CN105566264A - Livistona chinensis extract monomer, preparation method, and applications thereof - Google Patents
Livistona chinensis extract monomer, preparation method, and applications thereof Download PDFInfo
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- CN105566264A CN105566264A CN201610001351.3A CN201610001351A CN105566264A CN 105566264 A CN105566264 A CN 105566264A CN 201610001351 A CN201610001351 A CN 201610001351A CN 105566264 A CN105566264 A CN 105566264A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
- C07D307/77—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D307/78—Benzo [b] furans; Hydrogenated benzo [b] furans
- C07D307/79—Benzo [b] furans; Hydrogenated benzo [b] furans with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to carbon atoms of the hetero ring
- C07D307/80—Radicals substituted by oxygen atoms
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
- C07B2200/00—Indexing scheme relating to specific properties of organic compounds
- C07B2200/07—Optical isomers
Abstract
The invention discloses a livistona chinensis extract monomer, a preparation method, and applications thereof. The livistona chinensis extract monomer is obtained by extracting livistona chinensis by an organic solvent. The livistona chinensis extract monomer has the following functions: (1) inhibiting proliferation and/or growth of cervical cancer cells; (2) inhibiting the formation of clone of cervical cancer cells; (3) promoting the apoptosis of cervical cancer cells; (4) reducing the mitochondria membrane potential and damaging the integrity of membrane; (5) confining the cell period of cervical cancer cells in G1/S period; (6) increasing the expression level of cyclin CyclinA2 in the G1/S period and cyclin dependent kinase CDK2 protein, and at the same time increasing the expression level of P21 protein; (7) inhibiting the proliferation and/or growth of cervical cancer cells in naked rats; (8) treating cervical cancer. The invention provides a novel method for developing the pharmaceutical value of livistona chinensis, and livistona chinensis extract monomer has an important meaning for the treatment of cervical cancer.
Description
Technical field
The present invention relates to bioengineering field, particularly relate to a kind of Chinese fan palm extract monomer, its preparation method and its application.
Background technology
Chinese fan palm, belong to Palmae Livistona, Latin is called Livistonachinensis, likes environment that is warm, moistening, that face south, is distributed in tropical and subtropical region, and be commonly used to greening, in addition, Chinese fan palm also has pharmaceutical use.
Chinese fan palm is the tall and big arbor of a kind of perennial evergreen, and morphological specificity is as follows: go alone, and high 5-20 rice, diameter 20-30 centimetre, root often expands.Ye Kuo is that the kidney shape is fan-shaped, its diameter more than 1 meter, palmate drastic crack to middle part, sliver strip and lanceolar; The long 1-2 rice of petiole, both sides, bottom have ditch to sting.Inflorescence is coniform, is about 1 meter, and branch is many and evacuate; Calyx splits and becomes nearly pointed sliver to nearly base portion, and sliver has wide scariose edge; Have above ovary and deeply carve line, style is mutated into brill shape; Fruit chocolate ovalize, long 1.4-2.2 centimetre, diameter 1-1.2 centimetre; Seed is oval, long 1.5 centimetres, diameter 0.9 centimetre.
On tcm clinical practice, Chinese fan palm root has analgesic effect, and Chinese fan palm leaf has astringing to arrest bleeding anti-hidropoiesis, and Chinese fanpalm seed has pain relieving and the effect that is anticancer, the silt hemostasis that disappears that relieves internal heat.Its treatment leukemia conventional among the people, nasopharyngeal carcinoma, choriocarcinoma, esophagus cancer [Chen Yan, woods Xinhua, Li Shaoguang, Weng Shengmei, Yao Hong. the Anticancer Activity in vitro of Chinese fanpalm seed. Medical University Of Fujian's journal [J], 2008,2:93-95.; Zeng Chunhui Zheng Yang Ke writes a composition. the experimental study of the Contained Serum extracorporeal anti-tumor function of Chinese fanpalm seed. and Guangxi traditional Chinese medicine [J], 2007,30 (1): 58-59.].But there is no the report of other clinical application.
Cervical cancer is a kind of malignant tumour being only second to mammary cancer and large bowel cancer in female cancer patients, is the second common malignant tumour, is only second to mammary cancer in developing country.The concrete pathogenesis of cervical cancer it be unclear that, mainly relevant with the factors such as virus infection, transgenation, telomerase activation, hormone abnormality, malnutrition, immunologic dysfunction.At present, the treatment plan of cervical cancer mainly comprises excision and radiotherapy.Chemotherapy is an important treatment means, is specially adapted to late period and metastatic treatment.But in the process of chemotherapy, can there is resistance phenomenon in various degree in some cancer cells.In addition, the toxicity of most of chemotherapeutics is large, and after chemotherapy, a lot of untoward reaction appears in Most patients, and all these make chemotherapeutics apply and are subject to a definite limitation.Therefore, find a kind of newly effectively and the chemotherapeutics of low toxicity is significant.
Summary of the invention
Technical problem to be solved by this invention overcomes the deficiencies in the prior art, aims to provide a kind of Chinese fan palm extract monomer and its preparation method and application.
The technical scheme that Chinese fan palm extract monomer of the present invention adopts is that the structure of this monomer is as follows:
The technical scheme that the preparation method of the present invention's above-mentioned Chinese fan palm extract monomer adopts is: the method organic solvent extraction to be also separated with HPLC through silicagel column and to obtain Chinese fan palm extract monomer.
A preferred scheme is, the concrete steps of aforesaid method are:
(1) Chinese fan palm raw material is cleaned, dry, be crushed to 20-100 order, obtain dry powder;
(2) get the dry powder that step (1) obtains, add the aqueous ethanolic solution of 70%, leave standstill lixiviate 24 hours and pump around circuit under room temperature condition, the mass/volume of the dry powder wherein added and aqueous ethanolic solution is than being w/v=1:20;
(3) whole liquid step (2) obtained under the vacuum of 50 DEG C by organic solvent evaporation, obtain brown pulpous state crude extract, this crude extract is suspended in water, with chloroform extraction, said extracted thing is crossed 200-300 order silicagel column, with the composition on chloroform-methanol eluting silica gel post, namely obtain Chinese fan palm extract monomer by HPLC purifies and separates.
Further preferred scheme is, described Chinese fan palm raw material is the seed of Chinese fan palm, root or leaf.
The Chinese fan palm extract monomer prepared by aforesaid method is suppressing the application in cervical cancer cell.
Describedly be applied as following at least one:
(1) propagation and/or the growth of cervical cancer cell is suppressed;
(2) Clone formation of cervical cancer cell is suppressed;
(3) Hela Cell Apoptosis is promoted;
(4) reduce cervical cancer cell mitochondrial membrane potential and make film integrality impaired;
(5) by the Cyclin-dependent kinase of cervical cancer cell in the G1/S phase;
(6) raise the cyclin CyclinA2 of G1/S phase and the expression level of cell cycle dependent kinase CDK2 albumen, raise the expression level of P21 albumen simultaneously;
(7) cervical cancer cell also can be suppressed in nude mouse to breed and/or growth;
(8) cervical cancer is treated.
The invention has the beneficial effects as follows: the Chinese fan palm extract monomer that the present invention prepares significantly can suppress growth and the propagation of cervical cancer cell with time and dose-dependent mode; Significantly can reduce the mitochondrial membrane potential of cervical cancer cell and make that it is impaired, induced apoptosis sexual cell is dead; Significantly can block the expression level that cervical cancer cell rests on the G1/S phase, raises the expression level of CyclinA2 and the CDK2 albumen of G1/S phase, increases cancer suppressor protein P21; Also cervical cancer cell can be suppressed in nude mouse to breed and/or growth.The present invention is that the pharmaceutical use of exploitation Chinese fan palm opens new approach, has important value to the treatment of cervical cancer.
Accompanying drawing explanation
Fig. 1 is the structure iron of described Chinese fan palm extract monomer;
Fig. 2 is the result schematic diagram of MTT proliferation inhibition test;
Fig. 3 is the result schematic diagram of cell clonal formation experiment;
Fig. 4 is the result schematic diagram of the two transfect cell apoptosis rate of AnnexinV-PI;
Fig. 5 is the result schematic diagram of DAPI stained cells nuclear morphology;
Fig. 6 is the result schematic diagram that mitochondrial membrane potential detects;
Fig. 7 A is that Chinese fan palm extract affects schematic diagram to the cervical cancer cell cycle;
Fig. 7 B is that after the SiRNA adding CyclinA2, Chinese fan palm extract affects schematic diagram to the cervical cancer cell cycle;
Fig. 7 C is that after the inhibitor Flavopiridol adding CDK2, Chinese fan palm extract affects schematic diagram to the cervical cancer cell cycle;
Fig. 7 D is that Chinese fan palm extract affects schematic diagram to the relevant cell cyclin of cervical cancer cell;
Fig. 7 E be after adding SiRNA and Flavopiridol respectively Chinese fan palm extract cell cycle PROTEIN C yclinA2 and CDK2 affect schematic diagram;
Fig. 8 A is that Chinese fan palm extract affects schematic diagram on gross tumor volume in nude mouse;
Fig. 8 B is that Chinese fan palm extract affects schematic diagram on tumor weight in nude mouse.
Embodiment
Following embodiment is convenient to understand the present invention better, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.Test materials used in following embodiment, if no special instructions, is routine biochemistry reagent shop or company all can purchase available.Quantitative test in following examples, all arranges and repeats experiment for three times, results averaged.Cell cultures in embodiment all adopts the high sugared blood serum medium of DEME.
Hela cell (cervical cancer cell): purchased from China typical culture collection center (CCTCC), catalog number: 3142C0001000000009.Caski cell (cervical cancer cell): purchased from China typical culture collection center (CCTCC), catalog number: 3142C0001000000117.
The preparation of embodiment 1, Chinese fan palm extract
1, Chinese fan palm seed, root, leaf are cleaned, dry, are crushed to 60 orders (in practical application 20-100 order), obtain dry powder.
2, get the dry powder that 10kg step 1 obtains, add 200L70% aqueous ethanolic solution, under room temperature condition, leave standstill lixiviate 24 hours and pump around circuit.
3, by whole liquid of completing steps 2 under the vacuum of 50 DEG C by organic solvent evaporation, the brown pulpous state crude extract 1.8kg obtained, this crude extract is suspended in water, uses chloroform extraction.Said extracted thing is crossed 200-300 order silicagel column, with the composition on chloroform-methanol eluting silica gel post, can obtain 2-(3'-hydroxyl-5'-p-methoxy-phenyl)-3-methylol-7-methoxyl group-2,3-Dihydrobenzofuranes-5-carboxylic acid by HPLC purifies and separates, structure is shown in Fig. 1.In the dry powder in step 2, the concentration of Chinese fan palm extract is 10mg/mL.
Embodiment 2, Chinese fan palm extract suppress growth and the propagation of cervical cancer cell
Respectively Hela cell and Caski cell are tested as follows:
One, MTT proliferation inhibition test
By Hela cell suspension, (cell density is 1 × 10
4/ ml) be seeded in 96 orifice plates; Then Chinese fan palm extract prepared by embodiment 1 is added, make its final concentration in each hole be respectively 1.25 μ g/ml, 2.5 μ g/ml, 5 μ g/ml, 10 μ g/ml or each concentration of 20 μ g/ml(and 3 multiple holes are set), the blank replacing Chinese fan palm extract with equal-volume liquid nutrient medium is set, 37 DEG C of cultivations; Timing from adding Chinese fan palm extract, respectively at 24h, 48h(each treatment time, 3 multiple holes are set) often hole 20ul adds MTT solution (5mg/ml) afterwards, cultivate 4h for 37 DEG C, abandon supernatant, every hole adds the DMSO of 150 μ l, concussion 10min makes Viola crystallina all dissolve, and then surveys absorbance (OD490nm) in 490nm.Inhibiting rate=(OD of blank group
490nmthe OD of numerical value-experimental group
490nmnumerical value) OD of ÷ blank group
490nmnumerical value × 100%.
The inhibiting rate of each concentration different treatment time the results are shown in Figure 2 and table 1.
The inhibiting rate result (mean value) of table 1 each concentration process experimental group
Result shows, Chinese fan palm extract significantly suppress growth and the propagation of cervical cancer cell with time and dose-dependent mode.
Two, cell clonal formation experiment
By the logarithmic phase Caski cell suspension of configuration, (cell density is 1 × 10
3/ ml) be seeded to 6 orifice plate upper stratas (in each hole of 6 orifice plates, upper strata is 2 × DMEM substratum that 1ml contains the Chinese fan palm extract of 0.6g/100ml soft agar and embodiment 1 preparation, and lower floor is 2 × DMEM substratum that 1ml contains the Chinese fan palm extract of 1.2g/100ml soft agar and embodiment 1 preparation; The Chinese fan palm extract concentrations of the upper and lower is equal; In each hole, the final concentration of Chinese fan palm extract is respectively 1.25 μ g/ml, 2.5 μ g/ml, 5 μ g/ml, 10 μ g/ml or 20 μ g/ml, and each concentration arranges 3 multiple holes; The blank replacing Chinese fan palm extract with equal-volume substratum is set), 37 DEG C, cultivate 2 weeks, then fix with 1% paraformaldehyde and use 0.1% violet staining 15 minutes under 5%C02 and saturated humidity environment, counting clone's number is also taken pictures.
The results are shown in Figure 3.Chinese fan palm extract significantly suppress the Clone formation of cervical cancer cell in dose-dependent mode.
Embodiment 3, Chinese fan palm extract promote Hela Cell Apoptosis
Respectively Hela cell and Caski cell are tested as follows:
One, the two transfect cell apoptosis rate of AnnexinV-PI is analyzed
The Hela cell of taking the logarithm vegetative period to be seeded in 6 orifice plates (1 × 10
6individual/hole), after cell attachment, then Chinese fan palm extract prepared by embodiment 1 is added, make that its concentration in each hole is respectively 1.25 μ g/ml, 2.5 μ g/ml, 5 μ g/ml, 10 μ g/ml, each concentration of 20 μ g/ml(arranges 3 multiple holes), the blank replacing Chinese fan palm extract with equal-volume liquid nutrient medium is set, cultivates 24h for 37 DEG C; With sample collection 1 × 10 every after 0.25% tryptic digestion
6individual cell, with PBS buffer solution 2 times, add the re-suspended cell that 195 μ lAnnexinV-FITC binding buffer liquid are slowly soft, add 5 μ lAnnexinV-FITC, mix gently, room temperature lucifuge hatches 10 minutes.Centrifugal 5 minutes of 1000rpm, abandons supernatant, adds 190 μ lAnnexinV-FITC binding buffer liquid slowly soft re-suspended cell.Add 10 μ lPI staining fluids, mix gently, place drawer lucifuge.After 300 order nylon net filters, flow cytometer detects immediately.Experiment in triplicate, gets its average.
The results are shown in Figure 4, along with the rising of Chinese fan palm extract concentrations, the cell proportion of the AnnexinV-FITC positive increases gradually, illustrate Chinese fan palm extract induction of apoptotic generation and with dosage positive correlation.
Two, DAPI stained cells nuclear morphology is observed
The Hela cell of taking the logarithm vegetative period to be seeded in 6 orifice plates (1 × 10
6individual/hole), after cell attachment, then Chinese fan palm extract prepared by embodiment 1 is added, make that its concentration in each hole is respectively 5 μ g/ml, each concentration of 10 μ g/ml(arranges 3 multiple holes), the blank replacing Chinese fan palm extract with equal-volume liquid nutrient medium is set, cultivates 24h for 37 DEG C; Nutrient solution is abandoned in suction, with PBS buffer solution 2 times, fix 15min with 4% paraformaldehyde, with PBS buffer solution 2 times, to punch 5min with 0.1%Tritonx-100, with PBS buffer solution 2 times, add 100 μ LDAPI staining fluids, room temperature places 10min, absorbs DAPI staining fluid, with PBS buffer solution 3 times, then utilize fluorescence microscope cellular form.Apoptosis test kit used is purchased from Invitrogen company, and article No. is D1306.
The results are shown in Figure 5.In blank, nucleus presents circle, edge clear, and uniform coloring.After Chinese fan palm extract-treated, nucleus becomes irregular, and chromatin is fine and close dense dye, or is the fine and close dense dye of chunky shape.These results suggest that, Chinese fan palm extract is induction of the generation of Hela Cell Apoptosis.
Three, mitochondrial membrane potential detects
The Hela cell of logarithmic phase to be seeded in 6 orifice plates (1 × 10
6individual/hole), after cell attachment, then add Chinese fan palm extract prepared by embodiment 1, make that its concentration in hole is respectively 5 μ g/ml, 10 μ g/ml(arrange 3 multiple holes), the blank replacing Chinese fan palm extract with equal-volume liquid nutrient medium is set, cultivates 24h for 37 DEG C; Nutrient solution is abandoned in suction, with PBS buffer solution cell 2 times, adds 1ml liquid nutrient medium and 1mlJC-1 staining fluid, abundant mixing, hatches 20 minutes for 37 DEG C in cell culture incubator, washs 2 times with 1 × JC-1 dye solution of ice, add 2ml liquid nutrient medium, fluorescence microscopy Microscopic observation.Mitochondrial membrane potential detection kit used purchased from green skies company, article No.: C2006.
The results are shown in Figure 6.In blank group, cell can see the complete granular plastosome that red fluorescence marks, and show that cell mitochondrial film is complete, membrane potential is in normal value.After Chinese fan palm extract-treated, cell mitochondrial obviously sustains damage, and granular plastosome reduces, and mitochondrial membrane potential reduces, and fluorescence presents green.Result shows, the apoptosis of Chinese fan palm extract induction is by mitochondria pathway.
Embodiment 4, Chinese fan palm extract are on the impact of cervical cancer cell cycle and associated protein
A, use the flow cytometer showed cell cycle
The Hela cell of logarithmic phase to be seeded in 6 orifice plates (1 × 10
6individual/hole), after cell attachment, then Chinese fan palm extract prepared by embodiment 1 is added, make its concentration in each hole be respectively 1.25 μ g/ml, 2.5, each concentration of μ g/ml(arranges 3 multiple holes), the blank replacing Chinese fan palm extract with equal-volume liquid nutrient medium is set, cultivates 24h for 37 DEG C; Trypsin digestion cell, collects 1 × 10 of logarithmic phase
6individual cell, resuspended with 1mlPBS damping fluid, drop in 4mL precooling 70% aqueous ethanolic solution, 4 DEG C are fixedly spent the night, centrifugally abandon supernatant, with PBS buffer solution 2 times, resuspended and add RNaseA(and make its concentration be 20 μ g/ml with 1mlPBS damping fluid), hatch 30min for 37 DEG C, centrifugally abandon supernatant, with PBS buffer solution 1 time, resuspended and add 1 μ LPI staining fluid with 500 μ LPBS damping fluids, room temperature lucifuge dyeing 30min, carries out flow cytometry analysis with after 300 order nylon net filters.
B, detect the impact of Chinese fan palm extract cell cycle after knockdownCyclinA2 with FCM
The Hela cell of taking the logarithm vegetative period goes down to posterity, and spreads 6 orifice plates, after cell attachment, arranges four groups, a: blank group, only adds DMSO substratum; B: Chinese fan palm extract final concentration 2.5 μ g/ml; C: Chinese fan palm extract final concentration 2.5 μ g/ml+siRNA(CyclinA2 gene inhibitor); D:siRNA(CyclinA2 gene inhibitor.24h collecting cell does streaming, and streaming step is the same.
The analysis of C, FCM method adds the impact of cell cycle after Chinese fan palm extract and inhibitor
The Hela cell of taking the logarithm vegetative period goes down to posterity, and spreads 6 orifice plates, after cell attachment, makes the volume in every hole be 2ml, a: blank group, only to add DMSO substratum; B: Chinese fan palm extract final concentration 2.5 μ g/ml; C: Chinese fan palm extract final concentration 2.5 μ g/ml+50nMFlavopiridol(CDK2 inhibitor); D:50nMFlavopiridol(CDK2 inhibitor).Collecting cell after 24h, do the flow cytometer showed cell cycle, step is the same.
D, Westernblot analyze the impact of Chinese fan palm extract cell cycle albumen
Hela cell to be seeded in 6 orifice plates (1 × 10
6individual/hole), after cell attachment, then Chinese fan palm extract prepared by embodiment 1 is added, make that its concentration in each hole is respectively 1.25 μ g/ml, 2.5 μ g/ml, 5 μ g/ml, 10 μ g/ml, each concentration of 20 μ g/ml(arranges 3 multiple holes), the blank replacing Chinese fan palm extract with equal-volume liquid nutrient medium is set, cultivates 24h for 37 DEG C; Extract total protein of cell and carry out Wesernblot analysis (each swimming lane adopts equivalent total protein loading), primary antibodie 4 DEG C is spent the night, two anti-incubated at room 1h, X-mating plate exposures of HRP mark, development.Adopt β-actin as internal reference.
A described anti-antibody all purchased from Abcam company, CyclinA2 catalog number (Cat.No.) ab7956; CDK2 catalog number (Cat.No.) ab7954; CyclinD1 catalog number (Cat.No.) ab6152; CyclinE catalog number (Cat.No.) ab7959; P21 catalog number (Cat.No.) ab7960; β-actin catalog number (Cat.No.) ab3280.The goat-anti rabbit two of HRP mark is anti-purchased from green skies company, catalog number (Cat.No.) A0208.The sheep anti mouse two of HRP mark is anti-purchased from green skies company, A0216.
E, Westernblot analyze the impact adding Chinese fan palm extract cell cycle albumen after SiRNA and Flavopiridol inhibitor
Hela cell to be seeded in 6 orifice plates (1 × 10
6individual/hole), after cell attachment, blank group only adds DMSO substratum, and other each group adds in each hole by following requirements, 1.: Chinese fan palm extract final concentration 2.5 μ g/ml; 2.: Chinese fan palm extract final concentration 2.5 μ g/ml+50nMFlavopiridol; 3.: Chinese fan palm extract final concentration 2.5 μ g/ml+SiRNA.Cultivate 24h for 37 DEG C; Extract total protein of cell and carry out the expression of Wesernblot analysis (each swimming lane adopts equivalent total protein loading) cyclin CyclinA2 and CDK2, operation steps is the same.
The results are shown in Figure 7A-7E, Chinese fan palm extract, by raising CyclinA2 and CDK2 albumen, suppresses the cell cycle of cervical cancer cell in the G1/S phase, raises the expression level of cancer suppressor protein P21 simultaneously.
Embodiment 5, Chinese fan palm extract suppress cervical cancer cell to be bred and/or growth in nude mouse
By the Hela cell trysinization of logarithmic phase, collecting cell to be made into concentration be 5 × 10
8individual/ml.By every for 12 BALBc Female nude mice only subcutaneous injection 100 μ l cell suspension inside upper limbs, i.e. every Female nude mice inoculation 5 × 10
7individual cell.Be divided into two groups at random, often organize 6.Blank group injection DMSO, experimental group injection concentration is the Chinese fan palm extract (dissolving with DMSO) of 10 μ g/ml, and 48h injects once, each 100 μ l.By vernier caliper measurement knurl block length footpath L and minor axis D size before per injection, and according to formulae discovery gross tumor volume: V=LxD2/2, Chinese fan palm extract puts to death nude mice after injecting the 20th day, and careful stripping knurl block, weighs knurl block weight.
The results are shown in Figure 8A and 8B, Chinese fan palm extract also can suppress cervical cancer cell to be bred and/or growth in nude mouse.
The present invention can be applicable to bioengineering field.
Claims (7)
1. a Chinese fan palm extract monomer, is characterized in that, the structure of this monomer is as follows:
。
2. a preparation method for Chinese fan palm extract monomer as claimed in claim 1, it is characterized in that, the method is: to be separated with HPLC with organic solvent extraction and through silicagel column and to obtain Chinese fan palm extract monomer.
3. method according to claim 2, is characterized in that: described organic solvent comprises ethanol, methyl alcohol and chloroformic solution.
4. method according to claim 3, is characterized in that, the concrete steps of described method are as follows:
(1) Chinese fan palm raw material is cleaned, dry, be crushed to 20-100 order, obtain dry powder;
(2) get the dry powder that step (1) obtains, add the aqueous ethanolic solution of 70%, leave standstill lixiviate 24 hours and pump around circuit under room temperature condition, the mass/volume of the dry powder wherein added and aqueous ethanolic solution is than being w/v=1:20;
(3) whole liquid step (2) obtained under the vacuum of 50 DEG C by organic solvent evaporation, obtain brown pulpous state crude extract, this crude extract is suspended in water, with chloroform extraction, said extracted thing is crossed 200-300 order silicagel column, with the composition on chloroform-methanol eluting silica gel post, namely obtain Chinese fan palm extract monomer by HPLC purifies and separates.
5. method according to claim 4, is characterized in that: described Chinese fan palm raw material is the seed of Chinese fan palm, root or leaf.
6. the Chinese fan palm extract monomer prepared by method according to claim 2 is suppressing the application in cervical cancer cell.
7. application according to claim 6, is characterized in that, described in be applied as following at least one:
(1) propagation and/or the growth of cervical cancer cell is suppressed;
(2) Clone formation of cervical cancer cell is suppressed;
(3) Hela Cell Apoptosis is promoted;
(4) reduce cervical cancer cell mitochondrial membrane potential and make film integrality impaired;
(5) by the Cyclin-dependent kinase of cervical cancer cell in the G1/S phase;
(6) raise the cyclin CyclinA2 of G1/S phase and the expression level of cell cycle dependent kinase CDK2 albumen, raise the expression level of P21 albumen simultaneously;
(7) cervical cancer cell also can be suppressed in nude mouse to breed and/or growth;
(8) cervical cancer is treated.
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Citations (4)
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|---|---|---|---|---|
| CN101637550A (en) * | 2009-07-27 | 2010-02-03 | 林新华 | Chinese fanpalm seed polysaccharide extract and preparation method and purpose thereof |
| CN101698035A (en) * | 2009-07-27 | 2010-04-28 | 林新华 | Method for preparing anti-tumor active ingredients of Chinese fan palm |
| CN101845032A (en) * | 2010-06-03 | 2010-09-29 | 广东德鑫制药有限公司 | Phenolic compound and uses |
| US20110293753A1 (en) * | 2010-05-28 | 2011-12-01 | Louis Bellafiore | Tocotrienol Compositions |
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2016
- 2016-01-05 CN CN201610001351.3A patent/CN105566264A/en active Pending
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101637550A (en) * | 2009-07-27 | 2010-02-03 | 林新华 | Chinese fanpalm seed polysaccharide extract and preparation method and purpose thereof |
| CN101698035A (en) * | 2009-07-27 | 2010-04-28 | 林新华 | Method for preparing anti-tumor active ingredients of Chinese fan palm |
| US20110293753A1 (en) * | 2010-05-28 | 2011-12-01 | Louis Bellafiore | Tocotrienol Compositions |
| CN101845032A (en) * | 2010-06-03 | 2010-09-29 | 广东德鑫制药有限公司 | Phenolic compound and uses |
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Application publication date: 20160511 |