CN102964328B - Isopentene isoflavanone compound as well as preparation method and application of compound - Google Patents
Isopentene isoflavanone compound as well as preparation method and application of compound Download PDFInfo
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- CN102964328B CN102964328B CN201210546906.4A CN201210546906A CN102964328B CN 102964328 B CN102964328 B CN 102964328B CN 201210546906 A CN201210546906 A CN 201210546906A CN 102964328 B CN102964328 B CN 102964328B
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Abstract
The invention relates to a natural medicine, and in particular relates to a novel isoflavanone isoflavanone compound in wild peanuts as well as a preparation method and application of the compound. The novel isoflavanone isoflavanone compound provided by the invention has a structural formula shown in a formula (I). The preparation method comprises the following steps of: a, crushing; b, refluxing and extracting; c, concentrating; d, extracting; and e, separating. The novel isoflavanone isoflavanone compound shown in the formula (I) has a remarkable inhibition function to a human cervical carcinoma, human chronic myeloid leukemia or human acute myelogenous leukemia cell strain, has a better anti-cancer activity, and can be used as an anti-cancer active component or lead compound. The novel isoflavanone isoflavanone compound can be a natural organic compound, is wide in raw material source, is simple in preparation process, is high in purity, is easily realized in subsequent industrialized production, is used for preparing a medicament for preventing or treating tumors, and is used for clinically treating human cervical carcinoma, human chronic myeloid leukemia or human acute myelogenous leukemia.
Description
Technical field
The present invention relates to a kind of natural drug, be specifically related to a kind of new isopentene group isoflavanone compounds and its preparation method and application in wild kind beans.
Background technology
Cancer has developed into common disease, the frequently-occurring disease of serious harm people life, and same cardiovascular and cerebrovascular disease, diabetes are called as one of large Death in the world three together.The World Health Organization issues in February, 2010 and reports, the whole world will have 7,600,000 people to die from cancer to estimate the current year, if the half-hearted preventing cancer of taking measures of people, to the year two thousand thirty, the annual number of cancer deaths in the whole world may be doubled on existing basis, reach 1,700 ten thousand.This tissue statistic data also shows, and the current whole world on average just has 1 people to die from cancer in every 8 deaths, and this death toll summation caused than acquired immune deficiency syndrome (AIDS), tuberculosis and malaria is taller.Therefore find effective cancer therapy drug and become the task of top priority.In cancer therapy drug research, the studies and clinical application of chemotherapeutics obtains remarkable progress.As metal complexes platinum complex, organic germanium compounds etc., although have strong cancer resistance, there is toxic side effect, the defects such as poorly water-soluble in the cancer therapy drug of chemosynthesis.In recent years, China and foreign countries researchist turns one's attention to aboundresources, the herbal medicine of pure natural, ethnic drug gradually, ites is desirable to therefrom to find new cancer therapy drug.From herbal medicine, ethnic drug, find a series of natural anti-cancer drugs such as taxol, camptothecine, kind of harringtonine as medicinal materials, vincristine(VCR), podophyllotoxin, Elemenum Emulsion at present, and have many new product listings every year.These natural drugs have all showed good antitumour activity and have been widely used clinically, also day by day prove that screening antineoplastic drugs is a very promising direction from natural product.
Wild kind beans (Uraria clarkei Gagnep) are pulse family (Leguminosae) leopard cat tail bean plant (" Chinese Plants will " the 41st volume 72 pages).This platymiscium whole world about has 20 kinds, is distributed in Tropical Africa, Asia and Australia, and China has 9 kinds.According to document, (Chen Yan, thinks the elegant tinkling of pieces of jade, Wei Song, Xu Xuejian. the research [J] of rabbit tail grass chemical composition. Chinese patent medicine, 2009,31 (2): 266-269) report, this platymiscium has the effects such as treatment infantile malnutrition, puerperal lactation deficiency, hemoptysis, hemorrhoid, the tuberculosis of cervical lymph nodes, venomous snake bite.Its chemical composition is mainly flavonoid compound, comprises the structure types such as flavones, isoflavones, isoflavanone.Flavonoid compound has the multiple biological activitys such as antitumor, hypotensive, reducing blood-fat, Anti-bacterium, AntiHIV1 RT activity.The present invention attempts to do further further investigation to the antitumor action of isoflavanone compounds in wild kind beans, to finding cytotoxic activity natural product wherein, provides foundation for screening antitumor drug that is efficient, low toxicity.
Summary of the invention
The object of the present invention is to provide a kind of new isopentene group isoflavanone compounds.
Another object of the present invention is to provide a kind of method extracting the compounds of this invention from wild kind beans.
Further aim of the present invention is the application of the compounds of this invention in preparation prevention or tumor.
Further aim of the present invention is that the compounds of this invention prevents in preparation or treats the application in human cervical carcinoma, people's chronic myelogenous leukemia, human acute myeloid leukemia medicine.
Object of the present invention is achieved by following technical proposals.
Except as otherwise noted, the percentage ratio adopted in the present invention is mass percent.
The present invention isolates a kind of new isopentene group isoflavanone compounds from wild kind beans, and this compound has significant antitumour activity.
New isopentene group isoflavanone compounds of the present invention has the structural formula shown in formula I:
The called after of this compound: (3
r)-8,5 ′ – diisoamyl thiazolinyl-5,2 '-dihydroxyl-7,4 '-dimethoxy isoflavanone ((3
r)-8,5 ′ – di-(
γ,
γ-dimethylallyl)-5,2 '-dihydroxyl-7,4 '-dimethoxyl-isoflavanone).
The invention provides a kind of preparation method of formula compound, the method adopts following steps:
A. pulverize: obtain meal by after kind beanstalk dried and crushed of open country, for subsequent use;
B. refluxing extraction: by meal obtained in step a with alcohol reflux four times, merge ethanol extract, for subsequent use;
C. concentrate: carry out concentrating under reduced pressure after being filtered by ethanol extract obtained in step b and obtain medicinal extract, for subsequent use;
D. extract: medicinal extract obtained in step c is suspended in water, extracts with propyl carbinol by sherwood oil, ethyl acetate successively, then remove solvent, obtain sherwood oil, ethyl acetate and n-butyl alcohol extract respectively, for subsequent use;
E. be separated: by the petroleum ether extract that obtains in steps d through 100-200 order silica gel column chromatography, sherwood oil with volume ratio 30:1,20:1,10:1,5:1,2:1,1:1,0:1: ethyl acetate, gradient elution obtains Fr.1-Fr.7 totally 7 components, Fr.4 is through silica gel column chromatography, chloroform with volume ratio 98:2,97:3,96:4,95:5: methanol elution gradient, obtains component Fr.4A-4D.Fr.4C obtains formula I through sephadex LH-20 column chromatography methyl alcohol purifying.
The present invention has carried out cytotoxic activity test to formula compound, experimental result demonstrates good suppression human cervical carcinoma, people's chronic myelogenous leukemia, human acute myeloid leukemia cell strain activity, can be new compound or lead compound that medical industry provides using value.
beneficial effect
1, the formula I in the present invention has significant restraining effect to human cervical carcinoma, people's chronic myelogenous leukemia or human acute myeloid leukemia cell strain, points out it to have good antitumour activity, can as anticancer active constituent or lead compound.
2, the formula I in the present invention can be naturally occurring organic compound, and raw material sources are extensive, and compound preparation manipulation flow process is simple, and the compound purity obtained is high, and suitability for industrialized production subsequently easily realizes.
Accompanying drawing explanation
Fig. 1 is the structural formula of the compounds of this invention;
Fig. 2 is the compounds of this invention high resolution mass spectrum (HRESI-MS);
Fig. 3 be the compounds of this invention proton nmr spectra (
1h NMR);
Fig. 4 be the compounds of this invention carbon-13 nmr spectra (
13c NMR);
Fig. 5 is the DEPT spectrum of the compounds of this invention;
Fig. 6 is the HSQC Correlated Spectroscopy of the compounds of this invention;
Fig. 7 is the HMBC Correlated Spectroscopy of the compounds of this invention;
Fig. 8 is the ROESY Correlated Spectroscopy of the compounds of this invention;
Fig. 9 is the CD spectrum of the compounds of this invention.
Specific embodiment
By the following specific embodiment that provides and typically used embodiment, the present invention can be well understood to further.But this is not construed as limiting the scope of the present invention.
embodiment 1
The preparation of compound
Material source: wild kind beans are adopted in Kaiyuan, Yunnan, and be accredited as Uraria clarkei (Clarke) Gagnep. through Kunming Inst. of Botany, Chinese Academy of Sciences Tao Deding researcher, Saving specimen is in Hua Sheng institute of Yunnan Institute for nationalities specimen museum.
Adopt following steps:
A. pulverize: the meal wild for 10Kg kind beanstalk dried and crushed being become particle diameter 0.1cm size, for subsequent use;
B. refluxing extraction: by meal obtained in step a refluxing extraction 4 times at the temperature of 70-74 DEG C, each 2 hours, extract with the ethanol 60Kg of 95% concentration, merging ethanol extract at every turn, for subsequent use;
C, concentrated: the ethanol extract via hole diameter obtained by step b is the filter paper filtering of 80-120 micron and carries out concentrating under reduced pressure with Rotary Evaporators at the temperature of 50 DEG C, when being 1.2 to proportion, obtains medicinal extract 1025 g, for subsequent use;
D, extraction: 1025g medicinal extract is suspended in 3000ml water, extract with 3000ml sherwood oil, 3000ml ethyl acetate and 3000ml propyl carbinol successively, often kind of solvent extraction 5 times, boil off solvent with Rotary Evaporators again, obtain sherwood oil, ethyl acetate and n-butyl alcohol extract 100g, 70g, 200g respectively.
E. be separated: by the petroleum ether extract that obtains in steps d through 300 order silica gel column chromatographies, use volume ratio 30:1, 20:1, 10:1, 5:1, 2:1, 1:1, the sherwood oil of 0:1: ethyl acetate, gradient elution obtains Fr.1-Fr.7 totally 7 components, Fr.1 is 15.1g, Fr.2 is 8.9g, Fr.3 is 17.7g, Fr.4 is 7.9g, Fr.5 is 5.8 g, Fr.6 is 9.7g, Fr.7 is 14.3g, Fr.4 is through silica gel column chromatography, with volume ratio 98:2, 97:3, 96:4, the chloroform of 95:5: methanol-eluted fractions, obtain Fr.4A-4D totally 4 components, Fr.4A is 1.2g, Fr.4B is 0.3g, Fr.4C is 3.6g, Fr.4D is 0.8g.Fr.4C obtains faint yellow amorphous powder 138 mg through sephadex LH-20 column chromatography 2300ml methyl alcohol purifying.
embodiment 2:different concentration ethanol solution extracts wild kind beans sample contrast experiment
Due to few to the research report of wild kind beans chemical composition, therefore wish by using different concentration ethanol solution to extract wild kind beans sample, to find best ethanol solution concentration.This is not that hint cannot use other solvent extractions.
A, pulverizing: the meal being ground into particle diameter 0.1-0.5 cm size after being dried by wild kind beanstalk, for subsequent use;
B, refluxing extraction: the meal obtained with step a at the temperature of 70-74 DEG C makes Extraction solvent with the ethanol of 6 times amount 70% concentration, the ethanol of 80% concentration and the ethanol of 95% concentration respectively, refluxing extraction 4 times, each 2 hours, merge ethanol extract respectively, for subsequent use;
C, concentrated: the different concentration ethanol extracting solution via hole diameter obtained by step b is respectively the filter paper filtering of 80-120 micron and carries out concentrating under reduced pressure at the temperature of 50 DEG C, when being 1.2 to proportion, obtains medicinal extract, for subsequent use;
D, extraction: respectively the medicinal extract that step c is obtained is suspended in 3 times of water gagings, extract with water 1 times amount sherwood oil, water 1 times amount ethyl acetate and water 1 times amount propyl carbinol successively, often kind of solvent extraction 5 times, merge same solvent extract, use Rotary Evaporators pressure reducing and steaming solvent again, obtain sherwood oil, ethyl acetate and n-butyl alcohol extract respectively.
Experimental result shows that Extraction solvent made by ethanol, the ethanol of 80% concentration and the ethanol of 95% concentration by 70% concentration, open country kind beans acetic acid ethyl ester extract weight obtained respectively is close, therefore make Extraction solvent with the ethanol of the ethanol of 70% concentration, the ethanol of 80% concentration and 95% concentration, the composition containing formula I can be obtained equally.
The wild kind beans different solvents extract extract yield table of table 1.
embodiment 3
The Structural Identification of the faint yellow amorphous powder that embodiment 1 obtains
Described faint yellow amorphous powder (solvent is methyl alcohol), [
α] 22 D=-27.3 (
c=0.23, MeOH); UV (MeOH)
λ max(log ε): 291 (2.96) nm; CD (
c=0.101, MeOH) λ (Δ ε) 258.5 (+0.435), 346 (+0.059); IR (KBr)
ν max: 3360,1640,1601,1507,1453,1274,1108,1010 cm
-1; HRESI-MS (accompanying drawing 2) shows its quasi-molecular ion peak m/z 453.2158 [M+H]
+in conjunction with NMR spectrum, (calcd. 453.2232), determines that its molecular formula is C
27h
32o
6, degree of unsaturation is 12.
1h and
13c NMR (accompanying drawing 3, accompanying drawing 4 and accompanying drawing
5, attribution data in Table-
1) display molecule in have 2 phenyl ring (phenyl ring having 3 methyne double key carbons), 2 isopentene group (δ
c132.2,131.8,122.8,122.1,27.9,25.8,25.8,21.0,17.8,17.7), 1 carbonyl (δ
c197.0), 2 methoxyl group (δ
c55.9,55.4), 1 company Oxymethylene (δ
c69.5) and 1 methyne (δ
c45.0).In conjunction with HSQC Correlated Spectroscopy (accompanying drawing
6), and this compound of documents data susceptible of proof is the isoflavanone compounds containing 2 isopentene groups; Confirm that C-7, C-4 ' position is substituted by methoxyl group through HMBC relevant (accompanying drawing 7) further, C-5, C-2 ' position is substituted by hydroxyl, and C-8, C-5 ' position is substituted by isopentene group.The absolute configuration of compound by ROESY (accompanying drawing 8), CD spectrum (accompanying drawing 9) and with document (Tanaka T., Ohyama M., Iinuma M., Shirataki Y., Komatsu M., Burandt C.L. Isoflavonoids from
sophora secundiflora,
s. arizonicaand
s. gypsophila.
phytochemistry, 1998,48,1187-1193.; Slade D, Ferreira D, Marais JPJ. Circular dichroism, a powerful tool for the assessment of absolute configuration of flavonoids. Phytochemistry, 2005,66,2177-2215.) contrast turn out to be 3
r-configuration, the structure of compound is finally confirmed, and compound has the structural formula of formula I (as accompanying drawing 1).。Confirm that this compound is new compound by SCIFINDER database retrieval and literature query.
Table 2. formula I
1h-and
13c-NMR (CDCl
3) data.
embodiment 4
Described formula I anti-tumor activity detects
Adopt improvement mtt assay assessing compound to the increment restraining effect of tumour cell
Get tumor cell suspension for subsequent use and be inoculated in 96 well culture plates, 90 μ L/ holes, add the given the test agent of different concns, 10 μ L/ holes after continuing cultivation 24 h, make its final concentration be 100,10,1,0.1,0.01 μ g/mL, each concentration all establishes 3 multiple holes.Cis-platinum (DDP) is positive control, and equal-volume RPMI1640 substratum is negative control.When setting compound 100,10 μ g/mL, corresponding DMSO concentration is as Vehicle controls simultaneously, to eliminate the impact of DMSO cell growth.Continue after dosing to be placed in 37 DEG C, 5% CO
272 h cultivated by incubator, and every hole adds MTT(5 mg/mL) 20 μ L, continue cultivation 4 h, every hole adds three liquid [10%SDS-5% isopropylcarbinol-0.012 mol/L HCl(W/V/V)] 100 μ L Rong Xie formazans.Under 570 nm wavelength, the OD value in each hole is measured by microplate reader.
By following formulae discovery cell proliferation inhibiting rate:
Inhibiting rate (%)=(OD value control wells-OD value dosing holes)/OD value control wells × 100%
Data statistic analysis: adopt Microsoft Excel 2003 to calculate inhibiting rate, adopt logit method, SPSS11.5 software package calculation of half inhibitory concentration IC
50.
Calculation of half inhibitory concentration IC
50, to the half-inhibition concentration (IC of human cervical carcinoma (Hela), people's chronic myelogenous leukemia cell strain (k562) and human acute myeloid leukemia cell strain (HL60)
50) measurement result is respectively 12.7 ± 2.6 μ g/ml, 18.4 ± 3.7 μ g/ml, 15.9 ± 3.1 μ g/ml, shows that compound of the present invention has good anti-tumor activity.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, the equivalent replacement of all any amendments done within the spirit and principles in the present invention and improvement etc., all should be included within protection scope of the present invention.
Claims (1)
1. a preparation method for isopentene group isoflavanone compounds, isopentene group isoflavanone compounds has following structural formula (I):
，
It is characterized in that, preparation method adopts following steps:
A. pulverize: obtain meal by after kind beanstalk dried and crushed of open country, for subsequent use;
B. refluxing extraction: by meal obtained in step a with alcohol reflux four times, merge ethanol extract, for subsequent use;
C. concentrate: carry out concentrating under reduced pressure after being filtered by ethanol extract obtained in step b and obtain medicinal extract, for subsequent use;
D. extract: medicinal extract obtained in step c is suspended in water, extracts with sherwood oil, ethyl acetate and propyl carbinol successively, then remove solvent, obtain sherwood oil, ethyl acetate and n-butyl alcohol extract respectively, for subsequent use;
E. be separated: by the petroleum ether extract that obtains in steps d through 100-200 order silica gel column chromatography, Fr.1-Fr.7 totally 7 components are obtained with the petroleum ether-ethyl acetate gradient elution of volume ratio 30:1,20:1,10:1,5:1,2:1,1:1,0:1, Fr.4 is through silica gel column chromatography, chloroform with volume ratio 98:2 ~ 95:5: methanol elution gradient, obtain component Fr.4A-4D, Fr.4C obtains formula (I) compound through sephadex LH-20 column chromatography methyl alcohol purifying.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101002841A (en) * | 2006-12-27 | 2007-07-25 | 中国科学院新疆理化技术研究所 | Effective components of rose, its preparing method and use |
| CN101434592A (en) * | 2008-12-19 | 2009-05-20 | 沈阳药科大学 | Novel flavonoid extracted from Maackia amurensis |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101002841A (en) * | 2006-12-27 | 2007-07-25 | 中国科学院新疆理化技术研究所 | Effective components of rose, its preparing method and use |
| CN101434592A (en) * | 2008-12-19 | 2009-05-20 | 沈阳药科大学 | Novel flavonoid extracted from Maackia amurensis |
Non-Patent Citations (1)
| Title |
|---|
| Toshio,Fukai;等.Cytotoxic activity of low molecular weight polyphenols against human oral tumor cell lines.《Anticancer Research》.2000,第20卷(第4期),2525-2536. * |
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