CN101289453B - Ellagic acid compounds preparation method - Google Patents
Ellagic acid compounds preparation method Download PDFInfo
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- CN101289453B CN101289453B CN2008100182305A CN200810018230A CN101289453B CN 101289453 B CN101289453 B CN 101289453B CN 2008100182305 A CN2008100182305 A CN 2008100182305A CN 200810018230 A CN200810018230 A CN 200810018230A CN 101289453 B CN101289453 B CN 101289453B
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- 0 *C1C(Oc2cc(C(Oc3c-4c(C(O5)=O)cc(*)c3*=C)=O)c-4c5c2*)OC2(*)OC12 Chemical compound *C1C(Oc2cc(C(Oc3c-4c(C(O5)=O)cc(*)c3*=C)=O)c-4c5c2*)OC2(*)OC12 0.000 description 1
- OOHRLBCRGFLPSZ-UHFFFAOYSA-N COc(c(O1)c(c(C(Oc2c(c(O)c3)OC)=O)c4)-c2c3C1=C)c4O Chemical compound COc(c(O1)c(c(C(Oc2c(c(O)c3)OC)=O)c4)-c2c3C1=C)c4O OOHRLBCRGFLPSZ-UHFFFAOYSA-N 0.000 description 1
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Abstract
The invention discloses an ellagic acid compound, the preparation thereof and applications in anticancer drugs. The ellagic acid compound is an anticancer monomer compound obtained by extracting and separating from hylonoma euphorbia root (the root and the aerial part of the euphorbia hylonoma which belongs to euphorbia). The preparation method thereof is as follows: firstly, ethanol is used for extracting the crushed air dried hylonoma euphorbia root, and obtaining extractum by concentration; secondly, chloroform and ethyl acetate are used for extracting, passing a chromatography respectively, and an eluent is used for eluting to obtain two ellagic acid compounds. The two ellagic acid compounds can inhibit the growth of human hepatoma cells and gastric cancer cells obviously by anticancer pharmacological experiments, and have the advantages of the wide anticancer spectrum, the strong antitumor activity, etc., thus being capable of being used for preparing anti-hepatoma or anti-gastric cancer drugs after combined with the carriers and excipients accepted in pharmacy.
Description
Technical field
The present invention relates to a kind of cancer therapy drug and preparation method thereof, two kinds that specifically obtain for the raw material extraction separation with the Root of Hupeh Euphorbia have antitumour activity ellagic acid compounds and preparation method thereof and the application in cancer therapy drug.
Background technology
Cancer is the formidable enemy of harm humans health.The annual New Development cases of cancer of China has 1,600,000 people approximately, and that dies from cancer every year has 1,300,000 people approximately, rises year by year because of the dead number of cancer in the whole nation, and China is the zone occurred frequently of cancers such as liver cancer, cancer of the stomach, lung cancer, mammary cancer, and cancer mortality is high.Cancer has all caused for patient's body and mind to be difficult to the wound of repairing and curing.
Still do not have effective medicine at present and can cure most of cancer patientss, though chemotherapeutics has certain curative effect to cancer, toxic side effect is big, and Chinese patent medicine is generally lower to the curative effect of cancer, and curative effect is all undesirable.The cancer therapy drug of clinical application at present mostly is cytotoxic drug greatly, when suppressing tumor cell proliferation, the propagation of organism normal cell also is suppressed, as hemopoietic stem cell etc., thereby cause the marrow hemopoiesis function impaired, red corpuscle or leucocyte level are low, the propagation that has suppressed tumour often, but also destroyed patient's immunizing power, finally be difficult to obtain good therapeutic action, also limited the clinical application of chemotherapeutics.Therefore, exploitation high-efficiency low-toxicity, antitumor drug that target is strong become the antitumor drug hot of research and development.Also there are not these two kinds of ellagic acid compounds 3 of bibliographical information at present through data consultation, 3 '-di-O-methylellagic acid and 3,3 '-di-O-methylellagic acid-4 '-O-β-D-xylopyroside is used for treatment for cancer such as anti-liver cancer, cancer of the stomach.
Summary of the invention
The purpose of this invention is to provide a kind of ellagic acid compounds, be a kind of efficient, wide anticancer monomeric compound of anticancer spectrum, two kinds of ellagic acid compounds that specifically obtain for the raw material extraction separation and the application aspect preparation treatment liver cancer cancer of the stomach medicine thereof with the Root of Hupeh Euphorbia.
Technical scheme of the present invention is achieved in that
Its anticancer active substance mainly is an ellagic acid class material of the present invention in the medicinal extract of Root of Hupeh Euphorbia of the present invention.The present invention from Root of Hupeh Euphorbia (have another name called WUDUOYUN, overturn the heavens seal, thunder-crash bomb, Euphorbia hylonomaHand-Mazz.) in extraction separation two kinds of ellagic acid compounds, they are 3,3 '-di-O-methylellagic acid or 3,3 '-di-O-methylellagic acid-4 '-O-β-D-xylopyroside, its molecular formula is respectively C
16H
10O
8Or C
21H
18O
12, structure is as follows respectively:
3，3’-di-O-methylellagic?acid
Or
3，3’-di-O-methylellagic?acid-4’-O-β-D-xylopyroside
Two kinds of ellagic acid compounds 3 of the present invention, 3 '-di-O-methylellagic acid and 3, the preparation method of 3 '-di-O-methylellagic acid-4 '-O-β-D-xylopyroside the steps include:
A, Root of Hupeh Euphorbia is pulverized the back is 50～100% alcohol immersion, 3～12h with volumetric concentration, refluxing extraction 1～5 time, and each 1～6h, united extraction liquid is evaporated to medicinal extract;
B, in medicinal extract, add 1～10 times of water gaging, use sherwood oil, chloroform, ethyl acetate extraction more respectively, reclaim solvent, obtain sherwood oil part, chloroform part, ethyl acetate part respectively;
C, chloroform is partly carried out silica gel column chromatography, earlier with being that eluent carries out gradient elution with chloroform-methanol behind the petroleum ether-ethyl acetate, the compound that the centre elutes is 3,3 '-di-O-methylellagic acid-4 '-O-β-D-xylopyroside;
D, ethyl acetate part also being carried out column chromatography, is that eluent carries out gradient elution with the chloroform-methanol, and the compound that the centre elutes is 3,3 '-di-O-methylellagic acid.
Described C step eluent petroleum ether-ethyl acetate graded is 100: 1～1: 100, and the chloroform-methanol graded is 100: 1～1: 100.
Described D step eluent chloroform-methanol graded is 100: 1～1: 100.
3,3 '-di-O-methylellagic acid or 3, the application of 3 '-di-O-methylellagic acid-4 '-O-β-D-xylopyroside in anti-liver cancer of preparation or cancer of the stomach medicine.
Adopt mtt assay.The SMMC-7721 liver cancer cell in vegetative period of taking the logarithm, adjusting cell concn with complete culture solution is 3 * 10
4Individual/ml, be inoculated in 96 well culture plates, 200 μ l are inoculated in every hole, and adding the compound final concentration behind the conventional 24h of cultivation is 10,25,50,80,100 μ g/ml, other establishes no medicine (ethanol that contains compounding pharmaceutical) nutrient solution and blank hole, and each drug level is established 5 multiple holes.After the dosing 96 orifice plates are placed CO
237 ℃ of cultivations of thermostat container.24,48, the 72h different time points, take out cell plate respectively, it is the MTT of 5mg/ml that every hole adds 20 μ l mass concentrations, the conventional 4h that cultivates inhales and abandons supernatant, and every hole adds DMSO 150 μ l, mixing fully vibrates, precipitated crystal is fully dissolved, and is to detect wavelength with 490nm, utilizes microplate reader to detect each hole absorbancy (OD) value.
Inhibiting rate (%)=(1-treatment group OD value/control group OD value) * 100%
Test-results shows, along with 3,3 '-di-O-methylellagic acid and 3,3 '-di-O-methylellagic acid-4 '-O-β-D-xylopyroside rising of concentration separately, its inhibiting rate to human liver cancer cell also raises, when 3, when the concentration of 3 '-di-O-methylellagic acid is 100 μ g/ml to the inhibiting rate of human liver cancer cell up to 75.3%, when with 3, when the concentration of 3 '-di-O-methylellagic acid-4 '-O-β-D-xylopyroside is 100 μ g/ml to the inhibiting rate of human liver cancer cell up to 64.9%.
Two kinds of ellagic acid compounds application aspect the medicine of treatment cancer of the stomach among the present invention.
Adopt mtt assay.The SGC-7901 stomach cancer cell in vegetative period of taking the logarithm, adjusting cell concn with complete culture solution is 3 * 10
4Individual/ml, be inoculated in 96 well culture plates, 200 μ l are inoculated in every hole, and adding the compound final concentration behind the conventional 24h of cultivation is 10,25,50,80,100 μ g/ml, other establishes no medicine (ethanol that contains compounding pharmaceutical) nutrient solution and blank hole, and each drug level is established 5 multiple holes.After the dosing 96 orifice plates are placed CO
237 ℃ of cultivations of thermostat container.24,48, the 72h different time points, take out cell plate respectively, it is the MTT of 5mg/ml that every hole adds 20 μ l mass concentrations, the conventional 4h that cultivates inhales and abandons supernatant, and every hole adds DMSO150 μ l, mixing fully vibrates, precipitated crystal is fully dissolved, and is to detect wavelength with 490nm, utilizes microplate reader to detect each hole absorbancy (OD) value.
Inhibiting rate (%)=(1-treatment group OD value/control group OD value) * 100%
Test-results shows, along with 3,3 ' di-O-methylellagic acid and 3,3 '-di-O-methylellagic acid-4 '-O-β-D-xylopyroside rising of concentration separately, its inhibiting rate to gastric carcinoma cells also raises, when 3, when the concentration of 3 '-di-O-methylellagic acid is 100 μ g/ml to the inhibiting rate of gastric carcinoma cells up to 75.5%, when with 3, when the concentration of 3 '-di-O-methylellagic acid-4 '-O-β-D-xylopyroside is 100 μ g/ml to the inhibiting rate of gastric carcinoma cells up to 70.5%.
Two kinds of ellagic acid compounds 3 of the present invention, 3 '-di-O-methylellagic acid and 3,3 '-di-O-methylellagic acid-4 '-O-β-D-xylopyroside is the anticancer monomeric compound that extraction separation comes out from the Chinese medicinal materials Root of Hupeh Euphorbia, toxic side effect is little, anticancer spectrum is wide, and cancer cells is had clearly restraining effect.Two kinds of ellagic acid compounds 3 of the present invention, 3 '-di-O-methylellagicacid and 3,3 '-di-O-methylellagic acid-4 '-O-β-D-xylopyroside are through anticancer pharmacological testing, and the result shows its antitumour activity height.3,3 '-di-O-methylellagic acid and 3, when the concentration of 3 '-di-O-methylellagic acid-4 '-O-β-D-xylopyroside is 100 μ g/ml to the inhibiting rate of human liver cancer cell respectively up to 75.3% and 64.9%, to the inhibiting rate of gastric carcinoma cells respectively up to 75.5% and 70.5%.
According to the literature, ellagic acid class material has very strong antitumor and antimutagenic effect, experiment and people organize experiment in vitro to show that ellagic acid class material all has the good restraining effect to various tumour cells in the mouse body, and be particularly remarkable to tumour cell effects such as colorectal carcinoma, esophagus cancer, lung cancer, liver cancer, mammary cancer and skin carcinomas.Sehrawat Anuradha etc. studies show that 3,3 '-di-O-methylellagic acid produces liver cancer tumour promotion restraining effect after irritating hello mouse with the dosage of 50mg/kg body wt., infer the xyloside 3 of this compound in view of the above, 3 '-di-O-methylellagic acid-4 '-O-β-D-xylopyroside has the good anticancer activity in animal body too, therefore preliminary two kinds of ellagic acid compounds 3 of inferring among the present invention, 3 '-di-O-methylellagic acid and 3,3 '-di-O-methylellagic acid-4 '-O-β-D-xylopyroside has better action to various cancers especially liver cancer and cancer of the stomach, thus these two kinds of ellagic acid compounds with can be used to prepare anti-liver cancer or anti-cancer of the stomach medicine after pharmaceutically acceptable carrier and auxiliary material combine.
Description of drawings
Fig. 1 is the present invention 3,3 '-di-O-methylellagic acid's
1The H-NMR collection of illustrative plates;
Fig. 2 is the present invention 3,3 '-di-O-methylellagic acid's
13The C-NMR collection of illustrative plates;
Fig. 3 is the present invention 3,3 '-di-O-methylellagic acid-4 '-O-β-D-xylopyroside's
1The H-NMR collection of illustrative plates;
Fig. 4 is the present invention 3,3 '-di-O-methylellagic acid-4 '-O-β-D-xylopyroside's
13The C-NMR collection of illustrative plates;
Fig. 5 is the present invention 3, the DEPT collection of illustrative plates of 3 '-di-O-methylellagic acid-4 '-O-β-D-xylopyroside;
A is an aspect graph under the light microscopic of SMMC-7721 cell behind the blank group effect 48h among Fig. 6;
B is 3 among Fig. 6, and 3 '-di-O-methylellagic acid acts on aspect graph under the light microscopic of SMMC-7721 cell behind the 48h when concentration is 10 μ g/mL;
C is 3 among Fig. 6, and 3 '-di-O-methylellagic acid acts on aspect graph under the light microscopic of SMMC-7721 cell behind the 48h when concentration is 50 μ g/mL;
D is 3 among Fig. 6, and 3 '-di-O-methylellagic acid acts on aspect graph under the light microscopic of SMMC-7721 cell behind the 48h when concentration is 100 μ g/mL;
A is an aspect graph under the light microscopic of SGC-7901 cell behind the blank group effect 48h among Fig. 7;
B is 3 among Fig. 7, and 3 '-di-O-methylellagic acid-4 '-O-β-D-xylopyroside acts on aspect graph under the light microscopic of SGC-7901 cell behind the 48h when concentration is 10 μ g/mL;
C is 3 among Fig. 7, and 3 '-di-O-methylellagic acid-4 '-O-β-D-xylopyroside acts on aspect graph under the light microscopic of SGC-7901 cell behind the 48h when concentration is 50 μ g/mL;
D is 3 among Fig. 7, and 3 '-di-O-methylellagic acid-4 '-O-β-D-xylopyroside acts on aspect graph under the light microscopic of SGC-7901 cell behind the 48h when concentration is 100 μ g/mL.
Below in conjunction with accompanying drawing content of the present invention is described in further detail.
Embodiment
Embodiment 1:
Two kinds of ellagic acid compounds 3,3 '-di-O-methylellagic acid and 3, the extraction and separation method of 3 '-di-O-methylellagic acid-4 '-O-β-D-xylopyroside:
1. to pick up from the Taibai County, Shaanxi Province in October, 2005 domestic for the Root of Hupeh Euphorbia medicinal material, behind natural air drying, pulverize, with the Root of Hupeh Euphorbia 9kg that pulverizes with 85% alcohol immersion 12h, refluxing extraction 3 times, 4h at every turn, united extraction liquid is evaporated to medicinal extract;
2. in medicinal extract, add suitable quantity of water, use sherwood oil, chloroform, ethyl acetate extraction more respectively, reclaim solvent, obtain sherwood oil part, chloroform part, ethyl acetate part each 20g, 89g, 170g respectively;
3. chloroform is partly carried out silica gel column chromatography, earlier with being that eluent carries out gradient elution with chloroform-methanol behind the petroleum ether-ethyl acetate, equivalent is collected every part of 150ml of elutriant, collect 324 parts altogether, elutriant is done silica gel thin-layer chromatography respectively, the thin layer plate spray develops the color with 10% ethanol solution of sulfuric acid, merge identical elutriant, from wherein merge 212～240 parts, separated out crystal, obtain needle crystal with recrystallizing methanol again, be of the present invention 3,3 '-di-O-methylellagic acid-4 '-O-β-D-xylopyroside;
4. the ethyl acetate part is also carried out column chromatography, with the chloroform-methanol is that eluent carries out gradient elution, equivalent is collected every part of 150ml of elutriant, collect 350 parts altogether, elutriant is done silica gel thin-layer chromatography respectively, the thin layer plate spray develops the color with 10% ethanol solution of sulfuric acid, merge identical elutriant, from the 87-142 part that wherein merges, separated out crystal, again the crystal that obtains with acetone recrystallization be of the present invention 3,3 '-di-O-methylellagic acid.
Embodiment 2:
Two kinds of ellagic acid compounds 3,3 '-di-O-methylellagic acid and 3, the structure of 3 '-di-O-methylellagic acid-4 '-O-β-D-xylopyroside is identified
The crystal of separating out in the elutriant during ethyl acetate part column chromatography is the crystallization of pale yellow powder shape, and fusing point is 332-334 ℃, and MS shows that molecular weight is 344, and molecular formula is C
16H
10O
8By
1H-NMR and
13C-NMR collection of illustrative plates (seeing accompanying drawing 1 and accompanying drawing 2) is pointed out to such an extent that it is ellagic acid class material, and its chemical structural formula is as follows:
3，3’-di-O-methylellagic?acid
Accompanying drawing 1
1Spectral data and belong to as follows in the H-NMR collection of illustrative plates:
1H-NMR(DMSO-d
6)δppm：4.05(6H，s，2×OCH
3)，7.54(2H，s，H-5，H-5’)，10.79(2H，s，OH-4，OH-4’)。
Accompanying drawing 2
13C-NMR collection of illustrative plates spectral data and belong to as follows:
13C-NMR(DMSO-d
6)δppm：111.46(s，C-1，C-1’)，141.25(s，C-2，C-2’)，?140.23(s，C-3，C-3’)，152.23(s，C-4，C-4’)，111.69(d，C-5，C-5’)，112.18(s，C-6，C-6’)，158.52(s，C-7，C-7’)，61.00(q，2×OCH
3)。
1H-NMR,
13The C-NMR spectrum data determines that according to spectral data this compound is 3,3 '-di-o-methylellagic acid through contrasting basically identical with document.
The crystal of separating out in the elutriant during chloroform part column chromatography is the white powder crystallization, and fusing point is 227-229 ℃, and MS shows that molecular weight is 462, and molecular formula is C
21H
18O
12By
1H-NMR,
13C-NMR and DEPT collection of illustrative plates are pointed out to such an extent that it is ellagic acid class material, and its chemical structural formula is as follows:
3，3’-di-O-methylellagic?acid-4’-O-β-D-xylopyroside
Accompanying drawing 3
1Spectral data and belong to as follows in the H-NMR collection of illustrative plates:
1H-NMR(DMSO)δppm：δ7.48(1H，d，J＝5.3，H-5)，δ7.73(1H，d，J＝3.2，H-5)，δ4.05(1H，s，3-OCH
3)，δ4.08(1H，s，3’-OCH
3)，δ10.8(1H，s，4-OH)。
Accompanying drawing 4
13C-NMR collection of illustrative plates spectral data and belong to as follows:
13C-NMR(DMSO)δppm：111.07(C-1)，140.94(C-2)，140.19(C-3)，151.26(C-4)，111.65(C-5)，111.82(C-6)，158.37(C-7)，114.19(C-1’)，141.58(C-2’)，141.98(C-3’)，152.84(C-4’)，112.02(C-5’)，112.72(C-6’)，152.40(C-7’)，101.94(C-1”)，73.09(C-2”)，76.17(C-3”)，69.31(C-4”)，65.84(C-5”)，61.48(3-OCH
3)，61.02(3’-OCH
3)。
Accompanying drawing 5 DEPT collection of illustrative plates spectral datas and belong to as follows:
This compound of DEPT spectrum prompting has δ 111.89,111.60, and 101.84,76.18,73.08,69.28,61.68,61.02 places are methyl or methine carbon atom signals, and δ 65.84 is the mesomethylene carbon atom signals, and remaining is the quaternary carbon signal.
1H-NMR,
13C-NMR and DEPT spectrum data determine that according to spectral data this compound is 3,3 '-di-O-methylellagic acid-4 '-O-β-D-xylopyroside through contrasting basically identical with document.
Embodiment 3:
3,3 '-di-O-methylellagic acid and 3,3 '-di-O-methylellagic acid-4 '-O-β-D-xylopyroside antihepatocarcinoma effect
1. cell cultures and monomeric compound
Human hepatoma cell strain SMMC-7721 is provided by Xi'an Communications University's medical research test center, and cell cultures is cultivated at 37 ℃, 5%CO in the RPMI-1640 substratum that contains 10% calf serum
2The saturated humidity incubator in, when cell converged in per 2～3 days, go down to posterity with 0.25% tryptic digestion, take the logarithm vegetative period cell be used for the test.Two monomeric compounds are ellagic acid substances 3 in the Root of Hupeh Euphorbia that chemical structure is identified, 3 '-di-O-methylellagic acid and 3,3 '-di-O-methylellagic acid-4 '-O-β-D-xylopyroside.
2. test method
Adopt mtt assay to measure ellagic acid class material 3 in the Root of Hupeh Euphorbia, 3 '-di-O-methylellagic acid and 3,3 '-di-O-methylellagic acid-4 '-O-β-D-xylopyroside (dissolving with DMSO) is to the liver cancer cell growth restraining effect.
The SMMC-7721 liver cancer cell in vegetative period of taking the logarithm, adjusting cell concn with complete culture solution is 3 * 10
4Individual/ml, be inoculated in 96 well culture plates, 200 μ l are inoculated in every hole, and adding the compound final concentration behind the conventional 24h of cultivation is 10,25,50,80,100 μ g/ml, other establishes no medicine (ethanol that contains compounding pharmaceutical) nutrient solution and blank hole, and each drug level is established 5 multiple holes.After the dosing 96 orifice plates are placed CO
237 ℃ of cultivations of thermostat container.24,48, the 72h different time points, take out cell plate respectively, it is the MTT of 5mg/ml that every hole adds 20 μ l mass concentrations, the conventional 4h that cultivates inhales and abandons supernatant, and every hole adds DMSO150 μ l, mixing fully vibrates, precipitated crystal is fully dissolved, and is to detect wavelength with 490nm, utilizes microplate reader to detect each hole absorbancy (OD) value.
Inhibiting rate (%)=(1-treatment group OD value/control group OD value) * 100%
3. test-results
The result is as shown in table 1, along with 3,3 '-di-O-methylellagic acid and 3,3 '-di-O-methylellagic acid-4 '-O-β-D-xylopyroside rising of concentration separately, its inhibiting rate to human liver cancer cell also raises, when 3, when the concentration of 3 '-di-O-methylellagic acid is 100 μ g/ml to the inhibiting rate of human liver cancer cell up to 75.3%, when with 3, when the concentration of 3 '-di-O-methylellagic acid-4 '-O-β-D-xylopyroside is 100 μ g/ml to the inhibiting rate of human liver cancer cell up to 64.9%.
The growth inhibition ratio of 1,2 pairs of SSMC-7721 cells of table 1 sample
| Sample | Concentration (μ g/ml) | 24h? | 48h? | 72h? |
| 1? | 10 25 50 80 100 10 | 10.2±0.84 17.3±1.05 20.6±1.25 27.1±0.67 30.3±0.58 10.2±0.28 | 13.9±1.02 18.3±0.85 23.5±0.13 32.7±0.38 35.6±1.54 13.6±0.67 | 15.8±1.13 19.5±0.86 32.2±1.27 62.4±0.93 75.3±1.56 22.3±1.07 |
| 2? | 25 50 80 100 | 15.4±0.83 27.8±0.57 37.6±0.86 37.8±0.68 | 18.5±1.47 33.3±1.06 41.7±1.24 45.1±0.32 | 28.1±1.14 39.5±0.83 58.2±0.77 64.9±0.52 |
1-3，3’-di-O-methylellagic?acid
2-3，3’-di-O-methylellagic?acid-4’-O-β-D-xylopyroside
Wherein 3,3 '-di-O-methylellagic acid acts on the visible accompanying drawing 6 of form under the light microscopic of liver cancer cell behind the people SMMC-7721 liver cancer cell when different concns.By accompanying drawing 6 as can be seen, in the blank group, be viable cell all basically, and the dead cell of different ratios is all arranged in the administration group, and increase along with liquor strength, the shared ratio of dead cell is big more, and wherein when liquor strength reached 100 μ g/ml, the ratio of dead cell was up to about 3/4.
Embodiment 4:
3,3 '-di-O-methylellagic acid and 3, the anti-cancer of the stomach effect of 3 '-di-O-methylellagic acid-4 '-O-β-D-xylopyroside
1. cell cultures and monomeric compound
Human stomach cancer cell line SGC-7901 is provided by Xi'an Communications University's medical research test center, and cell cultures is cultivated at 37 ℃, 5%CO in the RPMI-1640 substratum that contains 10% calf serum
2The saturated humidity incubator in, when cell converged in per 2～3 days, go down to posterity with 0.25% tryptic digestion, take the logarithm vegetative period cell be used for the test.Two monomeric compounds are ellagic acid substances 3 in the Root of Hupeh Euphorbia that chemical structure is identified, 3 '-di-O-methylellagic acid and 3,3 '-di-O-methylellagic acid-4 '-O-β-D-xylopyroside.
2. test method
Adopt mtt assay to measure ellagic acid class material 3 in the Root of Hupeh Euphorbia, 3 '-di-O-methylellagic acid and 3,3 '-di-O-methylellagic acid-4 '-O-β-D-xylopyroside (dissolving with DMSO) is to the effect of stomach cancer cell growth-inhibiting.
The SGC-7901 stomach cancer cell in vegetative period of taking the logarithm, adjusting cell concn with complete culture solution is 3 * 10
4Individual/ml, be inoculated in 96 well culture plates, 200 μ l are inoculated in every hole, and adding the compound final concentration behind the conventional 24h of cultivation is 10,25,50,80,100 μ g/ml, other establishes no medicine (ethanol that contains compounding pharmaceutical) nutrient solution and blank hole, and each drug level is established 5 multiple holes.After the dosing 96 orifice plates are placed CO
237 ℃ of cultivations of thermostat container.24,48, the 72h different time points, take out cell plate respectively, it is the MTT of 5mg/ml that every hole adds 20 μ l mass concentrations, the conventional 4h that cultivates inhales and abandons supernatant, and every hole adds DMSO150 μ l, mixing fully vibrates, precipitated crystal is fully dissolved, and is to detect wavelength with 490nm, utilizes microplate reader to detect each hole absorbancy (OD) value.
Inhibiting rate (%)=(1-treatment group OD value/control group OD value) * 100%
3. test-results
The result is as shown in table 2, along with 3,3 '-di-O-methylellagic acid and 3,3 '-di-O-methylellagic acid-4 '-O-β-D-xylopyroside rising of concentration separately, its inhibiting rate to gastric carcinoma cells also raises, when 3, when the concentration of 3 '-di-O-methylellagic acid is 100 μ g/ml to the inhibiting rate of gastric carcinoma cells up to 75.5%, when with 3, when the concentration of 3 '-di-O-methylellagic acid-4 '-O-β-D-xylopyroside is 100 μ g/ml to the inhibiting rate of gastric carcinoma cells up to 70.5%.
The growth inhibition ratio of 1,2 pairs of SGC-7901 cells of table 2 sample
| Sample | Concentration (μ g/ml) | 24h? | 48h? | 72h? |
| 1? | 10? | 4.5±0.38? | 9.2±0.67? | 19.7±0.85? |
| 2? | 25 50 80 100 10 25 50 80 100 | 6.6±1.42 18.3±0.26 32.4±1.27 37.9±0.53 3.2±0.31 7.6±0.51 26.9±0.83 37.2±0.35 40.0±1.49 | 13.9±0.68 26.5±0.56 42.1±0.58 46±0.34 8.6±0.59 18.4±1.28 36.1±0.19 49.3±1.02 52.6±0.12 | 27.2±0.92 38.8±0.48 64.3±1.08 75.5±0.59 15.9±0.67 28.8±0.2 42.4±1.41 62±1.39 70.5±0.04 |
1-3，3’-di-O-methylellagic?acid
2-3，3’-di-O-methylellagic?acid-4’-O-β-D-xylopyroside
Wherein 3,3 '-di-O-methylellagic acid-4 '-O-β-D-xylopyroside acts on the visible accompanying drawing 7 of form under the light microscopic of stomach cancer cell behind the people SGC-7901 stomach cancer cell when different concns.By accompanying drawing 7 as can be seen, all be viable cell basically in the blank group, and viable count descends in the administration group, and increase along with liquor strength, the ratio row of viable cell are more and more littler, the shared ratio of corresponding dead cell is increasing, and wherein when liquor strength reached 100 μ g/ml, the ratio of dead cell can reach about 7/10.
Claims (1)
1. an anticancer ellagic acid compounds preparation method is characterized in that, comprises the following steps:
A, Root of Hupeh Euphorbia is pulverized the back is 50～100% alcohol immersion, 3～12h with volumetric concentration, refluxing extraction 1～5 time, and each 1～6h, united extraction liquid is evaporated to medicinal extract;
B, in medicinal extract, add 1～10 times of water gaging, use sherwood oil, chloroform, ethyl acetate extraction more respectively, reclaim solvent, obtain sherwood oil part, chloroform part, ethyl acetate part respectively;
C, chloroform is partly carried out silica gel column chromatography, use earlier the petroleum ether-ethyl acetate gradient elution, the back is that eluent carries out gradient elution with chloroform-methanol, and the compound that the centre elutes is 3,3 '-di-O-methylellagic acid-4 '-O-β-D-xylopyroside; Eluent petroleum ether-ethyl acetate graded is 100: 1～1: 100, and the chloroform-methanol graded is 100: 1～1: 100;
D, ethyl acetate partly being carried out column chromatography, is that eluent carries out gradient elution with the chloroform-methanol, and the compound that the centre elutes is 3,3 '-di-O-methylellagic acid, and eluent chloroform-methanol graded is 100: 1～1: 100;
Wherein, 3,3 '-di-O-methylellagic acid or 3,3 '-di-O-methylellagicacid-4 '-O-β-D-xylopyroside, its molecular formula is respectively C
16H
10O
8Or C
21H
18O
12, structure is as follows respectively:
3，3’-di-O-methylellagic?acid
Or
3，3’-di-O-methylellagic?acid-4’-O-β-D-xylopyroside。
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| CN112294800A (en) * | 2019-08-02 | 2021-02-02 | 四川大学 | Application of ellagic acid in preparing medicine for preventing and/or treating nonalcoholic fatty liver disease and liver injury |
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