CN112645911B - Compound separated from Botrytis cinerea and preparation method and application thereof - Google Patents
Compound separated from Botrytis cinerea and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to the field of pharmaceutical compounds, in particular to a compound separated from Yunnan mouse thorn and a preparation method and application thereof. The yunnan mouse is mainly subjected to systematic and deep chemical component research, 2-arylbenzo [ b ] furan compounds 1-4 are separated from the yunnan mouse, and the preparation method and the application of the compounds in preparing the liver cancer treatment drug are defined.
Description
Technical Field
The invention relates to the field of pharmaceutical compounds, in particular to a compound separated from Yunnan mouse thorn and a preparation method and application thereof.
Background
Liver cancer is one of the common malignant tumors, and is mainly Primary liver cancer (Primary liver cancer). About 81.4 thousands of new primary liver cancer cases around the world in 2018 are reported, and the cases live at the 6 th position of new malignant tumor cases; the death rate of liver cancer reaches 78.1 ten thousand, and the death rate of malignant tumor is 4 th [1]. Currently, surgical and chemical therapies are the main treatment strategies for liver cancer. Wherein, the operation treatment is only suitable for a small part of patients with liver cancer in the early stage, and the patients are easy to relapse for two times, and the five-year relapse rate after the operation is more than 70% [2-3]. Most patients with liver cancer are in the advanced stage after diagnosis, and systemic chemotherapy is mainly used. The current therapeutic drugs for liver cancer are mainly molecular targeted drugs such as sorafenib and lenvatinib, but the effect is limited, and the overall median survival time of patients is only about 12 months [4]. Although immunotherapy drugs represented by the nano Wu Liyou monoclonal antibody and avizumab show strong anti-liver cancer potential [5], low-toxicity and high-efficiency small-molecule anti-liver cancer drugs are in urgent need for development.
At present, no literature report on the separation compound 1-3 in the Yunnan rat poison and the pharmacological activity thereof exists at home and abroad, and no report related to the preparation method and the application of the Yunnan rat poison in the invention is found.
In order to overcome the technical problems, the team of the invention carries out systematic and deep chemical component research on the Yunnan rat thorn, and 2-arylbenzo [ b ] furan compounds 1-4 are separated from the Yunnan rat thorn, and the preparation method of the compounds and the application of the compounds in preparing the medicaments for treating liver cancer are determined.
Disclosure of Invention
The invention aims to provide a compound separated from Yunnan rat thorn;
the invention also aims to provide a preparation method of the compound in the Yunnan murraya paniculata;
the invention also aims to provide the application of the chinaroot greenbrier rhizome compound in preparing a medicament for treating liver cancer.
The compound separated from the Yunnan muricaria is a 2-aryl benzo [ b ] furan compound separated from the Yunnan muricaria, and the structural formula of the compound is as follows:
the preparation method of the compound comprises the following steps:
1) Soaking and extracting 15-21Kg of dried and pulverized medicinal material powder with ethanol at normal temperature for 2-4 times, each time for 4-10 days;
2) After extraction, the extract solutions are combined and concentrated under reduced pressure (55 ℃;0.08 Mpa) to obtain a crude extract;
3) Dispersing the total extract into 12L water, sequentially extracting with petroleum ether 18-30L for 2-6 times, ethyl acetate 18-30L for 2-6 times, and n-butanol 18-30L for 2-6 times; respectively mixing the extractive solutions, concentrating under reduced pressure to remove solvent to obtain petroleum ether fraction, ethyl acetate fraction and n-butanol fraction;
4) Separating the ethyl acetate part by silica gel column chromatography, and performing gradient elution by using petroleum ether/ethyl acetate according to the ratio of 50 → 1, v/v, and dividing into 8 components E1-E8;
5) Further subjecting the component E5 to silica gel column chromatography, eluting with petroleum ether/ethyl acetate at a ratio of 3:1 to obtain 10 subfractions E5A-E5J;
6) Subjecting the component E5E to silica gel column chromatography, eluting with dichloromethane/ethyl acetate at a ratio of 50 to obtain a component E5E1, and subjecting the component E to pure methanol crystallization to obtain a compound 1;
7) The fraction E5G was subjected to MCI column chromatography to 70% MeOH/H 2 Eluting with O to obtain a compound 2; subjecting the component E5H to silica gel column chromatography, and performing gradient elution with dichloromethane/ethyl acetate to obtain 8 components E5H1-E5H8; subfraction E5H4 is further chromatographed by a silica gel column, and 1 component is obtained by eluting with DCM/MeOH with the proportion of 90; the component is further purified and crystallized by methanol to obtain a compound 4; subjecting the component E6 to Sephadex LH-20 gel column chromatography, and eluting with DCM/MeOH at a ratio of 1:1 to obtain 7 components E6A-E6G; and (3) carrying out silica gel column chromatography on the component E6C, and eluting with DCM/MeOH with the ratio of 30.
The ethanol in the step 1) is 75-95% ethanol in an amount which is 2-6 times that of the ethanol.
The ethanol in the step 1) is preferably 4 times the amount of 95% ethanol.
The extraction frequency in the step 1) is preferably 3 times, and each time is 7 days.
In the step 3), 24L of petroleum ether is preferably used for extraction for 4 times, 24L of ethyl acetate is preferably used for extraction for 4 times, and 24L of n-butanol is preferably used for extraction for 4 times.
The compound of the invention is added with pharmaceutically acceptable auxiliary materials to prepare pharmaceutically acceptable dosage forms.
The pharmaceutically acceptable preparation provided by the invention is a solid preparation or a liquid preparation.
The solid preparation provided by the invention is granules, capsules, tablets, pills, powder and freeze-dried powder injection; the liquid preparation is injection preparation and oral liquid.
The compound disclosed by the invention is applied to preparation of a medicine for treating liver cancer.
The auxiliary materials are not limited and can be accepted in pharmacy.
Has the advantages that:
compared with the prior art, the invention has the following beneficial effects:
1. no literature report on the separation chemical 1-3 and the pharmacological activity thereof in the Yunnan muricatum is available at home and abroad, and no report related to the preparation method and the application of the compound in the Yunnan muricatum is available.
In order to overcome the technical problems, the team of the invention carries out systematic and deep chemical component research on the Yunnan rat thorn, separates the compounds 1-4 from the Yunnan rat thorn, and defines the preparation method of the compounds and the application of the compounds in preparing the medicine for treating liver cancer.
2. Evaluation by MTT methodValency 2-arylbenzo [ b]The anti-proliferation capacity of furan compounds shows that compounds 1-4 can inhibit the proliferation of human liver cancer cell SK-Hep-1, but the proliferation capacity of 1-2 is weaker than that of compounds 3 and 4, the proliferation inhibition rates of compounds 1 and 2 on human liver cancer cell SK-Hep-1 at the concentration of 20 mu M are 23% and 15%, respectively, and the IC corresponding to compounds 3 and 4 50 The values were 5.36. Mu.M and 6.01. Mu.M, respectively.
3. The present invention further evaluated the effect of compounds 3 and 4 on the clonality of SK-Hep-1. The results show that the three can obviously inhibit the clone formation of SK-Hep-1 cells under the concentration of 5 mu M. These results indicate that compounds 3 and 4 exhibit good inhibitory activity against the proliferation of hepatoma cells.
4. The influence of the compound 3 on the apoptosis of SK-Hep-1 cells was examined by flow cytometry. As a result, the compound 3 can remarkably induce the early apoptosis of SK-Hep-1 cells under the concentration of 10 mu M, and the early apoptosis rate is 22.1%.
5. The influence of Compound 3 on the expression level of ERK (RAS/ERK signaling pathway), AKT (PI 3K/AKT signaling pathway), STAT3 (JAK/STAT signaling pathway) and the phosphorylation level thereof in SK-Hep-1 cells was analyzed by immunoblotting experiments. As a result, the compound 3 (10 μ M) was found to significantly reduce the phosphorylation level of ERK without significant effect on other indicators. The compound 3 is shown to induce the apoptosis of the liver cancer cell by inhibiting RAS/ERK signal transduction path.
Drawings
FIG. 1 shows the effect of compounds (3 and 4) on the proliferation inhibitory activity (A) and clonogenic activity (B) of SK-Hep-1 cells.
FIG. 2 Effect of Compound 3 on apoptosis of SK-Hep-1 cells.
FIG. 3 immunoblot analysis of SK-Hep-1 cell lysates treated with Compound 3.
Detailed Description
The technical solution of the present invention will be further specifically described below by way of specific examples.
Example 1
2-aryl benzo [ b ] furan compounds separated from Yunnan mouse thorn, the structural formula of the compounds is as follows:
the preparation method comprises the following steps:
1) Soaking and extracting 18Kg of dried and crushed medicinal powder with 4 times of 95% ethanol at normal temperature for three times, each time for 7 days.
2) After extraction, the extract solutions are combined and concentrated under reduced pressure (55 ℃;0.08 Mpa) to obtain 1.25Kg of crude extract.
3) Dispersing the total extract in 12L water, sequentially extracting with petroleum ether (4 × 24L), ethyl acetate (4 × 24L) and n-butanol (4 × 24L), mixing extractive solutions, respectively, and concentrating under reduced pressure to remove solvent to obtain petroleum ether part 110.4g, ethyl acetate part 180.5g and n-butanol part 210.6g.
4) The ethyl acetate fraction was taken, separated by silica gel column chromatography, and eluted with a petroleum ether/ethyl acetate gradient (50 → 1, v/v) to divide into 8 fractions E1 to E8.
5) Fraction E5 (6.8 g) was further subjected to silica gel column chromatography eluting with petroleum ether/ethyl acetate (3:1) to give 10 subfractions E5A-E5J.
6) Fraction E5E (2.41 g) was subjected to silica gel column chromatography eluting with dichloromethane/ethyl acetate (50).
7) Fraction E5G (1.3G) was subjected to MCI column chromatography to 70% MeOH/H 2 Elution with O gave Compound 2 (5 mg). Fraction E5H (2.3 g) was subjected to silica gel column chromatography eluting with a dichloromethane/ethyl acetate gradient to give 8 fractions E5H1-E5H8. Subfraction E5H4 (0.37 g) was further chromatographed on a silica gel column eluting with DCM/MeOH (90. This fraction was further purified by methanol crystallization to give compound 4 (78 mg). Fraction E6 (2.77G) was chromatographed on Sephadex LH-20 gel column eluting with DCM/MeOH (1:1) to give 7 fractions E6A-E6G. Fraction E6C (73 mg) was chromatographed on a silica gel column eluting with DCM/MeOH (30).
Example 2
2-aryl benzo [ b ] furan compounds separated from Yunnan murinus, wherein the structural formula of the compounds is as follows:
the preparation method comprises the following steps:
1) Soaking and extracting 15Kg of dried and pulverized medicinal material powder with ethanol at normal temperature for 2 times, each time for 10 days;
2) After extraction, the extract solutions are combined and concentrated under reduced pressure (55 ℃;0.08 Mpa) to obtain a crude extract;
3) Dispersing the total extract into 12L water, sequentially extracting with petroleum ether 18L for 6 times, ethyl acetate 18L for 6 times, and n-butanol 18L for 6 times; respectively mixing the extractive solutions, concentrating under reduced pressure to remove solvent to obtain petroleum ether fraction, ethyl acetate fraction and n-butanol fraction;
4) Taking an ethyl acetate part, separating by silica gel column chromatography, and carrying out gradient elution by petroleum ether/ethyl acetate according to the proportion of 50 → 1, v/v, and dividing into 8 components E1-E8;
5) Further subjecting the component E5 to silica gel column chromatography, eluting with 3:1 petroleum ether/ethyl acetate to obtain 10 subfractions E5A-E5J;
6) Subjecting the component E5E to silica gel column chromatography, eluting with dichloromethane/ethyl acetate at a ratio of 50 to obtain a component E5E1, and subjecting the component E to pure methanol crystallization to obtain a compound 1;
7) The fraction E5G was subjected to MCI column chromatography to 70% MeOH/H 2 Eluting with O to obtain compound 2; subjecting the component E5H to silica gel column chromatography, and performing gradient elution with dichloromethane/ethyl acetate to obtain 8 components E5H1-E5H8; the subfraction E5H4 is further chromatographed by a silica gel column, and 1 component is obtained by eluting with the following materials according to the proportion of 90; the component is further purified and crystallized by methanol to obtain a compound 4; subjecting the component E6 to Sephadex LH-20 gel column chromatography, eluting with DCM/MeOH at a ratio of 1:1 to obtain 7 components E6A-E6G; and (3) carrying out silica gel column chromatography on the component E6C, and eluting with DCM/MeOH with the ratio of 30.
Example 3
2-aryl benzo [ b ] furan compounds separated from Yunnan mouse thorn, the structural formula of the compounds is as follows:
the preparation method comprises the following steps:
1) Soaking 21Kg of dried and pulverized medicinal material powder in ethanol at normal temperature for 4 times each for 4 days;
2) After extraction, the extract solutions are combined and concentrated under reduced pressure (55 ℃;0.08 Mpa) to obtain a crude extract;
3) Dispersing the total extract into 12L water, sequentially extracting with petroleum ether 30L for 2 times, ethyl acetate 30L for 2 times, and n-butanol 30L for 2 times; respectively mixing the extractive solutions, concentrating under reduced pressure to remove solvent to obtain petroleum ether fraction, ethyl acetate fraction and n-butanol fraction;
4) Taking an ethyl acetate part, separating by silica gel column chromatography, and carrying out gradient elution by petroleum ether/ethyl acetate according to the proportion of 50 → 1, v/v, and dividing into 8 components E1-E8;
5) Further subjecting the component E5 to silica gel column chromatography, eluting with 3:1 petroleum ether/ethyl acetate to obtain 10 subfractions E5A-E5J;
6) Subjecting the component E5E to silica gel column chromatography, eluting with dichloromethane/ethyl acetate at a ratio of 50 to obtain a component E5E1, and subjecting the component E to pure methanol crystallization to obtain a compound 1;
7) The fraction E5G was subjected to MCI column chromatography to 70% MeOH/H 2 Eluting with O to obtain a compound 2; performing silica gel column chromatography on the component E5H, and performing gradient elution by using dichloromethane/ethyl acetate to obtain 8 components E5H1-E5H8; the subfraction E5H4 is further chromatographed by a silica gel column, and 1 component is obtained by eluting with the following materials according to the proportion of 90; the component is further purified and crystallized by methanol to obtain a compound 4; subjecting the component E6 to Sephadex LH-20 gel column chromatography, and eluting with DCM/MeOH at a ratio of 1:1 to obtain 7 components E6A-E6G; and (3) carrying out silica gel column chromatography on the component E6C, and eluting with DCM/MeOH with the ratio of 30.
Example 4
Taking any one of the compounds 1-4 as a raw material, adding soluble starch (the proportion of the compound to the soluble starch is 1:8), mixing uniformly, and granulating to obtain granules.
Example 5
Taking any one of the compounds 1-4 as a raw material, adding soluble starch (the proportion of the compound to the soluble starch is 1.
Example 6
Taking any one of the compounds 1-4 as raw material, adding soluble starch (the ratio of the compound to the soluble starch is 1:5), mixing, and tabletting to obtain tablet.
Example 7
Taking any one of compounds 1-4 as raw material, adding soluble starch (the ratio of compound to soluble starch is 1:5), mixing, drying, and making into pill.
Example 8
Taking any one of the compounds 1-4 as a raw material, adding 25 times of water for injection, mixing uniformly, filtering, sterilizing and obtaining the injection.
In order to further verify the feasibility and the effectiveness of the invention, the inventor carries out a series of tests, which are as follows:
experimental example 1 isolation method test
Separation involving packing and manufacturer
Silica gel: 200-300 mesh, qingdao ocean chemical Co Ltd
Sephadex LH20: sweden GE Healthcare Bio-Sciences AB
And MCI: mitsubishi, japan
Solvent: commercially available analytical grade reagents.
Second, separation process
Soaking and extracting 18Kg of dried and crushed medicinal powder with 4 times of 95% ethanol at normal temperature for three times, each time for 7 days. After extraction, the extracting solutions are combined and concentrated under reduced pressure to obtain 1.25Kg of crude extract. Dispersing the total extract in 12L water, sequentially extracting with petroleum ether (4 × 24L), ethyl acetate (4 × 24L) and n-butanol (4 × 24L), mixing extractive solutions, and concentrating under reduced pressure to remove solvent to obtain petroleum ether part 110.4g, ethyl acetate part 180.5g and n-butanol part 210.6g.
The ethyl acetate fraction was taken, separated by silica gel column chromatography, and eluted with a petroleum ether/ethyl acetate gradient (50 → 1, v/v) to divide into 8 fractions E1 to E8. Component E5 (6.8 g) was further subjected to silica gel column chromatography with petroleum etherEthyl acetate (3:1) gave 10 subfractions E5A-E5J. Fraction E5E (2.41 g) was subjected to silica gel column chromatography, eluting with dichloromethane/ethyl acetate (50). Fraction E5G (1.3G) was subjected to MCI column chromatography to 70% MeOH/H 2 Elution with O gave Compound 2 (5 mg). Fraction E5H (2.3 g) was subjected to silica gel column chromatography eluting with a dichloromethane/ethyl acetate gradient to give 8 fractions E5H1-E5H8. Subfraction E5H4 (0.37 g) was further chromatographed on a silica gel column eluting with DCM/MeOH (90. This fraction was further purified by methanol crystallization to give compound 4 (78 mg). Fraction E6 (2.77G) was chromatographed on Sephadex LH-20 gel column eluting with DCM/MeOH (1:1) to give 7 fractions E6A-E6G. Fraction E6C (73 mg) was chromatographed on a silica gel column eluting with DCM/MeOH (30).
2-aryl benzo [ b ] was isolated and identified from the aerial part of Itea yunnanensis Franch
The furan compounds (representing new compounds) are 4, wherein 1-3 are new compounds.
Experimental example 2 Activity measurement
Reagent for activity determination
Fetal bovine serum: gibco Inc. of USA
DMEM medium: gibco Corp, USA
Annexin V-FITC apoptosis detection kit: biolegend, usa
Penicillin, streptomycin, DMSO, and crystal violet: sigma Co USA
MTT kit: MCE corporation of America
TBST buffer:
RIPA lysate:
BCA protein assay kit: bio-Rad Inc. of USA
Cellulose nitrate membrane: millipore Corp
And (3) skim milk powder: BD Co of USA
Antibodies ERK, p-ERK, AKT, p-AKT, STAT3 and p-STAT3: cell Signaling Technology, inc. of USA
Enhanced chemiluminescent liquid: saimei Fei Co Ltd
2. Activity assay
A. Cell culture at 37 ℃ in DMEM containing 10% fetal bovine serum based on 5% CO 2 SK-Hep-1 cells were cultured in an incubator. And (4) when the cell coverage rate is higher than 90%, carrying out passage, and selecting cells with good growth state for experiment.
B. Cell proliferation assay 96-well culture plates were seeded with SK-Hep-1 cells (100. Mu.L, density 2X 104 cells/mL) in logarithmic growth phase per well. After extraction, after culturing in DMEM medium containing 10% fetal calf serum and 1% penicillin/streptomycin for 24h, adding samples to be tested with different concentrations, and taking DMSO as negative control. After incubation at 37 ℃ for 72h in a 5% CO2 incubator, 20. Mu.L of MTT solution (5 mg/mL) was added to each well and incubation continued for 4h. After the extraction, the supernatant was carefully removed by aspiration, 150. Mu.L of DMSO was added to each well, and the mixture was placed on a microwell shaker and shaken for 10min to completely dissolve the purple crystals. And (3) measuring the absorption value at the 490nm wavelength by using an enzyme-labeling instrument, making a growth curve, and determining the IC50 value of the sample to be measured.
C. Cloning experiments 6-well culture plates were used to inoculate SK-Hep-1 cells (1000 cells/well) in the logarithmic growth phase. After extraction, the sample to be tested (5. Mu.M) was added after 24h of culture in DMEM medium containing 10% fetal bovine serum and 1% penicillin/streptomycin. After 7 days of incubation in a 5% CO2 incubator at 37 ℃ the cells were stained with 0.5% crystal violet for 20min. After extraction, the staining solution was removed and the image was recorded with a high resolution scanner. Subsequently, 2mL of 33% acetic acid solution was added to each well to suspend the cells, and the suspension was shaken on a micro-cell shaker and then measured for absorbance at a wavelength of 570nm using a microplate reader.
D. Apoptosis experiments 6-well culture plates were used, and 2X 105 SK-Hep-1 cells were inoculated per well. After extraction, the sample to be tested (10. Mu.M) is added into each hole for treatment for 36h. Subsequently, the cells were collected separately and washed once with pre-cooled PBS. The Annexin V-FITC-PI apoptosis detection kit is used for detecting live cells, early apoptotic cells and late apoptotic/dead cells. Specifically, fluorescein Isothiocyanate (FITC) and Propidium Iodide (PI) were added according to the instructions and incubated for 15min in the dark. And after the treatment and extraction are finished, analyzing the apoptosis condition of all the cells by adopting a flow cytometer.
E. Immunoblot experiments the treated cells were washed 2 times with physiological saline and subsequently lysed with RIPA lysate containing phosphatase and protease inhibitors. After the cell lysate was sufficiently lysed, the whole-cell protein lysate was centrifuged at 4 ℃ for 10min (12000 rpm), and the supernatant was collected to discard insoluble substances. Total protein concentration was determined by BCA protein assay kit. An equal amount of total protein was mixed with SDS-PAGE loading buffer and subjected to SDS-PAGE electrophoresis. After extraction, the proteins were transferred to nitrocellulose membranes at constant pressure. After membrane transfer, blocking with TBST buffer containing 5% skimmed milk powder for 30min. Subsequently, primary antibody (anti-RAS, anti-RAF-1, anti-p-RAF-1, anti-MEK 1/2, anti-p-MEK 1/2, anti-ERK, anti-p-ERK, anti-c-Jun, anti-p-c-Jun, anti-c-Fos, anti-p-c-Fos, anti-ELK-1, anti-p-ELK-1, anti-Caspase-3/-8/-9, anti-PARP, anti-GAPDH or anti-. Beta. -actin antibodies) labeling was performed, and incubation was performed overnight at 4 ℃. The next day, after three washes in TBST buffer, horseradish peroxidase conjugated secondary antibody labeling was performed at room temperature and incubated for 2h. After extraction, the protein was detected with enhanced chemiluminescence solution after three washes with TBST buffer.
As a result:
evaluation of the above-mentioned 2-Arylbenzo [ b ] by MTT method]The anti-proliferation capacity of furan compounds shows that compounds 1-4 can inhibit proliferation of human liver cancer cell SK-Hep-1, but the proliferation capacity of 1-2 is weaker than that of compounds 3 and 4, and the proliferation inhibition rates of compounds 1 and 2 to human liver cancer cell SK-Hep-1 at the concentration of 20 mu M are 23% and 15% respectively (the activity of compounds 1 and 2 is lower, and IC cannot be measured 50 Value, evaluation of compound by proliferation inhibition rate), IC corresponding to compounds 3, 4 50 The values were 5.36. Mu.M and 6.01. Mu.M, respectively. See fig. 1.
The effect of compounds 3 and 4 on the clonality of SK-Hep-1 was further evaluated. The results show that the three can obviously inhibit the clone formation of SK-Hep-1 cells under the concentration of 5 mu M. These results indicate that compounds 3 and 4 exhibit good inhibitory activity against the proliferation of hepatoma cells. See fig. 1.
The influence of the compound 3 on the apoptosis of SK-Hep-1 cells was examined by flow cytometry. As a result, the compound 3 can remarkably induce the early apoptosis of SK-Hep-1 cells under the concentration of 10 mu M, and the early apoptosis rate is 22.1%. See fig. 2.
The influence of Compound 3 on the expression level of ERK (RAS/ERK signaling pathway), AKT (PI 3K/AKT signaling pathway), STAT3 (JAK/STAT signaling pathway) and the phosphorylation level thereof in SK-Hep-1 cells was analyzed by immunoblotting experiments. It was found that compound 3 (10 μ M) significantly reduced the phosphorylation level of ERK without significant effect on other indicators. The compound 3 is shown to induce the apoptosis of the liver cancer cell by inhibiting RAS/ERK signal transduction path. See fig. 3.
Reference documents:
[1]Freddie B.B.M.,Jacques F.,Isabelle S.M.M.,et al.Global Cancer Statistics 2018:GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36Cancers in 185Countries[J].CA:A Cancer Journal for Clinicians,2018,68:394-424.
[2]Ma Z.-J.,Lu L.,Yang J.-J.,et al.Lariciresinol induces apoptosis in HepG2 cells via mitochondrial-mediated apoptosis pathway[J].European Journal of Pharmacology,2017,821:1-10.
[3] wang, xu Yixiang, han Xiao. Liver cancer treatment targets and related drug research progress [ J ]. Zhongnan pharmacology, 2018, 16 (149): 105-114.
[4]Llovet J.M.,Montal R.,Sia D.,et al.Molecular therapies and precision medicine for hepatocellular carcinoma[J].Nature Reviews Clinical Oncology,2018,15:599-616.
[5] Pan Tingting, cui Xiaonan. Pharmacotherapy of advanced primary liver cancer [ J ]. Journal of clinical hepatobiliary disease, 2020, 36 (1): 194-197.
While the invention has been described in detail in the foregoing by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that certain changes and modifications may be made therein based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (10)
1. The compound separated from the Yunnan rat poison is characterized in that the compound is a 2-aryl benzo [ b ] furan compound separated from the Yunnan rat poison, and the structural formula of the compound is as follows:
2. the compound of claim 1, wherein the compound is prepared by a method comprising:
1) Soaking and extracting 15-21Kg of dried and pulverized medicinal material powder with ethanol at normal temperature for 2-4 times, each time for 4-10 days;
2) After extraction, combining the extracting solutions, and keeping the temperature at 55 ℃; concentrating under reduced pressure of 0.08Mpa to obtain crude extract;
3) Dispersing the total extract into 12L water, sequentially extracting with petroleum ether 18-30L for 2-6 times, ethyl acetate 18-30L for 2-6 times, and n-butanol 18-30L for 2-6 times; respectively mixing the extractive solutions, concentrating under reduced pressure to remove solvent to obtain petroleum ether fraction, ethyl acetate fraction and n-butanol fraction;
4) Taking an ethyl acetate part, separating by silica gel column chromatography, and carrying out gradient elution by petroleum ether/ethyl acetate according to the proportion of 50 → 1, v/v, and dividing into 8 components E1-E8;
5) Further subjecting the component E5 to silica gel column chromatography, eluting with 3:1 petroleum ether/ethyl acetate to obtain 10 subfractions E5A-E5J;
6) Performing silica gel column chromatography on the component E5E, eluting by dichloromethane/ethyl acetate with the ratio of 50 to 1 to obtain a component E5E1, and performing pure crystallization on the component E by methanol to obtain a compound 1;
7) The fraction E5G was subjected to MCI column chromatography to 70% MeOH/H 2 Eluting with O to obtain a compound 2; subjecting the component E6 to Sephadex LH-20 gel column chromatography, eluting with DCM/MeOH at a ratio of 1:1 to obtain 7 components E6A-E6G; and (3) carrying out silica gel column chromatography on the component E6C, and eluting with DCM/MeOH with the ratio of 30.
3. The compound according to claim 2, wherein the ethanol in step 1) is 2-6 times the amount of 75-95% ethanol.
4. The compound of claim 3, wherein the ethanol in step 1) is 4-fold amount of 95% ethanol.
5. The compound of claim 2, wherein the number of extractions in step 1) is 3 for 7 days.
6. The compound of claim 2, wherein step 3) is performed by sequentially performing 24L petroleum ether extraction 4 times, 24L ethyl acetate extraction 4 times, and 24L n-butanol extraction 4 times.
7. The compound of claim 2, wherein the compound is formulated in a pharmaceutically acceptable dosage form with pharmaceutically acceptable excipients.
8. The compound of claim 7, wherein the pharmaceutically acceptable formulation is a solid formulation or a liquid formulation.
9. The compound of claim 8, wherein the solid preparation is a granule, a capsule, a tablet, a pill, a powder, a lyophilized powder for injection; the liquid preparation is injection preparation and oral liquid.
10. A compound according to any one of claims 1 to 9 for use in the manufacture of a medicament for the treatment of liver cancer.
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Non-Patent Citations (3)
| Title |
|---|
| 3-Formyl-2-arylbenzofurans from the aerial parts of Itea ilicifolia;Guoyong Luo et al;《Phytochemistry Letters》;20140614;19-22 * |
| Guoyong Luo et al.3-Formyl-2-arylbenzofurans from the aerial parts of Itea ilicifolia.《Phytochemistry Letters》.2014,19-22. * |
| LIGNANS AND NOR-NEOLIGNANS FROM KRAMERZA INTERIOR*;Xorge A. et al;《Phytochemistry》;19900831;2651-2653 * |
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