Panax notoginseng total saponin ophthalmic gel preparation as well as preparation method and application thereof

Abstract

The invention discloses a panax notoginseng total saponin ophthalmic gel preparation as well as a preparation method and application thereof. The ophthalmic gel preparation of panax notoginseng saponins comprises panax notoginseng saponins, EDTA-2Na, a gel matrix carbomer 980, mannitol, a preservative, a pH regulator, water for injection and the like. By optimizing and screening all the components and the content, the ophthalmic gel preparation containing total panax notoginseng saponins can deliver drugs to enter eyes for multiple times, the bioavailability of the eyes is improved, meanwhile, the drug effects of preventing and treating diseases such as diabetic retinopathy and diabetic cataract are improved, and the ophthalmic gel preparation is good in clinical applicability and druggability.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and particularly to a Panax notoginseng total saponin ophthalmic gel preparation, its preparation method and uses. Background Art

[0002] Diabetic retinopathy (DR) is a retinal complication of diabetes, which is caused by long-term hyperglycemia-induced damage to retinal microvessels and is a chronic progressive blinding eye disease. One of the main ocular complications in diabetic patients is cataract. The onset age of cataract in diabetic patients is earlier, and having DR is a risk factor for the occurrence of diabetic cataract. Currently, the number of diabetic patients in China exceeds 140 million, and the prevention and treatment of diabetes and its related complications are urgent. DR and diabetic cataract are the most serious complications of diabetes. Approximately 1 in every 3 diabetic patients has DR, and DR and diabetic cataract have become the main causes of vision loss and blindness in the working-age population.

[0003] Panax notoginseng total saponins are included in the first part of the Chinese Pharmacopoeia (2020 edition) and are the total saponins prepared from the main roots or rhizomes of the Panax notoginseng plant of the Araliaceae family. Panax notoginseng total saponins have the effects of promoting blood circulation to remove blood stasis and dredging meridians to activate collaterals. In 2015, it was reported that oral administration of Panax notoginseng total saponins could reduce the expression of glial fibrillary acidic protein and mRNA in the retinal tissues of rats and protect the retinas of streptozotocin-induced diabetic rats (Yao Qing, Ma Xiaodong, Zhang Qian, et al. Protective effect of Panax notoginseng total saponins on retinal function in early diabetic rats [J]. Ningxia Medical Journal, 2014, 36(03): 204-206.). In 2024, cell experiment studies showed that Panax notoginseng total saponins alleviated DR by inhibiting the activation of the cell NF-κB signaling pathway, thereby suppressing retinal inflammation (Yaru Wang, Xin Sun, Yumin Xie, Ao Du, Ming Chen, Shusheng Lai, Xiaohui Wei, Lili Ji, Changhong Wang. Panax notoginseng saponins alleviate diabetic retinopathy by inhibiting retinal inflammation: Association with the NF-κB signaling pathway. Journal of Ethnopharmacology [J]. 2024, 319: 117135-117148.). Thus, it can be seen that Panax notoginseng total saponins are highly effective drugs for treating DR.

[0004] Notoginsenosides need to be formulated into certain dosage forms before they can be used to treat eye diseases. Oral preparations, injection preparations, and ophthalmic preparations are common dosage forms of notoginsenosides. Among them, ophthalmic preparations cross the physiological and anatomical barriers of the eye and act directly on the eye, with the advantages of directness and high efficiency. They have been widely used to deliver drugs to the eye to treat eye diseases such as diabetic retinopathy. Preparations administered locally to the eye have high requirements for local release and absorption of drugs. The higher the concentration of the drug in the eye, the more likely it is to reach the therapeutic window range, and the more significant the therapeutic effect of the drug will be.

[0005] Eye drops and creams are the most commonly used dosage forms in clinical practice. Document 1 (Chinese Patent Application No. 201711262528.6 discloses an ophthalmic drug preparation of notoginsenoside extract, its preparation method and application. The components of the drug preparation are notoginseng extracts with a total saponin content of 45-95%. The preparation forms include various dosage forms such as eye drops, eye ointments, eye patches, and hydrogels. Among them, after the eye drops are instilled into the eye, the drug concentration will rapidly decrease, resulting in a very short residence time of the drug in the eye and poor bioavailability. Creams are viscous, and eye application affects vision, resulting in poor patient compliance. In the hydrogel preparation, the proportion of carbomer is too low. After being applied to the eye, it is diluted by tears, and the viscosity further decreases, resulting in a short residence time in the eye and still unsatisfactory bioavailability.

[0006] Ophthalmic gel is a new type of ophthalmic preparation. By adding bioadhesive materials to eye drops, the viscosity of the solution is increased, which can prolong the residence time of the liquid medicine in the eye, enhance corneal permeability, and promote drug penetration into the eye. Document 2 (Chinese Patent Application No. 201110453135.X) discloses a notoginsenoside eye drop and its preparation method. The viscosity regulator therein is one of sodium hyaluronate, methylcellulose, carbomer, or hydroxypropylcellulose. However, the viscosity and osmotic pressure of this preparation are not ideal, and the bioavailability is poor. The applicant has been committed to the research and development of ophthalmic preparations of notoginsenosides. Document 3 (Chinese Patent No. 202210010949.4) is an earlier application of the applicant, which discloses an ophthalmic gel preparation of notoginsenosides, its preparation method and uses. The bioadhesive material of this ophthalmic gel preparation is a low-viscosity cellulose polymer and an acrylic polymer. By using a combination of two polymers, the ocular bioavailability of the gel preparation is improved to a certain extent. However, the degree of improvement in ocular bioavailability of the gel preparation is not sufficient to meet the needs of clinical treatment. Moreover, the swelling process of the viscous excipient is difficult, the preparation process is complex, and the time cost is high, which hinders the application of this dosage form.

[0007] In view of the deficiencies and druggability defects of the existing panax notoginseng saponins ophthalmic preparations, there is still a great demand for panax notoginseng saponins ophthalmic preparations with high ocular bioavailability and effective treatment and / or prevention of diabetic retinopathy. There is also a great demand in the industry for a new preparation process of panax notoginseng saponins preparations with good stability, energy saving and high efficiency. Summary of the Invention

[0008] To solve some of the technical problems in the prior art, the inventors of the present invention, through in-depth research, optimized the formulation and preparation process of the preparation, and provided a panax notoginseng saponins ophthalmic gel with high ocular bioavailability and good effect in treating diabetic retinopathy. The present invention also provides a preparation method of the ophthalmic gel, which is convenient to operate, has high efficiency, and the obtained product has stable quality.

[0009] In the first aspect of the present invention, there is provided a panax notoginseng saponins ophthalmic gel preparation, comprising the following components:

[0010] 0.5 - 2.0 parts by weight of panax notoginseng saponins;

[0011] 0.05 - 0.06 parts by weight of EDTA-2Na;

[0012] 0.2 - 0.3 parts by weight of carbomer 980;

[0013] 3.7 - 5.0 parts by weight of mannitol;

[0014] 0.001 - 0.1 parts by weight of preservative;

[0015] An appropriate amount of pH regulator is used to adjust the pH of the ophthalmic gel preparation to 6.0 - 8.0;

[0016] Make up to 100 parts by weight with injection water.

[0017] Preferably, in the above-mentioned panax notoginseng saponins ophthalmic gel preparation, the parts by weight of panax notoginseng saponins are 0.5, 1.0 or 2.0; preferably, the parts by weight of EDTA-2Na are 0.05, 0.055 or 0.06; preferably, the parts by weight of carbomer 980 are 0.2, 0.25 or 0.3; preferably, the parts by weight of mannitol are 4.1; preferably, the parts by weight of the preservative are 0.005 - 0.05, more preferably 0.009 - 0.01, and most preferably 0.01.

[0018] The notoginsenoside ophthalmic gel preparation according to the present invention, wherein the notoginsenoside raw material can be a commercially available product. For example, the notoginsenoside purchased from Kunming Pharmaceutical Group Co., Ltd., batch number: JK2022041 is used as the raw material. Among them, the content of notoginsenoside R1 is 9.70%, the content of ginsenoside Rg1 is 31.00%, the content of ginsenoside Re is 4.20%, the content of ginsenoside Rb1 is 31.90%, the content of ginsenoside Rd is 8.80%, and the total content of total saponins exceeds 85%.

[0019] For the notoginsenoside ophthalmic gel preparation according to the present invention, preferably, the pH regulator includes sodium hydroxide or potassium hydroxide.

[0020] For the notoginsenoside ophthalmic gel preparation according to the present invention, preferably, the preservative includes benzalkonium chloride or benzalkonium bromide.

[0021] In the second aspect of the present invention, a preparation method of the notoginsenoside ophthalmic gel preparation according to the first aspect is provided, including the following steps:

[0022] (1) Under light-proof conditions, weigh notoginsenoside, EDTA-2Na and preservative and place them in injection water, heat and stir to dissolve to obtain a medicinal solution;

[0023] (2) Weigh mannitol and carbomer 980, disperse the two evenly, and slowly add them to injection water under high-speed stirring, and continuously heat and stir to obtain a clear matrix solution;

[0024] (3) Add the clear matrix solution obtained in step (2) to the medicinal solution obtained in step (1), stir, add a pH regulator, adjust the pH to 6.0 - 8.0, make up to 100% with injection water, and stir evenly to obtain a gel.

[0025] The gel prepared by the above method is preferably sterilized and then canned; preferably, high-pressure steam sterilization is used; the preferred sterilization conditions are 121 °C and 30 min.

[0026] According to the preparation method of the present invention, preferably, in step (1), the concentration of notoginsenoside in the medicinal solution is 6.25 - 100 g / L; the concentration of EDTA-2Na in the medicinal solution is 0.625 - 2.5 g / L; preferably, in step (1), the heating temperature is 50 - 60 °C, and the stirring speed is 200 rpm.

[0027] According to the preparation method of the present invention, preferably, in step (2), the concentration of mannitol in the matrix solution is 51-205 g / L; the concentration of carbomer 980 in the matrix solution is 3.1-12.5 g / L. Preferably, in step (2), the stirring speed is 350-400 rpm, and the heating temperature is 65-70 °C. More preferably, the heating and stirring time in step (2) is 3-5 hours, preferably 4 hours.

[0028] According to the preparation method of the present invention, preferably, in step (3), the stirring conditions for stirring after adding the matrix solution to the medicinal liquid are: the stirring speed is 40 rpm, and the stirring time is 5 minutes; the stirring conditions for stirring evenly after adding injection water to make up are: the stirring speed is 40 rpm, and the stirring time is 45 minutes;

[0029] According to the preparation method of the present invention, preferably, in step (3), adjusting the pH with an alkaline solution includes, but is not limited to, potassium hydroxide or sodium hydroxide solution.

[0030] According to the preparation method of the present invention, preferably, in step (4), the high-pressure steam sterilization is at 121 °C for 30 min.

[0031] In the third aspect of the present invention, there is provided the use of the total saponins of Panax notoginseng ophthalmic gel preparation according to the first aspect in the preparation of a drug for preventing and / or treating diabetic retinopathy or diabetic cataract.

[0032] According to the use of the present invention, preferably, the diabetic retinopathy includes changes in retinal thickness, changes in retinal vascular permeability, or damage to the integrity of the blood-retinal barrier.

[0033] According to the use of the present invention, more preferably, the total saponins of Panax notoginseng ophthalmic gel preparation can reduce retinal vascular permeability.

[0034] Advantages of the present invention:

[0035] Compared with Document 3, the present invention is a further improvement based on previous research. From the perspectives of process convenience and improvement of the bioavailability of the preparation, the hypromellose in the composite gel matrix in Document 3 is discarded in the present invention, eliminating the adverse effect of the time-consuming swelling of hypromellose. At the same time, the type of carbomer gel is changed, and carbomer 980 with a single gel matrix is used. Mannitol is added to adjust the osmotic pressure, and mannitol can also promote the dispersion and swelling of carbomer. The inventors originally thought that using a single gel matrix might reduce the properties such as the viscosity of the gel. However, surprisingly, through experiments, it is found that the ophthalmic gel formulation of total notoginsenosides of the present invention is more reasonable. Not only can the osmotic pressure and pH value of the gel be kept constant (Example 2), the component stability is good (Example 3), and there is no irritation (Example 6), but also the process is robust, the matrix swelling speed is fast, and the efficiency is increased by 33.3% (Examples 1 and 5). The gel with a single matrix of carbomer 980 has good properties. The ophthalmic pharmacokinetic results of the gel of the present invention show that under the action of the ophthalmic delivery of the gel formulation of the present invention, total notoginsenosides can be absorbed into the eye and can be reabsorbed into the eye through the systemic circulation, significantly increasing the eye drug concentration and bioavailability (Example 7); with the support of a specific gel carrier and combined with the enhancing effect of EDTA-2Na on the gel efficacy, the ophthalmic gel of total notoginsenosides not only significantly reduces the retinal thickness and the contents of VEGFA and AGEs in the retina of DR rats, but also significantly reduces the incidence rate of diabetic cataract and alleviates the degree of cataract condition (Example 8). The ophthalmic gel of total notoginsenosides of the present invention has significant process advantages, safety advantages, ophthalmic pharmacokinetic advantages and pharmacodynamic advantages. Description of the Drawings

[0036] Figure 1 : Number of acellular capillaries in the retina in Experimental Example 3

[0037] Figure 2 : Ratio of the length of pericytes lacking to the total length in Experimental Example 3

[0038] Figure 3 : Western blot of retinal tight junction proteins in Experimental Example 3

[0039] Figure 4 : Occludin protein expression in Experimental Example 3

[0040] Figure 5 : Notoginsenoside R in the rabbit ocular microdialysate in Experimental Example 6 1 Drug concentration-time curve

[0041] Figure 6 : Ginsenoside Rg in the rabbit ocular microdialysate in Experimental Example 6 1 Drug concentration-time curve

[0042] Figure 7 : Concentration-time curve of ginsenoside Re in the rabbit eye microdialysate in Experimental Example 6

[0043] Figure 8 : Incidence proportion of cataract in each group of rats during the administration intervention in Experimental Example 7

[0044] Figure 9 : Cataract scores of each group of rats 8 weeks after the administration intervention in Experimental Example 7. Note: Compared with the blank group, * indicates P < 0.05; compared with the model group, # indicates P < 0.05; compared with the Literature 3 gel - 940 group, Δ indicates P < 0.05; compared with the Xuesaitong for injection group, ▲ indicates P < 0.05

[0045] Figure 10 : Positive area expression rate of VEGFA in the retina of each group of rats in Experimental Example 7. Note: Compared with the blank group, * indicates P < 0.05; compared with the model group, # indicates P < 0.05; compared with the Literature 3 gel - 940 group, Δ indicates P < 0.05; compared with the Xuesaitong for injection group, ▲ indicates P < 0.05

[0046] Figure 11 : Positive area expression rate of AGEs in the retina of each group of rats in Experimental Example 7. Note: Compared with the blank group, * indicates P < 0.05; compared with the model group, # indicates P < 0.05, ## indicates P < 0.01; compared with the Literature 3 gel - 940 group, Δ indicates P < 0.05; compared with the Xuesaitong for injection group, ▲ indicates P < 0.05

[0047] Figure 12 : Detection of the retinal thickness of each group of rats by HE staining in Experimental Example 7. Note: Compared with the blank group, * indicates P < 0.05; compared with the model group, # indicates P < 0.05; compared with the Literature 3 gel - 940 group, Δ indicates P < 0.05; compared with the Xuesaitong for injection group, ▲ indicates P < 0.05 Detailed implementation manners

[0048] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0049] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that the upper and lower limits of the range and each intermediate value between them are specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0050] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although this invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the said documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0051] Those skilled in the art should understand that as long as the objectives of this invention can be achieved, other steps or operations may be included before, during, or after steps (1)-(4) of the method of this invention, such as further optimizing and / or improving the method described in this invention.

[0052] Example 1 Prescription and Preparation Process of 0.5% Total Notoginsenoside Ophthalmic Gel

[0053] 1. Prescription Composition of 0.5% Total Notoginsenoside Ophthalmic Gel:

[0054]

[0055] 2. Preparation Process of 0.5% Total Notoginsenoside Ophthalmic Gel:

[0056] (1) Under light-proof conditions, weigh total notoginsenoside, EDTA-2Na, and benzalkonium chloride and place them in 2000 mL of injection water. Heat and stir to dissolve, with the heating temperature: 50 - 60 °C and the stirring speed: 200 rpm, to obtain a medicinal solution.

[0057] (2) Weigh mannitol and carbomer 980 separately, disperse them evenly, and slowly add them to 2000 mL of injection water at a temperature of 65 - 70 °C while stirring at a speed of 350 - 400 rpm. Continuously heat and stir for 4 h to obtain a clear matrix solution.

[0058] (3) Finally, add the clear matrix solution to the medicinal solution, stir evenly for 5 minutes at a stirring speed of 40 rpm, adjust the pH to 6.5, make up the injection water to 5000 g, stir evenly for 45 minutes at a stirring speed of 40 rpm.

[0059] In the above preparation method, sodium hydroxide solution is used to adjust the pH. Preparation of sodium hydroxide solution: Weigh 4.0 g of sodium hydroxide, add water to 100 g, and stir to dissolve to obtain sodium hydroxide solution.

[0060] Autoclave the prepared gel at 121 °C for 30 min, take it out and wait for it to cool, and then fill it in a sterile environment.

[0061] Example 2 Prescription and Preparation Process of 1.0% Total Notoginsenoside Ophthalmic Gel

[0062] 1. Prescription composition of 1.0% total notoginsenoside ophthalmic gel:

[0063]

[0064] 2. Preparation process of 1.0% total notoginsenoside ophthalmic gel:

[0065] (1) Under light - avoiding conditions, weigh total notoginsenoside, EDTA - 2Na and benzalkonium chloride and place them in 2500 mL of injection water. Heat and stir to dissolve. Heating temperature: 50 - 60 °C, stirring speed: 200 rpm, to obtain a medicinal solution;

[0066] (2) Weigh mannitol and carbomer 980 separately, disperse them evenly, and slowly add them to 2000 mL of injection water at a temperature of 65 - 70 °C under a stirring speed of 350 - 400 rpm, and continuously heat and stir for 4 h to obtain a clear matrix solution;

[0067] (3) Finally, add the clear matrix solution to the medicinal solution, stir evenly for 5 minutes at a stirring speed of 40 rpm, adjust the pH to 6.5, make up the injection water to 5000 g, stir evenly for 45 minutes at a stirring speed of 40 rpm.

[0068] In the above preparation method, the pH is adjusted with sodium hydroxide solution. Preparation of sodium hydroxide solution: Weigh 4.0 g of sodium hydroxide, add water to 100 g, and stir to dissolve to obtain sodium hydroxide solution.

[0069] Sterilize the prepared gel in a high - pressure sterilizer by high - pressure steam sterilization (121 °C, 30 min), take it out and wait for it to cool, and fill it in a sterile environment.

[0070] Example 3 Prescription and Preparation Process of 2.0% Total Notoginsenoside Ophthalmic Gel

[0071] 1. Prescription composition of 2.0% total notoginsenoside ophthalmic gel:

[0072]

[0073]

[0074] 2. Preparation process of 2.0% total notoginsenoside ophthalmic gel:

[0075] (1) Under light - avoiding conditions, weigh total saponins of Panax notoginseng, EDTA - 2Na, and benzalkonium chloride and place them in 2900 mL of injection water. Heat and stir to dissolve them. Heating temperature: 50 - 60 °C, stirring speed: 200 rpm, to obtain a medicinal solution;

[0076] (2) Weigh mannitol and carbomer 980 separately, disperse them evenly, and slowly add them to 2000 mL of injection water at a temperature of 65 - 70 °C under a stirring speed of 350 - 400 rpm. Continuously heat and stir for 4 h to obtain a clear matrix solution;

[0077] (3) Finally, add the clear matrix solution to the medicinal solution, stir evenly for 5 minutes at a stirring speed of 40 rpm, adjust the pH to 6.5, make up the injection water to 5000 g, stir evenly for 45 minutes at a stirring speed of 40 rpm.

[0078] In the above preparation method, sodium hydroxide solution is used to adjust the pH. Preparation of sodium hydroxide solution: Weigh 4.0 g of sodium hydroxide, add water to 100 g, and stir to dissolve to obtain sodium hydroxide solution.

[0079] Sterilize the prepared gel in a high - pressure sterilizer by high - pressure steam sterilization (121 °C, 30 min). After taking it out and waiting for it to cool, fill it in a sterile environment.

[0080] Experimental Example 1 Optimization and Screening of Osmotic Pressure Regulators

[0081] In this experimental example, the total saponins of Panax notoginseng ophthalmic gels containing different types and proportions of osmotic pressure regulators were compared, their osmotic pressures were measured, and appropriate types and dosages of osmotic pressure regulators were selected.

[0082] Determine according to the method for determining osmotic molality in the Chinese Pharmacopoeia (General Rule 0632) in 2020 edition, and it should meet the requirements.

[0083] 1 Screening of Types of Osmotic Pressure Regulators

[0084] 1.1 Preparation of Samples

[0085] Precisely weigh 2.0 g of total saponins of Panax notoginseng, 0.05 g of EDTA - 2Na, and 0.01 g of benzalkonium chloride and place them in 40 mL of injection water, and heat to dissolve them;

[0086] Separately, disperse different types / proportions of osmotic pressure regulators (see Table 1) and 0.25 g of carbomer 980 evenly, and slowly add them to 40 mL of injection water at a temperature of 65 - 70 °C under high - speed stirring conditions (stirring speed: 350 - 400 rpm), and continuously heat and stir to obtain a clear solution.

[0087] Finally, add the clarified matrix solution to the medicinal liquid, stir evenly, add sodium hydroxide solution to adjust the pH, make up the volume to the prescription amount (100 g) with water, and stir evenly.

[0088] Table 1 Dosages of different osmotic pressure regulators

[0089]

[0090]

[0091] 1.2 Measurement results of osmotic pressure

[0092] The results are shown in Table 2. From the osmotic pressure measurement results and the gel forming state, it can be seen that the osmotic pressure of the total notoginsenoside ophthalmic gel with 4.1% mannitol added meets the requirements of the Chinese Pharmacopoeia, and the added amount meets the maximum requirements of the United States Pharmacopoeia; adding 4% - 5% glucose is not stable enough for osmotic pressure regulation; although KCl and NaCl can regulate the osmotic pressure, they will both affect the gel forming property, so mannitol is selected as the osmotic pressure regulator.

[0093] Table 2 Osmotic pressures at different osmotic pressure regulators and different contents

[0094]

[0095] Note: The osmotic pressure of the test standard is 292 mOsm2 Screening of mannitol dosage

[0096] Keep the dosages of other excipients in the prescription unchanged. Using mannitol as the osmotic pressure regulator, select mannitol dosages of 3.0%, 3.5%, 3.7%, 4.1%, 4.3%, and 5.0% (w / w) respectively to prepare gel preparations, and investigate the influence of different dosages of osmotic pressure regulators on the osmotic pressure.

[0097] 2.1 Preparation of samples

[0098] Precisely weigh 2.0 g of total notoginsenoside, 0.05 g of EDTA-2Na, and 0.01 g of benzalkonium chloride and place them in 40 mL of injection water, heat and dissolve it;

[0099] Separately, disperse different proportions of mannitol and 0.25 g of carbomer 980 evenly, and slowly add them to 40 mL of injection water at a temperature of 65 - 70 °C under high-speed stirring conditions (stirring speed: 350 - 400 rpm), continuously heat and stir to obtain a clarified solution.

[0100] Finally, add the clarified matrix solution to the medicinal liquid, stir evenly, add sodium hydroxide solution to adjust the pH, make up the volume to the prescription amount (100 g) with water, and stir evenly.

[0101] 2.2 Experimental results

[0102] Osmotic Pressure of Panax Notoginseng Saponins Ophthalmic Gel with Different Contents of Mannitol Added

[0103]

[0104] As can be seen from Table 3 after adding different contents of mannitol, the osmotic pressure of Panax notoginseng saponins ophthalmic gel with 3.7% - 5.0% mannitol added all meets the requirements of the Chinese Pharmacopoeia (General Rules 0632, Volume IV, 2020 Edition of the Chinese Pharmacopoeia, the osmotic molality should be 250 - 330 mOsmol / kg). Therefore, mannitol with a concentration of 3.7% - 5.0% is selected as the osmotic pressure regulator.

[0105] Experimental Example 2 Effect of EDTA-2Na on the Stability of Components in the Gel

[0106] 1 Experimental Method

[0107] EDTA-2Na is a strong chelating agent and preservative. Different amounts (0, 0.01%, 0.03%, 0.05%, 0.06%) of EDTA-2Na are added, and 3 samples of each concentration are prepared in parallel. The gels are placed in a constant temperature and humidity chamber and subjected to an accelerated experiment at a temperature of 30°C ± 2°C and a humidity of 65% RH ± 5% RH. Samples are taken for detection of the content of Panax notoginseng saponins after 1 month.

[0108] 2 Experimental Results

[0109] As can be seen from Table 4, EDTA-2Na has a certain effect on the stability of Panax notoginseng saponins. Without adding or adding a small amount (0.01%, 0.03%) of EDTA-2Na, the content change rate of Panax notoginseng saponins is relatively large after 1 month of acceleration, while 0.05% - 0.06% of EDTA-2Na has a significant promoting effect on the component stability, and the content change rate of Panax notoginseng saponins is very small after 1 month of acceleration. In ophthalmic preparations, the maximum addition amount of EDTA-2Na is 0.06%. Therefore, the optimal addition amount of EDTA-2Na is determined to be 0.05% - 0.06%.

[0110] Table 4 Content Change of Panax Notoginseng Saponins in the Gel with Different Concentrations of EDTA-2Na Added after 1 Month of Accelerated Experiment

[0111]

[0112]

[0113] Experimental Example 3 Pharmacodynamic Effect Experiment of EDTA-2Na on Panax Notoginseng Saponins Ophthalmic Gel

[0114] 1 Instruments

[0115] Electrophoresis apparatus (BioRad, USA); ultrasensitive chemiluminescence imager AI600 (Beijing Dequan Xingye Trading Co., Ltd.).

[0116] 2. Methods

[0117] 2.1 Method for establishing a diabetic mouse model

[0118] C57 mice were adaptively fed for 1 week, fasted for 4 h, and weighed. Except for the normal group, the remaining mice were intraperitoneally injected with STZ at a dose of 50 mg / kg body weight (prepared with 0.1 mol / L, pH 4.5 citrate buffer, stored in the dark on ice, and administered within 5 min). According to the above method, the drug was continuously administered for 5 days. On the 14th day after the start of drug administration, the mice were fasted for 6 h, and the fasting blood glucose was measured. A fasting blood glucose > 16.7 mmol / L was considered successful model establishment. If the blood glucose < 16.7 mmol / L, STZ was injected again, and the fasting blood glucose was measured 3 days later. The successfully modeled mice from the two injections were evenly used to ensure 6 animals in each group.

[0119] 2.2 Preparation method of Panax notoginseng saponin ophthalmic gel (PNS gel) with different prescriptions

[0120] The preparation method of 0.5% PNS gel containing EDTA-2Na was the same as that in Example 1, hereinafter referred to as the "low-dose PNS group containing EDTA"; the preparation method of 2.0% PNS gel containing EDTA-2Na was the same as that in Example 3, hereinafter referred to as the "high-dose PNS group containing EDTA"; for 0.5% PNS gel without EDTA-2Na, except for not adding EDTA-2Na, other components and preparation methods were the same as those in Example 1, hereinafter referred to as the "low-dose PNS group without EDTA";

[0121] For 2.0% PNS gel without EDTA-2Na, except for not adding EDTA-2Na, other components and preparation methods were the same as those in Example 3, hereinafter referred to as the "high-dose PNS group without EDTA".

[0122] 2.3 Grouping and dosing regimens

[0123] The experiment was divided into 6 groups, namely the normal group, the model group, the low-dose PNS group without EDTA, the low-dose PNS group with EDTA, the high-dose PNS group without EDTA, and the high-dose PNS group with EDTA.

[0124] In terms of drug administration, the low-dose PNS group without EDTA and the low-dose PNS group with EDTA were respectively administered 1 mL / kg by eye (equivalent to 1.56 mg / kg of ginsenoside Rg1 and 0.73 mg / kg of notoginsenoside R 1 0.73 mg / kg); the high-dose PNS group without EDTA and the high-dose PNS group with EDTA were respectively administered 1 mL / kg by eye (equivalent to ginsenoside Rg 16.67 mg / kg, Notoginsenoside R 1 1.69 mg / kg); Both were administered continuously for 8 weeks, once a day.

[0125] 2.4 Retinal acquisition

[0126] After the mice were anesthetized, the blood was perfused and rinsed clean with PBS. The eyeballs with optic nerves were removed. The sclera was incised circularly along the ora serrata from the posterior part of the ciliary body. The cornea and lens were removed. The posterior eye cup was cut into 3 pieces in a petal-like shape centered on the optic disc. The retina was carefully separated and refrigerated.

[0127] 2.5 Retinal digestion and spreading

[0128] The steps of retinal digestion and spreading are as follows:

[0129] A. The separated retina was rinsed with PBS and soaked in glycine buffer overnight; B. The retina was digested with 3% trypsin in a 37°C air bath oscillator for 2 - 4 h; C. The retina was transferred to 0.1 M PBS, and the inner limiting membrane was gently aspirated and blown off. Only a transparent vascular network remained. It was floated and flattened on an adhesive-free glass slide. After natural drying at room temperature, it was stored in a 4°C slide box for PAS staining.

[0130] 2.6 Retinal PAS staining

[0131] A. Transfer the retina from 4°C to RT and place for 10 min; B. Infiltrate with PBS (0.01 mol / L, pH 7.4) for 5 min; C. Pre-staining solution for 10 min; D. Rinse with deionized water; E. Mixed staining solution for 5 min; F. Differentiating solution for 5 min; G. Wash with tap water; H. Hematoxylin for 4 min; Wash with tap water; I. Differentiating solution for 1 sec; Wash with tap water; J. Blueing solution for 6 sec; Wash with tap water; K. 95% ethanol for several seconds; L. Absolute ethanol I and II for 2 min each; M. Xylene I and II for 2 min each; N. Mount with neutral gum.

[0132] 2.7 Desmin staining method

[0133] A. After the eyeball was fixed for half an hour, the retina was separated according to the above method, and the vitreous body and RPE were cleaned to ensure good permeability; B. The retina was placed in a 2 mL EP tube, and the PFA was washed with 1 mL PBS, 3×5 min; C. 1 mL of 1% Triton X-100 / PBS was added for punching and permeabilization, and it was placed on a shaker and incubated overnight at 4°C; D. It was blocked with 1 mL of 1% BSA and 0.5% Triton X-100 at 4°C and shaken for one day; E. Incubated with the primary antibody (desmin) for 2 days (the primary antibody was dissolved in 0.1% Triton X-100 and 1% BSA, 1:200); F. Washed with 1 mL of 0.1% Triton X-100 for 5×10 min, then added for 20 min, and shaken and washed at room temperature; Incubated with the secondary antibody at room temperature for 2 h (1:3000), and shaken, protected from light; G. Washed with 1 mL of 0.1% Triton X-100 for 4×30 min, shaken at room temperature, protected from light; H. Mounted the slices and sealed them (the sealing agent used was an anti-fluorescence quenching sealing agent containing DAPI); I. Observed the pericytes loss using a laser confocal microscope; J. Statistically analyzed the blood vessel length and the missing length using image J.

[0134] 2.8 Western blot analysis

[0135] (1) Total protein was extracted from retinal tissue; (2) The protein was quantitatively standardized to an equal amount using a BCA protein assay kit; (3) The protein (20 μg per lane) was separated by an 8% or 10% SDS-PAGE gel and transferred to a PVDF membrane; (4) Blocked with 5% BSA blocking buffer (SW3015, Solarbio) for 1 h; (5) Incubation with the primary antibody: The bands were soaked in the diluted primary antibodies Occludin (1:1000, abcam, ab216327) and β-actin (1:1000, CST, 3700S), and incubated overnight at 4°C with slow shaking on a shaker. (6) After incubation, the primary antibody was aspirated, TBST was added to a square fresh-keeping box, the membrane was placed in it, and TBST was made to cover the PVDF membrane, and shaken at low speed on a shaker for 10 min, and repeated 3 times; (7) Incubation with the secondary antibody: The PVDF membrane transferred with the protein was immersed in a square fresh-keeping box containing the secondary antibody (goat anti-rabbit IgG-HRP or goat anti-mouse IgG-HRP antibodies), and shaken and incubated for 1-2 h; (8) After incubation, the secondary antibody was aspirated, TBST was added to a square fresh-keeping box, the membrane was placed in it, and TBST was made to cover the PVDF membrane, and shaken at low speed on a shaker for 10 min, and repeated 3-5 times; (9) Chemiluminescence: Appropriate amount of luminescent solution was aspirated to cover the PVDF membrane, and exposed and imaged on an ECL luminescence instrument.

[0136] 3. Experimental Results

[0137] 3.1 Retinal Vascular Morphology

[0138] Figure 1 The results showed that compared with the retinal vascular network of normal group mice, in the model group mice, some capillaries degenerated, the lumen was blocked, acellular capillaries were formed, perfusion-free areas appeared, and the number of pericytes decreased. After 6 weeks of drug administration, compared with the model group, in the retinal capillaries of the PNS gel administration group mice, the number of pericytes increased to varying degrees, and the number of acellular capillaries decreased. Among them, at the same drug administration dose, compared with the group without EDTA, the number of acellular capillaries in the retina of the PNS gel administration group with EDTA added was significantly reduced (p < 0.05).

[0139] 3.2 Pericyte Loss in the Retina

[0140] Figure 2 The results showed that the pericyte loss in normal mice was not obvious, while the pericyte loss in the retinal blood vessels of the model group mice was obvious, with a significant difference compared with the normal group (p < 0.05). There was a significant difference between the PNS gel administration group and the model group (p < 0.05), and after adding EDTA, there was a significant difference in the pericyte loss in the retinal blood vessels of mice compared with the group without EDTA (p < 0.05). It can be seen that PNS gel has a significant inhibitory effect on the pericyte loss in blood vessels in diabetic retinopathy, and the effect is more significant after adding EDTA.

[0141] 3.3 Western Blot Result Analysis

[0142] Figure 3 The Western blot analysis results of the retinas of animals in each group were shown, and the expression of the tight junction protein Occludin was analyzed by Western blot. Figure 4 The results showed that compared with the normal group, the expression of the tight junction protein Occludin was down-regulated in the model group; compared with the model group, the expression of Occludin protein was up-regulated to varying degrees in the PNS gel administration group; among them, compared with the group without EDTA, the protein expression of tight junction Occludin was up-regulated in the group with EDTA added. These data indicate that PNS gel alleviates the damage of the retinal barrier permeability in diabetic mice, and the effect is stronger after adding EDTA.

[0143] As can be seen from the above experiments, in the prescription of the present invention, EDTA-2Na can cooperate with total saponins of Panax notoginseng to significantly reduce the number of acellular capillaries in the retina, significantly inhibit the pericytes loss of blood vessels in retinopathy, significantly up-regulate the protein expression of tight junction Occludin, and significantly repair the retinal permeability of diabetic mice. The synergistic effect of EDTA-2Na on the ophthalmic gel of total saponins of Panax notoginseng may be because EDTA-2Na can promote the absorption of total saponins of Panax notoginseng in the eye, increase the concentration of total saponins of Panax notoginseng in the eye and retina, or because EDTA-2Na can regulate the microenvironment of the eye, such as osmotic pressure, viscosity, pH value, etc., so as to form a synergistic effect with total saponins of Panax notoginseng and enhance the efficacy of the ophthalmic gel of total saponins of Panax notoginseng.

[0144] Experimental Example 4 Effect of Mannitol on the Swelling Time of Carbomer

[0145] 1 Experimental Method

[0146] Preparation Method 1: Prepare carbomer gel by using the preparation method of the present invention.

[0147] Precisely weigh 20.5 g of mannitol and 1.25 g of carbomer 980, disperse them evenly, and slowly add them to 200 mL of deionized water at a temperature of 65-70 °C under high-speed stirring (stirring speed: 350-400 rpm), continuously heat and stir to obtain a clear solution. Prepare 3 portions in parallel, record the swelling time of carbomer respectively, and take the average value.

[0148] Preparation Method 2:

[0149] Precisely weigh 1.25 g of carbomer 980, and slowly add it to 200 mL of deionized water at a temperature of 65-70 °C under high-speed stirring (stirring speed: 350-400 rpm), continuously heat and stir to obtain a clear solution. Prepare 3 portions in parallel, record the swelling time of carbomer respectively, and take the average value.

[0150] 2 Experimental Results

[0151] Dispersing mannitol and carbomer evenly can effectively shorten the swelling time of carbomer, and the production efficiency can be increased by 33.3%. The results of the average swelling time of carbomer are shown in Table 5.

[0152] Table 5 Results of the Effect of Mannitol on the Swelling Time of Carbomer (n = 3)

[0153]

[0154] Experimental Example 5 Irritation Study of Different Ophthalmic Gels of Total Saponins of Panax Notoginseng

[0155] 1 Instruments and Experimental Animals

[0156] 1.1 Instruments

[0157] Electronic balances (Changshu Shuangjie Testing Instrument Factory, model: TC30KHA, serial number: EQ02 - 0140; METTLER TOLEDO Instruments (Shanghai) Co., Ltd., model: PL023, serial number: EQ02 - 0205);

[0158] Portable barcode reader (Beijing Aimei Technology Development Co., Ltd., model: JD - C - 08, serial number: EQ03 - 3503)

[0159] Slit - lamp microscope (Kowa Company, Ltd., model: KOWA SL - 17, serial number: EQ03 - 8301)

[0160] Sodium fluorescein (Solarbio);

[0161] 1.2 Experimental animals

[0162] Forty - five Japanese white rabbits with large ears, half male and half female, weighing 2.5 - 2.9 Kg. After the adaptation period, 40 rabbits were selected for the experiment. When grouping, the weights of the selected animals were 2.870 - 3.150 kg (♂) and 2.960 - 3.185 kg (♀). They were provided by Tianjin Yuda Experimental Animal Breeding Co., Ltd., with animal certificate number: SCXK (Jin) 2021 - 0001, and the experimental animal quality certificate number: 120906231100068545; Breeding location: Experimental Animal Center of Tianjin University of Traditional Chinese Medicine; Temperature: 18℃ - 26℃; Humidity (RH): 40% - 70%; Relevant research was carried out in accordance with the principles of animal experiments.

[0163] 2 Sample preparation method

[0164] 2.0% PNS gel - Carbomer 940 test sample: Using the prescription and process of reference 3, 2.0% PNS gel was prepared as follows: Dissolve 2.0 g of total saponins of Panax notoginseng and 0.05 g of EDTA - 2Na in 40 mL of deionized water, then heat it to 70℃, add 0.4 g of HPMC under stirring conditions, disperse evenly, and then stir until clear under cooling conditions. Separately, slowly add 0.4 g of Carbomer 940 to 40 mL of deionized water under high - speed stirring, disperse evenly, and then place it in a refrigerator at 4℃ for more than 12 h until the polymer swells completely to obtain a clear solution. Finally, stir the two evenly, add an appropriate amount of 0.5 mol·L -1 Sodium hydroxide solution to adjust the pH to 6.5, and then add 20 ml of deionized water to make the total amount of deionized water in the prescription reach 100 ml of the prescription amount, thus obtaining the 2.0% PNS gel - Carbomer 940 test sample.

[0165] The 0.5% PNS gel - carbomer 980 test article was prepared according to the method of Example 1.

[0166] The 2.0% PNS gel - carbomer 980 test article was prepared according to the method of Example 3.

[0167] The 4.0% PNS gel - carbomer 980 test article, except that the dosage of total saponins of Panax notoginseng was 4.0%, other components and the preparation method were according to Example 3; the pH of the above 3 test articles was uniformly adjusted to 6.5.

[0168] 3 Irritation study of ophthalmic gels in rabbit eyes

[0169] 3.1 Grouping settings

[0170] On the day when the adaptation period ended, fluorescein sodium examination was performed on both eyes of each animal. After the fluorescein sodium examination, 40 animals (half male and half female) with good condition, moderate body weight and body weight growth rate were selected for the experiment. The animals screened and excluded were transferred to standby animals.

[0171] In this experiment, 10 groups were set up as shown in Table 6 below. For both single - dose and multiple - dose groups, the right eye was given the negative control or the test article. Using the method of random block by body weight, the animals were randomly divided into 10 groups, namely Experiment Groups 1 - 10, with 4 animals in each group, half male and half female.

[0172] Table 6 Grouping of irritation test

[0173]

[0174] 3.2 Dosage, concentration and administration method

[0175] Negative S group, Negative M group: 0.9% sodium chloride injection.

[0176] Test article S group, Test article M group: The administration dosage was 0.4 g / eye / day (0.1 g / eye / time, 4 times / day). Weigh 0.1 g (error < 10%) of the corresponding test drug using a balance, and administer the drug to rabbits by eye - dropping (smearing), then gently close the eyelids for about 10 seconds without rinsing the eyes. The interval between two administrations was 2 h (±5 min).

[0177] 3.3 Stimulation examination indicators

[0178] Clinical observation: Observe the appearance signs, behavioral activities, glandular secretions, respiration, etc. of the animals once a day.

[0179] Eye irritation observation: ① Single-dose administration: Observe the eye irritation reaction at 1, 2, 4, 24, 48, and 72 hours after the last dose on Day 1. ② Multiple-dose administration: Observe the eye irritation reaction before each daily dose and at 1, 2, 4, 24, 48, and 72 hours after the last dose on Day 7. Since eye irritation examination has been performed within 24 hours before animal dosing, no eye irritation examination is conducted before the first dose on Day 1 for both single-dose and multiple-dose groups. The eye irritation reaction examination is carried out using a slit lamp microscope, and fluorescein sodium staining examination should be performed throughout the observation process. Any abnormal eye reactions should be recorded for each examination, and the scores should be calculated according to Table 7, and then calculate the average score of eye irritation reaction for each group.

[0180] Data processing and result evaluation: Calculate the mean eye irritation score for each group at each observation time point, and combine the statistics for male and female. Evaluate the irritation intensity according to Table 8, and compare with the negative control to evaluate the irritation of the test article to the rabbit eyes.

[0181] Table 7 Scoring criteria for eye irritation reaction

[0182]

[0183] Table 8 Evaluation criteria for eye irritation intensity

[0184]

[0185]

[0186] 4 Results of the irritation study of ophthalmic gel in rabbit eyes

[0187] During the test period, no obvious abnormalities were observed in the clinical observation of the systemic reactions of the animals in each group. No irritation occurred at each detection time point in the negative S group and the negative M group.

[0188] After single-dose administration, the irritation scores of the animals in each test article group at the detection time points were all between 0 and 3, indicating that no irritation was observed in the eyes of each test article group after single-dose administration. The results are shown in Table 9.

[0189] Table 9 Results of eye irritation scores after single-dose administration (Mean)

[0190] Drug group D-1 D1(1h) D1(2h) D1(4h) D2(24h) D3(48h) D4(72h) Negative S group 0 0 0 0 0 0 0 940 - S group 0 1 3 1 0 0 0 L - 980 - S group 0 0 0 0 0 0 0 M - 980 - S group 0 0 0 0 0 0 0 H - 980 - S group 1 2 2 3 2 0 0

[0191] Note: D represents the number of test days.

[0192] After multiple-dose administration, the eye irritation scores before each daily dose in the 940-M group increased from 1 to 5, and the irritation scores increased with the increase of the number of dosing days; the eye irritation scores before each daily dose in the H-980-M group increased from 4 to 5, and the irritation scores increased with the increase of the number of dosing days. There was no irritation in the L-980-M group and the M-980-M group. The results are shown in Table 10.

[0193] Eye irritation score results before daily administration after multiple-dose administration (Mean), Table 10

[0194] Drug group D1 D2 D3 D4 D5 D6 D7 Negative M group 0 0 0 0 0 0 0 940 - M group 0 1 1 2 3 4 5 L - 980 - M group 0 0 0 0 0 0 0 M - 980 - M group 0 0 0 0 0 0 0 H - 980 - M group 0 4 4 5 6 6 7

[0195] Note: D represents the number of days of the test.

[0196] After multiple-dose administration, the ocular irritation score of the 940-M group decreased from 3 to 1 at 1 h - 24 h after administration on the 7th day, and the ocular irritation scores at 48 h and 72 h were 0, indicating that after administration on the 7th day, the irritation gradually decreased with the prolongation of time; the ocular irritation score of the H-980-M group decreased from 5 to 2 at 1 h - 24 h after administration on the 7th day, and the ocular irritation scores at 48 h and 72 h were 0, indicating that after administration on the 7th day, the irritation gradually decreased with the prolongation of time. The L-980-M group and the M-980-M group had no irritation. The results are shown in Table 11.

[0197] Eye irritation score results after the 7th day of multiple-dose administration (Mean), Table 11

[0198]

[0199] Note: D represents the number of days of the test.

[0200] In summary, none of the test article groups showed irritation after single-dose administration; after multiple-dose administration, the irritation scores of the 940-M group on the 6th and 7th days were 4 and 5, showing mild irritation; the H-980-M group had mild irritation from the 1st to the 7th day, and mild irritation within 1 h - 2 h after administration on the 7th day; the L-980-M group and the M-980-M group had no irritation. It can be seen that from the perspective of multiple-dose administration, the 2.0% total saponins of Panax notoginseng ophthalmic gel using carbomer 940 and HPMC as matrices in Document 3 had mild irritation, while the 2.0% total saponins of Panax notoginseng ophthalmic gel using carbomer 980 as the matrix in the present invention had no irritation, indicating that the gel preparation of the present invention has more advantages in ocular safety than the gel in Document 3.

[0201] At the same time, by comparing the irritation of 0.5%, 2% and 4% total saponins of Panax notoginseng ophthalmic gel, it can be seen that when the total saponins of Panax notoginseng reached 4%, the gel showed mild irritation, indicating that too high a content of total saponins of Panax notoginseng in the gel would cause ocular irritation, while the 0.5% and 2.0% total saponins of Panax notoginseng ophthalmic gel had no irritation. The above research shows that the total saponins of Panax notoginseng gel with a drug loading of 0.5% - 2.0% has good safety.

[0202] Experimental Example 6 Ocular pharmacokinetics study of different total saponins of Panax notoginseng ophthalmic gel

[0203] 1 Instruments and animals

[0204] 1.1 Instruments

[0205] Liquid chromatography - mass spectrometry (Agilent, USA, including 1290 high - performance liquid chromatograph, 6460 triple quadrupole mass spectrometer, MassHunter workstation)

[0206] XPR6UD5 one - millionth electronic balance (METTLER TOLEDO, Switzerland)

[0207] High - speed centrifuge (ThermoFisher, model: Heraeus Pico 17)

[0208] Milli Q ultrapure water system (Millipore, USA)

[0209] KQ - 400KDE high - power numerically controlled ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.)

[0210] CMA 30 linear microdialysis probe (CMA, Sweden);

[0211] 1.2 Animals

[0212] 24 New Zealand white rabbits, 12 males and 12 females, weighing 2.0 - 2.5 Kg, provided by Tianjin Yuda Experimental Animal Breeding Co., Ltd., animal certificate number: SCXK (Jin) 2021 - 0001; Breeding location: Experimental Animal Center of Tianjin University of Traditional Chinese Medicine; Temperature: 25 ± 1 °C; Humidity (RH): 45 - 55%; Related research was carried out in accordance with the principles of animal experiments.

[0213] 2 Methods

[0214] 2.1 Sample determination conditions

[0215] The contents of ginsenoside Rg 1 , notoginsenoside R 1 and ginsenoside Re in the samples were determined by UPLC - MS / MS.

[0216] 2.1.1 Preparation of mixed reference substance solution

[0217] Precision weighing of ginsenoside Rg 1 reference substance 3.6700 mg (purity: 98.5%), notoginsenoside R 1 reference substance 3.2390 mg (purity: 90.4%), ginsenoside Re reference substance 3.6810 mg (purity: 96.0%), were respectively placed in 25 mL volumetric flasks, dissolved with methanol and diluted to the scale, shaken well to obtain the reference substance stock solution. After calculating according to the purity, the concentrations were 117.12 μg / mL for notoginsenoside R 1 , 117.12 μg / mL for ginsenoside Rg 1144.60 μg / mL, ginsenoside Re 141.35 μg / mL, stored at 4 °C. Additionally, appropriate amounts of the reference substance stock solutions were taken and placed in 100 mL volumetric flasks, diluted to the mark with methanol to obtain the mixed reference substance stock solutions, with concentrations of notoginsenoside R 1 1171.24 ng / mL, ginsenoside Rg 1 1229.10 ng / mL, ginsenoside Re 1201.49 ng / mL, stored at 4 °C.

[0218] 2.1.2 Preparation of Samples

[0219] 2.0% total notoginsenoside ophthalmic gel - carbomer 940, prepared according to the prescription and process in Document 3, specifically referring to the description in the "2 Sample Preparation Method" section of Experimental Example 5 above, hereinafter referred to as the "Document 3 Gel - 940 Group".

[0220] 0.5% total notoginsenoside ophthalmic gel - carbomer 980, prepared the gel sample according to Example 1 of the present invention, hereinafter referred to as the "0.5% Gel - 980 of the Present Invention".

[0221] 2.0% total notoginsenoside ophthalmic gel - carbomer 980, prepared the gel sample according to Example 3 of the present invention, hereinafter referred to as the "2.0% Gel - 980 of the Present Invention".

[0222] Notoginsenoside for injection: Prepared a notoginsenoside for injection solution with a concentration of 100 mg / mL using 0.9% physiological sodium chloride injection as the solvent.

[0223] 2.2 Pharmacokinetics Study of Total Notoginsenoside Gel in Rabbit Eyes

[0224] Twenty - four rabbits were randomly divided into four groups, with six rabbits in each group, namely the Document 3 Gel - 940 Group, the 0.5% Gel - 980 of the Present Invention Group, the 2.0% Gel - 980 of the Present Invention Group, and the Notoginsenoside for Injection Group. The microdialysis probe was implanted into the eyes of the rabbits, and physiological saline was perfused into the probe at a flow rate of 1.5

[0225] μL·min -1 After 1 h of equilibration, 0.1 g of ophthalmic gel (i.e., ginsenoside Rg 1 0.014 mg·kg -1 , notoginsenoside R 1 0.005 mg·kg -1 , ginsenoside Re 0.002 mg·kg -1 ) was instilled into the eyes of the rabbits in the gel groups, and 9.34 mg / kg of notoginsenoside for injection solution (i.e., ginsenoside Rg 1 3.69 mg·kg -1 , notoginsenoside R 11.13 mg·kg -1 , ginsenoside Re 0.57 mg·kg -1 ), start collecting the dialysate. Collect a microdialysate sample every 30 min from 0 - 6 h and every 1 h from 6 - 8 h, continuously collect for 8 h, and determine the contents of ginsenoside Rg 1 , ginsenoside Re and notoginsenoside R 1 in the microdialysate by LC - MS / MS method. Use WinNonlin version 6.0 pharmacokinetic software for data processing and analysis.

[0226] Pharmacokinetics results of microdialysis in rabbit eyes

[0227] Ginsenoside Rg 1 , ginsenoside Re, notoginsenoside R 1 The drug concentration - time curves are shown in Figures 5 - 7 , and the calculated pharmacokinetic parameters are shown in Tables 12 - 14.

[0228] Table 12 Main pharmacokinetic parameters of notoginsenoside R after administration in different groups 1 (n = 6)

[0229]

[0230] Note: Compared with the gel - 940 group in reference 3, * indicates P < 0.05, ** indicates P < 0.01; compared with the Xuesaitong for injection group, # indicates P < 0.05, ## indicates P < 0.01

[0231]

[0232]

[0233] Table 13 Main pharmacokinetic parameters of ginsenoside Rg after administration in different groups 1 (n = 6)

[0234] Note: Compared with the gel - 940 group in reference 3, * indicates P < 0.05, ** indicates P < 0.01; compared with the Xuesaitong for injection group, # indicates P < 0.05, ## indicates P < 0.01

[0235] Table 14 Main pharmacokinetic parameters of ginsenoside Re after administration in different groups (n = 6)

[0236]

[0237]

[0238] Note: Compared with the gel - 940 group in Document 3, * indicates P < 0.05 and ** indicates P < 0.01; compared with the Xuesaitong for injection group, # indicates P < 0.05 and ## indicates P < 0.01

[0239] Table 12 and Figure 5 showed that the AUC of notoginsenoside R1 in the 2.0% gel - 980 group of the present invention 0-8h was significantly higher than that in the gel - 940 group of Document 3 and the Xuesaitong for injection group (P < 0.05, P < 0.01). The AUC of the 2.0% gel - 980 group of the present invention 0-∞ was also significantly higher than that in the gel - 940 group of Document 3 and the Xuesaitong for injection group (P < 0.05, P < 0.01). In the 2.0% gel - 980 group of the present invention, the concentration - time curve of notoginsenoside R1 in the rabbit eye microdialysate showed an obvious double - peak phenomenon. The first peak appeared at 1 h and the second peak appeared at 4 h. The gel preparation of the present invention significantly improved the bioavailability of notoginsenoside R1 through the above - mentioned pharmacokinetic behavior.

[0240] Table 13 and Figure 6 showed that the AUC of notoginsenoside Rg 1 in the 2.0% gel - 980 group of the present invention 0-8h was significantly higher than that in the Xuesaitong for injection group (P < 0.01). The AUC of notoginsenoside Rg 1 in the 2.0% gel - 980 group of the present invention 0-∞ was significantly higher than that in the gel - 940 group of Document 3 and the Xuesaitong for injection group (P < 0.05, P < 0.01). In the 2.0% gel - 980 group of the present invention, the concentration - time curve of notoginsenoside Rg 1 in the rabbit eye microdialysate showed an obvious triple - peak phenomenon. The first peak appeared at 1 h, the second peak appeared at 4 h, and the third peak appeared at 6 - 7 h. The gel preparation of the present invention significantly improved the bioavailability of notoginsenoside Rg 1 through the above - mentioned pharmacokinetic behavior.

[0241] Table 14 and Figure 7 showed that the AUC of notoginsenoside Re in the 2.0% gel - 980 group of the present invention 0-8h was significantly higher than that in the gel - 940 group of Document 3 and the Xuesaitong for injection group (P < 0.05, P < 0.01). The AUC of notoginsenoside Re in the 2.0% gel - 980 group of the present invention 0-∞, significantly higher than that of the gel-940 group in Document 3 and the Xuesaitong for injection group (P<0.05, P<0.01). In the 2.0% gel-980 group of rabbits in the present invention, the concentration-time curve of notoginsenoside Re in the ocular microdialysate showed an obvious bimodal phenomenon. The first peak appeared at 1 h, and the second peak appeared at 4 h. At the same time, in the 2.0% gel-980 group of rabbits in the present invention, the concentration-time curve of notoginsenoside Re in the ocular microdialysate had a large plateau period from 5 h to 8 h, and the drug concentration was significantly higher than that of the gel-940 group in Document 3 and the Xuesaitong for injection group. The gel preparation of the present invention significantly improved the bioavailability of notoginsenoside Re through the above pharmacokinetic behavior.

[0242] The ophthalmic gel of the present invention can effectively improve the bioavailability of total notoginsenosides in the eye. The ophthalmic gel of total notoginsenosides acts directly on the eye, has good bioadhesion and biocompatibility, and under the action of excipients, total notoginsenosides are absorbed into the eye through the corneal route and the conjunctiva-scleral route. The corneal route can successively pass through the anterior chamber, the lens and reach the posterior chamber and the retina. The conjunctiva-scleral route can either reach the vitreous body through the capillaries via the circulation, or be absorbed into the blood through the nasolacrimal duct system and then diffuse into the eye from the blood. It can be seen from the drug concentration-time curve graph that for the gel of the present invention, especially the 2.0% ophthalmic gel of total notoginsenosides-980 group, the drug concentration reached the first peak at 1 h after administration, and the drug concentration reached the second peak at 4 h. It is speculated that substances such as carbomer 980, mannitol, and EDTA-2Na in the prescription of the present invention may jointly play roles in promoting penetration, adhesion, regulation, etc., smooth the two routes of drug absorption in the eye, increase the residence time of the drug in the eye, regulate the microenvironment of the eye, increase the penetration of the drug in the eye, not only deliver the drug to act on the retinal part of the eye through the corneal route, but also through the conjunctiva-scleral route, be absorbed into the blood through the nasolacrimal duct system and reach the eye again through the systemic circulation. The drug overlaps with the drug absorbed through the corneal part of the eye tissue, increases the drug concentration and bioavailability in the eye, forms the second and third eye absorption peaks, and maintains a high drug concentration in the eye for 5-8 h. This is a phenomenon that does not occur in other ophthalmic preparations or ophthalmic gel preparations, and it is a unique and unexpected effect of the gel of the present invention. The Xuesaitong injection solution is administered by intravenous injection and can only enter the eye through the systemic circulation. At the same time, due to the effect of the blood-eye barrier, the drug concentration reaching the eye is relatively low, the drug absorption is poor, and there is only one absorption peak. Similarly, for the 2.0% ophthalmic gel of total notoginsenosides, there is only one absorption peak in the pharmacokinetics of the gel-940 group in Document 3 in the eyes of rats, that is, the absorption peak at 1 h, and the bioavailability is lower than that of the 2.0% gel-980 of the present invention, indicating that the carbomer 980 in the gel matrix of the present invention has a significantly higher promoting effect on the pharmacokinetics of total notoginsenosides in the eye than the mixed matrix of carbomer 940 and HPMC in Document 3.

[0243] Experimental Example 7 Pharmacodynamic Study of Different Ophthalmic Gels of Total Notoginsenosides

[0244] 1. Instrument

[0245] Analytical balance MCA2.7S-2CCN-M (Sartorius, Germany), electronic balance XY3000-2C (Changzhou Lucky Electronic Equipment Co., Ltd.), high-speed refrigerated centrifuge H1650R (Shanghai Lu Xiangyi Centrifuge Instrument Co., Ltd.), surgical microscope SM-201 (Shanghai Yuyan Scientific Instrument Co., Ltd.), handheld fundus camera Smartscope M5 (Ocebo, Finland), inverted microscope and camera system CKX41 (OLYMPUS, Japan), JY-Clear ECL luminometer (Shanghai Ruixi Instrument Equipment Co., Ltd.).

[0246] 2 Methods

[0247] 2.1 Diabetic rat modeling method

[0248] 2.1.1 Construction of rat type 2 diabetes model (DM model)

[0249] After SD rats were adaptively fed for 3-7 days, they were intraperitoneally injected with 30 mg / mL STZ solution at a dose of 30 mg / kg, and the normal group rats were intraperitoneally injected with an equal dose of 0.1 mol / L citric acid buffer. After all rats were injected, the DM group rats were fed with high-fat feed, and the normal group rats were fed with normal feed. After 5-7 days of feeding, random blood glucose values ​​were measured (without fasting).

[0250] ≥16.7mmol / L can be used to confirm that the type 2 diabetes rat model has been successfully established. Rats that have not developed type 2 diabetes can be injected intraperitoneally with STZ solution again.

[0251] 2.1.2 Construction of rat DR model

[0252] After the type 2 diabetes model was successfully established, the DM group rats were fed a mixture of ordinary feed and high-fat feed. During the period, funduscopy was performed to detect the retinopathy of the rats every 1 month. If abnormal new blood vessels or vascular tortuosity were observed in the rats under the funduscopy, the DR model was determined to be successful. If the funduscopy found that the animal image was blurred and preliminary lens opacity appeared, it indicated that the animal developed early cataract after the formation of diabetic retinopathy. If the rats reached the number of DR in the group, they began to be divided into groups for drug administration.

[0253] 2.2 Medication, Dosage and Schedule

[0254] 2.0% Panax notoginseng total saponins eye gel - Carbomer 940 was prepared using the prescription and process of Reference 3, for details, see the description of "2 Sample preparation method" in Experimental Example 5 above, hereinafter referred to as "Reference 3 Gel-940".

[0255] 0.5% Total Notoginseng Saponins Ophthalmic Gel - Carbomer 980, using the gel sample prepared in Example 1 of the present invention, hereinafter referred to as the 0.5% Gel - 980 of the present invention.

[0256] 2.0% Total Notoginseng Saponins Ophthalmic Gel - Carbomer 980, using the gel sample prepared in Example 3 of the present invention, hereinafter referred to as the 2.0% Gel - 980 of the present invention.

[0257] Xuesaitong for Injection: Prepared into an Xuesaitong for Injection solution with a concentration of 100 mg / mL using 0.9% physiological sodium chloride injection as the solvent.

[0258] The successfully established DR model rats were randomly grouped for drug administration according to body weight. There were 10 rats in the normal group and the model group, and no drug was given to either group; there were 12 rats in each of the Gel - 940 group in the literature, the 0.5% Gel - 980 group of the present invention, and the 2.0% Gel - 980 group of the present invention. The corresponding gel samples were administered, and the administration method was 100 μL / eye, both eyes were medicated, once a day, for 56 consecutive days; there were 10 rats in the Xuesaitong for Injection group, and 100 mg / mL Xuesaitong for Injection was administered at 100 mg / kg by intraperitoneal injection, once a day, for 56 consecutive days.

[0259] 2.3 Sample Collection and Detection Indicators

[0260] 2.3.1 Detection of Eye Cataract

[0261] After 8 weeks of drug intervention, all animals in each group were examined, the incidence of cataract was calculated, and cataract scores were made. The cataract scoring criteria are shown in Table 15.

[0262] Table 15 Cataract Scoring Criteria

[0263] Score Degree grading 0 Lens transparent (no cataract) 1 Peripheral vesicles and opacity 2 Central opacity 3 Diffuse central opacity 4 Mature cataract 5 Hypermature cataract

[0264] 2.3.2 Immunohistochemistry

[0265] After 8 weeks of drug intervention, the rats in each group were anesthetized and the eyeballs were collected. The left eye was used for immunohistochemistry: after being fixed in 4% paraformaldehyde for 30 min, dehydration was carried out using 5% - 15% - 30% gradient concentrations of sucrose (room temperature), and the dehydrated eyeballs were placed in a disposable embedding cassette and embedded and frozen using OCT. A cryostat was used to make frozen sections of the embedded frozen tissue. The specific operation steps are as follows:

[0266] 1) Using Anti - VEGF (1:50) and Anti - AGE (1:400) as the primary antibodies, immunohistochemical staining was performed on the frozen sections of the rat eyeball retina tissue.

[0267] 2) Antigen retrieval: Place the sections in antigen retrieval solution 1x EDTA 9.0 (Maixin, Fuzhou, MVS-0098, pH 9.0, 50×, dilution ratio 1:49 in pure water). Heat the sections in a microwave oven at high power for 10 min, then let them cool naturally at room temperature for about 30 min. Wash with 0.1% Triton TBS 3×3 min.

[0268] 3) Block endogenous catalase: Drain the water droplets on the slides, place the slides on a wet box, add 200 μL of 3% H2O2 to each slide, incubate at room temperature for 5 min, and wash with 0.1% Triton TBS 3×3 min.

[0269] 4) Block non-specific antigen epitopes: Drain the water droplets on the slides, add and incubate the blocking solution on the sections in a wet box at room temperature for 1 h (Solarbio, SL038, Blocking Goat Serum 20×, dilution ratio: 1:19 in PBS).

[0270] 5) Bind antibodies: Pour off the supernatant, place the sections in a wet box, and add 200 μL of primary antibody (primary antibody diluted in Signal Up immunostaining primary antibody diluent) to each section sample. After incubating at room temperature for 1 h, place it in 0.1% Triton TBS and wash on a shaker 3×5 min. Incubate with Peroxidase AffiniPure Goat Anti-Rabbit / Mouse IgG(H+L) (diluted 1:500 in PBS) at room temperature for 30 min, and place it in 0.1% Triton TBS and wash on a shaker 3×5 min.

[0271] 6) Substrate chromogenic reaction: Add about 200 μL of DAB chromogenic reagent (Maixin, Fuzhou, DAB-1031, DAB kit (20×), preparation method: Place 2 mL of pure water in a centrifuge tube, and add 2 drops of solution A, B, and C in sequence). Chromogenize at room temperature for 5 min, and immediately immerse in tap water to terminate the reaction.

[0272] 7) Nuclear counterstaining and dehydration and mounting: Counterstain the cell nuclei with hematoxylin for 1 min, then dehydrate with gradient ethanol (75% ethanol for 5 s - 95% ethanol for 5 s - absolute ethanol I for 1 min - absolute ethanol II for 5 min - xylene 3×5 min), and mount with neutral gum.

[0273] 8) Finish making the slides and air-dry the sections for more than 6 h. Digitally scan all the sections, and use the digital quantitative analysis software QuPath to statistically analyze the AGEs and VEGFA positive signals in the retinal tissue of the experimental samples respectively.

[0274] 2.3.3 HE staining:

[0275] After 8 weeks of drug intervention, the rats in each group were anesthetized and their eyeballs were collected. The right eyeballs were used for HE staining.

[0276] 1) Preparation of tissue sections: The right eyeballs of the rats were fixed in an eyeball fixative, embedded, and paraffin sections were cut parallel to the optic nerve to reach the cornea and retina at the same time.

[0277] 2) Dewaxing: The paraffin section samples baked on the staining rack in the oven were immersed in xylene I for 20 min, then transferred to xylene II and immersed for 20 min. After the wax on the samples was dissolved, the samples were transferred to absolute ethanol I and immersed for 5 min, then transferred to absolute ethanol II and immersed for 5 min. The samples were brushed with absolute ethanol for 20 s and then transferred to a water basin, and the alcohol on the samples was rinsed off with running tap water.

[0278] 3) HE staining and dehydration: The sections were stained in hematoxylin, a HE staining solution in the staining cylinder, for 3 - 5 min, and then taken out and washed with water in a staining cup until the sections were colorless. The sections were immersed in the differentiating solution, a HE staining solution in the staining cylinder, for 3 - 5 s, and quickly washed with water. The sections were immersed in the blueing solution, a HE staining solution in the staining cylinder, for 3 - 5 s, and quickly washed with water. The sections were successively immersed in 85% ethanol, 95% ethanol, eosin, a HE staining solution, absolute ethanol I, absolute ethanol II, absolute ethanol III, n-butanol, xylene I, and xylene II in the staining cylinders, and each cylinder was immersed for 3 - 5 min.

[0279] 4) Sealing the slides: After taking out the sections and quickly drying them in the air outlet, the slides were sealed with neutral balsam.

[0280] 5) Scanning: After the sections were scanned and imaged, 5 fields of view were randomly selected at 20.0× to measure the retinal thickness. 3 position points were measured for each retinal pathological photo, and Image-Pro Plus 6.0 software was used for calculation.

[0281] 3 Experimental results

[0282] 3.1 Cataract conditions of rats in each group

[0283] No rats in the normal group developed cataracts, so no data were shown in the figure. As Figure 8 shown, compared with the normal group, the incidence of cataracts in the model group, the literature 3 gel - 940 group, the 0.5% gel - 980 group of the present invention, the 2.0% gel - 980 group of the present invention, and the group injected with Xuesaitong showed an upward trend. In the first 2 weeks, the incidence of cataracts in the group injected with Xuesaitong was the lowest, but in the 3rd - 8th weeks, the incidence of cataracts in the 2.0% gel - 980 group of the present invention was the lowest. Especially in the 3rd - 6th weeks, the incidence of cataracts was significantly lower than that of other groups, indicating that the 2.0% gel - 980 of the present invention has significant advantages in reducing the incidence of diabetic cataracts.

[0284] AsFigure 9 As shown, compared with the normal group, the cataract score of the rats in the model group was significantly higher (P < 0.01); the cataract score of the 2.0% gel-980 group of the present invention was the lowest; the cataract scores of the Sanse Tong injection group and the 0.5% gel-980 group of the present invention were relatively high; the cataract score of the gel-940 group in Document 3 was the highest, close to the model group, indicating that the 2.0% gel-980 of the present invention has significant advantages in reducing the symptoms of diabetic cataract and delaying the disease progression.

[0285] Based on the above results, it is shown that the total saponins of Panax notoginseng ophthalmic gel of the present invention can delay the onset of diabetic cataract, reduce the incidence of cataract, and has a good effect against diabetic cataract.

[0286] 3.2 Positive area expression rates of VEGFA and AGEs in the retinas of rats in each group

[0287] VEGFA is the most common type of VEGF. VEGF stimulates the growth of new blood vessels and increases the permeability of existing blood vessels. VEGFA is overexpressed in the retinas of DR rats, which can cause abnormal blood vessel proliferation and blood vessel leakage, proving that DR rats have developed diabetic retinopathy. AGEs are deposited in the retinas of DR rats, aggravating the retinopathy and leading to the aggravation of DR.

[0288] As Figure 10 shown, compared with the normal group, the positive area expression rate of VEGFA in the retinas of rats in the model group increased significantly by 155.56%. Compared with the model group, the positive area expression rates of VEGFA in the retinas of rats in the 2.0% gel-980 group and the 0.5% gel-980 group of the present invention decreased significantly by 52.34% and 47.35% (P < 0.05, P < 0.05), while the positive area expression rate of VEGFA in the retinas of rats in the gel-940 group in Document 3 decreased by 24.61% (P < 0.05). Compared with the gel-940 group in Document 3, the positive area expression rates of VEGFA in the retinas of rats in the 2.0% and 0.5% gel-980 groups of the present invention decreased by 36.78% and 30.17% (P < 0.05, P < 0.05). Compared with the Sanse Tong injection group, the positive area expression rates of VEGFA in the retinas of rats in the 2.0% and 0.5% gel-980 groups of the present invention decreased by 40.23% and 33.98% (P < 0.05, P < 0.05). It can be seen that the 0.5% and 2.0% gel-980 of the present invention can significantly reduce VEGFA in the retinas of rats, which is significantly better than the gel-940 in Document 3 and the Sanse Tong injection.

[0289] As Figure 11As shown, compared with the normal group, the positive area expression rate of AGEs in the retinas of rats in the model group increased by 75.50%. Compared with the model group, the positive area expression rates of AGEs in the retinas of rats in the 2.0% gel-980 group of the present invention, the 0.5% gel-980 group of the present invention, the gel-940 group of Document 3, and the Xuesaitong for injection group decreased significantly by 73.79%, 39.89%, 30.20%, and 45.01% (P<0.01, P<0.05, P<0.05, P<0.05). Compared with the gel-940 group of Document 3 and the Xuesaitong for injection group, the positive area expression rate of AGEs in the retinas of rats in the 2.0% gel-980 group of the present invention decreased significantly by 52.33%. It can be seen that the 2.0% gel-980 of the present invention significantly reduces AGEs in the retinas of rats, and is superior to the gel-940 of Document 3 and Xuesaitong for injection.

[0290] From the above results, it can be known that the 2.0% and 0.5% gel-980 of the present invention can significantly reduce the content of VEGFA in the retinas of DR rats, and has the effect of inhibiting abnormal angiogenesis in the retina; it can reduce the content of AGEs in the retina and relieve the degree of retinal lesions. 3.3 Width of HE staining of retinas of rats in each group

[0291] As Figure 12 shown, compared with the normal group, the retinal thickness of rats in the model group increased significantly by 41.55% (P<0.05); compared with the model group, the retinal thicknesses of rats in the 2.0% gel-980 group of the present invention and the Xuesaitong for injection group decreased significantly by 38.50% and 26.61% respectively (P<0.05, P<0.05). Compared with the gel-940 group of Document 3, the retinal thickness of rats in the 2.0% gel-980 group of the present invention decreased significantly by 29.72% (P<0.05). Compared with the Xuesaitong for injection group, the retinal thickness of rats in the 2.0% gel-980 group of the present invention decreased significantly by 16.19% (P<0.05). It can be seen that the 2.0% gel-980 of the present invention significantly reduces the retinal thickness of rats, and is significantly superior to the gel-940 of Document 3 and Xuesaitong for injection in inhibiting the thickness of retinal lesions.

[0292] 4 Pharmacodynamic summary

[0293] The total notoginsenoside ophthalmic gel of the present invention can reduce the incidence rate of diabetic cataract and delay the onset process of cataract; significantly reduce the retinal thickness, significantly reduce the contents of VEGFA and AGEs in the retinas of DR rats, and effectively treat diabetic retinopathy, and its effect is superior to the gel-940 of Document 3 and Xuesaitong for injection.

Claims

1. A Panax notoginseng saponin ophthalmic gel preparation, characterized in that: In parts by weight, it includes the following ingredients: An appropriate amount of pH adjuster is used to adjust the pH of the ophthalmic gel preparation to 6.0-8.0; The amount of water for injection is made up to 100 parts by weight.

2. The Panax notoginseng saponins ophthalmic gel preparation according to claim 1, characterized in that: The content of Panax notoginseng total saponins is 0.5 parts by weight, 1.0 parts by weight or 2.0 parts by weight.

3. The Panax notoginseng total saponins ophthalmic gel preparation according to claim 1, characterized in that: The content of EDTA-2Na is 0.05 parts by weight, 0.055 parts by weight or 0.06 parts by weight.

4. The Panax notoginseng saponins ophthalmic gel preparation according to claim 1, characterized in that: The content of Carbomer 980 is 0.2 parts by weight, 0.25 parts by weight or 0.3 parts by weight.

5. The Panax notoginseng total saponins ophthalmic gel preparation according to claim 1, characterized in that: Wherein the content of mannitol is selected from 4.1 parts by weight.

6. The Panax notoginseng total saponins ophthalmic gel preparation according to claim 1, characterized in that: Wherein the preservative is selected from benzalkonium chloride or benzalkonium bromide.

7. The Panax notoginseng saponins ophthalmic gel preparation according to claim 1, characterized in that: The content of the preservative is selected from 0.005-0.05 parts by weight.

8. The Panax notoginseng total saponins ophthalmic gel preparation according to claim 1, characterized in that: The content of the preservative is selected from 0.009-0.01 parts by weight.

9. The Panax notoginseng total saponins ophthalmic gel preparation according to claim 1, characterized in that: The pH regulator is selected from sodium hydroxide or potassium hydroxide.

10. The Panax notoginseng total saponins ophthalmic gel preparation according to claim 1, characterized in that: The total content of total saponins in the Panax notoginseng total saponins exceeds 85%.

11. The Panax notoginseng total saponins ophthalmic gel preparation according to claim 1, characterized in that: The prescription composition is as follows: Sodium hydroxide is used to adjust the pH to 6.5; Make up to 5000g with water for injection.

12. The Panax notoginseng saponins ophthalmic gel preparation according to claim 1, characterized in that: The prescription composition is as follows: Add appropriate amount of sodium hydroxide to adjust the pH to 6.5; make up to 5000g with water for injection.

13. The Panax notoginseng saponins ophthalmic gel preparation according to claim 1, characterized in that: The prescription composition is as follows: Add appropriate amount of sodium hydroxide to adjust the pH to 6.5; make up to 5000g with water for injection.

14. The Panax notoginseng total saponins ophthalmic gel preparation according to any one of claims 1 to 13, wherein the content of notoginseng saponins R1 is 9.70%, the content of ginsenoside Rg1 is 31.00%, the content of ginsenoside Re is 4.20%, the content of ginsenoside Rb1 is 31.90%, and the content of ginsenoside Rd is 8.80%.

15. The method for preparing the Panax notoginseng total saponins ophthalmic gel preparation according to claim 1, characterized in that: The steps include: (1) Under light-proof conditions, weigh notoginseng total saponins, EDTA-2Na and preservatives, place them in water for injection, heat and stir to dissolve, and obtain a drug solution; (2) Weigh mannitol and carbomer 980, disperse them evenly, slowly add them into water for injection under high-speed stirring, continue heating and stirring to obtain a clear matrix solution; (3) Add the clear matrix solution obtained in step (2) to the drug solution obtained in step (1), stir, add a pH adjuster to adjust the pH to 6.0-8.0, add water for injection to 100 parts by weight, stir evenly, and obtain a gel.

16. The preparation method according to claim 15, characterized in that: The gel finally prepared is sterilized by high pressure steam, taken out, cooled, and then filled in a sterile environment.

17. The preparation method according to claim 15, characterized in that: In step (1), the concentration of Panax notoginseng total saponins in the medicinal solution is 6.25-100 g / L; the concentration of EDTA-2Na in the medicinal solution is 0.625-2.5 g / L, the heating temperature is 50-60° C., and the stirring speed is 200 rpm.

18. The preparation method according to claim 15, characterized in that: In step (2), the concentration of mannitol in the matrix solution is 51-205 g / L; the concentration of carbomer 980 in the matrix solution is 3.1-12.5 g / L, the stirring speed is 350-400 rpm, and the heating temperature is 65-70°C.

19. The preparation method according to claim 15, characterized in that: In step (2), the heating and stirring time is 3-5 hours.

20. The preparation method according to claim 19, characterized in that: In step (2), the heating and stirring time is 4 hours.

21. The preparation method according to claim 15, characterized in that: In step (3), the stirring conditions for stirring after the matrix solution is added to the drug solution are: stirring speed of 40 rpm, stirring time of 5 minutes; the stirring conditions for stirring evenly after replenishing the injection water are: stirring speed of 40 rpm, stirring time of 45 minutes.

22. The preparation method according to claim 15, characterized in that: The high pressure steam sterilization conditions are 121℃, 30min.

23. Use of the Panax notoginseng total saponin ophthalmic gel preparation according to any one of claims 1 to 14 in the preparation of a drug for preventing and / or treating diabetic retinopathy or diabetic cataract.

24. The use according to claim 23, wherein the diabetic retinopathy comprises changes in retinal thickness, changes in retinal vascular integrity and permeability, abnormal retinal vascular proliferation or retinal vascular leakage.

25. The use according to claim 23, characterized in that The Panax notoginseng total saponin ophthalmic gel preparation repairs retinal integrity and permeability.

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