A juniperane-type sesquiterpene, its preparation method and uses

Abstract

This invention discloses a juniperane-type sesquiterpene, its preparation method, and its uses. The juniperane-type sesquiterpene is isolated from cinnamon bark and has at least one of the structural formulas shown in formulas (I), (II), (III), and (IV). Hepatoprotective activity was evaluated using an alcohol-induced hepatocyte injury model, and it was found that the juniperane-type sesquiterpene of this invention can effectively alleviate alcohol-induced AML-12 hepatocyte injury and reduce fat accumulation, indicating that the juniperane-type sesquiterpene of this invention has anti-alcoholic liver disease activity and can be used in the preparation of anti-alcoholic liver disease drugs, showing good research and development prospects. It belongs to the field of natural product chemistry.

Description

Technical Field

[0001] This invention belongs to the field of natural product chemistry, specifically relating to a juniperane-type sesquiterpene, its preparation method, and its uses. Background Technology

[0002] Alcoholic liver disease (ALD) is a liver disease caused by long-term excessive alcohol consumption. Clinically, ALD ranges from simple steatosis to more severe pathological liver changes, and is mainly divided into: alcoholic fatty liver disease, alcoholic hepatitis, alcoholic liver fibrosis, and alcoholic cirrhosis. The pathogenesis of ALD is complex, and there are currently no definitively effective treatments. Most existing ALD medications play an adjunctive role in clinical treatment, with limited efficacy. Therefore, finding effective and safe anti-ALD drugs is of great significance.

[0003] Cinnamon (Cinnamomum cassia Presl.) is a plant belonging to the genus Cinnamomum in the family Lauraceae, and is widely distributed in tropical and subtropical regions of East and Southeast Asia. Cinnamon bark, a traditional Chinese medicine used in both food and medicine, is believed to have effects such as tonifying yang, guiding fire back to its source, dispelling cold and relieving pain, and warming and unblocking the meridians. Summary of the Invention

[0004] The first objective of this invention is to provide a class of juniperane-type sesquiterpenes that have anti-ALD activity.

[0005] The juniperane-type sesquiterpene provided by this invention is isolated from cinnamon bark; and its structural formula is shown as formula (I) and / or formula (II) and / or formula (III) and / or formula (IV):

[0006]

[0007] The aforementioned juniperane-type sesquiterpenes were isolated from the bark of *Cinnamomum cassia* Presl., a plant belonging to the genus *Cinnamomum* of the Lauraceae family. The cinnamon bark was purchased in April 2021 from the Qingping Traditional Chinese Medicine Market, Liwan District, Guangzhou City, Guangdong Province. The samples are preserved at the School of Biomedical Engineering, Guangdong University of Technology (ID: CC-202104, Location: School of Biomedical Engineering, Guangdong University of Technology, No. 100, Waihuan West Road, Guangzhou University Town, Guangzhou, China, 510006).

[0008] Another technical solution of this application is to provide a method for preparing the above-mentioned juniperane-type sesquiterpenes, wherein the juniperane-type sesquiterpenes are isolated from cinnamon bark.

[0009] Furthermore, in the above-mentioned method for preparing juniperane-type sesquiterpenes, the juniperane-type sesquiterpenes are obtained from cinnamon bark through extraction, concentration, extraction, column chromatography separation, and reversed-phase high-performance liquid chromatography.

[0010] Furthermore, in the above-mentioned method for preparing juniperine-type sesquiterpenes, the extraction is carried out by heating and reflux extraction using a 60% (v / v) ethanol-water solution.

[0011] Furthermore, in the above-mentioned method for preparing juniperane-type sesquiterpenes, the extraction is performed using dichloromethane to obtain a dichloromethane extract.

[0012] Furthermore, in the above-mentioned method for preparing juniperane-type sesquiterpenes, the specific method for separating the dichloromethane extract by column chromatography and reversed-phase high-performance liquid chromatography is as follows:

[0013] 1) The dichloromethane extract was subjected to silica gel column chromatography at atmospheric pressure, and eluted sequentially with cyclohexane-ethyl acetate eluents at volume ratios of 99:1, 97:3, 95:5, 90:10, 85:15, 80:20, 70:30, 60:40 and 0:100 to obtain sub-fractions C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16 and C17, a total of 17 sub-fractions;

[0014] 2) The C10 fraction obtained in step 1) was subjected to medium-low pressure ODS column chromatography, eluted sequentially with methanol-water-formic acid eluents at volume ratios of 35:65:0.1, 40:60:0.1, 45:55:0.1, 50:50:0.1, 60:40:0.1, 70:30:0.1, 80:20:0.1, and 100:0:0, to obtain C10.1, C10.2, C10.3, C10.4, C10.5, C10.6, C10.7, C10.8, C10.9, C10.10, C10.11, and C10. 0.12, C10.13, C10.14, C10.15, C10.16, C10.17, C10.18, C10.19, C10.20, C10.21, C10.22 and C10.23, a total of 23 sub-fractions;

[0015] 3) The sub-fraction C10.10 obtained in step 2) was processed by reverse-phase preparative HPLC using Cosmosil Packed C 18 The chromatographic column was prepared by elution with acetonitrile-water-formic acid at a flow rate of 12 mL / min and a volume ratio of 35:65:0.1, yielding eight sub-fractions: C10.10.1, C10.10.2, C10.10.3, C10.10.4, C10.10.5, C10.10.6, C10.10.7, and C10.10.8.

[0016] 4) The sub-fraction C10.10.5 obtained in step 3) was processed by reverse-phase preparative HPLC using Cosmosil Packed C 18 The chromatographic column was prepared by elution with methanol-water-formic acid eluent at a flow rate of 8 mL / min and a volume ratio of 45:55:0.1 to obtain compound (Ⅰ).

[0017] Furthermore, in the above-mentioned method for preparing juniperane-type sesquiterpenes, the specific method for separating the dichloromethane extract by column chromatography and reversed-phase high-performance liquid chromatography is as follows:

[0018] 1) The dichloromethane extract was subjected to silica gel column chromatography at atmospheric pressure, and eluted sequentially with cyclohexane-ethyl acetate eluents at volume ratios of 99:1, 97:3, 95:5, 90:10, 85:15, 80:20, 70:30, 60:40 and 0:100 to obtain sub-fractions C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16 and C17, a total of 17 sub-fractions;

[0019] 2) The C10 fraction obtained in step 1) was subjected to medium-low pressure ODS column chromatography, eluted sequentially with methanol-water-formic acid eluents at volume ratios of 35:65:0.1, 40:60:0.1, 45:55:0.1, 50:50:0.1, 60:40:0.1, 70:30:0.1, 80:20:0.1, and 100:0:0, to obtain C10.1, C10.2, and C10. 0.3, C10.4, C10.5, C10.6, C10.7, C10.8, C10.9, C10.10, C10.11, C10.12, C10.13, C10.14, C10.15, C10.16, C10.17, C10.18, C10.19, C10.20, C10.21, C10.22 and C10.23, a total of 23 sub-fractions;

[0020] 3) The sub-fraction C10.13 obtained in step 2) was subjected to reverse-phase preparative HPLC with Cosmosil Packed C 18 The chromatographic column was prepared by elution with acetonitrile-water-formic acid eluent at a flow rate of 8 mL / min and a volume ratio of 37:63:0.1 to obtain compound (II).

[0021] The sub-fraction C10.15 obtained in step 2) was subjected to reverse-phase preparative HPLC with Cosmosil Packed C 18The chromatographic column was prepared by elution with acetonitrile-water-formic acid eluent at a flow rate of 8 mL / min and a volume ratio of 40:60:0.1 to obtain compound (Ⅳ);

[0022] The sub-fraction C10.18 obtained in step 2) was subjected to reverse-phase preparative HPLC with Cosmosil Packed C 18 The chromatographic column was prepared by elution with acetonitrile-water-formic acid eluent at a flow rate of 8 mL / min and a volume ratio of 40:60:0.1 to obtain compound (III).

[0023] Furthermore, the preparation method of the above-mentioned juniperane-type sesquiterpenes specifically includes the following steps:

[0024] 1) Take cinnamon bark and extract it by heating and refluxing with 60% ethanol-water solution three times, each time for 2 hours. After filtration, concentrate the filtrate under reduced pressure to obtain concentrated solution.

[0025] 2) The concentrate was extracted with dichloromethane to obtain a dichloromethane extract;

[0026] 3) The dichloromethane extract was subjected to silica gel column chromatography at atmospheric pressure. The elution program was cyclohexane-ethyl acetate with volume ratios of 99:1, 97:3, 95:5, 90:10, 85:15, 80:20, 70:30, 60:4 and 0:100, yielding 17 sub-fractions: C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16 and C17.

[0027] 4) The C10 fraction obtained in step 3) was subjected to medium-low pressure ODS column chromatography. The elution program was sequentially applied with methanol-water-formic acid at volume ratios of 35:65:0.1, 40:60:0.1, 45:55:0.1, 50:50:0.1, 60:40:0.1, 70:30:0.1, 80:20:0.1, and 100:0:0 to obtain C10.1, C10.2, and C10. The distillates consist of 23 sub-fractions: C10.3, C10.4, C10.5, C10.6, C10.7, C10.8, C10.9, C10.10, C10.11, C10.12, C10.13, C10.14, C10.15, C10.16, C10.17, C10.18, C10.19, C10.20, C10.21, C10.22, and C10.23.

[0028] 5) The fraction C10.10 obtained in step 4) was subjected to reverse-phase preparative HPLC with Cosmosil Packed C 18The chromatographic column was eluted with acetonitrile-water-formic acid at a flow rate of 12 mL / min and a volume ratio of 35:65:0.1 to obtain eight sub-fractions: C10.10.1, C10.10.2, C10.10.3, C10.10.4, C10.10.5, C10.10.6, C10.10.7, and C10.10.8. Sub-fraction C10.10.5 was then analyzed by reversed-phase preparative HPLC using Cosmosil Packed C10.10.5. 18 The chromatographic column was eluted with methanol-water-formic acid elution buffer at a flow rate of 8 mL / min and a volume ratio of 45:55:0.1 to obtain compound (Ⅰ);

[0029] Furthermore, the preparation method of the above-mentioned juniperane-type sesquiterpenes specifically includes the following steps:

[0030] 1) Take cinnamon bark and extract it by heating and refluxing with 60% ethanol-water solution three times, each time for 2 hours. After filtration, concentrate the filtrate under reduced pressure to obtain concentrated solution.

[0031] 2) The concentrate was extracted with dichloromethane to obtain a dichloromethane extract;

[0032] 3) The dichloromethane extract was subjected to silica gel column chromatography at atmospheric pressure. The elution program was cyclohexane-ethyl acetate with volume ratios of 99:1, 97:3, 95:5, 90:10, 85:15, 80:20, 70:30, 60:4 and 0:100, yielding 17 sub-fractions: C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16 and C17.

[0033] 4) The C10 fraction obtained in step 3) was subjected to medium-low pressure ODS column chromatography. The elution program was sequentially applied with methanol-water-formic acid at volume ratios of 35:65:0.1, 40:60:0.1, 45:55:0.1, 50:50:0.1, 60:40:0.1, 70:30:0.1, 80:20:0.1, and 100:0:0 to obtain C10.1, C10.2, and C10. The distillates consist of 23 sub-fractions: C10.3, C10.4, C10.5, C10.6, C10.7, C10.8, C10.9, C10.10, C10.11, C10.12, C10.13, C10.14, C10.15, C10.16, C10.17, C10.18, C10.19, C10.20, C10.21, C10.22, and C10.23.

[0034] 5) The sub-fraction C10.13 obtained in step 4) was subjected to reverse-phase preparative HPLC with Cosmosil Packed C18 The chromatographic column was eluted with acetonitrile-water-formic acid eluent at a flow rate of 8 mL / min and a volume ratio of 37:63:0.1 to obtain compound (II).

[0035] The sub-fraction C10.15 obtained in step 4) was subjected to reverse-phase preparative HPLC with Cosmosil Packed C 18 The chromatographic column was eluted with acetonitrile-water-formic acid eluent at a flow rate of 8 mL / min and a volume ratio of 40:60:0.1 to obtain compound (Ⅳ).

[0036] The sub-fraction C10.18 prepared in step 4) was subjected to reverse-phase preparative HPLC with Cosmosil Packed C 18 The chromatographic column was prepared by elution with acetonitrile-water-formic acid eluent at a flow rate of 8 mL / min and a volume ratio of 40:60:0.1 to obtain compound (III).

[0037] Another technical solution of the present invention is the application of the above-mentioned juniperane-type sesquiterpenes in the preparation of drugs for the prevention or treatment of ALD.

[0038] Compared with the prior art, the technical solution provided by the present invention has the following technical advantages:

[0039] This invention isolated four juniperane-type sesquiterpenoid compounds from the bark of Cinnamomum cassia, a plant belonging to the Lauraceae family. Hepatoprotective activity was evaluated using an alcohol-induced hepatocyte injury model. The results showed that the juniperane-type sesquiterpenoids of this invention effectively alleviated alcohol-induced damage to normal mouse hepatocytes and reduced lipid accumulation, indicating that the juniperane-type sesquiterpenoids provided by this invention possess anti-ALD activity and can be used in the preparation of anti-ALD drugs, demonstrating promising research and development prospects. Attached Figure Description

[0040] Figure 1 This is the 400MHz 1H NMR spectrum of compound (I) in deuterated methanol;

[0041] Figure 2 This is the 100MHz carbon NMR spectrum of compound (Ⅰ) in deuterated methanol;

[0042] Figure 3 This is the 400MHz 1H NMR spectrum of compound (ⅠⅠ) in deuterated dimethyl sulfoxide;

[0043] Figure 4 This is the 100MHz carbon NMR spectrum of compound (ⅠⅠ) in deuterated dimethyl sulfoxide;

[0044] Figure 5This is the 400MHz 1H NMR spectrum of compound (ⅠⅠⅠ) in deuterated dimethyl sulfoxide;

[0045] Figure 6 This is the 100MHz carbon NMR spectrum of compound (ⅠⅠⅠ) in deuterated dimethyl sulfoxide;

[0046] Figure 7 This is the 600MHz 1H NMR spectrum of compound (IV) in deuterated dimethyl sulfoxide;

[0047] Figure 8 This is the 100MHz carbon NMR spectrum of compound (IV) in deuterated dimethyl sulfoxide;

[0048] Figure 9 This is a graph showing the anti-ALD activity of juniperane-type sesquiterpenes;

[0049] Wherein: (A) is the result of normal mouse hepatocyte survival rate (n=5); (B) is the result of Oil Red O staining quantification (n=3); (C) is the ratio of Oil Red O staining quantification to cell survival rate (n=3); (D) is the result of representative Oil Red O staining photographs. Compared with the control group, ### P < 0.001; compared with the model group, * P<0.05, ** P<0.01, *** P<0.001, no significant difference in ns. Detailed Implementation

[0050] The present invention will be further described below with reference to specific embodiments, but the implementation of the present invention is not limited thereto.

[0051] The equipment and materials used in the following embodiments are as follows:

[0052] The mass spectrometer was a Q-Exactive Orbitrap mass spectrometer manufactured by Thermo Fisher Scientific, USA. The superconducting nuclear magnetic resonance spectrometers were Bruker AV-400 and Bruker AV-600. The X-ray single-crystal diffractometer was a Bruker D8venture. The silica gel GF254 for thin-layer chromatography and the silica gel (200-300 mesh) for column chromatography were both products of Qingdao Ocean Chemical Plant. The reversed-phase ODS packing material (50μm) was a product of YMC Corporation, Japan. The medium- and low-pressure liquid chromatograph was a product of Shanghai Lishui Electronic Technology Co., Ltd. The multi-functional microplate reader was an Infinite F50 microplate reader from Tecan, Switzerland. The preparative-grade chromatographic column used in the liquid chromatography separation was a Cosmosil Packed C10 column. 18Column (20.0×250mm, 5μm). Acetonitrile or methanol was of chromatographic grade for liquid chromatography; all other reagents were of analytical grade. Normal mouse hepatocytes were purchased from the Cell Bank of the Chinese Academy of Sciences. Silymarin was a product of Aladdin (Shanghai) Co., Ltd.

[0053] Example 1: Preparation of compounds of formulas (I) to (IV)

[0054] 20.0 kg of dried cinnamon bark was extracted three times by heating and refluxing with 200 L of ethanol-water (60:40, v / v), each time for 2 hours. After filtration, the filtrate was concentrated under reduced pressure to obtain a concentrate (40 L). The concentrate was then extracted with dichloromethane to obtain a dichloromethane extract.

[0055] 434.3 g of dichloromethane extract was subjected to silica gel column chromatography at atmospheric pressure. The extract was eluted sequentially with cyclohexane-ethyl acetate eluents at volume ratios of 99:1, 97:3, 95:5, 90:10, 85:15, 80:20, 70:30, 60:40, and 0:100, yielding 17 sub-fractions: C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, and C17. The corresponding sub-fractions obtained by elution with different volume ratios of cyclohexane-ethyl acetate are shown in Table 1.

[0056] In the 80:20 cyclohexane-ethyl acetate elution stage, the eluent was subjected to column chromatography, and the fractions were collected stepwise based on the real-time thin-layer chromatography (TLC) monitoring results.

[0057] Sub-fraction C10: Sub-fraction C10 was collected during the initial stage of 80:20 cyclohexane-ethyl acetate elution. LC-MS analysis showed that it contained the characteristic quasi-molecular ion of the target compound (m / z 527.1 [2M+Na)). + Therefore, C10 was selected for subsequent reversed-phase column chromatography purification.

[0058] Sub-fraction C11: Sub-fraction C11 was collected during the later stage of 80:20 cyclohexane-ethyl acetate elution. LC-MS analysis showed no detection of the target compound, therefore it was not used in subsequent steps. Although C11 was collected under the same elution ratio in this step, it was discarded because it did not contain the target compound.

[0059] Table 1. Correlation table of sub-fractions obtained with cyclohexane-ethyl acetate eluent at different volume ratios.

[0060] Cyclohexane-ethyl acetate volume ratio Sub-fraction 99:1 C1, C2 97:3 C3, C4, C5 95:5 C6, C7 90:10 C8 85:15 C9 80:20 C10, C11 70:30 C12, C13, C14 60:40 C15, C16 0:100 C17

[0061] The fraction C10 (8.0 g) obtained by elution with cyclohexane-ethyl acetate at a volume ratio of 80:20 was then subjected to medium-low pressure ODS column chromatography, successively eluted with methanol-water-formic acid at volume ratios of 35:65:0.1, 40:60:0.1, 45:55:0.1, 50:50:0.1, 60:40:0.1, 70:30:0.1, 80:20:0.1, and 100:0:0, to obtain C10.1, C10.2, C10.3, and C10. 4, C10.5, C10.6, C10.7, C10.8, C10.9, C10.10, C10.11, C10.12, C10.13, C10.14, C10.15, C10.16, C10.17, C10.18, C10.19, C10.20, C10.21, C10.22, and C10.23, a total of 23 sub-fractions; the sub-fractions corresponding to methanol-water-formic acid elution with different volume ratios are shown in Table 2.

[0062] In the 45:55:0.1 methanol-water-formic acid elution stage, after the eluent was subjected to medium-low pressure reversed-phase column chromatography, the fractions were collected stepwise based on the real-time thin-layer chromatography (TLC) monitoring results.

[0063] Sub-fraction C10.9: Sub-fraction C10.9 was collected at the beginning of elution with methanol-water-formic acid at a ratio of 45:55:0.1. No target compound was found by LC-MS analysis, so it was not used in subsequent steps.

[0064] Sub-fraction C10.10: Sub-fraction C10.10 was collected during the middle stage of elution with methanol-water-formic acid at a ratio of 45:55:0.1. LC-MS analysis showed that it contained the characteristic quasi-molecular ion of the target compound (m / z 527.1 [2M+Na)). + Therefore, C10.10 was selected for subsequent reversed-phase preparative high-performance liquid chromatography preparation.

[0065] Sub-fraction C10.11: Sub-fraction C10.11 was collected in the later stage of elution with methanol-water-formic acid at a ratio of 45:55:0.1. No target compound was found by LC-MS analysis, so it was not used in subsequent steps.

[0066] In this step, C10.9 and C10.11 were collected at the same elution ratio, but were discarded because they did not contain the target compound.

[0067] Table 2. Correlation table of sub-fractions obtained with methanol-water-formic acid eluent at different volume ratios.

[0068] methanol-water-formic acid volume ratio Sub-fraction 35:65:0.1 C10.1, C10.2, C10.3, C10.4 40:60:0.1 C10.5, C10.6, C10.7, C10.8 45:55:0.1 C10.9, C10.10, C10.11 50:50:0.1 C10.12, C10.13 60:40:0.1 C10.14, C10.15, C10.16 70:30:0.1 C10.17, C10.18, C10.19 80:20:0.1 C10.20, C10.21, C10.22 100:0:0 C10.23

[0069] The fraction C10.10 (120.1 mg) obtained by elution with methanol-water-formic acid at a volume ratio of 45:55:0.1 was analyzed by reversed-phase preparative HPLC using Cosmosil Packed C. 18 The chromatographic column was prepared by elution with acetonitrile-water-formic acid at a flow rate of 12 mL / min and a volume ratio of 35:65:0.1, yielding a total of 8 sub-fractions: C10.10.1, C10.10.2, C10.10.3, C10.10.4, C10.10.5, C10.10.6, C10.10.7, and C10.10.8.

[0070] The fraction C10.10.5 (38.2 mg) was prepared by reverse-phase preparative HPLC using Cosmosil Packed C 18 Column preparation: Elution was performed using methanol-water-formic acid at a flow rate of 8 mL / min with a volume ratio of 45:55:0.1 to obtain compound (t) of formula (Ⅰ). R (95.4 min, 21.4 mg, purity 95%);

[0071]

[0072] The fraction C10.13 (77.1 mg) obtained by elution with methanol-water-formic acid at a volume ratio of 50:50:0.1 was analyzed by reversed-phase preparative HPLC using Cosmosil Packed C. 18 Column preparation was performed using acetonitrile-water-formic acid at a flow rate of 8 mL / min and a volume ratio of 37:63:0.1 to elute, yielding compound (t) of formula (II). R (27.9 min, 4.6 mg, purity 95%);

[0073]

[0074] The fraction C10.15 (124.1 mg) obtained by elution with methanol-water-formic acid at a volume ratio of 60:40:0.1 was analyzed by reversed-phase preparative HPLC using Cosmosil Packed C. 18 Column preparation was performed using acetonitrile-water-formic acid at a flow rate of 8 mL / min (v / v ratio 40:60:0.1) to obtain compound (t) of formula (Ⅳ). R (47.0 min, 15.4 mg, purity 95%);

[0075]

[0076] The fraction C10.18 (219.0 mg) obtained by elution with methanol-water-formic acid at a volume ratio of 70:30:0.1 was analyzed by reversed-phase preparative HPLC using Cosmosil Packed C. 18 Column preparation was performed using acetonitrile-water-formic acid at a flow rate of 8 mL / min and a volume ratio of 40:60:0.1 to obtain compound (t) of formula (III). R (90.1 min, 5.4 mg, purity 95%).

[0077]

[0078] The physicochemical constants are as follows:

[0079] Compound of formula (I): white needle-like crystals (methanol); mp 115.3-116.2℃; [α]25D+99.0 (c 0.5, methanol); UV (methanol)λ max (logε)203(3.30),284(1.66)nm; ECD(c 2.0×10 -3 M, methanol)λ max (Δε)305(22.41); IR(KBr)v max 3433,2961,2871,1710,1593,1376,1107cm –1 ESIMS (positive ion) m / z 527.1 [2M+Na] + HRESIMS (positive ion) m / z 275.1615 [M+Na] + (Calculated value [C]) 15 H 24 O3Na] + (275.1618), the molecular formula of the compound was determined to be C 15 H 24 O3; see the hydrogen spectrum. Figure 1 See the carbon spectrum. Figure 2 The NMR data are assigned in Table 3; the single-crystal diffraction data of compound (Ⅰ) are shown in Table 4.

[0080] Compound (II): White needle-like crystals (methanol); mp 137.1-138.8℃; [α]25D -57.5 (c 0.5, methanol); UV (methanol)λ max (logε)204(3.51),231(3.73),287(2.40)nm; ECD(c 2.0×10 -3 M, methanol)λ max (Δε)221(7.09),239(-13.80); IR(KBr)v max3444,2959,2871,1674,1593,1383,1057cm –1 ESIMS (positive ion) m / z 527.2 [2M+Na] + HRESIMS (positive ion) m / z 275.1616 [M+Na] + (Calculated value [C]) 15 H 24 O3Na] + (275.1618), the molecular formula of the compound was determined to be C 15 H 24 O3; see the hydrogen spectrum. Figure 3 See the carbon spectrum. Figure 4 The NMR data are assigned in Table 3; the single-crystal diffraction data of compound (II) are shown in Table 5.

[0081] Compound (III): Yellow oily substance; [α]25D-69.2 (c 0.5, methanol); UV (methanol)λ max (logε)205(3.88)nm; ECD(c 2.1×10 -3 M, methanol)λ max (Δε)203(-17.25); IR(KBr)v max 3422,2959,2871,1631,1595,1380,1064cm –1 ESIMS (positive ion) m / z 261.1 [M+Na] + HRESIMS (positive ion) m / z 261.1824 [M+Na] + (Calculated value [C]) 15 H 26 O2Na] + (261.1825), the molecular formula of this compound was determined to be C 15 H 26 O2; see the hydrogen spectrum. Figure 5 See the carbon spectrum. Figure 6 The NMR data attribution is shown in Table 3.

[0082] Compound (IV): Yellow oily substance; [α]25D-96.2 (c 0.5, methanol); UV(MeOH)λ max (logε)203(3.62),239(2.75)nm; ECD(c 2.1×10 -3 M, methanol)λ max (Δε)198(-18.23); IR(KBr)v max3431,2956,2875,1631,1600,1369,1064cm –1 ESIMS (positive ion) m / z 261.2 [M+Na] + HRESIMS (positive ion) m / z 261.1824 [M+Na] + (Calculated value [C]) 15 H 26 O2Na] + (261.1825), the molecular formula of this compound was determined to be C 15 H 26 O2; see the hydrogen spectrum. Figure 7 See the carbon spectrum. Figure 8 The data is shown in Table 3.

[0083] Table 3. Carbon and hydrogen spectral data of compounds of formulas (I) to (IV) and their attribution.

[0084]

[0085]

[0086] Signal 'a' was not labeled due to overlap or complex splitting.

[0087] b was tested in deuterated methanol (H1N1 spectrum at 400 MHz, C1N1 spectrum at 100 MHz).

[0088] c was tested in deuterated dimethyl sulfoxide (H 100 MHz, C 100 MHz).

[0089] d was tested in deuterated dimethyl sulfoxide (H 100 MHz, C 100 MHz).

[0090] Table 4 Single-crystal diffraction data of compound (I)

[0091]

[0092] Table 5 Single-crystal diffraction data of compound (II)

[0093]

[0094]

[0095] Experimental Example: Evaluation of the Activity of Juniperane-type Sesquiterpenes

[0096] The following demonstrates the in vitro anti-ALD activity of the juniperine-type sesquiterpenoids (I) to (IV) disclosed in this patent and the positive control drug silymarin.

[0097] The specific method is as follows:

[0098] Establishment of an alcoholic hepatocellular injury model: Normal mouse hepatocytes were injected with 1×10⁻⁶ cells. 4 and 5×10 4 Cells were seeded at a density of 100 μL / well in 96-well and 500 μL / well in 24-well plates and allowed to adhere for 24 hours. The culture medium was then removed from the wells. Treatment groups were added to DMEM / F-12 medium containing 10 μmol / L of the test monomer (the test monomer is a juniperane-type sesquiterpene represented by formulas (I)-(IV)) or 10 μmol / L of the positive control drug silymarin and 1000 mmol / L anhydrous ethanol. The model group was added to DMEM / F-12 medium containing 1000 mmol / L anhydrous ethanol, and the blank group was added to blank medium. Cells were cultured for another 24 hours. Cell viability was assessed using the CCK-8 assay in 96-well plates and Oil Red O staining was used in 24-well plates for qualitative imaging and quantitative analysis.

[0099] Cell viability assay: After 24 hours of cell culture following modeling and drug administration, the culture medium in the 96-well cells was aspirated, and 100 μL of DMEM / F-12 medium containing 10% CCK-8 reagent was added. The cells were then incubated at 37°C in the dark for 2.5 hours. The absorbance of the treated and control groups was then measured at 450 nm using a microplate reader. Cell viability was calculated as (absorbance of the treatment group / absorbance of the control group) × 100%.

[0100] Cellular lipid accumulation detection: Weigh 0.5g of Oil Red O solid in the dark, sonicate to dissolve in 100mL of isopropanol, and then filter twice through a 0.22μm microporous membrane to obtain the Oil Red O stock solution, which is stored at 4℃. The Oil Red O working solution is obtained by mixing the Oil Red O stock solution and ultrapure water in a 3:2 ratio, and filtering twice through a 0.22μm microporous membrane. After culturing cells in 24-well plates for the appropriate time, discard the original culture medium and wash twice with PBS (5 minutes each time). Then, fix the cells with 4% paraformaldehyde in the dark for 20 minutes, rinse twice with 60% isopropanol (5 minutes each time), and then stain with Oil Red O working solution in the dark for 30 minutes. After staining, wash the 24-well plates with PBS until no excess red is left, and then take pictures using a 40× inverted microscope. After taking pictures, discard the PBS in the 24-well plates, add 250μL of isopropanol to each well, and gently pipette. 200 μL was taken from each well and placed in a 96-well plate. The absorbance was measured at 492 nm using a microplate reader. The lipid accumulation rate was calculated as (absorbance of the treatment group / absorbance of the control group) × 100%.

[0101] This invention uses an alcohol-induced hepatocyte injury model to evaluate the anti-ALD activity of compounds of formulas (I) to (IV). Figure 9(A) shows that the cell viability in the control group was 100.0%, alcohol stimulation caused the cell viability to decrease to 55.3%, and further treatment with compounds of formulas (I) to (IV) (10 μM) significantly increased the cell viability to 83.0%, 81.3%, 69.3%, and 67.6%, respectively. Figure 9 (B) shows that the lipid accumulation rate in the control group was 100.0%, while alcohol stimulation led to an increase in the cellular lipid accumulation rate to 128.6%. Administration of 10 μM of compounds (I) through (III) significantly reduced alcohol-induced hepatocyte lipid accumulation to 98.0%, 92.4%, and 99.3%, respectively. Combining cell viability and lipid accumulation results, as shown... Figure 9 As shown in (C), the ratio of lipid accumulation to cell survival rate in the control group was 1.0. Alcohol caused this ratio to rise to 2.3. Treatment with compounds of formulas (I) to (IV) reduced this ratio to 1.2, 1.1, 1.4, and 1.7, respectively, all showing significant anti-ALD activity. Among them, the anti-ALD effect of compound (I) was close to that of the positive control drug silymarin, while the anti-ALD effect of compound (II) was superior to that of silymarin. In terms of improving cell survival rate, compounds of formulas (I) and (II) were superior to silymarin. These results indicate that the juniperane-type sesquiterpenes in this invention have a significant protective effect against alcohol-induced hepatocyte damage and can be used in the preparation of anti-ALD drugs, showing good research and development prospects.

[0102] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing juniperane-type sesquiterpenes, characterized in that, The steps are as follows: 1) Take dried cinnamon bark and extract it by heating and refluxing with 60% ethanol-water solution three times, each time for 2 hours. After filtration, concentrate the filtrate under reduced pressure to obtain concentrated solution. 2) The concentrate was extracted with dichloromethane to obtain a dichloromethane extract; 3) The dichloromethane extract was subjected to silica gel column chromatography at atmospheric pressure. The elution program was cyclohexane-ethyl acetate with volume ratios of 99:1, 97:3, 95:5, 90:10, 85:15, 80:20, 70:30, 60:4 and 0:100, yielding 17 sub-fractions: C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16 and C17. 4) The C10 fraction obtained in step 3) was subjected to medium-low pressure ODS column chromatography. The elution program was sequentially methanol-water-formic acid with volume ratios of 35:65:0.1, 40:60:0.1, 45:55:0.1, 50:50:0.1, 60:40:0.1, 70:30:0.1, 80:20:0.1, and 100:0:0 to obtain C10.1, C10.2, and C10.

3. C10.4, C10.5, C10.6, C10.7, C10.8, C10.9, C10.10, C10.11, C10.12, C10.13, C10.14, C10.15, C10.16, C10.17, C10.18, C10.19, C10.20, C10.21, C10.22, and C10.23, a total of 23 sub-fractions; 5) The sub-fraction C10.10 obtained in step 4) was subjected to reverse-phase preparative HPLC with Cosmosil Packed C 18 The chromatographic column was prepared by elution with acetonitrile-water-formic acid at a flow rate of 12 mL / min and a volume ratio of 35:65:0.1, yielding eight sub-fractions: C10.10.1, C10.10.2, C10.10.3, C10.10.4, C10.10.5, C10.10.6, C10.10.7, and C10.10.

8. 6) The sub-fraction C10.10.5 obtained in step 5) was subjected to reverse-phase preparative HPLC with Cosmosil Packed C 18 The chromatographic column was eluted with methanol-water-formic acid elution buffer at a flow rate of 8 mL / min and a volume ratio of 45:55:0.1 to obtain compound (Ⅰ); The juniperane-type sesquiterpenes have the following formula (I): ； or: The sub-fraction C10.13 obtained in step 4) was subjected to reverse-phase preparative HPLC with Cosmosil Packed C 18 The chromatographic column was prepared by elution with acetonitrile-water-formic acid eluent at a flow rate of 8 mL / min and a volume ratio of 37:63:0.1 to obtain compound (II); ； or: The sub-fraction C10.15 obtained in step 4) was subjected to reverse-phase preparative HPLC with Cosmosil Packed C 18 The chromatographic column was eluted with acetonitrile-water-formic acid elution buffer at a flow rate of 8 mL / min and a volume ratio of 40:60:0.1 to obtain compound (Ⅳ); ； or: The sub-fraction C10.18 obtained in step 4) was subjected to reverse-phase preparative HPLC with Cosmosil Packed C 18 The chromatographic column was eluted with acetonitrile-water-formic acid elution buffer at a flow rate of 8 mL / min and a volume ratio of 40:60:0.1 to obtain compound (III); 。 2. The use of the juniperane-type sesquiterpenes according to claim 1 in the preparation of drugs for the prevention or treatment of alcoholic liver disease.

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