.5％ 12.5％

Numeral is a number of animals in the table, and effective percentage and cure rate calculate gained according to criterion.

Statistics is used the X that spss software adopts a plurality of sample rates ²The method of inspection, clean degree variation of vagina and curative effect high dose group, middle dosage group, ofloxacin group zero difference (p＞0.10), but significant difference (p＜0.025) is arranged with adjuvant group, blank group

3.4.3 bacteriological detection:

3.4.3.1 three kinds of pathogenic bacterium selective mediums are cultivated after the modeling: get vaginal swab and cultivated 48 hours at three kinds of pathogenic bacterium selective mediums, 12 animal vaginal swabs of putting to death are coated with ware and all find all to have on three kinds of selective mediums a large amount of colony growths in flakes, illustrate that the vaginitis animal pattern is relevant with three kinds of pathogenic bacterium.

3.4.3.2 ne ar detects, and chooses the monoclonal smear on above-mentioned three kinds of selective mediums, Gram ' s dyeing, and mirror is observed ne ar down, so that the antibacterial from morphology evaluation clone.The result shows respectively, as seen a large amount of gram negative bacilli, a large amount of gram-positive staphylococcus and a large amount of gram-negative diplococci, from morphologic angle, coincide with the target of selective medium, further three kinds of pathogenic bacterium of proof actuating thing vaginitis model are present in the intravaginal of animal.

3.4.4 ordinary circumstance before and after the treatment:

3.4.4.1 animal subject ordinary circumstance and body weight change before and after the treatment

Enter all No. 48 animals survival of the overall process to treatment of test, none number death from modeling.The equal well-grown of animal subject, activity freely, hair sees that whole body is smooth.Each group of body weight all has increase slightly before and after the treatment, but no difference of science of statistics.Yet not statistically significant of the weight of animals increase between each group, side light bigeminy viable bacteria pudendal lotion does not have tangible toxic effect to animal subject, the results are shown in Table 10.

Table 10: the variation of body weight before and after the modeling (kg, X ± SD)

Grouping	Number of animals (n)	Before the treatment (kg)	Treatment back (kg)
				Dosage group high dose group in the blank group of the blank group adjuvant low dose group	8 8 8 8 8	2.0±0.2 1.9±0.3 2.1±0.3 2.2±0.3 2.1±0.4	2.2±0.2 2.1±0.2 2.2±0.3 2.3±0.2 2.2±0.4

Ofloxacin group

8

1.9±0.4

2.0±0.4

The ★ statistics is used the employing one-wayANOVA of spss statistical software and is analyzed, and treatment front and back body weight does not have significant difference (P＞0.05) between each group

3.4.4.2 animal heat changes before and after the treatment

Because this experimental model is a bacterial vaginitis.The variation of body temperature takes place judging the diseases associated with inflammation degree, and development has certain meaning.Table 11 is depicted as No. 48 variations before and after the animal subject treatment.No matter be that animal subject body temperature does not have significant change behind the peak period and healing of inflammation development.Animal subject is described to colpitis due to the pathogenic bacterium, body temperature changes not obvious.For this phenomenon is described, No. 20 healthy new zealand white rabbit anuses of this test determination and animal subject body temperature are approaching, about 39.5 ℃ of average out to.

Table 11: the variation of body temperature before and after the modeling (℃, X ± SD)

Grouping	Number of animals (n)	Before the treatment (kg)	Treatment back (kg)
				Dosage group high dose group ofloxacin group in the blank group of the blank group adjuvant low dose group	8 8 8 8 8 8	39.3±0.3 39.8±0.2 39.7±0.5 39.5±0.4 39.5±0.4 39.4±0.4	39.4±0.4 39.7±0.3 39.5±0.3 39.5±0.3 39.5±0.4 39.5±0.4

The ★ statistics is used the employing one-wayANOVA of spss statistical software and is analyzed, and treatment front and back body temperature does not have significant difference (P＞0.05) between each group

3.4.5 animal subject pudendum situation before and after the treatment

The scorching animal pattern pudendum of treatment provagina all has hyperemia in various degree, edema, and part has purulent secretion, minority that rotten to the corn phenomenon is arranged.The treatment back is except that adjuvant and blank treated animal pudendum mild hyperaemia edema, and bigeminy active bacteria formulation and ofloxacin treatment treated animal pudendum all recover normal outward appearance pink, totally do not have secretions.

3.4.6 bacteriology checking

3.4.6.1. bigeminy viable bacteria pudendal lotion treatment back vaginal secretions pathogenic bacterium and anaerobe detect.

On the basis of injecting pathogenic bacterium modeling success, for observing the bactericidal effect that presses down of bigeminy viable bacteria pudendal lotion, selective medium with three kinds of pathogenic bacterium enters the cultivation of vaginal secretions smear, checked that but whether effectively having killed pathogenic bacterium has reached therapeutic purposes. again because intravaginal normal flora major part is anaerobe, there is report to be referred to as bacterium vaginae, this test adopts the MRS culture medium anaerobism of cultivating the bigeminy viable bacteria to cultivate, observe and whether exist dysbacteriosis and treatment back dysbacteriosis can obtain correcting.

Observed result shows: high dose group and ofloxacin have the powerful effect of killing pathogenic bacterium that presses down, and do not turn out pathogenic bacterium.Bigeminy viable bacteria pudendal lotion can be rebuild the normal vaginal microbial flora of animal, and ofloxacin does not have this effect.But the bigeminy active bacteria formulation of low dosage very has obviously bactericidal action, and pathogenic bacterium colony obviously reduces.And the effect of rebuilding the intravaginal normal flora is arranged, and the control animals intravaginal still has a large amount of pathogenic bacterium survivals, no anaerobe.Normal intravaginal bacterial micro-ecological environment had suffered destruction after the generation vaginitis was described.

3.4.6.2. ne ar is identified.

For the flora of clearly selecting culture medium to cultivate is corresponding pathogenic bacterium.Get clone's microscopy and observe, the result shows that bacteriological form conforms to the purpose of corresponding selection culture medium.Show visible a large amount of gram-positive staphylococcus, gram negative bacilli, gram-negative diplococci, gauge rod shape and corynebacterium anaerobe respectively.

3.4.6.3. bigeminy viable bacteria pudendal lotion treatment back animal subject vaginal secretions pathogenic bacterium and anaerobe detect.

For comprehensive assessment bigeminy viable bacteria pudendal lotion is treated colpitic mechanism.Get all and respectively organize the animal subject vaginal secretions, pathogenic bacterium are detected, so that the therapeutic effect of proof bigeminy active bacteria formulation is to kill pathogenic bacterium or other mechanism by pressing down with selective medium.Cultivate the vaginitis normal flora with MRS culture medium anaerobism simultaneously, to prove that the vaginitis animal dis is in dysbacteriosis.Can reach the purpose of rebuilding the intravaginal dysbacteriosis after the treatment of bigeminy active bacteria formulation.Table 12 is the result show: bigeminy active bacteria formulation and ofloxacin local application can press down intravaginal pathogenic bacterium extremely effectively.The strongest with the ofloxacin effect.Bigeminy viable bacteria pudendal lotion to press down bactericidal action relevant with dosage, middle high dose effect is best.Blank and adjuvant does not have the bactericidal action of pressing down.Therefrom there is dysbacteriosis in the intravaginal of vaginitis animal pattern as can be seen.Can rebuild or correct the intravaginal dysbacteriosis of vaginitis animal pattern with bigeminy viable bacteria pudendal lotion.

The 6th day vaginal swab of table 12 treatment cultivated the plate number that colony growth was arranged after 48 hours

Grouping	The red blue agar of Yi Mei (cultivating escherichia coli)		Manitol salt agar (cultivating golden Portugal bacterium)		GC medium (cultivating gonococcus)		MRS culture medium (cultivation anaerobe)
									+	-	+	-	+	-	+	-
	Dosage group high dose group ofloxacin group in the blank group adjuvant group low dose group	8 8 4 1 * 1 * 0 *	0 0 4 7 7 8	8 8 4 1 * 0 * 0 *	0 0 4 7 8 8	8 8 4 1 * 1 * 1 *	0 0 4 7 7 7	1 1 5 6 + 7 + 1 +									7 7 3 2 1 7

Numeral is the plate number in the table ,+for bacteria growing is arranged ,-be asepsis growth.

After the treatment, vagina is wiped away to give and draw ware cultivation 48 hours on 4 kinds of selective medium, blank group and adjuvant group still have a large amount of varied bacteria growings, almost there is not the anaerobe growth. middle high dose group does not almost have the pathogenic bacterium growth, and a large amount of anaerobe growths are arranged. and ofloxacin group does not almost have the pathogenic bacterium growth not have the anaerobe growth yet. ^*P＜0.05 and blank group, the adjuvant group compares, ⁺P＜0.05 and blank group, the adjuvant group, ofloxacin group compares, and statistics is used the X that spss software adopts a plurality of sample rates ²The method of inspection is analyzed.

3.4.7 the pathology after the animal subject treatment change

The vaginal mucosa smooth in appearance of each group of treatment, no ulcer and erosion, structure is clear.In the treatment group, as seen two animal intravaginal of low dose group have heavy-gravity secretions slightly, and each animal vagina of blank group and adjuvant group is congestion and edema not, all the other all visible congestion and edema and secretions increase in various degree, thickness.During mirror is observed down and is found, the animal of the bigeminy active bacteria formulation treatment of high dose, histopathology is no abnormal.No. 2 animal vagina tissues of low dose group are seen congested companion's edema.Control animals is seen under the tangible mirror congested, edema, and 5 animal companion inflammatory cell infiltrations (as table 13) are arranged.

Table 13 treatment back vaginitis model vagina tissue pathological change

	Congested	Edema	Inflammatory cell infiltration	Mucosal erosion
					Dosage group high dose group ofloxacin group in the blank group adjuvant group low dose group	8 8 2 0 0 0	8 8 2 0 0 0	2 3 0 0 0 0	0 0 0 0 0 0

◆ numeral is a pathological abnormalities example number in the table, and every treated animal number is 8.The 5th day vagina tissue mirror of modeling seen down, and hyperemia, edema and inflammatory cell infiltration are arranged; Treat the 6th day vagina tissue mirror and see down, congested, edema obviously alleviates, and does not see inflammatory cell infiltration, do not have rotten to the corn, high dose group, middle dosage group, ofloxacin group zero difference, but significant difference (p＜0.05) is arranged with adjuvant group, blank group, statistics is used spss software and is adopted X ²The method of inspection is analyzed.

3.5 interpretation of result

As a result on the basis, observe the effect of bigeminy active bacteria formulation treatment vaginitis animal model at external efficiency assay.Directly inject the new zealand white rabbit intravaginal with three kinds of pathogenic bacterium, can successfully prepare the vaginitis animal model.Proved the colpitic existence of animal from outward appearance, vaginal secretions detection, bacteriological detection and vagina tissue pathological examination, its characteristics are leading indicators for the vaginal secretions pyocyte, can reflect colpitic existence well, and are identical with existing clinical diagnosis.Histopathology is based on the non-specific change of congestion and edema.Bacteriological detection has certain meaning.Do not find normal bacterium from vaginal secretions smear and the anaerobic culture of blank treatment group, prove that vaginitis the back intravaginal takes place has dysbacteriosis, promptly pathogenic bacterium can press down the normal bacterium of intravaginal extremely.

After the treatment of bigeminy active bacteria formulation, the redness and the secretions of vagina outward appearance are reduced to disappearance, recover normal appearance.Clinical being used to detected the method discovery that cleanliness factor is judged inflammation, and the animal model vaginal secretions after the treatment is clearly better, and almost all has towards clean direction and develops.Middle and high dosage group is evident in efficacy, and cure rate 88% obviously is better than adjuvant group and blank group, and is approaching with the therapeutic effect of ofloxacin.From vaginal smear and the explanation of pathogenic bacterium selective medium cultivation results, although the ofloxacin therapeutic effect clearly, the clean and abacterial vestige of secretions has reached therapeutic goal under the mirror, has also stayed the dysbacteriosis potential risk simultaneously.Can cause the recurrence of disease or infection again.Show that from the bacteria cultivation results of blank group and adjuvant matched group normal flora destroyed in the microbial vaginitis that causes a disease can cause leading the way.Bigeminy viable bacteria pudendal lotion treatment principle is to correct dysbacteriosis, and additional normal bacterium reaches.Therefore, the result shows middle and high dosage bigeminy live bacteria agent when reaching therapeutic effect, and keeping and having rebuild intravaginal normal flora microenvironment is the main advantage of this preparation.

3.6 conclusion

1, bigeminy viable bacteria pudendal lotion has the sure curative effect of treatment bacterial vaginitis.Approaching with ofloxacin local application effect, cure rate is 88%.Dosage is median dose (7 * 10 ⁴CFU/ml) for well.

2, the clean cloudy effervescent tablet of bigeminy viable bacteria is treated the colpitic mechanism of action and press down to be killed pathogenic bacterium and reach by setting up normal flora.

3, the clean cloudy effervescent tablet of bigeminy viable bacteria has the effect of keeping or rebuilding intravaginal normal flora microecological environment.

List of references:

[1] Xu Shuyun, Bian Rulian, old repairing, " pharmacological experimental methodology ", People's Health Publisher, version in 2002

[2] Shi Xinyou, " modern medicine experimental zoology ", Beijing, People's Medical Officer Press, version in 2000

Four, the clean cloudy effervescent lamellar body giving drugs into nose of bigeminy viable bacteria is imitated and is learned experiment (two)---treatment colpitis mycotica and gonococcal vaginitis experiment

4.1 material

5 number sword-shaped needles, 10ml syringe, aseptic paraffin oil.Trobicin (2g, lot number 020601 Lukang Medical Co., Ltd., Shandong produces).Fluconazole injection (lot number 020203025 Shanghai Hua Yuan Pharmacy stock Co., Ltd produces for 100ml, 0.2g).72 of female new zealand white rabbits are provided by Xiangye No. 2 Hospital of Central South University's Animal Lab..The gonococcus special culture media is available from the happy refreshed company in Guangzhou; Rose Bengal Sodium agar (cultivation Candida albicans) is available from China Import And Export Commodity Inspection Technology Institute (Beijing Luqiao Technology Co., Ltd.); MRS culture medium (cultivating lactobacillus and bacillus bifidus).Candida albicans clinical strain bacterium liquid (3.6 * 10 ⁷CFU/ml), gonococcus clinical strain bacterium liquid (6.3 * 10 ⁶CFU/ml) ^[3], provide and identify by clinical laboratory of Xiangye No. 2 Hospital of Central South University.Bigeminy viable bacteria mycopowder is by 2 preparations of previous experiments example, and adjuvant is provided by pharmaceutical college of Central South University.

4.2 method

4.2.1. mycotic and gonococcal vaginitis modelling: 72 female new zealand white rabbits, be divided into 2 groups at random, 36 every group, the modeling of one group of intravaginal injection Candida albicans liquid, another group injection gonococcus liquid modeling.Be coated with aseptic paraffin oil with 5 number sword-shaped needle silica gel tubes, slowly insert the White Rabbit vagina, the about 5-8cm of insertion depth, pumpback is determined to inject corresponding modeling bacterium liquid 0.25ml, Candida albicans liquid concentration 3.6 * 10 in intravaginal ⁷CFU/ml gonococcus liquid concentration 6.3 * 10 ⁶CFU/m, modeling the 6th day is put to death 6 White Rabbits for every group at random.Make the vaginal swab smear, Gram ' s dyeing microscopic examination, the clean degree classification of vagina, two kinds of selective mediums are cultivated and are carried out bacteriological detection.Get vagina tissue, 10% neutral formalin is fixed, the row histopathological examination.

4.2.2. zoopery: 30 Candida albicans vaginitis model White Rabbits are divided into 5 groups at random, 6 every group, are respectively low dose therapy group (7 * 10 ³CFU/ml), middle dosage treatment group (7 * 10 ⁴CFU/ml), the high-dose therapy group (7 * 10 ⁵CFU/ml), fluconazol group (14mg/kg), normal saline matched group ^[2]30 gonococcal vaginitis model White Rabbits are divided into 5 groups at random, 6 every group, are respectively low dose therapy group (7 * 10 ³CFU/ml), middle dosage treatment group (7 * 10 ⁴CFU/ml), the high-dose therapy group (7 * 10 ⁵CFU/ml), trobicin group (140mg/kg), normal saline matched group, every vaginitis animal pattern intravaginal liquid medicine injection 1ml.Inject little air subsequently and enter intracavity, treated 7 days to guarantee medicinal liquid.Respectively before treatment, treatment back observes the clean situation of growth of animal, active situation and pudendum, aseptic little cotton swab is got the vaginal swab smear, Gram ' s dyeing microscopic examination is checked the clean degree of vagina; Selective medium is cultivated and is carried out bacteriological detection; Put to death White Rabbit respectively, get vagina tissue 10% neutral formalin and fix, paraffin enclosed mass, section, HE dyeing row pathological examination.

4.3 result

4.3.1 doe vaginitis modeling result:

4.3.1.1 12 doe vaginitis modeling results that put to death: after the modeling the 6th day, put to death 12 White Rabbits at random, observe the pudendum outward appearance, make the vaginal swab smear, Gram ' s dyeing microscopic examination, the clean degree classification of vagina, bacteriological detection.Get the capable pathological examination of vagina tissue, whether successful to pass judgment on modeling.Found that the whole modelings successes of 12 does of execution, showing as the vagina outward appearance has in various degree redness, and secretions is unusual, clean degree III-IV level, vaginal mucosa hyperemia, edema.The result shows the whole modeling successes of White Rabbit, and success rate 100% sees Table 14.

12 White Rabbit modeling successes of table 14 judged result

Numbering	5	9	20	26	31	36	42	45	53	60	69	71
													The clean degree classification of project pudendum outward appearance vagina bacteriological detection pathologic finding	+ + + +	+ + + +	+ + + +	+ + + +	+ + + +	+ + + +	+ + + +	+ + + +	+ + + +	+ + + +	+ + + +	+ + + +

"+" number is according to the criterion gained in the table.Numeral is a number of animals.

5,9,20,26,31, No. 36 is Candida albicans modeling group, 42,45,53,60,69,71 gonococcus modeling groups.

4.3.1.2 do not put to death 60 animal vaginal secretions smears: the vaginal secretions Gram is checked.The result shows that the vagina cleanliness factor of animal is III level and IV level before the treatment, 29 animals of III level wherein, and 31 animals of IV level meet colpitic diagnosis.Proof is not put to death the yet modeling success of 60 animals.

4.3.2 the variation of the clean degree of vagina before and after the treatment

4.3.2.1 animal subject vaginal swab smear Gram mirror detects down:

See that a large amount of pyocytes are full of in the visual field in Candida albicans and the gonococcus animal model vaginal secretion before the treatment.Candida albicans animal model and trobicin treatment back gonococcus animal model vaginal secretions does not have pyocyte after the Fluconazole treating, and the visual field is cleaner.The gonococcus property of the bigeminy active bacteria formulation treatment of middle and high dosage and the most of no pyocyte of candida albicans vaginitis animal model vaginal secretions, a case each visible a little pyocyte of treatment group.Low dose group bigeminy active bacteria formulation treatment group, Candida albicans group 2 examples, gonococcus is organized visible a little pyocyte of 3 routine vaginal secretionies.

Wherein, the animal vaginal swab is checked before and after the treatment of Candida albicans modeling group: the vaginal swab smear was got in modeling on the 5th day, visible a large amount of pyocytes and pathogenic bacterium; The fluconazol group is not seen pyocyte and pathogenic bacterium, and epithelial cell produces; Pyocyte and pathogenic bacterium are not seen in high dose group treatment back the 8th day, visible vagina bacillus; Pyocyte and pathogenic bacterium are not seen, visible vagina bacillus in middle dosage group treatment back the 8th day; Normal saline group treatment back the 8th day, visible pyocyte and pathogenic bacterium do not see the vagina bacillus.

The animal vaginal swab is checked before and after the treatment of gonococcus modeling group: the vaginal swab smear was got in modeling on the 5th day, visible a large amount of pyocytes and pathogenic bacterium; B is a trobicin treatment group, does not see pyocyte and pathogenic bacterium, and visible epithelial cell produces; High dose group was treated the 8th day, did not see pyocyte and pathogenic bacterium, visible vagina bacillus; Middle dosage group was treated the 8th day, did not see pyocyte and pathogenic bacterium, visible vagina bacillus; The normal saline group was treated the 8th day, and visible pyocyte and pathogenic bacterium do not see the vagina bacillus.

4.3.2.2 bigeminy viable bacteria pudendal lotion treatment Candida albicans vaginitis clean degree of animal vagina and curative effect

The result shows that best with the curative effect of fluconazol, effective percentage and cure rate reach 100%.Bigeminy viable bacteria pudendal lotion curative effect certainly, effective percentage very reaches 100%, is that the cure rate of standard is 83% with the clean degree of vagina, than the weak curative effect of fluconazol.The cure rate of low dose therapy group very reaches 66%.Each treatment group of bigeminy active bacteria formulation all is better than the normal saline group, and statistical significance is arranged.Result such as table 15.

Table 15: the clean degree of vagina changes and curative effect before and after the treatment of Candida albicans modeling group

Grouping	n	Clean degree (model) (only) before the treatment				Treatment back clean degree (only)				Cure rate (%)	Effective percentage (%)
												I	II	III	IV	I	II	III	IV
		Dosage group high dose group fluconazol group normal saline group in the low dose group	6 6 6 6 6	0 0 0 0 0	0 0 0 0 0	3 3 2 3 3	3 3 4 3 3	4 5 5 6 0	2 1 1 0 1											0 0 0 0 3	0 0 0 0 2	66％ 83％ 83％ 100％ 0	100％ 100％ 100％ 100％ 17％

Numeral is a number of animals in the table, and effective percentage and cure rate calculate gained according to criterion.

Statistics is used the X that spss software adopts a plurality of sample rates ²The method of inspection, clean degree variation of vagina and curative effect high dose group, middle dosage group, fluconazol group zero difference (0.25＞p＞0.10), but significant difference (0.025＞p＞0.01) is arranged with adjuvant group, blank group

4.3.2.3 bigeminy viable bacteria pudendal lotion treatment clean degree of gonococcal vaginitis vagina and curative effect.

Shown in table 16 result, gonococcus property vaginitis is best with the curative effect with trobicin, and effective percentage and cure rate reach 100%.Bigeminy viable bacteria pudendal lotion curative effect certainly, effective percentage very reaches 100%, is that the cure rate of standard is 83% with the clean degree of vagina, than the weak curative effect of trobicin.The cure rate of low dose therapy group very reaches 66%.Each treatment group of bigeminy active bacteria formulation all is better than normal saline treatment group, and statistical significance is arranged.

Table 16: the clean degree of vagina changes and curative effect before and after the treatment of gonococcus modeling group

Grouping	n	Clean degree (model) (only) before the treatment				Treatment back clean degree (only)				Cure rate (%)	Effective percentage (%)
												I	II	III	IV	I	II	III	IV
		Dosage group high dose group trobicin group normal saline group in the low dose group	6 6 6 6 6	0 0 0 0 0	0 0 0 0 0	3 4 2 3 3	3 2 4 3 3	3 5 5 6 0	3 1 1 0 1											0 0 0 0 3	0 0 0 0 2	50％ 83％ 83％ 100％ 0	100％ 100％ 100％ 100％ 17％

Numeral is a number of animals in the table, and effective percentage and cure rate calculate gained according to criterion.

Statistics is used the X that spss software adopts a plurality of sample rates ²The method of inspection, high dose, middle dosage, trobicin be to gonococcal vaginitis curative effect zero difference (p＞0.10), but with low dose group, normal saline group significant difference (p＜0.025) is arranged.

4.3.3 bacteriological detection

The modeling animal pathogen is checked

Candida albicans modeling group: get vaginal swab and on the Rose Bengal Sodium agar culture medium, be coated with ware, cultivated 48 hours, a large amount of colony growths are arranged, illustrate that the vaginitis animal pattern is relevant with Candida albicans, the modeling success; And the MRS culture medium is not seen the viable bacteria growth.

Gonococcus modeling group: get vaginal swab and be coated with ware on the gonococcus special culture media, cultivated 48 hours, finding has a large amount of colony growths, illustrates that the vaginitis animal pattern is relevant with gonococcus, the modeling success; And the MRS culture medium is not seen the viable bacteria growth.

Ne ar detects: on above-mentioned two kinds of selective mediums, choose the monoclonal smear, and Gram ' s dyeing, mirror is observed ne ar down.Corresponding visible a large amount of gram-positive coccis and gram-negative diplococci.

4.3.4 animal ordinary circumstance and pudendum outward appearance before and after the treatment

Laboratory animal well-grown, activity freely, hair is next to the shin smooth, pudendum is clean before the modeling, there is not redness, no purulent secretion, modeling was observed on the 5th day, the pudendum redness all appears in laboratory animal in various degree, purulent secretion is arranged, and wherein No. 42, No. 44, No. 57 animals see festers, through after 5 days various dose Drug therapys, high, middle dosage group and fluconazol group, trobicin group treatment back animal pudendum redness all disappear, and purulent secretion obviously reduces.Low dose group treatment back pudendum does not have congestion and edema, and normal saline group treatment back animal pudendum has congestion and edema.

4.3.5 treatment back bacteriological detection

4.3.5.1 colpitis mycotica model treatment back bacteriological detection

Behind the bigeminy active bacteria formulation treatment candida albicans vaginitis animal subject, Candida albicans and anaerobe testing result such as table 17 in the vaginal secretions, fluconazol can effectively be killed Candida albicans, cultivate with Rose Bengal Sodium agar selectivity, all find no Candida albicans in all 6 animal vaginal secretionies of Fluconazole treating group, bigeminy active bacteria formulation treatment group high, middle dosage has in 5 animal vaginal secretionies and all finds no Candida albicans, illustrates that bigeminy viable bacteria pudendal lotion has the sure effect of killing Candida albicans that presses down.The bigeminy active bacteria formulation of low dose group has in 4 animal vaginal secretionies and all finds no albicans growth, and further specifying said preparation has the effect of killing Candida albicans that obviously presses down.And normal saline treatment group all can be turned out Candida albicans.Select to cultivate the bigeminy viable bacteria relatively with anaerobe MRS, table 17 result shows that the Fluconazole treating group is not turned out the bigeminy viable bacteria, and the bigeminy viable bacteria treatment group of each dosage all can be turned out anaerobic bigeminy viable bacteria.Each dosage group anaerobism is cultivated and is found simultaneously, and each 1 example of high, middle dosage group albicans growth occurs in the low dose group 2 routine culture dishs, and normal saline treatment group can be found a large amount of albicans growths.

Table 17 treatment back vaginal swab Candida albicans and anaerobe have the plate number of colony growth

Numeral is the plate number in the table ,+for bacteria growing is arranged ,-be asepsis growth.

Female white rabbit was treated the 8th day, vaginal swab is drawn ware and was cultivated 48 hours on 3 kinds of selective mediums, find that the normal saline group still has a large amount of pathogenic bacterium growths, almost there is not the anaerobe growth, and middle high dose group does not almost have the pathogenic bacterium growth, but a large amount of anaerobe growths are arranged, the fluconazol group does not almost have the pathogenic bacterium growth yet, there is not the anaerobe growth. ▲ vs ● in, high dose group and fluconazol group are antibacterial significantly, obviously be better than the normal saline group, among the ■ vs ★, high dose group anaerobe plate number is obviously more than the fluconazol group, and statistics is used the X that spss software adopts a plurality of sample rates ²The method of inspection is analyzed, and high dose group, middle dosage group, fluconazol group zero difference (0.25＞p＞0.10) have significant difference (0.025＞p＞0.01) with the normal saline group.

4.3.5.2 bigeminy viable bacteria pudendal lotion treatment back vaginal secretions pathogenic bacterium and anaerobe detect

On the basis of injecting pathogenic bacterium modeling success, for observing the bactericidal effect that presses down of bigeminy viable bacteria pudendal lotion, with Rose Bengal Sodium agar selective medium the vaginal secretions smear is cultivated, checked that but whether effectively having killed pathogenic bacterium has reached therapeutic purposes. again because intravaginal normal flora major part is anaerobe, there is report to be referred to as bacterium vaginae, this test adopts the MRS culture medium anaerobism of cultivating the bigeminy viable bacteria to cultivate, observe and whether exist dysbacteriosis and treatment back dysbacteriosis can obtain correcting.High dose group and fluconazol have the powerful effect of killing pathogenic bacterium that presses down, and do not turn out pathogenic bacterium.Bigeminy viable bacteria pudendal lotion can be rebuild the normal vaginal microbial flora of animal, and fluconazol does not have this effect.But the bigeminy active bacteria formulation of low dosage very has obviously bactericidal action, and pathogenic bacterium colony obviously reduces.And the effect of rebuilding the intravaginal normal flora is arranged, and the control animals intravaginal still has a large amount of pathogenic bacterium survivals, no anaerobe.Normal intravaginal bacterial micro-ecological environment had suffered destruction after the generation vaginitis was described.

4.3.5.3 gonococcal vaginitis model treatment back bacteriological detection

Behind the bigeminy active bacteria formulation treatment gonococcus property vaginitis animal subject, gonococcus and anaerobe testing result such as table 18 in the vaginal secretions, trobicin can effectively be killed gonococcus, cultivate with gonococcus special culture media selectivity, the trobicin treatment is organized in all 6 animal vaginal secretionies and is all found no gonococcus, bigeminy active bacteria formulation treatment group high, middle dosage has in 5 animal vaginal secretionies and all finds no gonococcus, illustrates that bigeminy viable bacteria pudendal lotion has the sure gonococcida effect that presses down.The bigeminy active bacteria formulation of low dose group has and all finds no gonococcus growth in 4 animal vaginal secretionies, and further specifying said preparation has the gonococcida effect that obviously presses down.And normal saline treatment group all can be turned out gonococcus.Select to cultivate the bigeminy viable bacteria relatively with anaerobe MRS, table 18 result shows that trobicin treatment group is not turned out the bigeminy viable bacteria, and the bigeminy viable bacteria treatment group of each dosage all can be turned out anaerobic bigeminy viable bacteria.Each dosage group anaerobism is cultivated and is found simultaneously, and the gonococcus growth appears in each 1 example of high, middle dosage group in the low dose group 2 routine culture dishs, and normal saline treatment group can be found a large amount of gonococcus growths.

The 8th day vaginal swab of table 18 treatment cultivated the plate number that colony growth was arranged after 48 hours

Numeral is the plate number in the table ,+for bacteria growing is arranged ,-be asepsis growth.

Female white rabbit was treated the 8th day, vaginal swab is drawn ware and was cultivated 48 hours on 3 kinds of selective mediums, find that the normal saline group still has a large amount of pathogenic bacterium growths, almost there is not the anaerobe growth, and middle high dose group does not almost have the pathogenic bacterium growth, but a large amount of anaerobe growths are arranged, the trobicin group does not almost have the pathogenic bacterium growth yet, there is not the anaerobe growth. ▲ vs ● in, high dose group and trobicin group are antibacterial significantly, obviously be better than the normal saline group, among the ■ vs ★, high dose group anaerobe plate number has significant difference obviously more than the trobicin group with the normal saline group, and statistics is used the X that spss software adopts a plurality of sample rates ²The method of inspection is analyzed.High dose group, middle dosage group, trobicin group zero difference (0.25＞p＞0.10) have significant difference (0.025＞p＞0.01) with the normal saline group.

4.3.5.4 bigeminy viable bacteria pudendal lotion treatment back vaginal secretions pathogenic bacterium and anaerobe detect

On the basis of injecting pathogenic bacterium modeling success, for observing the bactericidal effect that presses down of bigeminy viable bacteria pudendal lotion, select pool property culture medium that the vaginal secretions smear is cultivated with the gonococcus special use, checked that but whether effectively having killed pathogenic bacterium has reached therapeutic purposes, observe and whether exist dysbacteriosis and treatment back dysbacteriosis can obtain correcting.High dose group and trobicin have the powerful effect of killing pathogenic bacterium that presses down, and do not turn out pathogenic bacterium.Bigeminy viable bacteria pudendal lotion can be rebuild the normal vaginal microbial flora of animal, and trobicin does not have this effect.But the bigeminy active bacteria formulation of low dosage very has obviously bactericidal action, and pathogenic bacterium colony obviously reduces.And the effect of rebuilding the intravaginal normal flora is arranged, and the control animals intravaginal still has a large amount of pathogenic bacterium survivals, no anaerobe.Normal intravaginal bacterial micro-ecological environment had suffered destruction after the generation vaginitis was described.

4.3.6. animal subject histopathology

4.3.6.1. colpitic histopathology changes due to the bigeminy active bacteria formulation treatment Candida albicans.

The result of table 19 shows, middle high dose bigeminy active bacteria formulation and Fluconazole treating group, and the vagina tissue outward appearance does not have congestion and edema, and is smooth, and it does not have hyperemia, edema and inflammatory cell infiltration histological examination.The low dose therapy group has an exception to see the mild hyperaemia change, and pathological examination finds that 1 example is congested, and edema is accompanied a little inflammatory cell infiltration, congestion and edema under the 1 routine mirror.Normal saline.6 exceptions are seen all congestion and edema.Wherein rarely seen congestion and edema does not have inflammatory cell infiltration under the 3 routine mirrors.In addition 3 examples not only the mucous hyperemia edema all moisten with inflammatory cell.

Table 19 Candida albicans modeling group white rabbit treatment back vagina tissue pathological change

	Congested	Edema	Inflammatory cell infiltration	Mucosal erosion
					Dosage group high dose group fluconazol group in the normal saline group low dose group	6 2 0 0 0	6 2 0 0 0	3 0 0 0 0	0 0 0 0 0

◆ numeral is a pathological abnormalities example number in the table, and every treated animal number is 6

Candida albicans modeling group is treated the 8th day vagina tissue mirror and is seen down, and congested, edema obviously alleviates, and does not see inflammatory cell infiltration, and statistics is used spss software and adopted X ²The method of inspection is analyzed, high dose group, middle dosage, trobicin group zero difference, but significant difference (p＜0.05) is arranged with the normal saline group

4.3.6.2 colpitic histopathology changes due to the bigeminy active bacteria formulation treatment gonococcus

The result of table 20 shows, middle high dose bigeminy active bacteria formulation and trobicin treatment group, and the vagina tissue outward appearance does not have congestion and edema, and is smooth, and it does not have hyperemia, edema and inflammatory cell infiltration histological examination.The low dose therapy group has an exception to see the mild hyperaemia change, and pathological examination finds that 1 example is congested, and edema is accompanied a little inflammatory cell infiltration, congestion and edema under the 1 routine mirror.Normal saline 6 exceptions are seen all congestion and edema.Wherein rarely seen congestion and edema does not have inflammatory cell infiltration under the 3 routine mirrors.In addition 3 examples not only the mucous hyperemia edema with inflammatory cell infiltration.

Table 20 gonococcus modeling group white rabbit treatment back vagina tissue pathological change

	Congested	Edema	Inflammatory cell infiltration	Mucosal erosion
					Dosage group high dose group trobicin group in the normal saline group low dose group	6 2 0 0 0	6 2 0 0 0	2 1 0 0 0	0 0 0 0 0

◆ numeral is a pathological abnormalities example number in the table, and every treated animal number is 6

Gonococcus modeling group is treated the 8th day vagina tissue mirror and is seen down, and congested, edema obviously alleviates, and does not see inflammatory cell infiltration, does not have erosion, and statistics is used spss software and adopted X ²The method of inspection is analyzed, high dose group, middle dosage, trobicin group zero difference, but significant difference (p＜0.05) is arranged with the normal saline group

4.4 interpretation of result

Directly inject the new zealand white rabbit intravaginal with two kinds of pathogenic bacterium, can successfully prepare colpitis mycotica and gonococcal vaginitis animal model, from outward appearance, vaginal secretions detects, bacteriological detection, vagina tissue pathological examination have proved the colpitic existence of animal, and its characteristics are leading indicators for the vaginal secretions pyocyte, can reflect colpitic existence well, identical with existing clinical diagnosis.Histopathology is with the tangible non-specific change of congestion and edema.Bacteriological detection has certain meaning.Find also simultaneously that vaginitis the back intravaginal takes place has dysbacteriosis.

Through the ordinary circumstance of the animal subject of associating, the clean degree of vaginal secretions of vagina outward appearance simulation Clinical detection, the bacteriological detection of pathogenic bacterium and normal flora, multiple inspections such as histopathology, the curative effect of thoroughly evaluating bigeminy active bacteria formulation.The result shows that the bigeminy active bacteria formulation can be treated the vaginitis due to Candida albicans and the gonococcus effectively, and the cure rate of middle high dose has all reached more than 80%.Approaching with the curative effect of clinical main medicine fluconazol and trobicin.From the angle of bacteriological detection, bigeminy viable bacteria pudendal lotion has obvious suppression Candida albicans and gonococcal effect.In high dose effect and fluconazol and trobicin but bactericidal effect is approaching.Fluconazol and trobicin press down to kill Candida albicans and gonococcal ability is better than the bigeminy active bacteria formulation.Importantly, bigeminy viable bacteria pudendal lotion can be corrected intravaginal little disruption of ecological balance on the basis that produces effective therapeutical effect, set up the normal flora microenvironment.To prophylactic recurrence or infect again and can produce positive effect.Although and fluconazol and trobicin therapeutic effect and press down the bactericidal action curative effect have certainly also stayed simultaneously the unbalance hidden danger of intravaginal microecological environment.This stage is to be easy to period of infecting and recurring.Moreover fluconazol and trobicin are short action time for chemicals.And the intravaginal normal flora that the bigeminy active bacteria formulation is rebuild can continue to bring into play therapeutical effect.Secondly, fluconazol and trobicin find that in clinical practice it has apparent side effect, especially some breast-feeding female application are restricted, and these are the advantage place of bigeminy viable bacteria pudendal lotion just also.

4.5 conclusion:

1, the curative effect of bigeminy viable bacteria pudendal lotion treatment gonococcal vaginitis approaches trobicin.Press down gonococcida effect and be slightly poorer than trobicin, consumption is applicable to clinical treatment with middle dosage.

2, the bigeminy viable bacteria pudendal lotion treatment property read vaginitis curative effect in vain approaches fluconazol.Press down and kill the white effect of reading than fluconazol difference.Median dose is suitably for recommended amounts.

3, bigeminy viable bacteria pudendal lotion tool is significantly rebuild the intravaginal microecological environment, and replenishing normal flora correction dysbacteriosis is its outstanding advantage.

List of references:

[1] how to know blue or green health and epidemic prevention bacteriologic test [C], Beijing: Xinhua News Agency publishes, and 1989.

[2] Xu Shuyun, Bian Rulian, old repairing, " pharmacological experimental methodology ", People's Health Publisher, version in 2002

[3] Shi Xinyou, " modern medicine experimental zoology ", Beijing, People's Medical Officer Press, version in 2000.

Claims (38)

1, a kind of pharmaceutical composition that contains bacillus bifidus, it is characterized in that: contain the identification code for the treatment of effective dose and be 47757732 bacillus bifidus, it is 3.75-5.3 that an amount of acid, alkali and pharmacological-acceptable carrier, described an amount of acid, alkali make its mixed pharmaceutical composition pH value.

2, the described pharmaceutical composition of claim 1 is characterized in that: the pH value of described pharmaceutical composition is 4～5.

3, the described pharmaceutical composition of claim 1 is characterized in that: described acid is citric acid, tartaric acid, fumaric acid or adipic acid.

4, the described pharmaceutical composition of claim 1 is characterized in that: described alkali is sodium carbonate or sodium bicarbonate.

5, the described pharmaceutical composition of claim 1, it is characterized in that: described acid is tartaric acid, described alkali is sodium bicarbonate.

6, the described pharmaceutical composition of claim 5 is characterized in that: by weight percentage, described tartaric acid and sodium bicarbonate ratio are 0.8: 1～1.4: 1.

7, the described pharmaceutical composition of claim 6 is characterized in that: described tartaric acid and sodium bicarbonate ratio are 0.9: 1～1.2: 1.

8, the described pharmaceutical composition of claim 1, it is characterized in that: described pharmacological-acceptable carrier comprises filler and binding agent.

9, the described pharmaceutical composition of claim 8, it is characterized in that: described pharmacological-acceptable carrier also comprises dry adhesive.

10, the described pharmaceutical composition of claim 9, it is characterized in that: described pharmacological-acceptable carrier also comprises disintegrating agent.

11, a kind of pharmaceutical composition that contains bacillus bifidus is characterized in that: identification code is that the viable count content of 47757732 bacillus bifidus is not less than 1 * 10 in the described pharmaceutical preparation ²The CFU/ gram.

12, the described pharmaceutical composition of claim 11 is characterized in that: identification code is that the viable count content of 47757732 bacillus bifidus is 1 * 10 in the described pharmaceutical preparation ⁴CFU/ gram～1 * 10 ⁷The CFU/ gram.

13, identification code is 47757732 the purposes of bacillus bifidus in preparation treatment colpitis medicine.

14, identification code is 47757732 the purposes of bacillus bifidus in preparation treatment bacterial vaginitis medicine.

15, identification code is 47757732 the purposes of bacillus bifidus in preparation treatment colpitis mycotica medicine.

16, identification code is 47757732 the purposes of bacillus bifidus in preparation treatment gonococcal vaginitis medicine.

17, identification code is 47757732 the purposes of bacillus bifidus in preparation removing colon bacillus medicine.

18, identification code is 47757732 the purposes of bacillus bifidus in preparation removing staphylococcus aureus medicine.

19, identification code is 47757732 the purposes of bacillus bifidus in preparation removing gonococcus medicine.

20, identification code is 47757732 the purposes of bacillus bifidus in preparation removing Candida albicans medicine.

21, the purposes of any described pharmaceutical composition of claim 1-12 in preparation treatment colpitis medicine.

22, the purposes of the described pharmaceutical composition of claim 21 in preparation treatment bacterial vaginitis medicine.

23, the purposes of the described pharmaceutical composition of claim 21 in preparation treatment colpitis mycotica medicine.

24, the purposes of the described pharmaceutical composition of claim 21 in preparation treatment gonococcal vaginitis medicine.

25, the purposes of any described pharmaceutical composition of claim 1-12 in preparation removing colon bacillus medicine.

26, the purposes of any described pharmaceutical composition of claim 1-12 in preparation removing staphylococcus aureus medicine.

27, the purposes of any described pharmaceutical composition of claim 1-12 in preparation removing gonococcus medicine.

28, the purposes of any described pharmaceutical composition of claim 1-12 in preparation removing Candida albicans medicine.

29, a kind of preparation of drug combination method that contains bacillus bifidus is characterized in that comprising the steps:

(1) the preparation identification code is the lyophilizing mycopowder of 47757732 bacillus bifidus;

(2) with pharmacological-acceptable carrier drying, pulverizing back and an amount of acid, alkali mix homogeneously, the pH value that makes its mixture is 3.75-5.3;

(3) mycopowder that step (1) is made mixes the back press forming with the mixture that step (2) makes.

30, the described preparation of drug combination method of claim 29 is characterized in that described pharmacological-acceptable carrier comprises filler, dry adhesive and binding agent, and step (2) further comprises the steps:

(2-1) with filler, dry adhesive and binding agent drying, grinding and sieving;

(2-2) acid, filler, alkali, dry adhesive are mixed;

(2-3) in step (5), add binding agent, make the mixture soft material;

(2-4), make the mixture described in the step (2) with mixture soft material granulation and oven dry.

31, the described preparation of drug combination method of claim 30 is characterized in that described step (2-3) is that the mycopowder that earlier step (1) made mixes with a filler and mixes press forming afterwards again with mixture after grinding well dispersion.

32, any described preparation of drug combination method of claim 29-31 is characterized in that described acid is citric acid, tartaric acid, fumaric acid or adipic acid.

33, any described preparation of drug combination method of claim 29-31, it is characterized in that: described alkali is sodium carbonate or sodium bicarbonate.

34, any described preparation of drug combination method of claim 29-31, it is characterized in that: described acid is tartaric acid, described alkali is sodium bicarbonate.

35, the described preparation of drug combination method of claim 34 is characterized in that: by weight percentage, described tartaric acid and sodium bicarbonate ratio are 0.8: 1～1.4: 1.

36, the described preparation of drug combination method of claim 35, it is characterized in that: described tartaric acid and sodium bicarbonate ratio are 0.9: 1～1.2: 1.

37, the described preparation of drug combination method of claim 29, it is characterized in that described pharmacological-acceptable carrier comprises filler lactose, starch, dry adhesive, filler Celluloasun Microcrystallisatum, disintegrating agent, dry adhesive low-substituted hydroxypropyl cellulose, binding agent 5%PVPK ₃₀Alcoholic solution (95%), described acid is tartaric acid, and described alkali is sodium bicarbonate, and step (2) further comprises the steps:

(2-1) the tartaric acid adding starch with weighing mixes, and sodium bicarbonate and the lactose with weighing adds and mix homogeneously again;

(2-2) in step (4), add again behind Celluloasun Microcrystallisatum and the low-substituted hydroxypropyl cellulose mix homogeneously with 5%PVPK ₃₀Alcoholic solution (95%) is made binding agent, the system soft material;

(2-3) soft material is used granulation, baking in granulator, make blank granule;

Step (3) further comprises the steps:

(3-1) mycopowder of weighing is ground well dispersion with an amount of lactose respectively;

(3-2) again with the blank granule equivalent mix homogeneously that progressively increases,

(3-3) add the lubricant mixing, compacting in flakes.

38, claim 29-31,37 any described preparation of drug combination methods is characterized in that in the described lyophilizing mycopowder adding that content is arranged is 0.01%～0.1% cysteine.