Isoprenylated phenolic acid drupanin, single crystal and separation and purification method and application thereof

Abstract

The application discloses a kind of isoprenylated phenolic acid Drupanin, single crystal and its separation and purification method and application.The application adopts the combined purification process of dynamic axial column chromatography and preparative chromatography, realizes the efficient enrichment and accurate separation of Drupanin component in propolis;And for the first time, the complete three-dimensional spatial structure of Drupanin is determined by X-ray single crystal diffraction technology;Through in-vitro and in-vivo animal experiments, it is proved that the Drupanin prepared by the application can significantly improve neuroinflammation, oxidative stress and lipid metabolism disorder, and can effectively repair cognitive dysfunction, relieve depression and anxiety symptoms, has the advantages of multi-target, low side effect and significant efficacy, can be widely used in improving cognitive impairment, preventing or treating depression, anxiety and other nervous system diseases Functional products and innovative drug research and development, and has good clinical application value.

Description

Technical Field

[0001] This invention belongs to the field of natural product chemistry technology, specifically relating to an isopentenylated phenolic acid Drupanin, its single crystal, its separation and purification method, and its application. Background Technology

[0002] Neuroinflammation is the immune defense response of the central nervous system (CNS) to harmful stimuli such as infection, injury, metabolic abnormalities, or autoimmune reactions. Its core characteristics are the overactivation of microglia and astrocytes, and the infiltration of peripheral immune cells (Cookson MR, Cell. 2023). Existing research indicates that neuroinflammation is present in acute injuries ranging from stroke to CNS trauma, and in chronic neurodegenerative diseases such as cognitive impairment / Alzheimer's disease, depression, and anxiety (Shi FD, Science. 2025). Therefore, regulating the complex balance of neuroinflammation is crucial for the prevention and / or treatment of acute / chronic neurological diseases.

[0003] Currently, the clinical need for drugs to prevent and treat cognitive impairment, depression, and anxiety disorders remains unmet. Existing chemical drugs generally suffer from problems such as single-target action, limited efficacy, significant toxic side effects, and poor long-term safety. Compared to chemically synthesized drugs, traditional natural medicines and functional health products have long-term human experience, their safety is well-assured, and their effectiveness has been tested over a long period. Therefore, developing functional products and / or drugs with multiple effect targets and low toxicity and side effects for the prevention and treatment of neurological diseases based on traditional natural medicines / health products has significant clinical value and application prospects.

[0004] Propolis, as a traditional natural medicine, is widely used in the pharmaceutical and health product industries. Currently approved health functions include immune regulation (enhancing immunity), improving sleep, delaying aging, and anti-fatigue (relieving physical fatigue), among others, demonstrating high recognition of its medicinal and health value. Based on propolis's natural safety properties and diverse bioactive characteristics, screening and extracting natural active small molecules from propolis that can target and regulate neuroinflammation and improve cognitive function has significant clinical value and industrialization prospects for developing novel products for the prevention and treatment of nervous system diseases.

[0005] Existing research has identified over 300 chemical components from propolis, with flavonoids and phenolic acids being the core active ingredients. The purification of monomeric components such as pinocembrin, pinoside, guarban, coine, quercetin, galangin, caffeic acid phenethyl ester, and aspirin C has been studied and reported. However, current techniques lack methods for separating structurally similar isopentenylated phenolic acids in propolis. The systematic separation methods for propolis chemical components reported in the literature mostly employ solvent extraction combined with silica gel column chromatography and preparative thin-layer chromatography, which suffer from low separation efficiency, cumbersome steps, long cycles, significant loss of target components, and low yields. Furthermore, although X-ray single-crystal diffraction technology is mature, many small-molecule compounds in propolis are difficult to obtain qualified single crystals, resulting in unclear three-dimensional structures. This severely restricts the analysis of structure-activity relationships, pharmacophore screening, and in-depth development of crystalline drugs. Summary of the Invention

[0006] In view of the shortcomings of the prior art, the purpose of this invention is to provide isopentenylated phenolic acid Drupanin, single crystals, its separation and purification method and application.

[0007] This invention is achieved through the following technical solution:

[0008] On one hand, the present invention provides a method for the isolation and purification of isopentenylated phenolic acid Drupanin, comprising the following steps:

[0009] (1) Using green propolis as raw material, extracting with methanol or ethanol as solvent, filtering and concentrating the extract to obtain propolis alcohol extract;

[0010] (2) The propolis alcohol extract was initially separated by dynamic axial column chromatography. Combined with the retention time, the fraction rich in Drupanin was collected and concentrated to obtain crude Drupanin product.

[0011] The conditions for the dynamic axial column chromatography are as follows: reversed-phase silica gel C18 is used as the packing material; acetonitrile containing 10% methanol is used as mobile phase A and 0.1% formic acid aqueous solution is used as mobile phase B; gradient elution is performed with the following elution program: 0-10 min, 90%B→44%B; 17-21 min, 44%B→38%B; 21-32 min, 38%B→5%B; 32-35 min, 5%B→5%B; detection wavelength is 314 nm.

[0012] (3) The crude Drupanin product was purified by preparative liquid chromatography. The elution peaks at specific time points were collected, concentrated, and dried to obtain the final product.

[0013] The conditions for preparing the liquid chromatography were as follows: a C18 column was used; acetonitrile was used as mobile phase A and pure water containing 0.1% formic acid was used as mobile phase B; gradient elution was performed with the following elution program: 0–10 min, 45%–75% A; 10–20 min, 75%–90% A; 20–24 min, 90% A; 24–25 min, 95%–45% A; 25–30 min, 45% A; and the detection wavelength was 314 nm.

[0014] In a preferred embodiment of the present invention, in step (1), the extraction method is heating reflux extraction or ultrasonic extraction; the material-liquid ratio is 1:5-20.

[0015] As a further preferred embodiment of the present invention, the temperature of the heating reflux extraction is 80-90 ℃, the number of extractions is 1-2 times, and the extraction time is 1-4 h; the frequency of the ultrasonic extraction is 20-40 KHz, the number of extractions is 1-2 times, and the extraction time is 30-120 min.

[0016] In a preferred embodiment of the present invention, in step (3), the C18 chromatographic column has a specification of 250×21 mm and a packing particle size of 5~10 µm.

[0017] Secondly, the present invention provides a Drupanin single crystal, which is a monoclinic crystal with space group P 21 / n; cell dimensions: a=4.3764(1), b=14.2356(4), c=20.3173(6); its interplanar angles α / °=90, β / °=93.0250(10), γ / °=90, and unit lattice volume V=1264.02Å. 3 The number of molecules in a unit lattice is Z = 4.

[0018] Thirdly, the present invention provides a method for preparing the above-mentioned Drupanin single crystal, comprising the following steps:

[0019] The Drupanin compound is dissolved in a mixed solvent of acetonitrile and water at a concentration of 5-15 mg / mL. The solution is placed in a cool, ventilated place, and crystals are precipitated as the solvent slowly evaporates. The volume ratio of acetonitrile to water in the mixed solvent is 60:40-50:50.

[0020] Fourthly, the present invention provides the use of Drupanin, the above-mentioned Drupanin single crystal, or a pharmaceutically acceptable salt thereof in the preparation of drugs for improving neuroinflammation, enhancing cognitive function, and preventing or treating depression and anxiety.

[0021] As a preferred embodiment of the present invention, the application includes at least one of the following:

[0022] (1) Improves nerve inflammation;

[0023] (2) Reduce oxidative stress levels;

[0024] (3) Inhibits M1 polarization of microglia;

[0025] (4) Reduce the proportion of lipid droplet-associated glial cells;

[0026] (5) Improves lipid metabolism disorders in glial cells;

[0027] (6) Repairing cognitive impairment;

[0028] (7) Relieve depression and anxiety-like symptoms.

[0029] This invention constructs LPS-induced BV-2 microglia oxidative stress and neuroinflammation models. In vitro experimental results show that Drupanin has effects such as improving neuroinflammation, reducing ROS levels, inhibiting microglia M1 polarization, reducing the proportion of lipid droplet-associated glial cells, and improving glial cell lipid metabolism disorders.

[0030] This invention further establishes an LPS-induced depression and anxiety-like behavior model in C57BL / 6J mice. Animal behavioral experiments confirmed that Drupanin possesses excellent cognitive-improving, antidepressant, and anti-anxiety activities. In the Morris water maze test, Drupanin significantly increased the number of platform crossings and the time spent in the target quadrant in the model mice, effectively repairing learning and memory impairments caused by inflammatory damage. In the open field test, Drupanin significantly increased the total distance traveled, average speed, and percentage of activity time in the model mice, while prolonging the time spent in the middle area, effectively improving anxiety-like behavior and enhancing autonomous activity and exploration abilities. In the tail suspension test, Drupanin significantly shortened the time the model mice remained still and prolonged the effective movement time, significantly alleviating despair-like behavior in the mice.

[0031] Finally, the present invention also provides a pharmaceutical composition comprising an effective amount of Drupanin, the aforementioned Drupanin single crystal or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0032] The pharmaceutically acceptable salts described in this invention include hydrochloride, sulfate, phosphate, diphosphate, nitrate, hydrobromide, acetate, maleate, fumarate, tartrate, succinate, lactate, p-toluenesulfonate, hydrochloride, or oxalate, etc.

[0033] The pharmaceutically acceptable carriers described in this invention include conventional diluents, excipients (such as water), fillers (such as starch), binders (such as cellulose derivatives, gelatin, etc.), humectants (such as glycerin, etc.), disintegrants (such as agar, calcium carbonate, etc.), absorption promoters (such as quaternary ammonium compounds, etc.), surfactants (such as hexadecyl alcohol, etc.), adsorbent carriers (such as kaolin and soap clay, etc.), lubricants (such as talc, etc.), etc., in amounts that are conventional in the art.

[0034] In some embodiments, the pharmaceutical compositions of the present invention can be prepared according to conventional methods in the art to obtain any pharmaceutically acceptable dosage form, including but not limited to oral liquids, granules, powders, decoctions, pills, capsules or tablets, etc., and can also adopt formulations known in the modern pharmaceutical industry, such as nano, controlled-release, sustained-release formulations, etc.

[0035] Compared with the prior art, the present invention has the following advantages:

[0036] (1) This invention uses green propolis as raw material and adopts a purification process combining dynamic axial column chromatography and preparative chromatography to achieve efficient enrichment and precise separation of Drupanin in propolis, which greatly simplifies the preparation process, shortens the preparation cycle, and significantly improves the preparation yield and product purity of Drupanin.

[0037] (2) This invention is the first to clearly resolve and confirm the complete three-dimensional spatial structure of Drupanin through X-ray single crystal diffraction technology, which lays the core theoretical foundation for the subsequent in-depth analysis of the structure-activity relationship of Drupanin, precise screening of core pharmacophores, structural optimization and modification and crystal form drug development;

[0038] (3) Through in vitro and in vivo animal experiments, it has been confirmed that Drupanin prepared by this invention can significantly improve neuroinflammation, oxidative stress and lipid metabolism disorders. At the same time, it can effectively repair cognitive dysfunction and relieve depression and anxiety-like symptoms. It has the advantages of multiple targets, low side effects and significant efficacy. It can be widely used in the development of functional products and innovative drugs to improve cognitive impairment, prevent or treat depression, anxiety and other neurological diseases, and has good clinical application value. Attached Figure Description

[0039] Figure 1 The dynamic axial chromatography spectrum of propolis alcohol extract (red is the spectrum of Drupanin reference standard, black is the spectrum of propolis alcohol extract);

[0040] Figure 2 The chromatograms for the preparation of crude Drupanin are shown (black is the chromatogram of Drupanin reference standard, and blue is the chromatogram of crude product).

[0041] Figure 3This is an HPLC purity determination graph for Drupanin.

[0042] Figure 4 High-resolution mass spectrum for precise molecular weight determination of Drupanin;

[0043] Figure 5 For Drupanin 1 H-NMR spectrum;

[0044] Figure 6 For Drupanin 13 C-NMR spectrum;

[0045] Figure 7 The spectrum of DEPT135 for Drupanin;

[0046] Figure 8 For Drupanin 1 H- 1 H COSY spectrum;

[0047] Figure 9 For Drupanin 1 H- 1 H NOESY spectrum;

[0048] Figure 10 The HMQC spectrum of Drupanin;

[0049] Figure 11 The HMBC spectrum of Drupanin;

[0050] Figure 12 Here is the chemical structural formula of Drupanin;

[0051] Figure 13 This is a diagram of the naturally precipitated crystals of Drupanin.

[0052] Figure 14 The three-dimensional structure of Drupanin is obtained by X-ray single-crystal diffraction analysis;

[0053] Figure 15 The effect of Drupanin on LPS-induced NO content (A) and cell viability (B) in BV2 cells; compared with the Model group, ***p<0.001, **p<0.01 (n=6).

[0054] Figure 16 The effect of Drupanin on LPS-induced expression of inflammatory factors and CD86 mRNA in BV2 cells; compared with the Model group, ***: p < 0.001, **: p < 0.01, *: p < 0.05 (n=3).

[0055] Figure 17 The effect of Drupanin on LPS-induced oxidative stress (ROS) in BV2 microglia; compared with the Model group, ***: p < 0.001 (n = 6).

[0056] Figure 18 The effect of Drupanin on LPS-induced lipotoxicity in BV2 cells; compared with the Model group, ***: p < 0.001 (n = 6).

[0057] Figure 19 This is a staining analysis of Drupanin on LPS-induced NF-κB (p65) nuclear transport in BV2 cells.

[0058] Figure 20 Results and analysis of the Morris water maze test (n=6); compared with the Model group, *: p<0.05;

[0059] Figure 21 Results and analysis of open field tests (n=6); Compared with the Model group, ***: p<0.001, **: p<0.01, *: p<0.05;

[0060] Figure 22 Results and analysis of the tail suspension test (n=6); compared with the Model group, ***: p<0.001, **: p<0.01;

[0061] Figure 23 Results and analysis of qPCR for key genes in mouse hippocampus (n=3); compared with the Model group, ***: p<0.001, **: p<0.01. Detailed Implementation

[0062] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. The described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0063] Example 1: Isolation and Identification of Drupanin

[0064] 1. Raw material processing and crude extraction

[0065] Take 200 g of green propolis and place it in a 2 L conical flask. Add 1500 mL of 95% ethanol and extract with enhanced ultrasound at 20 kHz for 60 min (pause for 5 min every 20 min). Filter and add 1200 mL of 95% ethanol to the residue for a second ultrasonic extraction for 60 min (pause for 5 min every 20 min). Filter and combine the two filtrates. Filter the residue through a coarse filter with defatted cotton and a qualitative filter paper, and then filter under reduced pressure through a 0.45 µm PTFE membrane. Evaporate the filtrate at 55-60 ℃ and concentrate to obtain the propolis alcohol extract.

[0066] 2. Dynamic axial compression column separation in series for preparative chromatographic purification

[0067] A preparative chromatography system from Hanbang Technology Co., Ltd. was used, equipped with an NP7000 infusion pump and a NU3000 detector, and a DAC-50 dynamic axial compression column (50×600 mm) in series, packed with C18 bonded silica gel. The propolis ethanol extract was separated by dynamic axial compression column chromatography, and the separation was determined by the chromatographic retention time of Drupanin reference standard (11.9–12.1 min) and the target compound [MH]. - The mass-to-charge ratio (m / z = 231.1026) was confirmed. The fraction rich in Drupanin was collected and concentrated to obtain crude Drupanin product.

[0068] The dynamic axial compression column used C18 reverse-phase silica bonded phase as packing material; acetonitrile (containing 10% methanol) was used as mobile phase A and 0.1% formic acid aqueous solution was used as mobile phase B; gradient elution was used, with the elution program as follows: 0–10 min, 90%B → 44%B; 17–21 min, 44%B → 38%B; 21–32 min, 38%B → 5%B; 32–35 min, 5%B → 5%B; flow rate 75 mL / min. -1 The detection wavelength is 314 nm, and the single injection volume is 10 mL.

[0069] The crude Drupanin product was then purified by preparative liquid chromatography. The elution peak with a retention time of 12.25 min was collected, concentrated, and lyophilized to obtain the target compound Drupanin (purity >98.0%).

[0070] The conditions for the preparative liquid chromatography were as follows: C18 column (250×21 mm, 5 µm, Agilent Technologies); acetonitrile as mobile phase A and pure water containing 0.1% formic acid as mobile phase B; gradient elution; elution program: 0–10 min, 45%–75% A; 10–20 min, 75%–90% A; 20–24 min, 90% A; 24–25 min, 95%–45% A; 25–30 min, 45% A; flow rate: 16 mL / min; detection wavelength: 314 nm; injection volume: 1000 μL.

[0071] 3. Purity detection of compounds

[0072] The purity of the purified target compound was determined by HPLC normalization. A Waters 2695 HPLC system (Waters Corporation) was used, equipped with an analytical C18 column (250 mm × 4.6 mm, 5 µm, Kromasil). The mobile phase was 0.1% formic acid-water solution (A) and acetonitrile (B); the gradient elution program was: 0–20 min, 10% → 90% (B); 20–25 min, 90% (B); 25–27 min, 90% → 10% (B); 27–30 min, 10% (B). The detection wavelength was 314 nm; the injection volume was 10 µL. The results are as follows. Figure 3 As shown, its purity is 98.25%.

[0073] 4. Precise molecular weight determination of compounds

[0074] The precise molecular weight of the target compound was determined using a 6545 ESI-Q-TOF-MS (Agilent Technologies) high-resolution mass spectrometer. ESI-MS conditions: Dual AJS ion source (ESI), negative ion scan mode, nozzle voltage 1000 V; sheath gas temperature 350 °C, flow rate 11 L / min; desiccator temperature and flow rate 320 °C and 8 L / min, respectively. -1 The nebulizer gas pressure was 35 psi. The capillary voltage was 4000 V, and the MS collision energy was 10 eV; the scanning ion range was 100-1000 m / z. HRMS spectra ( Figure 4 The image shows a quasi-molecular ion peak [MH]. - m / z 231.1026.

[0075] 5. Structural characterization of compounds

[0076] An appropriate amount of the target compound was dissolved in deuterated chloroform (Chloroform-d), and then transferred to an NMR tube. 1D- and 2D-NMR data were acquired using a 600 MHz superconducting NMR spectrometer (AV-600, Bruker, Germany).

[0077] like Figures 5-11 As shown, according to [MH] - m / z 231.1026, combined with 1 H-、 13 C1-NMR data confirmed the molecular formula to be C10. 14 H 16 O3.

[0078] 1 H-NMR (600 MHz, Chloroform-d): δ 7.71 (d, J = 15.9 Hz, 1H, H-7), 7.32 (s, 1H, H-3), 7.36-7.28 (dd, 1H, H-5), 6.82 (d, J = 8.1 Hz, 1H, H-6), 6.30(d, J = 15.9 Hz, 1H, H-8), 5.35-5.28 (m, 1H, H-2'), 3.37 (d, J = 7.2 Hz, 2H, H-1'), 1.81–1.68 (m, 6H, H-4', 5').

[0079] 13 C-NMR (151 MHz, Chloroform-d): δ172.23 (C-9), 156.86 (C-1), 147.00 (C-7), 135.66 (C-3'), 130.43 (C-3), 128.23 (C-8), 127.48 (C-4), 126.93 (C-2), 120.96 (C-2'), 116.25 (C-6), 114.36 (C-8), 25.82 (C-4'), 17.93 (C-5').

[0080] According to the DEPT-135 spectrum, the compound has 5 quaternary carbons, 8 primary or tertiary carbons, and 1 secondary carbon. The HMQC spectrum was used to assign the compound to a direct carbon-hydrogen correlation. 1 H- 1H COSY showed correlations at δ1.80 and δ3.37, δ1.78 and δ5.52, δ3.37 and δ5.52, δ3.37 and δ7.32, δ6.81 and δ7.32, and δ6.30 and δ7.71, indicating a coupling effect. Combined with the HMBC spectrum, δ H 7.71 and δ C Correlation between 114.36, 128.24, 130.44, and 172.24, δ H 7.32 and δ C 29.58, 126.93, 128.28, 147.02, 156.88 correlation, δ H 6.81 and δ C Correlation between 126.93, 128.24, and 156.88, δ H 6.31 and δ C 127.27 related, δ H 3.37 and δ C 120.98, 126.93, 135.66, 156.88 correlation, δ H 1.78 and δ C Correlation at 17.93, 25.82, 120.98, and 135.66. NOESY shows δ H 7.71 and δ H Related to 6.30.

[0081] Based on the above spectral data, it can be inferred that the planar structure of the target compound prepared in this invention is 3-(4-hydroxy-3-(3-methylbut-2-en-1-yl)phenyl)acrylic acid, which is basically consistent with the data of Drupanin reported in the literature. Its structural formula is as follows: Figure 12 As shown.

[0082] Example 2: Preparation and Analysis of Drupanin Single Crystals

[0083] Drupanin was dissolved in a mixed solvent of acetonitrile and water (v:v, 55:45) to a concentration of 12 mg / mL; crystals were precipitated by slow evaporation of the solvent in a cool, ventilated place. Figure 13 ).

[0084] Diffraction data were collected using a single-crystal X-ray diffraction instrument (Bruker D8 VENTURE, Germany) equipped with a MicroMax-007 HF Cu rotating anode target and a Pilatus 300K surface detector, following the standard single-crystal X-ray diffraction analysis procedure.

[0085] The crystal structure was resolved using the ShelXT program and refined using the Olex2 software package based on F². The specific refinement work was performed using the SHELXL (2019 / 3 version) program via the least squares method.

[0086] Detailed information on crystallographic parameters, data collection, and structure refinement is summarized in Tables 1 and 2. Its three-dimensional structure is as follows: Figure 14 As shown.

[0087] Table 1. Single-crystal X-ray diffraction analysis data of Drupanin

[0088] Temperature K 293(2) Space group <![CDATA[P 21 / n]]> Cell parameters (Å) a 4.3764(1) b 14.2356(4) c 20.3173(6) α 90 β 93.0250(10) γ 90 <![CDATA[Cell volume (Å 3 )]]> 1264.02 Z, z' 4, 1 Data collection Radiation type Cu Kα <![CDATA[µ (mm -1 )]]> 0.690 Crystal size (mm) 0.168 × 0.178 × 0.184 mm Wavelength (Å) 1.54178 <![CDATA[(sinθ / λ) max (Oh -1 )]]> 0.638 θ range (º) 3.793‒79.513 θ_full, θ_max 0.999，0.995 Reflections collected / unique 33986 / 2737 <![CDATA[R int ]]> 0.0315 Refinement R_factor 0.0412 wR_factor (all) 0.1200 Goodness of fit 1.083 CCDC deposition number xxxxxx

[0089] Table 2. Atomic spacing and bond angle of Drupanin single crystals

[0090] O1–C10 1.306(2) O2–C10 1.235(2) O3–C11 1.361(1) C1–C2 1.504(2) C2–C3 1.317(2) C2–C14 1.496(2) C3–C4 1.500(2) C4–C5 1.510(2) C5–C6 1.384(1) C5–C11 1.397(1) C6–C7 1.398(1) C7–C8 1.456(1) C7–C13 1.396(2) C8–C9 1.327(2) C9–C10 1.454(2) C11–C12 1.388(2) C12–C13 1.376(2) Bond angles (º) C1–C2-C3 120.7(1) C1–C2-C14 114.8(1) C3–C2-C14 124.5(1) C2-C3-C4 128.1(1) C3–C4-C5 112.4(1) C4-C5–C6 121.90(9) C4-C5–C11 120.70(9) C6-C5–C11 117.37(9) C5-C6–C7 122.97(9) C6-C7–C8 119.12(9) C6-C7–C13 117.78(9) C8-C7–C13 123.1(1) C7-C8–C9 127.6(1) C8-C9–C10 123.1(1) O1-C10–O2 121.7(1) O1-C10–C9 116.0(1) O2-C10–C9 122.4(1) O3-C11–C5 116.79(9) O3-C11–C12 122.31(9) C5-C11–C12 120.90(9) C11-C12–C13 120.4(1) C7-C13–C12 120.5(1) Torsion angles (º) C1–C2–C3–C4 –179.3(1) C14–C2–C3–C4 –0.8(2) C2–C3–C4–C5 -108.2(1) C3–C4-C5-C6 166.2(2) C3–C4-C5-C11 –74.1(1) C4-C5-C6-C7 -177.3(1) C11-C5-C6-C7 0.8(1) C4-C5-C11-O3 –2.7(1) C4-C5-C11-C12 177.2(1) C6-C5-C11-O3 179.08(9) C6-C5-C11-C12 -0.9(1) C5-C6-C7-C8 178.4(1) C5-C6-C7-C13 -0.1(2) C6-C7-C8-C9 177.4(1) C13-C7-C8-C9 1.0(2) C6-C7-C13-C12 -0.6(2) C8-C7-C13-C12 179.0(1) C7-C8-C9-C10 177.9(1) C8-C9-C10-O1 -0.9(2) C8-C9-C10-O2 179.7(1) O3-C11-C12-C13 179.7(1) C5-C11-C12-C13 0.3(2) C11-C12-C13-C7 0.5(2)

[0091] Example 3: Evaluation of Drupanin's effect on improving neuroinflammation based on an LPS-stimulated BV2 cell oxidative stress and neuroinflammation model

[0092] Microglial BV2 cells were seeded in 96- or 12-well plates and cultured overnight at 37°C with 5% CO2. Working solutions of 5, 10, 20, and 40 μM Drupanin were prepared using DMEM / F12 medium, with 5 μM dexamethasone (DEX) as a positive control. The normal control group (Normal group) was cultured in DMEM / F12 medium (containing 0.04% DMSO); the model group (Model group) was treated with DMEM / F12 medium (containing 0.04% DMSO) for 1.5 h, followed by treatment with 100 ng / mL LPS; the Drupanin sample group and the positive control group were pre-treated with Drupanin and DEX working solutions of various concentrations for 1.5 h, followed by treatment with 100 ng / mL LPS and cultured for 24 h.

[0093] (1) Intracellular NO assay

[0094] The NO content in cells was measured according to the instructions of the nitric oxide assay kit. The results are as follows: Figure 15 As shown in Figure A, compared with the Normal group, the NO content in the Model group cells was significantly increased, while the NO content in the cells was significantly reduced after treatment with Drupanin (10, 20, 40 μM) and the positive control DEX, showing a dose-dependent trend.

[0095] (2) Cell viability assay

[0096] Cell viability was determined according to the CCK8 kit instructions. Results are as follows: Figure 15As shown in Figure B, 100 ng / mL of LPS, Drupanin (5, 10, 20, 40 μM) and 5 μM of DEX were not cytotoxic to BV2 cells, and Drupanin at concentrations of 10, 20, and 40 μM promoted the proliferation of BV2 cells.

[0097] (3) Expression of inflammation-related genes

[0098] Total RNA was extracted using RNAiso Easy (Takara). Reverse transcription was performed using PrimeScript™ FAST RT reagent Kit with gDNA Eraser (Takara). Changes in transcriptional levels of the target gene were examined using TB Green® Premix Ex Taq™ II FAST qPCR (Takara), with β-actin as an internal control. -ΔΔCT The relative expression level of the target gene can be calculated.

[0099] The corresponding PCR primer sequences are:

[0100] TNF-α: 5'-CTATGGCCCAGACCCTCACA-3' and 3'-TCTTGACGGCAGAGAGGAGG-5';

[0101] iL-6: 5'-TAGTCCTTCCTACCCAACTTCC-3' and 3'-TTGGTCCTTAGCCACTCCTTC-5';

[0102] iL-1β: 5′-TGGTGTGTGACGTTCCCATT-3′ and 3′-TCGTTGCTTGGTTCTCCTTG-5′;

[0103] iNOS: 5'-GGCAGCCTGTGAGACCTTTG-3' and 3'-GCATTGGAAGTGAAGCGTTTC-5'; COX-2: 5'-TTCAACACACTCTATCACTGGC-3' and 3'-AGAAGCGTTTGCGGTACTCAT-5'; CD86: 5'-GACCGTTGTGTGTGTTCTGG-3' and 3'-GATGAGCAGCATCACAAGGA-5';

[0104] β-actin: 5'-CCAGTTGGTAACAATGCCATGT-3' and 3'-GGCTGTATTCCCCTCCATCG-5'.

[0105] Based on qPCR results ( Figure 16 It was found that, compared with the Normal group, the relative expression levels of inflammatory factors such as TNF-α, IL-6, IL-1β, iNOS, and COX-2, as well as the mRNA of CD86 microglia M1 type markers, were significantly increased in the Model group, indicating that LPS treatment promoted the transformation of BV2 cells to the M1 pro-inflammatory phenotype. However, after treatment with Drupanin and the positive control DEX, the expression levels of the above-mentioned inflammation-related genes were significantly decreased, indicating that Drupanin has an inhibitory effect on the release of inflammatory factors induced by LPS in BV2 cells, thereby improving neuroinflammation.

[0106] (4) Intracellular ROS measurement

[0107] The 10 mM DCFH-DA (1:3000) fluorescent probe was diluted in DMEM / F12 medium, with 100 μL added to each well to replace the original cell culture medium. Cells were incubated for 30 min. Cells were then washed three times with DMEM / F12 medium. Images were acquired using an EVOS M5000 fluorescence imaging system (Thermo Scientific) and analyzed using ImageJ software. Results are as follows: Figure 17 As shown, compared with the Normal group, ROS accumulated significantly in the Model group cells, while the ROS level in the cells was significantly reduced after treatment with Drupanin and the positive control DEX, indicating that they can effectively improve LPS-induced oxidative stress in BV2 cells.

[0108] (5) Measurement of cell-associated lipids

[0109] Cells were stained using a Nile Red fluorescence detection kit, and images were acquired using an EVOS M7000 fluorescence imaging system (Thermo Scientific). The results were analyzed using ImageJ software. Figure 18 As shown, compared with the Normal group, lipid droplets accumulated significantly in the Model group cells, while treatment with 40 μM Drupanin significantly reduced lipid droplet aggregation in the cells, indicating that it can effectively improve LPS-induced lipid metabolism abnormalities in BV2 cells.

[0110] (6) Analysis of cellular NF-κB (p65) nuclear transport staining

[0111] BV2 cells were seeded in 24-well plates and cultured overnight at 37 °C with 5% CO2. A 40 μM working solution of Drupanin was prepared using DMEM / F12 medium. The normal control group (Normal group) was cultured with DMEM / F12 medium (containing 0.04% DMSO); the model group (Model group) was treated with DMEM / F12 medium (containing 0.04% DMSO) for 1.5 h and then treated with 100 ng / mL LPS; the sample group was pre-treated with the Drupanin working solution for 1.5 h and then treated with 100 ng / mL LPS. After 2 h of culture, the cells were fixed and stained using an NF-κB activation-nuclear translocation detection kit, and observed and photographed with an FV3000 laser confocal microscope (Olympus).

[0112] As Figure 19 shown, the red fluorescence in the nucleus of the Model group was significantly higher than that of the Normal group, while the red fluorescence in the nucleus of the Drupanin-treated group was significantly reduced, indicating that Drupanin can effectively inhibit the activation of NF-κB (p65) nuclear translocation in LPS-induced BV2 cells; thus inhibiting the inflammatory-related pathways.

[0113] Example 4: In vivo animal experiment research

[0114] 4.1 Experimental animals

[0115] This animal experiment was carried out in the Animal Experiment Center of Guangdong Pharmaceutical University. The license number for the experimental unit to use is: SYXK (Guangdong) 2017-0125. The animal experiment was approved and permitted by the Ethics Committee of the Experimental Animal Center of Guangdong Pharmaceutical University (gdpulac2022906). During the experiment, the "3R" principle was strictly followed while ensuring the scientificity and effectiveness of the experiment.

[0116] Twenty-five male SPF-grade C57BL / 6J mice, 8-10 weeks old, weighing 22-25 g, were purchased from the Guangdong Provincial Center for Medical Experimental Animals. The animal production license number is: SCXK (Guangdong) 2018-0002. All mice were housed in a clean-grade barrier environment in the Animal Center of Guangdong Pharmaceutical University. The environmental temperature was maintained at 22-26 °C, the humidity was 50-65%, and there was a 12 h light-dark cycle. All mice had free access to drinking water and food. The regular feed was provided by Beijing Keao Xieli Feed Co., Ltd.

[0117] After one week of acclimatization, 25 C57BL / 6J mice were randomly divided into three groups according to body weight: control group (Normal, n8), model group (Model, n9), and treatment group (Treated, n8). The Model and Treated groups were intraperitoneally injected with 500 µg / kg body weight of LPS (E. coli O55:B5) daily, while the Normal group was injected with an equal volume of physiological saline, for 7 consecutive days. Drupanin (50 mg / kg) was administered starting from day 3 and continued for 5 days. During this period, Morris water maze test, open field test, and tail suspension test were performed. At the end of the experiment, whole brain tissue of the mice was collected for histopathological analysis and qPCR analysis.

[0118] 4.2 Morris Water Maze Experiment The Morris water maze test was used to evaluate the effects of Drupanin on spatial learning and memory in mice. The experimental setup consisted of a circular pool (120 cm in diameter, 50 cm high) filled with drinking water (22 ± 1 ℃, with added titanium dioxide). A hidden platform (10 cm in diameter) was placed approximately 1 cm below the water surface. Spatial reference objects of different shapes were placed around the pool. The experiment consisted of three phases: adaptation training, orientation navigation test, and spatial exploration test. Adaptation training: Mice were allowed to swim freely in the platform-free pool for 2 minutes to adapt to the environment. Orientation navigation test: Training was conducted four times a day for four consecutive days at fixed times. Each time, mice were placed in the water facing the pool wall from different quadrants, and the time required to find the hidden platform within 90 seconds was recorded (escape latency). If the platform was not found within 90 seconds, the experimenter guided the mouse to the platform and held it for 15 seconds; this escape latency was recorded as 90 seconds. Each test was spaced approximately 20 minutes apart. Spatial exploration experiment: On day 5, the platform was removed, and mice were placed in water from the quadrant opposite to the platform. The number of times the mice crossed the original platform area, the time spent in the target quadrant, and the swimming path were recorded within 90 seconds. A shortened escape latency or an increased time spent in the target quadrant suggests that Drupanin has an effect on improving spatial learning and memory.

[0119] The effects of Drupanin on cognitive and memory impairment in LPS-induced neuroinflammation mice were investigated using a water maze test. Figure 20 shows the results: In the spatial exploration test, compared with the normal group, the LPS-induced model group mice had significantly reduced time spent in the target quadrant and the number of times they crossed the original platform location; compared with the model group, after Drupanin treatment, the treatment group mice showed a significant increasing trend in time spent in the target quadrant and the number of times they crossed the platform (p=0.083). These results demonstrate that LPS-induced neuroinflammation mice exhibit significant spatial memory impairment, and Drupanin can effectively improve the resulting spatial memory impairment.

[0120] 4.3 Open Field Experiment (OFT) The open field test is a classic method for assessing exploratory behavior, spontaneous activity, and emotional state in rodents. This experiment monitored the behavioral regulatory effects of Drupanin through spontaneous activity. Each mouse was placed individually in the center of a square open field (50×50×30 cm), which was divided into 25 equally sized squares by marked lines. The animals were observed for 5 minutes, and their free movement trajectory, the distance traveled within the central area, and the total distance traveled within the open field were recorded. After each animal's test, the experimental setup was cleaned with 75% ethanol to remove odor interference and ensure consistent testing conditions across all groups.

[0121] The motor activity, anxiety, and / or depression of LPS-induced neuroinflammation mice were evaluated using an open field assay. Figure 21 shows that, compared to the normal group, the LPS-induced model group mice exhibited significantly reduced total distance traveled, average speed, activity time percentage, and time spent in the middle zone. Compared to the model group, the treatment group mice showed significantly increased total distance traveled, average speed, activity time percentage, and time spent in the middle zone after Drupanin intervention. These results demonstrate that LPS-induced neuroinflammation mice exhibit decreased motor function and depressive behavior, and that Drupanin has a mitigating effect on these depressive changes.

[0122] 4.4 Suspended Tail Test (TST) The tail suspension test is a rapid behavioral test used to assess depressive-like behavior in rodents (primarily mice). The basic procedure involves securing the mouse's tail, leaving it suspended headfirst in the air, unable to escape. After initial struggle, the mouse gradually stops struggling and enters a state of "immobility"—this behavior is interpreted as "giving up" or "despair" at an unchangeable situation, and is an important indicator of depressive-like behavior.

[0123] The TST (Tail Suspension Test) was used to assess the antidepressant-like effects of Drupanin. Each mouse was secured to its tail approximately 1 cm from the tip with microporous tape and suspended in a test chamber at a height of approximately 50 cm above the ground to prevent contact with surrounding surfaces. Each mouse was tested for 6 minutes, recording both movement and resting time throughout the test. However, only the data from the last 4 minutes were used for scoring to exclude initial adaptation responses. A completely still, passively suspended state without struggling behavior was defined as immobile. A shorter resting time compared to the control group indicated antidepressant-like activity, consistent with the effective predictive criteria of the tail suspension test model.

[0124] The study investigated depressive-like despair behavior in mice treated with LPS-induced neuroinflammation using a tail suspension test. Figure 22 Experimental results showed that, compared with the normal group of mice, the resting time of the LPS-induced model group mice was significantly increased and the mobilization time was significantly decreased; compared with the model group, after Drupanin treatment, the resting time of the treated group mice was significantly decreased and the mobilization time was significantly increased. These results demonstrate that LPS-induced neuroinflammatory mice exhibit significant depressive-like hopelessness behavior, and that Drupanin can effectively alleviate LPS-induced depressive-like behavior in mice.

[0125] 4.5 qPCR analysis of inflammation-related genes in hippocampal tissue

[0126] Following the animal experimental protocol, after behavioral tests, mice were anesthetized with isoflurane, euthanized, and immediately their hippocampal tissue was dissected, flash-frozen in liquid nitrogen, and stored at -80 °C. Total RNA was extracted using RNAiso Easy (Takara). Reverse transcription was performed using the PrimeScript™ FAST RT reagent Kit with gDNA Eraser (Takara). Changes in the transcriptional level of the target gene were examined using TBGreen® Premix Ex Taq™ II FAST qPCR (Takara), with β-actin as an internal control. -ΔΔCT The relative expression level of the target gene was calculated using the method described in Example 3. The corresponding PCR primer sequences are the same as those in Example 3.

[0127] Based on qPCR results ( Figure 23 It was found that, compared with the Normal group, the relative expression levels of inflammatory factors such as TNF-α, IL-6, IL-1β, and iNOS, as well as CD86 microglia M1 marker mRNA, were significantly increased in the hippocampus of the Model group. This indicates that LPS treatment promoted the occurrence and development of neuroinflammation in the model group mice, and microglia in the hippocampus transformed into the M1 pro-inflammatory phenotype. However, after intervention with Drupanin, the expression levels of the above-mentioned inflammation-related genes decreased significantly, indicating that Drupanin has a good anti-neuroinflammatory effect, and its effect is related to the regulation of microglia.

Claims

1. A method for the isolation and purification of isopentenylated phenolic acid Drupanin, characterized in that, Includes the following steps: (1) Using green propolis as raw material, extracting with methanol or ethanol as solvent, filtering and concentrating the extract to obtain propolis alcohol extract; (2) The propolis alcohol extract was initially separated by dynamic axial column chromatography. Combined with the retention time, the fraction rich in Drupanin was collected and concentrated to obtain crude Drupanin product. The conditions for the dynamic axial column chromatography are as follows: reversed-phase silica gel C18 is used as the packing material; acetonitrile containing 10% methanol is used as mobile phase A and 0.1% formic acid aqueous solution is used as mobile phase B; gradient elution is performed with the following elution program: 0-10 min, 90%B→44%B; 17-21 min, 44%B→38%B; 21-32 min, 38%B→5%B; 32-35 min, 5%B→5%B; detection wavelength is 314 nm. (3) The crude Drupanin product was purified by preparative liquid chromatography. The elution peaks at specific time points were collected, concentrated, and dried to obtain the final product. The conditions for preparing the liquid chromatography were as follows: a C18 column was used; acetonitrile was used as mobile phase A and pure water containing 0.1% formic acid was used as mobile phase B; gradient elution was performed with the following elution program: 0–10 min, 45%–75% A; 10–20 min, 75%–90% A; 20–24 min, 90% A; 24–25 min, 95%–45% A; 25–30 min, 45% A; and the detection wavelength was 314 nm.

2. The separation and purification method according to claim 1, characterized in that, In step (1), the extraction method is heating reflux extraction or ultrasonic extraction; the material-liquid ratio is 1:5-20.

3. The separation and purification method according to claim 2, characterized in that, The temperature for the heating reflux extraction is 80-90 ℃, the number of extractions is 1-2 times, and the extraction time is 1-4 h; the frequency for the ultrasonic extraction is 20-40 KHz, the number of extractions is 1-2 times, and the extraction time is 30-120 min.

4. The separation and purification method according to claim 1, characterized in that, In step (3), the C18 chromatographic column has a specification of 250×21 mm and a packing particle size of 5~10 µm.

5. A Drupanin single crystal, characterized in that, The single crystal is monoclinic, space group: P 21 / n; cell size: a=4.3764(1), b=14.2356(4), c=20.3173(6); its interplanar angles α / °=90, β / °=93.0250(10), γ / °=90, and unit lattice volume V=1264.02Å. 3 The number of molecules in a unit lattice is Z = 4.

6. The method for preparing Drupanin single crystals according to claim 5, characterized in that, Includes the following steps: The Drupanin compound is dissolved in a mixed solvent of acetonitrile and water at a concentration of 5-15 mg / mL. The solution is placed in a cool, ventilated place, and crystals are precipitated as the solvent slowly evaporates. The volume ratio of acetonitrile to water in the mixed solvent is 60:40 to 50:

50.

7. The use of Drupanin, the Drupanin single crystal of claim 5, or a pharmaceutically acceptable salt thereof, in the preparation of remedies for improving neuroinflammation, enhancing cognitive function, and preventing or treating depression and anxiety.

8. The application according to claim 7, characterized in that, The application includes at least one of the following: (1) Improves nerve inflammation; (2) Reduce oxidative stress levels; (3) Inhibits M1 polarization of microglia; (4) Reduce the proportion of lipid droplet-associated glial cells; (5) Improves lipid metabolism disorders in glial cells; (6) Repairing cognitive impairment; (7) Relieve depression and anxiety-like symptoms.

9. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises an effective amount of Drupanin, the Drupanin single crystal of claim 5 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

Images