CN103739660B - A kind of compound, its extracting method, its application preparing antitumor drug and antitumor drug of preparation thereof - Google Patents
A kind of compound, its extracting method, its application preparing antitumor drug and antitumor drug of preparation thereof Download PDFInfo
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Abstract
The present invention relates to field of pharmaceutical chemistry technology, particularly to a kind of compound, its extracting method, its application preparing antitumor drug and antitumor drug of preparation thereof；The compound that the present invention provides extracts from oil tea root, and Structural Identification result shows, this compound is noval chemical compound, and structure is as shown in formula I；Cell assay in vitro shows, after compound effects 24h shown in formula I, has significantly the inhibitory action of (P < 0.05) to the growth of typeⅡ pneumocyte, B16 mouse black-in tumor cell, people's hepatocarcinoma BEL 7402 cell and human breast cancer cell MCF 7 cell；Mice-transplanted tumor model test shows, this compound is to rat liver cancer H22Transplanted tumor and mice S180The growth of sarcoma has significantly the inhibitory action of (P < 0.05), and dose-effect relationship is obvious.
Description
Technical field
The present invention relates to field of pharmaceutical chemistry technology, particularly to a kind of compound, its extracting method, its
Prepare application and the antitumor drug of preparation thereof of antitumor drug.
Background technology
Oil tea (CameLLia oLeifera AbeL) belongs to Theaceae (Theaceae) Camellia (CameLLia
Linn), it is a kind of evergreen dungarunga.Because of its seed can extract oil (Oleum Camelliae) edible, therefore named oil tea.Oil
Tea is distributed mainly on the torrid zone and subtropical zone, originates in the west and south and the southeast of China, spreads all over 17 provinces
District.The unsaturated fatty acid content of Oleum Camelliae is up to 90%, significantly larger than vegetable oil, Oleum Arachidis hypogaeae semen and Oleum Glycines, with
Olive oil doubles than content of vitamin E, has high nutritive value, and therefore Oleum Camelliae receives much concern.
Additionally, oil tea also has the highest medical value, described in Compendium of Material Medica: " camellia oleosa seed, bitter cold is fragrant
Poison (Saponin), cures mainly dyspnea with rapid respiration cough, removes disease dirt "；" China book on Chinese herbal medicine " is the most on the books, the root of oil tea and
Root bark has effect of heat-clearing and toxic substances removing, regulating QI to relieve pain, promoting blood circulation and detumescence, cures mainly laryngopharynx swelling and pain, stomachache, tooth
Bitterly, traumatic pain, burn due to hot liquid or fire, its curative effect is considerably beyond Oleum Camelliae.But the medical value of oil tea is not affected by
Enough attention.
In recent years, chemical composition and the biological activity of oil tea are studied by people, at present, from
Isolated saponins, flavonoid, fatty acid in the Semen Camelliae of oil tea, Oleum Camelliae, Residuum Camelliae Semen preparatum, Camellia Leaves
Class, tannin, the fragrance compound such as glycoside, alkaloid.Research finds, part saponins compound has
The intestines and stomach protection, blood fat reducing, fat-reducing, antiallergic or antineoplastic effect.Ma Liyuan etc. find, oil tea tea
In sub-cake, the Total oil tea saponins of more than 95% has significant anti-tumor activity.Therefore, Sasanguasaponin has
The highest medical value.Sasanguasaponin mainly extracts from oil tea, but oil tea research is focused mostly on by people before
In Semen Camelliae, Residuum Camelliae Semen preparatum, Camellia Leaves, Oleum Camelliae, less to the research of oil tea root, oil tea root may be deposited
At undiscovered tool bioactive Sasanguasaponin compounds, therefore, to saponin constituent in oil tea root
Research have great importance.
Summary of the invention
In view of this, the invention provides a kind of compound, its extracting method, its prepare antitumor drug
Application and preparation antitumor drug.The present invention is tested by cell assay in vitro and animal-transplanted tumor
Finding, the growth of the cancerous cell multiple to human body of compound shown in formula I has significant inhibitory action.
In order to realize foregoing invention purpose, the present invention provides techniques below scheme:
The invention provides a kind of compound of structure as shown in formula I:
Present invention also offers a kind of extracting method of the compound of structure as shown in formula I, comprise following step
Rapid:
Step 1: pulverized by oil tea root, take the first solvent extraction, concentrates, obtains fluid extract；
Step 2: take fluid extract and mix with water, filters, and collects filtrate, centrifugal, collects supernatant, then
Separate through D101 type macroporous resin column, take ethanol-water system gradient elution, collect ethanol and water volume ratio
For 70:30～95:5 eluting obtained component, separation, purification, to obtain final product；
First solvent is ethanol and water volume ratio is 0:100～95:5.
As preferably, the mesh number that oil tea root is pulverized is 10 mesh～60 mesh.
As preferably, the first solvent is ethanol and water volume ratio is 20:80～80:20.
It is furthermore preferred that the first solvent is ethanol and water volume ratio is 70:30.
As preferably, step 2 takes in fluid extract and water blend step, and fluid extract is 1 with the volume ratio of water:
1～10, the amount of water used reaches the purpose dissolved by fluid extract.
Preferably, the mesh number filtering filter cloth used is 100～300 mesh in effect.
As preferably, separation, the technology of purification are appointing in precipitating technology, crystallization technique or chromatographic technique
Anticipate a kind of or that both are above technology, but be not limited to above technology.
As preferably, separation, the technology of purification are chromatographic technique.
As preferably, separation, purification, particularly as follows: take component and separate through silicagel column, take chloroform-methanol system
Gradient elution, collects chloroform and methanol volume ratio is 80:20～60:40 eluting obtained component；Warp again
ODS post separates, and takes methanol-water system gradient elution, collects methanol and water volume ratio is 70:30～90:
10 eluting obtained components；Separate through dynamic axial compression column again, take methanol-water system gradient elution, receive
Integrate methanol and water volume ratio as 75:25 eluting obtained component；Separate finally by high performance liquid chromatography, adopt
Use C18Chromatographic column, the second solvent eluting, flow velocity is 2mL/min, collects 37.5min component, to obtain final product；
Second solvent is: first alcohol and water by volume for 72:28 composition mixed solution again with account for mix molten
Liquid weight/mass percentage composition is the mixture of the formic acid mixing gained of 0.2%.
C18The specification of chromatographic column is 250mm × 10mm, and filler granularity is 5 μm.
As preferably, silicagel column is decompression silicagel column.
As preferably, in silicagel column, the mesh number of silica gel is 60～100 mesh.
As preferably, the solvent of chloroform-methanol system gradient elution is followed successively by chloroform and methanol volume ratio is
90:10 → 80:20 → 70:30 → 60:40 → 50:50 → 35:65 → 20:80 → 0:100.
As preferably, ODS post is middle pressure anti-phase ODS post.
As preferably, the specification of middle pressure anti-phase ODS post is: 460mm × 26mm.
As preferably, the solvent of ODS post gradient elution is methanol and water volume ratio is 50:50～100:0.
As preferably, the solvent of ODS post gradient elution is methanol and water volume ratio is 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 μm, 1500g；
NewstyLe。
As preferably, the solvent of dynamic axial compression column gradient elution is followed successively by methanol 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 extracted described in step 1 is 30 DEG C～100 DEG C, extract described in step 1 time
Between be 8.5h～33h.
As preferably, the temperature that step 1 is extracted is 50 DEG C～85 DEG C, and the time that step 1 is extracted is
1.0h～2.5h.
As preferably, the temperature that step 1 is extracted is 70 DEG C～85 DEG C, the time that step 1 is extracted be 2h～
2.5h。
As preferably, extraction includes immersion, reflux, extract, filters and collect filtrate step.
As preferably, in soaking step, in terms of g/mL, oil tea root and the mass volume ratio of the first solvent
For 1:3～20.
It is furthermore preferred that in soaking step, in terms of g/mL, oil tea root and the mass volume ratio of the first solvent
For 1:5～15.
It is furthermore preferred that in soaking step, in terms of g/mL, oil tea root and the mass volume ratio of the first solvent
For 1:10.
During step 1 is extracted, as long as the first solvent excess all can reach close effect, all in the present invention
Protection domain in.
As preferably, the time of immersion is 8h～24h, and the temperature of immersion is 30 DEG C～80 DEG C.
As preferably, the temperature of reflux, extract, is 70 DEG C～100 DEG C.
It is furthermore preferred that the temperature of reflux, extract, is 80 DEG C.
As preferably, the time of reflux, extract, is 0.5h～3h.
It is furthermore preferred that the time of reflux, extract, is 2.0h.
As preferably, the number of times of reflux, extract, is 1～3 time.
It is furthermore preferred that the number of times of reflux, extract, is 2 times.
Filtration step can be carried out after each reflux, extract, it is also possible to carries out after reflux, extract, 2～3 times,
Collect whole filtrate, be extracting solution.
As preferably, filtration step is carried out after each reflux, extract, filters gained filtering residue molten with first again
Agent mixes, and carries out reflux, extract, next time.
As preferably, in terms of g/mL, the mass volume ratio of filtering residue and the first solvent is 1:3～20.
As preferably, the temperature that step 1 concentrates is 70 DEG C～100 DEG C, and the time that step 1 concentrates is 2.0
H～6.0h.
As preferably, the temperature that step 1 concentrates is 80 DEG C～100 DEG C, the time that step 1 concentrates be 3h～
5h。
As preferably, concentrate as concentrating under reduced pressure.
As preferably, the multiple of concentration is 6～10 times.
Present invention also offers the application in preparing antitumor drug of the compound of structure shown in formula I.This
Invention first passes through mtt assay and measures the inhibitory action of drug on tumor cell strain.Mtt assay, also known as
MTT colorimetry, is a kind of method detecting cell survival and growth.Its Cleaning Principle is living cells line grain
Succinate dehydrogenase in body can make exogenous MTT be reduced to water-insoluble bluish violet crystallization first a ceremonial jade-ladle, used in libation
(Formazan) and be deposited in cell, and dead cell is without this function.Dimethyl sulfoxide (DMSO)
The first a ceremonial jade-ladle, used in libation in cell can be dissolved, at 490nm wavelength, measure its absorbance value with enzyme-linked immunosorbent assay instrument,
Can indirectly reflect living cells quantity.In the range of certain cell number, the amount of MTT crystallization formation and cell number
It is directly proportional.The method is widely used in the Activity determination of some bioactie agents, large-scale antitumor
Drug screening, cell toxicity test and tumor radiosensitivity mensuration etc..Its feature be highly sensitive,
Economical.Therefore, this test uses mtt assay to screen test medicine.This research is by people's pulmonary carcinoma A549
Cell, B16 mouse black-in tumor cell, human hepatocellular carcinoma BEL-7402 cell and human breast cancer cell line Bcap-37,
As subject cell, and the action time of test medicine is set to 24h.Result of the test shows, the present invention
After compound effects 24h shown in the formula I provided, to typeⅡ pneumocyte, B16 mouse black-in lymphoma
Cell, human hepatocellular carcinoma BEL-7402 cell and human breast cancer cell line Bcap-37 cell IC50Value is respectively less than
20 μ g/mL, it can be seen that, the growth of above-mentioned 4 kinds of tumor cells is had significantly by compound shown in formula I
Inhibitory action.
The present invention tests the tumor killing effect of this compound also by mice-transplanted tumor model test, and result shows,
Compound shown in formula I is to rat liver cancer H22Transplanted tumor and mice S180The growth of sarcoma has significantly
The inhibitory action of (P < 0.05), dose-effect relationship is obvious, wherein to mice S180The suppression of growing sarcoma is made
With being significantly better than positive control CTX, therefore, the invention provides the compound of structure shown in formula I in system
Application in standby antitumor drug.
As preferably, tumor is pulmonary carcinoma, hepatocarcinoma, melanoma or breast carcinoma.Tumor can also be the present invention
The openest, but have the other kinds of tumor of inhibition.
Present invention also offers a kind of antitumor drug, its compound comprising structure shown in formula I and pharmacy
Upper acceptable adjuvant.
The dosage form of antitumor drug of the present invention can be the attainable any dosage form in this area, and adjuvant used is
The customary adjuvant of dosage form used, preparation method is the customary preparation methods of corresponding dosage form.
As preferably, the dosage form of antitumor drug be powder, tablet, granule, capsule, solution,
Emulsion, suspensoid, injection, injectable powder or spray.
The invention provides a kind of compound of structure as shown in formula I, this compound is extracted by oil tea root
Obtaining, Structural Identification result shows, this compound is noval chemical compound, named: 21 β, and 22 α-O-two work as
Return acyl group-15 α, 16 α, 28-trihydroxy olive-12-alkene-23-aldehyde 3 β-O-β-D-xylose-(1 → 2)-β-D-half
Lactose-(1 → 3)-[β-D-galactose-(1 → 2)]-β-D-Glucose aldehydic acid glycosides.The present invention uses mtt assay to enter
Row cell assay in vitro, result shows, after compound effects 24h shown in the formula I that the present invention provides, right
TypeⅡ pneumocyte, B16 mouse black-in tumor cell, human hepatocellular carcinoma BEL-7402 cell and human breast carcinoma
The IC of cell MCF-7 cell50Value is respectively less than 20 μ g/mL, and to typeⅡ pneumocyte, people's hepatocarcinoma
BEL-7402 cell, that the inhibitory action of growth of cell is significantly better than (P < 0.05) is positive
Comparison 5-FU.As can be seen here, the growth of above-mentioned 4 kinds of tumor cells is had significantly by compound shown in formula I
Inhibitory action.The present invention tests the tumor killing effect of this compound also by mice-transplanted tumor model test,
Test result shows, compound shown in formula I is to rat liver cancer H22Transplanted tumor and the life of mouse S 180 sarcoma
The long inhibitory action with notable (P < 0.05), dose-effect relationship is obvious, wherein to mouse S 180 sarcoma
The inhibitory action of growth is significantly better than positive control CTX, therefore, chemical combination shown in the formula I that the present invention provides
Thing can be additionally used in prepares antitumor drug.
Accompanying drawing explanation
Fig. 1 is 21 β, 22 α-O-two angeloyl groups-15 α, 16 α, 28-trihydroxy olive-12-alkene-
23-aldehyde 3 β-O-β-D-xylose-(1 → 2)-β-D-galactose-(1 → 3)-[β-D-galactose-(1 →
2)] the high resolution mass spectrum spectrogram of-β-D-Glucose aldehydic acid glycosides；
Fig. 2 is 21 β, 22 α-O-two angeloyl groups-15 α, 16 α, 28-trihydroxy olive-12-alkene-
23-aldehyde 3 β-O-β-D-xylose-(1 → 2)-β-D-galactose-(1 → 3)-[β-D-galactose-(1 →
2)] the hydrogen nuclear magnetic resonance spectrogram of-β-D-Glucose aldehydic acid glycosides；
Fig. 3 is 21 β, 22 α-O-two angeloyl groups-15 α, 16 α, 28-trihydroxy olive-12-alkene-
23-aldehyde 3 β-O-β-D-xylose-(1 → 2)-β-D-galactose-(1 → 3)-[β-D-galactose-(1 →
2)] the hydrogen nuclear magnetic resonance spectrogram (enlarged drawing) of-β-D-Glucose aldehydic acid glycosides；
Fig. 4 is 21 β, 22 α-O-two angeloyl groups-15 α, 16 α, 28-trihydroxy olive-12-alkene-
23-aldehyde 3 β-O-β-D-xylose-(1 → 2)-β-D-galactose-(1 → 3)-[β-D-galactose-(1 →
2)] the carbon-13 nmr spectra figure of-β-D-Glucose aldehydic acid glycosides；
Fig. 5 is 21 β, 22 α-O-two angeloyl groups-15 α, 16 α, 28-trihydroxy olive-12-alkene-
23-aldehyde 3 β-O-β-D-xylose-(1 → 2)-β-D-galactose-(1 → 3)-[β-D-galactose-(1 →
2)] the carbon-13 nmr spectra figure (enlarged drawing) of-β-D-Glucose aldehydic acid glycosides；
Fig. 6 is 21 β, 22 α-O-two angeloyl groups-15 α, 16 α, 28-trihydroxy olive-12-alkene-
23-aldehyde 3 β-O-β-D-xylose-(1 → 2)-β-D-galactose-(1 → 3)-[β-D-galactose-(1 →
2)] the carbon-13 nmr spectra figure (enlarged drawing) of-β-D-Glucose aldehydic acid glycosides；
Fig. 7 is 21 β, 22 α-O-two angeloyl groups-15 α, 16 α, 28-trihydroxy olive-12-alkene
-23-aldehyde 3 β-O-β-D-xylose-(1 → 2)-β-D-galactose-(1 → 3)-[β-D-galactose-(1 →
2)] the HSQC figure of-β-D-Glucose aldehydic acid glycosides；
Fig. 8 is 21 β, 22 α-O-two angeloyl groups-15 α, 16 α, 28-trihydroxy olive-12-alkene-
23-aldehyde 3 β-O-β-D-xylose-(1 → 2)-β-D-galactose-(1 → 3)-[β-D-galactose-(1 →
2)] the HMBC figure of-β-D-Glucose aldehydic acid glycosides；
Fig. 9 is 21 β, 22 α-O-two angeloyl groups-15 α, 16 α, 28-trihydroxy olive-12-alkene-
23-aldehyde 3 β-O-β-D-xylose-(1 → 2)-β-D-galactose-(1 → 3)-[β-D-galactose-(1 →
2)]-β-D-Glucose aldehydic acid glycosides1H-1H COSY schemes；
Figure 10 is 21 β, 22 α-O-two angeloyl groups-15 α, 16 α, 28-trihydroxy olive-12-alkene-
23-aldehyde 3 β-O-β-D-xylose-(1 → 2)-β-D-galactose-(1 → 3)-[β-D-galactose-(1 →
2)] the NOESY figure of-β-D-Glucose aldehydic acid glycosides.
Detailed description of the invention
The invention provides a kind of compound, its extracting method, its prepare antitumor drug application and
The antitumor drug of preparation.Those skilled in the art can use for reference present disclosure, is suitably modified technological parameter
Realize.Special needs to be pointed out is, all similar replacements and change are for a person skilled in the art
It will be apparent that they are considered as being included in the present invention.Method and the application of the present invention have been passed through relatively
Good embodiment is described, and related personnel substantially can be without departing from present invention, spirit and scope
Method described herein and application it is modified or suitably changes and combine, realize and apply the present invention
Technology.
The material used in the embodiment of the present invention and reagent all can be buied by market, 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
Micromass company of Q-TOF2(Britain)；(prunus mume (sieb.) sieb.et zucc. Teller-torr benefit instrument (Shanghai) has electronic balance
Limit company)；Polariscope 241 (PerkinElmer Inc., Waltham, MA, the U.S.)；Partly prepare high-efficient liquid
Chromatography (LC 20AT, SPD 20A, Shimadzu Corporation of Japan)；C18 half preparative hplc post (250
Mm × 10mm, 5 μm, Kromsil company of the U.S.)；Middle pressure preparative liquid chromatography (B ü chi chromatograph system
System, C-650 pump, middle compression leg (460mm × 26mm i.d., B ü chi Corp., Flawil, Swiss);Dynamic
State axial columns chromatograph (NP7000 pump (Newstyle), NU3000UV-VIS detector (Newstyle) and
ODS post (650mm × 100mm, 30 μm, 1500g;Newstyle)；Rotary Evaporators (Tokyo physics and chemistry
Apparatus individual proprietorship factory)；Chemical reagent (analytical pure, Chemical Reagent Co., Ltd., Sinopharm Group)；Thin layer color
Spectrum silica gel plate (HSGF254, Yantai City's Zhifu Huang business silica gel development experiments factory produces)；Various column chromatographies
It is Haiyang Chemical Plant, Qingdao with silica gel to produce.
TypeⅡ pneumocyte, B16 mouse black-in tumor cell, human hepatocellular carcinoma BEL-7402 cell and human milk
Adenocarcinoma cell MCF-7, is 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 mice: cleaning grade, male, body weight 18-22g, by the Shanghai limited public affairs of Si Laike laboratory animal
Department provides.
Mice: kind is ICR, Shanghai Slac Experimental Animal Co., Ltd. buy, laboratory animal is raw
Produce credit number SCXK (Shanghai) 2007-0005.
Complete medium: formula is the RPMI1640 culture medium containing 10% inactivated fetal bovine serum, produces
Producer is GIBCOBRL company of the U.S..
Remaining reagent and material are buied by market.
Below in conjunction with embodiment, the present invention it is expanded on further:
The extraction of embodiment 1 compound
Take dry oil tea root 2kg, be ground into the wood flour of 10-40 mesh with pulverizer, through the leaching of 40 DEG C of 6L water
After bubble 8h, being heated to reflux 0.5h at 100 DEG C, filter, filtering residue adds 3L water, at 100 DEG C
Being heated to reflux 3.0h, filter, filtering residue mixes with 3L water again, is heated to reflux 2h at 100 DEG C, filters,
Merge three times and filter gained filtrate, concentrating under reduced pressure 6h at 80 DEG C, obtain fluid extract 2000mL.
Take prepared fluid extract to mix with 20000mL water, 100 mesh filter-cloth filterings, centrifugal filtrate, take
Clear liquid through D101 type macroporous resin column separate, successively with water, 30% ethanol, 50% ethanol, 60% ethanol,
70% ethanol, 80% ethanol, 95% ethanol elution, collect 70% ethanol～the group of 95% ethanol elution gained
Point, concentrating under reduced pressure, obtain Total oil tea saponins.
Take the prepared Total oil tea saponins decompression silicagel column first through 60 mesh silica gel to separate, successively by chloroform and first
Alcohol volume ratio is 90:10 → 80:20 → 70:30 → 60:40 → 50:50 → 40:60 → 20:
80 → 0:100 gradient elution, obtains component 1～component 8, through lamellae analysis, merges chlorine therein
Imitative and methanol volume ratio 80:20～60:40 eluting obtained component (when developing solvent system is BAW system,
Rf value is 0.4-0.7), then warp presses anti-phase ODS post to separate, methanol-water system 50%-100% gradient is washed
De-, flow velocity 25mL/min, detection wavelength is 203nm, obtains 6 components, through efficient analysis liquid
Facies analysis, is 60:40～90:10 gradient elution 30min with methanol and water volume ratio, merges methanol and water body
The long-pending ratio component (peak is positioned at 20min-28min) for 70:30～90:10, separates through dynamic axial compression column,
Respectively with 60%, 75%, 80% and 90% methanol-eluted fractions, flow velocity 150mL/min, detect wavelength
203nm, there are five components, through efficient analysis liquid phase analysis, by 75% methanol-eluted fractions obtained component
(peak is positioned at 13.5-15.0min) is separated by partly preparing high-efficient liquid, methanol-water (72:28, v/v)-0.2%
Formic acid eluting eluting, sets flow velocity as 2mL/min, detects wavelength 203nm, obtains at 37.5min
White amorphous powder 150mg.
Take prepared white amorphous powder, detect development properties, hydrolysis properties, then warp1H-NMR,13C-NMR, HMBC, HSQC,1H-1H COSY, NOESY and HR-ESI-MS carry out wave spectrum
Resolving, spectral data figure is as shown in Fig. 1～Figure 10, and NMR data is as shown in table 1, structure elucidation process
As follows: through chromogenic assay, sulphuric acid ethanol displaing amaranth speckle, acetic anhydride-strong sulfuric acid response is positive, MoLish
Reacting positive, pointing out this compound may be saponins compound.Mass spectrum shown in Fig. 1 shows, this change
The quasi-quasi-molecular ions of the HR-ESI-MS of compound is m/z1315.5968 [M-H]-, show the molecule of this compound
Formula is C63H96O29(value of calculation [M-H]-, m/z1315.5959, value of calculation is according to molecular formula, by
High abundance exact value calculates).Obtaining monosaccharide with the complete acid hydrolysis of 2N TFA, sugar is laggard through derivatization
Row GC analyzes, and D-Glucose aldehydic acid, D-galactose, the existence of D-xylose detected.
By the compound of Fig. 4, Fig. 5, Fig. 613C-NMR spectrogram understands, and this compound shows altogether
63 carbon signals (as shown in table 1), the hsqc spectrum of the compound shown in Fig. 7 understands, this compound
Contain 10 methyl carbon, 10 mesomethylene carbons, 31 methine carbons and 12 quaternary carbons altogether.Enter one
Step is analyzed13C-NMR composes, and this spectrogram demonstrates four sugared end group carbon signal δ 104.3,101.8,107.9 Hes
103.1, it is D-Glucose aldehydic acid, D-galactose, D-xylose and another one D-galactose respectively
End group carbon.Additionally, an also aldehyde radical carbon signal being positioned at low place δ 210.3.By Fig. 2, Fig. 3
The hydrogen spectrogram of shown compound understands,1In H-NMR spectrum, high field region has six obvious triterpene saponin angles
Methyl 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)；One company Oxymethylene δ 3.57 (H-28, d, J=11.0Hz) and 3.82
(H-28,d,J=11.0Hz)；Five companies oxygen methine δ 4.09 (1H, dd, J=11.0,4.5Hz, H-3),
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)；One alkene Hydrogen Proton δ 5.58 (1H, brs, H-12) and an aldehyde
Base Hydrogen Proton δ 9.94 (1H, brs, H-23).Additionally, compound1H-NMR spectrum also demonstrates that two groups are worked as
Return acyl group signal δ [6.07 (1H, dq, J=7.5,1.5Hz, 21-O-Ang-3'), 2.18 (3H, d, J=7.5
Hz, 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 91H-1H COSY spectrum understands, the methine adjacent with even oxygen methine protons
δH4.26 (1H, brs) and δH4.54 (1H, brs) are correlated with, and illustrate that they are in ortho position and δH4.54 be H-16
Signal, δH4.26 is H-15 signal；δH6.78 (1H, d, J=10.5Hz) and δH6.41(1H,d,J
=10.5Hz) between coherent signal explanation they be in ortho position and δH6.78 is H-21 signal, δH6.41 it is
H-22 signal.
Two angeloyl groups being connected in respectively are learnt by the HMBC spectrum analysis of the compound shown in Fig. 8
The 21 of compound, on 22, because δ in HMBCH6.78 (1H, d, J=10.5Hz, H-21) and δC
168.0 (21-O-Ang-1') are correlated with, δH6.41 (1H, d, J=10.5Hz, H-22) and δC168.3(22-O-
Ang-1'') relevant.In conjunction with13C-NMR spectrum and1H-NMR modal data is comprehensively analyzed, and with literary composition
Offer the nuclear magnetic data contrast of the known aglycon that " Helvetica Chimica Acta2013.Vol.96 " reports,
The aglycone structure determining this compound is 21 β, 22 α-O-two angeloyl groups-15 α, the neat pier of 16 α, 28-trihydroxy
Really-12-alkene-23-aldehyde.
Compound hsqc spectrum figure as shown in Figure 7 is analyzed, belongs to the sugar of connection on this saponin
End group carbon, hydrogen signal, result is as follows: δH4.80(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), are connected in δ respectivelyC104.3 (glucal
Acid-C-1), 101.8 (galactose-C-1), 107.9 (xylose-C-1), on 103.1 (galactose '-C-1).Additionally should
Compound aglycon 3 is positioned at 12.6ppm and shows that sugar chain is connected on aglycon 3 to low field, and
In HMBC spectrum, glucuronic acid anomeric proton δH4.80 and 3 δ of aglyconC84.7 are correlated with, and enter one
Step confirms that sugar chain is connected in 3 of aglycon.Consulting literatures finds this compound sugar chain and document further
Report in " Bioorganic&medicinal chemistry letters2010,20,7435-7439 "
Camellenodiol sugar chain data are close, thus infer that this compound sugar chain is β-D-xylose
-β-D-galactose-(1 → 3) ,-(1 → 2)-[β-D-galactose-(1 → 2)]-β-D-Glucose aldehydic acid.The company of this sugar chain
Connect order to confirm by analyzing HMBC spectrum, glucuronic acid anomeric proton δ in HMBCH4.80
With 3 carbon δ of aglyconC84.7 are correlated with, galactose anomeric proton δH5.80 with 3 carbon of glucuronic acid
δC84.9 are correlated with, xylose anomeric proton δH5.09 and 2 carbon δ of galactoseC84.1 are correlated with, galactose
2 hydrogen δH4.57 and xylose end group carbon δC107.9 are correlated with, another galactose anomeric proton δH
5.95 and 2 carbon δ of glucuronic acidC78.2 are correlated with, 2 hydrogen δ of glucuronic acidH4.57 with this gala
Sugar end group carbon δC103.1 it is relevant.Additionally, four sugared anomeric proton coupling constants3JH-1,H-2It is relatively big,
Show that these four sugar are all beta configuration.
NOESY spectrum display shown in Figure 10, δH6.78 (1H, d, J=10.5Hz, H-21) and δH1.18
(3H, s, H-29) is correlated with, and prompting H-21 is α configuration；δH4.09(1H,dd,J=4.5,11.0Hz,H-3)
With δH9.94 (1H, brs, H-23) are correlated with, and prompting H-3 is α configuration；δH4.54 (1H, brs, H-16) with
δH3.57 (1H, d, J=11.0Hz, H-28), 3.82 (1H, d, J=11.0Hz, H-28) relevant prompting prompting
H-16 is beta comfiguration, δH6.41 (1H, d, J=10.5Hz, H-22) and δH1.41 (3H, s, H-30) are correlated with,
Prompting H-22 is beta comfiguration.
Therefore, named 21 β of this compound structure, 22 α-O-two angeloyl groups-15 α, 16 α, 28-trihydroxy
Olive-12-alkene-23-aldehyde 3 β-O-β-D-xylose-(1 → 2)-β-D-galactose-(1 → 3)-[β-D-galactose
-(1 → 2)]-β-D-Glucose aldehydic acid glycosides.Compound structure is as shown in formula I:
NMR (pyridine-d5,500MHz) data of compound shown in table 1 formula I
In table, chemical shift is in units of ppm, and coupling constant (J) is in units of Hz.
The extraction of embodiment 2 compound
Take dry oil tea root 2kg, be ground into the wood flour of 10 mesh with pulverizer, through 95% ethanol of 40L
After 40 DEG C are soaked 24h, being heated to reflux 0.5h at 80 DEG C, filter, filtering residue adds 95% ethanol 2L,
It is heated to reflux 3.0h at 80 DEG C, filters, merge twice and filter gained filtrate, concentrating under reduced pressure at 90 DEG C
2h, obtains fluid extract 4200mL.
Take prepared fluid extract to mix with 4200mL water, 200 mesh filter-cloth filterings, centrifugal filtrate, take supernatant
Liquid through D101 type macroporous resin column separate, successively with water, 30% ethanol, 50% ethanol, 60% ethanol,
70% ethanol, 80% ethanol, 95% ethanol elution, collect 70% ethanol～the group of 95% ethanol elution gained
Point, concentrating under reduced pressure, obtain Total oil tea saponins.
Take the prepared Total oil tea saponins decompression silicagel column first through 100 mesh silica gel to separate, successively with chloroform and
Methanol volume ratio is 90:10 → 80:20 → 70:30 → 60:40 → 50:50 → 40:60 → 20:
80 → 0:100 gradient elution, obtains component 1～component 8, through lamellae analysis, merges chlorine therein
Imitative and methanol volume ratio 80:20～60:40 eluting obtained component (when developing solvent system is BAW system,
Rf value is 0.4-0.7), then warp presses anti-phase ODS post to separate, methanol-water system 50%-100% gradient is washed
De-, flow velocity 25mL/min, detection wavelength is 203nm, obtains 6 components, through efficient analysis liquid
Facies analysis, is 60:40～90:10 gradient elution 30min with methanol and water volume ratio, merges methanol and water body
The long-pending ratio component (peak is positioned at 20min-28min) for 70:30～90:10, separates through dynamic axial compression column,
Respectively with 60%, 75%, 80% and 90% methanol-eluted fractions, flow velocity 150mL/min, detect wavelength
203nm, there are five components, through efficient analysis liquid phase analysis, by 75% methanol-eluted fractions obtained component
(peak is positioned at 13.5-15.0min) is separated by partly preparing high-efficient liquid, methanol-water (72:28, v/v)-0.2%
Formic acid eluting eluting, sets flow velocity as 2mL/min, detects wavelength 203nm, obtains when 37.5min
White amorphous powder 180mg, detects through proton nmr spectra, the data obtained and embodiment 1 gained
The hydrogen modal data of compound is consistent, accordingly, it is determined that this compound is 21 β, and 22 α-O-two angeloyl groups-15 α, 16 α,
28-trihydroxy olive-12-alkene-23-aldehyde 3 β-O-β-D-xylose-(1 → 2)-β-D-galactose-(1 → 3)-[β-D-
Galactose-(1 → 2)]-β-D-Glucose aldehydic acid glycosides, compound structure is as shown in formula I.
The extraction of embodiment 3 compound
Take dry oil tea root 2kg, be ground into the wood flour of 10-60 mesh with pulverizer, through the 70% of 20L
After ethanol 80 DEG C soaks 8h, at 85 DEG C, it is heated to reflux 3h, filters, collect filtrate, at 70 DEG C
Concentrating under reduced pressure 6h, obtains fluid extract 2000mL.
Take prepared fluid extract to mix with 6000mL water, 300 mesh filter-cloth filterings, centrifugal filtrate, take supernatant
Liquid through D101 type macroporous resin column separate, successively with water, 30% ethanol, 50% ethanol, 60% ethanol,
70% ethanol, 80% ethanol, 95% ethanol elution, collect 70% ethanol～the group of 95% ethanol elution gained
Point, concentrating under reduced pressure, obtain Total oil tea saponins.
Take the prepared Total oil tea saponins decompression silicagel column first through 80 mesh silica gel to separate, successively by chloroform and first
Alcohol volume ratio is 90:10 → 80:20 → 70:30 → 60:40 → 50:50 → 35:65 → 20:
80 → 0:100 gradient elution, obtains component 1～component 8, through lamellae analysis, merges chloroform therein
With methanol volume ratio 80:20～60:40 eluting obtained component (when developing solvent system is BAW system, Rf
Value is 0.4-0.7), then warp presses anti-phase ODS post to separate, methanol-water system 50%-100% gradient elution,
Flow velocity 25mL/min, detection wavelength is 203nm, obtains 6 components, divides through efficient analysis liquid phase
Analysis, is 60:40～90:10 gradient elution 30min with methanol and water volume ratio, merges methanol and water volume ratio
For the component (peak is positioned at 20min-28min) of 70:30～90:10, separate through dynamic axial compression column, point
Not with 60%, 75%, 80% and 90% methanol-eluted fractions, flow velocity 150mL/min, detect wavelength 203
Nm, there are five components, through efficient analysis liquid phase analysis, by 75% methanol-eluted fractions obtained component (peak
It is positioned at 13.5-15.0min) separated by partly preparing high-efficient liquid, methanol-water (72:28, v/v)-0.2% first
Pickling takes off eluting, sets flow velocity as 2mL/min, detects wavelength 203nm, obtains white when 37.5min
Color amorphous powder 160mg, detects through proton nmr spectra, the data obtained and embodiment 1 gained
The hydrogen modal data of compound is consistent, accordingly, it is determined that this compound is 21 β, and 22 α-O-two angeloyl groups-15 α, 16 α,
28-trihydroxy olive-12-alkene-23-aldehyde 3 β-O-β-D-xylose-(1 → 2)-β-D-galactose-(1 → 3)-[β-D-
Galactose-(1 → 2)]-β-D-Glucose aldehydic acid glycosides, compound structure is as shown in formula I.
The extraction of embodiment 4 compound
Take dry oil tea root 2kg, be ground into the wood flour of 10-40 mesh with pulverizer, through the 80% of 20L
After ethanol 30 DEG C soaks 8h, at 75 DEG C, it is heated to reflux 0.5h, at 80 DEG C, is heated to reflux 3.0h,
Being heated to reflux 2h at 80 DEG C, filter, collect filtrate, at 100 DEG C, concentrating under reduced pressure 3h, is flowed
Extractum 2000mL.
Take prepared fluid extract to mix with 6000mL water, 250 mesh filter-cloth filterings, centrifugal filtrate, take supernatant
Liquid through D101 type macroporous resin column separate, successively with water, 30% ethanol, 50% ethanol, 60% ethanol,
70% ethanol, 80% ethanol, 95% ethanol elution, collect 70% ethanol～the group of 95% ethanol elution gained
Point, concentrating under reduced pressure, obtain Total oil tea saponins.
Take the prepared Total oil tea saponins decompression silicagel column first through 60 mesh silica gel to separate, successively by chloroform and first
Alcohol volume ratio is 90:10 → 80:20 → 70:30 → 60:40 → 50:50 → 40:60 → 20:
80 → 0:100 gradient elution, obtains component 1～component 8, through lamellae analysis, merges chlorine therein
Imitative and methanol volume ratio 80:20～60:40 eluting obtained component (when developing solvent system is BAW system,
Rf value is 0.4-0.7), then warp presses 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 203
Nm, obtains 6 components, through efficient analysis liquid phase analysis, is 60:40～90:10 with methanol and water volume ratio
Gradient elution 30min, (peak is positioned to merge methanol and component that water volume ratio is 70:30～90:10
20min-28min), separate through dynamic axial compression column, respectively with 60%, 75%, 80% and 90%
Methanol-eluted fractions, flow velocity 150mL/min, detects wavelength 203nm, there are five components, through efficiently dividing
Analysis liquid phase analysis, by 75% methanol-eluted fractions obtained component (peak is positioned at 13.5-15.0min) by half preparation
High-efficient liquid is separated, methanol-water (72:28, v/v)-0.2% formic acid eluting eluting, sets flow velocity as 2mL/min,
Detection wavelength 203nm, obtains white amorphous powder 230mg, through nuclear magnetic resonance, NMR when 37.5min
Hydrogen spectrum detection, the data obtained is consistent with the hydrogen modal data of embodiment 1 gained compound, accordingly, it is determined that this
Compound is 21 β, 22 α-O-two angeloyl groups-15 α, 16 α, 28-trihydroxy olive-12-alkene-23-aldehyde
3 β-O-β-D-xyloses-(1 → 2)-β-D-galactose-(1 → 3)-[β-D-galactose-(1 → 2)]-β-D-Glucose aldehyde
Acid glycosides, compound structure is as shown in formula I.
Embodiment 5 cell assay in vitro
Take typeⅡ pneumocyte, B16 mouse black-in tumor cell, human hepatocellular carcinoma BEL-7402 cell and people
Breast cancer cell MCF-7, (separately adds GLu by the RPMI1640 culture medium containing 10% inactivation NBS respectively
0.03%, Hepes0.06%, NaHCO30.2%) in 37 DEG C, 5%CO2Under the conditions of cultivate, 3 days pass
In generation, trophophase cell of taking the logarithm is tested.
Regulate the concentration of the cell of the logarithm production period prepared with complete medium, make cell concentration be 5
×104Individual/mL, is inoculated in 96 well culture plates, and 100 μ L/ holes, at 37 DEG C, 5%CO2Under the conditions of
Cultivate after 24h, be divided into following groups:
Test group add embodiment 1 preparation formula I shown in compound, solubilizer 1%DMSO and
99%PBS, regulation final concentration 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, solubilizer 1%DMSO and 99%PBS, and regulation concentration is 50
μ g/mL, 10 μ L/ holes.
Negative control group adds complete medium, 10 μ L/ holes.
Often group is all provided with 3 multiple holes, cultivates 24h respectively.Before terminating cultivating, 4h adds concentration is 5mg/mL
MTT, 10 μ L/ holes, after cultivation terminates every hole add DMSO100 μ L, place shaking table 10min,
It is absorbance A value during 490nm in microplate reader detection wavelength.Calculate growth of tumour cell suppression ratio
(Inhibition Rate), computing formula is as follows:
Inhibition Rate(%)=[(A negative control group-A test group)/A negative control group] × 100%
IC is calculated according to growth of tumour cell suppression ratio50(half-inhibition concentration), result of the test such as table 2
Shown in:
The compound of the table 2 embodiment 1 preparation IC to 4 kinds of tumor cell 24h50(μ g/mL) value
Result of the test as shown in Table 2 understands, and shown in the formula I of embodiment 1 preparation, compound is to people's pulmonary carcinoma
A549 cell, B16 mouse black-in tumor cell, human hepatocellular carcinoma BEL-7402 cell and human breast cancer cell
IC after the 24h of MCF-750Value is respectively less than 20 μ g/mL, shows formula I shownization prepared by embodiment 1
Compound has significant inhibitory action to the growth of above-mentioned 4 kinds of tumor cells.
Process of the test finds, after positive control 5-FU acts on 24 hours when concentration is 50 μ g/mL,
People's pulmonary carcinoma A549 inhibition rate of tumor cell is only 32%, and chemical combination shown in the formula I of embodiment 1 preparation
After thing acts on 24h when concentration is 14.95 ± 0.40 μ g/mL, to people's pulmonary carcinoma A549 inhibiting tumour cells
Rate reaches 50%, it can be seen that, people's pulmonary carcinoma A549 is swollen by compound shown in the formula I of embodiment 1 preparation
The inhibitory action of oncocyte is significantly better than (P < 0.05) positive control 5-FU.
Also finding in process of the test, positive control 5-FU acts on 24 hours when concentration is 50 μ g/mL
After, the suppression ratio of Human hepatocarcinoma cell line SMMC-7721 tumor cell is only 24%, and the formula of embodiment 1 preparation
After compound shown in I acts on 24h when concentration is 15.76 ± 0.33 μ g/mL, to Human hepatocarcinoma cell line SMMC-7721
The suppression ratio of tumor cell reaches 50%, it can be seen that, compound pair shown in the formula I of embodiment 1 preparation
The inhibitory action of Human hepatocarcinoma cell line SMMC-7721 tumor cell is significantly better than (P < 0.05) positive control 5-FU.
Also finding in process of the test, positive control 5-FU acts on 24 hours when concentration is 50 μ g/mL
After, the suppression ratio to B16 mouse black-in tumor cell is 49%, it follows that the formula of embodiment 1 preparation
Compound shown in I is better than positive control 5-FU to the inhibitory action of B16 mouse black-in tumor cell.
Also finding in process of the test, positive control 5-FU acts on 24 hours when concentration is 50 μ g/mL
After, the suppression ratio to human breast cancer cell line Bcap-37 is 43%, it follows that embodiment 1 preparation
Compound shown in formula I is better than positive control 5-FU to the inhibitory action of human breast cancer cell line Bcap-37.
Compound shown in the formula I prepare the embodiment of the present invention 2～embodiment 4 carries out cell assay in vitro,
Test method is with embodiment 5, and acquired results is similar to Example 5, i.e. the embodiment of the present invention 2～embodiment
Shown in 4 formula I provided, compound is to typeⅡ pneumocyte, B16 mouse black-in tumor cell, people's hepatocarcinoma
The growth of BEL-7402 cell and human breast cancer cell line Bcap-37 cell has good inhibition, its
In to typeⅡ pneumocyte, human hepatocellular carcinoma BEL-7402 cell, MCF-7 Breast Cancer Cell growth
Inhibitory action is preferable.
Summary result of the test understands, and shown in the formula I that the embodiment of the present invention 1～4 provides, compound is to people
Lung cancer A549 cell, B16 mouse black-in tumor cell, human hepatocellular carcinoma BEL-7402 cell and human breast carcinoma are thin
The growth of born of the same parents' MCF-7 cell has good inhibition, wherein to typeⅡ pneumocyte, people liver
Cancer BEL-7402 cell, MCF-7 Breast Cancer Cell growth inhibited effect are preferable.
Embodiment 6 mice-transplanted tumor model test
Take H respectively22And S180Rat liver cancer (sarcoma) cell cryopreservation tube, is placed in 37 DEG C of waters bath with thermostatic control solution
Freeze, centrifugal collecting cell, wash twice with PBS liquid, then with PBS liquid re-suspended cell, little through ICR
Mus abdominal cavity passes 3 generations more than, takes the mice that abdominal part substantially expands, and dislocation is put to death, to abdominal part alcohol disinfecting,
Disposable sterilized injector extraction milky ascites, injects in sterilized centrifuge tube, cell counter meter
Number, adjusts cell density to 5 × 10 with normal saline6Individual/mL, to every mice right oxter inoculation 0.2mL
Cell suspension carries out modeling, and mice is randomly divided into next day following groups:
Blank group: the mice 8 of inoculation transplanted tumor, lumbar injection 0.9% normal saline 0.2mL,
Once a day, it is administered 10 days.
Positive controls: the mice 8 of inoculation transplanted tumor, intraperitoneal injection of cyclophosphamide (CTX), 20
Mg/kg, the next day 1 time, be administered 10 days.
Test group 1: the mice 8 of inoculation transplanted tumor, shown in the formula I of lumbar injection embodiment 1 preparation
Compound, 1.5mg/kg, once a day, successive administration 10 days.
Test group 2: the mice 8 of inoculation transplanted tumor, shown in the formula I of lumbar injection embodiment 1 preparation
Compound, 3.0mg/kg's, once a day, successive administration 10 days.
2h after last administration, peels off tumor body, weighs tumor weight, calculates tumour inhibiting rate, and computing formula is as follows:
Wherein administration group is positive controls, test group 1 or test group 2.
Result of the test is as shown in Table 3 and Table 4:
Shown in the formula I of table 3 embodiment 1 preparation, compound is to rat liver cancer H22The impact of transplanted tumor tumor weight
* P < 0.05, * * P < 0.01, vs blank group
Test data as shown in Table 3 understands, and compound shown in the formula I of the embodiment of the present invention 1 preparation exists
When dosage is respectively 1.5mg/kg and 3.0mg/kg, continuous use 10 days, to rat liver cancer H22Move
The suppression ratio planting tumor respectively reaches 32.1% and 43%, and compared with blank group, tumor killing effect is notable
(P < 0.05), and with the increase of dosage, tumour inhibiting rate is obviously enhanced；Positive controls when 20mg/kg,
To rat liver cancer H22The suppression ratio of transplanted tumor is 57.1%, compound shown in the formula I of embodiment 1 preparation
With positive controls ratio, to rat liver cancer H22The inhibition of transplanted tumor is not so good as positive controls.
As can be seen here, compound shown in the formula I of embodiment 1 preparation is to rat liver cancer H22The life of transplanted tumor
The long inhibitory action with notable (P < 0.05), dose-effect relationship is obvious.
Shown in the formula I of table 4 embodiment 1 preparation, compound is to mice S180The impact of sarcoma tumor weight
* P < 0.05, * * P < 0.01, vs blank group
Test data as shown in Table 4 understands, and compound shown in the formula I of the embodiment of the present invention 1 preparation exists
When dosage is respectively 1.5mg/kg and 3.0mg/kg, continuous use 10 days, to mice S180Sarcoma
Suppression ratio respectively reaches 30.3% and 55%, and compared with blank group, tumor killing effect is notable (P < 0.05),
And with the increase of dosage, tumour inhibiting rate is obviously enhanced；Positive controls is when 20mg/kg, to mice S180
The suppression ratio of sarcoma is only 37.1%, and shown in the formula I of embodiment 1 preparation, compound is to mice S180Sarcoma
Inhibition be significantly better than (P < 0.05) positive control CTX.
As can be seen here, compound shown in the formula I of embodiment 1 preparation is to mice S180The growth of sarcoma has
The significantly inhibitory action of (P < 0.05), tumor killing effect is significantly better than positive control CTX, and dose-effect relationship
Substantially.
Compound shown in the formula I prepare embodiment 2～embodiment 4 carries out mice-transplanted tumor model test,
Test method is with embodiment 6, and obtained experimental result is similar to Example 6, i.e. embodiment 2～embodiment 4
Shown in the formula I of preparation, compound is to rat liver cancer H22The growth of transplanted tumor has significantly (P's < 0.05)
Inhibitory action, dose-effect relationship is obvious；To mice S180The growth of sarcoma has significantly pressing down of (P < 0.05)
Making use, tumor killing effect is significantly better than positive control CTX, and dose-effect relationship is obvious.
In sum, compound shown in the formula I of the embodiment of the present invention 1～embodiment 4 preparation is to rat liver cancer
H22The growth of transplanted tumor has significantly the inhibitory action of (P < 0.05), and dose-effect relationship is obvious；To mice
S180The growth of sarcoma has significantly the inhibitory action of (P < 0.05), and tumor killing effect is significantly better than positive right
According to CTX, and dose-effect relationship is obvious.
The preparation of embodiment 7 medicine
Shown in the Formulas I of Example 1 preparation, the compound of structure adds the customary adjuvant of powder, according to often
Rule method makes powder.
The preparation of embodiment 8 medicine
Shown in the Formulas I of Example 2 preparation, the compound of structure adds the customary adjuvant of tablet, according to often
Rule method makes tablet.
The preparation of embodiment 9 medicine
Shown in the Formulas I of Example 3 preparation, the compound of structure adds the customary adjuvant of granule, according to
Conventional method makes granule.
The preparation of embodiment 10 medicine
Shown in the Formulas I of Example 4 preparation, the compound of structure adds the customary adjuvant of capsule, according to
Conventional method makes capsule.
The preparation of embodiment 11 medicine
Shown in the Formulas I of Example 2 preparation, the compound of structure adds the customary adjuvant of solution, according to
Conventional method makes solution.
The preparation of embodiment 12 medicine
Shown in the Formulas I of Example 1 preparation, the compound of structure adds the customary adjuvant of Emulsion, according to often
Rule method makes Emulsion.
The preparation of embodiment 13 medicine
Shown in the Formulas I of Example 3 preparation, the compound of structure adds the customary adjuvant of suspensoid, according to
Conventional method makes suspensoid.
The preparation of embodiment 14 medicine
Shown in the Formulas I of Example 4 preparation, the compound of structure adds the customary adjuvant of injection, according to
Conventional method makes injection.
The preparation of embodiment 15 medicine
Shown in the Formulas I of Example 2 preparation, the compound of structure adds the customary adjuvant of injectable powder, according to
Conventional method makes injectable powder.
The preparation of embodiment 16 medicine
Shown in the Formulas I of Example 1 preparation, the compound of structure adds the customary adjuvant of spray, according to
Conventional method makes spray.
The above is only the preferred embodiment of the present invention, it is noted that general for the art
For logical technical staff, under the premise without departing from the principles of the invention, it is also possible to make some improvement and profit
Decorations, these improvements and modifications also should be regarded as protection scope of the present invention.
Claims (4)
1. the extracting method of the compound of structure shown in a formula I, it is characterised in that comprise the steps of
Step 1: pulverized by oil tea root, take the first solvent extraction, concentrates, obtains fluid extract；
Step 2: take described fluid extract and mix with water, filters, and collects filtrate, centrifugal, collects supernatant,
Separate through D101 type macroporous resin column again, take ethanol-water system gradient elution, collect ethanol and water volume
Ratio is 70:30～95:5 eluting obtained component, and separation, purification to obtain final product；
Described first solvent is ethanol and water volume ratio is 0:100～95:5；
Described separation, purification, particularly as follows: take described component and separate through silicagel column, take chloroform-methanol system ladder
Degree eluting, collects chloroform and methanol volume ratio is 80:20～60:40 eluting obtained component；Again through ODS
Post separates, and takes methanol-water system gradient elution, collects methanol and water volume ratio is that 70:30～90:10 washes
De-obtained component；Separate through dynamic axial compression column again, take methanol-water system gradient elution, collect methanol
It is 75:25 eluting obtained component with water volume ratio；Separate finally by high performance liquid chromatography, use C18
Chromatographic column, the second solvent eluting, flow velocity is 2mL/min, collects 37.5min component, to obtain final product；
Described second solvent is: first alcohol and water by volume for 72:28 composition mixed solution again with account for institute
State the mixture of the formic acid mixing gained that mixed solution weight/mass percentage composition is 0.2%.
Extracting method the most according to claim 1, it is characterised in that gradient elution described in step 2
Solvent be followed successively by ethanol and water volume ratio be 0:100 → 30:70 → 50:50 → 60:40 → 70:30 →
80:20 → 95:5.
Extracting method the most according to claim 1, it is characterised in that the temperature extracted described in step 1
Degree is 30 DEG C～100 DEG C, and the time extracted described in step 1 is 8.5h～33h.
Extracting method the most according to claim 1, it is characterised in that the temperature concentrated described in step 1
Degree is 70 DEG C～100 DEG C, and the time concentrated described in step 1 is 2.0h～6.0h.
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| Tea Triterpenoidal Saponins from the Roots of Camellia sinensis Have Inhibitory Effects against Alcohol Dehydrogenase;Titto Varughese, et al.;《Planta Medica》;20110722;第77卷;第2029-2036页 * |
| Triterpenoids from the Roots of Camellia oleifera C.Abel and Their Cytotoxic Activities;Shou-Li Wang, et al.;《Helvetica Chimica Acta》;20131231;第96卷;第1126-1133页 * |
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