A sesquiterpenoid compound Callistephus G and its preparation method and application

Abstract

The present invention discloses a sesquiterpenoid compound Callistephus G and its preparation method and application, belonging to the technical field of natural medicinal chemistry. The sesquiterpenoid compound Callistephus G of the present invention is extracted and separated from aster flowers. It is a special sesquiterpenoid compound with a 6 / 7 fused structure as a skeleton obtained for the first time. It can effectively inhibit the fibrotic effect of TGF-β on hepatic stellate cells LX-2, that is, the obtained sesquiterpenoid compound has good anti-hepatic fibrosis activity. Therefore, the sesquiterpenoid compound Callistephus G of the present invention can be used to prepare drugs for treating and / or preventing liver fibrosis, and has good application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of natural medicinal chemistry, and particularly relates to a sesquiterpenoid compound Callistephus G, a preparation method and an application thereof. Background Art

[0002] Liver fibrosis is an intermediate stage in the progression of chronic liver disease to cirrhosis, characterized by massive fibrosis, excessive production and deposition of extracellular matrix in the liver. The World Health Organization (WHO) estimates that there are approximately 100 million patients with liver fibrosis and cirrhosis worldwide, resulting in more than 1 million deaths each year. Although great progress has been made in understanding the pathogenesis and molecular mechanisms of liver fibrosis in recent years, there is still a lack of effective therapeutic drugs for liver fibrosis and cirrhosis. Currently, the only treatment for patients with liver fibrosis is liver transplantation, but the limited number of available livers and the high cost make this surgery difficult for every patient. Therefore, there is a need to find an effective anti-liver fibrosis drug. An increasing number of drugs have been found to have the ability to inhibit liver fibrosis.

[0003] Sesquiterpenes are a group of compounds consisting of three isoprene units and containing 15 carbon atoms. They are classified by the number of carbon atoms in the ring, including five-, six-, and seven-membered ring sesquiterpenes. Sesquiterpenes are widely distributed in plants, animals, marine organisms, and microorganisms, with a particularly high concentration in the Asteraceae family. They exhibit a variety of biological activities, including antitumor cytotoxicity, antibacterial, anti-inflammatory, antiviral, and antioxidant activities. They also possess hepatoprotective and neuroprotective properties, and have therapeutic effects on diabetes. Sesquiterpenes are the primary and characteristic components of the ethnomedicinal plant Aster. Further exploration and discovery of novel sesquiterpenoids derived from Aster remains worthy of further investigation.

[0004] Plants in the Asteraceae family can be used to make teas and as medicine, earning them the reputation of "longevity enhancers." Asters, the only species in the genus Aster, possess a variety of pharmacological activities, including anti-inflammatory, hepatoprotective, and antihypertensive properties. Recent studies have confirmed that Asteraceae plants are rich in terpenes, flavonoids, and polysaccharides. However, current research on asters has primarily focused on cultivation and management, landscape applications, and the breeding of new varieties. In-depth studies on their material foundations and health benefits have been lacking. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a sesquiterpenoid compound Callistephus G, a preparation method and application thereof. The inventors successfully isolated a new sesquiterpenoid compound with a 6 / 7 fused structure as the skeleton, namely Callistephus G, from the ethanol extract of Aster flowers. It can effectively inhibit the fibrotic effect of TGF-β on hepatic stellate cells LX-2, that is, the prepared sesquiterpenoid compound has good anti-liver fibrosis activity.

[0006] The object of the present invention is to achieve the following goals:

[0007] The present invention provides a sesquiterpenoid compound Callistephus G, the structure of which is shown in the following formula:

[0008]

[0009] Another aspect of the present invention provides a method for preparing the above-mentioned sesquiterpenoid compound Callistephus G, comprising the following steps:

[0010] (1) adding aster flowers to ethanol for heating and refluxing extraction, and concentrating the obtained extract under reduced pressure to obtain aster ethanol extract;

[0011] (2) The aster ethanol extract obtained in step (1) is dispersed in hot water, extracted with dichloromethane, and separated to obtain a dichloromethane extract.

[0012] (3) The dichloromethane extract obtained in step (2) is loaded onto silica gel column chromatography, first eluted with an elution solvent having a dichloromethane-methanol volume ratio of 85 to 95:1, and then eluted with an elution solvent having a dichloromethane-methanol volume ratio of 45 to 55:1, collecting the eluate having a dichloromethane-methanol volume ratio of 45 to 55:1, loading it onto Sephadex LH-20 column chromatography, eluting with an elution solvent having a dichloromethane-methanol volume ratio of 1:1, loading the obtained product onto silica gel column chromatography, eluting with an elution solvent having a dichloromethane-methanol volume ratio of 9 to 11:1, loading the obtained eluate onto HPLC, the chromatographic column is C-18, and eluted with a mobile phase of 60 to 65% methanol aqueous solution to obtain the sesquiterpenoid compound Callistephus G.

[0013] Based on the above technical solution, further, the aster flowers described in step (1) are aster flower powder that has been shade-dried and crushed.

[0014] Based on the above technical solution, further, the mass volume ratio of the aster flowers to the ethanol in step (1) is 0.1 to 5 kg / L.

[0015] Based on the above technical solution, further, the extraction temperature of heating reflux in step (1) is 50-90° C., the extraction time is 1-5 hours, and the number of extractions is 1-3 times.

[0016] Based on the above technical solution, further, the temperature of the hot water in step (2) is 40-60°C.

[0017] Based on the above technical solution, further, the specific process in step (3) is: the dichloromethane extract obtained in step (2) is loaded onto silica gel column chromatography, first eluted with an elution solvent having a dichloromethane-methanol volume ratio of 90:1, then eluted with an elution solvent having a dichloromethane-methanol volume ratio of 50:1, collecting the eluate with a dichloromethane-methanol volume ratio of 50:1, loading it onto Sephadex LH-20 column chromatography, eluting with an elution solvent having a dichloromethane-methanol volume ratio of 1:1, loading the obtained product onto silica gel column chromatography, eluting with an elution solvent having a dichloromethane-methanol volume ratio of 10:1, and finally loading the eluate onto HPLC, the chromatographic column is YMC-C18, id10mm×25.0cm, 5μm, the flow rate is 2.0~3.0mL / min, and the mobile phase is 62% methanol aqueous solution for elution.

[0018] The present invention also provides use of the sesquiterpenoid compound Callistephus G in preparing a drug for treating and / or preventing liver fibrosis.

[0019] The present invention has the following beneficial effects compared to the prior art:

[0020] The sesquiterpenoid compound Callistephus G provided by the present invention is a stable and safe monomeric substance extracted from the functional food aster. It can effectively inhibit the fibrogenic effect of TGF-β on hepatic stellate cells LX-2. In other words, the obtained sesquiterpenoid compound has excellent anti-hepatic fibrosis activity. Therefore, the sesquiterpenoid compound Callistephus G of the present invention can be used to prepare a drug for treating and / or preventing hepatic fibrosis and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention, the drawings related to the embodiments are briefly introduced below.

[0022] Figure 1 is the structural formula of the compound prepared in Example 1 of the present invention;

[0023] Figure 2 This is the hydrogen nuclear magnetic resonance spectrum of Callistephus G prepared in Example 1 of the present invention;

[0024] Figure 3 This is the carbon NMR spectrum of Callistephus G prepared in Example 1 of the present invention;

[0025] Figure 4 HSQC spectrum of Callistephus G prepared in Example 1 of the present invention;

[0026] Figure 5 This is the HMBC spectrum of Callistephus G obtained in Example 1 of the present invention;

[0027] Figure 6 This is the HR-ESI-MS spectrum of Callistephus G prepared in Example 1 of the present invention;

[0028] Figure 7 This is a graph showing the cell viability of LX-2 cells using Callistephus G prepared in Example 1 of the present invention. DETAILED DESCRIPTION

[0029] The present invention is described in detail below with reference to the embodiments, but the embodiments of the present invention are not limited thereto. Obviously, the embodiments described below are only some embodiments of the present invention. For those skilled in the art, other similar embodiments obtained without creative work all fall within the scope of protection of the present invention.

[0030] If the manufacturer of the reagents or instruments used is not indicated, they are regarded as conventional products that can be purchased from the market.

[0031] Example 1

[0032] This embodiment provides a method for preparing a sesquiterpenoid compound Callistephus G, comprising the following steps:

[0033] A. Chrysanthemum aster flowers (7.13 kg) were extracted twice with ethanol (7 L, 70%) at 70°C under heating reflux (2.5 h). The extract was concentrated under reduced pressure to obtain the chrysanthemum aster ethanol extract (264.36 g).

[0034] B. Disperse the ethanol extract obtained in step A with hot water at 50° C., and extract and separate with dichloromethane to obtain a dichloromethane extract (81.36 g);

[0035] C. The dichloromethane extract obtained in step B was loaded onto silica gel column chromatography, first using an elution solvent of 90:1 dichloromethane-methanol volume ratio, and then eluted with an elution solvent of 50:1 dichloromethane-methanol volume ratio. The eluate of the silica gel column chromatography with a 50:1 dichloromethane-methanol volume ratio was collected and loaded onto Sephadex LH-20 column chromatography, eluted with an elution solvent of 1:1 dichloromethane-methanol volume ratio. The resulting product was loaded onto silica gel column chromatography, eluted with an elution solvent of 10:1 dichloromethane-methanol volume ratio. Finally, the eluate was loaded onto HPLC, the chromatographic column was YMC-C18 (id10 mm×25.0 cm, 5 μm), and eluted with a 62% methanol aqueous solution mobile phase at a flow rate of 3.0 mL / min to obtain the new compound of the present invention, Callistephus G (15 mg).

[0036] Example 2

[0037] This example identifies the structure of the compound obtained in Example 1

[0038] 1. Experimental methods:

[0039] In this example, the structure of the compound was identified by hydrogen nuclear magnetic resonance spectroscopy, carbon nuclear magnetic resonance spectroscopy, HSQC spectroscopy, HMBC spectroscopy, and HR-ESI-MS spectroscopy.

[0040] 2. Experimental results:

[0041] (1) Table 1 shows the Callistephus G obtained in Example 1 1 H and 13 C NMR signal assignments. Figure 2 is the hydrogen nuclear magnetic resonance spectrum of Callistephus G obtained in Example 1, 1H NMR spectrum (600 MHz, pyridine-d5) gave three methyl hydrogen proton signals δH 1.66 (3H, s, H3-12), δH 1.72 (3H, s, H3-13), δH 2.51 (3H, s, H3-15), three methylene hydrogen proton signals δH 1.44 (1H, d, J = 13.8 Hz, H1-7α), δH 1.82 (1H, d, J = 11.8 Hz, H1-7β), δH 1.04 (1H, d, J = 13.7 Hz, H1-8α), δH 1.40 (1H, d, J = 14.1 Hz, H1-8β), δH 1.59 (1H, d, J = 14.6 Hz, H1-9α), δH 1.79 (1H, d, J = 7.8 Hz, H1-9β), two para-position aromatic hydrocarbon hydrogen proton signals δH 7.08 (1H, s, H1-1), δH 7.18 (1H, s, H1-4). Figure 3 This is the carbon NMR spectrum of Callistephus G obtained in Example 1. 13 C NMR spectrum (150 MHz, pyridine-d5) gave 15 carbon signals. Figure 4 HSQC spectrum analysis included 3 methyl carbon signals δC 26.0 (C-12), δC 22.6 (C-13), δC 17.0 (C-15), 3 methylene carbon signals δC 35.9 (C-7), δC 22.7 (C-8), δC 37.3 (C-9), 1 quaternary carbon signal δC 45.9 (C-6), 1 oxygen-linked quaternary carbon signal δC 83.2 (C-10), 6 aromatic carbon signals δC 110.5 (C-1), δC 156.5 (C-2), δC 125.6 (C-3), δC 127.2 (C-4), δC 128.0 (C-5), δC 137.8 (C-11), and 1 carbonyl carbon signal δC 177.3 (C-14).

[0042] (2) Figure 5 This is the HMBC spectrum of Callistephus G prepared in Example 1. The HMBC spectrum shows correlations between H3-12 and C-9, C-10, and C-11, and between H3-13 and C-5, C-6, C-7, and C-14, suggesting a 6 / 7 fused skeleton in this compound. The positions of these two rings can be determined by the HMBC correlations between H1-1 and C-10, C-11, H1-4 and C-5, C-6, H3-12 and C-10, C-11, and H3-13 and C-5, C-6. C-5 and C-11 are shared carbon atoms.

[0043] (3) Figure 6This is the HR-ESI-MS spectrum of Callistephus G prepared in Example 1. HR-ESI-MS analysis shows that its quasi-molecular ion peak is m / z 247.1336 [M+H] + Calcd 247.1329, the molecular formula of the compound is determined to be C 15 H 18 O3, unsaturation is 7.

[0044] (4) Combining NMR and literature data, the compound was determined to be a new compound that had not been reported before and was named Callistephus G.

[0045] Table 1 Callistephus G 1 H and 13 C NMR signal assignments (δ in ppm, J in Hz).

[0046]

[0047] Example 3

[0048] Activity test of the Callistephus G compound of the present invention in the TGF-β-induced LX-2 cell fibrosis model

[0049] 1. Experimental methods:

[0050] (1) First, a TGF-β-induced LX-2 cell fibrosis model was established: LX-2 cell lines were cultured in a sterile cell culture incubator at 37°C and 5% CO2 using DMEM high-glucose medium containing 10% FBS and 1% double antibiotics (penicillin and streptomycin).

[0051] (2) LX-2 cells were seeded in 6-well plates and cultured for 24 hours. When the cells were in good condition and in the logarithmic growth phase, all cells except the CON group were treated with TGF-β (2, 5, and 10 ng / mL) for 24 hours. Commercial kits were used to detect various indicators of cell supernatants and cells, and the optimal dose of TGF-β for the LX-2 cell model was determined to be 5 ng / mL.

[0052] (3) The CCK-8 method was used to detect the effect of Callistephus G on the survival rate of LX-2 cells. When the cells grew to the logarithmic growth phase, the LX-2 cell suspension was inoculated into a 96-well plate and cultured for 24 hours before the experiment. Six replicate wells were set up for each group. Except for the CON group, all cells were treated with TGF-β (5ng / mL) inducer for 24 hours. Then, the cells were treated with different Callistephus G concentrations (1, 2, 2.5, 3, 4, 5, 6, 6.25μM) for 24 hours, and the cell viability was measured by CCK-8. The absorbance of each well at a wavelength of 450nm was then detected using an enzyme reader. The cell survival rate was calculated according to the formula: Cell survival rate = OD 给药组 / OD 空白组 ×100%.

[0053] 2. Test results

[0054] The results of Callistephus G on the cell survival rate of hepatic stellate cells LX-2 are shown in Figure 7 The results showed that Callistephus G could effectively inhibit the fibrotic effect of TGF-β on hepatic stellate cells LX-2, that is, the prepared sesquiterpenoid compound had good anti-liver fibrosis activity.

[0055] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A sesquiterpenoid compound Callistephus G, characterized in that The structure is shown below:

2. The method for preparing the sesquiterpenoid compound Callistephus G according to claim 1, characterized in that: The following steps are involved: (1) adding aster flowers to ethanol for heating and refluxing extraction, and concentrating the obtained extract under reduced pressure to obtain aster ethanol extract; (2) dispersing the aster ethanol extract obtained in step (1) into hot water, extracting with dichloromethane, and separating to obtain a dichloromethane extract; (3) The dichloromethane extract obtained in step (2) is loaded onto silica gel column chromatography, first eluted with an elution solvent having a dichloromethane-methanol volume ratio of 85 to 95:1, and then eluted with an elution solvent having a dichloromethane-methanol volume ratio of 45 to 55:1, collecting the eluate having a dichloromethane-methanol volume ratio of 45 to 55:1, loading it onto Sephadex LH-20 column chromatography, eluting with an elution solvent having a dichloromethane-methanol volume ratio of 1:1, loading the obtained product onto silica gel column chromatography, eluting with an elution solvent having a dichloromethane-methanol volume ratio of 9 to 11:1, loading the obtained eluate onto HPLC, the chromatographic column is C-18, and eluted with a mobile phase of 60 to 65% methanol aqueous solution to obtain the sesquiterpenoid compound Callistephus G.

3. The method for preparing the sesquiterpenoid compound Callistephus G according to claim 2, characterized in that: The aster flowers described in step (1) are aster flower powder that has been shade-dried and crushed.

4. The method for preparing the sesquiterpenoid compound Callistephus G according to claim 2, wherein: The mass volume ratio of the aster flowers to the ethanol in step (1) is 0.1-5 kg / L.

5. The method for preparing the sesquiterpenoid compound Callistephus G according to claim 2, characterized in that: The extraction temperature of heating reflux in step (1) is 50-90° C., the extraction time is 1-5 hours, and the number of extractions is 1-3 times.

6. The method for preparing the sesquiterpenoid compound Callistephus G according to claim 2, characterized in that: The temperature of the hot water in step (2) is 40-60°C.

7. The method for preparing the sesquiterpenoid compound Callistephus G according to claim 2, characterized in that: The specific process of step (3) is as follows: the dichloromethane extract obtained in step (2) is loaded onto a silica gel column chromatography, first eluted with an elution solvent having a dichloromethane-methanol volume ratio of 90:1, then eluted with an elution solvent having a dichloromethane-methanol volume ratio of 50:1, collecting the eluate having a dichloromethane-methanol volume ratio of 50:1, loading it onto a Sephadex LH-20 column chromatography, eluting with an elution solvent having a dichloromethane-methanol volume ratio of 1:1, loading the obtained product onto a silica gel column chromatography, eluting with an elution solvent having a dichloromethane-methanol volume ratio of 10:1, and finally loading the eluate onto an HPLC column, the chromatographic column being YMC-C18, id10mm×25.0cm, 5μm, the flow rate being 2.0~3.0mL / min, and eluting with a mobile phase of 62% methanol aqueous solution.

8. Use of the sesquiterpenoid compound Callistephus G according to claim 1 in the preparation of a medicament for treating and / or preventing liver fibrosis.

Images