A new preparation method of flavonoids and its application

Abstract

The present invention provides a new flavonoid compound and its preparation method and application, belonging to the field of medical technology. The flavonoid compound is isolated from the Calophyllum membranaceum Gardn.et Champ. of the Guttiferae family. The dried stems and leaves of Calophyllum membranaceum are taken, and a new flavonoid compound is obtained by extract extraction, MCI segmentation decolorization, silica gel column chromatography, open ODS column chromatography, Sephadex LH-20 gel column chromatography, and semi-preparative HPLC chromatography: the preparation method of the present invention is simple to operate and has a high degree of purification. The flavonoid compound of the present invention can target and inhibit CYP1A1 enzyme activity, can be used as a lead compound for developing drugs that inhibit CYP1A1 enzyme activity, is expected to prevent or treat cancers caused by pro-carcinogens / mutagens such as polycyclic aromatic hydrocarbons and heterocyclic aromatic amines, and has potential clinical application value in the development of drugs such as anti-lung cancer, anti-breast cancer, and anti-colon cancer.

Description

Technical Field

[0001] The invention belongs to the technical field of medicine, and particularly relates to a new flavonoid compound separated from the dried stems and leaves of the genus Echinops serrata, and a preparation method and application thereof. Background Art

[0002] CYP1A1 is an aromatic hydrocarbon hydroxylase that belongs to the cytochrome P450 1 family of enzymes and is widely present in extrahepatic tissues such as the placenta, lungs, lymph nodes, gastrointestinal tract, and skin. CYP1A1 not only participates in the metabolic activation of procarcinogens or mutagens such as polycyclic aromatic hydrocarbons and heterocyclic aromatic amines, but also CYP1A1 gene polymorphisms are closely associated with oral cancer, lung cancer, breast cancer, liver cancer, and colon cancer. For example, benzo[a]pyrene (B[a]P) is a Class 1 human carcinogen designated by the International Agency for Research on Cancer, and CYP1A1 plays a key role in its metabolic activation. First, B[a]P is oxidized by CYP1A1 to B[a]P-7,8-epoxide, which is then converted by epoxide hydrolase to (+ / -)-B[a]P-trans-7,8-dihydrodiol (BPD). BPD is then metabolically activated by CYP1A1 and ultimately converted to the ultimate carcinogen, B[a]P-trans-7,8-dihydrodiol-9,10-epoxide (BPDE). BPDE can covalently bind to DNA, proteins, and other molecules to form adducts, causing damage to biomacromolecules and potentially leading to cell carcinogenesis. Therefore, identifying inhibitors targeting CYP1A1 is a viable approach for discovering new anti-tumor drugs and holds great promise for clinical application. Studies have shown that natural products from medicinal plants, with their diverse chemical structures and high biocompatibility, are an important source for the discovery of CYP1A1 inhibitors, with flavonoids being a major type.

[0003] Calophyllum membranaceum (Gardn. et Champ.), also known as Dieda General, Thin-leaved Hutong, and Horizontal Jingxi, is a shrub to small tree in the genus Calophyllum of the Clusiaceae family. Its roots are used in folk medicine to treat injuries from falls, kidney deficiency, low back pain, rheumatic bone pain, dispelling blood stasis and relieving pain, and strengthening the kidneys and waist. Its leaves are also used to treat bleeding from trauma. Modern research shows that Calophyllum membranaceum contains a variety of compounds, including flavonoids, coumarins, and terpenes. Summary of the Invention

[0004] The first purpose of the present invention is to provide a novel method for preparing flavonoid compounds, and the second purpose is to provide the use of the flavonoid compounds in the preparation of CYP1A1 enzyme inhibitors.

[0005] The present invention isolates a new flavonoid compound from the dried stems and leaves of the genus Melanocarpa, which has the following structure:

[0006]

[0007] One object of the present invention is to provide a method for preparing the novel flavonoid compound, using the dried stems and leaves of Calophyllum membranaceum Gardn. et Champ., a plant of the genus Calophyllum in the family Guttiferae, as raw materials. The method comprises extract extraction, MCI segmentation decolorization, silica gel column chromatography, open ODS column chromatography, Sephadex LH-20 gel column chromatography, and semi-preparative HPLC chromatography. The method comprises the following steps:

[0008] (1) taking the dried stems and leaves of Boyehonghouke, crushing them, soaking and extracting them in alcohol or an alcohol-water solution, preferably using a 95% by volume ethanol-water solution at room temperature for 3 times, each time for 24 hours, and concentrating to obtain an extract;

[0009] (2) The crude extract obtained in step (1) was fully dissolved in water, filtered, and then eluted with an MCI gel CHP 20P column chromatography, first using a 70% by volume methanol-water solution to remove impurities, and then eluted with a 95% by volume methanol-water solution for 4 to 6 column volumes, and the 95% methanol-water solution elution fraction was collected and concentrated to obtain the extract Fr F;

[0010] (3) The extract Fr F obtained in step (2) was subjected to silica gel column chromatography, and gradient elution was performed using a petroleum ether-ethyl acetate mixed solvent with a volume ratio of 200:1, 100:1, 50:1, 30:1, 15:1, 8:1, 4:1, 2:1, 1:1, 1:2, 1:4, 1:8, 1:15, and 0:1, with each gradient elution lasting 4 to 6 column volumes, to obtain 18 component segments, Fr 1 to 18;

[0011] (4) Fr.12 obtained in step (3) was separated by open ODS column chromatography, using a methanol-water mixed solvent gradient elution with a volume ratio of 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10, and 100:0, with each gradient elution lasting 4 to 6 column volumes, to obtain 14 component segments, Fr.12-1 to 12-14;

[0012] (5) Fr.12-8 obtained in step (4) was subjected to Sephadex LH-20 gel column chromatography using a dichloromethane-methanol mixed solvent with a volume ratio of 3:1 for 2 column volumes to obtain 9 component segments, Fr.12-8-1 to 12-8-9;

[0013] (6) The Fr.12-8-5 component obtained in step (5) was subjected to semi-preparative HPLC chromatography using a 75% by volume acetonitrile-water mixture as the mobile phase, preferably using a Zorbax SB-C 18 (Agilent, 9.4 mm × 250 mm), with a flow rate of 3 mL / min and a detection wavelength of 254 nm, the chromatographic peak with a retention time of 20.1 min was collected to obtain compound I;

[0014] Another object of the present invention is to provide the use of the flavonoid compound in the preparation of a CYP1A1 enzyme inhibitor. The present invention measured the inhibitory activity of the compound against CYP1A1, CYP1A2, and CYP1B1 enzymes through an in vitro CYP1 enzyme activity inhibition assay. The results showed that the inhibition rate of compound I of the present invention against CYP1A1 enzyme was 31.31±3.26%, while its inhibitory effect on CYP1A2 and CYP1B1 enzymes was not statistically significant. Therefore, the flavonoid compound I of the present invention can target and inhibit CYP1A1 enzyme and can be used as a lead compound for the development of CYP1A1 enzyme inhibitory drugs. It can also be used to prepare drugs for preventing or treating DNA damage and cancer caused by the metabolic activation of procarcinogens / mutagens by CYP1A1 enzymes. The procarcinogens / mutagens include polycyclic aromatic hydrocarbons and heterocyclic aromatic amines, such as benzo[a]pyrene, 7H-methyldibenzocarbazole, and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine. The cancers include lung cancer, breast cancer, and colon cancer.

[0015] The present invention has the following advantages:

[0016] (1) The present invention uses the dried stems and leaves of the plant of the genus Glechoma of the family Garcinia family as raw materials, and obtains a new flavonoid compound through extract extraction, MCI segmentation decolorization, silica gel column chromatography, open ODS column chromatography, Sephadex LH-20 gel column chromatography, and semi-preparative HPLC chromatography. The preparation method is simple to operate, and the purity of the obtained flavonoid compound reaches more than 98%, providing a scientific reference basis for the preparation of flavonoid compounds.

[0017] (2) The flavonoid compound I described in the present invention can target and inhibit the activity of CYP1A1 enzyme, and can be used as a lead compound for developing drugs that inhibit the activity of CYP1A1 enzyme. It is expected to prevent or treat DNA damage and cancer caused by pro-carcinogens / mutagants such as polycyclic aromatic hydrocarbons and heterocyclic aromatic amines, and has potential good application prospects in the development of drugs for lung cancer, breast cancer, colon cancer, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the HR-ESI-MS spectrum of compound Ⅰ.

[0019] Figure 2 The NMR of compound Ⅰ 1 H-NMR spectrum.

[0020] Figure 3 The NMR of compound Ⅰ 13 C-NMR spectrum.

[0021] Figure 4 The NMR of compound Ⅰ 1 H- 1 H COSY spectrum

[0022] Figure 5 This is the NMR HSQC spectrum of compound Ⅰ.

[0023] Figure 6 This is the HMBC NMR spectrum of compound Ⅰ.

[0024] Figure 7 This is the NMR NOESY spectrum of compound Ⅰ.

[0025] Figure 8 These are the results of the inhibition experiment of compound I on CYP1A1, CYP1A2 and CYP1B1 enzyme activities. DETAILED DESCRIPTION

[0026] The present invention is further described below with reference to the following embodiments. However, the present invention is not limited to the following embodiments. It is foreseeable that various variations may occur in the implementation by those skilled in the art in combination with the prior art.

[0027] NMR spectra were measured using a Bruker AM-4Avance III 600 nuclear magnetic resonance spectrometer with TMS as the internal standard, δ represents the chemical shift (in ppm), and J represents the coupling constant (in Hz); UV spectra were measured using a Shimadzu UV-2700 ultraviolet-visible spectrophotometer; IR spectra were measured using a Nicolet iS10 Fourier transform mid-infrared spectrometer; specific rotation was measured using an AutopolVI polarimeter; and HR-ESI-MS was measured using an Agilent G6230 time-of-flight mass spectrometer.

[0028] The MCI packing material was MCI-gel CHP-20P; the reverse phase packing material was RP-18 (40-60 μm); the normal phase silica gel plates used for thin layer chromatography, the sample mixing silica gel (200-300 mesh) used for normal silica gel column chromatography, and the column chromatography silica gel (200-300 mesh) used were all produced by Qingdao Ocean Chemical Plant; the HPLC analyzer was a preparative 2000 high performance liquid chromatograph (Shanghai Kezhe Company), and the chromatographic column was Zorbax SB-C 18The reverse phase chromatography column was (Agilent, 9.4 mm×250 mm, 5 μm); the gel was Sephadex LH-20 (GE Healthcare); and the color developer was 10% H 2 SO 4 -ethanol solution.

[0029] Example 1

[0030] (1) 15.0 kg of dried stems and leaves of Boyehonghouke were ground and then soaked in 95% ethanol at room temperature for 3 times, each time for 24 hours. The extracts were combined and the ethanol was removed by vacuum distillation to obtain 1.2 kg of crude extract;

[0031] (2) The crude extract obtained in step (1) was fully dissolved in water, filtered, and separated by MCI gel CHP 20P column chromatography, and impurities were removed by elution with a 70% by volume methanol-water solution, followed by elution with a 95% by volume methanol-water solution for 4 to 6 column volumes, and the 95% methanol-water solution elution fraction was collected and concentrated to obtain the extract Fr F;

[0032] (3) The extract Fr.F obtained in step (2) was mixed with 200-300 mesh silica gel and subjected to normal silica gel column chromatography using petroleum ether:ethyl acetate (v:v, 200:1, 100:1, 50:1, 30:1, 15:1, 8:1, 4:1, 2:1, 1:1, 1:2, 1:4, 1:8, 1:15, 0:1) as the mobile phase for gradient elution, with each gradient elution lasting 4-6 column volumes. After TLC monitoring, the same components were combined to obtain 18 component segments, Fr.1-18;

[0033] (4) Fr.12 obtained in step (3) was separated by open ODS column chromatography, and gradient elution was performed using a methanol-water mixed solvent (v:v, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10, 100:0) as the mobile phase, with each gradient elution lasting 4 to 6 column volumes, monitored by TLC, and the same fractions were combined to obtain 14 fractions, Fr.12-1 to 12-14;

[0034] (5) Fr.12-8 obtained in step (4) was subjected to Sephadex LH-20 gel column chromatography using a dichloromethane-methanol mixed solvent (v:v, 3:1) as the eluent, and isocratically eluted for 2 column volumes. After TLC monitoring, the same components were combined to obtain 9 component segments, Fr.12-8-1 to 12-8-9;

[0035] (6) The Fr.12-8-5 component obtained in step (5) was chromatographed by semi-preparative HPLC using a Zorbax SB-C column. 18(Agilent, 9.4 mm × 250 mm), eluted with a 75% by volume acetonitrile-water mixed solution as the mobile phase at a flow rate of 3 mL / min, and the detection wavelength of the UV detector was 254 nm. The chromatographic peak with a retention time of 20.1 min was collected to obtain compound I; Compound I:

[0036] The compound I of the present invention is a yellow oil and is easily soluble in acetone and methanol; (c = 0.170, MeOH); HR-ESI-MS gives the quasi-molecular ion peak m / z: 475.1765 [MH] - (calcd.for C 28 H 27 O7 475.1762), its molecular formula is inferred to be C 28 H 28 O7, unsaturation is 15. NMR 1 H-NMR spectrum and 13 The C-NMR spectrum shows the chemical shifts of hydrogen and carbon atoms in the compound. 1 H- 1 H COSY, HSQC, and HMBC were used to determine the signal assignments of all hydrogen and carbon atoms and the chemical structure of the compound. 1 H-NMR and 13 C-NMR data are shown in Table 1.

[0037] Figure 1 This is the high-resolution mass spectrum of compound I, which illustrates the molecular weight of compound I. Figure 2 The NMR of compound Ⅰ 1 H-NMR spectrum showing the chemical shifts of hydrogen atoms in the structure of compound Ⅰ. Figure 3 The NMR of compound Ⅰ 13 C-NMR spectrum showing the chemical shifts of carbon atoms in the structure of compound Ⅰ. Figure 4 The NMR of compound Ⅰ 1 H- 1 H COSY spectrum, illustrating the connection pattern of relevant hydrogen atoms in the structure of compound Ⅰ. Figure 5 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 6 The HMBC NMR spectrum of compound I illustrates the connection positions of the substituents in the structure of compound I. Figure 7 This is the nuclear magnetic resonance NOESY spectrum of compound I, which further illustrates the connection mode of compound I.

[0038] Table 1 Compound Ⅰ 1H-NMR (600 MHz) and 13 C-NMR (150 MHz) NMR spectrum data (Acetone-d6)

[0039]

[0040] Note: δ in ppm; J in Hz

[0041] Example 2 Inhibition test of CYP1 enzyme activity of compound I of the present invention

[0042] 1. Experimental Materials

[0043] Nicotinamide adenine dinucleotide phosphate (NADPH), granisetron, phenacetin, α-naphthoflavone, β-estradiol, resveratrol, mouse liver microsomes (MLM), acetonitrile, and buffer (PBS).

[0044] 2. Experimental Methods

[0045] 2.1 Experimental reaction system

[0046] CYP1A1 reaction system: Contains MLM (20 mg / mL), NADPH (10 mM), granisetron (200 μM), α-naphthoflavone (1 mM) or compound (10 μM), and buffer (PBS, pH 7.4). After incubation, NADPH was added to allow the reaction to proceed. The reaction was terminated by centrifugation, and the supernatant was collected for testing. The incubation system was performed in triplicate. The incubation system was performed in triplicate. ① Positive control group: mouse liver microsomes with granisetron and α-naphthoflavone. ② Negative control group: mouse liver microsomes with granisetron, but without NADPH, with an equal volume of PBS. ③ Experimental group: mouse liver microsomes with granisetron and the test compound. ④ Blank group: mouse liver microsomes with granisetron alone.

[0047] CYP1A2 reaction system: The reaction system contains MLM (20 mg / mL), NADPH (10 mM), phenacetin (2 mM), α-naphthoflavone (100 μM) or the compound (10 μM), and buffer (PBS, pH 7.4). After incubation, the reaction system is incubated with NADPH. The reaction is terminated by centrifugation, and the supernatant is collected for analysis. The incubation system is performed in triplicate. ① Positive control: mouse liver microsomes in the presence of α-naphthoflavone and phenacetin. ② Negative control: mouse liver microsomes in the presence of an equal volume of PBS, but without NADPH. ③ Experimental group: mouse liver microsomes in the presence of phenacetin and the test compound. ④ Blank group: mouse liver microsomes in the presence of phenacetin alone.

[0048] CYP1B1 reaction system: Experimental reaction system: The reaction system contains β-estradiol (2mM), NADPH (10mM), resveratrol (1mM) or the test compound (1mM), MLM (20mg / mL), and buffer (PBS, pH=7.4). After incubation, NADPH is added to allow the reaction to proceed. After termination, the reaction is centrifuged and the supernatant is collected for analysis. The incubation system is performed in triplicate. ① Positive control: mouse liver microsomes with both resveratrol and estradiol. ② Negative control: mouse liver microsomes with β-estradiol but without NADPH, replaced with an equal volume of PBS. ③ Experimental group: mouse liver microsomes with both β-estradiol and the test compound. ④ Blank group: mouse liver microsomes with β-estradiol alone.

[0049] 2.2 UPLC-ESI-QTOFMS analysis

[0050] All microsomal samples were analyzed on an Agilent 1290 Series UPLC system equipped with a 1290 Quaternary Pump (Agilent, Santa Clara, CA). Drug metabolites were detected using an XDB-C18 column (2.1 × 100 mm, 1.8 mm, Agilent, Santa Clara, CA). The flow rate was 0.3 mL / min. Phase A consisted of 0.01% formic acid in water, and phase B consisted of acetonitrile containing 0.01% formic acid. The elution gradient was as follows: 2-98% B (0-12 min); 98% B (12-14 min); and 98% A (14-16 min). The column temperature was 45°C. Data were acquired in positive ion mode. The collision and drying gas flows were 9 L / min. The capillary voltage was 3.5 kV, the temperature was 350°C, and the nebulizer pressure was 35 psi. The target ions scanned were 273.1849 and 289.1798.

[0051] 2.3 Multivariate data analysis and statistical analysis

[0052] Multivariate Data Analysis and Statistical Analysis: Chromatographic and spectral data were analyzed using Mass Hunter Workstation data acquisition software (Agilent, Santa Clara, CA, USA). All values ​​were expressed as means, and statistical analysis was performed using Prism v.6. The enzyme activity inhibition rate of the target compound was calculated as follows: CYP1 enzyme activity inhibition rate = (positive control group or experimental group - blank group) / (negative control group - blank group) × 100%

[0053] 3. Experimental Results

[0054] The flavonoid compound I was subjected to CYP1A1, CYP1A2 and CYP1B1 enzyme activity inhibition test, and the results are shown in Table 2 and Figure 8As shown, the results showed that compound I had a good inhibitory effect on CYP1A1 enzyme, with an inhibition rate of 31.31±3.26%, while its inhibitory effect on CYP1A2 and CYP1B1 enzymes was not statistically significant. Therefore, flavonoid compound I can target and inhibit CYP1A1 enzyme activity.

[0055] Table 2 Compound I inhibits CYP1A1, CYP1A2, and CYP1B1 enzyme activities

[0056]

Claims

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

2. The method for preparing flavonoids according to claim 1, wherein The steps include: (1) Dry the stems and leaves of Boyehonghouke, crush them, extract them with alcohol or an alcohol aqueous solution, and concentrate them; (2) The crude extract obtained in step (1) was fully dissolved in water, filtered, and then separated using MCI gel CHP 20P column chromatography, first eluted with 70% by volume methanol-water solution to remove impurities, then eluted with 95% by volume methanol-water solution for 4 to 6 column volumes, and the 95% methanol-water solution elution fraction was collected and concentrated to obtain the extract Fr F; (3) The extract Fr F obtained in step (2) was subjected to silica gel column chromatography, using a petroleum ether-ethyl acetate mixed solvent gradient elution, with each gradient elution lasting 4 to 6 column volumes, to obtain 18 component segments, Fr 1 to 18; (4) Fr12 obtained in step (3) was separated by open ODS column chromatography using a methanol-water mixed solvent gradient elution with 4 to 6 column volumes per gradient elution to obtain 14 component segments, Fr.12-1 to Fr.12-14; (5) Fr.12-8 obtained in step (4) was subjected to Sephadex LH-20 gel column chromatography using a dichloromethane-methanol mixed solvent for isocratic elution for 2 column volumes to obtain 9 component segments, Fr.12-8-1 to Fr.12-8-9; (6) The Fr.12-8-5 component obtained in step (5) was subjected to semi-preparative HPLC chromatography, eluted with a 75% by volume acetonitrile-water mixed solution as the mobile phase, at a flow rate of 3 mL / min, a detection wavelength of 254 nm, and the chromatographic peak with a retention time of 20.1 min was collected to obtain the flavonoid compound according to claim 1.

3. The method for preparing flavonoids according to claim 2, wherein: Step (1) is to extract with 95% by volume ethanol aqueous solution; Step (3) is performed using a petroleum ether-ethyl acetate mixed solvent gradient elution using a petroleum ether-ethyl acetate mixed solvent with a volume ratio of 200:1, 100:1, 50:1, 30:1, 15:1, 8:1, 4:1, 2:1, 1:1, 1:2, 1:4, 1:8, 1:15, and 0:1 for 4 to 6 column volumes respectively; Step (4) uses a methanol-water mixed solvent gradient elution to use a methanol-water mixed solvent with a volume ratio of 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10, and 100:0 for 4 to 6 column volumes respectively; Step (5) uses a dichloromethane-methanol mixed solvent with a volume ratio of 3:1; The chromatographic column used in step (6) is Zorbax SB-C 18 , 9.4mm×250mm.

4. Use of the flavonoid compound according to claim 1 in the preparation of a CYP1A1 enzyme inhibitor.

5. The use according to claim 4, characterized in that The CYP1A1 enzyme inhibitor is a drug for preventing or treating cancer caused by procarcinogens or mutagens.

6. The use according to claim 5, characterized in that The pro-carcinogens or mutagens include polycyclic aromatic hydrocarbons and heterocyclic aromatic amines.

7. The use according to claim 5, characterized in that The cancer includes lung cancer, breast cancer or colon cancer.

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