Use of corydalis hendersoniana in the preparation of a medicine for treating liver fibrosis
Patent Information
- Application Number
- CN202510543778.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-28
- Publication Date
- 2026-10-30
AI Technical Summary
只有当上述病因完全消失后，肝纤维化才会发生自身逆转；而临床上仅靠病因治疗，大多数情况无法有效控制肝纤维化的发展
[0027]本发明首次发现毛黄堇的总提取物、总生物碱提取物、总生物碱、式I所示化合物可用于治疗肝纤维化，具体是可有缓解肝肿大、减少胶原等细胞外基质沉淀、改善转氨酶ALT和AST升高、改善肝功能，降低促炎细胞因子IL-1β、IL-6、TNF-α、TGF-β1、MCP-1，提升抗炎细胞因子IL-10水平的效果，通过调节PPAR信号通路来进行对肝脏代谢能力的调节与平衡，从而达到减轻肝星状细胞活化的效果，进而达到其治疗作用，具有广泛应用前景。
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Abstract
Description
Technical Field
[0001] This invention relates to the field of pharmaceutical technology, specifically to the application of Corydalis yanhusuo in the preparation of drugs for treating liver fibrosis. Background Technology
[0002] Hepatic fibrosis (HF) is a process in which hepatocytes are repeatedly damaged and regenerated during the development of chronic liver disease. It involves the diffuse excessive deposition and abnormal distribution of extracellular matrix such as collagen, glycoproteins, and proteoglycans in the liver. It is a pathological repair response of the liver to chronic damage and a key step in the progression of various chronic liver diseases to cirrhosis, as well as an important factor affecting the prognosis of chronic liver diseases.
[0003] Liver fibrosis (HF) is a pathophysiological process referring to the abnormal proliferation of connective tissue in the liver caused by various pathogenic factors. Specifically, it involves diffuse, excessive deposition of extracellular matrix (mainly collagen) within the liver, leading to interstitial fibrosis. Pathologically, it manifests as the formation of fibrous tissue and destruction of lobular structure within the liver.
[0004] In clinical practice, conventional treatment for liver fibrosis (HF) primarily targets the underlying causes such as liver injury, viral hepatitis, and fatty liver, rather than addressing the core process of liver fibrosis—the proliferation and activation of hepatic stellate cells (HSCs). Furthermore, the persistence of these causes continues to drive the progression of liver fibrosis. Only when these causes are completely eliminated can liver fibrosis spontaneously reverse; however, in most cases, treating the underlying cause alone is insufficient to effectively control the development of liver fibrosis. Therefore, current clinical medications cannot effectively reverse or halt the progression of liver fibrosis, necessitating targeted treatment.
[0005] Corydalis tomentella, also known as dried rockwort, is the dried aerial parts or dried whole herb of the plant Corydalis tomentella, belonging to the Papaveraceae family. Corydalis tomentella has a long history of medicinal use in my country. The whole herb is bitter and cool in nature. It is used to remove blood stasis, relieve pain, cool the blood, and stop bleeding. It is used for traumatic injuries, joint pain, hemoptysis, and hematemesis due to overexertion. However, there are no reports of the application of Corydalis tomentella extract in the preparation of drugs for treating liver fibrosis. Summary of the Invention
[0006] The present invention aims to provide the application of Corydalis yanhusuo in the preparation of a drug for treating liver fibrosis.
[0007] To achieve the above objectives, the present invention adopts the following technical solution:
[0008] The application of a species of Corydalis in the preparation of a drug for treating liver fibrosis.
[0009] The use of Corydalis yanhusuo or its extract in the preparation of a drug for treating liver fibrosis.
[0010] The term "hairy yellow violet" refers to the dried aerial parts, roots, or dried whole herb of *Corydalis tomentella*, a plant belonging to the Papaveraceae family.
[0011] The extract is one or more of the following: total extract, alkaloid extract, total alkaloids, or a single substance obtained by purifying the aforementioned extracts from Corydalis yanhusuo.
[0012] The total extract is obtained by taking the dried aerial parts, roots or whole herb of Corydalis pubescens, cutting them into small pieces, soaking them in an alcohol solution, heating and refluxing to extract them multiple times until the extract is relatively clear, combining the extracts and concentrating them into an extract, which is the total extract of Corydalis pubescens.
[0013] The total alkaloid extract of Corydalis yanhusuo is obtained by dissolving the total extract obtained above in water, extracting it with dichloromethane or ethyl acetate, recovering the solvent from the extract, and concentrating the solvent to obtain an extract.
[0014] The total alkaloids of Corydalis yanhusuo are obtained by dissolving the total extract obtained above in a dilute acid solution, extracting with dichloromethane or ethyl acetate, adjusting the pH of the acidic aqueous layer to alkaline, extracting with dichloromethane or ethyl acetate, and concentrating the extract to obtain a paste.
[0015] A monomeric compound obtained from the aforementioned Corydalis rhamnoides, the compound being dehydrocavidine, has the structural formula shown in Formula I.
[0016]
[0017] A method for preparing the compound described above:
[0018] The total extract of Corydalis rhamnoides was extracted with dichloromethane to obtain the extract, or the total alkaloids of Corydalis rhamnoides obtained above were subjected to silica gel column chromatography with gradient elution of dichloromethane / methanol. The 5:1 eluted fraction was further subjected to ODS column chromatography with methanol / water elution. The 40% methanol / water eluent was further solvent-treated to obtain the compound dehydrocavidine shown in Formula I.
[0019] An application of the compound, specifically its use in the preparation of a medicament for treating liver fibrosis.
[0020] A pharmaceutical composition comprising one or more of the following: a total extract of Corydalis yanhusuo, a total alkaloid extract, total alkaloids, or compounds represented by Formula I.
[0021] A pharmaceutical preparation comprising a total extract of Corydalis yanhusuo, a total alkaloid extract, a total alkaloid, a compound of Formula I, or a combination thereof as an active ingredient, and a pharmaceutically acceptable carrier thereof; wherein the active ingredient comprises 0.1%-99% of the mass of the preparation.
[0022] The dosage form of the pharmaceutical preparation is liquid, solid, semi-solid, or gaseous.
[0023] Liquid dosage forms include compound preparations, oral liquids, tinctures, syrups, and injections; solid dosage forms include powders, granules, tablets, capsules, drop pills, films, suppositories, and microcapsules; semi-solid dosage forms include ointments, pastes, extracts, and licks; and gaseous dosage forms include aerosols and inhalers.
[0024] This invention proposes that the total extract, total alkaloid extract, total alkaloids, and compounds shown in Formula I of Corydalis yanhusuo have the effect of inhibiting the proliferation and activation of hepatic stellate cells; and all of them have the effect of inhibiting the expression of α-SMA and Collagen1α1; at the same time, they can reduce the liver coefficient, eliminate hepatomegaly, and improve the deposition of extracellular matrix such as collagen caused by liver fibrosis.
[0025] In addition, the total extracts, total alkaloid extracts, total alkaloids, and compounds shown in Formula I of *Corydalis yanhusuo* all have the effect of improving liver function, reducing elevated transaminases ALT and AST caused by liver fibrosis, and can also improve the inflammatory response caused by liver fibrosis, reduce serum pro-inflammatory cytokines IL-1β, IL-6, TNF-α, TGF-β1, and MCP-1, and increase the level of anti-inflammatory cytokine IL-10. Furthermore, they can regulate and balance liver metabolism by modulating the PPAR signaling pathway, thereby reducing hepatic stellate cell activation. Therefore, they have the potential for use in the preparation of drugs for treating liver fibrosis.
[0026] Advantages of this invention:
[0027] This invention is the first to discover that the total extract, total alkaloid extract, total alkaloids, and compounds shown in Formula I of *Corydalis pubescens* can be used to treat liver fibrosis. Specifically, it can alleviate hepatomegaly, reduce the deposition of extracellular matrix such as collagen, improve elevated transaminases ALT and AST, improve liver function, reduce pro-inflammatory cytokines IL-1β, IL-6, TNF-α, TGF-β1, and MCP-1, and increase the level of anti-inflammatory cytokine IL-10. By regulating the PPAR signaling pathway, it regulates and balances the liver's metabolic capacity, thereby reducing the activation of hepatic stellate cells and achieving its therapeutic effect, which has broad application prospects. Attached Figure Description
[0028] Figure 1The hydrogen spectrum of the monomer compound dehydrocavidine in Example 1 of this invention;
[0029] Figure 2 This is the carbon spectrum of the monomer compound dehydrocavidine in Example 1 of the present invention;
[0030] Figure 3 This is a schematic diagram illustrating the effect of Corydalis yanhusuo extract on HSC cell proliferation in Example 3 of the present invention;
[0031] Figure 4 This is a schematic diagram illustrating the effect of Corydalis extract on the mRNA expression of Collagen1α1, an activation indicator of HSC cells, in Example 3 of the present invention.
[0032] Figure 5 This is a schematic diagram illustrating the effect of Corydalis extract on the mRNA expression of α-SMA, an activation indicator of HSC cells, in Example 3 of the present invention.
[0033] Figure 6 A schematic diagram illustrating the effect of the active monomer compound Dehydrocavidine from Corydalis yanhusuo on the protein expression of HSC cell activation indicators Collagen1α1 and α-SMA in Example 4 of this invention;
[0034] Figure 7 This is a schematic diagram illustrating the effect of Corydalis extract on liver coefficient in HF mice in Example 4 of the present invention;
[0035] Figure 8 This is a schematic diagram illustrating the effects of Corydalis extract on collagen deposition in the liver of HF mice and the protein expression of Collagen1α1 and α-SMA, indicators of HSC cell activation in vivo, in Example 4 of the present invention.
[0036] Figure 9 This is a schematic diagram illustrating the effect of Corydalis extract on ALT and AST liver function indicators in HF mice in Example 4 of the present invention.
[0037] Figure 10 This is a schematic diagram illustrating the effects of Corydalis yanhusuo extract on inflammatory markers IL-1β, IL-6, TNF-α, TGF-β1, MCP-1, and IL-10 in HF mice in Example 4 of the present invention.
[0038] Figure 11 This is a schematic diagram illustrating the effects of the active monomer compound Dehydrocavidine of Corydalis yanhusuo on liver gene expression and signaling pathways in HF mice in Example 4 of the present invention. Detailed Implementation
[0039] The application of Corydalis extract in the treatment of liver fibrosis is described in detail below through specific examples.
[0040] The Corydalis extract of this invention is extracted according to the extraction method of traditional Chinese medicine as recorded in "Extraction and Separation Technology of Traditional Chinese Medicine"; for example, the raw material (Corydalis) is extracted with solvents such as water and alcohol to obtain the extract, and the extracted components are not changed due to different extraction solvents; furthermore, in the following embodiments, the Corydalis extract can be obtained in the following manner: take the dried whole herb, root, or aerial parts of Corydalis, cut them into pieces, and soak them in a solvent (such as water, organic solvent, etc.) with a mass of 10-15 times its weight to extract, thereby obtaining the Corydalis extract and the monomeric compound dehydrocavidine extracted from the raw material, which are ready for use.
[0041] Example 1
[0042] The extraction method for obtaining the total extract of Corydalis pubescens is as follows:
[0043] Take 50 kg of dried whole herb of Corydalis pubescens, cut it into pieces, soak it in 750 L of ethanol and heat it under reflux to extract it three times until the extract is relatively clear. Combine the extracts and concentrate them into an extract, which is the total extract of Corydalis pubescens.
[0044] The total extract obtained above was dissolved in water and extracted three times with dichloromethane. The dichloromethane layer solution was recovered, combined and concentrated into an extract, which is the total alkaloid extract of Corydalis pubescens.
[0045] The total extract obtained above was dissolved in a 1% acid solution, extracted three times with ethyl acetate, and the combined aqueous layer was recovered and the pH was adjusted to 11. After adjustment, it was extracted with dichloromethane, and the dichloromethane layer solution was collected and concentrated to an extract, which is the total alkaloid extract of Corydalis pubescens.
[0046] The total extract obtained above was dissolved in water and extracted with dichloromethane. The dichloromethane layer extract was collected, and the extract was eluted onto a silica gel column (12×80cm) using a gradient elution of 20–5:1 (v / v) dichloromethane / methanol. The fraction with a dichloromethane / methanol ratio of 5:1 was collected. The collected fraction was then eluted with an ODS column using 40% methanol / water. The precipitate under the 40% methanol / water elution solvent was washed with methanol to obtain the compound shown in Formula I. Structural identification information can be found in [link to relevant documentation]. Figure 1 and 2 .
[0047]
[0048] 16,17-dimethoxy-12-methyl-5,7-dioxa-1-azoniapentacyclo[11.8.0.03,11.04,8.014,19]henicosa-1(13),2,4(8),9,11,14,16,18-octaene
[0049] (Dehydrocavidine)
[0050] Example 2 Formulation
[0051] The total extract, total alkaloid extract, total alkaloids, and compounds of Formula I obtained from the above embodiments are used as active ingredients and are formulated into pharmaceutical preparations with pharmaceutically acceptable carriers, excipients, or pharmaceutically acceptable excipients.
[0052] The dosage form of the pharmaceutical preparation is liquid, solid, etc.
[0053] Liquid dosage forms include oral liquids and injections; solid dosage forms include granules, tablets, and capsules.
[0054] 2.1 Preparation of oral liquid
[0055] Take 1g of the fine powder of total extract of Corydalis yanhusuo, dissolve it in distilled water, adjust the pH value to 7.5 with 2% sodium bicarbonate solution, add no more than 0.05% of potassium sorbate as a preservative in the total solvent, and then fill and sterilize.
[0056] Take 1g of fine powder of total alkaloid extract of Corydalis yanhusuo, dissolve it in distilled water, adjust the pH value to 7.5 with 2% sodium bicarbonate solution, add no more than 0.05% of potassium sorbate as a preservative in the total solvent, and then fill and sterilize.
[0057] Take 1g of the monomeric compound dehydrocavidine, dissolve it in distilled water, adjust the pH value to 7.5 with 2% sodium bicarbonate solution, add no more than 0.05% of potassium sorbate as a preservative in the total solvent, and then fill and sterilize.
[0058] 2.2 Tablet Preparation
[0059] Take 1g of total extract of Corydalis yanhusuo, make it into fine powder, add 4g of starch paste as binder and 4g of starch as diluent, and compress into tablets.
[0060] Take 1g of total alkaloid extract of Corydalis yanhusuo, make it into fine powder, add 4g of starch paste as binder and 4g of starch as diluent, and compress into tablets.
[0061] Take 1g of the monomeric compound dehydrocavidine, make it into a fine powder, add 4g of starch paste as a binder and 4g of starch as a diluent, and compress it into tablets.
[0062] Example 3: In vitro efficacy verification experiment of Corydalis yanhusuo
[0063] 3.1 Anti-HSC cell proliferation experiment
[0064] The anti-HSC cell proliferation activity was evaluated by inducing human HSC cell line LX-2 or mouse HSC cell line HSC-T6 with TGF-β1.
[0065] Take 5 × 10⁻⁶ cells cultured to the logarithmic growth phase and inoculate them separately. 4 Cells were seeded at 100 cells / mL (96-well plate) and cultured without FBS for 6 h until cell adhesion. Cells were then treated with TGF-β1 (10 ng / mL) to induce fibrosis. Each cell type was divided into 8 groups (3 wells per group): control group, model group (TGF-β1: 10 ng / mL), high-dose (ZT-H), low-dose (ZT-L) groups of total extract of Corydalis yanhusuo (ZT-L: 1 mg / mL, ZT-H: 2 mg / mL, TGF-β1), high-dose (ZJ-H), low-dose (ZJ-L: 1 mg / mL, ZJ-H: 2 mg / mL, TGF-β1) groups of total alkaloid extract of Corydalis yanhusuo (ZJ-H), and high-dose (DC-H), low-dose (DC-L) groups of the active monomeric compound Dehydrocavidine (ZJ-L: 4 μM, ZJ-H: 8 μM, TGF-β). All doses were administered at safe concentrations. The cytotoxicity of ZT and ZJ cells in each group was initially assessed at concentrations ranging from 0.1 to 10 mg / mL. The cytotoxicity of DC cells was initially assessed at concentrations ranging from 1 to 100 μM. The cell-killing activity of DC cells was detected using a cell counting kit (CCK-8 assay), see [link to documentation]. Figure 3 .
[0066] Depend on Figure 3 It can be seen that, compared with the control group, the proliferation capacity of HSC cells in the TGF-β1 model group was significantly increased. #### P < 0.0005); Compared with the model group, the proliferation capacity of HSC cells in each dose group was significantly decreased (**P < 0.05). This shows that various extracts and active monomeric compounds of Corydalis yanhusuo have a significant inhibitory effect on the proliferation capacity of HSC cells and have significant anti-liver fibrosis activity.
[0067] 3.2 Anti-HSC cell activation assay
[0068] The anti-HSC cell proliferation activity was evaluated by inducing LX-2 or HSC-T6 cell lines with TGF-β1.
[0069] Cells cultured to the logarithmic growth phase were taken separately at 5 × 10⁻⁶ cells per cell. 4Cells were seeded at 100 cells / mL (6-well plate) and cultured without FBS for 6 h until cell adhesion. The DC modeling method and drug dosage were the same as in "3.1 Anti-HSC Cell Proliferation Assay". Finally, protein and mRNA were collected from cell samples. The expression of α-SMA and Collagen1α1 mRNA and protein was examined by RT-PCR and Western Blot, see [link to relevant documentation]. Figure 4 , Figure 5 and Figure 6 .
[0070] Depend on Figure 4 It can be seen that, compared with the control group, the mRNA expression of Collagen1α1 in HSC cells of the TGF-β1 model group was significantly increased. #### P < 0.0005; Compared with the model group, the expression of Collagen1α1 in HSC cells was significantly decreased in each dose group (**P < 0.05). Figure 5 It can be seen that, compared with the control group, the expression of α-SMA in HSC cells of the TGF-β1 model group was significantly increased. #### P < 0.0005); Compared with the model group, the mRNA expression of α-SMA in HSC cells was significantly decreased in each dose group (**P < 0.05). Figure 6 It can be seen that, compared with the control group, the expression of α-SMA in HSC cells of the TGF-β1 model group was significantly increased. ### P < 0.005); Compared with the model group, the protein expression of α-SMA in HSC cells was significantly decreased in the high-dose monomer compound group (**P < 0.05). These results show that various extracts of Corydalis rhamnoides and the obtained monomer compounds can significantly reduce the expression of HSC cell activation markers Collagen1α1 and α-SMA, significantly inhibit the activation ability of HSC cells, and have significant anti-hepatic fibrosis activity.
[0071] Example 4: In vivo efficacy verification experiment of Corydalis yanhusuo
[0072] 4.1 Laboratory Animals and Materials
[0073] Experimental animals: 6-8 week old C57BL / 6J mice.
[0074] Experimental materials: carbon tetrachloride (CCl4), corn oil, silymarin, extracts and active monomers obtained from various sources in Example 1, paraformaldehyde, ethanol, Sirius red staining kit, Masson staining kit, Collagen 1α1 immunohistochemical staining kit, α-SMA immunohistochemical staining kit, enzyme-linked immunosorbent assay (ELISA) kit, RNAmini kit.
[0075] 4.2 Liver fibrosis model and drug administration grouping
[0076] Fifty-four male C57BL / 6J mice were acclimatized for one week and then randomly divided into nine groups (n=6) using a random number table: blank control group, model group, positive control (silymarin), high (ZT-H) and low (ZT-L) dose groups of total extract of Corydalis yanhusuo (ZT-L: 1 mg / kg, ZT-H: 2 mg / kg), high (ZJ-H) and low (ZJ-L) dose groups of total alkaloid extract of Corydalis yanhusuo (ZJ-L: 1 mg / kg, ZJ-H: 2 mg / kg), and high (DC-H) and low (DC-L) dose groups of the active monomer compound Dehydrocavidine (DC-L: 1 mg / kg, DC-H: 2 mg / kg).
[0077] During the experiment, except for the blank control group which was given distilled water, the other groups were injected intraperitoneally twice a week for 8 consecutive weeks with a corn oil solution containing 20% CCl4 (2 mL / kg) to induce the HF mouse model. After 28 days of continuous drug administration, the weights of the mice in each group before and after administration were weighed and marked, and then the results of each group were verified.
[0078] 4.3 Liver examination
[0079] To verify the therapeutic effect of *Corydalis yanhusuo* extract on CCl4-corn oil-induced liver fibrosis in mice, this invention evaluated mouse livers using the above-mentioned experiments, examining liver size. Mice were weighed and dissected, liver tissue was removed and weighed, and the liver coefficient was calculated as (liver weight / mouse body weight). See [reference needed]. Figure 7 .
[0080] Depend on Figure 7 It can be seen that, compared with the control group, the liver coefficient in the CCl4 corn oil model group was significantly increased. #### P < 0.0005); Compared with the model group, the liver coefficient in each high-dose group was significantly lower (P < 0.0005), which was better than the positive control group. This shows that Corydalis extract has a relieving effect on hepatomegaly caused by liver fibrosis and has significant anti-liver fibrosis activity.
[0081] 4.3 Pathological staining examination
[0082] Liver tissues from mice in each group were soaked in 4% paraformaldehyde for 48 hours, dehydrated, embedded in paraffin, and cut into 4 μm sections. The tissue sections were then dewaxed, hydrated, and stained according to the manufacturer's instructions using Sirius red staining kit, Masson staining kit, Collagen 1α1 immunohistochemical staining kit, and α-SMA immunohistochemical staining kit, respectively. The sections were observed under an Olympus optical microscope (Olympus, Tokyo, Japan, ×200). See [link to Olympus optical microscope]. Figure 8 .
[0083] Depend on Figure 8 As can be seen, compared with the control group, the Sirius red staining positive area and Masson staining positive area in the liver tissue of mice in the CCl4 corn oil model group were significantly larger. #### P < 0.0005; Compared with the model group, the Sirius red and Masson staining positive areas in the liver tissue of HF mice in each high-dose group were significantly reduced (P < 0.0005), which was better than the positive control drug; Compared with the control group, the Collagen 1α1 immunohistochemical staining positive area and α-SMA immunohistochemical staining positive area in the liver tissue of mice in the CCl4 corn oil model group were significantly increased (P < 0.0005); #### P < 0.0005; Compared with the model group, the areas of Collagen1α1 and α-SMA immunohistochemical staining positive in the liver tissue of HF mice in each dose group were significantly reduced (P < 0.0005). This shows that Corydalis extract can improve the pathological damage of liver tissue in mice with liver fibrosis, reduce collagen deposition and scar formation, inhibit HSC cell activation, and reduce the expression of HSC cell activation markers Collagen1α1 and α-SMA, and has significant anti-liver fibrosis activity.
[0084] 4.5 ELISA detection of serum transaminase levels
[0085] The levels of transaminases AST and ALT in the serum of mice in each group were measured using the ELISA method according to the kit instructions. (See attached image.) Figure 9 .
[0086] Transaminases AST and ALT are important indicators of liver function; elevated AST and ALT levels indicate abnormal liver function.
[0087] Depend on Figure 9 As can be seen, ELISA detection showed that compared with the control group, the serum AST and ALT levels in the CCl4 corn oil model group were significantly increased. #### P < 0.0005. Compared with the model group, the AST and ALT levels in mice in each high-dose group were significantly reduced. ****P < 0.0005, which fully demonstrates that the various extracts and active monomers of Corydalis yanhusuo obtained can effectively improve liver function abnormalities caused by liver fibrosis.
[0088] 4.6 ELISA detection of serum inflammatory factor levels
[0089] The levels of inflammatory factors IL-1β, IL-6, TNF-α, TGF-β1, MCP-1, and IL-10 in the serum of mice in each group were detected using ELISA according to the kit instructions. (See attached image.) Figure 10 .
[0090] IL-1β, IL-6, TNF-α, TGF-β1, and MCP-1 are important pro-inflammatory cytokines; their production further promotes the inflammatory response and triggers inflammation. IL-10 is an important anti-inflammatory cytokine; its production can inhibit the inflammatory response and eliminate inflammation.
[0091] Depend on Figure 10 As can be seen, serum IL-1β, IL-6, TNF-α, TGF-β1, MCP-1, and IL-10 levels were measured by ELISA. Compared with the control group, the CCl4-corn oil model group mice showed significantly increased levels of IL-1β, IL-6, TNF-α, TGF-β1, and MCP-1. #### P < 0.0005, IL-10 levels decreased significantly ( #### P < 0.0005. Compared with the model group, the levels of IL-1β, IL-6, TNF-α, TGF-β1, and MCP-1 in mice in each high-dose group were significantly reduced. **** P < 0.0005, L-10 levels were significantly elevated ( **** P < 0.0005. The results showed that various extracts and active monomers of Corydalis yanhusuo could significantly inhibit the inflammatory response in mice with liver fibrosis, exert anti-inflammatory effects, and alleviate the symptoms of liver fibrosis.
[0092] 4.7 Transcriptomics Detection and Analysis
[0093] Total RNA was isolated from each liver homogenate sample in each of the above groups using the RNAmini kit. RNA quality was assessed by gel electrophoresis and quantum bit analysis (Thermo, Waltham, MA, USA). For RNA sequencing, RNA samples obtained from 7 to 9 biological replicates at each time point (0, 12, 36, and 72 h) were isolated into three independent pools, each containing two or three equal numbers of different samples. Strand-specific libraries were constructed using the TruSeq RNA Sample Preparation Kit (Illumina, San Diego, CA, USA), and sequencing was performed using an Illumina Novaseq 6000 instrument. Raw data were processed using Skewer for quality checks. Read lengths were 2 × 150 bp. Alignment with the human genome hg38 was performed using STAR. Transcript expression was calculated using Perl via FPKM (Fragments Per Kilobase of exon model Per Million mapped reads). Differentially expressed transcripts (DETs) were determined using a MA plot-based method with a random sampling (MARS) model in the DEGseq package between different time points (12hpt vs. 0hpt, 36hpt vs. 0hpt, 72hpt vs. 0hpt). The threshold for determining DETs was P < 0.05, with an absolute fold change ≥ 2. DETs were then selected using the GO and KEGG databases for functional and pathway enrichment analysis. Significantly enriched pathways were identified when P < 0.05, and these pathways contained at least two related genes. (See [link to documentation]). Figure 11 .
[0094] Depend on Figure 11 It was observed that after treatment with dehydrocavidine, an active monomeric compound from Corydalis yanhusuo, gene transcriptional expression in the liver of mice was similar to that of the control group, but significantly different from that of the CCl4-corn oil model group. A total of 895 differentially expressed genes were identified. KEGG enrichment analysis of these genes revealed multiple metabolic pathways and the PPAR signaling pathway. The PPAR signaling pathway has the ability to regulate other metabolic pathways and can regulate the proliferation and differentiation of HSC cells.
[0095] The above indicates that the active monomeric compounds in Corydalis yanhusuo can alleviate liver metabolic dysfunction caused by liver fibrosis and inhibit the proliferation and differentiation of HSC cells through the PPAR signaling pathway, thereby exerting anti-liver fibrosis activity.
[0096] The above four examples demonstrate that various extracts and active monomers of *Corydalis zebrina* exhibit good anti-hepatic fibrosis activity. They can inhibit the proliferation and activation of hepatic stellate cells by regulating the PPAR signaling pathway, thus treating liver fibrosis. Furthermore, these extracts and active monomers can effectively improve symptoms such as hepatomegaly, abnormal liver function, and inflammatory responses caused by liver fibrosis. *Corydalis zebrina* shows promising application prospects in the treatment of liver fibrosis.
Claims
1. The application of Corydalis yanhusuo in the preparation of a drug for treating liver fibrosis.
2. The application according to claim 1, characterized in that: The use of Corydalis yanhusuo or its extract in the preparation of a drug for treating liver fibrosis.
3. The application according to claim 1, characterized in that: The term "hairy yellow violet" refers to the dried aerial parts, roots, or dried whole herb of *Corydalis tomentella*, a plant belonging to the Papaveraceae family.
4. The application according to any one of claims 1-3, characterized in that: The extract is one or more of the following: total extract, alkaloid extract, total alkaloids, or a single substance obtained by purifying the aforementioned extracts from Corydalis yanhusuo.
5. The application according to claim 4, characterized in that: The total extract is obtained by taking the dried aerial parts, roots or whole herb of Corydalis pubescens, cutting them into small pieces, soaking them in an alcohol solution, heating and refluxing to extract them multiple times until the extract is relatively clear, combining the extracts and concentrating them into an extract, which is the total extract of Corydalis pubescens. The total alkaloid extract of Corydalis yanhusuo is obtained by dissolving the total extract obtained above in water, extracting it with dichloromethane or ethyl acetate, recovering the solvent from the extract, and concentrating the solvent to obtain an extract. The total alkaloids of Corydalis yanhusuo are obtained by dissolving the total extract obtained above in a dilute acid solution, extracting with dichloromethane or ethyl acetate, adjusting the pH of the acidic aqueous layer to alkaline, extracting with dichloromethane or ethyl acetate, and concentrating the extract to obtain a paste.
6. A monomeric compound extracted from Corydalis yanhusuo according to claim 1, characterized in that: The compound is dehydrocavidine, and its structural formula is shown in Formula I.
7. A method for preparing the compound according to claim 6, characterized in that: The total extract of Corydalis rhamnoides was extracted with dichloromethane to obtain the extract, or the total alkaloids of Corydalis rhamnoides obtained above were subjected to silica gel column chromatography with gradient elution of dichloromethane / methanol. The 5:1 eluted fraction was further subjected to ODS column chromatography with methanol / water elution. The 40% methanol / water eluent was further solvent-treated to obtain the compound dehydrocavidine shown in Formula I.
8. An application of the compound according to claim 6, characterized in that: The compound is used in the preparation of drugs for treating liver fibrosis.
9. A pharmaceutical composition, characterized in that: The composition contains one or more of the following: total extract of Corydalis yanhusuo, total alkaloid extract, total alkaloids, or compounds represented by Formula I.
10. A pharmaceutical preparation, characterized in that: The formulation comprises a pharmaceutical preparation with a total extract of Corydalis pubescens, a total alkaloid extract, a total alkaloid, a compound of Formula I, or a composition of claim 9 as the active ingredient, and a pharmaceutically acceptable carrier; wherein the active ingredient accounts for 0.1%-99% of the mass of the formulation.