Eye drops containing L-ergothioneine and preparation method thereof

Abstract

The invention discloses eye drops containing L-ergothioneine and a preparation method of the eye drops. The eye drops are prepared from the following components in parts by weight: 0.0015 to 15 parts of L-ergothioneine, 0.001 to 0.01 part of a bacteriostatic agent, 0.05 to 15 parts of a buffering agent and the balance of normal saline, and the pH value of the eye drops is 5.0 to 7.0. Experiments show that L-ergothioneine can prevent and reverse denaturation of crystalline lens protein, the prepared eye drops containing L-ergothioneine can prevent and reduce opacity of crystalline lens protein, and the prevention and treatment effects on cataract are better than those of existing eye drops.

Description

Technical Field

[0001] The invention relates to the field of pharmaceutical preparations, and in particular to an eye drop containing L-ergothioneine and a preparation method thereof. Background Art

[0002] Cataract is a general term for a variety of diseases characterized by partial or complete turbidity of the lens structure in the eye due to various reasons, resulting in visual impairment. Cataract has always been the eye disease with the highest blindness rate in the world, accounting for more than 45% of the world's blind people, and the incidence of cataract is very high. With the increasing aging of the world, the trend of increasing cataract incidence will continue to intensify.

[0003] Up to now, cataract extraction combined with intraocular lens implantation is still the only effective treatment for cataracts. However, surgical treatment is not suitable for all types of cataracts, and although cataract surgery is relatively mature, complications are still inevitable, leading to decreased vision or even blindness. For example, shallow anterior chamber, epithelial implantation in the anterior chamber, persistent corneal edema, anterior chamber and vitreous hemorrhage, uveitis, suppurative endophthalmitis, glaucoma, abnormal position of intraocular lens, posterior capsule opacity, etc. These complications are also the main reasons why vision cannot return to normal after surgery. Surgical treatment alone cannot solve the increasingly serious problem of cataracts. Early detection and early treatment will become one of the main directions of cataract prevention and treatment in the future.

[0004] Although eye drops for cataracts are already available on the market, no drug has been proven to be effective in preventing and treating cataracts. Given the shortcomings of existing cataract treatments, there is an urgent need for eye drops that can effectively prevent and treat cataracts.

[0005] L-ergothioneine is a water-soluble amino acid found primarily in mushrooms. L-ergothioneine cannot be synthesized by the human body (or other vertebrates) and can only be supplemented through diet. It is a naturally occurring thiol / thione derivative of the essential amino acid histidine, with a molecular formula of C9H 15 N3O2S. So far, there is no precedent for using L-ergothioneine to treat cataracts. Summary of the invention

[0006] The invention aims to provide an eye drop containing L-ergothioneine and a preparation method thereof in view of the current situation that the efficacy of existing cataract drugs is poor.

[0007] To achieve the above object, the present invention provides an eye drop comprising L-ergothioneine, comprising the following components:

[0008] 0.0015-15 parts by weight of L-ergothioneine, 0.001-0.01 parts by weight of antibacterial agent, 0.05-15 parts by weight of buffer, and 100 parts by weight of physiological saline, wherein the pH value of the eye drops is 5.0-7.0.

[0009] Furthermore, the L-ergothioneine is 2 parts by weight.

[0010] Furthermore, the pH of the eye drops is 6.0.

[0011] Furthermore, the antibacterial agent is composed of one or more of thimerosal, benzalkonium chloride, and parabens mixed in any proportion.

[0012] Furthermore, the buffer is any one of a buffer pair of sodium dihydrogen phosphate monohydrate and disodium hydrogen phosphate, a buffer pair of boric acid and borax, and a buffer pair of citric acid and disodium hydrogen phosphate.

[0013] To achieve the above object, the present invention also provides a method for preparing the above-mentioned eye drops comprising L-ergothioneine, comprising the steps of:

[0014] (1) dissolving the antibacterial agent in physiological saline and filtering with a 0.22 μm microporous filter membrane to obtain solution I;

[0015] (2) Sterile L-ergothioneine was added to solution I, and ultrasonic stirring was performed for 30 min to obtain solution II;

[0016] (3) adding physiological saline to dilute solution II to 100 parts by weight to obtain solution III;

[0017] (4) adjusting the pH value of solution III to 5.0-7.0 with a buffer to obtain solution IV;

[0018] (5) sterilizing solution IV at 121-134° C., 102.9-137.2 kPa for 15-30 min to obtain eye drops containing L-ergothioneine.

[0019] To achieve the above object, the present invention also provides an application of the above-mentioned eye drops containing L-ergothioneine in the preparation of a drug for preventing and treating cataracts.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] (1) The eye drops containing L-ergothioneine of the present invention have a significant improvement effect on the H2O2-induced human lens epithelial cell oxidative damage model, and its improvement effect is reflected in protecting the activity of human lens epithelial cells;

[0022] (2) The eye drops containing L-ergothioneine of the present invention have a significant improvement effect on the lens organoid cataract model, and its improvement effect is reflected in slowing down the turbidity progression of the age-related cataract lens organoid model;

[0023] (3) the eye drops comprising L-ergothioneine of the present invention have a significant improvement effect on UVB-induced mouse cataract model, and its improvement effect is embodied in the occurrence of prevention of mouse lens anterior subcapsular opacity, and the early stage anterior subcapsular opacity that has occurred can be reversed at the same time, and the later stage progress of anterior subcapsular opacity can be alleviated to a certain extent;

[0024] (4) according to the results of the present invention, the eye drops comprising L-ergothioneine have obvious prevention and treatment of cataract, and there is obvious improvement for the cataract model in vitro, organoid and in vivo, and the improvement is embodied in protecting human lens epithelial cell activity, slowing down the turbidity progress of lens organoid, preventing and alleviating mouse lens anterior subcapsular turbidity. The results of the present invention show that the eye drops comprising L-ergothioneine will have good clinical efficacy for age-related cataract, can play the effect of prevention and early reversal, and control the late progress of cataract to a certain extent;

[0025] (5) The eye drops containing L-ergothioneine of the present invention have high stability and remarkable drug efficacy;

[0026] (6) The eye drops comprising L-ergothioneine of the present invention are safe and have no side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0028] Figure 1 A schematic diagram of the cell activity results of the eye drops containing L-ergothioneine prepared by the present invention in a H2O2-induced human lens epithelial cell oxidative damage model;

[0029] Figure 2 A schematic diagram of the slit lamp results of the eye drops containing L-ergothioneine prepared by the present invention for the purpose of preventing cataracts in a UVB-induced mouse cataract model;

[0030] Figure 3 A schematic diagram of the slit lamp results of the eye drops containing L-ergothioneine prepared by the present invention for the purpose of reversing early cataracts in a UVB-induced mouse cataract model;

[0031] Figure 4 A schematic diagram of the slit lamp results of using the eye drops containing L-ergothioneine prepared for the present invention for the purpose of treating cataracts in a UVB-induced mouse cataract model. DETAILED DESCRIPTION

[0032] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only partial embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0033] The preparation method of the eye drops comprising L-ergothioneine of the present invention, the specific steps are as follows:

[0034] (1) dissolving 0.001-0.01 parts by weight of an antibacterial agent in 70 parts by weight of physiological saline (0.9% sodium chloride injection), and filtering and sterilizing with a 0.22 μm microporous filter membrane to obtain a solution I; wherein the antibacterial agent is composed of one or more of thimerosal, benzalkonium chloride, and parabens mixed in any proportion;

[0035] (2) adding 0.0015-15.0 parts by weight of sterile L-ergothioneine to solution I, and stirring with ultrasonic waves for 30 min to obtain solution II;

[0036] (3) adding physiological saline (0.9% sodium chloride injection) to dilute solution II to 100 parts by weight to obtain solution III;

[0037] (4) adjusting the pH value of solution III to 5.0-7.0 with 0.05-15 parts by weight of a buffer to obtain solution IV; wherein the buffer is any one of a buffer pair of sodium dihydrogen phosphate monohydrate and sodium hydrogen phosphate, a buffer pair of boric acid and borax, and a buffer pair of citric acid and sodium hydrogen phosphate;

[0038] (5) sterilizing solution IV at 121-134° C. high temperature and 102.9-137.2 kPa high pressure for 15-30 min to obtain an eye drop containing L-ergothioneine.

[0039] Preferably, solution IV is sterilized at a temperature of 121.3° C. and a pressure of 103.4 kPa for 20 min to obtain an eye drop comprising L-ergothioneine.

[0040] (6) detecting the L-ergothioneine content, osmotic pressure, and pH value of the prepared eye drops, and packaging the eye drops into 0.4-10 mL portions in a sterile environment.

[0041] It should be understood that the characteristic of cataracts is that they are caused by the denaturation of lens proteins and the occurrence of turbidity due to a variety of reasons such as aging, heredity, local nutritional disorders, immune and metabolic abnormalities, trauma, poisoning, radiation, etc.

[0042] Embodiment 1: the preparation of L-ergothioneine eye drops

[0043] Weigh the following components in parts by weight: 0.2 part of L-ergothioneine, 0.003 part of antibacterial agent (benzalkonium chloride), 0.1 part of buffer (citric acid), 0.3 part of buffer (disodium hydrogen phosphate), 99.397 parts of normal saline, and add buffer to adjust pH to 5.0; Prepare the eye drops comprising L-ergothioneine according to the preparation method in the above-mentioned specific embodiment.

[0044] Embodiment 2: the preparation of L-ergothioneine eye drops

[0045] Weigh the following components in parts by weight: 2 parts of L-ergothioneine, 0.005 part of antibacterial agent (paraben), 2.4 parts of buffer (sodium dihydrogen phosphate monohydrate), 0.3 part of buffer (disodium hydrogen phosphate), 95.295 parts of normal saline, add buffer and adjust pH to 6.0; Prepare the eye drops comprising L-ergothioneine according to the preparation method in the above-mentioned specific embodiment.

[0046] Embodiment 3: the preparation of L-ergothioneine eye drops

[0047] Weigh the following components in parts by weight: 12 parts of L-ergothioneine, 0.001 part of antibacterial agent (thimerosal), 9.9 parts of buffer (boric acid), 1.1 parts of buffer (borax), 76.999 parts of normal saline, and add buffer to adjust pH to 7.0; Prepare the eye drops comprising L-ergothioneine according to the preparation method in the above-mentioned specific embodiment.

[0048] Example 4: Application of L-ergothioneine eye drops in H2O2-induced oxidative damage model of human lens epithelial cells

[0049] Human lens epithelial cells are used in the present embodiment, divided into blank control group, model group and experimental group, and cultured to cell density and then tested after reaching 80%. The eye drops comprising L-ergothioneine prepared by experimental group cell embodiment 1 are incubated for 1-72h and pre-treated, and then three groups of cells (blank control group, model group and experimental group) are replaced with 600 μM H2O2 culture medium after all cleaning with Hanks solution and incubated for 24h. Finally, the cell survival rate of each group is detected by cell proliferation / toxicity detection kit (Cell Counting Kit-8, CCK8).

[0050] Figure 1It is shown that in a H2O2-induced human lens epithelial cell oxidative damage model, the eye drops containing L-ergothioneine of the present invention have a protective effect on human lens epithelial cells, and pretreatment with the eye drops for 24h, 36h and 72h can significantly improve cell activity.

[0051] Example 5: Application of L-ergothioneine eye drops in lens organoid cataract model

[0052] In this example, the “fried egg” method was used to induce a lens organoid model, which was divided into a model group (n=3), a positive control group (n=3) and an experimental group (n=3). The lens organoids in the positive control group were incubated with commercially available lanosterol, and the lens organoids in the experimental group were incubated with the eye drops containing L-ergothioneine prepared in Example 1. The three groups (model group, positive control group and experimental group) of organoids were cultured for a long time to simulate the lens opacity process of age-related cataracts, and the turbidity of the lens organoids in each group was compared by microscopic observation.

[0053] The experimental results show that the eye drops containing L-ergothioneine of the present invention can slow down the progression of turbidity in the age-related cataract lens organoid model.

[0054] Example 6: Preventive effect of L-ergothioneine eye drops on UVB-induced cataract model in mice

[0055] Thirty-six C57BL / 6 male mice weighing 16-18 g and aged 6 weeks were used in this example. Before the start of the study, each eye of each mouse was examined with a slit lamp through a dilated pupil (1% atropine eye drops) to exclude any pre-existing abnormalities.

[0056] The mice were randomly divided into 4 groups: (1) blank control group (n=9); (2) model group (n=9); (3) positive control group (n=9); (4) prevention group (n=9).

[0057] From the first day, except for the blank control group, all mice in the other groups were irradiated with an ultraviolet lamp with a wavelength of 310 nm (TL20w / 01RS, Philips, Germany) for 30 minutes at a distance of 15 cm after mydriasis (1% atropine eye drops) for 5 minutes, once a day for 12 consecutive days.

[0058] Wherein blank control group and model group are given PBS (phosphate buffered saline) eye drops from (-1) day, positive control group is given commercially available Sha Pu Ai Si eye drops eye drops from (-1) day, and prevention group is given the eye drops eye drops comprising L-ergothioneine prepared by embodiment 2 from (-1) day.Each mouse eyes are all received eye drops, and eye drop amount is 5uL / eye / time, and frequency is 3 times / day.

[0059] The lens opacities were observed with a slit lamp on days 0, 4, 8, 10, and 12.

[0060] Figure 2 It is shown that the eye drops containing L-ergothioneine of the present invention can effectively prevent the occurrence of cataracts in mice, and the effect is significantly better than the eye drops of the positive control group. Figure 2 In the figure, the first row is the blank control group, the second row is the model group, the third row is the positive control group, and the fourth row is the prevention group; the column names represent the time before the construction of the UVB-induced mouse cataract model (day 0) and the 4th, 8th, 10th, and 12th days of model construction.

[0061] Example 7: Early reversal effect of L-ergothioneine eye drops in UVB-induced cataract model in mice

[0062] Thirty-six C57BL / 6 male mice weighing 16-18 g and aged 6 weeks were used in this example. Before the start of the study, each eye of each mouse was examined with a slit lamp through a dilated pupil (1% atropine eye drops) to exclude any pre-existing abnormalities.

[0063] The mice were randomly divided into 4 groups: (1) blank control group (n=9); (2) model group (n=9); (3) positive control group (n=9); (4) early reversal group (n=9).

[0064] From the first day, except for the blank control group, all mice in the other groups were irradiated with an ultraviolet lamp with a wavelength of 310 nm (TL20w / 01RS, Philips, Germany) for 30 minutes at a distance of 15 cm after mydriasis (1% atropine eye drops) for 5 minutes, once a day for 12 consecutive days.

[0065] Wherein blank control group and model group were given PBS (phosphate buffered saline) eye drops from the 9th day, and positive control group was given commercially available Sha Pu Ai Si eye drops eye drops from the 9th day, and early reversal group was given the eye drops comprising L-ergothioneine prepared by embodiment 2 from the 9th day.Each mouse eyes were all received eye drops, and the eye drop amount was 5uL / eyes / time, and the frequency was 3 times / day.

[0066] The lens opacities were observed with a slit lamp on the 8th, 10th, 11th, and 12th days.

[0067] Figure 3 It is shown that the eye drops containing L-ergothioneine of the present invention can effectively reverse early cataract in mice, and the effect is significantly better than the eye drops of the positive control group. Figure 3In the figure, the first row is the blank control group, the second row is the model group, the third row is the positive control group, and the fourth row is the early reversal group; the column names represent the 8th, 10th, 11th, and 12th days of constructing the UVB-induced mouse cataract model.

[0068] Example 8: Therapeutic effect of L-ergothioneine eye drops on UVB-induced cataract model in mice

[0069] Thirty-six C57BL / 6 male mice weighing 16-18 g and aged 6 weeks were used in this example. Before the start of the study, each eye of each mouse was examined with a slit lamp through a dilated pupil (1% atropine eye drops) to exclude any pre-existing abnormalities.

[0070] The mice were randomly divided into 4 groups: (1) blank control group (n=9); (2) model group (n=9); (3) positive control group (n=9); (4) treatment group (n=9).

[0071] From the first day, except for the blank control group, all mice in the other groups were irradiated with an ultraviolet lamp with a wavelength of 310 nm (TL20w / 01RS, Philips, Germany) for 30 minutes at a distance of 15 cm after mydriasis (1% atropine eye drops) for 5 minutes, once a day for 24 consecutive days.

[0072] Wherein blank control group and model group are given PBS (phosphate buffered saline) eye drops from the 13th day, positive control group is given commercially available Sha Pu Ai Si eye drops eye drops from the 13th day, and treatment group is given the eye drops eye drops comprising L-ergothioneine prepared by embodiment 2 from the 13th day.Each mouse eyes are all received eye drops, and eye drop amount is 5uL / eye / time, and frequency is 3 times / day.

[0073] The lens opacities were observed with a slit lamp on days 12, 14, 16, 18, 20, 22, and 24.

[0074] Figure 4 It is shown that the eye drops containing L-ergothioneine of the present invention can alleviate the already formed mouse cataract to a certain extent, and effectively control the progression of cataract, and the overall therapeutic effect is significantly better than the eye drops of the positive control group. Figure 4 In the data, the first row is the blank control group, the second row is the model group, the third row is the positive control group, and the fourth row is the treatment group; the column names represent the 12th, 14th, 16th, 18th, 20th, 22nd, and 24th days of constructing the UVB-induced mouse cataract model.

[0075] Although the present invention has been described in detail above with general instructions and specific implementation schemes, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention are within the scope of protection claimed by the present invention.

Claims

1. A purpose of an eye drop comprising L-ergothioneine, for: (i) preparing a drug for preventing and treating cataract; and / or (ii) prevention and treatment of cataracts; and / or (iii) Non-therapeutic improvement of H2O2-induced oxidative damage in human lens epithelial cells in vitro; in, The eye drops containing L-ergothioneine are composed of the following components: 2-15 parts by weight of L-ergothioneine, 0.001-0.01 parts by weight of an antibacterial agent, 0.05-15 parts by weight of a buffer, and 100 parts by weight of physiological saline. The pH of the eye drops is 5.0-7.

0.

2. The use according to claim 1, characterized in that The L-ergothioneine is 2 parts by weight.

3. The use according to claim 1, characterized in that The pH of the eye drops is 6.

0.

4. The use according to claim 1, characterized in that The antibacterial agent is composed of one or more of thimerosal, benzalkonium chloride and p-hydroxybenzoic acid esters mixed in any proportion.

5. The use according to claim 1, characterized in that: The buffer is any one of a buffer pair of sodium dihydrogen phosphate monohydrate and disodium hydrogen phosphate, a buffer pair of boric acid and borax, and a buffer pair of citric acid and disodium hydrogen phosphate.

6. The use according to claim 1, characterized in that: The eye drops containing L-ergothioneine are composed of the following components: 2 parts by weight of L-ergothioneine, 0.005 parts by weight of p-hydroxybenzoate, 2.4 parts by weight of sodium dihydrogen phosphate monohydrate, 0.3 parts by weight of disodium hydrogen phosphate, and 100 parts by weight of normal saline, and the pH of the eye drops is 6.

0.

7. The use according to any one of claims 1 to 6, characterized in that The cataract is UVB-induced cataract.

8. The use according to any one of claims 1 to 6, characterized in that The cataract is a lens organoid cataract.

9. The use according to any one of claims 1 to 6, characterized in that The cataract is age-related cataract.

10. The use according to claim 1, characterized in that The preparation method of the eye drops containing L-ergothioneine comprises the following steps: (1) dissolving the antibacterial agent in physiological saline and filtering with a 0.22 μm microporous filter membrane to obtain solution I; (2) Sterile L-ergothioneine was added to solution I, and ultrasonic stirring was performed for 30 min to obtain solution II; (3) adding physiological saline to dilute solution II to 100 parts by weight to obtain solution III; (4) adjusting the pH value of solution III to 5.0-7.0 with a buffer to obtain solution IV; (5) sterilizing solution IV at 121-134° C., 102.9-137.2 kPa for 15-30 min to obtain eye drops containing L-ergothioneine.

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