Trichilin-type limonoid compounds and extraction and separation methods and applications thereof

Abstract

The present invention discloses trichilin-type limonoid compounds and extraction and separation methods and applications thereof. The present invention extracts and separates trichilin-type limonoid compounds from partridge flowers and leaves: trichilitin G, trichilitin H, 12-hydroxyamoorastatin, azedarachin B and 12-O-deacetyltrichilin H. The limonoid compounds separated by the present invention have strong inhibitory activity on human colon cancer cells (HCT-116), human lung adenocarcinoma cells (NCl-H1975) and human neuroblastoma cells (SH-SY5Y), providing an alternative for broad-spectrum anticancer drugs. The limonoid compounds used in the present invention are derived from the natural plant partridge flower, have a wide range of sources, low production costs, and have good value and application prospects in the preparation of anti-tumor drugs.

Description

Technical Field

[0001] The invention relates to the field of natural products, in particular to Trichilin-type limonoid compounds and extraction and separation methods and applications thereof. Background Art

[0002] Cancer is the second most common cause of death worldwide and a major public health concern. Current cancer chemotherapy regimens have many shortcomings, the most obvious of which is the inability to adequately treat cancer. In addition, the implementation of these treatments in clinical settings also poses significant problems, such as the emergence of adverse effects and the development of multidrug resistance (MDR). For more than 50 years, natural products (NPs) have played an important role in the discovery of anticancer drugs, as evidenced by the increasing number of novel anticancer drugs derived from various natural sources. These naturally derived drugs exhibit their therapeutic effects through a variety of cellular mechanisms, some of which are novel or non-conventional. Therefore, the development of effective anticancer drugs from natural products (NPs) has become a widely accepted and promising approach.

[0003] Trichilia connaroides has a long history as a folk medicine, and its medicinal use has been recorded in Dai medicine, Yao medicine and Yi medicine. Its root is used as medicine, which is bitter and cool in nature, and has the effects of clearing away heat and detoxification, dispelling wind and relieving sore throat. It is mainly used in the treatment of rheumatoid arthritis, pharyngitis, tonsillitis and other inflammations in clinic. The study of its phytochemistry has found a variety of components including terpenes, phenols, steroids and fatty acids, among which the terpenes are mainly tetra-nortriterpenoid compounds, also known as limonin, which is a secondary metabolite unique to this plant. This type of component has long been the focus of domestic and foreign scholars due to its high degree of oxidation and variable skeleton. So far, at least 159 limonin compounds have been found in the roots, stems, bark, branches, leaves and fruits of Trichilia connaroides. There are many differences in the biological activities of different limonoid compounds. The present application extracts and separates new limonoid compounds from partridge flowers and finds that they have strong anti-cancer activity. Summary of the invention

[0004] The present invention aims to provide trichilin-type limonoid compounds in partridge flower: trichilitin G, trichilitin H, 12-hydroxyamoorastatin, azedarachin B, 12-O-deacetyltrichilin H, extraction and separation methods and applications thereof in preparing cancer drugs. The limonoid compounds provided by the present invention have strong inhibitory activity on human colon cancer cells (HCT-116), human lung adenocarcinoma cells (NCl-H1975) and human neuroblastoma cells (SH-SY5Y), providing an optional solution for broad-spectrum anticancer drugs.

[0005] In a first aspect of the present invention, a trichilin-type limonoid compound is provided, wherein the compound is trichilitin G or trichilitin H, and the structural formula is as follows:

[0006]

[0007] In a first aspect of the present invention, a trichilin-type limonoid compound is provided for use in the preparation of a drug for treating cancer, wherein the compound is trichilitin G, trichilitin H, 12-hydroxyamoorastatin, azedarachin B or 12-O-deacetyltrichilin H, and has the following structural formula:

[0008]

[0009] Preferably, the cancer is liver cancer, lung adenocarcinoma or neuroblastoma. The above limonoid compounds show good inhibitory activity on HCT-116 (human colon cancer cells), NCl-H1975 (human lung adenocarcinoma cells) and SH-SY5Y (human neuroblastoma cells).

[0010] As a further description of the above scheme: the limonin compound uses the compound shown in the structural formula or its pharmaceutically acceptable salt, ester, stereoisomer as a pharmaceutically active ingredient.

[0011] As a further description of the above scheme: the content of the active pharmaceutical ingredient is 0.1-99.9%.

[0012] As a further description of the above scheme: the limonoid compound is used in combination with a pharmaceutically acceptable carrier.

[0013] In a third aspect, the present invention provides a method for extracting and separating a trichilin-type limonoid compound. The method for extracting and separating the compound trichilitin G is as follows:

[0014] Step (1) using a methanol solution to reflux extract the leaves of the partridge flower, collecting the extract; concentrating the extract to obtain an extract; adding an appropriate amount of water to the extract for dispersion, and extracting with ethyl acetate to obtain an ethyl acetate fraction; dissolving the ethyl acetate fraction and performing chromatography on a 100-200 mesh normal phase silica gel column, and gradient eluting with petroleum ether-acetone and dichloromethane-methanol in sequence, collecting and obtaining 11 eluates, which are named Fr.A to Fr.K respectively; the volume ratio of petroleum ether to acetone is 40:1 to 5:1; the volume ratio of dichloromethane to methanol in the dichloromethane-methanol is 50:1 to 0:1;

[0015] Step (2) Fr.G is subjected to small pore resin chromatography, and gradient elution is performed using a methanol aqueous solution with a volume fraction of 50% to 100%, and 11 eluates are collected and named Fr.G-1 to Fr.G-11; Fr.G-3 is subjected to C 18 Reverse phase silica gel column chromatography separation, using a methanol aqueous solution with a volume fraction of 40% to 100% for gradient elution, collecting 10 eluents, named Fr.G-3a to Fr.G-3j in sequence; Sephadex LH-20 methanol gel column chromatography to elute the Fr.G-3e, collecting a chromatographic solution for every 5 mL, collecting 6 eluents, named Fr.G-3e1 to FrG-3e6 in sequence, the conditions of the Sephadex LH-20 methanol gel column chromatography include:

[0016] Eluent: analytical grade methanol;

[0017] Gel column size: 18mm*180mm;

[0018] The flow rate of the eluent: 0.5 mL / min;

[0019] The amount of eluent used: 2.0L;

[0020] Step (3) Fr.G-3e3 is subjected to normal phase silica gel column chromatography, eluted with petroleum ether-ethyl acetate, and three eluents are collected and named Fr.G-3e1a to Fr.G-3e1c, respectively, wherein the volume ratio of petroleum ether to ethyl acetate is 8:1 to 5:1; Fr.G-3e1b is subjected to preparative HPLC purification to obtain a limonoid compound trichilitin G, wherein the conditions of the preparative HPLC include:

[0021] Chromatographic column specifications: 4.6mm*250mm, 5μm;

[0022] Mobile phase: n-hexane-isopropanol 80:20;

[0023] Flow rate: 2mL / min.

[0024] As a further description of the above scheme: The extraction and separation method of the compound 12-hydroxyamoorastatin is as follows:

[0025] The Fr.G-3e6 was separated by normal phase silica gel column chromatography, eluted with petroleum ether-ethyl acetate, and two eluents were collected and named Fr.G-3e6a and Fr.G-3e6b, respectively, wherein the volume ratio of petroleum ether to ethyl acetate was 3:1 to 1:1; the Fr.G-3e6b was purified by preparative HPLC to obtain the limonoid compound 12-hydroxyamoorastatin, wherein the conditions of the preparative HPLC include:

[0026] Chromatographic column specifications: 4.6mm*250mm, 5μm;

[0027] Mobile phase: n-hexane-isopropanol 75:25;

[0028] Flow rate: 2mL / min.

[0029] As a further description of the above scheme: The extraction and separation methods of the compound azedarachin B and the compound trichilitin H are as follows:

[0030] Fr.G-7 was C 18 Reverse phase silica gel column chromatography separation, using 50% to 100% methanol aqueous solution for gradient elution, collecting 9 eluents, named Fr.G-7a to Fr.G-7i; Fr.G-7e is subjected to normal phase silica gel column chromatography separation, eluted with petroleum ether-ethyl acetate, collecting 5 eluents, named Fr.G-7e1 to Fr.G-7e5, the volume ratio of petroleum ether to ethyl acetate is 4:1 to 1:1; Fr.G-7e5 is subjected to preparative HPLC purification to obtain a limonoid compound azedarachin B, the conditions of the preparative HPLC include:

[0031] Chromatographic column specifications: 4.6mm*250mm, 5μm;

[0032] Mobile phase: 34% acetonitrile;

[0033] Flow rate: 2mL / min;

[0034] FrG-7g was separated by normal phase silica gel column chromatography, eluted with dichloromethane-acetone, and three eluents were collected and named FrG-7g-1 to FrG-7g-3, respectively, wherein the volume ratio of dichloromethane to acetone was 30:1 to 15:1; FrG-7g-2 was purified by preparative HPLC to obtain a limonoid compound trichilitin H, wherein the conditions of the preparative HPLC included:

[0035] Chromatographic column specifications: 4.6mm*250mm, 5μm;

[0036] Mobile phase: n-hexane-isopropanol 85:15;

[0037] Flow rate: 2mL / min.

[0038] As a further description of the above scheme: The extraction and separation method of the compound 12-O-deacetyltrichilin H is as follows:

[0039] Fr.F was separated by small pore resin chromatography, and gradient elution was performed with methanol aqueous solution with a volume fraction of 50% to 100%. Eight eluates were collected and named Fr.F-1 to Fr.F-8. Fr.F-5 was separated by C 18 Reverse phase silica gel column chromatography separation, using 50% to 100% methanol aqueous solution for gradient elution, collecting 10 eluents, named Fr.F-5a to Fr.F-5j in sequence; Sephadex LH-20 methanol gel column chromatography to elute Fr.F-5f, collecting a chromatographic solution for every 5 mL, collecting 4 eluents, named Fr.F-5f1 to Fr.F-5f4 in sequence, the conditions of the Sephadex LH-20 methanol gel column chromatography include:

[0040] Eluent: analytical grade methanol;

[0041] Gel column size: 18mm*180mm;

[0042] The flow rate of the eluent: 0.5 mL / min;

[0043] The amount of eluent used: 2.0L;

[0044] Fr.F-5f2 was separated by normal phase silica gel column chromatography, eluted with petroleum ether-acetone, and 6 eluents were collected and named Fr.F-5f2a to Fr.F-5f2f, respectively, wherein the volume ratio of petroleum ether to acetone was 30:1 to 10:1; Fr.F-5f2e was purified by preparative HPLC to obtain a limonoid compound 12-O-deacetyltrichilin H, wherein the conditions of the preparative HPLC included:

[0045] Chromatographic column specifications: 4.6mm*250mm, 5μm;

[0046] Mobile phase: n-hexane-isopropanol 90:10;

[0047] Flow rate: 2mL / min.

[0048] The present invention extracts and separates trichilin-type limonoid compounds having the structure shown in the above formula: trichilitin G, trichilitin H, 12-hydroxyamoorastatin, azedarachin B, mesendanins K and 12-O-deacetyltrichilin H. Studies have shown that the above limonoid compounds exhibit good inhibitory activity on HCT-116 (human colon cancer cells), NCl-H1975 (human lung adenocarcinoma cells) and SH-SY5Y (human neuroblastoma cells).

[0049] In comparison, trichilitin H has the highest inhibitory activity. The present invention studies the inhibitory mechanism of trichilitin H on cancer cells and finds that trichilitin H can induce S phase arrest and apoptosis of cancer cells by regulating the expression of cell cycle proteins (CDK1, CDK2 and Cyclin A) and apoptosis-related proteins (P53, Cleaved parp and Cleaved casp 9); at the same time, the compound trichilitin H can regulate downstream cycle (p21, cdc2) and apoptosis proteins (Bcl-2, Caspase 3) by inducing p53, promote S phase arrest and apoptosis of cancer cells, exert anti-tumor activity, and has a good application prospect in the preparation of anti-tumor drugs.

[0050] Compared with the prior art, the present invention has the following beneficial effects: the Trichilin-type limonoid compounds provided by the present invention show excellent inhibitory activity against HCT-116, NCl-H1975, SH-SY5Y and other cancer cells, and have good value and application prospects in the preparation of anti-tumor drugs. The limonoid compounds provided by the present invention are derived from partridge flowers and have low production costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 It is a limonoid compound trichilitin G 1 H NMR spectrum;

[0052] Figure 2 It is a limonoid compound trichilitin G 13 C NMR spectrum;

[0053] Figure 3 This is the HSQC spectrum of trichilitin G, a limonoid compound;

[0054] Figure 4 This is the HMBC spectrum of trichilitin G, a limonoid compound;

[0055] Figure 5 It is a limonoid compound trichilitin G 1 H- 1 H COSY spectrum;

[0056] Figure 6 This is the ROESY spectrum of trichilitin G, a limonoid compound;

[0057] Figure 7 This is the HR-ESI-MS spectrum of trichilitin G, a limonoid compound;

[0058] Figure 8 This is the IR spectrum of trichilitin G, a limonoid compound;

[0059] Fig. 9 This is the UV spectrum of trichilitin G, a limonoid compound;

[0060] Fig.10 This is the CD spectrum of trichilitin G, a limonoid compound;

[0061] Fig.11 The limonoid compound trichilitin H 1 H NMR spectrum;

[0062] Fig.12 The limonoid compound trichilitin H 13 C NMR spectrum;

[0063] Fig.13 This is the HSQC spectrum of trichilitin H, a limonoid compound;

[0064] Fig.14 This is the HMBC spectrum of trichilitin H, a limonoid compound;

[0065] Fig.15 The limonoid compound trichilitin H 1 H- 1 H COSY spectrum;

[0066] Fig.16 This is the ROESY spectrum of trichilitin H, a limonoid compound;

[0067] Fig.17 This is the HR-ESI-MS spectrum of trichilitin H, a limonoid compound;

[0068] Fig.18 This is the IR spectrum of trichilitin H, a limonoid compound;

[0069] Fig.19 This is the UV spectrum of trichilitin H, a limonoid compound;

[0070] Fig. 20 This is the CD spectrum of trichilitin H, a limonoid compound;

[0071] Fig.21 It is a limonoid compound 12-hydroxyamoorastatin 1 H NMR spectrum;

[0072] Fig. 22 It is a limonoid compound 12-hydroxyamoorastatin 13 C NMR spectrum;

[0073] Fig.23 This is the ESI-MS spectrum of the limonoid compound 12-hydroxyamoorastatin;

[0074] Fig.24 Azedarachin B is a limonoid compound. 1 H NMR spectrum;

[0075] Fig.25 Azedarachin B is a limonoid compound. 13 C NMR spectrum;

[0076] Fig.26This is the ESIMS spectrum of the limonoid compound azedarachin B;

[0077] Fig. 27 It is a limonoid compound 12-O-deacetyltrichilin H 1 H NMR spectrum;

[0078] Fig.28 It is a limonoid compound 12-O-deacetyltrichilin H 13 C NMR spectrum;

[0079] Fig.29 This is the ESI-MS spectrum of the limonoid compound 12-O-deacetyltrichilin H;

[0080] Fig.30 The cell cycle of HCT-116 cells was arrested in the S phase by the limonoid compound trichilitin H. A is the effect of different concentrations and time of the limonoid compound trichilitin H on the cell cycle distribution of HCT-116 cells detected by PI staining; BC is the quantitative histogram of different cell cycle stages in Figure A; D is the expression of cycle-related proteins detected by western blot after HCT-116 cells were treated with a specified concentration of the limonoid compound trichilitin H for different time periods; E is the statistical graph of the expression of each protein in Figure D.

[0081] Fig.31 The limonoid compound trichilitin H inhibits the proliferation of HCT-116 cells and induces their apoptosis. A shows the growth inhibitory effect of the limonoid compound trichilitin H on HCT-116 cells at different time points; B shows the apoptotic effect of the limonoid compound trichilitin H on HCT-116 cells after treatment with the specified concentration for 72 hours using the Annexin V-FITC / PI double staining method; C shows the statistical graph of the apoptosis rate of HCT-116 cells in Figure B; D shows the mRNA levels of various apoptotic genes detected by the qPCR method after treatment with the limonoid compound trichilitin H at the specified concentration for 24 hours; E shows the expression of apoptosis-related proteins detected by western blot after HCT-116 cells were treated with the limonoid compound trichilitin H at the specified concentration for 48 hours; F shows the statistical graph of the expression of various proteins in Figure E. DETAILED DESCRIPTION

[0082] The present invention provides five trichilin-type limonoid compounds separated and purified from partridge flowers: trichilitin G, trichilitin H, 12-hydroxyamoorastatin, azedarachin B and 12-O-deacetyltrichilin H, having a structure shown in formula I:

[0083]

[0084] Example 1 Extraction and separation of trichilitin G

[0085] (1) Using a methanol solution to reflux extract the leaves of the partridge flower, and collecting the extract; concentrating the extract to obtain an extract; adding an appropriate amount of water to the extract for dispersion, and then extracting with ethyl acetate to obtain an ethyl acetate fraction; dissolving the ethyl acetate fraction and then performing chromatography on a 100-200 mesh normal phase silica gel column, and performing gradient elution with petroleum ether-acetone and dichloromethane-methanol, respectively, to collect 11 eluates, which are named Fr.A to Fr.K in sequence; the volume ratio of petroleum ether to acetone is 40:1 to 5:1, and gradient elution is performed; the volume ratio of dichloromethane to methanol in the dichloromethane-methanol is 50:1 to 0:1, and gradient elution is performed;

[0086] (2) Fr.G 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 11 eluates were collected and named Fr.G-1 to Fr.G-11; Fr.G-3 was subjected to C 18 Reverse phase silica gel column chromatography separation, using a methanol aqueous solution with a volume fraction of 40% to 100% for gradient elution, collecting 10 eluents, named Fr.G-3a to Fr.G-3j in sequence; Sephadex LH-20 methanol gel column chromatography to elute the Fr.G-3e, collecting a chromatographic solution for every 5 mL, collecting 6 eluents, named Fr.G-3e1 to Fr.G-3e6 in sequence, the conditions of the Sephadex LH-20 methanol gel column chromatography include:

[0087] Eluent: analytical grade methanol;

[0088] Gel column size: 18mm*180mm;

[0089] The flow rate of the eluent: 0.5 mL / min;

[0090] The amount of eluent used: 2.0L;

[0091] (3) Fr.G-3e3 was separated by normal phase silica gel column chromatography and eluted with petroleum ether-ethyl acetate, and three eluents were collected and named Fr.G-3e1a to Fr.G-3e1c, respectively, wherein the volume ratio of petroleum ether to ethyl acetate was 6:1; Fr.G-3e1b was purified by preparative HPLC to obtain a limonoid compound trichilitin G, wherein the conditions of the preparative HPLC include:

[0092] Chromatographic column specifications: 4.6mm*250mm, 5μm;

[0093] Mobile phase: n-hexane-isopropanol 80:20;

[0094] Flow rate: 2 mL / min;

[0095] Retention time: 27min;

[0096] Structural characterization of limonoid compound trichilitin G:

[0097] Figure 1 It is a limonoid compound trichilitin G 1 H NMR spectrum; Figure 2 It is a limonoid compound trichilitin G 13 CNMR spectrum; Figure 3 This is the HSQC spectrum of trichilitin G, a limonoid compound; Figure 4 This is the HMBC spectrum of trichilitin G, a limonoid compound; Figure 5 It is a limonoid compound trichilitin G 1 H- 1 H COSY spectrum; Figure 6 This is the ROESY spectrum of trichilitin G, a limonoid compound; Figure 7 This is the HR-ESI-MS spectrum of trichilitin G, a limonoid compound; Figure 8 This is the IR spectrum of trichilitin G, a limonoid compound; Fig. 9 This is the UV spectrum of trichilitin G, a limonoid compound; Fig.10 This is the CD spectrum of trichilitin G, a limonoid compound.

[0098] Table 1 Limonoid compound trichilitin G 1 H-NMR and 13 C-NMR data (600 / 150 MHz, CDCl3 )

[0099]

[0100]

[0101] From Table 1 and Figures 1 to 10 It can be seen that the structural data of the limonoid compound trichilitin G are as follows: white amorphous powder. ECD(MeOH)λ max (Δε)222(4.82)nm; UV(MeOH)λ max (logε)216(2.93); IR(KBr)ν max 3441, 2928, 1721, 1257, 1063cm -1 . m / z in HR-EI-MS: 695.2690 [M+Na] + (calcd for C 35 H 44 O 13 Na: 695.2680); combined 13 C-NMR data confirmed the molecular formula to be C 35 H 44 O 13 , the unsaturation is 14. 1 H-NMR showed a characteristic furan substituted ring [δ H 7.32 (1H, s, H-23), 7.22 (1H, s, H-21), 6.53 (1H, s, H-22)], hemiacetal methine proton signal [δ H 5.81 (1H, s, H-29)], 7 methyl proton signals [δ H 2.12 (3H, s, 3-OAc), 2.03 (3H, s, 2-OAc), 1.86 (3H, s, H-5'), 1.83 (3H, d, J = 7.0 Hz, H-4'), 1.15 (3H, s, H-30), 1.14 (3H, s, H-18), 0.84 (3H, s, H-18)] and 8 oxygen-linked proton substitution signals [δ H 5.92(1H,t,J=4.6Hz,H-2), 5.58(1H,d,J=4.6Hz,H-3), 4.58(1H,s,H-1), 4.35(2 H,q,J=13.1Hz,H-19), 4.10(1H,s,H-12), 3.77(1H,s,H-15), 3.65(1H,s,H-7)]. 13C-NMR and HSQC showed 35 carbon signals, including 7 methyl, 3 methylene, 14 methine and 11 quaternary carbons. This indicates that the compound is trichilitin G, a limonoid 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- 1 Its stereostructure was preliminarily assigned by ROESY and finally confirmed by ECD calculation, including its absolute configuration.

[0102] Example 21 Extraction and Isolation of 2-hydroxyamoorastatin

[0103] The Fr.G-3e6 described in Example 1 was separated by normal phase silica gel column chromatography, eluted with petroleum ether-ethyl acetate, and two eluents were collected and named Fr.G-3e6a and Fr.G-3e6b, respectively, wherein the volume ratio of petroleum ether to ethyl acetate was 1.5:1; the Fr.G-3e6b was purified by preparative HPLC to obtain the limonoid compound 12-hydroxyamoorastatin, wherein the conditions of the preparative HPLC included:

[0104] Chromatographic column specifications: 4.6mm*250mm, 5μm;

[0105] Mobile phase: n-hexane-isopropanol 75:25;

[0106] Flow rate: 2 mL / min;

[0107] Retention time: 42min;

[0108] Structural characterization of limonoid compound 12-hydroxyamoorastatin:

[0109] The limonoid compound 12-hydroxyamoorastatin is a white amorphous powder. Figure 21-23 Its structural data are as follows: ESI-MS: m / z 555[M+Na] + (C 28 H 36 O 10 ); 1 H NMR (600 MHz, Acetone-d 6)δ: 7.38(1H,s,H-23), 7.33(1H,s,H-21), 6.61(1H,d,J=1.6Hz,H-22), 4. 91(1H,d,J=4.3Hz,H-3), 4.68(1H,s,H-29), 4.32(1H,t,J=4.6Hz,H-9), 4. 29(1H,d,J=2.2Hz,H-1), 4.24(2H,d,J=3.4Hz,H-19), 3.94(1H,d,J=2.4Hz ,H-12), 3.80(1H,s,H-15), 3.64(1H,d,J=2.2Hz,H-7), 2.91(1H,d,J=4.1H z,H-17), 2.86(1H,s,H-2α), 2.80(dt,J=15.9,4.9Hz,H-5), 2.21(1H,dd, J=12.9,6.5Hz,H-16α), 2.00(1H,m,H-16β), 1.98(3H,s,3-OAc), 1.95(1H, dd,J=7.6,5.5Hz,H-6β), 1.82(1H,d,J=15.9Hz,H-2β), 1.72(1H,dt,J=14. 2,3.9Hz,H-6α), 1.20(3H,s,H-18), 1.07(3H,s,H-30), 0.82(3H,s,H-28); 13 C NMR (150 MHz, Acetone-d 6 )δ: 214.4(C-11), 170.5(3'-OAc), 142.7(C-23), 141.5(C-21), 125.4(C-20), 114.0(C-22), 97.0(C-29), 79.8(C-12), 74.0(C-1), 73.7(C-7), 70.6(C-3), 70.5(C-4), 64.7(C-19), 59.1( C-15), 48.8(C-9), 47.2(C-13), 43.1(C-8), 42.5(C-10), 40.6(C-4), 40.2(C-17), 36.7(C-2) , 33.8(C-16), 28.9(C-5), 26.1(C-6), 23.1(C-30), 21.4(3'-OAc), 20.0(C-28), 14.7(C-18).

[0110] Example 3 Extraction and separation of azedarachin B

[0111] Fr.G-7 described in Example 1 was subjected to C 18Reverse phase silica gel column chromatography separation, using 50% to 100% methanol aqueous solution for gradient elution, collecting 9 eluents, named Fr.G-7a to Fr.G-7i; Fr.G-7e is subjected to normal phase silica gel column chromatography separation, eluted with petroleum ether-ethyl acetate, collecting 5 eluents, named Fr.G-7e1 to Fr.G-7e5, the volume ratio of petroleum ether to ethyl acetate is 2:1; Fr.G-7e5 is subjected to preparative HPLC purification to obtain a limonoid compound azedarachin B, the conditions of the preparative HPLC include:

[0112] Chromatographic column specifications: 4.6mm*250mm, 5μm;

[0113] Mobile phase: 34% acetonitrile;

[0114] Flow rate: 2mL / min;

[0115] Retention time: 96min;

[0116] Structural characterization of limonoid compound azedarachin B:

[0117] Compound azedarachin B is a white amorphous powder with structural data as shown in Figure 24-26 Shown: ESI-MS: m / z641[M+K] + (C 32 H 42 O 11 ); 1 H NMR (600 MHz, CDCl 3)δ: 7.34 (1H, m, H-23), 7.23 (1H, s, H-21), 6.52 (1H, m, H-22), 5.80 (1H, t, J = 4.1 Hz, H-3), 5.77 (1H, s, H-29), 4.67 (1H, m, H-1), 4.57 (1H, d, J = 5.0 Hz, H-9), 4.33 (1H, d, J = 14.3 Hz, H-19α), 4.09 (1H, d, J = 14.3 Hz, H-19β), 3.78 (1H, s, H-12), 3.67 (1H, s, H-15), 3.32 (1H, m, H-7), 3.01 (1H, m, H-17), 2.79 (1H, m, H-2β), 2.65 (1H, dd, J = 14.0, 7.0 Hz, H-5), 2.35 (1H, m, H-2'), 2.16 (3H, d, J = 3.0 Hz, 3-OAc), 2.05 (1H, m, H-16α), 1.93 (1H, m, H-16β), 1.87 (1H, m, H-2), 1.76 (1H, m, H-6α), 1.73 (3H, s, H-18), 1.22 (6H, m, H-3', H-4'), 1.18 (1H, s, H-6β), 1.14 (3H, d, J = 6.6 Hz, H-30), 1.03 (3H, d, J = 34.9 Hz, H-28); 13 C NMR (150 MHz, CDCl 3 )δ: 213.6 (C-11), 175.8 (C-1'), 169.8 (3-OAc), 142.3 (C-21), 140.7 (C-23), 123.5 (C-20), 112.7 (C-22), 94.2 (C-29), 78.8 (C-12), 73.0 (C-3), 72.7 (C-14), 70.9 (C-7), 70.0 (C-1), 64.0 (C-19), 59.1 (C-15), 47.7 (C-9), 46.1 (C-13), 42.4 (C-8), 41.5 (C-4), 38.8 (C-10), 38.7 (C-17), 34.2 (C-2), 34.1 (C-5), 33.1 (C-16), 27.9 (C-2'), 25.5 (C-6), 22.7 (C-18), 21.2 (3-OAc), 19.3 (C-28), 18.8 (C-3'), 18.7 (C-4'), 14.4 (C-30).

[0118] Example 4 Extraction and Isolation of Trichilitin H

[0119] The Fr.G-7g in Example 3 was subjected to normal phase silica gel column chromatography, eluted with dichloromethane-acetone, and three eluents were collected and named Fr.G-7g1 to Fr.G-7g3, respectively, wherein the volume ratio of dichloromethane to acetone was 20:1; the Fr.G-7g2 was subjected to preparative HPLC purification to obtain the limonoid compound trichilitin H, wherein the conditions of the preparative HPLC included:

[0120] Chromatographic column specifications: 4.6mm*250mm, 5μm;

[0121] Mobile phase: n-hexane-isopropanol 85:15;

[0122] Flow rate: 2mL / min;

[0123] Retention time: 34min;

[0124] Structural characterization of limonoid compound trichilitin H:

[0125] Fig.11 The limonoid compound trichilitin H 1 H NMR spectrum; Fig.12 The limonoid compound trichilitin H 13 CNMR spectrum; Fig.13 This is the HSQC spectrum of trichilitin H, a limonoid compound; Fig.14 This is the HMBC spectrum of trichilitin H, a limonoid compound; Fig.15 The limonoid compound trichilitin H 1 H- 1 H COSY spectrum; Fig.16 This is the ROESY spectrum of trichilitin H, a limonoid compound; Fig.17 This is the HR-ESI-MS spectrum of trichilitin H, a limonoid compound; Fig.18 This is the IR spectrum of trichilitin H, a limonoid compound; Fig.19 This is the UV spectrum of trichilitin H, a limonoid compound; Fig. 20 This is the CD spectrum of the limonoid compound trichilitin H.

[0126] Table 2 Limonoid compound trichilitin H 1 H-NMR and 13 C-NMR data (600 / 150 MHz, CDCl3 )

[0127]

[0128]

[0129] From Table 2 and Figures 11 to 20 It can be seen that the structural data of the limonoid compound trichilitin H are as follows: white amorphous powder. ECD(MeOH)λ max (Δε)221(8.22)nm; UV(MeOH)λ max (logε)211(3.23); IR(KBr)ν max 3442, 2931, 1713, 1258, 1064cm -1 ; m / z in HR-EI-MS: 653.2659 [M+Na] + (calcd for C 33 H 42 O 12 Na: 653.2574), combined 13 C-NMR data confirmed the molecular formula C 33 H 42 O 12 , the unsaturation is 13. 1 H-NMR showed a characteristic furan substituted ring signal [δ H 7.14 (s, H-21), 6.44 (s, H-22), 7.25 (s, H-23)], characteristic hemiacetal methine proton signals, 6 methyl proton signals [δ H 2.08 (3H, s, 2-OAc), 1.78 (3H, s, H-5'), 1.75 (3H, q, J = 6.9 Hz, H-4'), 1.07 (3H, s, H-30), 1.04 (3H, s, H-18), 0.91 (3H, s, H-28)], 1 olefin proton signal [δ H 6.91 (1H, q, J = 6.9 Hz, H-3')] and 8 oxygen proton signals [δ H 5.75(1H,s,1-OAc), 4.57(1H,s,H-3), 4.27(2H,q,J=13.1Hz,H-19), 4.1 4(1H,s,H-1), 4.01(1H,s,H-12), 3.70(1H,s,H-15), 3.59(1H,s,H-7)]. 13C-NMR and HSQC showed 33 carbon signals, including 6 methyl groups, 4 methylene groups, 13 methine groups and 10 quaternary carbon groups, indicating that the compound is trichilitin H, a limonoid compound having the structure shown in Formula I. The planar structure of the compound was finally determined by two-dimensional NMR techniques (HSQC, HMBC and 1 H- 1 Its stereostructure was preliminarily assigned by ROESY and finally confirmed by ECD calculation, including its absolute configuration.

[0130] Example 512 Extraction and Isolation of O-deacetyltrichilin H

[0131] (1) Fr.F in Example 1 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 8 eluates were collected and named Fr.F-1 to Fr.F-8 respectively; Fr.F-5 was subjected to C 18 Reverse phase silica gel column chromatography separation, using 50% to 100% methanol aqueous solution for gradient elution, collecting 10 eluents, named Fr.F-5a to Fr.F-5j; Sephadex LH-20 methanol gel column chromatography to elute the Fr.F-5f, collecting a chromatographic solution for every 5 mL, collecting 4 eluents, named Fr.F-5f1 to Fr.F-5f4, the conditions of the Sephadex LH-20 methanol gel column chromatography include:

[0132] Eluent: analytical grade methanol;

[0133] Gel column size: 18mm*180mm;

[0134] The flow rate of the eluent: 0.5 mL / min;

[0135] The amount of eluent used: 2.0L;

[0136] (2) Fr.F-5f2 was subjected to normal phase silica gel column chromatography, eluted with petroleum ether-acetone, and 6 eluents were collected and named Fr.F-5f2a to Fr.F-5f2f, respectively, wherein the volume ratio of petroleum ether to acetone was 30:1; Fr.F-5f2e was subjected to preparative HPLC purification to obtain a limonoid compound 12-O-deacetyltrichilin H, wherein the conditions of the preparative HPLC include:

[0137] Chromatographic column specifications: 4.6mm*250mm, 5μm;

[0138] Mobile phase: n-hexane-isopropanol 90:10;

[0139] Flow rate: 2mL / min;

[0140] Retention time: 58min;

[0141] Structural characterization of limonoid compound 12-O-deacetyltrichilin H:

[0142] Compound 12-O-deacetyltrichilin H is a white amorphous powder. Structural data are as follows Figure 27-29 Shown: ESI-MS: m / z 683[M+Na] + (C 34 H 44 O 13 ); 1 H NMR (600 MHz, CDCl 3 )δ: 7.33(1H,s,H-23), 7.22(1H,s,H-21), 6.45(1H,s,H-22), 5.89(1H,q,J=4.4Hz,H -2), 5.73(1H,s,H-29), 5.53(1H,d,J=4.6Hz,H-3), 4.57(1H,m,H-1), 4.55(1H,s,H-9 ), 4.36(1H,d,J=13.0Hz,H-19α), 4.31(1H,d,J=13.0Hz,H-19β), 4.11(1H,d,J=9.1H z,H-12), 3.77(1H,s,H-15), 3.65(1H,s,H-7), 3.01(1H,dd,J=10.9,6.1Hz,H-17), 2. 84(1H,dd,J=14.2,4.1Hz,H-5), 2.67(1H,m,H-2'), 2.45(1H,d,J=3.7Hz,H-16β), 2. 11(3H,d,J=16.2Hz,3-OAc), 2.03(3H,s,2-OAc), 1.99(1H,s,H-16α), 1.90(1H,dd,J= 13.1,11.2Hz,H-6α), 1.74(1H,m,H-6β), 1.21(3H,overlapped,H-3'), 1.21(3H,ove rlapped,H-4'), 1.32(3H,s,H-30), 1.14(3H,s,H-18), 0.81(3H,d,J=19.9Hz,H-28); 13 C NMR (150 MHz, CDCl 3)δ: 213.2(C-11), 175.8(C-1'), 170.2(2-OAc), 169.1(3-OAc), 142.3(C-23), 140.7(C-21), 123.5(C-20), 112.8( C-22), 93.5(C-29), 78.8(C-12), 72.9(C-14), 72.8(C-3), 71.6(C-1), 69.9(C-7), 68.6(C-2), 64.1(C-19), 59.2( C-15), 47.3(C-9), 46.1(C-13), 42.4(C-8), 42.3(C-10), 40.7(C-4), 38.6(C-17), 34.1(C-2'), 33.1(C-16), 27.7 (C-5), 25.3(C-6), 22.8(C-30), 21.0(3-OAc), 20.8(2-OAc), 18.8(C-28), 18.7(C-3'), 18.7(C-4'), 14.4(C-18).

[0143] Example 6 Antitumor Activity

[0144] MTT assay

[0145] Three cancer cells, HCT-116 (human colon cancer cells), NCl-H1975 (human lung adenocarcinoma cells) and SH-SY5Y (human neuroblastoma cells), were uniformly inoculated in 96-well plates (100 μL / well) at a density of 3,000 cells per well. After the cells adhered to the wall, the cells were treated with different concentrations of compounds, with three replicate wells for each concentration group, and cultured in a constant temperature incubator for 72 hours. After 72 hours, the 96-well plate was removed, the culture medium was discarded, MTT (100 μL / well) was added to develop color, and cultured in a constant temperature incubator for 4 hours. After 4 hours, the 96-well plate was removed, the supernatant was discarded, DMSO (100 μL / well) was added, and the absorbance was measured at 490 nm using an enzyme reader after shaking. After measuring the absorbance, the IC of the compound was calculated using IBM SPSS Statistics. 50 The values ​​are shown in Table 3.

[0146] Table 3 In vitro antitumor activity results of limonoid compounds with formula I (IC 50 value)

[0147]

[0148] It can be seen from Table 3 that the five compounds have strong cancer cell toxicity against the three cancer cells. Doxorubicin (adriamycin) is the positive control. The compound trichilitin H has high inhibitory activity against the three cancer cells, especially against colon cancer cells (HCT-116). The inhibitory activity is stronger than the positive control, and it is a potential highly active anti-tumor drug.

[0149] Example 7 Study on the anti-tumor mechanism of trichilitin H

[0150] (2) Cell cycle assay

[0151] HCT-116 cells were evenly seeded in 6-well plates at a density of 6000 cells per well; after the cells adhered to the wall, they were treated with different concentrations of compounds (0.02μM, 0.04μM, 0.08μM); after 48h, the cells were collected, washed with PBS, fixed in 70% ice ethanol, and kept in a -20℃ refrigerator overnight. The supernatant was then discarded, washed with pre-cooled PBS, and the cells were heated in a 37℃ water bath with 100μg / mL RNase A for 10min, then stained with 50μg / mL propidium iodide (PI) in the dark for 30min, and the cell DNA content was detected by flow cytometry.

[0152] (3) Cell apoptosis experiment

[0153] HCT-116 cells were evenly seeded in a 6-well plate at a density of 5000 cells per well. After the cells adhered to the wall, they were treated with different concentrations of compounds (0.02μM, 0.04μM, 0.08μM). After 48h, the cells were collected, washed 3 times with PBS, stained with Annexin V and propidium iodide (PI), incubated in the dark for 30min, and then flow cytometry was used to detect cell apoptosis.

[0154] (4) Protein immunoblotting analysis

[0155] Total protein extraction: After treating HCT-116 cells with different concentrations of trichilitin H (0.02 μM, 0.04 μM, 0.08 μM) for 48 h, remove the culture dish and discard the supernatant; wash with 2 mL of PBS buffer and discard the washing solution; add 150-200 μL of lysis solution to each dish and collect the lysis suspension with a scraper; transfer the lysis solution to a 1.5 mL centrifuge tube and place in a metal bath at 104°C for 10-20 min to fully lyse the cells.

[0156] Protein quantification: 10 μL of protein suspension was taken into a 1.5 mL centrifuge tube, purified with 1000 μL of trichloroacetic acid and centrifuged in a low-temperature centrifuge (4°C, 12000 rpm, 10 min); the supernatant was removed and discarded, 1000 μL of BCA solution was added to a constant temperature metal bath (60°C, 60 min), and then the suspension was removed and mixed. Three replicate wells were set for each concentration, with 200 μL in each well of a 96-well plate, and the protein concentration was measured at 562 nm.

[0157] Gel preparation: Select gel of appropriate concentration according to the molecular weight of the target protein, prepare separation gel according to the preparation ratio, add it to the glass template and flatten it with anhydrous ethanol. After it solidifies, drain the anhydrous ethanol, add the prepared concentrated gel, insert the comb, and use it after it solidifies.

[0158] Electrophoresis: According to the results of protein quantification, a protein suspension of a given concentration is drawn into the gel lane, and 80 V is used as the initial voltage. After the marker reaches the separation gel, the voltage is adjusted to 120 V for electrophoretic separation.

[0159] Transfer: After electrophoresis, the protein-blotted gel was assembled into a sandwich structure with a PVDF membrane of equivalent size and a sponge filter paper for transfer (110 V, 150 min).

[0160] Blocking: After the transfer is completed, remove the PVDF membrane, put it in 5% BSA or skimmed milk powder, place it on a shaker for blocking, and take it out after 60 minutes.

[0161] Incubation with primary antibody: After blocking, remove the membrane, wash it three times with TBST, 10 min each time, and then place it together with the corresponding primary antibody dilution in a 4°C refrigerator overnight.

[0162] Incubation with secondary antibody: After the primary antibody incubation is completed, the primary antibody is recovered and the membrane is washed three times with TBST, each time for 10 minutes, and then it is placed on a shaker and incubated with the corresponding secondary antibody dilution for 2 hours.

[0163] Development: After the secondary antibody incubation is completed, the secondary antibody is recovered and the membrane is washed three times with TBST, each time for 10 minutes, and then the developer is added and developed by the ChemiDoc TM MP imaging system, and the expression of related proteins is analyzed using Image J 1.6 software.

[0164] Fig.30The limonoid compound trichilitin H induces cell cycle arrest in the S phase of HCT-116 cells. A is the effect of different concentrations and time of the limonoid compound trichilitin H on the cell cycle distribution of HCT-116 cells detected by PI staining; BC is the quantitative histogram of different cell cycle stages in Figure A; D is the expression of cycle-related proteins (p-cdc2 / cdc2, p53 and p21) detected by western blot after HCT-116 cells were treated with the specified concentration of the limonoid compound trichilitin H for different time periods; E is the statistical graph of the expression of each protein in Figure D.

[0165] Fig.31 The limonoid compound trichilitin H inhibits the proliferation of HCT-116 cells and induces their apoptosis. A shows the growth inhibitory effect of the limonoid compound trichilitin H on HCT-116 cells at different time points; B shows the apoptotic effect of the limonoid compound trichilitin H on HCT-116 cells after treatment with the specified concentration for 72 hours using the Annexin V-FITC / PI double staining method; C shows the statistical graph of the apoptosis rate of HCT-116 cells in Figure B; D shows the mRNA levels of various apoptotic genes detected by the qPCR method after treatment with the limonoid compound trichilitin H at the specified concentration for 24 hours; E shows the expression of apoptosis-related proteins (P53, Caspase-3, Bcl-2) detected by western blot after HCT-116 cells were treated with the specified concentration of the limonoid compound trichilitin H for 48 hours; F shows the statistical graph of the expression of various proteins in Figure E.

[0166] Depend on Fig.30 and Fig.31 It can be seen that trichilitin H regulates downstream cycle (p21, cdc2) and apoptosis proteins (Bcl-2, Caspase-3) by inducing p53, causing S phase arrest and apoptosis of HCT-116 cancer cells.

[0167] 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. Trichilin-type limonoid compound, characterized in that: The compound is trichilitin G or trichilitin H, and the structural formula is as follows: 。 2. A use of the Trichilin-type limonoid compound according to claim 1 in the preparation of a drug for treating cancer, characterized in that: The cancer is liver cancer, lung adenocarcinoma or neuroblastoma.

3. The use of the Trichilin-type limonoid compound according to claim 2 in the preparation of a drug for treating cancer, characterized in that: The limonin compound uses the compound shown in the structural formula or a pharmaceutically acceptable salt thereof as a pharmaceutically active component.

4. The use of the Trichilin-type limonoid compound according to claim 3 in the preparation of a drug for treating cancer, characterized in that: The content of the active pharmaceutical ingredient is 0.1-99.9%.

5. The use of the Trichilin-type limonoid compound according to claim 2 in the preparation of a drug for treating cancer, characterized in that: The limonoid compound is used in combination with a pharmaceutically acceptable carrier.

6. A method for extracting and separating Trichilin-type limonoid compounds as claimed in claim 1, characterized in that: The extraction and separation methods are as follows: Step (1) using a methanol solution to reflux extract the leaves of the partridge flower, collecting the extract; concentrating the extract to obtain an extract; adding an appropriate amount of water to the extract for dispersion, and extracting with ethyl acetate to obtain an ethyl acetate fraction; dissolving the ethyl acetate fraction and performing chromatography on a 100-200 mesh normal phase silica gel column, and gradient eluting with petroleum ether-acetone and dichloromethane-methanol in sequence, collecting and obtaining 11 eluates, which are respectively named Fr. A to Fr. K; the volume ratio of petroleum ether to acetone is 40:1 to 5:1; the volume ratio of dichloromethane to methanol in the dichloromethane-methanol is 50:1 to 0:1; Step (2) Fr. G is subjected to small pore resin chromatography, and gradient elution is performed using a methanol aqueous solution with a volume fraction of 50% to 100%, and 11 eluates are collected and named Fr. G-1 to Fr. G-11; Fr. G-3 is subjected to C 18 Reverse phase silica gel column chromatography separation, using 40% to 100% methanol aqueous solution for gradient elution, collecting 10 eluents, named Fr. G-3a to Fr. G-3j; Sephadex LH-20 methanol gel column chromatography to elute Fr. G-3e, collecting a chromatographic solution for every 5 mL, collecting 6 eluents, named Fr. G-3e1 to FrG-3e6, the conditions of the Sephadex LH-20 methanol gel column chromatography include: Eluent: analytical grade methanol; Gel column size: 18 mm*180 mm; The flow rate of the eluent: 0.5 mL / min; The amount of the eluent: 2.0 L; Step (3) Fr. G-3e3 is subjected to normal phase silica gel column chromatography, eluted with petroleum ether-ethyl acetate, and three eluents are collected and named Fr. G-3e1a to Fr. G-3e1c, respectively, wherein the volume ratio of petroleum ether to ethyl acetate is 8:1 to 5:1; Fr. G-3e1b is subjected to preparative HPLC purification to obtain a limonoid compound trichilitin G, wherein the conditions of the preparative HPLC include: Column specifications: 4.6 mm*250 mm, 5 μm; Mobile phase: n-hexane-isopropanol 80:20; Flow rate: 2 mL / min; The extraction and separation method of the compound trichilitin H is as follows: Fr. G-7 was C 18 Reverse phase silica gel column chromatography separation, using 50% to 100% methanol aqueous solution for gradient elution, collecting 9 eluents, named Fr. G-7a to Fr. G-7i; FrG-7g is subjected to normal phase silica gel column chromatography separation, eluted with dichloromethane-acetone, collecting 3 eluents, named FrG-7g-1 to FrG-7g-3, wherein the volume ratio of dichloromethane to acetone is 30:1 to 15:1; FrG-7g-2 is subjected to preparative HPLC purification to obtain a limonoid compound trichilitin H, wherein the conditions of the preparative HPLC include: Column specifications: 4.6 mm*250 mm, 5 μm; Mobile phase: n-hexane-isopropanol 85:15; Flow rate: 2 mL / min.

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