A myocardial patch and its preparation method

Abstract

This invention provides a myocardial patch and its preparation method, addressing the obstacles to the use of naringin in myocardial patches, mainly including dissolution, storage, and bioavailability issues. The myocardial patch contains naringin, which acts as an active substance for anti-inflammatory and cardiac repair. This invention processes naringin into a myocardial patch, overcoming numerous obstacles to its use in myocardial patches, while leveraging the advantages of unsaturated fatty acids and naringin for myocardial health. Its application in advanced heart failure treatment products can improve the therapeutic effect of heart failure.

Description

Technical Field

[0001] This invention relates to the field of myocardial patch technology, specifically to a myocardial patch containing naringin and its preparation method. Background Technology

[0002] Naringin, a natural flavonoid compound, has shown multifaceted potential in the treatment of heart failure. It possesses antioxidant properties, neutralizing excess free radicals in the body, reducing oxidative stress damage to cardiomyocytes, and protecting myocardial tissue. Its anti-inflammatory effects can reduce inflammatory cytokine levels, inhibit myocardial inflammatory responses, slow the progression of myocardial fibrosis, and improve cardiac structure and function. It can also regulate cardiac energy metabolism, optimize energy supply to cardiomyocytes, and improve cardiac efficiency. Furthermore, it can regulate the neuroendocrine system to some extent, correcting the over-activated neuroendocrine mechanisms in heart failure, stabilizing cardiac electrophysiological activity, and reducing the risk of arrhythmias. These combined effects indicate that naringin has promising applications in the treatment of heart failure and is expected to become a potential drug for improving the condition and quality of life of heart failure patients.

[0003] Despite the wide range of pharmacological effects of naringin, its poor absorption and low bioavailability in the human body remain unresolved, limiting its clinical application and new drug development. Researchers have conducted extensive explorations to improve the bioavailability of naringin. Nanoparticle formulations are an effective method; by preparing naringin into nanoparticles, their surface area can be increased, improving the drug's dissolution rate and solubility, thereby promoting absorption. For example, naringin nanoparticles exhibit better dispersibility in vivo, enabling them to reach the site of action more quickly and improving bioavailability. Liposome technology is also widely used in the development of naringin formulations. Liposomes can encapsulate naringin, protecting it from the influence of the in vivo environment while enhancing its affinity for cell membranes, promoting drug entry into cells and improving efficacy. Although nanoparticle formulations and liposome technology have shown certain advantages, these technologies still face some challenges in practical applications. For example, the preparation process of nanoparticles is complex and costly, making large-scale production difficult; liposomes have poor stability and are prone to rupture during storage and transportation, leading to drug leakage. Therefore, finding more stable and efficient formulation excipients and optimizing formulation formulations and processes are key to improving the stability and bioavailability of naringin formulations.

[0004] To address the issue of low bioavailability of naringin, this invention provides a novel naringin preparation intermediate and its preparation method. The naringin preparation intermediate is then used in heart failure treatment formulations to obtain heart failure repair formulations, thereby achieving the goal of using naringin in the treatment of heart failure. Summary of the Invention

[0005] This invention provides a myocardial patch and its preparation method to overcome the obstacles to the use of naringin in myocardial patches, mainly including dissolution obstacles, storage obstacles and bioavailability obstacles.

[0006] A myocardial patch and its preparation method are disclosed, wherein the myocardial patch contains naringin, which is an active substance for anti-inflammatory and heart damage repair.

[0007] Furthermore, the naringin is a modified naringin or a liposome preparation intermediate.

[0008] Furthermore, the modified naringin is obtained by modifying naringin with unsaturated fatty acids, and the lipid formulation intermediate includes the modified naringin.

[0009] Furthermore, the unsaturated fatty acid is oleic acid, linoleic acid, γ-linoleic acid, or eicosapentaenoic acid.

[0010] Furthermore, the modified naringin has an octanol-water partition coefficient of 4-7.

[0011] Furthermore, the preparation method of the modified naringin is as follows: naringin, unsaturated fatty acids, lipase and solvent are placed in a reaction vessel and reacted. After the reaction, the lipase is separated by centrifugation. The remaining reaction solution is evaporated in a rotary evaporator to obtain a crude product. The crude product is dissolved in hexane, extracted with ethanol, and then rotary evaporated under reduced pressure to obtain the modified naringin. The lipase is Novozym® 435 or Lipozyme® RM IM.

[0012] Furthermore, the molar ratio of naringin to the unsaturated fatty acid is 1:3 to 1:5, the reaction temperature is between 40-50℃, the amount of lipase added is 10% of the sum of the weights of naringin and unsaturated fatty acid, and the reaction time is 6-8 hours.

[0013] Furthermore, the molar ratio of naringin to the unsaturated fatty acid is 1:3, the reaction temperature is between 40°C, the amount of lipase added is 10% of the sum of the weights of naringin and unsaturated fatty acid, and the reaction time is 6 hours.

[0014] Further, the preparation method of the liposome formulation intermediate is as follows: 800 mg of soybean lecithin and 200 mg of cholesterol are weighed and dissolved in 10 mL of chloroform solution; 200 mg of modified naringin is weighed and dissolved in 2 mL of methanol; the lipid solution and the modified naringin solution are mixed evenly in an in-situ flask, and evaporated on a rotary evaporator at a temperature of 45°C and a rotation speed of 400 r / min for 30-40 min until a clear lipid film is visible on the inner wall of the flask; 30 mL of pre-prepared PBS buffer solution is added to the round-bottom flask, and the mixture is stirred for 2 h to fully hydrate the lipid film, forming crude liposomes; the crude liposomes are homogenized by ultrasonic treatment; the mixture is centrifuged at 8000 r / min for 20 min, the supernatant is discarded, PBS buffer solution is added to the precipitate, and the mixture is centrifuged again. This process is repeated 2-3 times to obtain the liposome formulation intermediate.

[0015] Further, the preparation method of the myocardial patch is as follows: 10 mL of dichloromethane and 3.3 mL of N,N-dimethylformamide are poured into a beaker and stirred evenly; 1.5 g of PLGA is added to the above solvent and stirred continuously until the solution becomes clear to obtain a PLGA solution; 0.05 mL of Tween-80 is added to 5 mL of anhydrous ethanol and stirred evenly to obtain an ethanol solution; 1 g of lipid formulation intermediate is added to the ethanol solution and stirred to obtain a dispersion; the dispersion is slowly added dropwise to the PLGA solution and stirred continuously until fully mixed to obtain a spinning solution. The spinning solution is installed on a syringe pump, the distance between the nozzle and the receiving device is adjusted to 12 cm, the voltage to 15 kV, the solution flow rate to 1 mL / h, and the rotation speed of the rotating drum to 80 rpm. The myocardial patch obtained by spinning is placed in a drying oven and dried at 35°C for 18 h. The dried myocardial patch is then immersed in PBS buffer solution to wash away residues, obtaining the myocardial patch.

[0016] Compared with the prior art, the present invention has the following beneficial effects: This invention modifies naringin using unsaturated fatty acids. Unsaturated fatty acids have anti-inflammatory and myocardial nourishing effects. Applying them to heart failure preparations not only retains their inherent beneficial effects but also modifies naringin, resulting in higher lipid solubility and improved bioavailability. The modified naringin has an appropriate n-octanol-water partition coefficient, which not only improves lipid solubility but also enhances its hydrophilicity (pure naringin is poorly soluble in water), making it more suitable for the hydrophilic properties of heart failure preparations (beneficial for cell viscosity and growth). This invention establishes for the first time a positive correlation between esterification conditions, esterification rate, and hydrophilic lipid solubility, which is the key inventive aspect of this invention.

[0017] This invention prepares modified naringin into a pharmaceutical intermediate, thereby enhancing the stability of modified naringin and enabling it to remain stable for up to 6 months, providing a foundation for the subsequent preparation of heart failure preparations. In addition, processing it into a pharmaceutical intermediate also gives naringin a sustained-release effect, which is beneficial for heart failure preparations to have a long-term functional effect.

[0018] This invention is the first to process naringin into a myocardial patch, overcoming many obstacles in the use of naringin in myocardial patches. At the same time, it leverages the advantages of unsaturated fatty acids and naringin for myocardial benefit, and its use in products for the treatment of advanced heart failure is beneficial to improving the treatment effect of heart failure. Detailed Implementation

[0019] The extraction of naringin, one of the active ingredients from natural products such as Citrus reticulata peel, and its application as a drug is a recent achievement. Researchers extracted, separated, and purified the effective monomer of naringin from Citrus reticulata peel, and it took more than ten years to develop it into a new drug. Based on this, the research and development of naringin preparations has become the current focus.

[0020] To overcome the problem of low bioavailability of naringin mentioned in the background technology, researchers have made many attempts. For example, Xu Kunyong et al. used the gel addition method to prepare naringin microemulsion gel and used the modified Franz diffusion cell method to evaluate the in vitro release behavior of naringin microemulsion gel and conduct stability tests. The naringin microemulsion gel can be stably stored at 25°C for 6 months. In this process, naringin was added to ethyl oleate by mixing polyoxyethylene 35 castor oil and anhydrous ethanol as a mixed surfactant, and then added to the mixed surfactant. While stirring evenly, water was slowly added dropwise to the prescribed amount to obtain the naringin microemulsion intermediate.

[0021] The solubility of naringin is crucial in the preparation of naringin formulations or intermediates. Naringin has low solubility in water, only 0.1% at room temperature and only 10% at 75°C. It is soluble in methanol, acetone, ethanol, hot acetic acid, dilute alkaline solutions, pyridine, formamide, and dimethylformamide, as well as hot water, but insoluble in petroleum ether, benzene, diethyl ether, hexane, and chloroform. Naringin also has very low solubility in oils and fats; even after emulsification, it cannot form a stable emulsion when added to oils, limiting its application.

[0022] References: Li Peibo, Wang Yonggang, Wu Zhong, et al. Preclinical study of naringin, a new drug derived from Citrus reticulata peel [J]. Journal of Sun Yat-sen University (Natural Science Edition), 2015, 54(6): 1-5.

[0023] Xu Kunyong, Shi Yujie, Mei Yanfei, Yan Juan, Yun Guoping. "Preparation and In Vitro Release of Naringin Microemulsion Gel" "Observation." *Modern Chinese Medicine Research and Practice*, 5 (2020). Naringin is relatively stable under acidic and neutral conditions, but readily undergoes ring-opening reactions under alkaline conditions, leading to changes in its structure and activity. Naringin has low solubility in water, resulting in poor dispersion during direct gel preparation and preventing the formation of stable drug-gel systems (thus requiring the use of naringin intermediates). The presence of multiple hydroxyl groups in the chemical structure of naringin limits its lipophilicity, thus restricting its bioavailability. Commonly used methods for improving lipophilicity include acylation, alkylation, and glycosylation. Acylation, based on the dehydration condensation between organic acids or alcohols and the hydroxyl groups of naringin, offers relatively mild reaction conditions and can preserve the original structure and activity of naringin to some extent. In addition, etherification converts the hydroxyl groups in naringin into ether bonds, introducing lipophilic groups such as hydrocarbon groups. Although its reaction conditions are quite mild, its modification effect is limited. When applying naringin to myocardial repair products, such as myocardial injection gels, in addition to considering the bioavailability of naringin itself (achieved by enhancing its lipophilicity), it also needs to possess a certain degree of hydrophilicity to ensure uniform dispersion within the hydrophilic gel. The hydrophilic gel promotes cell migration and growth, thus better facilitating myocardial repair. Current research mostly focuses on optimizing esterification reaction conditions to achieve higher yields, but lacks insights into how to precisely control the hydrophilicity and lipophilicity of the product by managing the esterification rate to meet the requirements of myocardial repair products.

[0024] Therefore, the present invention aims to prepare a modified naringin with an appropriate esterification rate and to prepare naringin preparation intermediates in different dosage forms, making them suitable for addition to myocardial repair products.

[0025] Experimental materials: Naringin, purity ≥98%, purchased from Sigma-Aldrich; unsaturated fatty acids (oleic acid, linoleic acid, γ-linoleic acid, eicosapentaenoic acid EPA), purity ≥95%, purchased from Aladdin; lipases (Novozym435, LipozymeRM-IM), enzyme activity 10000 PLU / g or 275 IUN / g, purchased from Novozymes.

[0026] The esterification rate and hydrophilic-lipophilic solubility test methods used in this invention are as follows: Method for determining esterification rate: The esterification rate was determined by high-performance liquid chromatography (HPLC). The reaction sample was dissolved in acetonitrile and diluted to volume, filtered through a 0.22 μm organic membrane, and then injected into the HPLC system for testing. The chromatographic conditions were as follows: C18 reversed-phase column (4.6 mm × 250 mm, 5 μm); mobile phase A: water (containing 0.1% formic acid); mobile phase B: acetonitrile; gradient elution program: 0–10 min, 10%–30% B; 10–20 min, 30%–50% B; 20–30 min, 50%–80% B; 30–40 min, 80% B; flow rate: 1.0 mL / min; detection wavelength: 280 nm; column temperature: 30 °C.

[0027] Based on the change in the peak area of ​​naringin before and after the reaction, the esterification rate was calculated using the following formula:

[0028] Where A0 is the peak area of ​​naringin before the reaction, and A is the peak area of ​​naringin after the reaction.

[0029] Test methods for hydrophilicity and lipid solubility of modified naringin: The hydrophilicity and lipophilicity of the product were determined using the n-octanol-water partition coefficient method. The reaction product was added to a n-octanol-water (volume ratio 1:1) mixture and shaken at a constant temperature for 24 hours to allow the product dispersion in the two phases to reach equilibrium. Equal amounts of the aqueous phase and the n-octanol phase were then taken, and the concentration of the product in the two phases was determined by high performance liquid chromatography (HPLC). The n-octanol-water partition coefficient P was calculated using the following formula.

[0030]

[0031] Where C (n-octanol) is the concentration of the product in the n-octanol phase, and C (water) is the concentration of the product in the aqueous phase. The larger the P value, the stronger the lipophilicity of the product; conversely, the smaller the P value, the stronger the hydrophilicity of the product.

[0032] The following section will detail the method of modifying naringin by modifying it with unsaturated fatty acids.

[0033] Examples 1-4: Accurately weigh 5g of naringin and place it into four dry 1L reaction flasks. According to a molar ratio of naringin to unsaturated fatty acids of 1:3, weigh 8.9g, 9g, 9g, and 9.5g of oleic acid, linoleic acid, γ-linoleic acid, and eicosapentaenoic acid into each reaction flask, respectively. Add 10% (1.5g) of lipase Novozym® 435 (based on the total substrate weight) to each reaction flask, and add 300mL of anhydrous diethyl ether as the reaction solvent. Place the reaction flasks in a 40℃ thermostat with stirring and react for 6 hours. After the reaction, cool the reaction solution to room temperature and centrifuge at 4000 rpm for 10 minutes to separate the lipase. Transfer the centrifuged reaction solution to a rotary evaporator and distill off the reaction solvent at 40℃ under reduced pressure to obtain the crude product. Dissolve the crude product in n-hexane and extract three times with ethanol. Transfer the combined ethanol phases to a rotary evaporator and distill off the ethanol at 40℃ under reduced pressure to obtain the purified reaction product.

[0034] Due to differences in molecular weight and structure, unsaturated fatty acids react with naringin. Under the same reaction time (6 h), reaction temperature (40 °C), molar ratio (naringin:unsaturated fatty acid molar ratio) of 1:3, and catalysis by the same amount (10% of total substrate weight) of lipase Novozym® 435, the esterification rate and hydrophilic lipophilic solubility of the reaction products of different unsaturated fatty acids with naringin were tested. The test results are shown in the table below: Example Unsaturated fatty acids Esterification rate (%) n-Octanol-Water (P) Example 1 Oleic acid 63.47 6.83 Example 2 Linoleic acid 57.62 5.41 Example 3 γ-linoleic acid 53.93 5.26 Example 4 Eicosapentaenoic acid 50.06 5.18 The results show that oleic acid, due to its simple molecular structure and less steric hindrance, reacts more readily with naringin, resulting in a higher esterification rate. Linoleic acid, γ-linoleic acid, and eicosapentaenoic acid (EPA) have relatively complex molecular structures and greater steric hindrance, thus leading to a lower esterification rate. The partition coefficients of the products from the reaction of the four unsaturated fatty acids with naringin in the n-octanol-water region range from 4 to 7, exhibiting both lipophilicity and some hydrophilicity. A positive correlation exists between the esterification rate and the n-octanol-water partition coefficient; a higher esterification rate enhances the lipophilicity of the product. Although EPA and γ-linoleic acid have lower esterification rates compared to linoleic acid, their anti-inflammatory properties and myocardial nourishing effects are relatively better.

[0035] Examples 4-6: Accurately weigh 5g of naringin and place it into three dry 1L reaction flasks. Add 8.9g of oleic acid to each flask according to a 1:3 molar ratio of naringin to oleic acid. Add 5%, 10%, and 20% of the total substrate weight of Novozym® 435 lipase (0.75g, 1.5g, and 3g, respectively) to each flask, along with 300mL of anhydrous diethyl ether as the reaction solvent. Place the flasks in a 40℃ thermostat with stirring and react for 6 hours. After the reaction, cool the reaction solution to room temperature and centrifuge at 4000 rpm for 10 minutes to separate the lipase. Transfer the centrifuged reaction solution to a rotary evaporator and distill off the reaction solvent at 40℃ under reduced pressure to obtain the crude product. Dissolve the crude product in n-hexane and extract three times with ethanol. Transfer the combined ethanol phases to a rotary evaporator and distill off the ethanol at 40℃ under reduced pressure to obtain the purified reaction product. At a molar ratio of naringin to oleic acid of 1:3, a reaction temperature of 40℃, and a reaction time of 6 hours, the esterification rate gradually increased as the dosage of lipase Novozym® 435 increased from 5% to 20%, as shown in the table above. When the enzyme dosage was 5%, the esterification rate was approximately 30%; when the enzyme dosage increased to 10%, the esterification rate increased to 64%; and when the enzyme dosage increased to 20%, the esterification rate reached approximately 71%. It should be understood that with the increase of enzyme dosage, the number of active sites in the reaction system increases, and more reactants participate in the reaction, thus gradually increasing the esterification rate. From the corresponding n-octanol-water partition coefficient, the partition coefficient obtained with a 10% addition is more suitable for use as an intermediate in myocardial repair gel formulations.

[0036] Examples 7-9: Accurately weigh 5g of naringin and place it into three dry 1L reaction flasks. Add 8.9g of oleic acid to each flask according to a 1:3 molar ratio of naringin to oleic acid. Add 5%, 10%, and 20% of the total substrate weight of lipase Lipozyme® RM IM (0.75g, 1.5g, and 3g, respectively) to each flask, along with 300mL of anhydrous diethyl ether as the reaction solvent. Place the flasks in a 40℃ thermostat with stirring and react for 6 hours. After the reaction, cool the reaction solution to room temperature and centrifuge at 4000 rpm for 10 minutes to separate the lipase. Transfer the centrifuged reaction solution to a rotary evaporator and distill off the reaction solvent at 40℃ under reduced pressure to obtain the crude product. Dissolve the crude product in n-hexane and extract three times with ethanol. Transfer the combined ethanol phases to a rotary evaporator and distill off the ethanol at 40℃ under reduced pressure to obtain the purified reaction product. The same trend was observed for the lipase Lipozyme® RM IM, but its catalytic efficiency was relatively lower. When the enzyme dosage increased from 5% to 20%, the increase in esterification rate was relatively small. At 5% enzyme dosage, the esterification rate was approximately 22%; at 10% dosage, it was approximately 45%; and at 20% dosage, it reached approximately 53%. This may be due to differences in the contact rate between the active site and the reactant, and the catalytic mechanism of Lipozyme® RM IM.

[0037] Examples 10-13: Temperature is one of the important factors affecting esterification reactions. This invention also studies different reaction temperatures of 30℃, 40℃, 50℃, and 50℃. 5g of naringin was accurately weighed and placed into three dry 1L reaction flasks. 8.9g of oleic acid was weighed and added to each flask according to a 1:3 molar ratio of naringin to oleic acid. Lipozyme® RM IM (0.75g, 1.5g, and 3g) of the total substrate weight was added to each flask, along with 300mL of anhydrous diethyl ether as the reaction solvent. The flasks were placed in a thermostat at 30℃, 40℃, 50℃, and 60℃ with stirring, and reacted for 6 hours. After the reaction, the reaction solution was cooled to room temperature and centrifuged at 4000 rpm for 10 minutes to separate the lipase. The centrifuged reaction solution was transferred to a rotary evaporator, and the reaction solvent was removed by distillation under reduced pressure at 40℃ to obtain the crude product. The crude product was dissolved in n-hexane and extracted three times with ethanol. The test results are shown in the table below. The results showed that the esterification rate initially increased and then decreased with increasing temperature. At 30℃, the reaction rate was slow, and the esterification rate was relatively low, only about 26%. When the temperature rose to 40-50℃, the esterification rate increased significantly, reaching approximately 63%-69%. This is understandable, as the increased temperature intensifies molecular thermal motion, giving reactant molecules more energy to overcome the activation energy of the reaction, thus accelerating the reaction rate. Furthermore, within this temperature range, lipase activity is also at a high level, effectively catalyzing the esterification reaction. When the temperature further increased, especially above 60℃, the esterification rate decreased significantly, mainly due to the impact on enzyme activity, leading to a decrease in the esterification rate.

[0038] Examples 14-16: Reaction time primarily affects the properties of the esterification product and is therefore a crucial control factor in this invention. 5g of naringin was accurately weighed and placed into three separate dry 1L reaction flasks. Following a 1:3 molar ratio of naringin to oleic acid, 8.9g of oleic acid was added to each flask. Lipozyme® RM IM (0.75g, 1.5g, and 3g, respectively) was added to each flask, along with 300mL of anhydrous diethyl ether as the reaction solvent. The flasks were placed in a 40°C thermostat with stirring, and the reaction times were set to 3h, 6h, 8h, and 10h, respectively. After the reaction, the reaction solution was cooled to room temperature and centrifuged at 4000r / min for 10min to separate the lipase. The centrifuged reaction solution was transferred to a rotary evaporator, and the reaction solvent was removed by distillation under reduced pressure at 40°C to obtain the crude product. The crude product was dissolved in n-hexane and extracted three times with ethanol. The test results are shown in the table below. The results show that the esterification rate increases rapidly with increasing reaction time, reaching 30-40% within 2-3 hours. As the time continues, the esterification reaction slows down, which is understandable as the reaction gradually approaches equilibrium. However, when the reaction time exceeds 10 hours, a large number of byproducts are produced (which are also evident in the color change). Experiments indicate that the reaction time should be controlled within 3-8 hours.

[0039] Examples 17-19: The substrate molar ratio has a significant impact on the esterification rate and the hydrophilic-lipophilic solubility of the product. 5g of naringin was accurately weighed and placed into three separate dry 1L reaction flasks. Naringin was added to oleic acid at molar ratios of 1:1, 1:3, and 1:5, respectively (5g, 8.9g, and 14.8g of oleic acid). Lipozyme® RM IM (0.75g, 1.5g, and 3g, respectively) was added to each flask, along with 300mL of anhydrous diethyl ether as the reaction solvent. The flasks were placed in a 40℃ thermostat with stirring, and the reaction time was set to 6 hours. After the reaction, the reaction solution was cooled to room temperature and centrifuged at 4000 rpm for 10 minutes to separate the lipase. The centrifuged reaction solution was transferred to a rotary evaporator, and the reaction solvent was removed by distillation under reduced pressure at 40℃ to obtain the crude product. The crude product was dissolved in hexane and extracted three times with ethanol. The test results are shown in the table below. When the molar ratio of substrate is 1:1, the esterification rate is relatively low, at 33-35%; when the molar ratio is increased to 1:3, the esterification rate increases significantly, reaching 54-65%; when the molar ratio of substrate reaches 1:5 or higher, the esterification rate decreases, which is due to the limited diffusion of the system and the inhibition of lipase activity.

[0040] In summary, when the substrate molar ratio of naringin to unsaturated fatty acids (oleic acid, linoleic acid, γ-linoleic acid, and eicosapentaenoic acid) is maintained within the range of 1:3 to 1:5, and the reaction temperature is maintained within the range of 40-50℃, catalyzed by adding 10% of the total substrate weight of lipase Novozym® 435 and Lipozyme® RM IM, and reacting for 6-8 hours, a modified naringin product with an octanol-water partition coefficient in the range of 4-7 is obtained. This invention establishes for the first time the relationship between esterification reaction conditions and esterification rate, and further establishes the relationship with hydrophilic lipophilic solubility.

[0041] Modified naringin, obtained by modification with unsaturated fatty acids (oleic acid, linoleic acid, γ-linoleic acid, eicosapentaenoic acid), is not suitable for long-term storage due to the presence of double bonds. It is prepared into a pharmaceutical intermediate so that it can be stored for a long time. Then, it can be added to the preparation of myocardial injection gel or myocardial patch products, which is more conducive to industrialization.

[0042] Stability testing of pharmaceutical intermediates: The presence of pharmaceutical intermediates is for the next step of producing myocardial injection gels or myocardial patches, therefore, pharmaceutical intermediates need to be able to be stored stably for a long period (at least 6 months) under normal storage conditions.

[0043] Temperature test: The formulation intermediate and the control example (modified naringin prepared in Example 1) were placed in constant temperature incubators at 25℃, 30℃ and 40℃. Samples were taken out every month and observed continuously for 6 months to observe their appearance changes and whether there were any phenomena such as agglomeration, discoloration or deformation.

[0044] Humidity test: The formulation intermediate and the control example (modified naringin prepared in Example 1) were placed in a sealed container. 30% saturated potassium nitrate solution, 50% saturated sodium acetate solution and 75% saturated sodium chloride solution were placed in the sealed container, respectively. Samples were taken out and observed every month for 6 consecutive months to observe changes in appearance and whether there were any phenomena such as agglomeration, discoloration or deformation.

[0045] Lighting test: The formulation intermediate and the control example (modified naringin prepared in Example 1) were placed in light boxes with light intensities of 500 lx, 1000 lx, and 2000 lx. Samples were taken out monthly and observed continuously for 6 months to observe changes in appearance and whether there were any phenomena such as agglomeration, discoloration, or deformation.

[0046] According to the above method, the modified naringin prepared in Example 1 showed no significant changes within one month under 25°C, 30% saturated potassium nitrate solution and 500 lx light intensity. Regardless of the increase in temperature, humidity or light intensity, the sample could not be maintained for more than one month. In some cases, agglomeration and discoloration would occur within one week.

[0047] Example 20: This embodiment provides a method for preparing modified naringin into nanoparticles. 500 mg of modified naringin (the product of Example 1) was weighed and dissolved in 50 mL of deionized water; 1 g of polylactic acid-glycolic acid copolymer (molecular weight 10000-15000 Da) was dissolved in 10 mL of dichloromethane; the PLGA solution was slowly added dropwise to the aqueous solution, and the mixture was sonicated for 10 min to form a stable emulsion; the emulsion was placed in a round-bottom flask and rotary evaporated at 600 r / min and 40 °C to gradually precipitate nanoparticles, reacting for 2-3 h; the reacted solution was centrifuged, the precipitate was washed repeatedly three times, and finally the nanoparticles were dried in a vacuum drying oven at 30 °C to obtain a nanoparticle formulation intermediate.

[0048] Stability tests showed that the nanoparticle formulation intermediate could be stably stored for at least 6 months at temperatures below 40°C, 75% humidity, and 2000 lx light intensity, without significant changes during the process.

[0049] Example 21: This embodiment provides a second method for improving the storage stability of modified naringin. In this embodiment, modified naringin is prepared into microspheres. 300 mg of modified naringin is weighed and thoroughly mixed with 1.5 g of gelatin. 30 mL of deionized water is added, and the mixture is stirred evenly in a 50 °C water bath to form a mixed solution. The mixed solution is transferred to a spray dryer, and the inlet air temperature is adjusted to 190 °C, the outlet temperature is adjusted to 80 °C, the feed rate is 5 mL / min, and the atomization pressure is 0.3 MPa. The spray dryer is turned on to prepare microspheres, thus obtaining a microsphere formulation intermediate.

[0050] Stability tests showed that the microsphere formulation intermediate could be stably stored for at least 6 months at temperatures below 40°C, 75% humidity, and 2000 lx light intensity, with no significant changes in the nanoparticle formulation intermediate during the process.

[0051] Example 22: This embodiment provides a third method for improving the storage stability of modified naringin. In this embodiment, modified naringin is prepared into liposomes. 800 mg of soybean lecithin and 200 mg of cholesterol are weighed and dissolved in 10 mL of chloroform solution; 200 mg of modified naringin is weighed and dissolved in 2 mL of methanol; the lipid solution and the modified naringin solution are mixed evenly in an in-situ flask and evaporated on a rotary evaporator at 45°C and 400 rpm for 30-40 minutes until a clear lipid film is visible on the inner wall of the flask; 30 mL of pre-prepared PBS buffer solution is added to the round-bottom flask, and the mixture is stirred for 2 hours to fully hydrate the lipid film, forming crude liposomes; the crude liposomes are homogenized by ultrasonic treatment; the mixture is centrifuged at 8000 rpm for 20 minutes, the supernatant is discarded, PBS buffer solution is added to the precipitate, and the mixture is centrifuged again. This process is repeated 2-3 times to obtain the intermediate liposome formulation.

[0052] Stability tests showed that the liposome formulation intermediate could be stably stored for at least 6 months at temperatures below 40°C, 75% humidity, and 2000 lx light intensity, with no significant changes in the nanoparticle formulation intermediate during the process.

[0053] In addition to testing the stability of Examples 20, 21, and 22, the n-octanol-water partition coefficients of the three formulation intermediates were also tested. The results are shown in the table below.

[0054] Example Formulation intermediates n-Octanol-Water (P) Example 1 / 6.83 Example 20 Nanoparticles 6.41 Example 21 Microspheres 6.06 Example 22 Liposomes 7.05 The results show that after the modified naringin is prepared into a pharmaceutical intermediate, its n-octanol-water partition coefficient does not change much and is close to that of the modified naringin itself. Therefore, the modified naringin prepared into a pharmaceutical intermediate retains good lipid solubility and can be stored stably for a long time, which is beneficial for the subsequent preparation of heart failure injection gel or myocardial patch.

[0055] Example 23: This embodiment describes a method for preparing a myocardial patch from a liposomal formulation intermediate. Myocardial patches, as a treatment for heart failure, are implanted and fixed to the heart wall to prevent further ventricular dilation, thereby reducing the likelihood of further deterioration of heart failure. Simultaneously, the addition of anti-inflammatory, antioxidant, or myocardial tissue regeneration-promoting active substances to prepare functional myocardial patches is also a current research focus. However, there are currently no studies on the application of naringin in myocardial patches.

[0056] This invention uses existing technology to prepare myocardial patches, with electrospinning technology being preferred. The main focus is on exploring the conditions of electrospinning technology and the effect of the amount of naringin added on the myocardial patch, which is also an important inventive point of this invention.

[0057] The preparation method of the myocardial patch is as follows: 10 mL of dichloromethane and 3.3 mL of N,N-dimethylformamide are poured into a beaker and stirred evenly; 1.5 g of PLGA is added to the above solvent and stirred continuously until the solution is clear to obtain a PLGA solution; 0.05 mL of Tween-80 is added to 5 mL of anhydrous ethanol and stirred evenly to obtain an ethanol solution; 1 g of lipid formulation intermediate is added to the ethanol solution and stirred to obtain a dispersion; the dispersion is slowly added dropwise to the PLGA solution and stirred continuously until fully mixed to obtain a spinning solution. The spinning solution is installed on a syringe pump, the distance between the nozzle and the receiving device is adjusted to 12 cm, the voltage is 15 kV, the solution flow rate is 1 mL / h, and the rotation speed of the rotating drum is set to 80 rpm. The myocardial patch obtained by spinning is placed in a drying oven and dried at 35°C for 18 h. The dried myocardial patch is then immersed in PBS buffer solution to wash away residues, obtaining the myocardial patch.

[0058] Myocardial patch biocompatibility test: Biocompatibility testing of myocardial patches was performed using human umbilical vein endothelial cells (HUVECs). UV-sterilized myocardial patches were extracted in cell culture medium for 48 hours to obtain an extract. HUVECs were seeded into 96-well plates at a density of 8000 cells per well. After 24 hours, the cell culture medium was removed, and the extract was added for further culture. Cell proliferation rates were detected using CCK-8 staining after 24 and 72 hours. The results showed that this method was non-toxic to cells after 72 hours.

[0059] Test experiment on myocardial patch protection of cardiomyocytes from oxidative stress damage: The effect of rat cardiomyocytes H9C2 on protecting cardiomyocytes from oxidative stress damage using myocardial patches was evaluated. UV-sterilized myocardial patches were extracted in cell culture medium for 28 h to prepare an extract. H9C2 cells were seeded into 96-well plates at a density of 8000 cells per well. After 24 h, the cell culture medium was removed, and the cells were pretreated with hydrogen peroxide for 1 h to induce oxidative stress. The extract was then added back to the plates for further culture, and the results were analyzed using CCK-8 and FDA / PI staining methods. The results showed that hydrogen peroxide significantly reduced cell viability, but the addition of myocardial patches restored cell viability to some extent.

[0060] Animal experiments: A rat model of myocardial infarction was established based on existing literature. A myocardial patch was sutured to the ventricular wall, and ultrasound examinations were performed on the rats at 7 and 14 days. The tests showed that the rats in this embodiment exhibited good ventricular wall contraction and relaxation. Dissection revealed that the myocardial patch showed good fusion with the myocardial tissue starting at 7 days, with the edges gradually ingrained.

[0061] The comparative examples in the above tests were myocardial patches prepared without the addition of liposome formulation intermediates.

Claims

1. A myocardial patch and a method of preparing the same, characterized by, The myocardial patch contains naringin, which is an active substance that has anti-inflammatory and heart-damage-repairing properties.

2. The myocardial patch and its preparation method according to claim 1, characterized in that, The naringin mentioned is a modified naringin or an intermediate for liposome formulation.

3. A myocardial patch according to claim 2, wherein the collagen matrix is derived from a mammal. The modified naringin is obtained by modifying naringin with unsaturated fatty acids, and the lipid formulation intermediate includes the modified naringin.

4. A myocardial patch according to claim 3, wherein the collagen matrix is prepared by the method of claim 1 or 2. The unsaturated fatty acid mentioned is oleic acid, linoleic acid, γ-linoleic acid, or eicosapentaenoic acid.

5. A myocardial patch according to claim 4, wherein the collagen matrix is derived from a porcine small intestine submucosa (SIS) and the collagen matrix is cross-linked with a cross-linking agent. The modified naringin has an octanol-water partition coefficient of 4-7.

6. A myocardial patch according to claim 5, wherein the collagen matrix is derived from a porcine small intestine submucosa (SIS) and the collagen matrix is cross-linked with a cross-linking agent. The modified naringin is prepared by placing naringin, unsaturated fatty acids, lipase and solvent in a reaction vessel and reacting. After the reaction, the lipase is separated by centrifugation. The remaining reaction solution is evaporated in a rotary evaporator to obtain a crude product. The crude product is dissolved in hexane, extracted with ethanol, and then evaporated under reduced pressure to obtain modified naringin. The lipase is Novozym® 435 or Lipozyme® RM IM.

7. The myocardial patch and its preparation method according to claim 6, characterized in that, The molar ratio of naringin to unsaturated fatty acids is 1:3 to 1:5, the reaction temperature is between 40-50℃, the amount of lipase added is 10% of the sum of the weights of naringin and unsaturated fatty acids, and the reaction time is 6-8 hours.

8. The myocardial patch and its preparation method according to claim 7, characterized in that, The molar ratio of naringin to the unsaturated fatty acid is 1:3, the reaction temperature is between 40°C, the amount of lipase added is 10% of the sum of the weights of naringin and unsaturated fatty acid, and the reaction time is 6 hours.

9. A myocardial patch according to claim 7 or 8, characterized in that, The preparation method of the liposome formulation intermediate is as follows: Weigh 800 mg of soybean lecithin and 200 mg of cholesterol and dissolve them in 10 mL of chloroform solution; weigh 200 mg of modified naringin and dissolve it in 2 mL of methanol; mix the lipid solution and the modified naringin solution evenly in an in-situ flask, place it on a rotary evaporator and evaporate it at 45°C and 400 r / min for 30-40 min until a clear lipid film is visible on the inner wall of the flask; add 30 mL of pre-prepared PBS buffer solution to the round-bottom flask and stir for 2 h to fully hydrate the lipid film and form crude liposomes; homogenize the crude liposomes by ultrasonic treatment; centrifuge at 8000 r / min for 20 min, discard the supernatant, add PBS buffer solution to the precipitate, centrifuge again, and repeat 2-3 times to obtain the liposome formulation intermediate.

10. A myocardial patch according to any one of claims 6, 7, 8, 9, characterized in that, The preparation method of the myocardial patch is as follows: 10 mL of dichloromethane and 3.3 mL of N,N-dimethylformamide are poured into a beaker and stirred evenly; 1.5 g of PLGA is added to the above solvent and stirred continuously until the solution becomes clear to obtain a PLGA solution; 0.05 mL of Tween-80 is added to 5 mL of anhydrous ethanol and stirred evenly to obtain an ethanol solution; 1 g of lipid formulation intermediate is added to the ethanol solution and stirred to obtain a dispersion; the dispersion is slowly added dropwise to the PLGA solution and stirred continuously until fully mixed to obtain a spinning solution. The spinning solution is installed on a syringe pump, the distance between the nozzle and the receiving device is adjusted to 12 cm, the voltage is 15 kV, the solution flow rate is 1 mL / h, and the rotation speed of the rotating drum is set to 80 rpm. The myocardial patch obtained by spinning is placed in a drying oven and dried at 35°C for 18 h. The dried myocardial patch is then immersed in PBS buffer solution to wash away residues, thus obtaining the myocardial patch.

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