CN103739660A - Compound, extraction method thereof, application thereof to preparation of antitumor drugs, and antitumor drugs prepared by using compound - Google Patents
Compound, extraction method thereof, application thereof to preparation of antitumor drugs, and antitumor drugs prepared by using compound Download PDFInfo
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Abstract
The invention relates to the technical field of medicinal chemistry, in particular to a compound, an extraction method of the compound, application of the compound to the preparation of antitumor drugs, and the antitumor drugs prepared by using the compound. The compound is extracted from a camellia oleifera abel root; structure identification results show that the compound is a novel compound, and the structural formula is shown in the formula I; an in vitro cell test shows that after the compound shown in the formula I acts for 24 h, the compound has a remarkable inhibiting effect (P is less than 0.05) on the growth of human lung cancer A549 cells, B16 murine melanoma cells, human liver cancer BEL-7402 cells and human breast cancer MCF-7 cells; a murine transplantation tumor model test shows that the compound has a remarkable inhibiting effect (P is less than 0.05) on the growth of murine liver cancer H22 transplantation tumors and murine S180 sarcomas, and the dose-effect relationship is remarkable.
Description
Technical field
The present invention relates to pharmaceutical chemistry technical field, particularly a kind of compound, its extracting method, it prepares the antitumor drug of application and the preparation thereof of antitumor drug.
Background technology
Oil tea (CameLLia oLeifera AbeL) belongs to Theaceae (Theaceae) Camellia (CameLLia Linn), is 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 zone and subtropical zone, originates in the west and south and the southeast of China, spreads all over 17 provinces and regions.The unsaturated fatty acid content of tea oil, up to 90%, far away higher than rape oil, peanut oil and soya-bean oil, doubles than content of vitamin E with sweet oil, has high nutritive value, and therefore tea oil receives much concern.
In addition, oil tea also has very high pharmaceutical use, in < < Compendium of Materia Medica > >, records: " tea seed, the fragrant poison of bitter cold (saponin), cures mainly and breathe heavily anxious cough, removes disease dirt "; Herbal > > is also on the books in < < China, that the root of oil tea and root leatherware thereof have is clearing heat and detoxicating, the effect of regulating QI to relieve pain, activating blood circulation and reducing swelling, cure mainly swelling and pain in the throat, stomachache, toothache, traumatic pain, burn due to hot liquid or fire, its curative effect is considerably beyond tea oil.But the pharmaceutical use of oil tea is not subject to enough attention.
In recent years, chemical composition and the biological activity of people to oil tea is studied, at present, from the Semen Camelliae of oil tea, tea oil, camellia oleosa seed cake, Camellia Leaves, separate and obtain the compounds such as saponins, flavonoid, fatty acid, tannin, fragrant glycoside, alkaloid.Research discovery, part saponins compound has stomach protection, reducing blood-fat, fat-reducing, antianaphylaxis or antineoplastic effect.The discoveries such as Ma Liyuan, the oil tea total saponins in oil tea camellia oleosa seed cake more than 95% has significant anti-tumor activity.Therefore, Sasanguasaponin has very high pharmaceutical use.Sasanguasaponin mainly extracts from oil tea, but people focus mostly in Semen Camelliae, camellia oleosa seed cake, Camellia Leaves, tea oil to oil tea research before, less to the research of oil tea root, in oil tea root, may there is the bioactive Sasanguasaponin compounds of undiscovered tool, therefore, the research of saponin constituent in oil tea root is had great importance.
Summary of the invention
In view of this, the invention provides a kind of compound, its extracting method, it prepares the antitumor drug of application and the preparation thereof of antitumor drug.The present invention tests by cell in vitro and animal-transplanted tumor test is found, shown in formula I, compound has significant restraining effect to the growth of the multiple cancer cells of human body.
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:
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 fluid extract;
Step 2: get fluid extract and mix with water, filter, collect filtrate, centrifugal, collect supernatant liquor, then separate through D101 type macroporous resin column, get ethanol-water system gradient elution, collecting ethanol and water volume ratio is 70:30～95:5 wash-out obtained component, and separation, purifying, obtain;
The first solvent is that ethanol and water volume ratio are 0:100～95:5.
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 20:80～80:20.
Preferred, the first solvent is that ethanol and water volume ratio are 70:30.
As preferably, step 2 is got in fluid extract and water mixing step, and the volume ratio of fluid extract and water is 1:1～10, and the amount of institute's water reaches the object that fluid 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 is any one or both above technology in precipitating technology, crystallization technique or chromatographic technique, but is 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; Through ODS post, separate, get methanol-water system gradient elution, collecting methyl alcohol and water volume ratio is 70:30～90:10 wash-out obtained component; Through dynamic axial compression column, separate, get methanol-water system gradient elution, collecting methyl alcohol and water volume ratio is 75:25 wash-out obtained component; Finally by high performance liquid chromatography, separate, adopt C
18chromatographic column, the second solvent elution, flow velocity is 2mL/min, collects 37.5min component, obtains;
The second solvent is: first alcohol and water by volume for the mixing solutions of 72:28 composition again with the mixture that accounts for the formic acid mixing gained that mixing solutions quality percentage composition is 0.2%.
C
18the specification of chromatographic column 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 is followed successively by chloroform and methyl alcohol volume ratio is 90:10 → 80:20 → 70:30 → 60:40 → 50:50 → 35:65 → 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 is that methyl alcohol and water volume ratio are 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 thin layer plate 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 ℃～100 ℃, and the time of extracting described in step 1 is 8.5h～33h.
As preferably, the temperature that step 1 is extracted is 50 ℃～85 ℃, and the time that step 1 is extracted is 1.0h～2.5h.
As preferably, the temperature that step 1 is extracted is 70 ℃～85 ℃, and the time that step 1 is extracted is 2h～2.5h.
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 invention.
As preferably, the time of immersion is 8h～24h, and the temperature of immersion is 30 ℃～80 ℃.
As preferably, the temperature of refluxing extraction is 70 ℃～100 ℃.
Preferred, the temperature of refluxing extraction is 80 ℃.
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 whole filtrate, is extracting solution.
As preferably, filtration step is carrying out after each refluxing extraction, filter gained filter residue again with the first solvent, carry out 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, the concentrated temperature of step 1 is 70 ℃～100 ℃, and the concentrated time of step 1 is 2.0h～6.0h.
As preferably, the concentrated temperature of step 1 is 80 ℃～100 ℃, and the concentrated time of step 1 is 3h～5h.
As preferably, simmer down to concentrating under reduced pressure.
As preferably, concentrated multiple is 6～10 times.
The present invention also provides the compound of structure shown in formula I in the application of preparing in antitumor drug.First the present invention measures the restraining effect of medicine to tumor cell line by mtt assay.Mtt assay, claims again MTT colorimetry, is a kind of method that detects cell survival and growth.Its detection principle is that the succinodehydrogenase in viable cell plastosome can make exogenous MTT be reduced to water-insoluble bluish voilet crystallization first a ceremonial jade-ladle, used in libation (Formazan) and be deposited in cell, and dead cell is without this function.First a ceremonial jade-ladle, used in libation in dimethyl sulfoxide (DMSO) (DMSO) energy dissolved cell, measures its absorbance value with enzyme-linked immunosorbent assay instrument at 490nm wavelength place, can indirectly reflect viable cell quantity.Within the scope of certain cell count, the amount that MTT crystallization forms is directly proportional to cell count.The method has been widely used in the activity detection of some biologically active factorss, large-scale screening anti-tumor medicine, cell toxicity test and tumor radiosensitivity mensuration 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, and human hepatocellular carcinoma BEL-7402 cell and human breast cancer cell MCF-7, as subject cell, and will be made as 24h the action time of tested medicine.Test-results shows, shown in formula I provided by the invention after compound effects 24h, to 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 IC
50value is all less than 20 μ g/mL, and as can be seen here, shown in formula I, compound has significant restraining effect to the growth of above-mentioned 4 kinds of tumour cells.
The present invention also tests the tumor killing effect of this compound by mice-transplanted tumor model trial, result shows, shown in formula I, compound is to rat liver cancer H
22transplanted tumor and mouse S
180the growth of sarcoma has the significantly restraining effect of (P<0.05), and dose-effect relationship is obvious, wherein to mouse S
180the restraining effect of sarcoma growth is significantly better than positive control CTX, therefore, the invention provides the compound of structure shown in formula I in the application of preparing in antitumor drug.
As preferably, tumour is lung cancer, liver cancer, melanoma or mammary cancer.Tumour can also be not open for the present invention, but have the tumour of other kinds of inhibition.
The present invention also provides a kind of antitumor drug, the compound of structure and pharmaceutically acceptable auxiliary material shown in its contained I.
The formulation of antitumor drug of the present invention can be the attainable any formulation in this area, the conventional auxiliary material that auxiliary material used is formulation used, the conventional preparation method that preparation method is corresponding formulation.
As preferably, the formulation of antitumor drug is powder, tablet, granule, capsule, solution, emulsion, suspensoid, injection liquid, powder injection or sprays.
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; Structural Identification result shows; this compound is new compound; called after: 21 β; 22 α-O-, bis-angeloyl groups-15 α, 16 α, 28-trihydroxy-olea-12-alkene-23-aldehyde 3 β-O-β-D-wood sugar-(1 → 2)-β-D-semi-lactosi-(1 → 3)-[β-D-semi-lactosi-(1 → 2)]-β-D-Glucose aldehydic acid glycosides.The present invention adopts mtt assay to carry out cell in vitro test, and result shows, shown in formula I provided by the invention after compound effects 24h, to the IC 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
50value is all less than 20 μ g/mL, and the restraining effect of the growth to people's lung cancer A549 cell, human hepatocellular carcinoma BEL-7402 cell, cell is significantly better than (P<0.05) positive control 5-FU.As can be seen here, shown in formula I, compound has significant restraining effect to the growth of above-mentioned 4 kinds of tumour cells.The present invention also tests the tumor killing effect of this compound by mice-transplanted tumor model trial, test result shows, shown in formula I, compound is to rat liver cancer H
22the growth of transplanted tumor and mouse S 180 sarcoma has the significantly restraining effect of (P<0.05), dose-effect relationship is obvious, wherein the restraining effect of the growth to mouse S 180 sarcoma is significantly better than positive control CTX, therefore, shown in formula I provided by the invention, compound also can be used for preparing antitumor drug.
Accompanying drawing explanation
Fig. 1 is 21 β, 22 α-O-, bis-angeloyl groups-15 α, 16 α, the high resolution mass spectrum spectrogram of 28-trihydroxy-olea-12-alkene-23-aldehyde 3 β-O-β-D-wood sugar-(1 → 2)-β-D-semi-lactosi-(1 → 3)-[β-D-semi-lactosi-(1 → 2)]-β-D-Glucose aldehydic acid glycosides;
Fig. 2 is 21 β, 22 α-O-, bis-angeloyl groups-15 α, 16 α, the hydrogen nuclear magnetic resonance spectrogram of 28-trihydroxy-olea-12-alkene-23-aldehyde 3 β-O-β-D-wood sugar-(1 → 2)-β-D-semi-lactosi-(1 → 3)-[β-D-semi-lactosi-(1 → 2)]-β-D-Glucose aldehydic acid glycosides;
Fig. 3 is 21 β, 22 α-O-, bis-angeloyl groups-15 α, 16 α, the hydrogen nuclear magnetic resonance spectrogram (enlarged view) of 28-trihydroxy-olea-12-alkene-23-aldehyde 3 β-O-β-D-wood sugar-(1 → 2)-β-D-semi-lactosi-(1 → 3)-[β-D-semi-lactosi-(1 → 2)]-β-D-Glucose aldehydic acid glycosides;
Fig. 4 is 21 β, 22 α-O-, bis-angeloyl groups-15 α, 16 α, the carbon-13 nmr spectra figure of 28-trihydroxy-olea-12-alkene-23-aldehyde 3 β-O-β-D-wood sugar-(1 → 2)-β-D-semi-lactosi-(1 → 3)-[β-D-semi-lactosi-(1 → 2)]-β-D-Glucose aldehydic acid glycosides;
Fig. 5 is 21 β, 22 α-O-, bis-angeloyl groups-15 α, 16 α, the carbon-13 nmr spectra figure (enlarged view) of 28-trihydroxy-olea-12-alkene-23-aldehyde 3 β-O-β-D-wood sugar-(1 → 2)-β-D-semi-lactosi-(1 → 3)-[β-D-semi-lactosi-(1 → 2)]-β-D-Glucose aldehydic acid glycosides;
Fig. 6 is 21 β, 22 α-O-, bis-angeloyl groups-15 α, 16 α, the carbon-13 nmr spectra figure (enlarged view) of 28-trihydroxy-olea-12-alkene-23-aldehyde 3 β-O-β-D-wood sugar-(1 → 2)-β-D-semi-lactosi-(1 → 3)-[β-D-semi-lactosi-(1 → 2)]-β-D-Glucose aldehydic acid glycosides;
Fig. 7 is 21 β, 22 α-O-, bis-angeloyl groups-15 α, 16 α, the HSQC figure of 28-trihydroxy-olea-12-alkene-23-aldehyde 3 β-O-β-D-wood sugar-(1 → 2)-β-D-semi-lactosi-(1 → 3)-[β-D-semi-lactosi-(1 → 2)]-β-D-Glucose aldehydic acid glycosides;
Fig. 8 is 21 β, 22 α-O-, bis-angeloyl groups-15 α, 16 α, the HMBC figure of 28-trihydroxy-olea-12-alkene-23-aldehyde 3 β-O-β-D-wood sugar-(1 → 2)-β-D-semi-lactosi-(1 → 3)-[β-D-semi-lactosi-(1 → 2)]-β-D-Glucose aldehydic acid glycosides;
Fig. 9 is 21 β; 22 α-O-, bis-angeloyl groups-15 α; 16 α, 28-trihydroxy-olea-12-alkene-23-aldehyde 3 β-O-β-D-wood sugar-(1 → 2)-β-D-semi-lactosi-(1 → 3)-[β-D-semi-lactosi-(1 → 2)]-β-D-Glucose aldehydic acid glycosides
1h-
1h COSY figure;
Figure 10 is 21 β; 22 α-O-, bis-angeloyl groups-15 α; 16 α, the NOESY figure of 28-trihydroxy-olea-12-alkene-23-aldehyde 3 β-O-β-D-wood sugar-(1 → 2)-β-D-semi-lactosi-(1 → 3)-[β-D-semi-lactosi-(1 → 2)]-β-D-Glucose aldehydic acid glycosides.
Embodiment
The invention provides a kind of compound, its extracting method, it prepares the antitumor drug of application and the preparation thereof of antitumor drug.Those skilled in the art can use for reference content herein, suitably improve processing parameter and realize.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the artly, they are all deemed to be included in the present invention.Method of the present invention and application are described by preferred embodiment, related personnel obviously can change methods and applications as herein described in content of the present invention, spirit and scope or suitably change and combination not departing from, and realizes and apply 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 sources are specific as follows:
Nuclear magnetic resonance analyser 500Hz (Varian Inc., Palo Alto, CA, the U.S.); High resolution mass spectrum Q-TOF2(Britain Micromass company); Electronic balance (Mettler-Toledo Instrument (Shanghai) Co., Ltd.); Polarimeter 241 (PerkinElmer Inc., Waltham, MA, the U.S.); Half preparative high-performance liquid chromatographic instrument (LC-20AT, SPD-20A, Japanese Shimadzu company); C18 half preparative chromatography post (250mm × 10mm, 5 μ m, U.S. Kromsil company); Middle pressure preparative liquid chromatography (B ü chi chromatographic system, C-650 pump, middle compression leg (460mm × 26mm i.d., B ü chi Corp., Flawil, Swiss); Dynamic axial column chromatography (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 (analytical 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 factory produces); 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., laboratory animal production licence number SCXK (Shanghai) 2007-0005.
Perfect medium: fill a prescription as the RPMI1640 substratum containing 10% inactivated fetal bovine serum, manufacturer is 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 10-40 object wood chip with pulverizer, through 40 ℃, 6L water, soak after 8h, reflux 0.5h at 100 ℃, filters, and filter residue adds 3L water again, reflux 3.0h at 100 ℃, filter, filter residue mixes with 3L water again, reflux 2h at 100 ℃, filter, merge three times and filter gained filtrate, concentrating under reduced pressure 6h at 80 ℃, obtains fluid extract 2000mL.
Getting the fluid extract making mixes with 20000mL water, 100 order filter-cloth filterings, centrifugal filtrate, getting supernatant liquor separates through D101 type macroporous resin column, water, 30% ethanol, 50% ethanol, 60% ethanol, 70% ethanol, 80% ethanol, 95% ethanol elution successively, collect the component of 70% ethanol～95% ethanol elution gained, concentrating under reduced pressure, obtains oil tea total saponins.
Getting the oil tea total saponins making first separates through the decompression silicagel column of 60 order silica gel, by chloroform and methyl alcohol volume ratio, be 90:10 → 80:20 → 70:30 → 60:40 → 50:50 → 40:60 → 20:80 → 0:100 gradient elution successively, obtain component 1～component 8, through thin layer plate analysis, merge chloroform wherein and methyl alcohol volume ratio 80:20～60:40 wash-out obtained component (when developping agent system is BAW system, Rf value is 0.4-0.7), in warp, press again anti-phase ODS post to separate, methanol-water system 50%-100% gradient elution, flow velocity 25mL/min, detection wavelength is 203nm, obtain 6 components, through efficient analysis liquid phase analysis, with methyl alcohol and water volume ratio be 60:40～90:10 gradient elution 30min, merge the component (peak position is in 20min-28min) that methyl alcohol and water volume ratio are 70:30～90:10, through dynamic axial compression column, separate, use respectively 60%, 75%, 80% and 90% methanol-eluted fractions, flow velocity 150mL/min, detect wavelength 203nm, obtain altogether five components, through efficient analysis liquid phase analysis, 75% methanol-eluted fractions obtained component (peak position is in 13.5-15.0min) is separated by partly preparing high performance liquid phase, methanol-water (72:28, v/v)-0.2% formic acid wash-out wash-out, setting flow velocity is 2mL/min, detect wavelength 203nm, in 37.5min place, obtain white amorphous powder 150mg.
Get the white amorphous powder making, detect development properties, hydrolysis properties, then warp
1h-NMR,
13c-NMR, HMBC, HSQC,
1h-
1h COSY, NOESY and HR-ESI-MS carry out wave spectrum analysis, spectral data figure is as shown in Fig. 1～Figure 10, NMR data are as 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 positive, MoLish reacting positive, pointing out this compound may be saponins compound.Mass spectrum demonstration shown in Fig. 1, the accurate quasi-molecular ions of the HR-ESI-MS of this compound is m/z1315.5968[M-H]
-, the molecular formula that shows this compound is C
63h
96o
29(calculated value [M-H]
-, m/z1315.5959, calculated value is according to molecular formula, by high abundance exact value, is calculated).Obtain monose with the complete acid hydrolysis of 2N TFA, sugar carries out GC analysis after derivatize, the existence of D-Glucose aldehydic acid, D-semi-lactosi, D-wood sugar detected.
By the compound of Fig. 4, Fig. 5, Fig. 6
13c-NMR spectrogram is known, and this compound shows 63 carbon signals (as shown in table 1) altogether, and the hsqc spectrum of the compound shown in Fig. 7 is known, and this compound contains 10 methyl carbon altogether, 10 mesomethylene carbon, 31 methine carbons and 12 quaternary carbons.Further analyze
13c-NMR spectrum, this spectrogram demonstrates four sugared end group carbon signal δ 104.3,101.8,107.9 and 103.1, is respectively the end group carbon of D-Glucose aldehydic acid, D-semi-lactosi, D-wood sugar and another one D-semi-lactosi.In addition, also has an aldehyde radical carbon signal that is positioned at low place δ 210.3.From the hydrogen spectrogram of the compound shown in Fig. 2, Fig. 3,
1in H-NMR spectrum, high field region has six obvious triterpenoid saponin angular methyl(group) hydrogen signal δ 0.88 (Me-25), 1.04 (Me-26), 1.18 (Me-29), 1.41 (Me-30), 1.53 (Me-24) and 1.90 (Me-27); Even Oxymethylene δ 3.57 (H-28, d, J=11.0Hz) and 3.82 (H-28, d, J=11.0Hz); Five oxygen methyne δ 4.09 (1H, dd, J=11.0,4.5Hz, H-3) of company, 4.26 (1H, brs, H-15), 4.54 (1H, brs, H-16), 6.78 (1H, d, J=10.5Hz, H-21) and 6.41 (1H, d, J=10.5Hz, H-22); Alkene hydrogen proton δ 5.58 (1H, brs, H-12) and aldehyde radical hydrogen proton δ 9.94 (1H, brs, H-23).In addition compound,
1h-NMR spectrum also demonstrates two groups of angeloyl groups signal δ [6.07 (1H, dq, J=7.5,1.5Hz, 21-O-Ang-3'), 2.18 (3H, d, J=7.5Hz, 21-O-Ang-4') and 2.09 (3H, s, 21-O-Ang-5')]; δ [5.88 (1H, dq, J=7.5,1.5Hz, 22-O-Ang-3 "), 2.05 (3H, d, J=7.5Hz, 22-O-Ang-4 ") and 1.83 (3H, s, 22-O-Ang-5 ")].
As shown in Figure 9
1h-
1known in H COSY spectrum, with the methyne δ that even oxygen methine protons is adjacent
h4.26 (1H, brs) and δ
h4.54 (1H, brs) are relevant, illustrate that they are in ortho position and δ
h4.54 is H-16 signal, δ
h4.26 is H-15 signal; δ
h6.78 (1H, d, J=10.5Hz) and δ
hthey are in ortho position and δ coherent signal explanation between 6.41 (1H, d, J=10.5Hz)
h6.78 is H-21 signal, δ
h6.41 is H-22 signal.
By the HMBC spectrum analysis of the compound shown in Fig. 8 learn two angeloyl groups be connected in respectively 21,22 of compound upper because in HMBC δ
h6.78 (1H, d, J=10.5Hz, H-21) and δ
c168.0 (21-O-Ang-1') are relevant, δ
h6.41 (1H, d, J=10.5Hz, H-22) and δ
c168.3 (22-O-Ang-1'') are relevant.In conjunction with
13c-NMR spectrum and
1h-NMR spectrum data are comprehensively analyzed; and the nuclear magnetic data contrast with the known aglycon of document " Helvetica Chimica Acta2013.Vol.96 " report; the aglycon structure of determining this compound is 21 β; 22 α-O-, bis-angeloyl groups-15 α; 16 α, 28-trihydroxy-olea-12-alkene-23-aldehyde.
To compound, hsqc spectrum figure as shown in Figure 7 analyzes, and belongs to sugared end group carbon, the hydrogen signal that on this saponin(e, connect, and result is as follows: δ
h4.80 (1H, d, J=6.5Hz), 5.80 (1H, d, J=7.5Hz), 5.09 (1H, d, J=7.5Hz) and 5.95 (1H, d, J=7.5Hz), be connected in respectively δ
c104.3 (glucuronic acid-C-1), 101.8 (semi-lactosi-C-1), 107.9 (wood sugar-C-1), on 103.1 (semi-lactosi '-C-1).3 of this compound aglycons are positioned at 12.6ppm to show that sugar chain is connected in 3 of aglycons upper to low field in addition, and in HMBC spectrum, glucuronic acid anomeric proton δ
h4.80 with 3 δ of aglycon
c84.7 is relevant, further confirmed that sugar chain is connected in 3 of aglycon.Further By consulting literatures finds that the camellenodiol sugar chain data of report in this compound sugar chain and document " Bioorganic & medicinal chemistry letters2010; 20; 7435-7439 " are close, thereby infers that this compound sugar chain is β-D-wood sugar-(1 → 2)-β-D-semi-lactosi-(1 → 3)-[β-D-semi-lactosi-(1 → 2)]-β-D-Glucose aldehydic acid.The order of connection of this sugar chain also can confirm by analyzing HMBC spectrum, glucuronic acid anomeric proton δ in HMBC
h4.80 with 3 carbon δ of aglycon
c84.7 is relevant, semi-lactosi anomeric proton δ
h5.80 with 3 carbon δ of glucuronic acid
c84.9 is relevant, wood sugar anomeric proton δ
h5.09 with 2 carbon δ of semi-lactosi
c84.1 is relevant, 2 hydrogen δ of semi-lactosi
h4.57 with wood sugar end group carbon δ
c107.9 is relevant, another semi-lactosi anomeric proton δ
h5.95 with 2 carbon δ of glucuronic acid
c78.2 is relevant, 2 hydrogen δ of glucuronic acid
h4.57 with this semi-lactosi end group carbon δ
c103.1 relevant.In addition four sugared anomeric proton coupling constants,
3j
h-1,
h-2larger, show that these four sugar are all beta configuration.
NOESY spectrum shown in Figure 10 shows, δ
h6.78 (1H, d, J=10.5Hz, H-21) and δ
h1.18 (3H, s, H-29) are relevant, and prompting H-21 is α configuration; δ
h4.09 (1H, dd, J=4.5,11.0Hz, H-3) and δ
h9.94 (1H, brs, H-23) are relevant, and prompting H-3 is α configuration; δ
h4.54 (1H, brs, H-16) and δ
hthe relevant prompting prompting of 3.57 (1H, d, J=11.0Hz, H-28), 3.82 (1H, d, J=11.0Hz, H-28) H-16 is beta comfiguration, δ
h6.41 (1H, d, J=10.5Hz, H-22) and δ
h1.41 (3H, s, H-30) are relevant, and prompting 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-olea-12-alkene-23-aldehyde 3 β-O-β-D-wood sugar-(1 → 2)-β-D-semi-lactosi-(1 → 3)-[β-D-semi-lactosi-(1 → 2)]-β-D-Glucose aldehydic acid glycosides.Compound structure is suc as formula shown in I:
NMR (pyridine-d5, the 500MHz) data of compound shown in table 1 formula I
In table, chemical shift is take ppm as unit, and coupling constant (J) is take Hz as unit.
The extraction of embodiment 2 compounds
Get dry oil tea root 2kg, be ground into 10 object wood chips with pulverizer, after 40 ℃ of immersion 24h of 95% ethanol of 40L, reflux 0.5h at 80 ℃, filters, filter residue adds 95% ethanol 2L again, reflux 3.0h at 80 ℃, filters, and merges twice and filters gained filtrate, concentrating under reduced pressure 2h at 90 ℃, obtains fluid extract 4200mL.
Getting the fluid extract making mixes with 4200mL water, 200 order filter-cloth filterings, centrifugal filtrate, getting supernatant liquor separates through D101 type macroporous resin column, water, 30% ethanol, 50% ethanol, 60% ethanol, 70% ethanol, 80% ethanol, 95% ethanol elution successively, collect the component of 70% ethanol～95% ethanol elution gained, concentrating under reduced pressure, obtains oil tea total saponins.
Getting the oil tea total saponins making first separates through the decompression silicagel column of 100 order silica gel, by chloroform and methyl alcohol volume ratio, be 90:10 → 80:20 → 70:30 → 60:40 → 50:50 → 40:60 → 20:80 → 0:100 gradient elution successively, obtain component 1～component 8, through thin layer plate analysis, merge chloroform wherein and methyl alcohol volume ratio 80:20～60:40 wash-out obtained component (when developping agent system is BAW system, Rf value is 0.4-0.7), in warp, press again anti-phase ODS post to separate, methanol-water system 50%-100% gradient elution, flow velocity 25mL/min, detection wavelength is 203nm, obtain 6 components, through efficient analysis liquid phase analysis, with methyl alcohol and water volume ratio be 60:40～90:10 gradient elution 30min, merge the component (peak position is in 20min-28min) that methyl alcohol and water volume ratio are 70:30～90:10, through dynamic axial compression column, separate, use respectively 60%, 75%, 80% and 90% methanol-eluted fractions, flow velocity 150mL/min, detect wavelength 203nm, obtain altogether five components, through efficient analysis liquid phase analysis, 75% methanol-eluted fractions obtained component (peak position is in 13.5-15.0min) is separated by partly preparing high performance liquid phase, methanol-water (72:28, v/v)-0.2% formic acid wash-out wash-out, setting flow velocity is 2mL/min, detect wavelength 203nm, when 37.5min, obtain white amorphous powder 180mg, through proton nmr spectra, detect, the hydrogen spectrum data consistent of the data obtained and embodiment 1 gained compound, therefore, determine that this compound is 21 β, 22 α-O-, bis-angeloyl groups-15 α, 16 α, 28-trihydroxy-olea-12-alkene-23-aldehyde 3 β-O-β-D-wood sugar-(1 → 2)-β-D-semi-lactosi-(1 → 3)-[β-D-semi-lactosi-(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 10-60 object wood chip with pulverizer, through 80 ℃ of 70% ethanol of 20L, soak after 8h, reflux 3h at 85 ℃, filters, and collects filtrate, and concentrating under reduced pressure 6h at 70 ℃, obtains fluid extract 2000mL.
Getting the fluid extract making mixes with 6000mL water, 300 order filter-cloth filterings, centrifugal filtrate, getting supernatant liquor separates through D101 type macroporous resin column, water, 30% ethanol, 50% ethanol, 60% ethanol, 70% ethanol, 80% ethanol, 95% ethanol elution successively, collect the component of 70% ethanol～95% ethanol elution gained, concentrating under reduced pressure, obtains oil tea total saponins.
Getting the oil tea total saponins making first separates through the decompression silicagel column of 80 order silica gel, by chloroform and methyl alcohol volume ratio, be 90:10 → 80:20 → 70:30 → 60:40 → 50:50 → 35:65 → 20:80 → 0:100 gradient elution successively, obtain component 1～component 8, through thin layer plate analysis, merge chloroform wherein and methyl alcohol volume ratio 80:20～60:40 wash-out obtained component (when developping agent system is BAW system, Rf value is 0.4-0.7), in warp, press again anti-phase ODS post to separate, methanol-water system 50%-100% gradient elution, flow velocity 25mL/min, detection wavelength is 203nm, obtain 6 components, through efficient analysis liquid phase analysis, with methyl alcohol and water volume ratio be 60:40～90:10 gradient elution 30min, merge the component (peak position is in 20min-28min) that methyl alcohol and water volume ratio are 70:30～90:10, through dynamic axial compression column, separate, use respectively 60%, 75%, 80% and 90% methanol-eluted fractions, flow velocity 150mL/min, detect wavelength 203nm, obtain altogether five components, through efficient analysis liquid phase analysis, 75% methanol-eluted fractions obtained component (peak position is in 13.5-15.0min) is separated by partly preparing high performance liquid phase, methanol-water (72:28, v/v)-0.2% formic acid wash-out wash-out, setting flow velocity is 2mL/min, detect wavelength 203nm, when 37.5min, obtain white amorphous powder 160mg, through proton nmr spectra, detect, the hydrogen spectrum data consistent of the data obtained and embodiment 1 gained compound, therefore, determine that this compound is 21 β, 22 α-O-, bis-angeloyl groups-15 α, 16 α, 28-trihydroxy-olea-12-alkene-23-aldehyde 3 β-O-β-D-wood sugar-(1 → 2)-β-D-semi-lactosi-(1 → 3)-[β-D-semi-lactosi-(1 → 2)]-β-D-Glucose aldehydic acid glycosides, compound structure is suc as formula shown in I.
The extraction of embodiment 4 compounds
Get dry oil tea root 2kg, be ground into 10-40 object wood chip with pulverizer, after 30 ℃ of immersion 8h of 80% ethanol of 20L, reflux 0.5h at 75 ℃, reflux 3.0h at 80 ℃, reflux 2h at 80 ℃, filter, collect filtrate, concentrating under reduced pressure 3h at 100 ℃, obtains fluid extract 2000mL.
Getting the fluid extract making mixes with 6000mL water, 250 order filter-cloth filterings, centrifugal filtrate, getting supernatant liquor separates through D101 type macroporous resin column, water, 30% ethanol, 50% ethanol, 60% ethanol, 70% ethanol, 80% ethanol, 95% ethanol elution successively, collect the component of 70% ethanol～95% ethanol elution gained, concentrating under reduced pressure, obtains oil tea total saponins.
Getting the oil tea total saponins making first separates through the decompression silicagel column of 60 order silica gel, by chloroform and methyl alcohol volume ratio, be 90:10 → 80:20 → 70:30 → 60:40 → 50:50 → 40:60 → 20:80 → 0:100 gradient elution successively, obtain component 1～component 8, through thin layer plate analysis, merge chloroform wherein and methyl alcohol volume ratio 80:20～60:40 wash-out obtained component (when developping agent system is BAW system, Rf value is 0.4-0.7), in warp, press again 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, obtain 6 components, through efficient analysis liquid phase analysis, with methyl alcohol and water volume ratio be 60:40～90:10 gradient elution 30min, merge the component (peak position is in 20min-28min) that methyl alcohol and water volume ratio are 70:30～90:10, through dynamic axial compression column, separate, use respectively 60%, 75%, 80% and 90% methanol-eluted fractions, flow velocity 150mL/min, detect wavelength 203nm, obtain altogether five components, through efficient analysis liquid phase analysis, 75% methanol-eluted fractions obtained component (peak position is in 13.5-15.0min) is separated by partly preparing high performance liquid phase, methanol-water (72:28, v/v)-0.2% formic acid wash-out wash-out, setting flow velocity is 2mL/min, detect wavelength 203nm, when 37.5min, obtain white amorphous powder 230mg, through proton nmr spectra, detect, the hydrogen spectrum data consistent of the data obtained and embodiment 1 gained compound, therefore, determine that this compound is 21 β, 22 α-O-, bis-angeloyl groups-15 α, 16 α, 28-trihydroxy-olea-12-alkene-23-aldehyde 3 β-O-β-D-wood sugar-(1 → 2)-β-D-semi-lactosi-(1 → 3)-[β-D-semi-lactosi-(1 → 2)]-β-D-Glucose aldehydic acid glycosides, compound structure is suc as formula shown in I.
Get 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, with the RPMI1640 substratum containing 10% deactivation NBS, (separately add GLu0.03%, Hepes0.06%, NaHCO respectively
30.2%) in 37 ℃, 5%CO
2under condition, cultivate, within 3 days, go down to posterity, the vegetative period cell of taking the logarithm is tested.
The concentration that regulates the cell of the logarithm production phase making with perfect medium, makes cell concn be 5 × 10
4individual/mL, is inoculated in 96 well culture plates, and 100 μ L/ holes, at 37 ℃, 5%CO
2under condition, cultivate after 24h, be divided into following several groups:
Test group adds compound shown in formula I prepared by embodiment 1, and solubilizing agent 1%DMSO and 99%PBS regulate final concentration to be 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, solubilizing agent 1%DMSO and 99%PBS, and regulating concentration is 50 μ g/mL, 10 μ L/ holes.
Negative control group adds perfect 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, and after cultivation finishes, every hole adds DMSO100 μ L, places shaking table 10min, the absorbance A value when microplate reader detection wavelength is 490nm.Calculate growth of tumour cell inhibiting rate (Inhibition Rate), calculation formula is as follows:
Inhibition Rate(%)=[(A negative control group-A test group)/A negative control group] × 100%
According to growth of tumour cell inhibiting rate, calculate IC
50(half-inhibition concentration), test-results is as shown in table 2:
Compound prepared by table 2 embodiment 1 IC to 4 kinds of tumour cell 24h
50(μ g/mL) value
Test-results is as shown in Table 2 known, the IC of compound shown in formula I prepared by embodiment 1 after to the 24h 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
50value is all less than 20 μ g/mL, shows that shown in formula I prepared by embodiment 1, compound has significant restraining effect to the growth of above-mentioned 4 kinds of tumour cells.
In process of the test, find, positive control 5-FU acted on after 24 hours when concentration is 50 μ g/mL, to people's lung cancer A549 inhibition rate of tumor cell, be only 32%, and compound acts on after 24h when concentration is 14.95 ± 0.40 μ g/mL shown in formula I prepared by embodiment 1, people's lung cancer A549 inhibition rate of tumor cell is reached to 50%, as can be seen here, shown in the formula I that prepared by embodiment 1, compound is significantly better than (P<0.05) positive control 5-FU to the restraining effect of people's lung cancer A549 tumour cell.
In process of the test, also find, positive control 5-FU acted on after 24 hours when concentration is 50 μ g/mL, to the inhibiting rate of people's liver cancer BEL-7402 tumour cell, be only 24%, and compound acts on after 24h when concentration is 15.76 ± 0.33 μ g/mL shown in formula I prepared by embodiment 1, inhibiting rate to people's liver cancer BEL-7402 tumour cell reaches 50%, as can be seen here, shown in the formula I that prepared by embodiment 1, compound is significantly better than (P<0.05) positive control 5-FU to the restraining effect of people's liver cancer BEL-7402 tumour cell.
In process of the test, also find, positive control 5-FU acted on after 24 hours when concentration is 50 μ g/mL, to the inhibiting rate of B16 mouse black-in tumor cell, be 49%, hence one can see that, and shown in formula I prepared by embodiment 1, compound is better than positive control 5-FU to the restraining effect of B16 mouse black-in tumor cell.
In process of the test, also find, positive control 5-FU acted on after 24 hours when concentration is 50 μ g/mL, to the inhibiting rate of human breast cancer cell MCF-7, be 43%, hence one can see that, and shown in formula I prepared by embodiment 1, compound is better than positive control 5-FU to the restraining effect of human breast cancer cell MCF-7.
Shown in formula I prepared by the embodiment of the present invention 2～embodiment 4, compound carries out cell in vitro test, test method is with embodiment 5, acquired results is similar to embodiment 5, be that shown in the formula I that provides of the embodiment of the present invention 2～embodiment 4, compound has good inhibition to 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, wherein better to the restraining effect of people's lung cancer A549 cell, human hepatocellular carcinoma BEL-7402 cell, MCF-7 Breast Cancer Cell growth.
Comprehensive above-mentioned test-results is known, compound shown in the formula I that the embodiment of the present invention 1～4 provides has good inhibition to 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, wherein better to people's lung cancer A549 cell, human hepatocellular carcinoma BEL-7402 cell, the effect of MCF-7 Breast Cancer Cell growth-inhibiting.
Get respectively H
22and S
180rat liver cancer (sarcoma) cell cryopreservation tube, is placed in 37 ℃ of waters bath with thermostatic control and thaws, centrifugal collecting cell, by PBS liquid washed twice, use again PBS liquid re-suspended cell, more than ICR mouse peritoneal passed for 3 generations, get the mouse that belly obviously expands, dislocation is put to death, to belly alcohol disinfecting, disposable sterilized injector extracts oyster white ascites, injects sterilized centrifuge tube, cell counter counting, with physiological saline adjustment cell density to 5 × 10
6individual/mL, carries out modeling to every the right oxter inoculation of mouse 0.2mL cell suspension, and be at random divided into following several group by mouse next day:
Blank group: 8 of the mouse of inoculation transplanted tumor, abdominal injection 0.9% physiological saline 0.2mL, once a day, administration 10 days.
Positive controls: 8 of the mouse of inoculation transplanted 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 transplanted tumor, compound shown in formula I prepared by abdominal injection embodiment 1,1.5mg/kg, once a day, successive administration 10 days.
Test group 2: 8 of the mouse of inoculation transplanted tumor, compound shown in formula I prepared by abdominal 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 calculation formula is as follows:
The positive control group of wherein administration group, test group 1 or test group 2.
Test-results 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 H
22the impact that transplanted tumor knurl is heavy
* P<0.05, * * P<0.01, vs blank group
Testing data is as shown in Table 3 known, and compound shown in formula I prepared by the embodiment of the present invention 1 is when dosage is respectively 1.5mg/kg and 3.0mg/kg, and continuous use 10 days, to rat liver cancer H
22the inhibiting rate of transplanted tumor reaches respectively 32.1% and 43%, compared with blank group, and significantly (P<0.05) of tumor killing effect, and with the increase of dosage, tumour inhibiting rate significantly strengthens; Positive controls is when 20mg/kg, to rat liver cancer H
22the inhibiting rate of transplanted tumor is compound and positive controls ratio shown in the formula I prepared of 57.1%, embodiment 1, to rat liver cancer H
22the inhibition of transplanted 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 H
22the growth of transplanted tumor has the significantly restraining effect of (P<0.05), and dose-effect relationship is obvious.
Shown in formula I prepared by table 4 embodiment 1, compound is to mouse S
180the impact that sarcoma knurl is heavy
* P<0.05, * * P<0.01, vs blank group
Testing data is as shown in Table 4 known, and compound shown in formula I prepared by the embodiment of the present invention 1 is when dosage is respectively 1.5mg/kg and 3.0mg/kg, and continuous use 10 days, to mouse S
180the inhibiting rate of sarcoma reaches respectively 30.3% and 55%, compared with blank group, and significantly (P<0.05) of tumor killing effect, and with the increase of dosage, tumour inhibiting rate significantly strengthens; Positive controls is when 20mg/kg, to mouse S
180the inhibiting rate of sarcoma is only that compound shown in the formula I prepared of 37.1%, embodiment 1 is to mouse S
180the inhibition of sarcoma is significantly better than (P<0.05) positive control CTX.
As can be seen here, shown in the formula I that prepared by embodiment 1, compound is to mouse S
180the growth of sarcoma has the significantly restraining effect of (P<0.05), and tumor killing effect is significantly better than positive control CTX, and dose-effect relationship is obvious.
Shown in formula I prepared by embodiment 2～embodiment 4, compound carries out mice-transplanted tumor model trial, and test method is with embodiment 6, and obtained experimental result is similar to embodiment 6, and shown in the formula I that prepared by embodiment 2～embodiment 4, compound is to rat liver cancer H
22the growth of transplanted tumor has the significantly restraining effect of (P<0.05), and dose-effect relationship is obvious; To mouse S
180the growth of sarcoma has the significantly restraining effect of (P<0.05), and tumor killing effect is significantly better than positive control CTX, 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 H
22the growth of transplanted tumor has the significantly restraining effect of (P<0.05), and dose-effect relationship is obvious; To mouse S
180the growth of sarcoma has the significantly restraining effect of (P<0.05), and tumor killing effect is significantly better than positive control CTX, 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, according to ordinary method, makes powder.
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, according to ordinary method, makes tablet.
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 the agent of ordinary method granulation.
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 ordinary method, makes 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 ordinary method, makes 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, according to ordinary method, makes emulsion.
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 suspensoid, according to ordinary method, makes suspensoid.
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 ordinary method, makes 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 ordinary method, makes 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 sprays, according to ordinary method, makes sprays.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (10)
1. the compound suc as formula structure shown in I:
2. an extracting method for compound as claimed in claim 1, is characterized in that, comprises following steps:
Step 1: oil tea root is pulverized, got the first solvent extraction, concentrated, obtain fluid extract;
Step 2: get described fluid extract and mix with water, filter, collect filtrate, centrifugal, collect supernatant liquor, then separate through D101 type macroporous resin column, get ethanol-water system gradient elution, collecting ethanol and water volume ratio is 70:30～95:5 wash-out obtained component, and separation, purifying, obtain;
Described the first solvent is that ethanol and water volume ratio are 0:100～95:5.
3. extracting method according to claim 2, it is characterized in that, described separation, purifying are specially: get described 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; Through ODS post, separate, get methanol-water system gradient elution, collecting methyl alcohol and water volume ratio is 70:30～90:10 wash-out obtained component; Through dynamic axial compression column, separate, get methanol-water system gradient elution, collecting methyl alcohol and water volume ratio is 75:25 wash-out obtained component; Finally by high performance liquid chromatography, separate, adopt C
18chromatographic column, the second solvent elution, flow velocity is 2mL/min, collects 37.5min component, obtains;
Described the second solvent is: first alcohol and water by volume for the mixing solutions of 72:28 composition again with the mixture that accounts for the formic acid mixing gained that described mixing solutions quality percentage composition is 0.2%.
4. extracting method according to claim 2, is characterized in that, the solvent of 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 described in step 2.
5. extracting method according to claim 2, is characterized in that, the temperature of extracting described in step 1 is 30 ℃～100 ℃, and the time of extracting described in step 1 is 8.5h～33h.
6. extracting method according to claim 2, is characterized in that, concentrated temperature is 70 ℃～100 ℃ described in step 1, and the concentrated time is 2.0h～6.0h described in step 1.
One kind as claimed in claim 1 compound in the application of preparing in antitumor drug.
8. application according to claim 7, is characterized in that, described tumour is lung cancer, liver cancer, melanoma or mammary cancer.
9. an antitumor drug, is characterized in that, it comprises compound and pharmaceutically acceptable auxiliary material described in claim 1.
10. antitumor drug according to claim 9, is characterized in that, its formulation is powder, tablet, granule, capsule, solution, emulsion, suspensoid, injection liquid, powder injection or sprays.
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| CN107629104A (en) * | 2017-09-18 | 2018-01-26 | 昆明贝泰妮生物科技有限公司 | Triterpenoid saponin medical compounds and its preparation and application |
| CN113194910A (en) * | 2018-10-01 | 2021-07-30 | 株式会社爱茉莉太平洋 | Cosmetic composition for skin regeneration |
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| CN102548405A (en) * | 2009-07-16 | 2012-07-04 | 太平洋艾瑞有限公司 | Inhibiting the invasion and metastasis of cancer cells |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107629104A (en) * | 2017-09-18 | 2018-01-26 | 昆明贝泰妮生物科技有限公司 | Triterpenoid saponin medical compounds and its preparation and application |
| CN113194910A (en) * | 2018-10-01 | 2021-07-30 | 株式会社爱茉莉太平洋 | Cosmetic composition for skin regeneration |
| CN113194910B (en) * | 2018-10-01 | 2022-12-23 | 株式会社爱茉莉太平洋 | Cosmetic composition for skin regeneration |
| US11564875B2 (en) | 2018-10-01 | 2023-01-31 | Amorepacific Corporation | Cosmetic composition for skin regeneration |
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