CN1310952C - Extraction of polysaccharose of three bristle cudrania - Google Patents
Extraction of polysaccharose of three bristle cudrania Download PDFInfo
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- CN1310952C CN1310952C CNB2004100241998A CN200410024199A CN1310952C CN 1310952 C CN1310952 C CN 1310952C CN B2004100241998 A CNB2004100241998 A CN B2004100241998A CN 200410024199 A CN200410024199 A CN 200410024199A CN 1310952 C CN1310952 C CN 1310952C
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Abstract
The present invention relates to a method for extracting the polysaccharide of three-bristle cudrania, which comprises the following steps: immersing, extracting, centrifugating and filtering raw material; mixing filter liquor; processing the filter liquor by macroporous adsorptive resin; applying vacuum concentration, alcohol precipitation, deproteinization, dialysis, separation and purification to the processed filter liquor. The raw material is kept immersed for 6 to 8 hours and extracted at a temperature of 70DEG C; the adsorptive resin is 3520 resin whose pH value is 4; the deproteinization adopts a Sevag method; after dialysis, the filter liquor is added to a DEAE-SephadexA-50 gel column; the filter liquor is eluted by water and a NaCl solution; the eluent is dialyzed, cooled and dried to obtain the pure polysaccharide of three-bristle cudrania.
Description
Technical field:
The present invention relates to a kind of extracting method of vegetable polysaccharides, is to be the method for raw material extraction polysaccharide with the three-bristle cudrania tree root specifically.
Background technology:
Polysaccharide compound can participate in the transmission and the impression of information between immunocyte, and transformation, division and regeneration activity etc. have immunoregulatory function; Effect with antitumor, antiviral, anti-ageing, anticoagulation, Antiradiation injury; Have hypoglycemic, blood fat and cholesterol, antiatherogenic effect; The protection gi tract have antiulcer effect; Effect with the liver injury of preventing; Some mucopolysaccharides can control agent in anionic concentration, regulate the generation of collegen filament, stop the absorption of heavy metal ion in animal digestive tract etc.A large amount of pharmacology and clinical studyes show, polysaccharide compound is a kind of immunomodulator, it can activate T, bone-marrow-derived lymphocyte, M φ, immunocytes such as NKC, complement activation, promote cytokine generation etc., immunity system is brought into play many-sided regulating effect, thereby improve body's immunological function, in cancer therapy, it unlike chemotherapeutics, directly kill the cell of growing, but by regulating body's immunological function kill tumor cell, very little to normal impact cell, it is little to have toxic side effect, safe, advantages such as tumor killing effect is good, it is subject to people's attention in the superiority that treatment tumour and AIDS etc. demonstrate aspect viral day by day as immunomodulator.
Zhe Shu [Cudrania tricuspidata (Carr.) Bur.], plum, toothed oak tree, toothed oak cheek tree and toothed oak thorn are stung, deceived to another name three-bristle cudrania wood, three-bristle cudrania, three-bristle cudrania mulberry, three-bristle cudrania, is moraceae plants, and machaka or dungarunga are distributed in Central-South, East China, southwestern to the south, Hebei.In Compendium of Material Medica, " its root, root skin, stem, branches and leaves, fruit of record can be used as medicine in the Chinese pharmacopoeia, and it is sweet in flavor and warm in property nontoxic, can treat arthralgia, jaundice, internal lesion caused by overexertion hemoptysis, calculus, cough-relieving apophlegmatic etc.Zhejiang one was with and among the peoplely once three-bristle cudrania tree root decocting was treated tumour the end of the seventies, the three-bristle cudrania wood molasses that Shanghai Lei-Yun-Shang Pharmaceutical Plant and Shanghai Chinese medicine two factories produce is used for the treatment of digestive tract tumor such as cancer of the stomach, find that the three-bristle cudrania wood molasses not only has the effect that suppresses tumour, and can obviously alleviate the toxic side effect of chemotherapy with the time spent with chemotherapeutics, can resist the inhibition of chemotherapy, alleviate inhibition and the infringement of chemotherapy immunologic function to hemocyte.Xu Yutai etc. find that three-bristle cudrania tree Crude polysaccharides all increases to Turnover of Mouse Peritoneal Macrophages peroxidase activity, cytotoxicity and phagocytic function when the antitumor action of research three-bristle cudrania tree flavones, and can suppress the S180 tumour.And open report is not seen in the separation and purification of three-bristle cudrania tree root polysaccharide, physico-chemical property, structural analysis as yet.
Summary of the invention:
The purpose of this invention is to provide a kind of method of from the three-bristle cudrania tree root, extracting polysaccharide.
The objective of the invention is to realize as follows: the method for this extraction three-bristle cudrania tree polysaccharide, comprise raw material is soaked extraction, centrifugal, filtration, merging filtrate, filtrate is crossed macroporous adsorbent resin, concentrating under reduced pressure, alcohol is analysed, Deproteinization, dialysis and separation and purification.
The aforesaid method concrete operations are as follows:
A. the three-bristle cudrania tree root is pulverized, and the water logging that adds 6-8 times of volume was steeped 6 hours, and united extraction liquid is extracted in 70 ℃ of gradation;
B. said extracted liquid is centrifugal, filtration;
C. will go up the step extracting solution and cross 3520 macroporous adsorbent resins to remove flavones;
D. will go up step collection liquid and be evaporated to 1/3 of original volume, centrifugal;
E. will go up the alcohol chromatography of the filtrate in step, under 4 ℃ of environment, leave standstill 12 hours with 3 times of volumes 95%;
F. will go up step alcohol and analyse the thing centrifugal treating, remove supernatant liquor;
G. will go up step gained throw out and wash successively with 75% ethanol, dehydrated alcohol, ether, acetone, centrifugal;
H. will go up step gained throw out dissolved in distilled water, Sevag method Deproteinization, repeated multiple times does not have precipitation until intersection and occurs;
I. will go up step gained solution and remove organic solvent, the dry Crude polysaccharides that gets;
J. Crude polysaccharides is water-soluble, dialysed 48-72 hour;
K. it is centrifugal to go up the step dialyzate, and supernatant liquor is crossed the DEAE-SephadexA-50 post, uses H successively
2O, 0.1mol/L NaCl, 0.2mol/L NaCl, 0.3mol/L NaCl wash-out is collected mountain portions respectively;
L. will go up the solution dialysis of step collection respectively, and concentrate, lyophilize gets the pure product of three-bristle cudrania tree polysaccharide series.
Extracting in a step gradation is 2-3 time, the 1st time 2 hours, and the 2nd time 1 hour, the 3rd time 0.5-1 hour.
At the c 3520 resin pH=4 in step.
At d step concentrating under reduced pressure is to carry out at 40 ℃-50 ℃.
Throw out in the g step washs 2-3 time successively with 75% ethanol, dehydrated alcohol, ether, acetone successively.
H step by chloroform and propyl carbinol by volume 5 to the 1 Sevag reagent deproteinated processes that constitute be with the volume ratio adding of reagent and solution by 1: 5, shook 20-30 minute after adding reagent, make reagent and solution thorough mixing, after centrifugal, remove the sedimentary albumen of intersection, so repeated multiple times does not have the precipitation appearance until intersection.
Removing organic solvent in i step is to be evaporated to solution at 50 ℃ to add distilled water when a small amount of and continue to concentrate 3 times repeatedly.So-called solution is a small amount of, is meant the state that can see solution slightly.
The pure product of the polysaccharide that obtains are represented with following code name: CPS-1, CPS-2, CPS-3.Their IKI reaction is negative, and illustrates that the three is not starch-containing; Biuret reaction is negative, and illustrates that the three does not contain protein; UV spectrum UV scanning analysis shows CPS-1, and CPS-2, CPS-3 solution illustrate that at the no absorption peak of 260nm and 280nm place the three does not contain protein and nucleic acid; High-efficient liquid phase chromatogram HPLC analysis revealed CPS-1 is the homogeneous component, and number-average molecular weight is 4461, and weight-average molecular weight is 6572, and CPS-2 contains small amount of impurities, and number-average molecular weight is 24709, and weight-average molecular weight is 68110, and CPS-3 is a blending ingredients; GCMS analysis revealed CPS-1 contains rhamnosyl (Rha), pectinose (Ara), wood sugar (Xyl), seminose (Man), glucose (Glu), semi-lactosi (Gal), mole (Rha-Ara-Xyl-Man-Glu-Gal) is than being followed successively by 1.00: 14.02: 0.74: 2.62: 30.06: 17.72, contain rhamnosyl (Rha) among the CPS-2, pectinose (Ara), wood sugar (Xyl), seminose (Man), glucose (Glu), semi-lactosi (Gal), mole (Rha-Ara-Xyl-Man-Glu-Gal) is than being followed successively by 1.00: 2.68: 0.11: 0.16: 0.27: 3.83; Ultimate analysis shows that CPS-1, CPS-2 are the aminosugar of sulfur-bearing; IR,
1HNMR and
13The CNMR spectroscopic analysis shows that CPS-1 has the charateristic avsorption band of pyranose form sugar ring and the charateristic avsorption band of α-glycosidic link, C-2, and C-3 replace on a certain carbon among the C-4, and C-6 replaces; CPS-2 has the charateristic avsorption band of furan type sugar ring, and its glycosidic link may be a beta configuration.No matter having under the situation that does not have a mitogen ConA or LPS existence, CPS-1, CPS-2, CPS-3 all can significantly strengthen the propagation of mouse spleen lymphocyte, and clinical lentinan effect is more obvious than being used for; Turnover of Mouse Peritoneal Macrophages is being engulfed in the influence of toluylene red, CPS-1, CPS-2 is identical with the lentinan action effect in some concentration.
Embodiment:
Embodiment 1:
The three-bristle cudrania tree root is pulverized, and the water logging that adds 6 times of volumes was steeped 6 hours, and 70 ℃ are extracted 3 times, the 1st leaching 2 hours, and the 2nd order adds each lixiviate of water 1 hour 3 times, united extraction liquid, centrifugal 15 minutes of 4000r/min, the sand core funnel filters.With damping fluid balance 3520 adsorption columns to pH=4, filtrate is crossed 3520 posts, collect filtrate, 40 ℃ are evaporated to 1/3 of original volume, centrifugal 15 minutes of 4000r/min, collect the ethanol that supernatant liquor adds 3 times of volumes 95%, standing over night under 4 ℃ of environment, 4000r/min got throw out in centrifugal 15 minutes, with throw out 75% ethanol, dehydrated alcohol, ether, acetone washs 2-3 time successively, the throw out dissolved in distilled water that 4000r/min obtained in centrifugal 15 minutes, make it abundant redissolution, Sevag method Deproteinization, repeated multiple times does not have precipitation until intersection and occurs, gained solution is removed organic solvent, and lyophilize gets Crude polysaccharides; Crude polysaccharides is water-soluble, dialysed 48-72 hour, and centrifugal 15 minutes of dialyzate 4000r/min, supernatant liquor is crossed the DEAE-SephadexA-50 gel column, uses H successively
2O, 0.1mol/L NaCl, 0.2mol/L NaCl, 0.3mol/L NaCl wash-out is collected mountain portions respectively, through dialysis, concentrates, and lyophilize gets the pure product of three-bristle cudrania tree polysaccharide.
Embodiment 2:
The three-bristle cudrania tree root is pulverized, and adds the water logging bubble 6 hours of 8 times of volumes, and 70 ℃ are extracted 2 times, and the 1st leaching 2 hours added flooding 1 hour the 2nd time, united extraction liquid, and centrifugal 15 minutes of 4000r/min, the sand core funnel filters.With damping fluid balance 3520 adsorption columns to pH=4, filtrate is crossed 3520 posts, collect filtrate, 40 ℃ are evaporated to 1/3 of original volume, centrifugal 15 minutes of 4000r/min, collect the ethanol that supernatant liquor adds 3 times of volumes 95%, standing over night under 4 ℃ of environment, 4000r/min got throw out in centrifugal 15 minutes, with throw out 75% ethanol, dehydrated alcohol, ether, acetone washs 2-3 time successively, the throw out dissolved in distilled water that 4000r/min obtained in centrifugal 15 minutes, make it abundant redissolution, Sevag method Deproteinization, repeated multiple times does not have precipitation until intersection and occurs, gained solution is removed organic solvent, and lyophilize gets Crude polysaccharides; Crude polysaccharides is water-soluble, dialysed 48-72 hour, and centrifugal 15 minutes of dialyzate 4000r/min, supernatant liquor is crossed the DEAE-SephadexA-50 gel column, uses H successively
2O, 0.1mol/L NaCl, 0.2mol/L NaCl, 0.3mol/L NaCl wash-out is collected mountain portions respectively, through dialysis, concentrates, and lyophilize gets the pure product of three-bristle cudrania tree polysaccharide.
Claims (7)
1, a kind of method of extracting three-bristle cudrania tree polysaccharide comprises raw material is soaked extraction, centrifugal, filtration, and merging filtrate, filtrate is crossed macroporous adsorbent resin, concentrating under reduced pressure, alcohol is analysed, Deproteinization, dialysis and separation and purification is characterized in that being made of following concrete steps:
A. the three-bristle cudrania tree root is pulverized, and the water logging that adds 6-8 times of volume was steeped 6 hours, and united extraction liquid is extracted in 70 ℃ of gradation;
B. said extracted liquid is centrifugal, filtration;
C. will go up step filtrate and cross 3520 macroporous adsorbent resins to remove flavones;
D. will go up step collection liquid and be evaporated to 1/3 of original volume, centrifugal;
E. will go up the alcohol chromatography of the filtrate in step, under 4 ℃ of environment, leave standstill 12 hours with 3 times of volumes 95%;
F. will go up step alcohol and analyse the thing centrifugal treating, remove supernatant liquor;
G. will go up step gained throw out and wash centrifugal 15 minutes with 75% ethanol, dehydrated alcohol, ether, acetone successively;
H. will go up step gained throw out dissolved in distilled water, Sevag method Deproteinization, repeated multiple times does not have precipitation until intersection and occurs;
I. will go up step gained solution and remove organic solvent, the dry Crude polysaccharides that gets;
J. Crude polysaccharides is water-soluble, dialysed 48-72 hour;
K. it is centrifugal to go up the step dialyzate, and supernatant liquor is crossed the DEAE-SephadexA-50 post, uses H successively
2O, 0.1mol/L NaCl, 0.2mol/L NaCl, 0.3mol/L NaCl wash-out is collected mountain portions respectively;
1. will go up the solution dialysis of step collection respectively, and concentrate, lyophilize gets the pure product of three-bristle cudrania tree polysaccharide series.
2, the method for extraction according to claim 1 three-bristle cudrania tree polysaccharide is characterized in that: extract time-divisions 2-3 time 70 ℃ of a steps, and the 1st time 2 hours, the 2nd time 1 hour, the 3rd time 0.5-1 hour.
3, the method for extraction three-bristle cudrania tree polysaccharide according to claim 1 is characterized in that: at the c 3520 resin pH=4 in step.
4, the method for extraction three-bristle cudrania tree polysaccharide according to claim 1, it is characterized in that: at d step concentrating under reduced pressure is to carry out at 40 ℃-50 ℃.
5, the method for extraction three-bristle cudrania tree polysaccharide according to claim 1, it is characterized in that: the throw out in the g step washs 2-3 time successively with 75% ethanol, dehydrated alcohol, ether, acetone.
6, the method for extraction three-bristle cudrania tree polysaccharide according to claim 1, it is characterized in that: the Sevag reagent deproteinated process that was made of in 5: 1 by volume chloroform and propyl carbinol in h step is with reagent and the volume ratio adding of solution by 1: 5, shook 20-30 minute after adding reagent, make reagent and solution thorough mixing, the sedimentary albumen of intersection is removed in centrifugal back, and so repeated multiple times does not have the precipitation appearance until intersection.
7, the method for extraction according to claim 1 three-bristle cudrania tree polysaccharide is characterized in that: removing organic solvent in the i step is to be evaporated to solution at 50 ℃ to add distilled water when a small amount of and continue to concentrate 3 times repeatedly.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2004100241998A CN1310952C (en) | 2004-06-04 | 2004-06-04 | Extraction of polysaccharose of three bristle cudrania |
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| CNB2004100241998A CN1310952C (en) | 2004-06-04 | 2004-06-04 | Extraction of polysaccharose of three bristle cudrania |
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| CN1583798A CN1583798A (en) | 2005-02-23 |
| CN1310952C true CN1310952C (en) | 2007-04-18 |
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Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102311509A (en) * | 2011-06-17 | 2012-01-11 | 青阳县杰灵生物工程有限责任公司 | Extracting method of sweet potato stem leaf polysaccharide |
| CN104758197B (en) * | 2014-01-03 | 2018-02-09 | 伽蓝（集团）股份有限公司 | A kind of cudrania tricuspidata extract and its application |
| CN107549806B (en) * | 2017-08-31 | 2020-11-17 | 山东北柘药业有限公司 | Preparation method of natural flour additive |
| CN113549162A (en) * | 2021-07-07 | 2021-10-26 | 上海应用技术大学 | Preparation method of cudrania tricuspidata refined polysaccharide |
| CN115490776A (en) * | 2022-09-09 | 2022-12-20 | 上海应用技术大学 | Cudrania tricuspidata leaf polysaccharide extraction process |
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| CN1076859A (en) * | 1992-03-30 | 1993-10-06 | 河南省新乡市前卫制药厂 | The extraction of the flat effective ingredient of the cancer that disappears and dosage form preparation |
| CN1165148A (en) * | 1996-05-10 | 1997-11-19 | 中国科学院大连化学物理研究所 | Method for preparing dictyophora fungus polysaccharide |
| CN1343729A (en) * | 2000-09-20 | 2002-04-10 | 天津市贝特科技发展有限公司 | Liquorice polyose |
| CN1371920A (en) * | 2001-02-28 | 2002-10-02 | 中国科学院上海药物研究所 | Persimmon leaf polysaccharide and biological activity thereof |
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2004
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1076859A (en) * | 1992-03-30 | 1993-10-06 | 河南省新乡市前卫制药厂 | The extraction of the flat effective ingredient of the cancer that disappears and dosage form preparation |
| CN1165148A (en) * | 1996-05-10 | 1997-11-19 | 中国科学院大连化学物理研究所 | Method for preparing dictyophora fungus polysaccharide |
| CN1343729A (en) * | 2000-09-20 | 2002-04-10 | 天津市贝特科技发展有限公司 | Liquorice polyose |
| CN1371920A (en) * | 2001-02-28 | 2002-10-02 | 中国科学院上海药物研究所 | Persimmon leaf polysaccharide and biological activity thereof |
Non-Patent Citations (4)
| Title |
|---|
| 柘木根多糖对小鼠腹腔巨噬细胞活性的影响及其抑瘤作用 宫丽华，山东中医药大学学报，第26卷第2期 2002 * |
| 植物多糖提取分离及药理作用的研究进展 张晓静，时珍国医国药，第14卷第8期 2003 * |
| 银杏叶和柘树提取物的抗氧化作用 张可炜，山东大学学报(自然科学版)，第35卷第4期 2000 * |
| 银杏叶和柘树提取物的抗氧化作用 张可炜，山东大学学报(自然科学版)，第35卷第4期 2000;植物多糖提取分离及药理作用的研究进展 张晓静，时珍国医国药，第14卷第8期 2003;柘木根多糖对小鼠腹腔巨噬细胞活性的影响及其抑瘤作用 宫丽华，山东中医药大学学报，第26卷第2期 2002 * |
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