CN110876746A - Ginkgo diterpene lactone eye preparation and preparation method and application thereof - Google Patents

Abstract

The invention provides application of ginkgolide in preparing a medicament for treating optic nerve injury, wherein the ginkgolide is selected from a composition containing ginkgolide A, B and K, and the ginkgolide A: ginkgolide B: and (3) bilobalide K is (20-40): (50-75): (0.2-5.0). The ginkgo diterpene lactone provided by the invention has the effects of expanding blood vessels, increasing blood flow, repairing and protecting optic nerves, can be applied to treating optic nerve injury, retrobulbar neuritis and retinitis, and can also be applied to relieving visual fatigue. The invention prepares the ginkgo diterpene lactone into the eye preparation, overcomes the problem of low solubility of the ginkgo diterpene lactone, prolongs the contact time of the medicine and the cornea by increasing the viscosity, and can improve the bioavailability.

Description

Ginkgo diterpene lactone eye preparation and preparation method and application thereof

Technical Field

The invention relates to the technical field of medicaments, in particular to a ginkgo diterpene lactone ophthalmic preparation as well as a preparation method and application thereof.

Background

Treatment and functional recovery after optic nerve injury are historical challenges in the medical field. Due to the lack of microenvironment required for nerve repair and regeneration following optic nerve injury as part of the central nervous system. Effective neuroprotection, prevention of neuronal death, and promotion of neural repair are therefore of paramount importance. At present, about half of patients lose vision finally due to the lack of timely and effective treatment measures clinically.

Retrobulbar optic neuritis is one of multiple ophthalmic diseases, can cause irreversible damage to vision and even blindness, is commonly seen in inflammation of optic nerve center, mainly invades papillary macular fiber bundles, and has acute morbidity and poor healing. Clinically, the retrobulbar neuritis is generally treated by western medicine hormone systemic therapy, and a vasodilator and a neurotrophic agent are simultaneously applied. For those who are not effective in hormone therapy, maxillary sinus approach opening ethmoid sinus and sphenoid sinus can be selected, and the lower wall in the optic tube is cut under the operating microscope to reduce the pressure of optic nerve, improve nerve nutrition, and facilitate the recovery of optic nerve function. Generally, the traditional Chinese medicine composition can obtain good effect in acute stage, severe patients can cause optic nerve temporal atrophy and even total atrophy to cause blindness, the effect is not ideal enough, and the treatment cost is high.

Retinitis is mainly characterized by retinal tissue edema, exudation and hemorrhage, which cause different degrees of visual deterioration; typically secondary to choroiditis, resulting in choroidal retinal inflammation. Retinal tissue edema is treated with hormones, but the side effects of hormones limit its use; in addition, the western medicines include nicotinic acid, vitamin B1, B12, adenosine triphosphate, etc. Since chorioretinitis is susceptible to recurrence, treatment difficulty is increased.

Folium ginkgo (Ginkgobiloba) is a Chinese herbal medicine with wide use and obvious drug effect, has sweet, bitter, astringent and neutral nature and taste, and has good health care and treatment effects on blood vessels, nerves and other systems after entering heart and lung channels.

Disclosure of Invention

The invention aims to provide a medicine or preparation aiming at eye diseases based on traditional Chinese medicine sources. Specifically, the invention provides an application of ginkgolide in preparing a medicament for treating optic nerve injury. Wherein the ginkgolide is ginkgolide A, B or K, or any combination thereof, or an extract containing ginkgolide A, B or K.

Preferably, the ginkgolide is selected from the group consisting of ginkgolides A, B and K, wherein ginkgolide a: ginkgolide B: and (3) bilobalide K is (20-40): (50-75): (0.2-5.0). More preferably, the ratio of ginkgolide a: ginkgolide B: bilobalide K ═ 36: 60: 3.6.

preferably, the content of active ingredients contained in the extract containing the ginkgolide A, B or K is 90-99.5%.

Further, the medicine for optic nerve injury also comprises pharmaceutically acceptable auxiliary materials.

Specifically, the medicine for optic nerve injury is selected from oral preparations, injection preparations, eye preparations and inhalation preparations.

Further, the ophthalmic preparation includes eye drops, ophthalmic gel, eye ointment, ophthalmic liposome, ophthalmic microemulsion, and the like.

Specifically, the ophthalmic preparation comprises 0.05-2% of ginkgolide by weight and a pharmaceutic adjuvant of the ophthalmic preparation.

The invention also provides a ginkgo diterpenoid lactone ophthalmic preparation for optic nerve injury, wherein the ginkgo diterpenoid lactone is ginkgolide A, B or K, or any combination thereof, or an extract containing ginkgolide A, B or K.

Preferably, the ginkgolide is selected from the group consisting of ginkgolides A, B and K, wherein ginkgolide a: ginkgolide B: and (3) bilobalide K is (20-40): (50-75): (0.2-5.0). More preferably, the ratio of ginkgolide a: ginkgolide B: bilobalide K ═ 36: 60: 3.6.

further, when the ophthalmic preparation is selected from temperature-sensitive ophthalmic gels, the gels comprise 0.05-2% of ginkgo diterpene lactone, 10.5-40% of temperature-sensitive gels, 0.1-4.0% of pH regulators, 0.05-2% of bacteriostats, 0.1-0.9% of osmotic pressure regulators and a proper amount of water for injection, and the sum of the percentage contents of the components is one hundred percent.

Furthermore, the percentage content of the ginkgolide is preferably 0.1-1%.

Preferably, the temperature-sensitive gel is one or a mixture of poloxamer 407, poloxamer 188, pluronic, carbomer, hyaluronic acid, chitosan, xylan, sucrose acetate isobutyrate; more preferably, the temperature-sensitive gel is selected from the combination of poloxamer 407 and poloxamer 188, and more preferably a mixture of poloxamer 40724-28% and poloxamer 1882-6%. Most preferably, the concentrations of poloxamer 407 and poloxamer 188 are 24% and 2%, respectively.

The invention also provides a preparation method of the ginkgolide temperature-sensitive ophthalmic gel, which comprises the following steps:

step 1: weighing the ginkgo diterpene lactone and the pH regulator with the prescription amount, adding water for injection, and heating for dissolving; filtering, and cooling the filtrate;

step 2: slowly adding a surfactant into the filtrate obtained in the step 1 under magnetic stirring to wet and disperse the filtrate, refrigerating the filtrate in a refrigerator, and fully swelling the filtrate to obtain a solution 1;

and step 3: dissolving pH regulator, bacteriostatic agent and osmotic pressure regulator in water for injection, adding surfactant, wetting and dispersing, refrigerating in refrigerator, and swelling to obtain solution 2;

and 4, step 4: and (3) combining the solution 1 in the step (2) and the solution 2 in the step (3), uniformly mixing, filtering by using a 0.22 mu m microporous filter membrane, and subpackaging to obtain the composition.

In particular, the surfactant is selected from temperature sensitive gels.

Preferably, the heating temperature in the step 1 is 70-90 ℃;

preferably, the refrigeration temperature in the step 2 and the step 3 is 3-10 ℃.

Further, when the ophthalmic formulation is selected from eye drops, the eye drops include: 0.05-2% of ginkgolide, 1.5-6% of solubilizer, 0.1-2% of viscosity regulator, 0.1-7.0% of pH regulator, 0.1-2.0% of osmotic pressure regulator, 0.01-0.5% of bacteriostatic agent and a proper amount of water for injection, wherein the sum of the percentage contents of all the components is one hundred percent.

Furthermore, the percentage content of the ginkgolide is preferably 0.1-1%.

Specifically, the solubilizer is one or a mixture of hydroxypropyl- β -cyclodextrin, β -cyclodextrin and methyl cyclodextrin, wherein hydroxypropyl- β -cyclodextrin is preferred.

Further, the viscosity regulator is one or a mixture of more than two of sodium hyaluronate, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, povidone, polyethylene glycol, dextran, poloxamer, carbomer and glycerol; poloxamer and povidone are preferred.

The invention also provides a preparation method of the ginkgo diterpene lactone eye drops, which comprises the following steps:

step 1: dissolving solubilizer and pH regulator in water for injection, adding ginkgolide, and heating to dissolve;

step 2: adding a viscosity regulator, a bacteriostatic agent and an osmotic pressure regulator into the solution obtained in the step (1), and dissolving; cooling to room temperature, adding water for injection to full dose, and filtering with microporous membrane to obtain eye drop containing ginkgolide.

Preferably, the heating temperature in the step 1 is 75-85 ℃.

In the preparation, the pH regulator is one or a mixture of hydrochloric acid, sodium hydroxide, boric acid, borax, sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, citric acid and sodium citrate; among them, boric acid and borax are preferable.

Specifically, the osmotic pressure regulator is one or a mixture of more than two of sodium chloride, glucose, potassium chloride, magnesium chloride, calcium chloride, sodium lactate, bicarbonate, sodium phosphate, mannitol or boric acid; among them, sodium chloride and bicarbonate are preferable.

Further, the bacteriostatic agent is one or a mixture of methyl hydroxybenzoate, ethylparaben, propyl hydroxybenzoate, butyl hydroxybenzoate, benzalkonium chloride, benzalkonium bromide, chlorhexidine acetate, phenylmercuric nitrate, thimerosal, and chlorobutanol; among them, benzalkonium bromide is preferred.

Specifically, the pH value of the ginkgo diterpene lactone ophthalmic preparation is 6.0-8.5; wherein the preferable pH value is 6.5-7.5.

The invention also provides application of the ginkgolide in preparing medicaments for treating retrobulbar neuritis, retinitis or relieving visual fatigue and the like.

The ginkgo diterpene lactone provided by the invention has the effects of expanding blood vessels, increasing blood flow, repairing and protecting optic nerves, can be applied to treating optic nerve injury, retrobulbar neuritis and retinitis, and can also be applied to relieving visual fatigue.

The eye preparation has the specific viscosity of the viscosity regulator, and has special significance for eye drops, can reduce irritation and increase the residence time of a medicine solution in eyes.

The temperature-sensitive ophthalmic in-situ gel has the advantages of both liquid and gel preparations, is liquid at the storage temperature and is beneficial to storage and administration; can rapidly form semisolid gel in eyes, adhere to the surfaces of cornea and conjunctival sac, prolong the retention time, delay the release of the medicine, thereby improving the bioavailability. Therefore, the temperature-sensitive in-situ gel can be used as an excellent carrier of an eye preparation, and provides a high-efficiency administration mode for eye medication.

Detailed Description

The ginkgolide can be obtained by purchasing a commercially available product or prepared by separating and purifying by the existing method, and the ginkgolide conforms to the structure of a corresponding reference substance by inspection, and the purity of the ginkgolide is over 90 percent by HPLC (high performance liquid chromatography) detection. The ginkgolide composition can be prepared by combining corresponding monomer compounds. In the examples, the bilobalide K, bilobalide B and bilobalide composition (bilobalide A, B, K) were prepared by Jiangsu Kangyuan pharmaceutical industry GmbH.

It should be noted that, if the specific conditions are not specified, the procedures are carried out according to the conventional conditions or the conditions recommended by the manufacturer, and the raw materials or auxiliary materials used, and the reagents or equipment used are not specified by the manufacturer, and are conventional products commercially available. All percentages, ratios, proportions, or parts are by weight unless otherwise specified.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention.

The present invention is described in detail below with reference to specific test examples/embodiments, which are provided only to help understanding the technical solutions provided by the present invention, but not to limit the protection scope of the present invention; the invention can be implemented in a number of different ways, which are defined and covered by the claims.

Test example 1 comparison of pH regulators

The bilobalide composition (comprising bilobalide A, B, K in the amount of bilobalide A and bilobalide B and bilobalide K in the ratio of 36 to 60 to 3.6(w/w)), disodium hydrogen phosphate and borax are respectively weighed, added with water for injection, heated and stirred at 80 ℃ for dissolution, and the solution properties are observed, and the results are shown in table 1.

TABLE 1 comparison of pH adjusting Agents

The result shows that the borax and the boric acid are preferably used as the pH regulator, because the borax has better dissolving assisting effect on the ginkgolides under the condition of the same dosage of the pH regulator.

Experimental example 2 selection of proportion of temperature sensitive gel Poloxamer in prescription

1. Preparation of ginkgolide temperature-sensitive ophthalmic gel (examples 1 to 7)

The formula composition is shown in table 2, the balance is water for injection, and the ginkgolides in the examples 1 to 7 are ginkgolide compositions comprising ginkgolide A, B, K, the dosage ratio is ginkgolide A: ginkgolide B: bilobalide K ═ 36: 60: 3.6 (w/w).

TABLE 2 ginkgolide temperature sensitive ophthalmic gel formulations (by weight percent)

Examples 1 to 7 preparation methods: weighing bilobalide composition and borax in a prescription amount, adding water for injection, heating and stirring at 70 ℃ for dissolving; filtering, cooling the filtrate in a refrigerator at 4 ℃, slowly adding poloxamer 407 and poloxamer 188 under magnetic stirring to wet and disperse the filtrate, placing the filtrate in the refrigerator at 4 ℃ for more than 24 hours, fully swelling to obtain a clear and uniformly dispersed solution 1, dissolving borax in an equal amount of water for injection, slowly adding poloxamer P407 and poloxamer 188 under magnetic stirring to wet and disperse the solution, placing the solution in the refrigerator at 4 ℃ for more than 24 hours, fully swelling to obtain a clear and uniformly dispersed solution 2, equivalently combining the solution 1 and the solution 2, uniformly mixing, filtering with a 0.22 mu m microfiltration membrane, and subpackaging to obtain the finished product. 2. Measuring bilobalide temperature sensitive ophthalmic gel phase transition temperature, pH value and artificial tear diluted phase transition temperature

The method for measuring the gelling temperature comprises the following steps: respectively taking the ginkgolide temperature-sensitive ophthalmic gel prepared in the examples 1-7, filling 4ml of temperature-sensitive gel liquid and a magnetic stirrer into a penicillin bottle, punching the middle of a rubber plug, inserting a precision thermometer with the precision of 0.1 ℃, completely immersing a mercury ball of the thermometer into the gel liquid, putting the penicillin bottle into a low-temperature (10 ℃) water bath, and adjusting the rotating speed to 300 r.min^-1And keeping the water bath to slowly raise the temperature, wherein the temperature rise rate is about 1 ℃ every 2min, continuously inclining the penicillin bottle by 60 degrees, and taking the temperature when the magnetic stirring bar does not rotate and the solution does not flow as the gelling temperature. Each sample was assayed in 3 replicates and averaged.

6.78g of sodium chloride, 1.38g of potassium chloride, 2.18g of sodium bicarbonate and 0.084g of calcium chloride dihydrate are weighed and dissolved by adding ultrapure water to 1000ml, and the simulated tear is prepared. The ginkgolide temperature-sensitive ophthalmic gel prepared in the example 1-7 is respectively taken, fully mixed with simulated tears according to the volume ratio of 40:7, and the phase transition temperature of the ginkgolide temperature-sensitive ophthalmic gel diluted by the tears, namely the phase transition temperature under physiological conditions, is measured according to the method.

The pH of the ginkgolide temperature sensitive ophthalmic gel prepared in example 1-7 was measured using a Sartorius pH meter (PB-10).

TABLE 3 phase transition temperature (n-3) before and after tear dilution and pH of the gel solution for ginkgolide temperature sensitive ophthalmic gels

The results show that: the ideal temperature sensitive ophthalmic gel should be liquid at room temperature (not less than 25 ℃); quickly forming gel in the conjunctival sac (less than or equal to 34 ℃), namely diluting the tears; the ginkgolide temperature-sensitive ophthalmic gel prepared in the embodiments 1 to 7 has the advantages that the embodiments 4, 5 and 6 meet the requirements, and the preparation formula of the embodiment 4 is preferably selected from the consideration of reducing the using amount of auxiliary materials.

Experimental example 3 comparison of prescription preparations with different proportions of raw materials and raw and auxiliary materials

1. Preparation of ginkgolide temperature-sensitive ophthalmic gel (examples 8 to 14)

The formula composition is shown in table 4, and the balance is water for injection, wherein in the examples 8-12, the composition of the ginkgolide is a ginkgolide composition comprising ginkgolide A, B, K, and the dosage ratio is ginkgolide A: ginkgolide B: bilobalide K ═ 36: 60: 3.6 (w/w); the bilobalide in example 13 is bilobalide B; in example 14 the ginkgolide is ginkgolide K.

TABLE 4 ginkgolide temperature-sensitive ophthalmic gel formulations (by weight percent)

Examples 8 to 10 (same as examples 1 to 7).

Examples 11 to 12 preparation methods: weighing bilobalide composition and borax in a prescription amount, adding water for injection, heating and stirring at 80 ℃ for dissolving; filtering, cooling the filtrate in a refrigerator at 10 ℃, slowly adding poloxamer P407 and poloxamer 188 under magnetic stirring to wet and disperse the poloxamer, placing the filtrate in the refrigerator at 10 ℃ for more than 24 hours, fully swelling to obtain a clear and uniformly dispersed solution 1, dissolving borax in an equal amount of water for injection, slowly adding poloxamer P407 and poloxamer 188 under magnetic stirring to wet and disperse the poloxamer, placing the solution in the refrigerator at 10 ℃ for more than 24 hours, fully swelling to obtain a clear and uniformly dispersed solution 2, equivalently combining the solution 1 and the solution 2, uniformly mixing, filtering with a 0.22 mu m microfiltration membrane, and subpackaging to obtain the compound preparation.

Examples 13 to 14 preparation methods: weighing bilobalide and borax in a prescription amount, adding water for injection, heating and stirring at 90 ℃ for dissolving; filtering, cooling the filtrate in a refrigerator at 6 ℃, slowly adding poloxamer P407 and poloxamer 188 under magnetic stirring to wet and disperse the poloxamer, placing the filtrate in the refrigerator at 6 ℃ for more than 24 hours, fully swelling to obtain a clear and uniformly dispersed solution 1, dissolving borax in an equal amount of water for injection, slowly adding poloxamer P407 and poloxamer 188 under magnetic stirring to wet and disperse the poloxamer, placing the solution in the refrigerator at 6 ℃ for more than 24 hours, fully swelling to obtain a clear and uniformly dispersed solution 2, equivalently combining the solution 1 and the solution 2, uniformly mixing, filtering with a 0.22 mu m microfiltration membrane, and subpackaging to obtain the compound preparation. 2. Measuring the temperature and pH of the ginkgolide temperature sensitive ophthalmic gel and the phase transition temperature of the diluted artificial tear (same method as in test example 2)

TABLE 5 phase transition temperature (n-3) before and after tear dilution and pH of the gel solution for ginkgolide temperature sensitive ophthalmic gels

The results show that: the determination results of examples 8 to 14 show that different main components (bilobalide composition, bilobalide B and bilobalide K), different proportions of the main components and different proportions of the auxiliary materials in the formula have less influence on the phase transition temperature of the gel and the phase transition temperature of the diluted artificial tear, and the gel property is relatively stable.

Test example 4 preparation of eye drops containing ginkgolide (example 15)

The ginkgolide composition in the formula comprises ginkgolide A, B, K, and the dosage ratio is that ginkgolide A: ginkgolide B: bilobalide K ═ 36: 60: 3.6 (w/w).

TABLE 6 bilobalide eye drops prescription (by mass percent)

The preparation method comprises weighing HP- β -CD and borax according to the prescription amount, dissolving in 800mL injection water, heating in water bath to 85 deg.C, adding 10.0g bilobalide K, stirring, sequentially adding other adjuvants after completely dissolving, stirring to completely dissolve, adding injection water to 1000mL, filtering with 0.22 μm microporous membrane, and subpackaging in sealed plastic bottles according to dose.

Test example 5

The ginkgolide ophthalmic solutions prepared in examples 4, 13, 14 and 15 and the ginkgolide temperature-sensitive ophthalmic gel preparation prepared in example 5 were subjected to long-term tests according to the requirements related to stability in "Chinese pharmacopoeia" 2015 edition, and the results are shown in Table 7.

TABLE 7 Long-term test results of ophthalmic ginkgolide formulations

And (4) conclusion: the eye drop samples prepared in example 15 and the test items after being placed under the conditions of 25 ℃ +/-2 ℃ and RH 60% +/-10% (long-term test) for 12 months all meet the requirements, which indicates that the preparation is stable and the preparation method is reliable.

Test example 6 protective Effect of ophthalmic preparation of Ginkgo diterpene lactone on retinal ganglion cells after NMDA-induced excitatory injury of rat retina

1. Test materials

SD rats, male and female half, purchased in the center of Qinglong mountain experimental animals.

Tobramycin dexamethasone eye drops (folk medicine).

2. Test methods and results

80 healthy SD rats, half male and half female, weighing 180-. The cornea is transparent, the blood vessels of the iris are clear, the pupil is of the same size and the like, and the light reflection is sensitive. Experimental animals were divided into a blank group (blank 1 was a blank eye drop preparation group, blank 2 was a temperature-sensitive ophthalmic gel blank preparation group), a model group, a positive control tobramycin dexamethasone eye drop group, example 4 (a ginkgolide composition temperature-sensitive ophthalmic gel), example 13 (a ginkgolide B temperature-sensitive ophthalmic gel), example 14 (a ginkgolide K temperature-sensitive ophthalmic gel), and example 15 (a ginkgolide composition eye drop), each group contained 10 animals, NMDA (N-methyl-D-aspartic acid) was dissolved in Phosphate Buffer Solution (PBS) to 80mmol/L, and 2uL of the resulting solution was administered into the vitreous of rats of each group except the blank group to establish a rat retinal injury model. After the molding is finished, the rats in each group are applied with the medicine for two eyes, different eye drops are respectively applied for 8 times/d, 1 drop/time and 3 days continuously, the model group is applied with a blank solvent, and the tobramycin dexamethasone eye drops group is applied with 1.6 mg/kg; 16mg/kg of each drug was administered to each of the example 4 group, example 13 group, example 14 group and example 15 group, and after the administration on day 3, 100mg/kg of sodium pentobarbital was immediately intraperitoneally injected into rats in each group, general anesthesia was performed, and the eyeballs were removed. With 25% glutaraldehyde: 10% neutral buffered formalin 1: the mixed solution of 9 fixes the removed eyeball. After embedding the fixed eyeball with paraffin, the retina was sliced (3um thick) and stained with hematoxylin-eosin. The retinal slice of each eye was made into 8 slices at intervals of 45um so as to include optic nerve papilla. 3 sections were arbitrarily selected from the 8 sections, and a photograph of the retina at 700 to 900um from about 1.25mm from the optic nerve head was taken of the selected sections, and the number of cells in the retinal ganglion cell layer (GCL cell number) (average value) was calculated. From the number of GCL cells obtained, according to the formula: the GCL cell number reduction rate (%) was (number of cells in GCL administered group-number of GCL cells in blank group)/number of cells in GCL blank group) × 100%, and the cell number reduction rate of each administered group was calculated.

TABLE 8 Effect of ophthalmic Ginkgo diterpene lactone formulations on the number of GCL cells after injury of the optic nerve in rats

Note: p <0.05, P <0.01 compared to model group.

The results show that compared with the optic nerve injury model group, the ginkgolide temperature-sensitive ophthalmic preparation (examples 4, 13 and 14) and the eye drops (example 15) can effectively reduce the reduction of the number of retinal ganglion cells, and the improvement effect of the eye drops is better than that of the positive control tobramycin dexamethasone eye drops.

Experimental example 7 therapeutic action of ophthalmic preparation of ginkgolide on experimental optic neuritis in mouse induced by MOG35-55

1. Test materials

SPF grade C57BL/6 mice, half male and half female, were purchased from the center of Qinglong mountain laboratory animals.

Prednisolone acetate eye drops (Bailite).

2. Test methods and results

80C 57BL/6 mice weighing 22-28g were randomly divided into 8 groups, blank groups (blank 1 was eye drop blank preparation group, blank 2 was temperature sensitive eye gel blank preparation group), model group, positive control prednisolone acetate eye drop group, example 4 group, example 13 group, example 14 group, and example 15 group, each group containing 10 mice, and 800ug MOG was added_35-55(myelin oligodendrocyte glycoprotein polypeptide) is dissolved in 200ul PBS and then mixed with 200ul CFA, tubercle bacillus is added to make the final concentration of 2mg/ml, and the mixture is stirred for 5 to 10 minutes under ice bath by an electronic stirring rod to achieve full emulsification. Finally, whether the emulsification is complete or not needs to be identified by dropping a drop of the emulsion into water, wherein the emulsification is complete if the emulsion is not dispersed and floats on the water surface, and the emulsification is not sufficient if the emulsion is dispersed immediately. The operation is carried out under the conditions of light protection, low temperature and sterility as far as possible. The mixed emulsion is injected into the abdominal wall of the model group and the administration group at four points, and the mixed emulsion is directly injected into the blank group at four points through the abdominal wall of the both sides after 200ul PBS liquid and 200ul CFA are fully emulsified. Each group was injected with 100ul of pertussis solution (containing viable bacteria 2X 10) into the abdominal cavity of each mouse 24 hours before and 24 hours after immunization⁹One), establish MOG_35-55Mouse-induced experimental optic neuritis model. After molding, different eye drops were dropped for 8 times/d, 1 drop/time, and 7 consecutive days, and in addition to blank solvent administration to the model groups, 128mg/kg of each drug was administered to each prednisolone acetate eye drop group, and 128mg/kg of each drug was administered to each drug administration group, example 4 group, example 13 group, example 14 group, and example 15 group, respectively. The optic neuritis is diagnosed by using f-VEP, and the detection result of each eye of a blank group of mice is taken to establish the normal reference value of the latency and the amplitude of each wave of the f-VEP of the experimental mice. F-VEP examination was performed on each eye of each group of mice after the administration on day 7 after animal immunization, and VEP N1 wave and P wave were recordedLatency and P-wave amplitude as indices for evaluation of optic neuritis.

TABLE 9 Effect of ophthalmic ginkgolide formulations on the f-VEP index of optic neuritis in mice

Note: p <0.05, P <0.01 compared to model group.

The results show that compared with the optic neuritis model group, the ginkgolide temperature-sensitive ophthalmic preparation (examples 4, 13 and 14) and the eye drops (example 15) can effectively improve the latency change of the N1 wave and the P-predose of the f-VEP of the mice and the amplitude change of the P wave, and the improvement effect of the ginkgolide temperature-sensitive ophthalmic preparation is better than that of the positive control prednisolone acetate eye drops.

Test example 8 therapeutic Effect of ginkgolide ophthalmic preparation on viral chorioretinitis in mice 1. test Material

BALB/C mice, male and female, were purchased from the center of Qinglong mountain laboratory animals.

Prednisolone acetate eye drops (Bailite).

2. Test methods and results

128 BALB/C mice were randomly divided into 8 groups, a blank group (blank 1 is an eye drop blank preparation group, blank 2 is a temperature sensitive ophthalmic gel blank preparation group), a model group, a positive control prednisolone acetate eye drop group, an example 4 group, an example 13 group, an example 14 group, an example 15 group, and 16 mice in each group, and after the model group and the administration group were anesthetized by intraperitoneal injection of 2mg ketamine hydrochloride and 400ng mepivacaine hydrochloride, 1 × 10 mice were anesthetized under an operating microscope⁵A spot forming unit (PFU) herpes simplex virus-I type is injected into the right anterior chamber of the right eye of a BALB/C mouse to establish a mouse viral chorioretinitis model. After 1 day after herpes simplex virus-I infection, mice of each administration group are respectively dosed with different eye drops for 8 times/d, 1 drop/time and 7 days continuously, except that blank solvents are dosed to model groups, prednisolone acetate eye drops of each administration group are dosed to 128mg/kg, and drugs of each administration group are dosed to 128mg/kg respectively for example 4 groups, example 13 groups, example 14 groups and example 15 groups. SmallMice were routinely housed after infection, and eye changes were observed daily with a surgical microscope after infection and scored for symptoms. The inflammatory reaction of the anterior chamber, dilation of the vessels in the pupil and iris, formation of cataract and vitreous opacity with a white fundus reflection are scored as 1-4 points. Observed by two observers separately. And recording the number of mice in each group that exhibited inflammatory response in the non-injected eye according to the formula: incidence (%) non-injected mice exhibiting inflammatory response/total mice in each group x 100% the incidence of retinitis in each group was calculated

TABLE 10 Effect of Ginkgo diterpene lactone ophthalmic formulations on the incidence and clinical scores of retinitis in mice

Note: p <0.05, P <0.01 compared to model group.

The results show that compared with the retinitis model group, the ginkgolide temperature-sensitive ophthalmic preparation (examples 4, 13 and 14) and the eye drops (example 15) can effectively improve the morbidity and symptom score of mice, and the improvement effect of the ginkgolide temperature-sensitive ophthalmic preparation is better than that of the prednisolone acetate eye drops serving as a positive control.

The above description of the embodiments is only intended to facilitate the understanding of the method of the invention and its core idea. It should be noted that, for those skilled in the art, it is possible to make various improvements and modifications to the present invention without departing from the principle of the present invention, and those improvements and modifications also fall within the scope of the claims of the present invention.

Claims (10)

1. Application of ginkgolide in preparing medicine for treating optic nerve injury is provided.

2. The use of claim 1, wherein the ginkgolide is ginkgolide A, B or K, or any combination thereof, or an extract comprising ginkgolide A, B or K.

3. The use according to claim 1, wherein the ginkgolide is selected from the group consisting of compositions comprising ginkgolides A, B and K, wherein the ratio of ginkgolide A: ginkgolide B: and (3) bilobalide K is (20-40): (50-75): (0.2-5.0).

4. Use according to claims 1-3, wherein the ophthalmic formulation comprises eye drops, ophthalmic gels, eye ointments, ophthalmic liposomes, ophthalmic microemulsions.

5. An ophthalmic formulation of ginkgo diterpene lactones for optic nerve damage, wherein when said ophthalmic formulation is selected from the group consisting of temperature sensitive ophthalmic gels, the gels comprise: 0.05-2% of ginkgolide, 10.5-40% of temperature sensitive gel, 0.1-4.0% of pH regulator, 0.05-2% of bacteriostatic agent and 0.1-0.9% of osmotic pressure regulator.

6. The ophthalmic formulation according to claim 5, characterized in that the temperature sensitive gel is selected from the group consisting of 24-28% poloxamer 407 and 2-6% poloxamer 188 in combination.

7. A process for the preparation of an ophthalmic formulation as claimed in claim 5 or 6, comprising the steps of:

step 1: weighing the ginkgo diterpene lactone and the pH regulator with the prescription amount, adding water for injection, and heating for dissolving; filtering, and cooling the filtrate;

step 2: slowly adding the filtrate obtained in the step (1) into temperature-sensitive gel under magnetic stirring to wet and disperse the gel, refrigerating the gel in a refrigerator, and fully swelling the gel to obtain a solution 1;

and step 3: dissolving pH regulator, bacteriostatic agent and osmotic pressure regulator in water for injection, adding temperature sensitive gel, moistening and dispersing, refrigerating in refrigerator, and swelling to obtain solution 2;

and 4, step 4: and (3) combining the solution 1 in the step (2) and the solution 2 in the step (3), uniformly mixing, filtering by using a 0.22 mu m microporous filter membrane, and subpackaging to obtain the composition.

Preferably, the heating temperature in the step 1 is 70-90 ℃;

preferably, the refrigeration temperature in the step 2 and the step 3 is 3-10 ℃.

8. An ophthalmic preparation of ginkgo diterpene lactones for optic nerve damage, wherein when said ophthalmic preparation is selected from the group consisting of eye drops, the eye drops comprise: 0.05-2% of ginkgolide, 1.5-6% of solubilizer, 0.1-2% of viscosity regulator, 0.1-7.0% of pH regulator, 0.1-2.0% of osmotic pressure regulator and 0.01-0.5% of bacteriostatic agent.

9. A process for the preparation of an ophthalmic formulation as claimed in claim 8, comprising the steps of:

step 1: dissolving solubilizer and pH regulator in water for injection, adding ginkgolide, and heating to dissolve;

step 2: adding a viscosity regulator, a bacteriostatic agent and an osmotic pressure regulator into the solution obtained in the step (1), and dissolving; cooling to room temperature, adding water for injection to full dose, and filtering with microporous membrane to obtain eye drop containing ginkgolide.

Preferably, the heating temperature in the step 1 is 75-85 ℃.

10. Application of ginkgolide in preparing medicine for treating retrobulbar neuritis, retinitis or relieving asthenopia is provided.