CN113786388B - Method for improving natamycin by using bletilla striata polysaccharide and application - Google Patents
Method for improving natamycin by using bletilla striata polysaccharide and application Download PDFInfo
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- A61P31/10—Antimycotics
Abstract
The invention belongs to the technical field of medicine and material chemistry, and relates to a method for improving natamycin by bletilla striata polysaccharide and an application thereof, wherein the preparation method comprises 5 process steps, firstly, preparing a bletilla striata polysaccharide oxide solution, then adding ethylene glycol to stop an oxidation reaction, adding a natamycin methanol solution to react to form a bletilla striata polysaccharide natamycin oxide-natamycin mixture, dialyzing and freeze-drying to prepare a bletilla striata polysaccharide oxide-natamycin freeze-dried powder, and applying the prepared bletilla striata polysaccharide-natamycin oxide to ophthalmology to treat fungal keratitis.
Description
The technical field is as follows:
the invention belongs to the field of medicine and material chemistry, and relates to a method for modifying natamycin by using bletilla polysaccharide and application thereof, in particular to a method for preparing oxidized white and polysaccharide-natamycin by using natural bletilla polysaccharide modified natamycin, so as to enhance the water solubility and antifungal effect of natamycin, and the oxidized bletilla polysaccharide-natamycin is used for treating fungal keratitis, and the prepared eye drop has strong water solubility, small toxicity, and large antifungal and anti-inflammatory effects.
The background art comprises the following steps:
fungal keratitis is a corneal inflammatory pathological change caused by fungal infection, can be caused by ocular trauma, chronic inflammation of the eye, body resistance reduction and the like, is a blinding eye disease caused by blinding fungal infection, and is relatively troublesome in clinical treatment. Most of the antifungal eye drops clinically adopted at present are fat-soluble suspensions, and have the problems of poor local corneal osmosis, strong irritation, high toxicity, short half-life, frequent administration and the like, the blinding rate of the fungal keratitis caused by deficient treatment means is high, the voriconazole eye drops clinically used for the fungal keratitis at present have fast local metabolism after being dripped, the effective local action concentration cannot be maintained for a long time, and the poor compliance of patients and the eye irritation symptom are caused by frequent eye dripping; natamycin as a suspension preparation is an effective drug for treating fungal keratitis, but the solubility and dissolution speed of natamycin in tears are influenced by the secretion of tears, so that the effect of sustained release is difficult to achieve, and the defects of poor solubility, low corneal penetrability and the like limit the clinical application of natamycin.
Chinese patent with application number CN201910318888.6 discloses a lomefloxacin hydrochloride eye drop and a preparation process thereof, the eye drop is obtained by screening sodium alginate as a thickening excipient, swelling the thickening excipient with a solubilizing additive and water for injection to form a transparent solution, mixing the transparent solution with an isotonic regulator glycerol, an intraocular pressure regulator mannitol and a biodegradation aid, regulating the pH value by a buffer solution, filtering and carrying out aseptic packaging. Chinese patent with application number CN201010044868.3 discloses an eye drop and a preparation method thereof, wherein the eye drop comprises, by volume, 0.1-5% of bovine serum, 5-15% of a thickening agent, 1-5% of an acid-base adjusting solution, 0.5-2% of an antibiotic, 5-20% of a recombinant protein and the balance of a balanced salt solution, the thickening agent, the bovine serum, the antibiotic and the recombinant protein are added into the balanced salt solution, the pH value is adjusted by the acid-base adjusting agent, the osmotic pressure is adjusted by an osmotic pressure buffering agent, and bacteria are removed by membrane filtration; or preparing the recombinant protein into sterile micropowder, dissolving bovine serum in balanced salt solution, adjusting pH, performing membrane filtration sterilization, and dissolving the recombinant powder in the solution to obtain eye drop. The two prior arts use more raw materials and have more complex processes, and the prepared eye drops do not disclose the inhibition effect on fungi, nor do they disclose the treatment effect and curative effect on severe fungal keratitis. Therefore, the research and development of a novel medicament form with few raw material varieties, simple preparation process, antifungal property and good anti-inflammatory effect is very important.
The main improvement form of natamycin in the prior art is to prepare nano lipid particles loaded with drugs, the preparation of the lipid particles needs various core lipids and surfactants, the performance of the drug-loaded particles is improved by performing multi-layer chemical modification on the particle surfaces, the raw materials are various, the process is complex, and natamycin can act on the cornea only by processes of physical encapsulation, ocular surface release and the like. The existing developed carboxyvinyl polymer, guar gum and boric acid gel system loaded with natamycin polyethylene glycol nano-lipid particles only researches the corneal permeability of the gel system and the drug retention rate in the ocular surface and tears, but does not compare whether the gel system enhances the antifungal activity compared with natamycin or not, and does not research whether the gel system has a practical treatment effect on fungal keratitis or not. Chinese patent application No. CN201911030779.0 discloses a natamycin polymer micelle eye drop and a preparation method thereof, a polyethylene glycol-polymethacrylate glyceride block copolymer is placed in absolute ethyl alcohol to self-assemble into a cross-linked micelle to entrap natamycin to prepare the natamycin polymer micelle, the drug-loading rate can reach 10-30%, and the entrapment rate can reach more than 94%.
The bletilla polysaccharide is a water-soluble polysaccharide extracted from traditional Chinese medicine bletilla through a certain process, is used as a natural high-molecular carrier material, has the characteristics of excellent biocompatibility, biological adhesion, low toxicity and the like, has biological activities of bacteriostasis, anti-inflammation, procoagulant, anti-tumor, immunoregulation and the like, and has good development and utilization values in the field of medicine, so that a method for improving the natamycin by using the bletilla polysaccharide and an application thereof are sought, the native bletilla polysaccharide is used for improving the natamycin to prepare an oxidized bletilla polysaccharide-natamycin, namely natamycin, so that the water solubility and the antifungal effect of the natamycin are enhanced, the oxidized bletilla polysaccharide-natamycin is used for treating the fungal keratitis, and the prepared eye drops have strong water solubility, small toxicity, large antifungal and anti-inflammation effects, effectively increase the water solubility of the natamycin, reduce the toxicity of the natamycin and have multiple effects of resisting fungi, resisting inflammation and the like. The method for improving natamycin by using bletilla striata polysaccharide and the application thereof are not reported so far.
The invention content is as follows:
the invention aims to overcome the defects in the prior art, and designs and provides a method for improving natamycin by using bletilla polysaccharide and application thereof, so as to solve the problems of poor natamycin solubility and low corneal penetrability, solve the problems of various raw materials and complex process of the existing preparation method of the eye drops, and solve the problems of poor corneal local permeability, strong irritation and high toxicity of the existing antifungal eye drops; overcomes the defects of short drug effect action time and frequent administration of the existing eye drops, prolongs the half-life period of the ocular surface, and enhances the compliance of patients with drugs.
In order to achieve the above object, the method for improving natamycin by bletilla striata polysaccharide of the present invention comprises 5 process steps:
(1) Adding 2-10 g of bletilla polysaccharide into every 200ml of phosphate buffer solution to prepare a white and polysaccharide solution, adding 2-10 g of sodium periodate, and continuously stirring for reaction for 12 hours at room temperature in a dark condition to obtain an oxidized bletilla polysaccharide solution;
(2) Adding 2ml of ethylene glycol into the solution of the bletilla oxide and the polysaccharide prepared in the step (1), stirring for 5 minutes to quench sodium periodate in the reaction solution, and stopping the oxidation reaction;
(3) Dissolving 200-800 mg of natamycin in 40ml of methanol, then dripping the natamycin into the reaction solution prepared in the step (2), and continuously stirring for 12 hours at room temperature in a dark condition, wherein the reaction formulas of the step (2) and the step (3) are as follows:
(4) Adding 200-350 ml of absolute ethyl alcohol into the reaction liquid prepared in the step (3), fully stirring to separate out a white precipitate, standing for 4 hours to precipitate, and performing suction filtration on the separated precipitate, namely the mixture containing the oxidized bletilla striata polysaccharide-natamycin;
(5) Dialyzing the mixture containing the oxidized bletilla striata polysaccharide-natamycin precipitated in the step (4) with distilled water for 3 days to remove residual sodium periodate, ethylene glycol, methanol and ethanol to obtain purified oxidized bletilla striata polysaccharide-natamycin, standing the purified oxidized bletilla striata polysaccharide-natamycin at the temperature of-60 ℃ for 48 hours, freeze-drying to obtain white oxidized bletilla striata polysaccharide-natamycin freeze-dried powder, and storing at the constant temperature of 4 ℃.
The preparation method of the oxidized bletilla striata polysaccharide-natamycin eye drops comprises the following steps of: the content of the added bletilla oxide and polysaccharide-natamycin freeze-dried powder in each 10ml of phosphate buffer solution is 100-8000 ug. The eye drop can be used for treating fungal keratitis, and has the advantages of high water solubility, high corneal penetration, low toxicity, high antifungal and antiinflammatory effects, and rapid onset of drug action.
Compared with the prior art, the invention has the advantages of low raw material cost, simple and easy preparation process, high preparation efficiency, strong water solubility of the prepared eye drops, strong corneal penetration, low toxicity, and large antifungal and anti-inflammatory effects; the eye drop can directly act on the ocular surface without release and degradation, and has quick response; the half-life of the ocular surface is prolonged, the administration times are reduced, and the medication compliance of patients is enhanced; promoting the proliferation and migration of corneal epithelial cells of the eye and promoting the repair of corneal epithelium of the eye; the preparation process is simple, convenient to operate and wide in application.
Description of the drawings:
FIG. 1 shows a reaction scheme for producing oxidized bletilla striata and the polysaccharide natamycin (OBSP-NAT) according to the present invention.
FIG. 2 is a graph comparing the ultraviolet spectra of the oxidized form of the present invention and the polysaccharide natamycin (OBSP-NAT).
FIG. 3 is a comparison of the inhibitory concentrations of the oxidized bletillant polysaccharide natamycin (OBSP-NAT) and Natamycin (NAT) produced by the present invention.
FIG. 4 is a comparison of cytotoxicity of natamycin (OBSP-NAT) and Natamycin (NAT), both oxidized and produced by the present invention.
FIG. 5 is a graph comparing the levels of inflammatory factor (mRNA) in Mouse macrophages (Mouse macrophages, RAW 264.7) of oxidized bletilla striata polysaccharide-natamycin (OBSP-NAT) eye drops prepared according to the present invention and Natamycin (NAT).
FIG. 6 is a graph comparing the protein levels of oxidized bletilla striata polysaccharide natamycin (OBSP-NAT) eye drops prepared according to the present invention and Natamycin (NAT) in Mouse macrophages (Mouse macrophages, RAW 264.7).
Figure 7 is a comparison of the inflammatory scores of oxidized bletilla striata polysaccharide natamycin (OBSP-NAT) eye drops and Natamycin (NAT) prepared according to the invention in a mouse model of aspergillus fumigatus keratitis.
FIG. 8 is a graph comparing the levels of inflammatory factors (mRNA) in mouse A.fumigatus keratitis model between oxidized bletilla striata polysaccharide-natamycin (OBSP-NAT) eye drops and Natamycin (NAT) prepared according to the present invention.
The specific implementation mode is as follows:
the invention is further illustrated by the following specific examples in combination with the accompanying drawings.
Example 1:
the method for improving natamycin by using bletilla striata polysaccharide comprises 5 process steps:
(2) Adding 2-10 g of bletilla polysaccharide into every 200ml of phosphate buffer solution to prepare a white and polysaccharide solution, adding 2-10 g of sodium periodate, and continuously stirring for reaction for 12 hours at room temperature in a dark condition to obtain an oxidized bletilla polysaccharide solution;
(3) Adding 2ml of ethylene glycol into the solution of the bletilla oxide and the polysaccharide prepared in the step (1), stirring for 5 minutes to quench sodium periodate in the reaction solution, and stopping the oxidation reaction;
(3) Dissolving 200-800 mg of natamycin in 40ml of methanol, then dripping the natamycin into the reaction solution prepared in the step (2), and continuously stirring for 12 hours at room temperature in a dark condition, wherein the reaction formulas of the step (2) and the step (3) are as follows:
(4) Adding 200-350 ml of absolute ethyl alcohol into the reaction liquid prepared in the step (3), fully stirring to separate out a white precipitate, standing for 4 hours to precipitate, and performing suction filtration on the separated precipitate, namely the mixture containing the oxidized bletilla striata polysaccharide-natamycin;
(5) Dialyzing the mixture containing the oxidized bletilla striata polysaccharide-natamycin precipitated in the step (4) with distilled water for 3 days to remove residual sodium periodate, glycol, methanol and ethanol to obtain purified oxidized bletilla striata polysaccharide-natamycin, placing the purified oxidized bletilla striata polysaccharide-natamycin at the temperature of-60 ℃ for 48 hours, freeze-drying to obtain white oxidized bletilla striata polysaccharide-natamycin freeze-dried powder, and storing at the constant temperature of 4 ℃.
The oxidized bletilla striata polysaccharide-natamycin eye drops prepared by the embodiment are applied to ophthalmology to prepare the oxidized bletilla striata polysaccharide-natamycin eye drops, and the oxidized bletilla striata polysaccharide-natamycin eye drops comprise the following components in percentage by weight: the content of the added bletilla oxide and polysaccharide-natamycin freeze-dried powder is 100-8000 ug in each 10ml of phosphate buffer solution. The eye drop can be used for treating fungal keratitis, and has the advantages of high water solubility, high corneal penetration, low toxicity, good antifungal and anti-inflammatory effects, and rapid onset of drug action.
Example 2:
the method for modifying natamycin by using bletilla striata polysaccharide, which is related to the embodiment, is implemented according to the process steps of the embodiment 1:
(1) Adding 4g of rhizoma bletilla polysaccharide into per 200ml of phosphate buffer solution to obtain rhizoma bletilla polysaccharide solution, adding 4g of sodium periodate, and continuously stirring at room temperature in a dark place for reaction for 12 hours to obtain oxidized rhizoma bletilla polysaccharide solution;
(2) Adding 2ml of ethylene glycol into the solution of the bletilla oxide and polysaccharide prepared in the step (1), stirring for 5 minutes to quench sodium periodate in the reaction solution, and stopping the oxidation reaction;
(3) Dissolving 400mg of natamycin in 40ml of methanol, then dripping the natamycin into the reaction liquid prepared in the step (2), and continuously stirring the natamycin for 12 hours at room temperature in a dark condition, wherein the reaction formulas of the step (2) and the step (3) are as follows:
(4) Adding 300ml of absolute ethyl alcohol into the reaction solution prepared in the step (3), fully stirring to separate out a white precipitate, standing for 4 hours to precipitate, and performing suction filtration on the separated precipitate, namely the mixture containing the bletilla oxide polysaccharide-natamycin;
(5) Dialyzing the mixture containing the oxidized bletilla striata polysaccharide-natamycin precipitated in the step (4) with distilled water for 3 days to remove residual sodium periodate, ethylene glycol, methanol and ethanol to obtain purified oxidized bletilla striata polysaccharide-natamycin, standing the purified oxidized bletilla striata polysaccharide-natamycin at the temperature of-60 ℃ for 48 hours, freeze-drying to obtain white oxidized bletilla striata polysaccharide-natamycin freeze-dried powder, and storing at the constant temperature of 4 ℃.
The oxidized bletilla striata polysaccharide-natamycin eye drops prepared by the embodiment are applied to ophthalmology, and the oxidized bletilla striata polysaccharide-natamycin eye drops are prepared from the following components in percentage by weight: the content of the added bletilla oxide and polysaccharide-natamycin freeze-dried powder in each 10ml of phosphate buffer solution is 1600ug. The eye drop can be used for treating fungal keratitis, and has the effects of strong water solubility, strong corneal penetration, low toxicity, antifungal effect, high antiinflammatory effect, and rapid onset of drug action.
Example 3:
this example was prepared according to the procedure of example 2, and the resulting oxidized bletillae polysaccharide natamycin (OBSP-NAT) was analyzed and characterized by infrared spectroscopy and compared to Natamycin (NAT), bletillae polysaccharide (BSP), oxidized bletillae polysaccharide (OBSP): the infrared spectrum is detailed in figure 2 of the attached drawings of the specification: indicating that the oxidation reaction was successful and producing a new compound.
The prepared OBSP-NAT and NAT are compared with the minimum bacteriostatic and bactericidal concentration for inhibiting aspergillus fumigatus, and the detail is shown in figure 3: NAT can inhibit more than 90% of aspergillus fumigatus growth at 8 ug/ml; OBSP-NAT can inhibit more than 90% of Aspergillus fumigatus growth at 160 ug/ml.
The toxicity of corneal epithelial cells and mouse macrophages was compared between the prepared OBSP-NAT and NAT, which is shown in FIG. 4: NAT begins to show epithelial cytotoxicity at 16ug/ml, while OBSP-NAT begins to show epithelial cytotoxicity at 640ug/ml and begins to exhibit an effect of promoting epithelial cell growth at 160 ug/ml.
Comparison of the levels of inflammatory factors (mRNA) in Mouse macrophages (Mouse macrophages, RAW 264.7) was performed on the prepared OBSP-NAT eyedrops at a concentration of 160ug/ml and NAT eyedrops at a concentration of 8ug/ml, as detailed in fig. 5: both NAT and OBSP-NAT can inhibit the gene level expression of inflammatory factors IL-1 beta, TNF-alpha, IL-6 and LOX-1 in macrophage, and the inhibition effect of OBSP-NAT is superior to that of NAT.
The protein levels in Mouse macrophages (Mouse macrophages, RAW 264.7) were compared between the prepared OBSP-NAT eyedrops at 160ug/ml and NAT eyedrops at 8ug/ml, as shown in fig. 6: NAT does not significantly inhibit the levels of the inflammatory factors IL-1 beta, TNF-alpha and IL-6 in macrophages, while OBSP-NAT significantly reduces the levels of the inflammatory factors IL-1 beta, TNF-alpha and IL-6 in macrophages.
The prepared OBSP-NAT eye drops with the concentration of 160ug/ml and NAT eye drops with the concentration of 8ug/ml are subjected to inflammation score comparison in a mouse aspergillus fumigatus keratitis model, and detailed in figure 7: the clinical scores of the NAT and OBSP-NAT for treating the fungal keratitis of the mice on the 3 rd day are not obviously different; the OBSP-NAT group had significantly less corneal haze than the NAT group on day 5.
The prepared OBSP-NAT eye drops with the concentration of 160ug/ml and NAT eye drops with the concentration of 8ug/ml are compared with the level of inflammatory factors (mRNA) in a mouse aspergillus fumigatus keratitis model, and detailed results are shown in FIG. 8: on days 3 and 5 of the treatment of the mouse aspergillus fumigatus keratitis, both NAT and OBSP-NAT can inhibit the expression of inflammatory factors IL-1 beta, TNF-alpha, IL-6 and LOX-1 gene levels in the mouse cornea, and the inhibition effect of the OBSP-NAT is obviously superior to that of the NAT.
Compared with the adhesion of the OBSP-NAT eye drops with the concentration of 160ug/ml and the adhesion of NAT eye drops with the concentration of 8ug/ml, the adhesion of the OBSP-NAT eye drops is obviously better than that of the NAT eye drops.
Claims (8)
1. A preparation method of bletilla striata polysaccharide modified natamycin is characterized in that the preparation method of the bletilla striata polysaccharide modified natamycin comprises 5 process steps:
(1) Adding 2-10 g of bletilla polysaccharide into every 200ml of phosphate buffer solution to prepare a bletilla polysaccharide solution, adding 2-10 g of sodium periodate, and continuously stirring for reaction for 12 hours at room temperature in a dark condition to obtain an oxidized bletilla polysaccharide solution;
(2) Adding 2ml of ethylene glycol into the solution of the bletilla oxide and polysaccharide prepared in the step (1), stirring for 5 minutes to quench sodium periodate in the reaction solution, and stopping the oxidation reaction;
(3) Dissolving 200-800 mg of natamycin in 40ml of methanol, then dripping the natamycin into the reaction solution prepared in the step (2), and continuously stirring for 12 hours at room temperature in a dark condition, wherein the reaction formulas of the step (2) and the step (3) are as follows:
(4) Adding 200-350 ml of absolute ethyl alcohol into the reaction solution prepared in the step (3), fully stirring to separate out a white precipitate, standing for 4 hours to precipitate, and performing suction filtration on the separated precipitate, namely the mixture containing the bletilla oxide polysaccharide-natamycin;
(5) Dialyzing the mixture containing the oxidized bletilla striata polysaccharide-natamycin precipitated in the step (4) with distilled water for 3 days to remove residual sodium periodate, ethylene glycol, methanol and ethanol to obtain purified oxidized bletilla striata polysaccharide-natamycin, standing the purified oxidized bletilla striata polysaccharide-natamycin at the temperature of-60 ℃ for 48 hours, freeze-drying to obtain white oxidized bletilla striata polysaccharide-natamycin freeze-dried powder, and storing at the constant temperature of 4 ℃.
2. The process according to claim 1, wherein in the step (1), 4g of the bletilla polysaccharide is added into 200ml of phosphate buffer solution to prepare a bletilla polysaccharide solution, and 4g of sodium periodate is added; 400mg of natamycin was dissolved in 40ml of methanol in step (3), and 300ml of absolute ethanol was added to the reaction solution prepared in step (3) in step (4).
3. An eye drop of oxidized bletilla striata polysaccharide-natamycin is characterized in that the components and the contents of the oxidized bletilla striata polysaccharide-natamycin eye drop are as follows: the content of the added bletilla oxide and polysaccharide-natamycin freeze-dried powder in each 10ml of phosphate buffer solution is 100-8000 ug.
4. The oxidized bletilla striata-natamycin ophthalmic solution according to claim 3, wherein the oxidized bletilla striata-natamycin ophthalmic solution comprises the following components in percentage by weight: the content of added bletilla oxide and polysaccharide-natamycin freeze-dried powder in each 10ml of phosphate buffer solution is 1600ug; the eye drop has water solubility, corneal penetration, antifungal, and antiinflammatory effects.
5. An ophthalmic solution of bletilla striata and polysaccharide-natamycin as claimed in claim 3 or 4, wherein the ophthalmic solution is compared to the use of the natamycin for inhibiting Aspergillus fumigatus, and natamycin at 8ug/ml inhibits more than 90% of Aspergillus fumigatus growth; the oxidized bletilla striata polysaccharide-natamycin can inhibit the growth of more than 90 percent of aspergillus fumigatus at 160 ug/ml.
6. An oxidized bletilla striata polysaccharide-natamycin ophthalmic solution according to claim 3 or 4, characterized in that the ophthalmic solution is compared to corneal epithelial cell and mouse macrophage toxicity, natamycin starts to appear epithelial cytotoxic at 16ug/ml, and oxidized bletilla striata polysaccharide-natamycin starts to appear epithelial cytotoxic at 640ug/ml and starts to exhibit epithelial cell growth promoting effect at 160 ug/ml.
7. An ophthalmic solution of bletilla striata and polysaccharide-natamycin as claimed in claim 3 or 4, wherein the ophthalmic solution is compared with the level of inflammatory factors in mouse macrophages, and the bletilla striata and polysaccharide-natamycin has inhibitory effect; the oxidation and the polysaccharide-natamycin have the inhibiting effect by comparing the level of inflammatory factors in a mouse aspergillus fumigatus keratitis model.
8. An oxidized bletilla striata-natamycin ophthalmic solution according to claim 3 or 4, characterized in that the oxidized bletilla striata-polysaccharide-natamycin lowers the protein level of the inflammatory factors IL-1 β, TNF- α, IL-6 in macrophages when compared to the protein level of the ophthalmic solution in mouse macrophages.
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| CN104688689A (en) * | 2015-02-11 | 2015-06-10 | 青岛大学附属医院 | Natamycin cationic liposome eye drop and preparation method thereof |
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| CN104688689A (en) * | 2015-02-11 | 2015-06-10 | 青岛大学附属医院 | Natamycin cationic liposome eye drop and preparation method thereof |
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Effective date of registration: 20230905 Address after: No. 25 Xinyunhe Road, Shenyang Area, China (Liaoning) Pilot Free Trade Zone, Shenyang City, Liaoning Province, 110117 Patentee after: SHENYANG XINGQI PHARMACEUTICAL Co.,Ltd. Address before: No. 16, Jiangsu Road, Southern District, Shandong, Qingdao, Shandong Patentee before: THE AFFILIATED HOSPITAL OF QINGDAO University |