CN109206392B - Coumarin compound and preparation method and application thereof - Google Patents
Coumarin compound and preparation method and application thereof Download PDFInfo
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- CN109206392B CN109206392B CN201710522864.3A CN201710522864A CN109206392B CN 109206392 B CN109206392 B CN 109206392B CN 201710522864 A CN201710522864 A CN 201710522864A CN 109206392 B CN109206392 B CN 109206392B
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- ZYGHJZDHTFUPRJ-UHFFFAOYSA-N benzo-alpha-pyrone Natural products C1=CC=C2OC(=O)C=CC2=C1 ZYGHJZDHTFUPRJ-UHFFFAOYSA-N 0.000 title claims abstract description 55
- 235000001671 coumarin Nutrition 0.000 title claims abstract description 54
- -1 Coumarin compound Chemical class 0.000 title claims abstract description 51
- 229960000956 coumarin Drugs 0.000 title claims abstract description 50
- 238000002360 preparation method Methods 0.000 title claims description 18
- 150000001875 compounds Chemical class 0.000 claims abstract description 34
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 36
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 36
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- 239000000243 solution Substances 0.000 claims description 30
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Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
- C07D311/06—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2
- C07D311/08—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2 not hydrogenated in the hetero ring
- C07D311/16—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2 not hydrogenated in the hetero ring substituted in position 7
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Heterocyclic Carbon Compounds Containing A Hetero Ring Having Oxygen Or Sulfur (AREA)
Abstract
The invention provides a coumarin compound shown as a formula (I). Compared with the prior art, the compound can enhance H in SH-SY5Y cells2O2The cell activity is reduced, the SOD activity is improved, the MDA content is reduced, and the SOD activity and the cell protection activity are stronger, so that the SOD can be used for developing anti-aging and cardiovascular and cerebrovascular disease related medicines.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a coumarin compound as well as a preparation method and application thereof.
Background
Radix Angelicae Pubescentis, recorded in section of "Chinese pharmacopoeia" 2015 of the edition, is a dried root of Angelica gigas Maxim.f. biservata Shan et Yuan of Angelica gigas of Umbelliferae, and is pungent, bitter and slightly warm in nature and taste. It belongs to kidney and bladder channels, has effects of dispelling pathogenic wind, removing dampness, relieving arthralgia and relieving pain, and can be used for treating arthralgia due to wind-cold-dampness, pain of waist and knees, shaoyin wind-cold headache, and headache due to wind-cold with dampness. The pubescent angelica is listed as the top grade in Shen nong Ben Cao Jing, mainly distributed in Hubei, Sichuan and Jiangxi of China, and has a long history of medication, and is a common traditional Chinese medicinal material.
The main chemical components of radix angelicae pubescentis are coumarin compounds, including simple coumarin, furocoumarin, pyranocoumarin, coumarin glycoside and dicoumarin, and volatile oils and a small amount of alkyne alkene, sesquiterpene, steroid and alkaloid compounds. Modern pharmacological studies show that the radix angelicae pubescentis extract has the effects of calming, hypnotizing, easing pain and the like, and the radix angelicae pubescentis and parasitic loranthus decoction has an obvious anti-inflammatory effect; in addition, researches show that the radix angelicae pubescentis has the activities of regulating angiotensin receptors, calcium channel blocker receptors and the like, and has the effects of reducing blood pressure and resisting arrhythmia; and has lipolysis promoting and antitumor effects.
Cardiovascular and cerebrovascular diseases are the general names of cardiovascular and cerebrovascular diseases, and generally refer to ischemic or hemorrhagic diseases of heart, brain and systemic tissues caused by hyperlipidemia, blood hyperviscosity, atherosclerosis, hypertension and the like. Cardiovascular and cerebrovascular diseases have high prevalence rate, high disability rate and high death rate, and become the first killer seriously threatening human health worldwide. With the economic development, the living standard of people is gradually improved, the living rhythm is continuously accelerated, the incidence rate of cardiovascular and cerebrovascular diseases is continuously increased, and the medicine requirement for preventing and treating the cardiovascular and cerebrovascular diseases is increased. The activity of antioxidant SOD and the content of free radical metabolite MDA are important indexes for detecting antioxidant activity, scientific research shows that the occurrence of human diseases has a certain relation with oxygen free radicals, most of cancers, aging or other diseases are related to the generation of excessive free radicals, and the antioxidant can effectively overcome the harm brought by the antioxidant.
The invention provides a novel coumarin compound with strong antioxidant activity.
Disclosure of Invention
In view of this, the technical problem to be solved by the present invention is to provide a coumarin compound with a strong antioxidant activity, and a preparation method and an application thereof.
The invention provides a coumarin compound, which is shown as a formula (I):
the invention also provides a preparation method of the coumarin compound, which comprises the following steps:
s1) extracting radix angelicae pubescentis with an alcohol solvent to obtain an extract;
s2) separating the extractum by silica gel column chromatography, reversed phase column chromatography, gel column chromatography and preparative high performance liquid chromatography in sequence to obtain a compound shown in the formula (I);
preferably, the mass ratio of the radix angelicae pubescentis to the alcohol solvent is 1: (6-10).
Preferably, the eluent used for the silica gel column chromatography is one or more of petroleum ether, ethyl acetate, dichloromethane, acetone and methanol.
Preferably, the instrument used for the reversed phase column chromatography is medium and low pressure preparative chromatography, high performance liquid chromatography or dynamic axial compression chromatography; the mobile phase used for the reverse phase chromatography is an aqueous solution of an organic solvent; the organic solvent is methanol or acetonitrile.
Preferably, the elution of the reverse phase column chromatography is a gradient elution; the gradient elution degree is, in volume fraction: 0-20 min, 10-35% of water solution of organic solvent; 20-60 min, 35-65% of water solution of organic solvent; 60-80 min, 65-100% organic solvent water solution.
Preferably, the gel in the gel column chromatography is Sephadex LH-20, Sephadex G15 or Sephadex G50.
Preferably, the mobile phase of preparative high performance liquid chromatography is methanol-water solution or acetonitrile-water solution.
The invention also provides an application of the coumarin compound in preparing antioxidant drugs.
The invention also provides a pharmaceutical preparation which comprises the coumarin compound and a pharmaceutically acceptable carrier.
The invention provides a coumarin compound shown as a formula (I). Compared with the prior art, the compound can enhance H in SH-SY5Y cells2O2Resulting in decreased cell viability, increased SOD activity, and decreased MDA content, exhibiting strong antioxidant and cell differentiationHas protective activity, and can be used for developing anti-aging and improving cardiovascular and cerebrovascular diseases related drugs.
Drawings
FIG. 1 is an ESI-MS spectrum of coumarin compound shown in formula (I) obtained in example 1;
FIG. 2 shows coumarin compounds represented by formula (I) obtained in example 1 of the present invention1H-NMR spectrum;
FIG. 3 shows coumarin compounds represented by formula (I) obtained in example 1 of the present invention13A C-NMR spectrum;
FIG. 4 is DEPT spectrogram of coumarin compound shown in formula (I) obtained in example 1 of the present invention;
FIG. 5 is an HSQC spectrum of coumarin compound shown in formula (I) obtained in example 1;
FIG. 6 shows an HMBC spectrum of the coumarin compound shown in the formula (I) obtained in example 1 of the present invention;
FIG. 7 shows the NOESY spectrum of coumarin compound represented by formula (I) obtained in example 1.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention provides a coumarin compound, which is shown as (I):
the compound can enhance H in SH-SY5Y cells2O2Resulting in decreased cell activity, increased SOD activity, reduced MDA content, and strong antioxidant and cell protecting activity, and can be used for developing medicine for resisting aging and improving cardiovascular disease and cerebrovascular diseaseA compound (I) is provided.
The invention also provides a preparation method of the coumarin compound, which comprises the following steps:
s1) extracting radix angelicae pubescentis with an alcohol solvent to obtain an extract;
s2) separating the extractum by silica gel column chromatography, reversed phase column chromatography, gel column chromatography and preparative high performance liquid chromatography in sequence to obtain a compound shown in the formula (I);
according to the invention, firstly, the radix angelicae pubescentis is extracted by an alcohol solvent to obtain an extract; preferably, the radix angelicae pubescentis is mixed with a solvent for extraction, and an extract is obtained after an extracting solution is concentrated; the alcohol solvent is preferably ethanol, and is more preferably ethanol with the volume fraction of 50-95%; the mass ratio of the radix angelicae pubescentis to the alcohol solvent is preferably 1: (6-10), more preferably 1: (8-10); in some embodiments provided by the present invention, the ratio of the doubleteeth pubescent angilica root to the alcohol solvent is preferably 1: 6; in some embodiments provided by the present invention, the ratio of the pubescent angelica root to the alcohol solvent is preferably 1: 8; in other embodiments provided by the present invention, the mass ratio of the radix angelicae pubescentis to the alcohol solvent is preferably 1: 10; the extraction method is a method well known to those skilled in the art, and is not particularly limited, and the reflux method is adopted for extraction in the invention; in order to improve the yield, multiple times of extraction can be carried out; the extraction frequency is preferably 1-3 times, and more preferably 2-3 times; the extraction time of each time is preferably 1-3 h; in some embodiments provided herein, the time for each extraction is preferably 1.5 hours; in some embodiments provided herein, the time for each extraction is preferably 3 hours; in other embodiments of the present invention, the time for each extraction is preferably 2 h.
Separating the extract, preferably sequentially performing silica gel column chromatography, reversed phase column chromatography, gel column chromatography and preparative high performance liquid chromatography to obtain the compound shown in formula (I).
The silica gel column chromatography is well known to those skilled in the art, and is not particularly limited; the silica gel used for the column chromatography is preferably 80-100 meshes; the mass ratio of the extract to the silica gel sample is preferably 1: (1-2), more preferably 1: (1-1.5), preferably 1: 1.3; the eluent used for the silica gel column chromatography is preferably one or more of petroleum ether, ethyl acetate, dichloromethane, acetone and methanol, and more preferably a mixed solution of dichloromethane and acetone; the elution is preferably a gradient elution; the volume ratio of the dichloromethane to the acetone is preferably (1-50): 1, collecting dichloromethane and acetone in a volume ratio of (1-30): 1 to obtain a crude product after silica gel column chromatographic separation.
Separating the crude product after the silica gel column chromatographic separation by reverse phase column chromatography; the reverse phase column chromatography is the reverse phase column chromatography well known to those skilled in the art, and is not particularly limited, and the instrument used in the method is preferably medium-low pressure preparative chromatography, high performance liquid chromatography or dynamic axial compression chromatography, and more preferably dynamic axial compression chromatography; the mobile phase of the reversed phase column chromatography is preferably an aqueous solvent of an organic solvent; the organic solvent is preferably methanol or acetonitrile; in order to sufficiently separate the desired compound from impurities, it is preferable to perform gradient elution; the gradient elution procedure is preferably 0-20 min, and the gradient elution procedure is 10% -35% of an organic solvent aqueous solution; 20-60 min, 35-65% of water solution of organic solvent; 60-80 min, 65-100% organic solvent water solution; and collecting the eluted part for 35-60 min to obtain the crude product after reversed phase column chromatographic separation.
Then separating the crude product after the reverse phase column chromatographic separation by gel column chromatography; the gel column chromatography is a gel column chromatography well known to those skilled in the art, and is not particularly limited, and the gel in the gel column chromatography in the present invention is preferably Sephadex LH-20, Sephadex G15 or Sephadex G50, and more preferably Sephadex LH-20; the eluent of the gel column chromatography is preferably an alcohol solvent, more preferably methanol; eluting to obtain crude product after gel column chromatographic separation.
Finally, separating the crude product after the gel column chromatographic separation by using a preparative high performance liquid chromatography to obtain the coumarin compound shown in the formula (I); the preparative high performance liquid chromatography is not particularly limited as long as the preparative high performance liquid chromatography is well known to those skilled in the art, and the column in the invention is preferably a Polar phenyl column, more preferably Polar-rp; the specification of the chromatographic column is preferably 21.2X 250mm, 5 μm; the mobile phase of the preparative high performance liquid chromatography is preferably methanol-water solution or acetonitrile-water solution; the volume fraction of the organic solvent in the methanol-water solution or the acetonitrile-water solution is preferably 20 to 35 percent; the flow rate of the mobile phase is preferably 3-100 mL/min, more preferably 5-80 mL/min, still more preferably 10-60 mL/min, and most preferably 15-30 mL/min; the detection wavelength of the preparative high performance liquid chromatography is preferably 250 nm.
The coumarin compound shown in the formula (I) provided by the invention is colorless needle-shaped crystal, and the reaction of ferric trichloride is positive, and the reaction of ferric hydroxamate is positive. High resolution mass spectrum HR-ESI-MS gives M/z229.0939[ M-H [ ]]-，m/z253.0843[M+Na]+Molecular weight of the compound 230, binding element analysis and13the C-NMR spectrum and the DEPT spectrum conclude that the compound has the molecular formula of C14H14O3。
The invention carries out structure identification on the compound obtained by separation, and finally determines that the compound is (Z) -7-hydroxy-8- (2-methyllbut-2-enyl) coumarins, the structure of which is shown as the formula (I), and the coumarin compound is a novel coumarin compound. All hydrocarbon signal assignments are given in table 1, table 1 being the assignment of the individual carbons and hydrogens of the coumarins of formula (I).
TABLE 1 Nuclear magnetic data (CDCl) of coumarins of formula (I)3,1H-NMR 400MHz,13C-NMR 100MHz)
The invention also provides application of the coumarin compound shown in the formula (I) in preparation of antioxidant drugs.
The invention also provides a pharmaceutical preparation which comprises the coumarin compound shown in the formula (I) and a pharmaceutically acceptable carrier.
The pharmaceutically acceptable carrier can be properly selected according to the common auxiliary materials in the pharmaceutical field and according to the dosage form and the actual condition, for example, the common auxiliary materials comprise starch, low-substituted hydroxypropyl cellulose, aerosil, magnesium stearate, starch slurry, sucrose, dextrin, sodium carboxymethyl starch, talcum powder, polysorbate, polyethylene glycol, soybean phospholipid for injection, glycerol for injection and the like; when the coumarin compound shown in the formula (I) obtained by the invention is used for preparing various dosage forms of required medicines, the coumarin compound can be prepared according to the conventional production method in the field of pharmacy, for example, the extract is mixed with one or more carriers and then prepared into corresponding dosage forms. Preferably, the dosage form of the Chinese medicinal preparation comprises injection, tablets, suppositories, ointments, gels, pills, tablets, granules, capsules and mixtures.
The invention extracts the radix angelicae pubescentis medicinal material to obtain extract; then separating the obtained extract, selecting a compound with specific peak-out time to obtain the coumarin compound shown in the formula (I), and finding through cell experiments that the compound can enhance H in SH-SY5Y cells2O2Resulting in decreased cell viability, increased SOD activity, and decreased MDA content, showing stronger antioxidant and cytoprotective activity.
In order to further describe the present invention, the following embodiments are provided to describe a coumarin compound, its preparation method and its application in detail.
The reagents used in the following examples are all commercially available.
Example 1
1.1, 10kg of radix angelicae pubescentis medicinal material is added with 10 times of 50 percent ethanol water solution by volume fraction, the mixture is extracted for 3 times, each time lasts for 1.5h, and the filtrate is filtered, concentrated and the ethanol is recovered to obtain extract.
1.2 mixing the extract obtained in the step 1.1 with 80-100 mesh silica gel in a mass ratio of 1:1.3, performing silica gel column chromatographic separation, and performing gradient elution by using a dichloromethane-acetone mixed solution; and the volume ratio of dichloromethane to acetone is (1-50): 1, and the elution part with the volume ratio of dichloromethane to acetone being (1-30): 1 is collected to obtain the crude product after the chromatographic separation of the silica gel column.
1.3, separating the crude product obtained in the step 1.2 after the silica gel column chromatographic separation by reversed-phase dynamic axial compression column chromatography, and performing gradient elution by using acetonitrile-water solution, wherein the elution procedure is 0-20 min and is 10-35%; 35-65% of 20-60 min; collecting the elution part for 35-60 min after 60-80 min and 65-100% acetonitrile-water solution, and obtaining the crude product after reversed phase column chromatographic separation.
1.4 taking the crude product obtained in the step 1.3 after the reversed phase column chromatographic separation, carrying out Sephadex LH-20 gel column chromatographic separation, eluting with methanol, and purifying to obtain a purified sample.
1.5 taking the purified sample obtained in the step 1.4, and separating by preparative high performance liquid chromatography with Polar-rp 21.2X 250mm and 5 μm as a chromatographic column; eluting with 35% acetonitrile-water solution as mobile phase at flow rate of 15 mL/min; detecting wavelength at 250nm, collecting eluate, and drying under reduced pressure to obtain coumarin compound 7mg shown in formula (I) with purity of 98.5%.
The coumarin compound shown in the formula (I) obtained in the example 1 is colorless needle-shaped crystal, and the reaction of ferric trichloride is positive, and the reaction of ferric hydroxamate is positive. High resolution mass spectrum HR-ESI-MS gives M/z229.0939[ M-H [ ]]-，m/z253.0843[M+Na]+Molecular weight of the compound 230, binding element analysis and13the molecular formula of the compound is deduced to be C by C-NMR spectrum and DEPT spectrum14H14O3。
The compound obtained in example 1 is subjected to structure identification, the results are shown in figures 1-7, and figure 1 shows the incense shown in formula (I) obtained in example 1 of the inventionESI-MS spectrogram of coumarin compound, FIG. 2 is coumarin compound represented by formula (I) obtained in example 1 of the present invention1FIG. 3 is a H-NMR spectrum of a coumarin compound represented by the formula (I) obtained in example 1 of the present invention13A C-NMR spectrum, fig. 4 is a DEPT spectrum of the coumarin compound represented by formula (I) obtained in example 1 of the present invention, fig. 5 is an HSQC spectrum of the coumarin compound represented by formula (I) obtained in example 1 of the present invention, fig. 6 is an HMBC spectrum of the coumarin compound represented by formula (I) obtained in example 1 of the present invention, and fig. 7 is a NOESY spectrum of the coumarin compound represented by formula (I) obtained in example 1 of the present invention.
Analysis of FIGS. 1 to 7 shows that the compound obtained in example 1 has1H-NMR(CDCl3400MHz) spectrum (see fig. 2) giving a set of coumarin characteristic proton signals δ 6.25(1H, d, J ═ 9.4Hz, H-3), δ 7.65(1H, d, J ═ 9.4Hz, H-4), δ 7.23(1H, d, J ═ 8.4Hz, H-5), δ 6.86(1H, d, J ═ 8.4Hz, H-6); the compound is presumed to be 7, 8-disubstituted coumarin by combining the coupling constants of the 5-position proton signals and the 6-position proton signals. One methylene proton signal δ 3.60(2H, d, J ═ 7.2Hz, H-1'); a methine proton signal δ 5.28(1H, m, H-3'); two methyl proton signals delta 1.85(3H, s, 4' -CH)3)，δ1.73(3H，d,J＝0.9Hz,5′-CH3) And through 5' -CH3The proton coupling constant infers that 4 '-C and 5' -C are connected to two different C, respectively;13C-NMR(CDCl3100MHz) spectrum (see fig. 3), giving a total of 14 carbon signals, 9 coumarin nucleus carbon signals, wherein δ 162.2 is the characteristic carbon signal at position 2 of coumarin nucleus; the DEPT spectra (see FIG. 4) show δ 18.0 and δ 025.8 as two methyl carbon signals; δ 122.1 is a methylene carbon signal. In combination with the HMBC spectrum (see FIG. 6), the hydrocarbon signals with correlation are assigned, and delta 27.65(H-4) is related to delta 3162.2(C-2), delta 4153.2(C-9) and delta 5126.6 (C-5); delta 67.23(H-5) is related to delta 7144.5(C-4), delta 8158.6(C-7), delta 9153.2 (C-9); delta 6.86(H-6) is related to delta 0112.6(C-10), delta 1115.1 (C-8); delta 6.25(H-3) is related to delta 162.2(C-2), delta 112.6 (C-10); δ 3.60(H-1 ') is remotely related to δ 158.6(C-7), δ 153.2(C-9), 120.4(C-3 '), and 25.8(C-5 '); delta.5.28 (H-3 ') with 25.8 (C-5'), 18.0(C-4 ') and 22.1 (C-1') remoteCorrelation; delta.1.85 (4' -CH)3)、δ1.73(5′-CH3) With 120.4(C-3 ') and 135.4 (C-2') remote. NOESY spectra (see FIG. 7) show delta 5.28(H-3 ') vs. delta 1.73 (5' -CH)3) Has a correlation relationship, thereby determining that the connection directions of C-4 'and C-5' are opposite. The compound is determined to be (Z) -7-hydroxy-8- (2-methyllbut-2-enyl) coumarins by integrating the spectrogram data analysis, and is a novel coumarin compound.
Example 2
2.1 adding 10kg of radix angelicae pubescentis medicinal material into 6 times of 95% ethanol water solution by volume fraction, extracting for 2 times, 3h each time, filtering, concentrating the filtrate, and recovering ethanol to obtain extract.
2.2 mixing the extract obtained in the step 2.1 with 80-100 mesh silica gel in a mass ratio of 1:1.3, performing chromatographic separation on the mixture by using a silica gel column, and performing gradient elution by using a dichloromethane-acetone mixed solution; and the volume ratio of dichloromethane to acetone is (1-50): 1, and the elution part with the volume ratio of dichloromethane to acetone being (1-30): 1 is collected to obtain the crude product after the chromatographic separation of the silica gel column.
2.3, separating the crude product obtained in the step 2.2 after the silica gel column chromatographic separation by reversed-phase dynamic axial compression column chromatography, and performing gradient elution by using acetonitrile-water solution, wherein the elution procedure is 0-20 min and 10-35%; 35-65% for 20-60 min; and (3) collecting 35-60 min elution parts after 60-80 min and 65-100% acetonitrile-water solution to obtain a crude product after reverse phase column chromatographic separation.
2.4 taking the crude product obtained in the step 2.3 after the reverse phase column chromatographic separation, carrying out Sephadex LH-20 gel column chromatographic separation, eluting with methanol, and purifying to obtain a purified sample.
2.5 taking the purified sample obtained in the step 2.4, and separating by preparative high performance liquid chromatography by using Polar-rp 21.2X 250mm and 5 mu m as a chromatographic column; eluting with 20% acetonitrile-water solution as mobile phase at flow rate of 30 mL/min; detecting wavelength at 250nm, collecting eluate, and drying under reduced pressure to obtain coumarin compound 8mg shown in formula (I) with purity of 98.6%.
The structure of the compound obtained by the invention is shown as the formula (I) by analyzing the detection result.
Example 3
3.1 adding 10kg of pubescent angelica root into 8 times of 75 percent ethanol water solution by volume, extracting for 2 times, each time for 2 hours, filtering, concentrating the filtrate, and recovering ethanol to obtain extract.
3.2 mixing the extract obtained in the step 3.1 with 80-100 mesh silica gel in a mass ratio of 1:1.3, performing chromatographic separation on the mixture by using a silica gel column, and performing gradient elution by using a dichloromethane-acetone mixed solution; and the volume ratio of dichloromethane to acetone is (1-50): 1, and the elution part with the volume ratio of dichloromethane to acetone being (1-30): 1 is collected to obtain the crude product after the chromatographic separation of the silica gel column.
3.3, separating the crude product obtained in the step 3.2 after the silica gel column chromatographic separation by reversed-phase dynamic axial compression column chromatography, and performing gradient elution by using acetonitrile-water solution, wherein the elution procedure is 0-20 min and 10-35%; 35-65% for 20-60 min; and (3) collecting 35-60 min elution parts after 60-80 min and 65-100% acetonitrile-water solution to obtain a crude product after reverse phase column chromatographic separation.
3.4 taking the crude product obtained in the step 3.3 after the reverse phase column chromatographic separation, carrying out Sephadex LH-20 gel column chromatographic separation, eluting with methanol, and purifying to obtain a purified sample.
3.5 taking the purified sample obtained in the step 3.4, and separating by preparative high performance liquid chromatography with Polar-rp 21.2X 250mm and 5 μm as a chromatographic column; eluting with 30% acetonitrile-water solution as mobile phase at flow rate of 20 mL/min; detecting wavelength at 250nm, collecting eluate, and drying under reduced pressure to obtain coumarin compound 8mg shown in formula (I) with purity of 98.7%.
The structure of the compound obtained by the invention is shown as the formula (I) by analyzing the detection result.
Example 4 coumarin compound pairs H of formula (I)2O2Study on protective effect of SH-SY5Y cell injury
1. Material
1.1 medicine: coumarin compound shown in formula (I).
1.2 cells: SH-SY5Y human neuroblastoma cell line, Shanghai cell bank in the China academy of sciences type culture Collection.
The culture conditions are as follows: DMEM + 10% fetal bovine serum, 37 ℃, 5% CO2An incubator.
1.3 Instrument and reagents: super clean bench (sujing antai); carbon dioxide incubator (Thermo scientific); microplate reader (MD); centrifuge (Beijing Zhongyi neutralization Biotechnology Co., Ltd.); an automated cell counter (invitrogen); a high-pressure steam sterilizing boiler (BXM-30R vertical pressure steam sterilizing boiler); inverted microscope (OLYPUS), 96-well cell culture plate (Costar), 25cm2Cell culture flasks (Costar), pipette gun (eppendorf). DMEM medium (gibco), fetal bovine serum (Hyclone), trypsin (gibco), lps (sigma), dmso (sigma), total SOD activity assay kit (bio-technical institute in cloudy days).
2. Experimental methods and procedures
Preparing a liquid medicine: the drugs were dissolved in DMSO to prepare a 1mol/L stock solution. When in use, the coumarin compound liquid medicine is respectively diluted into coumarin compound liquid medicine with high, medium and low concentrations of 50, 25 and 12.5 mu mol/L shown in the formula (I).
The experimental method comprises the following steps: the cells were cultured at 1X 105The cells were plated at a concentration of 100. mu.L/well in 96-well cell culture plates at 37 ℃ with 5% CO2After culturing in a cell incubator for 24 hours, the supernatant is aspirated and randomly divided into a blank group, a model group and an administration group according to experimental requirements, wherein each group has 3 compound wells. The blank group was administered with 100. mu.L of serum-free DMEM medium, and the model group and the administration group were each administered with 100. mu.L of H at a concentration of 200. mu. mol/L2O2A serum-free medium; h2O2After 3 hours of oxidative damage, 100. mu.L of complete medium was administered to the blank group and the model group, and 100. mu.L of the compound represented by formula I was administered to the administration group at concentrations of 50. mu. mol/L, 25. mu. mol/L, and 12.5. mu. mol/L, respectively, to the high, medium, and low dose groups. PBS washing for 3 times, 3000 r.min-1Centrifuging for 5min, removing supernatant, adding cell lysate, and measuring SOD activity and MDA content according to kit instructions.
The experimental results are as follows: h2O2After the injury and the molding, the activity of SH-SY5Y cells is reduced, the SOD activity is obviously reduced, and the MDA content is increased. Adding high, medium and low dosage respectivelyAfter the compound shown in the formula I, the SOD activities of the high-dose and medium-dose groups are obviously improved compared with the SOD activities of the model group, the MDA content is reduced, and the compound has obvious difference (P)<0.01，P<0.05), there was no significant difference in the low dose group. Data results are shown in table 2.
TABLE 2 coumarin compound pair H shown in formula (I)2O2Damage to SH-SY5Y Effect of intracellular SOD Activity and MDA content (x + -s, n ═ 6)
# P <0.01 compared to blank group, # P <0.01, P <0.05 compared to model group.
4. Conclusion
The compound of the invention can enhance H in SH-SY5Y cells2O2Resulting in decreased cell viability, increased SOD activity, and decreased MDA content, showing stronger antioxidant and cytoprotective activity.
Example 5 preparation of tablet drug from coumarin compound represented by formula (I)
350g of the compound with the structure shown in the formula I, 50g of starch, 7.5g of sodium carboxymethyl starch, 0.8g of talcum powder, 50g of dextrin, 0.8g of magnesium stearate and a proper amount of 10% starch slurry are mixed appropriately, and 1000 tablets of the compound with the structure shown in the formula I are prepared according to a conventional method. The preparation is administered 1 tablet 3 times daily.
Example 6 preparation of a pill drug from coumarin compound represented by formula (I)
350g of the compound with the structure shown in the formula I, 12g of polyethylene glycol-6000, 80.5g of polysorbate-80 and a proper amount of liquid paraffin are mixed, and 1000 pills of the compound with the structure shown in the formula I are prepared according to a conventional method. The preparation is administered 1 granule 3 times daily.
Example 7 preparation of an injectable drug from coumarin compound represented by formula (I)
200g of the compound with the structure shown in the formula I, 15g of soybean lecithin for injection and 25g of glycerol for injection are mixed, the volume of water for injection is set to 1000mL, and 1000 compound injections with the structure shown in the formula I are prepared according to a conventional method. 1 time daily, 1 branch each time, at least 250mL 5% glucose injection diluted and intravenous drip.
Claims (7)
1. The preparation method of the coumarin compound is characterized by comprising the following steps:
s1) extracting radix angelicae pubescentis with an alcohol solvent to obtain an extract;
s2) separating the extractum by silica gel column chromatography, reversed phase column chromatography, gel column chromatography and preparative high performance liquid chromatography in sequence to obtain a compound shown in the formula (I);
2. the preparation method according to claim 1, wherein the mass ratio of the radix angelicae pubescentis to the alcohol solvent is 1: (6-10).
3. The preparation method according to claim 1, wherein the eluent used for the silica gel column chromatography is one or more of petroleum ether, ethyl acetate, dichloromethane, acetone and methanol.
4. The preparation method according to claim 1, wherein the apparatus for the reversed-phase column chromatography is medium-low pressure preparative chromatography, high performance liquid chromatography or dynamic axial compression chromatography; the mobile phase used for the reverse phase chromatography is an aqueous solution of an organic solvent; the organic solvent is methanol or acetonitrile.
5. The production method according to claim 1, wherein the elution by reverse phase column chromatography is gradient elution; the gradient elution degree is, in volume fraction: 0-20 min, 10-35% of water solution of organic solvent; 20-60 min, 35-65% of water solution of organic solvent; 60-80 min, 65-100% organic solvent water solution.
6. The method according to claim 1, wherein the gel in the gel column chromatography is Sephadex LH-20, Sephadex G15 or Sephadex G50.
7. The method according to claim 1, wherein the mobile phase of preparative high performance liquid chromatography is methanol-water solution or acetonitrile-water solution.
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