Cadinane type sesquiterpene as well as preparation method and application thereof

Abstract

The invention discloses a cadinane type sesquiterpene as well as a preparation method and application thereof. The cadinane type sesquiterpene is separated from cinnamon bark, and has at least one of structural formulas shown as a formula (I), a formula (II), a formula (III) and a formula (IV). Liver protection activity evaluation is carried out by applying an alcohol-induced hepatocyte injury model, and it is found that the cadinane type sesquiterpene can effectively relieve alcohol-induced AML-12 hepatocyte injury; the dumarane sesquiterpenes can inhibit the alcoholic liver disease and relieve fat accumulation, so that the dumarane sesquiterpenes have the anti-alcoholic liver disease activity, can be applied to preparation of the anti-alcoholic liver disease drugs, and have good research and development prospects; belongs to the field of natural medicinal chemistry.

Description

Technical Field

[0001] The present invention belongs to the field of natural medicinal chemistry, and in particular relates to a cadinane-type sesquiterpene, a preparation method thereof, and uses thereof. Background Art

[0002] Alcoholic liver disease (ALD) is a liver disease caused by long-term, excessive alcohol consumption. Clinically, the spectrum of ALD disease encompasses alcoholic fatty liver disease, alcoholic hepatitis, alcoholic liver fibrosis, and alcoholic cirrhosis, ranging from simple fatty degeneration to more severe pathological liver changes. The pathogenesis of ALD is complex, and no proven treatment is available. Currently available ALD treatments primarily serve as adjuvant therapies with limited efficacy. Therefore, the search for effective and safe anti-ALD drugs is of vital importance.

[0003] Cinnamon (Cinnamomum cassia) is a plant of the genus Cinnamomum in the Lauraceae family, widely distributed in tropical and subtropical regions of East and Southeast Asia. Cinnamon bark, a traditional Chinese medicine with both medicinal and edible properties, has the effects of tonifying fire and 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 object of the present invention is to provide a class of cadinane-type sesquiterpenes having anti-ALD activity.

[0005] The cadinane-type sesquiterpenes provided by the present invention are isolated from cinnamon bark; and their structural formulas are shown in formula (I) and / or formula (II) and / or formula (III) and / or formula (IV):

[0006]

[0007] The cadinane-type sesquiterpenes were isolated from the bark of Cinnamomum cassia Presl., a plant of the Lauraceae family. The bark was purchased from the Qingping Traditional Chinese Medicine Market in Liwan District, Guangzhou, Guangdong Province, in April 2021. The sample is stored at the School of Biomedicine, Guangdong University of Technology (No. CC-202104, Location: School of Biomedicine, Guangdong University of Technology, 100 Outer Ring West Road, Guangzhou University City, Guangzhou 510006, China).

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

[0009] Furthermore, in the preparation method of the above-mentioned cadinane-type sesquiterpenes, the cadinane-type sesquiterpenes are obtained from cinnamon bark by extraction, concentration, extraction, column chromatography separation, and reverse-phase high performance liquid chromatography separation.

[0010] Further, in the preparation method of the above-mentioned cembranoid, the extraction is carried out by heating reflux extraction with 60% ethanol-water solution.

[0011] Further, in the preparation method of the above-mentioned cembranoid, the extraction is carried out by heating reflux extraction with 60% ethanol-water solution.

[0012] Further, in the preparation method of the above-mentioned cembranoid, the extraction is carried out by heating reflux extraction with 60% ethanol-water solution.

[0013] 1) The dichloromethane extract is subjected to normal pressure silica gel column chromatography, and eluted with cyclohexane-ethyl acetate eluent with volume ratio of 99:1, 97:3, 95:5, 90:10, 85:15, 80:20, 70:30, 60:40 and 0:100 in sequence 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 sub-fraction C10 obtained in step 1) is subjected to medium-low pressure ODS column chromatography, and eluted with methanol-water-formic acid eluent with volume ratio 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, 100:0:0 in sequence to obtain C10.1, C10.2, 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;

[0015] 3) The sub-fraction C10.10 obtained in step 2) is subjected to reversed phase preparative HPLC Cosmosil Packed C 18 chromatography column preparation, and eluted with acetonitrile-water-formic acid eluent with volume ratio of 35:65:0.1 at a flow rate of 12 mL / min to obtain 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, a total of 8 sub-fractions;

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

[0017] Furthermore, in the above-mentioned preparation method of cadinane-type sesquiterpenes, the specific method of separating the dichloromethane extract by column chromatography and reverse-phase high-performance liquid chromatography is as follows:

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

[0019] 2) The subfraction C10 obtained in step 1) was subjected to medium-low pressure ODS column chromatography and eluted with methanol-water-formic acid eluents 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 C1 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;

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

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

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

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

[0024] 1) Extract cinnamon bark with a 60% ethanol-water solution by heating and refluxing for 3 times, each time for 2 hours. After filtering, the filtrate is concentrated under reduced pressure to obtain a concentrate;

[0025] 2) extracting the concentrate with dichloromethane to obtain a dichloromethane extract;

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

[0027] 4) The subfraction C10 obtained in step 3) was chromatographed on a medium-low pressure ODS column, and the elution procedure was 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 methanol-water-formic acid 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;

[0028] 5) The subfraction C10.10 obtained in step 4) was subjected to reverse phase preparative HPLC using Cosmosil Packed C 18The 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 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, a total of 8 sub-fractions; the obtained sub-fraction C10.10.5 was purified by reverse phase preparative HPLC Cosmosil Packed C 18 The column was eluted with methanol-water-formic acid at a flow rate of 8 mL / min and a volume ratio of 45:55:0.1 to obtain the compound of formula (I);

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

[0030] 1) Extract cinnamon bark with a 60% ethanol-water solution by heating and refluxing for 3 times, each time for 2 hours. After filtering, the filtrate is concentrated under reduced pressure to obtain a concentrate;

[0031] 2) extracting the concentrate with dichloromethane to obtain a dichloromethane extract;

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

[0033] 4) The subfraction C10 obtained in step 3) was chromatographed on a medium-low pressure ODS column, and the elution procedure was 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 methanol-water-formic acid 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;

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

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

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

[0037] Another technical solution of the present invention is the use of the above-mentioned cadinane-type sesquiterpenes in the preparation of drugs for preventing or treating ALD.

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

[0039] The present invention isolated four cadinane-type sesquiterpenoid compounds from the bark of cinnamon trees (Cinnamomum cassia), a plant of the Lauraceae family. Their hepatoprotective activity was evaluated using an alcohol-induced hepatocyte injury model. The results showed that the cadinane-type sesquiterpenoids of the present invention were able to effectively alleviate alcohol-induced damage to normal hepatocytes in mice and reduce lipid accumulation. This indicates that the cadinane-type sesquiterpenoids provided by the present invention have anti-ALD activity and can be used in the preparation of anti-ALD drugs, thus showing good research and development prospects. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

[0043] Figure 4 is the 100 MHz C NMR spectrum of the compound of formula (III) in deuterated dimethyl sulfoxide;

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

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

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

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

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

[0049] Among them: (A) is the result of normal mouse liver cell survival rate (n=5); (B) is the quantitative result of Oil Red O staining (n=3); (C) is the ratio of Oil Red O staining quantification to cell survival rate (n=3); (D) is the representative Oil Red O staining photo result. Compared with the control group, ### P<0.001; compared with the model group, * P<0.05, ** P<0.01, *** P<0.001, ns, no significant difference. DETAILED DESCRIPTION

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

[0051] The equipment and materials related to the following examples are as follows:

[0052] The mass spectrometer was a Q-Exactive Orbitrap mass spectrometer produced 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 Bruker D8venture. The silica gel GF254 for thin layer chromatography and the silica gel for column chromatography (200-300 mesh) were both products of Qingdao Ocean Chemical Plant. The reversed-phase ODS filler (50 μm) was a product of YMC Company, Japan. The medium and low pressure liquid chromatograph was a product of Shanghai Lisui Electronic Technology Co., Ltd. The multifunctional microplate reader was the Infinite F50 microplate reader produced by Tecan Company, Switzerland. The preparative-grade chromatographic column used for liquid phase separation was Cosmosil Packed C 18A column (20.0 × 250 mm, 5 μm) was used for liquid chromatography. Acetonitrile or methanol was chromatographically pure; all other reagents were analytically pure. Normal mouse hepatocytes were purchased from the Cell Bank of the Chinese Academy of Sciences. Silymarin was obtained from Shanghai Aladdin Company.

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

[0054] 20.0 kg of dried cinnamon bark was extracted with 200 L of ethanol-water (60:40, v / v) under reflux for three 2-hour cycles. 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 the dichloromethane extract was subjected to normal pressure silica gel column chromatography and eluted 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, respectively, to obtain C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16 and C17, a total of 17 sub-fractions. 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 eluate passed through the column chromatography and fractions were collected step by step according to the real-time thin layer chromatography (TLC) monitoring results.

[0057] Subfraction C10: Subfraction C10 was collected at the beginning of the elution with 80:20 cyclohexane-ethyl acetate. LC-MS analysis showed that it contained the characteristic quasi-molecular ion of the target compound (m / z 527.1 [2M+Na] + ), so C10 was selected for subsequent reverse phase column chromatography purification.

[0058] Subfraction C11: Subfraction C11 was collected late in the 80:20 cyclohexane-ethyl acetate elution. LC-MS analysis did not reveal the target compound and therefore was not used in the subsequent step. In this step, C11 was collected at the same elution ratio but was discarded because it did not contain the target compound.

[0059] Table 1 Correlation table of subfractions obtained with different volume ratios of cyclohexane-ethyl acetate eluents

[0060] Cyclohexane-ethyl acetate volume ratio sub-fractions 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] Then, the subfraction C10 (8.0 g) obtained by eluting with cyclohexane-ethyl acetate in a volume ratio of 80:20 was subjected to medium-low pressure ODS column chromatography and eluted with methanol-water-formic acid in a volume ratio 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. .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 the elution with different volume ratios of methanol-water-formic acid are shown in Table 2.

[0062] In the 45:55:0.1 methanol-water-formic acid elution stage, the eluate passed through a medium- and low-pressure reverse phase column chromatography, and fractions were collected step by step based on the real-time thin layer chromatography (TLC) monitoring results.

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

[0064] Subfraction C10.10: Subfraction C10.10 was collected in the middle of the 45:55:0.1 methanol-water-formic acid elution. LC-MS analysis showed that it contained the characteristic quasi-molecular ion of the target compound (m / z 527.1 [2M+Na] + ), therefore C10.10 was selected for subsequent reverse-phase preparative HPLC preparation.

[0065] Subfraction C10.11: Subfraction C10.11 was collected at the end of the elution phase with 45:55:0.1 methanol-water-formic acid. No target compound was found by LC-MS, so it was not used in subsequent steps.

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

[0067] Table 2 Correlation table of subfractions obtained with different volume ratios of methanol-water-formic acid eluents

[0068] Methanol-water-formic acid volume ratio sub-fractions 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 subfraction C10.10 (120.1 mg) obtained by eluting with methanol-water-formic acid in a volume ratio of 45:55:0.1 was purified by reverse phase preparative HPLC using Cosmosil Packed C 18 The column was prepared and eluted with acetonitrile-water-formic acid in a volume ratio of 35:65:0.1 at a flow rate of 12 mL / min to obtain 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, a total of 8 subfractions.

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

[0071]

[0072] The subfraction C10.13 (77.1 mg) obtained by eluting with methanol-water-formic acid in a volume ratio of 50:50:0.1 was purified by reverse phase preparative HPLC using Cosmosil Packed C 18 The column was prepared and eluted with acetonitrile-water-formic acid in a volume ratio of 37:63:0.1 at a flow rate of 8 mL / min to obtain the compound of formula (II) (t R :27.9min,4.6mg,purity 95%);

[0073]

[0074] The subfraction C10.15 (124.1 mg) obtained by eluting with methanol-water-formic acid in a volume ratio of 60:40:0.1 was purified by reverse phase preparative HPLC using Cosmosil Packed C 18 The chromatographic column was prepared and eluted with acetonitrile-water-formic acid in a volume ratio of 40:60:0.1 at a flow rate of 8 mL / min to obtain the compound of formula (IV) (t R :47.0min,15.4mg,purity 95%);

[0075]

[0076] The subfraction C10.18 (219.0 mg) obtained by eluting with methanol-water-formic acid in a volume ratio of 70:30:0.1 was purified by reverse phase preparative HPLC using Cosmosil Packed C 18 The chromatographic column was prepared and eluted with acetonitrile-water-formic acid in a volume ratio of 40:60:0.1 at a flow rate of 8 mL / min to obtain the compound of formula (III) (t R :90.1min,5.4mg,purity 95%).

[0077]

[0078] The physicochemical constants are as follows:

[0079] Compound of formula (I): white needle-shaped crystals (methanol); mp 115.3-116.2°C; [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; hydrogen spectrum see Figure 1 , carbon spectrum see Figure 2 , NMR data assignment is shown in Table 3; single crystal diffraction data of the compound of formula (Ⅰ) is shown in Table 4.

[0080] Compound of formula (II): white needle-like crystals (methanol); mp 137.1-138.8°C; [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; hydrogen spectrum see Figure 3 , carbon spectrum see Figure 4 , NMR data assignment is shown in Table 3; single crystal diffraction data of the compound of formula (II) is shown in Table 5.

[0081] Compound of formula (III): yellow oil; [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 the compound is determined to be C 15 H 26 O2; hydrogen spectrum see Figure 5 , carbon spectrum see Figure 6 , NMR data assignments are shown in Table 3.

[0082] Compound of formula (IV): yellow oil; [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 the compound is determined to be C 15 H 26 O2; hydrogen spectrum see Figure 7 , carbon spectrum see Figure 8 , data are shown in Table 3.

[0083] Table 3 C and H spectra data and their assignments of compounds of formula (I) to (IV)

[0084]

[0085]

[0086] aSignals are not labeled due to overlapping or complex splitting.

[0087] bTested in deuterated methanol (400 MHz for hydrogen spectrum and 100 MHz for carbon spectrum).

[0088] c was tested in deuterated dimethyl sulfoxide (400 MHz for hydrogen spectrum and 100 MHz for carbon spectrum).

[0089] d Measured in deuterated dimethyl sulfoxide (H spectrum at 600 MHz, C spectrum at 100 MHz).

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

[0091]

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

[0093]

[0094]

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

[0096] The following shows the in vitro anti-ALD activities of the cadinane-type sesquiterpenes of formula (I) to formula (IV) disclosed in this patent and the positive control drug silymarin.

[0097] The specific method is as follows:

[0098] Establishment of alcoholic hepatocyte injury model: normal mouse hepatocytes were plated at a density of 1 x 10 4 and 5 x 10 4 in 96-well plates (100 μL / well) and 24-well plates (500 μL / well) and adhered for 24 hours. The medium in the wells was aspirated, and the treatment groups were added with DMEM / F-12 medium containing 10 μmol / L of the monomer to be tested (the labdanoid sesquiterpenes shown in formula (I) to formula (IV)) or 10 μmol / L of silymarin and 1000 mmol / L of anhydrous ethanol; the model groups were added with DMEM / F-12 medium containing 1000 mmol / L of anhydrous ethanol, and the blank groups were added with blank medium, and incubated for another 24 hours. The cell survival rate was detected by CCK-8 experiment for the 96-well plates, and the cells in the 24-well plates were stained with oil red O, and qualitative photography and quantitative detection were performed.

[0099] Cell survival rate detection: after the cells in the 96-well plates were cultured for 24 hours after modeling and drug administration, the medium in the wells was aspirated, 100 μL of DMEM / F-12 medium containing 10% CCK-8 reagent was added, and the cells were incubated in a 37°C cell incubator for 2.5 hours in the dark. Subsequently, the absorbance values of the above treatment groups and blank groups were detected by a microplate reader at a wavelength of 450 nm. The cell survival rate was calculated as: (absorbance value of the treatment group / absorbance value of the control group) x 100%.

[0100] Cell lipid accumulation detection: 0.5 g of oil red O solid was weighed in the dark, ultrasonically dissolved in 100 mL of isopropyl alcohol, and then filtered twice through a 0.22 μm microporous filter to obtain an oil red O stock solution, which was stored at 4°C. The oil red O working solution was obtained by mixing the oil red O stock solution with ultrapure water at a ratio of 3:2, filtering twice through a 0.22 μm microporous filter, and then filtering twice through a 0.22 μm microporous filter. After the cells in the 24-well plates were cultured for a corresponding time, the original culture medium was discarded, and the cells were washed twice with PBS (5 minutes per time). Then, the cells were fixed with 4% paraformaldehyde in the dark for 20 minutes, washed twice with 60% isopropyl alcohol (5 minutes per time), and then stained with the oil red O working solution in the dark for 30 minutes. After staining, the 24-well plates were washed with PBS until no excess red color was present, and then photographed using a 40x inverted microscope. After photographing, the PBS in the 24-well plates was discarded, and 250 μL of isopropyl alcohol was added to each well and gently blown and sucked. 200 μL was taken from each well and placed in a 96-well plate, and the absorbance value was detected by a microplate reader at a wavelength of 492 nm. The lipid accumulation rate was calculated as: (absorbance value of the treatment group / absorbance value of the control group) x 100%.

[0101] The present application uses an alcohol-induced hepatocyte injury model to evaluate the anti-ALD activity of the compounds of formula (I) to formula (IV). From the results, it can be seen that the compounds of formula (I) to formula (IV) have good anti-ALD activity. Figure 9(A) It can be seen that the cell survival rate of the control group was 100.0%, and the cell survival rate decreased to 55.3% due to alcohol stimulation. Further treatment with the compounds of formula (I) to formula (IV) (10 μM) significantly increased the cell survival rate to 83.0%, 81.3%, 69.3% and 67.6%, respectively. Figure 9 (B) shows that the lipid accumulation rate of the control group was 100.0%, and alcohol stimulation caused the cell lipid accumulation rate to rise to 128.6%. Administration of 10 μM of the compounds of formula (I) to formula (III) significantly reduced alcohol-induced hepatocyte lipid accumulation to 98.0%, 92.4%, and 99.3%, respectively. The results of cell survival rate and lipid accumulation were combined, as shown in Figure 2. Figure 9 As shown in Figure (C), the ratio of lipid accumulation to cell viability 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, demonstrating significant anti-ALD activity. The anti-ALD effect of the compound of Formula (I) was close to that of the positive agent silymarin, while the anti-ALD effect of the compound of Formula (II) was superior to that of silymarin. In terms of improving cell viability, the compounds of Formulas (I) and (II) were superior to silymarin. These results demonstrate that the cadinane-type sesquiterpenes of the present invention have a significant protective effect against alcohol-induced liver cell damage and can be used in the preparation of anti-ALD drugs, showing promising research and development prospects.

[0102] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A cadinane-type sesquiterpene, characterized in that: The cadinane-type sesquiterpenes are isolated from cinnamon bark and have at least one of the structural formulas shown in formula (I), formula (II), formula (III), and formula (IV):

2. The method for preparing the cadinane-type sesquiterpenoid according to claim 1, characterized in that: The cadinane-type sesquiterpenoid is separated from cinnamon bark.

3. The method for preparing cadinane-type sesquiterpenes according to claim 2, characterized in that: The cadinane-type sesquiterpenes are obtained from cinnamon bark through extraction, concentration, extraction, column chromatography separation and reversed-phase high performance liquid chromatography separation.

4. The method for preparing cadinane-type sesquiterpenes according to claim 3, characterized in that: The extraction is carried out by heating and refluxing with a 60% volume fraction ethanol-water solution.

5. The method for preparing cadinane-type sesquiterpenes according to claim 3, characterized in that: The extraction is performed using dichloromethane to obtain a dichloromethane extract.

6. The method for preparing cadinane-type sesquiterpenes according to claim 5, characterized in that: The specific method for separating the dichloromethane extract by column chromatography and reverse-phase high performance liquid chromatography is as follows: 1) the dichloromethane extract was subjected to normal pressure silica gel column chromatography, and eluted with cyclohexane-ethyl acetate at volume ratios of 99:1, 97:3, 95:5, 90:10, 85:15, 80:20, 70:30, 60:40, and 0:100, in sequence, to obtain sub-fractions C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, and C17, for a total of 17 sub-fractions; 2) The subfraction C10 obtained in step 1) was subjected to medium-low pressure ODS column chromatography and eluted with methanol-water-formic acid eluents 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 C1 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; 3) The subfraction C10.10 obtained in step 2) was subjected to reverse phase preparative HPLC using Cosmosil Packed C 18 The column was prepared and eluted with acetonitrile-water-formic acid in a volume ratio of 35:65:0.1 at a flow rate of 12 mL / min to obtain 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, a total of 8 subfractions; 4) The subfraction C10.10.5 obtained in step 3) was subjected to reverse phase preparative HPLC Cosmosil Packed C 18 The chromatographic column was prepared and eluted with methanol-water-formic acid at a flow rate of 8 mL / min and a volume ratio of 45:55:0.1 to obtain the compound of formula (I).

7. The method for preparing cadinane-type sesquiterpenes according to claim 5, characterized in that: The specific method for separating the dichloromethane extract by column chromatography and reverse-phase high performance liquid chromatography is as follows: 1) the dichloromethane extract was subjected to normal pressure silica gel column chromatography, and eluted with cyclohexane-ethyl acetate at volume ratios of 99:1, 97:3, 95:5, 90:10, 85:15, 80:20, 70:30, 60:40, and 0:100, in sequence, to obtain sub-fractions C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, and C17, for a total of 17 sub-fractions; 2) The subfraction C10 obtained in step 1) was subjected to medium-low pressure ODS column chromatography and eluted with methanol-water-formic acid eluents 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 C1 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; 3) The subfraction C10.13 obtained in step 2) was subjected to reverse phase preparative HPLC Cosmosil Packed C 18 The chromatographic column was prepared and eluted with acetonitrile-water-formic acid at a flow rate of 8 mL / min and a volume ratio of 37:63:0.1 to obtain the compound of formula (II); The subfraction C10.15 obtained in step 2) was purified by reverse phase preparative HPLC using Cosmosil Packed C 18 The chromatographic column was prepared and eluted with acetonitrile-water-formic acid at a flow rate of 8 mL / min and a volume ratio of 40:60:0.1 to obtain the compound of formula (IV); The subfraction C10.18 obtained in step 2) was purified by reverse phase preparative HPLC using Cosmosil Packed C 18 The chromatographic column was prepared and eluted with acetonitrile-water-formic acid at a flow rate of 8 mL / min and a volume ratio of 40:60:0.1 to obtain the compound of formula (III).

8. The method for preparing the cadinane-type sesquiterpenoid according to any one of claim 6, characterized in that: The method includes the following steps in sequence: 1) Extracting dried cinnamon bark with a 60% ethanol-water solution by heating and refluxing for 3 times, each time for 2 hours, filtering, and concentrating the filtrate under reduced pressure to obtain a concentrate; 2) extracting the concentrate with dichloromethane to obtain a dichloromethane extract; 3) the dichloromethane extract was subjected to atmospheric pressure silica gel column chromatography, eluting with cyclohexane-ethyl acetate in volume ratios of 99:1, 97:3, 95:5, 90:10, 85:15, 80:20, 70:30, 60:4, and 0:100, to obtain 17 sub-fractions, including C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, and C17; 4) The subfraction C10 obtained in step 3) was subjected to medium-low pressure ODS column chromatography, eluting with methanol-water-formic acid in a volume ratio 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 subfraction C10.10 obtained in step 4) was subjected to reverse phase preparative HPLC Cosmosil Packed C 18 The column was prepared and eluted with acetonitrile-water-formic acid in a volume ratio of 35:65:0.1 at a flow rate of 12 mL / min to obtain 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, a total of 8 subfractions; 6) The subfraction C10.10.5 obtained in step 5) was subjected to reverse phase preparative HPLC Cosmosil Packed C 18 The column was eluted with methanol-water-formic acid at a flow rate of 8 mL / min and a volume ratio of 45:55:0.1 to obtain the compound of formula (I).

9. The method for preparing the cadinane-type sesquiterpenoid according to any one of claim 7, characterized in that: The method includes the following steps in sequence: 1) Extracting dried cinnamon bark with a 60% ethanol-water solution by heating and refluxing for 3 times, each time for 2 hours, filtering, and concentrating the filtrate under reduced pressure to obtain a concentrate; 2) extracting the concentrate with dichloromethane to obtain a dichloromethane extract; 3) the dichloromethane extract was subjected to atmospheric pressure silica gel column chromatography, eluting with cyclohexane-ethyl acetate in volume ratios of 99:1, 97:3, 95:5, 90:10, 85:15, 80:20, 70:30, 60:4, and 0:100, to obtain 17 sub-fractions, including C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, and C17; 4) The subfraction C10 obtained in step 3) was subjected to medium-low pressure ODS column chromatography, eluting with methanol-water-formic acid in a volume ratio 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 subfraction C10.13 obtained in step 4) was subjected to reverse phase preparative HPLC Cosmosil Packed C 18 The chromatographic column was prepared and eluted with acetonitrile-water-formic acid at a flow rate of 8 mL / min and a volume ratio of 37:63:0.1 to obtain the compound of formula (II); The subfraction C10.15 obtained in step 4) was purified by reverse phase preparative HPLC using Cosmosil Packed C 18 The column was eluted with acetonitrile-water-formic acid at a flow rate of 8 mL / min and a volume ratio of 40:60:0.1 to obtain the compound of formula (IV); The subfraction C10.18 obtained in step 4) was purified by reverse phase preparative HPLC using Cosmosil Packed C 18 The column was eluted with acetonitrile-water-formic acid at a flow rate of 8 mL / min and a volume ratio of 40:60:0.1 to obtain the compound of formula (III).

10. Use of the cadinane-type sesquiterpenoid according to claim 1 in the preparation of a drug for preventing or treating alcoholic liver disease.

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