Sesquiterpenoids as well as preparation method and application thereof

Abstract

The invention discloses sesquiterpenoids as well as a preparation method and application thereof. The structure of the sesquiterpenoids is shown in the specification. The sesquiterpenoids of # imgabs0 can be used for preparing drugs for preventing and / or treating cancers, and the cancers comprise at least one of breast cancer, liver cancer, lung cancer, colorectal cancer, leukemia, pancreatic cancer, glioma, osteosarcoma, cervical cancer, kidney cancer and skin cancer. According to the invention, various natural product extracts are subjected to activity screening, the sesquiterpenoids derived from the rattan chrysanthemum have excellent inhibitory activity on various different types of human cancer cells, and an optional scheme is provided for broad-spectrum anti-cancer drugs. The sesquiterpenoids applied in the invention are derived from rattan chrysanthemum, the source is wide, and the production cost is low.

Description

Technical Field

[0001] The present invention relates to the field of medicines, and in particular to a sesquiterpenoid compound and a preparation method and application thereof. Background Art

[0002] At present, cancer treatment is still one of the most difficult challenges to overcome worldwide. Cancer treatment adopts a multi-drug combination strategy, but it is easy to develop drug resistance during treatment, leading to treatment failure. my country is rich in plant resources, has many natural medicines, and has a long history of using plants as medicine. If we can extract extracts that are effective in treating cancer from existing Chinese herbal medicines, there is undoubtedly greater research potential.

[0003] Cissampelopsis volubilis, Cissampelopsis genus, Compositae, also known as passionflower. So far, no use of sesquiterpenoids extracted from Cissampelopsis volubilis for cancer treatment has been found. The present invention extracts a sesquiterpenoid compound from Cissampelopsis volubilis, and finds that it has an inhibitory effect on a variety of cancer cells. Summary of the invention

[0004] In view of the deficiencies of the prior art, the present invention provides a sesquiterpenoid compound and its use in the preparation of a drug for preventing and treating cancer. The sesquiterpenoid compound provided by the present invention has excellent inhibitory activity against a variety of different types of human cancer cells.

[0005] In order to solve the above technical problems, the technical solution of the present invention is as follows:

[0006] In a first aspect of the present invention, a sesquiterpene compound is provided, wherein the structure of the sesquiterpene compound is as shown in Formula I, II or III:

[0007]

[0008] Preferably, the structure of the sesquiterpenoid compound is as shown in Formula I.

[0009]

[0010] The second aspect of the present invention provides the use of the sesquiterpenoid compound in the preparation of a drug for preventing and / or treating cancer.

[0011] Preferably, the cancer is at least one of breast cancer, liver cancer, lung cancer, colorectal cancer, leukemia, pancreatic cancer, glioma, osteosarcoma, cervical cancer, kidney cancer and skin cancer. That is, the cancer cells inhibited by the compound are at least one of human breast cancer cells, human liver cancer cells, human leukemia cells, human cervical cancer cells, human lung cancer cells, human skin cancer cells, human colorectal cancer cells, human kidney cancer cells, human glioma cells, human pancreatic cancer cells and human osteosarcoma cells.

[0012] As further preferred, the cancer is breast cancer; the three sesquiterpenoid compounds all have a good inhibitory effect on breast cancer cells.

[0013] As a further description of the above scheme: the sesquiterpenoid compound is used in combination with a pharmaceutically acceptable carrier. That is, when the sesquiterpenoid compound is used as a medicine, it can be used directly or in the form of a pharmaceutical composition. It can be administered to a patient in need of treatment orally or by injection, and can be prepared into a variety of forms of medicine for use, and has a wide range of applications.

[0014] As a further description of the above scheme: the sesquiterpenoid compound uses a compound with a structure shown in Formula I, Formula II or Formula III, or a pharmaceutically acceptable salt, ester or stereoisomer thereof as a pharmaceutically active ingredient.

[0015] As a further description of the above scheme: the mass fraction of the active pharmaceutical ingredient is 0.1% to 99%. Preferably, the mass fraction of the active pharmaceutical ingredient is 0.1% to 20%.

[0016] The third aspect of the present invention provides a method for preparing the sesquiterpenoid compound. The method for preparing the sesquiterpenoid compound represented by formula I is as follows:

[0017] The step (1) uses a methanol solution to reflux extract the aerial part of the chrysanthemum, and collects the extract; concentrates the extract to obtain an extract; adds an appropriate amount of water to the extract and disperses it, and then extracts it with petroleum ether and ethyl acetate to obtain a petroleum ether extract and an ethyl acetate extract; after dissolving the petroleum ether extract, chromatographs it with a 100-200 mesh normal phase silica gel column, and performs gradient elution with petroleum ether-ethyl acetate and dichloromethane-methanol in sequence, and collects and obtains 9 parts of eluent, which are named Fr.A to Fr.I in sequence; the volume ratio of petroleum ether to ethyl acetate is 20:1-5:1, and gradient elution is performed; the volume ratio of dichloromethane to methanol in the dichloromethane-methanol is 50:1-3:1, and gradient elution is performed;

[0018] Step (2) The Fr.E component is separated by small pore resin chromatography, and a methanol aqueous solution with a volume fraction of 50% to 100% is used for gradient elution, and 7 eluates are collected and named Fr.E1 to Fr.E7; the Fr.E5 is subjected to C 18Reverse phase silica gel column chromatography separation, using 50% to 100% methanol aqueous solution for gradient elution, collecting 5 eluates, named Fr.E5a to Fr.E5e; Sephadex LH-20 methanol gel column chromatography was used to elute the Fr.E5c component, collecting a chromatographic solution for every 5 mL, collecting 4 eluates, named Fr.E5c1 to Fr.E5c4;

[0019] Step (3) purifying the Fr.E5c3 component by preparative HPLC to obtain sesquiterpenoid compound I.

[0020] As a further description of the above scheme: the preparation method of the hemiterpenoid compound represented by formula II is as follows:

[0021] The Fr.E component was separated by small pore resin chromatography, and a methanol aqueous solution with a volume fraction of 50% to 100% was used for gradient elution, and 7 eluents were collected and named Fr.E1 to Fr.E7. The Fr.E6 component was separated by C 18 Reverse phase silica gel column chromatography separation, using 50% to 100% methanol-water solution for gradient elution, collecting and obtaining 4 parts of eluent, named Fr.E6a to Fr.E6d; Sephadex LH-20 methanol gel column chromatography was used to elute the Fr.E6b component, collecting a chromatographic solution for every 5 mL, and obtaining 3 parts of eluent, named Fr.E6b1 to Fr.E6b3; the Fr.E6b2 component was purified by preparative HPLC to obtain a sesquiterpene compound of formula II.

[0022] As a further description of the above scheme: The preparation method of the hemiterpenoid compound represented by formula III is as follows: the Fr.E6b3 component is purified by preparative HPLC to obtain the hemiterpenoid compound represented by formula III.

[0023] Compared with the prior art, the present invention has the following beneficial effects: the present invention screens the activity of various natural product extracts and finds for the first time that the sesquiterpenoids extracted from chrysanthemum have excellent inhibitory activity against various types of human cancer cells, providing an alternative for broad-spectrum anticancer drugs. The sesquiterpenoids used in the present invention are derived from chrysanthemum, have a wide range of sources, and have low production costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 These are the structural formulas of three sesquiterpenoid compounds extracted from Chrysanthemum vulgare;

[0025] Figure 2 Sesquiterpenoid I 1H NMR spectrum;

[0026] Figure 3 Sesquiterpenoid I 13 C NMR spectrum;

[0027] Figure 4 is the HSQC spectrum of sesquiterpenoid compound I;

[0028] Figure 5 is the HMBC spectrum of sesquiterpenoid compound I;

[0029] Figure 6 Sesquiterpenoid I 1 H- 1 H COSY spectrum;

[0030] Figure 7 is the ROESY spectrum of sesquiterpenoid compound I;

[0031] Figure 8 is the HR-ESI-MS spectrum of sesquiterpenoid compound I;

[0032] Fig. 9 Sesquiterpenoid II 1 H NMR spectrum;

[0033] Fig.10 Sesquiterpenoid II 13 C NMR spectrum;

[0034] Fig.11 This is the HSQC spectrum of sesquiterpenoid II;

[0035] Fig.12 This is the HMBC spectrum of sesquiterpenoid II;

[0036] Fig.13 Sesquiterpenoid II 1 H- 1 H COSY spectrum;

[0037] Fig.14 This is the ROESY spectrum of sesquiterpenoid II;

[0038] Fig.15 HR-ESI-MS spectrum of sesquiterpenoid II;

[0039] Fig.16 Sesquiterpenoid III 1 H NMR spectrum;

[0040] Fig.17 Sesquiterpenoid III 13 C NMR spectrum;

[0041] Fig.18 This is the HSQC spectrum of sesquiterpenoid compound III;

[0042] Fig.19 This is the HMBC spectrum of sesquiterpenoid compound III;

[0043] Fig. 20 Sesquiterpenoid III 1 H- 1 H COSY spectrum;

[0044] Fig.21 This is the ROESY spectrum of sesquiterpenoid compound III;

[0045] Fig. 22 HR-ESI-MS spectrum of sesquiterpenoid compound III;

[0046] Fig.23 The inhibitory effects of sesquiterpenoids from three natural extracts on human breast cancer cell lines MDA-MB-231 (left) and HCC1806 (right); **p<0.01, ***p<0.001;

[0047] Fig.24 The in vivo inhibition results of compound I on human breast cancer xenograft tumors in mice; A. Tumor volume was measured every 2 days after the start of treatment and is presented as a line graph; B. Tumor weight of mice in the control group (DMSO) and the experimental group (27 mg / kg); C. Body weight changes of mice after administration of compound I; D. After 16 consecutive days of administration, the mice were euthanized, the tumors were removed and photographed; ***P<0.001, **P<0.01, ns represents no difference. DETAILED DESCRIPTION

[0048] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments, but the present invention is not limited to the following technical solution.

[0049] The human breast cancer cells described in the following examples are human breast cancer cells MDA-MB-231 and HCC1806, the human lung cancer cells are human lung cancer cells H460, the human liver cancer cells are human liver cancer cells sMMC-7721, the human leukemia cells are human leukemia cells Jurkat, the human cervical cancer cells are human cervical cancer cells Hela, the human skin cancer cells are human skin cancer cells A375, the human colorectal cancer cells are human colorectal cancer cells HT29, the human renal cancer cells are human renal cancer cells G401, the human glioma cells are human glioma cells U87, the human pancreatic cancer cells are human pancreatic cancer cells SW1990, and the human osteosarcoma cells are human osteosarcoma cells MG63. The conventional cancer cells used above are all from Kunming Medical University.

[0050] Unless otherwise specified, the reagents in the examples of the present invention can be purchased from commercial sources. Fetal bovine serum was purchased from ROyacel; RPMI1640 culture medium, DMEM culture medium, phosphate buffered saline (PBS) and 0.25% trypsin (containing EDTA) were purchased from Gibco.

[0051] The cell lines were cultured in DMEM or RPMI1640 medium containing 5% fetal bovine serum at 37°C, 5% CO2 and 90% humidity.

[0052] The structural formula of the sesquiterpenoid compounds used in the following examples is as follows: Figure 1 As shown, Ang in the structural formula represents a CH3CH=C(CH3)- group:

[0053]

[0054] Compounds I-III are all extracted from Chrysanthemum vulgare, and the specific extraction process is as follows:

[0055] Example 1 Extraction and separation of compound I (Formula I)

[0056] (1) using a methanol solution to reflux extract the aerial parts of the chrysanthemum, collecting the extract; concentrating the extract to obtain an extract; adding an appropriate amount of water to the extract for dispersion, respectively extracting with petroleum ether and ethyl acetate to obtain a petroleum ether extract and an ethyl acetate extract; dissolving the petroleum ether extract and performing gradient elution with a 100-200 mesh normal phase silica gel column, respectively using petroleum ether-ethyl acetate and dichloromethane-methanol, collecting and obtaining 9 eluates, which are named Fr.A to Fr.I in sequence; the volume ratio of petroleum ether to ethyl acetate is 20:1 to 5:1, and gradient elution is performed; the volume ratio of dichloromethane to methanol in the dichloromethane-methanol is 50:1 to 3:1, and gradient elution is performed;

[0057] (2) Fr.E was subjected to small pore resin chromatography, and gradient elution was performed using methanol aqueous solution with a volume fraction of 50% to 100%, and 7 eluates were collected and named Fr.E1 to Fr.E7; Fr.E5 was subjected to C 18 Reverse phase silica gel column chromatography separation, using 50% to 100% methanol aqueous solution for gradient elution, collecting 5 eluents, named Fr.E5a to Fr.E5e; Sephadex LH-20 methanol gel column chromatography to elute Fr.E5c, collecting a chromatographic solution for every 5 mL, collecting 4 eluents, named Fr.E5c1 to Fr.E5c4, the conditions of the Sephadex LH-20 methanol gel column chromatography include:

[0058] Eluent: analytical grade methanol;

[0059] Gel column size: 18mm*180mm;

[0060] The flow rate of the eluent: 0.5 mL / min;

[0061] The amount of eluent used: 2.0L;

[0062] (3) Purifying the Fr.E5c3 by preparative HPLC to obtain a sesquiterpenoid compound I, wherein the conditions of the preparative HPLC include:

[0063] Chromatographic column specifications: 4.6mm*250mm, 5μm;

[0064] Mobile phase: methanol-water 84:26;

[0065] Flow rate: 2mL / min;

[0066] Retention time: 20.4min;

[0067] Structural characterization of sesquiterpenoids I:

[0068] Figure 2 Sesquiterpenoid I 1 HNMR spectrum; Figure 3 Sesquiterpenoid I 13 CNMR spectrum; Figure 4 is the HSQC spectrum of sesquiterpenoid compound I; Figure 5 is the HMBC spectrum of sesquiterpenoid compound I; Figure 6 Sesquiterpenoid I 1 H- 1 HCOSY spectrum; Figure 7 is the ROESY spectrum of sesquiterpenoid compound I; Figure 8 is the HR-ESI-MS spectrum of sesquiterpenoid compound I;

[0069] From Table 1 and Figure 2 to Figure 8 It can be seen that the structural data of sesquiterpenoid compound I are as follows: white amorphous powder. High-resolution mass spectrometry HR-ESI-MS shows molecular ion peak m / z 571.3274 [M+Na] + , the molecular formula is C 31 H 48 O8Na (calculated value 571.3247), with 8 unsaturated groups. Infrared spectrum IR shows hydroxyl groups (3424 cm -1 ), ester carbonyl (1721cm -1 ) and double bond (1648cm -1 ) functional group absorption peak. 13 CNMR and DEPT spectra showed 8 methyl groups (2 singlet methyl groups and 2 doublet methyl groups), 5 methylene groups (one oxymethylene group), 12 methine groups (5 oxymethine groups) and 6 quaternary carbon groups (three sp 2 hybridized quaternary carbon, two carbonyl quaternary carbons, and one oxygen-containing quaternary carbon). 1 HNMR signal [δ H 6.15(1H,qd,J=7.2,1.6Hz), 1.99(3H,td,J=7.2,1.6Hz), 1.89(3H,t,J=1.6Hz); 6 .09(1H,qd,J=7.2,1.6Hz), 1.99(3H,td,J=7.2,1.6Hz), 1.89(3H,t,J=1.6Hz)] ​​and 13 CNMR signal [δ C 167.5,167.5,140.1,138.9,127.4,127.0,20.5,20.5,15.9,15.9], it can be seen that the compound contains two angelicae groups. 1 HNMR and 13 The δ H 4.55(1H,d,J=7.8Hz),[δ C (96.2, 74.7, 75.1, 71.2, 72.0, 62.9)] indicates that the compound contains a β-pyranose glucopyranose group. In addition to the above substituents, the aglycone also has 15 carbon signals, including 4 methyl groups, 4 methylene groups, 5 methine groups and 2 quaternary carbons (one oxygen-linked quaternary carbon). This indicates that the compound is a sesquiterpenoid compound with the structure shown in Formula I. The planar structure of the compound was finally determined by two-dimensional NMR technology (HSQC, HMBC and 1 H- 1Its stereostructure was preliminarily identified by ROESY, and its absolute configuration was confirmed.

[0070] Table 1 Compound 1 1 H-NMR and 13 C-NMR data (600 / 150 MHz, CDCl3)

[0071]

[0072]

[0073]

[0074] Example 2 Extraction and separation of compound II

[0075] (1) using a methanol solution to reflux extract the aerial parts of the chrysanthemum, collecting the extract; concentrating the extract to obtain an extract; adding an appropriate amount of water to the extract for dispersion, respectively extracting with petroleum ether and ethyl acetate to obtain a petroleum ether extract and an ethyl acetate extract; dissolving the petroleum ether extract and performing gradient elution with a 100-200 mesh normal phase silica gel column, respectively using petroleum ether-ethyl acetate and dichloromethane-methanol, collecting and obtaining 9 eluates, which are named Fr.A to Fr.I in sequence; the volume ratio of petroleum ether to ethyl acetate is 20:1 to 5:1, and gradient elution is performed; the volume ratio of dichloromethane to methanol in the dichloromethane-methanol is 50:1 to 3:1, and gradient elution is performed;

[0076] (2) Fr.E was subjected to small pore resin chromatography, and gradient elution was performed using a methanol aqueous solution with a volume fraction of 50% to 100%, and 7 eluates were collected and named Fr.E1 to Fr.E7; Fr.E6 was subjected to C 18 Reverse phase silica gel column chromatography separation, using 50% to 100% methanol aqueous solution for gradient elution, collecting 4 eluents, named Fr.E6a to Fr.E6d; Sephadex LH-20 methanol gel column chromatography to elute Fr.E6b, collecting a chromatographic solution for every 5 mL, collecting 3 eluents, named Fr.E6b1 to Fr.E6b3, the conditions of the Sephadex LH-20 methanol gel column chromatography include:

[0077] Eluent: analytical grade methanol;

[0078] Gel column size: 18mm*180mm;

[0079] The flow rate of the eluent: 0.5 mL / min;

[0080] The amount of eluent used: 2.0L;

[0081] (3) Purifying the Fr.E6b2 by preparative HPLC to obtain a sesquiterpenoid compound II, wherein the conditions of the preparative HPLC include:

[0082] Chromatographic column specifications: 4.6mm*250mm, 5μm;

[0083] Mobile phase: methanol-water 80:20;

[0084] Flow rate: 2mL / min;

[0085] Retention time: 26.3min;

[0086] Structural characterization of sesquiterpenoid II:

[0087] Fig. 9 Sesquiterpenoid II 1 HNMR spectrum; Fig.10 Sesquiterpenoid II 13 CNMR spectrum; Fig.11 This is the HSQC spectrum of sesquiterpenoid II; Fig.12 This is the HMBC spectrum of sesquiterpenoid II; Fig.13 Sesquiterpenoid II 1 H- 1 HCOSY spectrum; Fig.14 This is the ROESY spectrum of sesquiterpenoid II; Fig.15 This is the HR-ESI-MS spectrum of sesquiterpenoid compound II.

[0088] Table 2 Compound 3 1 H-NMR and 13 C-NMR data (600 / 150 MHz, CDCl3)

[0089]

[0090]

[0091] From Table 2 and Figures 9 to 15 It can be seen that the structural data of sesquiterpenoid compound II are as follows: colorless oil. HR-ESIMS at m / z 585.3043 [M+Na] + (calc.forC 31 H 46 O9Na,585.3040), combined 13 C-NMR data confirmed that the molecular formula is C 31 H46 O9, unsaturation is 9. 1 H-NMR and 13 C-NMR H 4.94(1H,t,J=9.2Hz,H-4′), 4.54(1H,d,J=7.6Hz,H-1′), 4.31(1H,dd,J=12.0,2.2Hz,H-6′a), 4.18( 1H,dd,J=12.0,7.0Hz,H-6′b), 3.76(1H,m,H-5′), 3.75(1H,m,H-3′), 3.49(1H,t,J=8.8Hz,H-2′)] and [δ C 96.2,75.4,74.5,72.1,71.1,62.8] is a pyranose glucose unit, [δ H 6.18 (1H, qd, J = 7.2, 1.4 Hz, H-3″), 6.11 (1H, qd, J = 7.2, 1.6 Hz, H-3″′), 1.99 (3H, td, J = 7.2, 1.8 Hz, H3-4″), 1.98 (3H, td, J = 7.2, 1.8 Hz, H3-4″′), 1.89 (6H, dd, t = 1.6 Hz, H3-5″ and H3-5″′)] and [δ C 167.6,140.5,127.3,20.5,16.0; δ C 167.4,139.1,126.9,20.5,15.9] are two angelica acyl groups, [δ H 1.88 (1H, m, H-11), 0.88 (6H, t, J = 6.8 Hz, H3-12 and H3-13)] and [δ C 27.8, 21.5, 15.6] is an isopropyl group. 13 31 carbon signals were observed in C-NMR and HSQC, including 8 methyl groups, 4 methylene groups, 12 methine groups and 7 quaternary carbons. In addition, among the 9 unsaturated groups, one pyranose glucose ring, two oxygen angelica acyl groups, one carbonyl group and one carbon-carbon double bond accounted for 7, indicating that the compound is a sesquiterpenoid compound II with the structure shown in formula II. The planar structure of the compound was finally determined by two-dimensional NMR technology (HSQC, HMBC and 1 H- 1 Its stereostructure was preliminarily identified by ROESY, and its absolute configuration was confirmed.

[0092] Example 3 Extraction and separation of compound III

[0093] (1) using a methanol solution to reflux extract the aerial parts of the chrysanthemum, collecting the extract; concentrating the extract to obtain an extract; adding an appropriate amount of water to the extract for dispersion, respectively extracting with petroleum ether and ethyl acetate to obtain a petroleum ether extract and an ethyl acetate extract; dissolving the petroleum ether extract and performing gradient elution with a 100-200 mesh normal phase silica gel column, respectively using petroleum ether-ethyl acetate and dichloromethane-methanol, collecting and obtaining 9 eluates, which are named Fr.A to Fr.I in sequence; the volume ratio of petroleum ether to ethyl acetate is 20:1 to 5:1, and gradient elution is performed; the volume ratio of dichloromethane to methanol in the dichloromethane-methanol is 50:1 to 3:1, and gradient elution is performed;

[0094] (2) Fr.E was subjected to small pore resin chromatography, and gradient elution was performed using a methanol aqueous solution with a volume fraction of 50% to 100%, and 7 eluates were collected and named Fr.E1 to Fr.E7; Fr.E6 was subjected to C 18 Reverse phase silica gel column chromatography separation, using 50% to 100% methanol aqueous solution for gradient elution, collecting 4 eluents, named Fr.E6a to Fr.E6d; Sephadex LH-20 methanol gel column chromatography to elute Fr.E6b, collecting a chromatographic solution for every 5 mL, collecting 3 eluents, named Fr.E6b1 to Fr.E6b3, the conditions of the Sephadex LH-20 methanol gel column chromatography include:

[0095] Eluent: analytical grade methanol;

[0096] Gel column size: 18mm*180mm;

[0097] The flow rate of the eluent: 0.5 mL / min;

[0098] The amount of eluent used: 2.0L;

[0099] (3) Purifying the Fr.E6b3 by preparative HPLC to obtain a sesquiterpenoid compound III, wherein the conditions of the preparative HPLC include:

[0100] Chromatographic column specifications: 4.6mm*250mm, 5μm;

[0101] Mobile phase: methanol-water 87:23;

[0102] Flow rate: 2mL / min;

[0103] Retention time: 23.1min.

[0104] Structural characterization of sesquiterpenoid III:

[0105] Fig.16 Sesquiterpenoid III 1 HNMR spectrum; Fig.17 Sesquiterpenoid III 13 CNMR spectrum; Fig.18 This is the HSQC spectrum of sesquiterpenoid compound III; Fig.19 This is the HMBC spectrum of sesquiterpenoid compound III; Fig. 20 Sesquiterpenoid III 1 H- 1 HCOSY spectrum; Fig.21 This is the ROESY spectrum of sesquiterpenoid compound III; Fig. 22 This is the HR-ESI-MS spectrum of sesquiterpenoid compound III.

[0106] Table 3 Compound III 1 H-NMR and 13 C-NMR data (600 / 150 MHz, CDCl3)

[0107]

[0108]

[0109]

[0110] From Table 3 and Figure 16 to Figure 22 It can be seen that the structural data of sesquiterpenoid compound III are as follows: colorless oil. HR-ESI-MS at m / z 573.3312 [M+Na] + (calc.forC 31 H 50 O8Na,573.3403), combined 13 C-NMR data confirmed that the molecular formula is C 31 H 50 O8, unsaturation is 7. 1 H-NMR and 13 C-NMR H 4.95(1H,t,J=9.2Hz,H-4′), 4.68(1H,d,J=7.8Hz,H-1′), 4.36(1H,dd,J=12.0,2.2Hz,H-6′a), 4.16( 1H,dd,J=12.0,6.8Hz,H-6′b), 3.76(1H,m,H-3′), 3.78(2H,m,H-5′), 3.46(1H,d,J=8.8Hz,H-2′)] and [δ C99.3,74.9,74.4,72.1,71.1,63.0] confirmed the presence of a pyranose glucose unit, [δ H 6.16(1H,qd,J=7.2,1.6Hz,H-3″), 6.11(1H,qd,J=7.2,1.6Hz,H-3″′), 2.00(3H,td,J=7.2,1.8Hz,,H-4 ″), 1.99(3H,td,J=7.2,1.8Hz,,H-4″′), 1.91(3H,t,J=1.6Hz,H-5″), 1.89(3H,t,J=1.6Hz,H-5″′)] and [δ C 167.4,140.1,127.3,20.5,15.9; δ C 167.4,139.2,127.0,20.6,16.0] confirmed the presence of two angelica acyl groups, [δ H 1.61 (1H, m, H-10), 0.95 (3H, d, J = 6.8 Hz, H-12), 0.91 (3H, d, J = 6.8 Hz, H-11)] and [δ C 33.6, 20.3, 19.6] confirmed the presence of an isopropyl unit. 13 31 carbon signals were observed in C-NMR and HSQC, including 9 methyl, 5 methylene, 11 methine and 6 quaternary carbons. In addition, among the 7 unsaturated groups, one pyranose ring and two oxygen angelica acyl groups accounted for 5, indicating that the compound was a sesquiterpenoid compound III with the structure shown in formula III. The planar structure of the compound was finally determined by two-dimensional NMR technology (HSQC, HMBC and 1 H- 1 Its stereostructure was preliminarily identified by ROESY, and its absolute configuration was confirmed.

[0111] Example 4 Test on the Anti-Breast Cancer Activity of Sesquiterpenoids

[0112] The CCK8 method was used to detect cell growth inhibition. The CCK-8 experiment, whose full name is Cell Counting Kit-8, is a commonly used method for detecting cell proliferation and cytotoxicity. The CCK-8 experimental principle is based on the compound WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonic acid phenyl)-2H-tetrazole monosodium salt). In the presence of the electron carrier 1-MethoxyPMS (menadione phosphate), WST-8 can be reduced by intracellular mitochondrial dehydrogenase to generate a highly water-soluble orange-yellow formazan product. The amount of formazan generated is proportional to the number of living cells, and the color depth can reflect the metabolic activity of the cells. The absorbance at a wavelength of 450nm can be measured by an enzyme reader to indirectly reflect the number of living cells. Therefore, CCK-8 can be used to evaluate cell proliferation, cytotoxicity or the cellular effects of drugs.

[0113] Three sesquiterpenoid compounds (Ⅰ, Ⅱ, Ⅲ) extracted from chrysanthemum Figure 1 ) were dissolved in DMSO (dimethyl sulfoxide) to prepare DMSO solutions with concentrations of 1000 μM, 100 μM, and 10 μM, respectively.

[0114] Breast cancer cells MDA-MB-231 and human breast cancer cells HCC1806 (6×10 3 / well) were inoculated in 96-well plates, with 100 μL of culture medium in each well. After culturing in a 37°C 5% CO2 constant temperature incubator for 24 hours, 1 μL of sesquiterpenoid compounds (Ⅰ, Ⅱ, Ⅲ) dissolved in DMSO at different concentrations were added to the cell culture in each well, so that the final concentration of each compound was one percent of the previous concentration. The control group only added the same volume of DMSO to the cell culture and incubated in an incubator for 72 hours. After the incubation, 10 μL of CCK8 solution was added to each well and gently mixed to avoid bubbles; continue to incubate in the incubator for 1 hour, use an enzyme reader to measure the absorbance of each well at a wavelength of 450nm, calculate the average OD value of each well, and analyze the changes in cell proliferation or toxicity by comparing with the control group (untreated cells). The percentage of the absorbance value of the experimental group relative to the absorbance value of the control group represents the cell survival rate or the cell proliferation level, that is, the control group defaults to 100% ( Fig.23 ).

[0115] The results are as follows Fig.23 As shown, among the three sesquiterpenoid compounds extracted from sesquiterpenes, compounds I, II, and III all exhibited breast cancer inhibitory activity at a concentration of 10 μM.

[0116] Example 5 Anti-breast cancer activity of sesquiterpenoids

[0117] The sesquiterpenoid compounds (Ⅰ, II, III) extracted from Chrysanthemum vulgare were dissolved in DMSO, and then prepared with DMSO to prepare 5000μM DMSO solution, 1000μM DMSO solution, 600μM DMSO solution, 300μM DMSO solution, 100μM DMSO solution, 60μM DMSO solution, 30μM DMSO solution, and 10μM DMSO solution, respectively. The above concentration solutions were used as test solutions.

[0118] Human breast cancer cells MDA-MB-231 and HCC1806 (6×10 3 / well) were inoculated in 96-well plates, cultured in a 37°C 5% CO2 constant temperature incubator for 24 hours, and then 1 μL of the test solution was added to the cell culture in each well, so that the final concentrations were 50 μM, 10 μM, 6 μM, 3 μM, 1 μM, 0.6 μM, 0.3 μM, and 0.1 μM, respectively. The control group only added 1 μL DMSO to the cell culture, and then placed it in an incubator for 72 hours, and 10 μL CCK8 solution was added to each well, gently mixed to avoid bubbles; continue to incubate in the incubator for 1 hour, use an enzyme reader to measure the absorbance of each well at a wavelength of 450nM, calculate the average OD value of each well, and analyze the changes in cell proliferation or toxicity by comparing with the control group (untreated cells). The results are shown in Table 4.

[0119] Table 4 IC50 of natural extract sesquiterpenoids (I, II, III) on human breast cancer cell lines MDA-MB-231 and HCC1806 50 (μM)

[0120]

[0121]

[0122] According to the results in Table 4, the natural extract sesquiterpenoid compounds (Ⅰ, II, III) have strong cytotoxic activity against human breast cancer cell line MDA-MB-231, IC 50 The natural extract sesquiterpenoids (Ⅰ, II, III) also have strong cytotoxic activity against human breast cancer cell line HCC1806, IC 50 They are 7.6 μM, 3.5 μM and 7.4 μM respectively.

[0123] Example 6

[0124] The human breast cancer cells MDA-MB-231 were replaced with the human lung cancer cells H460. The compound used was sesquiterpenoid compound I, and the rest was the same as in Example 5. 50 The results of the values ​​are shown in Table 5.

[0125] Table 5 IC50 of natural extract sesquiterpenoid compound I on human lung cancer cell line H460 50 (μM)

[0126]

[0127] According to the results in Table 5, the natural extract sesquiterpenoid compound Ⅰ has a strong cytotoxic activity against human lung cancer cell line H460, IC 50 It is 47.71μM.

[0128] Example 7

[0129] The human breast cancer cell MDA-MB-231 was replaced with the human liver cancer cell sMMC-7721. The compound used was sesquiterpenoid compound I, and the rest was the same as in Example 5. 50 The results of the values ​​are shown in Table 6.

[0130] Table 6 IC50 of natural extract sesquiterpenoid compound I on human hepatoma cell line sMMC-7721 50 (μM)

[0131]

[0132] According to the results in Table 6, the natural extract sesquiterpenoid compound Ⅰ has a strong cytotoxic activity against human hepatoma cell line sMMC-7721, IC 50 is 35μM.

[0133] Example 8

[0134] The human breast cancer cells MDA-MB-231 were replaced with human acute T lymphocytic leukemia cells Jurkat. The compound used was sesquiterpenoid compound I, and the rest was the same as in Example 5. 50 The results of the values ​​are shown in Table 7.

[0135] Table 7 IC50 of natural extract sesquiterpenoid compound I on human acute T lymphoblastic leukemia Jurkat cell line 50 (μM)

[0136]

[0137] According to the results in Table 7, the natural extract sesquiterpenoid compound I has a strong cytotoxic activity against Jurkat cell line of human acute T lymphoblastic leukemia cells, IC 50 It is 47.08μM.

[0138] Example 9

[0139] The human breast cancer cells MDA-MB-231 were replaced with human cervical cancer cells Hela cells. The compound used was sesquiterpenoid compound I, and the rest was the same as in Example 5. 50 The results of the values ​​are shown in Table 8.

[0140] Table 8 IC50 of natural extract sesquiterpenoid compound I on human cervical cancer cell Hela cell line 50 (μM)

[0141]

[0142] According to the results in Table 8, the natural extract sesquiterpenoid compound Ⅰ has a strong cytotoxic activity against the human cervical cancer cell Hela cell line, IC 50 It is 50.28μM.

[0143] Example 10

[0144] The human breast cancer cell MDA-MB-231 was replaced by the human colorectal cancer cell HT29. The compound used was sesquiterpenoid compound I, and the rest was the same as in Example 5. 50 The results of the values ​​are shown in Table 9.

[0145] Table 9 IC50 of natural extract sesquiterpenoid compound I on human colorectal cancer cell line HT29 50 (μM)

[0146]

[0147] According to the results in Table 9, the natural extract sesquiterpenoid compound I has a strong cancer cell toxicity against the human colorectal cancer cell line HT29, IC 50 It is 10.23μM.

[0148] Embodiment 11

[0149] The human breast cancer cell MDA-MB-231 was replaced with the human renal cancer cell G401. The compound used was sesquiterpenoid compound I, and the rest was the same as in Example 5. 50 The results of the values ​​are shown in Table 10.

[0150] Table 10 IC50 of natural extract sesquiterpenoid compound I on human renal cancer cell line G40150 (μM)

[0151]

[0152] According to the results in Table 10, the natural extract sesquiterpenoid compound I has a strong cytotoxic activity against human renal cancer cell line G401, IC 50 It is 47.50μM.

[0153] Example 12

[0154] The human breast cancer cells MDA-MB-231 were replaced by human glioma cells U87, human pancreatic cancer cells SW1990, human osteosarcoma cells MG63, and human skin cancer cells A375. The compound used was sesquiterpenoid compound I, and the rest was the same as in Example 5. 50 The results of the values ​​are shown in Table 11.

[0155] Table 11 IC50 of natural extract sesquiterpenoids Ⅰ on various cancer cells 50 (μM)

[0156]

[0157] According to the results in Table 11, the natural extract sesquiterpenoid compound I has strong cancer cell cytotoxicity against human glioma cells U87, human pancreatic cancer cells SW1990, human osteosarcoma cells MG63, and human malignant melanoma cells A375. 50 They are 28, 40, 33.72 and 16.26 μM respectively.

[0158] Example 13 Animal Experiment Results

[0159] Construction of subcutaneous tumor model in nude mice

[0160] HCC1806 cells in the logarithmic growth phase were digested, collected and counted. 1×10 6 After centrifugation of the cells to remove the supernatant, resuspend the cells in pre-cooled 1× PBS, then add Matrigel, mix thoroughly with a pre-cooled pipette tip, and inject into the subcutaneous tissue of the mouse abdomen with an insulin injection needle at a volume of 75 μL per point. One week after inoculation, observe the subcutaneous tumor formation of the mice. When the tumor volume grows to 50-70 mm 3The mice were randomly divided into groups, with 5 mice in each group, and different drug treatment concentration groups were set up. The drug injection solution was prepared according to the formula of 100 μL per mouse, 10% (DMSO or drug) drug mixture + 40% PEG300 + 8% Tween 80 + 42% saline. The drug was administered intraperitoneally once every 2 days, and the weight of the mice was weighed and recorded. At the same time, the mouse tumor was measured with a vernier caliper, and the tumor volume was calculated by the formula V = 0.5 × length × width 2. A total of 16 drug administration treatments were performed. After the drug treatment was completed, the mice were killed by cervical dislocation. The tumor tissue was separated using surgical instruments, weighed, and photographed; the drug described in this example is compound I, and the results are as follows Fig.24 shown.

[0161] Fig.24 The results are as follows: ... Fig.24 A is the tumor volume measured every 2 days after the start of treatment, represented by a line graph. Fig.24 A shows that compared with the control group, after administration of compound I, the tumor volume of mice was lower than that of the control group; Fig.24 B is the tumor weight of mice in the control group (DMSO) and the experimental group (27 mg / kg). The tumor weight of mice in the experimental group was lower than that in the control group; Fig.24 C shows the changes in the body weight of mice after administration of Compound I. There is basically no difference in the body weight of mice in the experimental group and the control group; Fig.24 D is subcutaneous administration of mice; after 16 consecutive days of administration, the mice were euthanized, the tumors were removed and photographed; ***P<0.001, **P<0.01, ns means no difference;

[0162] In summary, the sesquiterpenoid compound I extracted from Chrysanthemum vulgare has a good anti-cancer effect in vivo and can effectively inhibit the growth of breast cancer cells in mice.

[0163] It should be noted that the embodiments described above are only used to explain the present invention and do not constitute any limitation to the present invention. The present invention has been described with reference to typical embodiments, but it should be understood that the words used therein are descriptive and explanatory words, rather than restrictive words. The present invention may be modified as specified within the scope of the claims of the present invention, and the present invention may be revised without departing from the scope and spirit of the present invention. Although the present invention described therein relates to specific methods, materials and embodiments, it does not mean that the present invention is limited to the specific examples disclosed therein, but rather, the present invention extends to all other methods and applications with the same functions.

Claims

1. A sesquiterpenoid compound, characterized in that: The structure of the sesquiterpenoid compound is shown in Formula I, II or III:

2. Use of the sesquiterpenoid compound as claimed in claim 1 in the preparation of a drug for preventing and / or treating cancer.

3. Use of the sesquiterpenoid compound according to claim 2 in the preparation of a drug for preventing and / or treating cancer, characterized in that: The cancer is at least one of breast cancer, liver cancer, lung cancer, colorectal cancer, leukemia, pancreatic cancer, glioma, osteosarcoma, cervical cancer, kidney cancer and skin cancer.

4. Use of the sesquiterpenoid compound according to claim 2 in the preparation of a drug for preventing and / or treating cancer, characterized in that: The cancer is breast cancer.

5. Use of the sesquiterpenoid compound according to claim 2 in the preparation of a drug for preventing and / or treating cancer, characterized in that: The sesquiterpenoid compound is used in combination with a pharmaceutically acceptable carrier.

6. Use of the sesquiterpenoid compound according to claim 2 in the preparation of a drug for preventing and / or treating cancer, characterized in that: The sesquiterpenoid compound uses a compound with a structure shown in Formula I, Formula II or Formula III, or a pharmaceutically acceptable salt, ester or stereoisomer thereof as a pharmaceutically active component.

7. Use of the sesquiterpenoid compound according to claim 6 in the preparation of a drug for preventing and / or treating cancer, characterized in that: The mass fraction of the active pharmaceutical ingredient is 0.1% to 99%.

8. A method for preparing a sesquiterpenoid compound as claimed in claim 1, characterized in that: The preparation method of the hemiterpenoid compound represented by formula I is as follows: The step (1) uses a methanol solution to reflux extract the aerial part of the chrysanthemum, and collects the extract; concentrates the extract to obtain an extract; adds an appropriate amount of water to the extract and disperses it, and then extracts it with petroleum ether and ethyl acetate to obtain a petroleum ether extract and an ethyl acetate extract; after dissolving the petroleum ether extract, chromatographs it with a 100-200 mesh normal phase silica gel column, and performs gradient elution with petroleum ether-ethyl acetate and dichloromethane-methanol in sequence, and collects and obtains 9 parts of eluent, which are named Fr.A to Fr.I in sequence; the volume ratio of petroleum ether to ethyl acetate is 20:1-5:1, and gradient elution is performed; the volume ratio of dichloromethane to methanol in the dichloromethane-methanol is 50:1-3:1, and gradient elution is performed; Step (2) The Fr.E component is separated by small pore resin chromatography, and a methanol aqueous solution with a volume fraction of 50% to 100% is used for gradient elution, and 7 eluates are collected and named Fr.E1 to Fr.E7; the Fr.E5 is subjected to C 18 Reverse phase silica gel column chromatography separation, using 50% to 100% methanol aqueous solution for gradient elution, collecting 5 eluates, named Fr.E5a to Fr.E5e; Sephadex LH-20 methanol gel column chromatography was used to elute the Fr.E5c component, collecting a chromatographic solution for every 5 mL, collecting 4 eluates, named Fr.E5c1 to Fr.E5c4; Step (3) purifying the Fr.E5c3 component by preparative HPLC to obtain sesquiterpenoid compound I.

9. The method for preparing sesquiterpenoid compounds according to claim 8, characterized in that: The preparation method of the semiterpenoid compound represented by formula II is as follows: The Fr.E component was separated by small pore resin chromatography, and a methanol aqueous solution with a volume fraction of 50% to 100% was used for gradient elution, and 7 eluents were collected and named Fr.E1 to Fr.E7. The Fr.E6 component was separated by C 18 Reverse phase silica gel column chromatography separation, using 50% to 100% methanol-water solution for gradient elution, collecting and obtaining 4 parts of eluent, named Fr.E6a to Fr.E6d; Sephadex LH-20 methanol gel column chromatography was used to elute the Fr.E6b component, collecting a chromatographic solution for every 5 mL, and obtaining 3 parts of eluent, named Fr.E6b1 to Fr.E6b3; the Fr.E6b2 component was purified by preparative HPLC to obtain a sesquiterpene compound of formula II.

10. The method for preparing sesquiterpenoid compounds according to claim 9, characterized in that: The preparation method of the hemiterpenoid compound represented by formula III is as follows: the Fr.E6b3 component is purified by preparative HPLC to obtain the hemiterpenoid compound represented by formula III.

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