CN102335233A - Polyphenol extract of trembling poplar as well as extraction method and application thereof - Google Patents
Polyphenol extract of trembling poplar as well as extraction method and application thereof Download PDFInfo
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- liriodendron
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- 150000008442 polyphenolic compounds Chemical class 0.000 title claims abstract description 37
- 235000013824 polyphenols Nutrition 0.000 title claims abstract description 37
- 241000183024 Populus tremula Species 0.000 title abstract 5
- 238000000605 extraction Methods 0.000 title abstract 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 30
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims abstract description 24
- 239000002904 solvent Substances 0.000 claims abstract description 11
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 10
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- 239000000463 material Substances 0.000 abstract description 7
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- 239000000047 product Substances 0.000 description 7
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- Medicines Containing Plant Substances (AREA)
Abstract
The invention discloses a polyphenol extract of trembling poplar as well as an extraction method and application thereof. The extraction method of the polyphenol extract of trembling poplar comprises the following steps: baking a trembling poplar plant material at 35 DEG C, and crushing; extracting with 95% ethanol, distilling to remove a solvent to obtain residues; dissolving the residues with chloroform, filtering to obtain filtrate and a primary residue, and distilling the filtrate in reduced pressure to obtain the polyphenol extract; and dissolving the primary residue by use of methanol, and distilling the solvent at reduced pressure to obtain the polyphenol extract. The polyphenol extract of the trembling poplar is convenient to extract, has high content, can be applied to industrial large-scale production, has remarkable effects in removing oxygen radical of animals, inhibiting generation, proliferation and transfer of tumor cells of animals, is practical in treating cancer cells, and can generate good economical and social benefits.
Description
Technical field
The present invention relates to a kind of trees tissue extract and method for distilling and application, be specifically related to Polyphenols extract and method for distilling and the application of a kind of Liriodendron.
Background technology
The Magnoliaceae Liriodendron only has two kinds, and a kind of Cortex Liriodendri tulipiferae that is natural distributed in the eastern united states is another kind of for being distributed in the mandarin jacket wood of south China.These two kinds all have the pharmacy historical records in the U.S., China and world other countries.Cortex Liriodendri tulipiferae, promptly Radix Curcumae XIANGSHU or boxtree are a kind of tall trees kinds, can reach 100 feet high usually, are the distinctive seeds in eastern united states (Keeler, 1902).The bark of Cortex Liriodendri tulipiferae is used as tonic, analeptic and antipyretic by American Indian, and might be used to treat the intermittence fever (Rafinesque etc., 1828) that is caused by malaria.Cortex Liriodendri tulipiferae is used as anti-malaria medicaments in recent years by a large amount of confirmations (R. Graziosea, T. Rathinasabapathy, C. Lategan, et al. 2011) on American history.It is also migrated the succedaneum as the rare cinchonae cortex of import (ledger bark) (Thacher, 1967) by the U.S. afterwards.In India, Cortex Liriodendri tulipiferae is used to treat recovery, rheumatism, dyspepsia (S. K. Kuanar, 2006).In China, bark, leaf and the root of its sibling species mandarin jacket wood is used to treat rheumatism and because the cough (the new medical college in Jiangsu, calendar year 2001) that ailment said due to cold or exposure invasion (wind and cold) causes in traditional traditional Chinese medical science.1963, a new cenospecies, mandarin jacket wood * Cortex Liriodendri tulipiferae is successfully cultivated by Chinese scholar professor Ye Peizhong.No matter be in theory, proud achievement in research (Wang ZR, 2003 years) is still all arranged on Chinese garden planting in Forest Tree Genetic Breeding.Yet up to the present, the medical value of hybridization tree does not obtain research as yet, especially in the drug research of treatment tumor.Cancer causes the millions of people dead every year in the whole world.In reverse, inhibition even prophylaxis of cancer, plant amedica separately or the medicine intervention of associating with oncotherapy in played pivotal role (Siveen KS, Kuttan G. 2010).People are more and more interested in substituting medicine and nontoxic antitumour treatments.It is reported, some active component in ethnic drug or the functional food effective to cancer, and be safe (Hakimuddin et al., 2006) to normal zooblast.
Summary of the invention
Goal of the invention:, the purpose of this invention is to provide the Polyphenols extract of a kind of Liriodendron to the deficiency that exists in the prior art.Another object of the present invention provides a kind of method for distilling of said extracted thing.The present invention also has a purpose to provide a kind of said extracted in the application that is used for treating the cancerous cell medicine in preparation.
Technical scheme: in order to realize the foregoing invention purpose, the technical scheme that the present invention adopts is following:
The Polyphenols extract of a kind of Liriodendron is obtained by following method: get the Liriodendron vegetable material, 35 ℃ of oven dry are pulverized; Use 95% ethanol extraction, the evaporative removal solvent gets residue; Use the dissolved in chloroform residue, filter, must filtrate; Dissolve with methanol filtrating, distilling under reduced pressure gets the Polyphenols extract, merges the Polyphenols extract and both gets.
The polyphenol mass content of Polyphenols extract is 20-90%.
Leaf and bark that said Liriodendron vegetable material is a Liriodendron.
Said Liriodendron plant is Chinese mandarin jacket wood, Cortex Liriodendri tulipiferae, and hybridized Chinese tuliptree.
A kind of method of extracting the Polyphenols extract of Liriodendron may further comprise the steps:
(1) get the Liriodendron vegetable material, 35 ℃ of oven dry are pulverized;
(2) use 95% ethanol extraction, the evaporative removal solvent gets residue;
(3) use the dissolved in chloroform residue, filter, must filtrate, evaporate to dryness gets little polarity polyphenol extract;
(4) dissolve with methanol filtrating, distilling under reduced pressure gets high polarity and middle polarity polyphenol extract.
The Polyphenols extract of Liriodendron is used for treating the application of cancer cell medicine in preparation.
Described cancer comprises breast cancer cell MDA-MB-231, breast cancer cell MCF-7, stomach cancer cell SGC-7901, HCC Huh7 and colon cancer cell HCT15.
Beneficial effect: the Polyphenols extract of Liriodendron of the present invention; The outstanding advantage that has is: preparation technology is simple, and the product yield is high, prepared extract; Generation, propagation and transferance with tangible removing animal oxygen-derived free radicals, inhibition animal tumor cell; Be used for treating the purposes widely that has of cancer cell medicine in preparation, have good practicability, can produce good economic benefits and social benefit.
The specific embodiment
Below in conjunction with specific embodiment the present invention is done further explanation.
The employed material of following examples is: leaf and the bark of fresh Liriodendron are collected by Nanjing Forestry University, and the BIAO and BEN of all collections carries out system to be identified, dry BIAO and BEN is preserved and is Institutes Of Technology Of Nanjing.Breast cancer cell MDA-MB-231, breast cancer cell MCF-7, stomach cancer cell SGC-7901, HCC Huh7 and colon cancer cell HCT15 are all from No.1 Attached Hospital, Nanjing Medical Univ.
The employed medicine of following examples is: gallic acid (GA), forint phenol (2M), (3-[4 for MTT; 5-methyl-2-yl]-2; The 3-diphenyltetrazodium bromine), fluorouracil and amycin, all available from U.S. Sigma company, other all chemicals are analytical reagent.
Embodiment 1
Get the Liriodendron bark of fresh collection, 35 ℃ of oven dry in air.Siccative is ground to medium fine powder, about 40 orders, subsequent use.
Get fine powder 350g, use 95% ethanol extracting then, 500ml extracting 3 times; Filter, merging filtrate, evaporation removes and desolvates, and gets the about 35.0g of residue; Use the dissolved in chloroform residue, 250mL dissolving 3 times, chloroform solvent is removed in distillation, gets the 8.4g product LTB2 and first residue; With dissolve with methanol first residue, 250mL dissolving 3 times, the distilling under reduced pressure solvent gets 13.3g product LTB3.Each product is put into aseptic screw cap bottle ,-20 ℃ of preservations, subsequent use.When being used for bioassay, each extract is dissolved in dimethyl sulfoxide (DMSO), and concentration is 10mg/mL ,-20 ℃ of preservations.
The dry leaves of other Liriodendrons is identical with said method with the method that bark extracts.
The Polyphenols extract yield table of variant plant tissue is as shown in table 1.
The Polyphenols extract yield table of the different vegetable materials of table 1
In the table, LT: Cortex Liriodendri tulipiferae; LC: Chinese mandarin jacket wood; LX: hybridization mandarin jacket wood; B: bark; L: leaves; 0: ethanol extraction total extract for the first time; 1: chloroform extracting total extract: 2: extract obtained after the chloroform extracting with the methanol extracting.
Determining total phenol method: the sample of 300 μ L is dissolved in 10% (V/V) forint phenol aqueous solution of 2250 μ L, leaves standstill 5min under the room temperature, in the dark to the sodium bicarbonate solution that wherein adds 2250 μ L (60g/L), mixing; 30 ℃ of temperature controls, lucifuge leaves standstill 90min; At the 725nm place, measure absorbance.Adopt gallic acid (5.14 ~ 51.40 μ g/mL) to do standard curve, the total phenol content result in the Polyphenols extract is expressed as the percent by weight of gallic acid.Concrete mensuration result is as shown in table 2.
The table 2 Polyphenols extract composition table that extracting goes out from leaves of plants and bark
| Material | LTB0 | LTB1 | LTB2 | LCB0 | LCB1 | LCB2 | LXB0 | LXB1 | LXB2 |
| Total phenols (%) | 14.6 | 6.5 | 20.9 | 18.1 | 7.7 | 24.6 | 11.1 | 5.6 | 15.5 |
| Material | LTL0 | LTL1 | LTL2 | LCL0 | LCL1 | LCL2 | LXL0 | LXL1 | LXL2 |
| Total phenols (%) | 25.5 | 14.9 | 36.4 | 25.8 | 13.9 | 31.3 | 22.7 | 15.2 | 25.6 |
In the table, LT: Cortex Liriodendri tulipiferae; LC: Chinese mandarin jacket wood; LX: hybridization mandarin jacket wood; B: bark; L: leaves; 0: ethanol extraction total extract for the first time; 1: chloroform extracting total extract: 2: extract obtained after the chloroform extracting with the methanol extracting.
Embodiment 2
Get the Liriodendron bark of fresh collection, 35 ℃ of oven dry in air.Siccative is ground to medium fine powder, about 40 orders, subsequent use.Get fine powder 350g, use 90% ethanol extracting then, 500ml extracting 3 times; Filter, merging filtrate, evaporation removes and desolvates, and gets residue; Use the dissolved in chloroform residue, 250mL dissolving 3 times, chloroform solvent is removed in distillation, gets the product LTB2 and first residue; With dissolve with methanol first residue, 250mL dissolving 3 times, the distilling under reduced pressure solvent gets product LTB3.Each product is put into aseptic screw cap bottle ,-20 ℃ of preservations, subsequent use.The dry leaves of other Liriodendrons is identical with said method with the method that bark extracts.
The extract yield table of variant plant tissue is shown in 3.
The extract yield table of the different vegetable materials of table 3
In the table, LT: Cortex Liriodendri tulipiferae; LC: Chinese mandarin jacket wood; LX: hybridization mandarin jacket wood; *: the dried plant quality of materials; B: bark; L: leaves; 0: ethanol extraction total extract for the first time; 1: chloroform extracting total extract: 2: extract obtained after the chloroform extracting with the methanol extracting.
The determining total phenol method is with embodiment 1, and it is as shown in table 4 specifically to measure the result.
Polyphenol content in table 4 extract that extracting goes out from leaves of plants and bark
| Material | LTB0 | LTB1 | LTB2 | LCB0 | LCB1 | LCB2 | LXB0 | LXB1 | LXB2 |
| Total phenols (%) | 35.1 | 28.3 | 44.8 | 41.2 | 29.7 | 48.2 | 31.3 | 26.1 | 38.0 |
| Material | LTL0 | LTL1 | LTL2 | LCL0 | LCL1 | LCL2 | LXL0 | LXL1 | LXL2 |
| Total phenols (%) | 47.2 | 33.6 | 56.8 | 46.6 | 38.6 | 69.3 | 51.3 | 38.4 | 46.8 |
Total phenol content percent adopts GA to represent respectively.
Embodiment 3
The MCF-7 cell culture adopts the complete culture medium of ATCC; Comprise minimal medium EMEM (Eagle's Minimum Essential Medium), balanced salt solution EBSS (Earles Balanced Salt Solution), non essential amino acid, 2mM L-glutamine, 1 mM Sodium Pyruvate, the sodium bicarbonate solution of 1.5mg/ mL, 0.01mg/mL bovine insulin, 10% hyclone, contain the 25cm of 100U/mL penicillin and 100 mg/mL streptomycins
2Tissue culture flasks.37 ℃ of temperature controls are containing 5% (v/v) CO
2Saturated vapor in hatch.
Breast carcinoma MDA-MB-231 cell culture adopts the ATCC complete medium, comprises L-15 and 2 mM L-glutaminate, the 1.5mg/mL sodium bicarbonate, and 10% hyclone contains the 25cm of 100U/mL penicillin and 100mg/mL streptomycin
2Tissue culture flasks, 37 ℃ of temperature controls are containing 5% (v/v) CO
2Saturated vapor in hatch.
The condition of culture of other three-type-person's class tumor cell with above-mentioned two kinds identical, except breast cancer cell SGC7901 and colon cancer cell HCT15 use 1640 culture medium, LCT cell Huh7 uses the DMEM culture medium.
Adopt mtt assay to weigh the lethal effect of the extract of embodiment 1 to 5 kinds of tumor cells.The positive cellular control unit of 5-Fu and amycin.Adopt the extract (0.08,0.4,2,10,50 and 250 μ g/mL) of variable concentrations to handle cell 72h, undressed cultured cell is as negative control.1 * 10
4Individual cell inoculation behind the 6h, adds medicine to 96 orifice plates in 96 orifice plates continuously, through 72h, in every hole, adds the MTT of the 5mg/mL of 20 μ L.Behind the 4h, add the dimethyl sulfoxide of 150 μ L, dissolve every hole crystal.At 37 ℃, hatch 96 orifice plate 15min, read the result at the 590nm place with ELIASA then.IC50 is calculated as to have caused 50% inhibition cytoactive compound concentrations (μ g/mL).Concrete outcome is as shown in table 5.
Table 5 plant extract is to the toxicity test of cancerous cell in 5 table (IC50, μ g/mL) as a result
| Sample | LTB0 | LTB1 | LTB2 | LCB0 | LCB1 | LCB2 | LXB0 | LXB1 | LXB2 | 5-Fu |
| MDA-MB-231 | 8.0 | 1.3 | 5.2 | 20 | 6.0 | 17 | 14 | 12 | 23 | 1.2 |
| MCF-7 | 18 | 1.8 | 19 | 9.0 | 2.5 | 21 | 20 | 13 | 17 | 1.4 |
| HuH-7 | 1.8 | 0.42 | 16.9 | 6.5 | 2.4 | 14 | 2.1 | 7.0 | 15 | 0.51 |
| SGC-7901 | 5.5 | 0.5 | 22 | 37 | 1.8 | 13 | 11 | 0.62 | 22 | 0.19 |
| HCT-15 | 6.6 | 2.18 | 8.1 | 1.1 | 4.4 | 15 | 9.0 | 8.0 | 6.8 | 1.51 |
| Sample | LTL0 | LTL1 | LTL2 | LCL0 | LCL1 | LCL2 | LXL0 | LXL1 | LXL2 | A |
| MDA-MB-231 | 65 | 15 | - | 36 | 17 | 52 | 7 | 11 | 44 | 0.1 |
| MCF-7 | 21 | 0.4 | - | 20 | 16 | 23 | 32 | 21 | 52 | 0.11 |
| HuH-7 | 1.7 | 0.5 | 6.41 | 6.9 | 13 | 6.4 | 1.0 | 4.1 | 17 | 2.40 |
| SGC-7901 | 1.5 | 0.8 | 7.9 | 5.6 | 7.0 | 12 | 0.63 | 1.0 | 5.4 | 0.69 |
| HCT-15 | 4.9 | 1.9 | 8.7 | 3.0 | 3.0 | 2.9 | 1.1 | 0.61 | 3.4 | 3.05 |
In the table: A: amycin; 5-Fu: fluorouracil.
Claims (7)
1. the Polyphenols extract of a Liriodendron is obtained by following method: get the Liriodendron vegetable material, 35 ℃ of oven dry, pulverizing; Use 95% ethanol extraction, the evaporative removal solvent gets residue; Use the dissolved in chloroform residue, filter, must filtrate and first residue, distilling under reduced pressure filtrating gets the Polyphenols extract; Dissolve with methanol first residue, the distilling under reduced pressure solvent gets the Polyphenols extract, merges the Polyphenols extract and both gets.
2. the Polyphenols extract of Liriodendron according to claim 1, it is characterized in that: the polyphenol mass content of Polyphenols extract is 20-90%.
3. the Polyphenols extract of Liriodendron according to claim 1 is characterized in that: leaf and bark that said Liriodendron vegetable material is three kinds of Liriodendrons.
4. the Polyphenols extract of Liriodendron according to claim 1 is characterized in that: said Liriodendron plant is Chinese mandarin jacket wood, Cortex Liriodendri tulipiferae, and hybridized Chinese tuliptree.
5. a method of extracting the Polyphenols extract of Liriodendron is characterized in that, may further comprise the steps:
(1) get the Liriodendron vegetable material, 35 ℃ of oven dry are pulverized;
(2) use 95% ethanol extraction, the evaporative removal solvent gets residue;
(3) use the dissolved in chloroform residue, filter, must filtrate and first residue, distilling under reduced pressure filtrating gets the Polyphenols extract;
(4) dissolve with methanol first residue, distilling under reduced pressure filtrating gets the Polyphenols extract.
6. the Polyphenols extract of the described Liriodendron of claim 1 is used for treating the application of cancer cell medicine in preparation.
7. application according to claim 6 is characterized in that: described cancer cell comprises breast cancer cell MDA-MB-231, breast cancer cell MCF-7, stomach cancer cell SGC-7901, HCC Huh7 and colon cancer cell HCT15.
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| CN104125835A (en) * | 2012-02-16 | 2014-10-29 | 草堂药品工业株式会社 | Method for extracting treatment ingredients for gastrointestinal disease from bark of liriodendron tulipifera |
| CN104278068A (en) * | 2014-10-21 | 2015-01-14 | 浙江省林业科学研究院 | Method for increasing content of hyperoside in phellinus igniarius liquid fermentation product |
| CN105358171A (en) * | 2013-04-26 | 2016-02-24 | 草堂药品工业株式会社 | Pharmaceutical composition containing liriodendron tulipifera l. extract for treating chronic myelogenous leukemia |
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| CN104125835A (en) * | 2012-02-16 | 2014-10-29 | 草堂药品工业株式会社 | Method for extracting treatment ingredients for gastrointestinal disease from bark of liriodendron tulipifera |
| CN105358171A (en) * | 2013-04-26 | 2016-02-24 | 草堂药品工业株式会社 | Pharmaceutical composition containing liriodendron tulipifera l. extract for treating chronic myelogenous leukemia |
| CN104278068A (en) * | 2014-10-21 | 2015-01-14 | 浙江省林业科学研究院 | Method for increasing content of hyperoside in phellinus igniarius liquid fermentation product |
| CN104278068B (en) * | 2014-10-21 | 2017-02-01 | 浙江省林业科学研究院 | Method for increasing content of hyperoside in phellinus igniarius liquid fermentation product |
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