CN102617672B - 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 thering is preparation and the Application Areas of the new constituent of medical active in natural phant, be specifically related to a kind of Camellia nitidissima Chi flavone glycosides A, Preparation Method And The Use.
Background technology
Camellia nitidissima Chi (Camellia chrysantha (Hu) Tuyama) is Theaceae (Theaceae) Camellia (Camellia Linn.) Camellia nitidissima Chi group (Section chrysantha Chang) plant, is mainly distributed in Yongning county, Guangxi.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 having blood in stool, menoxenia etc.In early days the research of Camellia nitidissima Chi is mainly concentrated on to plant morphology, plant taxonomy aspect, in recent years the research of anti-oxidant, the reducing blood-fat of Camellia nitidissima Chi, anti-liver precancerous lesion isoreactivity is appeared in the newspapers, in addition the research of Camellia nitidissima Chi leaf chemical composition is also had to report.
Contriver finds in to the series of studies of Camellia Plants antitumor activity component, and 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 basic substance and the mechanism of action thereof of golden flower camellia anti-tumor activity, the present invention is studied the activity of the chemical composition of golden flower camellia and inducing apoptosis of tumour cell thereof.
Summary of the invention
The present invention has carried out separation to the chemical composition of golden flower camellia, and from the acetic acid ethyl ester extract of golden flower camellia 95% ethanol extraction, separated monomeric compound and the cut obtaining shows to be mainly flavones ingredient through qualitative, quantitative assay.Infer thus, the flavones ingredient in golden flower camellia may be the important substance basis of its anti-tumor activity.Therefore, the present invention is by flavones ingredient in golden flower camellia is carried out to isolation identification, and take inducing apoptosis of tumour cell as activity index, therefrom finds and screen new type antineoplastic medicine.Therefore, the present invention finds the monomer component with anti-tumor activity---Camellia nitidissima Chi flavone glycosides A from 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, is to provide 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:
By golden flower camellia with 95% industrial alcohol heating and refluxing extraction, solid-liquid ratio 1g: 2～5ml, 75～85 ℃ of temperature, ethanol extraction is condensed into after medicinal extract, in medicinal extract, adds water suspendible, is extracted with ethyl acetate, and reclaims acetic acid ethyl ester extract;
B. with the acetic acid ethyl ester extract of silica gel column chromatography separating step A gained, the volume ratio of chloroform-methanol of take is respectively 100: 1,50: 1,30: 1,20: 1,15: 1,10: 1 and 5: 1, carry out gradient elution, by the flow point eluting, after silica gel thin-layer plate inspection is known, merge identical flow point, collect standby by the flow point under 10: 1 volume ratio wash-outs of chloroform-methanol;
C. the flow point of step B gained, separated through ODS reversed-phase silica gel column chromatography, elutriant is methyl alcohol: acetonitrile: the volume ratio of water is 1: 1: 2, by each flow point of collecting through thin-layer chromatography aluminium foil reverse phase silica gel plate development, developping agent is methyl alcohol: acetonitrile: the volume ratio of water is 1: 1: 2, the spot of displaing yellow of take after flow point launches is principal spot, principal spot R
fvalue be 0.67 flow point merge, standby;
D. the flow point that step C obtains is separated through Sephadex LH-20 column chromatography, and with methanol-eluted fractions, by each flow point of collecting, through thin-layer chromatography aluminium foil reverse phase silica gel plate development, developping agent is methyl alcohol: acetonitrile: the volume ratio of water is 1: 1: 2, after flow point launches with spot R
fvalue is that 0.67 single flow point is finished product.
Concrete, in aforesaid method, in its steps A, in medicinal extract, add the water suspendible of 2～5 times of quality, and extract by 2 times of amount ethyl acetate.
Concrete, in aforesaid method, in its steps A, described solid-liquid ratio is 1g: 3ml.
Concrete, in aforesaid method, in its steps A, described temperature is 80 ℃.
Concrete, in aforesaid method, in its steps A, the number of times of described heating and refluxing extraction is 3 times, each 2 hours.
Another aspect of the present invention is, above-mentioned Camellia nitidissima Chi flavone glycosides A is in the purposes of preparing on antitumor drug.
Above-mentioned purposes, concrete, be by mix and make the medicine of appropriate dosage forms with pharmaceutical excipient using the Camellia nitidissima Chi flavone glycosides A of effective dose as effective constituent.
In embodiment in the present invention, having announced, by structural formula and systematic naming method, at SciFinder Scholar data base querying, proved that the above-mentioned Camellia nitidissima Chi flavone glycosides A of gained has novel structure, is new compound.
In embodiment, the activity of above-mentioned Camellia nitidissima Chi flavone glycosides A is detected: with the Camellia nitidissima Chi flavone glycosides A of different concns, intervene human leukemia U937 cell, adopt flow cytometer to detect it to apoptotic impact.
Through investigating, find, Camellia nitidissima Chi flavone glycosides A can suppress U937 cell proliferation, adopts Annexin V-FITC/PI couple to dye method Flow cytometry early apoptosis of cells rate result and shows, after Camellia nitidissima Chi flavone glycosides A is processed, U937 early apoptosis of cells rate significantly raises.
Accompanying drawing explanation
Fig. 1. the impact of Camellia nitidissima Chi flavone glycosides A on 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 processed U937 cell 24h, and mtt assay detects U937 cell survival rate, and result represents with the means standard deviation (n=6) of cell survival rate.Result shows, along with Camellia nitidissima Chi flavone glycosides A concentration increases, the survival rate of U937 cell obviously declines.
Fig. 2. apoptotic Annexin V-FITC/PI couple of Camellia nitidissima Chi flavone glycosides A induction U937 dyes analysis: Camellia nitidissima Chi flavone glycosides A is processed U937 cell 6h, flow cytometer Annexin V-FITC/PI double-staining detects apoptosis rate, and result represents with the means standard deviation (n=6) of apoptosis rate.Result shows, with the Camellia nitidissima Chi flavone glycosides A that concentration is 100,200 μ M, processes after U937 cell, and U937 apoptosis rate obviously raises.
Apoptotic Annexin V-FITC/PI couple of Fig. 3 Camellia nitidissima Chi flavone glycosides A induction U937 dyes analysis: Camellia nitidissima Chi flavone glycosides A (200 μ M) is processed U937 cell 6h, and flow cytometer Annexin V-FITC/PI double-staining detects apoptosis scatter diagram.Result shows, compares Camellia nitidissima Chi flavone glycosides A (200 μ M) process after U937 cell with control group, and U937 apoptosis rate obviously raises.
Fig. 4. the impact on Caspase-3 activation during Camellia nitidissima Chi flavone glycosides A induction U937 apoptosis: Camellia nitidissima Chi flavone glycosides A is processed U937 cell 6h, uses CaspGLOW
tMthe Caspase-3 of activation in activation Caspase-3 native staining kit detection cell, result represents with the means standard deviation (n=6) of Caspase-3 volume of activation.Result shows, with the Camellia nitidissima Chi flavone glycosides A that concentration is 100,200 μ M, processes after U937 cell, and in U937 cell, the Caspase-3 of activation obviously increases.
Fig. 5. the impact on Caspase-3 activation during Camellia nitidissima Chi flavone glycosides A induction U937 apoptosis: process after U937 cell 1h with 50 μ M Z-VAD, then add 200 μ M Camellia nitidissima Chi flavone glycosides As to process U937 cell 6h, then detect percentage of cell apoptosis.Result shows that the apoptotic activity of Camellia nitidissima Chi flavone glycosides A induction U937 is significantly suppressed.
Fig. 6. Camellia nitidissima Chi flavone glycosides A
1h-NMR composes entirely;
Fig. 7. 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 the present invention of those of ordinary skill in the art's comprehend, but does not limit the present invention in any way.In the mode of specific embodiment, the present invention is further illustrated below.
The various chemical reagent that use in the embodiment of the present invention if no special instructions, all obtain by conventional commercial sources;
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: Beijing tech electric light instrument plant;
Jasco V-560 type ultraviolet-visible spectrophotometer: Japan Spectroscopy Corporation;
AVANCE500Hz type nuclear magnetic resonance analyser: Switzerland Bruker company, TMS is interior mark;
UPLC/Q-TOF-MS system: U.S. Waters company;
Human lymphoma leukemia cell line U937:Human Sciences Research Resource Bank Japan mankind NSFC, Tokyo, Japan;
The preparation of embodiment 1 Camellia nitidissima Chi flavone glycosides A
The preparation method with the Camellia nitidissima Chi flavone glycosides A of inducing apoptosis of tumour cell activity of the present invention, the method comprises the steps:
A. the acetic acid ethyl ester extract of preparing golden flower camellia 95% ethanol extraction
Camellia nitidissima Chi dried floral 3kg, pulverizes, and by the 95% industrial alcohol heating and refluxing extraction of 9L, 80 ℃ of temperature, extract 3 times, each 2h, and concentrating under reduced pressure after filtering, obtains alcohol extract 588g.Get alcohol extract 500g with after 1500mL hot distilled water suspendible, by the ethyl acetate of 2 times of amounts, repeatedly extract, reclaim after solvent, partly 110g and water layer are partly total to 387g to the ethyl acetate obtaining altogether.
B. with the separated above-mentioned acetic acid ethyl ester extract of silica gel column chromatography
200-300 order silica gel with 1500g, wet method dress post, by the ethyl acetate part silica gel mixed sample of 75g, dry method loading, 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 stream is respectively with 1
#, 2
#, 3
#deng in turn numbering.Wherein, with chloroform-methanol (100: 1) wash-out, obtain flow point 1
#～110
#, with chloroform-methanol (50: 1) wash-out, obtain flow point 111
#～146
#, with chloroform-methanol (30: 1) wash-out, obtain flow point 147
#～200
#, with chloroform-methanol (20: 1) wash-out, obtain flow point 201
#～222
#, with chloroform-methanol (15: 1) wash-out, obtain flow point 223
#～249
#, with chloroform-methanol (10: 1) wash-out, obtain flow point 250
#～342
#, with chloroform-methanol (5: 1) wash-out, obtain flow point 343
#～440
#.Each flow point of collecting merges identical flow point after 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 after examining knowledge by silica gel thin-layer plate, merging same stream divides the component Fr. III (472mg) obtaining separated through ODS reversed-phase silica gel column chromatography, with methyl alcohol-acetonitrile-water (1: 1: 2) wash-out, collect flow point, it is developping agent that each flow point of collecting be take methyl alcohol-acetonitrile-water (1: 1: 2), through thin-layer chromatography aluminium foil reverse phase silica gel plate development, a visible obvious yellow color spot after each flow point launches, and the lower aobvious blackening of ultraviolet lamp (254nm), sulfuric acid ethanol displaing yellow, but be also shown in two other spot except above-mentioned yellow color spot, but all not obvious, and separate completely with principal spot.By 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, difference is called after Fr. III-1, Fr. III-2, Fr. III-3 successively, wherein component Fr. III-1 is total to 55mg, separated through Sephadex LH-20 column chromatography, with methanol-eluted fractions, collect flow point, it is developping agent that each flow point of collecting be take 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, and above-mentioned flow point is merged to obtain to Camellia nitidissima Chi flavone glycosides A (25mg).
The Structural Identification of embodiment 2 Camellia nitidissima Chi flavone glycosides As
Integrated using analytical technique of mass spectrum (ESI-MS), nmr analysis (
1h-NMR,
13c-NMR, HMBC, HMQC) etc. technology the structure of matter is analyzed.As Fig. 6 Camellia nitidissima Chi flavone glycosides A
1h-NMR composes entirely; As Fig. 7 Camellia nitidissima Chi flavone glycosides A
13c-NMR composes entirely; As the HMBC spectrum of Fig. 8 Camellia nitidissima Chi flavone glycosides A; As the high resolution positive ion electrospray ionization mass spectrum of Fig. 9 Camellia nitidissima Chi flavone glycosides A.
Measurement result: Camellia nitidissima Chi flavone glycosides A: yellow crystal (methyl alcohol), m.p.233-235 ℃, 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 and
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, judgement compound parent nucleus is Quercetin, and contains 1 coffee acyl and 1 glucosyl group.
1in H NMR spectrum, the coupling constant of glucose anomeric proton signal is 7.4Hz, illustrates that this glucose is beta comfiguration.At δ 6.24 (1H, d, J=15.8), δ 7.47 (1H, d, J=15.8) is 2 trans olefins hydrogen signals
[3].HMBC spectrum shows that glucose terminal hydrogen δ 5.08 has distant relation with Quercetin C-7 (δ 164.2), proves that 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 " there is distant relation ' (δ 169.1), illustrate glucose 6 " OH becomes ester with trans coffee acyl.Utilize
1h-NMR and
13c-NMR spectrum has belonged to the signal of hydrogen and carbon, and signal belongs in Table 1.
Table 1 Camellia nitidissima Chi flavone glycosides A
1h-and
13c-NMR data (CD
3oD)
By structural formula and systematic naming method, 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-Glucose glycosides, is a new compound, called after Camellia nitidissima Chi flavone glycosides A.There is following structural formula:
1 cell cultures
Human lymphoma leukemia cell line U937 is from Human Sciences Research Resource Bank (Japanese mankind NSFC, Tokyo, Japan) obtain, be incubated in the RPMI RPMI-1640 that contains 10% hot deactivation calf serum 37 ℃, 5%CO
2, under saturated humidity condition, cultivate.
2 impacts of Camellia nitidissima Chi flavone glycosides A on U937 cell proliferation
The U937 cell of collecting logarithmic phase, is diluted to 1 * 10
5cells/mL single cell suspension, be inoculated in 96 well culture plates, every hole 180 μ L, establish blank group, control group, drug treating group, wherein every group of Camellia nitidissima Chi flavone glycosides A 20 μ L that add different concns of drug treating 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 group replaces cell suspension and medicine with nutrient solution, and each group is established 6 parallel holes, put 37 ℃, 5%CO
2under condition, cultivate 24h.Cultivation finishes the every hole of front 4h and adds 20 μ L MTT solution (5mg/mL), continue to cultivate 4h, abandons supernatant liquor, adds the abundant dissolving crystallized thing of 200 μ L DMSO, puts and in microplate reader, surveys 490nm place absorbance (OD value).The average of cellular control unit survival rate of take is 100%, is calculated as follows drug treating group cell survival rate:
Average * 100% of drug treating group cell survival rate=drug treating group OD value/control group OD value
Result is with means standard deviation (n=6) expression of cell survival rate, and result shows that Camellia nitidissima Chi flavone glycosides A is to U937 cell proliferation inhibited (seeing Fig. 1), its IC
50be 140.1 μ M.
The two method Flow cytometry Camellia nitidissima Chi flavone glycosides A induction U937 apoptosis that dye of 3Annexin V-FITC/PI
In flow cytometry, adopt annexin V (Annexin the V) (Immunotech of propidium iodide (PI) and fluorescein isothiocyanate (FITC) mark, Marseille, France) detect the expression of early apoptosis end point indicator phosphatidylserine (Phosphatidylserine).The U937 cell of taking the logarithm vegetative period, is diluted to 1 * 10
6cells/mL single cell suspension, with the drug treating cell of different concns, cultivates 6h at 37 ℃.Centrifugal collecting cell, the centrifugal 3min of 1,200rpm after rinsing with 4 ℃ of PBS.Centrifugal rear cell with Annexin V-FITC test kit with binding buffer liquid be diluted to 10
5cells/mL, adds the Annexin V of 5 μ L FITC marks and 5 μ L PI to above-mentioned cell suspending liquid, mixes.4 ℃ of lucifuges, cultivate after 10min, with flow cytometer (Epics XL, Beckman-Coulter, Miami, FL), analyze.
Result shows, after Camellia nitidissima Chi flavone glycosides A is processed, and U937 early apoptosis of cells rate significantly raise (Fig. 2, Fig. 3)
The activation of 4 Camellia nitidissima Chi flavone glycosides As to caspase-3
Use CaspGLOW
tMactivation Caspase-3 native staining test kit (MBL, Nagoya, Japan) detects caspase-3 activity in cell.Using the caspase-3 inhibition substrate polypeptide DEVD (FITC-DEVD-FMK) of FITC mark as the fluorized marking of original position (in situ).Because the permeability of cell membrane of FITC-DEVD-FMK is fine, in apoptotic cell can with the irreversible combination of caspase-3 of activation, the fluorescent signal of launching in conjunction with rear FITC can use Flow cytometry, the reflection of its fluorescence volume activates the amount of capase-3.Add different concns drug treating cell, at 37 ℃, cultivate 6h, collecting cell, then by cell solution (1 * 10
6cells/ml) divide and install in sample hose, in every 300 μ L cell solutions, add 1 μ LFITC-DEVD-FMK, cultivate 0.5h for 37 ℃.At 3000rpm eccentric cell 5min, remove supernatant liquor, cell is dispersed in again after the lavation buffer solution of 0.5mL centrifugal.Again cell is dispersed in 500 μ L lavation buffer solutions again, on flow cytometer, with FL-1 passage, analyzes.
Result shows, compares Camellia nitidissima Chi flavone glycosides A can significantly activate caspase-3 activity (Fig. 4) with control group.
In order to confirm what whether Camellia nitidissima Chi flavone glycosides A induction U937 apoptosis was realized through Caspase Dependent, a kind of general Caspase inhibitor Z-VAD and Camellia nitidissima Chi flavone glycosides A join in U937 cell culture fluid simultaneously, with 50 μ M Z-VAD, process after U937 cell 1h, add again 200 μ M Camellia nitidissima Chi flavone glycosides As to process U937 cell 6h, then detect percentage of cell apoptosis.Result shows the Camellia nitidissima Chi flavone glycosides A induction apoptotic activity of U937 significantly suppressed (Fig. 5).
Through above-mentioned, experimental results show that: Camellia nitidissima Chi flavone glycosides A can suppress U937 cell proliferation, and by Caspase Dependent induction U937 apoptosis, this compound has the activity of obvious inducing apoptosis of tumour cell.
Camellia nitidissima Chi flavone glycosides A is in the purposes of preparing on 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 makes the formulations such as tablet, capsule, liposome and slow, controlled release preparation, for exploitation, the development for the treatment of 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 (4)
1. a preparation method for Camellia nitidissima Chi flavone glycosides A, is characterized in that: described Camellia nitidissima Chi flavone glycosides A, has following structural formula:
It is yellow crystal at normal temperatures, and fusing point is 233～235 ℃,
Described Camellia nitidissima Chi flavone glycosides A preparation method comprises the steps,
A. the method for preparing the acetic acid ethyl ester extract of golden flower camellia 95% ethanol extraction:
By golden flower camellia with 95% industrial alcohol heating and refluxing extraction, solid-liquid ratio 1g:2～5ml, 75～85 ℃ of temperature, ethanol extraction is condensed into after medicinal extract, in medicinal extract, adds water suspendible, is extracted with ethyl acetate, and reclaims acetic acid ethyl ester extract;
B. with the acetic acid ethyl ester extract of silica gel column chromatography separating step A gained, take respectively the chloroform of volume ratio as 100:1,50:1,30:1,20:1,15:1,10:1 and 5:1: methyl alcohol is developping agent, carry out gradient elution, by the flow point eluting, after silica gel thin-layer plate inspection is known, merge identical flow point, collect the chloroform that is 10:1 by volume ratio: the flow point under the elutriant wash-out of methyl alcohol is standby;
C. the flow point of step B gained, separated through ODS reversed-phase silica gel column chromatography, elutriant is that volume ratio is the methyl alcohol of 1:1:2: acetonitrile: water, by each flow point of collecting through thin-layer chromatography aluminium foil reverse phase silica gel plate development, developping agent is that volume ratio is the methyl alcohol of 1:1:2: acetonitrile: water, the spot of displaing yellow of take after flow point launches is principal spot, principal spot R
f value be 0.67 flow point merge, standby;
D. the flow point that step C obtains is separated through Sephadex LH-20 column chromatography, and with methanol-eluted fractions, by each flow point of collecting, through thin-layer chromatography aluminium foil reverse phase silica gel plate development, developping agent is that volume ratio is the methyl alcohol of 1:1:2: acetonitrile: water, after flow point launches with spot R
f value is that 0.67 single flow point is finished product.
2. method according to claim 1, is characterized in that: in steps A, described solid-liquid ratio is 1g:3ml.
3. method according to claim 1, is characterized in that: in steps A, described temperature is 80 ℃.
4. according to the either method described in claim 1～3, it is characterized in that: in steps A, the number of times of described heating and refluxing extraction is 3 times, each 2 hours.
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| CN103951723B (en) * | 2014-05-15 | 2015-12-30 | 大连大学 | A kind of Camellia nitidissima Chi flavonoid glycoside and its production and use |
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| 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 |
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| CN101270139A (en) * | 2008-04-17 | 2008-09-24 | 大连大学 | Common camellia flavone glycosides A with estrogen liveness, preparation method and application thereof |
| CN101284855A (en) * | 2008-05-29 | 2008-10-15 | 浙江大学 | Preparation method and use of camellin A and camellin B |
| FR2944794A1 (en) * | 2009-04-28 | 2010-10-29 | Rocher Yves Biolog Vegetale | Preparing flavonoid glycoside oligomer comprises solubilizing flavonoid glycoside in mixture comprising glycerin, ethanol and aqueous solution to obtain solution having flavonoid and polymerizing solubilized flavonoid glycoside by laccase |
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| JP5620660B2 (en) * | 2009-09-03 | 2014-11-05 | 学校法人近畿大学 | Fat metabolism improving agent, medicine and food containing the fat metabolism improving agent, and novel flavonoid compound |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101270139A (en) * | 2008-04-17 | 2008-09-24 | 大连大学 | Common camellia flavone glycosides A with estrogen liveness, preparation method and application thereof |
| CN101284855A (en) * | 2008-05-29 | 2008-10-15 | 浙江大学 | Preparation method and use of camellin A and camellin B |
| FR2944794A1 (en) * | 2009-04-28 | 2010-10-29 | Rocher Yves Biolog Vegetale | Preparing flavonoid glycoside oligomer comprises solubilizing flavonoid glycoside in mixture comprising glycerin, ethanol and aqueous solution to obtain solution having flavonoid and polymerizing solubilized flavonoid glycoside by laccase |
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| JP特开2011-51950A 2011.03.17 |
| Ying-Jun Zhang，等.Antiproliferative Activity of the Main Constituents from Phyllanthus emblica.《Biol. Pharm. Bull.》.2004,第27卷(第2期),251-255. * |
| 林华娟,等.金花茶茶花的化学成分及生理活性成分分析.《食品科技》.2010,第35卷(第10期),88-91. * |
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