CN110354247B

CN110354247B - Haematococin compound preparation for treating oral candidiasis and preparation method thereof

Info

Publication number: CN110354247B
Application number: CN201910739705.8A
Authority: CN
Inventors: 马晟利
Original assignee: Individual
Current assignee: Individual
Priority date: 2019-08-12
Filing date: 2019-08-12
Publication date: 2020-06-26
Anticipated expiration: 2039-08-12
Also published as: CN110354247A

Abstract

The invention relates to a compound preparation of streptococcin for treating oral candidiasis and a preparation method thereof, belonging to the technical field of microbial pharmacy. In order to solve the problem that the nisin can not accurately realize the drug effect on the affected part of the oral mucosa, the invention provides a nisin compound preparation for treating oral candida albicans, the preparation is a powder injection for oral mucosa injection, and an antifungal component nisin is dispersed in the powder injection in the form of drug-loaded compound microspheres, so that the activity and the stability of the nisin are improved, and the bioavailability of the nisin is improved. Honeysuckle, figwort root, dwarf lilyturf tuber and menthol can nourish yin and promote the production of body fluid, and relieve the symptoms of dry mouth and burning heat of a patient; the sodium alginate and the chitosan are safe, non-toxic, degradable and good in biocompatibility. The application can accurately realize drug effect through mucous membrane injection administration at an affected part, the streptostacin directly acts on the candida albicans, the local drug concentration is high, the adhesive force for reducing the candida albicans can be quickly achieved, and the effect of the candida albicans is inhibited and killed.

Description

Haematococin compound preparation for treating oral candidiasis and preparation method thereof

Technical Field

The invention belongs to the technical field of microbial pharmacy, and particularly relates to a nisin compound preparation for treating oral candidiasis and a preparation method thereof.

Background

In recent years, the incidence of candida has increased with the age due to abuse of antibiotics, frequent use of hormones, immunosuppressants and the like. Candida albicans is an opportunistic pathogen existing in the human oral environment, has extremely strong infectivity, and can cause various oral mucosa diseases under certain conditions. The change of the oral environment can cause the dysbacteriosis in the oral cavity, and the key factor of the generation of the periodontal disease is the dysbacteriosis. The long-term unhealthy oral candidiasis of human beings indicates that the body of a patient has local or systemic low immunity or is lost, and if treatment measures such as antifungal medicines are not timely taken, infection spreading or systemic candidiasis can be possibly caused. Currently, the drugs for clinically treating fungal infection diseases mainly comprise azoles, allylamines, polyenes and the like, and the fungi can be stimulated to generate corresponding drug resistance due to long-term use of broad-spectrum antibiotics, so that the selection of antifungal drugs in clinical diagnosis and treatment is extremely difficult.

Bacteriocin (Bacteriocin) is a polypeptide or precursor polypeptide with bacteriostatic activity generated by some bacteria through a ribosome synthesis mechanism in a metabolic process, and is favored by scholars at home and abroad because of narrow bacteriostatic spectrum, safety, high efficiency, no toxic or side effect and no drug resistance. The bacteriocin directly degrades the DNA of the target cell by perforating the target cell, inhibiting peptidoglycan synthesis, and inhibiting protein synthesis through interaction with ribosome or tRNA, thereby achieving the bacteriostatic effect.

Streptococcus sanguis is a gram-positive bacterium, can adhere to soft and hard tissues in the oral cavity, and is a well-known periodontal dominant bacterium. Streptococcus sanguis produces a bacteriocin, streptococci, which has bacteriostatic activity. The application of the invention patent with publication number CN106581638A on the preparation of medicines for inhibiting Candida albicans discloses that the streptococcus sanguis protein, namely the streptococcus sanguis can be used for treating the Candida albicans disease. However, the technical solution of this patent application is mainly in the theoretical research stage and does not disclose any specific therapeutic method for the treatment of oral candidiasis by streptococcin. In the actual oral candidiasis treatment process, the traditional administration modes such as spraying, buccal tablets and the like often cause that the effective components of the streptostacin can not accurately realize the drug effect on the affected part of the oral mucosa due to saliva dilution and dispersion. Therefore, how to accurately realize the drug effect of the streptostacin on the affected part of the oral mucosa in the treatment process becomes a problem to be solved urgently at the present stage.

Disclosure of Invention

In order to solve the problem that the nisin can not accurately realize the drug effect on the affected part of the oral mucosa, the invention provides a nisin compound preparation for treating oral candida albicans disease and a preparation method thereof.

The technical scheme of the invention is as follows:

a compound preparation of nisin for treating oral candidiasis is a powder injection for oral mucosa injection, and the nisin is uniformly dispersed in the powder injection in a drug-loaded composite microsphere form, and the preparation comprises the following components in parts by mass: 3-10 parts of nisin, 1-3 parts of honeysuckle extract, 1-3 parts of radix scrophulariae extract, 1-3 parts of radix ophiopogonis extract, 1-3 parts of menthol, 30-50 parts of sodium alginate, 60-100 parts of chitosan, 808-10 parts of tween-808 and 20-100 parts of glucose.

Further, the preparation comprises the following components in parts by mass: 6 parts of streptostacin, 2 parts of honeysuckle extract, 2 parts of figwort extract, 2 parts of dwarf lilyturf tuber extract, 2 parts of menthol, 40 parts of sodium alginate, 80 parts of chitosan, 809 parts of tween-809 and 80 parts of glucose.

Further, the particle size of the drug-loaded composite microspheres is 20-50 microns, the drug-loaded amount of the streptostacin in the drug-loaded composite microspheres is 2-9%, the drug-loaded amounts of the honeysuckle extract, the figwort extract, the ophiopogon root extract and the menthol are 0.5-3%, the powder injection for oral mucosa injection is packaged in a sterile cillin bottle, and the powder injection is fully suspended by sterile injection water before use and injected under the mucosa of an oral candida albicans affected part.

A preparation method of a nisin compound preparation for treating oral candidiasis comprises the following steps:

step one, preparing a drug-loaded composite microsphere:

weighing 30-50 parts of sodium alginate, preparing a sodium alginate solution with a certain mass concentration by using deionized water, adding 3-10 parts of nisin into the sodium alginate solution, and uniformly dispersing by using ultrasonic waves to obtain a drug-loaded sodium alginate solution for later use;

weighing 60-100 parts of chitosan, and preparing a chitosan solution with a certain mass concentration by taking acetic acid as a solvent; adding 1-3 parts of honeysuckle extract, 1-3 parts of radix scrophulariae extract, 1-3 parts of radix ophiopogonis extract and 1-3 parts of menthol into the chitosan solution, and uniformly dispersing by ultrasonic to obtain a drug-loaded chitosan solution for later use;

adding 8-10 parts of tween-80 into the drug-loaded chitosan solution, uniformly stirring, dropwise adding the obtained drug-loaded sodium alginate solution into the obtained drug-loaded chitosan solution under mechanical stirring at a certain rotating speed, continuously stirring at room temperature for 8-12 hours, then carrying out high-speed shearing on the obtained mixed system, centrifuging and collecting precipitates to obtain drug-loaded chitosan-sodium alginate composite microspheres;

step two, preparing a streptococcin compound preparation:

weighing 20-100 parts of glucose, dissolving the glucose in deionized water to prepare a glucose aqueous solution with a certain mass concentration, washing the drug-loaded chitosan-sodium alginate composite microspheres prepared in the step one with deionized water for three times, adding the drug-loaded chitosan-sodium alginate composite microspheres into the glucose aqueous solution for full suspension, carrying out vacuum freeze drying on the obtained suspension, and sterilizing to obtain the sterile nisin composite preparation.

Further, the extraction method of the haemaglobulin in the first step comprises the following steps:

performing proliferation culture on purified and identified streptococcus sanguis, inoculating the streptococcus sanguis into a BHI culture medium, performing constant-temperature static culture for 48 hours under the anaerobic condition of 37 ℃, centrifugally collecting culture solution under the condition of 4 ℃, and washing bacterial precipitation;

dispersing the obtained thalli in PBS buffer solution to prepare bacterial suspension, carrying out ultrasonic cell disruption on the bacterial suspension under the condition of ice-water bath, and centrifugally collecting cell disruption liquid supernatant under the condition of 4 ℃;

adding ammonium sulfate into the supernatant of the obtained cell disruption solution to enable the ammonium sulfate in the supernatant to reach 60% saturation degree, salting out at 4 ℃, centrifuging the obtained first salting-out solution at 4 ℃, respectively obtaining first salting-out supernatant and first salting-out precipitate, keeping the first salting-out precipitate properly, adding ammonium sulfate into the first salting-out supernatant again on the basis of the original ammonium sulfate of 60% saturation degree to adjust the saturation degree to 70%, salting out again at 4 ℃, centrifuging the obtained second salting-out solution at 4 ℃, collecting centrifugal second salting-out precipitate, combining the first salting-out precipitate and the second salting-out precipitate, dissolving by using PBS buffer solution, removing salt by passing through a Sephadex G-25 column, collecting the liquid passing through the column, dialyzing, concentrating, freeze-drying to obtain the streptostaphin, and storing at-80 ℃ for later use.

Further, the centrifugation for collecting the thallus precipitate is performed for 30min at 12000g/min, the thallus washing is performed by shaking and washing the thallus collected by centrifugation with 1 × PBS buffer solution, then the thallus is collected by centrifugation at 12000g/min for 30min, and the washing is repeated three times.

Further, the thallus concentration of the bacterial suspension is 50mg/mL, the frequency of ultrasonic disruption is 20-25 KHz, the ultrasonic power is 300W, the working time is 4s during ultrasonic treatment, the intermittent time is 8s, the total treatment time is 30min, and the centrifugation condition for centrifugally collecting the cell disruption supernatant is 15000g/min and centrifuging for 45 min.

Further, the salting-out time is 12h, the centrifugation conditions of the first salting-out solution and the second salting-out solution are 15000g/min for 45min, and the purity of the obtained streptostacin is 89.7%.

Further, the mass concentration of the sodium alginate solution in the step one is 3.0%, the mass concentration of the chitosan solution is 3.0%, the ultrasonic dispersion power is 300-1000W, and the ultrasonic dispersion treatment time is 20-30 min; the stirring speed is 200-300 rpm, the high-speed shearing rotating speed is 10000-13000 rpm, the high-speed shearing processing time is 5-20 min, and the centrifugation condition is 10000g/min for 25 min.

Further, the concentration of the glucose aqueous solution in the second step is 5-50%, and the sterilization is ethylene oxide sterilization for 48 hours.

The invention has the beneficial effects that:

the compound preparation of the haemostreptococcin for treating oral candidiasis provided by the invention takes the haemostreptococcin as a main antifungal component; the honeysuckle extract, the figwort extract, the ophiopogon root extract and the menthol can play a role in nourishing yin and promoting the production of body fluid and relieve the symptoms of dry mouth and burning sensation of a patient.

The compound preparation of the streptococcin is designed into powder injection for oral mucosa injection, wherein effective medicinal components are dispersed in the powder injection in the form of medicine-carrying compound microspheres, wherein the medicine-carrying compound microspheres are prepared by compounding honeysuckle extract, figwort extract, dwarf lilyturf tuber extract, menthol and chitosan to prepare a capsule layer, and are prepared by compounding the streptococcin and sodium alginate to prepare a core, so that the activity and the stability of the streptococcin are improved, and the bioavailability of the streptococcin is improved. The sodium alginate and the chitosan are degradable materials, the degradation products are safe and non-toxic, the biocompatibility is good, the sustained release effect is achieved in the degradation process, the drug effect period can be prolonged, and the pain of patients caused by frequent injection is avoided.

The compound preparation of the streptococcus sanguis is directly injected under the mucosa of an affected part of oral candida albicans by a mode of mucosal injection administration, so that the drug effect can be accurately realized, the streptococcus sanguis directly acts on the candida albicans, the local drug concentration is high, the adhesive force for reducing the candida albicans can be quickly reached, and the effect of the candida albicans can be inhibited and killed.

The preparation method is simple, the process is mild, a brand-new administration mode is provided for treating the oral candidiasis, the course of the oral candidiasis can be shortened, the pain of a patient is relieved, and a new thought is developed for treating the candida infection.

Drawings

FIG. 1 is a graph comparing the change in the number of Candida albicans colonies in the oral cavity of experimental mice in groups A, B, C and D at 0h, 12h, 24h, 36h, 48h, 60h and 72h of administration in animal experiments.

Detailed Description

The technical solutions of the present invention are further described below with reference to the following examples, but the present invention is not limited thereto, and any modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Example 1

The particle size of the drug-loaded composite microsphere is 20-50 μm, the drug-loaded amount of the streptostacin in the drug-loaded composite microsphere is 2-9%, the drug-loaded amounts of the honeysuckle extract, the figwort extract, the ophiopogon root extract and the menthol are 0.5-3%, the powder injection for oral mucosa injection is packaged in a sterile penicillin bottle, and the powder injection is fully suspended by sterile water for injection before use and injected under the mucosa of an affected part of candida albicans disease in an oral cavity.

Example 2

A compound preparation of nisin for treating oral candidiasis is a powder injection for oral mucosa injection, and the nisin is uniformly dispersed in the powder injection in a drug-loaded composite microsphere form, and the preparation comprises the following components in parts by mass: 6 parts of streptostacin, 2 parts of honeysuckle extract, 2 parts of figwort extract, 2 parts of dwarf lilyturf tuber extract, 2 parts of menthol, 40 parts of sodium alginate, 80 parts of chitosan, 809 parts of tween-809 and 80 parts of glucose.

The particle size of the drug-loaded composite microsphere is 20-50 μm, the drug-loading rate of the streptostacin in the drug-loaded composite microsphere is 4%, and the drug-loading rates of the honeysuckle extract, the figwort extract, the ophiopogon root extract and the menthol are all 1%. The powder injection for oral mucosa injection in the embodiment is packaged in a 15mL sterile penicillin bottle, and the powder injection is fully suspended by sterile water for injection before use and is respectively injected under mucosa at different positions of an affected part of candida albicans disease in an oral cavity.

Example 3

step one, preparing a drug-loaded composite microsphere:

step two, preparing a streptococcin compound preparation:

In the embodiment, the raw materials of menthol, sodium alginate, chitosan, tween-80, glucose and the like can be medical-grade commercial finished products of any brands.

Example 4

The embodiment provides a method for extracting nisin, which comprises the following specific steps:

the Streptococcus sanguis (Streptococcus sanguinis) strain ATCC10556 used in this example was purchased from important laboratories of department of oral biomedical engineering of the department of health of the oral medical institute of Wash, Sichuan, and was subjected to staining microscopy and biochemical identification after resuscitation and purification.

Resuscitation S.s strain ATCC 10556: inoculating the strain on a Columbia culture medium by a three-region streaking method, carrying out anaerobic culture at 37 ℃ for 48h, and then taking out the strain for passage.

Performing proliferation culture on purified and identified streptococcus sanguis, inoculating the streptococcus sanguis into a BHI culture medium, performing constant-temperature standing culture for 48h under the anaerobic condition at 37 ℃, centrifuging for 30min at 12000g/min by adopting a low-temperature high-speed centrifuge at 4 ℃, slowly taking out a centrifugal cylinder, removing supernatant, collecting thalli precipitate, washing thalli by using 1 × PBS buffer solution, centrifuging for 30min at 12000g/min, and repeatedly washing for three times.

And dispersing the collected thallus precipitate in 252mLPBS buffer solution to obtain a bacterial suspension with the thallus concentration of 50mg/mL, and carrying out cell disruption on the bacterial suspension in an ice-water bath by using an ultrasonic cell disrupter, wherein the ultrasonic frequency is 20-25 KHz, the ultrasonic power is 150W, the ultrasonic working time is 4s, the intermittent time is 8s, and the total treatment time is 30 min. The resulting cell disruption solution was centrifuged at 15000g/min at 4 ℃ for 45min to obtain 258mL of a supernatant of the cell disruption solution.

Adding 96.272g ammonium sulfate into the cell disruption liquid supernatant under ice water bath condition, stirring and dissolving to make the ammonium sulfate in the cell disruption liquid supernatant reach 60% saturation, and salting out the cell disruption supernatant at 4 deg.C for 12 h; to obtain a first salting-out solution.

Centrifuging the first salting-out solution at 15000g/min at 4 deg.C for 45min to obtain a first salting-out supernatant and a first salting-out precipitate, and keeping the first salting-out precipitate properly; 275mL of the first salting-out supernatant was added with 17.687g of ammonium sulfate based on the original 60% saturation of ammonium sulfate to adjust the saturation to 70% and the mixture was further salted out at 4 ℃ for 12 hours to obtain a second salting-out solution.

And centrifuging the second salting-out solution at the temperature of 4 ℃ for 45min at the speed of 15000G/min to obtain a second salting-out supernatant and a second salting-out precipitate, combining the first salting-out precipitate and the second salting-out precipitate, dissolving the combined precipitates by using a PBS (phosphate buffer solution) buffer solution, removing salt by using a Sephadex G-25 column, collecting the liquid passing through the column, dialyzing, concentrating and freeze-drying to obtain the streptostacin with the purity of 89.5-89.7%, and storing the streptostacin at the temperature of-80 ℃ for later use. The haemaglobulin contains a certain amount of impurity protein, and the drug effect of the haemaglobulin is not influenced.

In the embodiment, the secondary salting-out method is adopted to completely separate out the streptococcin in the streptococcus sanguis cell disruption solution, the extraction efficiency is high, and the purity of the prepared streptococcin is high. It has been demonstrated that streptostacin inhibits pathogenic candida species, represented by candida albicans, in five ways:

① Nisin may cause the destruction of cell membrane structure, form pore channels, release small and large molecular substances in the cell body. ② Nisin may destroy ergosterol formation, thus making the cell membrane frame of the cell body weak, and increase the fluidity of the cell membrane. ③ Nisin may adhere to the cell wall of the cell body and bind to the autolytic enzyme therein, activating and releasing the autolytic enzyme, and finally making the cell autolytic. ④ Nisin 1 (hyphalwallprotein 1, Hwp1) of Candida hyphae has a promoting function in Candida albicans adhesion and virulence, so Nisin may reduce the synthesis of Hwp 1.⑤ Nisin-like sequences (aglutin-lisequence, Als 1p and Als 5p) may be associated with Candida albicans adhesion, and Nisin may cause the alteration of Als 1p and Als 5p, thus reducing the adhesion of C.a.

Example 5

The embodiment provides a method for preparing a honeysuckle extract, a figwort extract and a radix ophiopogonis extract, which comprises the following specific steps:

selecting honeysuckle, figwort or dwarf lilyturf root as raw materials, cleaning, drying the raw materials in a vacuum drying oven at 65 ℃ for 15 hours, fully drying, and crushing the raw materials into 80 meshes to obtain honeysuckle, figwort or dwarf lilyturf root powder; adding 20 times volume of 60% ethanol solution into the obtained powder, and soaking at normal temperature for 3h for later use;

putting the ethanol solution soaked with the raw material powder into a pressure-resistant plastic bag, sealing, and putting into an extraction container of an ultrahigh-pressure extraction device; raising the pressure in the ultrahigh pressure extraction device to 500Mpa within 15min at normal temperature, maintaining the pressure for 20min, and then rapidly releasing the pressure in the ultrahigh pressure extraction device to normal pressure within 10-14 s; repeating the operation of boosting and relieving pressure for 3 times, taking out the plastic bag from an extraction container of an ultrahigh pressure extraction device, filtering the materials to remove filter residues, centrifuging the filtrate, taking the supernatant, repeatedly centrifuging for 1 time, and concentrating the supernatant to obtain a crude extract of honeysuckle, radix scrophulariae or radix ophiopogonis;

and respectively carrying out reduced pressure concentration and spray drying on the crude extracts of the honeysuckle, the figwort or the dwarf lilyturf tuber to obtain extracts of the honeysuckle, the figwort or the dwarf lilyturf tuber.

The honeysuckle, the radix scrophulariae or the radix ophiopogonis extract is extracted by the ultrahigh pressure extraction process, the pressure inside and outside the plant cells is quickly relieved after the pressure inside and outside the plant cells is balanced in the ultrahigh pressure extraction process, the osmotic pressure inside and outside the cells is suddenly increased, the structure of the cell membranes is changed, effective medicinal ingredients inside the cells can penetrate through the cell membranes and are transferred to an extracting solution outside the cells, the extraction is more sufficient, the utilization rate of the raw material medicines is improved, and the raw material cost is reduced.

Example 6

In this example, the nisin extracted in example 4 and the honeysuckle extract, the figwort extract and the ophiopogon root extract prepared in example 5 are used to prepare the nisin compound preparation for treating oral candidiasis, and the preparation method specifically comprises the following steps:

step one, preparing a drug-loaded composite microsphere:

weighing 30-50 parts of sodium alginate, preparing a sodium alginate solution with the mass concentration of 3.0% by using deionized water, adding 3-10 parts of nisin into the sodium alginate solution, and performing ultrasonic dispersion treatment at the power of 300-1000W for 20-30 min to obtain a drug-loaded sodium alginate solution for later use;

weighing 60-100 parts of chitosan, and preparing a solution with the mass concentration of the chitosan being 3.0% by taking acetic acid with the volume concentration of 3% as a solvent; the deacetylation degree of the chitosan used in the embodiment is 80-85%; adding 1-3 parts of honeysuckle extract, 1-3 parts of radix scrophulariae extract, 1-3 parts of radix ophiopogonis extract and 1-3 parts of menthol into the obtained chitosan solution, and performing ultrasonic dispersion treatment at 300-1000W for 20-30 min to obtain a drug-loaded chitosan solution for later use;

adding 8-10 parts of tween-80 into the drug-loaded chitosan solution, uniformly stirring, dropwise adding the obtained drug-loaded sodium alginate solution into the obtained drug-loaded chitosan solution under mechanical stirring of 200-300 rpm, continuously stirring at room temperature for 8-12 h, then carrying out high-speed shearing on the obtained mixed system, wherein the high-speed shearing rotation speed is 10000-13000 rpm, the high-speed shearing treatment time is 5-20 min, centrifuging at 10000g/min for 25min, and collecting precipitates to obtain drug-loaded chitosan-sodium alginate composite microspheres;

step two, preparing a streptococcin compound preparation:

weighing 20-100 parts of glucose, dissolving the glucose in deionized water to prepare a 5-50% glucose aqueous solution, washing the drug-loaded chitosan-sodium alginate composite microspheres prepared in the step one with deionized water for three times, adding the washed drug-loaded chitosan-sodium alginate composite microspheres into the glucose aqueous solution for full suspension, carrying out vacuum freeze drying on the obtained suspension, and sterilizing with ethylene oxide for 48 hours to obtain the sterile nisin composite preparation.

Example 7

step one, preparing a drug-loaded composite microsphere:

weighing 30g of sodium alginate, preparing a sodium alginate solution with the mass concentration of 3.0% by using deionized water, adding 3g of streptostacin into the sodium alginate solution, and performing ultrasonic dispersion treatment at 300W power for 30min to obtain a drug-loaded sodium alginate solution for later use;

weighing 60g of chitosan, and preparing a solution with the mass concentration of the chitosan being 3.0% by taking acetic acid with the volume concentration of 3% as a solvent; the deacetylation degree of the chitosan used in the embodiment is 80-85%; adding 1g of flos Lonicerae extract, 1g of radix scrophulariae extract, 1g of radix Ophiopogonis extract and 1g of Mentholum into the obtained chitosan solution, and performing ultrasonic dispersion treatment at 300 power for 30min to obtain drug-loaded chitosan solution;

adding 8g of tween-80 into the drug-loaded chitosan solution, uniformly stirring, dropwise adding the obtained drug-loaded sodium alginate solution into the obtained drug-loaded chitosan solution under mechanical stirring of 200rpm, continuously stirring for 12 hours at room temperature, and then carrying out high-speed shearing on the obtained mixed system, wherein the high-speed shearing rotation speed is 10000rpm, the high-speed shearing treatment time is 20 minutes, centrifuging for 25 minutes at 10000g/min and collecting precipitates to obtain the drug-loaded chitosan-sodium alginate composite microspheres;

step two, preparing a streptococcin compound preparation:

weighing 20g of glucose, dissolving the glucose in deionized water to prepare a 5% glucose aqueous solution, washing the drug-loaded chitosan-sodium alginate composite microspheres prepared in the step one with deionized water for three times, adding the washed drug-loaded chitosan-sodium alginate composite microspheres into the glucose aqueous solution for full suspension, carrying out vacuum freeze drying on the obtained suspension, and sterilizing with ethylene oxide for 48 hours to obtain the sterile nisin composite preparation.

The particle size of the drug-loaded composite microsphere is 20-50 μm, the drug-loading rate of the streptostacin in the drug-loaded composite microsphere is 2.5%, and the drug-loading rates of the honeysuckle extract, the figwort extract, the ophiopogon root extract and the menthol are all 0.9%.

625mg of powder injection for oral mucosa injection in the embodiment is packaged in a 15mL sterile penicillin bottle, 2mL sterile water for injection is used for fully suspending the powder injection before use, and 15mg of streptostacin contained in 2mL injection is respectively injected under mucosa at different positions of an oral candida albicans affected part.

Example 8

step one, preparing a drug-loaded composite microsphere:

weighing 35g of sodium alginate, preparing a sodium alginate solution with the mass concentration of 3.0% by using deionized water, adding 6g of streptostacin into the sodium alginate solution, and performing ultrasonic dispersion treatment at the power of 500W for 15min to obtain a drug-loaded sodium alginate solution for later use;

weighing 70g of chitosan, and preparing a solution with the mass concentration of the chitosan being 3.0% by taking acetic acid with the volume concentration of 3% as a solvent; the deacetylation degree of the chitosan used in the embodiment is 80-85%; adding 2g of honeysuckle extract, 2g of figwort extract, 2g of dwarf lilyturf tuber extract and 2g of menthol into the obtained chitosan solution, and performing ultrasonic dispersion treatment at 500W power for 15min to obtain a drug-loaded chitosan solution for later use;

adding 9g of tween-80 into the drug-loaded chitosan solution, uniformly stirring, dropwise adding the obtained drug-loaded sodium alginate solution into the obtained drug-loaded chitosan solution under mechanical stirring of 200rpm, continuously stirring for 12 hours at room temperature, and then carrying out high-speed shearing on the obtained mixed system, wherein the high-speed shearing rotation speed is 12000rpm, the high-speed shearing treatment time is 10 minutes, centrifuging for 25 minutes at 10000g/min, and collecting precipitates to obtain the drug-loaded chitosan-sodium alginate composite microspheres;

step two, preparing a streptococcin compound preparation:

weighing 40g of glucose, dissolving the glucose in deionized water to prepare a 20% glucose aqueous solution, washing the drug-loaded chitosan-sodium alginate composite microspheres prepared in the step one with deionized water for three times, adding the washed drug-loaded chitosan-sodium alginate composite microspheres into the glucose aqueous solution for full suspension, carrying out vacuum freeze drying on the obtained suspension, and sterilizing with ethylene oxide for 48 hours to obtain the sterile nisin composite preparation.

The particle size of the drug-loaded composite microsphere is 20-50 μm, the drug-loading rate of the streptostacin in the drug-loaded composite microsphere is 4.5%, and the drug-loading rates of the honeysuckle extract, the figwort extract, the ophiopogon root extract and the menthol are all 1.5%.

630mg of powder injection for oral mucosa injection in the embodiment is packaged in a 15mL sterile penicillin bottle, before use, 3mL sterile water for injection is used for fully suspending the powder injection, and 22.5mg of streptostacin contained in 3mL injection is respectively injected under mucosa at different positions of an affected part of candida albicans in the oral cavity.

Example 9

step one, preparing a drug-loaded composite microsphere:

weighing 40g of sodium alginate, preparing a sodium alginate solution with the mass concentration of 3.0% by using deionized water, adding 8g of streptostacin into the sodium alginate solution, and performing ultrasonic dispersion treatment at the power of 800W for 15min to obtain a drug-loaded sodium alginate solution for later use;

weighing 80g of chitosan, and preparing a solution with the mass concentration of the chitosan being 3.0% by taking acetic acid with the volume concentration of 3% as a solvent; the deacetylation degree of the chitosan used in the embodiment is 80-85%; adding 2g of honeysuckle extract, 2g of figwort extract, 2g of dwarf lilyturf tuber extract and 2g of menthol into the obtained chitosan solution, and carrying out ultrasonic dispersion treatment at the power of 800W for 15min to obtain a drug-loaded chitosan solution for later use;

adding 9g of tween-80 into the drug-loaded chitosan solution, uniformly stirring, dropwise adding the obtained drug-loaded sodium alginate solution into the obtained drug-loaded chitosan solution under mechanical stirring of 300rpm, continuously stirring for 10 hours at room temperature, and then carrying out high-speed shearing on the obtained mixed system, wherein the high-speed shearing rotation speed is 12000rpm, the high-speed shearing treatment time is 10 minutes, centrifuging for 25 minutes at 10000g/min, and collecting precipitates to obtain the drug-loaded chitosan-sodium alginate composite microspheres;

step two, preparing a streptococcin compound preparation:

weighing 80g of glucose, dissolving the glucose in deionized water to prepare a 30% glucose aqueous solution, washing the drug-loaded chitosan-sodium alginate composite microspheres prepared in the step one with deionized water for three times, adding the washed drug-loaded chitosan-sodium alginate composite microspheres into the glucose aqueous solution for full suspension, carrying out vacuum freeze drying on the obtained suspension, and sterilizing with ethylene oxide for 48 hours to obtain the sterile nisin composite preparation.

The particle size of the drug-loaded composite microsphere is 20-50 μm, the drug-loaded amount of the streptostacin in the drug-loaded composite microsphere is 5%, and the drug-loaded amounts of the honeysuckle extract, the figwort extract, the ophiopogon root extract and the menthol are all 1.3%.

The powder injection preparation 665mg for oral mucosa injection in the embodiment is packaged in a 15mL sterile penicillin bottle, 7mL sterile water for injection is used for fully suspending the powder injection preparation before use, and 35mg of streptostacin contained in 7mL injection is respectively injected under mucosa at different positions of an oral candida albicans affected part.

Example 10

step one, preparing a drug-loaded composite microsphere:

weighing 50g of sodium alginate, preparing a sodium alginate solution with the mass concentration of 3.0% by using deionized water, adding 10g of streptostacin into the sodium alginate solution, and performing ultrasonic dispersion treatment at the power of 1000W for 20min to obtain a drug-loaded sodium alginate solution for later use;

weighing 100g of chitosan, and preparing a solution with the mass concentration of the chitosan being 3.0% by taking acetic acid with the volume concentration of 3% as a solvent; the deacetylation degree of the chitosan used in the embodiment is 80-85%; adding 3g of honeysuckle extract, 3g of figwort extract, 3g of dwarf lilyturf tuber extract and 3g of menthol into the obtained chitosan solution, and performing ultrasonic dispersion treatment at 1000W power for 20min to obtain a drug-loaded chitosan solution for later use;

adding 10g of tween-80 into the drug-loaded chitosan solution, uniformly stirring, dropwise adding the obtained drug-loaded sodium alginate solution into the obtained drug-loaded chitosan solution under mechanical stirring of 300rpm, continuously stirring for 8 hours at room temperature, and then carrying out high-speed shearing on the obtained mixed system, wherein the rotating speed of the high-speed shearing is 13000rpm, the high-speed shearing treatment time is 5min, centrifuging for 25min at 10000g/min and collecting precipitates to obtain the drug-loaded chitosan-sodium alginate composite microspheres;

step two, preparing a streptococcin compound preparation:

weighing 100g of glucose, dissolving the glucose in deionized water to prepare a 50% glucose aqueous solution, washing the drug-loaded chitosan-sodium alginate composite microspheres prepared in the step one with deionized water for three times, adding the washed drug-loaded chitosan-sodium alginate composite microspheres into the glucose aqueous solution for full suspension, carrying out vacuum freeze drying on the obtained suspension, and sterilizing with ethylene oxide for 48 hours to obtain the sterile nisin composite preparation.

The particle size of the drug-loaded composite microsphere is 20-50 μm, the drug-loaded amount of the streptostacin in the drug-loaded composite microsphere is 5.5%, and the drug-loaded amounts of the honeysuckle extract, the figwort extract, the ophiopogon root extract and the menthol are all 1.6%.

637mg of powder injection for oral mucosa injection in the embodiment is packaged in a 15mL sterile vial, 7mL sterile water for injection is used for fully suspending the powder injection before use, and 35mg of streptococcin contained in 7mL injection is respectively injected under mucosa at different positions of an affected part of the oral candida albicans disease.

Comparative example 1

The spray containing the haemaglobulin prepared by the comparative example comprises the following steps:

step one, extracting the haemostreptocin:

Centrifuging the second salting-out solution at the temperature of 4 ℃ for 45min at the speed of 15000G/min to obtain a second salting-out supernatant and a second salting-out precipitate, combining the first salting-out precipitate and the second salting-out precipitate, dissolving the first salting-out precipitate and the second salting-out precipitate by using a PBS (phosphate buffer solution), removing salt by passing through a Sephadex G-25 column, collecting liquid passing through the column, dialyzing, concentrating and freeze-drying to obtain the streptostacin with the purity of 89.7%, and storing the streptostacin at the temperature of-80 ℃ for later use.

Step two, preparing spray containing nisin

2g of nisin is dissolved in 98mL of sterile water, and the mixture is shaken and mixed evenly to prepare a spray with the drug-loading rate of 2 percent.

Animal experiments

(I) test materials

Candida albicans: glabrata strain ATCC15126, purchased from Shanghai Fuxiang Biotechnology Ltd, recovered and purified, and then subjected to staining microscopy and biochemical identification.

60 Kunming mice, half of each male and female, 6-8 weeks, with weight of 20 +/-2 g, provided by northeast university of agriculture.

(II) Experimental method

60 Kunming mice were randomly divided into 4 groups of 15 mice each, namely, oral mucosa injection containing 7.5mg of nisin per ml prepared in example 7-group A, oral mucosa injection containing 5mg of nisin per ml prepared in example 9-group B, spray with 2% drug loading prepared in comparative example 1-group C and blank control-group D.

The amoxicillin is added into drinking water of 4 groups of mice to enable the concentration to reach 1%, and the amoxicillin concentration in the drinking water reaches 0.1% of the zingiberin until the experiment is finished after two weeks, the whole right buccal mucosa of the mice is used as an observation object because the area of the buccal mucosa on one side of the mice is smaller and is about 4mm × 4mm, 5% chloral hydrate is injected into the abdominal cavity every day to anaesthetize the mice, and then the amoxicillin is dipped with a sterile oil painting brush to obtain the concentration of 1 × 10⁹50 mu L of Candida albicans suspension of CFU/mL is smeared under the mucous membrane of the right cheek of the oral cavity of the mouse for three times, and the mucous membrane of the right cheek of the oral cavity of the mouse is scratched under the mucous membrane of the right cheek of the oral cavity of the mouse by a sterile probe every 3 days toThe mucous membrane is not scratched as a reference and is continued for 4 weeks until white spot sheet-shaped biofilm is formed on the mucous membrane of the right cheek of the mouse.

One mouse after modeling is selected from each group, white spot-shaped damage appears on mucous membrane of cheek on the right side of the oral cavity of the mouse observed clinically, and red festering face with serious hyperemia can be seen after the mouse is rubbed off by cotton swabs. After killing, the mucous membrane at the right buccal smear is taken for fixing, dehydrating, embedding and slicing, and HE staining and PAS staining are carried out conventionally. The secretion is smeared at the serious disease, and the yeast-like spores are seen under a light microscope after gram staining. Pathological exploration is carried out on a pathological part, and the PAS staining shows that a large number of hyphae and spores are arranged above a basal layer, a few chronic inflammatory cells infiltrate into the intrinsic layer, and the modeling is successful.

In successfully modeled mice, 5% chloral hydrate is injected intraperitoneally to anesthetize the mice, the oral mucosa injection containing 7.5mg of streptococcin per milliliter of injection prepared in example 7 and the oral mucosa injection containing 5mg of streptococcin per milliliter of injection prepared in example 9 are respectively injected under the right buccal mucosa of mice in groups A and B, 5 injection points are positioned at each mouse, 2mL are injected in total, the spray with the drug loading of 2% prepared in comparative example 1 is injected on the right buccal mucosa of the mice in group C, 0.9% of physiological saline is smeared on the right buccal mucosa of the mice in group D, and the candida albicans colony number of the mice in the oral cavity is detected by sampling every 12h after injection and is sampled for 72 h.

(III) results of the experiment

The colony number of Candida albicans in the oral cavity of the mice treated by the four groups of administration is shown in figure 1: as can be seen from fig. 1, 12h before administration, the mice treated with oral mucosa injection containing 7.5mg of nisin per ml of injection prepared in group a, example 7 and the mice treated with oral mucosa injection containing 5mg of nisin per ml of injection prepared in group B, in which the nisin in the injections prepared in examples 7 and 9 is wrapped in composite microspheres, did not act on candida albicans immediately, while the mice treated with oral mucosa injection directly sprayed with nisin in group C, were more rapid in effect.

But starting from 24h of administration, the sodium alginate and the chitosan in the composite microspheres start to be gradually degraded to release more nisin, the nisin accumulated at the mucous membrane affected part is gradually increased, the speed of killing candida albicans is also faster and faster, and the number of the candida albicans in the oral cavities of mice in the A group and the B group is respectively reduced by 98 percent and 92 percent after 72h of administration.

And the group C can not stay on the affected part for a long time after the group C is sprayed with the streptostacin, is easy to be diluted and dispersed by saliva, can not maintain the drug effect for a long time, and the number of candida albicans in the oral cavity is reduced by 70 percent after 72 hours.

Therefore, the compound preparation of the streptococcus sanguis is directly injected under the mucosa of an oral candida albicans disease affected part in a mucosa injection administration mode, the drug effect can be accurately realized, the streptococcus sanguis directly acts on candida albicans, the local drug concentration is high, the effects of reducing the adhesive force of the candida albicans and inhibiting and killing the candida albicans can be quickly achieved.

Claims

1. A preparation method of a nisin compound preparation for treating oral candidiasis is characterized by comprising the following steps:

step one, preparing a drug-loaded composite microsphere:

step two, preparing a streptococcin compound preparation:

2. The method for preparing the compound preparation of the streptococcus sanguis for treating the oral candidiasis according to the claim 1, wherein the extraction method of the streptococcus sanguis comprises the following steps:

3. The method for preparing the compound preparation of streptococcin for treating oral candidiasis according to claim 2, characterized in that the centrifugation is used for collecting thalli precipitates and is used for centrifugation at 12000g/min for 30min, the thalli washing is used for washing the thalli which are collected by centrifugation with 1 × PBS buffer solution in a shaking way, then the thalli are collected by centrifugation at 12000g/min for 30min, and the washing is repeated three times.

4. The preparation method of the compound preparation of the streptococcin for treating the oral candidiasis according to the claim 3, characterized in that the thallus concentration of the bacterial suspension is 50mg/mL, the frequency of the ultrasonic disruption is 20-25 KHz, the ultrasonic power is 300W, the working time of the ultrasonic treatment is 4s, the intermission time is 8s, the total treatment time is 30min, and the centrifugation condition for centrifugally collecting the cell disruption supernatant is 15000g/min and centrifugation is 45 min.

5. The method for preparing a compound preparation of streptococci for treating oral candida albicans according to claim 4, wherein the salting-out time is 12h, and the centrifugation conditions of the first salting-out solution and the second salting-out solution are 15000g/min and 45 min.

6. The preparation method of the compound preparation of the streptococcin for treating the oral candidiasis according to the claim 5, characterized in that the mass concentration of the sodium alginate solution in the step one is 3.0 percent, the mass concentration of the chitosan solution is 3.0 percent, the power of the ultrasonic dispersion is 300-1000W, and the time of the ultrasonic dispersion treatment is 20-30 min; the stirring speed is 200-300 rpm, the high-speed shearing rotating speed is 10000-13000 rpm, the high-speed shearing processing time is 5-20 min, and the centrifugation condition is 10000g/min for 25 min.

7. The method for preparing the compound preparation of nisin for treating oral candidiasis according to claim 6, wherein the concentration of the aqueous glucose solution in the step two is 5-50%, and the sterilization is ethylene oxide sterilization for 48 h.