A withanolide compound and a preparation method and application thereof
By extracting the solanine lactone compound withagulide F from bitter gourd, a drug for inhibiting the proliferation and activation of hepatic stellate cells was prepared, solving the problem of irreversible liver fibrosis in existing technologies and achieving a significant inhibitory effect on liver fibrosis.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- GUIZHOU MEDICAL UNIV
- Filing Date
- 2023-11-07
- Publication Date
- 2026-05-12
AI Technical Summary
The lack of effective drugs in the current technology to inhibit the activation and proliferation of hepatic stellate cells and promote their apoptosis makes liver fibrosis difficult to reverse.
Withagulide F, a lactone compound extracted and purified from bitter gourd, was used to prepare a drug that inhibits the proliferation and activation of hepatic stellate cells, promotes their apoptosis, and achieves anti-liver fibrosis effects by inhibiting α-SMA and collagen expression.
withagulide F significantly reduced the expression of α-SMA and collagen in hepatic stellate cells, inhibited their proliferation, induced apoptosis, and exhibited dose-dependent effects, demonstrating a good inhibitory effect on liver fibrosis.
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Figure CN117510569B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of pharmaceutical technology, specifically to a nightshade lactone compound, its preparation method, and its application. Background Technology
[0002] Liver fibrosis is a compensatory response during tissue repair following liver inflammation or injury caused by various pathogenic factors (such as HBV, HCV, and alcohol). It is a common pathological process in many chronic liver diseases. Liver fibrosis is characterized by hepatocyte damage, which activates hepatic stellate cells (HSCs) through paracrine signals, leading to the synthesis and secretion of large amounts of various extracellular matrix (ECM) components, including type I collagen, type III collagen, and fibronectin, which accumulate extensively in the liver. The continued development of liver fibrosis can lead to cirrhosis and even liver cancer, seriously threatening the patient's life and health.
[0003] Therefore, finding new ways to inhibit HSC activation and proliferation and promote HSC apoptosis has become an important approach and research direction for slowing down and reversing liver fibrosis. Summary of the Invention
[0004] In order to solve the above-mentioned technical problems in the prior art, the present invention provides a nightshade lactone compound, its preparation method and application.
[0005] Withagulide F, a sodium lycoside compound, has the following structural formula:
[0006]
[0007] Furthermore, the chemical name of the solanaceous lactone compound withagulide F is (17S,20R,22R)-18,20-epoxy 12β,14α,15β,28-tetrahydroxy-1-oxowitha-3,5,24-trienolide, and its molecular formula is C2. 28 H 34 O9 has a molecular weight of 514.57.
[0008] The method for preparing the solanaceous lactone compound withagulide F is characterized by the following specific steps:
[0009] The whole herb of *Smilax china* was pulverized and extracted twice with 30 L of 95% ethanol for 5 days each time. The extracts were combined and concentrated under reduced pressure to obtain an extract. The extract was suspended in 3 L of deionized water and extracted three times with 4 L of petroleum ether and chloroform each time to obtain a chloroform extract. The extract was then subjected to macroporous resin column chromatography with a methanol / water gradient elution (volume ratios of 30:70, 45:55, 60:40, 80:20, and 100:0) to obtain five fractions (F1-F5). F3 was further separated by silica gel column chromatography with a dichloromethane / methanol gradient elution (volume ratios of 50:1, 30:1, 20:1, 10:1, and 1:1) to obtain F34. F34 was further separated by high performance liquid chromatography (eluent: methanol / water, volume ratio 45:55, retention time t). R =22 min) to obtain the solanaceous lactone compound withagulide F.
[0010] The application of the aforementioned solanacolin compound withagulide F in the preparation of drugs for treating liver fibrosis.
[0011] Furthermore, the aforementioned drug for treating liver fibrosis is a drug that inhibits the proliferation or activation of hepatic stellate cells.
[0012] Furthermore, the aforementioned drug for treating liver fibrosis is a drug that inhibits the expression of hepatic stellate cell fibrosis marker proteins, thereby achieving an anti-liver fibrosis effect. Specifically, the inhibition of hepatic stellate cell fibrosis marker proteins involves inhibiting α-SMA and collagen I proteins.
[0013] Furthermore, the aforementioned drug for treating liver fibrosis is a drug that promotes apoptosis of hepatic stellate cells.
[0014] Furthermore, the medication for treating liver fibrosis includes oral or injectable formulations. The oral formulation may be any one or more of granules, capsules, tablets, powders, pellets, and sustained-release formulations.
[0015] Furthermore, in the treatment of liver fibrosis, the concentration of the solanine lactone compound is 0.75–3 μM.
[0016] Compared with the prior art, the technical effects of this invention are reflected in:
[0017] (1) The solanaceous lactone compound withagulide F in this application is a novel compound that has been extracted, isolated, and purified from Physalisangulata L. for the first time. It also possesses the potential to be developed as a drug for treating liver fibrosis. Currently, no relevant research literature has been found to report on the solanaceous lactone compound withagulide F, or on research into the application of withagulide F in the preparation of drugs for treating liver fibrosis.
[0018] (2) This invention provides for the first time the application of withagulide F in the preparation of drugs for treating liver fibrosis. Using human hepatic stellate cells (LX-2) as model cells, experiments demonstrated that withagulide F significantly reduced the expression levels of liver fibrosis markers such as α-SMA and collagen in LX-2 hepatic stellate cells, while effectively inhibiting their proliferation and inducing apoptosis. This indicates that withagulide F has the effect of inhibiting or reversing liver fibrosis, and its effect is dose-dependent, showing promising application prospects in the preparation of drugs for treating liver fibrosis. The novel application of withagulide F in the preparation of drugs for treating liver fibrosis provided by this invention is of great significance to addressing the challenges in the treatment of liver fibrosis. Attached Figure Description
[0019] Figure 1 This is a graph showing the effect of different concentrations of withagulide F on the proliferation activity of LX-2 cells.
[0020] Figure 2 This application uses ELISA to detect the effect of different concentrations of withagulide F on the protein expression level of collagen I;
[0021] Figure 3 This application uses Western blot to detect the effects of different concentrations of withagulide F on the expression levels of α-SMA and collagen I proteins;
[0022] Figure 4 A statistical graph showing the effect of different concentrations of withagulide F on the expression level of collagen I protein in this application;
[0023] Figure 5 A statistical graph showing the effect of different concentrations of withagulide F on α-SMA protein expression levels in this application;
[0024] Figure 6The image shows an inverted fluorescence microscope image of LX-2 cells under different concentrations of withagulide F treatment, observed by Hoechst 33258 staining.
[0025] In the attached figure: blue fluorescence indicates apoptotic or dead cells. *p<0.05, ***p<0.001 versus control. ### p<0.001 versus TGF-β1. Detailed Implementation
[0026] The technical solution of the present invention will be further defined below with reference to specific embodiments, but the scope of protection is not limited to the description made.
[0027] Experiment 1
[0028] Isolation, preparation and spectroscopic characteristics of compound withagulide F
[0029] Preparation method: The whole herb of *Smilax china* was pulverized and extracted twice with 30 L of 95% ethanol for 5 days each time. The extracts were combined and concentrated under reduced pressure to obtain an extract. The extract was suspended in 3 L of deionized water and extracted three times with 4 L of petroleum ether and chloroform each time to obtain a chloroform extract. The extract was subjected to macroporous resin column chromatography with a methanol / water gradient elution (volume ratios of 30:70, 45:55, 60:40, 80:20, and 100:0) to obtain five fractions (F1-F5). F3 was further separated by silica gel column chromatography with a dichloromethane / methanol gradient elution (volume ratios of 50:1, 30:1, 20:1, 10:1, and 1:1) to obtain F34. F34 was further separated by high performance liquid chromatography (eluent: methanol / water, volume ratio 45:55, retention time t). R =22 min) to obtain the solanaceous lactone compound withagulide F.
[0030] Spectral characteristics: White amorphous powder. 94.3(c 0.05,MeOH); ECD(MeOH)λ max (Δε):240(+14.3),308(-0.89)nm; IR(KBr)ν max :3422,2920,2851,1745,1709,1452,1405,1382,1248,1166,1125,941cm -1 ;HR ESIMS m / z:537.2108[M+Na] + The molecular formula is C 28 H 34 O9. 1H NMR (600MHz, CD3OD)δ H 6.10(1H,br d,J=9.9Hz,H-4),5.74(1H,m,H-6),5.64(1H,m,H-3),4.78(1H,dd,J=12.8,2.6Hz,H-22),4.40(1H,d,J=14.6Hz,H-28),4.28(1H,d ,J=14.6Hz,H-28),4.17(1H,dd,J=9.8,7.4Hz,H-15),3.62(1H,m,H-12),1.87(3H,s,H-27),1.56(3H,s,H-21),1.44(3H,s,H-19); 13 C NMR (150MHz, CD3OD)δ C 212.5(C-1),176.8(C-18),168.4(C-26),153.8(C-24),140.8(C-5),130.3(C-4),128.7(C- 6),122.7(C-3),122.6(C-25),85.9(C-20),82.5(C-14),80.8(C-22),76.1(C-15),74.0(C- 12),67.2(C-13),61.6(C-28),54.0(C-10),49.8(C-17),40.6(C-2),39.1(C-8),35.5(C-16 ),35.3(C-9),32.8(C-11),27.7(C-23),25.8(C-7),24.4(C-21),21.4(C-19),12.0(C-27).
[0031] Experiment 2
[0032] MTT assay for the effect of withagulide F on the proliferation activity of LX-2 cells stimulated by TGF-β1.
[0033] LX-2 cells were cultured in high-glucose DMEM medium containing 10% fetal bovine serum, 100 kU / L penicillin, and 100 mg / L streptomycin at 37°C in a 5% CO2 incubator. LX-2 cells in the logarithmic growth phase were harvested, digested with 0.25% trypsin, and then the cell density was adjusted to 5 × 10⁶ cells / year using medium (10% FBS + 1% penicillin-dextrin). 4Cells were inoculated at a density of 100 μL / mL into 96-well plates and divided into control, TGF-β1 (10 ng / mL) group, and withagulide F (0.75 μM, 1.5 μM, and 3 μM) groups. Each group had four replicates, with 100 μL of cell suspension per well, and a blank well was included for zeroing. 200 μL of sterile PBS was added around each well, and the cells were incubated at 37°C with 5% CO2 and saturated humidity for 24 h. Subsequently, 20 μL of MTT was added to each well, and the cells were incubated under the same conditions for another 3 h. The culture medium was aspirated, and 100 μL of DMSO was added. After shaking for 10 min, the absorbance (A) at 490 nm was measured using a microplate reader, and the cell proliferation rate was calculated. The results are shown below. Figure 1 As shown.
[0034] from Figure 1 It can be concluded that withagulide F can significantly reduce the proliferation of LX-2 cells in a concentration-dependent manner.
[0035] Experiment 3
[0036] ELISA kit to detect the effect of withagulide F on COL1A1 content in LX-2 cells
[0037] The culture conditions and grouping were the same as in Experiment 2. The cell density was adjusted to 5 × 10⁶ cells / year. 4 Samples were inoculated at a rate of 600 μL / well into 12-well plates and cultured for 24 h. The supernatant from each group was collected, centrifuged at 1000 rpm for 10 min at 4°C, and the supernatant was carefully aspirated. Each sample was processed strictly according to the ELISA kit instructions. Results are as follows: Figure 2 As shown.
[0038] from Figure 2 The results showed that TGF-β1 stimulation significantly increased the content of COL1A1; after intervention with agulide F, all concentration groups significantly reduced the abnormal increase in cellular COL1A1 content caused by TGF-β1 stimulation, and the effect was concentration-dependent.
[0039] Experiment 4
[0040] Western blot analysis of the effect of withagulide F on the expression of liver fibrosis markers α-SMA and collagen I in activated LX-2 cells.
[0041] After treating LX-2 cells with Withagulide F, 50 μL of lysis buffer was added to a 60 mm cell culture dish to fully lyse the cells. The cells were centrifuged at 12,000 rpm for 10 min at 4 °C, and the supernatant was collected. Protein concentration was determined using a BCA assay kit. After adjusting the protein concentration, the sample was mixed with 4× sample buffer and heated at 100 °C for 10 min to ensure complete protein denaturation. The extracted protein sample was then stored at -80 °C for later use. The target band was transferred to a PVDF membrane using 10% SDS-PAGE gel electrophoresis and blocked in 5% skim milk at room temperature for 2 h. The membrane was then incubated with primary antibody overnight at 4 °C. After incubation, the membrane was washed three times with TBST for 10 min each time. The secondary antibody was incubated at room temperature for 1.5 h, followed by washing three times with TBST for 10 min each time. The PVDF membrane was then immersed in ECL chromogenic solution for imaging and analysis using a gel imaging system.
[0042] The results are as follows Figure 3 , 4 As shown in Figure 5, withagulide F effectively inhibited the protein expression of LX-2 cell activation markers α-SMA and collagen I in a dose-dependent manner.
[0043] Experiment 5
[0044] Hoechst 33258 staining method was used to observe the effect of withagulide F on LX-2 cell apoptosis.
[0045] Log-phase LX-2 cells were harvested and the cell density was adjusted to 1 × 10⁻⁶ cells per milliliter. 5 1 mL of the solution was added to each well of a 6-well plate and incubated at 37°C with 5% CO2 for 24 h. The next day, different concentrations of withagulide F were added to treat LX-2 cells. After 24 h of intervention, the culture medium was removed, and the cells were washed three times with PBS. 600 μL of Hoechst 33258 staining solution was added to each well, and the cells were incubated for 5 min in the dark at room temperature. The cells were then washed three times with PBS for 5 min each time. The changes in fluorescence intensity were observed and photographed under an inverted fluorescence microscope.
[0046] from Figure 6 It was found that the number of apoptotic cells increased with increasing dose of withagulide F. The results indicate that withagulide F can induce apoptosis in LX-2 cells in a dose-dependent manner.
[0047] Therefore, withagulide F can significantly inhibit the proliferation of hepatic stellate cells (LX-2), reduce the protein expression of the main markers of liver fibrosis in LX-2 cells and thus inhibit their activation, induce apoptosis of LX-2 cells, and has the effect of inhibiting or reversing liver fibrosis. It has good application prospects in the preparation of drugs for the treatment of liver fibrosis.
[0048] In summary, this invention is the first to extract, isolate, and purify a novel compound, withagulide F, from Physalis angulata L., and also provides the first application of withagulide F in the preparation of drugs for treating liver fibrosis. This novel application of withagulide F in the preparation of drugs for treating liver fibrosis is of great significance in overcoming the bottleneck in liver fibrosis treatment.
[0049] Finally, it should be noted that the above embodiments are merely representative examples of the present invention. Obviously, the technical solution of the present invention is not limited to the above embodiments, and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the content disclosed in this invention should be considered within the scope of protection of this invention.
Claims
1. A nightshade lactone compound withagulide F, characterized in that, Its specific structural formula is as follows: Its chemical name is (17) S , 20 R , twenty two R -18,20-epoxy 12 β , 14 α , 15 β , 28-tetrahydroxy-1-oxowitha-3,5,24-trienolide, with the molecular formula C 28 H 34 O9 has a molecular weight of 514.
57.
2. The method for preparing the nightshade lactone compound withagulide F according to claim 1, characterized in that, Specifically as follows: The whole herb of *Smilax china* was pulverized and extracted twice with 30 L of 95% ethanol for 5 days each time. The extracts were combined and concentrated under reduced pressure to obtain an extract. The extract was suspended in 3 L of deionized water and then extracted three times with 4 L of petroleum ether and chloroform each time to obtain a chloroform extract. The extract was then eluted by macroporous resin column chromatography with methanol / water solutions of 30:70, 45:55, 60:40, 80:20, and 100:0 to obtain five fractions F1-F5. Fraction F3 was eluted by silica gel column chromatography with a dichloromethane / methanol gradient elution at volume ratios of 50:1, 30:1, 20:1, 10:1, and 1:1 to obtain F34. F34 was further separated by high performance liquid chromatography with methanol / water as the eluent at a volume ratio of 45:55 and a retention time t. R = 22 min, and the aforementioned nightshade lactone compound withagulide F was obtained.
3. The use of the solanine lactone compound withagulide F as described in claim 1 in the preparation of a drug for treating liver fibrosis.
4. The use of the solanine lactone compound withagulide F as described in claim 3 in the preparation of a drug for treating liver fibrosis, characterized in that, The aforementioned drug for treating liver fibrosis is a drug that inhibits the proliferation or activation of hepatic stellate cells.
5. The use of the solanine lactone compound withagulide F as described in claim 3 in the preparation of a drug for treating liver fibrosis, characterized in that, The aforementioned drug for treating liver fibrosis is a drug that inhibits the expression of hepatic stellate cell fibrosis marker proteins.
6. The use of the solanine lactone compound withagulide F as described in claim 5 in the preparation of a drug for treating liver fibrosis, characterized in that, The inhibition of hepatic stellate cell fibrosis marker proteins is achieved by inhibiting α-SMA and collagen I proteins.
7. The use of the solanine lactone compound withagulide F as described in claim 3 in the preparation of a drug for treating liver fibrosis, characterized in that, The aforementioned drug for treating liver fibrosis is a drug that promotes apoptosis of hepatic stellate cells.
8. The use of the solanine lactone compound withagulide F as described in claim 3 in the preparation of a drug for treating liver fibrosis, characterized in that, The medications for treating liver fibrosis include oral or injectable formulations.
9. The use of the solanine lactone compound withagulide F as described in claim 8 in the preparation of a drug for treating liver fibrosis, characterized in that, The oral preparation is any one or more of granules, capsules, tablets, powders, pellets, and sustained-release preparations.