CN105153187A - New diterpenoid as well as preparation method and medical application thereof - Google Patents
New diterpenoid as well as preparation method and medical application thereof Download PDFInfo
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- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
- C07D493/12—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains three hetero rings
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Abstract
The invention discloses a new diterpenoid as well as a preparation method and medical application thereof. The new diterpenoid is reported for the first time, serves as a diterpenoid adopting a new structure, and can be obtained through extraction from dried toona sinensis leaves, separation and purification. In vitro tests prove that the new diterpenoid can induce Molt4 cells to be subjected to remarkable cycle arrest and apoptosis after acting on the Molt4 cells, and enables the cell cycle to stay in S phase, so as to induce cell apoptosis and inhibit cell multiplication. The new diterpenoid with a structural formula shown in formula (I) has a potential application value in an aspect of treating acute T lymphocytic leukemia, and can be utilized for being developed into a drug for treating acute T lymphocytic leukemia.
Description
Technical field
The invention belongs to technical field of pharmaceuticals, be specifically related to from the dry leave of Chinese toon, be separated obtain a kind of and there is diterpene-kind compound for the treatment of Pancytopenia effect and preparation method thereof.
Background technology
Chinese toon
toonasinensis(A.Juss.) Roemor has another name called spring bud tree, Chinese toon, Ailanthus altissima, toon is called in " An Illustrated Book on Plants ", for Meliaceae (Meliaceae) Cedrela (Toona) deciduous tree, in the distinctive precious fast-growing medicinal material book of China, have the title of " Chinese mahogany ", mainly be distributed in the provinces such as the Shandong of China, Anhui, Henan, Hebei, wherein try to gain with Taihe county, Anhui, Jiaozhuo, Henan and Shan Dongxi the Chinese toon product produced again the most famous.
Chinese toon complete stool has special odor, and has very high pharmaceutical use because being rich in various bioactivators, and its leaf, bark and fruit all can be used as medicine, and applicating history is very long, begins to be loaded in Tang Materia Medica.Folium toonae sinensis bitter, warm in nature, there is heat-clearing convergence, eliminating inflammation and expelling toxin, go the effects such as eliminating dampness, cure mainly the disease such as enteritis, dysentery; In addition, Folium toonae sinensis rich vitamin C, amino acid, high-quality protein and the trace element such as phosphorus, iron, it is the famous traditional vegetables in season of China, the edible history of existing more than 2,000 year, once together offering or present imperial palace as tribute with lichee hired, is treasure rare in vegetables, the dark favor by consumers in general, be called as abroad " green health dish ", existing multiple exit is to Japan and country in Southeast Asia.Cortex toonae bitter and puckery flavor, cool in nature, there is effect of heat extraction, eliminating dampness, ease constipation hemostasis, desinsection, be clinically usually used in treatment dysentery, enteritis, urinary tract infection, have blood in stool, leukorrhea, lumbago, skelalgia of rheumatism etc.Fruit of Chinese Toona taste is arduous, warm in nature, and tool dispels the wind, effect of loose cold, pain relieving, is clinically used for the treatment of chill diseases caused by external factors, heart peratodynia, rheumatic arthritis, hernia and cold bone wind etc.
At present, the compound that in Chinese toon, isolation identification goes out mainly can be divided into flavonoid, phenols and derivative thereof, terpene (triterpene, sesquiterpene, diterpene), Phenylpropanoid Glycosides class, sulfocompound etc.Modern study shows, the chemical composition in Chinese toon has the multiple biological activitys such as antitumor, anti-oxidant, reducing blood-fat; Chinese scholars has carried out some comparatively deep researchs to the chemical composition in Chinese toon, finds the abundant species of compound contained by it, is a kind of medicinal and edible plant resource being worth studying further, DEVELOPMENT PROSPECT is good.
Summary of the invention
The object of this invention is to provide and a kind ofly from the Folium toonae sinensis of drying, be separated obtain a kind of there is diterpene-kind compound for the treatment of Pancytopenia effect and preparation method thereof.
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 Folium toonae sinensis that (a) is dry is pulverized, 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; Acetic acid ethyl ester extract macroporous resin removal of impurities in (b) step (a), first use 15% ethanol elution, 8 column volumes, then use 75% ethanol elution, 12 column volumes, collect 75% ethanol eluate, concentrating under reduced pressure obtains 75% ethanol elution thing medicinal extract; C in () step (b), 75% ethanol elution thing medicinal extract purification on normal-phase silica gel is separated, obtain 5 components successively with the methylene chloride-methanol gradient elution that volume ratio is 80:1,50:1,30:1,10:1 and 1:1; 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 20:1,10:1 and 5:1; E in () step (d), component 2 reverse phase silica gel of octadecylsilane bonding is separated, be the methanol aqueous solution isocratic elution of 70% by concentration expressed in percentage by volume, collect 8 ~ 10 column volume elutriants, elutriant concentrating under reduced pressure obtains pure compound (I).
In described step (a), extract with 85% alcohol heat reflux, united extraction liquid.
Described macroporous resin is AB-8 type macroporous adsorbent resin.
A kind of pharmaceutical composition, this pharmaceutical composition contains the compound according to claim 1 (I) for the treatment of significant quantity and pharmaceutically acceptable carrier.
The application of described compound (I) in the medicine of preparation treatment Pancytopenia.
The application of described pharmaceutical composition in the medicine of preparation treatment Pancytopenia.
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.
figure of description
Fig. 1 is compound (I) structural formula;
Fig. 2 is that the theoretical ECD value of compound (I) compares with experiment ECD value.
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.
Embodiment 1: compound (I) is separated preparation and structural identification
Reagent source: ethanol, sherwood oil, ethyl acetate, propyl carbinol, methylene dichloride are analytical pure, purchased from Shanghai Ling Feng chemical reagent company limited, methyl alcohol, analytical pure, purchased from Jiangsu Han Bang chemical reagent company limited.
Preparation method: the dry leave (8kg) of (a) Chinese toon is pulverized, (25L × 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 (355g) and n-butyl alcohol extract respectively; Acetic acid ethyl ester extract AB-8 type macroporous resin removal of impurities in (b) step (a), first use 15% ethanol elution, 8 column volumes, then use 75% ethanol elution, 12 column volumes, collect 75% ethanol eluate, concentrating under reduced pressure obtains 75% ethanol elution thing medicinal extract (136g); C in () step (b), 75% ethanol elution medicinal extract purification on normal-phase silica gel is separated, be 80:1(8 column volume successively by volume ratio), a 50:1(8 column volume), a 30:1(6 column volume), a 10:1(8 column volume) and 1:1(5 column volume) methylene chloride-methanol gradient elution obtain 5 components; Component 4(31g in (d) step (c)) be separated further by purification on normal-phase silica gel, be 20:1(8 column volume by volume ratio successively), a 10:1(10 column volume) and 5:1(6 column volume) methylene chloride-methanol gradient elution obtain 3 components; Component 2(11g in (e) step (d)) be separated with the reverse phase silica gel of octadecylsilane bonding, with the methanol aqueous solution isocratic elution that concentration expressed in percentage by volume is 70%, collect 8-10 column volume elutriant, elutriant concentrating under reduced pressure obtains pure compound (I) (35mg).
Structural identification: HR-ESIMS shows [M+Na]
+for m/z435.1806, can obtain molecular formula in conjunction with nuclear-magnetism feature is C
24h
28o
6, degree of unsaturation is 11.Hydrogen nuclear magnetic resonance modal data δ
h(ppm, DMSO-
d 6 , 600MHz): H-1(5.56, t,
j=1.8), H-2(1.79, m), H-2(2.04, m) and, H-3(1.08, m), H-3(1.83, m), H-6(5.77, d,
j=6.0), H-7(4.93, d,
j=6.0), H-11(6.90, s), H-15(6.76, d,
j=2.4), H-16(7.56, d,
j=2.4), H-17(2.47, s), H-18(1.14, s) and, H-19(1.14, s), H-20(1.52, s) and, 1-OCOCH
3(1.76, s), 6-OCOCH
3(2.11, s); Carbon-13 nmr spectra data δ
c(ppm, DMSO-
d 6 , 150Hz): 77.3(CH, 1-C), 22.6(CH
2, 2-C), 33.7(CH
2, 3-C), 39.6(C, 4-C), 80.9(C, 5-C), 81.2(CH, 6-C), 74.6(CH, 7-C), 128.2(C, 8-C), 140.8(C, 9-C), 50.4(C, 10-C), 104.8(CH, 11-C), 155.8(C, 12-C), 131.5(C, 13-C), 132.5(C, 14-C), 106.2(CH, 15-C), 146.4(CH, 16-C), 17.0(CH
3, 17-C), 29.9(CH
3, 18-C), 25.2(CH
3, 19-C), 29.7(CH
3, 20-C), 171.3(C, 1-OCOCH
3), 21.1(CH
3, 1-OCOCH
3), 173.0(C, 6-OCOCH
3), 22.2(CH
3, 6-OCOCH
3); Carbon atom mark is see Fig. 1.Infrared spectra shows that this compound contains aromatic carbon (3010nm, 1610nm).
1hNMR composes display 4 methyl signals δ H1.14, and 1.14,1.52,2.47.Two olefinic protons δ H6.76(H-15 intercoupled, d, J=2.4) and 7.56(H-16, d, J=2.4Hz) show that this compound contains a fused furan ring.
13cNMR shows 24 carbon signals, comprising 6 aromatic carbons (δ C104.8,128.2,131.5,132.5,140.8 and 155.8).These data show, this compound is the diterpene-kind compound condensed with furan nucleus.In HMBC spectrum, H-11(δ H6.90, s) and C-12(δ C155.8) and C-13(δ C131.5); H
3-17(δ H2.47, s) with C-8(δ C128.2), C-13(δ C131.5), C-14(δ C132.5) and C-15(δ C106.5) relevance verification phenyl ring and this inference of furan nucleus conjugation.In addition, the dependency of C-1 and C-6 and corresponding acetoxyl group shows that two acetoxyl groups lay respectively on C-1 and C-6 position.From H-1(5.56, t,
j=1.8) to H
3-19(δ H1.14, s) and H
3-20(δ H1.52, s) enhancing of NOE signal, show that the acetoxyl group of C-1 position is α configuration.?
1h-
1hCOSY composes, H-6(5.77, d,
j=6.0) and H-7(4.93, d,
j=6.0) dependency shows, C-6(δ C81.2) with C-7(δ C74.6) be all connected with oxygen.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
Pancytopenia Molt4 cell is so kind as to give by hemopathy institute of Ji'nan University; Chronic myeloid leukemia cell K562 cell is so kind as to give by Biochemistry and Molecular Biology teaching and research room by Ji'nan University.Compound (I) is self-control, and HPLC normalization method purity is greater than 98%, dissolves be mixed with 5.5 μ g/mL solution for standby with DMSO analytical pure.DMEM/F12 medium powder, hydroxyethyl piperazine ethanesulfonic acid (HEPES), the blue reagent of tetraphenyl nitrogen (MTT) are purchased from Gibeo company of the U.S..New-born calf serum (NewbomCalfSerum) is purchased from Hangzhou China folium ilicis chinensis company.Penicillin (Penicillin), Streptomycin sulphate (Streptomycin) are purchased from China of North China drugmaker.Iodate third eyelash (PT) is purchased from Sigma Co., USA.The two transfection reagent box of PI-AnnexinV is purchased from Bao Sai biotech company of BeiJing, China.ROS high-quality fluorimetric reagent box is purchased from Chinese green skies biotechnology research institute.Rhodamine123 is purchased from MolecularProbes company of the U.S..
CO
2incubator (U.S. ThermoForma), Bechtop (Chinese Suzhou purifying apparatus factory), pressure steam sterilization boiler (LDZX40BI) (Chinese Shanghai Shen An medical apparatus and instruments factory), TGL-16G type table model high speed centrifuge (Chinese Shanghai An Ting scientific instrument factory), MA260S type electronic analytical balance (Chinese Shanghai second balance equipment factory), automatic dual pure water distiller (Chinese Shanghai glassware one factory), SterivexTM, 0.22 μm of Millex Syringe Filters (Millipore company of the U.S.), XDS-1B optics inverted microscope (Chongqing in China optical instrument factory), full-automatic microplate reader (BIO-RID company of the U.S.), FACSCalibur flow cytometer (BDFACSAria company of the U.S.).QL-901 type vortex mixer (kylin medical apparatus factory of Jiangsu Haimen City).
Two, test method
1, cell cultures
People's Pancytopenia cell Molt4 cell culture system: DMEM/F12 nutrient solution containing 10% new-born calf serum, is put 37 DEG C, volume fraction is 5%CO
2incubator, every 2-3 days Secondary Culture.Select logarithmic phase, the cell of 0.2% trypan blue exclusion rate >95% tests.
2, mtt assay detects cell proliferation inhibition rate
3 × 10
4mLMolt4 cell is inoculated in 96 well culture plates, and final volume is 200 μ L/ holes, and often group establishes 5 multiple holes, and compound (I) establishes five concentration to be respectively 2.5 μ g/mL, 5 μ g/mL, 10 μ g/mL, 15 μ g//mL, 20 μ g//mL.Put 37 DEG C, 5%CO
2after cultivating 48h, 72h in incubator, every hole adds MTT solution (5mg/mL PBS<ph=7.4 joins) 20 μ L, continues to hatch 4 hours, stops cultivating, centrifugal, culture supernatant in hole is abandoned in careful suction, and every hole adds 150 μ LDMSO, vibrates 10 minutes, crystallisate is fully melted, microplate reader detects light absorption value (measuring wavelength 570nm, reference wavelength 690nm), calculates each group of proliferation inhibition rate.Each experiment repetition 3 times, gets its average.Proliferation inhibition rate (%)=(1-A experimental group/A control group) × 100%.
3, the mono-dye of PI detects the cell cycle
2 × l0
5/ mLMolt4 cell is inoculated in 12 well culture plates, every hole 1800 μ L, and compound (I) establishes three concentration to be respectively 5 μ g/mL, 10 μ g/mL15 μ g/mL, final volume 2500 μ L, if 3 multiple holes.Put 37 DEG C, 5%CO
2collecting cell after 48h is cultivated in incubator, the 0.01mol/LPBS washed cell of precooling 2 times, be that 70% ethanol 4 DEG C fixedly spends the night by volume fraction, centrifugally remove supernatant, add PI staining fluid (containing RNA enzyme), final concentration 50 μ g/mL, lucifuge dyeing 30min, flow cytometer analysis of cells DNA content after 300 order nylon net filters, each sample stochastic analysis 12000 cells, obtain each group of cell growth cycle ratio, U.S. BDFACSortCellQuest software analysis result.
4, the two dye of AnnexinV-PI measures apoptosis ratio
Cell process and adding method thereof the same, cultivate after 48h, adjustment cell concn is 5 × 106/mL, get lmL cell for each group, precooling 0.01mol/LPBS washs 3 times, exhausts supernatant, adds the binding buffer liquid that 200 μ L test kits provide, re-suspended cell, add 10 μ LAnnexinV-FITC and 5 μ L respectively, mix gently, 4 DEG C of lucifuge reaction 30min, add 300 μ L binding buffer liquid again, flow cytomery.The cell mass (i.e. LR cell mass) of AnnexinV-FITC+, PI-is viable apoptotic cell.
5, statistical study
Represent with mean scholar standard deviation (`X ± S), the process of application SPSS13.0 statistical software, adopts the one-way analysis of variance (one-wayANOVA) of completely randomized design to analyze the significance of group difference.
Three, result and conclusion
1, compound (I) is to the inhibited proliferation of Molt4 cell
Compound (I) all has obvious inhibited proliferation in different time points to Molt4 cell.DMSO(0.5%) group is 5.2% ± 0.8%, 6.40% ± 0.9% at the proliferation inhibition rate of 48h, 72h respectively.The proliferation inhibition rate of different concns compound (I) treatment group all has statistical significance (P<0.01) compared with DMSO group.Under same concentrations condition, comparatively 48h is high for the proliferation inhibition rate of 72h.Compound (I) is to the IC of Molt4 cell 48h, 72h
5019.4 ± 0.2 μ g/mL, 15.7 ± 0.1 μ g/mL respectively.The compound (I) of different concns all shows certain proliferation inhibiting effect, and there is the time, dosage relies relation.The results are shown in Table 1(note: * mark compares with Control group, P<0.01).
2, compound (I) is on the impact of Molt4 cell cycle
After compound (I) effect Molt4 cell 48h, blank group G1 phase cell proportion is 37.5% ± 0.2%, S phase cell proportion is 50.6% ± 0.1%, G2 phase cell proportion is 11.7% ± 0.1%, DMSO group G1 phase cell proportion is 39.6% ± 0.1%, S phase cell proportion is 51.6% ± 0.2%, G2 phase cell proportion is 8.7% ± 0.2%.Shared by compound (I) medicine 20 μ g/mL, 25 μ g/mL treatment group G1 phases, cell proportion is respectively 4.1% ± 0.1%, 65.8% ± 0.1%, ratio increase shared by S phase cell is respectively 85.1% ± 0.3%, 87.1% ± 0.25(P<0.01), G2 phase cell proportion is respectively 10.7% ± 0.2%, 7.0% ± 0.1%, the result display G1 phase have significant difference (P<0.05) to illustrate compound (I) retardance Molt4 cell is in the cycle S phase.The results are shown in Table 2(note: * mark compares with Control group, P<0.01).
3, the early apoptosis rate after compound (I) effect Molt4 cell
After compound (I) effect Molt4 cell 48h, AlmexinV/PI two dye flow cytomery early apoptosis rate.Early apoptosis rate 7.0% scholar 0.3% of blank group early apoptosis of cells rate 6.6% ± 0.4%, DMSO control group, not statistically significant (p>0.05) compared with blank.Compound (I) 10 μ g/mL group, 15 μ g/mL group early apoptosis rates are 9.5% ± 0.3%, 15.0% ± 0.5% respectively, have statistical significance (P<0.05) compared with blank.The results are shown in Table 3(note: * mark compares with Control group, P<0.05).
Conclusion, we can induce its significant Cycle Arrest and apoptosis after finding compound (I) effect Molt4 cell under study for action, and make cell cycle arrest in the S phase, thus cell death inducing, antiproliferative effect.Compound (I) has potential using value in treatment Pancytopenia.
Table 1 compound (I) is to the inhibited proliferation (%, `x ± s, n=3) of Molt4 cell
| Group | 48h inhibiting rate (%) | 72h inhibiting rate (%) |
| DMSO | 5.2±0.8 | 6.4±0.9 |
| Compound (I) 2.5 μ g/mL | 6.3±1.3 | 8.4±1.1 |
| Compound (I) 5 μ g/mL | 10.5±2.4 | 11.6±2.5 |
| Compound (I) 10 μ g/mL | 18.6±1.6 * | 26.9±2.7 * |
| Compound (I) 15 μ g/mL | 33.4±2.7 * | 49.4±2.2 * |
| Compound (I) 20 μ g/mL | 53.2±4.4 * | 65.4±1.6 * |
Table 2 is group cell cycle per-cent (%, `x ± s, n=3) respectively
Table 3 compound (I) is on the apoptotic impact of Molt4 (%, `x ± s, n=3)
| Group | Apoptosis rate |
| Contrast | 6.6±0.2 |
| DMSO | 7.0±0.4 |
| Compound (I) 10 μ g/mL | 9.5±0.3 * |
| Compound (I) 15 μ g/mL | 15.0±0.3 * |
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 1:9 in itself and excipient weight.
Embodiment 4
Prepared by 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, and 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 Folium toonae sinensis that (a) is dry is pulverized, 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; Acetic acid ethyl ester extract macroporous resin removal of impurities in (b) step (a), first use 15% ethanol elution, 8 column volumes, then use 75% ethanol elution, 12 column volumes, collect 75% ethanol eluate, concentrating under reduced pressure obtains 75% ethanol elution thing medicinal extract; C in () step (b), 75% ethanol elution thing medicinal extract purification on normal-phase silica gel is separated, obtain 5 components successively with the methylene chloride-methanol gradient elution that volume ratio is 80:1,50:1,30:1,10:1 and 1:1; 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 20:1,10:1 and 5:1; E in () step (d), component 2 reverse phase silica gel of octadecylsilane bonding is separated, be the methanol aqueous solution isocratic elution of 70% by concentration expressed in percentage by volume, collect 8 ~ 10 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: this pharmaceutical composition contains the compound according to claim 1 (I) for the treatment of significant quantity and pharmaceutically acceptable carrier.
6. the application of compound according to claim 1 (I) in the medicine of preparation treatment Pancytopenia.
7. the application of pharmaceutical composition according to claim 5 in the medicine of preparation treatment Pancytopenia.
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| CN105687179A (en) * | 2016-01-20 | 2016-06-22 | 温州统益生物医药科技有限公司 | Application of catclaw buttercup root tuber extract as composition in preparing medicine for treating cancer |
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