CN100528901C - Method for separating polysaccharide against growth of cancer cells from dictyophord - Google Patents
Method for separating polysaccharide against growth of cancer cells from dictyophord Download PDFInfo
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- CN100528901C CN100528901C CNB2007100675365A CN200710067536A CN100528901C CN 100528901 C CN100528901 C CN 100528901C CN B2007100675365 A CNB2007100675365 A CN B2007100675365A CN 200710067536 A CN200710067536 A CN 200710067536A CN 100528901 C CN100528901 C CN 100528901C
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- 150000004676 glycans Chemical class 0.000 title claims abstract description 47
- 229920001282 polysaccharide Polymers 0.000 title claims abstract description 41
- 239000005017 polysaccharide Substances 0.000 title claims abstract description 41
- 238000000034 method Methods 0.000 title abstract description 8
- 230000005907 cancer growth Effects 0.000 title 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 31
- 238000001035 drying Methods 0.000 claims abstract description 11
- 238000000926 separation method Methods 0.000 claims abstract description 11
- 239000002244 precipitate Substances 0.000 claims abstract description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 8
- 241001313734 Dictyophora Species 0.000 claims description 42
- 241000434037 Volva Species 0.000 claims description 41
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 14
- 239000007864 aqueous solution Substances 0.000 claims description 10
- 210000004881 tumor cell Anatomy 0.000 claims description 10
- 238000004458 analytical method Methods 0.000 claims description 7
- PHTQWCKDNZKARW-UHFFFAOYSA-N isoamylol Chemical compound CC(C)CCO PHTQWCKDNZKARW-UHFFFAOYSA-N 0.000 claims description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- 230000012010 growth Effects 0.000 claims description 5
- 239000002253 acid Substances 0.000 claims description 4
- 230000001476 alcoholic effect Effects 0.000 claims description 4
- 239000011259 mixed solution Substances 0.000 claims description 4
- 239000007983 Tris buffer Substances 0.000 claims description 3
- 238000010828 elution Methods 0.000 claims description 3
- 239000000706 filtrate Substances 0.000 claims description 3
- 239000012074 organic phase Substances 0.000 claims description 3
- 239000011780 sodium chloride Substances 0.000 claims description 3
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 2
- 229920002678 cellulose Polymers 0.000 claims 1
- 239000001913 cellulose Substances 0.000 claims 1
- 238000004587 chromatography analysis Methods 0.000 abstract description 5
- 235000017166 Bambusa arundinacea Nutrition 0.000 abstract description 2
- 235000017491 Bambusa tulda Nutrition 0.000 abstract description 2
- 241000233866 Fungi Species 0.000 abstract description 2
- 235000015334 Phyllostachys viridis Nutrition 0.000 abstract description 2
- 239000011425 bamboo Substances 0.000 abstract description 2
- 230000002401 inhibitory effect Effects 0.000 abstract description 2
- 241001330002 Bambuseae Species 0.000 abstract 1
- 238000000151 deposition Methods 0.000 abstract 1
- 230000004565 tumor cell growth Effects 0.000 abstract 1
- 241001313708 Dictyophora phalloidea Species 0.000 description 5
- 201000007270 liver cancer Diseases 0.000 description 4
- 208000014018 liver neoplasm Diseases 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- GUBGYTABKSRVRQ-WFVLMXAXSA-N DEAE-cellulose Chemical compound OC1C(O)C(O)C(CO)O[C@H]1O[C@@H]1C(CO)OC(O)C(O)C1O GUBGYTABKSRVRQ-WFVLMXAXSA-N 0.000 description 3
- 238000005033 Fourier transform infrared spectroscopy Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 230000000452 restraining effect Effects 0.000 description 3
- 238000001228 spectrum Methods 0.000 description 3
- 239000003643 water by type Substances 0.000 description 3
- 241000960391 Boletales Species 0.000 description 2
- 229920002307 Dextran Polymers 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 241000295597 Phallaceae Species 0.000 description 2
- 241000959233 Phallales Species 0.000 description 2
- 229920005654 Sephadex Polymers 0.000 description 2
- 239000012507 Sephadex™ Substances 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000002223 garnet Substances 0.000 description 2
- 230000009422 growth inhibiting effect Effects 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 125000000896 monocarboxylic acid group Chemical group 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 150000003214 pyranose derivatives Chemical class 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000011782 vitamin Substances 0.000 description 2
- 229940088594 vitamin Drugs 0.000 description 2
- 229930003231 vitamin Natural products 0.000 description 2
- 235000013343 vitamin Nutrition 0.000 description 2
- 150000003722 vitamin derivatives Chemical class 0.000 description 2
- 241000221198 Basidiomycota Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 206010020772 Hypertension Diseases 0.000 description 1
- 240000007880 Phyllostachys bambusoides Species 0.000 description 1
- 235000001550 Phyllostachys bambusoides Nutrition 0.000 description 1
- 244000082204 Phyllostachys viridis Species 0.000 description 1
- 206010044302 Tracheitis Diseases 0.000 description 1
- VQKFNUFAXTZWDK-UHFFFAOYSA-N alpha-methylfuran Natural products CC1=CC=CO1 VQKFNUFAXTZWDK-UHFFFAOYSA-N 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229940088623 biologically active substance Drugs 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 231100000263 cytotoxicity test Toxicity 0.000 description 1
- 230000003544 deproteinization Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000006806 disease prevention Effects 0.000 description 1
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- 239000000284 extract Substances 0.000 description 1
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- 230000008821 health effect Effects 0.000 description 1
- 229910017053 inorganic salt Inorganic materials 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 230000000050 nutritive effect Effects 0.000 description 1
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- 239000012071 phase Substances 0.000 description 1
- OQUKIQWCVTZJAF-UHFFFAOYSA-N phenol;sulfuric acid Chemical compound OS(O)(=O)=O.OC1=CC=CC=C1 OQUKIQWCVTZJAF-UHFFFAOYSA-N 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
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- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
Separation of polysaccharide in red bamboo fungus teleblemata is carried out by drying, crushing, extracting alcohol with water, depositing, separating out educt, drying, preparing dried educt into solution, deproteinizing in Sevag method to alcohol precipitate, separating out educt while drying to prepare into 12mg/mL solution, separating in chromatography column to obtain DRVP1. It has excellent inhibiting function for tumor-cell growth.
Description
Technical field
The present invention relates to a kind of edible fungi polysaccharide extracting method, the separation method of the polysaccharide that contains in particularly a kind of Dictyophora rubrovalvata volva.
Background technology
Malignant tumour is the great refractory disease of serious harm human health, and pharmacological agent still is the important means of treatment and prevention of tumour.There is abundant resources of medicinal plant in China, and the anti-cancer agent that utilizes the natural pharmaceutical resources exploitation to have independent intellectual property right has bright prospects.
Dictyophora rubrovalvata (Dictyophora rubrovalvata) is a kind of basidiomycetes, be subordinate to Mycophyta (Eumycota), Basidiomycotina (Basidiomycotina), Gasteromycete (Gasteromycetes), Phallales (Phallales), Phallaceae (Phallaceae), dictyophora phalloidea genus (Dictyophora), the high 20～33cm of Dictyophora rubrovalvata sporophore; The button oval, garnet, size are 4～5cm * 4～6cm, rhizomorph white can become bluish voilet after the wound; The volva garnet, the bell or blunt taper shape of cap, high 5～6cm, wide 3.5～4.5cm, the top is flat perforation, has grid all around, and there is darkcyan on the surface to the little smelly spore body of blue or green brown; Velum is bell, white, matter is crisp, from the sagging 7cm that reaches of cap, and hem width 4～8cm, tool polygon, the circular mesh of corner angle, diameter 1～1.5cm; Stem is cylindrical, long 11～20cm, diameter 3～5cm, white, spongioplasm, hollow.Be the dictyophora phalloidea kind of special product of China, mainly be distributed in Yunnan, the geographic cizu in contour mountain, Guizhou, sylvan life such as bamboo and Jin Zhu just.In the phyllostachys bambusoides woods of Linchuan, Jiangxi, also find to have distribution.
Dictyophora rubrovalvata is the treasure in the dictyophora phalloidea, contains multiple amino acids, VITAMIN, polysaccharide and plurality of inorganic salt etc.Except that its nutritive value, also have certain disease-prevention health effect simultaneously, take dictyophora phalloidea for a long time and have the medicinal functions such as cholesterol level for the treatment of chronic tracheitis, bring high blood pressure down and reducing in the blood.But in the Dictyophora rubrovalvata finished product course of processing, the producer's its stem commonly used and indusium part, and cap and volva are removed (weight ratio of the weight of stem and indusium and cap, volva 1: 1.3) as tankage, caused significant wastage, studies show that in the volva dry product, be rich in polysaccharide, amino acid, protein, VITAMIN and beneficial metallic elements.And so far, the Dictyophora rubrovalvata volva is had the polysaccharide that suppresses growth of tumour cell report is not arranged as yet.
Summary of the invention
The invention provides a kind of growth to tumour cell has the separation method of inhibiting Dictyophora rubrovalvata volva polysaccharide.
A kind of separation method of Dictyophora rubrovalvata volva polysaccharide comprises the steps:
(1) with Dictyophora rubrovalvata volva drying, pulverize after,, filter after 3～5 hours with the flooding of 10～30 times of volumes, filtrate is concentrated to 50% of original volume, add ethanol again and carry out alcohol and analyse, separate precipitate and dry;
(2) be made into the aqueous solution after the precipitate oven dry, at middle adding and isopyknic chloroform of the aqueous solution and primary isoamyl alcohol mixed solution, thorough mixing is after 3～4 hours, and centrifugal, layering obtains organic phase and water;
(3) water adds ethanol and carries out alcohol and analyse, and separates precipitate and oven dry, obtains drying thing;
(4) the oven dry thing that step (3) is obtained is made into the aqueous solution of 12mg/mL, in chromatography column, separate, Tris buffered soln wash-out with 0.1M NaCl obtains Dictyophora rubrovalvata volva polysaccharide DRVP1, elution time is 2hr, flow velocity is 3mL/min, and described chromatography column is DEAE-cellulose column 1.7cm * 20.3cm.
Alcohol described in step (1) and the step (3) is analysed, and behind the adding ethanol, the alcoholic acid mass percent concentration is 50%～80% in the system; Preferred 70%.
In the step (1), with Dictyophora rubrovalvata volva drying, pulverize after, with the flooding of 15 times of volumes 4 hours, its yield was the highest, is 12.03 ± 0.23%.If it is very few to add entry, before not reaching best boiling state, leach liquor becomes seldom, even is burned, thereby influences polysaccharide yield; If add water excess, then leach liquor is too much.
The volume of chloroform and primary isoamyl alcohol is 3: 1 in chloroform described in the step (2) and the primary isoamyl alcohol mixed solution.
Separate the Dictyophora rubrovalvata volva polysaccharide obtain by the inventive method, make molecular weight determination with HPLC, contrast with the dextran of the standard of T series, the molecular-weight average of Dictyophora rubrovalvata volva polysaccharide DRVP1 is respectively 1.47 * 10 as can be known
4
Dictyophora rubrovalvata volva polysaccharide DRVP1 sample is positioned at 3400～3500cm through the scanning of FT-IR spectrograph
-1Characteristic peak be the stretching vibration of glycan molecule O-H, 3000～2800cm
-1One group of stretching vibration that the peak is glycan molecule C-H, 1400～1200cm
-1The angle vibration that then is glycan molecule C-H of one group of peak.Can confirm that thus Dictyophora rubrovalvata volva polysaccharide DRVP1 is a compound of polysaccharide.Dictyophora rubrovalvata volva polysaccharide DRVP1 has-OH, COOR, C-O-C ehter bond ,-CHO/C=O, COOH, C-O-H ,-CH
2-,-CH
3, should belong to β-type pyranose glycosidic bond.
Dictyophora rubrovalvata volva polysaccharide DRVP1 at external S180 (mouse hydroperitoneum type liver cancer) tumour cell that acts on respectively, observes their growth-inhibiting effects to this tumour cell respectively 50,100 under the concentration of 200,500,1000 μ g/ml.Find in the experiment that DRVP1 is inhibited to S180 (mouse hydroperitoneum type liver cancer) tumour cell.
This its refuse volva of research and utilization extracts biologically active substance, and adopted method more easily, study its structure component and the effect that presses down tumour, for the anti-tumor medicinal preparation of developing low-cost provides basis and technology platform, can increase simultaneously the added value of dictyophora phalloidea, reach the purpose of comprehensive development and utilization.
Description of drawings
Fig. 1 is DEAE-cellulose column (Cl
-) separating spectrum
Fig. 2 be Dictyophora rubrovalvata volva polysaccharide DRVP1 Sephadex G-75 on separating spectrum
Fig. 3 is that Dictyophora rubrovalvata volva polysaccharide DRVP1 is through FT-IR spectrograph scanning spectra
Fig. 4 is the restraining effect figure of Dictyophora rubrovalvata volva polysaccharide DRVP1 to the growth of S180 cell, and X-coordinate is the concentration of DRVP1, and ordinate zou is relative cell growth rate
Embodiment
The separation of Dictyophora rubrovalvata volva polysaccharide
(1) with Dictyophora rubrovalvata volva drying, pulverize after,, filter after 4 hours with the flooding of 15 times of volumes, filtrate is concentrated to 50% of original volume, add ethanol again and carry out alcohol and analyse, separate precipitate and dry, obtain drying thing;
(2) with Sevag method deproteinated
To dry thing and be made into the aqueous solution, after adding was shaken 4 hours with the isopyknic Sevag agent of the aqueous solution (chloroform: primary isoamyl alcohol=3: 1, volume ratio) shaking table, centrifugal, layering obtained organic phase and water.By this method Deproteinization, effect is obvious, and not only clearance height, and technology is simple, need not big power and equipment, greatly reduces operating cost.
(3) the water adding ethanol that step (2) is obtained carries out alcohol analyses, and behind the adding ethanol, the alcoholic acid mass percent concentration is 70% in the system, separates the oven dry thing that precipitate and oven dry obtain.After testing, polysaccharide content is 81.20% in this oven dry thing.
(4) the oven dry thing that step (3) is obtained is made into the aqueous solution of 12mg/mL, in chromatography column, separate, Tris buffered soln wash-out with 0.1M NaCl obtains Dictyophora rubrovalvata volva polysaccharide DRVP1, elution time is 2hr, flow velocity is 3mL/min, and described chromatography column is DEAE-cellulose column (1.7cm * 20.3cm).
Purifying and the evaluation of Dictyophora rubrovalvata volva polysaccharide DRVP1
Dictyophora rubrovalvata volva polysaccharide DRVP1 is further purified through Sephadex G-75's, obtains polysaccharide products to be used for the research of following experiment.
The aqueous solution (1mg/ml) of Dictyophora rubrovalvata volva polysaccharide DRVP1, through the scanning of UV-Vis spectrophotometer, wavelength region is 200～400nm.The result shows that the DRVP1 component does not have absorption peak at the 260nm place, illustrates not contain nucleic acid; At the 280nm place, curve is level and smooth basically, shows that this does not contain protein.Behind the DRVP1 aqueous sample process phenolsulfuric acid Faxian look, through the scanning of UV-Vis spectrophotometer, wavelength region is 200～600nm again.The result shows after this component color reaction has remarkable absorption peak at the 490nm place, and curve is level and smooth on the whole, shows free from foreign meter substantially.
With HPLC Dictyophora rubrovalvata volva polysaccharide DRVP1 is made molecular weight determination, moving phase: 0.1MNaNO
3, flow velocity: 0.8mL/min, detector: Waters 2410 differential refractions, pump: Waters 515, pillar: Waters uktrahydrogel.1000,500,120 series connection, column temperature: 40 ℃.Dextran with the standard of T series contrasts, and the molecular-weight average of Dictyophora rubrovalvata volva polysaccharide DRVP1 is 1.47 * 10 as can be known
4
(W: W) ratio was ground evenly, then compressing tablet in 1: 20 to get 1mg Dictyophora rubrovalvata volva polysaccharide DRVP1 sample and KBr powder.(note: the thin slice of extrusion with more transparent be good) through Nexus670 type Fourier transformation infrared spectrometer scanning analysis, sweep limit: 4000cm
-1～400cm
-1
Dictyophora rubrovalvata volva polysaccharide DRVP1 sample as can be seen, is positioned at 3400～3500cm through the scanning of FT-IR spectrograph from scintigram
-1Characteristic peak be the stretching vibration of glycan molecule O-H, 3000～2800cm
-1One group of stretching vibration that the peak is glycan molecule C-H, 1400～1200cm
-1The angle vibration that then is glycan molecule C-H of one group of peak.Can confirm that thus Dictyophora rubrovalvata volva polysaccharide DRVP1 is a compound of polysaccharide.Dictyophora rubrovalvata volva polysaccharide DRVP1 has-OH, COOR, C-O-C ehter bond ,-CHO/C=O, COOH, C-O-H ,-CH
2-,-CH
3, should belong to β-type pyranose glycosidic bond.
The vitro cytotoxicity test (srb assay) of Dictyophora rubrovalvata volva polysaccharide DRVP1
Dictyophora rubrovalvata volva polysaccharide DRVP1 respectively 50,100, at external S180 (mouse hydroperitoneum type liver cancer) tumour cell that acts on respectively, is observed their growth-inhibiting effects to this tumour cell under the concentration of 200,500,1000 μ g/ml.Tumour cell has certain restraining effect to S180 (mouse hydroperitoneum type liver cancer) to find DRVP1 in the experiment, and Dictyophora rubrovalvata volva polysaccharide DRVP2 does not show restraining effect, the results are shown in Figure 4.
Claims (5)
1, has the separation method of the polysaccharide that suppresses growth of tumour cell in a kind of Dictyophora rubrovalvata volva, comprise the steps:
(1) with Dictyophora rubrovalvata volva drying, pulverize after,, filter after 3～5 hours with the flooding of 10-30 times of volume, filtrate is concentrated to 50% of original volume, add ethanol again and carry out alcohol and analyse, separate precipitate and oven dry;
(2) be made into the aqueous solution after the precipitate oven dry, and adding and isopyknic chloroform of the aqueous solution and primary isoamyl alcohol mixed solution, thorough mixing is after 3～4 hours, and centrifugal, layering obtains organic phase and water;
(3) water adds ethanol and carries out alcohol and analyse, and separates precipitate and oven dry, obtains drying thing;
(4) the oven dry thing that step (3) is obtained is made into the aqueous solution of 12mg/mL, in 1.7cm * 20.3cmDEAE-cellulose column, separate, Tris buffered soln wash-out with 0.1M NaCl obtains Dictyophora rubrovalvata volva polysaccharide DRVP1, and elution time is 2hr, and flow velocity is 3mL/min.
2, separation method as claimed in claim 1 is characterized in that: the alcohol described in step (1) and the step (3) is analysed, and behind the adding ethanol, the alcoholic acid mass percent concentration is 50%～80% in the system.
3, separation method as claimed in claim 2 is characterized in that: the alcohol described in step (1) and the step (3) is analysed, and behind the adding ethanol, the alcoholic acid mass percent concentration is 70% in the system.
4, separation method as claimed in claim 1 is characterized in that: the lixiviate described in the step (1) be with Dictyophora rubrovalvata volva drying, pulverize after, with the flooding of 15 times of volumes 4 hours.
5, separation method as claimed in claim 1 is characterized in that: the volume of chloroform and primary isoamyl alcohol is 3: 1 in chloroform described in the step (2) and the primary isoamyl alcohol mixed solution.
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| CN104068449B (en) * | 2014-06-17 | 2016-08-24 | 福建农林大学 | A kind of isolation and purification method of Cultivation of Dictyophora bacteriostatic activity monomer |
| CN104262500B (en) * | 2014-09-15 | 2017-04-05 | 华南理工大学 | It is a kind of that there is immunocompetent novel red support dictyophora fungus polysaccharide and its preparation method and application |
| CN104277134B (en) * | 2014-09-15 | 2016-10-05 | 华南理工大学 | A kind of preparation method and applications of the dictyophora fungus polysaccharide-chelates of zinc with anti-tumor activity |
| CN104479040A (en) * | 2014-12-21 | 2015-04-01 | 曾丹 | Process for extracting bamboo fungus polysaccharide |
| CN106432530B (en) * | 2016-11-24 | 2019-04-12 | 福建省农业科学院土壤肥料研究所 | A kind of extraction process of dictyophora phalloidea volva colloid polysaccharide |
| CN110144017B (en) * | 2019-06-05 | 2021-06-22 | 福建省农业科学院土壤肥料研究所 | Continuous preparation process of active ingredients in bamboo fungus spores |
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Non-Patent Citations (10)
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