CN104961791A - Novel triterpene compound in potentilla anserina, and preparation method and application thereof - Google Patents
Novel triterpene compound in potentilla anserina, and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a novel triterpene compound in potentilla anserina, and a preparation method and application thereof, belonging to the technical field of medicines. The compound is reported for the first time, is a cycloartane-type triterpene compound with a novel structure and can be extracted from rhizome of potentilla anserina and then prepared through separation and purification. In-vitro test results show that the compound can inhibit propagation of melanoma cells, so the compound can be developed into a drug used for treating melanoma.
Description
Technical field
The invention belongs to technical field of pharmaceuticals, be specifically related to from fern fiber crops, be separated a kind of Cycloartane type triterpene compound obtained, containing its pharmaceutical composition and its preparation method and application.
Background technology
Medicine fern fiber crops be Rosaceae potentilla plants potentilla anseriana (
potentilla anserinel.) block root, have promote the production of body fluid to quench thirst, invigorating spleen and reinforcing stomach, benefiting vital QI for enriching blood, astringing to arrest bleeding, antidiarrheal, cough-relieving, sharp phlegm effect, cure mainly the diseases such as haematemesis, lower blood, metrorrhagia, malaria carbuncle sore, insufficiency of the spleen diarrhoea, diarrhea.18 seed amino acids of fern fiber crops rich in starch, lipid acid and needed by human body and multivitamin, have higher medical treatment and nutritive value.The book such as " Chinese medicine voluminous dictionary ", " herbal medicine is commonly used in Tibet " is all on the books to this medicine.These product are very abundant at Qinghai Province's wild resource, and Medicinal for a long time, is particularly applied more in Tibetan medicine.
Triterpene compound removes by several isoprene the material formed that to join end to end after hydroxyl.Major part is 30 carbon atoms, and small part is containing the terpenoid of 27 carbon atoms.Triterpenes components is very wide in distributed in nature, has triterpene substance in the effective constituent of shark oil shai, Radix Glycyrrhizae, shizandra berry.Triterpene compound is functional group and different side chains contained by it, also can be divided into different basic frameworks.
Obtain triterpenoid in three pentacyclic triterpenes (Hong Xia etc., herbal medicine, the 37th volume the 2nd phase, 165 pages ~ 168 pages) except Hong Xia etc. is separated from fern snippings stem, there is not yet the report of other triterpenoids in fern fiber crops.
Summary of the invention
The object of this invention is to provide and a kind ofly from fern fiber crops, be separated a kind of Cycloartane type triterpene 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 triterpenoid I of following structural formula:
The preparation method of described chemical compounds I, comprise following operation steps: (a) fern snippings stem is pulverized, extract with 75% 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), use 15% ethanol and 70% ethanol elution successively, collect 70% elutriant, concentrating under reduced pressure obtains 70% ethanol elution enriched material; C in () step (b), 70% 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 40:1,20:1,10:1 and 5:1; 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 8:1,5:1 and 2:1; E in () step (d), component 2 reverse phase silica gel of octadecylsilane bonding is separated, be the methanol aqueous solution isocratic elution of 65% by concentration expressed in percentage by volume, collect 7-9 column volume elutriant, elutriant concentrating under reduced pressure obtains pure chemical compounds I.
Further, described macroporous resin is D101 type macroporous adsorbent resin.
Pharmaceutical composition, the described chemical compounds I wherein containing treatment significant quantity and pharmaceutically acceptable carrier.
The application of described chemical compounds I in the melanomatous medicine of preparation treatment.
The application of described pharmaceutical composition in the melanomatous medicine of preparation treatment.
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 chemical compounds I structural formula;
Fig. 2 is chemical compounds I two dimension hsqc spectrum;
Fig. 3 is chemical compounds I two dimension
1h-
1h COSY composes;
Fig. 4 is chemical compounds I two dimension HMBC spectrum;
Fig. 5 is chemical compounds I two dimension NOESY spectrum;
Fig. 6 is that chemical compounds 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.
People A375 melanoma cell strain is given by Third Military Medical University.Chemical compounds I, purity 99% is self-control.DMEM substratum, 0.25% pancreatin purchased from American Gibco company; MTT (3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide bromine salt; Methyl thiazoly tetrazolium assay, DMSO(dimethyl sulfoxide (DMSO)), BrdU purchased from American Sigma company; Mouse-anti BrdU polyclonal antibody purchased from American ABcam company; Goat against murine two anti-purchased from American Cell signaling company; Annexin V-FITC/PI cell apoptosis detection kit purchased from American Promega company.
Constant temperature CO
2incubator, general refrigerator ,-80 DEG C of refrigerators are Forma company of the U.S.; Bechtop is purchased from Chinese Suzhou cleaning project company; Inverted microscope, inverted fluorescence microscope is purchased from olympus company; Electro-heating standing-temperature cultivator to be leaped medical apparatus and instruments factory purchased from Chinese Shanghai; Automatic microplate reader is purchased from Japanese Wako company; UV detector purchased from American Beckman company; J6-HC supercentrifuge purchased from American Beckman company; Low-temperature trace whizzer is purchased from German Eppendorf company; Vibration shaking table purchased from American Forma company.
Embodiment 1: chemical compounds I is separated preparation and structural identification
A () fern snippings stem (10kg) is pulverized, (30L × 3 time) are extracted with 75% 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 D101 macroporous resin removal of impurities in (b) step (a), use 15% ethanol (8L) and 70% ethanol (12L) wash-out successively, collect 70% elutriant, concentrating under reduced pressure obtains 70% ethanol elution enriched material (125g); C in () step (b), 70% 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 40:1,20:1,10:1 and 5:1; Component 4(25g in (d) step (c)) be separated further by purification on normal-phase silica gel, obtain 3 components with the methylene chloride-methanol gradient elution that volume ratio is 8:1,5:1 and 2:1 successively; Component 2(12g 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 65%, collect 7-9 column volume elutriant, elutriant concentrating under reduced pressure obtains pure chemical compounds I (35mg).
Structural identification: faint yellow needle crystal, is soluble in chloroform, ethyl acetate, acetone and methyl alcohol; HR-ESIMS shows [M+Na]
+for m/z 489.6542, can obtain molecular formula in conjunction with nuclear-magnetism feature is C
30h
42o
4, degree of unsaturation is 10.Infrared IR is presented at 3442, and 1732 and 1715cm
-1having absorption, there is hydroxyl and carbonyl in prompting.Hydrogen nuclear magnetic resonance modal data δ
h(ppm, Pyridine-
d 5 , 400MHz): H-1(5.55, br, s), H-2(2.53, m) and, H-2(2.26, m), H-3(3.78, dd,
j=6.2,10.1), H-5(2.36, m), H-6(2.64, m) and, H-6(2.38, m), H-7(5.46, t,
j=6.4), H-11(5.38, br, d,
j=5.2), H-12(2.31, br, d,
j=17.4), H-12(2.16, dd,
j=17.4,6.0), H-15(2.41, d,
j=17.8), H-15(2.10, d,
j=17.8), H-17(2.39, d,
j=9.0), H-18(0.76, s), H-19(3.22, d,
j=13.5), H-19(3.13, d,
j=13.5), H-20(2.73, m), H-21(1.10, d,
j=6.6), H-22(4.09, br, d,
j=17.1), H-24(4.41, s), H-26(1.62, s) and, H-27(1.58, s), H-28(1.03, s) and, H-29(1.30, s), H-30(0.95, s); Carbon-13 nmr spectra data δ
c(ppm, Pyridine-
d 5 , 100MHz): 120.7(CH, 1-C), 33.2(CH
2, 2-C), 73.6(CH, 3-C), 39.0(C, 4-C) and, 51.1(CH, 5-C), 25.3(CH
2, 6-C), 125.6(CH, 7-C), 142.5(C, 8-C) and, 137.9(C, 9-C), 139.0(C, 10-C) and, 121.1(CH, 11-C), 36.6(CH
2, 12-C), 43.4(C, 13-C), 45.8(C, 14-C) and, 47.9(CH
2, 15-C), 218.3(C, 16-C), 60.0(CH, 17-C) and, 16.6(CH
3, 18-C), 43.8(CH
2, 19-C), 27.4(CH, 20-C), 20.4(CH
3, 21-C), 47.4(CH
2, 22-C), 214.3(C, 23-C), 84.4(CH, 24-C) and, 72.4(C, 25-C), 27.5(CH
3, 26-C), 26.3(CH
3, 27-C), 25.1(CH
3, 28-C), 25.1(CH
3, 29-C), 14.6(CH
3, 30-C); Carbon atom mark is see Fig. 1.Composed by DEPT known, in 30 carbon, have 7 methyl, 6 methylene radical, 8 methynes and 9 quaternary carbons.In hydrogen spectrum, typical δ H 0.76,1.62,1.58,1.03,1.30,0.95 six is connected to methyl signals on quaternary carbon and δ H 1.10(d, J=6.6) the methyl signals be connected on tertiary carbon points out this compound may be 9,19-Cycloartane type triterpene compound (cyclolanostane triterpenoid).Alkene hydrogen δ H 5.38 (1H, d, J=5.2 Hz in hydrogen spectrum, H-11), 5.46 (1H, t, J=6.4 Hz, H-7) and 5.55 (1H, s, H-1) corresponding to three couples of olefinic carbon δ C, i.e. 120.7 (C-1) and 139.0 (C-10) in carbon spectrum, 125.6 (C-7) and 142.5 (C-8), 121.1 (C-11) and 137.9 (C-9).In carbon spectrum, two carbonyl carbon δ C 218.3 (C-16), 214.3 (C-23) and three companies' oxygen carbon δ C 73.6 (C-3), 84.4 (C-24), 72.4 (C-25) point out this compound to be highly oxidized Fourth Ring 9,10-seco-9,19-cyclolanostane triterpene.The two dimensional structure of this compound can pass through HSQC(Fig. 2),
1h-
1h COSY(Fig. 3) and HMBC(Fig. 4) two-dimensional spectrum determines.In HMBC spectrum, H
3-29/30 is relevant to C-3, C-4, C-5, can determine that hydroxyl is positioned on C-3; H-1 and C-2, C-3, C-5 are correlated with, and H-7 and C-5, C-6, C-9, C-14 are correlated with, and H-11 and C-8, C-13 are relevant, H
2-19 is relevant to C-1, C-8, C-9, C-10, C-11, comprehensively
1h-
1h-1/H during H COSY composes
2-2/H-3, H
2-6/H-7, H-11/H
2-12 relevant positions can determining three double bonds (C-1 and C-10, C-7 and C-8, C-9 and C-11).H in being composed by HMBC
2-15 is relevant to C-16, H
2-22/H-24 the position of determining two carbonyls (C-16 and C-23) relevant to C-23.Relative configuration is determined (Fig. 5) by coupling constant and NOESY spectrum.From H-3(δ H 3.78, dd,
j=10.1,6.2Hz) the known C-3 of coupling constant is beta comfiguration.In NOESY spectrum, H-3/H
3-29 relevant and H-3/H-5 are correlated with, and to go out H-3 be α configuration in deducibility; Finally, by ECD(Fig. 6) determine that the absolute configuration of this compound is 3S, 5R, 13R, 14R, 17R, 20R, 24S, experimental value and theoretical value basically identical.Therefore, this compound structure can be determined as shown in Figure 1.
Embodiment 2: chemical compounds I pharmacological action is tested
One, test method
1, cell cultures
1.1 cell recovery
Frozen A375 melanoma cell in liquid nitrogen container taken out, put into rapidly the warm water of 37 DEG C, shake makes it melt as early as possible (about lmin) gently.Then sucking-off cell suspension, joins in the centrifuge tube being added with 2mL substratum, blows and beats mixing gently, 800r/min, and centrifugal 5min discards upper strata substratum.After making cell suspension with the DMEM substratum 8mL of the mould G/ Streptomycin sulphate containing 10%FBS and 1%, be seeded in 10cm culture plate.Then 5% CO is placed in
2, cultivate in 37 DEG C of constant incubators.
1.2 passage
Basis of microscopic observation cytogamy degree can go down to posterity when reaching 80%-90%.First use 2mLPBS washed cell 2 times, then add the pancreatin lmL of 0.25%.Horizontal wave and culture dish gently, makes Digestive system can cover the surface of cell, is then placed in 37 DEG C of incubators and digests about lmin.See that cell rounding bounces back under being placed in microscope, then add rapidly the substratum termination digestion that 1mL contains FBS.Blow and beat to cell dispersal even gently, then go down to posterity according to 1:2 or 1:3 according to cell density, be re-seeded into new culture plate, in 5%CO
2, cultivate in 37 DEG C of constant incubators.
1.3 cell cryopreservation
Get the cell that 1 dish is in logarithmic phase, trysinization is also collected in centrifuge tube, and the centrifugal 5min of 1000r/min, abandons supernatant.Then add lmL frozen storing liquid, dispel cell gently and make cell suspension, cell suspension is joined in the cryopreservation tube of 1.5mL.In the title of cryopreservation tube subscript clear-cells, shelf time, the name of preserver.First cryopreservation tube is put into 4 DEG C of refrigerators, place about 30min.Then be placed in-20 DEG C of refrigerators, place about 2h.Then be put in-80 DEG C of Ultralow Temperature Freezers to place and spend the night.Frozen cell was placed in the medium-term and long-term preservation of liquid nitrogen container in second day.
2, cell counting draws cell growth curve
(1) take the logarithm A375 melanoma cell in vegetative period, counting, adjustment cell density is 2 × 10
4/ mL.
(2) by cell suspension inoculation in 12 orifice plates, 1.5mL/ hole.
(3) after 12h, treat cell attachment, change 1mL serum-free DMEM substratum, and use 5 μm of ol/L chemical compounds Is and DMSO respectively (for the chemical compounds I control DMSO concentration of DMSO dilution below 1/1000, guarantee cell harmless) process, often organize cell and establish 3 parallel holes, be placed in 5%CO
2, cultivate in 37 DEG C of constant incubators.
(4) stop cultivating respectively at 0h, 24h, 48h, 72h, 96h, collect each group of cell, add 0.25% tryptic digestion, centrifugal, resuspended.
(5) cell counting: draw cell suspension 10 μ l, add isopyknic phenol blue area and divide viable cell, draw 10 μ L mixed solutions and add in cell counting count board groove gently, ensure tight and bubble.Under inverted microscope, the cell count in counting periphery four block plaid, substitutes into formula: archeocyte suspension concentration (cell count/mL)=(cell count/4 of 4 block plaid) × 10
4× extension rate.
(6) calculate number of viable cells, draw cell growth curve.
3, MTT method detects cell proliferation
(1) the A375 cell of taking the logarithm vegetative period, digestion, centrifugal, resuspended, counting, in adjustment cell suspension, the density of cell is 2 × 10
4/ mL.
(2) by each group of cell suspension inoculation in 96 well culture plates, the 200 every holes of μ L, often organize cell 3 parallel holes, establish blank control wells (only adding substratum) simultaneously.
(3) 12h is after cell attachment, changes 200 μ L serum-free DMEM substratum, and respectively with 5 μm of ol/L chemical compounds Is and DMSO process.
(4) stopped cultivating respectively at 0,1,2,3,4 day.
(5) every hole adds the Methyl thiazoly tetrazolium assay 20 μ L continuation culturing cell 4h that concentration is 5mg/mL.
(6) inhale the substratum abandoned in each aperture, add 200 μ LDMSO, microoscillator is vibrated 10min, and crystallisate is fully dissolved.
(7) return to zero with blank control wells, automatic microplate reader is adopted to measure the absorption photometric value (OD value) at nm place, each hole 570, ability of cell proliferation is represented with the OD value of correspondence, the each group of mean value getting 3 parallel holes, take time as transverse axis, with each absorbance for the longitudinal axis draws cell growth curve.
4, BrdU Immunofluorescence test cell proliferation
(1) creep plate is prepared: creep plate is put into 75% alcohol and soak 24h and disinfect.
(2) placed in 24 well culture plates by aseptic creep plate, the A375 melanoma cell in vegetative period of taking the logarithm, digestion, centrifugal, make cell suspension, 2 × 104cell/ hole, is inoculated in and is placed with in the hole of creep plate.
(3) 12h is after cell attachment, changes 1mL plasma-free DMEM medium, and respectively with 5 μm of ol/L chemical compounds Is and DMSO process.
(4) after 72h, add 10 μ lBrdU (1mg/mL) in the medium, cultivate 1h for 37 DEG C.
(5) take out 24 orifice plates, cold PBS washes 5min × 2 time, 4% paraformaldehyde fixed cell, room temperature 30min.
(6) PBS washes 5min × 3 time.
(7) 2mol/L HCl process, after room temperature 10min, 37 DEG C of 20min.
(8) the penetrating process in cell 1%Triton ×-100, washes 5min × 3 time.
(9) 10% lowlenthal serums are closed, room temperature, lh.
(10) BrdU monoclonal antibody (1:300 dilution) is added, 37 DEG C of 1.5h.
(11) l × PBST shakes and washes 5min × 3 time.
(12) add BrdU bis-anti-(1:300), after room temperature lucifuge 1h, l × PBST washes 3 times.
(13) DAPI staining fluid transfect cell core 15min.
(14) PBS washes 5min × 3 time.
(15) 1 anti-fluorescent quenching mounting liquid is added, neutral gum mounting, fluorescence microscope, photograph.
5, statistical study
Application SPSS 17.0 statistical analysis software, adopt two independent sample t inspections and one-way analysis of variance to carry out data analysis, data X ± S represents, P<0.05 is for there being statistical significance.
Two, data analysis
Cellular form is observed and counting display, and compared with DMSO control group, after chemical compounds I process A375 cell 24h, 48h, 72h, viable count significantly reduces and reaches 44.5%.The results are shown in Table 1(*P <0.05, * * P <0.01 versus DMSO group).
MTT result shows, and chemical compounds I can significantly suppress A375 cell proliferation, and OD value is that the decline of concentration (5 μm of ol/L, 10 μm of ol/L, 20 μm of ol/L) dependency reaches 27.3%, and difference has statistical significance.The results are shown in Table 2(*P <0.05, * * P <0.01 versus DMSO group).
Chemical compounds I energy check melanin tumor cell proliferation is confirmed further by BrdU immunofluorescence dyeing, 5 μm of ol/L chemical compounds I process A375 cells are after 3 days, compared with contrast (26.07 ± 2.59%), experimental group BrdU positive cell percentage (7.55 ± 2.76%) significantly reduces (P=0.000).
This experiment have detected the impact that chemical compounds I is bred melanoma cell, and cell counting and MTT analytical results all prove that chemical compounds I can remarkable check melanin tumor cell growth, and cell count and chemical compounds I concentration are dependency declines.After BrdU immunofluorescence dyeing also shows chemical compounds I process, BrdU positive cell percentage is significantly lower than control group, illustrates that chemical compounds I can suppress A375 melanoma cell to be bred.Chemical compounds I may be melanomatous efficient targeting medicine.
Table 1 determination of cell count chemical compounds I is to the suppression (× l0 of A375 melanoma cell
4/ ml)
Table 2 mtt assay measures chemical compounds I to the suppression of A375 melanoma cell vigor
Embodiment 3
The preparation of tablet: by embodiment 1 method first obtained chemical compounds 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 chemical compounds 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 chemical compounds 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 chemical compounds 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 chemical compounds 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.
Claims (6)
1. there is the triterpenoid I of following structural formula:
。
2. the preparation method of chemical compounds I according to claim 1, it is characterized in that comprising following operation steps: (a) fern snippings stem is pulverized, extract with 75% 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), use 15% ethanol and 70% ethanol elution successively, collect 70% elutriant, concentrating under reduced pressure obtains 70% ethanol elution enriched material; C in () step (b), 70% 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 40:1,20:1,10:1 and 5:1; 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 8:1,5:1 and 2:1; E in () step (d), component 2 reverse phase silica gel of octadecylsilane bonding is separated, be the methanol aqueous solution isocratic elution of 65% by concentration expressed in percentage by volume, collect 7-9 column volume elutriant, elutriant concentrating under reduced pressure obtains pure chemical compounds I.
3. preparation method according to claim 2, is characterized in that: described macroporous resin is D101 type macroporous adsorbent resin.
4. pharmaceutical composition, the chemical compounds I according to claim 1 wherein containing treatment significant quantity and pharmaceutically acceptable carrier.
5. the application of chemical compounds I according to claim 1 in the melanomatous medicine of preparation treatment.
6. the application of pharmaceutical composition according to claim 4 in the melanomatous medicine of preparation treatment.
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| CN105330716A (en) * | 2015-10-21 | 2016-02-17 | 淄博夸克医药技术有限公司 | New withanolides compound, and preparation method and medical application thereof |
| CN105198897A (en) * | 2015-10-26 | 2015-12-30 | 沈健龙 | New kaurane diterpenoid compound, preparation method and medical application of compound in treating liver cancer |
| CN105585609A (en) * | 2016-03-18 | 2016-05-18 | 温州高新技术产业开发区聚智汇科技信息咨询服务中心 | Aminophylline drug composition and medical application thereof |
| CN107434764A (en) * | 2016-05-29 | 2017-12-05 | 戴明虎 | It is a kind of to be used to treat new triterpene compound of melanoma and preparation method thereof and medical usage |
| CN106146315A (en) * | 2016-07-14 | 2016-11-23 | 朱正直 | A kind of medicinal compound and preparation method thereof |
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| CN106265623A (en) * | 2016-07-14 | 2017-01-04 | 朱正直 | Compound, cefoperazone pharmaceutical composition preparation treatment periodontitis medicine in application |
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