CN114831936A

CN114831936A - Preparation of glabridin eye medicine and its application in treating fungal keratitis

Info

Publication number: CN114831936A
Application number: CN202210587701.4A
Authority: CN
Inventors: 高晗
Original assignee: Individual
Current assignee: Individual
Priority date: 2022-05-27
Filing date: 2022-05-27
Publication date: 2022-08-02

Abstract

The invention provides a preparation method of a glabridin eye medicine and application of the glabridin eye medicine in treatment of fungal keratitis, and relates to the technical field of pharmacy. The glabridin eye medicine takes glabridin as an effective component, the concentration of the glabridin is 16 mug/mL, pathogenic fungi of fungal keratitis can be effectively killed, and meanwhile, corneal epithelium is not damaged to generate adverse effects; the glabridin eye drops can be used as a medicine for treating fungal keratitis: the fungal growth is inhibited by inhibiting fungal spore adhesion host cells, the expression of inflammatory response factors interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha) and high mobility group protein B1(HMGB1) is reduced, the keratitis reaction is relieved, the tissue repair is promoted, and the fungal keratitis treatment is greatly benefited.

Description

Preparation of glabridin eye medicine and its application in treating fungal keratitis

Technical Field

The invention relates to the technical field of pharmacy, in particular to a preparation method of a glabridin eye medicine and application of the glabridin eye medicine in fungal keratitis treatment.

Background

Fungal keratitis is a corneal inflammatory disease caused by pathogenic fungi; the disease has slow onset and long course of disease, symptoms such as red eye and ophthalmalgia, blurred vision, photophobia lacrimation, blepharospasm and the like can appear, and serious visual impairment and even intraocular infection can be caused by untimely treatment. Vegetative trauma, contact lens abuse, and long-term use of broad-spectrum antibiotics and glucocorticoids have all led to an increasing incidence of disease year by year. Aspergillis and fusarium are the main pathogenic bacteria of fungal keratitis. Fungal infection of the cornea produces a strong immune response: corneal vasodilation, recruitment of inflammatory cells to the site of injury, a cascade of immunologically active substances. The inflammatory response is beneficial to the elimination of pathogenic bacteria, but excessive inflammatory response can cause the aggravation of corneal tissue damage, delayed healing, and even blindness. At present, common medicaments for clinically treating the fungal keratitis comprise natamycin, voriconazole and amphotericin, but when the common medicaments are used for treating the fungal keratitis, the defects of low bioavailability, fungal drug resistance and the like exist, so that the search for a novel effective antifungal medicament is a difficult task for treating the fungal keratitis at present.

Glabridin, a flavonoid extracted from the traditional Chinese medicine glycyrrhiza glabra, has antibacterial, antifungal, antioxidant, anti-inflammatory, immunoregulatory and neuroprotective effects. The use of the traditional Chinese medicine liquorice has a long history in traditional Chinese medicine and is one of the most widely used herbs in the world.

Disclosure of Invention

Technical problem to be solved

Aiming at the defects of the prior art, the invention provides the preparation of the glabridin eye medicine and the application of the glabridin eye medicine in the treatment of fungal keratitis, and solves the problem of poor antibacterial effect of the existing ointment.

(II) technical scheme

In order to achieve the purpose, the invention is realized by the following technical scheme: glabridin eye drops, including glabridin, solvent and sterilized water for injection;

the solvent is capable of contacting eyes and dissolving glabridin.

Preferably, the solvent is dimethyl sulfoxide, and the volume concentration of the dimethyl sulfoxide is less than or equal to 0.5%.

Preferably, the concentration of the glabridin is 16 mug/mL.

The glabridin eye ointment includes glabridin and excipient.

Preferably, the concentration of the glabridin is 16 mug/mL.

Preferably, the excipient is one or more of liquid paraffin, lanolin and yellow vaseline.

A method for preparing glabridin eye drop comprises dissolving glabridin in dimethyl sulfoxide to obtain glabridin solution, adding glabridin solution into sterilized water for injection, and mixing.

The glabridin eye drops are applied to the treatment of fungal keratitis.

The glabridin eye ointment is used in treating fungal keratitis.

(III) advantageous effects

The invention provides a glabridin eye medicament (glabridin eye drops), which has the following beneficial effects:

1. under the condition of the concentration (16 mu g/mL), the pathogenic bacteria of the fungal keratitis can be effectively killed, and meanwhile, the corneal epithelium is not damaged, and the eye damage is not generated; and can inhibit fungal growth by destroying fungal cellular structure (99.9% reduction in fungal population); and can also reduce the cornea inflammatory reaction by reducing the expression of inflammatory factors IL-1 beta, TNF-alpha and HMGB1, which is greatly beneficial to the prognosis of patients with fungal keratitis.

2. As a medicine for treating the fungal keratitis, the medicine can be reasonably used without causing toxic damage to the eye surface, can effectively kill pathogenic fungi, obviously relieves the corneal inflammatory reaction, promotes the repair of tissues and is greatly beneficial to the prognosis of patients with the fungal keratitis.

3. The raw materials are easy to obtain, the formula is simple, the cost is low, the preparation method is simple and easy to control, the use method is simple, and the market prospect is huge.

Drawings

FIG. 1 is a diagram showing the results of cytotoxicity experiments of glabridin eye drops of the present invention;

FIG. 2 is a result chart of the minimal inhibitory experiment and the fungus inhibition degree of the glabridin eye drops of the present invention;

FIG. 3 is a diagram showing the results of spore adhesion experiments after the glabridin eye drops of the present invention are applied; in the figure, black arrows indicate transparent millet-shaped Aspergillus fumigatus conidia adhered to the cell surface;

FIG. 4 is a diagram of a mouse corneal slit lamp examination of the glabridin eye drops after application of the glabridin eye drops to mouse fungal keratitis;

FIG. 5 is a graph of clinical scoring results of glabridin eye drops after application in fungal keratitis in mice; in the figure, black squares represent the infection control group, and white circles represent the glabridin eye drop treatment group;

FIG. 6 is a PAS staining pattern of cornea after the glabridin eye drops are applied to the fungal keratitis of a mouse; in the figure, red arrows indicate the distribution of hyphae in the corneal stroma;

FIG. 7 shows the expression of corneal tissue inflammatory factor gene after the glabridin eye drops are applied to the fungal keratitis of mice.

In the above figures, "+" corresponds to p <0.05, indicating a statistical difference, "+" corresponds to p <0.01, indicating a significant statistical difference, and "+" corresponds to p <0.001, indicating a very significant statistical difference.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The first embodiment is as follows:

dissolving 16 μ g of glabridin powder in dimethyl sulfoxide to obtain glabridin solution, adding the glabridin solution into 1mL of sterilized water for injection, and mixing uniformly to obtain the glabridin eye drops, wherein the sterilized water for injection is water meeting the requirements of Chinese pharmacopoeia water for injection.

When the glabridin eye drops are applied as a medicine for treating fungal keratitis, the application and dosage are as follows: eye drop is applied 3-6 times per day, and the dosage is more than or equal to conjunctival sac volume. The volume of the human conjunctival sac is usually 20 muL, and when the dosage is more than 20 muL, the redundant eye drops can overflow, and waste is caused excessively.

Example two:

and mixing 16 μ g of glabridin powder with 1mL of liquid paraffin, lanolin and yellow vaseline to obtain the glabridin eye ointment. Wherein, the liquid paraffin, the lanolin and the yellow vaseline can be mixed according to any proportion.

When the glabridin eye ointment is applied as a medicine for treating fungal keratitis, the application and dosage are as follows: the ointment with the length of about 1 cm is directly applied to the conjunctival sac of the lower eyelid 2 times a day, and excessive eye ointment overflows, so that waste and inconvenience in opening the eyes are caused.

Cytotoxicity experiments:

a suspension of human corneal epithelial cells (1X 104/mL) was seeded in a 96-well plate at 100. mu.L per well and cultured for about 12 hours (37 ℃, 5% CO2) until the cells attached. Glabridin was added to the medium at final concentrations of 0, 1, 2, 4, 8, 16, 32. mu.g/mL. After 24 hours of incubation, 10. mu.l of CCK-8 reagent was added to each well, incubation was continued for 2 hours, and absorbance value (OD) was measured using a microplate reader (450 nm).

The results in figure 1 show that glabridin solution of no more than 16 mug/mL has no toxic effect on human corneal epithelial cells and does not affect the survival rate. The judgment basis is as follows: after the glabridin with the concentration of less than or equal to 16 mug/mL is incubated, the absorbance value of the corneal epithelial cells to light with the wavelength of 450nm is not obviously different from that of a normal control group.

And (3) determining the minimum inhibitory concentration:

the minimal inhibitory concentration of glabridin against A.fumigatus was measured according to the Clinical Laboratory Standards Institute (CLSI) broth microdilution method (M38-A2 document). The spore suspension concentration was adjusted and the suspension was spectrophotometrically adjusted to an absorbance value (OD) in the range of 0.09 to 0.13 at a wavelength of 530 nm. A96-well plate was used, and 100. mu.L of Sabouroud broth was added to each well of columns 2 to 8, and 200. mu.L of glabridin solution prepared from Sabouroud broth with a final concentration of 64. mu.g/mL was added to each well of column 9. And (3) taking 100 mu L of solution from each hole in the 9 th row by using a pipette, and adding the solution into the corresponding hole in the 8 th row to realize the gradient micro-dilution of the glabridin solution. It should be noted that 100. mu.L of the liquid taken out from column 4 was discarded. 2 μ L of the prepared spore suspension was added to each well in columns 3 to 9. Column 2 was set as a blank control group, and column 3 was set as an infection control group. After mixing gently, the mixture was incubated in an incubator at 37 ℃ for 36 hours. The OD of each well was measured with a microplate reader (620 nm).

The result of figure 2 shows that the OD value is obviously reduced after the glabridin solution with the concentration of more than or equal to 16 mug/mL is treated, and the minimum inhibitory concentration is 16 mug/mL. The results in FIG. 3 show that glabridin at concentrations of 2. mu.g/mL, 4. mu.g/mL, 8. mu.g/mL, 16. mu.g/mL, 32. mu.g/mL, and 64. mu.g/mL inhibited fungi by 8.44%, 20.08%, 59.22%, 99.9%, 99.98%, and 99.95%, respectively, as compared to the untreated control group.

Spore adhesion experiments:

human corneal epithelial cells were seeded onto chambered slides overnight until the cells grew adherent. The spore suspension and glabridin solution were mixed until the spore concentration was 1X 105/mL and the final concentration of glabridin was 16. mu.g/mL, and incubated at 37 ℃ for 3 hours. The slides were gently rinsed with PBS to wash away unadhered fungal spores. Staining the cells of each chamber by HE staining to prepare a sheet. The images were taken by bright field microscope observation. The number of spores adhering to the surface of 100 HCECs was counted, and the average value indicated the ability to adhere.

The results are shown in fig. 3, after the aspergillus fumigatus is infected, the aspergillus fumigatus conidia adhered to the surface of the human corneal epithelial cells treated by the glabridin are obviously reduced, which indicates that the glabridin can reduce the adhesive capacity of fungal spores so as to play a bacteriostatic action.

Establishing a mouse fungal keratitis model:

mice were given abdominal anesthesia (8% chloral hydrate, 0.4 ml/kg). Under an ophthalmic surgery microscope, a 30G disposable syringe needle was used to make a slight scratch on the cornea corresponding to the pupillary edge, and a 10 μ L LHamilton microsyrin was used to inject 2 μ L of spore suspension (1X 107/mL) into the central corneal stroma of the right eye of the mouse, and the left eye was left untreated and used as a blank control. After the intrastromal injection is completed for about 4 hours, the mice are subjected to gas anesthesia under a slit lamp to observe that the corneal opacity area of the right eye of the mice is less than 25%, the eyes are cloudy and the eye surfaces are slightly rough, at the moment, the right eye of an experimental group is treated by 16 mu g/mL glabridin eye drops in an eye drop mode, the right eye of a control group is treated by equivalent PBS at the same time, and the left eyes of the two groups are not treated. The eye drop treatment is a five-day treatment course, and is carried out three times a day. And 10 μ L of the corresponding drug was given to the right eye of the mouse as a subconjunctival injection treatment on the first and second evening after modeling. Mice were closely observed daily under slit lamps for changes in corneal disease and evaluated for clinical scores according to a score scale (table 1), as shown in fig. 5 and 6. Mice were sacrificed at the indicated times and the mice were harvested for subsequent experiments on the cornea or eyeball.

The result shows that compared with an infection control group, the glabridin eye drop with the concentration of 16 mu g/mL can obviously reduce the corneal ulcer area, reduce the inflammatory injury of the cornea, reduce the clinical score and improve the prognosis of keratitis.

TABLE 1 mouse model inflammation grading of fungal keratitis

Mouse corneal periodic acid-schiff (PAS) staining experiment:

the eyeballs of mice on day 3 after infection were collected and embedded with OCT embedding medium, snap frozen with liquid nitrogen, and then cut into 10 μm tissue sections with a cryomicrotome and fixed with acetone. Staining was performed according to the PAS staining kit instructions, neutral gum blocking. The observation was carried out using an optical microscope and photographed.

The results in fig. 6 show that, compared with the infected control group, after the glabridin eye drops with the concentration of 16 mug/mL are treated for 3 days, hypha in the corneal stroma of the mice are respectively obviously reduced, the corneal thickness is obviously recovered to be normal, and the stroma texture is relatively regular.

Glabridin anti-inflammatory action experiment:

and collecting the corneas of each group in the mouse fungal keratitis model, and detecting the mRNA relative expression level of IL-1 beta, TNF-alpha and HMGB1 by RT-PCR.

The results of fig. 7 show that glabridin can exert the effect of reducing the above gene expression of inflammatory factors by treatment for 1 day, and that the inhibitory effect is continued up to

treatment days

3 and 5. For uninfected cornea (N), no differences in the expression of the above inflammation-related molecules were detected between the two treatment groups. The results show that the glabridin eye drops can obviously reduce the level of proinflammatory factors generated by fungi induced cells.

In addition, the anti-inflammatory function experiment was performed on the fungal keratitis in mice by using the glabridin eye ointment of the same concentration instead of the glabridin eye drops. The experimental results show that: the glabridin eye ointment and the glabridin eye drops with the same concentration have equivalent inhibitory effect on proinflammatory factors.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Claims

1. Glabridin eye drops, which is characterized in that: comprises glabridin, solvent and sterilized water for injection;

the solvent is capable of contacting eyes and dissolving glabridin.

2. The glabridin eye drops according to claim 1, wherein: the solvent is dimethyl sulfoxide, and the volume concentration of the solvent is less than or equal to 0.5%.

3. The glabridin eye drops according to claim 1, wherein: the concentration of the glabridin is 16 mug/mL.

4. Glabridin eye ointment is characterized in that: comprises glabridin and excipient.

5. The glabridin eye ointment of claim 4, wherein: the concentration of the glabridin is 16 mug/mL.

6. The glabridin eye ointment of claim 4, wherein: the excipient is one or more of liquid paraffin, lanolin and yellow vaseline.

7. The method for preparing glabridin eye drops according to any one of claims 1 to 3, characterized in that: dissolving glabridin in dimethyl sulfoxide to obtain glabridin solution, adding glabridin solution into sterilized water for injection, and mixing.

8. The glabridin eye drops according to any one of claims 1 to 3, for use in the treatment of fungal keratitis.

9. The glabridin eye ointment according to any one of claims 4 to 6, for use in the treatment of fungal keratitis.