Sesquiterpenoids Callistephus G as well as preparation method and application thereof

Abstract

The invention discloses a sesquiterpenoids compound Callistephus G as well as a preparation method and application thereof, and belongs to the technical field of natural pharmaceutical chemistry. The sesquiterpenoids Callistephus G are extracted and separated from green chrysanthemum flowers, the sesquiterpenoids are the special sesquiterpenoids with 6 / 7 fused structures as frameworks for the first time, the fibrosis effect of TGF-beta on hepatic stellate cells LX-2 can be effectively inhibited, and the prepared sesquiterpenoids have good anti-hepatic fibrosis activity. Therefore, the sesquiterpenoid Callistephus G disclosed by the invention can be used for preparing the medicine for treating and / or preventing the hepatic fibrosis, and has a relatively good application prospect.

Description

Technical Field

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

[0002] Liver fibrosis is the intermediate stage of chronic liver disease progressing to cirrhosis, which is characterized by massive fibrosis, excessive production and deposition of extracellular matrix in the liver. The World Health Organization (WHO) estimates that there are about 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 the understanding of 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 number of available livers and the high price make it difficult to perform the surgery for every patient. Therefore, there is a need to find an effective anti-liver fibrosis drug. More and more drugs have been found to have the ability to inhibit liver fibrosis.

[0003] Sesquiterpenoids are a group of compounds with a structure consisting of three isoprene units and containing 15 carbon atoms. They are classified according to the number of carbon atoms in the ring into five-membered ring, six-membered ring, seven-membered ring sesquiterpenes, etc. Sesquiterpenoids are widely distributed in plants, animals, marine organisms and microorganisms, especially in Asteraceae plants. They have multiple biological activities such as anti-tumor cytotoxicity, antibacterial, anti-inflammatory, antiviral, and antioxidant activities. They also have the effects of protecting the liver, protecting nerves, and treating diabetes. Sesquiterpenoids are the main component type of the ethnic medicinal plant Aster, and are also its characteristic component type. Novel sesquiterpenoid components from Aster plants are still worth further exploration and discovery.

[0004] Asteraceae plants can be used to make tea and medicine, and are known as "longevity prolongers". Aster is the only plant in the genus Aster of the Asteraceae family, and has multiple pharmacological activities such as anti-inflammatory, liver protection, and blood pressure reduction. Recent studies have confirmed that Asteraceae plants are rich in terpenes, flavonoids, and polysaccharide compounds, but current research on aster is mostly focused on cultivation management, gardening applications, and new variety breeding. There has been no in-depth research on its material basis and health benefits. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide a sesquiterpenoid compound Callistephus G and a preparation method and application thereof. The inventors successfully separated a new sesquiterpenoid compound with a 6 / 7 fused structure as the skeleton, namely Callistephus G, from the ethanol extract of Aster flowers, which 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:

[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) dispersing the aster ethanol extract obtained in step (1) in hot water, extracting with dichloromethane, and separating to obtain a dichloromethane extract.

[0012] (3) 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 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 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 9 to 11:1, loading the obtained eluate onto an HPLC, the chromatographic column being C-18, and eluting with a mobile phase of 60 to 65% methanol aqueous solution, to obtain a sesquiterpene compound Callistephus G.

[0013] Based on the above technical solution, further, the aster flower described in step (1) is 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, in step (1), the extraction temperature of heating reflux 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 scheme, further, the specific process in step (3) is: the dichloromethane extract obtained in step (2) is loaded onto a silica gel column chromatography, firstly 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 HPLC, 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.

[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 beneficial effects of the present invention compared with the prior art are as follows:

[0020] The sesquiterpenoid compound Callistephus G provided by the present invention is a monomer substance with stable and safe components extracted from the functional food aster, which can effectively inhibit the fibrotic effect of TGF-β on hepatic stellate cells LX-2, that is, the prepared 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. 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 The carbon nuclear magnetic resonance 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 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 survival rate 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 partial embodiments of the present invention. For those skilled in the art, other similar embodiments obtained without creative labor all fall within the protection scope of the present invention.

[0030] If the manufacturer of the reagents or instruments used is not indicated, they are deemed to be conventional products that can be purchased commercially.

[0031] Example 1

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

[0033] A. Extract aster flowers (7.13 kg) with ethanol (7 L, 70%) under heating reflux at 70°C for 2.5 h, extract twice, and concentrate the extract under reduced pressure to obtain 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 having a dichloromethane-methanol volume ratio of 90:1, and then eluting with an elution solvent having a dichloromethane-methanol volume ratio of 50:1. The eluate having a dichloromethane-methanol volume ratio of 50:1 of the silica gel column chromatography was collected and loaded onto Sephadex LH-20 column chromatography, and eluted with an elution solvent having a dichloromethane-methanol volume ratio of 1:1. The obtained product was loaded onto silica gel column chromatography, and eluted with an elution solvent having a dichloromethane-methanol volume ratio of 10:1. Finally, the eluate was loaded onto HPLC, the chromatographic column was YMC-C18 (id10mm×25.0cm, 5μm), and the mobile phase was 62% methanol aqueous solution for elution at a flow rate of 3.0mL / min, to obtain the new compound Callistephus G (15mg) of the present invention.

[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 spectrum, carbon nuclear magnetic resonance spectrum, HSQC spectrum, HMBC spectrum, and HR-ESI-MS spectrum.

[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), 2 para-position aromatic hydrogen proton signals δH 7.08 (1H, s, H1-1), δH 7.18 (1H, s, H1-4). Figure 3 is the carbon NMR spectrum of Callistephus G obtained in Example 1, 13 C NMR spectrum (150 MHz, pyridine-d5) gave a total of 15 carbon signals. Figure 4 HSQC spectrum analysis, including 3 methyl carbon signals δC 26.0 (C-12), δC 22.6 (C-13), δC 17.0 (C-15), 3 methylene carbon signals δC35.9 (C-7), δC 22.7 (C-8), δC 37.3 (C-9), 1 quaternary carbon signal δC 45.9 (C-6), 1 oxygen-connected quaternary carbon signal δC83.2 (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 obtained in Example 1. The HMBC spectrum shows the correlation between H3-12 and C-9, C-10, C-11, H3-13 and C-5, C-6, C-7, and C-14, and it is inferred that the compound has a 6 / 7 fused skeleton. The positions of the two rings can be determined by the HMBC correlation between H1-1 and C-10, C-11, H1-4 and C-5, C-6, H3-12 and C-10, C-11, H3-13 and C-5, C-6, and C-5 and C-11 are shared carbons.

[0043] (3) Figure 6This is the HR-ESI-MS spectrum of Callistephus G obtained 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) Combined with 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 Callistephus G compound of the present invention in 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 cells were cultured in a sterile cell culture incubator at 37°C and 5% CO2 in DMEM high-glucose medium containing 10% FBS and 1% double antibodies (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, 10 ng / mL) inducer for 24 hours. The cell supernatant and various cell indicators were detected using commercial kits, and the optimal dose of TGF-β-induced 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 in a 96-well plate and cultured for 24 hours before the experiment. Six replicate wells were set up in 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 aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned 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) in hot water, extracting with dichloromethane, and separating to obtain a dichloromethane extract. (3) 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 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 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 9 to 11:1, loading the obtained eluate onto an HPLC, the chromatographic column being C-18, and eluting with a mobile phase of 60 to 65% methanol aqueous solution, to obtain a sesquiterpene 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 shade-dried and crushed aster flower powder.

4. The method for preparing the sesquiterpenoid compound Callistephus G according to claim 2, characterized in that: The mass volume ratio of the aster flower 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, firstly 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 HPLC, wherein 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.

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

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