CN108530505A - A kind of flavonoid glycoside compound and its preparation method and application - Google Patents
A kind of flavonoid glycoside compound and its preparation method and application Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
- C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
- C07H17/06—Benzopyran radicals
- C07H17/065—Benzo[b]pyrans
- C07H17/07—Benzo[b]pyran-4-ones
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
- C07H1/08—Separation; Purification from natural products
Abstract
The present invention provides a kind of flavonoid glycoside compounds and its preparation method and application; the flavonoid glycoside compound has structure shown in formula I; the compound can significantly reduce LDH and MDA contents in brain tissue, and improve SOD contents, have preferable protective effect to cerebral ischemia/reperfusion injury of rats.The compound can inhibit growth of tumour cell simultaneously, have certain antitumor activity.
Description
Technical field
The invention belongs to pharmaceutical technology fields, and in particular to a kind of flavonoid glycoside compound and its preparation method and application.
Background technology
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 steps up, and rhythm of life is constantly accelerated, and makes the 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, and act is occupied on international drug market
The status of sufficient weight, therefore research and develop one of the research field that cardiovascular and cerebrovascular disease medication becomes most popular now.
Ginkgo leaf be Ginkgoaceae Ginkgo plant Ginkgo biloba Ginkgo biloba L. dried leaf, main product in Jiangsu, Guangxi,
The ground such as Shandong are the peculiar rare tree in China.Ginkgo leaf is sweet, bitter, puckery, flat, distributed in heart and lung channels.With promoting blood circulation and removing blood stasis, dredging collateral stops
Bitterly, it astringes the lung and relievings asthma, change turbid lipid-loweringing and other effects.Modern pharmacological studies have shown that ginkgo leaf, which has, reduces cholesterol, the coronal blood of expansion
The effects that pipe, relaxation bronchial smooth muscle, it is usually used in preventing and treating the cardiovascular and cerebrovasculars diseases such as cerebrovascular deficiency, myocardial ischemia
Disease, experiment find that its main active is flavonoids and terpene lactones compound.
Chemical composition isolated from ginkgo leaf mainly has flavonoids, terpene lactones, polysaccharide, polypentenol at present
Class compound and organic acid, alkyl phenolic acid class and volatile oil etc..Wherein flavonoid content is higher, mainly there is flavones
The substances such as alcohol glycosides, biflavone and catechin, have it is apparent it is anti-oxidant, anti-inflammatory, remove free radical, adjust endocrine, is antitumor
And adjust a variety of physiological activity such as immune system.The relevant medicine of noval chemical compound and its resisting cardiovascular disease disclosed in this patent
Reason active function research yet there are no all reports.
Invention content
In view of this, the technical problem to be solved in the present invention is to provide a kind of flavonoid glycoside compound and preparation method thereof
And application, the flavone compound provided by the invention with Formulas I structure is with the relevant pharmacological activity of resisting cardiovascular disease
Effect, and there is certain antitumor activity.
The present invention provides a kind of flavone compounds, have Formulas I structure,
The present invention provides a kind of preparation methods of above-mentioned flavone compound, include the following steps：
Step 1：Ginkgo leaf is extracted, clear cream is obtained；
Step 2：Obtained clear cream is passed sequentially through into large pore resin absorption column chromatography, reverse phase C18Column chromatography, gel column chromatography
With compound shown in the isolated Formulas I of preparative liquid chromatography.
Preferably, the macroporous absorbent resin in the large pore resin absorption column chromatography be selected from D101 types macroporous absorbent resin,
HP-20 types macroporous absorbent resin, HPD-450 types macroporous absorbent resin, HPD-950 types macroporous absorbent resin and AB-8 type macropores are inhaled
One or more of attached resin；Eluent used in chromatographic isolation is ethanol-water solution in the large pore resin absorption column chromatography.
Preferably, described to pass through reversed-phase column C18Chromatography is dynamic axial compression chromatography, high performance liquid chromatography or mesolow system
Standby chromatography；Eluent used is methanol-water solution or acetonitrile-aqueous solution.
Preferably, the gel by gel column chromatography be Sephadex LH-20, Sephadex G15 or
Sephadex G50；Eluent used is methanol.
Preferably, it is described detached by preparative liquid chromatography in mobile phase used be methanol-water solution or acetonitrile-water
Solution.
Preferably, the step 1 is specially：
Ginkgo leaf is mixed, refluxing extraction with ethanol-water solution, filters, filtrate is concentrated to give clear cream.
The present invention also provides a kind of Formulas I compounds represented to prepare medicament against cardiovascular disease or antitumor drug
In application,
The present invention also provides a kind of Chinese medicine preparations, by the compound described in claim 1 with structure shown in formula I or as weighed
Profit requires the compound made from 2 to 7 any one of them preparation methods with structure shown in formula I and pharmaceutically acceptable carrier system
At,
Compared with prior art, the present invention provides a kind of compound with structure shown in formula I, which can be significantly
LDH and MDA contents in brain tissue are reduced, and improve SOD contents, there is preferable protection to make cerebral ischemia/reperfusion injury of rats
With.The compound can inhibit growth of tumour cell simultaneously, have certain antitumor activity.
Description of the drawings
Fig. 1 is the ESI-MS spectrograms of the compound of Formulas I structure made from the embodiment of the present invention 1；
Fig. 2 is the compound of Formulas I structure made from the embodiment of the present invention 11H-NMR spectrum；
Fig. 3 is the compound of Formulas I structure made from the embodiment of the present invention 113C-NMR spectrograms；
Fig. 4 is the hsqc spectrum figure of the compound of Formulas I structure made from the embodiment of the present invention 1；
Fig. 5 is the compound of Formulas I structure made from the embodiment of the present invention 11H-1H COSY spectrograms；
Fig. 6 is the HMBC spectrograms of the compound of Formulas I structure made from the embodiment of the present invention 1；
Fig. 7 is that the main HMBC of the compound of Formulas I structure made from the embodiment of the present invention 1 is related.
Specific implementation mode
The present invention provides one kind, the present invention provides a kind of compounds, have Formulas I structure,
The present invention provides a kind of preparation methods of compound shown in Formulas I, include the following steps：
Step 1：Ginkgo leaf is extracted, clear cream is obtained；
Step 2：Obtained clear cream is detached, compound shown in Formulas I is obtained.
According to the present invention, the present invention extracts ginkgo leaf, obtains clear cream；The present invention is not special to the method for extraction
Limitation, the method well known in the art for solvent extraction medicinal herb components, present invention preferably employs ethyl alcohol extractions；
Specifically, the present invention mixes ginkgo leaf with ethanol-water solution, filtrate is concentrated to give clearly by refluxing extraction, filtering
Cream；Wherein, the weight ratio of ginkgo leaf and ethyl alcohol is 1：The number of (6~10), the extraction is 1~3 time, preferably 2 times；It is described
The time of extraction is preferably 0.5~3 hour, more preferably 1.5~2.5 hours；The volume hundred of ethyl alcohol in the ethanol-water solution
Point content preferably 50~95%, more preferably 60%.
Then, the present invention detaches obtained clear cream, obtains compound shown in Formulas I；The separating step is preferably：
Obtained clear cream is passed sequentially through into large pore resin absorption column chromatography, reverse phase C18Column chromatography, gel column chromatography and preparative liquid phase color
Compose compound shown in isolated Formulas I；I.e.：
By obtained clear cream by large pore resin absorption column chromatography, the eluent is the ethanol-water solution of various concentration,
Obtain the crude product after large pore resin absorption column separation；
Crude product after large pore resin absorption column is detached is detached by reversed-phase column chromatography, the eluent be acetonitrile-water or
Methanol-water solution obtains the crude product after reversed-phase column chromatography separation；
The crude product obtained after reversed-phase column chromatography is detached is purified by gel column chromatography, and the eluent is methanol, is obtained
The crude product of gel column chromatography after purification；
By the crude product of gel column chromatography after purification by preparative liquid chromatography, compound shown in Formulas I is obtained.
Specifically, in the present invention, by obtained clear cream by large pore resin absorption column chromatographic isolation, macroreticular resin point is obtained
Crude product from after；Wherein, the macroporous absorbent resin be preferably D101 types macroporous absorbent resin, HP-20 types macroporous absorbent resin,
One kind or several in HPD-450 types macroporous absorbent resin, HPD-950 types macroporous absorbent resin and AB-8 type macroporous absorbent resins
Kind；The eluent is preferably ethanol-water solution；And in order to adequately detach impurity and required compound, the present invention selects ladder
Degree elution, the eluent of the gradient elution are preferably that the volume ratio of ethyl alcohol and water is (x)：The solution of (100-x), wherein 0≤
X≤95 more preferably use water, volumetric concentration to be eluted for (25~35) % ethanol-water solutions, collected volume concentration successively
Position is eluted for (25~35) % ethanol-water solutions, obtains the crude product after macroreticular resin separation.
In the present invention, the crude product after macroreticular resin is detached passes through reverse phase C18Pillar layer separation obtains reverse phase C18Column chromatography
Crude product after separation；Wherein, instrument is preferably dynamic axial compression chromatography；The eluent is preferably acetonitrile-aqueous solution,
And in order to more preferably detach impurity and required compound, the present invention selects gradient elution, the gradient elution string routine is 0~
10min, the acetonitrile-aqueous solution for being 10%~25% with volumetric concentration elute；10~70min, with volumetric concentration be 25%~
75% acetonitrile-aqueous solution elution；70~80min is eluted for 75%~100% acetonitrile-aqueous solution with volumetric concentration, collects 35
~45min elutes position, obtains reverse phase C18Crude product after pillar layer separation.
In the present invention, by reverse phase C18Crude product after pillar layer separation is detached by gel column chromatography, after obtaining gel separation
Crude product；The gel column chromatography separation is preferably Sephadex LH-20 with gel；The eluent is preferably methanol.
In the present invention, the crude product after gel is detached is detached by preparative liquid chromatography, obtains compound shown in Formulas I；
Wherein, it is (25~45) % acetonitrile-aqueous solutions that mobile phase used, which is volumetric concentration, and preferably volumetric concentration is (30~40) % second
Nitrile-aqueous solution；The flow velocity is 3~100mL/min, preferably 5~35mL/min；Detection wavelength is 340nm, obtains Formulas I institute
Show compound.
Flavone compound of the present invention with Formulas I structure is Yellow amorphous powder, and hydrochloric acid-magnesium powder reaction is in
The positive, Molish reactions are positive.High resolution mass spectrum HR-ESI-MS provides m/z755.1897 [M-H]-(calculated value is
755.1902), m/z779.1835 [M+Na]+(calculated value 779.1875), compound molecular weight 756, in conjunction with elemental analysis
And13C-NMR is composed and DEPT spectrums infer that the compound molecule formula is C36H36O18。
The present invention has carried out Structural Identification to the compound with Formulas I structure, the final structure for determining the compounds of this invention
For Keampferol-3-O- α-(6 " '-p-caffeoylglucosyl- β -1,2-rhamnoside), the entitled Kaempferol-of Chinese
3-O- α-(6 " '-p- coffee acyl glucose-β -1,2- rhamnoside), structure is shown in formula I, is a new flavonoid glycoside
Compound.All hydrocarbon signals assignments are shown in Table 1, and table 1 is each carbon of compound and the ownership of hydrogen shown in Formulas I.
Nuclear magnetic data (the DMSO-d of 1 compound of table6,1H-NMR 400MHz,13C-NMR 100MHz)
The present invention also provides a kind of Formulas I compounds represented in preparing medicament against cardiovascular disease or antitumor drug
Application,
The present invention also provides a kind of Chinese medicine preparation, prepare by the described compound with structure shown in formula I or as mentioned
The method compound obtained with structure shown in formula I is made with pharmaceutically acceptable carrier,
In the present invention, the pharmaceutically acceptable carrier can be according to the common auxiliary material of art of pharmacy, according to dosage form
It is appropriately selected with actual conditions, such as common auxiliary material has starch, low-substituted hydroxypropyl cellulose, superfine silica gel powder, tristearin
Sour magnesium, starch slurry, sucrose, dextrin, sodium carboxymethyl starch, talcum powder, polysorbate, polyethylene glycol, injection soybean lecithin and
Glycerol for injection etc.；When preparing the various dosage forms of required drug using the extract that the present invention obtains, it can be led according to pharmacy
It is prepared by the conventional production process in domain.The extract is mixed with one or more carriers such as, corresponding dosage form is then made.As
It is preferred that the dosage form of the Chinese medicine preparation includes injection, tablet, suppository, ointment, gelling agent, pill, tablet, granule, glue
Wafer and mixture.
Compared with prior art, the present invention provides a kind of compound of structure shown in formula I, which can significantly reduce
LDH and MDA contents in brain tissue, and SOD contents are improved, there is preferable protective effect to cerebral ischemia/reperfusion injury of rats.
The compound can inhibit growth of tumour cell simultaneously, have certain antitumor activity.
The present invention obtains clear cream by being extracted to ginkgo leaf；Then obtained clear cream is detached, is selected specific
The compound of appearance time finds that the compounds of this invention can significantly drop to get to compound shown in Formulas I, and by zoopery
LDH and MDA contents in low brain tissue, and SOD contents are improved, there is preferable protection to make cerebral ischemia/reperfusion injury of rats
With.The compound can inhibit growth of tumour cell simultaneously, have certain antitumor activity.
It is clearly and completely described below in conjunction with the technical solution of the embodiment of the present invention, it is clear that described implementation
Example is only a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, this field is common
The every other embodiment that technical staff is obtained without making creative work belongs to the model that the present invention protects
It encloses.
Embodiment 1：The preparation of the compound of structure shown in formula I
1) dry ginkgo leaf 10kg, the volumetric concentration that 10 times of amounts are added after pulverizing and sieving is 60% ethanol water, is carried
It takes 3 times, each 1.5h, filters, filtrate concentration, recycling ethyl alcohol are obtained into clear cream；
2) taking the clear cream of step 1) that purified water is added makes dissolving, is stored at room temperature, takes supernatant through HP-20 macroporous absorbent resins
Post separation is successively that 35% ethanol-water solution is eluted with purified water, volumetric concentration, 4 cylinders of each elution
Product collects 35% ethanol-water solution and elutes position；
3) 35% ethanol-water solution of step 2) is taken to elute position, inverted C18Dynamic axial compression column chromatographic isolation, is adopted
With gradient elution, the gradient elution program is：0~10min is that 10%~25% acetonitrile-aqueous solution carries out with volumetric concentration
Elution；10~70min is that 25%~75% acetonitrile-aqueous solution is eluted with volumetric concentration；70~80min uses volumetric concentration
It is eluted for 75%~100% acetonitrile-aqueous solution, flow velocity 100mL/min, collects 35~45min eluents；
4) eluent for taking step 3), through Sephadex LH-20 pillar layer separations, methanol elution is purified.
5) sample of step 4) after purification is taken, is detached through preparative HPLC, is that 25% acetonitrile-aqueous solution is with volumetric concentration
Mobile phase, Detection wavelength 340nm, flow velocity 15mL/min, separating obtained solution drying obtain structure shown in formula I of the present invention
Compound 10mg, purity 98.5%.
The compound that embodiment 1 obtains is Yellow amorphous powder.
Physics and chemistry and structural analysis are carried out to above compound, hydrochloric acid-magnesium powder reaction is positive, and Molish reactions are positive,
It may be flavonoid glycoside compound to prompt it.High resolution mass spectrum HR-ESI-MS provides m/z755.1897 [M-H]-(calculated value is
755.1902), m/z779.1835 [M+Na]+(calculated value 779.1875), it is 756 to prompt compound molecular weight, in conjunction with element
Analysis and13C-NMR is composed and DEPT spectrums infer that the compound molecule formula is C36H36O18。
Structural Identification is carried out to the compound that embodiment 1 obtains, the result is shown in Figure 1~7, Fig. 1 is made for the embodiment of the present invention 1
Formulas I structure compound ESI-MS spectrograms, Fig. 2 is the compound of Formulas I structure made from the embodiment of the present invention 11H-NMR
Spectrogram, Fig. 3 are the compound of Formulas I structure made from the embodiment of the present invention 113C-NMR spectrograms, Fig. 4 make for the embodiment of the present invention 1
The hsqc spectrum figure of the compound of the Formulas I structure obtained, Fig. 5 are the compound of Formulas I structure made from the embodiment of the present invention 11H-1H
COSY spectrograms, Fig. 6 are the HMBC spectrograms of the compound of Formulas I structure made from the embodiment of the present invention 1, and Fig. 7 is the embodiment of the present invention 1
The main HMBC of the compound of Formulas I structure obtained is related.
By analyzing Fig. 1~Fig. 7, this compound1H-NMR(DMSO-d6, 400MHz) and spectrum (see Fig. 2), provide 1
Group AA ' BB ' coupled system aromatic signal δ 7.75 (2H, d, J=8.6Hz, H-2 ', 6 '), 6.93 (2H, d, J=8.6Hz,
H-3′,5′)；1 group of ABX coupled system aromatic signal δ 6.96 (1H, d, J=1.7Hz, H-5 " "), 6.69 (1H, d, J=
8.1Hz, H-8 " "), 6.87 (1H, dd, J=8.1,1.7Hz, H-9 " ")；1 group of trans double bond signal δ 6.11 (1H, d, J=
15.9Hz, H-2 " "), 7.39 (1H, d, J=15.9Hz, H-3 " ")；2 singlet signal δ 6.19 (1H, s, H-6), 6.37
(1H,s,H-8)；In conjunction with13The compound of C-NMR modal datas inferring contains 1 glucose and 1 rhamnose, δ 4.33 (1H, d, J
=7.8Hz), 5.60 (1H, s) are respectively glucose and rhamnose anomeric proton signal, High-Field δ 0.89 (1H, d, J=7.8Hz)
For rhamnose methyl proton signal.
13C-NMR(DMSO-d6, 400MHz) spectrum in (see Fig. 3), provide 36 carbon signals, flavones parent nucleus carbon signal 15 altogether
A, wherein δ 178.2 is the feature carbon signal of flavones parent nucleus 4；The δ 17.9 of high field region is rhamnose methyl carbon signal, δ 63.4
For 6 carbon signals of glucose, δ 101.1 and 106.5 is 2 sugared end group carbon signals, and δ 145.6 and 114.1 is one group of olefinic carbon signal,
Hydrocinnamoyl segment there are one may containing is speculated in conjunction with hydrogen spectrum.
In conjunction with HSQC collection of illustrative plates (see Fig. 4) and HMBC collection of illustrative plates (see Fig. 6) to carbon signal and hydrogen signal with correlativity
Belonged to, wherein rhamnose anomeric proton δ 5.60 and 3 carbon δ 134.8 of flavones parent nucleus are long-range related, show rhamnose and
Aglycon 3 is connected；2 carbon δ 82.0 of glucose anomeric proton δ 4.33 and rhamnose are long-range related, show glucose 1 and sandlwood
Sugar 2 is connected；Olefinic proton 7.39 (1H, d, J=15.9Hz, H-3 " ") and ABX system carbon signals δ 115.5 and δ 11.4 and carbonyl
Base carbon signal δ 166.8 is related simultaneously, thus it is speculated that hydrocinnamoyl is coffee acyl；The coffee of glucose 6 proton signals and δ 166.8
Carbonyl carbon signals are remotely related on acyl group, while 6 carbon signals of glucose are more normally to about 2.2 units of low field displacement, it was demonstrated that
Glucose 6 is connected with coffee acyl segment.In conjunction with the data that 1D-NMR and 2D-NMR are provided, and portion similar to known compound
The comparison of point carbon modal data, it is determined that flavone aglycone, coffee acyl, rhamnose and glucose the order of connection.Data are shown in Table 1.
In summary data analysis, the final structure for determining the compounds of this invention are Keampferol-3-O- α-(6 " '-p-
Caffeoylglucosyl- β -1,2-rhamnoside), the entitled Kaempferol-O- α-(6 " '-p- coffee acyl grapes of Chinese
Sugar-β -1,2- rhamnoside), structure is shown in formula I, is a new flavonoid glycoside compound.All hydrocarbon signals assignments
It is shown in Table 1, table 1 is each carbon of compound and the ownership of hydrogen shown in Formulas I.
Nuclear magnetic data (the DMSO-d of 1 compound of table6,1H-NMR 400MHz,13C-NMR 100MHz)
By to Analysis of test results it is found that the structure for the compound that the present invention obtains be I compound represented of formula.
Embodiment 2：The preparation of the compound of structure shown in formula I
1) dry ginkgo leaf 10kg, the volumetric concentration that 6 times of amounts are added after pulverizing and sieving are 70% ethanol water, extraction
2 times, each 2h, filtrate concentration, recycling ethyl alcohol are obtained clear cream by filtering；
2) taking the clear cream of step 1) that purified water is added makes dissolving, is stored at room temperature, takes supernatant through D101 macroporous absorbent resins
Post separation is successively that 25% ethanol-water solution is eluted with purified water, volumetric concentration, 4 cylinders of each elution
Product, a concentration of 25% ethanol-water solution of collected volume elute position；
3) 25% ethanol-water solution of step 2) is taken to elute position, inverted C18Dynamic axial compression column chromatographic isolation, is adopted
With gradient elution, the gradient elution program is：0~10min is that 10%~25% acetonitrile-aqueous solution carries out with volumetric concentration
Elution；10~70min is that 25%~75% acetonitrile-aqueous solution is eluted with volumetric concentration；70~80min uses volumetric concentration
It is eluted for 75%~100% acetonitrile-aqueous solution, flow velocity 100mL/min, collects 35~45min eluents；
4) eluent for taking step 3), through Sephadex LH-20 pillar layer separations, methanol elution is purified.
5) sample of step 4) after purification is taken, is detached through preparative HPLC, is that 40% acetonitrile-aqueous solution is with volumetric concentration
Mobile phase, Detection wavelength 340nm, flow velocity 15mL/min, separating obtained solution drying obtain structure shown in formula I of the present invention
Compound 9mg, purity 98.6%.
The physics and chemistry and structure analysis method provided according to embodiment 1 is analyzed compound it is found that embodiment 2 obtained
The structure of compound is I compound represented of formula.
Embodiment 3：The preparation of the compound of structure shown in formula I
1) dry ginkgo leaf 10kg, 70% ethanol waters of 8 times of amounts of addition after pulverizing and sieving, extraction 2 times, every time
Filtrate concentration, recycling ethyl alcohol are obtained clear cream by 2h, filtering；
2) taking the clear cream of step 1) that purified water is added makes dissolving, is stored at room temperature, takes supernatant through D101 macroporous absorbent resins
Post separation is eluted with purified water, 25% ethanol-water solution successively, 4 column volumes of each elution, collects 25%
Ethanol-water solution elutes position；
3) 25% ethanol-water solution of step 2) is taken to elute position, inverted C18Dynamic axial compression column chromatographic isolation, is adopted
With gradient elution, the gradient elution program is：0~10min is that 10%~25% acetonitrile-aqueous solution carries out with volumetric concentration
Elution；10~70min is that 25%~75% acetonitrile-aqueous solution is eluted with volumetric concentration；70~80min uses volumetric concentration
It is eluted for 75%~100% acetonitrile-aqueous solution, flow velocity 100mL/min, collects 35~45min eluents；
4) eluent for taking step 3), through Sephadex LH-20 pillar layer separations, methanol elution is purified.
5) sample of step 4) after purification is taken, is detached through preparative HPLC, is that 30% acetonitrile-aqueous solution is with volumetric concentration
Mobile phase, Detection wavelength 340nm, flow velocity 15mL/min, separating obtained solution drying obtain structure shown in formula I of the present invention
Compound 9mg, purity 98.5%.
The physics and chemistry and structure analysis method provided according to embodiment 1 is analyzed compound it is found that embodiment 3 obtained
The structure of compound is I compound represented of formula.
Embodiment 4：Protective effect of the compounds of this invention to cerebral ischemia/reperfusion injury of rats
1. material
Compound shown in 1.1 drug Formulas I；
1.2 positive control drug nimotop vials；
Tonneau China experimental animal technology is tieed up in 1.3 animal SD rats 108, male, weight 180~200g, SPF grade, Beijing
Co., Ltd, credit number：(capital) 2012-0001.
1.4 instruments and reagent calcium current work station (MD companies)；Sigma 2-16PK centrifuges (Sigma companies)；MDA is detected
Kit, LDH detection kits, total SOD activity detection kits (green skies biotechnology research institute)；Ether (Chinese medicines group
Chemical reagent Co., Ltd)；Paraformaldehyde, TTC (amresco).
2. experimental method and step
108 SD rats are randomly divided into 6 groups by 2.1 groupings with administration, every group 18, respectively sham-operation group (tail vein
Injecting normal saline), model group (intraperitoneal injection of saline), high, medium and low 3 dosage groups of compound of formula I (gastric infusion 5,
2.5、1.25mg·kg-1·d-1) and Nimodipine group (tail vein injection 5mLkg-1·d-1, it is equivalent to Nimodipine 1mg
kg-1·d-1).Each group is administered once daily, in operation consent successive administration 7d.
Prepared by 2.2 cerebral ischemia re-pouring models prepares arteria cerebri media embolism (MCAO) model using line brush：10% water
Close chloral (3mLkg-1·d-1) after intraperitoneal administration anesthetized rat, separation right common carotid artery simultaneously cuts an osculum, by long 50mm, straight
Diameter 0.2mm, top are ironed to be inserted at smooth spherical nylon wire as embolus, and calculating inlet wire depth with arteria carotis communis crotch is
(18.5 ± 0.5) mm, until arteria cerebri media initial part to block blood supply completely.After ischemic 2h, line bolt is pulled out, Reperfu- sion is for 24 hours.It does evil through another person
Art group only carries out anesthesia and blood vessel exclusion, does not ligature blood vessel and imports nylon wire.Above procedure is in room temperature constant (24~25
DEG C) in the case of carry out.
In 2.3 brain tissues SOD, MDA, LDH detect rat in ischemia-reperfusion for 24 hours after, after etherization broken end take brain, entirely
It after brain is weighed, with ice-cold normal saline flushing, is put into homogenate tube, appropriate 4 DEG C of physiological saline is added and is homogenized, grinding system
It is homogenized at 10% tissue.With 3000rmin-110min is centrifuged, supernatant is taken, is divided into 8 parts.Coomassie Brilliant Blue measures total egg
In vain, illustrate respectively to be measured SOD, MDA and LDH in brain tissue homogenate according to kit.
3. experimental result
The experimental results showed that rat cerebral ischemia 2h Reperfu- sions for 24 hours after, cause the notable liter of brain tissue LDH and MDA level
Height, the significant decrease (P of SOD contents<0.01).Nimotop vial group, compound of formula I are high, middle dose group can significantly reduce
The amount of LDH and MDA in brain tissue increases the content of SOD, the statistically significant (P of difference<0.01, P<0.05), low dose group without
Significant difference (P ＞ 0.05).Data result such as table 2.
Influence of 2 compound of formula I of table to cerebral ischemia-reperfusion injury in rats brain tissue SOD, MDA and LDH
Compared with sham-operation group, ##P<0.01, compared with model group, * * P<0.01, * P<0.05
4. conclusion
The compounds of this invention can significantly reduce LDH and MDA contents in brain tissue, and improve SOD contents, to rat cerebral ischemia
Reperfusion injury has preferable protective effect.
Embodiment 5：Influence of the compounds of this invention to growth of tumour cell
1. material
Compound shown in 1.1 drug Formulas I；
1.2 positive control Cytoxans；
1.3 cell
Human liver cancer cell HepG2 (Chinese Academy of Sciences's Type Tissue Collection Shanghai cell bank).
1.4 instruments and reagent
Superclean bench (SuZhou Antai Air Tech Co., Ltd.), carbon dioxide incubator (Thermo
Scientific), inverted microscope (OLYPUS), high-pressure steam sterilizing pan (BOXUN), (many benefits in Beijing neutralize biology to centrifuge
Technology Co., Ltd.), microplate reader (Bio Tek), electronic balance (Switzerland's plum Teller)；RPMI-1640 culture mediums, fetal calf serum
(Gibco), DMSO (Sigma), trypsase (Amresco), MTT (Sigma), (Zhejiang Haizheng Pharmaceutical Co share has cyclophosphamide
Limit company).
2. experimental method and step
Cell culture：By human liver cancer cell HepG2 in 37 DEG C, 5%CO2Secondary culture is carried out in cell incubator, will be passed
Cell after generation is with 1 × 105The concentration of a/mL is inoculated in 96 porocyte culture plates, per 100 μ L of hole, in 37 DEG C, and 5%CO2Cell
It is cultivated 24 hours in incubator.After for 24 hours, 96 orifice plates are taken out, suction abandons supernatant, blank group is randomly divided into according to test requirements document, sun
Property control group and high, medium and low various concentration administration group, every group sets 5 multiple holes.
Blank group gives RPMI 1640 culture mediums 100 μ L；Positive drug group gives the cyclophosphamide of a concentration of 20 μm of ol/L
100μL；High, medium and low dosage group gives the 100 μ L of type I compound of a concentration of 50,25,12.5 μm of ol/L respectively；In 37 DEG C, 5%
CO2Continue culture in cell incubator for 24 hours.It inhales afterwards for 24 hours and abandons supernatant, add 100 μ L and MTT solution of RPMI 1640 culture mediums respectively
20 μ L continue to cultivate 4h after mixing.Absorbance (A) value is measured under 490nm.Inhibiting rate is calculated according to formula.
Inhibiting rate (%)=(A blank-A administrations)/A blank × 100%
3. experimental result
The experimental results showed that the administration group of various concentration can inhibit the growth of human liver cancer cell HepG2 to a certain extent, tool
There is certain antitumor activity.Data result is shown in Table 3.
Inhibiting effect of 3 compound of formula I of table to human liver cancer cell HepG2
Compared with blank group, * * P<0.01, * P<0.05
4. conclusion
The compounds of this invention can inhibit the growth of human liver cancer cell HepG2 to a certain extent, and upper with drug concentration
It rises, inhibiting effect enhancing has certain antitumor activity.
Embodiment 6：The compound of structure shown in Formulas I prepares granules medicine
The compound of structure shown in 350g Formulas I and 1000g sucrose and 500g dextrin are mixed, are conventionally made
The compound particle agent of structure shown in 1000 packet mode I.3 times a day, 1 tablet each time.
Embodiment 7：The compound of structure shown in Formulas I prepares tablet medicine
By the compound of structure shown in 350g Formulas I and 50g starch, 7.5g sodium carboxymethyl starches, 0.8g talcum powder, 50g pastes
The suitable mixing of essence, 0.8g magnesium stearates 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.
Embodiment 8：The compound of structure shown in Formulas I prepares pill medicine
By the compound of structure shown in 350g Formulas I and 12g polyethylene glycol-6000,80.5g Tween-80s, appropriate liquid
Paraffin mixes, and the compound pill 1000 of structure shown in Formulas I is conventionally made.3 times a day, 1 tablet each time.
Embodiment 9：The compound of structure shown in Formulas I prepares injection medicine
By the compound of structure shown in 200g Formulas I and 15g injections soybean lecithin, 25g glycerol for injection, water for injection is fixed
Hold to 1000mL, the compound injection agent 1000 of structure shown in Formulas I is conventionally made.One time a day, every time 1, until
Intravenous drip after using 250mL5% glucose injections to dilute less.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (9)
1. a kind of flavone compound has Formulas I structure,
2. a kind of preparation method of flavone compound as described in claim 1, which is characterized in that include the following steps：
Step 1：Ginkgo leaf is extracted, clear cream is obtained；
Step 2：Obtained clear cream is passed sequentially through into large pore resin absorption column chromatography, reverse phase C18Column chromatography, gel column chromatography and system
Compound shown in the isolated Formulas I of standby type liquid chromatogram.
3. preparation method according to claim 2, which is characterized in that the macropore in the large pore resin absorption column chromatography is inhaled
Attached resin is selected from D101 types macroporous absorbent resin, HP-20 types macroporous absorbent resin, HPD-450 types macroporous absorbent resin, HPD-
One or more of 950 type macroporous absorbent resins and AB-8 type macroporous absorbent resins；In the large pore resin absorption column chromatography
Eluent used in chromatographic isolation is ethanol-water solution.
4. preparation method according to claim 2, which is characterized in that described to pass through reversed-phase column C18Chromatography is dynamic axial pressure
Contracting chromatography, high performance liquid chromatography or mesolow prepare chromatography；Eluent used is methanol-water solution or acetonitrile-aqueous solution.
5. preparation method according to claim 2, which is characterized in that the gel by gel column chromatography is
Sephadex LH-20, Sephadex G15 or Sephadex G50；Eluent used is methanol.
6. preparation method according to claim 2, which is characterized in that it is described detached by preparative liquid chromatography in it is used
Mobile phase is methanol-water solution or acetonitrile-aqueous solution.
7. preparation method according to claim 2, which is characterized in that the step 1 is specially：
Ginkgo leaf is mixed, refluxing extraction with ethanol-water solution, filters, filtrate is concentrated to give clear cream.
8. a kind of application of Formulas I compound represented in preparing medicament against cardiovascular disease or antitumor drug,
9. a kind of Chinese medicine preparation, by the compound described in claim 1 with structure shown in formula I or such as any one of claim 2 to 7
The preparation method compound obtained with structure shown in formula I is made with pharmaceutically acceptable carrier,
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| CN112500444A (en) * | 2019-09-16 | 2021-03-16 | 江苏康缘药业股份有限公司 | Compound and preparation method and application thereof |
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| CN112500444A (en) * | 2019-09-16 | 2021-03-16 | 江苏康缘药业股份有限公司 | Compound and preparation method and application thereof |
| CN112500444B (en) * | 2019-09-16 | 2022-02-08 | 江苏康缘药业股份有限公司 | Compound and preparation method and application thereof |
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