Panax notoginseng saponins eye gel preparation, preparation method and use thereof

Abstract

The application discloses a panax notoginseng saponins ophthalmic gel preparation and a preparation method and application thereof. The panax notoginseng saponins ophthalmic gel preparation comprises panax notoginseng saponins, EDTA-2Na, a gel matrix carbomer 980, mannitol, a preservative, a pH regulator, water for injection and the like. Through optimization and screening of each component and content, the panax notoginseng saponins ophthalmic gel preparation can deliver the drug into the eye for multiple times, improve the bioavailability of the eye, and improve the pharmacodynamic effect of preventing and treating diseases such as diabetic retinopathy and diabetic cataract, and has good clinical applicability and drug property.

Description

Technical Field

[0001] This invention relates to the pharmaceutical field, specifically to a total saponin ophthalmic gel preparation of Panax notoginseng, its preparation method, and its uses. Background Technology

[0002] Diabetic retinopathy (DR) is a retinal complication of diabetes, caused by long-term hyperglycemia leading to damage to the microvessels of the retina. It is a chronic, progressive, and blinding eye disease. Cataracts are one of the major eye complications in diabetic patients. Cataracts tend to occur at a younger age in diabetic patients, and having DR is a risk factor for developing diabetic cataracts. Currently, there are over 140 million diabetic patients in my country, making the prevention and treatment of diabetes and its related complications an urgent priority. DR and diabetic cataracts are the most serious complications of diabetes, affecting approximately one in three diabetic patients. DR and diabetic cataracts have become leading causes of vision loss and blindness among working-age individuals.

[0003] Total saponins of Panax notoginseng are included in the 2020 edition of the Chinese Pharmacopoeia, and are the total saponins processed from the main root or rhizome of Panax notoginseng, a plant of the Araliaceae family. Total saponins of Panax notoginseng have the effects of promoting blood circulation, removing blood stasis, and clearing the meridians. In 2015, it was reported that total saponins of Panax notoginseng, when administered orally, can reduce the expression of acidic protein and mRNA in glial fibrils of rat retinal tissue and can protect the retina of streptozotocin-induced diabetic rats (Yao Qing, Ma Xiaodong, Zhang Qian, et al. Protective effect of total saponins of Panax notoginseng on retinal function in early diabetic rats [J]. Ningxia Medical Journal, 2014, 36(03):204-206.). A 2024 cellular experimental study showed that total saponins of Panax notoginseng alleviate diabetic retinopathy by inhibiting the activation of the NF-κB signaling pathway, thereby inhibiting retinal inflammation (YaruWang, 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.). Therefore, total saponins of Panax notoginseng are a highly effective drug for treating diabetic retinopathy.

[0004] Panax notoginseng saponins need to be formulated into specific dosage forms before they can be used to treat eye diseases. Oral, injectable, and ophthalmic formulations are commonly used dosage forms of Panax notoginseng saponins. Among them, ophthalmic formulations cross the physiological and anatomical barriers of the eye and act directly on the eye, offering the advantage of direct and high efficacy. They are widely used to deliver drugs to the eye to treat eye diseases such as diabetic retinopathy. Topical ophthalmic formulations require high levels of local drug release and absorption; the higher the drug concentration in the eye, the closer it is to reaching the therapeutic window, and the more significant the therapeutic effect.

[0005] Eye drops and creams are currently the most commonly used dosage forms in clinical practice. Reference 1 (Chinese Patent Application 201711262528.6) discloses an ophthalmic drug preparation of Panax notoginseng saponin extract, its preparation method, and its application. This drug preparation consists of Panax notoginseng extract with a total saponin content of 45-95%, and is available in various dosage forms including eye drops, ointments, eye patches, and hydrogels. However, eye drops experience a rapid decrease in drug concentration after instillation, resulting in a short residence time and poor bioavailability. Creams are viscous, affecting vision and causing poor patient compliance. Hydrogel formulations contain too low a carbomer content, which is diluted by tears after application, further reducing viscosity and resulting in a short residence time and unsatisfactory bioavailability.

[0006] Ophthalmic gels are a novel type of ophthalmic preparation. By adding bio-adhesive materials to eye drops, the viscosity of the solution increases, prolonging the retention time of the medication in the eye, enhancing corneal permeability, and promoting drug penetration into the eye. Reference 2 (Chinese Patent Application 201110453135.X) discloses an eye drop containing total saponins of Panax notoginseng and its preparation method. The viscosity modifier in this method is one of sodium hyaluronate, methylcellulose, carbomer, or hydroxypropyl cellulose. However, the viscosity and osmotic pressure of this preparation are not ideal, resulting in poor bioavailability. The applicant has been committed to the research and development of ophthalmic preparations of total saponins of Panax notoginseng. Document 3 (Chinese Patent 202210010949.4) is an early application of the applicant, which discloses an ophthalmic gel preparation of total saponins of Panax notoginseng, its preparation method and uses. The bioadhesive material of the ophthalmic gel preparation is a low-viscosity cellulose polymer and an acrylic polymer. By using the two polymers in combination, the ophthalmic bioavailability of the gel is improved to a certain extent. However, the degree to which the gel improves the ophthalmic bioavailability is not enough to meet the needs of clinical treatment. Moreover, the swelling process of the viscous excipients is difficult, the preparation process is complicated, and the time cost is high, which hinders the application of this dosage form.

[0007] Given the current shortcomings and drug-like defects of existing Panax notoginseng total saponins ophthalmic preparations, there is still a huge demand for Panax notoginseng total saponins ophthalmic preparations with high bioavailability and high efficacy for the treatment and / or prevention of diabetic retinopathy. There is also a huge industrial demand for new preparation processes of Panax notoginseng total saponins with good stability and energy efficiency. Summary of the Invention

[0008] To address some of the technical problems in existing technologies, the inventors, through in-depth research and optimization of the formulation and preparation process, have provided a Panax notoginseng total saponin ophthalmic gel with high ocular bioavailability and good therapeutic effect on diabetic retinopathy. This invention also provides a method for preparing the ophthalmic gel, which is convenient, efficient, and yields a product of stable quality.

[0009] In a first aspect, the present invention provides a Panax notoginseng total saponins ophthalmic gel formulation, comprising the following components:

[0010] 0.5-2.0 parts by weight of total saponins from Panax notoginseng;

[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] Use an appropriate amount of pH adjuster to adjust the pH of the ophthalmic gel preparation to 6.0-8.0;

[0016] Add water for injection to a total of 100 parts by weight.

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

[0018] According to the present invention, the total saponins ophthalmic gel preparation of Panax notoginseng can be a commercially available product, such as the total saponins of Panax notoginseng purchased from Kunming Pharmaceutical Group Co., Ltd., batch number: JK2022041, wherein the content of Panax notoginseng saponin 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] In the Panax notoginseng total saponin ophthalmic gel formulation according to the present invention, preferably, the pH adjuster includes sodium hydroxide or potassium hydroxide.

[0020] In the Panax notoginseng total saponin ophthalmic gel formulation according to the present invention, preferably, the preservative includes benzalkonium chloride or benzalkonium bromide.

[0021] A second aspect of the present invention provides a method for preparing a Panax notoginseng total saponins ophthalmic gel formulation according to the first aspect, comprising the following steps:

[0022] (1) Under light-protected conditions, weigh out total saponins of Panax notoginseng, EDTA-2Na and preservative and place them in water for injection. Heat and stir to dissolve them to obtain a drug solution.

[0023] (2) Weigh mannitol and carbomer 980, disperse them evenly, and slowly add them to water for injection under high-speed stirring. Continue heating and stirring to obtain a clear matrix solution.

[0024] (3) Add the clear matrix solution obtained in step (2) to the drug solution obtained in step (1), stir, add pH adjuster to adjust pH to 6.0-8.0, add water for injection to 100%, stir evenly to obtain gel.

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

[0026] According to the preparation method of the present invention, preferably, in step (1), the concentration of the total saponins of Panax notoginseng 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℃ 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; and 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 after the matrix solution is added to the drug solution are: stirring speed of 40 rpm and stirring time of 5 minutes; the stirring conditions after adding water for injection and stirring until uniform are: stirring speed of 40 rpm and stirring time of 45 minutes.

[0029] According to the preparation method of the present invention, preferably, in step (3), the pH is adjusted with an alkaline solution, including but 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 performed at 121°C for 30 minutes.

[0031] A third aspect of the invention provides the use of the Panax notoginseng total saponins ophthalmic gel formulation according to the first aspect in the preparation of a medicament for the prevention and / or treatment of diabetic retinopathy or diabetic cataract.

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

[0033] According to the uses described in this invention, it is further preferred that the Panax notoginseng total saponins ophthalmic gel preparation can reduce retinal vascular permeability.

[0034] The beneficial effects of this invention are:

[0035] Compared with Reference 3, this invention is a further improvement based on previous research. From the perspective of process convenience and improved bioavailability, this invention omits hydroxypropyl methylcellulose in the composite gel matrix of Reference 3, eliminating the adverse effects of the prolonged swelling time of hydroxypropyl methylcellulose. Simultaneously, it changes the carbomer gel type, using a single gel matrix, Carbomer 980, and adds mannitol to adjust osmotic pressure. Mannitol also promotes the dispersion and swelling of carbomer. The inventors initially thought that using a single gel matrix might reduce gel viscosity and other properties; however, through experiments, they were surprised to find that the Panax notoginseng total saponins ophthalmic gel formulation of this invention is more reasonable. Not only can the osmotic pressure and pH value of the gel remain constant (Example 2), and the components exhibit good stability (Example 3), and are non-irritating (Example 6), but the process is also robust, the matrix swelling speed is fast, and the efficiency is improved by 33.3% (Examples 1, 5). The carbomer 980 single matrix exhibits excellent gelling properties. Ocular pharmacokinetic results of the gel of this invention show that, under the ocular delivery of the gel formulation of this invention, total saponins of Panax notoginseng can be absorbed into the eye and reabsorbed into the eye through systemic circulation, significantly improving ocular drug concentration and bioavailability (Example 7). With the support of a specific gel carrier, combined with the enhancement effect of EDTA-2Na on gel efficacy, the ocular gel of total saponins of Panax notoginseng not only significantly reduces retinal thickness and the content of VEGFA and AGEs in the retina of DR rats, but also significantly reduces the incidence of diabetic cataracts and alleviates the severity of cataracts (Example 8). The ocular gel of total saponins of Panax notoginseng of this invention has significant advantages in process, safety, ocular pharmacokinetics, and pharmacodynamics. Attached Figure Description

[0036] Figure 1 The number of cellless capillaries in the retina in Experiment Example 3

[0037] Figure 2 The ratio of pericyte missing length to total length in Experiment Example 3

[0038] Figure 3 : Retinal tight junction protein imprint in Experiment Example 3

[0039] Figure 4 Occludin protein expression in Experiment Example 3

[0040] Figure 5 : Concentration-time curve of Panax notoginsenoside R1 in rabbit eye microdialysis solution in Experiment Example 6

[0041] Figure 6 : Concentration-time curve of ginsenoside Rg1 in rabbit eye microdialysis solution in Experiment Example 6

[0042] Figure 7: Concentration-time curve of ginsenoside Re in rabbit ocular microdialysis solution in Experiment Example 6

[0043] Figure 8 The incidence rate of cataracts in rats of each group during the drug intervention period in Experiment Example 7

[0044] Figure 9 Cataract scores of rats in each group after 8 weeks of drug intervention in Experiment Example 7. Note: * indicates P<0.05 compared with the blank group; # indicates P<0.05 compared with the model group; Δ indicates P<0.05 compared with the gel-940 group in Reference 3; ▲ indicates P<0.05 compared with the XueSaiTong injection group.

[0045] Figure 10 The expression rate of VEGFA-positive area in the retina of rats in Experiment Example 7. Note: Compared with the blank group, * indicates P<0.05; compared with the model group, # indicates P<0.05; compared with the gel-940 group in Reference 3, Δ indicates P<0.05; compared with the XueSaiTong injection group, ▲ indicates P<0.05.

[0046] Figure 11 : Expression rate of AGEs positive area in the retina of rats in each group in Experiment 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 gel-940 group in Reference 3, Δ indicates P<0.05; compared with the XueSaiTong injection group, ▲ indicates P<0.05.

[0047] Figure 12 In Experiment 7, HE staining was used to detect retinal thickness in each group of rats. Note: Compared with the blank group, * indicates P<0.05; compared with the model group, # indicates P<0.05; compared with the gel-940 group in Reference 3, Δ indicates P<0.05; compared with the injection of XueSaiTong group, ▲ indicates P<0.05. Detailed Implementation

[0048] Various exemplary embodiments of the present invention will now 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, features, and embodiments of the present invention.

[0049] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that the upper and lower limits of the range and each intermediate value between them are specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0050] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0051] Those skilled in the art should understand that, as long as the purpose of the present invention can be achieved, other steps or operations may be included in steps (1)-(4) of the method of the present invention, or before or after the steps, for example, to further optimize and / or improve the method of the present invention.

[0052] Example 1: Formulation and preparation process of 0.5% Panax notoginseng total saponins ophthalmic gel

[0053] 1. Composition of 0.5% Panax notoginseng total saponins ophthalmic gel:

[0054]

[0055] 2. Preparation process of 0.5% Panax notoginseng total saponins ophthalmic gel:

[0056] (1) Under light-protected conditions, weigh out total saponins of Panax notoginseng, EDTA-2Na and benzalkonium chloride and place them in 2000 mL of water for injection. Heat and stir to dissolve them. Heating temperature: 50-60℃, stirring speed: 200 rpm, to obtain the drug solution.

[0057] (2) Take mannitol and carbomer 980 separately, disperse them evenly, and slowly add them to 2000 mL of water for injection at a temperature of 65-70℃ while stirring at a speed of 350-400 rpm. Continue heating and stirring for 4 hours to obtain a clear matrix solution.

[0058] (3) Finally, add the clear matrix solution to the drug solution, stir evenly for 5 minutes, stirring speed: 40 rpm, adjust the pH to 6.5, add water for injection to 5000g, stir evenly, stirring speed: 40 rpm, stirring time: 45 minutes.

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

[0060] The prepared gel was sterilized by high-pressure steam in an autoclave (121℃, 30min), and after being removed and cooled, it was filled in a sterile environment.

[0061] Example 2: Formulation and preparation process of 1.0% Panax notoginseng total saponins ophthalmic gel

[0062] 1. Composition of 1.0% Panax notoginseng total saponins ophthalmic gel:

[0063]

[0064] 2. Preparation process of 1.0% Panax notoginseng total saponins ophthalmic gel:

[0065] (1) Under light-protected conditions, weigh out total saponins of Panax notoginseng, EDTA-2Na and benzalkonium chloride and place them in 2500 mL of water for injection. Heat and stir to dissolve them. Heating temperature: 50-60℃, stirring speed: 200 rpm, to obtain the drug solution.

[0066] (2) Take mannitol and carbomer 980 separately, disperse them evenly, and slowly add them to 2000 mL of water for injection at a temperature of 65-70℃ while stirring at a speed of 350-400 rpm. Continue heating and stirring for 4 hours to obtain a clear matrix solution.

[0067] (3) Finally, add the clear matrix solution to the drug solution, stir evenly for 5 minutes, stirring speed: 40 rpm, adjust the pH to 6.5, add water for injection to 5000g, stir evenly, stirring speed: 40 rpm, stirring time: 45 minutes.

[0068] In the above preparation method, sodium hydroxide solution is used to adjust the pH. The preparation of sodium hydroxide solution is as follows: weigh 4.0g of sodium hydroxide, add water to 100g, stir to dissolve and obtain sodium hydroxide solution.

[0069] The prepared gel was sterilized by high-pressure steam in an autoclave (121℃, 30min), and after being removed and cooled, it was filled in a sterile environment.

[0070] Example 3: Formulation and preparation process of 2.0% Panax notoginseng total saponins ophthalmic gel

[0071] 1. Composition of the 2.0% Panax notoginseng total saponins ophthalmic gel formula:

[0072]

[0073]

[0074] 2. Preparation process of 2.0% Panax notoginseng total saponins ophthalmic gel:

[0075] (1) Under light-protected conditions, weigh out total saponins of Panax notoginseng, EDTA-2Na and benzalkonium chloride and place them in 2900 mL of water for injection. Heat and stir to dissolve them. Heating temperature: 50-60℃, stirring speed: 200 rpm, to obtain the drug solution.

[0076] (2) Take mannitol and carbomer 980 separately, disperse them evenly, and slowly add them to 2000 mL of water for injection at a temperature of 65-70℃ while stirring at a speed of 350-400 rpm. Continue heating and stirring for 4 hours to obtain a clear matrix solution.

[0077] (3) Finally, add the clear matrix solution to the drug solution, stir evenly for 5 minutes, stirring speed: 40 rpm, adjust the pH to 6.5, add water for injection to 5000g, stir evenly, stirring speed: 40 rpm, stirring time: 45 minutes.

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

[0079] The prepared gel was sterilized by high-pressure steam in an autoclave (121℃, 30min), and after being removed and cooled, it was filled in a sterile environment.

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

[0081] This experiment compares the ophthalmic gels containing different types and proportions of osmotic pressure regulators, measures their osmotic pressure, and selects appropriate types and amounts of osmotic pressure regulators.

[0082] The determination should be performed in accordance with the osmolality determination method (General Rule 0632) of the 2020 edition of the Chinese Pharmacopoeia, and should comply with the regulations.

[0083] 1. Screening of Osmotic Pressure Regulators

[0084] 1.1 Sample Preparation

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

[0086] In addition, different types / proportions of osmotic pressure regulators (see Table 1) and 0.25g of carbomer 980 were uniformly dispersed and slowly added to 40mL of water for injection at a temperature of 65-70℃ under high-speed stirring (stirring speed: 350-400rpm). The mixture was heated and stirred continuously to obtain a clear solution.

[0087] Finally, add the clear matrix solution to the drug solution, stir well, add sodium hydroxide solution to adjust the pH, add water to the prescribed amount (100g), and stir well.

[0088] Table 1 Dosage of different osmotic pressure regulators

[0089]

[0090]

[0091] 1.2 Results of osmotic pressure measurement

[0092] The results are shown in Table 2. Based on the osmotic pressure measurement results and gel formation status, it can be seen that the osmotic pressure of the Panax notoginseng total saponins ophthalmic gel with 4.1% mannitol added meets the requirements of the Chinese Pharmacopoeia, and the amount added meets the maximum requirements of the United States Pharmacopoeia. Adding 4% to 5% glucose is not stable enough for osmotic pressure regulation. Although KCl and NaCl can regulate osmotic pressure, they both affect the gel formation. Therefore, mannitol was selected as the osmotic pressure regulator.

[0093] Table 2 Osmotic pressure under different osmotic pressure regulators and different contents.

[0094]

[0095] Note: The osmotic pressure of the test standard is 292 mOsm2, used for screening mannitol dosage.

[0096] With the dosage of other excipients in the fixed formulation unchanged, mannitol was used as the osmotic pressure regulator. Gel formulations were prepared with mannitol dosages of 3.0%, 3.5%, 3.7%, 4.1%, 4.3%, and 5.0% (w / w) to investigate the effect of different dosages of the osmotic pressure regulator on the osmotic pressure.

[0097] 2.1 Sample Preparation

[0098] Accurately weigh 2.0g of total saponins of Panax notoginseng, 0.05g of EDTA-2Na and 0.01g of benzalkonium chloride and place them in 40mL of water for injection, then heat to dissolve.

[0099] In another case, mannitol and 0.25 g of carbomer 980 were uniformly dispersed and slowly added to 40 mL of water for injection at 65-70 °C under high-speed stirring (stirring speed: 350-400 rpm) to obtain a clear solution.

[0100] Finally, add the clear matrix solution to the drug solution, stir well, add sodium hydroxide solution to adjust the pH, add water to the prescribed amount (100g), and stir well.

[0101] 2.2 Experimental Results

[0102] Table 3. Osmotic pressure of Panax notoginseng total saponins ophthalmic gel after adding different amounts of mannitol.

[0103]

[0104] Table 3 shows that the osmotic pressure of Panax notoginseng total saponins ophthalmic gels with added mannitol (3.7%–5.0% mannitol) all meet the requirements of the Chinese Pharmacopoeia (General Chapter 0632, Part IV, 2020 edition of the Chinese Pharmacopoeia, the osmotic molar concentration should be 250–330 mOsmol / kg). Therefore, mannitol with a concentration of 3.7%–5.0% was selected as the osmotic pressure regulator.

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

[0106] 1 Experimental Methods

[0107] EDTA-2Na is a powerful chelating agent and preservative. Different amounts (0, 0.01%, 0.03%, 0.05%, 0.06%) of EDTA-2Na were added, and three samples of each concentration were prepared in parallel. The gels were placed in a constant temperature and humidity chamber and accelerated experiments were carried out at a temperature of 30℃±2℃ and a humidity of 65%RH±5%RH. Samples were taken after one month to detect the content of total saponins in Panax notoginseng.

[0108] 2 Experimental Results

[0109] Table 4 shows that EDTA-2Na has a certain impact on the stability of total saponins of Panax notoginseng. With no addition or the addition of small amounts (0.01% and 0.03%) of EDTA-2Na, the content change rate of total saponins after one month of accelerated oxidation is relatively large. However, 0.05%-0.06% of EDTA-2Na significantly improves the stability of the components, and the content change rate of total saponins after one month of accelerated oxidation is very small. 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. Changes in the content of total saponins in Panax notoginseng after one month of gel-accelerated gelation experiments with different concentrations of EDTA-2Na.

[0111]

[0112]

[0113] Experimental Example 3: Pharmacodynamic Effect of EDTA-2Na on Panax notoginseng Total Saponins Ophthalmic Gel

[0114] 1 Instrument

[0115] Electrophoresis apparatus (BioRad, USA); AI600 ultra-sensitive chemiluminescence imager (Beijing Dequanxingye Trading Co., Ltd.).

[0116] 2. Methods

[0117] 2.1 Methods for establishing diabetic mouse models

[0118] C57 mice were acclimatized for one week, fasted for 4 hours, and weighed. Except for the normal control group, the remaining mice were intraperitoneally injected with STZ (prepared with 0.1 mol / L, pH 4.5 citrate buffer, stored on ice protected from light, and administered within 5 minutes) at a dose of 50 mg / kg body weight. This method was followed for 5 consecutive days. On the fourteenth day after the start of administration, the mice were fasted for 6 hours, and fasting blood glucose was measured. A fasting blood glucose level >16.7 mmol / L indicated successful model establishment; if the blood glucose level <16.7 mmol / L, STZ was injected again. Fasting blood glucose was measured again 3 days later. Mice with two successful model establishments were combined and used evenly, ensuring 6 animals per group.

[0119] 2.2 Preparation methods of Panax notoginseng total saponins ophthalmic gel (PNS gel) with different formulations

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

[0121] The 2.0% PNS gel without EDTA-2Na, except for the absence of EDTA-2Na, has the same components and preparation method as in Example 3, and is referred to as the "high-dose PNS without EDTA" group.

[0122] 2.3 Grouping and Dosing Regimen

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

[0124] For administration, the low-dose PNS group without EDTA and the low-dose PNS group with EDTA were administered 1 mL / kg (equivalent to ginsenoside Rg1 1.56 mg / kg and notoginsenoside R1 0.73 mg / kg) via ocular administration; the high-dose PNS group without EDTA and the high-dose PNS group with EDTA were administered 1 mL / kg (equivalent to ginsenoside Rg1 6.67 mg / kg and notoginsenoside R1 1.69 mg / kg) via ocular administration; all were administered continuously for 8 weeks, once daily.

[0125] 2.4 Retina Acquisition

[0126] After anesthetizing the mice, the blood was flushed out with PBS, the eyeball containing the optic nerve was removed, the sclera was cut in a ring 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 segments centered on the optic disc, the retina was carefully separated and refrigerated.

[0127] 2.5 Retinal digestive slices

[0128] The steps for retinal digestion and mounting are as follows:

[0129] A. Rinse the isolated retina with PBS and soak it in glycine buffer overnight; B. Digest the retina with 3% trypsin in a 37°C air bath with a shaker for 2-4 hours; C. Transfer the retina to 0.1M PBS, gently aspirate to remove the internal limiting membrane, leaving only a transparent vascular network. Rinse and flatten it on a detachable glass slide, allow it to air dry at room temperature, and then store it 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. Soak in 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. Separation solution for 5 min; G. Rinse with tap water; H. Hematoxylin for 4 min; Rinse with tap water; I. ​​Separation solution for 1 sec; Rinse with tap water; J. Reversion solution for 6 sec; Rinse with tap water; K. 95% ethanol for a few seconds; L. Anhydrous ethanol I and II for 2 min each; M. Xylene I and II for 2 min each; N. Mount with neutral resin.

[0132] 2.7 Desmin staining method

[0133] A. After fixing the eyeball for half an hour, separate the retina according to the above method, clean the vitreous body and RPE to ensure good permeability; B. Place the retina in a 2mL EP tube, wash the PFA with 1mL PBS for 3×5min; C. Add 1mL 1% Triton X-100 / PBS, perforate, place on a shaker, and incubate overnight at 4℃; D. Block with 1mL 1% BSA and 0.5% Triton X-100 at 4℃, and shake for one day; E. Incubate with primary antibody (desmin) for 2 days (primary antibody dissolved in 0.1% Triton X-100 and 1% BSA, 1:200); F. Wash with 1mL 0.1% Triton X-100 for 5×10min, then add 20min, and wash with shaking at room temperature; incubate with secondary antibody at room temperature for 2h (1:3000), shaking, and protected from light; G. Incubate with 1mL 0.1% Triton X-100... Wash with X-100 for 4×30 min, shake at room temperature, and protect from light; H. Spread and mount the slide (use anti-fluorescence quenching mounting medium containing DAPI); I. Observe the pericyte loss using a laser confocal microscope; J. Calculate the vessel length and loss length using ImageJ.

[0134] 2.8 Western blot analysis

[0135] (1) Extract total protein from retinal tissue; (2) Quantitatively normalize the protein to equal volume using the BCA protein analysis kit; (3) Separate the protein (20 μg per lane) by 8% or 10% SDS-PAGE gel and transfer it to a PVDF membrane; (4) Block with 5% BSA blocking buffer (SW3015, Solarbio) for 1 h; (5) Primary antibody incubation: Soak the strips in diluted primary antibodies Occludin (1:1000, abcam, ab216327) and β-actin (1:1000, CST, 3700S) and incubate overnight at 4°C with gentle shaking on a shaker. (6) After incubation, take the primary antibody, add TBST to a square food storage box, place the membrane in it, and make sure the TBST covers the PVDF membrane. Shake on a shaker at low speed for 10 minutes, and repeat the washing 3 times. (7) Secondary antibody incubation: Immerse the PVDF membrane with protein transfer into a square food storage box containing secondary antibody (goat anti-rabbit IgG-HRP or goat anti-mouse IgG-HRP antibodies) and incubate with shaking for 1-2 hours. (8) After incubation, take the secondary antibody, add TBST to a square food storage box, place the membrane in it, and make sure the TBST covers the PVDF membrane. Shake on a shaker at low speed for 10 minutes, and repeat the washing 3-5 times. (9) Chemiluminescence: Take an appropriate amount of luminescent solution to cover the PVDF membrane, expose it on an ECL luminescence analyzer and acquire images.

[0136] 3. Experimental Results

[0137] 3.1 Retinal vessel morphology

[0138] Figure 1 The results showed that, compared with the normal group of mice, the model group mice exhibited partial capillary degeneration, luminal closure, and the formation of cellless capillaries, resulting in non-perfusion areas and a reduced number of pericytes. After 6 weeks of administration, compared with the model group, the PNS gel-treated mice showed varying degrees of increase in the number of pericytes and a decrease in the number of cellless capillaries in the retinal capillaries. Specifically, at the same dosage, compared with the group without EDTA, the PNS gel-treated group with added EDTA showed a significant decrease in the number of cellless capillaries in the retina (p<0.05).

[0139] 3.2 Loss of retinal pericytes

[0140] Figure 2 The results showed that pericyte loss was not significant in normal mice, while significant pericyte loss was observed in the retinal vessels of the model group mice, with a statistically significant difference compared to the normal group (p<0.05). The PNS gel treatment group showed a significant difference compared to the model group (p<0.05), and the addition of EDTA significantly reduced pericyte loss in the retinal vessels compared to the group without EDTA (p<0.05). This indicates that PNS gel has a significant inhibitory effect on pericyte loss in blood vessels of diabetic retinopathy, and the effect is more pronounced after the addition of EDTA.

[0141] 3.3 Analysis of Western blot results

[0142] Figure 3 The results of retinal protein blot analysis for each group of animals are shown, and the expression of the tight junction protein Occludin is analyzed by protein blot analysis. Figure 4 The results showed that, compared with the normal group, the expression of the tight junction protein Occludin was downregulated in the model group; compared with the model group, the expression of Occludin protein was upregulated to varying degrees in the PNS gel-treated groups; and the expression of Occludin protein in tight junctions was upregulated in the EDTA-treated group compared with the group without EDTA. These data indicate that PNS gel reduces the damage to retinal barrier permeability in diabetic mice, and the effect is stronger after the addition of EDTA.

[0143] The above experiments show that EDTA-2Na in the formulation of this invention can synergistically reduce the number of acellular capillaries in the retina, significantly inhibit pericyte loss in blood vessels in retinopathy, significantly upregulate the protein expression of tight junction Occludin, and significantly repair retinal permeability in diabetic mice. The synergistic effect of EDTA-2Na on the ophthalmic gel containing total Panax notoginseng saponins may be because EDTA-2Na can promote the absorption of total Panax notoginseng saponins in the eye, increasing the concentration of total Panax notoginseng saponins in the eye and retina. It may also be because EDTA-2Na can regulate the microenvironment of the eye, such as osmotic pressure, viscosity, and pH, thereby forming a synergistic effect with total Panax notoginseng saponins and enhancing the therapeutic efficacy of the ophthalmic gel containing total Panax notoginseng saponins.

[0144] Experiment Example 4: Effect of Mannitol on Carbomer Swelling Time

[0145] 1 Experimental Methods

[0146] Preparation method 1: Carbomer gel was prepared using the preparation method of the present invention.

[0147] Accurately weigh 20.5g mannitol and 1.25g carbomer 980, disperse them evenly, and slowly add them to 200mL of deionized water at 65-70℃ under high-speed stirring (stirring speed: 350-400rpm). Continue heating and stirring to obtain a clear solution. Prepare 3 parallel solutions, record the swelling time of carbomer for each solution, and take the average value.

[0148] Preparation method 2:

[0149] Accurately weigh 1.25 g of carbomer 980 and slowly add it to 200 mL of deionized water at 65-70 °C under high-speed stirring (stirring speed: 350-400 rpm). Continue heating and stirring to obtain a clear solution. Prepare 3 parallel solutions and record the swelling time of carbomer for each solution. Take the average value.

[0150] 2 Experimental Results

[0151] Uniform dispersion of mannitol and carbomer can effectively shorten the swelling time of carbomer, increasing production efficiency by 33.3%. The average swelling time of carbomer is shown in Table 5.

[0152] Table 5. Effect of mannitol on carbomer swelling time (n=3)

[0153]

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

[0155] 1. Instruments and Laboratory 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 readers (Beijing Aimei Technology Development Co., Ltd., model: JD - C - 08, serial number: EQ03 - 3503)

[0159] Slit - lamp microscopes (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 - ear rabbits, with 22 males and 23 females, 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 conducted in accordance with the principles of animal experiments.

[0163] 2 Sample preparation methods

[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 is completely swollen 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 sample was prepared according to the method of Example 1.

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

[0167] The 4.0% PNS gel-carbomer 980 test sample, except that the amount of total saponins of Panax notoginseng was 4.0%, the other components and preparation methods were the same as in Example 3; the pH of the above three test samples was uniformly adjusted to 6.5.

[0168] Irritation study of ophthalmic gel in rabbit eyes

[0169] 3.1 Group Settings

[0170] On the day the adaptation period ended, each animal underwent a fluorescein sodium test in both eyes. After the fluorescein sodium test, 40 animals (half male and half female) with good condition and moderate weight and weight gain rate were selected to enter the experiment. Animals that were screened out were used as reserve animals.

[0171] This experiment consisted of 10 groups, as shown in Table 6 below. Both the single-dose and multiple-dose groups received a negative control or the test drug in the right eye. Animals were randomly divided into 10 groups (Experiment 1–10) using a weight-based randomized block designation method, with 4 animals in each group (half male and half female).

[0172] Table 6 Stimulation Test Grouping

[0173]

[0174] 3.2 Dosage, concentration and administration method

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

[0176] Test sample group S and test sample group M: The dosage was 0.4 g / eye / day (0.1 g / eye / dose, 4 times / day). Weigh 0.1 g (error <10%) of the corresponding test drug using a balance and administer to rabbits via eye drops (or topical application). Then gently close the eyelids for approximately 10 seconds; do not rinse the eyes. The interval between two administrations was 2 hours (±5 minutes).

[0177] 3.3 Stimulus Examination Indicators

[0178] Clinical observation: Observe the animal's appearance, behavior, glandular secretion, and respiration once a day.

[0179] Eye irritation observation: ① Single-dose administration: Eye irritation was observed at 1, 2, 4, 24, 48, and 72 hours after the last dose on Day 1. ② Multiple-dose administration: Eye irritation was observed before daily administration and at 1, 2, 4, 24, 48, and 72 hours after the last dose on Day 7. Since eye irritation was already assessed within 24 hours prior to administration, no eye irritation assessment was performed before the first dose on Day 1 in the single-dose and multiple-dose groups. Eye irritation was assessed using a slit-lamp microscope, and sodium fluorescein staining was performed throughout the observation process. Abnormal ocular reactions were recorded after each assessment, and scores were calculated according to Table 7. The average score for eye irritation in each group was calculated.

[0180] Data processing and result evaluation: The mean ocular stimulation score for each group at each observation time point was calculated, and the scores for males and females were combined for statistical analysis. The stimulation intensity was evaluated according to Table 8, and the irritation of the test sample to the rabbit eyes was evaluated by comparing with the negative control.

[0181] Table 7. Scoring Standards for Eye Irritation Response

[0182]

[0183] Table 8 Evaluation Criteria for Eye Irritation Intensity

[0184]

[0185]

[0186] Results of a 4-eye gel irritation study in rabbit eyes

[0187] During the experiment, no significant abnormalities were observed in the systemic reactions of any group of animals during clinical observation. No irritation was observed in the negative S group and the negative M group at any of the test time points.

[0188] After a single dose, the irritation scores of all animal groups at the test time points were between 0 and 3, indicating that no eye irritation was observed after a single dose of the test product. The results are shown in Table 9.

[0189] Table 9. Eye irritation score results after a single dose (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 indicates the number of days of the experiment.

[0192] After multiple administrations, the ocular irritation score in the 940-M group increased from 1 to 5 before daily administration, and the irritation score increased with the number of days of administration. In the H-980-M group, the ocular irritation score increased from 4 to 5 before daily administration, and the irritation score increased with the number of days of administration. No irritation was observed in the L-980-M and M-980-M groups. The results are shown in Table 10.

[0193] Table 10. Eye irritation score results before daily administration for multiple doses (Mean)

[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 indicates the number of days of the experiment.

[0196] After multiple administrations, in the 940-M group, the eye irritation score decreased from 3 to 1 within 1-24 hours after administration on day 7, and the eye irritation score was 0 at 48 hours and 72 hours, indicating that the irritation gradually decreased over time after administration on day 7. Similarly, in the H-980-M group, the eye irritation score decreased from 5 to 2 within 1-24 hours after administration on day 7, and the eye irritation score was 0 at 48 hours and 72 hours, indicating that the irritation gradually decreased over time after administration on day 7. No irritation was observed in the L-980-M and M-980-M groups. The results are shown in Table 11.

[0197] Table 11. Eye irritation scores after 7 days of multiple drug administration (Mean)

[0198]

[0199] Note: D indicates the number of days of the experiment.

[0200] In summary, none of the test groups showed irritation after a single dose. With multiple doses, the 940-M group showed irritation scores of 4 and 5 on days 6 and 7, respectively, indicating mild irritation. The H-980-M group showed mild irritation from days 1 to 7, with mild irritation occurring within 1-2 hours after administration on day 7. The L-980-M and M-980-M groups showed no irritation. Therefore, from the perspective of multiple doses, the 2.0% Panax notoginseng total saponins ophthalmic gel using carbomer 940 and HPMC as the matrix in Reference 3 showed mild irritation, while the 2.0% Panax notoginseng total saponins ophthalmic gel using carbomer 980 as the matrix in this invention showed no irritation. This indicates that the gel formulation of this invention has a greater advantage in ocular safety than the gel in Reference 3.

[0201] Meanwhile, a comparison of the irritation properties of 0.5%, 2%, and 4% Panax notoginseng total saponins ophthalmic gels showed that when the total saponin content reached 4%, the gel exhibited mild irritation, indicating that excessively high levels of Panax notoginseng total saponins in the gel could cause eye irritation. The 0.5% and 2.0% Panax notoginseng total saponins ophthalmic gels, however, showed no irritation. These studies demonstrate that Panax notoginseng total saponin gels with a drug loading of 0.5%-2.0% have good safety profiles.

[0202] Experimental Example 6: Ocular Pharmacokinetic Study of Different Panax notoginseng Total Saponins Ophthalmic Gels

[0203] 1. Instruments and Animals

[0204] 1.1 Instruments

[0205] Liquid chromatography - mass spectrometry instrument (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);<{0000567}>

[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 Rg1, notoginsenoside R1, and ginsenoside Re in the samples were determined by UPLC - MS / MS.

[0216] 2.1.1 Preparation of mixed reference substance solution

[0217] Accurately weigh 3.6700 mg of ginsenoside Rg1 reference standard (purity: 98.5%), 3.2390 mg of notoginsenoside R1 reference standard (purity: 90.4%), and 3.6810 mg of ginsenoside Re reference standard (purity: 96.0%), and place them separately in 25 mL volumetric flasks. Dissolve and dilute to the mark with methanol, shake well, and obtain the reference standard stock solutions. The concentrations after calculation according to purity are notoginsenoside R1 117.12 μg / mL, ginsenoside Rg1 144.60 μg / mL, and ginsenoside Re 141.35 μg / mL, respectively. Store at 4℃. In addition, take an appropriate amount of each reference stock solution into a 100 mL volumetric flask, dilute with methanol to the mark to obtain a mixed reference stock solution with concentrations of notoginsenoside R1 1171.24 ng / mL, ginsenoside Rg1 1229.10 ng / mL, and ginsenoside Re 1201.49 ng / mL, and store at 4℃.

[0218] 2.1.2 Sample Preparation

[0219] 2.0% Panax notoginseng total saponins ophthalmic gel-carbomer 940 was prepared according to the formulation and process of reference 3, as described in the "2 Sample Preparation Method" section of Experimental Example 5 above, hereinafter referred to as "Reference 3 Gel-940 Group".

[0220] 0.5% Panax notoginseng total saponins ophthalmic gel-carbomer 980, the gel sample was prepared according to Example 1 of the present invention, hereinafter referred to as "the 0.5% gel-980 of the present invention".

[0221] 2.0% Panax notoginseng total saponins ophthalmic gel-carbomer 980, the gel sample was prepared according to Example 3 of the present invention, hereinafter referred to as "the 2.0% gel-980 of the present invention".

[0222] Xuesaitong for Injection: A Xuesaitong solution with a concentration of 100 mg / mL was prepared using 0.9% physiological sodium chloride injection as the solvent.

[0223] 2.2 Pharmacokinetic Study of Panax notoginseng Total Saponins Gel in Rabbit Eyes

[0224] Twenty-four rabbits were randomly divided into four groups of six each: the gel-940 group (reference 3), the 0.5% gel-980 group (this invention), the 2.0% gel-980 group (this invention), and the injectable XueSaiTong group. A microdialysis probe was implanted into the rabbit's eye at a depth of 1.5...

[0225] μL·min -1 Physiological saline was instilled into the probe at a flow rate, and after equilibration for 1 hour, 0.1 g of ophthalmic gel (i.e., ginsenoside Rg1 0.014 mg·kg⁻¹) was instilled into the eyes of rabbits in the gel group. -1 Panax notoginseng saponin R1 0.005 mg·kg -1Ginsenoside Re 0.002 mg·kg -1 In the group receiving Xuesaitong injection, the marginal ear vein was injected with Xuesaitong solution at a dose of 9.34 mg / kg (equivalent to ginsenoside Rg 13.69 mg·kg). -1 Panax notoginseng saponins R11.13 mg·kg -1 Ginsenoside Re 0.57 mg·kg -1 The dialysate was collected starting from 0-6 hours, with one sample collected every 30 minutes. From 6-8 hours, one sample was collected every hour, for a total of 8 hours. The contents of ginsenoside Rg1, ginsenoside Re, and notoginsenoside R1 in the microdialysis solution were determined by LC-MS / MS. Data processing and analysis were performed using WinNonlin 6.0 pharmacokinetic software.

[0226] Pharmacokinetic results of microdialysis in rabbit eyes from 3 species

[0227] The concentration-time curves of ginsenoside Rg1, ginsenoside Re, and notoginsenoside R1 after ocular administration of Panax notoginseng total saponins ophthalmic gel and injectable XueSaiTong to rabbits are shown below. Figures 5-7 The calculated pharmacokinetic parameters are shown in Tables 12-14.

[0228] Table 12. Main pharmacokinetic parameters of Panax notoginseng saponin R1 after administration in different groups (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 injection group, # indicates P<0.05, ## indicates P<0.01.

[0231]

[0232]

[0233] Table 13. Main pharmacokinetic parameters of ginsenoside Rg1 after administration to different groups (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 injection group, # indicates P<0.05, ## indicates P<0.01.

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

[0236]

[0237]

[0238] Note: Compared with the gel-940 group in reference 3, * indicates P<0.05, ** indicates P<0.01; compared with the XueSaiTong injection group, # indicates P<0.05, ## indicates P<0.01.

[0239] Table 12 and Figure 5 The AUC of notoginsenoside R1 in the 2.0% gel-980 group of this invention is shown. 0-8h The AUC of the 2.0% gel-980 group in this invention was significantly higher than that of the gel-940 group and the injection of XueSaiTong group in reference 3 (P<0.05, P<0.01). 0-∞ The concentrations were also significantly higher than those in the gel-940 group and the injectable XueSaiTong group in Literature 3 (P<0.05, P<0.01). The concentration-time curve of Panax notoginseng saponin R1 in the rabbit eye microdialysis fluid of the 2.0% gel-980 group of this invention showed a clear bimodal phenomenon, with the first peak appearing at 1 h and the second peak appearing at 4 h. The gel formulation of this invention significantly improves the bioavailability of Panax notoginseng saponin R1 through the above pharmacokinetic behavior.

[0240] Table 13 and Figure 6 The AUC of notoginsenoside Rg1 in the 2.0% gel-980 group of this invention is shown. 0-8h The AUC of notoginsenoside Rg1 in the 2.0% gel-980 group was significantly higher than that in the injection group (P<0.01). 0-∞ The concentrations were significantly higher than those in the gel-940 group and the injectable XueSaiTong group in the literature (P<0.05, P<0.01). The concentration-time curve of Panax notoginseng saponin Rg1 in the rabbit eye microdialysis fluid of the 2.0% gel-980 group of this invention showed a clear three-peak phenomenon: the first peak appeared at 1 h, the second at 4 h, and the third at 6-7 h. The gel formulation of this invention significantly improves the bioavailability of Panax notoginseng saponin Rg1 through the above pharmacokinetic behavior.

[0241] Table 14 and Figure 7 The AUC of notoginsenoside Re in the 2.0% gel-980 group of this invention is shown. 0-8h The AUC of Panax notoginseng saponin Re in the 2.0% gel-980 group of this invention was significantly higher than that in the gel-940 group and the injection of Xuesaitong group in the literature (P<0.05, P<0.01). 0-∞The concentrations of notoginsenoside Re in the rabbit ocular microdialysis fluid of the 2.0% gel-980 group were significantly higher than those in the gel-940 group and the injection-of-Xuesaitong group in Literature 3 (P<0.05, P<0.01). The concentration-time curve of notoginsenoside Re in the rabbit ocular microdialysis fluid of the 2.0% gel-980 group of this invention showed a significant plateau in drug concentration between 5 and 8 hours, indicating a significantly higher drug concentration than that in the gel-940 group and the injection-of-Xuesaitong group in Literature 3. The gel formulation of this invention significantly improves the bioavailability of notoginsenoside Re through the aforementioned pharmacokinetic behavior.

[0242] The ophthalmic gel of this invention can effectively improve the bioavailability of Panax notoginseng total saponins in the eye. The Panax notoginseng total saponin ophthalmic gel acts directly on the eye, exhibiting good bioadhesion and biocompatibility. With the aid of excipients, Panax notoginseng total saponins are absorbed into the eye via the corneal and conjunctival-scleral pathways. The corneal pathway allows absorption sequentially through the anterior chamber and lens to the posterior chamber and retina. The conjunctival-scleral pathway allows absorption into the vitreous humor via capillary circulation, or absorption into the bloodstream through the nasolacrimal duct system and subsequent diffusion into the eye. As shown in the drug concentration-time curve, the gel of this invention, especially the 2.0% Panax notoginseng total saponins ophthalmic gel-980 group, reaches the first peak drug concentration 1 hour after administration and the second peak drug concentration 4 hours later. It is speculated that the carbomer 980, mannitol, EDTA-2Na and other substances in the formulation of this invention work together to promote penetration, adhesion and regulation, thus opening up two pathways for drug absorption in the eye, increasing the drug residence time in the eye, regulating the ocular microenvironment and increasing the drug permeability in the eye. It not only delivers the drug to the retina through the cornea but also through the conjunctiva-sclera pathway, is absorbed into the blood through the nasolacrimal duct system and reaches the eye again after systemic circulation. The drug overlaps with the drug absorbed through the cornea, which increases the drug concentration and bioavailability in the eye, forming the second and third ocular absorption peaks and maintaining a high drug concentration in the eye for 5-8 hours. This is a phenomenon not seen in other ophthalmic preparations or ophthalmic gel preparations and is an unexpected effect unique to the gel of this invention. The injectable XueSaiTong solution, administered intravenously, can only reach the eye via systemic circulation. Furthermore, due to the blood-eye barrier, the drug concentration reaching the eye is low, resulting in poor absorption and only one absorption peak. Similarly, the 2.0% Panax notoginseng total saponins ophthalmic gel in reference 3 (gel-940 group) showed only one absorption peak in the rat eye (the peak at 1 hour), and its bioavailability was lower than that of the 2.0% gel-980 of this invention. This indicates that the gel matrix Carbomer 980 of this invention significantly enhances the ophthalmic pharmacokinetics of Panax notoginseng total saponins compared to the Carbomer 940 and HPMC mixed matrix in reference 3.

[0243] Experimental Example 7: Pharmacodynamic Study of Ophthalmic Gels with Different Total Saponins from Panax notoginseng

[0244] 1. Instruments

[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 Luxiangyi Centrifuge Instrument Co., Ltd.), surgical microscope SM-201 (Shanghai Yuyan Scientific Instrument Co., Ltd.), handheld fundus camera Smartscope M5 (Eurovis, Finland), inverted microscope and imaging system CKX41 (Olympus Corporation, Japan), JY-Clear ECL luminescence analyzer (Shanghai Ruixi Instrument Equipment Co., Ltd.).

[0246] 2 methods

[0247] 2.1 Methods for establishing diabetic rat models

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

[0249] After acclimatizing SD rats for 3–7 days, they were intraperitoneally injected with a single dose of 30 mg / mL STZ solution at a dose of 30 mg / kg. Normal group rats were intraperitoneally injected with an equal dose of 0.1 mol / L citrate buffer. After all rats were injected, DM group rats were fed a high-fat diet, and normal group rats were fed a normal diet. After 5–7 days of continued feeding, random blood glucose levels were measured (without fasting).

[0250] A concentration ≥16.7 mmol / L confirms successful establishment of a type 2 diabetic rat model. Rats that have not developed type 2 diabetes can be injected intraperitoneally with STZ solution again.

[0251] 2.1.2 Construction of a rat DR model

[0252] After successful establishment of the type 2 diabetes model, rats in the DM group were fed a mixture of normal and high-fat diets. During this period, fundus examination was performed every month to assess retinopathy. If abnormal neovascularization or tortuous blood vessels were observed during fundus examination, the DR model was confirmed as successful. If fundus photography revealed blurred images and preliminary lens opacity, it indicated that the animal had progressed to early cataracts after developing diabetic retinopathy. Once the required number of rats for the DR group was reached, drug administration was initiated.

[0253] 2.2 Drug preparation, dosage and regimen

[0254] 2.0% Panax notoginseng total saponins ophthalmic gel-carbomer 940 was prepared using the formulation and process described in Reference 3, which can be found in the description of "2 Sample Preparation Method" in Experimental Example 5 above. It is referred to as "Reference 3 Gel-940".

[0255] 0.5% Panax notoginseng total saponins ophthalmic gel-carbomer 980, is a gel sample prepared using Example 1 of this invention, hereinafter referred to as 0.5% gel-980 of this invention.

[0256] 2.0% Panax notoginseng total saponins ophthalmic gel-carbomer 980, is a gel sample prepared using Example 3 of the present invention, hereinafter referred to as the 2.0% gel-980 of the present invention.

[0257] Xuesaitong for injection: Xuesaitong solution with a concentration of 100 mg / mL was prepared using 0.9% physiological sodium chloride injection as solvent.

[0258] Rats with successful DR model were randomly divided into groups according to body weight and given the drug. Ten rats were placed in the normal group and the model group, neither of which received any drug. Twelve rats were placed in each of the following groups: the gel-940 group (Reference 3), the 0.5% gel-980 group (of this invention), and the 2.0% gel-980 group (of this invention). Each group received the corresponding gel sample, administered at a dose of 100 μL / eye, once daily for 56 days. Ten rats were placed in the injection group (Xuesaitong), receiving 100 mg / mL injection of Xuesaitong (100 mg / kg) intraperitoneally once daily for 56 days.

[0259] 2.3 Sample Collection and Detection Indicators

[0260] 2.3.1 Cataract detection

[0261] Eight weeks after drug intervention, all animals in each group were tested, the proportion of cataracts was calculated, and cataract scores were calculated. The cataract scoring criteria are shown in Table 15.

[0262] Table 15 Cataract Scoring Criteria

[0263] score Degree Classification 0 The lens is clear (no cataracts). 1 Peripheral blisters and turbidity 2 Central turbidity 3 Diffuse central opacity 4 Mature cataracts 5 Overmature cataracts

[0264] 2.3.2 Immunohistochemistry

[0265] Eight weeks after drug intervention, rats in each group underwent anesthesia and eyeball collection. The left eye was used for immunohistochemistry: after fixation in 4% paraformaldehyde for 30 minutes, it was dehydrated using a gradient of 5%-15%-30% sucrose (at room temperature). The dehydrated eyeballs were then placed in disposable embedding cassettes and embedded and frozen using OCT. Cryosections were prepared from the embedded frozen tissue using a Leica cryostat. The specific procedures are as follows:

[0266] 1) Immunohistochemical staining of frozen sections of rat retinal tissue was performed using Anti-VEGF (1:50) and Anti-AGE (1:400) as primary antibodies.

[0267] 2) Antigen retrieval: Place the slides in 1x EDTA 9.0 (Fuzhou Maixin, MVS-0098, pH 9.0, 50×, dilution ratio 1:49 in pure water), heat the slides on high in a microwave oven for 10 min, and then allow them to cool naturally at room temperature for about 30 min; wash with 0.1% Triton TBS for 3×3 min.

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

[0269] 4) Blocking non-specific antigenic epitopes: Shake off the water droplets on the slide, and add and incubate the blocking solution (Solarbio, SL038, Blocking Goat Serum 20×, dilution ratio: 1:19 in PBS) to the slide in a humidified chamber at room temperature for 1 hour.

[0270] 5) Antibody binding: Discard the supernatant, place the slides in a humidified chamber, and add 200 μL of primary antibody (diluted in Signal Up immunostaining primary antibody dilution buffer) to each slide sample. After incubating at room temperature for 1 h, wash with shaking in 0.1% Triton TBS for 3 × 5 min. Incubate with Peroxidase AffiniPure Goat Anti-Rabbit / Mouse IgG (H+L) (1:500 diluted in PBS) at room temperature for 30 min, and wash with shaking in 0.1% Triton TBS for 3 × 5 min.

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

[0272] 7) Nuclear counterstaining and dehydration mounting: After counterstaining cell nuclei with hematoxylin for 1 min, dehydrate them with a gradient of ethanol (75% ethanol 5 s - 95% ethanol 5 s - anhydrous ethanol I 1 min - anhydrous ethanol II 5 min - xylene 3 × 5 min), and mount them with neutral resin.

[0273] 8) After slide preparation, the slides were dried for more than 6 hours. All slides were digitally scanned, and the positive signals of AGEs and VEGFA in the retina of the experimental sample tissue were statistically analyzed using the digital quantitative analysis software QuPath.

[0274] 2.3.3 HE staining:

[0275] Eight weeks after drug intervention, rats in each group were anesthetized and their eyeballs were collected. The right eyeball was used for HE staining.

[0276] 1) Preparation of tissue sections: The right eyeball of the rat was fixed in an ocular fixative solution, embedded, and paraffin sections were made by cutting the cornea and retina parallel to the optic nerve.

[0277] 2) Dewaxing: Place the baked paraffin sections on the staining rack in the oven into xylene I and soak for 20 minutes. Then, transfer the samples to xylene II and soak for 20 minutes. After the wax on the samples has dissolved, transfer the samples to anhydrous ethanol I and soak for 5 minutes. Then, transfer the samples to anhydrous ethanol II and soak for 5 minutes. Brush with anhydrous ethanol for 20 seconds and transfer to a basin of water to rinse the samples with running tap water to remove the alcohol.

[0278] 3) HE staining and dehydration: Immerse the sections in HE staining solution-hematoxylin in the staining tank for 3-5 minutes. Remove the sections and wash them with water in a staining cup until they are colorless. Immerse the sections in HE staining solution-differentiation solution in the staining tank for 3-5 seconds, then wash quickly with water. Immerse the sections in HE staining solution-blueing solution in the staining tank for 3-5 seconds, then wash quickly with water. Immerse the sections in the following staining tanks in sequence: 85% ethanol, 95% ethanol, HE staining solution-eosin, anhydrous ethanol I, anhydrous ethanol II, anhydrous ethanol III, n-butanol, xylene I, and xylene II, soaking each tank for 3-5 minutes.

[0279] 4) Mounting: After removing the slides and quickly drying them in an air vent, mount them with neutral resin.

[0280] 5) Scanning: After the slides are scanned and imaged, 5 fields of view are randomly selected at 20.0× to measure retinal thickness. 3 locations are measured for each retinal pathology photograph, and the results are calculated using Image-Pro Plus 6.0 software.

[0281] 3 Experimental Results

[0282] 3.1 Cataract status in rats of each group

[0283] No rats in the normal control group developed cataracts, therefore their data are not shown in the figure. Figure 8 As shown, compared with the normal group, the incidence of cataracts in rats in the model group, the gel-940 group (reference 3), the 0.5% gel-980 group of this invention, the 2.0% gel-980 group of this invention, and the injection of Xuesaitong group all showed an increasing trend. In the first two weeks, the incidence of cataracts in the injection of Xuesaitong group was the lowest, but from weeks 3 to 8, the incidence of cataracts in the 2.0% gel-980 group of this invention was the lowest, especially from weeks 3 to 6, where the incidence of cataracts was significantly lower than in other groups, indicating that the 2.0% gel-980 of this invention has a significant advantage in reducing the incidence of diabetic cataracts.

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

[0285] The results above show that the Panax notoginseng total saponins ophthalmic gel of the present invention can delay the onset of diabetic cataracts, reduce the severity of cataracts, and has a good anti-diabetic cataract effect.

[0286] 3.2 Expression rate of VEGFA and AGEs positive area in the retina 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. Overexpression of VEGFA in the retina of DR rats can cause abnormal angiogenesis and vascular leakage, proving that DR rats have developed diabetic retinopathy. AGEs are deposited in the retina of DR rats, aggravating retinopathy and leading to the worsening of DR.

[0288] like Figure 10 As shown, compared with the normal group, the VEGFA-positive area expression rate in the retina of rats in the model group was significantly increased by 155.56%. Compared with the model group, the VEGFA-positive area expression rate in the retina of rats in the 2.0% gel-980 group and the 0.5% gel-980 group of this invention was significantly decreased by 52.34% and 47.35% respectively (P<0.05, P<0.05), while the VEGFA-positive area expression rate in the retina of rats in the gel-940 group of reference 3 was decreased by 24.61% respectively (P<0.05). Compared with the gel-940 group of reference 3, the VEGFA-positive area expression rate in the retina of rats in the 2.0% and 0.5% gel-980 groups of this invention was decreased by 36.78% and 30.17% respectively (P<0.05, P<0.05). Compared with the group treated with XueSaiTong injection, the expression rate of VEGFA-positive area in the rat retina of the 2.0% and 0.5% gel-980 groups of this invention decreased by 40.23% and 33.98%, respectively (P<0.05, P<0.05). It is evident that the 0.5% and 2.0% gel-980 of this invention can significantly reduce VEGFA in the rat retina, which is significantly superior to gel-940 in reference 3 and XueSaiTong injection.

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

[0290] The results above show that the 2.0% and 0.5% gel-980 of this invention can significantly reduce the VEGFA content in the retina of DR rats, inhibiting abnormal retinal angiogenesis; it can also reduce the content of AGEs in the retina and alleviate the severity of retinal lesions. 3.3 HE staining width of rat retina in each group.

[0291] like Figure 12 As shown, compared with the normal group, the retinal thickness of rats in the model group was significantly increased by 41.55% (P<0.05); compared with the model group, the retinal thickness of rats in the 2.0% gel-980 group and the Xuesaitong injection group were significantly decreased by 38.50% and 26.61%, respectively (P<0.05, P<0.05). Compared with the gel-940 group in reference 3, the retinal thickness of rats in the 2.0% gel-980 group was significantly decreased by 29.72% (P<0.05). Compared with the Xuesaitong injection group, the retinal thickness of rats in the 2.0% gel-980 group was significantly decreased by 16.19% (P<0.05). It is evident that the 2.0% gel-980 of this invention significantly reduces retinal thickness in rats and is significantly superior to gel-940 and Xuesaitong injection in inhibiting retinal lesion thickness in reference 3.

[0292] 4. Summary of Pharmacodynamics

[0293] The Panax notoginseng total saponins ophthalmic gel of the present invention can reduce the incidence of diabetic cataracts and delay the progression of cataracts; significantly reduce retinal thickness and significantly reduce the content of VEGFA and AGEs in the retina of DR rats, effectively treating diabetic retinopathy, and its effect is superior to that of gel-940 in reference 3 and Xuesaitong for injection.

Claims

1. A Panax notoginseng total saponins ophthalmic gel preparation, characterized in that, Based on parts by weight, it includes the following components: Apply an appropriate amount of pH adjuster to adjust the pH of the ophthalmic gel preparation to 6.0-8.0; Add water for injection to a total of 100 parts by weight.

2. The Panax notoginseng total saponins ophthalmic gel preparation according to claim 1, characterized in that, The content of total saponins of Panax notoginseng 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 total 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, The mannitol content was selected from 4.1 parts by weight.

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

7. The Panax notoginseng total saponins ophthalmic gel preparation according to claim 1, characterized in that, The preservative content 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 preservative content 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 adjuster 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 saponins exceeds 85%.

11. The Panax notoginseng total saponins ophthalmic gel preparation according to claim 1, characterized in that, The prescription consists of the following components: 25.0g of total saponins from Panax notoginseng; Add an appropriate amount of sodium hydroxide to adjust the pH to 6.5; Add water for injection to a total of 5000g.

12. The Panax notoginseng total saponins ophthalmic gel preparation according to claim 1, characterized in that, The prescription consists of the following components: 50.0g of total saponins from Panax notoginseng; Add an appropriate amount of sodium hydroxide to adjust the pH to 6.5; add water for injection to a total of 5000g.

13. The Panax notoginseng total saponins ophthalmic gel preparation according to claim 1, characterized in that, The prescription consists of the following components: Total saponins of Panax notoginseng 100.0g; Add an appropriate amount of sodium hydroxide to adjust the pH to 6.5; add water for injection to a total of 5000g.

14. The Panax notoginseng total saponin ophthalmic gel preparation according to any one of claims 1-13, wherein the content of Panax notoginseng saponin 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 formulation according to claim 1, characterized in that, Includes the following steps: (1) Under light-protected conditions, weigh out total saponins of Panax notoginseng, EDTA-2Na and preservative and place them in water for injection. Heat and stir to dissolve them to obtain a drug solution. (2) Weigh mannitol and carbomer 980, disperse them evenly, and slowly add them to 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 pH adjuster to adjust pH to 6.0-8.0, add water for injection to make up to 100 parts by weight, stir evenly to obtain gel.

16. The preparation method according to claim 15, characterized in that, The final prepared gel was sterilized by autoclaving, then removed and cooled before being filled in a sterile environment.

17. The preparation method according to claim 15, characterized in that, In step (1), the concentration of total saponins of Panax notoginseng 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℃; 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℃.

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 after the matrix solution is added to the drug solution are: stirring speed of 40 rpm and stirring time of 5 minutes; the stirring conditions after adding water for injection and stirring evenly are: stirring speed of 40 rpm and stirring time of 45 minutes.

22. The preparation method according to claim 15, characterized in that, The autoclaving conditions are 121℃ for 30 minutes.

23. Use of the Panax notoginseng total saponins ophthalmic gel formulation according to any one of claims 1-14 in the preparation of a medicament for the prevention and / or treatment of diabetic retinopathy or diabetic cataracts.

24. The use according to claim 23, wherein the diabetic retinopathy includes 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 total saponins of Panax notoginseng ophthalmic gel preparation repairs retinal integrity and permeability.

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