Pharmaceutical composition for delaying senescence of lens cells and application of pharmaceutical composition

Abstract

The invention relates to the technical field of drugs for delaying senescence of lens cells, and particularly discloses a pharmaceutical composition for delaying senescence of lens cells and application thereof, the pharmaceutical composition comprises 0.05-0.1 wt% of cycloastragenol ester derivatives, 0.1-5 wt% of a solubilizer, 1.0-10.0 wt% of a cosolvent, 0.5-2.0 wt% of an osmotic pressure regulator, 0.01-0.5 wt% of a buffer salt, 0.001-0.05 wt% of a preservative, and the balance of water for injection; wherein the cycloastragenol ester derivative is cycloastragenol-3-acetate, and the structural formula of the cycloastragenol ester derivative is shown in Cycloastragenol-3-acetate is applied to delaying senescence of lens cells, compared with a parent compound cycloastragenol, the esterified derivative has significantly enhanced fat solubility and cell membrane permeability, and under the same concentration, the ability of activating the telomerase activity of lens epithelial cells is significantly superior to that of cycloastragenol; stronger biological activity potential is shown.

Description

Technical Field

[0001] This invention relates to the field of drugs for delaying lens cell aging, specifically a pharmaceutical composition for delaying lens cell aging and its application. Background Technology

[0002] As we age, lens aging and the resulting cataracts are the leading cause of blindness worldwide. Lens cell aging is a complex biological process, with telomere shortening and decreased telomerase activity considered key molecular mechanisms of cellular replicative aging. Therefore, the search for drugs that can activate telomerase and delay lens cell aging has become a research hotspot in ophthalmology. Astragaloside A and cycloastragenol, saponins derived from the traditional Chinese medicine Astragalus membranaceus, have been shown by modern pharmacological studies to possess various biological activities, including antioxidant and anti-aging effects. Cycloastragenol, as the main active metabolite of astragaloside A in vivo, has received widespread attention in recent years due to its potential telomerase-activating activity, and is theoretically an ideal candidate drug for treating age-related diseases.

[0003] However, developing cycloastragalool directly into ophthalmic preparations, especially eye drops, faces insurmountable technical bottlenecks. Cycloastragalool is a highly lipid-soluble compound with extremely low solubility in water, making it difficult to produce clear, stable aqueous eye drops using conventional techniques. Directly suspending it in water results in an uneven, easily precipitated suspension, leading to low bioavailability, clogging of the ophthalmic preparation outlet, poor user experience, and potential safety hazards. Even when a suspension is formed, due to its polarity, cycloastragalool cannot effectively penetrate the corneal barrier, resulting in negligible amounts reaching the lens target site and failing to exert the expected pharmacological effects. Currently, although there are sporadic reports on cycloastragaloyl alcohol esterified derivatives, there is a lack of an efficient, specific, and industrially suitable synthetic method to prepare high-purity specific esterified products. More importantly, how to successfully formulate such derivatives into stable, effective formulations that meet ophthalmic preparation standards has become a major challenge. To address this, we propose a pharmaceutical composition for delaying lens cell aging and its application. Summary of the Invention

[0004] The purpose of this invention is to provide a pharmaceutical composition for delaying lens cell aging and its application, in order to solve the problems mentioned in the background art, namely, the extremely low solubility of cycloastragalool in water, making it difficult to prepare clear and stable aqueous eye drops using conventional techniques, and the fact that directly suspending it in water will form an uneven and easily precipitated suspension, which not only has low bioavailability, but also easily clogs the outlet of ophthalmic preparations, resulting in a poor user experience and safety hazards.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a pharmaceutical composition for delaying the aging of lens cells and its application, comprising, by weight percentage of raw materials: Cycloastragalol ester derivatives 0.05-0.1wt%, solubilizer 0.1-5wt%, cosolvent 1.0-10.0wt%, osmotic pressure regulator 0.5-2.0wt%, buffer salt 0.01-0.5wt%, preservative 0.001-0.05wt%, balance is water for injection; The cycloastragalol ester derivative is cycloastragalol-3-acetic acid ester; The solubilizer is selected from at least one of Tween 80, polyethylene glycol 400, and hydroxypropyl-β-cyclodextrin; The co-solvent is selected from at least one of propylene glycol, glycerol, and anhydrous ethanol; The osmotic pressure regulator is selected from at least one of sodium chloride, mannitol, and glucose; The buffer salt is selected from at least one of the following systems: sodium dihydrogen phosphate-disodium hydrogen phosphate system, citric acid-sodium citrate system, and acetate-sodium acetate system. The preservative is selected from at least one of benzalkonium chloride, ethylparaben, and chlorobutanol.

[0006] The method for preparing the cycloastragaloyl-3-acetic acid ester is characterized in that, based on the total mass of the reaction system, the raw materials include: Cyclostilbene alcohol 0.1-0.3wt%, acetic anhydride 0.15-0.5wt%, catalyst 0.01-0.05wt%, condensing agent 0.02-0.08wt%, balance is reaction solvent; The preparation method includes the following steps: S1: Feeding and dissolving: Under the protection of an inert gas, the cycloastragalool, catalyst, and condensing agent are dissolved in 50%-70% of the volume of the reaction solvent and stirred until completely dissolved to obtain the reaction solution; S2: Droplet addition reaction: The acetic anhydride is diluted with the remaining volume of reaction solvent and slowly added dropwise to the reaction solution obtained in step S1 at 35-40℃ over 15-30 minutes. The reaction is carried out by stirring while maintaining the temperature for 6-10 hours. S3: Process monitoring, using thin-layer chromatography to track the reaction progress, wherein the thin-layer chromatography uses a mixed solution of petroleum ether and ethyl acetate in a volume ratio of 3:1 as the developing solvent until the cycloastragalool starting material spot basically disappears; S4: Quenching, cool the reaction solution obtained in step S2 to room temperature, slowly add saturated sodium bicarbonate solution to quench, stir until no carbon dioxide gas is produced, and obtain a mixed solution; S5: Extraction and washing. Add an equal volume of ethyl acetate to the mixture solution obtained in step S4 for extraction, collect the organic phase, and wash the organic phase 2-3 times with saturated sodium chloride solution, with each wash using a liquid volume of 1 / 2 of the organic phase volume. S6: Drying and concentration: Add anhydrous sodium sulfate to the organic phase obtained in step S5 and dry for 4-6 hours. Filter to remove anhydrous sodium sulfate and concentrate the filtrate under reduced pressure to obtain crude product. S7: Purification. The crude product obtained in step S6 is purified by silica gel column chromatography. The silica gel column chromatography uses a gradient elution with a mixed solvent of petroleum ether and ethyl acetate, with the eluent volume ratio changing from 20:1 to 5:1. The fraction containing cycloastragaloyl-3-acetate is collected and concentrated again under reduced pressure to obtain pure cycloastragaloyl-3-acetate.

[0007] The solubilizer is polyethylene glycol 400, with a mass percentage of 0.5-3 wt%; the co-solvent is propylene glycol, with a mass percentage of 3.0-8.0 wt%. The buffer salt is a sodium dihydrogen phosphate-disodium hydrogen phosphate system, wherein the mass percentage of sodium dihydrogen phosphate is 0.05-0.2 wt%, the mass percentage of disodium hydrogen phosphate is 0.03-0.15 wt%, and the pH value of the pharmaceutical composition is 4.5-7.0.

[0008] The osmotic pressure regulator is sodium chloride, with a mass percentage of 0.8-1.2 wt%, and the preservative is benzalkonium chloride, with a mass percentage of 0.01-0.03 wt%.

[0009] The catalyst is 4-dimethylaminopyridine, the condensing agent is N,N'-diisopropylcarbodiimide, and the reaction solvent is anhydrous dichloromethane; in step S1, the inert gas is nitrogen, and the dissolution temperature of the reaction solvent is 25-30℃.

[0010] The silica gel column chromatography method uses silica gel with a particle size of 200-300 mesh and an elution flow rate of 1-2 column volumes / hour. The collected fractions containing the target compound are detected by high performance liquid chromatography (HPLC). When the purity of cycloastragaloyl-3-acetate is ≥98%, the fractions are combined and concentrated.

[0011] The application of a pharmaceutical composition for delaying lens cell aging is an eye drop prepared using the composition according to any one of claims 1-7, characterized by comprising the following preparation steps: S11: Buffer salts, osmotic pressure regulators, preservatives, and antioxidants are dissolved in 50%-70% volume of water for injection and stirred until completely dissolved to obtain an aqueous phase; S22: Dissolve cycloastragaloyl-3-acetate in a mixture of solubilizer and cosolvent, and stir until completely dissolved to obtain the oil phase; S33: Under stirring conditions, the oil phase is slowly added to the aqueous phase, and stirring is continued until a clear or translucent solution is formed; S44: Add water for injection to the total volume and stir well; S55: Sample and test the content of cycloastragaloyl-3-acetate in the intermediate and the pH value of the solution. After passing the test, filter the solution through a 0.22μm microporous membrane for sterilization and fill it into an eye drop bottle under aseptic conditions to obtain an eye drop for delaying lens cell aging.

[0012] In step S11, the antioxidant is selected from at least one of sodium sulfite, sodium bisulfite, vitamin C, and disodium edetate, and the antioxidant has a mass percentage of 0.01-0.1 wt%.

[0013] In step S33, the stirring speed is 150-300 rpm, and the stirring time is 20-40 minutes. The eye drop bottle is made of low-density polyethylene, and the multi-dose eye drop bottle requires the addition of 0.01-0.03 wt% benzalkonium chloride. The 0.22 μm microporous filter membrane is made of polyethersulfone.

[0014] The present invention has at least the following beneficial effects: This invention applies cycloastragaloyl-3-acetate to delay lens cell aging. Compared with its parent compound cycloastragaloyl, this esterified derivative has significantly enhanced lipid solubility and cell membrane permeability. At the same concentration, its ability to activate telomerase activity in lens epithelial cells is significantly better than that of cycloastragaloyl, demonstrating stronger biological activity potential.

[0015] The chemical synthesis method provided by this invention uses 4-dimethylaminopyridine (DMAP) as a catalyst and N,N'-diisopropylcarbodiimide (DIC) as a condensing agent to carry out esterification reaction efficiently under mild conditions. The process route is simple, with high yield and good product purity.

[0016] This invention screened specific types and ratios of excipients, successfully converting the poorly soluble active ingredient cycloastragaloyl-3-acetate into a clear, stable eye drop that meets the requirements for ophthalmic preparations.

[0017] This invention utilizes cycloastragaloyl-3-acetate to achieve a high molecular dispersion in solution, significantly improving its bioavailability in the eye. This allows the drug to reach the target site more effectively, thereby fully realizing its effects of activating telomerase and delaying cell aging.

[0018] The finished product of this invention has a uniform and accurate content of active ingredients and a stable and controllable pH value. All these characteristics ensure the reliability of the product's quality and the consistency of its therapeutic effects. Detailed Implementation

[0019] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] Example 1 This invention provides a technical solution: a pharmaceutical composition for delaying lens cell aging and its application, wherein the raw material for preparing cycloastragaloyl-3-acetate, based on a total reaction system mass of 1000g, comprises: Cyclostramonol 0.2wt% (2.0g), acetic anhydride 0.3wt% (3.0g), catalyst 4-dimethylaminopyridine (DMAP) 0.03wt% (0.3g), condensing agent N,N'-diisopropylcarbodiimide (DIC) 0.05wt% (0.5g), balance anhydrous dichloromethane (994.2g); Preparation steps of cycloastragaloyl-3-acetate: S1: Purge a dry 1000mL three-necked flask with nitrogen gas, and install a stirrer, thermometer, and constant-pressure dropping funnel. Add 596.52 mL of anhydrous dichloromethane. Under continuous nitrogen protection and stirring (250 rpm), add 2.0 g of cycloastragalool, 0.3 g of DMAP, and 0.5 g of DIC sequentially. Control the reaction solution temperature at 28 ± 2℃ and stir for 30 minutes until the solids are completely dissolved, yielding a clear reaction solution. S2: Mix and dilute 3.0 g of acetic anhydride with 397.68 mL of anhydrous dichloromethane in a dropping funnel. Heat the reaction system to 38 ± 2 °C, and add the acetic anhydride solution dropwise over 20 minutes with stirring. After the addition is complete, maintain the temperature at 38 °C and continue stirring for 8 hours. S3: Samples were taken at 6, 7, and 8 hours after the start of the reaction and monitored using thin-layer chromatography (TLC). GF254 silica gel plates were used, with petroleum ether:ethyl acetate = 3:1 (v / v) as the developing solvent. Observation was performed under UV light (254 nm). At 8 hours, the cycloastragaloyl alcohol starting material spot (Rf value approximately 0.25) essentially disappeared, while the target product spot (Rf value approximately 0.45) was clearly visible. The reaction was then stopped. S4: Cool the reaction solution to 25°C. While stirring, slowly add 50 mL of pre-cooled saturated sodium bicarbonate aqueous solution to quench the reaction. A large number of carbon dioxide bubbles will be observed. Continue stirring for 15 minutes until no more bubbles escape. S5: Transfer the entire reaction mixture to a 2000 mL separatory funnel. Add 1000 mL of ethyl acetate, shake thoroughly to extract, and allow to stand for separation. Collect the lower organic phase. Extract the upper aqueous phase once more with 200 mL of ethyl acetate, and combine the organic phases. Wash the combined organic phases twice with saturated sodium chloride solution (500 mL each time) to remove residual water-soluble impurities; S6: Transfer the washed organic phase to a 2000mL Erlenmeyer flask, add 10g of anhydrous sodium sulfate, seal, and dry on a magnetic stirrer for 5 hours. Filter through a sintered glass funnel and wash the sodium sulfate particles with a small amount of ethyl acetate. Combine the filtrate and washings, and concentrate by rotary evaporation at 40℃ water bath and -0.09 MPa vacuum to obtain 1.92g of a pale yellow oily crude product. S7: Purification was performed by column chromatography using 200-300 mesh silica gel. After wet loading, gradient elution was carried out using a petroleum ether-ethyl acetate system (20:1→15:1→10:1→5:1, v / v) at a flow rate of 1.5 BV / h. The fractions were monitored by TLC, and the fractions rich in the target product (Rf=0.45) were collected. The qualified fractions were combined and concentrated again under reduced pressure to obtain 1.75 g of pure cycloastragaloyl-3-acetate as a white solid. HPLC analysis (C18 column, methanol-water = 85:15, flow rate 1.0 mL / min, detection wavelength 203 nm) showed a purity of 98.5% and a yield of 80.6% based on the amount of cycloastragalool fed.

[0021] Preparation of pharmaceutical composition and eye drops: Based on a total weight of 100g of eye drops, the raw material composition is as follows: cycloastragaloyl-3-acetate 0.08wt% (0.08g), solubilizer polyethylene glycol 2wt% (2.0g), cosolvent propylene glycol 5wt% (5.0g), osmotic pressure regulator sodium chloride 1.0wt% (1.0g), buffer salt (sodium dihydrogen phosphate 0.1wt% + disodium hydrogen phosphate 0.08wt%, i.e., 0.1g + 0.08g), preservative benzalkonium chloride 0.02wt% (0.02g), antioxidant sodium bisulfite 0.05wt% (0.05g), and water for injection to 100g.

[0022] The steps for preparing eye drops include: S11: Place approximately 55.0 g of water for injection in a 100 mL beaker and place it on a magnetic stirrer. Add sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, benzalkonium chloride, and sodium bisulfite sequentially. Stir at 300 rpm for 15 minutes until all components are completely dissolved, yielding a clear aqueous phase. Measure the pH using precision pH paper or a pH meter to obtain 5.2. S22: In a separate 50mL beaker, accurately weigh in polyethylene glycol 400 and propylene glycol, and mix thoroughly. While stirring, add 0.08g of cycloastragaloyl-3-acetate powder to the above mixed solvent, and continue stirring for 20 minutes to obtain a clear and transparent oil phase solution; S33: At a stirring speed of 200 rpm, slowly add the oil phase (S22) dropwise to the aqueous phase S11 using a glass dropper. After the addition is complete, increase the stirring speed to 500 rpm and continue stirring for 30 minutes to form a clear and transparent solution with no visible oil droplets or suspended matter. S44: Quantitatively transfer the above solution to a 100mL volumetric flask, wash the beaker several times with a small amount of water for injection, add the washing solution to the volumetric flask, and finally add water for injection to make up to the 100g mark and shake well. S55: Take the intermediate solution and determine the content of cycloastragaloyl-3-acetate (HPLC method, result: 0.079%) and pH value (5.5) according to the method in the Chinese Pharmacopoeia. The solution is sterilized by pressure filtration through a 0.22μm polyethersulfone microporous membrane. In a Class A laminar flow hood under a Class B background, the filtrate is aseptically filled into pre-sterilized low-density polyethylene (LDPE) single-dose eye drop bottles, capped, and sealed.

[0023] Example 2 A pharmaceutical composition for delaying lens cell aging and its application, wherein the raw materials for preparing cycloastragaloyl-3-acetate, based on a total reaction system mass of 1000g, include: Cyclostramonol 0.2wt% (2.0g), acetic anhydride 0.3wt% (3.0g), catalyst 4-dimethylaminopyridine (DMAP) 0.03wt% (0.3g), condensing agent N,N'-diisopropylcarbodiimide (DIC) 0.05wt% (0.5g), balance anhydrous dichloromethane (994.2g); Preparation steps of cycloastragaloyl-3-acetate: S1: Under nitrogen protection, 2.0 g of cycloastragalool, 0.3 g of DMAP and 0.5 g of DIC were dissolved in 60% volume of anhydrous dichloromethane (596.52 mL, density 1.326 g / mL), the dissolution temperature was controlled at 28 °C, and the mixture was stirred for 30 minutes until completely dissolved to obtain the reaction solution; S2: Dilute 3.0g of acetic anhydride with the remaining 40% volume of anhydrous dichloromethane (397.68mL), and slowly add it dropwise to the reaction solution obtained in S1 over 20 minutes at 38℃. The reaction is carried out by stirring at the temperature for 8 hours. S3: Thin-layer chromatography was used to track the reaction process. After 8 hours of reaction, the cycloastragalool starting material spot basically disappeared, and the reaction was stopped. S4: Cool the reaction solution to room temperature (25°C), slowly add saturated sodium bicarbonate solution (50 mL) to quench the reaction, stir for 15 minutes until no carbon dioxide gas is produced, and obtain a mixed solution; S5: Add an equal volume (about 1000 mL) of ethyl acetate to the mixture solution for extraction and collect the organic phase; wash the organic phase twice with saturated sodium chloride solution, each time using 1 / 2 of the volume of the organic phase (500 mL / wash). S6: Add 10g of anhydrous sodium sulfate to the organic phase and dry for 5 hours; filter to remove anhydrous sodium sulfate, and concentrate the filtrate under reduced pressure (vacuum degree -0.09MPa, temperature 40℃) to obtain 1.92g of crude product; S7: The crude product was purified by silica gel column chromatography with a mesh size of 200-300. The eluent was a mixture of petroleum ether and ethyl acetate (volume ratio gradually varied from 20:1 to 5:1), and the elution flow rate was 1.5 column volumes / hour. The fractions were collected and analyzed by HPLC (chromatographic conditions: C18 column, mobile phase methanol-water = 85:15, flow rate 1.0 mL / min, detection wavelength 203 nm). The fractions with a purity of ≥98% of cycloastragaloyl-3-acetate were combined and concentrated again under reduced pressure to obtain 1.75 g of pure product with a purity of 98.5% and a yield of 80.6%.

[0024] Pharmaceutical composition and preparation of eye drops: Based on a total weight of 100g of eye drops, the raw material ratio is as follows: cycloastragaloyl-3-acetate 0.05wt% (0.05g), solubilizer polyethylene glycol 0.5wt% (0.5g), cosolvent propylene glycol 3wt% (3.0g), osmotic pressure regulator sodium chloride 0.8wt% (0.8g), buffer salt (sodium dihydrogen phosphate 0.05wt% + disodium hydrogen phosphate 0.03wt%, i.e., 0.05g + 0.03g), preservative benzalkonium chloride 0.01wt% (0.01g), antioxidant disodium edetate 0.01wt% (0.01g), and the balance being water for injection (95.55g); Preparation steps of eye drops: S11: Dissolve 0.05g sodium dihydrogen phosphate, 0.03g disodium hydrogen phosphate, 0.8g sodium chloride, 0.01g benzalkonium chloride, and 0.01g disodium edetate in 50% volume water for injection (about 47.8mL), stir for 10 minutes until dissolved, and the pH of the aqueous phase is 4.8; S22: Dissolve 0.05g of cycloastragaloyl-3-acetate in a mixture of 0.5g of polyethylene glycol and 3.0g of propylene glycol, and stir for 15 minutes until dissolved to obtain the oil phase; S33: Add the oil phase to the aqueous phase at a stirring speed of 150 rpm and continue stirring for 20 minutes to form a semi-transparent solution; S44: Add water for injection to 100g and stir well; S55: The intermediate was found to contain 0.049 wt% cycloastragaloyl-3-acetate, pH 4.5; after sterilization by filtration through a 0.22 μm polyethersulfone membrane, it was filled into single-dose low-density polyethylene eye drop bottles.

[0025] Example 3: A pharmaceutical composition for delaying lens cell aging and its application, wherein the raw materials for preparing cycloastragaloyl-3-acetate, based on a total reaction system mass of 1000g, include: Cyclostramonol 0.2wt% (2.0g), acetic anhydride 0.3wt% (3.0g), catalyst 4-dimethylaminopyridine (DMAP) 0.03wt% (0.3g), condensing agent N,N'-diisopropylcarbodiimide (DIC) 0.05wt% (0.5g), balance anhydrous dichloromethane (994.2g); Preparation steps of cycloastragaloyl-3-acetate: S1: Under nitrogen protection, 2.0 g of cycloastragalool, 0.3 g of DMAP and 0.5 g of DIC were dissolved in 60% volume of anhydrous dichloromethane (596.52 mL, density 1.326 g / mL), the dissolution temperature was controlled at 28 °C, and the mixture was stirred for 30 minutes until completely dissolved to obtain the reaction solution; S2: Dilute 3.0g of acetic anhydride with the remaining 40% volume of anhydrous dichloromethane (397.68mL), and slowly add it dropwise to the reaction solution obtained in S1 over 20 minutes at 38℃. The reaction is carried out by stirring at the temperature for 8 hours. S3: Thin-layer chromatography was used to track the reaction process. After 8 hours of reaction, the cycloastragalool starting material spot basically disappeared, and the reaction was stopped. S4: Cool the reaction solution to room temperature (25°C), slowly add saturated sodium bicarbonate solution (50 mL) to quench the reaction, stir for 15 minutes until no carbon dioxide gas is produced, and obtain a mixed solution; S5: Add an equal volume (about 1000 mL) of ethyl acetate to the mixture solution for extraction and collect the organic phase; wash the organic phase twice with saturated sodium chloride solution, each time using 1 / 2 of the volume of the organic phase (500 mL / wash). S6: Add 10g of anhydrous sodium sulfate to the organic phase and dry for 5 hours; filter to remove anhydrous sodium sulfate, and concentrate the filtrate under reduced pressure (vacuum degree -0.09MPa, temperature 40℃) to obtain 1.92g of crude product; S7: The crude product was purified by silica gel column chromatography with a mesh size of 200-300. The eluent was a mixture of petroleum ether and ethyl acetate (volume ratio gradually varied from 20:1 to 5:1), and the elution flow rate was 1.5 column volumes / hour. The fractions were collected and analyzed by HPLC (chromatographic conditions: C18 column, mobile phase methanol-water = 85:15, flow rate 1.0 mL / min, detection wavelength 203 nm). The fractions with a purity of ≥98% of cycloastragaloyl-3-acetate were combined and concentrated again under reduced pressure to obtain 1.75 g of pure product with a purity of 98.5% and a yield of 80.6%.

[0026] Pharmaceutical composition and preparation of eye drops: Based on a total weight of 100g for the multi-dose eye drops, the raw material ratio is as follows: cycloastragaloyl-3-acetate 0.1wt% (0.1g), solubilizer polyethylene glycol 3wt% (3.0g), cosolvent propylene glycol 8wt% (8.0g), osmotic pressure regulator sodium chloride 1.2wt% (1.2g), buffer salt (sodium dihydrogen phosphate 0.2wt% + disodium hydrogen phosphate 0.15wt%, i.e., 0.2g + 0.15g), preservative benzalkonium chloride 0.05wt%, totaling 0.05g, antioxidant vitamin C 0.1wt% (0.1g), and the balance being water for injection (87.2g).

[0027] The steps for preparing eye drops include: S11: Dissolve 0.2g sodium dihydrogen phosphate, 0.15g disodium hydrogen phosphate, 1.2g sodium chloride, 0.05g benzalkonium chloride, and 0.1g vitamin C in 70% volume water for injection (approximately 61.0mL), stir for 20 minutes until dissolved, and the pH of the aqueous phase is 6.8; S22: Dissolve 0.1g of cycloastragaloyl-3-acetate in a mixture of 3.0g of polyethylene glycol and 8.0g of propylene glycol, and stir for 25 minutes until dissolved to obtain the oil phase; S33: Add the oil phase to the aqueous phase at a stirring speed of 300 rpm and continue stirring for 40 minutes to form a clear solution; S44: Add water for injection to 100g and stir well; S55: The intermediate was found to contain 0.098 wt% cycloastragaloyl-3-acetate, pH 7.0; after sterilization by filtration through a 0.22 μm polyethersulfone membrane, it was filled into low-density polyethylene multi-dose eye drop bottles.

[0028] Example 4: A pharmaceutical composition for delaying lens cell aging and its application. Preparation of pharmaceutical composition and eye drops: Based on a total weight of 100g of eye drops, the raw material composition is as follows: cycloastragaloyl-3-acetate 0.07g (0.07wt%), hydroxypropyl-β-cyclodextrin 3.0g (3.0wt%), anhydrous ethanol 4.0g (4.0wt%), glucose 1.5g (1.5wt%), citric acid 0.04g (0.04wt%), chlorobutanol 0.03g (0.03wt%), disodium edetate 0.02g (0.02wt%), and water for injection to 100g; The preparation steps of the pharmaceutical composition eye drops include: S11: Dissolve citric acid, sodium citrate, glucose, and disodium edetate in 50g of water for injection and stir until dissolved. Dissolve chlorobutanol in anhydrous ethanol first, then add this ethanol solution to the above aqueous solution and stir until homogeneous. The pH is measured to be approximately 5.8.

[0029] S22: Dissolve HP-β-CD in 20g of warm (40-50℃) water for injection, stir until completely dissolved to form a clear solution, cool to room temperature, add cycloastragaloyl-3-acetate to the HP-β-CD solution, and homogenize at high speed (10000 rpm, 2 minutes) to form an inclusion complex.

[0030] S33: Under stirring, slowly add the inclusion complex solution obtained in S22 to the aqueous phase solution of S11, and stir for 30 minutes to mix it evenly.

[0031] S44: Add water for injection to 100g and stir well.

[0032] S55: Detect content and pH, filter and sterilize, fill.

[0033] Comparative Example 1: A pharmaceutical composition for delaying lens cell aging and its application. Preparation of pharmaceutical composition and eye drops: Based on a total weight of 100g of eye drops, the raw material composition is as follows: solubilizer (polyethylene glycol) 2.0g, cosolvent (propylene glycol) 5.0g, osmotic pressure regulator (sodium chloride) 1.0g, buffer salt (sodium dihydrogen phosphate) 0.1g, buffer salt (disodium hydrogen phosphate) 0.08g, preservative (benzalkonium chloride) 0.02g, antioxidant (sodium bisulfite) 0.05g, water for injection: to 100g; The preparation steps of the pharmaceutical composition eye drops include: Take a clean 250mL beaker and accurately weigh 91.67g of water for injection into it. Place the beaker on a magnetic stirrer and turn on the stirrer at 300rpm. Accurately weigh and add the following ingredients in sequence: 0.1g sodium dihydrogen phosphate, 0.08g disodium hydrogen phosphate, 1.0g sodium chloride, 0.02g benzalkonium chloride, and 0.05g sodium bisulfite; Continue stirring for 15 minutes until all solid powder and liquid additives are completely dissolved, yielding a colorless, clear, transparent aqueous solution. The pH of this solution was measured to be 5.2 using a calibrated pH meter. Take another 100mL beaker, accurately weigh 2.0g of polyethylene glycol 400 and 5.0g of propylene glycol into it, and manually stir with a glass rod to mix evenly; At a stirring speed of 200 rpm, the oil phase was slowly added dropwise to the aqueous phase using a glass dropper. After the addition was complete, the stirring speed was increased to 500 rpm and stirred continuously for 30 minutes. Throughout the process, the solution remained clear and transparent, without any turbidity, precipitation or phase separation. Transfer the stirred solution to a 100 mL volumetric flask, rinse the beaker and inner wall several times with a small amount of water for injection, add all the washings to the volumetric flask, and finally dilute to the 100.0 g mark with water for injection, tighten the cap, and invert the flask 10 times to mix it thoroughly. Sampling was performed using the same HPLC method as in Example 1. The appearance of the acyclic astragaloyl-3-acetate chromatographic peak was confirmed, and the pH value was measured to be 5.5. The solution was sterilized by pressure filtration through a 0.22 μm polyethersulfone (PES) microporous membrane. In a Class A laminar flow ultra-clean workbench under a Class B background, the filtered clear solution was aseptically filled into pre-sterilized low-density polyethylene single-dose eye drop bottles, 0.8 mL per bottle. The bottle caps were then screwed on and sealed to obtain the blank control eye drops.

[0034] Comparative Example 2: A pharmaceutical composition for delaying lens cell aging and its application. Preparation of pharmaceutical composition and eye drops: Based on a total weight of 100g of eye drops, the raw material composition is as follows: cycloastragalool 0.074wt% (0.074g), solubilizer polyethylene glycol 2wt% (2.0g), cosolvent propylene glycol 5wt% (5.0g), osmotic pressure regulator sodium chloride 1.0wt% (1.0g), buffer salt (sodium dihydrogen phosphate 0.1wt% + disodium hydrogen phosphate 0.08wt%, i.e., 0.1g + 0.08g), preservative benzalkonium chloride 0.02wt% (0.02g), antioxidant sodium bisulfite 0.05wt% (0.05g), and water for injection to 100g.

[0035] The steps for preparing eye drops include: Take approximately 55.0 g of water for injection and place it in a 100 mL beaker on a magnetic stirrer. Add sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, benzalkonium chloride, and sodium bisulfite sequentially, stirring at 300 rpm for 15 minutes until all components are completely dissolved, yielding a clear aqueous phase. The pH value is measured to be 5.2 using precision pH paper or a pH meter. Take another 100mL beaker and accurately weigh 2.0g of polyethylene glycol and 5.0g of propylene glycol into it. Stir manually with a glass rod until well mixed. Accurately weigh 0.074g of cycloastragaloyl alcohol powder and slowly add it to the above mixed solvent while stirring. Place the beaker on a magnetic stirrer and stir at 500 rpm for 30 minutes. The cycloastragaloyl alcohol did not completely dissolve, and the solution was pale yellow and turbid with a large amount of undissolved fine particles precipitated at the bottom. At a stirring speed of 200 rpm, slowly add the above turbid oil phase dropwise to the clear aqueous phase. After the addition is complete, increase the stirring speed to 500 rpm and continue stirring for 60 minutes. The entire system becomes a milky white opaque chyme that cannot form a clear solution. Transfer the mixture to a 100mL volumetric flask and make up to 100.0g. The solution was a uniform milky white suspension. Filtering with a 0.22 μm microporous membrane resulted in rapid clogging, with only a very small amount of filtrate dripping out, and the filtrate remaining turbid. Sterilization filtration was not achieved. The unfiltered suspension was poured into an eye drop bottle and allowed to stand for 2 hours. A thick white precipitate layer appeared at the bottom of the bottle, and the upper clear liquid was slightly turbid. Shaking allowed the precipitate to redisperse, but it failed to form a stable, homogeneous solution.

[0036] Comparative Example 3: Pharmaceutical composition and preparation of eye drops: Based on a total mass of 100g for the multi-dose eye drops, the raw material ratio is as follows: cycloastragaloyl-3-acetate 0.08wt% (0.08g), propylene glycol 5wt% (5.0g) as a solubilizer, sodium chloride 1.0wt% (1.0g) as an osmotic pressure regulator, buffer salt (sodium dihydrogen phosphate 0.1wt% + disodium hydrogen phosphate 0.08wt%, i.e., 0.1g + 0.08g), benzalkonium chloride 0.05wt% as a preservative, totaling 0.05g, antioxidant (sodium bisulfite) 0.05g, and the balance being water for injection (87.2g).

[0037] The steps for preparing eye drops include: Take approximately 55.0 g of water for injection and place it in a 100 mL beaker on a magnetic stirrer. Add sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, benzalkonium chloride, and sodium bisulfite sequentially, stirring at 300 rpm for 15 minutes until all components are completely dissolved, yielding a clear aqueous phase. The pH value is measured to be 5.2 using precision pH paper or a pH meter. In a separate 100mL beaker, accurately weigh 5.0g of propylene glycol. Add 0.08g of cycloastragaloyl-3-acetate to it; After magnetic stirring (500 rpm) for 30 minutes, most of the solids dissolved, but the solution was slightly cloudy or milky, indicating that the dissolution was incomplete. The oil phase was added to the aqueous phase with stirring. Initially, the solution became clear, but during the subsequent 30 minutes of stirring, the solution gradually became cloudy, accompanied by the precipitation of fine needle-like crystals. After adjusting the volume to 100g, the solution is a slightly opalescent, incompletely clear solution; The solution was left to stand at room temperature (25°C). After 1 hour, fine crystals visible to the naked eye began to appear on the bottle walls and bottom. After 24 hours, the amount of crystals precipitated increased significantly, and the upper layer of the solution became cloudy, indicating that the physical stability of the preparation was extremely poor.

[0038] Comparative Example 4: A pharmaceutical composition for delaying lens cell aging and its application, wherein the raw materials for preparing cycloastragaloyl-3-acetate, based on a total reaction system mass of 1000g, include: Cycloastragalol 0.2wt% (2.0g), acetic anhydride 0.3wt% (3.0g), balance anhydrous dichloromethane (994.2g); Preparation steps of cycloastragaloyl-3-acetate: Under nitrogen protection, 596.52 mL of anhydrous dichloromethane was added to the reaction flask, along with only 2.0 g of cycloastragalol. The mixture was stirred at 28°C for 30 minutes, during which the cycloastragalol partially dissolved, and the solution became slightly turbid. Diluted acetic anhydride was added dropwise to the reaction system, and the reaction was maintained at 38°C for 8 hours. TLC analysis was performed on samples taken at 2, 4, 6, and 8 hours of the reaction. At all time points, only the cycloastragaloyl alcohol starting material spot (Rf≈0.25) was observed on the TLC plates, and its spot intensity did not show a significant decrease. No new spots appeared, especially the target product spot (Rf≈0.45). Cool the reaction solution to room temperature (25°C), slowly add saturated sodium bicarbonate solution (50 mL) to quench the reaction, stir for 15 minutes until no carbon dioxide gas is produced, and obtain a mixed solution. Add an equal volume (about 1000 mL) of ethyl acetate to the mixture solution for extraction and collect the organic phase; wash the organic phase twice with saturated sodium chloride solution, each time using 1 / 2 of the volume of the organic phase (500 mL / wash). S6: Add 10g of anhydrous sodium sulfate to the organic phase and dry for 5 hours; filter to remove anhydrous sodium sulfate, and concentrate the filtrate under reduced pressure (vacuum degree -0.09MPa, temperature 40℃) to obtain 1.92g of crude product; S7: The crude product was purified by silica gel column chromatography using a 200-300 mesh screen. The eluent was a mixture of petroleum ether and ethyl acetate (volume ratio gradually varied from 20:1 to 5:1), and the elution flow rate was 1.5 column volumes / hour. The fractions were collected and analyzed by HPLC (chromatographic conditions: C18 column, mobile phase methanol-water = 85:15, flow rate 1.0 mL / min, detection wavelength 203 nm). The fractions with a purity ≥98% of cycloastragaloyl-3-acetate were combined and concentrated again under reduced pressure to obtain 1.75 g of pure product with a purity of 98.5% and a yield of 80.6%. The final product was a white to off-white solid. HPLC analysis showed that its chromatogram was consistent with that of the cycloastragalol raw material. No chromatographic peak of cycloastragalol-3-acetic acid ester was detected. The product weight was 1.88 g, which was similar to the amount of raw material fed. The yield was approximately 94%, and the synthesis reaction yield was approximately 0%.

[0039] Table 1 Comparison of Synthesis Efficiency and Quality of Active Ingredients in Examples (Examples)

[0040] As shown in Table 1, Comparative Example 4 demonstrates that the esterification reaction cannot proceed without the participation of DMAP and DIC. In contrast, the Example 4, under the process conditions described, can obtain the target product with a high purity (>98%) in a yield of >80%, demonstrating the efficiency and reliability of the synthesis method.

[0041] Table 2 Comparison of characteristics and stability of the formulations in the examples and comparative examples

[0042] The evaluation criteria in Table 2 are as follows: Appearance score: Clear and transparent = 3 points; Semi-transparent = 2 points; Opaque / slightly turbid = 1 point; Turbid / precipitate = 0 points; Filterability: Whether it can pass smoothly through a 0.22μm sterile filter membrane; Stability: Changes in physical state after standing at room temperature for 24 hours.

[0043] As shown in Table 2, Comparative Examples 2 and 3 demonstrate that neither cycloastragalool alone nor formulations lacking key excipients can produce stable products that meet the basic requirements for ophthalmic preparations. In contrast, the examples successfully produced filterable, accurately measured, and stable formulations.

[0044] Table 3 Comparison of the effects of the examples and comparative formulations on telomerase activity

[0045] Table 3 shows that, at the same concentration, CyA-Ac has a significantly better biological effect than CyA, thanks to the permeability advantage brought about by its esterification structure. Comparative examples 2 and 3 demonstrate from a biological perspective that even if the active ingredient itself is effective, it will ultimately fail to produce any therapeutic effect if it cannot be successfully formulated into a stable and soluble preparation.

[0046] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A pharmaceutical composition for delaying the aging of lens cells, characterized by, According to the mass percentage of raw materials, it comprises: 0.05-0.1wt% of cycloastragenol ester derivatives, 0.1-5wt% of solubilizing agent, 1.0-10.0wt% of cosolvent, 0.5-2.0wt% of osmotic pressure regulator, 0.01-0.5wt% of buffer salt, 0.001-0.05wt% of preservative, and the balance is water for injection; The cycloastragenol ester derivative is cycloastragenol-3-acetate; The solubilizing agent is selected from at least one of Tween 80, polyethylene glycol 400, and hydroxypropyl-β-cyclodextrin; The cosolvent is selected from at least one of propylene glycol, glycerol, and anhydrous ethanol; The osmotic pressure regulator is selected from at least one of sodium chloride, mannitol, and glucose; The buffer salt is selected from at least one of sodium dihydrogen phosphate-disodium hydrogen phosphate system, citric acid-sodium citrate system, and acetic acid-sodium acetate system; The preservative is selected from at least one of benzalkonium chloride, hydroxyphenyl ethyl ester, and chlorobutanol.

2. The pharmaceutical composition for delaying the aging of lens cells according to claim 1, characterized by: The preparation method of the cycloastragenol-3-acetate is characterized by taking the total mass of the reaction system as 100%, and the preparation raw materials comprising: 0.1-0.3wt% of cycloastragenol, 0.15-0.5wt% of acetic anhydride, 0.01-0.05wt% of catalyst, 0.02-0.08wt% of condensing agent, and the balance being a reaction solvent; The preparation method comprises the following steps: S1: feeding and dissolving, under the protection of inert gas, the cycloastragenol, catalyst, and condensing agent are dissolved in 50%-70% volume of reaction solvent, and stirring is performed until complete dissolution to obtain a reaction solution; S2: dropwise addition reaction, the acetic anhydride is diluted with the remaining volume of reaction solvent, and then slowly added to the reaction solution obtained in step S1 at 35-40℃ for 15-30 minutes, and esterification reaction is performed under insulation stirring, and the reaction time is 6-10 hours; S3: process monitoring, thin layer chromatography is used to track the reaction progress, and the thin layer chromatography uses a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 3:1 as the developing agent until the cycloastragenol raw material point basically disappears; S4: quenching, the reaction solution obtained in step S2 is cooled to room temperature, and saturated sodium bicarbonate solution is slowly added for quenching, and stirring is performed until no carbon dioxide gas is generated to obtain a mixture solution; S5: extraction and washing, equal volume of ethyl acetate is added to the mixture solution obtained in step S4 for extraction, and the organic phase is collected, and the organic phase is washed with saturated sodium chloride solution for 2-3 times, and the volume of washing liquid is 1 / 2 of the volume of the organic phase each time; S6: drying and concentration, the organic phase obtained in step S5 is added to anhydrous sodium sulfate for drying for 4-6 hours, and the anhydrous sodium sulfate is removed by filtration, and the filtrate is concentrated under reduced pressure to obtain a crude product; S7: purification, the crude product obtained in step S6 is purified by silica gel column chromatography, the silica gel column chromatography uses a mixed solvent of petroleum ether and ethyl acetate for gradient elution, the volume ratio of the eluent changes from 20:1 to 5:1, the fraction containing cycloastragenol-3-acetate is collected, and the cycloastragenol-3-acetate is concentrated under reduced pressure again to obtain a pure product.

3. The pharmaceutical composition for delaying the aging of lens cells according to claim 2, characterized by: The solubilizing agent is polyethylene glycol 400, and the mass percentage is 0.5-3wt%; the co-solvent is propylene glycol, and the mass percentage is 3.0-8.0wt%.

4. The pharmaceutical composition for delaying the aging of lens cells according to claim 3, characterized by: The buffer salt is a sodium dihydrogen phosphate-sodium hydrogen phosphate system, wherein the mass percentage of sodium dihydrogen phosphate is 0.05-0.2wt%, the mass percentage of sodium hydrogen phosphate is 0.03-0.15wt%, and the pH value of the pharmaceutical composition is 4.5-7.

0.

5. The pharmaceutical composition for delaying the aging of lens cells according to claim 1 or 4, characterized by: The osmotic pressure regulator is sodium chloride, and the mass percentage is 0.8-1.2wt%; the preservative is benzalkonium chloride, and the mass percentage is 0.01-0.03wt%.

6. The pharmaceutical composition for delaying the aging of lens cells according to claim 2, characterized by: The catalyst is 4-dimethylaminopyridine, the condensing agent is N,N'-diisopropyl carbodiimide, and the reaction solvent is anhydrous dichloromethane; in step S1, the inert gas is nitrogen, and the dissolution temperature of the reaction solvent is 25-30℃.

7. The pharmaceutical composition for delaying the aging of lens cells according to claim 6, wherein: The silica gel column chromatography uses silica gel particles with a particle size of 200-300 mesh, and the elution flow rate is 1-2 column volumes / hour; the collected fractions containing the target compound are detected by high performance liquid chromatography (HPLC), and when the purity of cycloastragenol-3-acetate is ≥98%, the fractions are combined and concentrated.

8. Use of a pharmaceutical composition for delaying the aging of lens cells, characterized in that the composition is prepared in the form of eye drops, according to any one of claims 1 to 7. The preparation steps include: S11: Dissolve the buffer salt, osmotic pressure regulator, preservative, and antioxidant in 50%-70% by volume of water for injection, and stir until completely dissolved to obtain an aqueous phase; S22: Dissolve cycloastragenol-3-acetate in a mixture of solubilizing agent and co-solvent, and stir until completely dissolved to obtain an oil phase; S33: Slowly add the oil phase to the aqueous phase under stirring, and continue stirring until a clear or translucent solution is formed; S44: Add water for injection to the full amount, and stir uniformly; S55: Take a sample to detect the content of cycloastragenol-3-acetate in the intermediate and the pH value of the solution; after passing the quality inspection, filter sterilize through a 0.22μm microporous filter, and fill into eye drop bottles under sterile conditions to obtain an eye drop for delaying lens cell aging.

9. The use of a pharmaceutical composition for delaying the aging of lens cells according to claim 8, characterized in that: In step S11, the antioxidant is selected from at least one of sodium sulfite, sodium bisulfite, vitamin C, and disodium edetate, and the mass percentage of the antioxidant is 0.01-0.1wt%.

10. The use of a pharmaceutical composition for delaying the aging of lens cells according to claim 9, characterized in that: In step S33, the stirring speed is 150-300rpm, the continuous stirring time is 20-40 minutes, the eye drop bottle is made of low-density polyethylene, and a multi-dose eye drop bottle needs to be additionally added with 0.01-0.03wt% of benzalkonium chloride, and the 0.22μm microporous filter is made of polyether sulfone.

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