CN105175481A - Highly-oxidized diterpenoid compound and preparation method and medical application thereof - Google Patents
Highly-oxidized diterpenoid compound and preparation method and medical application thereof Download PDFInfo
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- CN105175481A CN105175481A CN201510704754.XA CN201510704754A CN105175481A CN 105175481 A CN105175481 A CN 105175481A CN 201510704754 A CN201510704754 A CN 201510704754A CN 105175481 A CN105175481 A CN 105175481A
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- -1 diterpenoid compound Chemical class 0.000 title abstract description 6
- 150000001875 compounds Chemical class 0.000 claims abstract description 57
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- 238000000605 extraction Methods 0.000 claims abstract description 8
- 238000000746 purification Methods 0.000 claims abstract description 7
- 230000009441 vascular protection Effects 0.000 claims abstract description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 44
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 20
- 238000010828 elution Methods 0.000 claims description 18
- 239000007788 liquid Substances 0.000 claims description 16
- 241000628997 Flos Species 0.000 claims description 15
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical class CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims description 15
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 12
- 239000000284 extract Substances 0.000 claims description 12
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- 238000000034 method Methods 0.000 description 13
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- Medicinal Preparation (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses a highly-oxidized diterpenoid compound and a preparation method and medical application thereof and belongs to the technical field of medicines. The compound is disclosed for the first time, is a diterpenoid compound with a novel structure and can be prepared from dried flower buds of common coltsfoot flowers through extraction, separation and purification. An in-vitro test proves that the compound has the protection effect on vascular endothelial cells suffering from H2O2 oxidative damage, further has free radical scavenging and reactive oxygen resisting characteristics and can be used for developing vascular protection drugs.
Description
Technical field
The invention belongs to technical field of pharmaceuticals, be specifically related to from the dry flower of Flos Farfarae, be separated a kind of diterpene compound obtained, containing its pharmaceutical composition and its preparation method and application.
Background technology
Flos Farfarae another name coltsfoot, Rhizome of Japanese Butterbur or coltsfoot taraxacum, be the bud of composite family tussilago farfara genus plant coltsfoot (TussilagofarfaruL.), be distributed in the provinces and regions such as China Hebei, Xinjiang.The Hua Xianye of coltsfoot is open, and Hua Ting is several, has flakey bract 10 multi-disc, spends female, 2-March of florescence.The medicinal part of coltsfoot is its dry flower, distributes in irregular club-like, and the outer face length of fresh idea has many fish scale-shaped bracts, usually excavates when flower is not yet unearthed in late October to late December.Medicinal material smell delicate fragrance, taste is slightly bitter.Flos Farfarae is warm in nature, and taste is pungent, micro-hardship, moistening lung to lower QI, relieving cough and reducing sputum, cures mainly dyspnea and cough with excessive sputum, labor coughs the diseases such as spitting of blood, chronic and acute tracheitis.Record in " herbal classic ": to " the drink heresy of cold bundle lung channel is breathed heavily, cough the most suitable ".
Now report that the chemical composition type of Flos Farfarae mainly comprises flavones, terpene, phenols, alkaloids and volatile oil both at home and abroad.
Flos Farfarae has cough-relieving, relievings asthma, boosts, anti-oxidant, anti-inflammatory, the pharmacologically active such as antitumor.Coltsfoot Cough remedy granules obviously can extend strong aqua and draw the latent period coughing rear mouse cough, reduces cough number of times, increases mouse tracheae section phenols contents.Farfaratin, methylbutyric Flos Farfarae ester and 14-remove acetoxy-3, and 14-dehydrogenation-2-Methyl Butyric Acid Flos Farfarae ester has restraining effect to the platelet aggregation that platelet activation factor causes.Flos Farfarae flavones is to O
2 -, OH, H
2o
33 kinds of free radicals have good Scavenging activity, and at 0.38 ~ 47.65mg/L
-1a certain amount of effect relationship is presented, the Scavenging activity size to 3 kinds of free radicals: H in scope
2o
3> O
2 -> OH.Flos Farfarae ethanol extraction obviously can reduce the swollen and carrageenin induced mice foot sole of the foot of mice caused by dimethylbenzene xylene ear and swell; Flos Farfarae ethanol extraction obviously can reduce Viscotrol C, sennae induced mice diarrhoea, reduces ulcer caused by ulcer caused by water logging straining lasering type, hydrochloric acid and indomethacin-ethanol.Flos Farfarae extract 1,2-di-(3 ', 4 '-dihydroxycinnamoyl)-cychopenta-3-ol, kaempferol and Quercetin all demonstrate certain restraining effect to the increment of murine lung cancer cell LA795, and wherein the restraining effect of Quercetin to lung carcinoma cell LA795 is the most remarkable.Have document to show, Quercetin also can suppress the growth of human umbilical vein endothelial cell cell, and makes its apoptosis.Quercetin also demonstrates stronger restraining effect to other tumours.
Summary of the invention
The object of this invention is to provide and a kind ofly from the dry flower of Flos Farfarae, be separated a kind of diterpene compound obtained, containing its pharmaceutical composition and its preparation method and application.
Above-mentioned purpose of the present invention is achieved by technical scheme below:
There is the compound (I) of following structural formula:
The preparation method of described compound (I), comprise following operation steps: the dry flower of Flos Farfarae is pulverized by (a), extract with 80 ~ 90% alcohol heat reflux, united extraction liquid, be concentrated into without alcohol taste, use sherwood oil, ethyl acetate and water saturated n-butanol extraction successively, obtain petroleum ether extract, acetic acid ethyl ester extract and n-butyl alcohol extract respectively; B in () step (a), the removal of impurities of thing macroporous resin got by propyl carbinol, first use 5% ethanol elution, 6 column volumes, then use 75% ethanol elution, 8 column volumes, collect 75% elutriant, concentrating under reduced pressure obtains 75% ethanol elution enriched material; C in () step (b), 75% ethanol elution enriched material purification on normal-phase silica gel is separated, obtain 4 components successively with the methylene chloride-methanol gradient elution that volume ratio is 65:1,30:1,15:1 and 8:1; D in () step (c), component 4 is separated further by purification on normal-phase silica gel, obtain 3 components successively with the methylene chloride-methanol gradient elution that volume ratio is 15:1,8:1 and 5:1; E in () step (d), component 2 reverse phase silica gel of octadecylsilane bonding is separated, with the methanol aqueous solution isocratic elution that concentration expressed in percentage by volume is 75%, collect 8 ~ 12 column volume elutriants, elutriant concentrating under reduced pressure obtains pure compound (I).
Further, in step (a), extract with 85% alcohol heat reflux, united extraction liquid.
Further, described macroporous resin is AB-8 type macroporous adsorbent resin.
Pharmaceutical composition, the described compound (I) wherein containing treatment significant quantity and pharmaceutically acceptable carrier.
Described compound (I) application in the medicine preparing vascular protection.
The application of described pharmaceutical composition in the medicine preparing vascular protection.
When the compounds of this invention is used as medicine, directly can uses, or use with the form of pharmaceutical composition.
This pharmaceutical composition contains the compounds of this invention (I) for the treatment of significant quantity, and all the other are acceptable on pharmacology, nontoxic to humans and animals and pharmaceutically acceptable carrier of inertia and/or vehicle.
Described pharmaceutically acceptable carrier or vehicle are that one or more are selected from solid, semisolid and liquid diluent, filler and pharmaceutical preparation assistant agent.Pharmaceutical composition of the present invention is used with the form of per weight dose.Medicine of the present invention is applied to by form that is oral or injection the patient needing treatment.For time oral, tablet, slow releasing tablet, controlled release tablet, capsule, dripping pill, micropill, suspensoid, emulsion, powder or granule, oral liquid etc. can be made into; During for injecting, can be made into water-based or oily solution, aseptic powder injection, liposome or the emulsion etc. of sterilizing.
Accompanying drawing explanation
Fig. 1 is compound (I) structural formula;
Fig. 2 is that compound (I) calculates ECD and experiment ECD figure.
Embodiment
Further illustrate essentiality content of the present invention below in conjunction with embodiment, but do not limit scope with this.Although be explained in detail the present invention with reference to preferred embodiment, those of ordinary skill in the art should be appreciated that and can modify to technical scheme of the present invention or equivalent replacement, and does not depart from essence and the scope of technical solution of the present invention.
Main raw, reagent source and instrument type:
Ethanol, sherwood oil, ethyl acetate, propyl carbinol, methylene dichloride are analytical pure, and purchased from Shanghai Ling Feng chemical reagent company limited, methyl alcohol, analytical pure, purchased from Jiangsu Han Bang chemical reagent company limited.
Embodiment 1: compound (I) is separated preparation and structural identification
A the dry flower (8kg) of Flos Farfarae is pulverized by (), (30L × 3 time) are extracted with 85% alcohol heat reflux, united extraction liquid, be concentrated into without alcohol taste (6L), use sherwood oil (6L × 3 time), ethyl acetate (6L × 3 time) and water saturated propyl carbinol (6L × 3 time) to extract successively, obtain petroleum ether extract, acetic acid ethyl ester extract (375g) and n-butyl alcohol extract respectively; Acetic acid ethyl ester extract AB-8 macroporous resin removal of impurities in (b) step (a), first use 5% ethanol elution, 6 column volumes, use 75% ethanol elution, 8 column volumes again, collect 75% elutriant, concentrating under reduced pressure obtains 75% ethanol elution enriched material (129g); C in () step (b), 75% ethanol elution enriched material purification on normal-phase silica gel is separated, successively with volume ratio be 65:1 (8 column volumes), the methylene chloride-methanol gradient elution of 30:1 (8 column volumes), 15:1 (8 column volumes) and 8:1 (10 column volumes) obtains 4 components; D component 4 (27g) is separated further by purification on normal-phase silica gel in () step (c), successively with volume ratio be 12:1 (8 column volumes), the methylene chloride-methanol gradient elution of 8:1 (10 column volumes) and 5:1 (8 column volumes) obtains 3 components; E in () step (d), component 2 (15g) reverse phase silica gel of octadecylsilane bonding is separated, with the methanol aqueous solution isocratic elution that concentration expressed in percentage by volume is 75%, collect 8 ~ 12 column volume elutriants, elutriant concentrating under reduced pressure obtains pure compound (I) (40mg).
Structural identification: HR-ESIMS shows [M+Na]
+for m/z355.1900, can obtain molecular formula in conjunction with nuclear-magnetism feature is C
20h
28o
4, degree of unsaturation is 7.Hydrogen nuclear magnetic resonance modal data δ
h(ppm, DMSO-d
6, 600MHz): H-2 (1.99, m), H-2 (1.55, m), H-3 (1.59, m), H-3 (1.23, m), H-5 (2.09, dd, J=3.1, 14.6), H-6 (2.48, dd, J=3.1, 14.6), H-6 (2.38, m), H-11 (2.35, m), H-11 (2.64, m), H-12 (1.59, m), H-12 (1.48, m), H-14 (4.22, s), H-15 (4.19, t, J=7.8), H-16 (4.09, t, J=7.8), H-16 (3.55, t, J=7.8), H-17 (0.91, s), H-18 (0.93, s), H-19 (0.95, s), H-20 (1.11, s), carbon-13 nmr spectra data δ
c(ppm, DMSO-d
6, 150MHz): 216.4 (C, 1-C), 25.2 (CH
2, 2-C), 33.0 (CH
2, 3-C), 32.3 (C, 4-C), 42.9 (CH, 5-C), 34.5 (CH
2, 6-C), 198.6 (C, 7-C), 128.8 (C, 8-C), 168.1 (C, 9-C), 44.2 (C, 10-C), 20.8 (CH
2, 11-C), 21.7 (CH
2, 12-C), 40.4 (C, 13-C), 76.1 (CH, 14-C), 79.3 (CH, 15-C), 70.2 (CH
2, 16-C), 17.8 (CH
3, 17-C), 31.9 (CH
3, 18-C), 20.6 (CH
3, 19-C), 17.8 (CH
3, 20-C), carbon atom mark is see Fig. 1.Infrared spectra shows this compound and contains hydroxyl (3217cm
-1) and carbonyl (1685cm
-1) group.
1the display of HNMR spectrum is containing four methyl signals δ H0.91 (s, H
3-17), 0.93 (s, H
3-18), 0.95 (s, H
3-19) and 1.11 (s, H
3-20).
13cNMR stave is bright contains four methyl signals, six methylene radical, and (two containing oxygen methyne δ C76.1 for three methynes, 79.3), seven quaternary carbons [comprise two carbonyl carbon (δ C216.4,198.6), two alkene carbon (δ C128.8,168.1)].Above-mentioned data show that this compound is highly oxidized diterpene-kind compound.H in HMBC spectrum
3with the dependency of C-1 (δ C216.4) ,-20 (δ H1.11) show that C-1 is ketone carbonyl.In addition, and H-5 (2.09, dd, J=3.1,14.6Hz) and H-14 (4.22, s) show with the dependency of C-7 (δ C198.6) that C-7 is also ketone carbonyl.H-6 and C-8, H-14 and C-8, H in being composed by HMBC
3be double bond between-20 and the dependency C-8 of C-9 and C-9.Low field, C-14 position carbon signal (δ C76.1), then infer to there is ehter bond between C-14 and C-16 in conjunction with degree of unsaturation.H-14 and C-16 in HMBC spectrum; H-14 and C-15; And the above-mentioned supposition of the relevance verification of H-16 and C-13.H-14 and H in NOESY spectrum
3-17; H-15 and H-12 α; H-15 and H
3the dependency of-17 shows H-14, H-15 and H
3-17 is beta comfiguration.Comprehensive hydrogen spectrum, carbon spectrum, HMBC spectrum and NOESY spectrum, and document is about correlation type nuclear magnetic data, can substantially determine this compound as shown in Figure 1, steric configuration is determined further by ECD test, theoretical value and experimental value basically identical (Fig. 2).
Embodiment 2: compound (I) pharmacological action is tested
One, material and instrument
Vascular endothelial cell (ECV-304) is presented by immunity teaching and research room of medical college of Shandong University.Compound (I) is made by oneself, and preparation method is shown in that embodiment 1, HPLC normalization method purity is greater than 98%.RPMI-1640 dehydrated medium, trypsinase are purchased from Gibeo company of the U.S..Foetal calf serum is purchased from Tianjin TBD company.MTT, agarose, AnnexinV-FITC are purchased from Sigma Co., USA.LDH, MDA, SOD, GSH-Px mensuration test kit is purchased from Nanjing and builds up Bioengineering Research Institute.DMSO is purchased from Chinese Medicine (group) Solution on Chemical Reagents in Shanghai company limited.Hematorylin is purchased from Foochow and steps true tumor Technology Co., Ltd..Rhodamine123 is purchased from Solarbic company of the U.S..Positive drug Ligustrazine is purchased from Yuan Ye bio tech ltd, Shanghai.
Inverted light microscope (Japanese OlymPus company), CO2gas incubator (FormaScientific company of the U.S.), electronic analytical balance (ER-182A type) (Japanese A & D company), Bechtop (ZHJH-1209 type) (Shanghai ZHICHENG Anaiytical Instrument Manufacturing Co., Ltd.), flow cytometer (FACSVantage type) (BeetonDiehnson company of the U.S.), constant temperature oscillator (Taicang, Jiangsu experimental installation factory), 1420Vietor
3multiple labeling analyser (PerkinElmerLifescience company of the U.S.), the visible ultraviolet grating spectrophotometer of 721 type (Shanghai exact science company limited), electric heating constant-temperature water-bath tank (production of Shanghai Medical instrument three factory), enzyme-linked immunosorbent assay instrument (MK3Multiskan) (Shanghai ThermoLabsystem analyzes).
Two, test method
1, cell cultures
ECV-304 cell is cultivated based on 37 DEG C with RPMI-1640,5%CO
2secondary Culture in incubator.Microscopic observation, goes down to posterity with tryptic digestion when cell confluency rate reaches more than 70%, prepares cell suspension to grow stable 3-5 for cell.
2, cell grouping and process
Get well-grown ECV-304 cell and make cell suspension, be inoculated in 96 holes, 24 holes, 6 porocyte culture plates or culture dish, 37 DEG C, 5%CO
2cultivate 24h.Be divided into six groups at random: 1. normal group; 2. H
2o
2model group (150 μm of ol/L); 3. Ligustrazine (TMP) (50 μm of ol/L)+H
2o
2group; 4. compound (I) (10 μm of ol/L)+H
2o
2group; 5. compound (I) (50 μm of ol/L)+H
2o
2group; 6. compound (I) (100 μm of ol/L)+H
2o
2group.
3, experimental project and Testing index
3.1MTT method detects cell viability
Well-grown ECV-304 cell is prepared into 5 × 10
4the cell suspension of/mL, is inoculated in 96 orifice plates by every hole 100 μ L, puts 37 DEG C, 5%CO
2hatch 24h.Cell divides into groups and processes.After continuing to cultivate 6h and 12h, every hole adds 10 μ LMTT solution (final concentration 0.5mg/L) and puts 37 DEG C, 5%CO
2after incubator hatches 4h, every hole adds 100 μ LDMSO, and the built-in enzyme-linked immunosorbent assay instrument of 60s, 30min that vibrates after leaving standstill 10min detects 570nm place absorbance (OD
570).
By formula: cell injury inhibiting rate=(medication group OD
570-model group OD
570)/(normal group OD
570-model group OD
570) × 100%, calculates cell injury inhibiting rate.
3.2LDR release rate assay
Get well-grown ECV-304 cell and make l × 10
5the cell suspension inoculation of/mL, in 24 orifice plates, puts 37 DEG C, 5%CO
2hatch 24h.Cell divides into groups and processes the same.Continue to cultivate 6h and 12h, collect culture supernatant.After PBS washs 2 times, every hole adds 0.5mL cell pyrolysis liquid (150mmol/LNaCl, 150mmol/LTris-HCI, lmmol/LEDTA, 1%TritonX-100), vibrate after 4 DEG C of standing 15min several minutes, 10000rpm, 4 DEG C of centrifugal 10min collecting cell lysates, measure the LDH activity in supernatant liquor and cell pyrolysis liquid respectively with reference to LDH test kit specification sheets.
By formula: LDH activity in LDH release rate=supernatant liquor/(in supernatant liquor in LDH activity+cell pyrolysis liquid LDH activity) ` × 100%, calculates LDH release rate.
3.3 enzyme biochemical process measures MDA content, SOD vigor, GSH-Px vigor
Get well-grown ECV-304 cell and make 1 × 10
5the cell suspension inoculation of/mL, in 24 orifice plates, puts 37 DEG C, 5%CO
2hatch 24h.Cell divides into groups and processes the same.Continue to cultivate 12h, collect culture supernatant.Cell MDA content, SOD vigor, GSH-Px vigor is measured by MDA, SOD, GSH-Px detection kit specification sheets.
4, data processing
The statistical software that MicrosoftExcel carries processes, experimental data with
represent, group difference t checks.
Three, result and conclusion
1, compound (I) is to H
2o
2the impact of damage ECV-304 cells survival rate
Produce succinodehydrogenase in normal live cells mitochondrial process, faint yellow MTT can be reduced into the cured crystallization of water-fast hepatic first, crystallization quantity is directly proportional to viable count.This experimental result shows: ECV-304 cell is through H
2o
2after (150 μm of ol/L) oxidative damage 6h and 12h, cell OD value obviously reduces and have statistical significance (P<0.01) compared with normal group, shows that cell viability declines.And compound (I) has provide protection to cell, significantly H can be suppressed
2o
2to the oxidative damage of cell, improve survival rate.Effect 6h, 50,100 μm of ol/L compounds (I) are respectively 16.67% (P<0.05) and 28.21% (P<0.01) to cell injury inhibiting rate.Effect 12h, 10,50,100 μm of ol/L compounds (I) rise to 24.85% (P<0.05) to cell injury inhibiting rate, 29.64% (P<0.01) and 38.47% (P<0.01).In table 1 (* * P<0.01vsNormal;
#p<0.05,
#p<0.01vsH
2o
2group;
▲p<0.05,
▲ ▲p<0.01vsTMPgroup, lower same).
2, compound (I) is to H
2o
2the impact of damage ECV-304 cell LDH release rate
Serum lactic dehydrogenase energy catalysis lactic acid generates pyruvic acid, and pyruvic acid and 2,4 dinitrophenyl hydrazine react and generate pyruvic acid dinitrophenylhydrazone, in red-brown in basic solution, indirectly can obtain Ldh Activity by colorimetric estimation reaction product.Result shows: compared with normal group, and model group ECV-304 cell LDH release rate significantly increases (P<0.01).With model group ratio, compound (I) respectively group cell LDH release rate all reduces: 10,50,100 μm of ol/L compound (I) effect 6h, and the LDH release rate of cell reduces to 20.54% (P<0.01) and 21.22% (P<0.01); 50,100 μm of ol/L compound (I) effect 12h, the LDH release rate of cell reduces to 33.64% (P<0.01) and 29.53% (P<0.01).Compound (I), with the increase of concentration, also strengthens gradually the provide protection of oxidative damage ECV-304 cell, presents certain dose-effect relationship.The results are shown in Table 2.
3, compound (I) is to H
2o
2the impact of damage ECV-304 cell MDA content, SOD, GSH-Px vigor
Experimental result shows: significantly increase (P<0.01) than MDA content in model group ECV-304 cell culture fluid with normal group.Compared with model group, 10,50,100 μm of ol/L compound (I) group cell MDA growing amounts all obviously reduce, be respectively 3.00 ± 0.79nmol/mL (P<0.05), 2.86 ± 0.75nmol/mL (P<0.05) and 2.69 ± 0.45nmol/mL (P<0.01).Contrast display between compound (I) each concentration group group, with the increase of concentration, the provide protection of compound (I) to ECV-304 cytolipin peroxide injury also strengthens gradually, presents certain dose-effect relationship.The results are shown in Table 3.
Experimental result shows: compared with normal group, and model group ECV-304 cell SOD is active significantly reduces (P<0.01).Compared with model group, 50,100 μm of ol/L compounds (I) all can to strengthen the SOD of ECV-304 cell active, SOD activity rises to 17.9 ± l.34U/mL (P<0.05) and 19.25 ± 0.81U/mL (P<0.01) respectively.And with the increase of concentration, the SOD activity of cell also improves gradually, shows that compound (I) can strengthen the antioxygen free action of ECV-304 cell, a certain amount of effect relationship of tool; 10 μm of ol/L compounds (I), though can improve SOD activity, do not have remarkable statistical significance yet.In table 3.
Experimental result shows: compared with normal group, and in model group ECV-304 cell culture fluid, GSH-Px is active significantly reduces (P<0.01).Compared with model group, 10,50,100 μm of ol/L compound (I) group cell GSH-Px activity are significantly increased, be respectively 73.8 ± 10.3U/mL (P<0.05), 92.2 ± 8.5U/mL (P<0.01) and 102.5 ± 10.3U/mL (P<0.01).And with the increase of compound (I) concentration, the GSH-Px activity of ECV-304 cell also improves gradually, shows that the ability of cellular anti-oxidant effect also strengthens gradually, presents certain dose-effect relationship.The results are shown in Table 3.
Sum up: this experiment adopts H
2o
2as exogenous free radical generation system, can induction of vascular endothelial cell (ECV-304) oxidativestress damage, promote apoptosis.Detected by mtt assay and LDH activity and confirm that compound (I) is to H
2o
2oxidative damage cell has provide protection; By cell conditioned medium liquid MDA content, SOD is active, GSH-Px is active mensuration, confirm that compound (I) has the anti-oxidation characteristics of scavenging free radicals and active oxygen.
Table 1 compound (I) is to H
2o
2the impact of damage ECV-304 cells survival rate (
n=8)
Table 2 compound (I) is to H
2o
2the impact of damage ECV-304 cell LDH release rate (
n=8)
Table 3 compound (I) is to H
2o
2the impact of damage ECV-304 cell MDA content, SOD, GSH-Px vigor (
n=8)
Embodiment 3
The preparation of tablet: by embodiment 1 method first obtained compound (I), and the salt utilizing organic acid to make as tartrate or citric acid or formic acid or oxalic acid etc., mineral acid example hydrochloric acid or sulfuric acid or phosphoric acid, vehicle is added, pelletizing press sheet than the ratio for 1:7 in itself and excipient weight.
Embodiment 4
The preparation of oral liquid: by embodiment 1 method first obtained compound (I), and the salt utilizing organic acid to make as tartrate or citric acid or formic acid or oxalic acid etc., mineral acid example hydrochloric acid or sulfuric acid or phosphoric acid, oral liquid method for making makes oral liquid routinely.
Embodiment 5
The preparation of capsule or granule: by embodiment 1 method first obtained compound (I), and the salt utilizing organic acid to make as tartrate or citric acid or formic acid or oxalic acid etc., mineral acid example hydrochloric acid or sulfuric acid or phosphoric acid, add vehicle in itself and excipient weight than the ratio for 1:9, make capsule or granule.
Embodiment 6
The preparation of injection liquid: by embodiment 1 method first obtained compound (I), and the salt utilizing organic acid to make as tartrate or citric acid or formic acid or oxalic acid etc., mineral acid example hydrochloric acid or sulfuric acid or phosphoric acid, inject with water routinely, essence filter, injection liquid is made in embedding sterilizing.
Embodiment 7
The preparation of aseptic powder injection: by embodiment 1 method first obtained compound (I), and the salt utilizing organic acid to make as tartrate or citric acid or formic acid or oxalic acid etc., mineral acid example hydrochloric acid or sulfuric acid or phosphoric acid, be dissolved in sterile water for injection, stirring makes molten, filter with aseptic suction funnel, aseptic essence filter again, be sub-packed in ampoule, after frozen drying, aseptic sealing by fusing obtains powder injection.
The effect of above-described embodiment is essentiality content of the present invention is described, but does not limit protection scope of the present invention with this.Those of ordinary skill in the art should be appreciated that and can modify to technical scheme of the present invention or equivalent replacement, and does not depart from essence and the protection domain of technical solution of the present invention.
Claims (7)
1. there is the compound (I) of following structural formula:
2. the preparation method of compound according to claim 1 (I), it is characterized in that comprising following operation steps: the dry flower of Flos Farfarae is pulverized by (a), extract with 80 ~ 90% alcohol heat reflux, united extraction liquid, be concentrated into without alcohol taste, use sherwood oil, ethyl acetate and water saturated n-butanol extraction successively, obtain petroleum ether extract, acetic acid ethyl ester extract and n-butyl alcohol extract respectively; B in () step (a), the removal of impurities of thing macroporous resin got by propyl carbinol, first use 5% ethanol elution, 6 column volumes, then use 75% ethanol elution, 8 column volumes, collect 75% elutriant, concentrating under reduced pressure obtains 75% ethanol elution enriched material; C in () step (b), 75% ethanol elution enriched material purification on normal-phase silica gel is separated, obtain 4 components successively with the methylene chloride-methanol gradient elution that volume ratio is 65:1,30:1,15:1 and 8:1; D in () step (c), component 4 is separated further by purification on normal-phase silica gel, obtain 3 components successively with the methylene chloride-methanol gradient elution that volume ratio is 15:1,8:1 and 5:1; E in () step (d), component 2 reverse phase silica gel of octadecylsilane bonding is separated, with the methanol aqueous solution isocratic elution that concentration expressed in percentage by volume is 75%, collect 8 ~ 12 column volume elutriants, elutriant concentrating under reduced pressure obtains pure compound (I).
3. the preparation method of compound according to claim 2 (I), is characterized in that: in step (a), extracts, united extraction liquid with 85% alcohol heat reflux.
4. the preparation method of compound according to claim 2 (I), is characterized in that: described macroporous resin is AB-8 type macroporous adsorbent resin.
5. a pharmaceutical composition, is characterized in that: the compound according to claim 1 (I) wherein containing treatment significant quantity and pharmaceutically acceptable carrier.
6. compound according to claim 1 (I) application in the medicine preparing vascular protection.
7. the application of pharmaceutical composition according to claim 5 in the medicine preparing vascular protection.
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| CN105237604A (en) * | 2015-09-12 | 2016-01-13 | 徐建立 | New limonin compound, preparation method and medical uses thereof |
| CN105560223A (en) * | 2016-02-26 | 2016-05-11 | 温州统益生物医药科技有限公司 | Application of highly oxidized diterpene compound in preparation of myocardial preservation medicine |
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| CN105237604A (en) * | 2015-09-12 | 2016-01-13 | 徐建立 | New limonin compound, preparation method and medical uses thereof |
| CN105693657A (en) * | 2016-01-12 | 2016-06-22 | 王尧尧 | New isocoumarin compound and preparing method and medical application thereof |
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| CN108578458A (en) * | 2018-06-08 | 2018-09-28 | 山西大学 | A kind of tussilago active component of anti influenza and its preparation method and application |
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Effective date of registration: 20170313 Address after: 274319 Shandong city of Heze province Shanxian County Gao Lao Jia Xiang Cao Po in Nanjie Village Administrative Village No. 53 Patentee after: Fan Ying Address before: Longwan Waterfront Street Wenzhou city Zhejiang province 325024 Sand Street No. 228 second floor Patentee before: WENZHOU HONGCHENGXIANG TECHNOLOGY CO., LTD. Patentee before: Huang Jiawen |
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Granted publication date: 20160824 Termination date: 20171026 |