CN106138064B - Application of the potentilla discolor triterpenoid in antitumor - Google Patents
Application of the potentilla discolor triterpenoid in antitumor Download PDFInfo
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Abstract
The present invention proposes that a kind of triterpene compound as shown in formula (I), the compound have the purposes for preparing anti-tumor drug.
Description
Technical field
A kind of application the present invention relates to potentilla discolor triterpenoid and its in antitumor.
Background technique
Chinese medicine potentilla discolor is the drying of rosaceae potentilla plants potentilla discolor (Potentilla discolor Bunge)
Herb, first recorded in herbal for Relief of Famines.Potentilla discolor is distributed widely in the temperate zone and frigid zone in the Northern Hemisphere, and China is with Shandong, Liaoning, Anhui
The three provinces root yield is maximum, in addition, the provinces such as Hebei, Henan, the Inner Mongol, Hunan, Jiangsu and Guangxi are also distributed.Its property
It is sweet, bitter, flat, there is the effect of clearing heat and detoxicating, hemostasia and detumescence, can be used for treating the diseases such as dysentery, malaria, pulmonary abscess.
In terms of the therapeutic evaluation for concentrating on single link to the research of potentilla discolor both at home and abroad at present, for the chemistry of potentilla discolor
The systematic Study of ingredient, separation and Extraction, pharmacological activity, effective substance etc. is less, needs further deeply.
Summary of the invention
The present invention provides a kind of -3 alpha-hydroxy-2 9- dimethoxy -27- Betulinic Acid of potentilla discolor triterpenoid (20S),
Shown in structure such as formula (I):
The present invention provides structure -3 alpha-hydroxy-2 9- dimethoxy of potentilla discolor triterpene compound (20S)-as shown in formula (I)
The application of 27- Betulinic Acid, specifically, referring to the compound application in preparation of anti-tumor drugs.
Further, the anti-tumor drug is for treating breast cancer, uterine cancer, liver cancer etc..
Detailed description of the invention
Fig. 1 is that separation process schematic diagram is extracted in potentilla discolor；
Fig. 2-1,2-2 are (20S)-3 alpha-hydroxy-2 9- dimethoxy-27- Betulinic Acid1H NMR spectra；
Fig. 3-1,3-2,3-3 are (20S)-3 alpha-hydroxy-2 9- dimethoxy-27- Betulinic Acid13C NMR spectra；
Fig. 4-1,4-2 are (20S)-3 alpha-hydroxy-2 9- dimethoxy-27- Betulinic Acid 2D-NMR spectrogram；
Fig. 5-1,5-2,5-3 are (20S)-3 alpha-hydroxy-2 9- dimethoxy-27- Betulinic Acid ROESY spectrogram.
Specific embodiment
(1) extraction of compound
1, experimental method
1.1 laboratory apparatus and material
Fusing point is not with X-4 type binocular micro melting point apparatus (temperature corrects, Beijing Tyke Instrument Ltd. manufacture)
Measurement；UV with Shimadzu UV-2501PC type it is visible-ultraviolet specrophotometer measure；NMR:Bruker AV-500 type nuclear-magnetism
Resonate instrument, δ ppm, J Hz.
Tlc silica gel H, thin-layer chromatography silica G F254, column chromatography silica gel (100-200 mesh, 200-300 mesh, Qing Daohai
Foreignize factory)；Reverse phase C18 column, i.e. ODS column (40-60 μm, Fuji company), Sephadex LH-20 sephadex (is purchased from
Pharmacia company)；MCI column (Mitsubishi Chemical Co., Ltd).
Agents useful for same is the pure grade of analysis.
Unless otherwise specified, the signified ratio of the present invention and percentage refer both to volume.
1.2 plant origin
Potentilla discolor (Potentilla discolor Bunge) medicinal material picks up from Guangxi, and plant specimen is identified as rosaceae
Potentilla plants potentilla discolor, sample deposit in Analysis of Chinese Traditional Medicine teaching and research room of China Medicine University.
1.3 extract separation
The drying potentilla discolor herb 14kg for picking up from Guangxi is taken, as shown in Figure 1, with 90% ethanol water heating and refluxing extraction
3 times, each 3h, combined extract, decompression boils off most of ethyl alcohol, is suspended with water, then three times with petroleum ether extraction, recycles molten
Petroleum ether moiety 160g is obtained after agent, is then extracted with ethyl acetate three times, after recycling design, obtains ethyl acetate portion 300g.
Ethyl acetate portion (300g) mixes sample with 400g silica gel (100-300 mesh), and 3.0kg (200-300 mesh) carries out silica gel
Column chromatography, chloroform-methanol (1:0-0:1, v:v) gradient elution, the identical flow point of thin-layer chromatography (TLC) combining data detection.
It is dried under reduced pressure after the flow point of 30-50 volume % and 50-70 volume % is merged, carries out aperture resin (MCI) column layer
Analysis is collected the ingredient of 70%-90% methanol elution, solvent is recovered under reduced pressure with water-methanol (10:0-0:10, v:v) gradient elution
To medicinal extract shape, active component is obtained.A small amount of active component is taken, it is anti-through color reaction, Libermann-Burchard, Molish
It answers, prompts to contain more triterpene compound (potentilla discolor total triterpene).Again with methanol dissolves active component, carries out reversed C18 column
Chromatography, acetone-water (1:0-0:1, v:v) gradient elution are purified repeatedly, are obtained compound 1 (17mg), 2 (25mg), 3 (8mg), and 4
(20mg), 5 (8mg), 6 (100mg), 7 (5mg), 8 (4mg), 9 (5mg), 10 (120mg).
Compound 5 is more from the flow point of the 50-70 volume % acetone-water elution of reverse phase C18 column chromatography.
Potentilla discolor separation process is as shown in Figure 1.
According to compared with reference substance TLC and various spectral datas [IR, MS,1H NMR、13C NMR and 2D NMR (HMQC,
HMBC, COSY)] finally confirm obtained compound and is respectively as follows: euscaphic acid (euscaphic acid, 1), 2 Alpha-hydroxies crow
Soviet Union's acid (corosolic acid, 2), 2 α, 3 β, 19 α-trihydroxy-ursolic acid (tormentic acid, 3), 3 α, 30- dihydroxy
Base -20 (29)-alkene -27- Betulinic Acid (3 α, 30-dihydroxylup-20 (29)-en-27-oic acid, 4), (20S) -3 α -
Hydroxyl -29- dimethoxy -27- Betulinic Acid ((20S) -3 α-hydroxy-29-dimethoxy-lupan-27-oic acid, 5)
With ursolic acid (ursolic acid, 6), hawthorn acid (maslinic acid, 7), 19a-Hydroxyursolic Acid (pomolic acid,
8), 2 α, 3 alpha-dihydroxy -12- alkene -28- ursolic acids (2 α, 3 α-dihydroxyurs-12-en-28-oic acid, 9), neat pier
Tartaric acid (oleanolic acid, 10).
Wherein, -3 alpha-hydroxy-2 9- dimethoxy -27- Betulinic Acid ((20S) -3 α-hydroxy-29- of compound (20S)
Dimethoxy-lupan-27-oic acid, 5) have no document report, it is noval chemical compound.
2, Structural Identification
Compound 5: character: white amorphous powder (MeOH) is soluble in methanol；
Color reaction: 10% ethanol solution of sulfuric acid reacts displaing amaranth, and Libermann-Burchard reaction is positive,
Molish reaction negative, information above prompt for triterpene compound.HR-TOF-MS provides molecular weight m/z [M-H]-
517.3900(calcd for C32H53O5, 517.3898), in conjunction with13C-NMR and1H-NMR spectrum speculates that its molecular formula is
C32H54O5。
IR(KBr)νmax cm-1 3447,1685；
1H-NMR(C5D5N),δ(ppm):1.79–1.56(m,2H,H-1),2.00–1.72(m,2H,H-2),3.54(s,H-
3),1.73(m,1H,H-5),1.49–1.20(m,2H,H-6),1.99–1.96(m,2H,H-7),2.21(m,1H,H-9),
1.71–1.36(m,2H,H-11),2.71–1.82(m,2H,H-12),1.89(m,1H,H-13),2.29–1.68(m,2H,H-
15),1.76–1.72(m,2H,H-16),1.61(m,1H,H-18),1.93(m,1H,H-19),2.50(m,1H,H-20),
1.72–1.50(m,2H,H-21),1.41–1.05(m,2H,H-22),1.04(s,3H,CH3-23),0.87(s,3H,CH3-24),
0.97(s,3H,CH3-25),1.21(s,3H,CH3-26),0.88(s,3H,CH3- 28), 4.18 (d, J=8.15Hz, 1H, H-
29), 1.05 (d, J=5.6Hz, 3H, CH3-30),3.30(s,3H,CH3-31),3.38(s,3H,CH3-32)；
13C-NMR(C5D5N),δ(ppm):34.23(C-1),26.63(C-2),75.06(C-3),38.91(C-4),
49.57(C-5),18.75(C-6),38.21(C-7),40.92(C-8),51.23(C-9),37.41(C-10),21.20(C-
11),28.22(C-12),38.38(C-13),60.79(C-14),25.73(C-15),38.04(C-16),42.60(C-17),
50.57(C-18),39.38(C-19),38.07(C-20),22.66(C-21),40.98(C-22),29.10(C-23),22.78
(C-24),16.96(C-25),17.59(C-26),178.31(C-27),18.96(C-28),108.75(C-29),9.31(C-
30),53.01(C-31),53.58(C-32).
1On H H NMR spectroscopy there are six angular methyl signal (such as Fig. 2-1): δ 0.87,0.88,0.97,1.04,1.21 (s, 3H,
Each) and 1.05 (d, J=5.60Hz), in addition near δ 3.30 there are two apparent methyl singlets signal 3.30 (3H, s),
3.38 (3H, s) are prompted containing there are two methoxyl groups.Proton signal δ 5.30 (H, s), 4.18 (H, d, J=8.15Hz), 3.54 (H,
S) it implies and is connected (such as Fig. 2-2) with oxygen-containing groups such as hydroxyls.
13There are 32 carbon signals, including six angular methyl carbon signal δ in C H NMR spectroscopyC9.31、16.61、17.59、18.75、
18.96 and 21.20 (such as Fig. 3-1), two methoxyl group carbon signal δC53.01,53.58 (such as Fig. 3-2), two company oxygen carbon signal δC
A 75.06 and 60.79 and carboxyl carbon signal δC178.31 (such as Fig. 3-2 and 3-3).
In summary information, estimating the compound is 3 alpha-hydroxy-2 9- dimethoxy -27- Betulinic Acid (3 α-hydroxy-
29-dimethoxy-lupan-27-oic acid), shown in structure such as formula (I).
Confirmation and full ownership can be carried out to its structure according to 2D-NMR, as follows:
?1H–12H-1 (δ 1.56/1.79) is related to 2H-2 (δ 1.72/2.00) in H COSY spectrum, and the latter while and H-3
(δ 3.54) is related；H-5 (δ 1.73) is related to 2H-6 (δ 1.49/1.40), at the same the latter also with 2H-7 (δ 1.96/1.99) signal
It couples；H-9 (δ 2.21) is related to 2H-11 (δ 1.71/1.36), 2H-12 (δ 2.71/1.82) and H-13 (δ 1.89) phase
It closes, and 2H-11 (δ 1.71/1.36) is also related simultaneously to 2H-12 (δ 2.71/1.82).H-13 (δ 1.89) and H-19 (δ 1.93)
Correlation, the latter is related to 2H-21 (δ 1.50/1.72) simultaneously, while 2H-21 is also related to 2H-22 (δ 1.05/1.41).
?1In HMBC spectrum (Fig. 4-1,4-2), carboxyl carbon signal δc178.31 is related to H-13 (δ 1.89)；δ1.21(3H-
26), δ 1.89 (H-13) and δ 1.61 (H-18) are related to C-14 (δ 60.78)；C-28 (δ 18.96) and H-18 (δ 1.61), H-22
(δ 1.41) is related.
(Fig. 5-1,5-2,5-3), δ 0.88 (3H-28) and 13 β, 15 β, 16 β, 19 β, 21 β and 22 β H phases in ROESY spectrum
It closes.In conjunction with document, it is C-27 carboxyl that information above, which confirms the compound,.
?1In H-NMR, H-3 (δ 3.54, s) shows that H-3 is on e key.(Fig. 5-1,5-2,5-3) H-3 (δ in ROESY
3.54) related to H-1 β (δ 1.79), H-2 β (δ 2.0), 3H-24 β (δ 0.87), the H on A ring can be belonged to, while also confirming this
Configuration.
In addition, (Fig. 4-1,4-2), 3H-31 (δ 3.30, s), 3H-32 (δ 3.38, s) and 108.7 (C-29) phases in HMBC spectrum
It closes, shows to connect on C-29 that there are two methoxyl groups.
In addition, the absolute configuration of C-20 is determined by the chemical shift size of C-30, because if C-20 is S configuration,
Then the chemical shift of C-30 is δc10.3；If C-20 is R configuration, it is δ that the chemical shift of C-30, which is in compared with low field,c17.7。
Therefore, C-20 is S configuration in compound 5.
Comprehensive institute is above-mentioned, and the structure determination of compound 5 is (20S) -3 α-hydroxy-29-dimethoxy-lupan-27-
oic acid.By literature search, compound 5 is a noval chemical compound, and spectral data is following (table 1).
1 compound 5 of table1H-NMR、13C-NMR data (C5D5N,δppm,J Hz).
According to Literature Consult, isolated triterpene compound is nearly all free aglycon from Potentilla, mainly
It is pentacyclic triterpene, by its classification, mainly there is Ursane, oleanane type and lupinane type；Have mostly in its structure double bond,
Hydroxyl and carboxyl, and the position of double bond is nearly all 12 (13) positions, the position of carboxyl is all 28 mostly；The variation of its structure is just
Be mainly reflected in the variation of hydroxyl quantity, the position of substitution and spatial chemistry, the most common hydroxy substitution pattern be 2 α, 2 β, 3 α,
3 β, 19 α, 23,24, other positions are then rare.
It is noted that there are two be lupin alkane type triterpenoid in 10 triterpene compounds that inventor obtains
(one of them is noval chemical compound), and carboxyl is all present in C-27.According to literature search, most of naturally occurring lupins
Alkane type triterpenoid is mostly C-28 carboxyls, and the native compound of C-27 carboxyls only has seven, is from Lamiaceae plant respectively
It is isolated in Hyptis suaveolens, Rhamnaceae plant Gouania microcarpa and the potentilla discolor of this plant.Now
Inventor's also lupin alkane type triterpenoid of isolated one new C-27 carboxyl from potentilla discolor again, is greatly enriched this
The compound of type.
Two, compound activity is studied
(1) sugar consumption
AMP protein kinase (AMPK, AMP, that is, adenosine-phosphoric acid) mainly controls cell metabolism: it can be determined in body
Fat be storage or burning, this depends primarily on gross energy in cell.When cellular energy levels are higher, exist in cell
The kinetomeres ATP (atriphos) of high concentration, therefore AMPK can promote cell to carry out " anabolism ", and excess energy is made
It is stored for fat；When ATP is horizontal lower, AMPK will close anabolism, excite catabolism instead, allow fat combustion
For energy.Existing research shows that AMPK can be as a kind of effective means for the treatment of diabetes B.When AMPK is detected in cell
When ATP concentration is lower, they can make the glucose in cell become ATP using various mechanism.Experiment for rodent
It has been shown that AMPK is effective for reducing blood glucose.
Can this experiment be exactly to explore potentilla discolor total triterpene position and triterpene monomer therein using sugar consumption experiment lead to
It crosses AMPK approach and therapeutic effect is realized to diabetes B.
1, experimental material
1.1 sample
1. 3 α, 30- dihydroxy -20 (29)-alkene -27- Betulinic Acid, 2. novel compound of present invention (i.e. (20S) -3 α-hydroxyl
Base -29- dimethoxy -27- Betulinic Acid), 3. Corosolic acid, 4. potentilla discolor total triterpene (compound 1-10), wherein 4. sample is used
A small amount of DMSO (dimethyl sulfoxide) dissolution (ensuring final concentration of the DMSO in cultivating system no more than 0.1%), then with PBS (phosphoric acid
Salt buffer) dilution, so that sample ultimate density in cell supernatant is followed successively by 10 μm of ol/L, 10 μm of ol/L, 10 μm of ol/L and 100
μ g/ml, melbine are dissolved with PBS, make the final concentration of 1mmol/L of sample in cell supernatant.
1.2 reagent
DMEM culture medium (Gibco company, lot number: 1115806)；
Separately add 0.06g/L penicillin (800,000 unit) and 0.1g/L streptomycin sulphate (1,000,000 unit) and 3.7g/ using liquid
L sodium bicarbonate, pH are adjusted to 7.2；
Benzylpenicillin sodium for injection (Suzhou Erye Pharmaceutical Co., Ltd., lot number 100501)；
Streptomycin sulphate (Nanjing Sheng Xing Bioisystech Co., Ltd, lot number: 0E100E10)；
Sodium bicarbonate (Nanjing Chemistry Reagent Co., Ltd., lot number: 11022610211)；
Newborn calf serum (Newborn calfserum, NCS) (Shanghai Wei Ke biochemical reagents Co., Ltd, lot number:
A10110-0904)；
Trypsase (Sunshine company, lot number: 1C221H04)；
DMSO (Snshine company, lot number: 2A022A02)；
Melbine (ALEXIS BIOCHEMICALS, lot number: L20508/a)
Glucose (Sinopharm Chemical Reagent Co., Ltd., lot number: F20101115)；
Glucose estimation kit (ShangHai RongSheng Biology Pharmacy Co., Ltd, lot number: 20120701)；
1.3 cell strain
PECTORAL LIMB SKELETON 3T3-L1, small source of mouse.
1.4 animal
ICR mouse, male, 18-22g are provided by Nanjing Qinglongshan experimental animal farm.
2, experimentation
The preparation of 2.1 conditioned medium CM (conditioned-medium)
It takes mouse cervical dislocation to put to death, impregnates 3-5min in 75% alcohol, move in super-clean bench, PBS5ml/ is injected intraperitoneally
Only, the abdomen 2min of mouse is gently rubbed, is sucked out with liquid-transfering gun with 6 × 105It is inoculated in 6 orifice plates, in 5%CO2, 37 DEG C of cell culture
It is incubated in case, is inhaled after 4h and abandon non-attached cell, washed 2 times with PBS, attached cell is changed with KRH (Krebs'-Ringer's-
Henseleit) liquid culture, every hole adds KRH liquid 2ml, and the LPS (whole solubility is 5 μ g/ml) of 10 μ l is added in every hole, receives after 48h
Collect supernatant be used as macrophage conditioned medium (conditioned-medium, CM), bacteriological filtration, dispense, store in -70 DEG C it is ultralow
It is saved backup in temperature refrigerator.
2.2 cell recoveries and culture
It takes out the 3T3-L1 cell frozen to set in 37 DEG C of water-baths, makes its fast melt, be transferred to 5ml containing the 10% new blood that calves
In clear DMEM culture medium, cell is resuspended, then be transferred in 75ml culture bottle in 5%CO2, it cultivates in 37 DEG C of cell incubator,
Liquid is changed daily.PECTORAL LIMB SKELETON 3T3-L1 cell is attached cell, 1-2 days after inoculation, shows as single layer dense growth, cell is in
Shuttle shape has branch.It when cell reaches 80% fusion, is cleaned twice, 0.25% trypsin digestion 30s with PBS, is passed by 1:6
Generation.The cell of logarithmic growth phase, trypan blue exclusion carry out viable count, and adjusting cell concentration is 106/ ml is inoculated in 48
Well culture plate, 5%CO2, 37 DEG C of cell incubators are interior to be incubated for for 24 hours, when cell merges substantially, is cleaned twice with KRH and is used KRH instead
Liquid is hungry, is tested after every 200 μ L starvation 4h of hole.
2.3 drug-treateds and measurement
2.3.1 being screened under physiological condition
Experiment sets blank group, sample sets and positive control (melbine) group.First hungry good cell is cleaned with KRH liquid
Twice, product then are loaded with KRH system, the sample that predetermined concentration is separately added into cultivation plate hole is 1. 2. 3. 4. (1. 2. 3. dense eventually
10 μm of ol/L are spent, 4. 100 μ g/ml of final concentration) and melbine (25 μm of ol/L of final concentration), the KRH of blank group addition same volume
Liquid；Glucose solution is added in each group, makes its final concentration of 11mmol/L, 5%CO2, 37 DEG C of cell incubators are interior to be incubated for 4h, uses
Glucose kit measurement.
2.3.2 being screened under inflammatory conditions
Experiment sets blank group, model group, sample sets and positive control (melbine) group.It is separately added into cultivation plate hole pre-
Determine the sample of concentration 1. 2. 3. 4. (1. 2. 3. 10 μm of ol/L of final concentration, 4. 100 μ g/ml of final concentration) and melbine (final concentration
1mmol/L), 5%CO2, 37 DEG C of cell incubators are interior to be incubated for 0.5h, and model group, sample sets and positive control add in cultivation plate hole
Enter the CM of same volume, the KRH liquid of same volume is added in blank group, and each group is added glucose solution, keeps its final concentration of
11mmol/L, 5%CO2, 37 DEG C of cell incubators are interior to be incubated for 4h, is measured with glucose kit.
2.4 experimental result
2.4.1 the selection result under physiological condition
After cell incubation 4h, OD value is surveyed under 492nm wavelength with Glucose estimation kit, is calculated on cell as a result,
Sugared content in clear, and then obtain sugar consumption amount.It the results are shown in Table 2-1.
Sugar consumption amount (n=6) under table 2-1 physiological condition
* P < 0.05 compared to the blank group
2.4.2 the selection result under inflammatory conditions
After cell incubation 4h, OD value is surveyed under 492nm wavelength with Glucose estimation kit, is calculated on cell as a result,
Sugared content in clear, and then obtain sugar consumption amount.It the results are shown in Table 2-2.
Sugar consumption amount (n=8) under table 2-2 inflammatory conditions
* P < 0.05 compared with model group
3, conclusion
It can be seen that by table 2-1, there were significant differences with blank group for positive controls, has prompted the reliability of experiment.Three kinds of samples
Product compared to the blank group, though sugar consumption has raising, without significant difference, prompt drug to make in terms of activating AMPK without significant
With.Melbine is AMPK specific inhibitor, inhibits consumption sugared under physiological condition, and prompting this approach to activate with AMPK has
Relationship.
It can be seen that by table 2-2, there were significant differences with blank group for model group, and display modeling success prompts the reliability of experiment.
In four kinds of samples, 1., 2., 3. the sugar consumption amount of number sample is increased, and there were significant differences compared with model group, illustrates them in inflammation
Intake of the 3T3-L1 to glucose is remarkably promoted under the conditions of property, prompts these three samples that may activate AMPK under inflammatory conditions,
And then promote sugar consumption, 2. the sugar consumption amount of number sample is close to positive control melbine.Melbine is quick as an insulin
Perception has AMPK activity, while also having anti-inflammatory activity (may lead to AMPK approach to be mediated), significant to increase as a positive drug
Sugaring consumption, has prompted the participation of AMPK approach.
Sugar consumption experiment performance is that fat cell utilizes the intake of sugar under non-insulin state, this process is mainly
It is mediated by AMPK approach.Inflammatory stimulus can be by inhibiting the activation of AMPK that cell is inhibited to utilize the oxidation of sugar.
(2) cytotoxic activity is studied
Tumour is the hot spot of whole world medical research, and anti-tumor drug used at present is mostly the life by interfering cell
The relevant mechanism such as long, dead, differentiation and function, to achieve the purpose that inhibit the growth of cell even to kill cell.It is anti-swollen
The screening of tumor medicine includes internal and external two methods.When few for sample size, generally swollen with sample in vitro culture
The inhibiting effect of tumor cell growth is as primary dcreening operation.Since growth of cancer cells has opposite independence, therefore tool can be established in vitro
There is the cell line of unlimited multiplication capacity, which has amount of consumption of drugs few (2-10mg), the features such as period is short, and expense is low.In the experiment
We study -3 alpha-hydroxy-2 9- dimethoxy of Triterpenoid compound (20S)-isolated from potentilla discolor using mtt assay
27- Betulinic Acid and 3 α, 30- dihydroxy -20 (29)-alkene -27- Betulinic Acid is to human body HepG2, MCF-7 and Hela cancer cell strain
Cytotoxicity.
1, experimental material
1.1 instruments and equipment
Superclean bench (Wu County experimental animal equipment factory) constant temperature CO2Incubator (German Heraeus) 96 well culture plates
(NUNCLON) the full-automatic distilled water machine of microplate reader (Tecan Sunrise) inverted biologic microscope (Chongqing optical instrument factory) (on
Extra large one factory of glass apparatus) ultracentrifuge OPTIMAXL-100K (Bake Mann).
1.2 reagents and compound
RPMI1640 culture medium (GIBCO)；Trypsase (SIGMA)；Fetal calf serum (HYCLONE)；MTT(SIGMA)；
DMSO(SIGMA)。
3 α, 30- dihydroxy -20 (29)-alkene -27- Betulinic Acid are separated by this laboratory and are identified, compound is analyzed through HPLC,
Purity >=98%.
(20S) -3 alpha-hydroxy-2 9- dimethoxy -27- Betulinic Acid is separated by this laboratory and is identified, compound is through HPLC points
Analysis, purity >=98%.
1.3 cell strains and culture
Breast cancer lines (MCF-7), people's uterine cancer cells strain (Hela) and human hepatoma cell strain (HepG2) (by
Beijing consonance drug is provided) it is incubated in RPMI1640 culture medium, containing 10% calf serum, 37 DEG C, 5%CO2。
2, experimental section
2.1 experimental principle
MTT colorimetric method is the method for a kind of classical detection cell survival and growth.Its testing principle is living cells line grain
Succinate dehydrogenase in body can make exogenous MTT be reduced to the blue Jie Jing formazan of water-insoluble and be deposited in cell, and
Dead cell is without this function.Dimethyl alum (DMSO) can dissolve formazan in cell, with enzyme-linked immunosorbent assay instrument in 490nm wavelength
Place measures its absorbance value, can reflect living cells quantity indirectly.Within the scope of certain cell number, MTT crystallizes the amount to be formed and thin
Born of the same parents' number is directly proportional.This method oneself be widely used in the Activity determinations of some bioactie agents, large-scale screening anti-tumor medicine,
Cell toxicity test etc..
2.2 experimentation
Take respectively adherent Breast cancer lines (MCF-7) in logarithmic growth phase, people's uterine cancer cells strain (Hela),
Human hepatoma cell strain (HepG2), after being digested with pancreatin, with containing 10% calf serum RPMI1640 culture medium be made into 5000/
The cell suspension of ml is seeded in 96 porocyte culture plates with 8 × 103/hole, every hole inoculation 200 μ l, and 37 DEG C, 5%CO2Training
It supports 24 hours.A series of various concentrations are set, while control group is changed containing isometric PBS culture medium, every group sets 3 parallel holes,
37 DEG C, 5%CO2Culture 2 days.1500r/min centrifugation, discards supernatant liquid, and working containing MTT for the fresh configuration of 5mg/ml is added in every hole
20 μ l of liquid, continues culture 4 hours by 37 DEG C.Liquid carefully is discarded supernatant, after being digested with pancreatin, 150 μ l DMSO dissolution is added in every hole
MTT formazan precipitating after being mixed with miniature ultrasonic oscillator, measures OD value with tested wavelength in microplate reader for 490nm.It presses
Following formula calculates drug to the inhibiting rate of growth of tumour cell: growth of tumour cell inhibiting rate=(1- medicine group A490Value/control group
A490) × 100%.Then the half casualty-producing concentrations IC of the compound is acquired50。
3, result
It is tested by preliminary cytotoxic activity, calculates to obtain 3 α, 30- dihydroxy -20 (29)-alkene -27- Betulinic Acid is to human milk
Adenocarcinoma cell strain (MCF-7), people's uterine cancer cells strain (Hela), human hepatoma cell strain (HepG2) IC50Respectively 24.9,
17.5 and 20.1mmol/L, (20S) -3 alpha-hydroxy-2 9- dimethoxy -27- Betulinic Acid to Breast cancer lines (MCF-7),
People's uterine cancer cells strain (Hela), human hepatoma cell strain (HepG2) IC50Respectively 21.7,13.8 and 14.9mmol/L.Prompt
(20S) -3 alpha-hydroxy-2 9- dimethoxy -27- Betulinic Acid is to Breast cancer lines (MCF-7), people's uterine cancer cells strain
(Hela), human hepatoma cell strain (HepG2) has good inhibitory activity, can be used for breast cancer, uterine cancer, the clinic of liver cancer and controls
It treats.
The cytotoxic activity (n=8) of 3 potentilla discolor triterpenoid of table
Claims (1)
1. the potentilla discolor triterpene compound application in preparation of anti-tumor drugs as shown in formula (I), the tumour be breast cancer,
Uterine cancer, liver cancer
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102908422A (en) * | 2012-09-29 | 2013-02-06 | 暨南大学 | Potentilla discolor extract for reversing multidrug resistance of tumor, preparation method and application of potentilla discolor extract |
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2016
- 2016-07-01 CN CN201610510756.XA patent/CN106138064B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102908422A (en) * | 2012-09-29 | 2013-02-06 | 暨南大学 | Potentilla discolor extract for reversing multidrug resistance of tumor, preparation method and application of potentilla discolor extract |
Non-Patent Citations (2)
| Title |
|---|
| Antitumor Activity of Leaves from Potentilla discolor on Human Hepatocellular Carcinoma Cell Line HepG-2;JIN Quan等;《Chinese Journal of Natural Medicines》;20110131;第9卷(第1期);0061-0064 |
| Structural determination of two new triterpenoids from Potentilla discolor Bunge by NMR techniques;Jie Yang等;《Magn. Reson. Chem.》;20080528;第46卷;794-797 |
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