Crystal form X of resveratrol as well as preparation method and application of crystal form X

Abstract

The invention discloses a resveratrol crystal form X and a preparation method and application thereof, an X-ray powder diffraction pattern of the resveratrol crystal form X has characteristic peaks at 2 theta angles of 27.4 degrees, 31.8 degrees, 45.6 degrees and 56.5 degrees, and the 2 theta angle error range is + / -0.2 degrees; meanwhile, the invention discloses a preparation method and application in a medicine for treating pneumonia. According to the crystal form X provided by the invention, the saturation solubility of resveratrol is increased by 35.1 times (up to 1793.8 + / -36.8 mu g / mL); the tablet has good dissolution capacity in a slightly acidic inflammation environment; meanwhile, the compound has higher affinity to a protein receptor on a respiratory syncytial virus (RSV), shows a more excellent inhibition effect on the RSV in vitro and in vivo, and has better anti-inflammatory and antioxidant activity; meanwhile, the crystal form X of the resveratrol is more beneficial to cellular uptake, the uptake amount is 3.5 times of the original amount compared with common resveratrol, retention in the lung can be improved, aerosol inhalation for treating the RSV pneumonia has safety and effectiveness, and it is shown that the resveratrol can be used for preparing the medicine for treating the pneumonia, especially the RSV pneumonia.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical crystals, and in particular to a crystal form X of resveratrol, its preparation method, and its uses. Background Technology

[0002] Resveratrol (3,4',5-trihydroxystilbene) is a natural compound synthesized by plants after exposure to external influences. It possesses anti-inflammatory, antioxidant, anti-aging, and neuroprotective properties and is commonly found in grapes, peanut shells, and mulberries. It is currently widely used as a dietary supplement. However, its poor water solubility (30 μg / mL) and low bioavailability (<1%) hinder its application. In recent years, research on nanocrystalline drugs has gradually become a hot topic in nanoparticle formulation research. Nanocrystalline drugs are pure drug particles with a particle size of 1–1000 nm, requiring no carrier material. Due to their large specific surface area, drug nanocrystals can enhance adhesion to biological membranes while improving drug solubility, dissolution rate, and bioavailability. Nanocrystal technology can be used as an intermediate formulation technology and is widely applied in the preparation of formulations via various routes of administration (oral, intravenous, pulmonary, transdermal, and ocular administration, etc.). Summary of the Invention

[0003] The purpose of this invention is to provide a crystal form X of resveratrol, its preparation method and uses, in order to solve the problems of poor water solubility and low bioavailability of resveratrol, and to provide a new crystal form X of resveratrol that is safe, stable, has good water solubility and good therapeutic effect.

[0004] The present invention provides a crystal form X of resveratrol, whose X-ray powder diffraction pattern has characteristic peaks at 2θ angles of 27.4°, 31.8°, 45.6° and 56.5°, wherein the error range of the 2θ angle is ±0.2°.

[0005] As a further improvement, its X-ray powder diffraction pattern is basically the same as... Figure 1 Consistent with the above.

[0006] As a further improvement, its X-ray powder diffraction pattern also has characteristic peaks at 2θ angles of 6.6°, 13.2°, 16.5°, 19.2° and 28.3°.

[0007] As a preferred option, the microstructure of resveratrol crystal form X is a regular spherical shape with a particle size of 178.1±5.68 nm, a PDI of 0.137±0.037, and a Zeta potential of -13.4±1.52 mV.

[0008] Meanwhile, the present invention provides a method for preparing the crystal form X of resveratrol, comprising the following steps: S1. dissolving resveratrol in dimethyl sulfoxide to form a resveratrol solution; S2. rapidly injecting the resveratrol solution in S1 into deionized water under ultrasonic conditions for crystallization; S3. separating the solid part by physical methods and drying it to obtain the crystal form X.

[0009] As a further improvement, the resveratrol concentration in step S1 exceeds 5 mM, preferably 5 to 20 mM, and most preferably 10 mM.

[0010] As another improvement, the ultrasonic conditions described in step S2 are 50-150 Hz, preferably 100 Hz.

[0011] As another improvement, the volume ratio of the resveratrol solution to the deionized water in step S2 is 1:25-200, preferably 1:25-100, and most preferably 1:50.

[0012] The present invention provides a pharmaceutical composition comprising a therapeutically effective amount of the crystal form X of the above-mentioned resveratrol and one or more pharmaceutically acceptable carriers, diluents or excipients.

[0013] The present invention provides the use of the crystal form X of the above-mentioned resveratrol or the above-mentioned pharmaceutical composition in the preparation of a medicament for treating pneumonia, wherein the pneumonia is preferably respiratory syncytial virus (RSV) pneumonia or acute lung injury.

[0014] Beneficial effects

[0015] The resveratrol crystal form X provided by this invention has significantly improved water solubility (1793.8±36.8 μg / mL) compared to untreated resveratrol (water solubility 47.9±1.97 μg / mL); it also exhibits good dissolution ability in a slightly acidic inflammatory environment; simultaneously, it has a stronger affinity for the protein receptor on respiratory syncytial virus (RSV), showing significantly enhanced inhibitory effects on RSV both in vitro and in vivo compared to resveratrol, with the inhibitory effect on RSV being dose-dependent; it also possesses good anti-inflammatory and antioxidant activities; furthermore, resveratrol crystal form X is more conducive to cellular uptake, with an uptake 3.5 times greater than that of ordinary resveratrol, which can improve retention in the lungs. Nebulized inhalation therapy for RSV pneumonia is safe and effective, indicating that it can be used to prepare drugs for treating pneumonia, especially RSV pneumonia. Attached Figure Description

[0016] Figure 1 This is the X-ray powder diffraction (XRD) pattern of resveratrol crystal form X.

[0017] Figure 2This is a comparison of the XRD patterns of resveratrol crystal form X and ordinary resveratrol.

[0018] Figure 3 This is a comparison of the differential scanning calorimetry (DSC) spectra of resveratrol crystal form X and ordinary resveratrol.

[0019] Figure 4 These are TEM images (A), particle size distribution (B), and potential change diagrams (C) of resveratrol crystal form X.

[0020] Figure 5 This is a laser-irradiated photograph of a solution of resveratrol crystal form X.

[0021] Figure 6 The saturated solubility of resveratrol crystal form X in different solvents.

[0022] Figure 7 These are the physical formulations and methods that scientists have used over the past 15 years to improve the solubility of resveratrol.

[0023] Figure 8 The crystal form of resveratrol is represented by the cumulative in vitro dissolution rate; data are expressed as mean ± SEM, representing the results of three independent experiments, *P<0.05, **P<0.01, ***P<0.001.

[0024] Figure 9 This is the result of the in vitro antiviral activity study of resveratrol crystal form X. A shows a typical image of RSV-infected host cells; B is a fluorescence image; C and D are quantitative fluorescence measurements of RSV protein expression during RSV infection using immunofluorescence; EC50 values ​​of resveratrol (E) and resveratrol crystal form X (F); G is a schematic diagram of RSV entry into host cells; H is a schematic diagram of the host cell cycle during RSV infection, including attachment, invasion, replication, and release phases; I shows the effects of resveratrol and resveratrol crystal form X on RSV infection in different RSV infection cycles. Effect of resveratrol on SV proliferation; J represents the relative expression of RSV protein mRNA in A549 cells cultured for 24 hours with 25 μM resveratrol or resveratrol crystal form X as determined by RT-qPCR; K represents the molecular docking of resveratrol with the M protein receptor; L represents the heatmap of docking binding energy; data are expressed as mean ± SEM and represent the results of three independent experiments. Compared with the control group, ##P<0.01, ##P<0.001; compared with the model group or the indicated group, *P<0.05, **P<0.01, ***P<0.001.

[0025] Figure 10The results of the antiviral phase investigation of resveratrol crystal form X are shown below. A represents the antiviral results during the adsorption phase; B represents the antiviral results during the entry phase; C represents the antiviral results during the replication phase; and D represents the antiviral results during the release phase. Data are expressed as mean ± SEM and represent the results of three independent experiments. Compared with the group shown, *P<0.05, **P<0.01, ***P<0.001.

[0026] Figure 11 The results are from molecular docking simulations of resveratrol oligomers (dimers, trimers, and tetramers) with protein receptors.

[0027] Figure 12 This section presents the results of in vitro anti-inflammatory and antioxidant activity studies of resveratrol crystal form X. A shows a schematic diagram of LPS-induced RAW264.7 cells; B shows the NO level in LPS-induced RAW264.7 cells after treatment with resveratrol or resveratrol crystal form X; CF shows the expression of CD86 and CD206 in LPS-induced RAW264.7 cells treated with resveratrol or resveratrol crystal form X by flow cytometry; GJ shows the expression of IL-6, IL-1β, and TNF-α in the supernatant of LPS-induced RAW264.7 cells; and KN shows qRT-PCR results. The expression of IL-6, IL-1β, and TNF-α mRNA in LPS-induced RAW264.7 cells treated with resveratrol or resveratrol crystal form X was analyzed; fluorescence images (O) and quantitative changes in ROS (P) in RSV-infected RAW264.7 cells treated with resveratrol or resveratrol crystal form X were analyzed; data are expressed as mean ± SEM and represent the results of three independent experiments; compared with the control group, ##P<0.01, ##P<0.001 by t-test; compared with the model group or the indicated group, *P<0.05, **P<0.01, ***P<0.001.

[0028] Figure 13 The results show the cellular uptake of resveratrol and resveratrol crystal form X by A549 cells, where A represents fluorescence microscopy observations; B represents the average fluorescence intensity of resveratrol and resveratrol crystal form X entering the cells; and C and D represent the cellular uptake of resveratrol and resveratrol crystal form X under different temperatures, times (E), and different endocytosis inhibitors (F). Data are expressed as mean ± SEM and represent the results of three independent experiments. Compared with the group shown, *P<0.05, **P<0.01, ***P<0.001.

[0029] Figure 14The results of lung retention of resveratrol crystal form X in mice after nebulized inhalation are shown. A shows in vivo imaging images and average fluorescence intensity of resveratrol crystal form X labeled with Cy7.5 fluorescent dye at 1 hour, 3 hours and 6 hours in different organs (B); C shows H&E stained sections of tissues. Compared with the group shown, **P<0.01, ***P<0.001.

[0030] Figure 15 The results of treating RSV-infected mouse viral pneumonia with resveratrol crystal form X are shown in Figure A, which is a schematic diagram of the mouse viral pneumonia model and drug administration; B is the appearance of fresh lung tissue; C is the change in mouse body weight; D is the viral load in the lungs of different groups of mice measured by RT-qPCR; E is the change in pro-inflammatory factors in different groups of mice measured by RT-qPCR; F is the micro-CT image of the lung microenvironment before and after viral infection in different groups of mice; G is the pulmonary function test of different groups of mice; H is the representative H&E staining image of the lungs of different groups of mice (scale bar, 200 μm); blue arrows indicate bronchiectasis, yellow arrows indicate pulmonary consolidation, and black arrows indicate inflammatory infiltration; n=8, **P<0.01, ***P<0.001, and t-test was used for comparison with the shown groups.

[0031] Figure 16 The images show lung function tests in mice after treatment with RSV-infected viral pneumonia using resveratrol crystal form X. A represents the expiratory time variation; B represents the inspiratory time variation; C represents the peak inspiratory flow rate variation; D represents the peak expiratory flow rate variation; E represents the tidal volume variation; F represents the expiratory volume variation; G represents the relaxation time variation; and H represents the 50% expiratory flow rate variation.

[0032] The test sample "resveratrol crystal form X" in the above figure is "resveratrol crystal form X obtained according to the preparation method in Example 3 of this invention" (except for...). Figure 7 , 11 outside) Detailed Implementation

[0033] The technical solution of the present invention will be further described below by means of the attached drawings and examples, but it is not a limitation to the present invention. The resveratrol used in the examples was purchased from Shanghai Yuanye Bio-Technology Co., Ltd. The protein primers of F, G, M, NS1, NS2, IL-6, IL-1β and TNF-α were purchased from Shanghai Sangon Biotech Co., Ltd. Cy7.5 NHS was purchased from Lumiprobe Corporation (USA). Trizo, Hi-Scrper Mix for qPCR and gDNAEraser (R223) and SYBR Green PCR MasterMix (Q331-02) were purchased from Vazyme Co., Ltd. (Nanjing, China). Dulbecco's Modified Eagle Medium (DMEM, C11995500BT / 8123412) and fetal bovine serum (FBS) were purchased from Gibco Invitrogen. Phosphate Buffered Saline (PBS) was provided by Servicebio. Lipopolysaccharide (LPS), DAPI, NO detection kit, reactive oxygen species detection kit and Radio-Immunoprecipitation Assay (RIPA) were purchased from Beyotime Biotechnology Co., Ltd.

[0034] The human respiratory syncytial virus A2 strain was purchased from the Institute of Virology, Wuhan University (A2 strain, Wuhan, China). Human laryngeal epithelial cells (HEp-2), human lung cancer cells (A549) and mouse macrophages (RAW 264.7) were purchased from ATCC (Manassas, VA, USA). In all cell experiments, resveratrol was prepared in DMEM containing 0.01% DMSO.

[0035] BALB / c mice (female, 6 weeks old, body weight 16 - 18 g) were purchased from Shanghai Bikai Biotechnology Co., Ltd. (Shanghai, China, SCXKK). (Shanghai, China; SCXK (Hu) 2018-0006). All animal experiments were approved by the Animal Ethics Committee of Nanjing University of Chinese Medicine (202312A054).

[0036] XRD analysis was performed using a D8 QUEST X-ray diffractometer (Bruker Corporation, Germany) to generate CuKa radiation at 40 KV and 30 mV. The scanning range of the diffraction angle (2θ) was from 5° to 60°, and the scanning speed was 5° / min. When performing DSC analysis, 6 mg of the powder was placed in an aluminum pan, sealed and protected with nitrogen. The temperature was raised from 25°C to 600°C at a rate of 10°C per minute and a flow rate of 30 mL per minute. A DSC instrument (DZ-DSC300C, Nanjing, China) was used to evaluate the thermal stability.

[0037] Other instrument equipment and reagent materials not described were also obtained through commercial channels, and the test methods not described in detail were all conventional methods in the art.

[0038] Example 1 Crystal Form X of Resveratrol

[0039] X-ray powder diffraction (XRD) spectroscopy shows that resveratrol crystal form X has a sharp diffraction peak, indicating that it is in a well-crystallized state. Figure 1 It exhibits characteristic peaks at 2θ angles of 27.4°, 31.8°, 45.6°, and 56.5°, with an error range of ±0.2° for the 2θ angles. Simultaneously, diffraction peaks are maintained at 6.6°, 13.2°, 16.5°, 19.2°, and 28.3°. Compared to resveratrol, the crystal structure of resveratrol crystal form X has changed (…). Figure 2 This indicates that the packing pattern of small molecules in crystal form X has changed.

[0040] DSC detection was used. Figure 3 It was observed that the main endothermic peak of resveratrol was 273℃ and the initial melting temperature was 267℃, while the main endothermic peak of resveratrol crystal form X was 267℃ and the initial melting temperature was 258℃, which further indicates that the lattice energy of resveratrol crystal form X is reduced and the crystal form has changed.

[0041] Example 2 Characterization of resveratrol crystal form X

[0042] The resveratrol crystal form X was characterized, and the results are as follows: TEM showed that it is a regular spherical shape; DLS indicated that its particle size was 178.1±5.68 nm, PDI was 0.137±0.037, and Zeta potential was -13.4±1.52 mV. Figure 4 The research report indicates that when PDI < 0.3, the larger the absolute value of the Zeta potential, the more uniform the particle size distribution. Simultaneously, under laser irradiation, resveratrol crystal form X exhibits a significant Tyndall effect, confirming the formation of nanocrystals. Figure 5 ).

[0043] Example 3: Preparation method of resveratrol crystal form X

[0044] S1. Resveratrol is dissolved in dimethyl sulfoxide (DMSO) to obtain the resveratrol organic phase. S2. The resveratrol organic phase is rapidly injected into deionized water under ultrasonic conditions to induce crystallization. S3. The solid fraction is separated using physical methods and dried to obtain resveratrol crystal form X. In step S1, the concentration of the resveratrol organic phase exceeds 5 mM, preferably 5–20 mM, and most preferably 10 mM; the ultrasonic conditions in step S2 are 50–150 Hz, preferably 100 Hz; the volume ratio of the resveratrol solution to the deionized water is 1:25–200, preferably 1:25–100, and most preferably 1:50.

[0045] Example 4: Optimization of the preparation method of resveratrol crystal form X – Investigation of the concentration of resveratrol organic phase

[0046] In Example 3, the resveratrol organic phase prepared in step S1 was configured at concentrations of 80, 40, 20, 10, and 5 mM. The particle size, PDI, and potential of the obtained resveratrol crystal form X were measured using a DLS instrument at 25°C. The results are shown in Table 1. In all groups with resveratrol organic phase concentrations ≥ 5 mM, resveratrol particles were formed. The particle size was stable at 150-200 nm at 5-20 mM, with the smallest particle size at 10 mM. The PDI and potential results showed that resveratrol crystal form X was most stable under these conditions.

[0047] Table 1. Results of the investigation of final drug concentrations (n=3)

[0048] Concentration (mM) Particle size (nm) PDI Potential (mV) 80 443.1±46.4 0.434±0.010 -4.62±0.33 40 450.6±15.9 0.435±0.045 -5.96±0.60 20 181.5±2.27 0.084±0.075 -12.6±0.85 10 178.1±5.68 0.137±0.037 -13.4±1.52 5 178.2±1.52 0.118±0.016 -12.99±0.45

[0049] Example 5: Optimization of the preparation method of resveratrol crystal form X – Investigating the optimal ratio of DMSO to aqueous phase

[0050] In Example 3, the ratio of resveratrol organic phase to aqueous phase in step S2 was 1:10, 1:25, 1:50, 1:100, and 1:200, respectively. The particle size, PDI, and potential of the obtained resveratrol crystal form X were measured using a DLS instrument at 25°C. The results are shown in Table 2. Resveratrol nanoparticles were obtained at organic phase to aqueous phase ratios of 1:25-200. The particle size was stable at 150-300 nm at ratios of 1:25-100, and the smallest (178.1±5.68 nm) was observed at 1:50. Furthermore, the PDI and potential results showed that resveratrol crystal form X was most stable under these conditions.

[0051] Table 2 Results of the investigation on the ratio of organic phase to aqueous phase (n=3)

[0052] Proportion Particle size (nm) PDI Potential (mV) 1:10 1784±460 1 -7.3±0.26 1:25 300.1±6.90 0.402±0.079 -7.11±0.01 1:50 178.1±5.68 0.137±0.037 -13.4±1.52 1:100 301.4±10.6 0.101±0.028 -11.4±0.95 1:200 512.5±10.6 0.354±0.132 -7.3±0.47

[0053] Example 6: A pharmaceutical composition comprising resveratrol crystal form X

[0054] The composition comprises resveratrol crystal form X prepared in Example 3 at a therapeutically effective amount or higher, and one or more pharmaceutically acceptable carriers, diluents, or excipients. The resveratrol crystal form X at a therapeutically effective amount of 1 mg / kg or higher, the carrier comprising other small molecule compounds or high molecular weight polymers, the diluent comprising water, ethanol, DMSO, and SDS, etc., and the excipient comprising starch, dextrin, etc.

[0055] Example 7: Saturated solubility of resveratrol crystal form X

[0056] The saturated solubility of resveratrol and resveratrol crystal form X in different solvents was compared using the saturated solubility method. The specific method was as follows: Excess resveratrol / resveratrol crystal form X powder was dissolved in various solvents (water, ethanol, DMSO, and 1% SDS) to a final volume of 4 mL. The mixture was then incubated in a constant-temperature shaker at 37±2℃ for 48 hours to assess its solubility. After incubation, the precipitate was separated by centrifugation, and the concentration of resveratrol in the supernatant was quantified using fluorescence spectrophotometry.

[0057] The results are as follows Figure 6 As shown, compared with resveratrol (water solubility 47.9±1.97 μg / mL), the water solubility of resveratrol crystal form X (1793.8±36.8 μg / mL) increased by 35.1 times (P<0.001). Furthermore, the solubility in methanol increased by 4.5 times, in DMSO by 2.1 times, and in 1% SDS by 1.8 times. This indicates that the prepared resveratrol crystal form X can significantly improve the water solubility of resveratrol. Compared with other studies, the resveratrol crystal form X prepared by this method has significant advantages (see literature review results for details). Figure 7 ).

[0058] Studies have shown that substances with poor solubility are often difficult to dissolve in biological fluids due to limitations in solubility and particle size, thus significantly reducing their efficacy. Therefore, a PBS buffer solution containing 1% SDS was used as the dissolution medium to evaluate the solubility of drugs and nanocrystals in vitro. The results are as follows: Figure 8 As shown, after 48 hours at pH 7.4, the cumulative dissolution rate of resveratrol was only 17.8%, while that of resveratrol crystal form X was 41.6%. This indicates that the small particle size, high solubility, and excellent water dispersibility of resveratrol crystal form X contribute to the slow and sustained dissolution of the active molecule. Furthermore, under slightly acidic inflammatory conditions (pH 5.5), resveratrol crystal form X exhibited rapid drug dissolution within 0–4 hours, reaching a cumulative dissolution rate of 58.1%, which then gradually increased to 78.9% over 48 hours. This dissolution profile is significantly higher than the dissolution rates of 28.1% (4 hours) and 41.2% (48 hours) at pH 7.4 without inflammation, indicating that resveratrol crystal form X has a strong ability to effectively dissolve the drug in slightly acidic inflammatory environments.

[0059] Example 8: In vitro viral inhibitory effect of resveratrol crystal form X

[0060] Cell morphology was observed using an inverted microscope, and the results are as follows: Figure 9As shown in Figure A, RSV-infected cells exhibited significant cytopathic effects (CPE) and syncytia. Immunofluorescence analysis of RSV infection (MOI = 0.1) showed decreased fluorescence intensity in the resveratrol and resveratrol crystal form X treatment groups, indicating that RSV amplification was inhibited. Figure 9 (B). In particular, compared with the free resveratrol group, the inhibitory effect of resveratrol crystal form X on RSV amplification was more significant (P<0.001). Figure 9 (C), which may be due to differences in the level of resveratrol uptake by cells. Figure 9 (D). Figure 9 qRT-PCR experiments in E and F confirmed consistent results, revealing a dose-dependent inhibitory effect of resveratrol and its crystal form X on RSV. The 50% effective concentration (EC50) of resveratrol was determined. 50 The concentration of resveratrol was 31.8 μM, and that of resveratrol crystal form X was 21.3 μM. Notably, at a concentration of 100 μM (below the saturation solubility of resveratrol crystal form X), the inhibitory effect was close to 90%, while the same concentration of free resveratrol (soluble in 0.5% DMSO) showed only a 60% inhibitory effect on RSV. Furthermore, selection index (SI) calculated based on the 50% cytotoxic concentration (CC50) revealed that resveratrol crystal form X (SI = 25.8) exhibited significantly improved antiviral efficacy compared to free resveratrol (SI = 16).

[0061] like Figure 9 As shown in G, the viral life cycle involves several key stages. Initially, RSV attaches to receptors on the apical surface of ciliated epithelial cells via G proteins. Subsequently, the fusion protein (F) promotes the fusion of the viral envelope with the host membrane, resulting in the release of the nucleocapsid into the host cell's cytoplasm. Following this, the mRNA is translated and replicated within the host cell. Finally, matrix proteins (M) and non-domain proteins (NS1 and NS2) interact with the complex, allowing RSV to be released from the cell via budding. To elucidate the potential antiviral mechanism of resveratrol crystal form X, the effects of resveratrol and its crystal form X on the four stages of the viral life cycle (attachment, invasion, replication, and release) were investigated, and the results are shown below. Figure 9 H and Figure 10 (As shown). Throughout all stages of RSV entry into host cells, resveratrol and resveratrol crystal form X exhibited concentration-dependent inhibitory effects on RSV proliferation. During viral invasion and replication, the inhibitory abilities of resveratrol and resveratrol crystal form X on RSV mRNA expression differed significantly (P<0.001). Figure 9 (I).

[0062] Similarly, the same results were observed when the expression of RSV-related proteins was suppressed. G and F proteins are extracellular RSV proteins, while M, NS1, and NS2 proteins are intracellular RSV proteins. Figure 9 The results in J showed that, compared with free resveratrol, crystalline form X of resveratrol significantly inhibited the replication of intracellular RSV proteins (M, NS1, and NS2), thereby affecting the increase of the RSV genome. Molecular docking simulations yielded consistent conclusions.

[0063] As is well known, the oligomers produced by the oxidation of resveratrol are collectively known as glucosamine, an antitoxin produced by plants in response to environmental stress. To further simulate the crystal form X of resveratrol, molecular docking simulations were performed using resveratrol oligomers (dimers, trimers, and tetramers) with protein receptors. For example... Figure 9 K and Figure 11 As shown, resveratrol significantly increased the binding energies of M, NS1, and NS2 protein receptors (-6.6, -7.1, and -6.9 kcal / mol, respectively).

[0064] The study found that resveratrol interacts with M receptors ALA53, LYS50, ILE49, VAL5, ASP46, MET1, and PHE0 via hydrogen bonds, Pi alkyl groups, and Pi-Pi amino acid residues. Importantly, the binding energy of the protein-receptor interaction decreases with increasing resveratrol molecule count, indicating enhanced potency. Figure 9 These results further support the idea that resveratrol crystal form X, formed through the self-assembly of multiple small molecules, has a stronger affinity for the protein receptor on RSV, thereby inhibiting RSV replication.

[0065] Example 9: In vitro anti-inflammatory and antioxidant activity of resveratrol crystal form X

[0066] An inflammation model was established using LPS stimulation of Raw 264.7 cells. Figure 12 (A). The effects of different doses (high, 50 μM; medium, 25 μM; low, 12.5 μM) of resveratrol and its crystal form X on cellular NO levels were evaluated. Figure 12 (B). The results showed that, compared with resveratrol alone, resveratrol crystal form X significantly reduced cellular NO levels in a dose-dependent manner. Furthermore, compared with free resveratrol, resveratrol crystal form X significantly reduced intracellular NO concentration.

[0067] To further explore the anti-inflammatory properties of resveratrol crystal form X, flow cytometry was used to detect macrophage polarization, and the results are as follows: Figure 12As shown in the CF, compared with normal Raw 264.7 cells, LPS stimulation significantly increased CD86 expression and promoted macrophage polarization from M0 to M1. Compared with the LPS group, each treatment group inhibited CD86 expression to varying degrees, with the resveratrol crystal form X group showing half the inhibition rate of the resveratrol group. Furthermore, compared with the resveratrol group, the resveratrol crystal form X group not only promoted macrophage polarization from M1 to M2 but also increased CD206 expression from 0.35% to 4.59%. These findings suggest that resveratrol crystal form X enhances the immunomodulatory effects of the drug, thereby leading to an anti-inflammatory response.

[0068] The expression of pro-inflammatory cytokines in cells was detected using ELISA and RT-qPCR. Results are as follows: Figure 10 As shown in the GJ, the levels of IL-6, IL-1β, and TNF-α cytokines were significantly increased after LPS stimulation, indicating the effectiveness of the cell model. Compared with resveratrol, resveratrol crystal form X significantly reduced the expression of these cytokines. Furthermore, RT-qPCR results showed that treatment with resveratrol crystal form X significantly reduced the expression of IL-6 and TNF-α mRNA. Figure 12 (KN).

[0069] ROS production was induced in Raw 264.7 cells using RSV to assess the pharmacological differences between resveratrol and resveratrol crystal form X in alleviating oxidative stress. Results are as follows: Figure 12 As shown in the O and P values, RSV stimulation of A549 cells leads to excessive ROS production, while the amount of ROS production is reduced when the same concentration (25 μM) of resveratrol or resveratrol crystal form X is applied. These results indicate that nanoscale resveratrol crystal form X not only has antiviral effects but also inhibits RSV-induced ROS.

[0070] Example 10 Cellular uptake of resveratrol crystal form X

[0071] The effects of resveratrol and resveratrol crystal form X on cellular uptake were observed using fluorescence microscopy. Results are as follows: Figure 13 As shown in Figures A and B, resveratrol crystal form X was more favorable for cellular uptake compared to the free drug (green fluorescence). Since cellular uptake is influenced by various factors, different incubation times, concentrations, temperatures, and inhibitors were tested. The results showed that in both the resveratrol group and the resveratrol crystal form X group, the concentration-dependent (…) Figure 13 C and D) and time dependence ( Figure 13 Both E) showed increased cellular uptake. Specifically, the intracellular content of resveratrol gradually increased over time in a time-dependent manner, and there was no significant change in intracellular content after 6 hours.

[0072] Since low temperatures may inhibit the activity of cellular transport proteins, the effect of temperature on cellular uptake was investigated, with temperatures decreasing from 37°C to 4°C. Figure 13 As shown in C, for the resveratrol group, no significant difference was observed between 4 °C and 37 °C, indicating that absorption mainly depends on passive diffusion. Conversely, for the resveratrol crystal form X group (… Figure 13 The uptake of resveratrol (D) at 37°C was four times that at 4°C. This significant increase from low to high temperatures suggests that cellular uptake of resveratrol crystal form X may involve transport proteins and may be related to an energy-dependent process.

[0073] The uptake mechanism of resveratrol crystal form X was investigated by adding different inhibitors. Figure 13 (F). Cellular internalization occurs via multiple pathways. The typical effects of inhibitors on the uptake of resveratrol crystal form X were investigated, including the clathrin inhibitor chlorpromazine (10 μg / mL), the caveolin inhibitors nystatin and methyl-β-cyclodextrin (30 μg / mL), the macrocytosis inhibitor amiloride (30 μg / mL), and the mitotic inhibitor colchicine (40 μg / mL). The presence of chlorpromazine resulted in the most significant decrease in uptake (50%) compared to the control group without inhibitors. This indicates that the cellular uptake pathway of resveratrol crystal form X is a lectin-mediated endocytosis process.

[0074] Example 11 Biodistribution and biocompatibility of resveratrol crystal form X via nebulized inhalation

[0075] Nebulized inhalation is a method of direct lung administration, and due to its high compliance, it is particularly suitable for treating respiratory infections, especially in pediatric patients. Cy7.5-labeled resveratrol and resveratrol crystal form X were delivered to the lungs of mice using a micronebulizer, and their biodistribution was investigated using fluorescence imaging. Figure 14 The results described in sections A and B showed that the deposition levels in the lungs at 1, 3, and 6 hours post-administration differed compared to the free Cy7.5 dye group. Notably, there was no significant difference in fluorescence intensity between the resveratrol group and the resveratrol crystal form X group at 1 hour. Over time, the fluorescence intensity of the resveratrol group decreased, while the resveratrol crystal form X group maintained a consistent intensity, indicating that resveratrol crystal form X improved lung retention and may enhance the treatment of RSV pneumonia. Furthermore, histological analysis ( Figure 14 (C) The results showed no obvious abnormalities or lesions in the lungs or other tissues, indicating that the nebulized inhalation of nano-sized resveratrol crystal form X was well-tolerated. These results support the safety and efficacy of nebulized inhalation of resveratrol crystal form X for the treatment of RSV pneumonia.

[0076] Example 12: Pharmacodynamic evaluation of resveratrol crystal form X in vivo.

[0077] Resveratrol crystal form X exhibited significant therapeutic effects in vitro against viruses and inflammation. The in vivo antiviral effects of resveratrol and resveratrol crystal form X on a mouse model of RSV-infected pneumonia were then evaluated. Figure 15 As shown in Figure A, mice infected with RSV were administered the drug via the pulmonary route using a microneedler once daily. After three days of continuous treatment, the mice were euthanized to assess viral load and inflammation levels. Figure 15 Lung tissue imaging analysis in mice B showed that, compared to the uninfected RSV control group, RSV-infected mice exhibited pulmonary capillary congestion and edema (indicated by red arrows), appearing deep red. The ribavirin-treated group showed essentially normal lung tissue appearance with milder pulmonary capillary congestion and edema, indicating less lung tissue damage. Conversely, the free resveratrol group showed mild pulmonary capillary congestion and edema, while the combined resveratrol group showed a normal lung tissue appearance, appearing pale pink, without significant pulmonary capillary congestion, edema, or hemorrhage. Figure 15 As shown in Figure C, compared with the control group without RSV infection, each treatment group exhibited different abilities to inhibit weight loss. Interestingly, the resveratrol crystal form X group was significantly more effective than the resveratrol group in this regard. Figure 15 Further studies D and E showed that, compared with resveratrol alone, resveratrol crystal form X more effectively inhibited viral load in bronchoalveolar lavage fluid (50%, P<0.001) and reduced the expression of pro-inflammatory factors IL-6 and TNF-α. Notably, although the dosage of resveratrol crystal form X was only 1 mg / kg, one-fifth that of ribavirin (5 mg / kg), it showed comparable efficacy in both antiviral and anti-inflammatory aspects. Ribavirin is a broad-spectrum antiviral drug and is not specific to RSV; therefore, a dose of 1 mg / kg in vivo was insufficient to effectively reduce RSV viral load. The significant reduction in viral titer and inflammation levels confirms the outstanding advantages of resveratrol crystal form X in the treatment of viral pneumonia.

[0078] Each mouse underwent two micro-CT scans before and after RSV infection. The model group exhibited typical symptoms of viral pneumonia, such as severe bronchiectasis, inflammatory infiltration, and exudation. Furthermore, compared to the control group, no significant diffuse white consolidation (yellow arrow) or bronchiectasis (blue arrow) was observed in the lungs of mice treated with resveratrol (crystalline X-ray). Figure 13 (F). For example Figure 15 G and Figure 16 As shown, lung function is a definitive indicator of lung injury in RSV-infected pneumonia mice. Compared with the free drug group, resveratrol crystal form X significantly reduced bronchoconstriction, apnea, maximal inspiratory volume, and maximal expiratory volume in mice (P<0.001). Histopathological analysis of lung tissue ( Figure 15 Similar results were obtained in the H group, showing that the lung lesions in the resveratrol crystal form X treatment group were reduced, with only mild alveolar collapse and inflammatory infiltration (black arrows), while severe lung damage was observed in the model group. In conclusion, the use of resveratrol crystal form X in RSV-induced viral pneumonia is not only effective in antiviral and anti-inflammatory treatment, but also protects lung function and provides good prevention.

[0079] Therefore, the resveratrol crystal form X prepared in this invention is a regular spherical shape with a particle size of 178.1±5.68 nm, a PDI of 0.137±0.037, and a Zeta potential of -13.4±1.52 mV, exhibiting reliable stability without the use of stabilizers. Compared with untreated resveratrol (water solubility of 47.9±1.97 μg / mL), it has significantly improved water solubility, reaching 1793.8±36.8 μg / mL. It also exhibits good dissolution capacity in a slightly acidic inflammatory environment. Compared with resveratrol, it shows significantly enhanced inhibitory effects on RSV both in vitro and in vivo, and the inhibitory effect on RSV is dose-dependent. It also has good anti-inflammatory and antioxidant activities. The resveratrol crystal form X is more conducive to cellular uptake and can improve retention in the lungs, making nebulized inhalation therapy for RSV pneumonia safe and effective.

[0080] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A crystal form X of resveratrol, characterized in that: Its X-ray powder diffraction pattern has characteristic peaks at 2θ angles of 27.4°, 31.8°, 45.6° and 56.5°, with an error range of ±0.2° for the 2θ angle.

2. The crystal form X of resveratrol as described in claim 1, characterized in that: Its X-ray powder diffraction pattern is basically consistent with that shown in Figure 1.

3. The crystal form X of resveratrol as described in claim 1 or 2, characterized in that: Its X-ray powder diffraction pattern also shows characteristic peaks at 2θ angles of 6.6°, 13.2°, 16.5°, 19.2° and 28.3°.

4. The method for preparing crystal form X of resveratrol according to any one of claims 1-3, characterized in that, Includes the following steps: S1. Resveratrol is dissolved in dimethyl sulfoxide to form a resveratrol solution. S2. The resveratrol solution in S1 is rapidly injected into deionized water under ultrasonic conditions to induce crystallization. S3. The solid part is separated by physical methods and dried to obtain the crystal form X.

5. The preparation method according to claim 4, characterized in that: The resveratrol concentration in step S1 exceeds 5 mM, the ultrasonic conditions in step S2 are 50-150 Hz, and the volume ratio of the resveratrol solution to the deionized water is 1:25-200.

6. The preparation method according to claim 5, characterized in that: The resveratrol concentration in step S1 is 5–20 mM.

7. The preparation method according to claim 5, characterized in that: The ultrasonic conditions described in step S2 are 100 Hz.

8. The preparation method according to claim 5, characterized in that: In step S2, the volume ratio of resveratrol solution to deionized water is 1:25-100.

9. The preparation method according to claim 8, characterized in that: In step S2, the volume ratio of the resveratrol solution to the deionized water is 1:

50.

10. The crystal form X of resveratrol prepared by any one of the preparation methods of claims 4-9.

11. A pharmaceutical composition, characterized in that: The product comprises a therapeutically effective amount of crystal form X of resveratrol as described in any one of claims 1-3, 10, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

12. Use of resveratrol crystal form X as described in any one of claims 1-3 and 10, or the pharmaceutical composition as described in claim 11, in the preparation of a medicament for treating pneumonia.

13. The use as described in claim 12, characterized in that, The pneumonia in question is respiratory syncytial virus pneumonia, or acute lung injury.

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