A sesquiterpene compound with lipid-lowering activity and a preparation method thereof

Abstract

The application relates to a sesquiterpene compound with lipid-lowering activity and a preparation method thereof, a structural formula (I) of which is as follows, the preparation method is as follows: taking the rootstock of Morinda officinalis How, alcohol extraction and concentration are carried out to obtain a crude extract extract, silica gel column chromatography is carried out, petroleum ether and acetone gradient elution is adopted, fractions with the same thin layer chromatography results are combined, 8 fractions are obtained according to thin layer chromatography, methanol and water gradient elution is adopted, and the fractions are combined according to thin layer chromatography, 17 sub-fractions are obtained, silica gel column chromatography is carried out according to TLC color development and LC-MS detection, dichloromethane and ethyl acetate gradient elution and purification are carried out to obtain 7 sub-fractions, Fr.2g-3 contains a sesquiterpene structure according to TLC color development and LC-MS detection, and the Fr.2g-3 is further purified through silica gel column chromatography to obtain the sesquiterpene compound. The preparation method is simple, rapid, high in purity and easy to obtain raw materials.

Description

Technical Field

[0001] This invention belongs to the pharmaceutical field, specifically relating to a lipid-lowering sesquiterpene compound, and also to a method for preparing the sesquiterpene compound. Background Technology

[0002] Non-alcoholic fatty liver disease (NAFLD) is an acquired metabolic disease characterized by hepatic steatosis caused by metabolic stress, excluding alcohol and other clearly defined liver-damaging factors. The prevalence of NAFLD in the general adult population is as high as 20%–40%, posing a serious threat to human health. Studies have shown that the development of NAFLD is closely related to total cholesterol (TC) and triglycerides (TG). Abnormally elevated levels and accumulation of TC and TG lead to lipid metabolism disorders, thereby triggering NAFLD. Therefore, the discovery of drugs that inhibit TC and TG is of significant research importance and value. Although a series of anti-NAFLD drugs are available on the market, such as atorvastatin, fenofibrate, and metformin, these drugs have significant side effects. Natural products have become a popular area for the treatment of NAFLD; therefore, the discovery of new anti-NAFLD drugs from natural products has attracted great interest from pharmacologists.

[0003] Belamcanda chinensis (L.) DC., belonging to the genus Belamcanda of the family Iridaceae, is a perennial herb with a wide distribution and is a traditional Chinese medicine. The *Shennong Bencao Jing* (Shennong's Classic of Materia Medica) records its effects of clearing heat and detoxifying, reducing swelling and relieving pain, and resolving phlegm and soothing the throat; it has been used since ancient times to treat sore throat and phlegm. Its dried rhizome is often used medicinally to treat sore throat, excessive phlegm, cough, and asthma. Modern phytochemical studies show that it mainly contains flavonoids, volatile oils, phenolic acids, steroids, and triterpenoids. Modern pharmacological studies show that it mainly has anti-tumor, anti-inflammatory, antioxidant, and neuroprotective biological activities. Discovering drug lead compounds with lipid-lowering activity from the traditional Chinese medicine Belamcanda chinensis is of great significance. Summary of the Invention

[0004] The purpose of this invention is to provide a lipid-lowering sesquiterpene compound that is simple and rapid to prepare, has high purity, and uses readily available raw materials.

[0005] Another object of the present invention is to provide a method for preparing the lipid-lowering sesquiterpene compound.

[0006] The objective of this invention and the main technical problem it solves are achieved through the following technical solutions:

[0007] The present invention discloses a lipid-lowering sesquiterpene compound with the following structural formula:

[0008]

[0009] The present invention discloses a method for preparing a lipid-lowering sesquiterpene compound, comprising the following steps:

[0010] (1) Take 50 parts of Belamcanda chinensis rhizome, crush it, use 200 parts of 95% ethanol, extract it 3 times at room temperature for 4 days each time, recover the ethanol and concentrate it to obtain 10 parts of extract.

[0011] (2) The crude extract was subjected to silica gel column chromatography with a gradient elution of petroleum ether to acetone in a volume ratio of 95:5 to 0:100. Each gradient was 200 fractions. The different eluted fractions were detected by silica gel thin-layer chromatography. Fractions with the same thin-layer chromatographic results were combined and eight fractions were obtained according to their thin-layer chromatographic performance: Fr.1, Fr.2, Fr.3, Fr.4, Fr.5, Fr.6, Fr.7 and Fr.8.

[0012] (3) Fr.2 was separated by MCI using a gradient elution of methanol to water at a volume ratio of 6:4 to 9:1. The fractions were combined according to their thin-layer chromatographic characteristics, yielding 17 subfractions: Fr.2a, Fr.2b, Fr.2c, Fr.2d, Fr.2e, Fr.2f, Fr.2g, Fr.2h, Fr.2i, Fr.2j, Fr.2k, Fr.2l, Fr.2m, Fr.2n, Fr.2o, Fr.2p, and Fr.2q. Based on TLC colorimetry and LC-MS detection, Fr.2g contained... Fr.2g was purified by silica gel column chromatography using a gradient elution of dichloromethane to ethyl acetate in a volume ratio of 100:0 to 90:10 to obtain seven subfractions: Fr.2g-1, Fr.2g-2, Fr.2g-3, Fr.2g-4, Fr.2g-5, Fr.2g-6, and Fr.2g-7. Based on TLC color development and LC-MS detection, Fr.2g-3 contained a sesquiterpene structure. Further purification of Fr.2g-3 by silica gel column chromatography yielded norosesquiterpenoid compounds.

[0013] Compared with existing technologies, this invention has significant advantages. As can be seen from the above technical solution, this invention obtains the compound of this invention by ethanol extraction at room temperature from Belamcanda chinensis rhizome powder, followed by extract concentration, silica gel column chromatography, and MCI column chromatography. The extraction and separation process is simple, and the raw materials are abundant and readily available. Using a triglyceride (TG) content detection kit, the sesquiterpenoid compounds of this invention were detected, which can effectively downregulate the accumulation of TG in oleic acid-treated human liver cancer cells (HepG2). Experimental results show that the sesquiterpenoid compounds of this invention have a good inhibitory effect on TG enrichment at low concentrations (2.5 μM), which is superior to the positive control drug atorvastatin. This invention has good lipid-lowering activity, providing a new approach for developing highly effective and low-toxicity lipid-lowering drugs. Attached Figure Description

[0014] Figure 1 The proton NMR spectrum of the sesquiterpenoid compounds of the present invention;

[0015] Figure 2 This is the carbon NMR spectrum of the sesquiterpenoid compounds of the present invention. Detailed Implementation

[0016] The following detailed description, in conjunction with preferred embodiments and experimental examples, illustrates the specific implementation of a sesquiterpene compound and its preparation method according to the present invention.

[0017] Example:

[0018] A method for preparing a sesquiterpene compound with lipid-lowering activity includes the following steps:

[0019] (1) Take 29.4 kg of Belamcanda chinensis rhizome, crush it, and extract it three times at room temperature using 170 L of 95% industrial ethanol for 4 days each time. The ethanol was recovered and concentrated to obtain 4.2 kg of extract.

[0020] (2) The crude extract was subjected to silica gel column chromatography with gradient elution using petroleum ether to acetone in volume ratios of 97:3, 9:1, 8:2, 7:3, 6:4, 5:5, and 4:6, with 80 L of each gradient. Different eluted fractions were detected using silica gel thin-layer chromatography. Fractions with the same thin-layer chromatographic results were combined, and eight fractions were obtained based on their thin-layer chromatographic performance: Fr.1 (10 g), Fr.2 (117 g), Fr.3 (118 g), Fr.4 (800 g), Fr.5 (200 g), Fr.6 (100 g), Fr.7 (400 g), and Fr.8 (450 g).

[0021] (3) Fr.2 (117g) was separated by MCI using a gradient elution with methanol to water volume ratios of 60:40, 65:35, 70:30, 80:20, 85:15, and 90:10. The fractions were combined according to their thin-layer chromatographic characteristics, yielding 17 subfractions: Fr.2a (89.2mg), Fr.2b (56.6mg), Fr.2c (57.4mg), Fr.2d (742.3mg), F... r.2e(473.1mg), Fr.2f(190.2mg), Fr.2g(519.2mg), Fr.2h(540.7mg), Fr.2i(1.5g), Fr.2j(10 .7g), Fr.2k(3.7g), Fr.2l(12.3g), Fr.2m(3.1g), Fr.2n(1.5g), Fr.2o(3.4g), Fr.2p(34.1g) and Fr.2q (30.8 g); analysis of different fractions using TLC and LC-MS techniques showed that Fr.2g was rich in sesquiterpenoids. Therefore, Fr.2g (519.2 mg) was purified by silica gel column chromatography using a gradient elution with dichloromethane to ethyl acetate in volume ratios of 100:0, 97:3, 92:8, and 90:10, yielding seven subfractions: Fr.2g-1, Fr.2g-2, Fr.2g-3, Fr.2g-4, Fr.2g-5, Fr.2g-6, Fr.2g-7, Fr.2g-8, Fr.2g-9, Fr.2g-1, Fr.2g-2, Fr.2g-3 ... Fr.2g-3, Fr.2g-4, Fr.2g-5, Fr.2g-6, and Fr.2g-7 were analyzed by TLC and LC-MS. The results showed that Fr.2g was rich in sesquiterpenoids. Therefore, Fr.2g-3 was further purified by silica gel column chromatography with dichloromethane to acetone in volume ratios of 100:0, 98:2, and 95:5 to obtain norosesquiterpenoids (compound 1) (26.0 mg).

[0022] Structural identification of sesquiterpenoid compound (compound 1):

[0023] Compound 1 was analyzed by high-resolution mass spectrometry and nuclear magnetic resonance spectroscopy. 1 H NMR, 13 The structural formula of compound 1 was determined by comprehensive analysis of data from C NMR, 2D-NMR, ultraviolet spectroscopy, infrared spectroscopy, etc., as shown below:

[0024]

[0025] Compound 1: A colorless oily substance with the molecular formula C 14 H 22 O2;UV(MeOH)λ max (logε)277(2.81)nm; IR(KBr)ν max3452, 2925, 1676, 1632, 1359, 1201 and 971cm -1 ;(+)-HRESIMS:m / z 223.1687[M+H] + (The calculated value is C) 14 H 23 O2, 223.1693). 1 H-NMR (CDCl3, 600MHz) δ H 9.43(1H,s,H-1),6.86(1H,d,J=11.2Hz,H-3),6.74(1H,dd,J=15.1,11.2Hz,H-4),6.24(1H,d,J=15.1Hz,H-5),5.10(1H,t,J=7. 2Hz,H-9),2.00-2.06(2H,m,H-8),1.85(3H,s,H-13),1.66(1H,s,H-12),1.63(2H,m,H-7),1.57(3H,s,H-11),1.34(3H,s,H-14). 13 C NMR (150MHz, CDCl3)δ C 195.3 (C-1), 150.5 (C-5), 148.6 (C-3), 137.5 (C-2), 132.6 (C-10), 124.0 (C-9), 122.8 (C-4), 73.8 (C-6), 42.2 (C-7), 28.3 (C-14), 25.8 (C-12), 23.0 (C-8), 17.8 (C-11), 9.6 (C-13). Compound 1... 1 HNMR, 13 See CNMR spectrum Figure 1 and Figure 2 .

[0026] Experimental Example: The inhibitory effect of compound 1 obtained in Example 1 on triglyceride (TG) accumulation in oleic acid-treated human hepatocellular carcinoma cells (HepG2).

[0027] 1. Test Methods

[0028] Oleic acid-induced hyperlipidemia in HepG2 cells was selected as a screening model. In this model, oleic acid stimulation leads to lipid deposition in the stem cells, mimicking the pathogenesis of fatty liver caused by lipid metabolism disorders, resulting in a large accumulation of triglycerides (TG). The TG content in the control and experimental groups was measured using a triglyceride (TG) content detection kit to determine the inhibitory effect of the samples on triglyceride accumulation.

[0029] 2. Experimental Materials and Procedures

[0030] (1) Cell culture

[0031] The human liver-derived cell line HepG2 was obtained from the Chinese Academy of Sciences (Shanghai, China). Cells were seeded in DMEM and supplemented with 10% fetal bovine serum and 100 IU / mL... -1 Penicillin and 100 IU mL -1 Streptomycin was administered, and the culture medium was then placed in a 37°C, 5% CO2 incubator for cell digestion experiments during the logarithmic growth phase.

[0032] (2) Cytotoxicity evaluation (MTT)

[0033] HepG2 cells were seeded into 96-well cell culture plates (1 × 10⁶ cells per well). 4 Cells were incubated at 37°C for 12 h, then treated with 0.5 mM oleic acid for 24 h. Different concentrations of compound 1 (5, 10, 20 μM) were added to 96-well plates and cultured for 24 h. Afterward, 5 μL of MTT (5 mg / ml) solution was added to the remaining 100 μL of cell culture medium in the 96-well plate, and the plates were incubated in a 5% CO2 incubator for 4 hours. Then, 150 μL of DMSO was added and reacted for 10 min. The absorbance was measured at 490 nm using a microplate reader.

[0034] (3) Evaluation of triglyceride (TG) accumulation

[0035] HepG2 cells in the logarithmic growth phase were seeded into 6-well cell culture plates (3 × 10⁶ cells per well). 5 Cells were cultured (1,25, 2.5, 5 μM) for 12 hours. Afterward, the cells were washed with PBS, and a 0.5 mM oleic acid-bovine serum albumin (OA-BSA) complex was added. Then, different concentrations of compound 1 (1.25, 2.5, 5 μM) and a positive control (atorvastatin 5, 10, 20 μM) were added to the culture medium, and the cells were incubated for 24 hours. A BSA-only culture medium was used as a control. The accumulation of TG in the cells was measured using a triglyceride (TG) assay kit to determine the inhibitory effect of the compounds on TG.

[0036] Cell viability (%) = ([A 490nm ] OA+样品 / [A 490nm ] 空白 )×100

[0037] 3. Test Results

[0038] Following the experimental procedure described above, the cytotoxicity of compound 1 against oleic acid (OA)-induced human liver cancer cells (HepG2) was first determined. The results showed that the compound exhibited certain cytotoxicity at 10 and 20 μM, but no significant cytotoxicity was observed at 5 μM (see Table 1). Therefore, three low sample concentrations (1.25 μM, 2.5 μM, and 5 μM) were selected for subsequent experiments. The inhibitory effect of compound 1 on triglyceride (TG) accumulation in oleic acid (OA)-activated human liver cancer cells (HepG2) was determined, and the results are shown in Table 2. Compound 1 showed significant inhibitory effects on TG accumulation at low concentrations (2.5 μM and 5 μM), which were superior to the positive control drug atorvastatin, especially showing good inhibitory activity at 2.5 μM. The experimental results demonstrate that the compound of this invention has good lipid-lowering activity, providing a new drug and a new approach for the development and utilization of *Iris tectorum* plant and the development of drugs for the treatment of non-alcoholic fatty liver disease.

[0039] Table 1. Cytotoxicity of Compound 1 on Oleic Acid (OA)-Induced HepG2 Cells

[0040]

[0041] Note: * p<0.05 and ** p<0.001 indicates a comparison with the OA group. - No corresponding sample was added, resulting in no activity results.

[0042] Table 2. Inhibitory effect of compound 1 on oleic acid (OA)-induced TG accumulation in HepG2 cells.

[0043]

[0044] Note: ### p<0.001 indicates a comparison with DMSO; ** p<0.01 and *** p<0.001 indicates a comparison with the OA group.

[0045] - No activity results were obtained without the addition of the corresponding sample.

[0046] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, equivalent changes, and alterations made to the above embodiments without departing from the technical essence of the present invention shall still fall within the scope of the present invention.

Claims

1. A sesquiterpene compound with lipid-lowering activity, the structural formula of which is as follows: 。 2. A method for preparing a lipid-lowering sesquiterpene compound as described in claim 1, comprising the following steps: (1) Take 50 parts of Belamcanda chinensis rhizome, crush it, use 200 parts of 95% ethanol, extract it 3 times at room temperature for 4 days each time, recover the ethanol and concentrate it to obtain 10 parts of extract. (2) The crude extract was subjected to silica gel column chromatography with a gradient elution of petroleum ether to acetone in a volume ratio of 95:5 to 0:

100. Each gradient was 200 fractions. The different eluted fractions were detected by silica gel thin-layer chromatography. Fractions with the same thin-layer chromatographic results were combined and eight fractions were obtained according to their thin-layer chromatographic performance: Fr. 1, Fr. 2, Fr. 3, Fr. 4, Fr. 5, Fr. 6, Fr. 7 and Fr.

8. (3) Fr. 2 was separated by MCI using a gradient elution of methanol to water in a volume ratio of 6:4 to 9:

1. The fractions were combined according to their thin-layer chromatographic properties to obtain 17 subfractions: Fr.2a, Fr.2b, Fr.2c, Fr.2d, Fr.2e, Fr.2f, Fr.2g, Fr.2h, Fr.2i, Fr.2j, Fr.2k, Fr.2l, Fr.2m, Fr.2n, Fr.2o, Fr.2p, and Fr.2q. Based on TLC colorimetry and LC-MS detection, Fr.2g contained sesquiterpenoid structures. Fr.2g was further analyzed by silica gel column chromatography with a dichloromethane to ethyl acetate volume ratio of 100: The solution was purified by gradient elution with an eluent ratio of 0 to 90:10 to obtain seven subfractions: Fr.2g-1, Fr.2g-2, Fr.2g-3, Fr.2g-4, Fr.2g-5, Fr.2g-6, and Fr.2g-7. According to TLC color development and LC-MS detection, Fr.2g-3 contained sesquiterpenoid structures. Fr.2g-3 was further purified by silica gel column chromatography with dichloromethane to acetone volume ratios of 100:0, 98:2, and 95:5 to obtain no-sesquiterpenoid compounds.

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