Preparation method and use of a furoquinoline alkaloid

Abstract

The present invention relates to a preparation method and use of furoquinoline alkaloids. The method uses the dried medicinal material of the whole herb of Rutaceae graveolens as the raw material, extracts with an organic solvent, and separates by normal-phase silica gel column chromatography, reverse-phase silica gel column chromatography, macroporous resin column chromatography, and Sephadex LH-20 column chromatography of hydroxypropyl dextran gel. Two or three of the preparative high-performance liquid chromatography methods are used for separation, and the furoquinoline alkaloids obtained are 4,8-dimethoxy-7-((3-methylbut-2-en-1-yl)oxy)furo[2,3-b]quinoline and ( R )-1-((4,8-dimethoxyfuro[2,3-b]quinolin-7-yl)oxy)-3-ethoxy-3-methylbutan-2-ol. The furoquinoline alkaloids obtained by the said method have a certain inhibitory effect on the proliferation of human colon cancer cells (HT-29), and have potential value for the development of drugs for treating colon cancer.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to a method for preparing furanoquinoline alkaloid compounds from the medicinal plant Rutagraveolens and the application of the compounds as anti-colorectal cancer drugs or lead compounds. Background Art

[0002] Colorectal cancer is a cancer that begins in the large intestine (colon), occurring in the digestive tract of the colon region. Colorectal cancer is a disease in which the growth of colon or rectal cells gets out of control, and it can develop circumferentially along the intestinal wall, spread longitudinally along the intestinal tract or infiltrate deep into the intestinal wall. In addition to lymphatic, blood stream metastasis and local invasion, it can also metastasize by intraperitoneal implantation or spread along the suture line and incision surface. Colorectal cancer can be effectively treated by surgical resection in the early stage. However, for patients in the middle and late stages, cancer cells are prone to metastasis, and the mortality rate is still relatively high in chemotherapy treatment. Therefore, developing safe, stable and effective anti-colorectal cancer drugs has great clinical significance.

[0003] Rutagraveolens is a perennial rhizomatous herb of the genus Rutagraveola in the family Rutaceae, distributed in the northern regions of Xinjiang, China and the central and western regions of Asia. Plants of the genus Rutagraveola including Rutagraveolens are widely used in folk medicine for treating burns, toothaches, oral ulcers, stomachaches, respiratory diseases and skin diseases. The use of furanoquinoline alkaloid compounds isolated from Rutagraveolens involved in the present invention for combating colorectal cancer has not been reported in patents and literature so far. Summary of the Invention

[0004] The object of the present invention is to provide a method for preparing and using furanoquinoline alkaloids. The method uses the dried medicinal material of the whole herb of Rutagraveolens as the raw material, extracts with an organic solvent, and separates by normal-phase silica gel column chromatography, reversed-phase silica gel column chromatography, macroporous resin column chromatography, Sephadex LH-20 column chromatography of hydroxypropyl dextran gel, and two or three of preparative high performance liquid chromatography, thus obtaining the furanoquinoline alkaloids 4,8-dimethoxy-7-((3-methylbut-2-en-1-yl)oxy)furo[2,3-b]quinoline and (R)-1-((4,8-dimethoxyfuro[2,3-b]quinolin-7-yl)oxy)-3-ethoxy-3-methylbutan-2-ol. The furanoquinoline alkaloids obtained by the method have a certain inhibitory effect on the proliferation of human colon cancer cells (HT-29), and have potential value for the development of anti-colorectal cancer drugs.

[0005] A method for preparing furanoquinoline alkaloids according to the present invention uses the dried medicinal material of the whole herb of Rutagraveolens as the raw material and extracts with an organic solvent. The specific operation is carried out according to the following steps:

[0006] a. Take the dried whole herb of Rutaceae graveolens, crush it, and then perform percolation, cold maceration extraction or heating reflux extraction at room temperature with ethanol with a volume concentration of 50 - 99%, absolute ethanol, acetone, methanol aqueous solution, or absolute methanol. Combine the extraction solutions, concentrate under reduced pressure, and dry to obtain the crude extract;

[0007] b. Disperse the crude extract obtained in step a in water, and extract it 3 - 6 times successively with petroleum ether or n - hexane, dichloromethane or chloroform, ethyl acetate, and n - butanol respectively, or perform silica gel column chromatography, and elute it with petroleum ether, ethyl acetate, dichloromethane or chloroform, and n - butanol for 3 - 6 times the column volume respectively to obtain petroleum ether extract, ethyl acetate extract, dichloromethane or chloroform extract, and n - butanol extract;

[0008] c. Subject the ethyl acetate extract and the dichloromethane or chloroform extract obtained in step b to normal - phase silica gel column chromatography, reverse - phase silica gel column chromatography, macroporous resin column chromatography, Sephadex LH - 20 column chromatography, and two or three of preparative high - performance liquid chromatography for separation. Among them, the normal - phase silica gel column chromatography method used is atmospheric or pressurized column chromatography, the filler used is normal - phase silica gel, and the eluent used is a mixture of at least two solvents selected from petroleum ether, n - hexane, acetone, chloroform, dichloromethane, ethyl acetate, and methanol, and isocratic elution or gradient elution is adopted; the reverse - phase silica gel column chromatography method used is atmospheric or pressurized column chromatography, the eluent is methanol aqueous solution with a volume concentration of 10 - 90% or acetonitrile aqueous solution with a volume concentration of 10 - 90%, and isocratic elution or gradient elution is adopted; the Sephadex LH - 20 column chromatography method used is atmospheric column chromatography, the eluent is methanol or methanol aqueous solution with a volume concentration of 50% - 100%, and isocratic elution or gradient elution is adopted; the macroporous resin chromatography method used is atmospheric or pressurized column chromatography, the eluent is methanol aqueous solution with a volume concentration of 30 - 99% or acetonitrile aqueous solution with a volume concentration of 20 - 99%, and isocratic elution or gradient elution is adopted; the preparative high - performance liquid chromatography method used is pressurized column chromatography, the eluent is methanol aqueous solution with a volume concentration of 40 - 99% or acetonitrile aqueous solution with a volume concentration of 30 - 99%, and isocratic elution or gradient elution is adopted, and then the furoquinoline alkaloid 4,8 - dimethoxy - 7 - ((3 - methylbut - 2 - en - 1 - yl)oxy)furo[2,3 - b]quinoline and the compound (R)-1 - ((4,8 - dimethoxyfuro[2,3 - b]quinoline - 7 - yl)oxy)-3 - ethoxy - 3 - methylbutan - 2 - ol are obtained.

[0009] In step c, the two separations are as follows: the elution components in step b are separated and purified by normal-phase silica gel column chromatography and Sephadex LH-20 column chromatography with hydroxypropyl dextran respectively. The collected ethyl acetate eluate is subjected to normal-phase silica gel column chromatography and gradient eluted with n-hexane-ethyl acetate with a volume ratio of 100:1 - 0:1. The fractions are analyzed by silica gel thin-layer chromatography (TLC) and developed with Dragendorff's reagent. The fractions showing positive red and orange-red color are combined to obtain the total alkaloid fraction. The total alkaloid fraction is then subjected to Sephadex LH-20 column chromatography with hydroxypropyl dextran and gradient eluted with 50 - 100% methanol-water to obtain the furoquinoline alkaloid 4,8-dimethoxy-7-((3-methylbut-2-en-1-yl)oxy)furo[2,3-b]quinoline and the compound (R)-1-((4,8-dimethoxyfuro[2,3-b]quinoline-7-yl)oxy)-3-ethoxy-3-methylbutan-2-ol;

[0010] Or they are separated and purified by macroporous resin column chromatography and semi-preparative liquid chromatography respectively. The ethyl acetate extract is subjected to macroporous resin column chromatography and gradient eluted with an acetonitrile-water solution with a volume ratio of 20% - 90%. The fractions with an acetonitrile-water volume ratio of 40 - 60% are collected. Then the collected fractions are subjected to semi-preparative high performance liquid chromatography using a reverse-phase C18 column at a detection wavelength of 245 nm and a flow rate of 3 mL / min. The mobile phase is a gradient elution with an acetonitrile-water solution with a volume ratio of 50 - 80% to obtain the furoquinoline alkaloid 4,8-dimethoxy-7-((3-methylbut-2-en-1-yl)oxy)furo[2,3-b]quinoline and the compound (R)-1-((4,8-dimethoxyfuro[2,3-b]quinoline-7-yl)oxy)-3-ethoxy-3-methylbutan-2-ol.

[0011] In step c, the three separations are as follows: the ethyl acetate extract obtained in step b is purified by reverse-phase silica gel column chromatography, Sephadex LH-20 column chromatography, and semi-preparative high-performance liquid chromatography. For the reverse-phase silica gel column chromatography, the eluent is a 10-90% methanol aqueous solution, and the fraction eluted with 80% methanol-water is collected. Then, it is further purified by Sephadex LH-20 column chromatography with isocratic elution using 50% methanol-water. The different fractions are collected and separated by semi-preparative high-performance liquid chromatography using an XSelect CSH C18 column at a detection wavelength of 245 nm and a flow rate of 3 mL / min. The mobile phase is a gradient elution of acetonitrile-water solution with a volume ratio of 40%-47%, resulting in the furoquinoline alkaloid 4,8-dimethoxy-7-((3-methylbut-2-en-1-yl)oxy)furo[2,3-b]quinoline and the compound (R)-1-((4,8-dimethoxyfuro[2,3-b]quinoline-7-yl)oxy)-3-ethoxy-3-methylbutan-2-ol;

[0012] Alternatively, the eluted fractions in step b are separated and purified by normal-phase silica gel column chromatography, macroporous resin chromatography, and semi-preparative liquid chromatography. The collected eluate is subjected to normal-phase silica gel column chromatography and gradient eluted with petroleum ether-ethyl acetate at a volume ratio of 100:1 - 0:1. The fractions are analyzed by silica gel thin-layer chromatography and visualized with Dragendorff's reagent, and the same fractions are combined to obtain four fractions F1 - F4. Fraction F2 is subjected to macroporous resin chromatography and eluted with a methanol aqueous solution with a volume ratio of 30 - 99%. The fraction with a methanol-water volume ratio of 50 - 60% is collected. The collected fraction is then further separated by semi-preparative high-performance liquid chromatography using an XSelect CSH C18 column at a detection wavelength of 245 nm and a flow rate of 3 mL / min with isocratic elution using 50% acetonitrile-water, resulting in the furoquinoline alkaloid 4,8-dimethoxy-7-((3-methylbut-2-en-1-yl)oxy)furo[2,3-b]quinoline and the compound (R)-1-((4,8-dimethoxyfuro[2,3-b]quinoline-7-yl)oxy)-3-ethoxy-3-methylbutan-2-ol;

[0013] Alternatively, the elution components in step b are separately separated and purified by normal-phase silica gel column chromatography, reverse-phase silica gel column chromatography, and semi-preparative liquid chromatography. The ethyl acetate extract collected is subjected to normal-phase silica gel column chromatography and gradient eluted with petroleum ether-ethyl acetate with a volume ratio of 100:1 - 0:1. The fractions are analyzed by silica gel thin-layer chromatography and developed with Dragendorff's reagent. The fractions showing positive red and orange-red color development are combined to obtain the total alkaloid fraction. The total alkaloid fraction is subjected to reverse-phase silica gel column chromatography and gradient eluted with an acetonitrile-aqueous solution with a volume ratio of 20 - 99%. The fractions with an acetonitrile-water volume ratio of 50 - 70% are collected. The collected fractions are subjected to semi-preparative high-performance liquid chromatography on a reverse-phase C18 column at a detection wavelength of 245 nm and a flow rate of 3 mL / min. The mobile phase is isocratically eluted with a methanol-aqueous solution with a volume ratio of 60% to obtain the furoquinoline alkaloids 4,8-dimethoxy-7-((3-methylbut-2-en-1-yl)oxy)furo[2,3-b]quinoline and the compound (R)-1-((4,8-dimethoxyfuro[2,3-b]quinoline-7-yl)oxy)-3-ethoxy-3-methylbutan-2-ol;

[0014] Alternatively, the elution components in step b are separately separated and purified by normal-phase silica gel column chromatography, macroporous resin column chromatography, and Sephadex LH-20 column chromatography. The ethyl acetate eluate collected is subjected to normal-phase silica gel column chromatography and gradient eluted with petroleum ether-ethyl acetate with a volume ratio of 100:1 - 0:1. The fractions are analyzed by silica gel thin-layer chromatography and developed with Dragendorff's reagent, and then combined to obtain the total alkaloid fraction. The total alkaloid fraction is subjected to macroporous resin column chromatography and gradient eluted with a methanol-aqueous solution with a volume ratio of 30 - 99%. The fractions with a methanol-water volume ratio of 50% - 70% are collected. The collected fractions are subjected to Sephadex LH-20 column chromatography and isocratically eluted with a 60% methanol-water solution. Different components are collected and recrystallized to obtain the furoquinoline alkaloids 4,8-dimethoxy-7-((3-methylbut-2-en-1-yl)oxy)furo[2,3-b]quinoline and the compound (R)-1-((4,8-dimethoxyfuro[2,3-b]quinoline-7-yl)oxy)-3-ethoxy-3-methylbutan-2-ol.

[0015] Use of the furoquinoline alkaloids obtained by the said method in the preparation of anti-colorectal cancer drugs.

[0016] Use of the furoquinoline alkaloids obtained by the said method as lead compounds in the preparation of anti-colorectal cancer drugs.

[0017] The present invention discloses a method for preparing a furanoquinoline alkaloid and its use, wherein the furanoquinoline alkaloid is 4,8-dimethoxy-7-((3-methylbut-2-en-1-yl)oxy)furan[2,3-b]quinoline and the compound is (R)-1-((4,8-dimethoxyfuran[2,3-b]quinolin-7-yl)oxy)-3-ethoxy-3-methylbutan-2-;

[0018] The structural formula is:

[0019]

[0020] The preparation method and use of furanquinoline alkaloids of the present invention have the following advantages:

[0021] The furanoquinoline alkaloids of structural formula 1 and 2 separated from the medicinal plant Rutaecarpa by normal phase silica gel column chromatography, reverse phase silica gel column chromatography, hydroxypropyl dextran gel Sephadex LH-20 column chromatography and preparative high performance liquid chromatography have a certain inhibitory effect on the proliferation of human colon cancer cells (HT-29), and have potential value for the development of drugs for the treatment of colon cancer. DETAILED DESCRIPTION

[0022] The present invention is described in detail below through specific examples, but the use and purpose of these exemplary implementation modes are only used to illustrate the present invention, and do not constitute any form of limitation on the actual protection scope of the present invention, nor limit the protection scope of the present invention to them.

[0023] Example 1

[0024] Three separation methods:

[0025] a. Take 5 kg of dried Haplophyllum acutifolium medicinal material, use 50 L of 50% ethanol aqueous solution, cold-immerse and extract 6 times, combine the extracts, concentrate under reduced pressure, and dry to obtain a crude extract;

[0026] b. The crude extract in step a was subjected to normal phase silica gel column chromatography, and gradient elution was performed with petroleum ether, ethyl acetate and n-butanol for 3 column volumes respectively, the ethyl acetate eluate was collected, and the solvent was evaporated to obtain ethyl acetate extract;

[0027] c. The ethyl acetate extract obtained in step b was purified by reverse phase silica gel column chromatography, hydroxypropyl dextran gel (Sephadex LH-20) column chromatography and semi-preparative high performance liquid chromatography, wherein the reverse phase silica gel column chromatography used was an eluent of 10-90% methanol-water solution, and the methanol-water portion with an eluent of 80% was collected, and then eluted isocratically with 50% methanol-water on a hydroxypropyl dextran gel (Sephadex LH-20) column, and different components were collected and subjected to semi-preparative high performance liquid chromatography, and XSelect CSH The method is characterized in that the method is carried out on a C18 chromatographic column with a detection wavelength of 245 nm and a flow rate of 3 mL / min, and the mobile phase is a gradient elution of acetonitrile-water solution with a volume ratio of 40%-47%, to obtain the furanoquinoline alkaloids of 4,8-dimethoxy-7-((3-methylbut-2-en-1-yl)oxy)furan[2,3-b]quinoline and the compound of (R)-1-((4,8-dimethoxyfuran[2,3-b]quinolin-7-yl)oxy)-3-ethoxy-3-methylbutan-2-ol.

[0028] Example 2

[0029] a. Take 5 kg of dried Haplophyllum acutifolium medicinal material, use 50 L of 99% ethanol aqueous solution, heat reflux extraction 3 times, combine the extracts, concentrate under reduced pressure, and dry to obtain a crude extract;

[0030] b. Extract the crude extract in step a with petroleum ether, dichloromethane, ethyl acetate and n-butanol three times respectively, collect the ethyl acetate eluate, and evaporate the solvent to obtain ethyl acetate extract;

[0031] c. Collect the eluted components in step b, separate and purify them by normal phase silica gel column chromatography, small pore resin chromatography (MCI) and semi-preparative liquid chromatography, and subject the collected eluate to normal phase silica gel column chromatography, and gradient elute with petroleum ether-ethyl acetate in a volume ratio of 100:1-0:1. The fractions are analyzed by silica gel thin layer chromatography (TLC) and developed with potassium bismuth iodide, and the same fractions are combined to obtain 4 fractions F1-F4. The F2 fraction is eluted by small pore resin chromatography (MCI) with a methanol-water solution in a volume ratio of 30%-99%, and the fractions with a methanol-water volume ratio of 50%-60% are collected. The collected fractions are then subjected to semi-preparative high performance liquid chromatography, and XSelect CSH C18 chromatographic column, detection wavelength of 245nm, flow rate of 3mL / min, 50% acetonitrile-water solution isocratic elution, to obtain furanoquinoline alkaloids such as 4,8-dimethoxy-7-((3-methylbut-2-en-1-yl)oxy)furan[2,3-b]quinoline and the compound (R)-1-((4,8-dimethoxyfuran[2,3-b]quinolin-7-yl)oxy)-3-ethoxy-3-methylbutan-2-ol.

[0032] Example 3

[0033] a. Take 5 kg of dried Haplophyllum acutifolium medicinal material, extract it with 50 L acetone for 6 times, combine the extracts, concentrate under reduced pressure, and dry to obtain a crude extract;

[0034] b. Extract the crude extract in step a with petroleum ether, chloroform, ethyl acetate and n-butanol for 6 times respectively, collect the ethyl acetate eluate, and evaporate the solvent to obtain ethyl acetate extract;

[0035] c. Collect the eluted components in step b, separate and purify them by normal phase silica gel column chromatography, reverse phase silica gel column chromatography and semi-preparative liquid chromatography, respectively; subject the collected ethyl acetate extract to normal phase silica gel column chromatography, and gradient elute with petroleum ether-ethyl acetate in a volume ratio of 100:1-0:1; analyze the fractions according to silica gel thin layer chromatography (TLC) and develop color with potassium bismuth iodide, and the color-positive red and orange-red fractions are combined to obtain the total alkaloid fraction; subject the total alkaloid fraction to reverse phase silica gel column chromatography, and gradient elute with acetonitrile-water solution in a volume ratio of 20%-99%, and collect the acetonitrile-water solution in a volume ratio of 50%-70%. The collected fractions were subjected to semi-preparative high performance liquid chromatography on a reverse phase C18 column (C18, 5 μm, 10×150 mm), with a detection wavelength of 245 nm and a flow rate of 3 mL / min, and the mobile phase was a 60% by volume methanol-water solution for isocratic elution to obtain the furanoquinoline alkaloids 4,8-dimethoxy-7-((3-methylbut-2-en-1-yl)oxy)furan[2,3-b]quinoline and the compound (R)-1-((4,8-dimethoxyfuran[2,3-b]quinolin-7-yl)oxy)-3-ethoxy-3-methylbutan-2-ol.

[0036] Example 4

[0037] Two separation methods:

[0038] a. Take 5 kg of dried Haplophyllum acutifolium medicinal material, extract it with 50 L of anhydrous ethanol for 3 times, combine the extracts, concentrate under reduced pressure, and dry to obtain a crude extract;

[0039] b. Extract the crude extract in step a with petroleum ether, chloroform, ethyl acetate and n-butanol for 5 times respectively, collect the ethyl acetate eluate, and evaporate the solvent to obtain ethyl acetate extract;

[0040] c. Collect the eluted components in step b, separate and purify them by normal phase silica gel column chromatography and hydroxypropyl dextran gel (Sephadex LH-20) column chromatography, respectively; the collected ethyl acetate eluate is chromatographed by normal phase silica gel column, and gradient eluted with n-hexane-ethyl acetate in a volume ratio of 100:1-0:1; the fractions are analyzed by silica gel thin layer chromatography (TLC) and color development with potassium bismuth iodide; the red and orange-red fractions are combined to obtain the total alkaloid fraction; the total alkaloid fraction is purified by hydroxypropyl dextran gel (Sephadex LH-20) column chromatography; LH-20) column chromatography, with 50-100% methanol-water gradient elution, to obtain the furanoquinoline alkaloids 4,8-dimethoxy-7-((3-methylbut-2-en-1-yl)oxy)furan[2,3-b]quinoline and the compound (R)-1-((4,8-dimethoxyfuran[2,3-b]quinolin-7-yl)oxy)-3-ethoxy-3-methylbutan-2-ol.

[0041] Example 5

[0042] a. Take 5 kg of dried Haplophyllum acutifolium medicinal material, extract it with 50 L of 50% methanol-water under reflux, concentrate under reduced pressure, and dry it to obtain a crude extract;

[0043] b. Extract the crude extract in step b with n-hexane, chloroform, ethyl acetate and n-butanol for 6 times respectively, collect the ethyl acetate eluate, and evaporate the solvent to obtain ethyl acetate extract;

[0044] c. Collect the eluted components in step b, separate and purify them by small-pore resin column chromatography and semi-preparative liquid chromatography, respectively. The ethyl acetate extract collected in step b is chromatographed by small-pore resin, eluted by gradient elution of acetonitrile-water solution with a volume ratio of 20%-90%, collect the flow fraction with a volume ratio of acetonitrile-water of 40%-60%, and perform semi-preparative high performance liquid chromatography on a reversed-phase C18 column (C18, 5μm, 10×150mm) at a detection wavelength of At 245 nm and a flow rate of 3 mL / min, the mobile phase is an acetonitrile-water solution with a volume ratio of 50%-80% for gradient elution to obtain the furanoquinoline alkaloids 4,8-dimethoxy-7-((3-methylbut-2-en-1-yl)oxy)furan[2,3-b]quinoline and the compound (R)-1-((4,8-dimethoxyfuran[2,3-b]quinolin-7-yl)oxy)-3-ethoxy-3-methylbutan-2-ol.

[0045] Example 6

[0046] a. Take 5 kg of dried Haplophyllum acutifolium medicinal material, extract it with 50 L of 99% methanol-water, concentrate it under reduced pressure, and dry it to obtain a crude extract;

[0047] b. The crude extract in step a was subjected to normal phase silica gel column chromatography, and gradient elution was performed with petroleum ether, ethyl acetate and n-butanol for 6 column volumes, respectively, the ethyl acetate eluate was collected, and the solvent was evaporated to obtain ethyl acetate extract;

[0048] c. Collect the eluted components in step b, separate and purify them by normal phase silica gel column chromatography, small pore resin column chromatography and hydroxypropyl dextran gel (Sephadex LH-20) column chromatography, respectively; subject the collected ethyl acetate eluate to normal phase silica gel column chromatography, and perform gradient elution with petroleum ether-ethyl acetate in a volume ratio of 100:1-0:1; the fractions are analyzed by silica gel thin layer chromatography (TLC) and developed with potassium bismuth iodide to obtain the total alkaloid fraction; the total alkaloid fraction is subjected to small pore resin column chromatography, and gradient eluted with a methanol-water solution in a volume ratio of 30%-99%; the fractions in a methanol-water volume ratio of 50%-70% are collected; the collected fractions are subjected to hydroxypropyl dextran gel (Sephadex LH-20) column chromatography, and the total alkaloid fractions are subjected to 1% to 20% of the total alkaloid fraction; ... LH-20) column chromatography, isocratically eluted with 60% methanol-water, collected different components, and recrystallized to obtain the furanoquinoline alkaloids 4,8-dimethoxy-7-((3-methylbut-2-en-1-yl)oxy)furan[2,3-b]quinoline and the compound (R)-1-((4,8-dimethoxyfuran[2,3-b]quinolin-7-yl)oxy)-3-ethoxy-3-methylbutan-2-ol.

[0049] Example 7

[0050] MTT assay was used to detect the inhibitory effects of furanoquinoline alkaloids, 4,8-dimethoxy-7-((3-methylbut-2-en-1-yl)oxy)furan[2,3-b]quinoline and compound (R)-1-((4,8-dimethoxyfuran[2,3-b]quinolin-7-yl)oxy)-3-ethoxy-3-methylbutan-2-ol, on the proliferation of colon cancer cells HT-29:

[0051] 1. Materials:

[0052] Human colon cancer cell HT-29 cells (purchased from Wuhan Punosai Biotechnology Co., Ltd.) were cultured in a DMEM high-glucose medium (purchased from Logan Hyclone, USA) containing 10% fetal bovine serum (FBS) (purchased from Giboco, USA) in an incubator at 37°C and 5% CO2;

[0053] The test samples are compounds of Structural Formula 1 and Structural Formula 2, which are dissolved in dimethyl sulfoxide (DMSO) at the time of use and diluted with culture medium to the required concentration, so that the final concentration of dimethyl sulfoxide (DMSO) is 1%;

[0054] 2. Experimental method:

[0055] For HT-29 cells growing in the logarithmic growth phase, aspirate the culture medium, wash once with phosphate buffer solution (PBS), digest with trypsin, terminate with culture medium, gently pipette, count, and inoculate in a 96-well plate (100 μL / well) at the corresponding cell density, and culture overnight; add the compounds of Structural Formula 1 and Structural Formula 2 (20 μL / well), set a concentration gradient for each compound, set 3 replicates for each concentration, and culture in an incubator with CO2 at a temperature of 37 °C;

[0056] 3. Detection by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method:

[0057] Add 100 μL of MTT, and continue to culture for 2 hours. After incubating at 37 °C for 2 hours, use an MB microplate reader to detect the optical absorbance value (OD) at 570 nm;

[0058] 4. Statistical analysis:

[0059] The calculation formulas for the percentage of cell viability and the cell inhibition rate are as follows:

[0060]

[0061] Cell inhibition rate % = 1 - cell viability percentage %

[0062] For the primary screening, under the condition of a single concentration, the monomer compound concentration is 50 μM, and the activity of the sample is tested. The compounds of Structural Formula 1 and Structural Formula 2 show an inhibition rate % (Inhibition) greater than 50%; further test the dose-dependent relationship of the activity, that is, the IC 50 value. The IC 50 value is obtained by non-linear fitting of the sample activity to the sample concentration; each sample is set with replicates (n≥3) in the test, and is expressed as the standard deviation (Standard Deviation, SD) in the results; a statistical significance (P value) < 0.05 is considered to be statistically significant;

[0063] 5. Experimental results:

[0064] The experimental results are shown in Table 1:

[0065] At a concentration of 50 μM, the compounds of Structural Formula 1 and Formula 2 have a certain inhibitory effect on the proliferation of human colon cancer cell line HT-29. The inhibition rate of the compound of Structural Formula 1 is 77.00%, and the inhibition rate of the compound of Formula 2 is 52.00%. The positive control, doxorubicin, has an inhibition rate of 40.46% on human colon cancer cell line HT-29 at a concentration of 1 μM;

[0066] To further test the activity dose-dependence relationship, the IC 50 value of the compound of Structural Formula 1 is 16.25 ± 3.04 μM, and the IC 50 value of the compound of Formula 2 is 67.77 ± 5.91 μM. The IC 50 value of the positive control, doxorubicin, is 2.85 ± 0.12 μM;

[0067] Table 1 Inhibition rates of Structural Formula 1 and Formula 2 on human colon cancer cell line HT-29

[0068]

[0069] In summary, Structural Formula 1 and Formula 2 have a strong inhibitory effect on the growth of human colon cancer cell line HT-29 and show a certain dose-dependence relationship; Structural Formula 1 and Formula 2 have potential value for the development of drugs for treating colon cancer.

Claims

1. A preparation method of a furoquinoline alkaloid, characterized in that: The method uses the dried medicinal material of the whole herb of Haplophyllum acutifolium as the raw material and an organic solvent as the extractant for extraction. The specific operation is carried out according to the following steps: Three separation methods: a. Take 5 kg of dry Ruta graveolens L. herbs, extract them 6 times by cold maceration with 50 L of 50% ethanol aqueous solution, combine the extracts, concentrate them under reduced pressure, and then dry to obtain the crude extract; Haplophyllum acutifolium ​ b. The crude extract in step a is subjected to normal-phase silica gel column chromatography, and gradient elution is carried out with petroleum ether, ethyl acetate and n-butanol for 3 column volumes respectively. The ethyl acetate eluate is collected and the solvent is evaporated to obtain an ethyl acetate extract; c. The ethyl acetate extract obtained in step b was purified by reverse-phase silica gel column chromatography, Sephadex LH-20 column chromatography of hydroxypropyl dextran gel, and semi-preparative high-performance liquid chromatography. For the reverse-phase silica gel column chromatography used, the eluent was an aqueous solution of methanol at 10−90%, and the fraction with an eluent of 80% methanol-water was collected. Then, it was subjected to Sephadex LH-20 column chromatography of hydroxypropyl dextran gel and isocratically eluted with 50% methanol-water. Different components were collected and separated by semi-preparative high-performance liquid chromatography using an XSelect CSH C18 column. At a detection wavelength of 245 nm and a flow rate of 3 mL / min, the mobile phase was a gradient elution of acetonitrile−aqueous solution with a volume ratio of 40%-47%, and the furoquinoline alkaloids obtained were 4,8-dimethoxy-7-((3-methylbut-2-en-1-yl)oxy)furo[2,3-b]quinoline and ( R )-1-((4,8-dimethoxyfuro[2,3-b]quinolin-7-yl)oxy)-3-ethoxy-3-methylbutan-2-ol.

2. A method for preparing furoquinoline alkaloids, characterized in that: The method uses the dried medicinal material of the whole herb of Haplophyllum acutifolium as the raw material and an organic solvent as the extractant for extraction. The specific operation is carried out according to the following steps: Three separation methods: a. Take the dried Heracleum moellendorffii Hance Haplophyllum acutifolium medicinal materials (5 kg), extract them 3 times by hot reflux with 50 L of 99% ethanol aqueous solution, combine the extracts, concentrate under reduced pressure, and dry to obtain the crude extract; b. The crude extract in step a is successively extracted 3 times with petroleum ether, dichloromethane, ethyl acetate and n-butanol respectively. The ethyl acetate eluate is collected and the solvent is evaporated to obtain an ethyl acetate extract; c. Collect the eluted components in step b and separate and purify them by normal-phase silica gel column chromatography, macroporous resin chromatography MCI, and semi-preparative liquid chromatography. Subject the collected eluent to normal-phase silica gel column chromatography and perform gradient elution with petroleum ether-ethyl acetate in a volume ratio of 100:1 - 0:

1. The fractions are analyzed by silica gel thin-layer chromatography TLC and developed with Dragendorff's reagent, and the same fractions are combined to obtain 4 fractions F1 - F4. Subject fraction F2 to macroporous resin chromatography MCI and elute with a methanol-aqueous solution in a volume ratio of 30% - 99%, and collect the fractions with a methanol-water volume ratio of 50% - 60%. Then subject the collected fractions to semi-preparative high-performance liquid chromatography. Using an XSelect CSH C18 chromatographic column, with a detection wavelength of 245 nm and a flow rate of 3 mL / min, perform isocratic elution with a 50% acetonitrile-aqueous solution to obtain the furoquinoline alkaloids 4,8-dimethoxy-7-((3-methylbut-2-en-1-yl)oxy)furo[2,3-b]quinoline and ( R )-1-((4,8-dimethoxyfuro[2,3-b]quinolin-7-yl)oxy)-3-ethoxy-3-methylbutan-2-ol.

3. A method for preparing furoquinoline alkaloids, characterized in that: The method uses the dried medicinal material of the whole herb of Haplophyllum acutifolium as the raw material and an organic solvent as the extractant for extraction. The specific operation is carried out according to the following steps: Three separation methods: a. Take 5 kg of dry Heracleum moellendorffii Hance, Haplophyllum acutifolium extract it 6 times by cold soaking with 50 L of acetone, combine the extracts, concentrate under reduced pressure, and dry to obtain a crude extract; b. The crude extract in step a is successively extracted 6 times with petroleum ether, chloroform, ethyl acetate and n-butanol respectively. The ethyl acetate eluate is collected and the solvent is evaporated to obtain an ethyl acetate extract; c. Collect the eluted components in step b and separate and purify them by normal-phase silica gel column chromatography, reverse-phase silica gel column chromatography and semi-preparative liquid chromatography. Subject the collected ethyl acetate extract to normal-phase silica gel column chromatography and perform gradient elution with petroleum ether - ethyl acetate in a volume ratio of 100:1 - 0:

1. The fractions are analyzed by silica gel thin-layer chromatography (TLC) and developed with Dragendorff's reagent. The fractions showing positive red and orange-red coloration are combined to obtain the total alkaloid fraction. The total alkaloid fraction is subjected to reverse-phase silica gel column chromatography and gradient eluted with acetonitrile - aqueous solution in a volume ratio of 20% - 99%. Collect the fractions with an acetonitrile - water volume ratio of 50% - 70%. Subject the collected fractions to semi-preparative high-performance liquid chromatography using a reverse-phase C18 column (C18, 5 µm, 10 × 150 mm), a detection wavelength of 245 nm, and an isocratic elution with a mobile phase of methanol - aqueous solution in a volume ratio of 60% at a flow rate of 3 mL / min to obtain the furoquinoline alkaloids 4,8-dimethoxy-7-((3-methylbut-2-en-1-yl)oxy)furo[2,3-b]quinoline and ( R -1-((4,8-dimethoxyfuro[2,3-b]quinolin-7-yl)oxy)-3-ethoxy-3-methylbutan-2-ol).

4. A method for preparing furoquinoline alkaloids, characterized in that: The method uses the dried medicinal material of the whole herb of Haplophyllum acutifolium as the raw material and an organic solvent as the extractant for extraction. The specific operation is carried out according to the following steps: Two separation methods: a. Take the dried Heracleum moellendorffii Hance Haplophyllum acutifolium medicinal materials (5 kg), extract them with 50 L of anhydrous ethanol by cold maceration for 3 times, combine the extraction solutions, concentrate them under reduced pressure, and then dry to obtain the crude extract; b. The crude extract in step a is successively extracted 5 times with petroleum ether, chloroform, ethyl acetate and n-butanol respectively. The ethyl acetate eluate is collected and the solvent is evaporated to obtain an ethyl acetate extract; c. Collect the eluted components in step b and separate and purify them by normal-phase silica gel column chromatography and Sephadex LH-20 column chromatography. Subject the collected ethyl acetate eluate to normal-phase silica gel column chromatography and perform gradient elution with n-hexane-ethyl acetate in a volume ratio of 100:1 - 0:

1. The fractions are analyzed by silica gel thin-layer chromatography (TLC) and developed with Dragendorff's reagent. The fractions showing positive red or orange-red color are combined to obtain the total alkaloid fraction. The total alkaloid fraction is then subjected to Sephadex LH-20 column chromatography and eluted with a gradient of 50 - 100% methanol-water to obtain the furoquinoline alkaloids 4,8-dimethoxy-7-((3-methylbut-2-en-1-yl)oxy)furo[2,3-b]quinoline and ( R -1-((4,8-dimethoxyfuro[2,3-b]quinolin-7-yl)oxy)-3-ethoxy-3-methylbutan-2-ol.

5. A method for preparing furoquinoline alkaloids, characterized in that: The method uses the dried medicinal material of the whole herb of Haplophyllum acutifolium as the raw material and an organic solvent as the extractant for extraction. The specific operation is carried out according to the following steps: Two separation methods: a. Take 5 kg of dry Rutaceae graveolens and extract it by hydrothermal reflux with 50 L of 50% methanol-water solution. After concentration under reduced pressure, dry it to obtain the crude extract; Haplophyllum acutifolium ​ b. The crude extract in step b is successively extracted 6 times with n-hexane, chloroform, ethyl acetate and n-butanol respectively. The ethyl acetate eluate is collected and the solvent is evaporated to obtain an ethyl acetate extract; c. Collect the eluted components in step b and separate and purify them by small pore resin column chromatography and semi-preparative liquid chromatography respectively. Subject the ethyl acetate extract collected in step b to small pore resin column chromatography, elute it with an acetonitrile - aqueous solution gradient with a volume ratio of 20% - 90%, and collect the fractions with an acetonitrile - water volume ratio of 40% - 60%. Subject the collected fractions to semi-preparative high performance liquid chromatography using a reversed-phase C18 column C18, 5 µm, 10 × 150 mm, with a detection wavelength of 245 nm and a flow rate of 3 mL / min. Elute with an acetonitrile - aqueous solution gradient with a volume ratio of 50% - 80% to obtain the furoquinoline alkaloids 4,8-dimethoxy-7-((3-methylbut-2-en-1-yl)oxy)furo[2,3-b]quinoline and ( R -1-((4,8-dimethoxyfuro[2,3-b]quinolin-7-yl)oxy)-3-ethoxy-3-methylbutan-2-ol.

6. A method for preparing furoquinoline alkaloids, characterized in that: The method uses the dried medicinal material of the whole herb of Haplophyllum acutifolium as the raw material and an organic solvent as the extractant for extraction. The specific operation is carried out according to the following steps: Two separation methods: a. Take the dried Heracleum moellendorffii Hance Haplophyllum acutifolium 5 kg of the medicinal material is extracted by cold maceration with 50 L of methanol-water with a concentration of 99%. After concentration under reduced pressure, it is dried to obtain a crude extract; b. The crude extract in step a is subjected to normal-phase silica gel column chromatography, and gradient elution is carried out with petroleum ether, ethyl acetate and n-butanol for 6 column volumes respectively. The ethyl acetate eluate is collected and the solvent is evaporated to obtain an ethyl acetate extract; c. Collect the eluted components in step b and separate and purify them by normal-phase silica gel column chromatography, macroporous resin column chromatography, and Sephadex LH-20 column chromatography. Subject the collected ethyl acetate eluate to normal-phase silica gel column chromatography and perform gradient elution with petroleum ether-ethyl acetate at a volume ratio of 100:1 - 0:

1. The fractions are analyzed by silica gel thin-layer chromatography (TLC) and developed with Dragendorff's reagent, and the total alkaloid fraction is obtained by combining them. The total alkaloid fraction is subjected to macroporous resin column chromatography and gradient eluted with a methanol-water solution at a volume ratio of 30% - 99%. Collect the fractions with a methanol-water volume ratio of 50% - 70%. Subject the collected fractions to Sephadex LH-20 column chromatography and perform isocratic elution with a 60% methanol-water solution. Collect different components, and 4,8-dimethoxy-7-((3-methylbut-2-en-1-yl)oxy)furo[2,3-b]quinoline and ( R -1-((4,8-dimethoxyfuro[2,3-b]quinolin-7-yl)oxy)-3-ethoxy-3-methylbutan-2-ol can be obtained as furoquinoline alkaloids by recrystallization.

7. Use of the furoquinoline alkaloids obtained by the method according to claim 1, 2, 3, 4, 5 or 6 in the preparation of anti-colorectal cancer drugs.

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