Method for separating sesquiterpenoids from euphorbia ebracteolata and application of sesquiterpenoids

Abstract

The invention relates to a method for separating sesquiterpenoids from euphorbia euphorbia and application of the sesquiterpenoids, which comprises the following steps: crushing the overground part of euphorbia euphorbia, extracting by using ethanol at room temperature, evaporating a solvent under reduced pressure to dryness to obtain an euphorbia euphorbia crude extract, dispersing the crude extract by using water, adding ethyl acetate for extraction until an organic phase is colorless, and filtering to obtain the euphorbia euphorbia sesquiterpenoids. Combining organic phase layers, and evaporating the solvent to dryness under reduced pressure to obtain ethyl acetate extract; the method comprises the following steps of: firstly, performing normal-phase silica gel column chromatography and reversed-phase C18 column chromatography to obtain six carrot alkane type sesquiterpenoids, and performing anti-inflammatory and anti-tumor activity determination on the six compounds, and the result shows that the six separated carrot alkane type sesquiterpenoids have different degrees of anti-inflammatory and anti-tumor activity and have the potential for preparing related medicines.

Description

Technical Field

[0001] The present invention relates to the technical fields of phytochemistry and medicine, and particularly relates to a method for separating sesquiterpenoid compounds from Euphorbia esula and applications thereof. Background Art

[0002] The genus Euphorbia contains approximately 2,000 plant species globally. Euphorbia esula L. belongs to the Euphorbiaceae family and is a perennial herb that can grow to a certain height. It has typical appearance characteristics, with green leaves, and when the plant is injured, it secretes milky white juice, hence its name. This plant is widely distributed in many provinces and municipalities in China and is also found in other regions of the world. In traditional Chinese medicine, Euphorbia esula has been used to treat various diseases, including cancer, swelling, and warts. Its extracts and some compounds are considered to have anti-cancer, anti-inflammatory, and antibacterial activities. In addition to its medicinal use, Euphorbia esula can also be used in the preparation of natural pesticides and dyes.

[0003] Carotane-type sesquiterpenoids are a class of bicyclic sesquiterpenoid compounds, and their research has progressed rapidly. They are not only widely distributed in Umbelliferae plants but also the most abundant metabolites in Trichoderma viride. Earlier phytochemical and biological studies have shown that carotane compounds have various pharmacological activities, including anti-inflammatory, anti-tumor, antibacterial, anti-HIV, proliferation promotion, contraception, and inhibitory effects on certain marine plankton.

[0004] In the present invention, carotane-type sesquiterpenoid compounds are isolated and identified from Euphorbia esula. Among them, the compound of formula (I) is a new compound, and the compounds of formula (II)-(VI) are isolated from this genus of plants for the first time. At the same time, studies have shown that these compounds not only have significant anti-inflammatory activities but also exhibit obvious anti-tumor activities. Therefore, it is of great significance to conduct a systematic study on the sesquiterpenoid components in Euphorbia esula, clarify the material basis of their pharmacological activities, and discover sesquiterpenoid compounds with specific activities. Summary of the Invention

[0005] The object of the present invention is to provide a method for separating sesquiterpenoid compounds from Euphorbia esula and applications thereof. The aerial parts of Euphorbia esula are crushed and then extracted with ethanol at room temperature, and the solvent is evaporated under reduced pressure to obtain an extract paste of Euphorbia esula crude extract. Then, the crude extract paste is dispersed in water, ethyl acetate is added for extraction until the organic phase is colorless, the organic phase layers are combined, and the solvent is evaporated under reduced pressure to obtain an extract paste of ethyl acetate extract; then, through a normal-phase silica gel column, a reversed-phase C 18Column chromatography was used to obtain the carotane-type sesquiterpenoids of formulas (I)-(VI), and their anti-inflammatory and anti-tumor activities were measured. The results showed that the isolated carotane-type sesquiterpenoids of formulas (I)-(VI) all had anti-inflammatory and anti-tumor activities to varying degrees and had the potential to be used in the preparation of related drugs.

[0006] The sesquiterpenoid compound isolated from Euphorbia esula described in the present invention has the following structural formula:

[0007]

[0008] Among them: Compound (I) is (1R,4R,5S,6S)-4-hydroxy-6-(4'-hydroxy-5'-methoxybenzoyloxy)-carot-7-en-9-one;

[0009] Compound (II) is (1R,4R,5S,6S)-4-hydroxy-6-(4'-methoxy-5'-hydroxybenzoyloxy)-carot-7-en-9-one;

[0010] Compound (III) is (1R,4R,5S,6S)-4-hydroxy-6-(4',5'-dimethoxybenzoyloxy)-carot-7-en-9-one;

[0011] Compound (IV) is (1R,4R,5S,6S)-6-p-hydroxybenzoyloxy-carot-8-en-4-ol;

[0012] Compound (V) is (1R,4R,5S,6S,8S)-6-p-hydroxybenzoyloxy-carot-9-en-4,8-diol;

[0013] Compound (VI) is (1R,4R,5S,6S)-6-(4'-hydroxy-5'-methoxybenzoyloxy)-carot-8-en-4-ol. The specific operation is carried out according to the following steps:

[0014] a. Extraction: The above-ground part of Euphorbia esula was crushed according to a material-liquid ratio of 1:5 and extracted 5 times with 80% ethanol at room temperature for 24 hours each time, and then extracted 3 times with 70% ethanol for 24 hours each time. The solvent was evaporated under reduced pressure to obtain the crude extract of Euphorbia esula. The crude extract was dispersed with water and extracted with ethyl acetate until the organic phase was colorless. The organic phase layers were combined and the solvent was evaporated under reduced pressure to obtain the ethyl acetate extract.

[0015] b. Separation: The ethyl acetate extract obtained in step a was separated by a normal-phase silica gel column, and gradient elution was carried out with petroleum ether - ethyl acetate with a volume ratio of 100:1 - 0:1 as the solvent. The fractions were analyzed by silica gel thin-layer chromatography, and the same fractions were combined to obtain 8 fractions Fr.1 - Fr.8. Fraction Fr.2 was separated by reverse-phase C 18 column chromatography, and gradient elution was carried out with methanol - water with a volume ratio of 10:90 - 100:0 as the solvent to obtain 13 fractions Fr.2 - 1 - Fr.2 - 13. Fraction Fr.2 - 9 was further separated by reverse-phase column C 18 5μm 150×10mm, and an isocratic elution was carried out with an acetonitrile - water mixed solution with a volume ratio of 45:55 as the eluent to obtain the compound of formula (I) (1R,4R,5S,6S)-4-hydroxy-6-(4'-hydroxy-5'-methoxybenzoyloxy)-carotene-7-en-9-one and the compound of formula (II) (1R,4R,5S,6S)-4-hydroxy-6-(4'-methoxy-5'-hydroxybenzoyloxy)-carotene-7-en-9-one; Fraction Fr.2 - 10 was separated by reverse-phase column C 18 5μm 150×10mm, and an isocratic elution was carried out with an acetonitrile - water mixed solution with a volume ratio of 53:47 as the eluent to obtain the compound of formula (III) (1R,4R,5S,6S)-4-hydroxy-6-(4',5'-dimethoxybenzoyloxy)-carotene-7-en-9-one; Fraction Fr.3 was separated by reverse-phase C 18 column chromatography, and gradient elution was carried out with methanol - water with a volume ratio of 10:90 - 100:0 as the solvent to obtain 7 fractions Fr.3 - 1 - Fr.3 - 7. Fraction Fr.3 - 5 was further separated by reverse-phase column C 18 5μm 150×10mm, and gradient elution was carried out with an acetonitrile - water mixed solution with a volume ratio of 40:60 - 100:0 as the eluent to obtain the compound of formula (IV) (1R,4R,5S,6S)-6-p-hydroxybenzoyloxy-carotene-8-en-4-ol; Fraction Fr.3 - 4 was further separated by reverse-phase column C 18 5μm 150×10mm, and an isocratic elution was carried out with an acetonitrile - water mixed solution with a volume ratio of 53:47 as the eluent to obtain the compound of formula (V) (1R,4R,5S,6S,8S)-6-p-hydroxybenzoyloxy-carotene-9-en-4,8-diol and the compound of formula (VI) (1R,4R,5S,6S)-6-(4'-hydroxy-5'-methoxybenzoyloxy)-carotene-8-en-4-ol.

[0016] Use of the sesquiterpene compounds isolated from Euphorbia esula by the said method in the preparation of anti-inflammatory drugs.

[0017] The application of the sesquiterpenoid compound isolated from Euphorbia esula L. obtained by the said method in the preparation of drugs for anti-tumor human cervical cancer cells (HeLa), human colon cancer cells (HT-29), and human breast cancer cells (MCF-7).

[0018] A method for isolating sesquiterpenoid compounds from Euphorbia esula L. and its application as described in the present invention, and the structural identification of the isolated sesquiterpenoid compounds:

[0019] Compound of formula (I) (1R,4R,5S,6S)-4-hydroxy-6-(4'-hydroxy-5'-methoxybenzoyloxy)-carota-7-ene-9-one; is a yellow oil, and its molecular formula was determined to be C – by (–)-HRESIMS (m / z 401.1975 [M-H] 23 H 30 O6; according to 1 H, 13 C and two-dimensional nuclear magnetic resonance data, its structure was determined, the skeleton type is carotane type, and its 1 H NMR and 13 C NMR data attribution is shown in Table 1 [600 MHz ( 1 H), 150 MHz ( 13 C), solvent: CD3OD]; Compound of formula (II) (1R,4R,5S,6S)-4-hydroxy-6-(4'-methoxy-5'-hydroxybenzoyloxy)-carota-7-ene-9-one; is a yellow oil, and its molecular formula was determined to be C – by (–)-HRESIMS (m / z 401.1975 [M-H] 23 H 30 O6; according to 1 H, 13 C and two-dimensional nuclear magnetic resonance data, its structure was determined, the skeleton type is carotane type, and its 1 H NMR and 13 C NMR data attribution is shown in Table 1 [600 MHz ( 1 H), 150 MHz ( 13 C), solvent: CD3OD];

[0020] Compound of formula (III) (1R,4R,5S,6S)-4-hydroxy-6-(4',5'-dimethoxybenzoyloxy)-carota-7-ene-9-one; is a yellow oil, and its molecular formula was determined to be C + by (+)-HRESIMS (m / z 417.2280 [M+H] 23 H 32O6; Based on 1 H, 13 C and two-dimensional nuclear magnetic resonance data, its structure was determined, and the skeleton type is carotane. Its 1 H NMR and 13 C NMR data assignments are shown in Table 1 [600 MHz ( 1 H), 150 MHz ( 13 C), solvent: CD3OD].

[0021] Table 1. 1 H NMR and 13 C NMR data of compounds of formula (I), (II), and (III)

[0022]

[0023] The compound of formula (IV), (1R,4R,5S,6S)-6-(4-hydroxybenzoyloxy)-carot-8-en-4-ol, is a yellow oil. Its molecular formula was determined by (-)-HRESIMS (m / z 357.2079 – , theoretical value 357.2060) to be C 22 H 30 O4; Based on 1 H, 13 C and two-dimensional nuclear magnetic resonance data, its structure was determined, and the skeleton type is carotane. Its 1 H NMR and 13 C NMR data assignments are shown in Table 2 [600 MHz ( 1 H), 150 MHz ( 13 C), solvent: CD3OD];

[0024] The compound of formula (V), (1R,4R,5S,6S,8S)-6-(4-hydroxybenzoyloxy)-carot-9-ene-4,8-diol, is a pale yellow oil. Its molecular formula was determined by (-)-HRESIMS (m / z 373.2026 – , theoretical value 373.2010) to be C 22 H 30 O5; Based on 1 H, 13 C and two-dimensional nuclear magnetic resonance data, its structure was determined, and the skeleton type is carotane. Its 1 H NMR and 13 C NMR data assignments are shown in Table 2 [600 MHz ( 1 H), 150 MHz ( 13 C), solvent: CD3OD];

[0025] The compound of formula (VI), (1R,4R,5S,6S)-6-(4'-hydroxy-5'-methoxybenzoyloxy)-carotene-8-en-4-ol, is a pale yellow oil. Its molecular formula was determined to be C – 23 H 32 O5 by (–)-HRESIMS (m / z 387.2183 [M-H] 1 , theoretical value 387.2166); its structure was determined based on 13 H, 1 C and two-dimensional nuclear magnetic resonance data. The skeleton type is carotane. The 13 H NMR and 1 C NMR data assignments are shown in Table 2 [600 MHz ( 13 H), 150 MHz (

[0026] Table 2. 1 H NMR and 13 C NMR data of compounds of formula (IV), (V), and (VI)

[0027]

[0028] The application of the sesquiterpene compounds isolated from Euphorbia esula in the fields of anti-inflammatory and anti-tumor refers to the determination of the anti-inflammatory and anti-tumor activities of the isolated sesquiterpene compounds, which show varying degrees of activity and can be used to prepare anti-inflammatory and anti-tumor related drugs.

[0029] The sesquiterpene compounds isolated from Euphorbia esula described in the present invention can currently only be isolated and purified from plants, and no other chemical methods have been found to obtain them. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 is the 1 H NMR spectrum of the compound of formula (I) described in the present invention;

[0031] Figure 2 is the 13 C NMR spectrum of the compound of formula (I) described in the present invention;

[0032] Figure 3 is the 1 H NMR spectrum of the compound of formula (II) described in the present invention;

[0033] Figure 4 is the 13 C NMR spectrum of the compound of formula (II) described in the present invention;

[0034] Figure 5 ​For the compound of formula (III) of the present invention 1 1H NMR spectrum;

[0035] Figure 6 For the compound of formula (III) of the present invention 13 13C NMR spectrum;

[0036] Figure 7 For the compound of formula (IV) of the present invention 1 1H NMR spectrum;

[0037] Figure 8 For the compound of formula (IV) of the present invention 13 13C NMR spectrum;

[0038] Figure 9 For the compound of formula (V) of the present invention 1 1H NMR spectrum;

[0039] Figure 10 For the compound of formula (V) of the present invention 13 13C NMR spectrum;

[0040] Figure 11 For the compound of formula (VI) of the present invention 1 1H NMR spectrum;

[0041] Figure 12 For the compound of formula (VI) of the present invention 13 13C NMR spectrum. Detailed implementation mode

[0042] All reagents used are of analytical purity. Acetonitrile in high-performance liquid chromatography is of HPLC grade (Merk, USA). Column chromatography silica gel (100 - 200 mesh, 200 - 300 mesh): produced by Qingdao Ocean Chemical Factory; thin-layer chromatography silica gel is HSGF 254 , produced by Yantai Huangwu Silica Gel Development and Test Factory. High-performance liquid chromatography (DIONEX, USA): P680 HPLC pump, ASI-100 autoinjector, TCC-100 column oven, UVD170U ultraviolet detector (four wavelengths), quaternary solvent system, online degasser, Chromeleon chromatography workstation. Preparative high-performance liquid chromatography (DIONEX, USA): P680 HPLC pump, UVD170U ultraviolet detector (four wavelengths), quaternary solvent system, online degasser, Chromeleon chromatography workstation. Mass spectrometry was determined using a QSTAR Elite mass spectrometer (Applied Biosystems / MDS Sciex); nuclear magnetic resonance was determined using a Varian Vnmrs 600 / 400 type nuclear magnetic resonance spectrometer (Varian, USA).

[0043] Example 1

[0044] a. Extraction: 10 kg of the above-ground part of Euphorbia esula L. was crushed at a material-liquid ratio of 1:5 and extracted 5 times with 80% ethanol at room temperature for 24 hours each time, and then extracted 3 times with 70% ethanol for 24 hours each time. The solvent was evaporated under reduced pressure to obtain an extract of the crude extract of Euphorbia esula L. The crude extract was dispersed in water, and ethyl acetate was added for extraction until the organic phase was colorless. The organic phase layers were combined, and the solvent was evaporated under reduced pressure to obtain an extract of the ethyl acetate extract;

[0045] b. Separation: The ethyl acetate extract obtained in step a was separated by a normal-phase silica gel column, and gradient elution was carried out with a solvent of petroleum ether-ethyl acetate with a volume ratio of 100:1 - 0:1. The fractions were analyzed by silica gel thin-layer chromatography, and the same fractions were combined to obtain 8 fractions Fr.1 - Fr.8. Fraction Fr.2 was separated by reverse-phase C 18 column chromatography, and gradient elution was carried out with a solvent of methanol-water with a volume ratio of 10:90 - 100:0 to obtain 13 fractions Fr.2-1 - Fr.2-13. Fraction Fr.2-9 was further separated by reverse-phase column C 18 5μm 150×10mm, and isocratic elution was carried out with a mixed solution of acetonitrile-water with a volume ratio of 45:55 to obtain the compound of formula (Ⅰ) (1R,4R,5S,6S)-4-hydroxy-6-(4'-hydroxy-5'-methoxybenzoyloxy)-carotene-7-ene-9-one and the compound of formula (Ⅱ) (1R,4R,5S,6S)-4-hydroxy-6-(4'-methoxy-5'-hydroxybenzoyloxy)-carotene-7-ene-9-one; Fraction Fr.2-10 was separated by reverse-phase column C 18 5μm 150×10mm, and isocratic elution was carried out with a mixed solution of acetonitrile-water with a volume ratio of 53:47 to obtain the compound of formula (Ⅲ) (1R,4R,5S,6S)-4-hydroxy-6-(4',5'-dimethoxybenzoyloxy)-carotene-7-ene-9-one; Fraction Fr.3 was separated by reverse-phase C 18 column chromatography, and gradient elution was carried out with a solvent of methanol-water with a volume ratio of 10:90 - 100:0 to obtain 7 fractions Fr.3-1 - Fr.3-7. Fraction Fr.3-5 was further separated by reverse-phase column C 18 5μm 150×10mm, and gradient elution was carried out with a mixed solution of acetonitrile-water with a volume ratio of 40:60 - 100:0 as the eluent to obtain the compound of formula (Ⅳ) (1R,4R,5S,6S)-6-p-hydroxybenzoyloxy-carotene-8-en-4-ol; Fraction Fr.3-4 was further separated by reverse-phase column C 18Separation was carried out with a 5 μm, 150×10 mm column, using an acetonitrile - water mixed solution with a volume ratio of 53:47 as the eluent for isocratic elution to obtain the compound of formula (Ⅴ), (1R,4R,5S,6S,8S)-6-p-hydroxybenzoyloxy-carotene-9-ene-4,8-diol, and the compound of formula (Ⅵ), (1R,4R,5S,6S)-6-(4'-hydroxy-5'-methoxybenzoyloxy)-carotene-8-ene-4-ol.

[0046] Example 2

[0047] Application of the sesquiterpenoid compounds isolated from Euphorbia esula in the fields of anti-inflammatory and anti-tumor:

[0048] Anti-inflammatory experiment:

[0049] Measuring cell viability by MTT method:

[0050] (1) When the resuscitated RAW264.7 cells reached 80 - 90% confluence, after subculturing according to the cell status, take cells in good condition, stain with trypan blue. Add 10 μL of trypan blue to 10 μL of cell suspension, mix well, then add to the cell counting chamber and count with a cell counter.

[0051] (2) Uniformly seed the cells in a 96-well plate at a density of 1×10 4 cells / well, and incubate overnight in a constant temperature incubator at 37 °C with 5% CO2.

[0052] (3) The next day, add the test sample, set 3 replicate wells, after incubating for 1 h, add 1 μg / mL LPS, and co-incubate in a constant temperature incubator at 37 °C with 5% CO2 for 16 h.

[0053] (4) Aspirate the liquid in the wells, add 100 μL of 0.5 mg / mL MTT to each well, continue to incubate in a 37 °C, 5% CO2 incubator for 3 - 4 h, and then terminate the culture.

[0054] (5) Aspirate the liquid in the wells, add 150 μL of dimethyl sulfoxide (DMSO) to each well, shake for 10 min to fully dissolve the crystals inside the cells, and measure the absorbance of each well at a wavelength of 490 nm with an enzyme-linked immunosorbent assay (ELISA) reader.

[0055] The cell survival rate is calculated according to the following formula:

[0056] Cell proliferation viability (%) = (A 样品 - A 零孔 ) / (A 对照 - A 零孔 ) × 100%;

[0057] Measuring the intracellular NO release amount by Griess method:

[0058] (1) Cytotoxicity was measured by MTT assay. After confirming that the drug concentration had no significant effect on cell viability, samples of different concentrations were added and incubated for 1 h. 1 μg / mL LPS was then added and incubated for 16 h. After incubation, the supernatant was collected and the nitric oxide content in the cell supernatant was determined by Griess assay. Before the assay, Griess Reagent I and II were removed and allowed to return to room temperature.

[0059] (2) Dilute the standard (1-100 μM) using complete culture medium. The concentration of the standard can be 0, 1, 2, 5, 10, 20, 40, 60, or 100 μM.

[0060] (3) Add the standard sample and the collected culture supernatant to the 96-well plate at 50 μL / well;

[0061] (4) Add 50 μL of Griess Reagent I and 50 μL of Griess Reagent II, which have been restored to room temperature, to the wells in sequence;

[0062] (5) After vortexing and mixing for 5 minutes, measure the absorbance at 540 nm. Create a standard curve and calculate the NO content in the culture supernatant based on the standard curve.

[0063] The inhibition rate was calculated according to the following formula: Inhibition rate (%) = [(A LPS -A 样品 ) / A LPS ]×100%;

[0064] The experimental results are shown in Table 3:

[0065] Table 3. Anti-inflammatory activity of sesquiterpenoids from Euphorbia pulegosa

[0066]

[0067] The above results indicate that the sesquiterpenoid compounds (I)-(III) described in the present invention have different degrees of anti-inflammatory activity.

[0068] Example 3

[0069] Anti-tumor experiments of sesquiterpenoids:

[0070] The MTT assay was used to measure cell viability in the same manner as the anti-inflammatory assay. For the initial screening, a single concentration was selected, for example, a monomer compound concentration of 50 μM and an extract concentration of 50 μg / μL. The activity of the samples was tested. Samples with inhibition rates greater than 50%, 60%, or 70% were selected for further testing of the activity-dose dependence, i.e., IC 50 The values were obtained by nonlinear fitting of sample activity to sample concentration, and the software used for calculation was Graphpad Prism 4;

[0071] Table 4. Antitumor activities of sesquiterpenoids from Euphorbia esula

[0072]

[0073] The above results indicate that the sesquiterpenoids (I), (III)-(VI) of the present invention have varying degrees of activities against human cervical cancer cells (HeLa), human colon cancer cells (HT-29), and human breast cancer cells (MCF-7). Among them, the compound of formula (I) is a new compound and has both anti-inflammatory and antitumor activities.

Claims

1. A method for separating sesquiterpenoids from Euphorbia esula, characterized in that The structural formula of the compound is as follows: Among them: Compound (I) is (1R,4R,5S,6S)-4-hydroxy-6-(4'-hydroxy-5'-methoxybenzoyloxy)-carotene-7-ene-9-one; Compound (II) is (1R,4R,5S,6S)-4-hydroxy-6-(4'-methoxy-5'-hydroxybenzoyloxy)-carotene-7-ene-9-one; Compound (III) is (1R,4R,5S,6S)-4-hydroxy-6-(4',5'-dimethoxybenzoyloxy)-carotene-7-ene-9-one; Compound (IV) is (1R,4R,5S,6S)-6-p-hydroxybenzoyloxy-carotene-8-ene-4-ol; Compound (V) is (1R,4R,5S,6S,8S)-6-p-hydroxybenzoyloxy-carotene-9-ene-4,8-diol; Compound (VI) is (1R,4R,5S,6S)-6-(4'-hydroxy-5'-methoxybenzoyloxy)-carotene-8-ene-4-ol. The specific operation is carried out according to the following steps: a. Extraction: The above-ground part of Euphorbia esula is crushed according to a material-liquid ratio of 1:5, and at room temperature, extracted 5 times with 80% ethanol and 3 times with 70% ethanol. The extraction time for each time is 24 hours. The solvent is evaporated under reduced pressure to obtain an extract of the crude extract of Euphorbia esula. Then, the crude extract is dispersed with water, and ethyl acetate is added for extraction until the organic phase is colorless. The organic phase layers are combined, and the solvent is evaporated under reduced pressure to obtain an extract of the ethyl acetate extract; b. Separation: The ethyl acetate extract obtained in step a was separated by a normal-phase silica gel column, and gradient elution was carried out with petroleum ether-ethyl acetate with a volume ratio of 100:1 - 0:1 as the solvent. The fractions were analyzed by silica gel thin-layer chromatography, and the same fractions were combined to obtain 8 fractions Fr.1 - Fr.

8. Fraction Fr.2 was separated by reverse-phase C 18 column chromatography, and gradient elution was carried out with methanol-water with a volume ratio of 10:90 - 100:0 as the solvent to obtain 13 fractions Fr.2-1 - Fr.2-13. Fraction Fr.2-9 was further separated by reverse-phase column C 18 5μm 150×10mm, and an isocratic elution was carried out with an acetonitrile-water mixed solution with a volume ratio of 45:55 as the eluent to obtain the compound of formula (Ⅰ) (1R,4R,5S,6S)-4-hydroxy-6-(4'-hydroxy-5'-methoxybenzoyloxy)-carotene-7-en-9-one and the compound of formula (Ⅱ) (1R,4R,5S,6S)-4-hydroxy-6-(4'-methoxy-5'-hydroxybenzoyloxy)-carotene-7-en-9-one; Fraction Fr.2-10 was separated by reverse-phase column C 18 5μm 150×10mm, and an isocratic elution was carried out with an acetonitrile-water mixed solution with a volume ratio of 53:47 as the eluent to obtain the compound of formula (Ⅲ) (1R,4R,5S,6S)-4-hydroxy-6-(4',5'-dimethoxybenzoyloxy)-carotene-7-en-9-one; Fraction Fr.3 was separated by reverse-phase C 18 column chromatography, and gradient elution was carried out with methanol-water with a volume ratio of 10:90 - 100:0 as the solvent to obtain 7 fractions Fr.3-1 - Fr.3-7. Fraction Fr.3-5 was further separated by reverse-phase column C 18 5μm 150×10mm, and gradient elution was carried out with an acetonitrile-water mixed solution with a volume ratio of 40:60 - 100:0 as the eluent to obtain the compound of formula (Ⅳ) (1R,4R,5S,6S)-6-p-hydroxybenzoyloxy-carotene-8-en-4-ol; Fraction Fr.3-4 was further separated by reverse-phase column C 18 5μm 150×10mm, and an isocratic elution was carried out with an acetonitrile-water mixed solution with a volume ratio of 53:47 as the eluent to obtain the compound of formula (Ⅴ) (1R,4R,5S,6S,8S)-6-p-hydroxybenzoyloxy-carotene-9-en-4,8-diol and the compound of formula (Ⅵ) (1R,4R,5S,6S)-6-(4'-hydroxy-5'-methoxybenzoyloxy)-carotene-8-en-4-ol.

2. Use of the sesquiterpene compound separated from Euphorbia esula obtained by the method according to claim 1 in the preparation of an anti-inflammatory drug.

3. Use of the sesquiterpene compound separated from Euphorbia esula obtained by the method according to claim 1 in the preparation of a drug for treating tumors of human cervical cancer cells (HeLa), human colon cancer cells (HT-29), and human breast cancer cells (MCF-7).

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