CN103554289A - Rhizoma atractylodis sinensis polysaccharide and extraction method and applications thereof in preparing anti-tumor medicaments - Google Patents
Rhizoma atractylodis sinensis polysaccharide and extraction method and applications thereof in preparing anti-tumor medicaments Download PDFInfo
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Abstract
The invention discloses rhizoma atractylodis sinensis polysaccharide and an extraction method and applications thereof in preparing anti-tumor medicaments. The extraction method comprises the steps: degreasing rhizoma atractylodis sinensis to obtain loose degreased rhizoma atractylodis sinensis powder; adding cellulase, hemicellulase, pectinase or complex enzyme and a buffer solution, performing enzymolysis and enzyme deactivation, supplementing distilled water for ultrasonic extraction, filtering, centrifuging filtrate, concentrating supernate, adding ethanol into the concentrated solution, centrifuging, precipitating and washing, and drying to obtain the rhizoma atractylodis sinensis polysaccharide. According to the method, not only can the extraction rate of effective ingredients in the rhizoma atractylodis sinensis be increased, the energy consumption be reduced and the medicine resources be fully utilized, but also no special instrument equipment is needed, and thus the extraction method is easy to implement in production. The method is simple and feasible, dispense with a great amount of organic solvents, safe and low-toxicity, and suitable for industrial production. The yield of the rhizoma atractylodis sinensis polysaccharide extracted by adopting the method can achieve 32%-42%, and the purity of the rhizoma atractylodis sinensis polysaccharide which is subjected to the follow-up purification can achieve 80%-95%.
Description
Technical field
The present invention relates to Atractylis chinensis polysaccharide and extracting method thereof and purposes, belong to Effective Component of Chinese Medicine and extract separation and utilisation technology.
Background technology
Polysaccharide is extensively present in animal cell membrane, plant and microorganism wall, is the abundantest biological ployose of nature content, is also the important component part of all Living organisms, and relevant with the required different physiological roles that sustains life.Polysaccharide originates in nineteen forty-three as drug research, but to after the fifties, people just start to recognize gradually the complicacy of polysaccharide structures and the importance of function, and find gradually that polysaccharide has and permitted many-sided biological activity, as effects such as immunostimulant and adjusting, antitumor, antiviral, radioprotective, anticoagulation, anti-ageing, fat-reducing.
Tumour is serious harm human life and healthy common disease and frequently-occurring disease, and its case fatality rate occupies first of various diseases.At present, the method for the treatment of malignant tumour has operation, radiotherapy and chemotherapy, and chemotherapy mainly be take synthetic drugs as main, although tumor killing effect certainly, toxic side effect is extensive and serious, and has resistance problem.Treatment by Chinese herbs tumour system considers from integral body, diagnosis and treatment based on an overall analysis of the illness and the patient's condition, and strengthening vital QI to eliminate pathogenic factors, suppresses tumour by improving the number of ways such as body's immunity.
Atractylis chinensis is feverfew Atractylis chinensis Atractylodes chinensis(DC.) dry rhizome of Koidz., one of two kinds of Original plants of rhizoma atractylodis that record for < < Chinese Pharmacopoeia > > 2010 version (), it is a kind of drying damp and strengthening spleen conventional Chinese medicine, be applied in many compound preparations, as three wonderful balls, the SHUJIN WAN of dispeling the wind, two wonderful balls, JINGFUKANG KELI etc.Modern pharmacology research shows, Atractylis lancea polysaccharide has antiviral, antitumor, reduces blood sugar, reduces protein, anti-oxidant, the effect such as delay senility, still, less to the research of Atractylis chinensis polysaccharide, the research of especially active aspect.Up to now, only with the research of extraction with aqueous solution Atractylis chinensis polysaccharide, not yet find to extract Atractylis chinensis polysaccharide and for its antineoplastic report be used for the treatment of the record of cancer with enzymolysis process.
Most domestic Chinese medicine preparation is to extract as pickling process, percolation, decocting method and continuous backflow method with traditional method at present, and aforesaid method power consumption is higher, and effective component extraction rate is low.Some new technology is constantly come out in recent years, as microwave extract method, ultrasonic extracting method, super critical extraction etc., although new extractive technique demonstrates higher extraction effect, but, due to the limitation of some specific equipment and application, make these new technologies present stages also there is no large-scale application in producing.
Summary of the invention
The object of this invention is to provide a kind of Atractylis chinensis polysaccharide with anti-tumor activity.
Second object of the present invention is to provide a kind of extracting method of simple, with low cost, Atractylis chinensis polysaccharide that extraction yield is high.
The 3rd object of the present invention is to provide a kind of purposes of Atractylis chinensis polysaccharide.
An extracting method for Atractylis chinensis polysaccharide, comprises the steps:
(1) the aqueous ethanolic solution degreasing that is 95%～99.5% by sherwood oil and volumetric concentration successively by the Atractylis chinensis of pulverizing, cross 40～80 mesh sieves, described Atractylis chinensis, sherwood oil and volumetric concentration are that the ratio of 95%～99.5% aqueous ethanolic solution is 1g:4～6mL:2～5mL, volatilize the shape degreasing Atractylis chinensis powder that must loosen after sherwood oil and ethanol;
(2) add the cellulase of described degreasing Atractylis chinensis opaque amount 0.8%～3%, hemicellulase, polygalacturonase or prozyme, and add the buffered soln of pH=4～6, described degreasing Atractylis chinensis powder is 1g: 4mL～10mL with the ratio of described buffered soln, in 30～60 ℃ of water enzyme digestion 30～60min, the boiling water enzymic activity of going out, supplementing distilled water to degreasing Atractylis chinensis powder is 1g:15mL～25mL with the ratio of total mixed solution, in 40～60 ℃ of supersound extraction 30～50min, filter, filtrate is centrifugal, supernatant concentration to every ml soln is equivalent to 0.4～1.0g crude drug, in concentrated solution, add ethanol to reaching 70%～80% containing alcohol quality, place 12～48h, centrifugal, precipitation is used dehydrated alcohol successively, washing with acetone 2～3 times, the dry Atractylis chinensis polysaccharide that both obtained,
Prozyme is to be (1～3) in mass ratio: the ratio of (3～1) is comprised of cellulase and hemicellulase; Or formed by hemicellulase and polygalacturonase; Or formed by cellulase and polygalacturonase; Or be (1～2) in mass ratio: 1: the ratio of (1～2) is comprised of cellulase, hemicellulase and polygalacturonase.
Degreasing Atractylis chinensis powder is preferably 1g: 5mL～8mL with the ratio of described buffered soln.
Buffered soln optimization citric acid-sodium citrate buffer, acetic acid-sodium-acetate buffer.
Cellulase, hemicellulase, polygalacturonase or prozyme add-on are 0.8%～2% of degreasing Atractylis chinensis opaque amount.
The temperature of water enzyme digestion is preferably 40～50 ℃, and the time of water enzyme digestion is preferably 40～50min.
The electric power of supersound extraction is preferably 500W, and calibration is 40KHz, and the temperature of supersound extraction is preferably 50～60 ℃, and the time of supersound extraction is preferably 40～50min.
The Atractylis chinensis polysaccharide that aforesaid method extracts.
The purposes of Atractylis chinensis polysaccharide in the anti-curing oncoma medicine of preparation.
Tumour is Hela cell, liver cancer cell or ovarian cancer cell.
Advantage of the present invention
Method of the present invention can not only improve the extraction yield of effective constituent in Atractylis chinensis, reduces power consumption, makes full use of herb resource, and does not need special plant and instrument, easily implements aborning.Method of the present invention is simple, does not need a large amount of organic solvents, and safe toxicity is little, is suitable for industrial production.The Atractylis chinensis polysaccharide yield that the inventive method is extracted can reach 32%～42%, follow-up after dialysis membrane dialysis, DEAE-Sepharose, SephadexG-150 column chromatography purifying its purity 80%～95%.Enzyme can optionally destroy plant cell wall, is conducive to dissolving, the diffusion of the multiple bioactive ingredients such as the interior polysaccharide of vegetable cell, can improve the extraction yield of effective constituent.
Accompanying drawing explanation
Fig. 1 is Atractylis chinensis polysaccharide DEAE-Sepharose Fast Flow elution curve.
Fig. 2 is Atractylis chinensis polysaccharide SephadexG-150 gel column elution curve.
Embodiment
The following examples can make the present invention of those skilled in the art comprehend, but do not limit the present invention in any way.
The degreasing of embodiment 1 Atractylis chinensis medicinal material
To pulverize, cross the Atractylis chinensis powder 100g of 40 mesh sieves, add 400mL sherwood oil (60～90 ℃ of boiling ranges) in 50～60 ℃ of reflux 4h, suction filtration while hot, wave the sherwood oil in the loose dregs of a decoction, it is that 95% aqueous ethanolic solution is in 60 ℃ of reflux 2h that the dregs of a decoction continue to add 400mL volumetric concentration, suction filtration, waves loose ethanol in medicine residue while hot, is dried to obtain loose shape degreasing Atractylis chinensis powder 81.62g.
The degreasing of embodiment 2 Atractylis chinensis medicinal materials
To pulverize, cross the Atractylis chinensis powder 100g of 60 mesh sieves, add 500mL sherwood oil (60～90 ℃ of boiling ranges) in 50～60 ℃ of reflux 4h, suction filtration while hot, wave the sherwood oil in the loose dregs of a decoction, it is that 95% aqueous ethanolic solution is in 60 ℃ of reflux 2h that the dregs of a decoction continue to add 500mL volumetric concentration, suction filtration, waves loose ethanol in medicine residue while hot, is dried to obtain loose shape degreasing Atractylis chinensis powder 80.54g.
The degreasing of embodiment 3 Atractylis chinensis medicinal materials
To pulverize, cross the Atractylis chinensis powder 100g of 80 mesh sieves, add 600mL sherwood oil (60～90 ℃ of boiling ranges) in 50～60 ℃ of reflux 4h, suction filtration while hot, wave the sherwood oil in the loose dregs of a decoction, it is that 99.5% aqueous ethanolic solution is in 60 ℃ of reflux 2h that the dregs of a decoction continue to add 200mL volumetric concentration, suction filtration, waves loose ethanol in medicine residue while hot, is dried to obtain loose shape degreasing Atractylis chinensis powder 83.75g.
The extraction of embodiment 4 Atractylis chinensis Crude polysaccharides
Precision takes the loose shape degreasing Atractylis chinensis powder 20g of embodiment 1 preparation, add the cellulase of its quality 1.0% to be dissolved in the citric acid-sodium citrate buffer of 80mL pH=5, 50 ℃ of Water Unders are bathed after enzymolysis 40min with the boiling water enzyme 10min that goes out, distilled water is supplemented to 300mL, 60 ℃ of supersound extraction 40min, 8 layers of gauze filtered while hot, filtrate is with the centrifugal 10min of 4000r/min rotating speed, 55 ℃ of supernatant liquors are evaporated to 40mL, slowly add 213mL95% ethanol limit edged gently jolting to separate out flocks, standing 12h under 4 ℃ of conditions, centrifugal 10min, precipitation is used dehydrated alcohol successively, washing with acetone 2 times, be dried to constant weight, calculating Atractylis chinensis Crude polysaccharides yield is 35.20%, and to adopt phenol-sulphoacid method to record its Atractylis chinensis polysaccharide content be 23.72%.
The extraction of embodiment 5 Atractylis chinensis Crude polysaccharides
Precision takes the loose shape degreasing Atractylis chinensis powder 25g of embodiment 2 preparations, add the hemicellulase of its quality 0.8% to be dissolved in acetic acid-sodium-acetate buffer of 150mL pH=4, 45 ℃ of Water Unders are bathed after enzymolysis 30min with the boiling water enzyme 10min that goes out, distilled water is supplemented to 450mL, 60 ℃ of supersound extraction 40min, 8 layers of gauze filtered while hot, filtrate is with the centrifugal 10min of 4000r/min rotating speed, 55 ℃ of supernatant liquors are evaporated to 45mL, slowly add 200mL95% ethanol, limit edged gently jolting to separate out flocks, standing 14h under 4 ℃ of conditions, centrifugal 10min, precipitation is used dehydrated alcohol successively, washing with acetone 3 times, be dried to constant weight, calculating Atractylis chinensis Crude polysaccharides yield is 33.64%, and to adopt phenol-sulphoacid method to record its polysaccharide content be 21.48%.
The extraction of embodiment 6 Atractylis chinensis Crude polysaccharides
Precision takes the loose shape degreasing Atractylis chinensis powder 30g of embodiment 3 preparations, add the polygalacturonase of its quality 2.0% to be dissolved in the citric acid-sodium citrate buffer of 180mL pH=6, 60 ℃ of Water Unders are bathed after enzymolysis 60min with the boiling water enzyme 10min that goes out, distilled water is supplemented to 750mL, 50 ℃ of supersound extraction 50min, 8 layers of gauze filtered while hot, filtrate is with the centrifugal 10min of 4000r/min rotating speed, 55 ℃ of supernatant liquors are evaporated to 50mL volume, slowly add 250mL95% ethanol, limit edged gently jolting to separate out flocks, standing 13h under 4 ℃ of conditions, centrifugal 10min, precipitation is used dehydrated alcohol successively, washing with acetone 3 times, be dried to constant weight, calculating Atractylis chinensis Crude polysaccharides yield is 34.06%, and to adopt phenol-sulphoacid method to record its polysaccharide content be 23.33%.
The extraction of embodiment 7 Atractylis chinensis Crude polysaccharides
Precision takes the loose shape degreasing Atractylis chinensis powder 20g of embodiment 1 preparation, what add its quality 0.8% take by cellulase and hemicellulase the prozyme that mass ratio formed as 1: 2, be dissolved in acetic acid-sodium-acetate buffer of 200mL pH=5, 40 ℃ of Water Unders are bathed after enzymolysis 50min with the boiling water enzyme 10min that goes out, distilled water is supplemented to 500mL, 40 ℃ of supersound extraction 40min, 8 layers of gauze filtered while hot, filtrate is with the centrifugal 10min of 4000r/min rotating speed, 55 ℃ of supernatant liquors are evaporated to 20mL volume, slowly add 120mL95% ethanol, limit edged gently jolting to separate out flocks, standing 24h under 4 ℃ of conditions, centrifugal 10min, precipitation is used dehydrated alcohol successively, washing with acetone 3 times, be dried to constant weight, calculating Atractylis chinensis Crude polysaccharides yield is 39.91%, and to adopt phenol-sulphoacid method to record its polysaccharide content be 25.08%.
The extraction of embodiment 8 Atractylis chinensis Crude polysaccharides
Precision takes the loose shape degreasing Atractylis chinensis powder 25g of embodiment 1 preparation, what add its quality 1.2% take by hemicellulase and polygalacturonase the prozyme that mass ratio forms as 1: 3 ratio, be dissolved in acetic acid-sodium-acetate buffer of 200mL pH=4, 55 ℃ of Water Unders are bathed after enzymolysis 50min with the boiling water enzyme 10min that goes out, distilled water is supplemented to 500mL, 60 ℃ of supersound extraction 40min, 8 layers of gauze filtered while hot, filtrate is with the centrifugal 10min of 4000r/min rotating speed, 55 ℃ of supernatant liquors are evaporated to 50mL volume, slowly add 200mL95% ethanol, limit edged gently jolting to separate out flocks, standing 12h under 4 ℃ of conditions, centrifugal 10min, precipitation is used dehydrated alcohol successively, washing with acetone 2 times, be dried to constant weight, calculating Atractylis chinensis Crude polysaccharides yield is 40.12%, and to adopt phenol-sulphoacid method to record its polysaccharide content be 26.93%.
The extraction of embodiment 9 Atractylis chinensis Crude polysaccharides
Precision takes the loose shape degreasing Atractylis chinensis powder 30g of embodiment 1 preparation, what add its quality 1.2% take by cellulase and polygalacturonase the prozyme that ratio that mass ratio is 1: 1 forms, be dissolved in the citric acid-sodium citrate buffer of 175mL pH=5, 50 ℃ of Water Unders are bathed after enzymolysis 60min with the boiling water enzyme 10min that goes out, distilled water is supplemented to 500mL, 60 ℃ of supersound extraction 40min, 8 layers of gauze filtered while hot, filtrate is with the centrifugal 10min of 4000r/min rotating speed, 55 ℃ of supernatant liquors are evaporated to 38mL volume, slowly add 180mL95% ethanol, limit edged gently jolting to separate out flocks, standing 12h under 4 ℃ of conditions, centrifugal 10min, precipitation is used dehydrated alcohol successively, washing with acetone 3 times, be dried to constant weight, calculating Atractylis chinensis Crude polysaccharides yield is 41.23%, and to adopt phenol-sulphoacid method to record its polysaccharide content be 32.29%.
The extraction of embodiment 10 Atractylis chinensis Crude polysaccharides
Precision takes the loose shape degreasing Atractylis chinensis powder 30g of embodiment 3 preparations, what add its quality 3% take by hemicellulase and polygalacturonase the prozyme that ratio that mass ratio is 3: 1 forms, be dissolved in acetic acid-sodium-acetate buffer of 150mL pH=5.5, 30 ℃ of Water Unders are bathed after enzymolysis 30min with the boiling water enzyme 10min that goes out, distilled water is supplemented to 450mL, 40 ℃ of supersound extraction 30min, 8 layers of gauze filtered while hot, filtrate is with the centrifugal 10min of 4000r/min rotating speed, 55 ℃ of supernatant liquors are evaporated to 50mL volume, slowly add 250mL95% ethanol, limit edged gently jolting to separate out flocks, standing 48h under 4 ℃ of conditions, centrifugal 10min, precipitation is used dehydrated alcohol successively, washing with acetone 2 times, be dried to constant weight, calculating Atractylis chinensis Crude polysaccharides yield is 40.47%, and to adopt phenol-sulphoacid method to record its polysaccharide content be 35.07%.
The extraction of embodiment 11 Atractylis chinensis Crude polysaccharides
Precision takes the loose shape degreasing Atractylis chinensis powder 20g of embodiment 1 preparation, add its quality 0.8% by cellulase, hemicellulase and polygalacturonase be take the prozyme that ratio that mass ratio is 1: 1: 1 forms, be dissolved in the citric acid-sodium citrate buffer of 140mL pH=4, 40 ℃ of Water Unders are bathed after enzymolysis 60min with the boiling water enzyme 10min that goes out, distilled water is supplemented to 450mL, 60 ℃ of supersound extraction 50min, 8 layers of gauze filtered while hot, filtrate is with the centrifugal 10min of 4000r/min rotating speed, 55 ℃ of supernatant liquors are evaporated to 50mL volume, slowly add 200mL95% ethanol, limit edged gently jolting to separate out flocks, standing 12h under 4 ℃ of conditions, centrifugal 10min, precipitation is used dehydrated alcohol successively, washing with acetone 2 times, be dried to constant weight, calculating Atractylis chinensis Crude polysaccharides yield is 40.64%, and to adopt phenol-sulphoacid method to record its polysaccharide content be 27.40%.
The extraction of embodiment 12 Atractylis chinensis Crude polysaccharides
Precision takes the loose shape degreasing Atractylis chinensis powder 20g of embodiment 2 preparations, add its quality 1.5% by cellulase, hemicellulase and polygalacturonase be take the prozyme that ratio that mass ratio is 2: 1: 2 forms, be dissolved in the citric acid-sodium citrate buffer of 120mL pH=5, 35 ℃ of Water Unders are bathed after enzymolysis 45min with the boiling water enzyme 10min that goes out, distilled water is supplemented to 400mL, 50 ℃ of supersound extraction 40min, 8 layers of gauze filtered while hot, filtrate is with the centrifugal 10min of 4000r/min rotating speed, 55 ℃ of supernatant liquors are evaporated to 40mL volume, slowly add 112mL95% ethanol, limit edged gently jolting to separate out flocks, standing 20h under 4 ℃ of conditions, centrifugal 10min, precipitation is used dehydrated alcohol successively, washing with acetone 3 times, be dried to constant weight, calculating Atractylis chinensis Crude polysaccharides yield is 30.58%, and to adopt phenol-sulphoacid method to record its polysaccharide content be 24.76%.
Embodiment 13 Crude polysaccharides are separated, purifying
The method of separation of polysaccharides, purifying has a lot, as method for removing protein has Sevag method, trichloroacetic acid method, papain enzymolysis-trichloroacetic acid method and trichoroacetic acid(TCA)-propyl carbinol method etc.; The dark general method decolourings such as gac or hydrogen peroxide that adopt of polysaccharide color, the further separation and purification of attached gel column chromatography simultaneously.This experiment adopts following methods purifying Atractylis chinensis polysaccharide:
A. deproteinated precision takes the Atractylis chinensis Crude polysaccharides 800mg that embodiment 5 extracts, with distilled water, be made into the Atractylis chinensis Crude polysaccharides aqueous solution of 4mg/mL, mass concentration 20% trichloroacetic acid solution that adds polysaccharide soln volume l doubly to measure, after magnetic agitation 30min, standing 14h in 0～4 ℃ of refrigerator, 4000rmin
-1centrifugal 10min, separates supernatant liquor.
B. polysaccharide soln after deproteinated is got in decolouring, adds the gac of massfraction 2% in 40 ℃ of ultrasonic decolouring 15min, is evaporated to 1/10 volume.
C. dialyse and pack decolouring deproteinated polysaccharide soln into handle well dialysis tubing (diameter 36mm, hold back relative molecular mass 6000～8000D) in, tighten dialysis tubing two ends, putting into distilled water dialyses, every 4h changes water once, dialysis treatment 2d, the polysaccharide soln after dialysis is through concentrating under reduced pressure, and lyophilize obtains 274.7mg Atractylis chinensis polysaccharide.
D.DEAE-Sepharose Fast Flow anion exchange chromatography is got the rear Atractylis chinensis polysaccharide 274.7mg of dialysis, be dissolved in 10mL Tris-HCl buffered soln, pack above-mentioned sample solution into DEAE-Sepharose Fast Flow anion-exchange chromatography post, then use respectively 0, 0.1, 0.2, the NaCl aqueous solution stepwise elution of 0.3mol/L, adopt phenol-sulphoacid detection method, each manages all tracking monitor absorbancys under wavelength 488nm of solution, draw elution curve, merge the corresponding elutriant of main peak, distill water dialysis, concentrating under reduced pressure, lyophilize, elution curve is shown in Fig. 1.
The e.SephadexG-150 gel filtration chromatography Atractylis chinensis polysaccharide 130.4mg after DEAE column purification that learns from else's experience, is dissolved in 5mL distilled water, packs above-mentioned polysaccharide solution into SephadexG-150 gel column, with distilled water wash-out, collect respectively elutriant, every pipe 3mL, follows the tracks of detection method with phenol-sulphoacid, each is managed solution and all under wavelength 488nm, measures absorbancy, draw elution curve, see Fig. 2, merge elutriant corresponding to main peak, concentrating under reduced pressure, lyophilize must be refined Atractylis chinensis polysaccharide 88.3mg.
F. with phenol-sulphoacid method, record refining Atractylis chinensis purity of polysaccharide and reach 92.68%, by High Performance Gel Permeation Chromatography chromatographic determination, through " detecting gel chromatography data processing " method, process more, measuring its molecular weight is 1.17 * 10
4d, through 2mol/LH
2sO
4, the hydrolysis of 100 ℃ of tube sealings, uses BaCO after cold
3neutralization, the centrifugal supernatant liquor that obtains, after concentration, forms through efficient liquid phase chromatographic analysis monose, with differential refraction detector, detects, and proves the homopolysaccharide that refining Atractylis chinensis polysaccharide is comprised of glucose.
Embodiment 14MTS method is measured the restraining effect of Atractylis chinensis polysaccharide to ovarian cancer cell Skov3 cell proliferation
1 experiment material
1.1 the Atractylis chinensis polysaccharide of Atractylis chinensis polysaccharide: embodiment 4～13 preparations.
1.2 tumour cells: Proliferation of Human Ovarian Cell Skov3, purchased from Ai Yan bio tech ltd, Shanghai.
1.3 reagent
FBS(embryo bovine serum): U.S. Hyclone company product, lot number: 1115.
MTS: Beijing Ding Guo Bioisystech Co., Ltd.
DMEM cell culture medium: Beijing Ding Guo Bioisystech Co., Ltd.
1.4 laboratory apparatus
Micro sample adding appliance: Thermo Fisher Scientific Inc..
Precious special microplate reader: the U.S. produces, model: ELX800.
Shellab CO
2incubator: the U.S. produces, model: 3517-2.
2. test method
By Skov3 cell with 3 * 10
3/ hole density is inoculated in 96 orifice plates, after adherent 24h, add respectively Atractylis chinensis Crude polysaccharides and refining Atractylis chinensis polysaccharide (100 μ g/mL), blank group adds the PBS of same volume, each dosage is established 4 secondary holes, cultivate every hole after 4 days and add 20 μ L MTS, put into after incubator continues to cultivate 90min and by microplate reader, in 490nm place, detect absorbancy, by the propagation level of absorbance A value representation ovarian cancer cell Skov3.Adopt SPSS16.0 to analyze experimental data.According to formula below, calculate inhibiting rate,
Average cell number * 100% of inhibiting rate=(the average cell number of the average cell number-experimental group of control group)/control group.
Experimental result is in Table 1, known in table, with regard to ovarian cancer cell, most of dosing groups are compared difference and are had statistical significance (P<0.001 with control group, P<0.01, P<0.05), illustrate that Atractylis chinensis polysaccharide can effectively suppress the propagation of ovarian cancer cell Skov3.
The restraining effect of table 1 Atractylis chinensis polysaccharide to ovarian cancer cell Skov3 cell proliferation
Embodiment 15 trypan blue methods are measured the impact of Atractylis chinensis polysaccharide on Hela cell, liver cancer cell HepG2 and 7721
1 experiment material
The Atractylis chinensis polysaccharide of 1.1 Atractylis chinensis polysaccharide: embodiment 4～13 preparations.
1.2 tumour cells: human cervical carcinoma Hela cell, human liver cancer cell HepG2 and 7721 are purchased from Ai Yan bio tech ltd, Shanghai.
1.3 reagent
FBS(embryo bovine serum): U.S. Hyclone company product, lot number: 1115.
Trypan blue: Beijing Ding Guo Bioisystech Co., Ltd.
DMEM cell culture medium: Beijing Ding Guo Bioisystech Co., Ltd.
1.4 laboratory apparatus
With embodiment 14.
2. test method
By Hela cell, human liver cancer cell HepG2 and 7721 cells with 3 * 10
5/ hole density is inoculated in 24 orifice plates, and after inoculation, 24h dosing is cultivated, and adds respectively Atractylis chinensis Crude polysaccharides and refining Atractylis chinensis polysaccharide (100 μ g/mL), and blank group adds the PBS of same volume, every group of 3 secondary holes.The 4th day collecting cell after dosing, utilizes platform to expect blue dyeing, and optics is just being put and under microscope, carried out cell counting.Adopt SPSS16.0 to analyze experimental data.According to formula below, calculate inhibiting rate,
Average cell number * 100% of inhibiting rate=(the average cell number of the average cell number-experimental group of control group)/control group
Experimental result is in Table 2～4, from table, can find, compare with control group, most of Atractylis chinensis polysaccharide show statistical significance (P<0.05 to the restraining effect of Hela cell, liver cancer cell HepG2 and 7721, P<0.01, P<0.001), particularly evident to the restraining effect of human liver cancer cell HepG2, inhibiting rate is up to 78.40%, and Crude polysaccharides also has certain restraining effect to three kinds of cells.
The impact of table 2 Atractylis chinensis polysaccharide on Hela cell proliferation
The impact of table 3 Atractylis chinensis polysaccharide on liver cancer cell HepG2 cell proliferation
The impact of table 4 Atractylis chinensis polysaccharide on 7721 hepatocellular carcinoma propagation
The restraining effect of embodiment 16 Atractylis chinensis polysaccharide to Proliferation of Human Ovarian Cell Skov3 nude mice knurl body
1. material
1.1. laboratory animal: BALB/C nude mouse, female, mouse age 5～7 week age, body weight 20～25g, by Fukang, Beijing, biotech inc provides, laboratory animal occupancy permit number: SCXK(capital) 2013-0005.Experimental animal feeding under SPF condition, free diet.
1.2. the preparation of Atractylis chinensis polysaccharide sample liquid: the Atractylis chinensis polysaccharide of embodiment 10 preparations is dissolved in PBS, and concentration is 100 μ g/mL.
2. experimental technique Skov3 cell is incubated at according to a conventional method and goes in the RPMI1640 substratum of new-born calf serum of hormone containing 10%, in 37 ℃, 5% CO
2under saturated humidity, cultivate, within every 2～3 days, go down to posterity 1 time.Logarithmic phase cell is collected in conventional digestion, living cell counting number, and to adjust cell concn be 2 * 10
7/ mL, extracts cell suspension 200uL with syringe, is inoculated under 2 right ribs of sterilization nude mice.When the successful primary transplanted tumor of cell inoculation grows to diameter 8～10mm, cervical vertebra dislocation method is put to death nude mice, strips Subcutaneous tumor, and being cut into diameter is the knurl piece of 1～2mm.With No. 12 trochars, draw a tubercle piece, be seeded under the right ribbed hide of nude mice, seeded process completes in 30min after tumour is in vitro, inoculates altogether 30 nude mices.In inoculation latter 12 days, get wherein 20 nude mices, the diameter of its tumour is about 6～7mm on average, is divided at random two groups, and 10 every group, each group is indifference (P>0.05) in size of tumor, body weight.
Grouping: (1) control group: physiological saline (containing 0.001% dehydrated alcohol), intraperitoneal injection
(2) administration group: Atractylis chinensis polysaccharide group: 1mg/kg, tail vein injection
Injection in three days once, is injected 6 times altogether.After medication every 3 days with the maximum radial line (a) of vernier caliper measurement transplanted tumor and minimum radial line (b), until treatment finishes.According to following formula, calculate nude mice gross tumor volume (V), V=π * ab
2/ 6.Administration finishes rear animal and is condemned to death, and takes out each tumor tissues and weighs, and according to formula below, calculates tumour inhibiting rate.
The average knurl heavy * 100% of tumour inhibiting rate=(the average knurl weight of the average knurl weight-experimental group of control group)/control group
3. all data acquisitions of statistical procedures SPSS16.0 software processes, measurement data is used
represent, between group, relatively adopt t check, the results are shown in Table 5～6.
After table 5 medication, respectively organize the variation of Skov3 nude mice gross tumor volume
P<0.05, vs. control group
The impact that table 6 Atractylis chinensis polysaccharide is heavy on Skov3 nude mice knurl
P<0.01, vs. control group
Our experiments show that, the Atractylis chinensis polysaccharide group of embodiment 10 preparations is obviously dwindled compared with control group nude mice gross tumor volume, and tumor weight also obviously alleviates.Illustrate that Atractylis chinensis polysaccharide can obviously suppress the growth of Skov3 nude mice tumour.
The impact of embodiment 17 Atractylis chinensis polysaccharide on HepG2 liver cancer mouse knurl body
1. material
1.1. laboratory animal HepG2 liver cancer mouse, female, body weight 20～24g, and by Fukang, Beijing, biotech inc provides, laboratory animal occupancy permit number: SCXK(capital) 2013-0005.Experimental animal feeding under SPF condition, free diet.
1.2. the preparation of Atractylis chinensis polysaccharide sample liquid: the refining Atractylis chinensis polysaccharide of embodiment 13 preparations is dissolved in PBS, and high, medium and low dosage group is respectively: 150 μ g/mL, 100 μ g/mL, 50 μ g/mL.
2. experimental technique is selected 6～8 days well-grown HepG2 liver cancer mouses of intraperitoneal inoculation, and cervical vertebra dislocation is put to death, and aseptic extraction ascites, becomes 5.4 * 10 with stroke-physiological saline solution by 1: 2 dilution proportion
7/ mL tumor cell suspension, is inoculated in healthy mice right fore armpit subcutaneous, and every 0.2mL inoculates 50 altogether.Inoculate next day, by body weight, be divided at random 5 groups, gavage gives the tested medicine of 0.6mL high, medium and low dosage, every day 1 time, continuous 9 days.Control group gives physiological saline, every 0.3mL.Positive controls abdominal injection cis-platinum (5mgkg
-1d
-1), each administration 0.2mL, administration every other day 1 time, administration is 3 times altogether.Observe the situation such as general activity, fur, ight soil of mouse every day.After last administration, mouse is put to death in the dislocation of 24h cervical vertebra, strips tumor tissues, weighs.According to formula below, calculate tumour inhibiting rate.
The average knurl heavy * 100% of tumour inhibiting rate=(the average knurl weight of the average knurl weight-experimental group of control group)/control group
3. data processing data acquisition SPSS16.0 software processes, measurement data is used
represent, between group, relatively adopt t check, the results are shown in Table 7.
The impact that table 7 Atractylis chinensis polysaccharide is heavy on HepG2 liver cancer mouse knurl
P<0.05, vs. control group
Our experiments show that, the Atractylis chinensis polysaccharide of embodiment 13 preparations heavily has obvious restraining effect to HepG2 liver cancer mouse knurl.Illustrate that Atractylis chinensis polysaccharide has stronger restraining effect to liver cancer.
The impact of embodiment 18 Atractylis chinensis polysaccharide on cervical cancer Hela cells nude mice knurl body
1. material
1.1. laboratory animal: BALB/C nude mouse, female, mouse age 5～7 week age, body weight 20～25g, by Fukang, Beijing, biotech inc provides, laboratory animal occupancy permit number: SCXK(capital) 2013-0003.Experimental animal feeding under SPF condition, free diet.
1.2. the preparation of Atractylis chinensis polysaccharide sample liquid: the refining polysaccharide of the Atractylis chinensis of embodiment 13 preparations is dissolved in PBS, and concentration is 100 μ g/mL,
2. experimental technique is HeLa cell 0.25% trysinization of logarithmic phase, collecting cell and centrifugal after, with the DMEM substratum of serum-free, wash 2 times, then by the resuspended one-tenth concentration of cell, be 5 * 10 with the DMEM substratum of serum-free
8individual/mL, blows even and fine born of the same parents.By every subcutaneous injection 100uL cell suspension inside upper limbs of 12 female nude mices of BALB/C, i.e. every female nude inoculation 5 * 10
7individual cell.The female nude mice of BALB/C after 12 inoculations is divided into two groups at random, 6 every group.Blank group injection PBS, administration group injection concentration is the Atractylis chinensis polysaccharide soln of 100 μ g/mL, 48h injects once, each 100 μ L.Drug injection was put to death nude mice after the 20th day, carefully peeled off knurl piece, weighed knurl piece weight.According to formula below, calculate tumour inhibiting rate.
The average knurl heavy * 100% of tumour inhibiting rate=(the average knurl weight of the average knurl weight-experimental group of control group)/control group
3. data processing data acquisition SPSS16.0 software processes, measurement data is used
represent, between group, relatively adopt t check, the results are shown in Table 8.
The impact that table 8 Atractylis chinensis polysaccharide is heavy on cervical cancer Hela cells mouse tumor
P<0.05, vs. control group
Our experiments show that, the refining polysaccharide group of Atractylis chinensis of embodiment 13 preparations heavily has obvious restraining effect to cervical cancer Hela cells mouse tumor.Illustrate that Atractylis chinensis polysaccharide has stronger restraining effect to cervical cancer.Atractylis chinensis polysaccharide and pharmaceutically acceptable carrier are made to the medicine of the formulations such as tablet, capsule, granule, microcapsule, injection, powder pin.
Claims (9)
1. an extracting method for Atractylis chinensis polysaccharide, is characterized in that comprising the steps:
(1) the aqueous ethanolic solution degreasing that is 95%～99.5% by sherwood oil and volumetric concentration successively by the Atractylis chinensis of pulverizing, cross 40～80 mesh sieves, described Atractylis chinensis, sherwood oil and volumetric concentration are that the ratio of 95%～99.5% aqueous ethanolic solution is 1g: 4～6mL: 2～5mL, volatilize the shape degreasing Atractylis chinensis powder that must loosen after sherwood oil and ethanol;
(2) add the cellulase of described degreasing Atractylis chinensis opaque amount 0.8%～3%, hemicellulase, polygalacturonase or prozyme, and add the buffered soln of pH=4～6, described degreasing Atractylis chinensis powder is 1g: 4mL～10mL with the ratio of described buffered soln, in 30～60 ℃ of water enzyme digestion 30～60min, the boiling water enzymic activity of going out, supplementing distilled water to degreasing Atractylis chinensis powder is 1g: 15mL～25mL with the ratio of total mixed solution, in 40～60 ℃ of supersound extraction 30～50min, filter, filtrate is centrifugal, supernatant concentration to every ml soln is equivalent to 0.4～1.0g crude drug, in concentrated solution, add ethanol to reaching 70%～80% containing alcohol quality, place 12～48h, centrifugal, precipitation is used dehydrated alcohol successively, washing with acetone 2～3 times, the dry Atractylis chinensis polysaccharide that both obtained,
Described prozyme is to be (1～3) in mass ratio: the ratio of (3～1) is comprised of cellulase and hemicellulase; Or formed by hemicellulase and polygalacturonase; Or formed by cellulase and polygalacturonase; Or be (1～2) in mass ratio: 1: the ratio of (1～2) is comprised of cellulase, hemicellulase and polygalacturonase.
2. the extracting method of a kind of Atractylis chinensis polysaccharide according to claim 1, is characterized in that pH=4～5 of buffered soln, and described degreasing Atractylis chinensis powder is 1g: 5mL～8mL with the ratio of described buffered soln.
3. the extracting method of a kind of Atractylis chinensis polysaccharide according to claim 1 and 2, is characterized in that described buffered soln is citric acid-sodium citrate buffer, acetic acid-sodium-acetate buffer.
4. the extracting method of a kind of Atractylis chinensis polysaccharide according to claim 1, is characterized in that cellulase, hemicellulase, polygalacturonase or prozyme add-on are 0.8%～2% of degreasing Atractylis chinensis opaque amount.
5. the extracting method of a kind of Atractylis chinensis polysaccharide according to claim 1, the temperature that it is characterized in that water enzyme digestion is 40～50 ℃, the time of water enzyme digestion is 40～50min.
6. the extracting method of a kind of Atractylis chinensis polysaccharide according to claim 1, the electric power that it is characterized in that supersound extraction is 500W, and frequency is 40KHz, and the temperature of supersound extraction is 50～60 ℃, and the time of supersound extraction is 40～50min.
7. the Atractylis chinensis polysaccharide that the method for one of claim 1～6 is extracted.
Atractylis chinensis polysaccharide claimed in claim 7 in preparation the purposes in anti-curing oncoma medicine.
9. according to the purposes described in right 8, it is characterized in that described tumour is Hela cell, liver cancer cell or ovarian cancer cell.
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| CN113106138A (en) * | 2021-03-16 | 2021-07-13 | 中国农业科学院农产品加工研究所 | Preparation method for extracting and separating anti-tumor active protein from shiitake mushrooms |
| CN114767654A (en) * | 2022-03-24 | 2022-07-22 | 燕山大学 | Piezoelectric catalysis-based anti-tumor nano-drug and preparation method thereof |
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| CN113106138A (en) * | 2021-03-16 | 2021-07-13 | 中国农业科学院农产品加工研究所 | Preparation method for extracting and separating anti-tumor active protein from shiitake mushrooms |
| CN114767654A (en) * | 2022-03-24 | 2022-07-22 | 燕山大学 | Piezoelectric catalysis-based anti-tumor nano-drug and preparation method thereof |
| CN114767654B (en) * | 2022-03-24 | 2023-04-21 | 燕山大学 | Anti-tumor nano-drug based on piezoelectric catalysis and preparation method thereof |
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