CN103739658B - A kind of compound, its extracting method, it prepares the antineoplastic of application and the preparation thereof of antineoplastic - Google Patents
A kind of compound, its extracting method, it prepares the antineoplastic of application and the preparation thereof of antineoplastic Download PDFInfo
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Abstract
The present invention relates to pharmaceutical chemistry technical field, particularly a kind of compound, its extracting method, it prepares the antineoplastic of application and the preparation thereof of antineoplastic; Compound provided by the invention extracts from oil tea root, the demonstration of Structural Identification result, and this compound is noval chemical compound, structure is suc as formula shown in I; Cell in vitro test shows, shown in formula I, after compound effects 24h, the growth of people's lung cancer A549 cell, B16 mouse black-in tumor cell, human hepatocellular carcinoma BEL-7402 cell and human breast cancer cell MCF-7 cell is had to the significantly inhibitory action of (P<0.05); Mice-transplanted tumor model test shows, this compound is to rat liver cancer H22Transplantable tumor and mouse S180The growth of sarcoma has the significantly inhibitory action of (P<0.01), and dose-effect relationship is obvious.
Description
Technical field
The present invention relates to pharmaceutical chemistry technical field, particularly a kind of compound, its extracting method, it prepares antitumorThe application of medicine and the antineoplastic of preparation thereof.
Background technology
Oil tea (CameLLiaoLeiferaAbeL) belongs to Theaceae (Theaceae) Camellia (CameLLiaLinn), be a kind of evergreen dungarunga. Because of its seed can extract oil (tea oil) edible, therefore named oil tea. Oil tea is mainly distributed in the torrid zoneAnd subtropical zone, originate in the west and south and the southeast of China, spread all over 17 provinces and regions. The unsaturated fatty acid content of tea oil up to90%, far away higher than rape oil, peanut oil and soya-bean oil, double than content of vitamin E with olive oil, there is high nutritive value,Therefore tea oil receives much concern.
In addition, oil tea also has very high medical value, in Compendium of Material Medica, records: " tea seed, the fragrant poison of bitter cold (saponin), mainControl and breathe heavily anxious cough, remove disease dirt "; " China book on Chinese herbal medicine " is also on the books, that the root of oil tea and root leatherware thereof have is clearing heat and detoxicating, regulating qi-flowing for relieving pain,Effect of activating blood circulation and reducing swelling, cures mainly abscess of throat, stomachache, toothache, injuries from falls as well, scald, and its curative effect is considerably beyond tea oil. ButThe medical value of oil tea is not subject to enough attention.
In recent years, chemical composition and the biologically active of people to oil tea is studied, at present, and from tea of oil teaIn the heart, tea oil, camellia oleosa seed cake, Camellia Leaves, separate and obtain saponins, flavonoids, fatty acid, tannin, fragrant glycoside, alkaloid etc.Compound. Research discovery, part saponins compound has stomach protection, reducing blood lipid, fat-reducing, antiallergy or antineoplastic meritEffect. The discoveries such as Ma Liyuan, the total saponin(e of oil tea in oil tea camellia oleosa seed cake more than 95% has significant antitumor activity. Therefore, oil teaSaponin(e has very high medical value. Sasanguasaponin mainly extracts from oil tea, but people focus mostly at tea to oil tea research beforeIn the sub-heart, camellia oleosa seed cake, Camellia Leaves, tea oil, less to the research of oil tea root, in oil tea root, may exist undiscovered tool biological aliveThe Sasanguasaponin compounds of property, therefore, has great importance to the research of saponin constituent in oil tea root.
Summary of the invention
In view of this, the invention provides a kind of compound, its extracting method, its prepare antineoplastic application andThe antineoplastic of preparation. The present invention tests by cell in vitro and animal-transplanted tumor test is found, shown in formula I, compound is to peopleThe growth of the multiple cancer cell of body has significant inhibitory action.
In order to realize foregoing invention object, the invention provides following technical scheme:
The invention provides a kind of compound suc as formula structure shown in I:
Formula I.
The present invention also provides a kind of extracting method of the compound suc as formula structure shown in I, comprises following steps:
Step 1: oil tea root is pulverized, got the first solvent extraction, concentrated, obtain liquid extract;
Step 2: get liquid extract and mix with water, filter, collect filtrate, centrifugal, collect supernatant, then set through D101 type macroporeFat post separates, and gets ethanol-water system gradient elution, and collecting ethanol and water volume ratio is 70:30～95:5 wash-out obtained component, pointFrom, purifying, to obtain final product;
The first solvent is that ethanol and water volume ratio are 30:70～95:5.
As preferably, the order number that oil tea root is pulverized is 10 order～60 orders.
As preferably, the first solvent is that ethanol and water volume ratio are 40:60～80:20.
Preferred, the first solvent is that ethanol and water volume ratio are 70:30.
As preferably, step 2 is got in liquid extract and water blend step, and the volume ratio of liquid extract and water is 1:3～20, usedThe amount of water reaches the object that liquid extract is dissolved.
Preferably, the order number that filters filter cloth used is 100～300 orders in effect.
As preferably, separate, the technology of purifying be in precipitating technology, crystallization technique or chromatographic technique any one or twoTechnology more than person, but be not limited to above technology.
As preferably, separate, the technology of purifying is chromatographic technique.
As preferably, separate, purifying is specially: get component and separate through silicagel column, get chloroform-methanol system gradient elution,Collecting chloroform and methyl alcohol volume ratio is 80:20～60:40 wash-out obtained component; Separate through ODS post again, get methanol-water system ladderDegree wash-out, collecting methyl alcohol and water volume ratio is 70:30～90:10 wash-out obtained component; Separate through dynamic axial compression column again, getMethanol-water system gradient elution, collecting methyl alcohol and water volume ratio is 75:25 wash-out obtained component; Finally pass through high-efficient liquid phase colorSpectrum separates, and adopts C18Chromatographic column, the second solvent elution, setting flow velocity is 2mL/min, collects 43.3min component, to obtain final product;
The second solvent is: first alcohol and water by volume for the mixed solution of 72:28 composition again with account for mixed solution quality hundredDivide the mixture of the formic acid mixing gained that content is 0.2%.
C18Chromatographic column specification is 250mm × 10mm, and filler granularity is 5 μ m.
As preferably, silicagel column is decompression silicagel column.
As preferably, in silicagel column, the order number of silica gel is 60～100 orders.
As preferably, the solvent of chloroform-methanol system gradient elution be followed successively by chloroform and methyl alcohol volume ratio be 90:10 →80：20→70：30→60：40→50：50→40：60→20：80→0：100。
As preferably, ODS post is the anti-phase ODS post of middle pressure.
As preferably, the specification of the anti-phase ODS post of middle pressure is: 460mm × 26mm.
As preferably, the solvent of ODS post gradient elution is that methyl alcohol and water volume ratio are 50:50～100:0.
As preferably, the solvent of ODS post gradient elution be methyl alcohol and water volume ratio be 50:50 → 60:40 → 70:30 →80：20→90：10。
As preferably, ODS column flow rate is 25mL/min.
As preferably, the detection wavelength of ODS post is 203nm.
As preferably, dynamic axial compression column is ODS post.
As preferably, the specification of dynamic axial compression column is: 650mm × 100mm, 30 μ m, 1500g; NewstyLe.
As preferably, the solvent of dynamic axial compression column gradient elution is followed successively by methyl alcohol and water volume ratio is 60:40 → 75:25→80：20→90：10。
As preferably, the flow velocity of dynamic axial compression column is 150mL/min.
As preferably, the detection wavelength of dynamic axial compression column is 203nm.
As preferably, high performance liquid chromatography is half preparative high-performance liquid chromatographic.
As preferably, the detection wavelength of half preparative high-performance liquid chromatographic is 203nm.
As preferably, compound test method comprises lamellae analysis, efficient liquid phase chromatographic analysis.
As preferably, the detection wavelength of the compound of structure shown in formula I is 203nm.
As preferably, the solvent of step 2 gradient elution is followed successively by ethanol and water volume ratio is 0:100 → 30:70 → 50:50→60：40→70：30→80：20→95：5。
As preferably, the temperature of extracting described in step 1 is 30 DEG C～100 DEG C, the time of extracting described in step 1 be 1h～15.5h。
As preferably, the temperature that step 1 is extracted is 80 DEG C～85 DEG C, and the time that step 1 is extracted is 2h～3h.
As preferably, extract and comprise immersion, refluxing extraction, filtration and collection filtrate step.
As preferably, in soaking step, in g/mL, the mass volume ratio of oil tea root and the first solvent is 1:3～20.
Preferred, in soaking step, in g/mL, the mass volume ratio of oil tea root and the first solvent is 1:5～15.
Preferred, in soaking step, in g/mL, the mass volume ratio of oil tea root and the first solvent is 1:10.
In step 1 leaching process, the first solvent needs only the excessive close effect that all can reach, all in protection scope of the present inventionIn.
As preferably, the time of immersion is 4h～12h, and the temperature of immersion is 30 DEG C～50 DEG C.
As preferably, the temperature of refluxing extraction is 80 DEG C～85 DEG C.
Preferred, the temperature of refluxing extraction is 80 DEG C.
As preferably, the time of refluxing extraction is 0.5h～3h.
Preferred, the time of refluxing extraction is 2.0h.
As preferably, the number of times of refluxing extraction is 1～3 time.
Preferred, the number of times of refluxing extraction is 2 times.
Filtration step can carry out after each refluxing extraction, also can after refluxing extraction 2～3 times, carry out, and collects completePortion's filtrate, is extract.
As preferably, filtration step is carrying out after each refluxing extraction, filter gained filter residue again with the first solvent, enterRow refluxing extraction next time.
As preferably, in g/mL, the mass volume ratio of filter residue and the first solvent is 1:3～20.
As preferably, concentrated temperature is 70 DEG C～100 DEG C described in step 1, described in step 1 the concentrated time be 3h～6h。
As preferably, the concentrated temperature of step 1 is 80 DEG C～90 DEG C, and the concentrated time of step 1 is 3h～4h.
As preferably, simmer down to reduced pressure concentration.
As preferably, concentrated multiple is 20～100 times.
The present invention also provides the compound of structure shown in formula I in the application of preparing in antineoplastic. The present invention firstMeasure the inhibitory action of medicine to tumor cell line by mtt assay. Mtt assay, claims again MTT colorimetric method, is that a kind of cell that detects is depositedThe method of living and growing. Its detection principle is that the succinate dehydrogenase in living cells mitochondria can make exogenous MTT be reduced to waterInsoluble bluish violet crystallization first a ceremonial jade-ladle, used in libation (Formazan) is also deposited in cell, and dead cell is without this function. Dimethyl sulfoxide (DMSO)(DMSO) the first a ceremonial jade-ladle, used in libation in can dissolved cell, measures its absorbance value at 490nm wavelength place with enzyme-linked immunosorbent assay instrument, can be indirectly anti-Reflect living cells quantity. Within the scope of certain cell number, the amount that MTT crystallization forms is directly proportional to cell number. The method is extensively usedIn the activity detection of some bioactie agents, large-scale screening anti-tumor medicine, cell toxicity test and tumour radiotherapySensitivity testing etc. Its feature is highly sensitive, economical. Therefore, this test adopts mtt assay to screen tested medicine.This research is people's lung cancer A549 cell, B16 mouse black-in tumor cell, human hepatocellular carcinoma BEL-7402 cell and human breast cancer cell MCF-7, as subject cell, and the action time of tested medicine is made as to 24h. Result of the test shows, shown in formula I provided by the inventionAfter compound effects 24h, to people's lung cancer A549 cell, B16 mouse black-in tumor cell, human hepatocellular carcinoma BEL-7402 cell and people's mammary glandCancer cell MCF-7 cell IC50Value is all less than 15 μ g/mL, and to people's lung cancer A549 cell, human hepatocellular carcinoma BEL-7402 cell's lifeLong inhibitory action is significantly better than (P < 0.05) positive control 5-FU. As can be seen here, compound shown in formula I is to above-mentioned 4 kinds of tumoursThe growth of cell has significant inhibitory action.
The present invention also tests the tumor killing effect of this compound by mice-transplanted tumor model test, result shows, shown in formula ICompound is to rat liver cancer H22Transplantable tumor and mouse S180The growth of sarcoma has the significantly inhibitory action of (P < 0.05), and dose-effect closesSystem is obvious, wherein to mouse S180The inhibitory action of sarcoma growth is significantly better than positive control CTX, therefore, the invention provides formulaThe compound of structure shown in I is in the application of preparing in antineoplastic.
As preferably, tumour is lung cancer, liver cancer, melanoma or breast cancer. Tumour can also be not open for the present invention, butThere is the tumour of other kinds of inhibition.
The present invention also provides a kind of antineoplastic, the compound of structure and pharmaceutically acceptable shown in its contained IAuxiliary material.
The formulation of antineoplastic of the present invention can be the attainable any formulation in this area, and auxiliary material used is formulation usedConventional auxiliary material, the conventional preparation method that preparation method is corresponding formulation.
As preferably, the formulation of antineoplastic is powder, tablet, granule, capsule, solution, emulsion, suspendibleAgent, parenteral solution, powder-injection or spray.
The invention provides a kind of compound suc as formula structure shown in I, this compound is by extracting and obtain in oil tea root, structureQualification result demonstration, this compound is noval chemical compound, called after: 21 β, 22 α-O-, bis-angeloyl groups-15 α, 16 α, 28-trihydroxyOlive-12-alkene 3 β-O-β-D-wood sugar-(1 → 2)-β-D-galactolipin-(1 → 3)-[β-D-galactolipin-(1 → 2)]-β-D-PortugalGrape glycuronide. The present invention adopts mtt assay to carry out cell in vitro test, and result shows, compound shown in formula I provided by the inventionAfter effect 24h, to people's lung cancer A549 cell, B16 mouse black-in tumor cell, human hepatocellular carcinoma BEL-7402 cell and human breast cancer cellThe IC of MCF-7 cell50Value is all less than 15 μ g/mL, and the inhibition that people's lung cancer A549 cell, human hepatocellular carcinoma BEL-7402 cell are grownEffect is significantly better than (P < 0.05) positive control 5-FU. As can be seen here, the life of compound shown in formula I to above-mentioned 4 kinds of tumour cellsLength has significant inhibitory action. The present invention also tests the tumor killing effect of this compound by mice-transplanted tumor model test, surveyTest result shows, shown in formula I, compound is to rat liver cancer H22Transplantable tumor and mouse S180The growth of sarcoma has significantly (P < 0.01)Inhibitory action, dose-effect relationship is obvious, wherein to mouse S180The inhibitory action of sarcoma growth is significantly better than positive control CTX, because ofThis, shown in formula I provided by the invention, compound also can be used for preparing antineoplastic.
Brief description of the drawings
Fig. 1 is 21 β, 22 α-O-, bis-angeloyl groups-15 α, and 16 α, 28-trihydroxy olive-12-alkene 3 β-O-β-D-wood sugar-The high resolution mass spectrum spectrum of (1 → 2)-β-D-galactolipin-(1 → 3)-[β-D-galactolipin-(1 → 2)]-β-D-Glucose aldehydic acid glycosidesFigure;
Fig. 2 is 21 β, 22 α-O-, bis-angeloyl groups-15 α, and 16 α, 28-trihydroxy olive-12-alkene 3 β-O-β-D-wood sugar-The proton nmr spectra of (1 → 2)-β-D-galactolipin-(1 → 3)-[β-D-galactolipin-(1 → 2)]-β-D-Glucose aldehydic acid glycosidesFigure;
Fig. 3 is 21 β, 22 α-O-, bis-angeloyl groups-15 α, and 16 α, 28-trihydroxy olive-12-alkene 3 β-O-β-D-wood sugar-The hydrogen nuclear magnetic resonance spectrogram of (1 → 2)-β-D-galactolipin-(1 → 3)-[β-D-galactolipin-(1 → 2)]-β-D-Glucose aldehydic acid glycosides(enlarged drawing);
Fig. 4 is 21 β, 22 α-O-, bis-angeloyl groups-15 α, and 16 α, 28-trihydroxy olive-12-alkene 3 β-O-β-D-wood sugar-The hydrogen nuclear magnetic resonance spectrogram of (1 → 2)-β-D-galactolipin-(1 → 3)-[β-D-galactolipin-(1 → 2)]-β-D-Glucose aldehydic acid glycosides(enlarged drawing);
Fig. 5 is 21 β, 22 α-O-, bis-angeloyl groups-15 α, and 16 α, 28-trihydroxy olive-12-alkene 3 β-O-β-D-wood sugar-The carbon-13 nmr spectra of (1 → 2)-β-D-galactolipin-(1 → 3)-[β-D-galactolipin-(1 → 2)]-β-D-Glucose aldehydic acid glycosidesFigure;
Fig. 6 is 21 β, 22 α-O-, bis-angeloyl groups-15 α, and 16 α, 28-trihydroxy olive-12-alkene 3 β-O-β-D-wood sugar-The carbon-13 nmr spectra figure of (1 → 2)-β-D-galactolipin-(1 → 3)-[β-D-galactolipin-(1 → 2)]-β-D-Glucose aldehydic acid glycosides(enlarged drawing);
Fig. 7 is 21 β, 22 α-O-, bis-angeloyl groups-15 α, and 16 α, 28-trihydroxy olive-12-alkene 3 β-O-β-D-wood sugar-The carbon-13 nmr spectra figure of (1 → 2)-β-D-galactolipin-(1 → 3)-[β-D-galactolipin-(1 → 2)]-β-D-Glucose aldehydic acid glycosides(enlarged drawing);
Fig. 8 is 21 β, 22 α-O-, bis-angeloyl groups-15 α, and 16 α, 28-trihydroxy olive-12-alkene 3 β-O-β-D-wood sugar-The DEPT figure of (1 → 2)-β-D-galactolipin-(1 → 3)-[β-D-galactolipin-(1 → 2)]-β-D-Glucose aldehydic acid glycosides;
Fig. 9 is 21 β, 22 α-O-, bis-angeloyl groups-15 α, and 16 α, 28-trihydroxy olive-12-alkene 3 β-O-β-D-wood sugar-The DEPT figure of (1 → 2)-β-D-galactolipin-(1 → 3)-[β-D-galactolipin-(1 → 2)]-β-D-Glucose aldehydic acid glycosides (amplifiesFigure);
Figure 10 is 21 β, 22 α-O-, bis-angeloyl groups-15 α, 16 α, 28-trihydroxy olive-12-alkene 3 β-O-β-D-woodThe HSQC figure of sugar-(1 → 2)-β-D-galactolipin-(1 → 3)-[β-D-galactolipin-(1 → 2)]-β-D-Glucose aldehydic acid glycosides;
Figure 11 is 21 β, 22 α-O-, bis-angeloyl groups-15 α, 16 α, 28-trihydroxy olive-12-alkene 3 β-O-β-D-woodThe HMBC figure of sugar-(1 → 2)-β-D-galactolipin-(1 → 3)-[β-D-galactolipin-(1 → 2)]-β-D-Glucose aldehydic acid glycosides;
Figure 12 is 21 β, 22 α-O-, bis-angeloyl groups-15 α, 16 α, 28-trihydroxy olive-12-alkene 3 β-O-β-D-woodSugar-(1 → 2)-β-D-galactolipin-(1 → 3)-[β-D-galactolipin-(1 → 2)]-β-D-Glucose aldehydic acid glycosides1H-1HCOSYFigure;
Figure 13 is 21 β, 22 α-O-, bis-angeloyl groups-15 α, 16 α, 28-trihydroxy olive-12-alkene 3 β-O-β-D-woodThe NOESY figure of sugar-(1 → 2)-β-D-galactolipin-(1 → 3)-[β-D-galactolipin-(1 → 2)]-β-D-Glucose aldehydic acid glycosides.
Detailed description of the invention
The invention provides a kind of compound, its extracting method, it prepares the anti-of the application of antineoplastic and preparation thereofTumour medicine. Those skilled in the art can use for reference content herein, suitably improve technological parameter and realize. Of particular noteBe, all similar replacements and change apparent to those skilled in the artly, they are all deemed to be included in thisInvention. Method of the present invention and application are described by preferred embodiment, and related personnel obviously can not depart from thisIn summary of the invention, spirit and scope, methods and applications as herein described changed or suitably changed and combination, realize andApplication the technology of the present invention.
The material of using in the embodiment of the present invention and reagent all can be buied by market, and in embodiments of the present invention, raw material comesSource is specific as follows:
NMR 500Hz (VarianInc., PaloAlto, CA, the U.S.); High resolution mass spectrum Q-TOF2(BritainMicromass company); Electronic balance (Mettler-Toledo Instrument (Shanghai) Co., Ltd.); Polarimeter 241(PerkinElmerInc., Waltham, MA, the U.S.); Half preparative high-performance liquid chromatographic instrument (LC-20AT, SPD-20A, dayThis Shimadzu company); C18 half preparative chromatography post (250mm × 10mm, 5 μ m, Kromsil company of the U.S.); Middle pressure preparative liquid chromatography(B ü chi chromatographic system, C-650 pump, middle compression leg (460mm × 26mmi.d., B ü chiCorp., Flawil, Swiss); DynamicallyAxial columns chromatogram (NP7000 pump (Newstyle), NU3000UV-VIS detector (Newstyle) and ODS post (650mm ×100mm, 30 μ m, 1500g; Newstyle); Rotary Evaporators (Tokyo physics and chemistry apparatus individual proprietorship factory); Chemical reagent (it is analyze pure,Chemical Reagent Co., Ltd., Sinopharm Group); Thin-layer chromatography silica gel plate (HSGF254, the yellow business of Yantai City's Zhifu silica gel development experiments factoryProduce); Various column chromatographys are Haiyang Chemical Plant, Qingdao with silica gel and produce.
People's lung cancer A549 cell, B16 mouse black-in tumor cell, human hepatocellular carcinoma BEL-7402 cell and human breast cancer cell MCF-7, be all purchased from Shanghai cell institute of the Chinese Academy of Sciences.
H22 rat liver cancer (sarcoma) cell: be purchased from Chinese Academy of Sciences's Shanghai cell bank.
S180 rat liver cancer (sarcoma) cell: be purchased from Chinese Academy of Sciences's Shanghai cell bank.
ICR mouse: clean level, male, body weight 18-22g, is provided by Shanghai Slac Experimental Animal Co., Ltd..
Mouse: kind is ICR, is buied by Shanghai Slac Experimental Animal Co., Ltd., animal used as test production licence numberSCXK (Shanghai) 2007-0005.
Complete medium: fill a prescription as the RPMI1640 culture medium containing 10% inactivated fetal bovine serum, manufacturer is the U.S.GIBCOBRL company.
All the other reagent and material are buied by market.
Below in conjunction with embodiment, further set forth the present invention:
The extraction of embodiment 1 compound
Get dry oil tea root 2kg, be ground into 10-60 object wood chip with pulverizer, through 50 DEG C of immersions of 30% ethanol of 6LAfter 12h, add hot reflux 0.5h at 90 DEG C, filter, filter residue adds 30% ethanol of 10L again, adds hot reflux 3.0h at 85 DEG C,Filter, filter residue mixes with 30% ethanol of 30L again, adds hot reflux 2h at 85 DEG C, merges filtration gained filtrate three times, at 80 DEG CReduced pressure concentration 4h, obtains liquid extract 500mL.
Get the liquid extract making and mix with 5.5L water, 100 order filter-cloth filterings, centrifugal filtrate, gets supernatant large through D101 typeHole resin column separates, and water, 30% ethanol, 50% ethanol, 60% ethanol, 70% ethanol, 80% ethanol, 95% ethanol elution successively receivedCollect the component of 70% ethanol～95% ethanol elution gained, reduced pressure concentration, obtains the total saponin(e of oil tea.
Get the total saponin(e of the oil tea making and first separate through the decompression silicagel column of 60 order silica gel, use successively chloroform and methyl alcohol volume ratioFor 90:10 → 80:20 → 70:30 → 60:40 → 50:50 → 40:60 → 20:80 → 0:100 gradient elution, obtain component 1～Component 8, through lamellae analysis, merges chloroform and methyl alcohol volume ratio 80:20～60:40 wash-out obtained component (solvent whereinWhen system is BAW system, Rf value is 0.4-0.7), then in warp, press anti-phase ODS post to separate, methanol-water system 50%-100% gradientWash-out, flow velocity 25mL/min, detection wavelength is 203nm, obtains 6 components, through efficient analysis liquid phase analysis, uses first alcohol and waterVolume ratio is 60:40～90:10 gradient elution 30min, merges the component (peak position that methyl alcohol and water volume ratio are 70:30～90:10In 20min-28min), separate through dynamic axial compression column, use respectively 60%, 75%, 80% and 90% methanol-eluted fractions, flow velocity150mL/min, detects wavelength 203nm, obtains altogether five components, through efficient analysis liquid phase analysis, by 75% methanol-eluted fractions gained groupDivide (peak position is in 13.5-15.0min) to separate by partly preparing high efficiency liquid phase, methanol-water (72:28, v/v)-0.2% formic acid wash-outWash-out, setting flow velocity is 2mL/min, detects wavelength 203nm, obtains white amorphous powder 72mg in 43.3min place.
Get the white amorphous powder making, detect development properties, hydrolysis properties, then warp1H-NMR，13C-NMR，HMBC，HSQC，1H-1HCOSY, NOESY and HR-ESI-MS carry out wave spectrum analysis, spectral data figure as shown in Fig. 1～Figure 13, NMR dataAs shown in table 1, structure elucidation process is as follows: through chromogenic assay, and sulfuric acid ethanol displaing amaranth spot, aceticanhydride-strong sulfuric acid response sunProperty, Molish reacting positive, pointing out this compound may be saponins compound. Mass spectrum demonstration shown in Fig. 1, this compoundThe accurate quasi-molecular ions m/z1301.6198[M-H of HR-ESI-MS]-, the molecular formula that shows this compound is (calculated value [M-H]-,m/Z1301.6166, calculated value is according to molecular formula, is calculated by high abundance exact value). Obtain with the complete acid hydrolysis of 2NTFATo monose, sugar carries out GC analysis after derivatization, the existence of D-Glucose aldehydic acid, D-galactolipin, D-wood sugar detected.
The compound of Fig. 5, Fig. 6, Fig. 7, Fig. 8 and Fig. 913C-NMR spectrum and the demonstration of DEPT spectrum, this compound has 63 carbon lettersNumber, more known in conjunction with the hsqc spectrum analysis shown in Figure 10, this compound contains 11 methyl carbon altogether, 10 mesomethylene carbons, 30Methine carbon and 12 quaternary carbons. Further analyze13C-NMR spectrum, this spectrogram demonstrates four sugared end group carbon signal δ 105.5,101.9,107.5 and 103.2, be respectively the end of D-Glucose aldehydic acid, D-galactolipin, D-wood sugar and another one D-galactolipinBase carbon. Compound shown in Fig. 2, Fig. 3, Fig. 41H-NMR composes demonstration,1In H-NMR spectrum, high field region has seven obvious triterpene soapsGlycosides angular methyl hydrogen signal δ 0.93 (Me-25), 1.10 (Me-26), 1.19 (Me-29), 1.23 (Me-24), 1.36 (Me-23),1.42 (Me-30) He 1.94 (Me-27); Even Oxymethylene δ 3.59 (H-28, d, J=11.0Hz) and 3.85 (H-28, d, J=10.0Hz); Five oxygen methine δ 3.34 (1H, dd, J=11.0,4.5Hz, H-3) of company, 4.30 (1H, brs, H-15), 4.52(1H, brs, H-16), 6.79 (1H, d, J=10.5Hz, H-21) and 6.40 (1H, d, J=10.5Hz, H-22) and an alkene hydrogenProton δ 5.61 (1H, brs, H-12). In addition compound,1H-NMR spectrum also demonstrates two groups of angeloyl groups signal δ [6.06(1H, dq, J=7.5,1.5Hz, 21-O-Ang-3'), 2.18 (3H, d, J=7.5Hz, 21-O-Ang-4') and 2.10 (3H, s, 21-O-Ang-5')]；δ[5.88(1H,dq,J=7.5,1.5Hz,22-O-Ang-3″),2.06(3H,d,J=7.5Hz,22-O-Ang-4 ") and 1.84 (3H, s, 22-O-Ang-5 ")].
As shown in Figure 121H-1HCOSY is known, with the methine δ that even oxygen methine protons is adjacentH4.30(1H,brs)With δH4.52 (1H, brs) are relevant, illustrate that they are in ortho position and δH4.52 is H-16 signal, δH4.30 is H-15 signal; δH6.79 (1H, d, J=10.5Hz) and δHThey are in ortho position and δ coherent signal explanation between 6.40 (1H, d, J=10.5Hz)H6.79 is H-21 signal, δH6.40 is H-22 signal.
Learn that by the HMBC spectrum analysis of the compound shown in Figure 11 two angeloyl groups are connected in respectively Compound C21,22 are upper because in HMBC δH6.79 (1H, d, J=10.5Hz, H-21) and δC167.9 (21-O-Ang-1') are relevant, δH6.40 (1H, d, J=10.5Hz, H-22) and δC168.3 (22-O-Ang-1'') are relevant. In conjunction with13C-NMR spectrum and1H-NMR composes numberAccording to comprehensively analyzing, and with the known aglycon 21 of document " Chem.Pharm.Bull.1984,32,3378-3384 " reportβ, 22 α-O-diangeloyl-15 α, 16 α, 28-trihydroxyolean-12-ene and document " PlantaMed2013,79,353-360.11 " report 21 β, 22 α-O-diangeloyl-15 α, 16 α, 28-trihydroxy-olean-12-ene3β-O-β-D-xylopyranosyl-(1→2)-α-L-arabinopyranosyl-(1→3)-[β-D-glucopyranosyl-(1 → 2)] contrast of-β-D-glucuronopyranoside nuclear magnetic data, the aglycon structure of determining this compound is 21 β, 22 α-O-bis-angeloyl groups-15 α, 16 α, 28-trihydroxy olive-12-alkene.
To compound, hsqc spectrum figure as shown in figure 10 analyzes, belonged to the sugared end group carbon that connects on this saponin(e,Hydrogen signal, as follows: δH4.86 (1H, d, J=6.5Hz), 5.79 (1H, d, J=7.0Hz), 5.15 (1H, d, J=7.5Hz) and 5.89(1H, d, J=7.5Hz), is connected in respectively δC105.5 (glucuronic acid-C-1), 101.9 (galactolipin-C-1), 107.5 (wood sugar-C-1), on 103.2 (galactolipin '-C-1). 3 of this compound aglycons show that to low the about 16.3ppm in position sugar chain is connected in glycosides in addition3, unit is upper, and in HMBC spectrum, glucuronic acid anomeric proton δH4.86 with 3 δ of aglyconC89.7 is relevant, further confirmsSugar chain be connected in 3 of aglycon. Further By consulting literatures is found this compound sugar chain and document " Bioorganic&medicinalChemistryletters2010,20,7435-7439 " the middle camellenodiol sugar chain data of reporting are close, thus deductionGoing out this compound sugar chain is β-D-wood sugar-(1 → 2)-β-D-galactolipin-(1 → 3)-[β-D-galactolipin-(1 → 2)]-β-D-PortugalGrape uronic acid. The order of connection of this sugar chain also can confirm by analyzing HMBC spectrum, glucuronic acid anomeric proton δ in HMBCH4.86 with 3 carbon δ of aglyconC89.7 is relevant, galactolipin anomeric proton δH5.79 with 3 carbon δ of glucuronic acidC84.5 is relevant, woodSugar anomeric proton δH5.15 with 2 carbon δ of galactolipinC83.6 is relevant, 2 hydrogen δ of galactolipinH4.58 with wood sugar end group carbon δC107.5 phaseClose another galactolipin anomeric proton δH5.89 with 2 carbon δ of glucuronic acidC79.0 is relevant, 2 hydrogen δ of glucuronic acidH4.58With this galactolipin end group carbon δC103.2 relevant. In addition four sugared anomeric proton coupling constants,3JH-1,H-2Larger, show this fourIndividual sugar is all beta configuration.
NOESY spectrum shown in Figure 13 shows, δH6.79 (1H, d, J=10.5Hz, H-21) and δH1.19 (3H, s, H-29) phaseClose, prompting H-21 is α configuration; δH3.34 (1H, dd, J=4.5,11.0Hz, H-3) and δH1.36 (3H, s, H-23) are relevant, promptingH-3 is α configuration; δH4.52 (1H, brs, H-16) and δH3.59(1H,d,J=11.0Hz,H-28)、3.85(1H,d,J=10.0Hz,H-28) relevant prompting H-16 is beta comfiguration, δH6.40 (1H, d, J=10.5Hz, H-22) and δH1.42 (3H, s, H-30) are relevant, carryShow that H-22 is beta comfiguration.
Therefore, this compound structure called after 21 β, 22 α-O-, bis-angeloyl groups-15 α, 16 α, 28-trihydroxy olive-12-alkene 3 β-O-β-D-wood sugar-(1 → 2)-β-D-galactolipin-(1 → 3)-[β-D-galactolipin-(1 → 2)]-β-D-Glucose aldehydeAcid glycosides. Compound structure is suc as formula shown in I:
Formula I.
NMR (the pyridine-d of compound shown in table 1 formula I5, 500MHz) and data
In table, chemical shift is taking ppm as unit, and coupling constant (J) is taking Hz as unit, and nd represents to exist but be undetectedSignal.
The extraction of embodiment 2 compounds
Get dry oil tea root 2kg, be ground into 10-40 object wood chip with pulverizer, through 30 DEG C of immersions of 95% ethanol of 40LAfter 8h, add hot reflux 0.5h at 80 DEG C, filter, filter residue adds 95% ethanol 10L again, adds hot reflux 3.0h, mistake at 80 DEG CFilter, merges twice and filters gained filtrate, and reduced pressure concentration 6h at 70 DEG C, obtains liquid extract 612mL.
Get the liquid extract making and mix with 5.8L water, 200 order filter-cloth filterings, centrifugal filtrate, gets supernatant large through D101 typeHole resin column separates, and water, 30% ethanol, 50% ethanol, 60% ethanol, 70% ethanol, 80% ethanol, 95% ethanol elution successively receivedCollect the component of 70% ethanol～95% ethanol elution gained, reduced pressure concentration, obtains the total saponin(e of oil tea.
Get the total saponin(e of the oil tea making and first separate through the decompression silicagel column of 100 order silica gel, use successively chloroform and methyl alcohol volumeThan for 90:10 → 80:20 → 70:30 → 60:40 → 50:50 → 40:60 → 20:80 → 0:100 gradient elution, obtain component 1～component 8, through lamellae analysis, the chloroform and the methyl alcohol volume ratio 80:20～60:40 wash-out obtained component that merge wherein (launchWhen agent system is BAW system, Rf value is 0.4-0.7), then in warp, press anti-phase ODS post to separate, methanol-water system 50%-100% ladderDegree wash-out, flow velocity 25mL/min, detections wavelength is 203nm, obtains 6 components, through efficient analysis liquid phase analysis, with methyl alcohol andWater volume ratio is 60:40～90:10 gradient elution 30min, merges the component (peak that methyl alcohol and water volume ratio are 70:30～90:10Be positioned at 20min-28min), separate through dynamic axial compression column, use respectively 60%, 75%, 80% and 90% methanol-eluted fractions, flow velocity150mL/min, detects wavelength 203nm, obtains altogether five components, through efficient analysis liquid phase analysis, by 75% methanol-eluted fractions gained groupDivide (peak position is in 13.5-15.0min) to separate by partly preparing high efficiency liquid phase, methanol-water (72:28, v/v)-0.2% formic acid wash-outWash-out, setting flow velocity is 2mL/min, detects wavelength 203nm, obtains white amorphous powder 75mg, through nuclear-magnetism in the time of 43.3minResonance hydrogen spectrum detects, and the hydrogen spectrum data consistent of the data obtained and embodiment 1 gained compound, therefore, determines that this compound is 21β, 22 α-O-, bis-angeloyl groups-15 α, 16 α, 28-trihydroxy olive-12-alkene 3 β-O-β-D-wood sugar-(1 → 2)-β-D-galaSugar-(1 → 3)-[β-D-galactolipin-(1 → 2)]-β-D-Glucose aldehydic acid glycosides, compound structure is suc as formula shown in I.
The extraction of embodiment 3 compounds
Get dry oil tea root 2kg, be ground into 10-20 object wood chip with pulverizer, through 40 DEG C of immersions of 70% ethanol of 20LAfter 8h, add hot reflux 3h at 85 DEG C, filter, collect filtrate, reduced pressure concentration 3h at 100 DEG C, obtains liquid extract 592mL.
Get the liquid extract making and mix with 6.0L water, 300 order filter-cloth filterings, centrifugal filtrate, gets supernatant large through D101 typeHole resin column separates, and water, 30% ethanol, 50% ethanol, 60% ethanol, 70% ethanol, 80% ethanol, 95% ethanol elution successively receivedCollect the component of 70% ethanol～95% ethanol elution gained, reduced pressure concentration, obtains the total saponin(e of oil tea.
Get the total saponin(e of the oil tea making and first separate through the decompression silicagel column of 80 order silica gel, use successively chloroform and methyl alcohol volume ratioFor 90:10 → 80:20 → 70:30 → 60:40 → 50:50 → 40:60 → 20:80 → 0:100 gradient elution, obtain component 1～Component 8, through lamellae analysis, merges chloroform and methyl alcohol volume ratio 80:20～60:40 wash-out obtained component (solvent whereinWhen system is BAW system, Rf value is 0.4-0.7), then in warp, press anti-phase ODS post to separate, methanol-water system 50:50 → 60:40→ 70:30 → 80:20 → 90:10 gradient elution, flow velocity 25mL/min, detection wavelength is 203nm, obtains 6 components, through too highEffect analytic liquid facies analysis, with methyl alcohol and water volume ratio be 60:40～90:10 gradient elution 30min, merging methyl alcohol and water volume ratioFor the component (peak position is in 20min-28min) of 70:30～90:10, separate through dynamic axial compression column, use respectively 60%, 75%,80% and 90% methanol-eluted fractions, flow velocity 150mL/min, detects wavelength 203nm, obtains altogether five components, through efficient analysis liquid phaseAnalyze, 75% methanol-eluted fractions obtained component (peak position is in 13.5-15.0min) is separated to methanol-water by partly preparing high efficiency liquid phase(72:28, v/v)-0.2% formic acid wash-out wash-out, setting flow velocity is 2mL/min, detects wavelength 203nm, in the time of 43.3min, obtainsWhite amorphous powder 81mg, detects through proton nmr spectra, the hydrogen spectrum data one of the data obtained and embodiment 1 gained compoundCause, therefore, determine that this compound is 21 β, 22 α-O-, bis-angeloyl groups-15 α, 16 α, 28-trihydroxy olive-12-alkene 3 β-O-β-D-wood sugar-(1 → 2)-β-D-galactolipin-(1 → 3)-[β-D-galactolipin-(1 → 2)]-β-D-Glucose aldehydic acid glycosides, compoundStructure is suc as formula shown in I.
The extraction of embodiment 4 compounds
Get dry oil tea root 2kg, be ground into 20-30 object wood chip with pulverizer, through 30 DEG C of immersions of 80% ethanol of 30LAfter 12h, at 85 DEG C, add hot reflux 0.5h, at 80 DEG C, add hot reflux 3.0h, at 80 DEG C, add hot reflux 2h, filter, collectFiltrate, reduced pressure concentration 4h at 85 DEG C, obtains liquid extract 615mL.
Get the liquid extract making and mix with 6.0L water, 250 order filter-cloth filterings, centrifugal filtrate, gets supernatant large through D101 typeHole resin column separates, and water, 30% ethanol, 50% ethanol, 60% ethanol, 70% ethanol, 80% ethanol, 95% ethanol elution successively receivedCollect the component of 70% ethanol～95% ethanol elution gained, reduced pressure concentration, obtains the total saponin(e of oil tea.
Get the total saponin(e of the oil tea making and first separate through the decompression silicagel column of 60 order silica gel, use successively chloroform and methyl alcohol volume ratioFor 90:10 → 80:20 → 70:30 → 60:40 → 50:50 → 40:60 → 20:80 → 0:100 gradient elution, obtain component 1～Component 8, through lamellae analysis, merges chloroform and methyl alcohol volume ratio 80:20～60:40 wash-out obtained component (solvent whereinWhen system is BAW system, Rf value is 0.4-0.7), then in warp, press anti-phase ODS post to separate, methanol-water system 50%-100% gradientWash-out, flow velocity 25mL/min, detection wavelength is 203nm, obtains 6 components, through efficient analysis liquid phase analysis, uses first alcohol and waterVolume ratio is 60:40～90:10 gradient elution 30min, merges the component (peak position that methyl alcohol and water volume ratio are 70:30～90:10In 20min-28min), separate through dynamic axial compression column, use respectively 60%, 75%, 80% and 90% methanol-eluted fractions, flow velocity150mL/min, detects wavelength 203nm, obtains altogether five components, through efficient analysis liquid phase analysis, by 75% methanol-eluted fractions gained groupDivide (peak position is in 13.5-15.0min) to separate by partly preparing high efficiency liquid phase, methanol-water (72:28, v/v)-0.2% formic acid wash-outWash-out, setting flow velocity is 2mL/min, detects wavelength 203nm, obtains white amorphous powder 68mg, through nuclear-magnetism in the time of 43.3minResonance hydrogen spectrum detects, and the hydrogen spectrum data consistent of the data obtained and embodiment 1 gained compound, therefore, determines that this compound is 21β, 22 α-O-, bis-angeloyl groups-15 α, 16 α, 28-trihydroxy olive-12-alkene 3 β-O-β-D-wood sugar-(1 → 2)-β-D-galaSugar-(1 → 3)-[β-D-galactolipin-(1 → 2)]-β-D-Glucose aldehydic acid glycosides, compound structure is suc as formula shown in I.
Embodiment 5 cell in vitro tests
Get people's lung cancer A549 cell, B16 mouse black-in tumor cell, human hepatocellular carcinoma BEL-7402 cell and human breast cancer cellMCF-7, respectively with containing the RPMI1640 culture medium of 10% deactivation NBS (separately add GLu0.03%, Hepes0.06%,NaHCO30.2%) in 37 DEG C, 5%CO2Under condition, cultivate, within 3 days, go down to posterity, the growth period cell of taking the logarithm is tested.
The concentration that regulates the cell of the logarithm campaign making with complete medium, makes cell concentration be 5 × 104Individual/ML, is inoculated in 96 well culture plates, and 100 μ L/ holes, at 37 DEG C, 5%CO2Under condition, cultivate after 24h, be divided into following several groups:
Test group adds compound shown in formula I prepared by embodiment 1, and solubilizer 1%DMSO and 99%PBS regulate final concentrationBe respectively 6.25 μ g/mL, 9.375 μ g/mL, 12.5 μ g/mL, 18.25 μ g/mL, 25.00 μ g/mL, 10 μ L/ holes.
Positive controls adds 5-FU, solubilizer 1%DMSO and 99%PBS, and regulating concentration is 50 μ g/mL, 10 μ L/ holes.
Negative control group adds complete medium, 10 μ L/ holes.
All establish 3 multiple holes, cultivate respectively 24h for every group. It is the MTT of 5mg/mL that the front 4h of termination cultivation adds concentration, 10 μ L/ holes,After cultivation finishes, every hole adds DMSO100 μ L, places shaking table 10min, the absorbance in the time that ELIASA detection wavelength is 490nmA value. Calculate growth of tumour cell inhibiting rate (InhibitionRate), computing formula is as follows:
InhibitionRate(%)=[(A negative control group-A test group)/A negative control group] × 100%
Calculate IC according to growth of tumour cell inhibiting rate50(half-inhibition concentration), result of the test is as shown in table 2:
Compound prepared by table 2 embodiment 1 IC to 4 kinds of tumour cell 24h50(μ g/mL) value
Result of the test is as shown in Table 2 known, compound shown in formula I prepared by embodiment 1 to people's lung cancer A549 cell,IC after the 24h of B16 mouse black-in tumor cell, human hepatocellular carcinoma BEL-7402 cell and human breast cancer cell MCF-750Value is all less than 15μ g/mL, wherein for the IC of A549 cell50Value is 11.37 ± 0.14 μ g/mL, shows chemical combination shown in formula I prepared by embodiment 1Thing has the significantly inhibitory action of (P < 0.05) to the growth of above-mentioned 4 kinds of tumour cells, to pressing down of people's lung cancer A549 cell growthEffect processed is the most remarkable.
In process of the test, find, positive control 5-FU acted on after 24 hours in the time that concentration is 50 μ g/mL, to people's lung cancer A549Inhibition rate of tumor cell is only 32%, and compound shown in formula I prepared by embodiment 1 is in the time that concentration is 11.37 ± 0.14 μ g/mLAfter effect 24h, people's lung cancer A549 inhibition rate of tumor cell is reached to 50%, as can be seen here, chemical combination shown in formula I prepared by embodiment 1Thing is significantly better than (P < 0.05) positive control 5-FU to the inhibitory action of people's lung cancer A549 tumour cell.
In process of the test, also find, positive control 5-FU acts on after 24 hours, to people's liver cancer in the time that concentration is 50 μ g/mLThe inhibiting rate of BEL-7402 tumour cell is only 24%, and compound shown in formula I prepared by embodiment 1 be 12.02 in concentration ±When 0.16 μ g/mL, act on after 24h, the inhibiting rate of people's liver cancer BEL-7402 tumour cell is reached to 50%, as can be seen here, embodiment 1Shown in the formula I of preparation, compound is significantly better than (P < 0.05) positive control to the inhibitory action of people's liver cancer BEL-7402 tumour cell5-FU。
In process of the test, also find, positive control 5-FU acts on after 24 hours, to B16 mouse in the time that concentration is 50 μ g/mLThe inhibiting rate of melanoma cells is 46%, and hence one can see that, and shown in formula I prepared by embodiment 1, compound is thin to B16 mouse black-in lymphomaBorn of the same parents' inhibitory action is better than positive control 5-FU.
In process of the test, also find, positive control 5-FU acts on after 24 hours, to human breast carcinoma in the time that concentration is 50 μ g/mLThe inhibiting rate of cell MCF-7 is 53%, and hence one can see that, and shown in formula I prepared by embodiment 1, compound is to human breast cancer cell MCF-7Inhibitory action be better than positive control 5-FU.
Shown in formula I prepared by the embodiment of the present invention 2～embodiment 4, compound carries out cell in vitro test, test methodWith embodiment 5, acquired results is similar to embodiment 5, compound pair shown in the formula I that embodiment of the present invention 2～embodiment 4 providesPeople's lung cancer A549 cell, B16 mouse black-in tumor cell, human hepatocellular carcinoma BEL-7402 cell and human breast cancer cell MCF-7 cellGrowth has good inhibition, the wherein inhibition to people's lung cancer A549 cell, human hepatocellular carcinoma BEL-7402 cell, Growth of CellsEffect is significantly better than (P < 0.05) positive control 5-FU.
Comprehensive above-mentioned result of the test is known, and shown in the formula I that the embodiment of the present invention 1～4 provides, compound is to people's lung cancer A549The growth of cell, B16 mouse black-in tumor cell, human hepatocellular carcinoma BEL-7402 cell and human breast cancer cell MCF-7 cell has goodGood inhibition, wherein to the inhibitory action of people's lung cancer A549 cell, human hepatocellular carcinoma BEL-7402 cell's growth be significantly better than (P <0.05) positive control 5-FU.
Embodiment 6 mice-transplanted tumor model tests
Get respectively H22And S180Rat liver cancer (sarcoma) cell cryopreservation tube, is placed in 37 DEG C of waters bath with thermostatic control and thaws, centrifugal collectionCell, by PBS liquid washed twice, then uses PBS liquid re-suspended cell, more than ICR mouse peritoneal passed for 3 generations, gets belly and obviously expandsMouse, dislocation is put to death, to belly alcohol disinfecting, disposable sterilized injector extracts milky ascites, inject sterilized fromIn core barrel, cell counter counting, with physiological saline adjustment cell density to 5 × 106Individual/mL, connects to every right oxter of mousePlant 0.2mL cell suspension and carry out modeling, be at random divided into following several group by mouse next day:
Blank group: 8 of the mouse of inoculation transplantable tumor, lumbar injection 0.9% physiological saline 0.2mL, once a day, givesMedicine 10 days.
Positive controls: 8 of the mouse of inoculation transplantable tumor, intraperitoneal injection of cyclophosphamide (CTX), 20mg/kg, the next day 1 time,Administration 10 days.
Test group 1: 8 of the mouse of inoculation transplantable tumor, compound shown in formula I prepared by lumbar injection embodiment 1,1.5mg/Kg, once a day, successive administration 10 days.
Test group 2: 8 of the mouse of inoculation transplantable tumor, compound shown in formula I prepared by lumbar injection embodiment 1,3.0mg/Kg's, once a day, successive administration 10 days.
2h after last administration, peels off knurl body, takes knurl weight, calculates tumour inhibiting rate, and computing formula is as follows:
The positive control group of wherein administration group, test group 1 or test group 2.
Result of the test is as shown in Table 3 and Table 4:
Shown in formula I prepared by table 3 embodiment 1, compound is to rat liver cancer H22The impact that transplantable tumor knurl is heavy
* P < 0.05, * * P < 0.01, vs blank group
Test data is as shown in Table 3 known, and shown in formula I prepared by the embodiment of the present invention 1, compound is respectively at dosageWhen 1.5mg/kg and 3.0mg/kg, continuous use 10 days, to rat liver cancer H22The inhibiting rate of transplantable tumor reaches respectively 33.0% He56.7%, compared with blank group, tumor killing effect highly significant (P < 0.01), and with the increase of dosage, tumour inhibiting rate significantly increasesBy force; Positive controls is in the time of 20mg/kg, to rat liver cancer H22The inhibiting rate of transplantable tumor prepared by formula I by 57.1%, embodiment 1Show compound and positive controls ratio, to rat liver cancer H22The inhibition of transplantable tumor is not as positive controls.
As can be seen here, shown in the formula I that prepared by embodiment 1, compound is to rat liver cancer H22The growth of transplantable tumor has significantly (P< 0.01) inhibitory action, dose-effect relationship is obvious.
Shown in formula I prepared by table 4 embodiment 1, compound is to mouse S180The impact that sarcoma knurl is heavy
* P < 0.05, * * P < 0.01, vs blank group
Test data is as shown in Table 4 known, and shown in formula I prepared by the embodiment of the present invention 1, compound is respectively at dosageWhen 1.5mg/kg and 3.0mg/kg, continuous use 10 days, to mouse S180The inhibiting rate of sarcoma reaches respectively 33.3% and 54.7%,Compared with blank group, tumor killing effect highly significant (P < 0.01), and with the increase of dosage, tumour inhibiting rate significantly strengthens; PositiveControl group is in the time of 20mg/kg, to mouse S180The inhibiting rate of sarcoma is only 37.1%.
As can be seen here, shown in the formula I that prepared by embodiment 1, compound is to mouse S180The growth of sarcoma have highly significant (P <0.01) inhibitory action, and dose-effect relationship is obvious.
Shown in formula I prepared by embodiment 2～embodiment 4, compound carries out mice-transplanted tumor model test, test methodWith embodiment 6, obtained experimental result is similar to embodiment 6, and shown in the formula I that prepared by embodiment 2～embodiment 4, compound is to littleMouse liver cancer H22The growth of transplantable tumor has the inhibitory action of highly significant (P < 0.01), and dose-effect relationship is obvious; To mouse S180SarcomaGrowth there is the inhibitory action of highly significant (P < 0.01), and dose-effect relationship is obvious.
In sum, shown in the formula I that prepared by the embodiment of the present invention 1～embodiment 4, compound is to rat liver cancer H22Transplantable tumorGrowth there is the inhibitory action of highly significant (P < 0.01), dose-effect relationship is obvious; To mouse S180The growth of sarcoma has veryThe significantly inhibitory action of (P < 0.01), and dose-effect relationship is obvious.
The preparation of embodiment 7 medicines
The compound of getting structure shown in formula I prepared by embodiment 1 adds the conventional auxiliary material of powder, makes according to conventional methodPowder.
The preparation of embodiment 8 medicines
The compound of getting structure shown in formula I prepared by embodiment 2 adds the conventional auxiliary material of tablet, makes according to conventional methodTablet.
The preparation of embodiment 9 medicines
The compound of getting structure shown in formula I prepared by embodiment 3 adds the conventional auxiliary material of granule, according to conventional method systemBecome granule.
The preparation of embodiment 10 medicines
The compound of getting structure shown in formula I prepared by embodiment 4 adds the conventional auxiliary material of capsule, according to conventional method systemBecome capsule.
The preparation of embodiment 11 medicines
The compound of getting structure shown in formula I prepared by embodiment 2 adds the conventional auxiliary material of solution, according to conventional method systemBecome solution.
The preparation of embodiment 12 medicines
The compound of getting structure shown in formula I prepared by embodiment 1 adds the conventional auxiliary material of emulsion, makes according to conventional methodEmulsion.
The preparation of embodiment 13 medicines
The compound of getting structure shown in formula I prepared by embodiment 3 adds the conventional auxiliary material of supensoid agent, according to conventional method systemBecome supensoid agent.
The preparation of embodiment 14 medicines
The compound of getting structure shown in formula I prepared by embodiment 4 adds the conventional auxiliary material of injection, according to conventional method systemBecome injection.
The preparation of embodiment 15 medicines
The compound of getting structure shown in formula I prepared by embodiment 2 adds the conventional auxiliary material of powder-injection, according to conventional method systemBecome powder-injection.
The preparation of embodiment 16 medicines
The compound of getting structure shown in formula I prepared by embodiment 1 adds the conventional auxiliary material of spray, according to conventional method systemBecome spray.
The above is only the preferred embodiment of the present invention, it should be pointed out that the ordinary skill people for the artMember, under the premise without departing from the principles of the invention, can also make some improvements and modifications, and these improvements and modifications also shouldBe considered as protection scope of the present invention.
Claims (4)
1. suc as formula an extracting method for the compound of structure shown in I, it is characterized in that, comprise following steps:
Step 1: oil tea root is pulverized, got the first solvent extraction, concentrated, obtain liquid extract;
Step 2: get described liquid extract and mix with water, filter, collect filtrate, centrifugal, collect supernatant, then set through D101 type macroporeFat post separates, and gets ethanol-water system gradient elution, and collecting ethanol and water volume ratio is 70:30～95:5 wash-out obtained component, pointFrom, purifying, to obtain final product;
Described the first solvent is that ethanol and water volume ratio are 30:70～95:5;
Described separation, purifying are specially: get described component and separate through silicagel column, get chloroform-methanol system gradient elution, collect chlorineImitative and methyl alcohol volume ratio is 80:20～60:40 wash-out obtained component; Separate through ODS post again, get methanol-water system gradient elution,Collecting methyl alcohol and water volume ratio is 70:30～90:10 wash-out obtained component; Separate through dynamic axial compression column again, get methanol-waterSystem gradient elution, collecting methyl alcohol and water volume ratio is 75:25 wash-out obtained component; Finally separate by high performance liquid chromatography,Adopt C18Chromatographic column, the second solvent elution, setting flow velocity is 2mL/min, collects 43.3min component, to obtain final product;
Described the second solvent is: first alcohol and water by volume for the mixed solution of 72:28 composition again with account for described mixed solution matterThe mixture of the formic acid mixing gained that amount percentage composition is 0.2%;
2. extracting method according to claim 1, is characterized in that, the solvent of gradient elution is followed successively by second described in step 2Alcohol and water volume ratio is 0:100 → 30:70 → 50:50 → 60:40 → 70:30 → 80:20 → 95:5.
3. extracting method according to claim 1, is characterized in that, the temperature of extracting described in step 1 is 30 DEG C～100DEG C, the time of extracting described in step 1 is 1h～15.5h.
4. extracting method according to claim 1, is characterized in that, concentrated temperature is 70 DEG C～100 described in step 1DEG C, the concentrated time is 3h～6h described in step 1.
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| Triterpenoid saponins from the roots of tea plant (Camellia sinensis var. assamica);Lu Y, et al.;《Phytochemistry》;20000413;第53卷(第8期);第941-946页 * |
| Triterpenoids from the roots of Camellia oleifera C.abel and their cytotoxic activities;Wang SL, et al.;《Helvetica Chimica Acta》;20130619;第96卷(第6期);第1126-1133页 * |
| 油茶根化学成分的研究;佟小静，等.;《中草药》;20111031;第42卷(第10期);第1936-1938页 * |
| 油茶根化学成分研究;佟小静;《中国优秀硕士学文论文全文数据库（医药卫生科技辑）》;20120615(第06期);第7、63、67-70、82-83页 * |
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