CN102617672A - Camellia nitidissima flavonoid glycoside, and preparation method and application thereof - Google Patents
Camellia nitidissima flavonoid glycoside, and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to an anticancer compound camellia nitidissima flavonoid glycoside A separated from camellia nitidissima, and a preparation method and application thereof. By qualitative and quantitative determination, a monomeric compound and fraction which are obtained by separation from an ethyl acetate extract of a 95% ethanol extract of the camellia nitidissima are mainly flavonoid components. The chemical structure of the compound is determined by analysis of a modern spectrum technology, and the chemical name is quercetin-7-O-(6''-O-E- dicaffeoyl group)-beta-D-glucoside. An in vitro anti-tumor experiment proves that the compound can remarkably restrain proliferation of tumor cells and has activity of inducing apoptosis of the tumor cells. An anticancer compound camellia nitidissima flavonoid glycoside A product has the developing potential of antitumor medicines.
Description
Technical field
The invention belongs to preparation and Application Areas, be specifically related to a kind of Camellia nitidissima Chi flavone glycosides A, Preparation Method And The Use the new constituent that has medical active in the natural phant.
Background technology
Camellia nitidissima Chi (Camellia chrysantha (Hu) Tuyama) is Theaceae (Theaceae) Camellia (Camellia Linn.) Camellia nitidissima Chi group (Section chrysantha Chang) plant, mainly is distributed in Yongning, Guangxi county.Camellia nitidissima Chi not only has higher ornamental value, and has certain pharmaceutical use and nutritive value, is traditional Zhuang medication among the people.Camellia nitidissima Chi tealeaves is mainly used in pharyngolaryngitis, dysentery, ephritis, oedema, urinary tract infections, icterohepatitis, hepatic ascites, hypertension, prophylaxis of tumours etc.; Flower is mainly used in and has blood in stool, menoxenia etc.The research to Camellia nitidissima Chi in early days mainly concentrates on plant morphology, plant taxonomy aspect, and the research to pathology isoreactivity before anti-oxidant, the reducing blood-fat of Camellia nitidissima Chi, the anti-liver cancer in recent years appears in the newspapers, in addition the Camellia nitidissima Chi leaf The Chemical Constituents is also had report.
The contriver finds that in the series of studies to Camellia plant antitumor activity component the different plant parts of Camellia nitidissima Chi (flower, leaf, seed) all have the activity of stronger inhibition human leukemia cell line U937 cell proliferation.In order to illustrate the basic substance and the mechanism of action thereof of golden flower camellia anti-tumor activity, the present invention studies the chemical ingredients of golden flower camellia and the activity of inducing apoptosis of tumour cell thereof.
Summary of the invention
The present invention separates the chemical ingredients of golden flower camellia, from the acetic acid ethyl ester extract of golden flower camellia 95% ethanol extraction, separates the monomeric compound obtain and cut and shows through qualitative, quantitatively determined and be mainly flavones ingredient.Infer that thus the flavones ingredient in the golden flower camellia possibly be the important substance basis of its anti-tumor activity.Therefore, the present invention is through carrying out isolation identification to flavones ingredient in the golden flower camellia, and is activity index with the inducing apoptosis of tumour cell, therefrom seeks and screen new type antineoplastic medicine.Therefore, the present invention seeks the monomer component with anti-tumor activity---Camellia nitidissima Chi flavone glycosides A from the golden flower camellia, and it has following structural formula:
It is yellow crystal at normal temperatures, and fusing point is 233～235 ℃.
Another object of the present invention provides a kind of preparation method of above-mentioned Camellia nitidissima Chi flavone glycosides A, and it comprises the steps,
A. the method for preparing the acetic acid ethyl ester extract of golden flower camellia 95% ethanol extraction:
With the golden flower camellia with 95% industrial alcohol heating and refluxing extraction, solid-liquid ratio 1g: 2～5ml, 75～85 ℃ of temperature after ethanol extraction is condensed into medicinal extract, add the entry suspendible in medicinal extract, use ethyl acetate extraction, reclaim acetic acid ethyl ester extract;
B. with the acetic acid ethyl ester extract of silica gel column chromatography separating step A gained; Volume ratio with chloroform-methanol is 100: 1,50: 1,30: 1,20: 1,15: 1,10: 1 and 5: 1 respectively; Carry out gradient elution; With the flow point that elutes, to know the back through silica gel thin-layer plate inspection and merge identical flow point, collection is subsequent use by the flow point under 10: 1 volume ratio wash-outs of chloroform-methanol;
C. the flow point of step B gained; Separate through the ODS reversed-phase silica gel column chromatography; Elutriant is a methyl alcohol: acetonitrile: the volume ratio of water is 1: 1: 2, and through thin-layer chromatography aluminium foil reverse phase silica gel plate development, developping agent is a methyl alcohol: acetonitrile with each flow point of collecting: the volume ratio of water is 1: 1: 2; Spot with displaing yellow after flow point launches is a principal spot, principal spot R
fValue be 0.67 flow point merge, subsequent use;
D. the flow point that obtained of step C separates through Sephadex LH-20 column chromatography, and with methanol-eluted fractions, through thin-layer chromatography aluminium foil reverse phase silica gel plate development, developping agent is a methyl alcohol: acetonitrile with each flow point of collecting: the volume ratio of water is 1: 1: 2, and flow point launches the back with spot R
fValue is that 0.67 single flow point is a finished product.
Concrete, in aforesaid method, in its steps A, in medicinal extract, add the water suspendible of 2～5 times of quality, and with 2 times of amount ethyl acetate extractions.
Concrete, in aforesaid method, in its steps A, said solid-liquid ratio is 1g: 3ml.
Concrete, in aforesaid method, in its steps A, said temperature is 80 ℃.
Concrete, in aforesaid method, in its steps A, the number of times of said heating and refluxing extraction is 3 times, each 2 hours.
Another aspect of the present invention is, the purposes of above-mentioned Camellia nitidissima Chi flavone glycosides A on the preparation antitumor drug.
Above-mentioned purposes, concrete, be through the Camellia nitidissima Chi flavone glycosides A of effective dose is mixed and processes the medicine of appropriate dosage forms with pharmaceutical excipient as effective constituent.
Having announced in the embodiment among the present invention, at SciFinder Scholar data base querying, proved that the above-mentioned Camellia nitidissima Chi flavone glycosides A of gained has novel structure through structural formula and systematic naming method, is new compound.
Activity to above-mentioned Camellia nitidissima Chi flavone glycosides A among the embodiment detects: intervene human leukemia U937 cell with the Camellia nitidissima Chi flavone glycosides A of different concns, adopt flow cytometer to detect the influence of its pair cell apoptosis.
Find that through investigating the Camellia nitidissima Chi flavone glycosides A can suppress U937 cell proliferation, V-FITC/PI couple of employing Annexin dyes method Flow cytometry early apoptosis of cells rate result and shows, after the Camellia nitidissima Chi flavone glycosides A was handled, U937 early apoptosis of cells rate significantly raise.
Description of drawings
Fig. 1. the Camellia nitidissima Chi flavone glycosides A is to the influence of U937 cell survival rate:
The Camellia nitidissima Chi flavone glycosides A that is respectively 10,25,50,100,200 and 400 μ M with final concentration is handled U937 cell 24h, and mtt assay detects the U937 cell survival rate, and the result is with means standard deviation (n=6) expression of cell survival rate.The result shows that along with Camellia nitidissima Chi flavone glycosides A concentration increases, the survival rate of U937 cell obviously descends.
Fig. 2. the Camellia nitidissima Chi flavone glycosides A induces the apoptotic Annexin V-FITC/PI of U937 pair to dye analysis: the Camellia nitidissima Chi flavone glycosides A is handled U937 cell 6h; The two stainings of flow cytometer Annexin V-FITC/PI detect apoptosis rate, and the result is with means standard deviation (n=6) expression of apoptosis rate.The result shows that use concentration is that the U937 apoptosis rate obviously raise after the Camellia nitidissima Chi flavone glycosides A of 100,200 μ M was handled the U937 cell.
Fig. 3 Camellia nitidissima Chi flavone glycosides A induces the apoptotic Annexin V-FITC/PI of U937 pair to dye analysis: Camellia nitidissima Chi flavone glycosides A (200 μ M) is handled U937 cell 6h, and the two stainings of flow cytometer Annexin V-FITC/PI detect the apoptosis scatter diagram.The result shows, after comparing Camellia nitidissima Chi flavone glycosides A (200 μ M) with control group and handling the U937 cell, the U937 apoptosis rate obviously raises.
Fig. 4. when the Camellia nitidissima Chi flavone glycosides A is induced the U937 apoptosis Caspase-3 activatory is influenced: the Camellia nitidissima Chi flavone glycosides A is handled U937 cell 6h, uses CaspGLOW
TMActivation Caspase-3 original position staining kit detects activatory Caspase-3 in the cell, and the result is with means standard deviation (n=6) expression of Caspase-3 volume of activation.The result shows that use concentration is that activatory Caspase-3 obviously increased in the U937 cell after the Camellia nitidissima Chi flavone glycosides A of 100,200 μ M was handled the U937 cell.
Fig. 5. when the Camellia nitidissima Chi flavone glycosides A is induced the U937 apoptosis Caspase-3 activatory is influenced: after handling U937 cell 1h with 50 μ M Z-VAD, add 200 μ M Camellia nitidissima Chi flavone glycosides As again and handle U937 cell 6h, detect percentage of cell apoptosis then.The result shows that the Camellia nitidissima Chi flavone glycosides A induces the apoptotic activity of U937 significantly to be suppressed.
Fig. 6. the Camellia nitidissima Chi flavone glycosides A
1H-NMR composes entirely;
Fig. 7. the Camellia nitidissima Chi flavone glycosides A
13C-NMR composes entirely;
Fig. 8. the HMBC spectrum of Camellia nitidissima Chi flavone glycosides A;
Fig. 9. the high resolution positive ion electrospray ionization mass spectrum of Camellia nitidissima Chi flavone glycosides A.
Embodiment
Following non-limiting example can make those of ordinary skill in the art more fully understand the present invention, but does not limit the present invention in any way.Mode with specific embodiment is further described the present invention below.
Employed various chemical reagent all obtain through conventional commercial sources like no specified otherwise in the embodiment of the invention;
Thin-layer chromatography aluminium foil reverse phase silica gel plate: RP-18F254, German Merck company, No.1.05559.0001;
XT4A type micro melting point apparatus: appearance electric light instrument plant of Beijing section;
Jasco V-560 type ultraviolet-visible spectrophotometer: Japanese beam split Co., Ltd.;
AVANCE500Hz type NMR: Switzerland Bruker company, TMS are interior mark;
UPLC/Q-TOF-MS system: U.S. Waters company;
The human NSFC of human lymphoma leukemia cell line U937:Human Sciences Research Resource Bank Japan, Tokyo, Japan;
The preparation of embodiment 1 Camellia nitidissima Chi flavone glycosides A
Preparing method with the active Camellia nitidissima Chi flavone glycosides A of inducing apoptosis of tumour cell of the present invention, this method comprises the steps:
A. the acetic acid ethyl ester extract for preparing golden flower camellia 95% ethanol extraction
Camellia nitidissima Chi dried floral 3kg pulverizes, and with the 95% industrial alcohol heating and refluxing extraction of 9L, 80 ℃ of temperature are extracted 3 times, and each 2h filters the back concentrating under reduced pressure, gets alcohol extract 588g.Get alcohol extract 500g with 1500mL hot distilled water suspendible after, extract repeatedly with the ETHYLE ACETATE of 2 times of amounts, reclaim solvent after, the ETHYLE ACETATE that obtains partly altogether 110g partly be total to 387g with water layer.
B. separate above-mentioned acetic acid ethyl ester extract with silica gel column chromatography
With the 200-300 order silica gel of 1500g, wet method dress post is with the ETHYLE ACETATE part silica gel mixed sample of 75g; Appearance on the dry method; With the volume ratio of chloroform-methanol 100: 1,50: 1,30: 1,20: 1,15: 1,10: 1 and 5: 1, carry out gradient elution, collect flow point; Every part of 100mL, every part of flow point is not with 1
#, 2
#, 3
#Deng in order the numbering.Wherein, with chloroform-methanol (100: 1) wash-out, obtain flow point 1
#～110
#,, obtain flow point 111 with chloroform-methanol (50: 1) wash-out
#～146
#,, obtain flow point 147 with chloroform-methanol (30: 1) wash-out
#～200
#,, obtain flow point 201 with chloroform-methanol (20: 1) wash-out
#～222
#,, obtain flow point 223 with chloroform-methanol (15: 1) wash-out
#～249
#,, obtain flow point 250 with chloroform-methanol (10: 1) wash-out
#～342
#,, obtain flow point 343 with chloroform-methanol (5: 1) wash-out
#～440
#Each flow point of collecting merges identical flow point after the silica gel thin-layer plate inspection is known, name successively respectively, and wherein flow point 250
#～278
#Merge into component Fr. I, flow point 279
#～300
#Merge into component Fr. II, flow point 301
#～316
#Merge into component Fr. III, flow point 317
#～331
#Merge into component Fr. IV, flow point 332
#～342
#Merge into component Fr. V, flow point 343
#～360
#Merge into component Fr. VI, flow point 361
#～387
#Merge into component Fr. VII.
C. above-mentioned by chloroform-methanol (10: 1) wash-out, and know the back through silica gel thin-layer plate inspection and merge the component Fr. III (472mg) that the same stream branch obtains and separate through the ODS reversed-phase silica gel column chromatography, with methyl alcohol-acetonitrile-water (1: 1: 2) wash-out; Collect flow point, each flow point of collecting is a developping agent with methyl alcohol-acetonitrile-water (1: 1: 2), through thin-layer chromatography aluminium foil reverse phase silica gel plate development; All visible tangible yellow color spot after each flow point launches; And uv lamp (254nm) is apparent blackening down, sulfuric acid ethanol displaing yellow, but except that above-mentioned yellow color spot, go back visible two other spot; But all not obvious, and separate fully with principal spot.With above-mentioned after thin-layer chromatography launches principal spot R
fValue is that 0.67 flow point is merged into component 1, principal spot R
fValue is that 0.56 flow point is merged into component 2, principal spot R
fValue is that 0.44 flow point is merged into component 3, called after Fr. III-1, Fr. III-2, Fr. III-3 successively respectively, wherein component Fr. III-1 55mg altogether; Separate through Sephadex LH-20 column chromatography; With methanol-eluted fractions, collect flow point, each flow point of collecting is a developping agent with methyl alcohol-acetonitrile-water (1: 1: 2); Through thin-layer chromatography aluminium foil reverse phase silica gel plate development, visible R
fValue is the flow point of 0.67 single spot, with above-mentioned flow point merge Camellia nitidissima Chi flavone glycosides A (25mg).
The structure of embodiment 2 Camellia nitidissima Chi flavone glycosides As is identified
Comprehensive use analytical technique of mass spectrum (ESI-MS), nmr analysis (
1H-NMR,
13C-NMR, HMBC, HMQC) etc. technology the structure of matter is analyzed.Like Fig. 6 Camellia nitidissima Chi flavone glycosides A
1H-NMR composes entirely; Like Fig. 7 Camellia nitidissima Chi flavone glycosides A
13C-NMR composes entirely; HMBC spectrum like Fig. 8 Camellia nitidissima Chi flavone glycosides A; High resolution positive ion electrospray ionization mass spectrum like Fig. 9 Camellia nitidissima Chi flavone glycosides A.
Measure the result: the Camellia nitidissima Chi flavone glycosides A: yellow crystal (methyl alcohol), m.p.233-235 ℃, the ultraviolet full wavelength scanner shows λ
Max(methyl alcohol) is respectively 369,337 and 254nm, and HR-ESI-MS provides quasi-molecular ion peak 627.1425 [M+H]
+, in conjunction with
1H NMR with
13C NMR infers that its molecular formula is C
30H
26O
15 1H NMR (500MHz, CD
3OD) and
13C NMR (125MHz, CD
3OD) data and document
[1,2]Contrast is judged that the compound parent nucleus is a Quercetin, and is contained 1 coffee acyl and 1 glucone.
1The coupling constant of glucose anomeric proton signal is 7.4Hz in the H NMR spectrum, explains that this glucose is beta comfiguration.(J=15.8), (1H, d J=15.8) are 2 trans olefins hydrogen signals to δ 7.47 for 1H, d at δ 6.24
[3]It is long-range relevant that the HMBC spectrum shows that glucose terminal hydrogen δ 5.08 and Quercetin C-7 (δ 164.2) have, and proves that the C-7 position is connected with glucose.The carbonyl C-9 of the H-6a of glucose (δ 4.61), H-6b (δ 4.31) and trans coffee acyl " ' (δ 169.1) have long-range relevant, explain glucose 6 " OH becomes ester with trans coffee acyl.Utilize
1H-NMR with
13The C-NMR spectrum has belonged to the signal of hydrogen and carbon, and the signal ownership is seen table 1.
Table 1 Camellia nitidissima Chi flavone glycosides A
1H-with
13C-NMR data (CD
3OD)
At SciFinder Scholar data base querying, the structure of to sum up analyzing this compound is Quercetin-7-O-(6 " O-E-coffee acyl)-β-D-glucoside, is a new compound, called after Camellia nitidissima Chi flavone glycosides A through structural formula and systematic naming method.Have following structural formula:
1 cell cultures
Human lymphoma leukemia cell line U937 obtains from Human Sciences Research Resource Bank (Japanese human NSFC, Tokyo, Japan), is incubated in the RPMI RPMI-1640 that contains 10% hot deactivation calf serum 37 ℃, 5%CO
2, cultivate under the saturated humidity condition.
2 Camellia nitidissima Chi flavone glycosides As are to the influence of U937 cell proliferation
Collect the U937 cell of logarithmic phase, be diluted to 1 * 10
5The cells/mL single cell suspension is inoculated in 96 well culture plates, every hole 180 μ L; If blank control group, control group, drug-treated group, wherein every group of Camellia nitidissima Chi flavone glycosides A 20 μ L that add different concns of drug-treated group make Camellia nitidissima Chi flavone glycosides A final concentration be respectively 10,25,50,100,200 and 400 μ M; Control group replaces medicine with DMSO, and blank control group replaces cell suspension and medicine with nutrient solution, and each group is established 6 parallel holes; Put 37 ℃, 5%CO
2Cultivate 24h under the condition.The every hole of 4h adds 20 μ L MTT solution (5mg/mL) before cultivating end, continues to cultivate 4h, abandons supernatant, adds the abundant dissolving crystallized thing of 200 μ L DMSO, puts and surveys 490nm place's absorbance (OD value) on the ELIASA.Average with the cellular control unit survival rate is 100%, is calculated as follows drug-treated group cell survival rate:
Average * 100% of drug-treated group cell survival rate=drug-treated group OD value/control group OD value
The result is with means standard deviation (n=6) expression of cell survival rate, and the result shows that the Camellia nitidissima Chi flavone glycosides A is to the inhibited (see figure 1) of U937 cell proliferation, its IC
50Be 140.1 μ M.
3Annexin V-FITC/PI pair is dyed method Flow cytometry Camellia nitidissima Chi flavone glycosides A and induces the U937 apoptosis
Adopt annexin V (Annexin the V) (Immunotech of propidium iodide (PI) and fluorescein isothiocyanate (FITC) mark in the flow cytometry; Marseille France) detects the expression of early apoptosis end point indicator phosphatidylserine (Phosphatidylserine).The U937 cell of taking the logarithm vegetative period is diluted to 1 * 10
6The cells/mL single cell suspension with the drug-treated cell of different concns, is cultivated 6h at 37 ℃.Centrifugal collecting cell is with 4 ℃ of PBS flushing backs 1, the centrifugal 3min of 200rpm.Centrifugal back cell is diluted to 10 with the binding buffer liquid that Annexin V-FITC test kit has
5Cells/mL, the Annexin V and the 5 μ L PI that add 5 μ L FITC marks to above-mentioned cell suspending liquid, mixing.After 4 ℃ of lucifuges were cultivated 10min, (Miami FL) analyzed for Epics XL, Beckman-Coulter with flow cytometer.
The result shows, after the Camellia nitidissima Chi flavone glycosides A is handled, and U937 early apoptosis of cells rate significantly raise (Fig. 2, Fig. 3)
4 Camellia nitidissima Chi flavone glycosides As are to the activation of caspase-3
Use CaspGLOW
TM(MBL, Nagoya Japan) detect caspase-3 activity in the cell to activation Caspase-3 original position staining kit.With the caspase-3 inhibition substrate polypeptide DEVD (FITC-DEVD-FMK) of FITC mark fluorized marking as original position (in situ).Because the permeability of cell membrane of FITC-DEVD-FMK is fine, in apoptotic cell, can combine with activatory caspase-3 is irreversible, can use Flow cytometry in conjunction with back FITC institute fluorescent signal emitted, the amount of its fluorescence volume reflection activation capase-3.Add different concns drug-treated cell, cultivate 6h at 37 ℃, collecting cell is then with cell solution (1 * 10
6Cells/ml) branch installs in the sample hose, adds 1 μ LFITC-DEVD-FMK in per 300 μ L cell solutions, cultivates 0.5h for 37 ℃.At 3000rpm eccentric visual cell 5min, remove supernatant, cell is dispersed in again behind the lavation buffer solution of 0.5mL centrifugal.Again cell is dispersed in the 500 μ L lavation buffer solutions again, on flow cytometer, analyzes with the FL-1 passage.
The result shows, compares the Camellia nitidissima Chi flavone glycosides A with control group and can significantly activate caspase-3 activity (Fig. 4).
Induce the U937 apoptosis whether to realize in order to confirm the Camellia nitidissima Chi flavone glycosides A through Caspase dependence approach; A kind of general Caspase suppressor factor Z-VAD and Camellia nitidissima Chi flavone glycosides A join in the U937 cell culture fluid simultaneously; Behind 50 μ M Z-VAD processing U937 cell 1h; Add 200 μ M Camellia nitidissima Chi flavone glycosides As again and handle U937 cell 6h, detect percentage of cell apoptosis then.The result shows that the Camellia nitidissima Chi flavone glycosides A induces the apoptotic activity of U937 significantly to be suppressed (Fig. 5).
Prove through above-mentioned experiment: the Camellia nitidissima Chi flavone glycosides A can suppress U937 cell proliferation, and induces the U937 apoptosis through Caspase dependence approach, and promptly this compound has the activity of tangible inducing apoptosis of tumour cell.
The purposes of Camellia nitidissima Chi flavone glycosides A on the preparation antitumor drug
According to the present invention, new compound Camellia nitidissima Chi flavone glycosides A can be used as bulk drug or forms compound preparation with other drug, and processes formulations such as tablet, capsule, liposome and slow, controlled release preparation, is used for exploitation, the development of treating malignant tumor medicine.
Reference:
[1]S.Bloor，J.Bradley，D.Lewis，K.Davies，Identification?of?flavonol?and?anthocyanin?metabolites?in?leaves?of?Petunia‘Mitchill’and?its?LC?transgenic.，Phytochemistry.49(1998)1427-1430.
[2]S.Vitalini，A.Braca，G.Fico，Study?on?secondary?metabolite?content?of?Helleborus?niger?L.leaves，Fitoterapia.82(2011)152-154.
[3]K.G.Fiasson，J.L.Fiasson，H.Waton，Quercetin?glycosides?from?European?aquatic?Ranunculus?species?of?subgenus?Batrachium，Phytochemistry.45(1997)1063-1067.
Claims (8)
1. Camellia nitidissima Chi flavone glycosides A is characterized in that: have following structural formula:
It is yellow crystal at normal temperatures, and fusing point is 233～235 ℃.
2. the preparation method of Camellia nitidissima Chi flavone glycosides A as claimed in claim 1 is characterized in that: comprises the steps,
A. the method for preparing the acetic acid ethyl ester extract of golden flower camellia 95% ethanol extraction:
With the golden flower camellia with 95% industrial alcohol heating and refluxing extraction, solid-liquid ratio 1g: 2～5ml, 75～85 ℃ of temperature after ethanol extraction is condensed into medicinal extract, add the entry suspendible in medicinal extract, use ethyl acetate extraction, reclaim acetic acid ethyl ester extract;
B. with the acetic acid ethyl ester extract of silica gel column chromatography separating step A gained; Volume ratio with chloroform-methanol is 100: 1,50: 1,30: 1,20: 1,15: 1,10: 1 and 5: 1 respectively; Carry out gradient elution; With the flow point that elutes, to know the back through silica gel thin-layer plate inspection and merge identical flow point, collection is by chloroform: the flow point under 10: 1 wash-outs of the volume ratio of methyl alcohol is subsequent use;
C. the flow point of step B gained; Separate through the ODS reversed-phase silica gel column chromatography; Elutriant is a methyl alcohol: acetonitrile: the volume ratio of water is 1: 1: 2, and through thin-layer chromatography aluminium foil reverse phase silica gel plate development, developping agent is a methyl alcohol: acetonitrile with each flow point of collecting: the volume ratio of water is 1: 1: 2; Spot with displaing yellow after flow point launches is a principal spot, principal spot R
fValue be 0.67 flow point merge, subsequent use;
D. the flow point that obtained of step C separates through Sephadex LH-20 column chromatography, and with methanol-eluted fractions, through thin-layer chromatography aluminium foil reverse phase silica gel plate development, developping agent is a methyl alcohol: acetonitrile with each flow point of collecting: the volume ratio of water is 1: 1: 2, and flow point launches the back with spot R
fValue is that 0.67 single flow point is a finished product.
3. method according to claim 2 is characterized in that: in the steps A, in medicinal extract, add the water suspendible of 2～5 times of quality, and with 2 times of amount ethyl acetate extractions.
4. method according to claim 2 is characterized in that: in the steps A, said solid-liquid ratio is 1g: 3ml.
5. method according to claim 2 is characterized in that: in the steps A, said temperature is 80 ℃.
6. according to the described arbitrary method of claim 2～5, it is characterized in that: in the steps A, the number of times of said heating and refluxing extraction is 3 times, each 2 hours.
7. the purposes of Camellia nitidissima Chi flavone glycosides A as claimed in claim 1 on the preparation antitumor drug.
8. purposes as claimed in claim 7 is characterized in that: the medicine that the Camellia nitidissima Chi flavone glycosides A of effective dose is mixed and processes appropriate dosage forms as effective constituent with pharmaceutical excipient.
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| CN109464541A (en) * | 2019-01-15 | 2019-03-15 | 北京工商大学 | A kind of extracting method and its neuroprotection purposes of golden camellia tea extract |
| CN110776542A (en) * | 2019-11-28 | 2020-02-11 | 海南大学 | Compound extracted from golden camellia and application of compound in preparing anti-tumor medicine |
| CN110818755A (en) * | 2019-11-28 | 2020-02-21 | 海南大学 | Compound extracted from golden camellia and pharmaceutical application thereof |
| CN110818755B (en) * | 2019-11-28 | 2022-11-11 | 海南大学 | Compound extracted from golden camellia and pharmaceutical application thereof |
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