Haitesan type diterpenoid alkaloid as well as preparation method, application and pharmaceutical composition thereof

Abstract

The invention provides Haitesan type diterpenoid alkaloid as well as a preparation method, application and a pharmaceutical composition thereof. After rats are treated by the Haitesan diterpenoid alkaloid provided by the invention, the quality reduction trend is obviously relieved, and the foot swelling degree and the arthritis score are obviously reduced; bone loss induced by AIA can be effectively inhibited, and the structural integrity of bone trabecula is protected; cartilage matrix degradation can be inhibited, and cartilage erosion damage is relieved; the anti-inflammatory effect can be achieved by inhibiting the expression of key proinflammatory factors in bone tissues.

Description

Technical Field

[0001] This disclosure relates to the field of natural product chemistry, and in particular to hydantoin-type diterpenoid alkaloids, their preparation methods and applications, and pharmaceutical compositions. Background Technology

[0002] Rheumatoid arthritis (RA) is an autoimmune disease characterized by persistent chronic inflammation of the synovium, progressive cartilage erosion, and bone destruction. It progresses slowly and recurs frequently, severely impairing joint function and quality of life. The adjuvant-induced arthritis (AIA) mouse model closely resembles human RA in pathological manifestations such as synovial inflammation, bone loss, and joint dysfunction, making it a classic animal model for preclinical efficacy evaluation of anti-arthritis drugs. *Delphinium zhongdianense*, a traditional Chinese medicinal plant, is commonly used to treat inflammatory pain-related conditions; however, the core pharmacological activity and anti-arthritis molecular mechanism of its medicinal properties remain unclear. Summary of the Invention

[0003] The embodiments of this disclosure provide a hydantoin-type diterpenoid alkaloid, the structural formula of which is shown below: Where Ac is an acetyl group and Bz is a benzoyl group.

[0004] Another embodiment of this disclosure provides a pharmaceutical composition comprising the above-described hyterazoid diterpenoid alkaloid, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or a prodrug molecule thereof.

[0005] Another embodiment of this disclosure provides a method for preparing hydatigenous diterpenoid alkaloids. The method includes: weighing delphinium flowers, extracting them by heating and reflux with ethanol as solvent to obtain an extract, concentrating the extract to obtain a paste; separating the paste by gradient elution with methanol-water as eluent to obtain an eluent fraction corresponding to the highest methanol concentration; coarsely separating the eluent fraction by gradient elution with dichloromethane-methanol as eluent to obtain a first target fraction; gradient eluting the first target fraction by silica gel column chromatography to obtain a second target fraction; gradient eluting the second target fraction by a Sephadex LH-20 dextran gel column to obtain a third target fraction; and purifying the third target fraction by preparative high-performance liquid chromatography to obtain hydatigenous diterpenoid alkaloids.

[0006] In some embodiments, the extract is separated by gradient elution with methanol-water as the eluent to obtain the eluent corresponding to the highest methanol concentration, including: preliminary separation by an imino microsphere (MCI) column for chromatography, followed by gradient elution with water, 30 wt%, 60 wt%, and 90 wt% methanol as the eluent to obtain the eluent corresponding to the 90 wt% methanol concentration.

[0007] In some embodiments, the eluent fraction is coarsely separated by gradient elution using a dichloromethane-methanol eluent system to obtain the first target fraction, which includes: coarsely separating the eluent fraction by silica gel column chromatography, using a dichloromethane-methanol eluent system to perform gradient elution, with each gradient washing for 5-6 column volumes to obtain the first target fraction.

[0008] In some embodiments, the second target fraction is obtained by gradient elution of the first target fraction by silica gel column chromatography, which includes: using dichloromethane-methanol as the eluent system, gradient elution is performed by silica gel column chromatography, with each gradient washing for 5-6 column volumes to obtain the second target fraction.

[0009] In some embodiments, the second target fraction is eluted with a dextran gel LH-20 (Sephadex LH-20) column to obtain the third target fraction by gradient elution. This includes using a dichloromethane-methanol system with a volume ratio of 1:1 as the eluent, and eluting the second target fraction with a dextran gel LH-20 column to obtain the third target fraction.

[0010] In some embodiments, the eluent system used in preparative high-performance liquid chromatography is acetonitrile-water with a mass ratio of 25.5:74.5.

[0011] Another embodiment of this disclosure provides the use of the above-described hydantoin-type diterpenoid alkaloids, the above-described pharmaceutical compositions, or any of the above-described preparation methods of hydantoin-type diterpenoid alkaloids in the preparation of medicaments for anti-inflammatory or treatment of inflammation-related diseases.

[0012] In some embodiments, inflammation-related diseases include rheumatoid arthritis.

[0013] The compound DY4 disclosed herein significantly alleviated the decline in rat weight after treatment, and significantly reduced paw swelling and arthritis scores; it can effectively inhibit AIA-induced bone loss and protect the integrity of bone trabecular structure; it can inhibit cartilage matrix degradation and reduce cartilage erosion damage; and it can exert anti-inflammatory effects by inhibiting the expression of key pro-inflammatory factors in bone tissue. Attached Figure Description

[0014] Figure 1 The structural formula of compound DY4 is shown.

[0015] Figure 2 The high-resolution electrospray ionization mass spectrum (HR-ESI-MS) of compound DY4 is shown.

[0016] Figure 3 The ultraviolet (UV) spectrum of compound DY4 is shown.

[0017] Figure 4 The infrared (IR) spectrum of compound DY4 is shown.

[0018] Figure 5 The compound DY4 is shown. 1 H nuclear magnetic resonance (NMR) spectrum.

[0019] Figure 6 The compound DY4 is shown. 13 C NMR spectrum.

[0020] Figure 7 The distortion-free enhancement (DEPT-135) spectrum of polarization transfer at 135 degrees is shown for compound DY4.

[0021] Figure 8 The compound DY4 is shown. 1 H- 1 H-correlation (COSY) spectrum.

[0022] Figure 9 The heteronuclear single quantum coherence (HSQC) spectrum of compound DY4 is shown.

[0023] Figure 10 The heteronuclear multibond correlation (HMBC) spectrum of compound DY4 is shown.

[0024] Figure 11 The nuclear Overhauser effect (NOESY) spectrum of compound DY4 is shown.

[0025] Figure 12 The compound DY4 is shown. 1 H- 1 H COSY, HMBC, and NOESY are key correlations.

[0026] Figure 13 A schematic diagram of the X-single crystal ORTEP of compound DY4 is shown.

[0027] Figure 14 The study showed that compound DY4 reduced the morphology of the hind paws in different groups of AIA rats.

[0028] Figure 15 Representative Micro-CT images of rats with DY4 alleviating AIA are shown.

[0029] Figure 16The histopathology of compound DY4 in AIA rats is shown, specifically in H&E stained joint tissue sections (100x).

[0030] Figure 17 The histopathology of compound DY4 in AIA rats is shown, specifically in SF-stained joint tissue sections (100x).

[0031] Figure 18 The Western blot results show the effects of compound DY4 on the protein expression levels of INOS, COX2, TNF-α, and IL-1β in AIA rats. Detailed Implementation

[0032] To enable those skilled in the art to better understand the technical solutions of this disclosure, the technical solutions of this disclosure will be described in detail below with reference to the accompanying drawings.

[0033] Exemplary embodiments will be described more fully below with reference to the accompanying drawings; however, these exemplary embodiments may be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will enable those skilled in the art to fully understand the scope of this disclosure.

[0034] Where there is no conflict, the various embodiments of this disclosure and the features thereof in the embodiments may be combined with each other.

[0035] As used herein, the term “and / or” includes any and all combinations of one or more related enumerated entries.

[0036] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit this disclosure. As used herein, the singular forms “a” and “the” are also intended to include the plural forms unless the context clearly indicates otherwise. It will also be understood that when the terms “comprising” and / or “made of” are used in this specification, the presence of the stated feature, integral, step, operation, element, and / or component is specified, but the presence or addition of one or more other features, integrals, steps, operations, elements, components, and / or groups thereof is not excluded.

[0037] Unless otherwise specified, all terms used herein (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art. It will also be understood that terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant art and this disclosure, and will not be interpreted as having an idealized or overly formal meaning, unless expressly so defined herein.

[0038] This disclosure discloses the isolation of a novel compound, DY4, from *Delphinium zhongdianense*, and hypothesizes that it may exert its anti-arthritis effect by regulating inflammatory responses and bone metabolism balance. DY4 is a key inflammatory mediator in the pathological process of rheumatoid arthritis (RA), accelerating articular cartilage erosion and bone destruction by promoting synovial hyperplasia, inflammatory cell infiltration, and osteoclast activation. Based on this, this disclosure evaluates the anti-arthritis efficacy of the novel compound DY4 using an AIA rat model, elucidating the anti-arthritis mechanism of compound DY4.

[0039] Extraction and Separation (1) Collection of medicinal materials: The Zhongdian Delphinium was collected from Zhongdian area, Yunnan Province. It was identified by Professor Duan Lixin of Guangzhou University of Chinese Medicine as Zhongdian Delphinium (Delphinium yuanum Chen) of Ranunculaceae family and genus Delphinium. The specimen is stored at Guangzhou University of Chinese Medicine.

[0040] (2) Extraction: 25 kg of dried and pulverized Daphne odora flowers from Zhongdian were extracted three times by heating and refluxing (90°C) with 95 wt% ethanol as solvent (enough to submerge the Daphne odora flowers). The three extracts were combined and concentrated to obtain the extract, namely, the extract of Daphne odora flowers.

[0041] (3) Separation: After extraction, the extract was initially separated by chromatography using an imino microsphere (MCI) column (a high-performance gel permeation chromatography column with imino-modified styrene-divinylbenzene microspheres as packing material). Methanol-water was used as the eluent, and the extract was eluted sequentially with water, 30%, 60%, and 90% methanol to obtain three crude fractions, namely the 30% methanol fraction, the 60% methanol fraction, and the 90% methanol fraction (850g).

[0042] (4) The 90% methanol elution fraction was selected for further study. First, the 90% methanol fraction was crudely separated by silica gel column chromatography using dichloromethane-methanol (1:0) chromatography. A 0:1 eluent system was used, with each gradient wash consisting of 5-6 column volumes. Simultaneously, the collected fractions were identified using thin-layer chromatography (TLC), and the spots were analyzed. f Regions with similar values ​​were merged, resulting in 8 fractions (AH) (fraction A 43g, fraction B 57g, fraction C 75g, fraction D 25g, fraction E 57g, fraction F 43g, fraction G 60g, fraction F 40g). Fraction E was selected for further separation based on the TLC identification results.

[0043] Fraction E (57g) was separated by silica gel column chromatography (DCM / MeOH v / v 100:0 - 0:100) gradient elution, with 5-6 column volumes of washing per gradient. After TLC analysis, fractions with similar Rf values ​​were combined to obtain 6 fractions E1~E6 (E1 5g, E2 10g, E3 18g, E4 12g, E5 8g, E6 4g). Fraction E4 was separated using a Sephadex LH-20 column with DCM:MeOH = 1:1 as the eluent. After TLC analysis, fractions with similar Rf values ​​were combined to obtain 3 fractions E4-1~E4-3 (E4-1 1.5g, E4-2 8g, E4-3 2.5g). E4-2 was purified by preparative high-performance liquid chromatography under the conditions of acetonitrile:water = 25.5:74.5 to obtain compound DY4 (114 mg, t). R =24min).

[0044] LC / MS analysis Mass spectrometry analysis of the monomeric compounds was performed using an Agilent 6540 high-resolution mass spectrometer (HPLC-Q-TOF). The mobile phase consisted of water containing 0.1% formic acid (A) and acetonitrile (B), with a flow rate of 0.4 mL / min. A Waters ACQUITYUPLC HSS T3 column (1.8 μm, 2.1 mm × 100 mm) was used, with the column temperature controlled at 45 °C. Mass spectrometry acquisition was performed in positive ionization mode. Ion source parameters were: gas temperature 320 °C, dry gas flow rate 8 L / min, nebulizer 35 psig, sheath gas temperature 350 °C, sheath gas flow rate 11 L / min, and voltage 3.5 kV.

[0045] Analysis of physical constants of compounds Specific rotation was measured using a Rudolph Autopol VI (Rudolph Research Analytical, Hackettstown, NJ, USA) polarimeter; infrared spectroscopy data were processed using a Thermo Scientific Nicolet iS50 FTIR with ATR attachment; 1D and 2D NMR were measured using a Bruker Avance III 400MHz (Bruker BioSpin AG, Fällanden, Switzerland) nuclear magnetic resonance spectrometer.

[0046] Electronic circular dichroism (ECD) calculation Before performing ECD calculations, the structure with the determined relative configuration was first subjected to energy minimization under the MMFF94 force field using a software tool. Then, a conformational search was performed, and after the search, information on all conformations was reviewed. Conformations with a Boltzmann distribution greater than 5% were selected from all possible conformations. The selected conformations were then imported into another software tool for conformational optimization at the B3LYP / 6-31G(d) level. Next, frequency analysis was performed on the optimized conformations at the B3LYP / 6-31G(d) level to exclude conformations with imaginary frequencies calculated from the structure, ensuring that each conformation was stable. Finally, another software tool was used to perform energy calculations using density functional theory (TDDFT), setting the B3LYP / 6-31+G(d,p) parameter level, selecting a conductor-like polarized continuum model (CPCM) as the solvent, and methanol as the solvent. After the calculation is completed, another software tool is used to extract the spectrum and energy of the calculation results, based on the Boltzmann distribution theory and its relative Gibbs free energy (…). G) Average the spectrum of the simulated conformation to obtain the theoretically calculated ECD data, and export the data; finally, visualize the exported calculated values ​​and the measured CD spectrum, analyze and determine the final absolute configuration.

[0047] Statistical analysis All experimental data were analyzed using statistical software. Quantitative data were expressed as mean ± standard deviation (x ± s). One-way ANOVA was used for comparisons among multiple groups, and LSD-t test was used for pairwise comparisons between groups. P < 0.05 was considered statistically significant.

[0048] Structural analysis of compound DY4 Figure 1 The structural formula of compound DY4 is shown; Figure 2 HR-ESI-MS of compound DY4 is shown; Figure 3 The UV spectrum of compound DY4 is shown; Figure 4 The IR spectrum of compound DY4 is shown; Figure 5 The compound DY4 is shown. 1 H NMR spectrum; Figure 6 The compound DY4 is shown. 13 C NMR spectrum; Figure 7 The DEPT-135 spectrum of compound DY4 is shown; Figure 8 The compound DY4 is shown. 1 H- 1 H COSY spectrum; Figure 9 The HSQC spectrum of compound DY4 is shown; Figure 10The HMBC spectrum of compound DY4 is shown; Figure 11 The NOESY spectrum of compound DY4 is shown; Figure 12 The compound DY4 is shown. 1 H- 1 H COSY, HMBC, and NOESY are key correlations; Figure 13 A schematic diagram of the X-single crystal ORTEP of compound DY4 is shown.

[0049] Compound DY4 is a white amorphous powder that crystallizes into colorless crystals after incubation in a methanol-water system; it shows a positive result in modified bismuth potassium iodide staining, suggesting it may be an alkaloid component; optical rotation value... -4.0 (c 0.13, MeOH); HR-ESI-MS showed a quasi-molecular ion peak at m / z 508.2330 [M+H]. + (C) 29 H 34 NO7 + (508.2330), its molecular formula is inferred to be C 29 H 33 NO7, its degree of unsaturation is calculated as =12. The UV spectrum shows a maximum absorption at 230 nm, and the IR spectrum shows absorption at 1730, 1453, 1274, 1077, 952, and 724 cm⁻¹. -1 The presence of an absorption peak suggests that the molecule may contain structures such as conjugated benzene rings or ester groups.

[0050] Compound DY4 1 H-NMR spectrum ( Figure 5 Three sets of aromatic proton signals can be observed in the low-field region. δ H The integrals (8.03, 7.60, 7.47) show a total of 5 protons, indicating the presence of a single-substituted benzene ring in the molecule; two sets of characteristic methyl signals can be observed in the high-field region. δ H 1.11,2.02,both3H,s) indicates the presence of two methyl groups in the molecule.

[0051] 13 C-NMR spectrum ( Figure 6 There is an acetyl carbonyl carbon signal in the low field region. δ C 170.3), a benzoyl carbonyl carbon signal ( δ C 165.8); Six benzene ring carbon signals are present in the aromatic region ( δ C 133.0, 129.9, 129.2×2, 128.2×2) and a pair of double-bonded carbon signals ( δC 164.8, 106.4). The above information indicates that compound DY4 may be a C20 type diterpenoid alkaloid. Comparing the NMR spectrum of compound DY4 with that of compound DY1 reveals that in the NMR spectrum of compound DY4, vakogavines-type C... 20 The characteristic aldehyde signal at position 19 in the diterpene alkaloid skeleton disappears, according to δ H 2.02 and δ C The HMBC-related signal of 170.3 is inferred. δ H 2.02 is an acetylated methyl group signal rather than an N-CH3 signal, meaning that compound DY4 lacks the characteristic 19-position aldehyde signal and N-methyl signal of the vakogavines type. It is speculated that compound DY4 may be a hetisines-type C-type compound. 20 Diterpenoid alkaloids.

[0052] The above speculation can be made through 1 H- 1 H-COSY spectrum and HMBC spectrum ( Figure 8 , Figure 10 Further confirmation, 1 H- 1 The HCOSY spectrum reveals four spin-coupled systems, including, in addition to the benzene ring spin-coupled system: δ H 3.25 / 2.07(H2-1) δ H 5.49(H-2) δ H 6.16(H-3), δ H 1.95 (H-5) δ H 3.58 (H-6) δ H 1.73(H2-7), δ H 1.99 (H-9) δ H 4.24(H-11) δ H 2.35 (H-12) δ H 4.11(H-13) δ H 2.21(H-14) δ H 4.00 (H-20). In the HMBC spectrum, H-2 and quaternary carbon C-4 ( δ C47.7), C-10 ( δ C 50.1) is related to H2-1 and C-10, C-5 ( δ C 62.6) is related, H-3 is related to C-4 and C-5, indicating the presence of a six-membered ring A in the molecule; H-6 ( δ H 3.58) and quaternary carbon C-8 ( δ C 43.9), related to C-10, H-5 related to C-10, C-9 ( δ C 54.6) is related, H-7 is related to C-8 and C-9, indicating the existence of a six-membered ring B that couples with ring A through C-5-C-10; H-11 is related to C-8 and C-16 ( δ C Related to 146.8), H-9 is related to C-8 and C-15 ( δ C 33.5) is related, H-12 is related to C-16 and C-15, indicating the existence of a six-membered ring C and B coupled through C-8-C-9; according to the key H-19 ( δ H 5.27) and C-4 ( δ C 447.7), C-3 ( δ C 775.3), C-5 ( δ C 662.6), C-6 ( δ C 660.5) and C-20 ( δ C Based on the HMBC correlation signal of 64.2), it was inferred that C-6, C-20, and C-19 are linked by N atoms, thus confirming that the skeleton of compound DY4 is a hetisines-type C. 20 Diterpenoid alkaloids. Based on H-2 and... δ C 170.3 (-OAc) related, H-3 and δ C Based on the correlation with 165.8(-OBz), it is speculated that the acetyl and benzoyl groups are attached at the C-2 and C-3 positions, respectively; finally, based on the chemical shift value at C-11 ( δ H 4.24, δ C 75.0)C-13( δ H 4.11, δ C 71.3), C-19 ( δ H 5.27, δ C Based on the molecular formula (88.3), it is deduced that there is a hydroxyl substitution at positions C-11, C-13, and C-19. Therefore, the planar structure of compound DY4 was determined. The NMR data of compound DY4 are shown in Table 5.

[0053] The relative configuration of compound DY4 was determined by NOESY spectra ( Figure 11 Based on the NOESY correlations between H-2, H-3, and H-5, it is determined that H-2 and H-3 are β-configurations, meaning 2-OAc and 3-OBz are α-configurations. Based on the NOESY correlations between H-9 and H-15β, and between H-15β and H-11, it is inferred that H-11 is a β-configuration, meaning 11-OH is an α-configuration. Based on the NOESY correlations between H-14 and H-15α, it is determined that H-14 is an α-configuration. The NOESY correlations between H-20 and H-19 indicate that H-19β and 19-OH are α-configurations. Therefore, the relative configuration of compound DY4 is determined to be... , , , , , , , , , , , , , .

[0054] Single crystals of compound DY4 were successfully cultured using a methanol-water system, and their absolute configuration was determined by single-crystal diffraction analysis [CuKα radiation type (λ=1.54184), Frank parameter = -0.03(4)]. In summary, compound DY4 is identified as a novel hetisines-type C20 diterpenoid alkaloid.

[0055] Compound DY4: colorless crystals; -4.0 (c 0.1, MeOH); UV(MeOH) λ max 230 nm; IR (KBr)ν max 1730, 1453, 1274, 1231, 1124, 1077, 952, 724 cm –1 HR-ESI-MS (positive) m / z508.2333 [M+ H] + (C 29 H 33 NO7 +:508.2330).

[0056] Table 1 shows the crystal data and structural refinement of compound DY4.

[0057] Table 1 Table 2 shows the NMR data of compound DY4 ( δ The unit for J is ppm, and the unit for J is Hz.

[0058] Table 2 Animal model establishment and grouping Twenty-four 6-8 week old SPF-grade SD rats were randomly divided into four groups (n=6 per group): normal control group, AIA model group, DY4 treatment group, and methotrexate (MTX) positive drug group. Arthritis models were established by intradermal injection of 0.1 mL of incomplete Freund's adjuvant into the right hind paw of rats in the AIA model group and DY4 treatment group; the normal control group received an equal volume of physiological saline. After successful modeling (7 days post-modeling, arthritis score ≥2), the DY4 treatment group received 20 mg / kg daily via gavage, while the normal control group and AIA model group received an equal volume of physiological saline, for 21 consecutive days.

[0059] General indicator testing Rats' body weight changes were recorded every 3 days. Swelling of the right hind paw was measured using calipers (expressed as plantar thickness). The degree of joint inflammation was assessed according to the arthritis scoring criteria (0-4 points): 0 points = no inflammation; 1 point = mild erythema or swelling; 2 points = swelling from the plantar to the ankle; 3 points = swelling of the entire hind limb; 4 points = severe joint deformity or dysfunction.

[0060] like Figure 14 As shown, compared with the normal control group, the body weight of rats in the AIA model group continued to decrease during the intervention period (P<0.05), and the paw swelling and arthritis scores were significantly increased (P<0.01); while the trend of body weight decline in the DY treatment group was significantly alleviated, and after 21 days of intervention, the body weight was significantly higher than that in the AIA model group (P<0.05), and the paw swelling and arthritis scores were significantly reduced (P<0.01), and were close to the level of the normal control group (P>0.05).

[0061] Micro-CT Detection and Analysis After the experiment, rats were euthanized by cervical dislocation, and the right hind leg ankle joint was separated and fixed in 4% paraformaldehyde for 24 hours. Scanning was performed using a Micro-CT system (scanning parameters: voltage 70kV, current 114μA, pixel size 18μm). Three-dimensional images of bone tissue were reconstructed using the accompanying analysis software. The region of interest (ROI) was selected as the proximal tibia cancellous bone region, and bone structural parameters such as bone mineral density (BMD), trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular separation (Tb.Sp) were analyzed.

[0062] like Figure 15 Micro-CT results showed that in the normal control group, the trabeculae of the rats were neatly arranged, dense, and structurally intact. In the AIA model group, the trabeculae were sparse and significantly fractured, with significantly decreased BMD, Tb.Th, and Tb.N (P<0.01) and significantly increased Tb.Sp (P<0.01), indicating severe bone tissue damage. The DY treatment group showed significant improvement in the disordered and fractured trabecular structure, with significantly increased BMD, Tb.Th, and Tb.N (P<0.05) and significantly decreased Tb.Sp (P<0.05) compared to the AIA model group, indicating that compound DY4 can effectively inhibit AIA-induced bone loss and protect the integrity of the trabecular structure.

[0063] Histopathological staining Ankle joint tissue was fixed in 4% paraformaldehyde, decalcified with 10% EDTA, dehydrated with graded ethanol, and embedded in paraffin to prepare serial sections with a thickness of 5 μm. Hematoxylin-eosin (HE) staining was used to observe synovial tissue proliferation and the degree of inflammatory cell infiltration; Safranin O-Fix Green (SO) staining was used to assess the preservation of proteoglycans in the cartilage matrix and the degree of cartilage erosion. Pathological sections were observed under an optical microscope, and images were acquired. Appropriate software tools were used to quantitatively analyze the synovial tissue thickness and the percentage of positively stained cartilage area.

[0064] like Figure 16 Hematoxylin-eosin (H&E) staining results showed that the synovial tissue in the normal control group was thin and smooth, with no inflammatory cell infiltration; the synovial tissue in the AIA model group showed significant hyperplasia and thickening, narrowing of the synovial cavity, and infiltration of a large number of inflammatory cells (neutrophils and lymphocytes). The degree of synovial hyperplasia in the DY treatment group was significantly reduced, the synovial thickness was significantly lower than that in the AIA model group, and the number of infiltrating inflammatory cells was significantly reduced. Figure 17Safranin and Fast Green staining (S&F) results showed that the cartilage matrix in the normal control group was dark red and uniformly stained; the cartilage surface in the AIA model group was eroded and damaged, and the dark red stained area was significantly reduced (P<0.01), indicating a large loss of proteoglycan; the cartilage surface in the DY treatment group was more intact, and the proportion of positive cartilage staining area was significantly higher than that in the AIA model group (P<0.05), indicating that compound DY4 can inhibit cartilage matrix degradation and reduce cartilage erosion damage.

[0065] Western Blot (WB) detection Tibial bone tissue from the right hind limb of rats was isolated and lysed with RIPA lysis buffer (Servicebio) containing a protease inhibitor (phenylmethylsulfonyl fluoride, PMSF) and a phosphatase inhibitor (Phosphatase Inhibitor Cocktail II). The tissue was homogenized on ice and centrifuged at 12,000 rpm for 30 minutes at 4°C to extract total protein. Protein concentration was determined using the BCA method. 50 μg of total protein was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane. Block the membrane with 5% skim milk at room temperature for 2 hours, then add primary antibody (iNOS, COX2, TNF-α, IL-1β, and internal control GAPDH (Proteintech, diluted 1:1000) and incubate overnight at 4°C. Wash the membrane three times with TBST (20 mM Tris, 150 mM Sodium Chloride, 0.1% Tween-20, pH approximately 7.4), then add HRP-labeled secondary antibody (diluted 1:5000) and incubate at room temperature for 1 hour. Develop the membrane using an ECL chemiluminescence kit, and quantitatively analyze the gray values ​​of the target protein bands. The relative protein expression level is expressed as the ratio of the gray values ​​of the target protein to those of GAPDH.

[0066] like Figure 18 Western blot results showed that, compared with the normal control group, the relative expression levels of iNOS, COX2, TNF-α, and IL-1β proteins in the bone tissue of rats in the AIA model group were significantly increased (P<0.01); after treatment with DY, the expression levels of the above four pro-inflammatory proteins were significantly downregulated (P<0.05), among which the expression levels of iNOS and TNF-α were close to those of the normal control group, indicating that compound DY4 can exert anti-inflammatory effects by inhibiting the expression of key pro-inflammatory factors in bone tissue.

[0067] Example embodiments have been disclosed herein, and while specific terminology has been used, it is for illustrative purposes only and should be construed as such, and is not intended to be limiting. In some instances, it will be apparent to those skilled in the art that features, characteristics, and / or elements described in connection with particular embodiments may be used alone, or in combination with features, characteristics, and / or elements described in connection with other embodiments, unless otherwise expressly indicated. Therefore, those skilled in the art will understand that various changes in form and detail may be made without departing from the scope of this disclosure as set forth by the appended claims.

Claims

1. A hydatigen-type diterpenoid alkaloid, characterized in that, The structural formula of the hydatigenous diterpenoid alkaloid is shown below: Where Ac is an acetyl group and Bz is a benzoyl group.

2. A pharmaceutical composition, characterized in that, Includes the hydantoin-type diterpenoid alkaloids as described in claim 1, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or a prodrug molecule thereof.

3. A method for preparing hydatigen-type diterpenoid alkaloids, characterized in that, The preparation method includes: Weigh out the larkspur flowers, extract them by heating and reflux with ethanol as solvent to obtain the extract, and concentrate it to obtain the extract paste; The extract was separated by gradient elution using methanol-water as the eluent to obtain the eluent fraction corresponding to the highest methanol concentration. The eluted fraction was coarsely separated by gradient elution using a dichloromethane-methanol eluent system to obtain the first target fraction. The first target fraction was subjected to gradient elution by silica gel column chromatography to obtain the second target fraction; The second target fraction was subjected to gradient elution using a dextran gel LH-20 (Sephadex LH-20) column to obtain the third target fraction; The third target fraction was purified by preparative high-performance liquid chromatography to obtain the hydatisen-type diterpenoid alkaloid.

4. The preparation method according to claim 3, characterized in that, The extract was separated by gradient elution using methanol-water as the eluent to obtain the eluent corresponding to the highest methanol concentration. The eluent was initially separated by an imino microsphere (MCI) column, and then eluted sequentially with water, 30 wt%, 60 wt%, and 90 wt% methanol using methanol-water as the eluent to obtain the eluent corresponding to the 90 wt% methanol concentration.

5. The preparation method according to claim 3, characterized in that, The process of coarsely separating the eluted components and performing gradient elution using a dichloromethane-methanol eluent system to obtain the first target fraction includes: coarsely separating the eluted components by silica gel column chromatography, performing gradient elution using a dichloromethane-methanol eluent system, and washing for 5-6 column volumes for each gradient to obtain the first target fraction.

6. The preparation method according to claim 3, characterized in that, The second target fraction was obtained by gradient elution of the first target fraction using silica gel column chromatography, which involved using a dichloromethane-methanol eluent system and performing gradient elution with each gradient elution consisting of 5-6 column volumes.

7. The preparation method according to claim 3, characterized in that, The second target fraction was eluted using a Sephadex LH-20 dextran gel column to obtain the third target fraction. This process involved using a 1:1 volume ratio of dichloromethane to methanol as the eluent, and eluting the second target fraction using a Sephadex LH-20 dextran gel column to obtain the third target fraction.

8. The preparation method according to claim 3, characterized in that, The eluent system used in preparative high performance liquid chromatography is acetonitrile-water with a mass ratio of 25.5:74.

5.

9. The use of the hydantoin-type diterpenoid alkaloid according to claim 1, the pharmaceutical composition according to claim 2, or the hydantoin-type diterpenoid alkaloid obtained by any one of claims 3 to 8 in the preparation of a medicament for anti-inflammatory or treatment of inflammation-related diseases.

10. The application according to claim 9, characterized in that, The inflammation-related diseases mentioned include rheumatoid arthritis.

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