CN102101878B - Hellebore petunidin space isomer compound and preparation and application thereof - Google Patents
Hellebore petunidin space isomer compound and preparation and application thereof Download PDFInfo
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- CN102101878B CN102101878B CN 200910248445 CN200910248445A CN102101878B CN 102101878 B CN102101878 B CN 102101878B CN 200910248445 CN200910248445 CN 200910248445 CN 200910248445 A CN200910248445 A CN 200910248445A CN 102101878 B CN102101878 B CN 102101878B
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Abstract
The invention provides a hellebore petunidin space isomer compound and preparation and application thereof. The hellebore petunidin space isomer compound is prepared from the conventional Chinese medicinal toad skin by using macroporous resin and a two-dimensional preparation chromatographic separation technology. The compound is prepared by mainly comprising the following steps of: cutting the toad skin into pieces; extracting with ethanol; separating a total extract by using the macroporous resin to obtain a toad bufadienolides extract; and performing two-dimensional preparation chromatographic separation and purification to obtain the compound. An in-vitro experiment proves that the compound has a good tumor cell suppression effect.
Description
Technical field
The present invention relates to the bufadienolides compounds, a kind of hellebore petunidin space isomer new compound and preparation method thereof specifically, it is a kind ofly to prepare the new compound of separating by macroporous resin treatment and two-dimentional preparative chromatography isolation technique from traditional Chinese medicine toad skin.
Background technology
Toad is that tcm clinical practice is used medicine early, and it is historical that oneself has the clinical application of more than one thousand years in China.Its medicinal part is: skin, the dried venom of toads, dry all.The toad skin has the effect of clearing heat and detoxicating, sharp water dissipate-swelling, and hot, cool, little poison cure mainly ulcer, pyogenic infections, China's multiplex primary hepatocarcinoma, lung cancer etc. for the treatment of among the people, have preferably clinical effectiveness (Dai Liping etc. Acta Pharmaceutica Sinica, 2007,42 (8): 858-861).
The bufadienolides compounds is the main activeconstituents in the toad skin, modern pharmacology studies show that its have cardiac stimulant, the effect such as antibiotic, antitumor (Jia Caiyun etc. herbal medicine, 2005,36 (2): 244; Enomoto A, et al.J Nat Prod, 2004,67 (12): 2070; Cunha Filho GA, et al.Toxicon, 2005,45 (6): 777; Usanova IV, et al.Eksp Klin Farmakol, 2002,65 (4): 23).
This compounds of report has kind more than 250 at present, but the report of steric isomer is few.Yet steric isomer, especially chiral isomer may have larger difference on biological activity, are one of study hotspots of pharmacy field.
Summary of the invention
Given this, the inventor extracts separation and obtains a kind of new bufadienolide compound through further investigation from the dry skin of bufo gargarizans Cantor, prove that through experiment in vitro it has the preferably effect of inhibition tumor cell.
The invention provides a kind of hellebore petunidin space isomer new compound, described compound has one of three kinds of structural formulas as follows,
The invention provides the extracting method of bufadienolide extract: toad skin extraction using alcohol, macroporous resin on the concentrated solution behind the Recycled ethanol, elder generation's water and low-concentration ethanol wash-out, use again the high concentration ethanol wash-out, collect elutriant, decompression and solvent recovery namely gets the bufadienolide extract, and the weight percentage of bufadienolide is 60%～90% in the bufadienolide extract.
Wherein said low-concentration ethanol refers to 5%～50% ethanol, and high concentration ethanol refers to 70%～100% ethanol.
Wherein said macroporous resin can be selected nonpolar or polar macroporous resin, and said non-polar macroporous resin can be selected AB-8, D-101, XAD-4, X-5, HPD-100; Said polar macroporous resin can be selected HPD-400, S-8, NAK-9.
The present invention also provides the preparation method of bufadienolide compound: get the bufadienolide extract, can obtain high-purity monomer after adopting two-dimentional HPLC preparative chromatography purifying, the first dimension adopts reverse-phase chromatographic column, and the second dimension adopts the cyclodextrin chromatographic column.
The concrete preparation method of described compound is:
1) get the bufadienolide extract, separate through preparation HPLC, chromatographic condition is: anti-phase Xterra C18 chromatographic column, A:0.1% (v/v) formic acid water; B:0.1% (v/v) formic acid acetonitrile, gradient elution: 5%B～95%B, 60min.Adopt UV-detector to detect in the sepn process, detect wavelength 300nm, collect respectively each cut by the retention time at different samples peak in the testing process, collect altogether 20 cuts;
2) get Fraction 5 and reclaim solvent, separate through HPLC, chromatographic condition is: cyclodextrin chromatographic column, A: normal hexane; B: Virahol, gradient elution: 5%B～30%B, 55min collects each chromatographic peak, reclaims respectively solvent, determines through the nuclear-magnetism experiment, obtains to contain the cut of compound I;
Get Fraction 6 and reclaim solvent, separate through HPLC, chromatographic condition is: cyclodextrin chromatographic column, A:0.1% (v/v) formic acid water; B:0.1% (v/v) formic acid acetonitrile, gradient condition: 95%B～60%B, 40min collects each chromatographic peak, reclaims respectively solvent, determines through the nuclear-magnetism experiment, obtains to contain the cut of compound I I;
Get Fraction 10 and reclaim solvent, separate through HPLC, chromatographic condition is: cyclodextrin chromatographic column, A: water; B: methyl alcohol, gradient elution: 5%B～50%B, 35min collects each chromatographic peak, reclaims respectively solvent, determines through the nuclear-magnetism experiment, obtains to contain the cut of compound III.
Described compound can be in the medicine of preparation treatment and prevention solid tumor application, such as cervical cancer, mammary cancer etc.
The invention provides a kind of method that from traditional Chinese medicine toad skin, prepares the hellebore petunidin space isomer new compound by macroporous resin and two-dimentional preparative chromatography isolation technique.Prove that through experiment in vitro it has the preferably effect of inhibition tumor cell.
Specific embodiments
Now in conjunction with example, the present invention will be further described.Example only limits to illustrate the present invention, but not limitation of the invention.
Embodiment 1: the preparation of bufadienolide extract
Get toad skin 5Kg with 95% extraction using alcohol 3 times, be 10 times with the alcohol amount, each extraction time is 120 minutes, merging filtrate, filtrate decompression is concentrated into 5L, upper macroporous resin washes with water first, uses 5%～50% ethanol elution again, use again 70%～100% ethanol elution, collect the latter, decompression and solvent recovery, drying obtains bufadienolide extract 62g.
Embodiment 2: the preparation of bufadienolide cut
Get embodiment 1 resulting bufadienolide extract, separate (XterraC18, A:0.1% (v/v) formic acid water through preparation HPLC; B:0.1% (v/v) formic acid acetonitrile, gradient condition: 5%B～95%B, 60min; ), adopt UV-detector to detect in the sepn process, detect wavelength 300nm.Retention time by different samples peak in the testing process is collected respectively each cut; Collect altogether 20 cuts (Fraction1～20).
Embodiment 3: the preparation of compound I
Fraction 5 reclaims solvent, separates (cyclodextrin chromatographic column, A: normal hexane through HPLC; B: Virahol, gradient condition: 5%B～30%B, 55min), collect wherein main chromatographic peak, reclaim respectively solvent, determine through the nuclear-magnetism experiment, acquisition contains the cut 3mg at compound I (peak 3), the HPLC detection level is 99%, measures through physics and chemistry, and data are as follows: white powder, HR-MS, [M+H]+(m/z): 417.2275, UV:298nm
1HNMR (CD
3OD, 400MHz) δ: 10.12 (1H, s), 8.01 (1H, dd, J=9.6,1.6), 7.56 (1H, s), 6.43 (1H, d, J=9.6), 3.55 (1H, m), 2.17 (1H, d, J=5.6), 0.78 (1H, s) etc.;
13CNMR (CD
3OD, 100MHz) δ: 210.2,167.9,155.4,145.1,133.2,117.5,86.2,70.8,69.3,55.7,51.2,47.3,43.2,42.5,35.7,36.8,31.2,31.6,26.3,25.2,23.9,22.4,19.7.
Embodiment 4: the preparation of compound I I
Fraction 6 reclaims solvent, separates (cyclodextrin chromatographic column, A:0.1% (v/v) formic acid water through HPLC; B:0.1% (v/v) formic acid acetonitrile, gradient condition: 95%B～60%B, 40min), collect wherein main chromatographic peak, reclaim respectively solvent, determine through the nuclear-magnetism experiment, acquisition contains the cut 2mg at compound I (peak 4), the HPLC detection level is 98%, measures through physics and chemistry, and data are as follows: white powder, HR-MS, [M+H]+(m/z): 417.2281, UV:300nm
1HNMR (CD
3OD, 400MHz) δ: 9.92 (1H, s), 7.89 (1H, dd, J=10,2.4), (7.34 1H, s), 6.18 (1H, d, J=10), 3.83 (1H, m), (2.46 1H, dd, J=8.8,6.4), 0.58 (1H, s) etc.;
13CNMR (CD
3OD, 100MHz) δ: 208.5,163.4,149.2,147.9,123.5,114.1,84.2,74.4,66.4,53.8,50.5,42.2,41.8,40.1,38.9,37.0,30.9,30.5,28.6,28.2,24.0,21.0,21.8.
Embodiment 5: the preparation of compound III
Fraction 10 reclaims solvent, separates (cyclodextrin chromatographic column, A: water through HPLC; B: methyl alcohol, gradient condition: 5%B～50%B, 35min), collect wherein main chromatographic peak, reclaim respectively solvent, determine through the nuclear-magnetism experiment, acquisition contains the cut 5mg at compound I (peak 1), the HPLC detection level is 99%, measures through physics and chemistry, and data are as follows: white powder, HR-MS, [M+H]+(m/z): 417.2264, UV:299nm
1HNMR (CD
3OD, 400MHz) δ: 9.14 (1H, s), 8.67 (1H, dd, J=9.7,2.2), (8.34 1H, s), 7.18 (1H, d, J=9.5), 4.43 (1H, d=4.2), (3.78 1H, dd, J=7.3,5.5), 1.01 (1H, s) etc.;
13CNMR (CD
3OD, 100MHz) δ: 211.4,175.3,151.2,149.5,128.3,119.0,90.8,85.6,68.3,57.4,56.2,46.4,45.2,43.9,39.2,38.3,34.2,35.6,27.3,29.2,25.9,20.7,20.3.
Embodiment 6: hellebore petunidin space isomer new compound I～III is to HeLa cell and MCF cytotoxic activity
Material: Cervical Cancer HeLa Cells and human breast carcinoma MCF are available from Chinese The 2nd Army Medical College
Compound I～III that embodiment 3～5 obtains
HUACHANSU ZHUSHEYE: Anhui Jinchan Biochemical Co., Ltd.
Respectively take Cervical Cancer HeLa Cells and human breast carcinoma MCF as cell model, primary dcreening operation compound I～V.Experimental design is as follows:
Respectively logarithmic phase HeLa and MCF cell are adjusted to 5 * 10 with 10% calf serum RPMI1640 substratum
4Individual/ml, set up control group, HUACHANSU ZHUSHEYE group and experimental group separately, 3 every group multiple holes, every hole 10
4Individual, be connected in 96 orifice plates, place 37 ℃, contain the incubator of 5%CO2, after 24h is adherent, change 10% calf serum RPMI-1640 substratum; Experimental group adds respectively and contains 4 * 10
-4((amount to dry maxima skin concentration is 6 * 10 to the methanol solution of compound I～III) to each compound of mg/ml
-3G/ml), the HUACHANSU ZHUSHEYE group adds identical HUACHANSU ZHUSHEYE of amounting to concentration, and control group adds the methanol solution of same amount.Take out behind the 48h, after physiological saline is washed cell, violet staining half an hour, flush away Viola crystallina, and with the dissolve with methanol of 100 μ l, measure the absorption value of 490nm with microplate reader.Cell mortality %=(A
490Contrast-A
490Sample)/A
490Contrast * 100% the results are shown in Table 1.
Table 1 compound I～III is to the mortality ratio (%) of HeLa and MCF cell
By data in the table as can be known, compound I～III is to HeLa and the MCF cell mortality HUACHANSU ZHUSHEYE under the identical dry maxima skin concentration.
Claims (3)
1. the preparation method of a hellebore petunidin space isomer compound is characterized in that:
1) get the bufadienolide extract, separate through preparation HPLC, chromatographic condition is: anti-phase Xterra C18 chromatographic column, A:0.1%v/v formic acid water; B:0.1%v/v formic acid acetonitrile, gradient elution: 5%B～95%B, 60min; Adopt UV-detector to detect in the sepn process, detect wavelength 300nm, collect respectively each cut by the retention time at different samples peak in the testing process, collect altogether 20 cuts;
2) get Fraction 5 and reclaim solvent, separate through HPLC, chromatographic condition is: cyclodextrin chromatographic column, A: normal hexane; B: Virahol, gradient elution: 5%B～30%B, 55min collects each chromatographic peak, reclaims respectively solvent, determines through the nuclear-magnetism experiment, obtains to contain the cut of compound I;
Get Fraction 6 and reclaim solvent, separate through HPLC, chromatographic condition is: cyclodextrin chromatographic column, A:0.1%v/v formic acid water; B:0.1%v/v formic acid acetonitrile, gradient condition: 95%B～60%B, 40min collects each chromatographic peak, reclaims respectively solvent, determines through the nuclear-magnetism experiment, obtains to contain the cut of compound I I;
Get Fraction 10 and reclaim solvent, separate through HPLC, chromatographic condition is: cyclodextrin chromatographic column, A: water; B: methyl alcohol, gradient elution: 5%B～50%B, 35min collects each chromatographic peak, reclaims respectively solvent, determines through the nuclear-magnetism experiment, obtains to contain the cut of compound III;
2. preparation method according to claim 1 is characterized in that:
The preparation process of described bufadienolide extract is: toad skin extraction using alcohol, macroporous resin on the concentrated solution behind the Recycled ethanol, first water and low-concentration ethanol wash-out are used the high concentration ethanol wash-out again, collect elutriant, decompression and solvent recovery namely gets the bufadienolide extract;
Low-concentration ethanol refers to 10%～50% ethanol, and high concentration ethanol refers to 70%～100% ethanol.
3. according to claim 2 preparation method, it is characterized in that: the weight percentage of bufadienolide is 60%～90% in the described bufadienolide extract.
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Non-Patent Citations (2)
| Title |
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| 刘俊珊等.蟾酥及其活性成分抗肿瘤作用研究进展.《国际药学研究杂志》.2009,第36卷(第2期),115-120. * |
| 釜野德明等.ガマbufadienolideの構造と抗腫瘍活性.《symposium on the chemistry of natural products》.1996,第38卷349-354. * |
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