A forsythiaside phospholipid complex and its preparation method and application

Abstract

The present invention discloses a forsythiaside phospholipid complex and its preparation method and application, which belong to the field of medical technology. The forsythiaside phospholipid complex includes forsythiaside and phospholipid, and its preparation method includes dissolving a certain proportion of forsythiaside and phospholipid in an organic solvent and stirring the reaction in a constant temperature water bath, and then subjecting the reaction solution to reduced pressure rotary evaporation and vacuum drying to obtain the forsythiaside phospholipid complex. The forsythiaside phospholipid complex prepared by the present invention can be prepared as an oral preparation on the one hand to improve oral bioavailability and drug distribution in lung tissue, and on the other hand, it can be prepared into a suspension by hydration, and the drug can be delivered to the lungs by intravesical instillation to treat interstitial cystitis, or delivered to the lungs by atomized inhalation to prolong the lung residence time and lung drug exposure. Compared with conventional original drug preparations, the forsythiaside phospholipid complex of the present invention significantly improves the efficacy of treating interstitial cystitis and the anti-inflammatory effect of inflammation caused by lung infection viruses and bacteria.

Description

Technical Field

[0001] The present invention relates to a forsythiaside phospholipid complex, its preparation method, and its use. Specifically, the present invention relates to a forsythiaside phospholipid complex using a phospholipid as an excipient, its preparation method, and its use in preparing a drug for preventing and / or treating urinary or respiratory diseases. The present invention belongs to the field of pharmaceutical technology. Background Art

[0002] Forsythia suspensa is a renowned Traditional Chinese Medicine (TCM) with benefits such as clearing heat and detoxifying, reducing swelling and dispersing stagnation, and dispersing wind-heat. It is often used to treat ailments such as respiratory inflammation and oral ulcers, earning it the nickname "the holy elixir for sores." Forsythia suspensa is one of the main active ingredients in the herb. The Chinese Pharmacopoeia stipulates that the content of forsythia suspensa must not be less than 0.15%. Numerous studies have demonstrated its anti-inflammatory, antiviral, and antioxidant properties. However, its poor solubility in water, low dissolution rate, and limited oral bioavailability have hindered the development and application of forsythia suspensa products.

[0003] In the treatment of localized diseases, such as those affecting the respiratory and urinary systems, tissue drug distribution is directly correlated with local efficacy. Improving drug distribution can significantly enhance local efficacy. Furthermore, local administration can increase the tissue-to-plasma concentration ratio by 10-100 times, suggesting that local administration can enhance target tissue exposure and prolong local anti-inflammatory effects. Currently, nanoparticles containing polymer excipients are commonly used to enhance the solubility of poorly soluble drugs and achieve local sustained release. However, the biocompatibility of polymer excipients themselves is uncertain, leading to potential in vivo toxicity. In comparison, phospholipids exhibit excellent biocompatibility, and numerous liposome formulations using phospholipids as excipients have entered clinical use. However, liposomes often suffer from disadvantages such as low drug loading, complex preparation processes, and poor stability. Therefore, it is necessary to develop novel forsythiaside formulation technologies and delivery systems with potential clinical applications. Summary of the Invention

[0004] The present invention aims to provide a forsythiaside phospholipid complex with phospholipid as an excipient and a preparation method thereof. The preparation can improve the lung and bladder bioavailability of forsythiaside, thereby enhancing its anti-inflammatory effect in the lung / bladder and prolonging the duration of action.

[0005] In order to achieve the above object, the present invention adopts the following technical means:

[0006] The present invention discloses a forsythiaside phospholipid complex, comprising forsythiaside and phospholipid, wherein the forsythiaside and phospholipid are compounded via intermolecular non-covalent bonds, and the mass ratio of the forsythiaside to the phospholipid is 1:(1-10), preferably 1:(1-4); the forsythiaside phospholipid complex is prepared by the following method: weighing forsythiaside and phospholipid at a mass ratio of 1:(1-10), preferably 1:(1-4), adding an organic solvent to dissolve them, and uniformly mixing them; compounding is performed at a temperature of 30-60°C for a reaction time of 0.5-4 hours, preferably 1.5-2.5 hours; after the reaction is completed, cholesterol is added until dissolved; then, reduced pressure rotary evaporation is performed, a redissolution solvent is added, filtering is performed through an organic microporous filter membrane, rotary evaporation is performed again, and vacuum drying is performed to obtain the forsythiaside phospholipid complex.

[0007] Among them, preferably, the phospholipid is one of soybean lecithin, egg yolk lecithin, dipalmitoylphosphatidylcholine (DPPC), and distearoylphosphatidylcholine (DSPC), the organic solvent is a methanol-chloroform solution mixed in a volume ratio of 1:1, and the re-dissolution solvent is dichloromethane.

[0008] Among them, preferably, the mass ratio of cholesterol to phospholipid is 1:2.

[0009] Furthermore, the present invention also proposes the use of the forsythiaside phospholipid complex in the preparation of a drug for treating interstitial cystitis or inflammation caused by viruses or bacteria in lung infections.

[0010] Preferably, the drug is an oral preparation, a suspension for topical administration, or an inhalable aerosol.

[0011] Furthermore, the present invention provides an oral preparation containing the forsythiaside phospholipid complex, which is prepared into an oral preparation by conventional pharmaceutical techniques.

[0012] Furthermore, the present invention provides a suspension containing forsythiaside phospholipid complex, which is prepared by the following method:

[0013] The forsythiaside phospholipid complex is added to physiological saline or PBS for hydration, and the particle size is controlled by ultrasonic dispersion, high-pressure homogenization, microfluidics or other particle size control methods, and then high-pressure sterilization or filtration is performed to form a forsythiaside phospholipid complex suspension;

[0014] Wherein, preferably, the mass ratio of forsythiaside phospholipid complex to normal saline or PBS is 1:100;

[0015] Preferably, the particle size distribution range of the suspension of the forsythiaside phospholipid complex is 50-1000 nm, and the median particle size range is 100-350 nm, preferably 100-250 nm.

[0016] Furthermore, the present invention provides an inhalable aerosol containing the forsythiaside phospholipid complex, which is formed by passing a suspension of the forsythiaside phospholipid complex through an air compression atomizer or a vibrating mesh atomizer to form the inhalable aerosol.

[0017] Furthermore, the present invention proposes the use of the oral preparation, the suspension, and the inhalable aerosol in the preparation of a drug for treating interstitial cystitis or inflammation caused by viruses or bacteria in lung infections.

[0018] The forsythiaside phospholipid complex suspension can be instilled via syringe or formed into an inhalable aerosol using a commercial nebulizer without affecting the physical and chemical properties of the complex itself. The preparation significantly improves the drug's pulmonary bioavailability after pulmonary administration and, compared to the original drug suspension, significantly enhances its anti-inflammatory effect against viral and bacterial lung infections. Intravesical instillation of the forsythiaside phospholipid complex suspension significantly increases the drug's bioavailability in bladder tissue, enhancing its efficacy against interstitial cystitis.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The present invention discloses a method for preparing a forsythiaside phospholipid complex. The forsythiaside phospholipid complex is directly prepared into an oral preparation, or the complex can be prepared into a suspension. After sterilization, the suspension can be used as a suspension for aerosol inhalation and efficiently delivered to the lungs, or can be delivered to the bladder via intravesical instillation.

[0021] Although numerous literature reports describe the preparation of drug-phospholipid complexes, which typically involve dissolving the drug and phospholipid in an organic solvent and reacting to form a complex, followed by vacuum drying to obtain a dry powder, different phospholipid complexes have varying stabilities, and only stable phospholipid complexes hold promise for future applications.

[0022] Forsythiaside phospholipid complexes have not been previously reported in the literature. The inventors unexpectedly discovered that the prepared forsythiaside phospholipid complex powder, after stirring and particle size control during the hydration process, can produce a storage-stable suspension. This suspension, after autoclaving, retains its particle size and the properties of the phospholipid complex. The sterilized suspension is suitable for intravesical instillation for the treatment of interstitial cystitis. The sterilized suspension is also suitable for aerosol inhalation, and the aerosolization process similarly does not affect the particle size or the properties of the phospholipid complex.

[0023] The poorly soluble drug forsythin typically has low bioavailability due to its low solubility, limiting its efficacy in interstitial cystitis. When the original drug suspension is administered intravesically, forsythin has poor affinity for bladder tissue, resulting in limited distribution and poor efficacy. However, the phospholipid complex enhances the drug's affinity for bladder tissue, thereby improving its distribution within bladder tissue cells and, in turn, enhancing its efficacy in treating interstitial cystitis.

[0024] Similarly, forsythin's poor affinity for lung tissue restricts its drug distribution and therapeutic efficacy in the lungs, thus limiting its clinical application in treating respiratory diseases. The present invention unexpectedly discovered that an oral formulation of forsythin phospholipid complex can improve its anti-inflammatory efficacy in the lungs. Indeed, in vivo pharmacokinetic and tissue distribution results showed that the plasma bioavailability (AUC) of the phospholipid complex was 2.03 times that of the original drug, while the AUCs of the phospholipid complex in lung tissue and immune cells in the lung epithelial lining fluid were 3.66 times and 7.51 times that of the original drug, respectively. This suggests that the forsythin phospholipid complex enhances the drug's affinity for lung tissue, thereby improving its lung distribution and local therapeutic efficacy.

[0025] The present invention further discovered that compared to an oral formulation, pulmonary administration of the phospholipid complex significantly increased and prolonged drug concentration in lung tissue. The latter's AUCs on immune cells in lung tissue and lung epithelial lining fluid were 39.61 and 197.86 times higher than those of the oral phospholipid complex group. The efficacy of low-dose pulmonary administration in anti-inflammatory efficacy in mouse lungs and improved survival in mice with acute respiratory distress syndrome could not be replicated by increasing the oral or injection dose. This suggests that the pulmonary administration of the forsythiaside phospholipid complex is irreplaceable to other systemic routes of administration.

[0026] Furthermore, the present invention also found that compared with the forsythiaside phospholipid complex suspension, the forsythiaside stock suspension had a weaker effect on pneumonia caused by viral or bacterial infections after pulmonary administration. This suggests that the anti-inflammatory effect of forsythiaside after pulmonary administration is due to the lung tissue affinity of the forsythiaside phospholipid complex, rather than local administration to the lungs.

[0027] Compared to liposome preparations, the phospholipid complex-based drug formulations prepared in this invention offer advantages such as high drug loading, excellent stability, and simple preparation methods. Drug-phospholipid complexes are novel phospholipid nanoparticles produced by reacting phospholipids with natural products in an organic solvent. While they share many similarities with traditional liposomes, they also exhibit unique advantages in physical stability, encapsulation efficiency, and technological scale-up. Furthermore, the forsythiaside phospholipid complex can be prepared into inhalable microparticles through spray drying and delivered to the lungs using a dry powder inhaler for therapeutic effects, while maintaining the physical and chemical properties of the phospholipid complex. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The particle size distribution results and transmission electron microscopy images of the forsythiaside phospholipid complex (FPC) suspension after hydration dispersion / sterilization and then atomization by a nebulizer.

[0029] Figure 2 The in vitro release curves of forsythiaside (FOR) suspension, forsythiaside phospholipid complex (FPC) and forsythiaside physical mixture suspension in 1% SDS-PBS (n=3, mean ± SD).

[0030] Figure 3A The uptake kinetic curves of forsythiaside (FOR) and forsythiaside phospholipid complex (FPC) in RAW264.7 cells.

[0031] Figure 3B Figure 3 is the change of the lung tissue affinity constant (Kp) of FPC and FOR on isolated lung tissue over time.

[0032] Figure 3C The desorption kinetic curves of FPC and FOR on isolated lung tissue (n=3, mean ± SD).

[0033] Figure 4A This is the inhibition rate curve of FPC and FOR at different concentrations on the inflammatory factor IL-6 in RAW264.7 cells after lipopolysaccharide (LPS) stimulation.

[0034] Figure 4B The figure shows the inhibition rate curve of inflammatory factor IL-6 by FPC and FOR at different concentrations on RAW264.7 cells after stimulation with polyinosinic-polycytidylic acid complex (PIC) (n=3, mean ± standard deviation).

[0035] Figure 5 Comparison of the NO inhibition rate (A), antioxidant capacity (B) and ROS fluorescence images (C) of FPC and FOR at different concentrations in RAW264.7 cells after LPS stimulation (n=3, mean ± SD).

[0036] Figure 6 Drug distribution in (A) pulmonary epithelial lining fluid (ELF), (B) immune cells in the ELF, (C) lung tissue, and (D) plasma at different time points in mice with acute lung injury following intrapulmonary and intraperitoneal administration of a forsythiaside phospholipid complex suspension (FPC) or intraperitoneal administration of a forsythiaside (FOR) suspension. (E) Lung / plasma drug concentration ratios at 1, 3, and 5 hours after intrapulmonary and intraperitoneal administration of FPC or FOR in mice with acute lung injury. (n = 6, mean ± SD)

[0037] Figure 7This study evaluated the dose-dependent anti-inflammatory efficacy of forsythiaside phospholipid complex suspension (FPC) and forsythiaside (FOR) suspension in mice with lipopolysaccharide (LPS)-induced acute lung injury after intrapulmonary and intraperitoneal administration. (A) Changes in total protein content in mouse bronchoalveolar lavage fluid (BALF) after administration. (B) Immune cell counts in mouse bronchoalveolar lavage fluid after administration. (C) Changes in cyclic adenosine monophosphate (cAMP) content in mouse lung tissue after administration. Relative levels of inflammatory factors (D) TNF-α, (E) IL-6, and (F) IL-1β in mouse bronchoalveolar lavage fluid after administration. (G) HE staining of lung tissue sections (10x and 20x magnification). (H) Lung injury score. (n = 6, mean ± SD)

[0038] Figure 8 This study evaluated the time-dependent anti-inflammatory efficacy of forsythiaside phospholipid complex suspension (FPC) and forsythiaside (FOR) suspension administered intraperitoneally and intrapulmonaryally in lipopolysaccharide (LPS)-induced acute lung injury. (A) Time chart of drug administration and sample collection in mice. (B) Changes in total protein content in bronchoalveolar lavage fluid (BALF) of mice after drug administration. (C) Immune cell counts in bronchoalveolar lavage fluid of mice after drug administration. (D) Lung injury score. Relative levels of inflammatory factors (E) TNF-α, (F) IL-6, and (G) IL-1β in bronchoalveolar lavage fluid of mice after drug administration. (H) HE staining of lung tissue sections (magnification 10x and 20x). (n = 6, mean ± SD)

[0039] Figure 9 This study evaluated the dose-dependent anti-inflammatory efficacy of forsythiaside phospholipid complex suspension (FPC) and forsythiaside (FOR) suspension administered intrapulmonary or intragastrically in mice with acute lung injury induced by polyinosinic-polycytidylic acid (PIC). (A) Changes in total protein content in bronchoalveolar lavage fluid (BALF) of mice after administration. (B) Immune cell counts in bronchoalveolar lavage fluid of mice after administration. (C) Lung injury score. Relative levels of inflammatory factors (D) TNF-α, (E) IL-6, and (F) IL-1β in bronchoalveolar lavage fluid of mice after administration. (G) HE staining of lung tissue sections (magnification 10x and 20x). (n = 6, mean ± SD)

[0040] Figure 10 Effects of intrapulmonary and intraperitoneal injection of forsythiaside phospholipid complex suspension (FPC) and intraperitoneal injection of forsythiaside (FOR) suspension on mouse survival in an LPS-induced acute respiratory distress syndrome (ARDS) model. (A) 7-day survival curve. (B) 7-day body weight changes. (C) Lung injury score. (D) HE staining of lung tissue sections (10x and 20x magnification). (n = 6, mean ± SD)

[0041] Figure 11To investigate the effects of oral and intravesical administration of forsythiaside (FOR) suspension and forsythiaside phospholipid complex suspension (FPC) on bladder wall permeability in rats in a cyclophosphamide (CYP)-induced interstitial cystitis (IC) model. (n=5, mean ± SD) DETAILED DESCRIPTION

[0042] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are merely exemplary and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solutions of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, and such modifications and replacements fall within the scope of protection of the present invention.

[0043] Example 1 Preparation and Optimization of Forsythiaside Phospholipid Complex

[0044] Forsythiaside and lecithin or DPPC were dissolved in an organic solvent in different mass ratios and heated under reflux to produce a dry powder of forsythiaside phospholipid complex. Since forsythiaside is insoluble in dichloromethane, while dipalmitoylphosphatidylcholine (DPPC) is soluble in dichloromethane, the phospholipid complex formed by the two is also soluble in dichloromethane. Therefore, dichloromethane was selected as the re-dissolution solvent to extract the forsythiaside phospholipid complex and calculate the reconstitution rate. After re-dissolution, extraction and filtration in dichloromethane, the ratio of the phospholipid complex content in the filtrate to the total input was determined. The results showed that:

[0045] 1) Because forsythin is slightly soluble in organic solvents such as methanol and ethanol, but completely soluble in a 1:1 methanol-chloroform mixture, a mixture in which phospholipids are also completely soluble, a 1:1 methanol-chloroform mixture was selected as the reaction solvent. The recombination rate in a 1:1 methanol-chloroform solution at a mass ratio of 1:1 (1-10) was examined after a 2-hour reaction at 45°C. The recombination rate was higher at a mass ratio of 1:1 (1-4), with the highest recombination rate occurring at a mass ratio of 1:3.

[0046] 2) When the mass ratio of forsythin to DPPC was 1:3, the reaction time in methanol-chloroform (1:1) solution at 45°C was 0.5, 1, 2, 3, and 4 hours, respectively. The recombination rate first increased and then decreased with the extension of reaction time. The recombination rate was higher in 1-2 hours, and the highest was in 2 hours.

[0047] 3) When the mass ratio of forsythiaside to DPPC was 1:3, the recombination rate increased first and then decreased with the increase of reaction temperature in methanol-chloroform (1:1) solution at 30, 45, and 60°C for 2 hours, and the recombination rate was the highest at 45°C.

[0048] Example 2 Preparation of Forsythiaside Phospholipid Complex Suspension (FPC)

[0049] 8 mg of forsythin and 24 mg of DPPC (or soybean lecithin) were weighed in a mass ratio of 1:3, dissolved in 4 mL of methanol-chloroform (volume ratio 1:1), and stirred in the dark at 45°C and 500 rpm for 2 hours. After the reaction was completed, 12 mg of cholesterol (the mass ratio of cholesterol to phospholipid was 1:2) was added. After ultrasonication for 30 seconds, the mixture was rotary evaporated at 100 rpm for 15 minutes, 4 mL of dichloromethane was added, and the mixture was filtered through a 0.22 μm organic microporous membrane and rotary evaporated again at 100 rpm for 15 minutes. After vacuum drying overnight, the forsythin phospholipid complex dry powder was obtained.

[0050] 1) Add 4 mL of normal saline or PBS to the DPPC phospholipid complex dry powder (the mass ratio of dry powder to normal saline or PBS is 1:100), heat and ultrasonicate at 60°C for hydration, and then ultrasonicate with a 300W probe in an ice bath for 10 minutes (3 seconds on and 7 seconds off in one cycle) to obtain a suspension. After the suspension is sterilized in an autoclave for 1 hour, a forsythiaside phospholipid complex suspension for aerosol inhalation is obtained.

[0051] The average particle size of the suspension was 119.0±6.9nm, the PDI was 0.228±0.027, the potential was -10.0±0.5mV, the encapsulation efficiency was 85.15%, and the drug loading was 21.33%. After sterilization and atomization with an Aerogen Solo nebulizer, the particle size of the suspension collected also showed no significant change. TEM images also showed that the phospholipid complex maintained a similar nanostructure before and after atomization ( Figure 1 Furthermore, after 14 days of storage at 4°C, the particle size and PDI of the suspension remained unchanged, reaching 116.3±1.3 nm and 0.204±0.042, respectively. The potential decreased to -16.1±0.8 mV. The increase in the absolute value of the potential indicated that the structure of the suspension stabilized. The phospholipid complex remained well dispersed after gentle mixing in water, indicating good storage stability.

[0052] 2) DPPC phospholipid complex dry powder was added with physiological saline or PBS and heated at 60°C for ultrasonic hydration. High-pressure homogenization was then performed 10 times at 500 bar to obtain a suspension. The average particle size of the suspension was 157.6 ± 10.1 nm. Furthermore, after 14 days of storage at 4°C, the particle size decreased to 139.0 ± 1.4 nm. Compared to the probe ultrasonic method, the suspension obtained by high-pressure homogenization exhibits an equilibrium period.

[0053] 3) Physiological saline or PBS was added to the DPPC phospholipid complex dry powder and ultrasonically hydrated at 60°C. The suspension was then ultrasonicated for 10 minutes (3 seconds on, 7 seconds off per cycle) using a 300W probe in an ice bath to obtain a suspension. The suspension had an average particle size of 139.6±3.5nm, a PDI of 0.200±0.006, a potential of -2.6±0.1mV, an encapsulation efficiency of 97.08%, and a drug loading of 24.57%. Furthermore, after 14 days of storage at 4°C, the particle size and PDI results did not show significant changes. The phospholipid complex remained well dispersed after gentle mixing in water, demonstrating good storage stability.

[0054] Experimental Example 1 Characterization and in vitro release evaluation of forsythiaside phospholipid complex (FPC, prepared in Example 2) suspension

[0055] (1) The aerodynamic characteristics of the phospholipid complex suspension aerosol were verified using a next generation pharmaceutical impactor (NGI). Specifically, the NGI was first placed in a 4°C refrigerator for pre-cooling for more than 1.5 hours. The NGI with glass fiber filter paper was assembled according to the instructions, and the flow rate was measured and adjusted to 15 L / min using an airflow meter. Subsequently, 2 mL of FPC saline dilution or forsythiaside (FOR) suspension (forsythiaside original drug was diluted with saline) was added to the nebulizer cup, with a concentration of 0.5 mg / mL. The nebulizer adapter was connected to the L-shaped artificial throat of the NGI. The nebulizer and vacuum pump were turned on at the same time. After the test was completed within the specified time, the vacuum pump and nebulizer were turned off. The NGI was turned on and the state of the droplets at each level of the NGI was observed to be normal. Then, 10 mL of 50% methanol-water was used to rinse the NGI and the forsythiaside active substance remaining in the nebulizer components. After centrifugation at 12,000 rpm for 15 minutes, the supernatant was analyzed by HPLC to determine the forsythin content of each fraction. The results were then imported into the CITDAS data analysis system to calculate the total dose delivered (TD), fine particle fraction (FPF), mass median diameter (MMAD), and geometric standard deviation (GSD) to characterize the aerodynamic performance differences between the forsythin phospholipid complex and the forsythin physical suspension. The results showed that the forsythin phospholipid complex exhibited superior aerodynamic properties compared to the physical mixture. Compared with the FOR, the FPC aerosol exhibited a higher TD, smaller MMAD, greater GSD, and higher FPF, indicating that the nanoscale phospholipid complex forms a smaller aerodynamic droplet size than the larger physical suspension, thus facilitating deposition in the deep lung (Table 1).

[0056] Table 1 Aerodynamic properties of aerosols after nebulization of forsythiaside phospholipid complex (FPC) and forsythiaside (FOR) suspensions (Mean±SD, n=3).

[0057]

[0058]

[0059] (2) The in vitro release of forsythiaside phospholipid complex suspension (FPC) was evaluated by dialysis method, and the differences in drug release between FPC suspension and forsythiaside (FOR) suspension and physical mixture of forsythiaside, DPPC and cholesterol in the ratio of 1:3:2 were compared. Specifically, 2 mL of a 1 mg / mL forsythiaside phospholipid complex suspension, a forsythiaside suspension, and a physical mixture suspension were added to a 3500 Da dialysis bag (n=3), and 200 mL of 1% SDS-PBS was used as the release medium. Release was carried out at 37°C and 200 rpm. 0.5 mL of the drug-containing release medium was removed at 0, 0.25, 0.5, 1, 2, 4, 6, 8, 12, and 24 hours after the start of release, and the same volume was immediately replenished with a blank release medium. The removed drug-containing release medium was diluted with 0.5 mL of methanol and centrifuged at 12000 rpm for 15 minutes. The supernatant was used to determine the drug amount at each time point on HPLC and the release curve was plotted. The results showed that the average cumulative release rate of forsythiaside API in 24 hours was only 49.21%, which may be related to the strong hydrophobicity of forsythiaside API and the large particle size of drug. The average cumulative release rate of physical mixture increased to 56.00%, which may be due to the solubilization effect of excipients phospholipids and cholesterol. The average cumulative release rate of forsythiaside phospholipid complex FPC in 6 hours was 84.93%, which significantly improved the release rate and cumulative release of forsythiaside ( Figure 2 After the poorly soluble drug forsythiaside is prepared as a phospholipid complex, the particle size is significantly reduced. According to the Ostwald-Freundlich equation, when the drug particle size is less than 1 μm, the drug solubility increases with decreasing particle size. Furthermore, according to the Noyes-Whitney equation, the smaller the drug particle size, the larger the specific surface area, and the faster the drug dissolution rate. Therefore, compared with the forsythiaside raw material and physical mixture, the forsythiaside phospholipid complex has higher solubility, faster release rate, and greater cumulative dissolution rate.

[0060] Experimental Example 2 Determination of the uptake of forsythiaside phospholipid complex (FPC, prepared in Example 2) suspension on RAW 264.7 cells and adsorption and desorption kinetics on isolated lung tissue

[0061] (1) Before conducting the cell experiment, the safe concentrations of forsythiaside (FOR) and forsythiaside phospholipid complex (FPC) on RAW264.7 cells were first determined by the MTT method. The results showed that FPC had no cytotoxicity at concentrations of 0.05-100 μM, so the maximum dose of 100 μM was selected for the uptake experiment.

[0062] (2) Comparison of the uptake kinetics of forsythiaside phospholipid complex (FPC) and forsythiaside (FOR) in monocyte macrophage RAW 264.7 cells after lipopolysaccharide (LPS) stimulation was conducted to verify the difference between the phospholipid complex and the API in terms of cellular uptake behavior. Specifically, RAW 264.7 cells were cultured at a concentration of 1×10 6 / well were seeded in 6-well plates and cultured at 37°C, 5% CO2 for 24 hours. They were stimulated with 100 ng / mL LPS for 24 hours, then the original culture medium was aspirated and fresh drug-containing culture medium was added (the concentrations of forsythiaside phospholipid complex and forsythiaside were both 100 μM). After incubation for 0.5, 1, 2, and 4 hours, the supernatant was discarded, and the cells were washed three times with PBS buffer. RIPA cell lysis buffer was added, and all cells were collected with a cell scraper, transferred to a centrifuge tube, and methanol was added. The cells were sonicated for 1 minute with a 100w power probe, shaken for 3 minutes, and then centrifuged at 12000 rpm for 10 minutes. The supernatant was taken for HPLC detection. The results showed that after forsythiaside was prepared into a phospholipid complex, the uptake amount and uptake rate of RAW 264.7 cells were higher than those of the raw drug forsythiaside, indicating that the phospholipid complex may have the effect of improving cellular uptake. The reason may be that the lipophilicity of the drug is increased after preparation into a phospholipid complex ( Figure 3A ).

[0063] (3) Compare the affinity of forsythiaside phospholipid complex (FPC) and forsythiaside (FOR) on isolated lung tissue to verify the difference between the phospholipid complex and the API in terms of lung tissue affinity. The isolated lung tissue explant model was used to evaluate the tissue to buffer partition coefficient (Kp), Kp (C lung / C Buffer ) is used to reflect the distribution of drugs in lung tissue. The higher the Kp value, the greater the adsorption amount of the drug and the better the affinity with lung tissue. First, the adsorption kinetics experiment on isolated lung tissue was carried out. Specifically, fresh rat lung tissue was cut into about 1mm 3About 100 mg of lung pieces and 0.9 mL of KREBS-RINGER buffer were added to each well of a 24-well plate. 100 μL of 1.5 mg / mL FPC or FOR suspension was then added to the wells and pipetted evenly to make the final drug concentration in the wells 150 μg / mL. The 24-well plate was incubated at 37°C and taken out after incubation for 15, 30, 45, 60 or 120 minutes, and 0.2 mL of buffer and lung pieces in the wells were collected respectively. The lung pieces were washed with physiological saline to remove the drugs attached to the surface, and then homogenized in a 50% methanol-water solution to extract the drugs inside. 0.2 mL of methanol was added to the buffer, and after sonication, the supernatant was centrifuged at 12,000 rpm for 5 minutes and analyzed by HPLC to obtain the buffer (C buffer , μg / mL) and lung tissue (C lung , μg / g lung tissue), and calculated the Kp value and plotted its time-dependent curve. Subsequently, a desorption kinetics experiment on isolated lung tissue was conducted. Specifically, lung pieces were first incubated in a buffer containing FPC or FOR at a drug concentration of 150 μg / mL for 60 minutes. After washing with saline to remove surface-adsorbed drug, the pieces were transferred to 1 mL of blank buffer. After incubation at 37°C for 15, 30, 60, or 120 minutes, lung pieces were collected from each well, washed with saline to remove surface-adherent drug, and then homogenized in a 50% methanol-water solution to extract the drug. The supernatant was then centrifuged at 12,000 rpm for 5 minutes, and analyzed by HPLC. The drug concentration in the lung tissue was calculated and plotted as a function of time. The results showed that after the drug was prepared into a phospholipid complex, its average Kp value at different time points increased from 0.76-1.42 to 1.17-1.51, and the desorption half-life was extended from 20.64 minutes of forsythiaside to 25.07 minutes. This result shows that preparing forsythiaside into a phospholipid complex improves its affinity for lung tissue and prolongs the retention time of forsythiaside in the lungs ( Figure 3B , C).

[0064] Experimental Example 3 Anti-inflammatory and antioxidant capacity test of forsythiaside phospholipid complex (FPC, prepared in Example 2) suspension on RAW 264.7 cells

[0065] (1) The inflammatory factor interleukin-6 (IL-6) level in the supernatant of RAW 264.7 cells stimulated with lipopolysaccharide (LPS) or polyinosinic-polycytidylic acid (PIC) complex (PIC) was measured after adding forsythin phospholipid complex (FPC) or forsythin (FOR) to compare the difference in the anti-inflammatory effect of FPC with that of FOR. Specifically, RAW264.7 cells in the logarithmic phase with good growth status were seeded into 96-well cell culture plates (cell density: 1×10 5Cells / well), after incubation in the incubator for 12 hours to adhere to the wall, the culture medium was aspirated and culture medium containing different concentrations of FOR or FPC (62.5, 12.5, 2.5, 0.5, 0.1, 0.02, 0.004 μM) was added. After 1 hour, 20 μL of LPS culture medium containing 1 μg / mL (incubation concentration 100 ng / mL) or 20 μL of PIC culture medium containing 200 μg / mL (incubation concentration 20 μg / mL) was added. After incubation for 12 hours, the supernatant was taken and diluted appropriately and tested according to the instructions of the IL-6 ELISA kit. The four-parameter fitting method was then used to draw the inhibition rate curve of the drug on IL-6 at each concentration, and the half-inhibitory concentration (IC50) was calculated. The results showed that under the LPS model, the IC50 of FOR and FPC on the inflammatory factor IL-6 were 775.2 and 145.7 nM, respectively ( Figure 4A ), while the IC50 of FOR and FPC against inflammatory factor IL-6 in PIC model were 504.4 and 144.8 nM respectively ( Figure 4B ), the IC50 values ​​of FPC in both models were smaller than those of FOR, indicating that the anti-inflammatory effect of forsythin was improved after it was prepared into phospholipid complex.

[0066] (2) The inflammatory mediator nitric oxide (NO) level in the supernatant of monocyte macrophage RAW 264.7 cells stimulated with lipopolysaccharide (LPS) was measured after adding forsythiaside phospholipid complex (FPC) or forsythiaside (FOR) to compare the difference in the anti-inflammatory effect of FPC with that of FOR. Specifically, RAW264.7 cells with good growth status were seeded in 12-well plates (cell density: 5×10 5 Cells were treated with different concentrations (4, 20, 100 μM) of FOR, FPC and the positive drug dexamethasone (DEX, 100 μM) for 2 hours, and then stimulated with LPS (concentration 100 ng / ml) for 24 hours. The supernatant was collected and centrifuged at 10,000 rpm for 5 minutes. The NO level was detected using a NO kit. The results showed that the inhibitory ability of FPC on NO was better than that of FOR at all concentrations, and there was a significant difference at 4 and 20 μM (P<0.05). In addition, the inhibitory ability of FPC at 100 μM was comparable to that of the positive drug DEX. This may be because the preparation of forsythin as a phospholipid complex increased the cellular uptake, thereby showing better in vitro anti-inflammatory activity ( Figure 5 A).

[0067] (3) The reactive oxygen species (ROS) levels in lipopolysaccharide (LPS)-stimulated mononuclear macrophage RAW 264.7 cells were measured after adding forsythiaside phospholipid complex (FPC) or forsythiaside (FOR) suspension to compare the differences in cellular antioxidant capacity between FPC and FOR. RAW264.7 cells were treated with different concentrations of FPC or FOR (4, 20, 100 μM) and the positive drug N-acetylcysteine ​​(NAC) for 2 h, then stimulated with LPS for 30 min and washed three times with blank culture medium. Subsequently, the cells were incubated with the fluorescent probe DCFH-DA in the dark at 37°C for 30 min, washed three times with blank culture medium, and observed and photographed with a fluorescence microscope. The cells were then transferred to a black 96-well plate and the intracellular ROS levels were quantitatively detected using a fluorescence spectrophotometer. The results showed that the antioxidant capacity of FPC was better than that of FOR at all concentrations, and there was a significant difference at 4 and 20 μM (P < 0.05). This may be because the cellular uptake of forsythin increased after it was prepared as a phospholipid complex, thus showing better in vitro antioxidant activity ( Figure 5 B, C).

[0068] Experimental Example 4 Pulmonary Pharmacokinetic Evaluation of Forsythiaside Phospholipid Complex (FPC, Prepared in Example 2) Suspension

[0069] For drugs that exert anti-inflammatory effects in the lungs, their anti-inflammatory effects are more closely correlated with their pulmonary bioavailability than their plasma bioavailability. Therefore, it is necessary to clarify the pulmonary bioavailability of drugs after pulmonary administration. This study compared the pulmonary pharmacokinetics of a forsythin phospholipid complex suspension administered intraperitoneally and intrapulmonary in a mouse model of LPS-induced acute lung injury (ALI). The results were compared with those of a forsythin physical suspension administered intraperitoneally, aiming to explore the differences in pulmonary bioavailability between the two administration routes and the two dosage forms.

[0070] The specific experimental process is:

[0071] (1) Experimental operation of lung tissue distribution: First, the ALI model of mice was induced by tracheal instillation of LPS (0.5 mg / kg). The tracheal instillation method was improved on the basis of our previous method. Specifically, isoflurane was first poured into the anesthesia device, the air pump was turned on and the flow rate was adjusted to 250 mL / min, the concentration was adjusted to 3, and the mouse was placed in the induction box. The mouse was anesthetized for about 2-3 minutes (it could not be turned over), and was quickly placed on the tracheal drug delivery plate. The angle of the lamp was adjusted, and a plastic tube containing isoflurane was used to fumigate the tip of the mouse's nose for about 5 seconds. The mouse's tongue was pinched with tweezers to find the trachea. The tracheal drug delivery needle (22g) was immediately inserted into the trachea, the inner needle was pulled out, and 50 μL of the drug solution was dripped into the blue needle sleeve. If the trachea was inserted, the liquid level would drop. If the liquid level did not move, it meant that the trachea was not inserted and it needed to be reinsed and inserted. After the drug delivery was completed, the mouse was rotated 3 times to ensure that the drug solution was evenly distributed. Mice were then randomly divided into three groups. Two hours after modeling, the first group received a 2 mg / kg (calculated as forsythiaside) suspension of forsythiaside phospholipid complex (FPC) via intratracheal instillation (IT); two hours after modeling, the second group received a 20 mg / kg (calculated as forsythiaside) FPC via intraperitoneal injection (IP); and two hours after modeling, the third group received a 20 mg / kg (calculated as forsythiaside) suspension of forsythiaside via intraperitoneal injection. At different time points after administration, mice were euthanized. Approximately 200 μL of blood was collected from the heart and immediately centrifuged at 13,000 rpm for 15 minutes. 50 μL of the supernatant was collected. After endotracheal intubation, bronchoalveolar lavage fluid was collected and lavaged three times with 0.8 mL of normal saline to a total volume of 2.0 mL. The bronchoalveolar lavage fluid was centrifuged at 3,000 rpm for 15 minutes to separate cells and lavage fluid. Lung tissue was removed, cleaned, and weighed. All samples were aliquoted and stored at −80°C for further analysis.

[0072] (2) Sample processing:

[0073] ① Bronchoalveolar lavage fluid: centrifuge the bronchoalveolar lavage fluid at 3000 rpm for 15 minutes, take 200 μL of the supernatant and add 200 μL of methanol, vortex for 5 minutes, centrifuge at 13000 rpm for 15 minutes, and take 150 μL of the supernatant to detect the concentration by LC-MS / MS.

[0074] ② Cells in alveolar lavage fluid: After discarding the supernatant of the alveolar lavage fluid, add 300 μL of methanol to the cell pellet at the bottom, disrupt the cells by ultrasonication at 100w for 15 minutes, vortex for 5 minutes, centrifuge at 13000 rpm for 15 minutes, and take 150 μL of the supernatant to detect the concentration by LC-MS / MS.

[0075] ③ Lung tissue: Add 500 μL of saline to the removed lung tissue, then add approximately 1 mL of methanol to a constant volume of 2 mL. Grind the mixture with grinding beads using a grinder. Vortex the resulting homogenate for 5 minutes and centrifuge at 13,000 rpm for 15 minutes. Collect 100 μL of the supernatant, add 30 μL of the internal standard (50 ng / mL formononetin) and 170 μL of methanol, centrifuge at 13,000 rpm for 15 minutes, and collect 120 μL of the supernatant for LC-MS / MS analysis.

[0076] ④ Plasma: Add 30 μL of internal standard (formononetin 50 ng / mL) to the plasma sample, then add 220 μL of methanol, vortex for 5 minutes, centrifuge at 13000 rpm for 15 minutes, and take 120 μL of the supernatant to LC-MS / MS for concentration detection.

[0077] (3) Result analysis: After the acute lung injury model mice were given forsythiaside suspension or forsythiaside phospholipid complex suspension according to different administration methods, the pharmacokinetic data of drug content in plasma, alveolar lavage fluid, lavage fluid cells and lung tissue after lavage were shown in Figure 6 The results showed that the exposure of intraperitoneally injected FPC to lung tissue, immune cells in the lung epithelial lining fluid, and plasma was 3.66, 7.51, and 2.03 times that of the original drug FOR, respectively. This indicates that the phospholipid complex is beneficial for improving the plasma bioavailability of the drug, achieving long-term drug circulation, and thus improving the drug's bioavailability in the inflamed lung. Pulmonary administration of the phospholipid complex significantly prolonged the drug's exposure to immune cells in the lung tissue and lung epithelial lining fluid, which was 39.61 and 197.86 times that of the intraperitoneal administration of the phospholipid complex group, respectively. This indicates that pulmonary administration of forsythiaside phospholipid complex can significantly improve the pulmonary bioavailability of forsythiaside and prolong the drug's duration of action in the lungs. The ratio of drug concentrations in lung tissue and plasma (lung / blood ratio) can reflect the affinity of the drug to lung tissue. The results showed that there was almost no difference in the lung / blood ratio between FPC and FOR 1, 3, and 5 hours after intraperitoneal administration, but the lung / blood ratio after tracheal administration of FPC was 11.00-78.08 times that of intraperitoneal injection of FPC, indicating that tracheal administration can significantly increase the affinity of the drug to lung tissue.

[0078] Experimental Example 5 Evaluation of the anti-inflammatory efficacy of forsythiaside phospholipid complex (FPC, prepared in Example 2) suspension (FPC) in lipopolysaccharide (LPS)-induced acute lung injury (ALI)

[0079] The present invention uses an ALI model induced by LPS instillation in mice to comparatively evaluate the pulmonary anti-inflammatory efficacy of FPC and forsythin (FOR) suspensions administered by tracheal instillation and intraperitoneal injection. Specifically, acute lung injury (ALI) was induced by LPS instillation in mice. First, to evaluate the dose-dependent anti-inflammatory effects in vivo, mice were divided into a saline control group (Ctrl), a model group (LPS), a budesonide positive control group (BUD), an FPC tracheal instillation group (IT-FPC), a forsythin suspension tracheal instillation group (IT-FOR), an FPC intraperitoneal injection group (IP-FPC), and an FOR intraperitoneal injection group (IP-FOR). Each mouse in the Ctrl group received 50 μL of normal saline. The BUD group received a 0.5 mg / kg intratracheal instillation of a commercially available budesonide suspension for inhalation (Pulmicort Respules). The FPC group received intratracheal instillation of 0.5, 1, and 2 mg / kg intratracheal doses, while the IP group received 10, 20, and 40 mg / kg doses. The FOR group received intratracheal instillation of 2 mg / kg intratracheal doses, while the IP group received 20 and 40 mg / kg doses. Three hours after administration, all mice, except those in the NS group, received an intratracheal instillation of LPS (10 μg / mouse).

[0080] Subsequently, to evaluate the time dependence of the anti-inflammatory effect of the drug, mice were given intratracheal instillation of FPC (2 mg / kg) or intraperitoneal injection of FPC (20 mg / kg) or FOR (20 mg / kg), and then LPS (10 μg / mouse) was administered 3, 6, and 12 hours later. Finally, to evaluate the therapeutic anti-inflammatory effect, mice were given intratracheal instillation of LPS (10 μg / mouse), followed by tracheal instillation of FPC (2 mg / kg) or intraperitoneal injection of FPC (20 mg / kg) or FOR (20 mg / kg) 2 hours after LPS stimulation. All mice in all groups were euthanized 6 hours after LPS stimulation. The time sequence of drug administration and sampling is shown in Figure 8A. Subsequently, bronchoalveolar lavage fluid was immediately collected and centrifuged at 3000 rpm for 15 minutes to separate the supernatant and cell pellet. The cell pellet was resuspended in 60 μL of red blood cell lysis buffer, shaken for 5 minutes, and centrifuged again at 3000 rpm for 15 minutes. The pellet was resuspended in PBS and the total cell count was determined using a cell counter. The cell supernatant was diluted appropriately to measure the secretion of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β), as well as the total protein content in the supernatant. The contents of TNF-α, IL-6, and IL-1β were detected by ELISA kits according to the manufacturer's instructions, and the total protein content was detected by BCA kits. At the same time, part of the mouse lung tissue was collected, fixed with tissue fixative for more than 24 hours, and then embedded in paraffin. After sectioning, HE staining was performed: the sections were sequentially placed in xylene I for 20 minutes - xylene II for 20 minutes - anhydrous ethanol I for minutes - anhydrous ethanol II for 5 minutes - 75% alcohol for 5 minutes, rinsed with tap water, and then stained with hematoxylin: the sections were stained with hematoxylin solution for 3-5 minutes, washed with tap water, differentiated with differentiation solution, washed with tap water, blued with blueing solution, rinsed with running water, and then stained with eosin: the sections were sequentially placed in 85% and 95% gradient elution. Dehydrate with ethanol for 5 minutes each, stain in eosin solution for 5 minutes, and finally dehydrate and seal the sections: the sections are sequentially placed in anhydrous ethanol I for 5 minutes - anhydrous ethanol II for 5 minutes - anhydrous ethanol III for 5 minutes - xylene I for 5 minutes - xylene II for 5 minutes to make them transparent, seal the sections with neutral gum, examine under a microscope, and perform image acquisition and analysis. Subsequently, tissue damage is assessed using a standardized lung injury scoring system to obtain a histological score for lung injury; 100 mg of mouse lung tissue was taken, 0.5 mL of normal saline was added, and ground with a tissue grinder. The homogenate was then centrifuged at 12,000 rpm for 15 minutes, and the supernatant was diluted appropriately and the cAMP content in the lung tissue was detected using a cyclic adenosine monophosphate (cAMP) ELISA kit.

[0081] The anti-inflammatory effects of FPC and FOR at different administration methods and dosages on LPS-induced ALI are shown in Figure 4. First, the total protein content and total cell count of the alveolar lavage fluid are two key indicators for measuring the severity of lung inflammation. Compared with the control group, the total protein content in the alveolar lavage fluid of LPS-induced ALI mice ( Figure 7 A) and total cell number ( Figure 7 B) increased significantly by 3 times and 12 times, indicating that the degree of pulmonary edema and inflammation has deepened; at the same time, the pulmonary inflammation in ALI is mediated by proinflammatory cytokines, which may cause ALI. Common proinflammatory cytokines include TNF-α, IL-6, and IL1β. Compared with the control group, the levels of these three indicators in the alveolar lavage fluid were also significantly higher than those in the control group ( Figure 7DF); In addition, cyclic adenosine monophosphate (cAMP) is an intracellular second messenger that can reduce the activity of multiple signaling pathways involved in inflammation. The cAMP level in the lung tissue of the LPS model group was reduced by 33.66% compared with the control group, reflecting the generation of inflammation ( Figure 7 C) After administration of budesonide or forsythin, the total protein content, total cell number, and various inflammatory factors decreased compared with the LPS model group. Forsythin, as a natural phosphodiesterase inhibitor, increased cAMP levels in lung tissue by inhibiting PDE4. Among them, the anti-inflammatory effect of intratracheal administration of FPC at doses of 0.5, 1, and 2 mg / kg was dose-dependent, with the best anti-inflammatory effect at a dose of 2 mg / kg. Compared with the LPS model group, the total protein content and total cell number were reduced by 38.75% and 59.29%, respectively, TNF-α, IL-6, and IL1β were reduced by 52.85%, 62.00%, and 49.36%, respectively, and cAMP increased by 17.70%. The results were significantly different from those of intratracheal administration of FOR at the same dose (P<0.001), indicating that the formation of nano-sized phospholipid complexes is more conducive to the anti-inflammatory effect of forsythin in the lungs. This may be related to the fact that the smaller particle size of phospholipid complexes reduces mucus clearance and increases the uptake of pulmonary immune cells and epithelial cells. The physical suspension formed by simply adding phospholipids and cholesterol to forsythin only has a limited solubilization effect on forsythin and cannot effectively reduce the particle size of forsythin crystals, resulting in its limited bioavailability. Therefore, its effect after inhalation is not as good as that of phospholipid complexes. Compared with tracheal administration, the anti-inflammatory effect of FPC injected intraperitoneally was weaker, but still better than FOR injected intraperitoneally. In terms of total protein content, total cell number, TNF-α, IL-6, and IL1β, FPC injected intraperitoneally at 20 mg / kg and 40 mg / kg showed similar anti-inflammatory effects, indicating that the maximum platform range of the effective dose had been reached. Compared with the LPS model group, the total protein content and total cell number of 40 mg / kg FPC (IP) decreased by 55.8% and 33.38%, respectively, TNF-α, IL-6, and IL1β decreased by 35.88%, 40.41%, and 43.84%, respectively, and cAMP increased by 5.94%, all of which were not more significant than FPC (IT) 2 mg / kg, indicating that tracheal administration can exert better lung anti-inflammatory effects than intraperitoneal injection, and the high-dose intraperitoneal injection group cannot achieve the efficacy of the low-dose tracheal administration group, proving the irreplaceable nature of tracheal administration. The lung injury protection results shown by the pathological sections are consistent with the results reflected by the above indicators ( Figure 7 G, H).

[0082] To further clarify the difference in duration of the pharmacodynamic effect of the phospholipid complex FPC compared with the original drug FOR after intratracheal and intraperitoneal injection, a time-dependent study of the anti-inflammatory effect was conducted. The results are as follows: Figure 8 The results showed that intraperitoneal injection of FOR for 12 hours showed no significant difference from the LPS model group in reducing total cell count and inhibiting TNF-α, IL-6, and IL1β levels in bronchoalveolar lavage fluid (P>0.05), indicating that its anti-inflammatory effect can only last for 6 hours at most. Intraperitoneal administration of FPC was effective at 3, 6, and 12 hours of prevention, and still showed significant differences from the LPS model group at 12 hours (P<0.001). In addition, there was a significant difference from FOR in reducing total cell count and inhibiting TNF-α and IL-6 levels in bronchoalveolar lavage fluid (P<0.01). The efficacy of FPC administered intratracheally also lasted for 12 hours, and at 12 hours, there was a significant difference from the FOR intraperitoneal injection group (P<0.001). In addition, in terms of therapeutic effect, tracheal administration of FPC showed the best anti-inflammatory effect among all indicators, and intraperitoneal injection of FPC had a better therapeutic effect than FOR. The lung injury protection effect shown by the pathological section results is consistent with the results reflected by the above indicators. Figure 6 As shown in Table 2, the improvement of lung bioavailability significantly enhanced the intensity of the anti-inflammatory effect of forsythin in the lungs and prolonged the duration of action.

[0083] Experimental Example 6 Evaluation of the anti-inflammatory efficacy of forsythiaside phospholipid complex (FPC, prepared in Example 2) suspension (FPC) in virus-like acute lung injury (ALI) induced by polyinosinic-polycytidylic acid complex Poly (I:C) (PIC)

[0084] The present invention used a virus-like ALI model induced by tracheal instillation of PIC in mice to compare and evaluate the pulmonary anti-inflammatory efficacy of FPC and FOR after tracheal instillation and oral gavage. Specifically, the mice were divided into a normal saline control group (Ctrl), a model group (PIC), a dexamethasone positive control group (DEX), an FPC tracheal instillation group (IT-FPC), a forsythin tracheal instillation group (IT-FOR), an FPC oral gavage group (IG-FPC), and a FOR oral gavage group (IG-FOR). Each mouse in the Ctrl group was given 50 μL of normal saline and the positive drug dexamethasone was intraperitoneally injected at 5 mg / kg. The FPC IT group was given at doses of 0.02, 0.1, 0.5, and 1 mg / kg, and the IG group was given at a dose of 20 mg / kg. The FOR IT group was given at a dose of 1 mg / kg, and the IG group was given at a dose of 20 mg / kg. Three hours after administration, except for the NS group, PIC (120 μg / mouse) was instilled into the trachea. Mice in all groups were euthanized 24 hours after PIC stimulation. The time sequence of drug administration and sampling is shown in the table. Figure 6A. Subsequently, the alveolar lavage fluid was immediately collected and centrifuged at 3000 rpm for 15 minutes to separate the supernatant and cell pellet. The cell pellet was resuspended in 60 μL of red blood cell lysis buffer, shaken for 5 minutes, and then centrifuged at 3000 rpm for 15 minutes, resuspended in PBS, and the total cell count was performed using a cell counter; the cell supernatant was diluted appropriately to determine the secretion and total protein content of tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), and interleukin 1β (IL-1β) in the supernatant. The content of TNF-α, IL-6, and IL-1β was detected by the ELISA kit according to the manufacturer's instructions, and the total protein content was detected by the BCA kit; at the same time, part of the mouse lung tissue was collected, fixed with tissue fixative for more than 24 hours, and then embedded in paraffin. After sectioning, HE staining and sealing were performed, microscopic examination, and image acquisition and analysis were performed. Subsequently, tissue damage was evaluated using a standardized lung injury scoring system to obtain a histological score for lung injury. The results are as follows Figure 9 As shown in the results, consistent with the in vitro data, the inhibitory onset concentration of forsythin on the mouse PIC model was lower than that on the LPS model. The total protein concentration, total cell number and TNF-α, IL-6 and IL1β levels in the alveolar lavage fluid of 0.1 mg / kg FPC administered intratracheally were significantly different from those in the model group (P<0.001). The anti-inflammatory effects were dose-dependent at doses of 0.02, 0.1 and 0.5 mg / kg, and the anti-inflammatory effects of 0.5 and 1 mg / kg were similar. Compared with the model group, the total protein concentration and total cell count in the alveolar lavage fluid of 1 mg / kg group were reduced by 60.16% and 44.11%, and the levels of TNF-α, IL-6, and IL1β in the alveolar lavage fluid were reduced by 64.69%, 56.60%, and 57.93%, respectively. However, the physical suspension administered by trachea at the same dose only reduced the total protein concentration and total cell count in the alveolar lavage fluid by 11.48% and 14.73%, respectively, and the levels of TNF-α, IL-6, and IL1β in the alveolar lavage fluid were reduced by 11.48% and 14.73%, respectively. 6. IL1β levels were only reduced by 27.01%, 19.93%, and 23.07%, respectively. There were significant differences between the two groups in all indicators (P < 0.001). Compared with intratracheal administration, the anti-inflammatory effect of FPC administered orally was weaker, but still superior to that of FOR administered orally. Oral administration of FPC reduced TNF-α and IL-6 levels by 33.83% and 44.57%, respectively, significantly superior to the 15.27% and 0.69% reductions achieved by FOR administered orally (P < 0.05). The protective effect of lung injury shown by pathological sections was consistent with the results reflected by the above indicators. Therefore, similar to the LPS model, intratracheal administration of FPC still exhibited the best anti-inflammatory ability in the PIC-induced ALI model.

[0085] Experimental Example 7 Study on the Effect of Forsythiaside Phospholipid Complex (FPC, Prepared in Example 2) Suspension (FPC) on the Survival Rate of LPS-Induced Acute Respiratory Distress Syndrome (ARDS) Mice

[0086] The present invention uses a mouse tracheal instillation of high-dose LPS to induce an ARDS model, and compares and evaluates the effects of FPC and forsythiaside FOR on the survival rate of mice after tracheal instillation and intraperitoneal administration. Specifically, the mice were divided into a normal saline control group (Ctrl), a model group (LPS), a dexamethasone positive control group (DEX), an FPC tracheal instillation group (IT-FPC), an FPC intraperitoneal injection group (IP-FPC), and a FOR intraperitoneal injection group (IP-FOR). Each mouse in the Ctrl group was given 50 μL of normal saline, and the positive drug dexamethasone was administered intraperitoneally at 5 mg / kg. The FPC IT group was administered at a dose of 2 mg / kg, the IP group was administered at a dose of 20 mg / kg, and the FOR IP group was administered at a dose of 20 mg / kg. Three hours after administration, LPS (600 μg / mouse) was instilled into the trachea of ​​mice except the NS group. The survival of the mice was then observed and their weight was recorded every 12 hours. After 7 days of continuous observation and recording (168 hours), the mice were euthanized and part of their lung tissue was collected. The lung tissue was fixed with a tissue fixative for more than 24 hours, then embedded in paraffin, sliced, stained with HE, and sealed. The sections were examined under a microscope and image acquisition and analysis were performed. Tissue damage was then assessed using a standardized lung injury scoring system to obtain a histological score for lung injury. The resulting survival curve is shown in Figure 2. Figure 10 As shown in A, the results showed that the 7-day survival rate of the FOR intraperitoneal injection group in this ARDS model was only 30%, which was not significantly different from the model group (10%) (P>0.05), while the 7-day survival rate of the FPC intraperitoneal injection group was 40%, which was significantly different from the model group (P<0.05), indicating that compared with FOR, the improved bioavailability of FPC was beneficial to the relief of ARDS symptoms in mice, and the survival rate of the FPC tracheal administration group was 60%, showing a better protective effect than the intraperitoneal injection group, with a significant difference compared with the model group (P<0.01). This may be due to the increased bioavailability and prolonged retention time of FPC in lung immune cells and lung tissues through tracheal administration, resulting in a stronger anti-inflammatory effect and the function of repairing lung epithelium. The weight of mice recovered faster after treatment with tracheally administered FPC, which may be due to the advantages of small side effects and faster onset of pulmonary administration ( Figure 10 B). Pathological sections showed that mice receiving tracheal administration of FPC showed more significant improvements in alveolar wall thickening, pulmonary fibrosis, inflammatory cell infiltration, and alveolar hemorrhage compared to the other groups, which was consistent with the survival rate. Figure 10 C).

[0087] Experimental Example 8 Study on the Effects of Forsythin Physical Suspension (FOR) and Phospholipid Complex Suspension (FPC, Prepared in Example 2) on CYP-Induced Interstitial Cystitis (IC)

[0088] The present invention uses a rat intraperitoneal injection of high-dose CYP to induce an IC model, and compares and evaluates the effects of FPC and forsythin FOR on the bladder wall permeability and bladder edema of rats after oral and intravesical administration. Specifically, the rats were divided into a normal saline control group (Ctrl), a model group (CYP), a FOR oral group (IG-FOR), an SPC-FPC oral group (IG-SPC-FPC), a DPPC-FPC intravesical group (IDD-DPPC-FPC), and an SPC-FPC intravesical group (IDD-SPC-FPC). Each rat was intraperitoneally injected with 150 mg / kg CYP to induce an IC model. 24 hours after modeling, except for the NS group, the FOR IG group was orally administered at 40 mg / kg, the FPC IG group was administered at a dose of 40 mg / kg, and the IDD group was administered at a dose of 2 mg / kg. 23 hours after administration, Evans blue was used to measure the permeability of the bladder inflammation barrier. Specifically, 2% Evans blue dye (3 mL / kg) was injected into the tail vein. One hour after injection, the rats were euthanized. The bladders were removed, weighed, and incubated with 1 ml of formamide at 56°C overnight with shaking. The absorbance (620 nm) was measured using a spectrophotometer, and the Evans blue content in the bladder tissue was calculated as pg Evans blue / μg tissue, and the degree of bladder edema was assessed as mg of rat bladder weight / g of body weight. Figure 11 As shown, the results showed that the FPC intravesical administration group could reduce the Evans blue content in the rat bladder tissue under this IC model, with a significant difference compared with the model group (CYP) (P<0.05), while the Evans blue content in the rat bladder tissue of the FOR and FPC oral groups was not significantly different from that of the model group (P>0.05). This shows that compared with oral administration, intravesical administration is beneficial to enhance the therapeutic effect of FPC on IC rats, with a certain significant difference compared with the model group (P<0.05). This may be because intravesical administration can directly deliver the drug to the lesion site, so a smaller drug dose can be used to achieve the therapeutic effect.

Claims

1. A forsythiaside phospholipid complex, characterized in that: The invention comprises forsythiaside and phospholipid, wherein the forsythiaside and phospholipid are compounded through intermolecular non-covalent bonds, and the mass ratio of the forsythiaside to the phospholipid is 1:(1-10). The forsythiaside phospholipid complex is prepared by the following method: forsythiaside and phospholipid are weighed in a mass ratio of 1:(1-10), an organic solvent is added to dissolve them, and the mixture is evenly mixed; the compounding is carried out at a temperature of 30-60°C for a reaction time of 0.5-4 hours, cholesterol is added after the reaction is completed until it is dissolved, and then vacuum rotary evaporation is carried out, a redissolution solvent is added, and the mixture is filtered through an organic microporous filter membrane, rotary evaporation is performed again, and vacuum drying is carried out to obtain the forsythiaside phospholipid complex.

2. The forsythiaside phospholipid complex according to claim 1, characterized in that The mass ratio of the forsythiaside to the phospholipid is 1:(1-4).

3. The forsythiaside phospholipid complex according to claim 1, characterized in that The reaction time is 1.5-2.5 hours.

4. The forsythiaside phospholipid complex according to claim 1, characterized in that The phospholipid is one of soybean lecithin, egg yolk lecithin, dipalmitoylphosphatidylcholine (DPPC), and distearoylphosphatidylcholine (DSPC); the organic solvent is a methanol-chloroform solution mixed in a volume ratio of 1:1; and the re-dissolution solvent is dichloromethane.

5. The forsythiaside phospholipid complex according to claim 1, characterized in that The mass ratio of cholesterol to phospholipids is 1:

2.

6. An oral preparation containing forsythiaside phospholipid complex, characterized in that: The forsythiaside phospholipid complex according to any one of claims 1 to 5 is prepared into an oral preparation by conventional pharmaceutical techniques.

7. A suspension containing forsythiaside phospholipid complex, characterized in that: Prepared by the following method: The forsythiaside phospholipid complex according to any one of claims 1 to 5 is added to physiological saline or PBS for hydration, and the particle size is controlled by ultrasonic dispersion, high-pressure homogenization or microfluidics, and then high-pressure sterilization or filtration is performed to form a forsythiaside phospholipid complex suspension.

8. The suspension according to claim 7, wherein The mass ratio of the forsythiaside phospholipid complex to physiological saline or PBS is 1:100; the particle size distribution range of the forsythiaside phospholipid complex suspension is 50-1000 nm, and the median particle size range is 100-350 nm.

9. The suspension according to claim 8, wherein The median particle size ranges from 100 to 250 nm.

10. An inhalable aerosol containing forsythiaside phospholipid complex, characterized in that: The suspension of the forsythiaside phospholipid complex according to any one of claims 7 to 9 is passed through an air compression nebulizer or a vibrating mesh nebulizer to form an inhalable aerosol.

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