CN102675330B - Furan-flavone compound in arundina graminifolia, preparation method and application - Google Patents
Furan-flavone compound in arundina graminifolia, preparation method and application Download PDFInfo
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- CN102675330B CN102675330B CN201210131014.8A CN201210131014A CN102675330B CN 102675330 B CN102675330 B CN 102675330B CN 201210131014 A CN201210131014 A CN 201210131014A CN 102675330 B CN102675330 B CN 102675330B
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Abstract
The invention discloses a furan-flavone compound in arundina graminifolia, a preparation method and application, belongs to the field of plant chemical research, and in particular relates to a new furan-flavone compound separated from arundina graminifolia, a preparation method for the compound and application of the compound in anti-cancer medicaments. The preparation method for the compound comprises the following steps of: crushing an arundina graminifolia sample, performing ultrasonic extraction by using 95 weight percent ethanol, and combining extracting solutions; and evaporating the solvent from the extracting solution under reduced pressure to obtain extract, stirring the extract by using silica gel, performing chromatography and primary separation by using a silica gel column, performing secondary separation by adopting high performance liquid chromatography, and thus obtaining the required compound. The compound has high activity for human liver cancer cells (Be1-7402), has certain activity for human chronic granulocytic leukemia cell strains (K562) and gastric cancer cell strains (MKN-28), and can be used as a pilot compound for developing the anti-cancer medicaments.
Description
Technical field
The invention belongs to fitochemical studies field, in particular, the present invention relates in a kind of Purpleback Murdannia to separate the furoflavone new compound obtaining, the preparation method of this compound with and application in cancer therapy drug.
Background technology
Purpleback Murdannia belongs to the orchid family Purpleback Murdannia platymiscium, and happiness is warm wet, barren-resistant, happiness light, not resistance to shade.Purpleback Murdannia is found in Nepal at first, and the plant of this genus has 8 kinds at present, distributes very extensive, can both see its trace from Nepal, Sri Lanka, Thailand, Laos, Vietnam, Cambodia to China, Japan, Malaysia.In China Xishuangbanna, the local compatriot of the Dai nationality is called " agriculture still " the Purpleback Murdannia of opening beautiful flower, is a kind of good medicine common to all.Purpleback Murdannia has clearing heat and detoxicating, dispels rheumatism, and blood stasis removing analgesic, anti-inflammatory, the effect of diuresis, is used for the treatment of jaundice, and heat is drenched, oedema, beriberi oedema, hernia stomachache, rheumatic arthralgia, stomachache, urinary tract infections, venomous snake bite, sore and toxic, wound etc.; Current research also shows that Purpleback Murdannia ethanol extraction has obvious anti-tumor activity.Main active ingredient in Purpleback Murdannia has luxuriant and rich with fragrance compounds, Bibenzyl compound, terpenoid, flavonoid compound, sterol etc.
Two of flavonoid compound general references have a series of compounds that the phenyl ring (A-and B-encircle) of phenolic hydroxyl group links mutually by central thricarbon atom, and its basic parent nucleus is 2-phenyl chromone.Flavones is the abundantest class polyphenolic substance of content in human diet, is extensively stored in fruit, vegetables, cereal, rhizome, bark, flowers, tealeaves and red wine.Except outstanding anti-oxidation characteristics, flavonoid also has that prevention of arterial is atherosis, anti-inflammatory, antianaphylaxis, ntiviral characteristic, the multiple biological activity such as antitumor.
Summary of the invention
The object of the present invention is to provide a kind of new furan-flavone compounds obtaining that separates from Purpleback Murdannia (Arundina graminifolia).
Another object of the present invention is to provide a kind of method that obtains described compound that separates from Purpleback Murdannia (Arundina graminifolia) from Purpleback Murdannia.
Further object of the present invention is the cytotoxic activity of described compound and the application in antitumor drug thereof.
Object of the present invention is achieved by following technical proposals.
Except as otherwise noted, the percentage ratio adopting in the present invention is mass percent.
A. compound of the present invention is to separate and obtain from Purpleback Murdannia (Arundina graminifolia), has following structural formula:
The called after of this compound
5-hydroxy-8-(2-hydroxyethyl)-3-methoxy-2-(4-methoxyphenyl)-4H-furo[2，3-h]chromen-4-one。
B. in Purpleback Murdannia of the present invention, the preparation method of furan-flavone compounds adopts following steps:
(1) Purpleback Murdannia sample is pulverized, then with the ethanol supersound extraction of 95wt%, and united extraction liquid;
(2) extracting solution evaporated under reduced pressure solvent obtains medicinal extract, and medicinal extract silica gel mixed sample just divides with silica gel column chromatography, then adopts preparative high-performance liquid chromatographic further to separate, and obtains required compound.
The concrete scheme that the present invention prepares furan-flavone compounds in described Purpleback Murdannia is:
1. Purpleback Murdannia complete stool is crushed to 20～30 orders, gets ethanol supersound extraction 4 times of the sample 95wt% after pulverizing, and the supersound extraction time is 30～60min, merges each extracting solution, standing filtering throw out;
2. to extracting solution, adopt pressure reducing mode by whole solvent evaporates to dryness, obtain medicinal extract; Medicinal extract is with after dissolve with methanol, with 80～100 object silica gel mixed samples, then with 100～200 order silica gel column chromatographies, separate, adopt chloroform-acetone gradient elution, chloroform-acetone gradient elution respectively is: pure chloroform, 20: 1,9: 1,8: 2,3: 2,1: 1,20: 1, pure acetone, wherein 8: 2 wash-out parts of chloroform-acetone separate with high performance liquid preparative chromatography, and the crude product of resulting separation is purified with gel filtration chromatography again, obtains required pure compounds.
C. the compounds of this invention is by proton nmr spectra, carbon spectrum, two-dimensional spectrum, and the technical evaluation structure such as infrared spectra, UV spectrum, mass spectrum.
D. the present invention has carried out cytotoxic activity screening to described new compound, this compound has good activity to human liver cancer cell (Bel-7402), and human chronic polymorpho nuclear leukemia cells strain (K562) cell and stomach cancer cell line (MKN-28) are also had to certain activity.
The invention has the beneficial effects as follows: the compounds of this invention is simple in structure, synthetic is easily realized, industrialization prospect is good, this compound has good cytotoxic activity to Bel-7402 cell, K562 cell and MKN-28 cell are also had to certain activity, can be used as the guiding compound of antitumor drug exploitation.
Accompanying drawing explanation
Fig. 1 be the compounds of this invention proton nmr spectra (
1h NMR).
Fig. 2 be the compounds of this invention carbon-13 nmr spectra (
13c NMR).
Fig. 3 is that the main HMBC of the compounds of this invention is relevant.
Embodiment
The separation preparation of embodiment 1---compound
Purpleback Murdannia sample is got complete stool and is crushed to 20～30 orders, gets the sample 3.5kg after pulverizing, with ethanol supersound extraction 4 times of 95wt%; Add extracting solution 5L, ultrasonic time 30～60min at every turn.The sedimentary extracting solution evaporated under reduced pressure of standing filtering solvent is condensed into medicinal extract, obtains medicinal extract 136g.Medicinal extract is with after dissolve with methanol, mix sample with 80～100 object silica gel 200g, then with 100～200 order silicagel column 1.5kg dress posts, carry out chromatographic separation, adopt chloroform-acetone gradient elution, chloroform: acetone gradient elution respectively is: pure chloroform, 20: 1,9: 1,8: 2,3: 2,1: 1,20: 1, pure acetone.Wherein chloroform-acetone (8: 2) wash-out part 11.5g further separates with preparative high-performance liquid chromatographic, and the methyl alcohol take 38% is moving phase, C
18preparative chromatography post (20 × 250mm, 5 μ m) semipreparative column are stationary phase, and UV-detector detects, and detection wavelength is 254nm, each sample introduction 200 μ L, the chromatographic peak of collection 23.5min, repeatedly cumulative rear evaporate to dryness.Gained crude product is used pure dissolve with methanol again, then take methyl alcohol as moving phase, is further purified with Sephadex LH-20 gel filtration chromatography, gets final product to obtain this new compound.
The Structural Identification of embodiment 2---compound
Described compound is yellow jelly; UV spectrum (solvent is methyl alcohol), λ
max(log ε) 370 (2.90), 257 (3.22), 210 (3.87) nm; Infrared spectra (pressing potassium bromide troche) v
max3415,2920,1665,1559,1451,1357,1223,1158,1006,766,683cm
-1; HRESIMS shows its quasi-molecular ion peak m/z 405.0958[M+Na]
+(calculated value 405.0950), determines that in conjunction with NMR spectrum its molecular formula is C
21h
18o
7, degree of unsaturation is 13.Compound
1h and
13c NMR spectrum (accompanying drawing-1 and accompanying drawing-2, attribution data is in Table-1) shows that this compound is furan-flavone structure.By main HMBC relevant (accompanying drawing-3) can determine a 2-hydroxyethyl be substituted in furan nucleus 2 " position, two methoxyl groups are substituted in respectively 3 and 4 of phenyl ring ' position, phenolic hydroxyl group is substituted in 5 of phenyl ring; The structure of compound is finally confirmed.By SCIFINDER database retrieval and literature query, confirm that this compound is new compound.
Table 1 the compounds of this invention
1h,
13c NMR data
Embodiment 3---cytotoxicity assay
Cell strain: MKN-28 (stomach cancer cell line), K562 (human chronic polymorpho nuclear leukemia cells strain), A549 (Non-small cell lung carcinoma cell), Bel-7402 (human liver cancer cell) provides by Shanghai Pharmaceutical Inst., Chinese Academy of Sciences.
Experimental design: above cell and different concns compound incubation 72 hours, the experiment of every strain cell all repeats once, carry out data processing by the result of twice experiment, adopt the inhibition degree of improvement mtt assay and srb assay assessing compound on cell proliferation, calculate inhibiting rate, according to inhibiting rate, adopt Logit method to calculate IC
50, the anti tumor activity in vitro of comparative compound.
Proliferation inhibition rate=(the OD value of blank OD value-medicine feeding hole)/blank OD value × 100% of cell.
(a) improvement mtt assay
Get the suspension cell in logarithmic phase: K562, cem cell, cell concn is adjusted into 4 × 10
4/ ml, adds 96 well culture plates, 90 μ L/ holes.Positive control is cis-platinum, uses physiological saline solution.Every hole adds respectively the sample (No. 1 test solution-No. 5 test solution) of 10 μ l different concns.Application of sample group and control group are all established 4 multiple holes, and the high density group of application of sample group, positive controls is also established the dosing parallel hole of substratum, and every block of plate is equipped with 4 blank holes (only adding substratum).The final concentration of sample is respectively 10
-2, 10
-1, 1,10 and 10
2μ g/mL, the final concentration of corresponding DMSO is respectively 0.1%, 0.01%, 0.001%, 0.0001%, 0.00001%.Sample is at final concentration 10
2during μ g/mL, with 0.1%DMSO, as solvent control, all the other concentration are all made negative control with physiological saline.The final concentration of positive control drug cis-platinum is 10
-1, 1,10 μ g/mL.Cell is at 37 ℃, 5%CO
2in incubator, hatch respectively after 48h, add MTT (5mg/ml, Sigma), 10 μ L/ holes.Continue to cultivate after 4h, add three liquid [10%SDS-5% isopropylcarbinol-0.012mol/L HCL (w/v/v)], 100 μ L/ holes, place the OD value of measuring each hole after spending the night by microplate reader under 570nm, 630nm dual wavelength.
(b) srb assay
Get the attached cell strain in logarithmic phase: HCT, MKN-28, CNE, CA, Tca8113, T-24, SCaBER, with after the conventional digestion of 25% pancreatin, then be adjusted into 5 × 10 by cell concn with the complete RPMI-1640 substratum of 15% calf serum
4/ mL, adds 96 well culture plates, 90 μ L/ holes.Cell is at 37 ℃, 5%CO
2in incubator, hatch respectively the same mtt assay of each tested concentration (10 μ L/ hole) that adds positive control, negative control and given the test agent after 24h, the final concentration of sample is respectively 10
-2, 10
-1, 1,10,10
2μ g/mL, the final concentration of corresponding DMSO is respectively 0.1%, 0.01%, 0.001%, 0.0001%, 0.00001%.To the mensuration of knurl strain CNE, SCaBER, T-24, sample is at final concentration 10
2, during 10 μ g/mL with 0.1%, 0.01%DMSO is as solvent control, all the other concentration are all made negative control with physiological saline; To the mensuration of other knurl strain, sample is at final concentration 10
2during μ g/mL, use 0.1%DMSO as solvent control, all the other concentration are all made negative control with physiological saline.The final concentration of positive control drug cis-platinum is 10
-1, 1,10 μ g/mL, negative control is isopyknic physiological saline.Application of sample group and control group are all established 4 multiple holes, and the high density group of application of sample group, positive controls is also established the dosing parallel hole of substratum, and every block of plate is equipped with 4 blank holes (only adding substratum).96 well culture plates are placed in to 37 ℃, 5%CO
2in incubator, hatch after (cell and sample effect) 48h, add 4 ℃, 50% TCA (trichoroacetic acid(TCA)) 50 μ L/ holes.Add after TCA, 96 well culture plates are placed in to 4 ℃ and hatch 1 hour, take out culture plate, liquid in the plate that inclines gently.Rinse gently (by tap water by pouring into gently in beaker in plate, after light rolling, again water being gone) 5 times with tap water, be placed in air air-dry to loseing washmarking.Then add the 0.4%SRB for preparing (with 1% acetic acid dilution), 50 μ L/ holes, under room temperature, SRB solution is removed in standing dyeing hypsokinesis in 30 minutes, with 1% acetic acid flushing 4 times, to remove not and the dyestuff of protein bound.Be placed in air air-dry extremely without after washmarking, add 10mM not cushion Tris (slow blood ammonia acid) solution 150 μ L/ holes (PH10, with tri-distilled water prepare), after dyestuff is dissolved, on vibrator, vibrate 5 minutes, by microplate reader, under 570nm wavelength, read each hole OD value.
(c) experimental result
Experimental result shows: carry out 5 tests and average, the compounds of this invention is 2.18 μ M, to the IC50 value of K562 cell, is 3.47 μ M the IC50 value of MKN-28, and the IC50 value of Bel-7402 cell is 0.62 μ M, to the IC50 value of A-549 cell, is 15.8 μ M.Compound has good cytotoxic activity to Bel-7402 cell, and K562 cell and MKN-28 cell are also had to certain activity.
Claims (3)
1. the furan-flavone compounds in Purpleback Murdannia, it is characterized in that from Purpleback Murdannia (
arundina graminifolia) in separate obtain, structural formula is
Called after 5-hydroxy-8-(the 2-hydroxyethyl)-3-methoxy-2-(4-methoxyphenyl)-4 of this compound
h-furo[2,3-h] chromen-4-one.
2. by the preparation method of the furan-flavone compounds in Purpleback Murdannia claimed in claim 1, it is characterized in that concrete technology step is as follows:
1. Purpleback Murdannia complete stool is crushed to 20～30 orders, gets ethanol supersound extraction 4 times of the sample 95wt% after pulverizing, and the supersound extraction time is 30～60 min, merges each extracting solution, standing filtering throw out;
2. to extracting solution, adopt pressure reducing mode by whole solvent evaporates to dryness, obtain medicinal extract; Medicinal extract is with after dissolve with methanol, with 80～100 object silica gel mixed samples, then with 100～200 order silica gel column chromatographies, separate, adopt chloroform-acetone gradient elution, chloroform-acetone gradient elution respectively is: pure chloroform, 20:1,9:1,8:2,3:2,1:1,20:1, pure acetone, wherein the 8:2 wash-out part of chloroform-acetone separates with high performance liquid preparative chromatography, and the crude product of resulting separation is purified with gel filtration chromatography again, obtains required pure compounds.
3. the application of the furan-flavone compounds in a Purpleback Murdannia claimed in claim 1 in anti-cancer of the stomach, human chronic myelogenous leukemia and people's liver-cancer medicine.
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| WO2008141074A1 (en) * | 2007-05-10 | 2008-11-20 | Salk Institute For Biological Studies | Identification of compounds that protect against amyloid diseases |
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| 李晓芬等.大孔吸附树脂对竹叶兰中总黄酮的分离纯化.《湖北农业科学》.2011,第50卷(第14期), * |
| 邵伟艳等.3个新呋喃黄酮的NMR研究.《分析测试学报》.2001,第20卷(第1期), * |
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