Flavonoid diglycoside compound and application thereof

Abstract

The invention relates to a preparation method and application of a flavonoid diglycoside compound which is separated from a dry whole plant of daphne tenuifolia plant and has antineoplastic activity. The preparation method comprises the following steps: taking a dry whole plant of a daphne plant daphne gemma E.Pritz. Ex Diels as a raw material, and preparing a raw material from the dry whole plant, namely the daphne gemma E.Pritz. Ex Diels, of the daphne gemma E.Pritz. Ex Diels, of the daphne gemma E.Pritz. Ex Diels; the preparation method comprises the following steps: extracting an extract, extracting and concentrating, and then carrying out various separation methods such as silica gel column chromatography, reversed-phase ODS column chromatography and Sephadex LH-20 to obtain the flavonoid diglycoside compound: # imgabs0 #. The cytotoxic effects of the compounds I-III on three human cancer cell strains of lung cancer A549, breast cancer MDA-MB-231 and colon cancer SW480 are evaluated by adopting an MTS method; the compounds have strong anti-lung cancer and anti-breast cancer activity, and have potential anti-tumor drug research and development and application values.

Description

Technical Field

[0001] The invention relates to the field of medical technology, and in particular to a preparation method and application of a class of flavonoid disodium compounds separated from the dried whole plant of Daphne koreana plant in western Sichuan. Background Art

[0002] Cancer, also known as malignant tumor, refers to a disease in which cells lose control during growth and division, forming abnormal proliferation and invading surrounding tissues. The mechanisms of cancer occurrence are diverse and complex. From gene mutations to the influence of environmental factors to the failure of the immune system, these processes may lead to cancer. At the molecular level, the occurrence of cancer is usually related to uncontrolled cell proliferation, blocked apoptosis, abnormal gene expression, and abnormal cell metabolism. Obtaining natural products with anti-tumor activity from traditional medicinal plants is still an important source for the discovery of lead compounds for anti-tumor drugs.

[0003] Daphne plants are rich in resources and have high medicinal value. Studies have shown that Daphne plants are rich in flavonoid glycoside compounds, which have novel and diverse structures and have anti-tumor, anti-inflammatory, antioxidant and other pharmacological activities. They are one of the hot spots in the study of natural active products in recent years. Daphne gemmata E.Pritz.ex Diels, also known as Daphne gemmata, is a deciduous shrub of the genus Daphne in the family Thymelaeaceae, 0.3 to 1 meter high, distributed in northwest to western Sichuan, and is endemic to Sichuan. It is a traditional Chinese medicinal material with the effects of clearing heat and detoxifying, dispelling wind and dampness. At present, there are few reports on the chemical composition of Daphne gemmata, so it is of great significance to study the flavonoid glycoside components in Daphne gemmata and evaluate their anticancer activity. Summary of the invention

[0004] In order to better utilize the resources of Daphne koreana and discover lead compounds with biological activity, the present invention systematically studies the chemical composition and activity of the dried whole plant of Daphne koreana from western Sichuan, and isolates a class of flavonoid disoside compounds having the following structure:

[0005]

[0006] One object of the present invention is to provide a method for preparing the flavonoid disoside compounds described in the present invention, which uses the dried whole plant of Daphne gemmata E. Pritz. ex Diels, a plant of the genus Daphne in the family Thymelaeaceae, as a raw material, and obtains the flavonoid disoside compounds by extracting and concentrating the extract, and then separating and obtaining the flavonoid disoside compounds by normal phase silica gel column chromatography, reverse phase ODS column chromatography, Sephadex LH-20 gel column chromatography, and the like.

[0007] Preferably, the method comprises the following steps:

[0008] (1) The whole plant of Daphne koreana is dried and crushed, and the alcohol or alcohol aqueous solution is preferably one or more of water, methanol, and ethanol, and more preferably 95% ethanol is used for cold soaking extraction three times, each time for 24 hours;

[0009] (2) extracting the extract obtained in step (1) using ethyl acetate;

[0010] (3) The ethyl acetate extract obtained in step (2) is concentrated to obtain an extract, and the sample is mixed with silica gel (200-300 mesh) at a ratio of 1:1 (g / g), and subjected to normal phase silica gel column chromatography, and the mobile phase is a dichloromethane-methanol mixed solvent with a volume ratio of 100:1, 50:1, 10:1, 8:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:5, 1:10, 0:1 as a gradient elution for 3 to 6 column volumes (preferably 5 column volumes), and the same components are combined by TLC detection to obtain 14 components, namely Fr.1 to 14;

[0011] (4) subjecting Fr.12 obtained in step (3) to Sephadex LH-20 gel column chromatography, using methanol as the eluent for 2 to 4 column volumes (preferably 3 column volumes), and combining the same components for TLC detection to obtain the components Fr.12-1 to 12-12;

[0012] (5) Fr.12-7 obtained in step (4) was subjected to reverse phase ODS column chromatography, and gradient eluted with a methanol-water mixed solvent with a volume ratio of 45:55→95:5 for 3 to 6 column volumes (preferably 5 column volumes), and the same components were combined by TLC detection to obtain the components Fr.12-7-1 to 12-7-3;

[0013] (6) Fr.12-7-3 obtained in step (5) was separated by normal phase silica gel column chromatography, and a petroleum ether-acetone mixed solvent with a volume ratio of 3:1→1:1 was used as the mobile phase for elution for 3 to 6 column volumes (preferably 5 column volumes). After the sample was completely eluted, TLC was synchronously detected to obtain compound I;

[0014] (7) Fr.13 obtained in step (3) was separated by normal phase silica gel chromatography, and eluted with a gradient of petroleum ether-ethyl acetate in a volume ratio of 3:1→0:1 for 3 to 6 column volumes (preferably 5 column volumes), and the same components were combined by TLC detection to obtain Fr.13-1 to 13-8 components;

[0015] (8) Fr.13-6 obtained in step (7) was subjected to reverse phase ODS column chromatography, and gradient eluted with a methanol-water mixed solvent with a volume ratio of 40:60→90:10 for 3 to 6 column volumes (preferably 5 column volumes), and the same components were combined by TLC detection to obtain each component of Fr.13-6-1 to 13-6-4;

[0016] (9) The Fr.13-6-3 obtained in step (8) was subjected to Sephadex LH-20 gel chromatography, and methanol was used as the eluent to elute for 2 to 4 column volumes (preferably 3 column volumes), and the same components were detected by TLC to obtain the components of Fr.13-6-3-1 to 13-6-3-4; (10) The Fr.13-6-3-3 obtained in step (9) was subjected to normal phase silica gel column chromatography, and a petroleum ether-acetone mixed solvent with a volume ratio of 5:1→1:1 was used for gradient elution for 3 to 6 column volumes (preferably 5 column volumes), and the same components were detected by TLC to obtain compound II;

[0017] (11) Fr.13-6-3-4 obtained in step (9) was subjected to Sephadex LH-20 gel chromatography, eluted with methanol for 2 to 4 column volumes (preferably 3 column volumes), and the same components were combined by TLC detection to obtain compound III.

[0018] Another object of the present invention is to provide the use of the flavonoid disoside compounds with anti-tumor activity in the preparation of anticancer drugs. The present invention uses the MTS method to evaluate the cytotoxic effects of the compounds I to III of the present invention on three human cancer cell lines: lung cancer A549, breast cancer MDA-MB-231, and colon cancer SW480. The results show that compound I exhibits strong cytotoxic activity (IC 50 The values ​​were 10.81 μM and 11.07 μM, respectively). Therefore, the flavonoid bioside compounds of the present invention can be used to prepare drugs for preventing or treating cancer, including lung cancer, breast cancer, and colon cancer, and may provide active new compounds or lead compounds for the development of new drugs.

[0019] Advantages of the present invention:

[0020] (1) The present invention uses the dried whole plant of Daphnegemmata E. Pritz. ex Diels, a plant of the genus Daphne in the family Thymelaeaceae, as a raw material, extracts the extract, concentrates the extract, and separates the flavonoid disoside compounds by normal phase silica gel column chromatography, reverse phase ODS column chromatography, Sephadex LH-20 gel column chromatography, etc. The preparation method is easy to operate, is one of the sources for obtaining safe and effective anti-tumor drugs, and may provide active new compounds or lead compounds for the research and development of new drugs.

[0021] (2) Compound I of the present invention exhibits strong cytotoxic activity against lung cancer A549 and breast cancer MDA-MB-231 cells (IC 50The values ​​are 10.81μM and 11.07μM respectively), which can be used to provide active small molecules or lead compounds in the development of drugs for the prevention or treatment of lung cancer and breast cancer, and has potential development and application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the HR-ESI-MS spectrum of compound Ⅰ.

[0023] Figure 2 This is the UV spectrum of compound Ⅰ.

[0024] Figure 3 is the IR spectrum of compound Ⅰ.

[0025] Figure 4 NMR of compound Ⅰ 1 H NMR spectrum.

[0026] Figure 5 NMR of compound Ⅰ 13 C NMR spectrum.

[0027] Figure 6 This is the NMR HSQC spectrum of compound Ⅰ.

[0028] Figure 7 This is the HMBC NMR spectrum of compound Ⅰ.

[0029] Figure 8 COSY NMR spectrum of compound Ⅰ.

[0030] Fig. 9 This is the ROESY NMR spectrum of compound Ⅰ.

[0031] Fig.10 This is the TOCSY NMR spectrum of compound Ⅰ.

[0032] Fig.11 This is the HR-ESI-MS spectrum of compound II.

[0033] Fig.12 This is the UV spectrum of compound II.

[0034] Fig.13 is the IR spectrum of compound II.

[0035] Fig.14 NMR of compound II 1 H NMR spectrum.

[0036] Fig.15 NMR of compound II 13 C NMR spectrum.

[0037] Fig.16 This is the NMR HSQC spectrum of compound II.

[0038] Fig.17 This is the HMBC NMR spectrum of compound II.

[0039] Fig.18 This is the COSY NMR spectrum of compound II.

[0040] Fig.19 This is the ROESY NMR spectrum of compound II.

[0041] Fig. 20 This is the HR-ESI-MS spectrum of compound III.

[0042] Fig.21 This is the UV spectrum of compound III.

[0043] Fig. 22 This is the IR spectrum of compound III.

[0044] Fig.23 NMR of compound III 1 H NMR spectrum.

[0045] Fig.24 NMR of compound III 13 C NMR spectrum.

[0046] Fig.25 This is the NMR HSQC spectrum of compound III.

[0047] Fig.26 This is the HMBC NMR spectrum of compound III.

[0048] Fig. 27 This is the COSY NMR spectrum of compound III.

[0049] Fig.28 This is the ROESY NMR spectrum of compound III. DETAILED DESCRIPTION

[0050] The present invention is further described below in conjunction with embodiments, but the present invention is not limited to the following embodiments. It can be foreseen that those skilled in the art may make various changes in the implementation in combination with the prior art.

[0051] The specific rotation was determined by a Japanese JASCO P-1020 fully automatic digital polarimeter; the UV spectrum was determined by a Shimadzu UV-2401PC ultraviolet spectrometer; the IR spectrum was determined by a Bruker Tensor-27 Fourier transform infrared spectrometer, and KBr was pressed into a pellet; the HR-ESI-MS was determined by an Agilent G6230 time-of-flight mass spectrometer; and the NMR was determined by a Bruker Avance III 600 nuclear magnetic resonance instrument, with TMS as the internal standard, δ representing chemical shift (in ppm), and J representing coupling constant (in Hz).

[0052] Normal phase silica gel plates for thin layer chromatography, silica gel for sample mixing (80-100 mesh) and silica gel for column chromatography (200-300 mesh) were all produced by Qingdao Ocean Chemical Plant; the reverse phase filling material RP-18 was 40-60 μm, produced by Merk; Sephadex LH-20 dextran gel was produced by GE Healthcare; MCI filling material was 75-100 μm, produced by Mitsubishi Materials; the color developer was 10% H2SO4-ethanol solution, homemade.

[0053] Example 1

[0054] (1) 11.5 kg of dried whole plant of Daphne koreana was taken, crushed and extracted with 95% ethanol cold soaking for 3 times, each time for 24 hours, and the extracts were combined to obtain 4.5 kg of total extract;

[0055] (2) extracting the extract obtained in step (1) using ethyl acetate;

[0056] (3) The ethyl acetate extraction portion obtained in step (2) was concentrated to obtain an extract (612.0 g), and the sample was mixed with silica gel (200-300 mesh) in a 1:1 ratio, and subjected to normal phase silica gel column chromatography. The mobile phases were dichloromethane-methanol with a volume ratio of 100:1, 50:1, 10:1, 8:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:5, 1:10, and 0:1, respectively, and eluted for 5 column volumes. After TLC detection, the same components were combined to obtain 14 components, Fr.1 to 14;

[0057] (4) Fr.12 (104.4 g) obtained in step (3) was subjected to Sephadex LH-20 gel column chromatography, with methanol as the eluent for 3 column volumes, at a flow rate of 1 d / s, 1 bottle / 15 min, and the same components were combined by TLC detection to obtain the components Fr.12-1 to 12-12;

[0058] (5) Fr.12-7 (6.3 g) obtained in step (4) was subjected to reverse phase ODS column chromatography, and gradient eluted with a methanol-water mixed solvent with a volume ratio of 45:55→95:5 for 5 column volumes. The same components were combined by TLC detection to obtain each component of Fr.12-7-1 to 12-7-3;

[0059] (6) Fr.12-7-3 (0.7 g) obtained in step (5) was separated by normal phase silica gel column chromatography, and a petroleum ether-acetone mixed solvent with a volume ratio of 3:1→1:1 was used as a mobile phase for gradient elution for 5 column volumes. After the sample was completely eluted, TLC was synchronously detected to obtain compound I (20.0 mg).

[0060]

[0061] (7) Fr.13 (48.2 g) obtained in step (3) was separated by normal phase silica gel chromatography, and gradient eluted with a petroleum ether-ethyl acetate mixed solvent with a volume ratio of 3:1→0:1 for 5 column volumes. The same components were combined by TLC detection to obtain Fr.13-1 to 13-8 components;

[0062] (8) Fr.13-6 (3.9 g) obtained in step (7) was subjected to reverse phase ODS column chromatography, and gradient eluted with a methanol-water mixed solvent with a volume ratio of 40:60→90:10 for 5 column volumes. The same components were detected by TLC and combined to obtain components Fr.13-6-1 to 13-6-4; (9) Fr.13-6-3 (1.7 g) obtained in step (8) was subjected to Sephadex LH-20 gel chromatography, and eluted with methanol for 3 column volumes at a flow rate of 1 d / s, 1 bottle / 15 min. The same components were detected by TLC and combined to obtain components Fr.13-6-3-1 to 13-6-3-4;

[0063] (10) Fr.13-6-3-3 (66.0 mg) obtained in step (9) was subjected to normal phase silica gel column chromatography, and gradient eluted with a petroleum ether-acetone mixed solvent with a volume ratio of 5:1→1:1 for 5 column volumes. The same components were combined by TLC detection to obtain compound II (28.5 mg).

[0064]

[0065] (11) Fr.13-6-3-4 (407.0 mg) obtained in step (9) was subjected to Sephadex LH-20 gel chromatography, with methanol as the eluent for 3 column volumes, a flow rate of 1 d / s, 1 bottle / 15 min, and TLC detection was performed to combine the same components to obtain compound III (16.8 mg).

[0066]

[0067] Structure identification:

[0068] Using nuclear magnetic resonance spectroscopy ( 1 H-NMR, 13 The structures of the compounds were identified by C-NMR, COSY, HSQC, HMBC, ROESY) and mass spectrometry (HR-ESI-MS).

[0069] (1) Compound I of the present invention is a light yellow powder, slightly soluble in methanol, and easily soluble in DMSO. (c = 0.100, MeOH). Its molecular formula was determined to be C by negative ion mode ESI high-resolution mass spectrometry. 42 H 42 O 20 (m / z865.2200[MH] - , calculated value: 865.2197), indicating that the structure has 22 unsaturations. UV(MeOH)λmax(log∈)215(3.55), 283(3.20), 324(2.86), indicating that it is a flavanol compound. Infrared spectrum at 3412, 3217cm -1 1657, 1622 cm-1 is the stretching vibration of hydroxyl group. -1 The strong absorption peak at is the stretching vibration of the unsaturated double bond. The signal attribution of all hydrogen atoms and carbon atoms and the chemical structure of the compound were determined by measuring the two-dimensional nuclear magnetic resonance spectra HSQC, COSY, HMBC, ROESY, and TOCSY. 1 H NMR and 13 C NMR data are shown in Tables 1 and 2.

[0070] Figure 1 This is the high-resolution mass spectrum of compound Ⅰ, which illustrates the molecular weight of compound Ⅰ. Figure 2 This is the UV spectrum of compound I, which provides information on the different electronic structures in the compound I molecule. Figure 3 This is the IR spectrum of compound I, which illustrates the characteristic vibration frequencies of the functional groups or chemical bonds of compound I. Figure 4 NMR of compound Ⅰ 1 H NMR spectrum illustrates the assignment of hydrogen atoms in the structure of compound Ⅰ. Figure 5 NMR of compound Ⅰ 13 C NMR spectrum illustrates the assignment of carbon atoms in the structure of compound Ⅰ. Figure 6 This is the NMR HSQC spectrum of compound Ⅰ, which illustrates the assignment of relevant carbon atoms and hydrogen atoms in the structure of compound Ⅰ. Figure 7 The HMBC NMR spectrum of compound Ⅰ illustrates the connection position of each substituent in the structure of compound Ⅰ. Figure 8This is the COSY NMR spectrum of compound Ⅰ, which illustrates the structural fragments of the relevant hydrogen atoms in the structure of compound Ⅰ. Fig. 9 This is the ROESY NMR spectrum of compound Ⅰ, which illustrates the spatial connection mode of hydrogen atoms in compound Ⅰ. Fig.10 This is the TOCSY spectrum of compound I, which illustrates the coupling relationship of all hydrogen atoms in the same spin system of sugar.

[0071] (2) Compound II of the present invention is a light yellow powder, slightly soluble in methanol, and easily soluble in DMSO. (c = 0.100, MeOH). Its molecular formula was determined to be C by negative ion mode ESI high-resolution mass spectrometry. 35 H 44 O 19 (m / z767.2414[MH] - , theoretical calculation value: 767.2404), indicating that the structure has 14 unsaturated degrees. UV (MeOH) λmax (log∈) 205 (3.67), 282 (3.21), 361 (2.53), indicating that it is a dihydroflavonoid compound. Infrared spectrum at 3423cm -1 Hydroxyl stretching vibration, 2922, 1726, 1629, 1444 cm -1 The absorption peak at indicates the presence of benzene ring. At the same time, the signal attribution of all hydrogen atoms and carbon atoms and the chemical structure of the compound were determined by measuring the two-dimensional nuclear magnetic resonance spectra HSQC, COSY, HMBC, and ROESY. 1 H NMR and 13 C NMR data are shown in Tables 1 and 2.

[0072] Fig.11 This is the high-resolution mass spectrum of compound II, which illustrates the molecular weight of compound II. Fig.12 This is the UV spectrum of compound II, which provides information on the different electronic structures in the compound II molecule. Fig.13 This is the IR spectrum of compound II, which illustrates the characteristic vibration frequency of the functional groups or chemical bonds of compound II. Fig.14 NMR of compound II 1 H NMR spectrum illustrates the assignment of hydrogen atoms in the structure of compound II. Fig.15 NMR of compound II 13 C NMR spectrum illustrates the assignment of carbon atoms in the structure of compound II. Fig.16 This is the NMR HSQC spectrum of compound II, which illustrates the assignment of relevant carbon atoms and hydrogen atoms in the structure of compound II. Fig.17 The HMBC NMR spectrum of compound II illustrates the connection positions of each substituent in the structure of compound II. Fig.18This is the COSY NMR spectrum of compound II, which illustrates the structural fragments of the relevant hydrogen atoms in the structure of compound II. Fig.19 This is the NMR ROESY spectrum of compound II, which further illustrates the connection mode of compound II.

[0073] (3) Compound III of the present invention is a yellow powder, slightly soluble in methanol, and easily soluble in DMSO. (c = 0.100, MeOH). Its molecular formula was determined to be C by negative ion mode ESI high-resolution mass spectrometry. 34 H 40 O 20 (m / z767.2046[MH] - , calculated value: 767.2040), indicating that the structure contains 15 unsaturated units. UV(MeOH)λmax(log∈)205(3.37), 267(3.18), 330(2.51), indicating that it is a flavonol compound. Infrared spectrum at 3428cm -1 is the stretching vibration of hydroxyl group, 1726cm -1 The strong absorption peak at is the stretching vibration of the unsaturated double bond. At the same time, the signal attribution of all hydrogen atoms and carbon atoms and the chemical structure of the compound were determined by measuring the two-dimensional nuclear magnetic resonance spectra HSQC, COSY, HMBC, and ROESY. 1 H NMR and 13 C NMR data are shown in Tables 1 and 2.

[0074] Fig. 20 This is the high-resolution mass spectrum of compound III, which illustrates the molecular weight of compound III. Fig.21 This is the UV spectrum of compound III, which provides information on the different electronic structures in the compound III molecule. Fig. 22 This is the IR spectrum of compound III, which illustrates the characteristic vibration frequency of the functional groups or chemical bonds of compound III. Fig.23 NMR of compound III 1 H NMR spectrum illustrates the assignment of hydrogen atoms in the structure of compound III. Fig.24 NMR of compound III 13 C NMR spectrum illustrates the assignment of carbon atoms in the structure of compound III. Fig.25 This is the NMR HSQC spectrum of compound III, which illustrates the assignment of relevant carbon atoms and hydrogen atoms in the structure of compound III. Fig.26 This is the HMBC NMR spectrum of compound III, which illustrates the connection positions of each substituent in the structure of compound III. Fig. 27 This is the COSY NMR spectrum of compound III, which illustrates the structural fragments of the relevant hydrogen atoms in the structure of compound III. Fig.28This is the NMR ROESY spectrum of compound III, which further illustrates the connection mode of compound III.

[0075] Table 1 Compounds I, II, and III 1 H NMR data (DMSO-d6)

[0076]

[0077]

[0078] Note: δ in ppm, J in Hz. 1 H-NMR:600MHz

[0079] Table 2 Compounds I, II, and III 13 C NMR data (DMSO-d6)

[0080]

[0081] Note: δ in ppm, J in Hz. 1 H-NMR: 600 MHz.

[0082] Example 2

[0083] The tumor cell inhibition activities of the compounds I to III of the present invention were investigated, and the cytotoxic effects were evaluated on three human cancer cell lines, namely A549 (lung cancer), MDA-MB-231 (breast cancer) and SW480 (colon cancer).

[0084] 1. Experimental instruments

[0085] Biological safety cabinet (Model 1287, Thermo Corporation), -80℃ ultra-low temperature refrigerator (DW-86L829BP, Haier Biomedicine Co., Ltd.), desktop centrifuge (Sorvall ST16R, Thermo Corporation), fluorescence inverted microscope (Model IX50, Olympus China Company), multifunctional microplate reader (Multiskan-FC, Thermo Corporation), water bath (DK-600, Shanghai Feiyue Experimental Instrument), carbon dioxide incubator (CR-3018, Thermo Corporation), and high pressure sterilizer (LDZM-80KCS-II, Shanghai Shen'an Medical Equipment Co., Ltd.).

[0086] 2. Experimental Materials

[0087] 10% fetal bovine serum (500 mL, Vivacell), penicillin-streptomycin (100 mL, Suzhou Xinsaimei Biotechnology Co., Ltd.), RPMI1640 liquid culture (500 mL, White Shark Biotechnology Co., Ltd.), PBS phosphate buffer (500 mL, White Shark Biotechnology Co., Ltd.), trypsin-EDTA 0.25% trypsin (100 mL, Yingjie Biotechnology Co., Ltd.), MTS cell proliferation and toxicity detection kit (Yingjie Biotechnology Co., Ltd.), dimethyl sulfoxide (DMSO, Solebao Biotechnology Co., Ltd.), cisplatin (DDP, purity ≥ 98%, Beijing Zhongke Quality Inspection Biotechnology Co., Ltd.), paclitaxel (Taxol, purity ≥ 98%, Beijing Zhongke Quality Inspection Biotechnology Co., Ltd.), A549 human lung adenocarcinoma cell line, MDA-MB-231 breast cancer cell line, SW480 colon cancer cell line (provided by Beijing Zhongyuan Heju Biotechnology Co., Ltd.), DMSO (analytical grade, Solebao Biotechnology Co., Ltd.).

[0088] 3. Experimental methods

[0089] MTS reduction method

[0090] 3.1 Experimental principle of MTS method

[0091] MTS is a new MTT analogue, the full name is 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxy phenyl)-2-(4-sulfopheny)-2H-tetrazolium, a yellow dye. Succinate dehydrogenase in the mitochondria of living cells can metabolize and reduce MTS to generate soluble formazan compounds, and the content of formazan can be measured at 490nm using a microplate reader. Under normal circumstances, the amount of formazan generated is proportional to the number of living cells, so the number of living cells can be inferred based on the optical density OD value.

[0092] 3.2 Operation steps

[0093] 3.2.1 Cell inoculation

[0094] A single cell suspension was prepared with culture medium (DMEM or RMPI1640) containing 10% fetal bovine serum, and 3000 to 5000 cells were inoculated into a 96-well plate with a volume of 100 μL per well. The cells were inoculated and cultured 12 to 24 hours in advance.

[0095] 3.2.2 Add the test compound solution

[0096] The compounds were dissolved in DMSO and initially screened at a concentration of 40 μM. The final volume of each well was 200 μL, and three replicate wells were set for each treatment.

[0097] 3.2.3 Color Rendering

[0098] After culturing at 37°C for 48 hours, discard the culture medium in the wells of adherent cells, add 20 μL of MTS solution and 100 μL of culture medium to each well; set up 3 blank replicate wells (a mixture of 20 μL MTS solution and 100 μL culture medium), continue incubation for 2 to 4 hours, and measure the light absorbance value after the reaction is fully carried out.

[0099] 3.2.4 Colorimetry

[0100] Select 492nm wavelength, read the light absorption value of each well with a multifunctional microplate reader, and record the results. Cisplatin (DDP) is set as the positive compound in each experiment, and the IC value of the compound is calculated using the two-point method (Reed and Muench method). 50 value.

[0101]

[0102] 3.3 Statistical analysis

[0103] GraphPad Prism 7.0 and Image J software were used for data analysis and processing, and the measurement results were expressed as mean ± standard deviation (X ± S). One-way ANOVA was used for comparison among multiple groups, and P < 0.05 was considered statistically significant.

[0104] 4. Experimental Results

[0105] As shown in Table 3, compound I showed strong cytotoxic activity against A549 and MDA-MB-231 cells (IC 50 The values ​​were 10.81 μM and 11.07 μM, respectively). Therefore, the flavonoid glycoside compounds with anti-tumor activity in Daphne koreana of the present invention have the prospect of further developing anti-cancer drugs.

[0106] Table 3 Cytotoxic activity IC 50 (μM)

[0107]

Claims

1. A flavonoid bioside compound, characterized in that It has the following structure:

2. Use of the flavonoid bioside compound according to claim 1 in the preparation of a drug for preventing or treating cancer.

3. The use according to claim 2, characterized in that The cancers include lung cancer, breast cancer, and colon cancer.

4. The method for preparing flavonoid bioside compounds according to claim 1, characterized in that The steps include: (1) taking the whole dried plant of Daphne koreana, crushing it, and extracting it with alcohol or an alcohol aqueous solution; (2) extracting the extract obtained in step (1) with ethyl acetate, and taking the ethyl acetate phase; (3) The ethyl acetate extract obtained in step (2) was concentrated to obtain an extract, and the sample was mixed with silica gel 1:1, g / g, and eluted with dichloromethane-methanol mobile phases with volume ratios of 100:1, 50:1, 10:1, 8:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:5, 1:10, and 0:1 for 3 to 6 column volumes respectively. The same components were combined by TLC detection to obtain Fr.1 to 14 components; (4) subjecting Fr.12 obtained in step (3) to Sephadex LH-20 gel column chromatography, eluting with methanol for 2 to 4 column volumes, and combining the same components for TLC detection to obtain Fr.12-1 to 12-12 components; (5) Fr.12-7 obtained in step (4) was subjected to reverse phase ODS column chromatography, and gradient eluted with a methanol-water mixed solution with a volume ratio of 45:55→95:5 for 3 to 6 column volumes. The same components were combined by TLC detection to obtain Fr.12-7-1 to 12-7-3 components; (6) Fr.12-7-3 obtained in step (5) was subjected to normal phase silica gel chromatography, with a petroleum ether-acetone mixed solvent of a volume ratio of 3:1→1:1 as the mobile phase for gradient elution for 3 to 6 column volumes to obtain compound I; (7) Fr.13 obtained in step (3) was separated by normal phase silica gel column chromatography, and gradient eluted with a mixed solution of petroleum ether and ethyl acetate in a volume ratio of 3:1→0:1 for 3 to 6 column volumes to obtain Fr.13-1 to 13-8 components; (8) Fr.13-6 obtained in step (7) was subjected to reverse phase ODS column chromatography and gradient eluted with a methanol-water mixed solution with a volume ratio of 40:60→90:10 for 3 to 6 column volumes to obtain Fr.13-6-1 to 13-6-4 components; (9) Fr.13-6-3 obtained in step (8) was subjected to Sephadex LH-20 gel chromatography and eluted with methanol for 2 to 4 column volumes to obtain Fr.13-6-3-1 to 13-6-3-4 components; (10) Fr.13-6-3-3 obtained in step (9) was subjected to normal phase silica gel column chromatography, and gradient eluted with a petroleum ether-acetone mixed solution with a volume ratio of 5:1→1:1 for 3 to 6 column volumes to obtain compound II; (11) Fr.13-6-3-4 obtained in step (9) was subjected to Sephadex LH-20 gel chromatography using methanol as the eluent for 2 to 4 column volumes to obtain compound III.

5. The preparation method according to claim 4, characterized in that: Step (1) using 95% ethanol solution for cold extraction 3 times, each time for 24 hours; Step (3) each gradient elution was 5 column volumes; Step (4) elution for 3 column volumes; Step (5) each gradient elution is 5 column volumes; Step (6) each gradient elution is 5 column volumes; Step (7) each gradient elution is 5 column volumes; Step (8) each gradient elution is 5 column volumes; Step (9) elution for 3 column volumes; Step (10) each gradient elution is 5 column volumes; Step (11) was eluted for 3 column volumes.

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