CN103356736B - Dyetree fruit polyphenol extract with antineoplastic effect and preparation method thereof - Google Patents
Dyetree fruit polyphenol extract with antineoplastic effect and preparation method thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of natural medicines, and particularly relates to an adyetree fruit polyphenol extract with antineoplastic effect and a preparation method thereof. The preparation method comprises the following steps of: by taking dyetree infructescence as the raw material, treating by degreasing, extracting, leaching and drying to obtain the adyetree fruit polyphenol extract (see figure 1). The prepared adyetree fruit polyphenol extract is light in color and high in purity, shows remarkable effect on inhibiting the growth of tumor cells, and can be applied to preparation of an antitumor drug.
Description
Technical field
The invention belongs to natural medicine technical field, be specifically related to a kind of change Oleum Linderae polyphenol extract with antitumor action and preparation method thereof.
Background technology
Malignant tumor is the common disease of serious threat human health, and in China's urban population, tumor is first cause of the death, accounts for 1/4th of whole mortality rate, and tumor is also a great problem of current medical circle.In three large therapies of malignant tumor, Drug therapy occupies an important position, development speed is also the fastest, the chemical classes inhibiting cancer medicine overwhelming majority of synthesis is for lead compound with natural antitumor active component, the exploitation of anticancer, antitumor drug, comprises computer mould and fits Bionic Design all based on natural anti-cancer, anti-tumor active ingredient.In order to tackle grave danger that cancer causes to human health, countries in the world malignant tumor medicine developers particularly pay attention to the native compound in plant with anti-tumor activity.Therefore, from each kind of plant, the natural antitumor active component monomer of screening safety, efficient, low toxicity or component become the emphasis problem of recent domestic scientist research.
Fructus Platycaryae strobilaceac preface be juglandaceae plant Platycarya strobilacea (
plotytarya strohilaceasieb et Zuce) dry infructescence, infructescence strobiloid, ovum shape is oval cylindrical to long ellipticity, long 2.5 ~ 5 cm, diameter 2 ~ 3 cm, and bag sheet place is deposited, large matter, brown; Pyrene is flat, the narrow wing of both sides tool.Seed is avette, seed coat film quality.The ground such as NORTHWEST CHINA, East China, Central China, south China all produce.Platycarya strobilacea is wide and with a long history in the application that China is among the people, is used as medicine mainly with leaf, infructescence.Fructus Platycaryae strobilaceac preface has heat-clearing and toxic substances removing. and loose wind pain relieving, effect of blood circulation promoting and blood stasis dispelling, sensible evacuation of pus, is mainly used in nasal sinusitis, headache, internal injury breast is swollen, the disease such as stomachache, bones and muscles pain, carbuncle, scabies.Change containing more rich polyphenol compound in Oleum Linderae sequence, its special biology had and pharmacological activity are the bases of its medical value.Therefore, the extraction preparation of development Oleum Linderae sequence polyphenol and bioactivity research, to better exploitationization Oleum Linderae sequence resource, have important directive significance.Known domestic and foreign literature is to the research changing Oleum Linderae sequence extract and pharmacologically active, focus mostly at Platycarya strobilacea and infructescence extract thereof to inhibitory action, the antioxidation of harmful bacteria in cytotoxicity, intestinal, and Platycarya strobilacea treats the report of acute and chronic rhinitis, sinusitis as herbal mixture, but the research and development of Fructus Platycaryae strobilaceac preface polyphenol extract anti-tumor activity are still belonged to blank.
Summary of the invention
The object of this invention is to provide a kind of change Oleum Linderae polyphenol extract with antitumor action and preparation method thereof.
Technical scheme of the present invention is summarized as follows:
The preparation method of the change Oleum Linderae polyphenol extract that the present invention proposes, comprises the steps:
(1) with Fructus Platycaryae strobilaceac preface that is dry, that pulverize for raw material, add petroleum ether solution room temperature lixiviate 1 ~ 10 h ungrease treatment by mass volume ratio 1 g: 2.5 mL ~ 1 g: 3.0 mL.Filter, filtering residue room temperature is dried or 40 ~ 50 DEG C of oven dry;
(2) the Fructus Platycaryae strobilaceac preface raw material through ungrease treatment adds 1% ~ 100% methanol solution by mass volume ratio 1 g: 2 mL ~ 1 g: 5 mL, room temperature or heating extraction, or utilize atmospheric pressure reflux extraction element to extract, or microwave or ultrasound assisted extraction 1 ~ 5 time, each 10 min ~ 10 h, filter, filtrate merges.Reduction vaporization removing methanol solvate under 40 ~ 60 ° of C, aqueous phase obtains crude extract through lyophilization;
(3) crude extract adds pure water, stirring and dissolving by mass volume ratio 1 g: 5 mL ~ 1 g: 10 mL, adds same volume extraction into ethyl acetate 1 ~ 5 time, is separated, obtains extraction into ethyl acetate phase and aqueous phase;
(4) extraction into ethyl acetate phase solvent removed by evaporation at reduced pressure, obtains light yellowization Oleum Linderae polyphenol extract through lyophilization.
Preferably: the concentration expressed in percentage by volume of extraction methanol aqueous solution is 70%.
Extraction time is preferably 3 times.
Adopt total phenol content in Folin-Ciocalteu method mensurationization Oleum Linderae polyphenol extract, in change Oleum Linderae polyphenol extract prepared by the present invention, total phenol content is 68% ~ 72.5%.Change Oleum Linderae polyphenol extract and carry out chemical composition qualification through LC-MS, its LC-MS spectrogram is shown in Fig. 2.Qualification shows by analysis, in change Oleum Linderae polyphenol extract prepared by the present invention, main component is: ellagic acid, two-hexahydroxy dibenzoyl-pyrans (type) glucose (bis-HHDP-glucopyranose), 3, 4, 5-trioxybenzol formyl-two-hexahydroxy dibenzoyl-pyrans (type) glucose (galloyl-bis-HHDP-glucopyranose), three galloyls-hexahydroxy dibenzoyl-pyrans (type) glucose (trigalloy-HHDP-glucopyranose), Penta-O-galloyl-D-glucopyranose (penta-galloyl-glucopyranose), the polyphenolic substances such as two galloyl-two-hexahydroxy dibenzoyl-pyrans (type) glucoses (digalloyl-bis-HHDP-glucopyranose).
Change Oleum Linderae polyphenol extract prepared by the present invention has the features such as of light color, purity is high, and preparation method technique is simple.
Change the display of Oleum Linderae polyphenol extract anti tumor activity in vitro result of the test: change Oleum Linderae polyphenol extract and matched group compare non-small cell lung cancer cell A549, human liver cancer cell HepG2, human neuroblastoma cells SH-SY-5Y, human colon cancer cell HCT116, osteosarcoma cell U2Os-NKFB growth there is remarkable inhibitory action, can be used for preparing antitumor drug.
Accompanying drawing explanation
The preparation flow figure of being of Fig. 1 Oleum Linderae polyphenol extract;
Fig. 2 analyzes chromatogram for changing Oleum Linderae polyphenol extract chemical composition LC-MS.
Detailed description of the invention
With reference to the following example by easier, comprehend the present invention, providing embodiment is to illustrate the present invention, instead of limits the present invention by any way.
Embodiment 1
(1) with 150 g Fructus Platycaryae strobilaceac prefaces for raw material, dry, pulverize after, add 400 mL petroleum ether solution room temperature lixiviate 10 h ungrease treatments.Filter, filtering residue 45 DEG C oven dry;
(2) the Fructus Platycaryae strobilaceac preface raw material through ungrease treatment adds 400 mL 70% methanol solutions, and 40 ° of C water-baths extract 3 times, each 1 h, filters, and filtrate merges.Filtrate reduce pressure under 40 ° of C rotary evaporation removing methanol solvate, aqueous phase obtains crude extract 14.6 g through lyophilization;
(3) crude extract is scattered in the pure water of 100 mL, adds same volume extraction into ethyl acetate 3 times, is separated, obtains extraction into ethyl acetate phase and aqueous phase;
(4) by extraction into ethyl acetate phase solvent removed by evaporation at reduced pressure, light yellowization Oleum Linderae polyphenol extract 3.42 g is obtained through lyophilization.
Embodiment 2
(1) with 120 g Fructus Platycaryae strobilaceac prefaces for raw material, dry, pulverize after, add 300 mL petroleum ether solution room temperature lixiviate 4 h ungrease treatments.Filter, filtering residue 45 DEG C oven dry;
(2) the Fructus Platycaryae strobilaceac preface raw material through ungrease treatment adds 400 mL 80% methanol solutions, and 45 ° of C water-baths extract 3 times, each 1 h, filters, and filtrate merges.Filtrate reduce pressure under 50 ° of C rotary evaporation removing methanol solvate, aqueous phase obtains crude extract through lyophilization;
(3) crude extract is scattered in the pure water of 100 mL, adds same volume extraction into ethyl acetate 3 times, is separated, obtains extraction into ethyl acetate phase and aqueous phase;
(4) by extraction into ethyl acetate phase solvent removed by evaporation at reduced pressure, light yellowization Oleum Linderae polyphenol extract 1.91 g is obtained through lyophilization.
Embodiment 3
(1) with 200 g Fructus Platycaryae strobilaceac prefaces for raw material, dry, pulverize after, add 400 mL petroleum ether solution room temperature lixiviate 2 h ungrease treatments.Filter, filtering residue room temperature is dried;
(2) the Fructus Platycaryae strobilaceac preface raw material through ungrease treatment adds 700 mL 60% methanol solutions, and 40 ° of C water-baths extract 3 times, each 1 h, filters, and filtrate merges.Filtrate reduce pressure under 50 ° of C rotary evaporation removing methanol solvate, aqueous phase obtains crude extract through lyophilization;
(3) crude extract is scattered in the pure water of 100 mL, adds same volume extraction into ethyl acetate 3 times, is separated, obtains extraction into ethyl acetate phase and aqueous phase;
(4) by extraction into ethyl acetate phase solvent removed by evaporation at reduced pressure, light yellowization Oleum Linderae polyphenol extract 4.10 g is obtained through lyophilization.
Embodiment 4 changes Oleum Linderae polyphenol extract active Immunotherapy of Cancer Induced
(1) experiment material and medicine
(1) for the preparation of reagent thing: the change Oleum Linderae polyphenol extract prepared by the embodiment of the present invention 1 adopts phosphate buffer PBS to be dissolved as the mother solution of concentration 1 mg/mL, is stored in 4 DEG C of refrigerators.Desired concn is mixed with cell culture fluid dilution before experiment.
(2) the selecting and cultivation of tumor cell line: tumor cell line selects non-small cell lung cancer cell A549, human liver cancer cell HepG2, human neuroblastoma cells SH-SY-5Y, human colon cancer cell HCT116, osteosarcoma cell U2Os-NKFB.DMEM culture medium, 5A culture medium ultra-pure water are made into cell culture fluid.Containing DMEM culture medium, 5A culture medium 10.4 g, Hepes 4.8 g, NaHCO in often liter of cell culture fluid
32.4 g, each 100 mg, the pH 7.2 of penicillin, streptomycin, after degerming with 0.22 μm of membrane filtration, subpackage, 4 DEG C of Refrigerator stores are for subsequent use.Add calf serum before use, make its final concentration be 10%.
(2) experimental technique
Mtt assay is adopted to measure cytotoxicity: non-small cell lung cancer cell A549, human liver cancer cell HepG2, human neuroblastoma cells SH-SY-5Y, osteosarcoma cell U2Os-NKFB add DMEM culture fluid and 10 μMs of glutamine respectively, human colon cancer cell HCT116 adds 5A culture fluid, at 37 DEG C, 5%CO
2cellar culture in the incubator of saturated humidity.48 h change culture fluid, during cell confluency, go down to posterity with 0.25% trypsinization.Collect exponential phase cell, trypan exclusion stain shows cell viability > 95%, and cell is made 5 × 10
4/ mL suspension, is seeded in 96 well culture plates, every hole 200 μ L, administration after cell attachment.Solvent control group and administration group are established in experiment respectively, often organize 6 holes, after the dilution of administration group cell culture fluid is mixed with change Oleum Linderae polyphenol extract effect 24 h of desired concn, every hole adds 5 mg/mL MTT 20 μ L, continue cultivation 4 h, suck supernatant, every hole adds DMSO 200 μ L, and fully the rear enzyme-linked immunosorbent assay instrument of vibration mixing measures the absorbance (OD in each hole at 570 nm places
570nm), experiment repetition 3 times, averages.Calculate suppression ratio and the IC of medicine cell growth
50.Suppression ratio (%)=(1-OD
administration group/ OD
matched group) × 100.
(3) experimental result
(1) inhibitory action that Oleum Linderae polyphenol extract grows non-small cell lung cancer cell A549 is changed
Change Oleum Linderae polyphenol extract to non-small cell lung cancer cell A549 half growth concentration IC
50be 40.1 μ g/mL, each acute drug all shows significant inhibitory action (see table 1) to non-small cell lung cancer cell A549 growth.
Table 1 Oleum Linderae polyphenol extract is to the growth inhibited effect of non-small cell lung cancer cell A549
Average ± SD,
n=3,24 h.
* p<0.05,
* p<0.01, compared with matched group.
(2) inhibitory action that Oleum Linderae polyphenol extract grows human liver cancer cell HepG2 is changed
Change Oleum Linderae polyphenol extract to human liver cancer cell HepG2 half growth concentration IC
50be 42.6 μ g/mL, each acute drug all shows significant inhibitory action (see table 2) to human liver cancer cell HepG2 growth.
Table 2 Oleum Linderae polyphenol extract is to the growth inhibited effect of human liver cancer cell HepG2
Average ± SD,
n=3,24 h.
* p<0.05,
* p<0.01, compared with matched group.
(3) inhibitory action that Oleum Linderae polyphenol extract grows human neuroblastoma cells SH-SY-5Y is changed
Change Oleum Linderae polyphenol extract to human neuroblastoma cells SH-SY-5Y half growth concentration IC
50be 77.4 μ g/mL, for each concentration of reagent thing, significant inhibitory action (see table 3) revealed to human neuroblastoma cells SH-SY-5Y growth table.
Table 3 Oleum Linderae polyphenol extract is to the growth inhibited effect of human neuroblastoma cells SH-SY-5Y
Average ± SD,
n=3,24 h.
* p<0.05,
* p<0.01, compared with matched group.
(4) inhibitory action that Oleum Linderae polyphenol extract grows human colon cancer cell HCT116 is changed
Change Oleum Linderae polyphenol extract to human colon cancer cell HCT116 half growth concentration IC
50be 40.1 μ g/mL, for each concentration of reagent thing, significant inhibitory action (see table 4) revealed to human colon cancer cell HCT116 growth table.
Table 4 Oleum Linderae polyphenol extract is to the growth inhibited effect of human colon cancer cell HCT116
Average ± SD,
n=3,24 h.
* p<0.05,
* p<0.01, compared with matched group.
(5) inhibitory action that Oleum Linderae polyphenol extract grows osteosarcoma cell U2Os-NKFB is changed
Change Oleum Linderae polyphenol extract to osteosarcoma cell U2Os-NKB half growth concentration IC
50be 40.1 μ g/mL, for each concentration of reagent thing, significant inhibitory action (see table 5) revealed to osteosarcoma cell U2Os-NKFB growth table.
Table 5 Oleum Linderae polyphenol extract is to the growth inhibited effect of osteosarcoma cell U2Os-NKFB
Average ± SD,
n=3,24 h.
* p<0.05,
* p<0.01, compared with matched group.
Visible to table 5 by above table 1, change Oleum Linderae polyphenol extract, to non-small cell lung cancer cell, human liver cancer cell, human neuroblastoma cells, human colon cancer cell, osteosarcoma cell, all there is good inhibitory action, therefore may be used for preparing anti-tumor drug.
Claims (1)
1. there is a preparation method for the change Oleum Linderae polyphenol extract of antitumor action, it is characterized in that, comprise the steps: in order
(1) with Fructus Platycaryae strobilaceac preface that is dry, that pulverize for raw material, add petroleum ether solution room temperature lixiviate 1 ~ 10 h ungrease treatment by mass volume ratio 1 g: 2.5 mL ~ 1 g: 3.0 mL, filter, filtering residue room temperature is dried or 40 ~ 50 DEG C of oven dry;
(2) the Fructus Platycaryae strobilaceac preface raw material through ungrease treatment adds 1% ~ 100% methanol solution by solid-liquid mass volume ratio 1 g: 2 mL ~ 1 g: 5 mL, room temperature or heating extraction, or utilize atmospheric pressure reflux extraction element to extract, or microwave or ultrasound assisted extraction 1 ~ 5 time, each 10 min ~ 10 h, filter, and filtrate merges, reduction vaporization removing methanol solvate under 40 ~ 60 ° of C, aqueous phase obtains crude extract through lyophilization;
(3) crude extract adds pure water, stirring and dissolving by mass volume ratio 1 g: 5 mL ~ 1 g: 10 mL, adds same volume extraction into ethyl acetate 1 ~ 5 time, is separated, obtains extraction into ethyl acetate phase and aqueous phase;
(4) extraction into ethyl acetate phase solvent removed by evaporation at reduced pressure, obtains light yellowization Oleum Linderae polyphenol extract through lyophilization.
2. the preparation method with the change Oleum Linderae polyphenol extract of antitumor action according to claim 1, is characterized in that extracting front Fructus Platycaryae strobilaceac preface raw material ungrease treatment solvent for use is petroleum ether.
3. the preparation method with the change Oleum Linderae polyphenol extract of antitumor action according to claim 1, is characterized in that described extraction solvent is methanol aqueous solution.
4. the preparation method with the change Oleum Linderae polyphenol extract of antitumor action according to claim 1, is characterized in that the concentration expressed in percentage by volume of described extraction methanol aqueous solution is 1% ~ 100%.
5. the preparation method with the change Oleum Linderae polyphenol extract of antitumor action according to claim 1, is characterized in that the method for described extraction comprises extract at room temperature, heating extraction, utilizes the extraction of atmospheric pressure reflux extraction element, microwave radiation exaraction and ultrasonic assistant to extract.
6. the preparation method with the change Oleum Linderae polyphenol extract of antitumor action according to claim 1, is characterized in that described antitumor Oleum Linderae polyphenol extract is extraction into ethyl acetate layer.
7. the change Oleum Linderae polyphenol extract that the preparation method according to claim 1 with the change Oleum Linderae polyphenol extract of antitumor action obtains is preparing the application in the medicine inhibited to tumor cell.
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| CN105237589A (en) * | 2015-11-04 | 2016-01-13 | 中国科学院过程工程研究所 | Method for simultaneously extracting polyphenol and hemicellulose in grape pips |
| CN111944165B (en) * | 2020-07-15 | 2022-04-01 | 汕头大学 | Polyphenol tyrosinase inhibitor and extraction method and application thereof |
| CN112979725B (en) * | 2021-03-05 | 2022-07-29 | 中国林业科学研究院林产化学工业研究所 | Method for separating and preparing 1,2,3,4,6-O-pentagalloyl glucose from passion fruit |
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| CN102807570B (en) * | 2012-08-17 | 2015-09-02 | 中国林业科学研究院林产化学工业研究所 | The fragrant fruit of a kind ofization prepares the method for ellagic acid |
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