CN113081956A - Natamycin eye drops modified by oxidized sodium alginate and preparation method thereof - Google Patents
Natamycin eye drops modified by oxidized sodium alginate and preparation method thereof Download PDFInfo
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- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
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- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
- C08B37/0084—Guluromannuronans, e.g. alginic acid, i.e. D-mannuronic acid and D-guluronic acid units linked with alternating alpha- and beta-1,4-glycosidic bonds; Derivatives thereof, e.g. alginates
Abstract
The invention relates to the technical field of medical materials, and relates to natamycin eye drops modified by oxidized sodium alginate and a preparation method thereof, wherein the natamycin eye drops comprise the following components in percentage by weight: 1mL of sterilized deionized water contains 0.5-1.5mg of oxidized sodium alginate-natamycin freeze-dried powder, wherein the oxidized sodium alginate-natamycin is a product of Schiff base reaction between the oxidized sodium alginate and natamycin; the preparation method comprises the following specific steps: preparing oxidized sodium alginate solid, preparing oxidized sodium alginate-natamycin medicine powder and preparing eye drops; because the property of the oxidized sodium alginate is similar to that of the extracellular matrix of a human body, the oxidized sodium alginate has good histocompatibility and can be degraded into nontoxic polysaccharide which does not participate in metabolism; the water solubility and the permeability of the natamycin are improved, the eye irritation and the discomfort of the natamycin are reduced, and the treatment effect of the fungal keratitis is improved; the preparation process is relatively simple, the cost is low, and the storage is easy.
Description
The technical field is as follows:
the invention relates to the technical field of medical materials, and relates to natamycin eye drops modified by oxidized sodium alginate and a preparation method thereof.
Technical background:
fungal Keratitis (FK) is a corneal disease caused by infection with pathogenic fungi. As a developing country taking industrial and agricultural industries as main industries, China is a high incidence area of fungal keratitis, corneal trauma, particularly vegetal trauma, in production activities provides opportunities for the fungal keratitis to occur, and in addition, people wearing corneal contact lenses are continuously expanded and antibiotics and hormones are abused, in recent years, the incidence rate of the fungal keratitis is obviously increased, patients mostly use green and strong labor force working at first line, and the blindness rate is high. The clinical treatment of the fungal keratitis is relatively troublesome. At present, no satisfactory antifungal medicine exists clinically, most of common antifungal eye drops are fat-soluble, the problems of poor corneal local osmosis, strong irritation, high toxicity, short half-life period, complicated administration mode and the like exist, and the blindness rate of the fungal keratitis is high due to the lack of treatment means.
Natamycin is a tetraene antibiotic extracted from streptomyces. The action mechanism is that the medicine molecule is combined with sterol part of fungus cell membrane to form polyene sterol compound, so as to change permeability of cell membrane and make the basic cell component in fungus cell flow out to kill fungus. The natamycin suspension used clinically can be used for treating fungal keratitis, but natamycin has larger particle size and poorer solubility, and is influenced by lacrimal secretion, so that the effect of sustained release is difficult to achieve. Researchers are always dedicated to improving the administration effect of natamycin, for example, a cationic adhesion type natamycin nano eye drop disclosed in chinese patent CN200810140337.7, which comprises natamycin, a proper amount of osmotic pressure regulator and pH regulator, and further comprises chitosan, pluronic F-68 and phospholipid, wherein the mass percent concentration of phospholipid in the eye drop is 0.01% -2%, the mass percent concentration of pluronic F-68 in the eye drop is 0.1% -20%, the mass percent concentration of chitosan in the eye drop is 0.01% -4%, the molecular weight of chitosan is 5000-300000, and the degree of deacetylation is > 85%; the natamycin common suspension is prepared into suspension particles with the diameter of 10-1000nm, the solubility of the drug is increased, the safety and the effectiveness are improved, the natamycin drug particles of the common suspension are modified by phospholipid and chitosan, so that the natamycin drug particles are positively charged, the corneal absorption of the drug is improved, the drug effect is enhanced, the drug action time is prolonged, and the chitosan has a biological adhesion effect, so that the corneal adhesion of the drug particles is enhanced; but the raw materials are various, and the preparation process is complex; chinese application patent CN201911030779.0 discloses natamycin polymer micelle eye drops, which comprise natamycin polymer micelles and phosphate buffer solution with a certain mass-volume ratio, wherein the natamycin in the natamycin polymer micelles accounts for 10-30% by mass, and the balance is polyethylene glycol-poly (glyceryl methacrylate) block copolymer; the natamycin polymer micelle eye drops obtained by putting the polyethylene glycol-poly (glyceryl methacrylate) segmented copolymer into ethanol to self-assemble cross-linked micelles and entrapping natamycin can prolong the release time of natamycin and reduce the times of medication, but the treatment effect is not improved.
Sodium alginate is a byproduct after iodine and mannitol are extracted from brown algae such as kelp or gulfweed, the molecule of the sodium alginate is formed by connecting beta-D-mannuronic acid (beta-D-mannuronic acid, M) and alpha-L-guluronic acid (alpha-L-guluronic acid, G) according to a (1 → 4) bond, and the sodium alginate is a natural polysaccharide and has stability, solubility, viscosity and safety required by pharmaceutical preparation auxiliary materials. Sodium alginate has been widely used in the food industry and in the medical field. But the degradation rate of the alginate is very low, and in order to improve the degradation performance of the sodium alginate, the sodium alginate is oxidized, so that the hydroxyl of partial uronic acid units of the sodium alginate is converted into aldehyde groups, and the purpose of improving the degradation performance of the sodium alginate is achieved; because the aldehyde group has relatively high reactivity, the natamycin eye drops can be coupled with bioactive substances such as active protein, polypeptide, specific amino acid sequence and the like, and can improve the interaction between materials and cells, so that the natamycin eye drops modified by oxidized sodium alginate and the preparation method thereof need to be designed, the oxidized sodium alginate prepared by oxidizing sodium alginate is used for improving natamycin, the water solubility and the permeability of natamycin are effectively improved, the eye irritation and the discomfort of natamycin are reduced, and the treatment effect of fungal keratitis is improved.
The invention content is as follows:
aiming at the defects in the prior art, the invention provides the natamycin eye drops modified by oxidized sodium alginate and the preparation method thereof, which are used for treating fungal infection of eyes.
In order to achieve the aim, the invention provides natamycin eye drops modified by oxidized sodium alginate, which comprises the following components in percentage by weight: the sterilized deionized water of 1mL contains 0.5-1.5mg of oxidized sodium alginate-natamycin freeze-dried powder, wherein the oxidized sodium alginate-natamycin is a product of Schiff base reaction between the oxidized sodium alginate and natamycin.
The preparation method of the sodium alginate oxide modified natamycin eye drops comprises the following specific steps:
firstly, preparing oxidized sodium alginate solid, dissolving sodium alginate in water, adding sodium periodate, reacting in the dark, then adding ethylene glycol to terminate the reaction, generating oxidized sodium alginate, adding absolute ethyl alcohol to precipitate, and obtaining pure oxidized sodium alginate solid through reduced pressure suction filtration, dialysis and freeze drying;
secondly, preparing oxidized sodium alginate-natamycin medicine powder: dissolving the oxidized sodium alginate prepared in the first step in water, adding natamycin, stirring in a dark environment, carrying out Schiff base reaction on the oxidized sodium alginate and the natamycin to generate oxidized sodium alginate-natamycin, and then dialyzing, freeze-drying and grinding to obtain oxidized sodium alginate-natamycin solid powder;
step three, preparing eye drops: 0.5-1.5mg of sodium alginate oxide-natamycin powder is added into 1mL of sterilized deionized water to prepare the sodium alginate oxide modified natamycin eye drops with the concentration of 0.5-1.5 mg/mL.
The first step of preparing the oxidized sodium alginate solid comprises the following specific steps: (1) weighing 10g of sodium alginate by using a precision electronic balance, adding 200mL of sterilized deionized water, magnetically stirring at room temperature for 3-10 hours to prepare a sodium alginate aqueous solution with the mass concentration of 0.05g/mL, adding 1-10g of sodium periodate, reacting for 3-9 hours in a dark place, adding 0.260-2.60mL of ethylene glycol, magnetically stirring for 0.5 hour, stopping the reaction to generate an oxidized sodium alginate mixture solution, adding 400mL of absolute ethyl alcohol to precipitate, and then performing vacuum filtration by using a circulating water vacuum pump to obtain a mixed product of the oxidized sodium alginate;
(2) dissolving the mixed product containing sodium alginate oxide in sterilized deionized water, dialyzing in a dialysis bag for 1-3 days in sterilized double distilled water, changing the double distilled water every 6 hours, and freeze-drying the dialyzed solution at-70 ℃ to obtain pure sodium alginate oxide solid.
Further, 5g of sodium periodate was added to the aqueous solution of sodium alginate, and the reaction was carried out for 6 hours in the absence of light.
The second step of oxidizing the sodium alginate-natamycin medicine powder comprises the following specific steps: dissolving the oxidized sodium alginate solid powder prepared in the first step in sterilized deionized water to prepare an oxidized sodium alginate aqueous solution with the mass concentration of 0.01g/mL, adding 0.5-5g natamycin into 100mL of the oxidized sodium alginate aqueous solution, stirring for 24-48 hours in a dark environment at 40 ℃, then dialyzing in sterilized double distilled water for 1-3 days by using a dialysis bag for removing unreacted natamycin, freeze-drying the dialyzed solution at-70 ℃ to obtain oxidized sodium alginate-natamycin solid, grinding the oxidized sodium alginate-natamycin solid into powder by using a quartz mortar, and placing the powder in a dry sterile glass bottle for dark storage;
compared with the prior art, the invention has the beneficial effects that:
1) the natamycin eye drops taking the oxidized sodium alginate as the matrix have good histocompatibility and can be degraded into nontoxic polysaccharide which does not participate in metabolism because the oxidized sodium alginate is similar to the extracellular matrix of a human body in property.
2) The natamycin eye drops taking the oxidized sodium alginate as the matrix have the advantages of relatively simple preparation process, low cost and easy storage.
3) The natamycin eye drops taking oxidized sodium alginate as a matrix change the suspension property of natamycin, improve the water solubility and the permeability of natamycin, reduce the eye irritation and the uncomfortable feeling of natamycin and improve the treatment effect of fungal keratitis.
Description of the drawings:
FIG. 1 is a standard curve diagram of natamycin solution measured by UV spectrophotometer in example 2 according to the present invention.
FIG. 2 is a physical characterization spectrum of Fourier infrared detection of the sodium alginate oxide modified natamycin eye drops of example 5 in accordance with the present invention.
FIG. 3 is a schematic diagram of the cytotoxicity test results of natamycin eyedrops modified with sodium alginate oxide in example 7.
FIG. 4 is a schematic diagram showing the results of in vitro bacteriostatic experiments for the oxidized sodium alginate modified natamycin eye drops of example 8.
FIG. 5 is a schematic diagram showing the experimental results of the application of the oxidized sodium alginate modified natamycin eye drops of example 10 in the fungal keratitis of mice.
The specific implementation mode is as follows:
the invention is further described with reference to the following specific embodiments and the accompanying drawings.
Example 1:
the embodiment relates to natamycin eye drops modified by oxidized sodium alginate, wherein 1mL of sterilized deionized water contains 0.5-1.5mg of oxidized sodium alginate-natamycin freeze-dried powder, and the specific preparation process is as follows:
step one, preparing oxidized sodium alginate:
(1) weighing 10g of sodium alginate by using a precision electronic balance, adding 200mL of sterilized deionized water, magnetically stirring at room temperature for 3-10 hours to prepare a sodium alginate aqueous solution with the mass concentration of 0.05g/mL, adding 1-10g of sodium periodate, reacting for 3-9 hours in a dark place, adding 0.260-2.60mL of ethylene glycol, magnetically stirring for 0.5 hour, stopping the reaction to generate an Oxidized Sodium Alginate (OSA) mixture solution, adding 400mL of absolute ethyl alcohol to precipitate out, and then carrying out vacuum filtration by using a circulating water vacuum pump to obtain a mixed product containing OSA.
(2) Dissolving the mixed product containing OSA in sterilized deionized water, dialyzing in sterilized double distilled water with dialysis bag (MWCO 7000D) for 1-3 days, changing the double distilled water every 6 hours for removing small molecular substances such as sodium periodate and ethylene glycol, and freeze-drying the dialyzed solution at-70 deg.C to obtain pure OSA solid;
secondly, preparing oxidized sodium alginate-natamycin medicine powder: dissolving 1g of OSA solid powder prepared in the step (2) in 100mL of sterilized deionized water to prepare an OSA solution with the mass concentration of 0.01g/mL, then adding 0.5-5g of Natamycin (NATA), stirring for 36 hours at 40 ℃ in a dark environment, then dialyzing for 1-3 days in sterilized double distilled water by using a dialysis bag (MWCO ═ 7000D) for removing unreacted natamycin, freeze-drying the dialyzed solution at-70 ℃ to obtain an OSA-NATA solid, grinding the OSA-NATA solid into powder by using a quartz mortar, and placing the powder in a dry sterile glass bottle to be stored in a dark environment;
the preparation process of the OSA-NATA is expressed by the following chemical reaction equation:
step three, preparing eye drops: 0.5-1.5mg of OSA-NATA powder is added into 1mL of sterilized deionized water to prepare 0.5-1.5mg/mL of oxidized sodium alginate modified natamycin eye drops (OSA-NATA eye drops for short).
Example 2:
the embodiment relates to a preparation method of natamycin eye drops modified by oxidized sodium alginate, which comprises the following specific preparation processes:
step one, preparing oxidized sodium alginate:
weighing 10g of sodium alginate by a precision electronic balance, adding 200mL of sterilized deionized water, magnetically stirring at room temperature for 6 hours to prepare a sodium alginate aqueous solution with the mass concentration of 0.05g/mL, adding 5g of sodium periodate, reacting for 6 hours in a dark place, adding 1.430mL of ethylene glycol, magnetically stirring for 0.5 hour, terminating the reaction to generate an Oxidized Sodium Alginate (OSA) mixture solution, adding 400mL of absolute ethyl alcohol to precipitate, and then carrying out vacuum filtration by a circulating water vacuum pump to obtain a mixed product containing OSA.
The OSA-containing mixed product was dissolved in sterilized deionized water, and then dialyzed against a dialysis bag (MWCO 7000D) in sterilized double distilled water for 2 days, the double distilled water was changed every 6 hours, and the dialyzed solution was freeze-dried at-70 ℃ to obtain an OSA solid.
Secondly, preparing oxidized sodium alginate-natamycin medicine powder: dissolving 1g of OSA solid powder prepared in the step (2) in 100mL of sterilized deionized water to prepare an OSA solution with the mass concentration of 0.01g/mL, adding 2.5g of Natamycin (NATA), stirring for 36 hours at 40 ℃ in a dark environment, dialyzing for 2 days in sterilized double distilled water by using a dialysis bag (MWCO 7000D), freeze-drying the dialyzed solution at-70 ℃ to obtain OSA-NATA solid, grinding the OSA-NATA solid into powder by using a quartz mortar, and storing the powder in a dry sterile glass bottle in a dark environment.
Step three, preparing eye drops: 1mg/mL of OSA-NATA eye drops are prepared by adding 1mL of sterilized deionized water to 1mg/mL of OSA-NATA powder.
The effective content of natamycin in the eye drops is measured: the results show that: the natamycin standard curve is measured at 303nm by using an ultraviolet spectrophotometer, the absorbance measured at 303nm in the 1mg/mL OSA-NATA eye drops is 0.578, and the effective natamycin content in the 1mLOSA-NATA eye drops is about 8 mu g.
Example 3:
the embodiment relates to a preparation method of natamycin eye drops modified by oxidized sodium alginate, which comprises the following specific preparation processes: step one, preparing oxidized sodium alginate:
(1) weighing 10g of sodium alginate by using a precision electronic balance, adding 200mL of sterilized deionized water, magnetically stirring at room temperature for 3 hours to prepare a sodium alginate aqueous solution with the mass concentration of 0.05g/mL, adding 1g of sodium periodate, reacting for 3 hours in a dark place, adding 0.260mL of ethylene glycol, magnetically stirring for 0.5 hour, terminating the reaction to generate an Oxidized Sodium Alginate (OSA) mixture solution, adding 400mL of absolute ethyl alcohol to precipitate, and then carrying out vacuum filtration by using a circulating water vacuum pump to obtain a mixed product containing OSA.
(2) Dissolving the mixed product containing OSA in sterilized deionized water, dialyzing in sterilized double distilled water with dialysis bag (MWCO 7000D) for 1 day, changing the double distilled water every 6 hr for removing small molecular substances such as sodium periodate and ethylene glycol, and freeze-drying the dialyzed solution at-70 deg.C to obtain pure OSA solid;
secondly, preparing oxidized sodium alginate-natamycin medicine powder: dissolving 1g of OSA solid powder prepared in the step (2) in 100mL of sterilized deionized water to prepare an OSA solution with the mass concentration of 0.01g/mL, adding 0.5g of Natamycin (NATA), stirring for 36 hours at 40 ℃ in a dark environment, dialyzing for 1 day in sterilized double distilled water by using a dialysis bag (MWCO 7000D) to remove unreacted natamycin, freeze-drying the dialyzed solution at-70 ℃ to obtain an OSA-NATA solid, grinding the OSA-NATA solid into powder by using a quartz mortar, and placing the powder in a dry sterile glass bottle to be stored in a dark environment;
step three, preparing eye drops: 0.5mg of OSA-NATA powder is added into 1mL of sterilized deionized water to prepare 0.5mg/mL of natamycin eye drops modified by oxidized sodium alginate (OSA-NATA eye drops for short).
Example 4:
the embodiment relates to a preparation method of natamycin eye drops modified by oxidized sodium alginate, which comprises the following specific preparation processes: step one, preparing oxidized sodium alginate:
(1) weighing 10g of sodium alginate by using a precision electronic balance, adding 200mL of sterilized deionized water, magnetically stirring at room temperature for 10 hours to prepare a sodium alginate aqueous solution with the mass concentration of 0.05g/mL, adding 10g of sodium periodate, reacting for 9 hours in a dark place, adding 2.60mL of ethylene glycol, magnetically stirring for 0.5 hour, terminating the reaction to generate an Oxidized Sodium Alginate (OSA) mixture solution, adding 400mL of absolute ethyl alcohol to precipitate, and then carrying out vacuum filtration by using a circulating water vacuum pump to obtain a mixed product containing OSA.
(2) Dissolving the mixed product containing OSA in sterilized deionized water, dialyzing with dialysis bag (MWCO 7000D) in sterilized double distilled water for 3 days, changing the double distilled water every 6 hours for removing small molecular substances such as sodium periodate and ethylene glycol, and freeze-drying the dialyzed solution at-70 deg.C to obtain pure OSA solid;
secondly, preparing oxidized sodium alginate-natamycin medicine powder: dissolving 1g of OSA solid powder prepared in the step (2) in 100mL of sterilized deionized water to prepare an OSA solution with the mass concentration of 0.01g/mL, adding 5g of Natamycin (NATA), stirring for 36 hours at 40 ℃ in a dark environment, dialyzing for 3 days in sterilized double distilled water by using a dialysis bag (MWCO 7000D) to remove unreacted natamycin, freeze-drying the dialyzed solution at-70 ℃ to obtain an OSA-NATA solid, grinding the OSA-NATA solid into powder by using a quartz mortar, and storing the powder in a dry sterile glass bottle in a dark environment;
step three, preparing eye drops: 1.5mg of OSA-NATA powder is added into 1mL of sterilized deionized water to prepare 1.5mg/mL of natamycin eye drops (OSA-NATA eye drops for short) modified by oxidized sodium alginate.
Example 5:
in the embodiment, physical characteristics of sodium alginate, oxidized sodium alginate, natamycin and oxidized sodium alginate-natamycin in the preparation process of the oxidized sodium alginate modified natamycin eye drops in the embodiment 2 are detected by Fourier infrared analysis, and an infrared spectrum is shown in figure 2.
As can be seen from FIG. 2, the infrared spectrum of oxidized sodium alginate was 1655cm as compared to that of sodium alginate-1A new peak appears nearby and is supposed to be an aldehyde carbonyl symmetric vibration absorption peak, which indicates that the sodium alginate generates aldehyde groups after being oxidized; 1655cm of infrared spectrum in OSA-NATA-1No absorption peak appears nearby, and a new peak appears at 1632cm, which indicates that OSA and natamycin have Schiff base reaction to generate-C-N-bond.
Example 6:
this example is a pharmacokinetic test of the oxidized sodium alginate modified natamycin eye drops prepared in example 2, and a high performance liquid chromatograph is used to detect the concentration changes of the aqueous humor and the cornea after administration, and the specific test process is as follows:
1. preparation of natamycin standard curve: at room temperature, the natamycin preparation concentrations of the standard solutions of 0.01, 0.1, 0.2, 0.4, 0.8, 1.0, 2.0, 4.0 and 8.0 mug/ml are precisely weighed, the absorbance of the wavelength at 303nm is detected by using a high performance liquid chromatograph through ultraviolet, the sample amount is 50 muL, and a natamycin concentration-absorbance standard curve is prepared.
2. 30 New Zealand white rabbits with unlimited male and female bodies and weight of 2.0-2.9kg are selected. Dripping 50 μ L of OSA-NATA eye drop into two eyes of rabbit with microsyringe, killing 3 rabbits with air embolism at 0, 5, 10, 15, 30, 60, 120, 180, 240 and 360 min after administration, respectively, collecting 150 μ L aqueous humor and 200 μ L corneal stroma, and storing in-20 deg.C refrigerator.
Adding 200 mu L acetonitrile into 100 mu L aqueous humor, oscillating, centrifuging, taking supernatant, detecting the ultraviolet absorption light intensity at 303nm, and calculating the corresponding natamycin drug concentration through a standard curve; grinding the corneal tissue in 1ml of phosphate buffer solution, adding ethyl acetate, shaking, centrifuging, taking the organic phase, adding 200 mu L of acetonitrile, centrifuging, taking the supernatant, detecting the ultraviolet absorption light intensity at 303nm, and calculating the corresponding natamycin drug concentration.
The result shows that after single eye drop of the OSA-NATA eye drops, the change trend of the drug concentration of the aqueous humor is consistent with the change trend of the drug concentration of the cornea, the maximum drug concentration is reached 30 minutes after eye drop application, and the half-life period of the drug is 2 hours.
Example 7:
this example is a cytotoxicity experiment of the oxidized sodium alginate modified natamycin eye drops prepared in example 2, specifically a CCK8 cell proliferation-toxicity test, and includes the following steps:
2, inoculating immortalized human corneal epithelial cell suspension into a 96-well plate, and placing the culture plate in an incubator for pre-culture for 24 hours; adding oxidized sodium alginate modified natamycin eye drops (OSA-NATA) with different concentrations into a 96-well plate culture solution, and simultaneously adding natamycin solutions (NATA) with different concentrations into another 96-well plate culture solution; after incubation in a constant temperature incubator for 24 hours, the culture medium was removed and 100. mu.L of fresh culture medium and 10. mu.L of CCK8 solution were added per well and placed in the incubator for incubation for 3 hours; the absorbance at 450nm was measured with a microplate reader, and the results are shown in FIG. 3.
As can be seen from fig. 3: after 24 hours, the natamycin has an inhibitory effect on HCEC at 16 mug/ml compared with the non-added drug group (natamycin concentration is 0); meanwhile, compared with the non-drug-added drug group (the concentration of OSA-NATA is 0), when the concentration of the OSA-NATA solution is less than 1024 mu g/mL, the activity of HCEC cells is not obviously abnormal, which indicates that 1mg/mL of OSA-NATA eye drops have no obvious cytotoxicity.
Example 8:
this example relates to an in vitro bacteriostatic experiment of the oxidized sodium alginate modified natamycin eye drops prepared in example 2:
culturing the aspergillus fumigatus strain in a Sabouraud's medium at 35 ℃ for 7 days to promote the generation of microconidia, flushing bacterial colonies by using 1mL of sterile phosphate buffer solution, collecting flushing liquid, and taking uniform suspension to count the spores; diluting natamycin and OSA-NATA eye drops with Sabouraud's medium to different concentration gradients, packaging in sterilized EP tubes, and adding Aspergillus fumigatus spore suspension into each tube to make the final concentration of spores in the tube about 0.4 × 104-5×104CFU/mL, the culture plate loaded with the EP tube is placed in a constant temperature incubator at 35 ℃, incubated for 48 hours, and then the growth of bacteria under different drug concentrations is detected by a microplate reader under the wavelength of 570nm, and the result is shown in FIG. 4.
As can be seen from FIG. 4, the MIC for Aspergillus fumigatus, natamycin, is comparable to the unadditized drug groups (natamycin and OSA-NATA concentrations are 0, respectively)902 μ g/mL, MIC 998 mug/mL; MIC of oxidized sodium alginate modified natamycin eye drops5064 μ g/mL, MIC 70128. mu.g/mL, MIC99512 μ g/mL. Therefore, the OSA-NATA has good bacteriostatic effect on the aspergillus fumigatus.
Example 9:
this example is a stability test experiment of oxidized sodium alginate modified natamycin eye drops:
the oxidized sodium alginate modified natamycin eye drops prepared in example 2 were stored at room temperature 15-28 ℃ and 4 ℃ for 1, 2, 3, 4, 5, 6, 7, 8 and 9 months respectively before in vitro bacteriostasis experiments, the procedure of the in vitro bacteriostasis experiments was as in example 8.
The experimental results are as follows: the OSA-NATA is stored at room temperature for 2 months, the dosage and in-vitro sterilization efficacy of the OSA-NATA are not obviously changed, and the dosage and in-vitro sterilization efficacy start to gradually decrease after 2 months; while OSA-NATA stored at 4 ℃ did not change significantly in potency within half a year and started to decline after 6 months. It is demonstrated that the sodium alginate oxide-modified natamycin eye drops prepared in example 2 can be stored for a long period of time, which provides favorable conditions for mass production, storage, transportation, wide application and the like of the sodium alginate oxide-modified natamycin eye drops.
Example 10:
the embodiment relates to an application experiment of sodium alginate oxide modified natamycin eye drops in treating mouse fungal keratitis:
24 healthy female mice C57BL/6 with the age of 8 weeks are taken to establish mouse aspergillus fumigatus keratitis animal models, and the right eyes are taken as experimental eyes. After the model is successfully established, the model is randomly divided into four groups, 6 models in each group are respectively: the first group was the fungus only group (AF) without any eye treatment administered to the mice; the second group is an oxidized sodium alginate solution group (AF + OSA), and the mice are instilled with the oxidized sodium alginate solution for 6 times every day, and each time is 5 mu L; the third group is a natamycin eye drop group (AF + OSA-NATA) modified by oxidized sodium alginate, and the mice are instilled with the natamycin eye drop group modified by the oxidized sodium alginate for 6 times every day, and each time is 5 mu L; the fourth group was a natamycin eyedrops group (AF + NATA) in which mice were instilled 6 times a day with 5. mu.L of natamycin eyedrops. The eye dropping time was 5 days. Corneal ulcer scoring criteria were performed according to the O' Day criteria, and the scoring of corneal ulcers in mice was observed and recorded daily under a slit lamp. The results of the experiment are shown in FIG. 5.
The results show that the natamycin eye drop group taking oxidized sodium alginate as the matrix has lower corneal ulcer score than the single fungus infection group, the oxidized alginic acid solution group and the natamycin eye drop group, the lower the score is, the lower the degree of corneal ulcer is, and further the treatment effect of the natamycin eye drop modified by the oxidized sodium alginate on the aspergillus fumigatus keratitis is better than that of the other three groups
Example 11:
the embodiment relates to application of natamycin eye drops modified by oxidized sodium alginate in treating fungal keratitis.
31 patients with fungal keratitis treated in affiliated hospitals of Qingdao university from 3 months to 2020 months in 2019 were collected, wherein 18 of the patients were 18 of the patients, 13 of the patients were 13 of the patients, the ages of the patients were 26-61 years, and the average (36.25 +/-6.07) of the patients was.
All patients received OSA-NATA eye drop treatment 6 times a day for 3 weeks.
And (3) evaluating the clinical effect of the patient after treatment according to relevant standards, and curing: after treatment, the corneal infiltration and edema condition subsides, the conjunctival congestion condition disappears, the fungus culture is negative, the corneal ulcer is healed, and no hypha is seen after the confocal microscope examination; the effect is shown: after treatment, the relevant detection result of the patient is negative, all indexes are improved, and only the conjunctival congestion or corneal pannus condition exists; the method has the following advantages: after treatment, the relevant examination results of the patients are negative, and only 1-2 indexes are improved. And (4) invalidation: before and after treatment, all indexes are unchanged or have aggravation. The total effective rate of the treatment is (cure + significant effect + effective)/the total number of cases is multiplied by 100%. The symptoms of the patients are evaluated according to relevant standards, each symptom has a score of 5 at most, and the higher the score is, the more serious the symptom is.
After statistics, after 31 patients receive treatment, 28 patients have treatment effects, wherein 17 patients are cured, 6 patients have obvious effects, 5 patients have effective effects, 3 patients have no effects, and the treatment effective rate is 90.3% (28/31).
The statistical results of relevant parameters before and after treatment are shown in table 1.
TABLE 1 statistics of relevant parameters before and after treatment
Claims (5)
1. The sodium alginate oxide modified natamycin eye drops are characterized by comprising the following components in percentage by weight: the sterilized deionized water of 1mL contains 0.5-1.5mg of oxidized sodium alginate-natamycin freeze-dried powder, wherein the oxidized sodium alginate-natamycin is a product of Schiff base reaction between the oxidized sodium alginate and natamycin.
2. The oxidized sodium alginate modified natamycin eye drops as claimed in claim 1, characterized in that the preparation method comprises the following steps:
firstly, preparing oxidized sodium alginate solid, dissolving sodium alginate in water, adding sodium periodate, reacting in the dark, then adding ethylene glycol to terminate the reaction, generating oxidized sodium alginate, adding absolute ethyl alcohol to precipitate, and obtaining pure oxidized sodium alginate solid through reduced pressure suction filtration, dialysis and freeze drying;
secondly, preparing oxidized sodium alginate-natamycin medicine powder: dissolving the oxidized sodium alginate prepared in the first step in water, adding natamycin, stirring in a dark environment, carrying out Schiff base reaction on the oxidized sodium alginate and the natamycin to generate oxidized sodium alginate-natamycin, and then dialyzing, freeze-drying and grinding to obtain oxidized sodium alginate-natamycin solid powder;
step three, preparing eye drops: 0.5-1.5mg of sodium alginate oxide-natamycin powder is added into 1mL of sterilized deionized water to prepare the sodium alginate oxide modified natamycin eye drops with the concentration of 0.5-1.5 mg/mL.
3. An oxidized sodium alginate modified natamycin eye drop as claimed in claim 2, wherein the specific steps for preparing oxidized sodium alginate in the first step are: (1) weighing 10g of sodium alginate by using a precision electronic balance, adding 200mL of sterilized deionized water, magnetically stirring at room temperature for 3-10 hours to prepare a sodium alginate aqueous solution with the mass concentration of 0.05g/mL, adding 1-10g of sodium periodate, reacting for 3-9 hours in a dark place, adding 0.260-2.60mL of ethylene glycol, magnetically stirring for 0.5 hour, stopping the reaction to generate an oxidized sodium alginate mixture solution, adding 400mL of absolute ethyl alcohol to precipitate, and then performing vacuum filtration by using a circulating water vacuum pump to obtain a mixed product containing oxidized sodium alginate;
(2) dissolving the mixed product containing sodium alginate oxide in sterilized deionized water, dialyzing in a dialysis bag for 1-3 days in sterilized double distilled water, changing the double distilled water every 6 hours, and freeze-drying the dialyzed solution at-70 ℃ to obtain pure sodium alginate oxide solid.
4. An oxidized sodium alginate modified natamycin eye drop according to claim 3, characterized in that 5g of sodium periodate is added to the aqueous solution of sodium alginate and reacted for 6 hours in the absence of light.
5. An oxidized sodium alginate modified natamycin eye drop as claimed in claim 2, wherein the specific steps of the second step are: dissolving the oxidized sodium alginate solid powder prepared in the first step in sterilized deionized water to prepare an oxidized sodium alginate aqueous solution with the mass concentration of 0.01g/mL, adding 0.5-5g natamycin into 100mL of the oxidized sodium alginate aqueous solution, stirring for 24-48 hours in a dark environment at 40 ℃, dialyzing in sterilized double distilled water for 1-3 days by using a dialysis bag to remove unreacted natamycin, freeze-drying the dialyzed solution at-70 ℃ to obtain oxidized sodium alginate-natamycin solid, grinding the oxidized sodium alginate-natamycin solid into powder by using a quartz mortar, and placing the powder in a dry sterile glass bottle to be stored in a dark environment.
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