Triterpene alcohol ferulate compound as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of chemical medicines, and particularly relates to a novel triterpene alcohol ferulate compound as well as a preparation method and medical application thereof. According to the invention, oryzanol is used as a raw material, and the new triterpene alcohol ferulate compound is obtained through reversed-phase preparative high performance liquid chromatography and SFC chiral separation and purification. A method and a tool of network pharmacology are used for researching the action mechanism of potential anti-inflammatory and anti-infection related treatment of the new compound. Hierarchical analysis from a global network to a core target reveals a possible action mechanism of the compound in anti-inflammatory and anti-infection, and highlights a regulation mode of a key acting protein. Wherein IGF1R, CTSS, MMP2, BCL2 and other proteins may be core regulatory factors of compound mediated host immune regulation and bacterial drug resistance inhibition, and an important theoretical basis is provided for further research on molecular mechanisms and potential therapeutic targets of compounds in anti-inflammatory and anti-infection related treatment.

Description

Technical Field

[0001] The present invention belongs to the technical field of chemical medicine, and in particular relates to a new triterpene alcohol ferulate compound, a preparation method and medical use thereof. Background Art

[0002] Oryzanol, a white to pale yellow crystalline powder, is a mixture of triterpenoid and sterol ferulates. It exhibits antioxidant, anti-inflammatory, anti-cancer, lipid-lowering, anti-gastric acid secretion, mood and sleep improvement, and blood sugar regulation. The major components of oryzanol are cycloartenol ferulate (25-30%), 24-methylenecycloartenol ferulate (35-40%), campesterol ferulate (10-12%), cycloartenol ferulate (8-10%), β-sitosterol ferulate (6-8%), cyclofurfuryl ferulate (2-3%), stigmasterol ferulate (1-2%), 24-methylcycloartenol ferulate (0.2-0.5%), and 24-methylcycloartenol ferulate (0.5-1.0%). According to statistics, there are more than 19 types of oryzanol in rice. However, due to the trace amount of some oryzanol and insufficient preparation, many oryzanol components cannot be accurately detected.

[0003] Inflammation is the body's defensive response to damaging factors. However, it is a double-edged sword. Excessive or uncontrolled inflammation (overactivation of the innate immune system and overexpression of inflammatory cytokines) can lead to tissue damage and contribute to the development of numerous diseases, including sepsis, obesity, and metabolic diseases such as diabetes and its complications. Therefore, drugs that inhibit excessive inflammation hold promise for treating a variety of inflammation-related diseases.

[0004] Oysterol has good anti-inflammatory activity and is favored by researchers. In mice treated with oryzanol, it was observed that the mRNA expression of TNF-α and IL-1β was significantly reduced, and the infiltration of inflammatory cell tissues was reduced (British Journal of Pharmacology, 2008, 154 (4): 812-824). Studies have shown that γ-oryzanol can prevent the phosphorylation of IkB, causing NF-kB to remain in a trimer state, in an inactive stage and inhibit the occurrence of inflammation (Chen Yaya. Oysterol inhibits the expression of inflammatory factors in macrophages induced by endotoxin and its molecular mechanism [D]. Central South University of Forestry and Technology, 2016). Liu Yuanjin et al. studied the effect of γ-oryzanol on the expression of inflammatory factors in macrophages. The results showed that γ-oryzanol can significantly inhibit the secretion and expression of inflammatory factors (interleukins) and tumor necrosis factor (Food Science, 2015, 36 (19): 238-243). Chen Yaya et al. allowed mice to drink sodium glucose sulfate continuously for 12 consecutive days to establish an ulcerative colitis model, and then added γ-oryzanol to explore its anti-inflammatory effect. The results showed that the content of malondialdehyde in the mouse group supplemented with γ-oryzanol was improved, and the content of nitrite was reduced, which could significantly reduce the colon inflammation caused by DSS (Modern Food Science and Technology, 2016, 32(05):14-21).

[0005] Cycloartenyl ferulate, one of the main components of oryzanol, is a typical triterpene alcohol with the structural formula shown in Formula I below. Many studies have shown that cycloartenyl ferulate also has good anti-inflammatory activity: Reiko Nagasaka et al. demonstrated through experiments that cycloartenyl ferulate can significantly reduce lipopolysaccharide-induced NO production and iNOS and COX-2 mRNA expressions (Biochem Biophys Res Commun. 2007, 358(2): 615-619); Md. Shafiqul Islam et al. studied the anti-inflammatory effect mechanism of cycloartenyl ferulate on lipopolysaccharide-induced macrophages. The results showed that cycloartenyl ferulate downregulated iNOS mRNA expression by inhibiting the NF-kB signaling pathway (Asian J. Med. Biol. Res. 2016, 2(4), 523-531).

[0006]

[0007] Currently, all clinically used steroidal or non-steroidal anti-inflammatory drugs target the cyclooxygenase / prostaglandin system and inflammatory vascular responses, rather than the inflammatory signaling pathways caused by overexpression of inflammatory factors. They have a single target and strong limitations, and cannot be used to treat chronic inflammatory diseases. Antibody drugs targeting inflammatory factors and their receptors have targets downstream of inflammation, and only a single type of inhibitory effect on inflammatory factors. They have limited therapeutic effects on inflammatory-related diseases and metabolic diseases with a large number of complex inflammatory factors. Summary of the Invention

[0008] In view of the deficiencies in the prior art, the present invention aims to provide a novel triterpene alcohol ferulate compound, a preparation method and medical use thereof.

[0009] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0010] A new triterpenoid ferulic acid ester compound with the molecular formula C 40 H 56 O4, the structural formula is as follows:

[0011]

[0012] A method for preparing the novel triterpene alcohol ferulate compound comprises the following steps:

[0013] (1) Oryzanol was dissolved in anhydrous ethanol and separated by reverse phase preparative high performance liquid chromatography. Fractions were collected according to time periods to obtain 12 fractions, named GWS-P1 to GWS-P12. The separation conditions were as follows:

[0014]

[0015]

[0016] (2) Fraction GWS-P1 (fraction collected at 30-35 min) was purified by preparative SFC chiral separation under the following conditions:

[0017]

[0018] The residue was concentrated under reduced pressure and dried to obtain a new compound (Rt=4.5 min).

[0019] Network pharmacology studies of the above compounds have shown that they have potential medical uses in anti-inflammatory and anti-infection related treatments.

[0020] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0021] This study, for the first time, used SFC chromatography to isolate a new triterpenoid ferulate compound from oryzanol. Network pharmacology methods and tools were then applied to investigate the compound's potential anti-inflammatory and anti-infective therapeutic mechanisms. The findings revealed the compound's potential mechanisms of action in these settings and highlighted the regulatory mechanisms of key proteins. Proteins such as IGF1R, CTSS, MMP2, and BCL2 were identified as key regulatory factors in the compound's ability to mediate host immune regulation and bacterial resistance inhibition. This study provides an important theoretical basis for further investigation of the compound's molecular mechanisms and potential therapeutic targets in these settings. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is the UV spectrum of the compound obtained in Example 1;

[0023] Figure 2 is the IR spectrum of the compound obtained in Example 1;

[0024] Figure 3 The compound obtained in Example 1 1 H-NMR spectrum (400 MHz);

[0025] Figure 4 The compound obtained in Example 1 13 C-NMR spectrum (400 MHz);

[0026] Figure 5 HSQC spectrum of the compound obtained in Example 1;

[0027] Figure 6 is the HMBC spectrum of the compound obtained in Example 1;

[0028] Figure 7 COSY spectrum of the compound obtained in Example 1;

[0029] Figure 8 is the NOESY spectrum of the compound obtained in Example 1;

[0030] Figure 9 is the TOCSY spectrum of the compound obtained in Example 1;

[0031] Figure 10-1 A map of disease-related targets; Figure 10-2 VENN diagram of compound targets and disease targets; Figure 10-3 The complete protein interaction network in which the compound acts on anti-inflammatory and anti-infective effects; Figure 10-4 The original PPI map obtained by importing the intersection targets into the STRING database; Figure 10-5 The PPI (protein-protein interaction) network of the compound and CRE E.coli intersection genes; Figure 10-6 To optimize through median selection, the core gene PPI network with high connectivity was retained; Figure 10-7 Enrichment analysis of KEGG (Kyoto Encyclopedia of Genes and Genomes) signaling pathways for compounds acting on anti-inflammatory and anti-infective functions; Figure 10-8 GO (Gene Ontology) functional enrichment analysis of compounds acting on anti-inflammatory related genes; Figure 10-9 A three-dimensional structural model of the β-secretase 1 protein into which the compound is embedded; Figure 10-10 This is an enlarged view of the compound binding site to β-secretase 1; Figure 10-11 Embed the compound into the three-dimensional structural model of the insulin-like growth factor 1 receptor protein; Figure 10-12 This is an enlarged view of the compound's binding site to the insulin-like growth factor 1 receptor; Figure 10-13 A three-dimensional structural model of the compound embedded in the monoamine oxidase B protein; Figure 10-14 This is an enlarged view of the compound's binding site to monoamine oxidase B; Figure 10-15 A three-dimensional structural model of the compound embedded in the matrix metalloproteinase-2 protein; Figure 10-16 This is an enlarged view of the compound binding site to matrix metalloproteinase 2; Figure 10-17 A three-dimensional structural model of the compound embedded in the cathepsin S protein; Figure 10-18 This is an enlarged view of the compound's binding site to cathepsin S. DETAILED DESCRIPTION

[0032] In order to make the purpose and content of the invention of this application more clear, the applicant will provide a clear and complete description of the technical solution of the present invention in conjunction with specific embodiments below.

[0033] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0034] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0035] Example 1: Isolation, preparation and structural identification of compounds

[0036] (1) Extraction of triterpenoid ferulate and sterol ferulate fractions: 200 g of oryzanol was dissolved in anhydrous ethanol and separated by reverse phase preparative high performance liquid chromatography. Fractions were collected according to time periods to obtain 12 fractions, named GWS-P1 to GWS-P12. The separation conditions were as follows:

[0037]

[0038] (2) Fraction GWS-P1 (fraction collected at 30-35 min, 1.2 g) was purified by preparative SFC chiral separation under the following conditions:

[0039]

[0040] The residue was concentrated under reduced pressure and dried to obtain a new compound (Rt=4.5 min).

[0041] Structural identification of the new compound obtained:

[0042] The compound is a white powder. The mass spectrum ESI-MS gives a molecular weight m / z: 1202.05 [2M+2H] + , its molecular formula is speculated to be C 40 H 56 O4. The UV spectrum (CHCl3) shows that the compound has strong absorption peaks at 242, 295, and 320 nm. The infrared spectrum (KBr pellet method) shows that the peak at 3528 cm -1 and 1152cm -1 It is speculated that it is the characteristic absorption peak of hydroxyl group, 2958cm -1 and 2865cm -1 It is speculated to be the characteristic absorption peak of methoxy, 1709cm -1 and 1633cm -1 It is speculated that the characteristic absorption peak of the ester group is 1604 cm -1 and 1519cm -1 It is speculated to be the characteristic absorption peak of benzene ring, 1266cm -1 and 1097cm -1 It is speculated to be the characteristic absorption peak of ether group.

[0043] After dissolving the compound in CDCl3, transfer it to a nuclear magnetic resonance tube and detect the hydrogen spectrum, carbon spectrum and two-dimensional spectrum on a nuclear magnetic resonance instrument. 1 H and 13 C-NMR and HMBC data are shown in Table 1.

[0044] Table 1: Compounds 1 H and 13 C-NMR and HMBC data

[0045]

[0046]

[0047] Example 2: Using network pharmacology methods and tools to study the medical use mechanism of the compound obtained in Example 1

[0048] 1 Experimental Methods

[0049] 1.1 Drug Target Prediction

[0050] According to the compound structure diagram, the corresponding structural formula was drawn in chemdraw23 to obtain its SMILES number and SDF file (for docking), and the compound targets were predicted in SwissTargetPrediction, totaling 100.

[0051] 1.2 Acquisition of disease-related targets

[0052] The disease target gene information was obtained by entering the disease keyword inflammation in GeneCards and filtering with a correlation score > 1. The selected disease targets were converted into standardized gene names using the Uniprot (UniProt) database. After searching the OMIM (Home-OMIM) database by entering the disease keyword inflammation, a total of 2142 relevant target genes were obtained after deduplication and merging using the VENN package in R language. Figure 10-1 ).

[0053] 1.3 Drug-Disease Target Prediction Results

[0054] After removing duplicate targets, the obtained drug component targets were compared with the disease targets using the Venn package in the Bioconductor package in R language to obtain 52 intersection genes and construct a VENN diagram ( Figure 10-2 ). The 52 intersection genes were imported into the STRING database, and the protein interaction map was constructed with a confidence threshold of ≥0.4 as the screening basis ( Figure 10-4 ), using 6 indicators including Betweenness, Closeness, Degree, Eigenvector, LAC and Network to perform cyclic median analysis and identify the key nodes in the network.

[0055] Based on these key nodes, we hypothesized that the compound could interfere with the regulation of CRE E. coli, thereby inhibiting bacterial infection. CRE E. coli, a carbapenem-resistant Enterobacteriaceae, can cause gastrointestinal disease, pneumonia, wound infections, and invasive infections of the bloodstream or other organs. Using the aforementioned method, we generated a PPI network of targets intersecting the compound and CRE E. coli, and screened for core targets.

[0056] Figure 10-5The PPI (protein-protein interaction) network of genes interacting with the compound and CRE E. coli is presented. The network contains numerous targets, demonstrating complex interactions. These genes may play important roles in bacterial infection, drug resistance, and host immune regulation. Some of these genes are closely linked to multiple pathways and may play a key regulatory role in CRE E. coli infection and compound intervention mechanisms.

[0057] Figure 10-6 The PPI network was optimized by median screening, retaining only core genes with high connectivity, forming a tighter interaction network. Figure 10-5 The network more clearly displays the key regulatory factors, among which genes such as IGF1R, CTSS, MMP2, and BCL2 may play a key role in the drug resistance regulation, cell apoptosis, and immune response of CRE E. coli infection. The intervention of compounds may achieve the inhibitory effect on bacterial infection through these genes.

[0058] This hierarchical screening analysis revealed the key regulatory gene network of the compound in CRE E. coli infection, providing an important molecular basis for further exploring its mechanism of inhibiting bacterial resistance and mediating host immune regulation.

[0059] 1.4GO and KEGG enrichment

[0060] KEGG signaling pathway enrichment analysis: Figure 10-7 The KEGG (Kyoto Encyclopedia of Genes and Genomes) signaling pathway enrichment analysis of the compounds in anti-inflammatory and anti-infective activities was presented, revealing their possible molecular mechanisms: highly enriched pathways included the PI3K-Akt signaling pathway, the MAPK signaling pathway, and the EGFR tyrosine kinase inhibition resistance pathway, indicating that the compounds may affect the anti-inflammatory and anti-infective processes by regulating cell survival, proliferation, and drug resistance mechanisms. Inflammation and immune-related pathways, such as the IL-17 signaling pathway, the HIF-1 signaling pathway, and the TNF signaling pathway, were also significantly enriched, suggesting that the compounds may reduce infection-induced inflammatory damage by regulating the host immune response. Bacterial infection-related pathways, such as apoptosis and oxidative stress response, indicate that the compounds may intervene in the cell damage process during anti-inflammatory and anti-infective activities, thereby inhibiting the development of infection.

[0061] GO function enrichment analysis: Figure 10-8The GO (Gene Ontology) functional enrichment analysis of the compounds acting on anti-inflammatory related genes was presented, covering three major categories: biological process (BP), cellular component (CC), and molecular function (MF): In terms of BP (biological process), genes are enriched in response to oxidative stress, inflammatory signal regulation, apoptosis signaling pathways, etc., indicating that the compounds may affect the infection process by regulating cell apoptosis and antioxidant defense mechanisms. In terms of CC (cellular components), genes are mainly distributed in cell membrane microdomains (membrane rafts), cytoskeleton, and signal transduction complexes, suggesting that compounds may intervene in anti-inflammatory and anti-infection by affecting membrane protein function and signal transduction. In terms of MF (molecular function), genes are enriched in protein tyrosine kinase activity, HSP90 binding, and DNA binding transcription factor activity, indicating that compounds may affect the effects of bacterial infection on host cells by regulating protein kinase signaling and transcriptional regulation.

[0062] 1.5 Molecular docking

[0063] Core targets identified after PPI screening were searched in Uniport for human-proven proteins and their corresponding IDs were obtained. The IDs were then entered into the PDB database to download the corresponding protein structure files in PDB format. The protein structures were then imported into Autodock for dehydrogenation optimization. Molecular docking was then performed using Autodock VINA with the ligand compound structure files optimized for minimum free energy using the CHEM3D23 MM2 force field. Finally, the top five ligand-protein pairs with the highest binding energies were selected for visualization using Pymol.

[0064] Figure 10-9 The three-dimensional structural model of the BACE1 (β-secretase 1) protein is shown. The protein is presented in light purple and consists of multiple α-helices and β-folds, forming a stable conformation. At the binding site on the right side of the protein, the compound (yellow stick-shaped structure) can be clearly observed embedded in the active pocket of BACE1 and forming specific interactions with surrounding amino acid residues. This binding site is located in the catalytic core region of BACE1, where the compound forms a hydrogen bond interaction with GLU-134 (marked in green).

[0065] Figure 10-10 A magnified view of the BACE1-compound binding site is provided, further revealing the interaction pattern of molecular docking. In this view, the compound (yellow) forms a hydrogen bond interaction (indicated by the yellow dashed line) with GLU-134 (green residue) with an interaction distance of These hydrogen-bonding interactions may play an important role in stabilizing the binding of compounds to BACE1 and may affect the enzymatic activity of BACE1.

[0066] Figure 10-11The three-dimensional structural model of the IGF1R (insulin-like growth factor 1 receptor) protein is shown. The protein is presented in light purple and consists of multiple α-helices and β-folds, forming a stable conformation. At the right binding site of the protein, it can be clearly observed that the compound (yellow stick structure) is embedded in the active pocket of IGF1R and forms a specific interaction with the key amino acid residues. The compound forms a hydrogen bond interaction with PHE-418 (amino acid marked in green) at a distance of It may play an important role in stabilizing the binding of compounds to IGF1R.

[0067] Figure 10-12 A magnified view of the IGF1R-compound binding site is provided, showing the spatial relationship of the compound (yellow) to the IGF1R active pocket. Although it interacts directly with the IGF1R through only one hydrogen bond, the compound's position suggests that its binding to the IGF1R may be stabilized by non-covalent forces, such as hydrophobic interactions.

[0068] Figure 10-13 The three-dimensional structural model of the MAOB (monoamine oxidase B) protein is shown. The protein, shown in light purple, is composed of multiple α-helices and β-sheets, forming a stable globular conformation. At the binding site on the right side of the protein, the compound (yellow stick-like structure) can be clearly observed embedded in the active pocket of MAOB. Despite the lack of obvious hydrogen bonding, the compound still forms a certain spatial coordination with the MAOB active site.

[0069] Figure 10-14 A magnified view of the MAOB-compound binding site is provided, showing the spatial relationship of the compound (yellow) to the MAOB active pocket. The position of the compound suggests that its binding to MAOB may be stabilized by non-covalent forces, such as hydrophobic interactions.

[0070] Figure 10-15 The three-dimensional structural model of the MMP2 (matrix metalloproteinase 2) protein is shown. The protein is presented in light purple and consists of multiple α-helices and β-folds, forming a stable globular conformation. At the right binding site of the protein, it can be clearly observed that the compound (yellow stick structure) is embedded in the active pocket of MMP2 and forms a specific interaction with the key amino acid residues. The compound forms a hydrogen bond interaction with ALA-402 (amino acid marked in green) with a distance of This may play an important role in stabilizing the binding of the compound to MMP2.

[0071] Figure 10-16A magnified view of the MMP2-compound binding site is provided, showing the spatial relationship of the compound (yellow) to the MMP2 active pocket. The position of the compound suggests that it may stably bind to MMP2 through non-covalent interactions, such as hydrophobic interactions.

[0072] Figure 10-17 The three-dimensional structural model of the CTSS (cathepsin S) protein is shown. The protein is presented in light purple and is composed of multiple α-helices and β-sheets, forming a stable globular conformation. At the binding site on the right side of the protein, the compound (yellow stick-like structure) can be clearly observed embedded in the active pocket of CTSS. Although there is no obvious hydrogen bonding, the compound still forms a certain spatial coordination with the active site of CTSS.

[0073] Figure 10-18 A magnified view of the CTSS-compound binding site is provided, showing the spatial relationship of the compound (yellow) to the active pocket of the CTSS. Although no direct hydrogen bonds or other obvious interactions are shown, the position of the compound suggests that it may have non-covalent hydrophobic or other weak interactions with the CTSS domain.

Claims

1. A method for preparing a triterpene alcohol ferulic acid ester compound, characterized in that: The chemical structural formula of the triterpene alcohol ferulate compound is: The preparation method comprises the following steps: (1) Oryzanol was dissolved in anhydrous ethanol and separated by reverse phase preparative high performance liquid chromatography. Fractions were collected according to time periods to obtain 12 fractions, named GWS-P1 to GWS-P12. The separation conditions were as follows: (2) Fraction GWS-P1 (fraction collected at 30-35 min) was purified by preparative SFC chiral separation under the following conditions: The component with a retention time of 4.5 min was collected, concentrated under reduced pressure, and dried to obtain the triterpene alcohol ferulic acid ester compound.

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