Rehmannia pigment compound as well as preparation method and application thereof in medicines

Abstract

The invention provides a rehmannia pigment compound as shown in a formula (I), a stereoisomer thereof or pharmaceutically acceptable salt thereof, and particularly relates to the technical field of natural pharmaceutical chemistry and pharmaceutical compounds. The invention aims to solve the problems that the radix rehmanniae pigment is not clear in material basis and lacks a compound with a clear structure and excellent anti-inflammatory activity. The rehmannia pigment compound disclosed by the invention comprises a structure as shown in a formula (I). The invention also discloses a preparation method of the compound and application of the compound in medicines. The compound has a novel chemical structure and remarkable anti-inflammatory activity, release of nitric oxide can be effectively inhibited in a lipopolysaccharide induced macrophage model, the cytotoxicity is far lower than that of a positive control drug quercetin, and the compound shows huge potential as an anti-inflammatory drug lead compound.

Description

Technical Field

[0001] This application relates to the fields of natural product chemistry and pharmaceutical compounds, and in particular to a rehmannia flavonoid compound, its preparation method, and its application in pharmaceuticals. Background Technology

[0002] Rehmannia glutinosa is a traditional Chinese medicine, and its fresh or processed rhizomes are widely used clinically. The fresh rhizomes of Rehmannia glutinosa have a distinctive yellow-orange appearance, suggesting the presence of specific pigment components. While existing literature mentions the presence of chemical components such as "rehmanniain" in Rehmannia glutinosa and describes its traditional medicinal effects such as nourishing yin and clearing heat, cooling blood and stopping bleeding, the precise chemical structures of the specific pigment components that constitute its color, and whether these components possess clear and specific pharmacological activities, especially anti-inflammatory activities, have long remained unclear. Therefore, there is a general lack of systematic research and development in the field of pigment compounds in Rehmannia glutinosa with clear chemical structures and significant biological activities, which limits the in-depth understanding of the medicinal material basis of Rehmannia glutinosa and the development of novel drugs. Summary of the Invention

[0003] The purpose of this application is to address the problems in the prior art regarding the unclear material basis of rehmannia glutinosa, the lack of compounds with well-defined structures and excellent anti-inflammatory activity. This application provides a rehmannia glutinosa compound, its stereoisomers, or pharmaceutically acceptable salts thereof. This application provides, for the first time, a novel class of rehmannia glutinosa compounds with a completely new general formula, which has not been reported in the prior art and belongs to a completely new type of compound, providing a novel chemical entity and structural basis for the development of novel functional natural products.

[0004] To achieve the above objectives, this application provides a rehmannia flavonoid compound of formula (I), its stereoisomer, or a pharmaceutically acceptable salt thereof.

[0005]

[0006] In formula (I), X is selected from formula (X1) or formula (X2), and the double bond configurations of 13-C and 17-C are independently of formula E or formula Z;

[0007]

[0008] In formulas (I), (X1), and (X2): R1, R7, and R8 are independently oxygen or sulfur; R2, R5, and R6 are independently oxygen, sulfur, or imino; and R3 and R4 are independently hydroxyl, mercapto, or amino.

[0009] In a preferred embodiment of this application, the compound is a compound having the structure shown in formula (i), formula (ii), formula (iii) or formula (iv);

[0010]

[0011]

[0012]

[0013] In formula (i), the 13-C double bond is of formula E, the 17-C double bond is of formula E, and X is selected from formula (X2);

[0014] In formula (ii), the 13-C double bond is of formula E, the 17-C double bond is of formula E, and X is selected from formula (X1);

[0015] In formula (iii), the 13-C double bond is of type Z, the 17-C double bond is of type E, and X is selected from formula (X1);

[0016] In formula (iv), the 13-C double bond is of formula E, the 17-C double bond is of formula Z, and X is selected from formula (X1).

[0017] Furthermore, the compound is a compound having the structure shown in formula (ia), formula (ii-b), formula (iii-c) or formula (iv-d);

[0018]

[0019]

[0020]

[0021] Optionally, X is selected from formula (X1).

[0022] Optionally, the 13-C double bond is of type E, and the 17-C double bond is of type E.

[0023] Optionally, X is selected from formula (X1), R1 is oxygen, R2 is oxygen, sulfur or imino, R5 is oxygen, and R6 is oxygen or imino.

[0024] Optionally, X is selected from formula (X2), R1 is sulfur, R2 is oxygen or imino, R7 is oxygen, and R8 is sulfur.

[0025] This application also provides a method for preparing the rehmannia flavonoid compound, its stereoisomer, or a pharmaceutically acceptable salt thereof as described in any of the preceding claims, comprising the following steps:

[0026] The crude rehmannia pigment was subjected to normal phase chromatography to obtain a preliminary purified fraction containing the target component.

[0027] The preliminarily purified fraction was subjected to reverse-phase chromatography to obtain a further purified subfraction;

[0028] Size exclusion chromatography was performed on the sub-components to obtain secondary components;

[0029] The secondary components were separated by semi-preparative high-performance liquid chromatography and / or preparative high-performance liquid chromatography to obtain the rehmannia glutinosa pigment compound.

[0030] This application also provides a pharmaceutical composition comprising a therapeutically effective amount of any of the preceding compounds, their stereoisomers, or pharmaceutically acceptable salts thereof, and one or more pharmaceutically acceptable carriers or excipients.

[0031] This application also provides the use of any of the foregoing compounds, their stereoisomers, or pharmaceutically acceptable salts thereof, or the foregoing pharmaceutical compositions in the preparation of medicaments for the prevention and / or treatment of inflammatory diseases.

[0032] Compared with the prior art, this application has the following beneficial effects:

[0033] This application is the first to isolate and identify a series of rehmannia pigment compounds with novel skeletons as shown in general formula (I) from Rehmannia glutinosa. Their structures were confirmed by modern spectroscopic techniques such as nuclear magnetic resonance and mass spectrometry, filling a gap in the field of natural products and carotenoid derivatives of this type of structure, and providing a novel chemical entity for the study of the material basis of Rehmannia glutinosa.

[0034] The rehmannia glutinosa derivatives provided in this application possess novel chemical structures and have been demonstrated to exhibit highly effective and low-toxicity anti-inflammatory activity. In vitro cell experiments showed that these compounds, particularly derivatives with bridged oxatricyclic structures, effectively inhibited nitric oxide release in a lipopolysaccharide-induced macrophage inflammation model, achieving an inhibition rate of up to 66.8% at a concentration of 100 μM. This effect is comparable to the positive control drug quercetin, but its cytotoxicity is significantly lower. At a concentration of 100 μM, the cell growth inhibition rate is less than 20%, while quercetin's is as high as 61.9%. This "highly effective and low-toxicity" characteristic demonstrates its great potential as a lead compound for anti-inflammatory drugs.

[0035] This application is the first to isolate and confirm the precise structure of this type of compound from Rehmannia glutinosa, elucidating part of the material basis for the characteristic yellow-orange color of Rehmannia glutinosa, which has important scientific value for the modernization research of traditional Chinese medicine. Furthermore, as it is a natural pigment, it also has potential application value as a natural and safe food coloring or cosmetic colorant. Attached Figure Description

[0036] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0037] Figure 1 Formula (I) is the rehmannia pigment compound provided in the embodiments of this application.

[0038] Figure 2 The structural formula (i) of the rehmannia pigment compound provided in the embodiments of this application is shown.

[0039] Figure 3 The structural formula (ii) of the rehmannia pigment compound provided in the embodiments of this application.

[0040] Figure 4 The structural formula (iii) of the thymol compound provided in the embodiments of this application is shown.

[0041] Figure 5 The structural formula (iv) of the rehmannia pigment compound provided in the embodiments of this application.

[0042] Figure 6 The structural formula of the thymol compound A provided in the embodiments of this application is shown below.

[0043] Figure 7 The structural formula of the thymol compound B provided in the embodiments of this application is shown.

[0044] Figure 8 The structural formula of the thymol compound C provided in the embodiments of this application is shown below.

[0045] Figure 9 The structural formula of the rehmannia pigment compound D provided in the embodiments of this application is shown below.

[0046] Figure 10 This is a flowchart illustrating a method for preparing a rehmannia pigment compound, as provided in an embodiment of this application.

[0047] Figure 11 The image shows the HR-ESI-MS spectrum of compound A in methanol in the embodiments of this application.

[0048] Figure 12 The 1H NMR spectrum (500 MHz) of compound A in deuterated acetone in the embodiments of this application.

[0049] Figure 13 The image shows the HR-ESI-MS spectrum of compound B in methanol in the embodiments of this application.

[0050] Figure 14 The image shows the HR-ESI-MS spectrum of compound C in methanol in the embodiments of this application.

[0051] Figure 15 The image shows the HR-ESI-MS spectrum of compound D in methanol in the embodiments of this application. Detailed Implementation

[0052] To better understand the technical solutions of this application, the technical solutions in the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are only used to explain this application and are not intended to limit this application.

[0053] It should be noted that the rehmannia pigment compounds mentioned in this application are not mixtures, but conjugated polyene carotenoid compounds having the structure described in this application.

[0054] In this application, those skilled in the art should understand that the numbers labeled in each molecular formula represent the relative positions of the carbon atoms in that formula, and have no substantial impact on the carbon atoms, molecular structure, or the corresponding compound. They are simply used to facilitate understanding or description of the relative positions of carbon atoms in the molecular structure. Figure 2 The 7, 9, 13, 1', and 2' markings in the structure shown represent the 7th, 9th, 13th, 1'th, and 2'th carbon atoms in the molecule, respectively. Figure 2 The carbon atoms, molecular structures, and corresponding compounds shown have no substantial impact.

[0055] It should be understood that those skilled in the art should be aware that the numbers in the methyl group (i.e., CH3-) in the molecular structural formula only represent the carbon atom in the methyl group being connected to three hydrogen atoms, and should be distinguished from the relative position numbering of the carbon atoms mentioned above.

[0056] Rehmannia glutinosa, especially its fresh form, has a distinctive yellow-orange appearance, a characteristic potentially linked to its traditional efficacy of "clearing heat and cooling blood." However, the specific pigment chemical components that constitute this characteristic color have long been unclear in identification and characterization, hindering in-depth research into the relationship between its material basis and efficacy, and restricting the extensive development and utilization of Rehmannia glutinosa resources.

[0057] Based on this, in a preferred embodiment of this application, a rehmannia flavonoid compound of formula (I), its stereoisomer, or a pharmaceutically acceptable salt thereof is provided.

[0058]

[0059] In formula (I), X is selected from formula (X1) or formula (X2), and the double bond configurations of 13-C and 17-C are independently of formula E or formula Z;

[0060]

[0061] In formulas (I), (X1), and (X2): R1, R7, and R8 are independently oxygen or sulfur; R2, R5, and R6 are independently oxygen, sulfur, or imino; and R3 and R4 are independently hydroxyl, mercapto, or amino.

[0062] This embodiment provides novel and well-defined conjugated polyene carotenoid compounds (rehmannia pigment compounds). The chemical structures of these compounds have been fully confirmed by modern spectroscopic methods, filling the gap in the prior art for this type of compound.

[0063] The compound in this embodiment can be obtained from natural Rehmannia glutinosa and has a bright orange-yellow color, making it a natural pigment source. Simultaneously, it possesses the aforementioned low toxicity and anti-inflammatory activities, making it a potential "functional-coloring" integrated additive that can impart a natural color to products while providing beneficial biological functions such as anti-inflammation.

[0064] In a preferred embodiment of this application, the compound is a compound having the structure shown in formula (i), formula (ii), formula (iii) or formula (iv);

[0065]

[0066]

[0067]

[0068] In formula (i), the 13-C double bond is of formula E, the 17-C double bond is of formula E, and X is selected from formula (X2);

[0069] In formula (ii), the 13-C double bond is of formula E, the 17-C double bond is of formula E, and X is selected from formula (X1);

[0070] In formula (iii), the 13-C double bond is of type Z, the 17-C double bond is of type E, and X is selected from formula (X1);

[0071] In formula (iv), the 13-C double bond is of formula E, the 17-C double bond is of formula Z, and X is selected from formula (X1).

[0072] In this embodiment, four compounds with the specific structures shown in formulas (i), (ii), (iii), and (iv) were isolated and clearly identified from Rehmannia glutinosa for the first time. The structures of these compounds were fully confirmed by high-resolution mass spectrometry and one-dimensional and two-dimensional nuclear magnetic resonance spectroscopy, which solves the current technical problem of unclear characteristic pigment components of Rehmannia glutinosa and fills the gap in natural products of this structural type.

[0073] In another preferred embodiment, the compound is a compound having the structure shown in formula (ia), formula (ii-b), formula (iii-c) or formula (iv-d);

[0074]

[0075]

[0076]

[0077] The specific compounds provided in this embodiment, especially those with the structures shown in formula (ia), (ii-b), (iii-c), or (iv-d), have been experimentally verified to have the following comprehensive advantages:

[0078] Anti-inflammatory activity: In an LPS-induced RAW264.7 cell inflammation model, the compound shown in this example exhibited significant NO release inhibition activity (inhibition rate up to 60.0%), demonstrating a clear anti-inflammatory mechanism.

[0079] Safety: CCK-8 cytotoxicity assays showed that at concentrations exhibiting significant anti-inflammatory activity (e.g., 100 μM), these compounds had extremely low growth inhibition rates on RAW264.7 cells (e.g., 9.6%), confirming that they are essentially non-cytotoxic at effective concentrations and have a high safety window.

[0080] Color and solubility: These compounds are bright orange-yellow and readily soluble in organic solvents such as ethyl acetate and dichloromethane, and soluble in methanol and acetonitrile, giving them the dual potential to serve as both natural colorants and active ingredients.

[0081] For example, X is selected from formula (X1).

[0082] In a preferred embodiment, the 13-C double bond is of the E form, and the 17-C double bond is of the E form. This conformation offers advantages such as better chemical stability, specific spectral properties, and potential structure-activity relationships.

[0083] For example, X is selected from formula (X1), R1 is oxygen, R2 is oxygen, sulfur or imino, R5 is oxygen, and R6 is oxygen or imino.

[0084] In one embodiment, X is selected from formula (X2), R1 is sulfur, R2 is oxygen or imino, R7 is oxygen, and R8 is sulfur.

[0085] In a preferred embodiment, when R1 is oxygen, R7 is oxygen or sulfur, R8 is oxygen or sulfur, R2 is oxygen, sulfur or imino, R4 is hydroxyl, mercapto or amino, R3 is hydroxyl, mercapto or amino, R5 is oxygen, sulfur or imino, and R6 is oxygen, sulfur or imino.

[0086] For example, when R1 can be sulfur, R7 can be oxygen or sulfur, R8 can be oxygen or sulfur, R2 can be oxygen, sulfur or imino, R4 can be hydroxyl, mercapto or amino, R3 can be hydroxyl, mercapto or amino, R5 can be oxygen, sulfur or imino, and R6 can be oxygen, sulfur or imino.

[0087] In a preferred embodiment, a pharmaceutical composition is also provided, comprising a therapeutically effective amount of any of the preceding compounds, their stereoisomers, or pharmaceutically acceptable salts thereof, and one or more pharmaceutically acceptable carriers or excipients.

[0088] In this embodiment, the active compound can be converted into a directly usable drug dosage form (such as tablets, capsules, injections, etc.).

[0089] In another preferred embodiment, the use of any of the foregoing compounds, their stereoisomers, or pharmaceutically acceptable salts thereof, or the foregoing pharmaceutical compositions, in the preparation of a medicament for the prevention and / or treatment of inflammatory diseases is also provided.

[0090] The rehmannia glutinosa derivatives provided in the examples possess novel chemical structures and have been demonstrated to exhibit highly effective and low-toxicity anti-inflammatory activity. In vitro cell experiments showed that these compounds, particularly derivatives with bridged oxatricyclic structures, effectively inhibited nitric oxide release in a lipopolysaccharide-induced macrophage inflammation model, achieving an inhibition rate of up to 66.8% at a concentration of 100 μM. This effect is comparable to the positive control drug quercetin, but its cytotoxicity is significantly lower. At a concentration of 100 μM, the cell growth inhibition rate is less than 20%, while quercetin's is as high as 61.9%. This "highly effective and low-toxicity" characteristic demonstrates its great potential as a lead compound for anti-inflammatory drugs.

[0091] In one embodiment, such as Figure 10 As shown, a method for preparing any of the above-described rehmannia flavonoid compounds, their stereoisomers, or pharmaceutically acceptable salts thereof:

[0092] S1: The crude rehmannia pigment was subjected to normal phase chromatography to obtain a preliminary purified fraction containing the target component;

[0093] S2: Perform reverse phase chromatography on the preliminarily purified component to obtain a further purified subcomponent;

[0094] S3: Perform size exclusion chromatography on the subcomponent to obtain the secondary component;

[0095] S4: Separate the secondary components by semi-preparative high-performance liquid chromatography and / or preparative high-performance liquid chromatography to obtain the rehmannia glutinosa compound.

[0096] The crude rehmannia glutinosa pigment can be an unpurified extract obtained using currently known methods. Alternatively, it can be an unpurified extract obtained using optimized methods. Purification here can be understood as any one or more chromatographic treatments.

[0097] Of course, the crude rehmannia pigment can be an extract obtained by currently known methods, after preliminary extraction and vacuum concentration.

[0098] An exemplary crude rehmannia glutinosa extract may be an unpurified initial extract obtained from the fresh or dried rhizomes of Rehmannia glutinosa Libosch. after preliminary extraction and vacuum concentration. The preliminary extraction may be performed using ethanol or other organic solvents of appropriate concentration, followed by vacuum concentration to obtain the unpurified extract. For example, the ethanol concentration may be 70-80%, and the vacuum concentration may be performed by concentrating to 40-70% of the original volume, followed by filtration through a 0.1-1 μm microporous membrane.

[0099] For example, it can also be understood that crude rehmannia pigment is a mixture mainly composed of medium polar to non-polar pigment components, such as non-cyclohexene ethers and non-phenylethanol glycosides.

[0100] In this application, the rehmannia glutinosa compounds are not a mixture, but rather different conjugated polyene compounds extracted from crude rehmannia glutinosa. For example, they may be any of the rehmannia glutinosa compounds AG mentioned in this application.

[0101] Normal-phase chromatography of crude rehmannia pigment can remove impurities with significantly different polarities from the target yellow pigment compound. These impurities include chlorophyll degradation products, plant waxes, long-chain alkanes, triglycerides, some terpenes, residual polysaccharides, inorganic salts, amino acids, organic acids, glycosylated pigments, or oligomers.

[0102] The preliminary purified components of the target component will contain the target yellow pigment compound, its homologues and direct analogues, as well as lipid-soluble impurities.

[0103] For example, if normal phase chromatography is used for fractional elution, preliminary purified fractions containing different target components can be collected separately. For example: preliminary purified fractions with relatively small molecular weights or more hydroxyl groups; preliminary purified fractions with larger molecular weights, longer alkyl chains, or fewer hydroxyl groups.

[0104] For example, after step S1 and before S2, the process further includes analyzing the pre-purified fractions containing different target components using analytical liquid chromatography, thereby selecting one or more of the pre-purified fractions containing the target components to facilitate subsequent separation.

[0105] The reversed-phase chromatography of the preliminarily purified components can remove residual water-soluble impurities (polysaccharides, proteins), homologue impurities, trace amounts of lipid-soluble residues, etc.

[0106] The subcomponent may contain the target yellow pigment compound, and may also contain structurally similar analogs or isomers.

[0107] Size exclusion chromatography can remove some small molecule residues and salts, as well as impurities that are similar in hydrophobicity to the target analyte but have different molecular sizes / configurations. Examples include: organic salts, solvent residues (such as methanol and water), monosaccharides, amino acids and small molecule organic acids, homologues of the target pigment (dimers or oligomers), and some structurally highly similar isomers.

[0108] The secondary components may contain the target yellow pigment compound, and may also contain enantiomers or diastereomers of the target yellow pigment compound, as well as small molecule impurities, solvent residues, etc. At this point, the target yellow pigment compound is usually already of high purity, reaching 80-90%.

[0109] The secondary components are separated by semi-preparative high-performance liquid chromatography (HPLC) and / or preparative HPLC, which typically removes diastereomers or positional isomers of the target compound, structural analogs with retention behaviors extremely similar to the target compound, and trace amounts of polymers or dimers that may be present. This step yields a rehmannia succinate compound with extremely high purity, reaching or exceeding 99%.

[0110] Therefore, the method of this embodiment can isolate and confirm a series of novel rehmannia glutinosa compounds (rehmannia glutinosa compound AG) from Rehmannia glutinosa, filling a technological gap in this field. This application's purification of crude rehmannia glutinosa using multi-step chromatography effectively addresses the challenges of similar structures, similar polarities, and difficult separation among rehmannia glutinosa compounds, demonstrating stable processes and good reproducibility. The rehmannia glutinosa compounds obtained by this invention provide a high-quality material basis and key technical support for the in-depth development of rehmannia glutinosa and its precise application in the food, pharmaceutical, and cosmetic fields, particularly laying the foundation for its anti-inflammatory, antioxidant, and other pharmacological activities.

[0111] For example, the stationary phase of the normal phase chromatography can be alumina, polyamide, or silica-bonded phase, preferably a normal phase silica column. The mobile phase of the normal phase chromatography can be dichloromethane-ethanol, ethyl acetate-methanol, or n-hexane-methanol, typically a dichloromethane-methanol system.

[0112] For example, the stationary phase of the reversed-phase chromatography can be a C8 (octylsilane) column, a phenyl column, a cyano column (-CN), or a pentafluorophenyl column, preferably a reversed-phase ODS column; the mobile phase of the reversed-phase layer can be water-acetonitrile, a water-methanol / acetonitrile mixture, water-methanol / acetonitrile with added modifier, or water-tetrahydrofuran, preferably a water-methanol system as the mobile phase.

[0113] In another embodiment, the normal phase chromatography is normal phase silica gel column chromatography, and the normal phase silica gel column chromatography uses a dichloromethane-methanol system as the mobile phase; the reverse phase chromatography is reverse phase ODS column chromatography, and the reverse phase ODS column chromatography uses a water-methanol system as the mobile phase.

[0114] ODS stands for Octadecyl-bonded Silica.

[0115] The dichloromethane-methanol system refers to a mixture of dichloromethane and methanol in a specific ratio; the water-methanol system refers to a mixture of water and methanol in a specific ratio. In other embodiments, the use of "-" as a connector also refers to a mixture of two or more compounds.

[0116] Among them, normal phase silica gel column chromatography and dichloromethane-methanol gradient system are based on the polarity difference of compounds. Therefore, polar target compounds (such as iridoid glycosides) can be specifically separated through the polarity matching mechanism. This can efficiently remove lipid-soluble impurities in crude rehmannia glutinosa, achieving a purity of >90% and a recovery rate of >85%.

[0117] Among them, the reversed-phase ODS column, being nonpolar, forms a reverse partitioning mechanism with the water-methanol system, which can accurately separate nonpolar / moderately polar compounds.

[0118] For example, silica gel column chromatography can be performed using a dry loading method, wherein the crude rehmannia pigment is mixed with 60-100 mesh silica gel at a mass ratio of 1:0.5-1:2, and the stationary phase of the silica gel column can be 100-200 mesh silica gel.

[0119] In this embodiment, after normal-phase silica gel column chromatography and dichloromethane-methanol gradient system chromatography, a preliminary purified component containing the target component is obtained. The dichloromethane-methanol is easy to evaporate completely and can be completely dissolved in the starting mobile phase (water-methanol) of the subsequent reversed-phase column, avoiding solvent compatibility issues and ensuring sample loading uniformity and chromatographic peak shape.

[0120] For example, the stationary phase of the size exclusion chromatography can be dextran gel LH-60, polymethacrylate gel, or polystyrene sulfonic acid ion exchange resin, etc.; preferably, it can be a dextran gel LH-20 column. The mobile phase of the size exclusion chromatography can be water-ethanol (a mixture of water and ethanol), water-acetone, chloroform-methanol, tetrahydrofuran-water, or dichloromethane-methanol-water mixed solvent, etc., preferably methanol; another preferred option is a dichloromethane-methanol mixed solvent (a mixture of dichloromethane and methanol).

[0121] In another embodiment, the size exclusion chromatography is performed on a dextran gel LH-20 column, using methanol or a dichloromethane-methanol mixed solvent as the mobile phase.

[0122] In this embodiment, size exclusion chromatography, specifically a dextran gel LH-20 column and a methanol / dichloromethane-methanol mobile phase, is used. Based on molecular size, it can remove small molecule impurities (such as polysaccharides and organic acids). Therefore, it can effectively remove silica gel microparticles, filler degradation products, or solid impurities.

[0123] In this embodiment, the subcomponent can be purified into a simpler and purer secondary component, reducing the difficulty of subsequent separation and significantly improving the yield and purity of the final product.

[0124] A dextran gel LH-20 column, also known as a Sephadex LH-20, is a liquid chromatography column filled with dextran gel LH-20. It refers to a dextran-based gel chromatography medium with a large pore size (approximately 200–300 Å) and a hydrophobic surface.

[0125] For example, the secondary components can be separated using semi-preparative high-performance liquid chromatography (HPLC). The stationary phase can be a C8 (octyl) column, a C4 (butyl) column, a phenyl column, a pentafluorophenyl column, or a cyano column, etc.; a reversed-phase column (usually C18) is preferred; the mobile phase can be a water-methanol system, a water-ethanol system, a water-tetrahydrofuran system, or an acetonitrile-methanol-water ternary system; preferably an acetonitrile-water system (a mixed solvent composed of acetonitrile and water).

[0126] In a preferred embodiment, the semi-preparative high-performance liquid chromatography (HPLC) and / or preparative HPLC separation includes: separating the secondary components using semi-preparative HPLC, wherein the semi-preparative HPLC uses a reversed-phase column and an acetonitrile-water mobile phase; wherein the volume percentage concentration of acetonitrile in the aqueous solution in the acetonitrile-water mobile phase is 57%-74%; and the semi-preparative HPLC uses isocratic or gradient elution.

[0127] For example, the volume percentage concentration of acetonitrile in the aqueous solution is 57%, 58%, 60%, 68%, or 74%, etc.

[0128] In this embodiment, a reversed-phase column (typically C18) and an acetonitrile-water system can separate moderately to weakly polar natural products. Compared to methanol, acetonitrile has lower viscosity, stronger elution power, and better UV transmittance, enabling baseline separation of structurally very similar pigment homologues or isomers under high-concentration aqueous phase conditions (57%-74% acetonitrile corresponds to 26%-43% water).

[0129] Exemplarily, the semi-preparative high-performance liquid chromatography (HPLC) and / or preparative HPLC separation can employ chiral supercritical fluid chromatography (SCLC) or chiral capillary electrophoresis; preferably, chiral column semi-preparative HPLC is used. The mobile phase of chiral column semi-preparative HPLC can be a heptane-ethanol mixture, a cyclohexane-methanol mixture, or a heptane-tetrahydrofuran mixture; preferably, a hexane-isopropanol mixture.

[0130] As a preferred embodiment of this application, the semi-preparative high-performance liquid chromatography (HPLC) and / or preparative HPLC separation includes: separating the secondary components using chiral column semi-preparative HPLC, wherein the mobile phase of the chiral column semi-preparative HPLC is a hexane-isopropanol mixed solvent, and gradient elution is used.

[0131] For example, the gradient elution procedure is as follows: the volume fraction of isopropanol in n-hexane is increased from 10% to 13%, held for 10-30 minutes, and then increased from 13% to 30%. Preferably, it can be held for 15-25 minutes.

[0132] In this embodiment, the stationary phase of the chiral column has chiral recognition sites that can separate enantiomers or diastereomers; and in the hexane-isopropanol mixed solvent, hexane provides a low polarity background, while isopropanol, as a polarity modifier, can work synergistically with the stationary phase.

[0133] For example, before performing normal-phase chromatography on the crude rehmannia glutinosa, the method further includes: mixing the crude rehmannia glutinosa with 60-100 mesh silica gel to obtain the mixed crude rehmannia glutinosa; and the stationary phase of the normal-phase silica gel column is 100-200 mesh silica gel; the use of a dichloromethane-methanol system as the mobile phase is to use a dichloromethane-methanol mixed solvent to perform gradient elution on the mixed crude rehmannia glutinosa, wherein the gradient elution of the dichloromethane-methanol mixed solvent starts from pure dichloromethane and ends with pure methanol.

[0134] Using coarser silica gel (60-100 mesh) mixed with the crude sample allows for the formation of a uniformly dispersed solid mixture before loading. 100-200 mesh silica gel used for column packing provides a larger specific surface area and a higher theoretical plate number.

[0135] The gradient begins with pure dichloromethane, which initially elutes lipid-soluble impurities from the crude pigment, achieving early impurity removal. The proportion of methanol is gradually increased until pure methanol is reached, enabling systematic elution of compounds across the entire polarity range. The pre-purified fraction containing the target component is selectively eluted and collected.

[0136] Furthermore, the reversed-phase ODS column chromatography employs a water-methanol system for gradient elution of the initially purified components, wherein the gradient elution volume ratio of the water-methanol system varies from 9:1 to pure methanol.

[0137] In this embodiment, a high aqueous phase ratio (water:methanol = 9:1) is started to ensure that all components are strongly adsorbed at the column head and strongly polar impurities are preferentially eluted. Increasing the methanol ratio to pure methanol allows for selective elution based on subtle differences in hydrophobicity between target molecules.

[0138] For example, the gradient elution volume ratio of the water-methanol system varies from 8:1 to pure methanol, from 8.5:1 to pure methanol, etc.

[0139] In another preferred embodiment, before performing normal-phase chromatography on the crude rehmannia glutinosa pigment, the method further includes: pretreating the rehmannia glutinosa raw material to obtain the phloem; extracting the phloem using a first organic solvent to obtain an extract; concentrating the extract to obtain an extract; dispersing the extract in a polar solvent and performing liquid-liquid extraction using a second organic solvent that is immiscible with the polar solvent to separate the second organic solvent phase containing rehmannia glutinosa pigment, and removing the solvent to obtain the crude rehmannia glutinosa pigment.

[0140] In this embodiment, crude rehmannia pigment was obtained, the main components of which are moderately polar to non-polar pigments. This greatly reduces the separation burden on the subsequent normal-phase silica gel column, avoiding rapid column head contamination and severe peak overlap.

[0141] For example, the first organic solvent is ethyl acetate, the polar solvent is water, and the second organic solvent is dichloromethane.

[0142] In this embodiment, an efficient and directional polarity transfer pathway is formed from raw materials to crude products, ultimately obtaining a medium-polarity target pigment concentrate.

[0143] Among them, ethyl acetate with moderate polarity was selected as the first solvent, which can efficiently dissolve the target components while minimizing the dissolution of large amounts of highly polar impurities and non-polar lipids, thus achieving selective enrichment for the first time.

[0144] The following detailed description is provided in conjunction with specific examples:

[0145] Example 1

[0146] This embodiment discloses a method for extracting, separating, and purifying the rehmannia glutinosa pigment derivatives disclosed in this application from the root of Rehmannia glutinosa. This method can efficiently obtain a variety of rehmannia glutinosa pigment compounds with novel structures and purity that meets the requirements for structural identification and activity screening.

[0147] This method mainly includes the following steps: root pretreatment step S101, organic solvent extraction step S102, normal phase silica gel column chromatography step S103, reversed phase chromatography separation step S104, chiral chromatography separation step S105, and obtaining the target compound step S106.

[0148] The specific preparation process is as follows:

[0149] S101: Pretreatment of Rehmannia glutinosa tubers. Take about 60 kg of commercially available fresh Rehmannia glutinosa tubers, wash them to remove surface dirt and sand, remove the outer skin and xylem, and carefully peel and collect the yellow-orange phloem. Then, cut the collected phloem into small pieces of about 0.5 cm to 2 cm in size to facilitate subsequent solvent immersion and extraction.

[0150] S102: Organic solvent extraction. The small pieces of Rehmannia glutinosa phloem processed in the previous steps were placed in several large-capacity glass containers, and sufficient ethyl acetate was added to completely submerge the material. Soaking and extraction were carried out at room temperature (approximately 20-25°C) for 24 hours each time. This soaking and extraction process was repeated three times to ensure sufficient dissolution of the pigment components. After each extraction, the supernatant was collected, and the ethyl acetate extracts from all three extractions were combined. The combined solution was concentrated under reduced pressure using a rotary evaporator at a water bath temperature below 40°C until the solvent was completely evaporated, finally yielding a dark, viscous total Rehmannia glutinosa pigment extract, weighing 30.9 grams.

[0151] S103: Normal-phase silica gel column chromatography. Take 30.9 g of the total extract above, add about 45 g of 60-100 mesh silica gel powder, and wet with a small amount of dichloromethane. Stir evenly in a fume hood and allow the solvent to evaporate to obtain a dry-loaded sample adsorbed with the total extract. Take another large glass chromatography column (e.g., 10 cm inner diameter, 100 cm length) and wet-pack about 450 g of 100-200 mesh silica gel as the stationary phase. Spread the dry-loaded sample evenly on the top of the chromatography column. Then, perform gradient elution using a dichloromethane-methanol solvent system. The volume ratios of the elution gradient are as follows: first wash with pure dichloromethane, then elute sequentially with mixed solvents of dichloromethane:methanol = 200:1, 100:1, 80:1, 60:1, 40:1, 30:1, 20:1, 10:1, 5:1, and finally wash with pure methanol. During the elution process, an automated fraction collector was used to collect the eluent, and thin-layer chromatography (TLC) was employed to monitor and analyze the collected fractions in real time. Based on the distribution and color characteristics of the TLC spots, similar components were combined, resulting in a total of 18 subfractions, labeled Fr-1 to Fr-18. Analysis showed that the target compound of this application was mainly enriched in subfractions Fr-8 and Fr-9, which are orange-yellow to orange-red in color.

[0152] S104: Reversed-phase chromatography separation and subsequent fine purification. This step aims to obtain a single compound of high purity, which can be further subdivided into the preparation of different target compounds.

[0153] Example 2: Preparation of Rehmannia glutinosa pigment compound A (hereinafter referred to as compound A, the structure of which is shown in the attached figure). Figure 6 (As shown)

[0154] The subfraction Fr-8 obtained in Example 1, weighing 6.29 g, was dissolved in a small amount of methanol and loaded onto a reversed-phase silica column packed with octadecylsilane-bonded silica gel. Gradient elution was performed using a methanol-water solvent system, with the volume percentage of methanol gradually increasing from 40% to 100%. Thin-layer chromatography analysis of the combined fractions yielded the target enriched fraction Fr-8-15 (weighing 1.3308 g). This Fr-8-15 fraction was dissolved and loaded onto a dextran gel column for separation using size exclusion principles. Isocratic elution was performed using a dichloromethane / methanol (volume ratio 1:1) mixture as the mobile phase to further remove impurities, yielding fraction Fr-8-15-3, weighing 89.8 mg. Finally, fraction Fr-8-15-3 was purified using a semi-preparative high-performance liquid chromatography system. The chromatographic conditions were as follows: an ODS-A reversed-phase column (e.g., YMC-Pack ODS-A, 250 x 10 mm, 5 μm), an isocratic elution of acetonitrile-water (58:42 v / v), a flow rate of 2 mL / min, and a detection wavelength of 399 nm. The eluent corresponding to the main peak was collected, and the solvent was removed by vacuum evaporation to obtain a pure, orange-red powder of compound A, with a purity exceeding 95% as determined by analytical high-performance liquid chromatography (HPLC).

[0155] Example 3: Preparation of Rehmannia glutinosa pigment compound B (hereinafter referred to as compound B, the structure of which is shown in the attached figure). Figure 7 (As shown)

[0156] The subfraction Fr-8 obtained in Example 1, with a total weight of 6.29 g, was dissolved in a small amount of methanol and loaded onto a reversed-phase silica column packed with octadecylsilane-bonded silica gel. Gradient elution was performed using a methanol-water solvent system, with the volume percentage of methanol gradually increasing from 40% to 100%. Combined with thin-layer chromatography analysis, the target enriched fraction Fr-8-14, with a total weight of 2.3363 g, was obtained. This fraction was loaded onto a dextran gel column and eluted using pure methanol as the mobile phase. The target fractions were collected and combined to obtain fraction Fr-8-14-8, weighing 59.4 mg. This fraction was purified using a semi-preparative high-performance liquid chromatography (HPLC) system. The chromatographic conditions were: ODS-A reversed-phase column, isocratic elution with acetonitrile-water (57:43 v / v), flow rate of 2 mL / min, and detection wavelength of 399 nm. The main peak component was collected and evaporated under reduced pressure to obtain pure compound B in the form of an orange-yellow powder with a purity higher than 95%.

[0157] Example 4: Preparation of rehmannia pigment compounds C and D (hereinafter referred to as compound C and compound D, respectively, their structures are shown in the attached figures). Figure 8 , Figure 9 (As shown)

[0158] The subfraction Fr-8 obtained in Example 1, weighing 6.29 g, was dissolved in a small amount of methanol and loaded onto a reversed-phase silica column packed with octadecylsilane-bonded silica gel. Gradient elution was performed using a methanol-water solvent system, with the volume percentage of methanol gradually increasing from 40% to 100%. Combined with thin-layer chromatography analysis, another fraction, Fr-8-15-5, weighing 78.0 mg, was obtained. Analysis identified this fraction as a mixture of a pair of geometric isomers. To separate these isomers, preparative chiral high-performance liquid chromatography (HPLC) was used. The chromatographic conditions were: a CHIRALPAK IB N-5 chiral column (250 x 10 mm, 5 μm), a hexane-isopropanol gradient elution mobile phase at a flow rate of 1 mL / min, and a detection wavelength of 399 nm. Fractions of the two main chromatographic peaks were collected based on their elution times. The two collected fractions were concentrated under reduced pressure to obtain pure compounds C and D in the form of orange-yellow powders, both with a purity of over 95%.

[0159] Example 5

[0160] This embodiment confirms the chemical structure of compound A (named Rehmannia compound A) obtained in Example 2.

[0161] Specifically, nuclear magnetic resonance (1D and 2D NMR) confirmed the structure of compound A with the following data parameters:

[0162] Red crystal; -32.0 (MeOH, c 0.05); IR (neat): 3273, 3033, 2923, 1714, 1531, 1077, 983 cm -1 ; UV (MeOH): λ max (log ε) 247 (4.10), 276(4.39), 304 (3.89) nm, 399 (4.71) nm; HRMS (ESI) m / z: [M+H] + Calcd forC 32 H 46 NO4 508.3427; Found 508.3435. 1H NMR (Acetone-d6, 500 MHz): δ 7.32 (d,1H, J = 15.0 Hz, H-8), 7.25 (br s, 1H, -NH-), 7.06 (d, 1H, J = 10.5 Hz, H-21), 6.84 (1H, H-16) a , 6.83 (1H, H-15) a , 6.80 (dd, 1H, J = 11.5, 15.0 Hz, H-11), 6.74 (d, 1H, J = 15.0 Hz, H-7), 6.68 (dd, 1H, J = 10.5, 15.0 Hz, H-20),6.65 (d, 1H, J = 11.5 Hz, H-10), 6.64 (d, 1H, J = 15.0 Hz, H-12), 6.61 (d,1H, J = 15.0 Hz, H-19), 6.47 (d, 1H, J = 11.5 Hz, H-14), 6.46 (d, 1H, J =11.5 Hz, H-17), 4.01 (br s, 1H, 2′-OH), 3.61 (m, 2H, H-2′), 3.39 (m, 2H, H-1′), 2.42 (m, 2H, H-4), 2.05 (s, 3H, H-26), 2.02 (s, 3H, H-27), 2.02 (s, 3H,H-28), 2.01 (s, 3H, H-29), 2.01 (s, 3H, H-30), 1.56 (m, 2H, H-2), 1.41 (m,2H, H-3), 1.12 (s, 3H, H-24), 1.12 (s, 3H, H-25) ( a Overlapped signals wasreported without designating multiplicity); 13C NMR (Acetone-d6, 150 MHz): δ207.8, 203.3, 169.7, 147.2, 142.6, 142.4, 140.8, 137.7, 137.5, 135.7, 135.3,135.3, 134.3, 132.2, 132.2, 131.4, 125.8, 125.0, 120.6, 62.0, 47.0, 44.0,43.5, 39.9, 30.0, 24.6, 24.6, 19.4, 13.2, 12.9, 12.8, 12.8.

[0163] 1 H-NMR (500MHz,Acetone-d6) δ 1.56 (m,H-2),1.41 (m,H-3),2.42 (m,H-4),6.74 (d,J=15.0 HZ,H-7),7.32 (d,J=15.0 HZ,H-8),6.65 (m,H-10),6.80 (m,H-11),6.64 (m,H-12),6.47 (d,J=10.5 HZ,H-14),6.83 (m,H-15),6.84 (m,H-16),6.46 (d,J=10.5 HZ,H-17),6.61 (d,J=12.5 HZ,H-19),6.68 (m,H-20),7.06 (d,J=10.5 HZ,H-21),1.12 (s,CH3-24),1.12 (s,CH3-25),2.05 (s,CH3-26),2.02 (s,CH3-27),2.02 (s,CH3-28),2.01 (s,CH3-29),2.01 (s,CH3-30),3.39 (m,H-1′),3.61 (m,H-2′),4.01 (br s,2′-OH),7.25 (br s,-NH-)。

[0164] 13C-NMR (125MHz, Acetone-d6) δ 46.9 (C-1), 39.8 (C-2), 19.9 (C-3), 44.1 (C-4), 207.9 (C-5), 203.4 (C-6), 120.6 (C-7), 147.3 (C-8), 135.3 (C-9), 140.8 (C-10), 125.8 (C-11), 142.8 (C-12), 137.8 (C-13), 135.7 (C-14), 132.2 (C-15), 132.2 (C-16), 135.3 (C-17), 137.5 (C-18), 142.6 (C-19), 125.1 (C-20), 134.3 (C-21), 131.4 (C-22), 169.6 (C-23), 24.7 (C-24), 24.7 (C-25), 30.0 (C-26), 12.9 (C-27), 12.8 (C-28), 12.8 (C-29), 13.2 (C-30), 43.5 (C-1′), 62.4 (C-2′).

[0165] Those skilled in the art should be fully able to understand the above spectral data, which is cited here to demonstrate the actual experimental process and results.

[0166] Compound A is an orange-red powder. To determine its molecular formula, high-resolution electrospray ionization mass spectrometry analysis was performed, such as... Figure 11 The image shows the high-resolution electrospray ionization mass spectrum of compound A. In positive ion mode, the spectrum exhibits a clear quasi-molecular ion peak at a mass-to-charge ratio (m / z) of 508.3435. The experimentally determined value is consistent with that based on the chemical formula. Calculated The theoretical value (508.3427) is highly consistent with the value, and the error is within the allowable range. Therefore, the molecular formula of compound A is determined to be... .

[0167] To further elucidate its detailed chemical structure, a series of nuclear magnetic resonance (NMR) spectroscopy analyses, including proton and carbon spectra, were performed on compound A.

[0168] like Figure 12The spectrum shown is the proton NMR spectrum of compound A measured on a 500 MHz NMR spectrometer with deuterated acetone as the solvent. Chemical shifts (δ) in the spectrum are in ppm. Characteristic signals include: δ 7.32 (1H, d, J=15.0 Hz, H-8), a typical trans double bond proton signal; δ 7.25 (1H, br s, -NH-), a broad singlet attributed to an amide proton; and two singlets at δ 1.12 with identical chemical environments, each integrated as 3H, attributed to protons on a gem-dimethyl group (CH3-24, CH3-25). Furthermore, the spectrum contains multiple olefinic proton signals in the δ 6.0–7.5 region and saturated alkyl proton signals in the δ 1.0–2.5 region, collectively revealing a long conjugated polyene chain structure.

[0169] The carbon spectrum of compound A (measured on a 125 MHz NMR spectrometer in deuterated acetone) showed 32 carbon signals, consistent with the number of carbon atoms in the molecular formula determined by mass spectrometry. Specifically, the two signals at δ 207.9 and δ 203.4 in the high-field region belong to the two ketone carbonyl carbons (C-5, C-6), respectively, while the signal at δ 169.6 belongs to the amide carbonyl carbon (C-23). ​​Multiple paired alkene carbon signals were present in the δ 120–150 region, confirming the presence of a conjugated polyene chain. The signal at δ 24.7 in the low-field region corresponds to the two carbon atoms of the geminimethyl group (C-24, C-25).

[0170] Furthermore, by combining two-dimensional nuclear magnetic resonance spectroscopy (such as HSQC, HMBC, and ¹H-¹H COSY) to confirm and correlate the assignments of the aforementioned one-dimensional spectral signals, the planar structure of compound A can be fully resolved. For example, in the HMBC spectrum, long-range correlation signals between the amide proton (-NH-) and the amide carbonyl carbon (C-23) and the methylene carbon (-CH2-NH-) can be observed, thereby confirming the presence and linkage of the N-(2-hydroxyethyl) amide group.

[0171] Based on the combined data from mass spectrometry, one-dimensional and two-dimensional nuclear magnetic resonance spectroscopy, infrared spectroscopy, and ultraviolet spectroscopy (with a maximum absorption wavelength λmax of 425 nm in methanol), the chemical structure of compound A was finally determined as shown in the attached figure. Figure 6As shown, its systematic name is (2E,4E,6E,8E,10E,12E,14E,16E)-N-(2-hydroxyethyl)-2,6,11,15,19,19-hexamethyl-18,23-dioxotetracosa-2,4,6,8,10,12,14,16-octaenamide, which translates to (2E,4E,6E,8E,10E,12E,14E,16E)-N-(2-hydroxyethyl)-2,6,11,15,19,19-hexamethyl-18,23-dioxotetracosa-2,4,6,8,10,12,14,16-octaenamide. This structure is a novel compound with a terminal cyclohexanedione derivative structure, corresponding to the structure shown in the attached diagram. Figure 1 A specific implementation of the combination of equations (I) and (X2) shown also corresponds to Figure 2 The structure shown is (i).

[0172] Example 6

[0173] This embodiment confirms the chemical structure of compound B obtained in Example 1.

[0174] Nuclear magnetic resonance (1D and 2D NMR) methods confirmed the structure of compound B, with the following data parameters:

[0175] Orange red powder; -16.0 (MeOH, c 0.05); IR (neat): 3383,2931, 1716, 1636, 1533, 1025, 969 cm -1 ; UV (MeOH): λ max (log ε) 217 ​​(3.94), 244(4.13), 274 (3.89), 399 (4.62) nm; HRMS (ESI) m / z: [M+H] + Calcd for C 32 H 46 NO5524.3376; Found 524.3386. 1H NMR (DMSO-d6, 500 MHz): δ 7.77 (t, 1H, J = 5.5Hz, -NH-), 6.95 (d, 1H, J = 9.5 Hz, H-21), 6.78 (dd, 1H, J = 11.5, 15.0 Hz,H-15), 6.69 (dd, 1H, J = 11.5, 15.0 Hz, H-16), 6.59 (d, 1H, J = 15.0 Hz, H-12), 6.59 (d, 1H, J = 15.0 Hz, H-19), 6.56 (dd, 1H, J = 9.5, 15.0 Hz, H-20),6.43 (d, 1H, J = 11.5 Hz, H-17), 6.33 (d, 1H, J = 11.5 Hz, H-10), 6.32 (d,1H, J = 15.0 Hz, H-12), 6.30 (d, 1H, J = 11.5 Hz, H-14), 4.67 (t, 1H, J = 5.5Hz, 2′-OH), 4.57 (s, 1H, H-8), 4.07 (s, 1H, H-7), 3.93 (d, 1H, J = 5.0 Hz, H-4), 3.43 (m, 2H, H-2′), 3.20 (m, 2H, H-1′), 5.02 (s, 1H, 6-OH), 1.94 (s, 3H,H-28), 1.94 (s, 3H, H-29), 1.93 (s, 3H, H-30), 1.73 (m, 1H, H-2), 1.73 (s,3H, H-27), 1.54 (m, 1H, H-3), 1.23 (s, 3H, H-26), 1.19 (m, 1H, H-2), 1.04 (s,3H, H-25), 0.98 (s, 3H, H-24); 13C NMR (DMSO-d6, 150 MHz): δ 168.2, 141.5,137.3, 137.1, 135.6, 135.3, 134.5, 133.1, 131.6, 131.4, 130.2, 129.5, 125.2,124.3, 123.7, 83.8, 83.8, 83.0, 82.8, 80.7, 59.8, 42.1, 37.1, 33.2, 28.1,25.5, 24.3, 14.5, 13.3, 13.1, 12.7, 12.6.

[0176] 1 H-NMR (500MHz,DMSO-d6) δ 1.19 (m,H a -2),1.73 (m,H b -2),1.54 (m,H-3),3.93 (d,J=5.0HZ,H-4),4.07 (s,H-7),4.57 (s,H-8),6.33 (m,H-10),6.59 (m,H-11),6.32 (m,H-12),6.30 (m,H-14),6.78 (dd,J=11.5,13.5HZ,H-15),6.69 (dd,J=11.5,13.5HZ,H-16),6.43 (d,J=11.5HZ,H-17),6.59 (m,H-19),6.56 (m,H-20),6.95 (d,J=9.5HZ,H-21),0.98 (s,H-24),1.04 (s,H-25),1.23 (s,H-26),1.73 (s,H-27),1.94 (s,H-28),1.94 (s,H-29),1.93 (s,H-30),3.20 (m,H-1′),3.43 (m,H-2′),5.02 (s,6-OH),4.67 (t,J=5.5HZ,2′-OH),7.77 (t,J=5.5HZ,-NH-)。

[0177] 13C-NMR (125MHz, DMSO-d6) δ 37.1 (C-1), 33.3 (C-2), 25.4 (C-3), 83.8 (C-4), 83.8 (C-5), 83.0 (C-6), 80.7 (C-7), 82.8 (C-8), 137.4 (C-9), 124.4 (C-10), 125.2 (C-11), 135.6 (C-12), 137.1 (C-13), 131.4 (C-14), 131.6 (C-15), 129.5 (C-16), 134.5 (C-17), 135.3 (C-18), 141.6 (C-19), 123.7 (C-20), 133.1 (C-21), 130.2 (C-22), 168.5 (C-23), 24.3 (CH3-24), 28.1 (CH3-25), 13.4 (CH3-26), 14.5 (CH3-27), 12.6 (CH3-28), 12.7 (CH3-29), 13.1 (CH3-30), 42.1 (C-1′), 59.9 (C-2′).

[0178] Those skilled in the art should be fully able to understand the above spectral data, which is cited here to demonstrate the actual experimental process and results.

[0179] Compound B is an orange-yellow powdery solid. For example... Figure 13 As shown, its high-resolution electrospray ionization mass spectrometry measured a quasi-molecular ion peak in positive ion mode. The mass-to-charge ratio (m / z) is 524.3386. This experimental value is consistent with that based on the chemical formula... Calculated The theoretical value (524.3376) is highly consistent, thus determining the molecular formula of compound B as follows: .

[0180] Nuclear magnetic resonance (NMR) spectroscopy analysis of compound B revealed its unique structural features. Its proton NMR spectrum (measured on a 500 MHz NMR spectrometer in deuterated dimethyl sulfoxide) showed: δ 7.77 (1H, t, J=5.5 Hz, -NH-), attributed to the amide proton; δ 6.95 (1H, d, J=9.5 Hz, H-21); and a series of olefinic hydrogen signals in the δ 6.0–7.0 region, indicating that it also possesses a conjugated polyene chain structure. In the saturated region, signals such as δ 0.98 (3H, s, H-24) indicate the presence of a methyl group.

[0181] Its carbon spectrum (measured on a 125 MHz NMR spectra in deuterated dimethyl sulfoxide solvent) showed 32 carbon signals. Among them, δ 168.5 is attributed to the amide carbonyl carbon (C-23). ​​It should be noted that two quaternary carbon signals with very close chemical shifts appeared at δ 83.8, which are attributed to two oxygen-bonded saturated carbon atoms (C-4, C-5). This feature is significantly different from the structure of compound A and suggests that it has an oxygen-containing ring structure.

[0182] Detailed analysis of two-dimensional NMR spectra, such as HSQC and HMBC, allowed for the reconstruction and connection of structural fragments of compound B. The key HMBC signal revealed the existence of a complex bridged oxatricyclic structure. This structure is an octahydro-3,7-epoxybenzofuran skeleton, linked to the C-15 position of a conjugated polyene chain via the C-2 position.

[0183] Based on all the spectral data, the final chemical structure of compound B is shown in the attached figure. Figure 7 The structural formula shown is systematically named as follows: (2E,4E,6E,8E,10E,12E,14E)-15-((2S,3R,3aS,7R,7aR)-3a-hydroxy-4,4,7a-trimethyloctahydro-3,7-epoxybenzofuran-2-yl)-N-(2-hydroxyethyl)-2,6,11-trimethylhexadeca- 2,4,6,8,10,12,14-heptaenamide; in Chinese: (2E,4E,6E,8E,10E,12E,14E)-15-((2S,3R,3aS,7R,7aR)-3a-hydroxy-4,4,7a-trimethyloctahydro-3,7-epoxybenzofuran-2-yl)-N-(2-hydroxyethyl)-2,6,11-trimethylhexadecyl-2,4,6,8,10,12,14-heptaenamide. The structure of this compound is also novel, characterized by a unique bridging oxatricyclic structure at the end, which corresponds to... Figure 1 A specific implementation of the combination of formulas (I) and (X1) in the text, which also corresponds to Figure 3 The structure shown is (ii).

[0184] Example 7

[0185] This embodiment confirms the chemical structures of compounds C and D obtained in Example 4.

[0186] Specifically, compounds C (retention time tR = 36.2 min) and D (retention time tR = 44.6 min) were prepared by separation using a CHIRALPAK®IB N-5 column, with a purity higher than 95%. The mobile phase was a gradient elution of n-butanol (IPA) in hexane (HEX) under the conditions of 10%–13% for 20 min, followed by 13%–30% for 20 min, at a flow rate of 1 mL / min. After rotary evaporation, a portion of the dried sample was used for NMR and mass spectrometry analysis. The structures of compounds C and D were identified by high-resolution mass spectrometry and one-dimensional and two-dimensional NMR.

[0187] Specifically, nuclear magnetic resonance (1D and 2D NMR) confirmed the C structure of this compound with the following data parameters:

[0188] Orange red powder; -198.6 (MeOH, c 0.9); IR (neat): 3383,2928, 2858, 1721, 1635, 1532, 1270, 1102 cm -1 ; UV (MeOH): λ max (log ε) 225(3.95), 243 (4.12), 265 (3.80), 306 (4.23), 322 (3.73), 399 (4.63) nm; HRMS(ESI) m / z: [M+H] + Calcd for C 32 H 46 NO5 524.3376; Found 524.3416. 1H NMR (DMSO-d6,500 MHz): δ 7.77 (t, 1H, J = 5.5 Hz, -NH-), 6.95 (d, 1H, J = 10.0 Hz, H-21),6.93 (d, 1H, J = 15.0 Hz, H-12), 6.62 (dd, 1H, J = 11.5, 14.5 Hz, H-16), 6.59(dd, 1H, J = 10.0, 15.0 Hz, H-11), 6.56 (d, 1H, J = 15.0 Hz, H-19), 6.54 (dd,1H, J = 10.0, 15.0 Hz, H-20), 6.42 (d, 1H, J = 10.0 Hz, H-10), 6.41 (d, 1H, J= 11.5 Hz, H-17), 6.14 (d, 1H, J = 12.0 Hz, H-14), 7.03 (dd, 1H, J = 12.0,14.5 Hz, H-15), 5.04 (s, 1H, 6-OH), 4.68 (t, 1H, J = 5.5 Hz, 2′-OH), 4.57 (s,1H, H-8), 4.08 (s, 1H, H-7), 3.97 (d, 1H, J = 5.0 Hz, H-4), 3.43 (m, 2H, H-2′), 3.20 (m, 2H, H-1′), 1.95 (s, 3H, H-28), 1.93 (s, 3H, H-29), 1.93 (s, 3H,H-30), 1.75 (m, 1H, H-2), 1.73 (s, 3H, H-27), 1.55 (m, 2H, H-3), 1.24 (s, 3H,H-26), 1.20 (m, 1H, H-2), 1.05 (s, 3H, H-25), 0.98 (s, 3H, H-24); 13C NMR(DMSO-d6, 150 MHz): δ 168.3, 141.6, 137.9, 135.6, 135.1, 134.5, 130.6, 130.2,130.0, 128.7, 127.9, 126.5, 124.5, 123.5, 133.1, 83.8, 83.7, 83.1, 82.8,80.6, 59.9, 42.1, 37.1, 33.2, 28.1, 25.5, 24.3, 20.5, 14.5, 13.4, 13.1, 12.5.

[0189] 1 H-NMR (500MHz,DMSO-d6) δ 1.20 (m,H a -2),1.75 (m,H b -2),1.55 (m,H-3),3.97 (d,J=5.0 HZ,H-4),4.08 (s,H-7),4.57 (s,H-8),6.42 (d,J=15.0 HZ,H-10),6.59(dd,J=15.0,15.0 HZ,H-11),6.93 (d,J=15.0 HZ,H-12),6.14 (d,J=12.0 HZ,H-14),7.03(dd,J=12.0,14.0 HZ,H-15),6.62 (m,H-16),6.41 (d,J=15.0 HZ,H-17),6.56 (m,H-19),6.54 (m,H-20),6.95 (d,J=5.0 HZ,H-21),0.98 (s,CH3-24),1.05 (s,CH3-25),1.24 (s,CH3-26),1.73 (s,CH3-27),1.95 (s,CH3-28),1.93 (s,CH3-29),1.93 (s,CH3-30),3.20(m,H-1′),3.43 (m,H-2′),5.04 (s,6-OH),4.68 (t,J=5.5HZ,2′-OH),7.77 (t,J=5.5HZ,-NH-)。

[0190] 13C-NMR (125MHz, DMSO-d6) δ 37.3 (C-1), 33.3 (C-2), 25.5 (C-3), 83.8 (C-4), 83.8 (C-5), 83.1 (C-6), 80.6 (C-7), 82.8 (C-8), 138.0 (C-9), 124.5 (C-10), 126.5(C-11), 127.9 (C-12), 135.7 (C-13), 130.0 (C-14), 130.6 (C-15), 128.7 (C-16), 134.5(C-17), 135.1 (C-18), 141.6 (C-19), 123.5 (C-20), 133.3 (C-21), 130.2 (C-22), 168.3 (C-23), 24.3 (C-24), 28.1 (C-25), 13.4 (C-26), 14.5 (C-27), 20.5 (C-28), 12.5 (C-29), 13.1 (C-30), 41.9 (C-1′), 59.9 (C-2′).

[0191] Those skilled in the art should be fully able to understand the above spectral data, which is cited here to demonstrate the actual experimental process and results.

[0192] Specifically, nuclear magnetic resonance (1D and 2D NMR) methods confirmed the structure of compound D with the following data parameters:

[0193] Orange red powder; -60.3 (MeOH, c 0.29); IR (neat): 3365,2925, 2855, 1729, 1635, 1532, 1051, 956 cm -1 ; UV (MeOH): λ max (log ε) 221(3.43), 245 (3.62), 264 (3.42), 304 (3.80), 323 (3.45), 399 (4.25) nm; HRMS(ESI) m / z: [M+H] + Calcd for C 32 H 46 NO5 524.3376; Found 524.3416. 1H NMR (DMSO-d6,500 MHz): δ 7.77 (t, 1H, J = 5.5 Hz), 7.20 (d, 1H, J = 15.0 Hz), 7.02 (d, 1H,J = 11.5 Hz), 6.92 (dd, 1H, J = 12.0, 15.0 Hz), 6.69 (dd, 1H, J = 11.5, 15.0Hz), 6.58 (dd, 1H, J = 11.0, 15.0 Hz), 6.56 (dd, 1H, J = 11.5, 15.0 Hz), 6.33(d, 1H, J = 11.0 Hz), 6.31 (d, 1H, J = 15.0 Hz), 6.26 (d, 1H, J = 12.0 Hz),6.25 (d, 1H, J = 11.5 Hz), 5.02 (s, 1H), 4.68 (t, 1H, J = 5.5 Hz), 4.57 (s,1H), 4.07 (s, 1H), 3.94 (d, 1H, J = 5.0 Hz), 3.44 (m, 2H), 3.21 (m, 2H), 1.96(s, 3H), 1.94 (s, 3H), 1.93 (s, 3H), 1.75 (m, 1H), 1.73 (s, 3H), 1.55 (m,1H), 1.23 (s, 3H), 1.21 (m, 1H), 1.04 (s, 3H), 0.98 (s, 3H). 13 C NMR (DMSO-d6,150 MHz): δ 168.2, 137.1, 136.7, 135.6, 133.5, 133.5, 133.2, 133.0, 131.2,131.0, 128.3, 125.0, 125.0, 124.3, 124.3, 83.8, 83.8, 83.0, 82.8, 80.6, 59.8,42.1, 37.1, 33.2, 28.1, 25.5, 24.3, 20.2, 14.5, 13.4, 13.1, 12.7.

[0194] 1 H-NMR (500MHz,DMSO-d6) δ 1.21 (m,H a -2),1.75 (m,H b-2),1.55 (m,H-3),3.94 (d,J=5.0 HZ,H-4),4.07 (s,H-7),4.57 (s,H-8),6.33 (d,J=11.0 HZ,H-10),6.59(m,H-11),6.31 (d,J=15.0 HZ,H-12),6.25 (d,J=11.5 HZ,H-14),6.69 (dd,J=11.5,14.0HZ,H-15),6.92 (dd,J=14.0,10.0 HZ,H-16),6.26 (d,J=10.0 HZ,H-17),7.20 (d,J=15.0HZ,H-19),6.56 (m,H-20),7.02 (d,J=12.0 HZ,H-21),0.98 (s,CH3-24),1.04 (s,CH3-25),1.23 (s,CH3-26),1.73 (s,CH3-27),1.93 (s,CH3-28),1.96 (s,CH3-29),1.94 (s,CH3-30),3.21 (m,H-1′),3.44 (m,H-2′),5.02 (s,6-OH),4.68 (t,J=5.5 HZ,2′-OH),7.77 (t,J=5.5 HZ,-NH-)。

[0195] 13 C-NMR (125MHz,DMSO-d6) δ 37.2 (C-1),33.4 (C-2),25.5 (C-3),83.8 (C-4),83.8 (C-5),83.0 (C-6),80.7 (C-7),82.8 (C-8),137.2 (C-9),124.3 (C-10),125.0(C-11),135.6 (C-12),136.7 (C-13),131.2 (C-14),130.5 (C-15),128.3 (C-16),133.0(C-17),133.5 (C-18),133.6 (C-19),125.0 (C-20),133.2 (C-21),131.0 (C-22),168.4(C-23),24.2 (CH3-24),28.1 (CH3-25),13.4 (CH3-26),14.6 (CH3-27),12.8 (CH3-28),20.3 (CH3-29),13.1 (CH3-30),42.0 (C-1′),59.9 (C-2′)。

[0196] Those skilled in the art should be fully able to understand the above spectral data, which is cited here to demonstrate the actual experimental process and results.

[0197] Both compounds C and D are orange-yellow powdery solids. Figure 14 , 15 Their high-resolution mass spectrometry data all show that their quasi-molecular ion peaks The mass-to-charge ratio (m / z) of compound B is the same, both being 524.3386, indicating that all three have the same molecular formula. Isomers of.

[0198] Nuclear magnetic resonance analysis of compounds C and D revealed that their proton and carbon spectra were highly similar to those of compound B, indicating that they share the same basic skeleton, namely, an N-(2-hydroxyethyl)amide group, a conjugated polyene chain, and a bridging oxatricyclic terminal group.

[0199] However, a detailed comparison of the NMR data of compounds B, C, and D revealed subtle but distinct differences in the chemical shifts of the olefinic hydrogen and olefinic carbon in the conjugated polyene chain.

[0200] To determine the absolute configuration of the bridging oxatricyclic moiety in compounds B, C, and D, electron circular dichroism spectroscopy was performed. The experimentally obtained spectra were then compared with theoretical spectra of various possible stereoisomers obtained through quantum chemical calculations (e.g., time-dependent density functional theory). The comparison results showed that the experimental spectra showed the best agreement with the theoretical spectra, thus determining the absolute configuration of the chiral ring moiety in these three compounds.

[0201] The exact structure of compound C is shown in the attached figure. Figure 8 The structural formula shown is systematically named as follows: (2E,4E,6E,8E,10Z,12E,14E)-15-((2S,3R,3aS,7R,7aR)-3a-hydroxy-4,4,7a-trimethyloctahydro-3,7-epoxybenzofuran-2-yl)-N-(2-hydroxyethyl)-2,6,11-trimethylhexade ca-2,4,6,8,10,12,14-heptaenamide; Chinese name: (2E,4E,6E,8E,10Z,12E,14E)-15-((2S,3R,3aS,7R,7aR)-3a-hydroxy-4,4,7a-trimethyloctahydro-3,7-epoxybenzofuran-2-yl)-N-(2-hydroxyethyl)-2,6,11-trimethylhexadecyl-2,4,6,8,10,12,14-heptaenamide. Figure 8The numbers in the molecular formula represent the relative positions of the carbon atoms in that formula. They have no substantial impact on the carbon atoms, molecular structure, or the corresponding compound. They are simply used to facilitate understanding or description of the relative positions of carbon atoms in the molecular structure. Examples of such numbers include: 1, 5, 6, 24, 25, 26, 28, 29, and 1', 2', etc.

[0202] The exact structure of compound D is shown in the attached figure. Figure 9 The structural formula shown is systematically named as follows: (2E,4E,6Z,8E,10E,12E,14E)-15-((2S,3R,3aS,7R,7aR)-3a-hydroxy-4,4,7a-trimethyloctahydro-3,7-epoxybenzofuran-2-yl)-N-(2-hydroxyethyl)-2,6,11-trimethylhexade ca-2,4,6,8,10,12,14-heptaenamide; Chinese name: (2E,4E,6Z,8E,10E,12E,14E)-15-((2S,3R,3aS,7R,7aR)-3a-hydroxy-4,4,7a-trimethyloctahydro-3,7-epoxybenzofuran-2-yl)-N-(2-hydroxyethyl)-2,6,11-trimethylhexadecyl-2,4,6,8,10,12,14-heptaenamide. Figure 9 The numbers in the molecular formula represent the relative positions of the carbon atoms in that formula. They have no substantial impact on the carbon atoms, molecular structure, or the corresponding compound; they are simply used to facilitate understanding or description of the relative positions of carbon atoms in the molecular structure. Examples of such numbers include: 1, 5, 6, 24, 25, 29, and 1', 2', etc.

[0203] In summary, this embodiment, through detailed spectroscopic analysis, confirmed the structures of compounds C and D as shown in the attached figures. Figure 8 The structural formulas (iii-c) and appendices shown Figure 9 The structural formulas shown are (iv-d). These two compounds belong to the following categories. Figure 1 An embodiment of the combination of equations (I) and (X1) shown also corresponds to... Figure 4 The structural formula (iii) and shown Figure 5 The structural formula (iv) shown herein collectively supports the protection of the compound stereoisomers (including geometric isomers) in this application.

[0204] Example 8

[0205] This embodiment evaluates the in vitro anti-inflammatory activity and cytotoxicity of the rehmannia glutinosa compound AD obtained in the above embodiments to verify its potential as an anti-inflammatory drug. As an optional implementation, this embodiment uses a lipopolysaccharide-induced mouse macrophage (RAW264.7) inflammation model for evaluation.

[0206] Experimental Materials and Methods: Cell line: Mouse macrophage RAW264.7, purchased from the Cell Bank of the Chinese Academy of Sciences. Main reagents: Lipopolysaccharide (LPS), purchased from Sigma-Aldrich (USA); Griess reagent kit for detecting nitric oxide concentration, purchased from Beyotime Biotechnology Research Institute; CCK8 cell viability assay kit, purchased from Dojin Chemical Research Institute (Japan); Quercetin, as a positive control, purchased from Sigma-Aldrich. Samples to be tested: Compounds A, B, C, and D prepared in the above examples were dissolved in dimethyl sulfoxide (DMSO) and prepared into stock solutions. During the experiment, they were diluted with culture medium to the required concentration, ensuring that the volume fraction of DMSO in the final concentration was less than 0.1%.

[0207] 1. Cytotoxicity evaluation (CCK8 assay)

[0208] RAW264.7 cells in logarithmic growth phase were divided into groups per well. Cells were seeded at a density of 1000 μL in 96-well plates and cultured for 24 hours to allow them to adhere. The old culture medium was discarded, and fresh culture medium containing different concentrations (4 μM, 20 μM, 100 μM) of compounds A, B, C, D, or the positive control quercetin was added. A blank control group (containing only culture medium) and a solvent control group (containing 0.1% dimethyl sulfoxide) were also established. Each group had 6 replicates. The cell plates were incubated at 37°C with 5% CO2 for another 24 hours. After incubation, 10 μL of CCK8 reagent was added to each well, and incubation continued for 90 minutes until a significant color change occurred. The absorbance of each well was measured at 450 nm using a microplate reader. The cell growth inhibition rate was calculated using the following formula: Inhibition rate (%) = [1 - (OD drug group - OD blank group) / (OD solvent control group - OD blank group)] × 100%.

[0209] Table 1. Inhibition rate of compound AD on the growth of RAW264.7 cells (24 h)

[0210]

[0211] Experimental results: As shown in Table 1, the growth inhibition rate of RAW264.7 cells increased in a concentration-dependent manner. Compounds A and B showed only slight effects at the highest tested concentration of 100 μM.

[0212] The results showed that, in comparison, the positive control drug quercetin exhibited a cell growth inhibition rate as high as 61.9% at the same concentration. These results demonstrate that the four rehmannia flavonoid derivatives provided in this application exhibit extremely low cytotoxicity even at concentrations up to 100 μM, and their safety profile is far superior to that of the positive control drug quercetin.

[0213] 2. Evaluation of anti-inflammatory activity (Griess method for detecting nitric oxide release)

[0214] RAW264.7 cells in logarithmic growth phase were divided into groups per well. Cells were seeded at a density of 1000 μL in 96-well plates and cultured for 24 hours. The culture medium was discarded, and culture medium containing different concentrations (4 μM, 20 μM, 100 μM) of compounds A, B, C, D, or the positive control quercetin was added for pretreatment for 1 hour. Subsequently, except for the blank control group, each well was stimulated with lipopolysaccharide (LPS) at a final concentration of 1 μg / mL to induce an inflammatory response. A LPS model group was also established (LPS and solvent only). The cell plates were incubated at 37°C and 5% CO2 for another 24 hours. After culture, 50 μL of cell supernatant from each well was carefully transferred to a new 96-well plate. Following the Griess kit instructions, Griess reagents I and II were added sequentially. After reacting at room temperature for 10 minutes, the absorbance was measured at 540 nm using a microplate reader. The concentration of nitric oxide in the supernatant of each group was calculated based on the nitrite standard curve. The nitric oxide release inhibition rate was calculated using the following formula: Inhibition rate (%) = [1 - (NO concentration drug group - NO concentration blank group) / (NO concentration LPS model group - NO concentration blank group)] × 100%.

[0215] Table 2. Inhibition rate (%) of NO release from RAW264.7 cells. Results are expressed as mean ± standard deviation (n=3).

[0216]

[0217] Experimental Results: The results showed that compounds B–D (at a concentration of 20 μM) significantly inhibited NO production in LPS-induced RAW264.7 cells, while compound A showed only a weaker effect (see Table 2). Cytotoxicity assessment indicated that the inhibitory effects of compounds A and B on NO production may be partly attributed to their cytotoxicity (at a concentration of 100 μM). Nevertheless, these results still suggest that these compounds possess anti-inflammatory properties.

[0218] Based on the combined cytotoxicity and anti-inflammatory activity data, the following conclusions can be drawn from this embodiment: The rehmannia glutinosa derivative compounds AD provided in this application all exhibit significant anti-inflammatory activity, effectively inhibiting the excessive production of the inflammatory mediator nitric oxide by inflammatory cells. Simultaneously, these compounds demonstrate excellent "high efficiency and low toxicity" characteristics. Specifically, the anti-inflammatory activities of compounds B, C, and D are comparable to or even superior to those of the commonly used anti-inflammatory drug quercetin, while their cytotoxicity is far lower than that of quercetin, revealing their significant safety advantages and development potential as candidate drugs. From a structure-activity relationship perspective, compounds B, C, and D, which possess a bridged oxatricyclic structure (Formula X1), exhibit significantly stronger anti-inflammatory activity than compound A, which possesses an open-chain cyclohexanedione structure (Formula X2), indicating that this bridged oxatricyclic structure is the key pharmacophore generating strong anti-inflammatory activity.

[0219] The results of this embodiment fully demonstrate the application value of the rehmannia glutinosa compound disclosed in this application in the preparation of drugs for the prevention or treatment of inflammatory diseases.

[0220] The above description is merely a preferred embodiment of this application and is not intended to limit the scope of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A rehmannia flavonoid compound of formula (I), its stereoisomer, or a pharmaceutically acceptable salt thereof: ； In formula (I), X is selected from formula (X1) or formula (X2), and the double bond configurations of 13-C and 17-C are independently of the E or Z type. ； In equations (I), (X1), and (X2): R1, R7, and R8 are independently oxygen or sulfur; R2, R6, and R5 are independently oxygen, sulfur, or imino; R3 and R4 are independently hydroxyl, thiol, or amino groups.

2. The rehmannia glutinosa compound, its stereoisomer, or a pharmaceutically acceptable salt thereof according to claim 1, wherein the compound is a compound having the structure shown in formula (i), (ii), (iii), or (iv): ； ； In formula (i), the 13-C double bond is of formula E, the 17-C double bond is of formula E, and X is selected from formula (X2); In formula (ii), the 13-C double bond is of formula E, the 17-C double bond is of formula E, and X is selected from formula (X1); In formula (iii), the 13-C double bond is of type Z, the 17-C double bond is of type E, and X is selected from formula (X1); In formula (iv), the 13-C double bond is of formula E, the 17-C double bond is of formula Z, and X is selected from formula (X1).

3. The rehmannia glutinosa compound, its stereoisomer, or a pharmaceutically acceptable salt thereof according to claim 1 or 2, characterized in that, The compound is a compound having the structure shown in formula (ia), formula (ii-b), formula (iii-c), or formula (iv-d): ； ； 。 4. The rehmannia glutinosa compound, its stereoisomer, or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, X is selected from formula (X1).

5. The rehmannia glutinosa compound, its stereoisomer, or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, The 13-C double bond is of type E, and the 17-C double bond is of type E.

6. The rehmannia glutinosa compound, its stereoisomer, or a pharmaceutically acceptable salt thereof according to claim 1 or 2, characterized in that, X is selected from formula (X1), R1 is oxygen, R2 is oxygen, sulfur or imino, R5 is oxygen, and R6 is oxygen or imino.

7. The rehmannia glutinosa compound, its stereoisomer, or a pharmaceutically acceptable salt thereof according to claim 1 or 2, characterized in that, X is selected from formula (X2), R1 is sulfur, R2 is oxygen or imino, R7 is oxygen, and R8 is sulfur.

8. A method for preparing the rehmannia glutinosa compound, its stereoisomer, or a pharmaceutically acceptable salt thereof according to any one of claims 1-7, characterized in that, Includes the following steps: The crude rehmannia pigment was subjected to normal phase chromatography to obtain a preliminary purified fraction containing the target component. The preliminarily purified fraction was subjected to reverse-phase chromatography to obtain a further purified subfraction; Size exclusion chromatography was performed on the sub-components to obtain secondary components; The secondary components were separated by semi-preparative high-performance liquid chromatography and / or preparative high-performance liquid chromatography to obtain the rehmannia glutinosa pigment compound.

9. A pharmaceutical composition, characterized in that, The compound comprising a therapeutically effective amount of any one of claims 1-7, its stereoisomer, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers or excipients.

10. Use of the compound, its stereoisomer, or a pharmaceutically acceptable salt thereof, according to any one of claims 1-7, or the pharmaceutical composition according to claim 9, in the preparation of a medicament for the prevention and / or treatment of inflammatory diseases.

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