Construction of a long-circulating liposome of aesculin and its anti-enteritis effect

Abstract

The present invention discloses the construction of a long-circulating quinquercetin liposome and the study of its anti-enteritis effect, which belongs to the field of pharmaceutical preparations and aims to provide a long-circulating quinquercetin liposome with increased drug loading, so as to improve the solubility and oral bioavailability of quinquercetin and improve its enteritis effect. The key points of its technical solution are the construction of a long-circulating quinquercetin liposome and the study of its anti-enteritis effect. The liposome consists of a liposome bilayer and quinquercetin and is prepared by a thin film dispersion method. The liposome bilayer contains phospholipids, cholesterol and PEG modifiers. The preparation method is simple, easy to operate and has good reproducibility. The prepared long-circulating quinquercetin liposome has a smaller particle size and a higher drug encapsulation rate, which enhances its in vitro solubility and in vivo bioavailability, and enhances its anti-enteritis therapeutic effect. It provides preliminary information for the clinical application of quinquercetin and other similar water-insoluble compounds.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical preparations, and in particular relates to the construction of a long-circulating liposome of aesculin and the study of its anti-enteritis effect. Technical Background

[0002] Fraxinus cortex is a commonly used Chinese herbal medicine. First recorded in the Shennong's Herbal Classic, it originates from the bark or stems of Fraxinus cortex, Fraxinus microphylla, or Fraxinus chinensis, native to the Qinling Mountains. Its primary effects include clearing heat and dampness, relieving asthma and cough, and improving eyesight. It is clinically used to treat enteritis, leucorrhea, chronic bronchitis, bacillary dysentery, and psoriasis. The main chemical constituents of Fraxinus cortex are coumarins, with aesculin being the primary coumarin monomer.

[0003] Fraxinusin is a natural coumarin compound named 7,8-dihydroxy-6-methoxycoumarin. It is a flaky crystal with a normal pale yellow color. It is highly soluble in organic solvents such as ethanol and methanol, but has low solubility in water. As a precious Chinese herbal medicine, fraxinusin has a wide range of clinical applications, including anti-tumor, antibacterial, anti-inflammatory, anti-diabetic, antioxidant, and neuroprotective effects.

[0004] Liposomes have shown great promise as an encapsulation medium. Compared to other encapsulation technologies, liposomes offer key advantages such as high solubility, excellent stability, enhanced drug targeting, and enhanced therapeutic efficacy. Liposomes, as microcapsules, are encapsulated by a phospholipid bilayer, which is typically composed of phospholipids and cholesterol. The non-polar ends of phospholipids are linked to form the phospholipid bilayer, while cholesterol can regulate the fluidity, stability, and permeability of the phospholipid bilayer. Because the phospholipid bilayer is structurally similar to biological membranes, liposomes have excellent biocompatibility and biosafety. Unlike other nanocarriers that only encapsulate specific payloads, liposomes facilitate the regulation of hydrophobic, hydrophilic, and amphiphilic compounds due to the amphiphilic nature of phospholipids.

[0005] Inflammatory bowel disease (IBD), which primarily includes ulcerative colitis (UC) and Crohn's disease (CD), is a group of intestinal inflammatory diseases characterized by chronic, recurrent, nonspecific tissue inflammation. The primary manifestations are long-term, recurrent abdominal pain, diarrhea, and bloody, mucus-purulent stools. Its pathogenesis remains unclear, but it is generally believed that multiple factors, including intestinal infection, impaired intestinal mucosal barrier, dysregulation of the intestinal mucosal immune system, genetics, and environmental factors, contribute to the development of IBD. Among these, dysregulation of the immune system plays a significant role in the pathogenesis of IBD.

[0006] Ulcerative colitis (UC) is a chronic systemic disease characterized by inflammation of the colonic mucosa and submucosal membranes. It is characterized by recurrent diarrhea, mucus or pus in the stool, and abdominal pain. In recent years, the incidence of UC has been increasing year by year due to improvements in living standards and changes in dietary plans and habits. Furthermore, UC is difficult to cure and prone to recurrence after recovery. It is also associated with the incidence of colon cancer.

[0007] Currently, the main drug treatments for inflammatory bowel disease (IBD) are 5-aminosalicylic acid preparations, glucocorticoids, immunosuppressants (6-mercaptopurine, methotrexate), and biologics (anti-tumor necrosis factor). However, traditional treatments have numerous drawbacks, including low sustained resolution rates, a high risk of serious infection, and significant side effects, resulting in poor clinical efficacy. For example, sulfasalazine-salicylic acid preparations are the primary treatments for inflammatory bowel disease or ulcerative colitis. Commonly used corticosteroids are prednisone or dexamethasone, but there is currently no evidence that long-term steroid maintenance can prevent relapses. Intravenous infusions of hydrocortisone or dexamethasone can also be used during acute attacks, as can nightly retention enemas with hydrocortisone added to normal saline. While the value of steroid therapy during acute attacks is undeniable, there is disagreement regarding the continued use of steroids during the chronic phase. Due to their potential side effects, most advocates do not recommend long-term use. Furthermore, the value of immunosuppressants in ulcerative colitis remains questionable. Rosenberg et al reported that azathioprine has no effect on disease control during exacerbations, but it helps reduce the use of corticosteroids in chronic cases. Summary of the Invention

[0008] The purpose of the present invention is to provide a method for constructing long-circulating liposomes of quercetin and studying its anti-enteritis effect in order to address the problems of poor water solubility, low in vitro release and bioavailability of quercetin. The present invention prepares quercetin with poor water solubility into long-circulating liposomes, which can significantly improve the solubility and in vitro release of quercetin, thereby further improving the efficacy of quercetin pharmaceutical preparations, and its preparation method should be simple.

[0009] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0010] The construction and anti-enteritis effects of long-circulating esculin liposomes were studied. The liposomes consisted of a liposome bilayer and esculin; the bilayer was made of phospholipids, cholesterol, and a PEG modifier. The weight percentages of the components were as follows: a phospholipid to cholesterol ratio of 8:1-12:1; a drug to phospholipid ratio of 1:4-1:8; and a PEG modifier of 6%-10%.

[0011] Preferably, a long-circulating fraxinusin liposome is constructed and its anti-enteritis effect is studied, which is composed of the following components in mass percentage: the ratio of phospholipid to cholesterol is 10:1, the ratio of fraxinusin to phospholipid is 1:8, and the amount of PEG modifier is 8%.

[0012] The phospholipid is lecithin; the PEG modification is DSPE-PEG 2000; and the purity of fraxinusin is 98%.

[0013] A method for constructing a long-circulating esculin liposome and studying its anti-enteritis effect comprises the following preparation steps:

[0014] (1) According to the ratio of the components, lecithin, cholesterol, DSPE-PEG 2000 and quercetin were weighed and dissolved in 20 mL of anhydrous ethanol and placed in a round-bottom flask.

[0015] (2) The mixture formed in step (1) was ultrasonically treated to obtain a transparent solution, and the ethanol was removed by rotary evaporation at 55° C. until a thin film was formed at the bottom of the bottle.

[0016] (3) Then, 5 mL of double-distilled water was added at a constant speed at 55°C to obtain long-circulating fraxinus liposomes.

[0017] Beneficial effects of the present invention:

[0018] The present invention discloses a method for constructing a long-circulating esculin liposome and studying its anti-enteritis effect. The preparation method is simple, easy to operate, and low in cost. The particle size of the prepared long-circulating esculin liposome is 166.65±8.75nm, and the encapsulation efficiency is 92.18±0.17%. In different dissolution media (pH 1.2 HCl, pH 7.0 DDW, and pH 7.4 PBS), the cumulative release of esculin and its long-circulating liposome preparation in different dissolution media all reaches a plateau at 12 hours. The difference is that the cumulative release of the long-circulating esculin liposome is about 1.5 times that of esculin, reaching more than 85%, and the drug is basically released completely. The solubility, in vitro release, and bioavailability of esculin are significantly improved.

[0019] The invention discloses a long-circulating facitin liposome prepared by the invention and studies its anti-enteritis effect. In actual application, the facitin can be directly taken orally, or can be prepared into freeze-dried powder or other dosage forms as needed to enhance its storage stability and facilitate transportation. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In the present invention Figure 1 Characterization of long-circulating afraxetin liposomes: A) particle size distribution of long-circulating afraxetin liposomes; B) transmission electron microscopy (TEM) image of long-circulating afraxetin liposomes.

[0021] In the present invention Figure 2 Comparison of the in vitro release curves of free fraxinusin and fraxinusin long-circulating liposomes in different media: A) in vitro release curves of free fraxinusin and fraxinusin long-circulating liposomes in hydrochloric acid solution (pH 1.2); B) in vitro release curves of free fraxinusin and fraxinusin long-circulating liposomes in PBS (pH 7.4); C) in vitro release curves of free fraxinusin and fraxinusin long-circulating liposomes in double distilled water (Ph 7.0) (Fraxinusin long-circulating liposomes 2), (mean±SD, n=3).

[0022] In the present invention Figure 3 The plasma concentration distributions of free aesculin and aesculin long-circulating liposomes (Mean±SD, n=6).

[0023] In the present invention Figure 4 The expression of IL-1β, IL-6, TNF-α, CRP, PEG2 and IL-10 in the serum of rats in each group.

[0024] In the present invention Figure 5 The expression of IL-1β, IL-6, TNF-α, CRP, PEG2 and IL-10 in the colon tissue of rats in each group.

[0025] In the present invention Figure 6 Histopathological analysis of liver of rats treated with different treatments.

[0026] In the present invention Figure 7 Histopathological analysis of liver of rats treated with different treatments.

[0027] ( Figure 1-4 NC: normal control group; M: model control group; P: positive control group; FH: high-dose fraxetine group; FM: medium-dose fraxetine group; FL: low-dose fraxetine group; F-LC-Lipo-H: high-dose fraxetine long-circulating liposome group; F-LC-Lipo-H: medium-dose fraxetine long-circulating liposome group; F-LC-Lipo-L: low-dose fraxetine long-circulating liposome group; F-LC-Lipo: long-circulating liposome group) DETAILED DESCRIPTION

[0028] The following examples describe embodiments of the present invention in detail. However, those skilled in the art will appreciate that the following examples are intended to illustrate the present invention only and should not be construed as limiting its scope. Where specific conditions are not specified in the examples, conventional conditions or manufacturer's recommended conditions were used. Reagents and instruments used without manufacturer's indication are commercially available conventional products.

[0029] Example 1

[0030] Construction of a long-circulating esculin liposome and study of its anti-enteritis effect Determination of influencing factors such as the mass ratio of phospholipids to cholesterol (A), the ratio of drug to phospholipids (B), and the dosage of DSPE-PEG 2000 (C) in the formulation:

[0031] Using the phospholipid-to-cholesterol mass ratio (A), the drug-to-phospholipid ratio (B), and the DSPE-PEG 2000 dosage (C) as influencing factors, an orthogonal experiment was conducted to optimize the formulation of a long-circulating esculin liposome and its anti-enteritis efficacy research. Secondly, the quality of the long-circulating esculin liposome and its anti-enteritis efficacy research was evaluated using particle size and encapsulation efficiency as indicators. The factor level table is shown in Table 1, and the experimental results are shown in Table 2.

[0032] Table 1 Influencing factors and levels of orthogonal experiment

[0033]

[0034] Table 2 Orthogonal test results

[0035]

[0036]

[0037] Example 2

[0038] Construction of a long-circulating liposome of aesculin and its preparation for anti-enteritis research:

[0039] Through orthogonal experimental design, the formulation factors that affect EE% and particle size were determined. In the experimental design, K1, K2 and K3 were used to represent the sum of each level (Table 2). The order of influence of each factor on the EE% and particle size of the long-circulating liposomes of quercetin was B>A>C>D, and the best formulation was A2B1C2. The results showed that the long-circulating liposomes of quercetin prepared at this time had the smallest particle size and the highest EE%.

[0040] Long-circulating liposomes were successfully prepared using a thin film dispersion method. Phosphatidylcholine, cholesterol, DSPE-PEG, and quercetin were dissolved in 20 mL of anhydrous ethanol and placed in a round-bottom flask. The phospholipid-to-cholesterol ratio was 10:1, the quercetin-to-phospholipid ratio was 1:8, and the DSPE-PEG2000 dosage was 8%. After ultrasonic treatment, a clear solution was obtained. Ethanol was removed by rotary evaporation at 55°C until a thin film formed at the bottom of the flask. Then, 5 mL of DDW was added at a constant rate at 55°C.

[0041] Morphological observation: The long-circulating esculin liposomes prepared in Example 2 were diluted with water and then dropped onto a copper mesh covered with a support film. The mixture was allowed to air dry and then stained with a 2% phosphotungstic acid solution. The mesh was naturally dried and then observed under a transmission electron microscope. The long-circulating esculin liposomes diluted with water were spherical in shape under the transmission electron microscope. They were not adhered to each other and were evenly dispersed. Figure 1 shown.

[0042] Determination of in vitro release: The long-circulating liposomes of quercetin and quercetin of Example 2 were taken, respectively, and in vitro release studies were conducted in different media (pH 1.2 HCl, pH 7.0 DDW, and pH 7.4 PBS) on a constant temperature oscillator using dialysis. The quercetin solution (1 mg / mL, 1 mL) and the quercetin liposomes (containing equal amounts of quercetin) were appropriately placed in dialysis bags, tied at both ends under trough conditions, and then placed in different release media (pH 1.2 hydrochloric acid, pH 7.0 DDW, and pH 7.4 PBS). The release test was carried out using a constant temperature water bath oscillator (37±0.5°C, 100 rpm), and the total volume of the release medium was 100 mL. Samples (1 mL each) were taken from each culture medium at 0.08, 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8, 10, 12, and 24 h, and replaced with fresh culture medium of the same volume and type to maintain the sink state. The results showed that compared with esculin, the dissolution of esculin long-circulating liposomes was more rapid and complete, and could effectively improve the in vitro dissolution rate of esculin. Figure 2 shown.

[0043] Example 3

[0044] Study on the pharmacokinetics of long-circulating liposomes of aesculin

[0045] (1) In vivo studies

[0046] Sprague-Dawley (SD) rats (200 ± 20 g, male) were provided by the Animal Experimental Center of Zhangjiagang Hospital of Traditional Chinese Medicine, affiliated to Nanjing University of Chinese Medicine. All rats were fasted for 12 hours prior to the experiment but were allowed free access to water. Twelve rats were randomly divided into two groups: a free aesculin group and a long-circulating aesculin liposome group, with six rats in each group. Both groups were administered the same dose (200 mg / kg) of free aesculin (suspended in 0.5% sodium carboxymethylcellulose) and long-circulating aesculin liposomes. Whole blood samples (0.5 mL each) were collected at various time points after administration (0.08, 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8, 10, 12, and 24 hours). Plasma was collected after centrifugation (3700 rpm, 10 min) and stored at −20°C for further analysis.

[0047] (2) Blood sample processing

[0048] Fraxinus quinquefolius was extracted from rat plasma using a liquid-liquid extraction method. A dihydromyricetin solution was used as an internal standard. Plasma (200 μL) was mixed with acetanilide (20 μL, 50 μg / mL) and ethyl acetate (800 μL) was added to purposefully extract the drug from the plasma. After repeated extraction twice, the ethyl acetate layer was carefully removed and combined. After drying under nitrogen and gentle heating, the residue was dissolved in methanol (400 μL) and centrifuged at 10,000 rpm for 10 minutes. The quinquefolius content in the supernatant was analyzed by high-performance liquid chromatography (HPLC).

[0049] (3) In vivo pharmacokinetics of long-circulating liposomes containing quercetin

[0050] Key pharmacokinetic parameters, including maximum peak concentration (Cmax), time to peak concentration (Tmax), mean residence time (MRT), and area under the concentration-time curve (AUC0-∞), were calculated using BAPP2.3 pharmacokinetic software (provided by the Drug Metabolism Center of China Pharmaceutical University). The equation for calculating the relative oral bioavailability (Fr) of the drug is as follows:

[0051]

[0052] Among them, AUC T and AUC R They represent the areas under the concentration-time curves of aesculin long-circulating liposomes and aesculin aqueous solution, respectively.

[0053] (4) In vivo pharmacokinetic analysis of long-circulating liposomes of quercetin

[0054] Figure 3 The plasma drug concentration-time curves of rats after oral administration of a single dose (200 mg / kg) of free aesculin and aesculin long-circulating liposomes are described, and the calculated pharmacokinetic variables are shown in Table 3. It can be observed that the plasma drug concentration of rats orally administered with aesculin long-circulating liposomes was significantly higher than that of free aesculin. Similarly, the observed distribution of aesculin showed that the plasma drug concentration of rats orally administered with aesculin long-circulating liposomes was significantly higher than that of free aesculin.

[0055] Table 3 Pharmacokinetic parameters of free aesculin and aesculin long-circulating liposomes (F-LC-Lipo) after oral administration in rats (n=6)

[0056] Parameters Free fraxetin F-LC-Lipo <![CDATA[AUC 0-36h (μg / mL)]]> 9.03±0.84 <![CDATA[39.96±2.48 ** ]]> <![CDATA[C max (μg / mL)]]> 1.84±0.24 <![CDATA[7.69±0.43 ** ]]> <![CDATA[T 1 / 2 (h)]]> 5.44±0.61 <![CDATA[7.42±0.70 ** ]]> <![CDATA[MRT 0-t (h)]]> 6.03±0.91 <![CDATA[9.17±1.21 ** <!-- 5 -->]]> <![CDATA[T max (h)]]> 1.13±0.31 1.58±0.38

[0057] ** p<0.01, compared with free aesculin

[0058] C of free aesculin max (1.84±0.24μg / mL) and AUC 0-24h The results showed that the bioavailability of long-circulating liposomes of quercetin (9.03±0.84μg / mL) was significantly lower than that of long-circulating liposomes of quercetin (7.69±0.43μg / mL and 39.96±2.48μg / mL, respectively). The results showed that long-circulating liposomes could significantly promote the absorption of quercetin in vivo (p<0.01) and was positively correlated with the in vitro release results. After a single oral administration, the bioavailability of long-circulating liposomes of quercetin was significantly higher than that of unprescribed quercetin, and its relative bioavailability increased by 4.43 times.

[0059] AUC 0-24h and C max The values showed that the in vivo absorption of quinacrin long-circulating liposomes was significantly higher than that of the unprescribed drug (p<0.01). In addition, the MRT of quinacrin long-circulating liposomes (9.17±1.21h) was significantly longer than that of the unprescribed quinacrin long-circulating liposomes (6.03±0.91h), which means that the prepared quinacrin long-circulating liposomes can increase the systemic circulation of quinacrin in the body. These data indicate that long-term circulating lipids can significantly increase and prolong the absorption and duration of action of quinacrin, and improve the in vivo availability of quinacrin. The enhanced oral absorption of quinacrin in long-circulating liposomes may be attributed to the ability of the lipid layer of quinacrin long-circulating liposomes to overcome the absorption barrier of the intestinal mucosa and increase the bioavailability of the drug through lymphatic absorption. This phenomenon reduces the first-pass effect of the liver and improves the total bioavailability of the drug in the body.

[0060] Example 4

[0061] Study on the anti-enteritis effect of long-circulating liposome preparation of esculin:

[0062] 1. Establishment of rat enteritis model

[0063] Sixty male Sprague-Dawley rats were randomly divided into 10 groups (n=6): normal control group (NC), model control group (M), positive control group (P), low-dose esculetin group (FL, 25 mg / kg), medium-dose esculetin group (FM, 50 mg / kg), high-dose esculetin group (FH, 100 mg / kg), low-dose esculetin long-circulating liposome group (F-LC-Lipo-L, 25 mg / kg), medium-dose esculetin long-circulating liposome group (F-LC-Lipo-M, 50 mg / kg), high-dose esculetin long-circulating liposome group (F-LC-Lipo-H, 100 mg / kg), and LC-Lipo group (LC-Lipo). Except for the NC group, which did not receive TNBS, all other groups received TNBS solution and underwent drug intervention for 4 weeks. After fasting for 24 hours, the M group was anesthetized, and a well-lubricated 2-mm diameter rubber tube was gently inserted into the anus to a depth of approximately 8 cm. After the enema (4 mL dose), administer TNBS 50% ethanol solution (25 mg / mL). Subsequently, while holding the anus with your hand, slowly remove the plastic tube. Simultaneously, lift the rat's tail and invert it for 1 minute to allow the modeling agent to completely penetrate the rat's intestinal cavity.

[0064] 2. Detection indicators and methods

[0065] (1) Physical and chemical index testing

[0066] The rats were sacrificed after blood sampling, and the colon and liver were removed for further experiments.

[0067] To explore the possible mechanism of aesculin in treating enteritis, we measured the levels of inflammatory factors in rat serum and colon. The activities of interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-10 (IL-10), tumor necrosis factor-α (TNF-α), malondialdehyde (MDA), superoxide dismutase (SOD), C-reactive protein (CRP), and prostaglandin E2 (PEG2) in serum and colon tissue were detected using commercially available kits. All measurements were performed according to the manufacturer's instructions.

[0068] Figure 4 The serum levels of IL-1β, IL-6, TNF-α, CRP, and PEG2 in the M group were higher than those in the NC group (p < 0.01), and the IL-10 level was lower than that in the NC group (p < 0.01). Compared with the M group, the serum levels of IL-1β, IL-6, TNF-α, CRP, and PEG2 in the free aesculin group and the aesculin long-circulating liposome group were significantly decreased, while the IL-10 level was increased. The levels of inflammatory factors in the aesculin long-circulating liposome-H group were similar to those in the PC group, and there were no significant changes in the aesculin long-circulating liposome group compared with the M group.

[0069] like Figure 5 As shown, compared with the NC group, the levels of IL-1β, IL-6, MDA, SOD, and TNF-α in the colon tissue of the M group were significantly increased (p<0.01), while the level of IL-10 was decreased (p<0.01). Compared with the M group, the IL-10 level in the colon tissue of the free aesculin group and the aesculin long-circulating liposome group was significantly increased (p<0.01), while the IL-10 levels of IL-1β, IL-6, MDA, SOD, and TNF-α were significantly decreased (p<0.01). The IL-10 content in the three groups was higher than that in the free aesculin group. At the same time, the levels of IL-1β, IL-6, MDA, SOD, and TNF-α in the three groups were lower than those in the free drug group in a dose-dependent manner. The levels of these factors in the aesculin long-circulating liposome group were similar to those in the M group. The results show that aesculin long-circulating liposomes can significantly inhibit the expression of these cytokines.

[0070] (2) Histopathological analysis

[0071] After the rats were sacrificed, the colon and liver were removed and fixed with 4% paraformaldehyde solution. The samples were then washed with anhydrous ethanol before being embedded in paraffin, sectioned at 5 μm, and stained with hematoxylin and eosin. The prepared samples were observed under a microscope (Nikon, Japan).

[0072] The morphological changes of liver tissues of rats in each group were observed. Figure 6 As shown in the figure. Compared with the NC group, the liver tissue of rats in the M group showed obvious pathological changes such as edema, necrosis, and degeneration, suggesting that enteritis may have damaged the rat liver. However, the degree of tissue morphological recovery of rat organs in the free aesculin group and the aesculin long-circulating liposome group was different. More importantly, the tissue morphology of the FLC-Lipo-H group was close to that of the NC group, indicating that the aesculin liposome preparation has a better organ repair effect than free aesculin.

[0073] The histological morphology of the colon in each group was as follows Figure 7 The colonic mucosa structure of the NC group was intact and smooth, with clear boundaries between the mucosa, submucosa, muscularis, and serosa. The cell morphology was normal, the glands in the lamina propria were arranged regularly, and there was no erosion or ulceration ( Figure 7 A), while the M group had transmural inflammation and deep ulcers, and glandular structure disorder. At the same time, as the ulcer healing began, the degree of inflammation in the free quercetin group and the quercetin long-circulating liposome group improved to a certain extent, but showed fibrosis. The quercetin long-circulating liposome repair effect was better and dose-dependent. The results showed that the quercetin long-circulating liposome delivery system can effectively enhance the organ protective effect of quercetin in rats with enteritis.

[0074] 3. Construction of a long-circulating liposome of aesculin and its anti-enteritis study

[0075] In this study, long-circulating esculin liposomes were successfully prepared using a thin film dispersion method. The formulation was optimized using an orthogonal design. Characterization of the long-circulating esculin liposomes demonstrated that the drug exhibited a small particle size and a high drug EE%. In vitro release profiles and in vivo pharmacokinetic studies in rats demonstrated that the long-circulating esculin liposomes improved the solubility and oral bioavailability of esculin. They also significantly reduced IL-1β, IL-6, MDA, SOD, TNF-α, CRP, and PEG2 levels in the serum and colonic tissues of rats with enteritis, and increased IL-10 levels in the serum and colonic tissues of both groups of rats. Furthermore, the therapeutic effect of the long-circulating esculin liposomes was significantly superior to that of the free esculin group in a dose-dependent manner. Furthermore, compared with free esculin, the long-circulating esculin liposomes exhibited superior multi-organ protective effects. Taken together, these results provide preliminary information for the clinical application of esculin and other similar water-insoluble compounds.

Claims

1. A method for preparing long-circulating esculin liposomes, characterized by: It is made of liposome bilayer and anti-enteritis drug aesculin; the liposome bilayer is made of phospholipids, cholesterol and PEG modifiers; The mass ratio of phospholipid to cholesterol is 10:1; the mass ratio of quercetin to phospholipid is 1:8; the mass proportion of PEG modification in the liposome is 8%; The phospholipid is lecithin; The PEG modification is DSPE-PEG 2000; The preparation method is a thin film dispersion method, and the specific steps are as follows: 1) Dissolve lecithin, cholesterol, DSPE-PEG 2000, and fraxetin in 20 mL of anhydrous ethanol and place in a round-bottom flask; 2) After ultrasonic treatment, a clear solution was obtained, and the ethanol was removed by rotary evaporation at 55°C until a thin film formed at the bottom of the bottle; 3) Then, 5 mL of double-distilled water was added at a constant speed at 55° C. to obtain the long-circulating esculentin liposomes.

2. The method for preparing a esculin long-circulating liposome according to claim 1, characterized in that: The fraxinin is a natural coumarin compound derived from the bark or stem of Fraxinus fraxinus, Fraxinus microphylla or Fraxinus chinensis in the Qinling Mountains. It is normally a light yellow flaky crystal.

3. The method for preparing a esculin long-circulating liposome according to claim 1, characterized in that: The purity of the fraxetin is 98%.

4. Use of the long-circulating aesculin liposomes prepared by the method for preparing the long-circulating aesculin liposomes according to any one of claims 1 to 3 in the preparation of anti-enteritis drugs.

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