CN109206392A - A kind of coumarin kind compound and the preparation method and application thereof - Google Patents
A kind of coumarin kind compound and the preparation method and application thereof Download PDFInfo
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- CN109206392A CN109206392A CN201710522864.3A CN201710522864A CN109206392A CN 109206392 A CN109206392 A CN 109206392A CN 201710522864 A CN201710522864 A CN 201710522864A CN 109206392 A CN109206392 A CN 109206392A
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- 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
Abstract
The present invention provides a kind of coumarin kind compounds, as shown in formula (I).Compared with prior art, which can enhance H in SH-SY5Y cell2O2Caused cell viability decline, improves SOD activity, and reduce MDA content, shows compared with strong anti-oxidation and cell protection activity, can be used for developing anti-aging and improves cardiovascular and cerebrovascular disease related drugs.
Description
Technical field
The invention belongs to pharmaceutical technology fields more particularly to a kind of coumarin kind compound and the preparation method and application thereof.
Background technique
Radix Angelicae Pubescentis, 2015 version " Chinese Pharmacopoeia " one record, be Umbelliferae archangel Angelica pubescens Angelica
The dry root of pubescens Maxim.f.biserrata Shan et Yuan, nature and flavor are pungent, bitter, tepor.Return kidney, bladder meridian,
With dispelling wind and eliminating dampness, the effect of numbness relieving and pain relieving, it is used for treating arthralgia due to cold and dampness, lumbocrural pain, few the moon volt wind headache, headache caused by cold and dampness etc.
Disease.Radix Angelicae Pubescentis is listed in top grade first recorded in Shennong's Herbal, is distributed mainly on the ground such as China Hubei, Sichuan, Jiangxi, and medication is gone through
History is long, to commonly use traditional Chinese medicine.
The main chemical compositions of Radix Angelicae Pubescentis are coumarin kind compound, including simple cumarin, furocoumarin, pyrans
Cumarin and coumarin and bicoumarin etc., except this there are also volatile oil and a small amount of ene-alkyne class, sesquiterpenoids, steroidal and
Alkaloid compound.Modern pharmacology research shows that Radix Angelicae Pubescentis medicinal extract has the effects that calmness, hypnosis, analgesia, duhuo jisheng decoction
With obvious anti-inflammatory effect；Separately some researches show that Radix Angelicae Pubescentis to have adjusting angiotensin receptor, calcium channel blocker receptor etc. living
Property, have the function of decompression and arrhythmia；Also have simultaneously and promotes lipolysis, antitumor isoreactivity.
Cardiovascular and cerebrovascular disease is the general designation of cardiovascular and cranial vascular disease, refers to due to hyperlipidemia, blood viscousness, moves
The ischemic or hemorrhagic disease that heart caused by pulse atherosclerosis, hypertension etc., brain and body tissue occur.Cardiovascular and cerebrovascular
Disease illness rate is high, disability rate is high, the death rate is high, has become and seriously threatens the No.1 of human health in worldwide and kill
Hand.With economic development, people's living standard is stepped up, and rhythm of life is constantly accelerated, and makes the disease incidence of cardiovascular and cerebrovascular disease
It is constantly soaring, and the pharmaceutical requirements for preventing and treating cardiovascular and cerebrovascular disease are consequently increased.Antioxidant SOD activity and free radical
Metabolite MDA content be detect antioxidant activity important indicator, scientific investigations showed that, the generation of human body diseases all with oxygen from
There is certain relationship by base, cancer, aging or Other diseases are mostly relevant with the generation of excessive free radicals, and antioxidant can
Effectively to overcome harm brought by it.
The present invention provides a kind of new coumarin kind compound with stronger antioxidant activity.
Summary of the invention
In view of this, the technical problem to be solved in the present invention is that providing a kind of tonka-bean with stronger antioxidant activity
Chlorins compound and the preparation method and application thereof.
The present invention provides a kind of coumarin kind compounds, as shown in formula (I):
The present invention also provides a kind of preparation methods of coumarin kind compound, comprising the following steps:
S1 Radix Angelicae Pubescentis is extracted with alcoholic solvent), obtains medicinal extract；
S2 the medicinal extract) is passed sequentially through into silica gel column chromatography, reversed-phase column chromatography, gel column chromatography and preparative efficient liquid phase
Chromatography is separated, and formula (I) compound represented is obtained；
Preferably, the mass ratio of the Radix Angelicae Pubescentis and alcoholic solvent is 1:(6~10).
Preferably, eluent used in the silica gel column chromatography is petroleum ether, ethyl acetate, methylene chloride, acetone and first
One of alcohol is a variety of.
Preferably, instrument used in the reversed-phase column chromatography is that mesolow prepares chromatography, high performance liquid chromatography or dynamic shaft
To compression chromatography；Mobile phase used in the reverse-phase chromatography is the aqueous solution of organic solvent；The organic solvent is methanol or second
Nitrile.
Preferably, the elution of the reversed-phase column chromatography is gradient elution；With volume fraction, the gradient elution degree
Are as follows: 0~20min, the aqueous solution of 10%~35% organic solvent；20~60min, the aqueous solution of 35%~65% organic solvent；
60~80min, the aqueous solution of 65%~100% organic solvent.
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-aqueous solution.
The present invention also provides a kind of above-mentioned coumarin kind compounds to prepare the application in anti-oxidant class drug.
The present invention also provides a kind of pharmaceutical preparations, including above-mentioned coumarin kind compound and pharmaceutically acceptable load
Body.
The present invention provides a kind of coumarin kind compounds, as shown in formula (I).Compared with prior art, the compound energy
Enough enhance H in SH-SY5Y cell2O2Caused cell viability decline, improves SOD activity, and reduce MDA content, shows relatively strong
Anti-oxidant and cell protection activity can be used for developing anti-aging and improve cardiovascular and cerebrovascular disease related drugs.
Detailed description of the invention
The ESI-MS spectrogram of coumarin kind compound shown in the formula (I) that Fig. 1 obtains for the embodiment of the present invention 1；
Coumarin kind compound shown in the formula (I) that Fig. 2 obtains for the embodiment of the present invention 11H-NMR spectrum；
Coumarin kind compound shown in the formula (I) that Fig. 3 obtains for the embodiment of the present invention 113C-NMR spectrogram；
The DEPT spectrogram of coumarin kind compound shown in the formula (I) that Fig. 4 obtains for the embodiment of the present invention 1；
The hsqc spectrum figure of coumarin kind compound shown in the formula (I) that Fig. 5 obtains for the embodiment of the present invention 1；
The HMBC spectrogram of coumarin kind compound shown in the formula (I) that Fig. 6 obtains for the embodiment of the present invention 1；
The NOESY spectrogram of coumarin kind compound shown in the formula (I) that Fig. 7 obtains for the embodiment of the present invention 1.
Specific embodiment
Below in conjunction with the embodiment of the present invention, technical scheme in the embodiment of the invention is clearly and completely described,
Obviously, described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.Based in the present invention
Embodiment, every other embodiment obtained by those of ordinary skill in the art without making creative efforts, all
Belong to the scope of protection of the invention.
The present invention provides a kind of coumarin kind compounds, such as shown in (I):
The compound can enhance H in SH-SY5Y cell2O2Caused cell viability decline, improves SOD activity, and reduce
MDA content is shown compared with strong anti-oxidation and cell protection activity, be can be used for developing anti-aging and is improved cardiovascular and cerebrovascular disease correlation
Drug.
The present invention also provides a kind of preparation methods of above-mentioned coumarin compound, comprising the following steps:
S1 Radix Angelicae Pubescentis is extracted with alcoholic solvent), obtains medicinal extract；
S2 the medicinal extract) is passed sequentially through into silica gel column chromatography, reversed-phase column chromatography, gel column chromatography and preparative efficient liquid phase
Chromatography is separated, and formula (I) compound represented is obtained；
According to the present invention, Radix Angelicae Pubescentis is extracted with alcoholic solvent first, obtains medicinal extract；Preferably, specially by Radix Angelicae Pubescentis with
Solvent mixing, extracts, after extracting solution is concentrated, obtains medicinal extract；The alcoholic solvent is preferably ethyl alcohol, is further preferably volume point
The ethyl alcohol that number is 50%~95%；The mass ratio of the Radix Angelicae Pubescentis and alcoholic solvent is preferably 1:(6~10), more preferably 1:(8~
10)；In some embodiments provided by the invention, the mass ratio of the Radix Angelicae Pubescentis and alcoholic solvent is preferably 1:6；It is provided in the present invention
Some embodiments in, the mass ratio of the Radix Angelicae Pubescentis and alcoholic solvent is preferably 1:8；In other embodiments provided by the invention
In, the mass ratio of the Radix Angelicae Pubescentis and alcoholic solvent is preferably 1:10；The method of the extraction is side well known to those skilled in the art
Method has no special limitation, is extracted in the present invention using the method for reflux；In order to improve yield, can carry out more
Secondary extraction；The number of the extraction is preferably 1~3 time, and more preferably 2~3 times；Each extraction time is preferably 1~3h；?
In some embodiments provided by the invention, the time extracted every time is preferably 1.5h；In some implementations provided by the invention
In example, the time extracted every time is preferably 3h；In other embodiments provided by the invention, it is described extract every time when
Between preferably 2h.
The medicinal extract is separated, silica gel column chromatography, reversed-phase column chromatography, gel column chromatography and preparation are preferably passed sequentially through
Type high performance liquid chromatography is separated, and formula (I) compound represented is obtained.
The silica gel column chromatography is silica gel column chromatography well known to those skilled in the art, has no special limitation；Institute
Stating column chromatography used silica gel is preferably 80~100 mesh；Medicinal extract and silica gel mixed sample mass ratio are preferably 1:(1~2), more preferably 1:
(1~1.5) is further preferably 1:1.3；Eluent used in the silica gel column chromatography is preferably petroleum ether, ethyl acetate, dichloromethane
One of alkane, acetone and methanol are a variety of, more preferably the mixed solution of methylene chloride and acetone；The elution is preferably ladder
Degree elution；The volume ratio of the methylene chloride and acetone is preferably (1~50): 1, it collects methylene chloride and acetone volume ratio is (1
~30): 1 elution position, the crude product after obtaining silica gel column chromatography separation.
Crude product after silica gel column chromatography is separated again is separated by reversed-phase column chromatography；The reversed-phase column chromatography is ability
Reversed-phase column chromatography known to field technique personnel, has no special limitation, instrument used in its in the present invention be preferably in it is low
Suppress standby chromatography, high performance liquid chromatography or dynamic axial compression chromatography, more preferably dynamic axial compression chromatography；The reversed-phase column
The mobile phase of chromatography is preferably the aqueous solvent of organic solvent；The organic solvent is preferably methanol or acetonitrile；In order to sufficiently by institute
Compound is needed to separate with impurity, preferably progress gradient elution；The program of the gradient elution is preferably 0~20min, 10%~
The aqueous solution of 35% organic solvent；20~60min, the aqueous solution of 35%~65% organic solvent；60~80min, 65%~
The aqueous solution of 100% organic solvent；35~60min elution fraction is collected, the crude product after obtaining reversed-phase column chromatography separation.
Then the crude product after reversed-phase column chromatography being separated is separated by gel column chromatography；The gel column chromatography is this
Gel column chromatography known to the technical staff of field has no special limitation, solidifying in heretofore described gel column chromatography
Glue is preferably Sephadex LH-20, Sephadex G15 or Sephadex G50, more preferably Sephadex LH-20；It is described
The eluent of gel column chromatography is preferably alcoholic solvent, more preferably methanol；Crude product after obtaining gel column chromatography separation after elution.
The crude product after gel column chromatography separation is separated by preparative high performance liquid chromatography finally, obtains formula
(I) coumarin kind compound shown in；The preparative high performance liquid chromatography is that preparative well known to those skilled in the art is high
Effect liquid phase chromatogram has no special limitation, its chromatographic column is preferably polarity phenyl column, more preferably Polar- in the present invention
rp；The specification of the chromatographic column is preferably 21.2 × 250mm, and 5 μm；The mobile phase of the preparative high performance liquid chromatography is preferably
Methanol-water solution or acetonitrile-aqueous solution；The volume fraction of organic solvent is preferred in the methanol-water solution or acetonitrile-aqueous solution
It is 20%~35%；The flow velocity of the mobile phase is preferably 3~100mL/min, more preferably 5~80mL/min, further preferably for
10~60mL/min, most preferably 15~30mL/min；The Detection wavelength of the preparative high performance liquid chromatography is preferably
250nm。
Coumarin kind compound shown in formula (I) provided by the invention is colorless needle crystals, and ferric chloride reaction is in sun
Property, the reaction of hydroxamic acid iron is positive.High resolution mass spectrum HR-ESI-MS provides m/z229.0939 [M-H]-, m/z253.0843
[M+Na]+, compound molecular weight 230, in conjunction with elemental analysis and13C-NMR spectrum and DEPT spectrum infer that the compound molecule formula is
C14H14O3。
The present invention carries out Structural Identification to isolated compound, final to determine that the compounds of this invention is (Z) -7-
Hydroxy-8- (2-methylbut-2-enyl) coumarin is a new Coumarins chemical combination shown in structure such as formula (I)
Object.All hydrocarbon signals assignments are shown in Table 1, and table 1 is each carbon of coumarin kind compound and the ownership of hydrogen shown in formula (I).
Nuclear magnetic data (the CDCl of coumarin kind compound shown in 1 formula of table (I)3,1H-NMR 400MHz,13C-NMR
100MHz)
The present invention also provides coumarin kind compounds shown in a kind of above-mentioned formula (I) in preparing anti-oxidant class drug
Using.
The present invention also provides a kind of pharmaceutical preparation, including coumarin kind compound shown in formula (I) with can pharmaceutically connect
The carrier received.
The pharmaceutically acceptable carrier can according to the common auxiliary material of art of pharmacy, according to dosage form and actual conditions into
The appropriate selection of row, for example, common auxiliary material have starch, low-substituted hydroxypropyl cellulose, superfine silica gel powder, magnesium stearate, starch slurry,
Sucrose, dextrin, sodium carboxymethyl starch, talcum powder, polysorbate, polyethylene glycol, injection soybean lecithin and glycerol for injection etc.；
It, can be according to medicine when the various dosage forms of drug needed for prepared by coumarin kind compound shown in the formula (I) obtained using the present invention
Prepared by the conventional production process in agent field, such as mix the extract with one or more carriers, corresponding agent is then made
Type.Preferably, the dosage form of the Chinese materia medica preparation include injection, tablet, suppository, ointment, gelling agent, pill, tablet,
Granula, capsule and mixture.
The present invention obtains medicinal extract by extracting to Radix Angelicae Pubescentis medicinal material；Then obtained medicinal extract is separated, selection is special
It makes the compound of peak time to find to get to coumarin kind compound shown in formula (I), and by cell experiment, the chemical combination
Object can enhance H in SH-SY5Y cell2O2Caused cell viability decline, improves SOD activity, and reduce MDA content, shows
Compared with strong anti-oxidation and cell protection activity.
In order to furtherly of the invention, with reference to embodiments to a kind of coumarin kind compound provided by the invention and its
Preparation method is described in detail with application.
Reagent used in following embodiment is commercially available.
Embodiment 1
1.1 Radix Angelicae Pubescentis medicinal material 10kg, be added 10 times amount volume fraction be 50% ethanol water, extract 3 times, every time
Filtrate concentration, recycling ethyl alcohol are obtained medicinal extract by 1.5h, filtering.
1.2 take medicinal extract obtained in step 1.1 and 80~100 mesh silica gel to carry out mixing sample with the ratio of mass ratio 1:1.3, warp
Silica gel column chromatography separation carries out gradient elution using dichloromethane-acetone mixed solution；The dichloromethane-acetone volume ratio
For (1~50): 1, collecting dichloromethane-acetone volume ratio is (1~30): 1 elution position, thick after obtaining silica gel column chromatography separation
Product.
1.3 take silica gel column chromatography obtained in step 1.2 separate after crude product, inverted dynamic axial compression column chromatography into
Row separation, with acetonitrile-aqueous solution carry out gradient elution, elution program be 0~20min, 10%~35%；20~60min, 35%
~65%；60~80min, 65%~100% acetonitrile-aqueous solution collect 35~60min and elute position, obtain reversed-phase column chromatography point
Crude product from after.
1.4 take the crude product after the separation of reversed-phase column chromatography obtained in step 1.3, through Sephadex LH-20 gel column chromatography
Separation, methanol elution, is purified, obtains sample after purification.
1.5 take the sample obtained in step 1.4 after purification, are separated by preparative high performance liquid chromatography, with
21.2 × 250mm of Polar-rp, 5 μm are chromatographic column；It is eluted using 35% acetonitrile-aqueous solution as mobile phase, flow velocity 15mL/
min；Detection wavelength is 250nm, collects eluent and is dried under reduced pressure, obtains coumarin kind compound 7mg shown in formula (I), purity
It is 98.5%.
Coumarin kind compound shown in the formula (I) that embodiment 1 obtains is colorless needle crystals, and ferric chloride reaction is in sun
Property, the reaction of hydroxamic acid iron is positive.High resolution mass spectrum HR-ESI-MS provides m/z229.0939 [M-H]-, m/z253.0843
[M+Na]+, compound molecular weight 230, in conjunction with elemental analysis and13C-NMR spectrum and DEPT spectrum infer that the compound molecule formula is
C14H14O3。
Structural Identification is carried out to the compound that embodiment 1 obtains, the result is shown in Figure 1~7, Fig. 1 is that the embodiment of the present invention 1 obtains
Formula (I) shown in coumarin kind compound ESI-MS spectrogram, Fig. 2 is perfume shown in the obtained formula (I) of the embodiment of the present invention 1
Beans chlorins compound1H-NMR spectrum, Fig. 3 are coumarin kind compound shown in the obtained formula (I) of the embodiment of the present invention 113C-NMR spectrogram, Fig. 4 are the DEPT spectrogram of coumarin kind compound shown in the obtained formula (I) of the embodiment of the present invention 1, and Fig. 5 is
The hsqc spectrum figure of coumarin kind compound shown in the formula (I) that the embodiment of the present invention 1 obtains, Fig. 6 are that the embodiment of the present invention 1 obtains
Formula (I) shown in coumarin kind compound HMBC spectrogram, Fig. 7 is tonka-bean shown in the obtained formula (I) of the embodiment of the present invention 1
The NOESY spectrogram of chlorins compound.
By analyzing FIG. 1 to FIG. 7, compound obtained in embodiment 11H-NMR(CDCl3, 400MHz) and spectrum
(see Fig. 2) provides one group of cumarin diagnostic protons signal 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)；In conjunction with 5 and 6 protons
The coupling constant of signal, thus it is speculated that the compound is 7,8 two substituted cumarins.One methene proton signal δ 3.60 (2H, d, J=
7.2Hz,H-1′)；One methine proton signal δ 5.28 (1H, m, H-3 ')；Two methyl proton signal δ 1.85 (3H, s, 4 '-
CH3), the (- CH of 3H, d, J=0.9Hz, 5 ' of δ 1.733), and pass through 5 '-CH3Proton coupling constant infer 4 '-C and 5 '-C respectively with
Two difference C are connected；13C-NMR(CDCl3, 100MHz) and spectrum (see Fig. 3), 14 carbon signals, 9 cumarin parent nucleus carbon are provided altogether
Signal, the feature carbon signal that wherein δ 162.2 is cumarin parent nucleus 2；DEPT composes (see Fig. 4) and shows that δ 18.0 and δ 25.8 is two
A methyl carbon signal；δ 22.1 is a mesomethylene carbon signal.(see Fig. 6) is composed in conjunction with HMBC, to the hydrocarbon letter with correlativity
Number belonged to, δ 7.65 (H-4) is related to δ 162.2 (C-2), δ 153.2 (C-9), δ 126.6 (C-5)；δ 7.23 (H-5) and δ
144.5 (C-4), δ 158.6 (C-7), δ 153.2 (C-9) are related；δ 6.86 (H-6) and δ 112.6 (C-10), δ 115.1 (C-8) phase
It closes；δ 6.25 (H-3) is related to δ 162.2 (C-2), δ 112.6 (C-10)；δ 3.60 (H-1 ') and δ 158.6 (C-7), δ 153.2
(C-9), 120.4 (C-3 ') are long-range related to 25.8 (C-5 ')；δ 5.28 (H-3 ') and 25.8 (C-5 '), 18.0 (C-4 ') and
22.1 (C-1 ') are long-range related；δ1.85(4′-CH3)、δ1.73(5′-CH3) with 120.4 (C-3 ') and the long-range phase of 135.4 (C-2 ')
It closes.NOESY composes (see Fig. 7) and shows δ 5.28 (H-3 ') and 1.73 (5 '-CH of δ3) there is correlativity, thereby determine that C-4 ' and C-
5 ' connection directions are opposite.In summary spectral data is analyzed, and determines that the compound is (Z) -7-hydroxy-8- (2-
Methylbut-2-enyl) coumarin is a new coumarin kind compound.
Embodiment 2
2.1 Radix Angelicae Pubescentis medicinal material 10kg, the volume fraction that 6 times of amounts are added is 95% ethanol water, is extracted 2 times, each 3h, mistake
Filtrate concentration, recycling ethyl alcohol are obtained medicinal extract by filter.
2.2 take medicinal extract obtained in step 2.1 and 80~100 mesh silica gel to carry out mixing sample with mass ratio 1:1.3 ratio, through silicon
Rubber column gel column chromatographic isolation carries out gradient elution using dichloromethane-acetone mixed solution；The dichloromethane-acetone volume ratio is
(1~50): 1, collecting dichloromethane-acetone volume ratio is (1~30): 1 elution position, thick after obtaining silica gel column chromatography separation
Product.
2.3 take silica gel column chromatography obtained in step 2.2 separate after crude product, inverted dynamic axial compression column chromatography into
Row separation, with acetonitrile-aqueous solution carry out gradient elution, elution program be 0~20min, 10~35%；20~60min, 35~
65%；60~80min, 65~100% acetonitrile-aqueous solutions collect 35~60min and elute position, after obtaining reversed-phase column chromatography separation
Crude product.
2.4 take the crude product after the separation of reversed-phase column chromatography obtained in step 2.3, through Sephadex LH-20 gel column chromatography
Separation, methanol elution, is purified, obtains sample after purification.
2.5 take the sample obtained in step 2.4 after purification, are separated by preparative high performance liquid chromatography, with
21.2 × 250mm of Polar-rp, 5 μm are chromatographic column；It is eluted using 20% acetonitrile-aqueous solution as mobile phase, flow velocity 30mL/
min；Detection wavelength is 250nm, collects eluent and is dried under reduced pressure, obtains coumarin kind compound 8mg shown in formula (I), purity
It is 98.6%.
By to Analysis of test results it is found that shown in the obtained compound structure such as formula (I) of the present invention.
Embodiment 3
3.1 Radix Angelicae Pubescentis medicinal material 10kg, the volume fraction that 8 times of amounts are added is 75% ethanol water, is extracted 2 times, each 2h, mistake
Filtrate concentration, recycling ethyl alcohol are obtained medicinal extract by filter.
3.2 take medicinal extract obtained in step 3.1 and 80~100 mesh silica gel to carry out mixing sample with mass ratio 1:1.3 ratio, through silicon
Rubber column gel column chromatographic isolation carries out gradient elution using dichloromethane-acetone mixed solution；The dichloromethane-acetone volume ratio is
(1~50): 1, collecting dichloromethane-acetone volume ratio is (1~30): 1 elution position, thick after obtaining silica gel column chromatography separation
Product.
3.3 take silica gel column chromatography obtained in step 3.2 separate after crude product, inverted dynamic axial compression column chromatography into
Row separation, with acetonitrile-aqueous solution carry out gradient elution, elution program be 0~20min, 10~35%；20~60min, 35~
65%；60~80min, 65~100% acetonitrile-aqueous solutions collect 35~60min and elute position, after obtaining reversed-phase column chromatography separation
Crude product.
3.4 take the crude product after the separation of reversed-phase column chromatography obtained in step 3.3, through Sephadex LH-20 gel column chromatography
Separation, methanol elution, is purified, obtains sample after purification.
3.5 take the sample obtained in step 3.4 after purification, are separated by preparative high performance liquid chromatography, with
21.2 × 250mm of Polar-rp, 5 μm are chromatographic column；It is eluted using 30% acetonitrile-aqueous solution as mobile phase, flow velocity 20mL/
min；Detection wavelength is 250nm, collects eluent and is dried under reduced pressure, obtains coumarin kind compound 8mg shown in formula (I), purity
It is 98.7%.
By to Analysis of test results it is found that shown in the obtained compound structure such as formula (I) of the present invention.
Coumarin kind compound shown in 4 formula of embodiment (I) is to H2O2The protective effect of caused SH-SY5Y cellular damage is ground
Study carefully
1. material
1.1 drugs: coumarin kind compound shown in formula (I).
1.2 cells: SH-SY5Y human neuroblastoma cells' strain, Chinese Academy of Sciences's Type Tissue Collection Shanghai
Cell bank.
Condition of culture: DMEM+10% fetal calf serum, 37 DEG C, 5%CO2Incubator.
1.3 instruments and reagent: superclean bench (Su Jing is safe and sound)；Carbon dioxide incubator (Thermo scientific)；
Microplate reader (MD)；Centrifuge (many benefits in Beijing neutralize Bioisystech Co., Ltd)；Auto-counting of Cells instrument (invitrogen)；It is high
It presses steam sterilization pan (BXM-30R vertical pressure steam sterilization pot)；Inverted microscope (OLYPUS), 96 porocyte culture plates
(Costar), 25cm2Tissue Culture Flask (Costar), liquid-transfering gun (eppendorf).DMEM culture medium (gibco), fetal calf serum
(Hyclone), trypsase (gibco), LPS (sigma), DMSO (sigma), (the green skies are raw for total SOD activity detection kit
Object technical research institute).
2. experimental method and step
The preparation of medical fluid: drug is dissolved with DMSO, and the stock solution of 1mol/L is made.Face the used time be diluted to respectively it is high, in,
Low concentration is coumarin kind compound medical fluid shown in the formula (I) of 50,25,12.5 μm of ol/L.
Experimental method: cell is with 1 × 105The concentration of a/mL is inoculated in 96 porocyte culture plates, every 100 μ L of hole, in 37
DEG C, 5%CO2After being cultivated 24 hours in cell incubator, inhale abandon supernatant, according to requirement of experiment be randomly divided into blank group, model group,
Administration group, every group of 3 multiple holes.Blank group gives 100 μ L plasma-free DMEM mediums, and model group and administration group give 100 μ respectively
The H that L is 200 μm of ol/L containing concentration2O2Serum free medium；H2O2After oxidative damage 3h, blank group and model group are given respectively
100 μ L complete mediums, administration group give 100 μ L concentration be respectively 50 μm of ol/L, 25 μm of ol/L, 12.5 μm of ol/L height, in,
Compound shown in the formula I of low dose group.PBS is cleaned 3 times, 3000rmin-1It is centrifuged 5min, inhales and abandons supernatant, cell is added and splits
After solving liquid, illustrate to measure SOD activity and MDA content according to kit.
Experimental result: H2O2After damaging modeling, SH-SY5Y cell viability decline, SOD activity is substantially reduced, MDA content liter
It is high.After being separately added into high, medium and low dosage compound of formula I, it is seen that high, middle dose group SOD activity is significantly raised compared with model group,
MDA content decreases, and has significant difference (P < 0.01, P < 0.05), there was no significant difference for low dose group.Data result is such as
Table 2.
Coumarin kind compound shown in 2 formula of table (I) is to H2O2Damage the intracellular SOD activity of SH-SY5Y and MDA content shadow
It rings (x ± s, n=6)
Compared with blank group, ##P < 0.01, compared with model group, P < 0.05 * * P < 0.01, *.
4. conclusion
The compounds of this invention can enhance H in SH-SY5Y cell2O2Caused cell viability decline improves SOD activity, and
MDA content is reduced, is shown compared with strong anti-oxidation and cell protection activity.
Coumarin kind compound shown in 5 formula of embodiment (I) prepares tablet medicine
By the compound of structure shown in 350g Formulas I and 50g starch, 7.5g sodium carboxymethyl starch, 0.8g talcum powder, 50g paste
The suitable mixing of essence, 0.8g magnesium stearate and appropriate 10% starch slurry, is conventionally made the compound tablet of structure shown in Formulas I
1000.3 times a day, 1 tablet once.
Coumarin kind compound shown in 6 formula of embodiment (I) prepares pill medicine
By the compound of structure shown in 350g Formulas I and 12g polyethylene glycol-6000,80.5g Tween-80, appropriate liquid
Paraffin mixing, is conventionally made compound pill 1000 of structure shown in Formulas I.3 times a day, 1 tablet each time.
Coumarin kind compound shown in 7 formula of embodiment (I) prepares injection medicine
By the compound of structure shown in 200g Formulas I and 15g injection soybean lecithin, 25g glycerol for injection, water for injection is fixed
Hold to 1000mL, compound injection agent 1000 of structure shown in Formulas I are conventionally made.One time a day, every time 1, until
Less using intravenous drip after the dilution of 5% glucose injection of 250mL.
Claims (10)
1. a kind of coumarin kind compound, which is characterized in that as shown in formula (I):
2. a kind of preparation method of coumarin kind compound, which comprises the following steps:
S1 Radix Angelicae Pubescentis is extracted with alcoholic solvent), obtains medicinal extract；
S2 the medicinal extract) is passed sequentially through into silica gel column chromatography, reversed-phase column chromatography, gel column chromatography and preparative high performance liquid chromatography
It is separated, obtains formula (I) compound represented；
3. preparation method according to claim 2, which is characterized in that the mass ratio of the Radix Angelicae Pubescentis and alcoholic solvent be 1:(6~
10)。
4. preparation method according to claim 2, which is characterized in that eluent used in the silica gel column chromatography is petroleum
One of ether, ethyl acetate, methylene chloride, acetone and methanol are a variety of.
5. preparation method according to claim 2, which is characterized in that instrument used in the reversed-phase column chromatography is mesolow
Prepare chromatography, high performance liquid chromatography or dynamic axial compression chromatography；Mobile phase used in the reverse-phase chromatography is organic solvent
Aqueous solution；The organic solvent is methanol or acetonitrile.
6. preparation method according to claim 2, which is characterized in that the elution of the reversed-phase column chromatography is gradient elution；
With volume fraction, the gradient elution degree are as follows: 0~20min, the aqueous solution of 10%~35% organic solvent；20~
60min, the aqueous solution of 35%~65% organic solvent；60~80min, the aqueous solution of 65%~100% organic solvent.
7. preparation method according to claim 2, which is characterized in that the gel in the gel column chromatography is Sephadex
LH-20, Sephadex G15 or Sephadex G50.
8. preparation method according to claim 2, which is characterized in that the mobile phase of preparative high performance liquid chromatography is first
Alcohol-water solution or acetonitrile-aqueous solution.
9. Coumarins chemical combination prepared by coumarin kind compound described in claim 1 or claim 2~8 any one
Object is preparing the application in anti-oxidant class drug.
10. a kind of pharmaceutical preparation, which is characterized in that including coumarin kind compound described in claim 1 or claim 2~
Coumarin kind compound prepared by 8 any one and pharmaceutically acceptable carrier.
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| CN113754625A (en) * | 2020-06-01 | 2021-12-07 | 沈阳药科大学 | Sesquiterpene coumarin compound and preparation method and application thereof |
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|---|---|---|---|---|
| CN102584763A (en) * | 2012-02-22 | 2012-07-18 | 北京大学 | 4-methyl-7-hydroxy-8-(1-hydroxyethyl) coumarin as well as preparation method and application thereof |
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| CN102584763A (en) * | 2012-02-22 | 2012-07-18 | 北京大学 | 4-methyl-7-hydroxy-8-(1-hydroxyethyl) coumarin as well as preparation method and application thereof |
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| CN113754625A (en) * | 2020-06-01 | 2021-12-07 | 沈阳药科大学 | Sesquiterpene coumarin compound and preparation method and application thereof |
| CN113754625B (en) * | 2020-06-01 | 2023-04-18 | 沈阳药科大学 | Sesquiterpene coumarin compound and preparation method and application thereof |
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