Leonurine hydrochloride and ursolic acid self-assembled nano preparation and application thereof in preparation of medicine for treating acute lung injury

Abstract

The invention discloses a leonurine hydrochloride and ursolic acid self-assembled nano preparation and application thereof in preparation of a medicine for treating acute lung injury. The invention provides a leonurine and ursolic acid self-assembled nano-preparation. The leonurine and ursolic acid self-assembled nano-preparation is obtained by self-assembling leonurine pharmaceutically acceptable salts and ursolic acid, wherein the amounts of the leonurine pharmaceutically acceptable salts and the ursolic acid are equal. The self-assembled nanoparticles have an obvious anti-lung inflammation effect, and the anti-inflammatory effect is obviously superior to that of a composition of the leonurine pharmaceutically acceptable salt and ursolic acid with the same dosage. Therefore, the leonurine and ursolic acid self-assembled nano preparation provided by the invention has the prospect of being developed into the medicine for treating the acute lung injury.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of nano-preparation, and relates to preparation and application of a nano-preparation, in particular to a self-assembled nano-preparation of Leonurine hydrochloride and ursolic acid and application of the self-assembled nano-preparation in preparation of a drug for treating acute lung injury. BACKGROUND

[0002] Acute lung injury (ALI) is usually caused by inflammatory factors accumulating in the lungs under the conditions of severe infection, shock, trauma, etc., inducing inflammatory reaction to damage the capillary barrier and alveoli, causing pulmonary edema and leading to lung injury. The clinical symptoms mainly include cough, coughing and wheezing, and dyspnea, which is a common critical illness. The mortality of sepsis-related acute lung injury ALI / acute respiratory distress syndrome (ARDS) is higher, and the onset is rapid, with a mortality rate of up to 40%.

[0003] At present, the clinical treatment of ALI mainly includes respiratory support and drug treatment, and there is a lack of specific drugs and methods in general. However, the self-assembled nano-system without carrier effectively solves the inherent defects of traditional drugs, and provides a new treatment option for treating acute lung injury due to its simple preparation process, strong drug loading capacity, pharmacological effect amplification, and small side effects.

[0004] Leonurine hydrochloride (LN) is a biologically active alkaloid extracted from Leonurus japonicus Houtt. and has curative effects on acute liver injury, acute kidney injury, rheumatoid arthritis and other diseases. Ursolic acid (UA) is extracted from various medicinal plants such as Ligustrum lucidum, Crataegus pinnatifida and Pyrus pyrifolia, and belongs to ursolic acid type pentacyclic triterpenoids. It is water-insoluble, has good biocompatibility and low toxicity. It is reported that both leonurine hydrochloride and ursolic acid have anti-inflammatory, anti-allergic, antioxidant, antitumor and other biological activities, can reduce oxidative stress and inflammatory response, and have the potential to treat acute lung injury. However, the therapeutic effect of both on acute lung injury is not satisfactory.

[0005] In order to overcome the shortcomings of the prior art, the present application is proposed. SUMMARY

[0006] The purpose of the present application is to overcome the shortcomings of the prior art, and to provide a self-assembled nano-preparation of leonurine hydrochloride and ursolic acid and application of the self-assembled nano-preparation in preparation of a drug for treating acute lung injury.

[0007] The above-mentioned purpose of the present application is achieved by the following technical solutions.

[0008] A self-assembled nano-preparation of leonurine and ursolic acid is obtained by self-assembly of equal amounts of pharmaceutically acceptable salts of leonurine and ursolic acid.

[0009] Preferably, the self-assembly comprises the following steps: preparing an aqueous solution of a pharmaceutically acceptable salt of Leonurine; preparing a solution of Ursolic acid by dissolving Ursolic acid in tetrahydrofuran; adding the solution of Ursolic acid dropwise into the aqueous solution of the pharmaceutically acceptable salt of Leonurine under stirring and ultrasonic, so that the amount of substance of the pharmaceutically acceptable salt of Leonurine is equal to that of Ursolic acid, adjusting the pH of the solution to neutral using an inorganic base, continuing the ultrasonic, centrifuging and discarding the supernatant, thereby obtaining the self-assembly nano-preparation of Leonurine and Ursolic acid.

[0010] Preferably, the pharmaceutically acceptable salt of Leonurine is hydrochloric acid Leonurine.

[0011] More preferably, the concentration of the aqueous solution of the pharmaceutically acceptable salt of Leonurine is 2 mM.

[0012] More preferably, the concentration of the solution of Ursolic acid is 20 mM.

[0013] More preferably, the pH of the solution is adjusted to neutral using an inorganic base, such as sodium carbonate, sodium hydroxide or sodium bicarbonate aqueous solution.

[0014] More preferably, the ultrasonic frequency is 40 kHz.

[0015] More preferably, after adjusting the pH of the solution to neutral, the ultrasonic is continued for 15 min.

[0016] More preferably, after centrifuging for 30 min at a speed of 13000 rmp, the supernatant is discarded.

[0017] The self-assembly nano-preparation of Leonurine and Ursolic acid described above is used for preparing a drug for treating acute lung injury.

[0018] Advantages:

[0019] The self-assembly nano-preparation of Leonurine and Ursolic acid provided by the present application is obtained by self-assembly of equal amount of substance of a pharmaceutically acceptable salt of Leonurine and Ursolic acid; the self-assembly nanoparticles have obvious anti-lung inflammation effect, and the anti-inflammatory effect is significantly better than that of a composition of the same amount of pharmaceutically acceptable salt of Leonurine and Ursolic acid. Therefore, the self-assembly nano-preparation of Leonurine and Ursolic acid provided by the present application has the prospect of being developed into a drug for treating acute lung injury. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 Scanning electron microscope image of the self-assembly nanoparticles LUNP of hydrochloric acid Leonurine and Ursolic acid synthesized in Example 1;

[0021] Figure 2 Particle size distribution graph of the self-assembly nanoparticles LUNP of hydrochloric acid Leonurine and Ursolic acid synthesized in Example 1;

[0022] Figure 3 The potential distribution diagram of the LUNP of the self-assembly of the leonurine hydrochloride synthesized in Example 1 and ursolic acid;

[0023] Figure 4 The UV-visible absorption spectrum of the LUNP of the self-assembly of the leonurine hydrochloride, ursolic acid and the leonurine hydrochloride and ursolic acid synthesized in Example 1;

[0024] Figure 5 The FTIR spectrum of the LUNP of the self-assembly of the leonurine hydrochloride, ursolic acid and the leonurine hydrochloride and ursolic acid synthesized in Example 1;

[0025] Figure 6 The NMR spectrum of the LUNP of the self-assembly of the leonurine hydrochloride, ursolic acid and the leonurine hydrochloride and ursolic acid synthesized in Example 1;

[0026] Figure 7 The FTIR spectrum of the LUNP of the self-assembly of the leonurine hydrochloride synthesized in Example 1 and ursolic acid;

[0027] Figure 8 The pathological section of the HE staining of the lung tissue of the acute lung injury mice in each group;

[0028] Figure 9 The expression level of the inflammatory factor IL-1β in the BALF of the acute lung injury mice in each group (n=5);

[0029] Figure 10 The expression level of the inflammatory factor IFN-γ in the BALF of the acute lung injury mice in each group (n=5);

[0030] Figure 11 The expression level of the inflammatory factor TNF-α in the BALF of the acute lung injury mice in each group (n=5);

[0031] Figure 12 The expression level of the inflammatory factor IL-6 in the BALF of the acute lung injury mice in each group (n=5). DETAILED DESCRIPTION

[0032] The substantial content of the application will be specifically introduced below in combination with examples, but the protection scope of the application is not limited by this.

[0033] Example 1: Preparation and characterization of leonurine hydrochloride-ursolic acid nanoparticles (LUNP)

[0034] A preparation method of a leonurine hydrochloride and ursolic acid self-assembly nano-preparation LUNP, the specific steps are as follows:

[0035] A 2 mM aqueous solution of Leonurine hydrochloride (LN) was prepared and used as a stock solution; Ursolic acid (UA) was dissolved in tetrahydrofuran to prepare a 20 mM solution. The UA solution was added dropwise into the aqueous solution of Leonurine hydrochloride under stirring and ultrasonication to obtain a 1:1 molar ratio of the two components, and the pH of the solution was adjusted to 7 using a 100 mM inorganic base sodium carbonate (or sodium hydroxide, sodium bicarbonate, etc.). The solution was ultrasonicated for another 15 min at a frequency of 40 kHz, and then centrifuged at 13000 rpm for 30 min. The supernatant was discarded, and LUNP was obtained.

[0036] Figure 1 Scanning electron microscope image of the self-assembled nanoparticles of Leonurine hydrochloride and Ursolic acid synthesized in Example 1. The LUNP sample was dropped onto a silicon wafer, and then observed under a scanning electron microscope after natural drying. As can be seen from Figure 1 the image, the synthesized nanoparticles are uniformly distributed and have a spherical morphology.

[0037] Figure 2 Particle size distribution of the self-assembled nanoparticles of Leonurine hydrochloride and Ursolic acid synthesized in Example 1. The LUNP obtained in Example 1 was resuspended in ultrapure water, and the hydrated particle size of the LUNP was measured using a particle size analyzer. The experimental results show that the particle size of the LUNP prepared in Example 1 is normally distributed, with a narrow particle size distribution and a diameter of 208.57 ± 1.76 nm (n = 3).

[0038] Figure 3 Zeta potential distribution of the self-assembled nanoparticles of Leonurine hydrochloride and Ursolic acid synthesized in Example 1. The LUNP obtained in Example 1 was resuspended in ultrapure water, and the zeta potential of the LUNP was measured using a particle size analyzer. The experimental results show that the zeta potential of the LUNP prepared in Example 1 is normally distributed, with a narrow zeta potential distribution and a value of -26.07 ± 2.55 mV (n = 3).

[0039] Figure 4 UV-Vis absorption spectra of Leonurine hydrochloride, Ursolic acid, and LUNP synthesized in Example 1. Ursolic acid, Leonurine hydrochloride, and LUNP samples were dissolved in aqueous solution, and the absorbance at 200-800 nm was measured on a UV spectrophotometer. As can be seen from Figure 4 the figure, LUNP shows obvious absorption peaks at 288 nm and 298 nm, which is a significant red shift compared to the maximum absorption wavelength of LN at 276 nm. This red shift phenomenon indicates an enhanced molecular electron conjugated system, further supporting the existence of π-π stacking interactions within the assembly structure.

[0040] Figure 5Fourier transform infrared spectroscopy (FTIR) spectra of LUNP synthesized in Example 1. According to the FTIR spectra analysis, the C-H stretching vibration of the saturated alkyl chain in UA shows multiple absorption peaks in the range of 2800-3000 cm⁻¹. After the formation of LUNP, these absorption peaks are significantly broadened, indicating that the molecular stacking of the molecular skeleton between UA molecules has occurred. The C=O stretching vibration peak of UA red shifts from 1685 cm⁻¹ to 1689 cm⁻¹, while the N-H bending vibration peak of LN blue shifts from 1515 cm⁻¹ to 1552 cm⁻¹, proving that strong hydrogen bond interactions are formed between the carboxyl group of UA and the guanidino group of LN.

[0041] Figure 6 Powder X-ray diffraction (PXRD) spectra of LUNP synthesized in Example 1. The PXRD pattern shows that both LN and UA have obvious sharp diffraction peaks, indicating that both have crystalline structures. However, after the formation of LUNP, most of the diffraction peaks disappear, and only residual diffraction signals are observed at 10.82°, 14.26°, and 21.82°, indicating that the co-assembly of UA and LN destroys the original crystal order.

[0042] Figure 7 Nuclear magnetic resonance (NMR) spectra of LUNP synthesized in Example 1. 1 H-NMR). The guanidino hydrogen position in LN is at 7.8 ppm, and the characteristic C-H of UA is at 5.0 ppm. The H integral further proves that the molar ratio of LN to UA in LUNP is 1:1.

[0043] Example 2: Therapeutic effect of LUNP on acute lung injury mice

[0044] I. Model grouping and administration

[0045] BALB / c mice (6 weeks old, 20 g) were adaptively fed for one week before the experiment, and randomly divided into PBS (control group), LPS (model group), NAC (acetylcysteine, positive drug group), LUNP, LN, UA, LN+UA, a total of seven groups, 10 in each group. At 0.5 h, except for the control group, the trachea of the mice was dropped with LPS (5 mg / kg) to induce acute lung injury model, and the control group was given the same volume of PBS. The drug administration group inhaled the corresponding treatment drug at 0 h and 12.5 h. The positive drug group was given a dose of 150 mg / kg, the LUNP group was given a dose of 7.5 mg / kg, the LN and UA groups were given a dose of 3.4 mg / kg and 4.5 mg / kg respectively, and the LN+UA group was given 3.4 mg / kg LN and 4.5 mg / kg UA. The model group and the control group were given the same volume of PBS. At 24.5 h, all mice were sacrificed, and the bronchoalveolar lavage fluid (BALF) was separated, and the lung tissue of the mice was obtained.

[0046] Dose explanation of drug administration group except positive drug group: The molecular weight of ursolic acid (UA) is about 450 g / moL, the molecular weight of leonurine hydrochloride (LN) is about 340 g / moL, and the molecular weight of leonurine is about 310 g / moL; Since the molar ratio of LN to UA in LUNP is 1:1, and the pH is adjusted to neutral during the preparation of LUNP, it is not in the form of hydrochloride salt, so the molecular weight of LUNP is about 310 g / moL+450 g / moL=760 g / moL. As can be seen from the above, the drug administration group except the positive drug group can ensure the comparability of each group from the design of the administration dose.

[0047] II. Detection index

[0048] 1. Hematoxylin-eosin (HE) staining of lung tissue

[0049] 24.5 h, 5 mice in each group were sacrificed for tissue and organ separation, the left lung of the mice was taken out, washed with normal saline to remove residual blood, fixed in 4% paraformaldehyde solution, trimmed, dehydrated, embedded in paraffin, cut into 5 μm thick paraffin sections, deparaffinized with xylene, rehydrated, and HE stained, and the pathological changes of lung tissue were observed under a light microscope.

[0050] 2. Take bronchoalveolar lavage fluid (BALF)

[0051] 5 mice in each group were sacrificed at 24.5 h, the trachea of the mice was intubated and fixed with a disposable 1 mL syringe, and 1 mL of PBS was used to perform alveolar lavage, repeated 2 times, and the BALF was recovered, with a recovery rate of about 75%. The BALF was centrifuged at 4 ℃ and 1000 rpm for 20 min, and the supernatant and cell precipitate were collected.

[0052] 3. Determination of the levels of inflammatory factors in BALF

[0053] The BALF supernatant was taken, and the contents of various indexes were detected by double antibody sandwich enzyme-linked immunoassay. The operation steps were strictly followed according to the instructions of the kit. The levels of IL-1β, IFN-γ, TNF-α and IL-6 in the BALF of mice were detected by using an ELISA kit. The absorbance at 450 nm was measured by using an enzyme-labeled instrument. The levels of the above-mentioned inflammatory factors in each group of samples were calculated, and the experimental results were analyzed by one-way ANOVA.

[0054] III. Experimental results

[0055] 1. Effect of LUNP on lung tissue of acute lung injury mice

[0056] Figure 8 The figure is a pathological section of HE staining of lung tissue of acute lung injury mice. According to the results of HE, the alveolar cavity structure of the normal group of mice is complete, the alveolar wall is thin, and no obvious interstitial congestion and inflammatory infiltration is observed. Compared with the control group, the alveolar wall of the model group of mice is significantly thickened, and congestion and edema appear in the alveolar cavity. At the same time, inflammatory cell infiltration around the alveolar wall can be observed, indicating that the modeling is successful. Compared with the model group, NAC, LUNP, LN, UA and LN+UA groups can improve the inflammation and congestion and edema of the alveoli of mice to different extents, and improve the pathological changes, but compared with the LUNP group, the NAC, LN, UA and LN+UA groups are more serious, the lesion sites are relatively more, the alveolar wall thickening sites are more, the inflammation is more serious, and the LN+UA group is relatively relieved compared with the LN and UA groups. The results show that compared with other administration groups, the lung lesions of the LUNP administration group of mice are significantly smaller, and the drug efficacy for treating acute lung injury is the best.

[0057] 3. Effect of LUNP on inflammatory factors IL-1β, IFN-γ, TNF-α and IL-6 of acute lung injury mice

[0058] Figures 9-12 The figure is the analysis results of inflammatory factors IL-1β, IFN-γ, TNF-α and IL-6 in the BALF of mice. The results show that compared with the control group, the expression levels of IL-1β, IFN-γ, TNF-α and IL-6 in the BALF of the model group of mice are significantly increased, indicating that the modeling is successful. Compared with the model group, LUNP has the strongest inhibitory effect on inflammatory factors such as IL-1β, IFN-γ, TNF-α and IL-6 in the BALF of mice, which is better than that of the NAC positive group and the LN, UA and LN+UA treatment groups.

[0059] In summary, the self-assembled nanoparticles of leonurine hydrochloride and ursolic acid provided in Example 1 have obvious anti-lung inflammation effect, and the anti-inflammatory effect is significantly better than that of the combination of leonurine hydrochloride and ursolic acid with the same amount of administration. Therefore, the self-assembled nanoparticles of leonurine hydrochloride and ursolic acid provided in Example 1 have the prospect of developing into a drug for treating acute lung injury.

[0060] The above examples serve to specifically introduce the essential content of the present application, but those skilled in the art should know that the protection scope of the present application should not be limited to the specific examples.

Claims

1. A self-assembled nanoformulation of Leonurine and Ursolic acid characterized in that: The self-assembly of the Leonurine and the Urosolic acid is prepared by using equal amount of the pharmaceutically acceptable salt of the Leonurine and the Urosolic acid.

2. The self-assembled nanoformulation of Leonurine with ursolic acid as claimed in claim 1, wherein, The self-assembly includes the following steps: preparing an aqueous solution of the pharmaceutically acceptable salt of the Leonurine; dissolving the Urosolic acid in tetrahydrofuran to prepare a Urosolic acid solution; adding the Urosolic acid solution into the aqueous solution of the pharmaceutically acceptable salt of the Leonurine under stirring and ultrasonic, so that the amount of the pharmaceutically acceptable salt of the Leonurine is equal to that of the Urosolic acid; adjusting the pH of the solution to neutral by using an inorganic base; continuing the ultrasonic treatment; centrifuging and discarding the supernatant.

3. The self-assembly nanoformulation of Leonurine with ursolic acid as claimed in claim 1 or 2, wherein: The pharmaceutically acceptable salt of the Leonurine is the hydrochloride of the Leonurine.

4. The self-assembled nanoformulation of orobanchin and ursolic acid as claimed in claim 2, wherein: The concentration of the aqueous solution of the pharmaceutically acceptable salt of the Leonurine is 2 mM.

5. The self-grown nanoformulation of Leonurine with ursolic acid as claimed in claim 2, wherein: The concentration of the Urosolic acid solution is 20 mM.

6. The self-grown nanoformulation of orobanchol with ursolic acid as claimed in claim 2, wherein: The inorganic base used for adjusting the pH of the solution to neutral is sodium carbonate, sodium hydroxide or sodium bicarbonate aqueous solution.

7. The self-grown nanoformulation of orobanchol with ursolic acid as claimed in claim 2, wherein: The ultrasonic frequency is 40 kHz.

8. The self-grown nanoformulation of orobanchol and ursolic acid as claimed in claim 2, wherein: After adjusting the pH of the solution to neutral, the ultrasonic treatment is continued for 15 min.

9. The self-grown nanoformulation of Leonurine with ursolic acid as claimed in claim 2, wherein: After centrifuging at 13000 rpm for 30 min, the supernatant is discarded.

10. The use of the self-assembly of the Leonurine and the Urosolic acid for preparing a drug for treating acute lung injury.

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