CN102532339A - Method for selenizing pilose asiabell root polysaccharides and application of product - Google Patents
Method for selenizing pilose asiabell root polysaccharides and application of product Download PDFInfo
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- CN102532339A CN102532339A CN2012100319889A CN201210031988A CN102532339A CN 102532339 A CN102532339 A CN 102532339A CN 2012100319889 A CN2012100319889 A CN 2012100319889A CN 201210031988 A CN201210031988 A CN 201210031988A CN 102532339 A CN102532339 A CN 102532339A
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- radix codonopsis
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Abstract
The invention provides a method for selenizing pilose asiabell root polysaccharides. The method comprises the following steps of: adding selenous acid into an aqueous solution of the pilose asiabell root polysaccharides with stirring, adding a catalyst, reacting at the temperature of between 50 and 70 DEG C for 4 to 8 hours, cooling, separating, and purifying to obtain a selenized pilose asiabell root polysaccharide product, wherein the catalyst is barium chloride. By the method, the content of selenium in the pilose asiabell root polysaccharides can be improved to 500-5,000mu g/g, the anti-tumor activity and immunity improving activity of the selenized pilose asiabell root polysaccharides are obviously improved compared with those of non-selenized pilose asiabell root polysaccharides, and the product can be used for preparing anti-tumor medicines or medicines or health care products for improving immunity.
Description
Technical field
The invention belongs to the synthetic field of medicine, be specifically related to the selenizing method and the application thereof of Radix Codonopsis polysaccharide.
Background technology
Radix Codonopsis is a conventional Chinese medicine, and the sweet property of distinguishing the flavor of is flat, has invigorating the spleen and replenishing QI, and the function of invigorating the spleen benefit lung is used to treat that the spleen deficiency syndrome of the lung is weak, the palpitaition of breathing hard, anorexia and loose stool, void are breathed with cough the disease and the substitute of Chang Zuowei genseng such as cough, interior heat is quenched one's thirst.According to bibliographical information, contained chemical ingredients more complicated in the Radix Codonopsis comprises polyose, xylogen glycoside, phenolic acids, terpene, steroid class etc.
Radix Codonopsis polysaccharide is one of effective constituent in the Radix Codonopsis, and tradition control and evaluation Radix Codonopsis quality all are through measuring the wherein content of polysaccharide.Modern pharmacology research confirms that Radix Codonopsis polysaccharide has the various biological activity, wherein with antitumor, anti-oxidant and to improve the immunizing power activity research more also comparatively deep.
Selenium is the necessary trace element of human body, is the main active ingredient of Selenoperoxidase, and human body lacks selenium can suffer from Keshan disease, Kaschin-Beck disease etc., and selenium confirms to have the effect of anti-multiple carcinogenic substance in animal experiment.The existence form of selenium is divided into two kinds of inorganic selenium and organoselenium, compares with inorganic selenium, and organoselenium has higher biological activity because of it and lower toxicity is prone to by the organism utilization.Organoselenium comprises selenium polysaccharide, seleno-protein, seleno nucleic acid etc.Selenium polysaccharide is as important organoselenium kind, and the activity that not only has inorganic selenium also has the various physiological functions of polysaccharide.
Selenium and Radix Codonopsis polysaccharide are organically combined the effect that not only can improve Radix Codonopsis polysaccharide, can also improve the selenium absorption in vivo.But existing research shows that natural selenium content is extremely low in the Radix Codonopsis polysaccharide, is approximately 12ng/g, so how to improve the selenium content in the Radix Codonopsis polysaccharide, the pharmaceutical use of at utmost bringing into play Radix Codonopsis polysaccharide and selenium is the problem that needs solution in the Radix Codonopsis polysaccharide drug development.
Summary of the invention
The present invention seeks to solve the low problem of selenium content in the Radix Codonopsis polysaccharide, a kind of method and application thereof of Radix Codonopsis polysaccharide selenizing are provided.
The present invention realizes that the technical scheme of above-mentioned purpose is following:
A kind of method of Radix Codonopsis polysaccharide selenizing, this method comprise the steps: under agitation, selenous acid are joined in the aqueous solution of Radix Codonopsis polysaccharide; Add catalyzer again; Reacted 4～8 hours down at 50～70 ℃ then, cooling, separation and purification obtains the Radix Codonopsis polysaccharide product of selenizing; Wherein, said catalyzer is a bariumchloride.
Further, the weight ratio of selenous acid and Radix Codonopsis polysaccharide is (0.5～1.5): 1.
Further, the weight ratio of bariumchloride and Radix Codonopsis polysaccharide is (1～2): 1.
Further, in the reaction, press 0.2%～0.6% of reaction solution volume and add nitric acid, can improve the productive rate of selenylation reaction.
Further, the method that said separation and purification can adopt alcohol precipitation to combine with dialysis, concrete steps are in reaction solution, to add ethanol to appropriate amount; Hold over night; Obtain throw out, throw out is used water dissolution again, and gained solution is again through the dialysis purifying; Solution behind the purifying concentrates down in 40～70 ℃, obtains the Radix Codonopsis polysaccharide product of selenizing again through lyophilize.
Further, ethanol volumetric concentration to 60～85% in the adding ethanol conditioned reaction liquid.
Further, the ethanol volumetric concentration to 80% in the adding ethanol conditioned reaction liquid.
For prevent that the catalyzer bariumchloride in the reaction solution from together precipitating in above-mentioned alcohol precipitation step, can before the alcohol precipitation step, add an amount of sodium sulfate, after the barium ion deposition is removed, carry out the separation and purification of Radix Codonopsis polysaccharide selenizing product again.
Further, the selenium content that adopts aforesaid method to obtain product is 500～5000 μ g/g.
Further, the application of product in the preparation antitumor drug of adopting aforesaid method to obtain.
Further, the product that adopts aforesaid method to obtain improves the medicine of immunizing power or the application in the healthcare products in preparation.
Beneficial effect: the present invention adopts selenous acid that Radix Codonopsis polysaccharide is modified; Can the selenium content of Radix Codonopsis polysaccharide be increased to 500～5000 μ g/g; The Radix Codonopsis polysaccharide of selenizing is compared with the Radix Codonopsis polysaccharide of not selenizing, and its anti-tumor activity all is significantly improved with the raising immunocompetence.
Description of drawings
Fig. 1 is the response surface chart.
Embodiment
Below in conjunction with embodiment and accompanying drawing the present invention is described further.
The extraction of embodiment 1 Radix Codonopsis polysaccharide
Adopt decocting-alcohol deposition method to extract Radix Codonopsis polysaccharide, detailed process is: get codonopsis pilosula 1kg, pulverize, place round-bottomed flask, with 85 ~ 95% (V/V) alcohol reflux 2 times; Each 2 hours, the decocting that the dregs of a decoction dry 5～12 times of amounts of back adding naturally boiled 4 times, and each 2 hours, merge soup, water-bath concentrates; Cycles of concentration is 4, and hold over night discards deposition, adds 95% ethanol and makes alcohol concn reach 80%, hold over night; The centrifugal deposition that obtains, deposition is used washing with alcohol again, and drying obtains the coarse tuning gracilis polysaccharide.The coarse tuning gracilis polysaccharide obtains polysaccharide content and reaches the Radix Codonopsis polysaccharide more than 50% through depigmentation, except that after the albumen processing.
The modification of embodiment 2 Radix Codonopsis polysaccharides
This experiment exam the influences of selenous acid and Radix Codonopsis polysaccharide mass ratio, reaction times, three factors of temperature of reaction to the Radix Codonopsis polysaccharide selenizing.Adopt the Box-Behnken method; Three variablees choosing are respectively selenous acid and Radix Codonopsis polysaccharide mass ratio (X1), reaction times (X2) and temperature of reaction (X3), by equation ci=(Xi-X0)/△ X independent variable(s) are encoded, wherein; Ci is the encoded radio of variable; Xi is the actual value of variable, and X0 is the actual value of experimental center point variable, and △ X is the change step of variable.Each variable and level see the following form
Experimentation: Radix Codonopsis polysaccharide is dissolved in processes saturated aqueous solution in the zero(ppm) water, be added in the three-necked bottle of being furnished with prolong and TM, use magnetic stirrer, slowly add selenous acid again, and add BaCl down in stirring
2The stirring that solution does not stop adds 10% HNO behind the 2h
3Solution reacts reasonable time under the temperature of reaction of setting, after question response stops, and cooling; Add 95% (V/V) ethanol, the ethanol volumetric concentration reaches 80% in the conditioned reaction liquid, hold over night, the centrifugal throw out that obtains; Throw out with dissolved in distilled water after, the dialysis tubing of packing into, first flowing water dialysis 1～4d uses distill water dialysis 1～3d again; Concentrate in 40～70 ℃, lyophilize obtains the Radix Codonopsis polysaccharide product of selenizing, is the Radix Codonopsis selenium polysaccharide again.
Adopt atomic fluorescence spectrum to measure Radix Codonopsis polysaccharide and modify the selenium content in the after product.
Adopt tabulation of Box-Behnken method plan and experimental result
Adopt Response Surface Method that above-mentioned experimental result is analyzed, be decided to be 0.60 with product amount n weight, selenium content m weight location 0.40, overall target Y is the comprehensive of two indexs, promptly Y is 0.6 * n
i/ n
Max+ 0.4 * m
i/ m
Max, carry out multinomial logistic regression by following formula:
Wherein Y representes response value, β
KoBe intercept, β
Ki, β
KiiAnd β
KijBe respectively once item, interactive coefficient between quadratic term and each variable.With Y is 0.6 * n
i/ n
Max+ 0.4 * m
i/ m
MaxFor response is optimized, the polynomial regression equation of its response is following:
Y=15.71+1.00X1+0.74X2+0.64X3-0.74X1*X1+0.15X1*X2-0.13X1*X3+0.26X2*X2+0.02X2*X3-1.08X3*X3
Its variance is 0.808978, and coefficient variation is 5.794829, R2=0.941003, explain this equation preferably response variable and the response between relation.According to its response surface chart (see figure 1), best selenizing condition when confirming in the product selenium content the highest (being 4906.6 μ g/g): the mass ratio of selenous acid and Radix Codonopsis polysaccharide is 1.5, the reaction times is that 7 hours, temperature of reaction are 65 ℃.
The biological activity of the Radix Codonopsis polysaccharide product of embodiment 3 selenizings (Radix Codonopsis selenium polysaccharide)
Make the Radix Codonopsis selenium polysaccharide according to embodiment 2 determined best selenizing conditions, selenium content is 4906.6 μ g/g in the products therefrom, and is used for following biological activity test.
(1) improves immunizing power
Laboratory animal: six age in week 6 of BALB/C mices
Experimental technique: (1) Radix Codonopsis selenium polysaccharide with taking out medullary cell down in aseptic condition after the mouse orbit sacrificed by exsanguination, is processed cell suspension with complete culture solution DMEM to bone marrow cells in mice increment influence, and counting makes cell concn reach 5 * 10
5/ hole, behind the cultivation 2h, the centrifugal supernatant that goes adds complete culture solution DMEM, adds phytohemagglutinin and Radix Codonopsis selenium polysaccharide or Radix Codonopsis polysaccharide again, puts CO
2Cultivate 3d in the incubator, the back is detected with mtt assay.
(2) the Radix Codonopsis selenium polysaccharide is got mouse spleen and thymus gland respectively to spleen and thymic lymphocytes increment influence and in substratum, is torn to shreds and make into cell suspension, and counting makes cell concn reach 2 * 10
6/ hole adds polyhydroxyalkanoate (PHA) and Radix Codonopsis selenium polysaccharide or Radix Codonopsis polysaccharide, puts CO
2Cultivate 3d in the incubator, the back is detected with mtt assay.
Seven groups are established in experiment, and the grouping situation is following:
First group: control group neither adds Radix Codonopsis polysaccharide and does not also add Radix Codonopsis selenizing polysaccharide;
The second～four group: the Radix Codonopsis polysaccharide group, establish high, normal, basic three concentration respectively, 6 parts of each concentration parallel tests;
The five～seven group: Radix Codonopsis selenium polysaccharide group (the prepared Radix Codonopsis selenium polysaccharide of embodiment), establish high, normal, basic three concentration respectively, 6 parts of each concentration parallel tests; Experimental result is following,
Radix Codonopsis polysaccharide and Radix Codonopsis selenium polysaccharide are to influence (
± s), the n=6 of medullary cell, spleen cell, thymocyte proliferation
* expression and blank control group relatively P 0.05,
aThe Radix Codonopsis selenium polysaccharide group of expression same dose and the Radix Codonopsis polysaccharide group of same dose be P relatively<0.05.
The dosage Radix Codonopsis polysaccharide was compared with the normal control group bone marrow cells in mice, spleen cell, thymocyte proliferation rate and is increased significantly during experimental result showed, high dose group does not but have proliferation function, and this maybe be relevant with the cytotoxicity of Radix Codonopsis polysaccharide when the high dosage.The high, normal, basic dose groups of Radix Codonopsis selenium polysaccharide has all shown significant cel l proliferation, and compares with the Radix Codonopsis polysaccharide group of same dose and all to have significant difference, so this experimental result shows that the Radix Codonopsis selenium polysaccharide has significant raising immunization.
(2) antitumor
Experimental cell: liver cancer cell
TP: the tumour cell of adherent growth is inoculated in the orifice plate after with 0.25% tryptic digestion, and test group adds the Radix Codonopsis selenium polysaccharide or the Radix Codonopsis polysaccharide of each concentration, and the blank group adds the RPMI-1640 substratum, in CO
2Cultivate 2d in the incubator, the back is detected with mtt assay.
Seven groups are established in experiment, and the grouping situation is following:
First group: control group neither adds Radix Codonopsis polysaccharide and does not also add Radix Codonopsis selenizing polysaccharide;
The second～four group: the Radix Codonopsis polysaccharide group, establish high, normal, basic three concentration respectively, 6 parts of each concentration parallel tests;
The five～seven group: Radix Codonopsis selenium polysaccharide group (the prepared Radix Codonopsis selenium polysaccharide of embodiment), establish high, normal, basic three concentration respectively, 6 parts of each concentration parallel tests; Experimental result is following,
The restraining effect that the Radix Codonopsis polysaccharide of different concns and Radix Codonopsis selenium polysaccharide are grown to liver cancer cell (SMMC-7721) [(
± s) %], n=6
* expression and blank control group relatively P 0.05,
aThe Radix Codonopsis selenium polysaccharide group of expression same dose and the Radix Codonopsis polysaccharide group of same dose be P relatively<0.05.
The result shows that the restraining effect to liver cancer cell (SMMC-7721) obviously is better than not selenizing Radix Codonopsis polysaccharide of same dose after the Radix Codonopsis polysaccharide selenizing.
Claims (10)
1. the method for a Radix Codonopsis polysaccharide selenizing is characterized in that, this method comprises the steps: under agitation; Selenous acid is joined in the aqueous solution of Radix Codonopsis polysaccharide, add catalyzer again, reacted 4～8 hours down at 50～70 ℃ then; Cooling; Separation and purification obtains the Radix Codonopsis polysaccharide product of selenizing, and wherein, said catalyzer is a bariumchloride.
2. the method for Radix Codonopsis polysaccharide selenizing according to claim 1 is characterized in that: the weight ratio of selenous acid and Radix Codonopsis polysaccharide is (0.5～1.5): 1.
3. the method for Radix Codonopsis polysaccharide selenizing according to claim 1 is characterized in that: the weight ratio of bariumchloride and Radix Codonopsis polysaccharide is (1～2): 1.
4. the method for Radix Codonopsis polysaccharide selenizing according to claim 1 is characterized in that: in the reaction, press 0.2%～0.6% of reaction solution volume and add nitric acid.
5. the method for Radix Codonopsis polysaccharide selenizing according to claim 1; It is characterized in that: the detailed process of said separation and purification is for adding ethanol to appropriate amount in reaction solution, hold over night obtains throw out; Throw out is used water dissolution again; Gained solution is again through the dialysis purifying, and the solution behind the purifying concentrates down in 40～70 ℃, obtains the Radix Codonopsis polysaccharide product of selenizing again through lyophilize.
6. the method for Radix Codonopsis polysaccharide selenizing according to claim 5 is characterized in that: add ethanol volumetric concentration to 60～85% in the ethanol conditioned reaction liquid.
7. the method for Radix Codonopsis polysaccharide selenizing according to claim 6 is characterized in that: add the ethanol volumetric concentration to 80% in the ethanol conditioned reaction liquid.
8. according to the method for the described Radix Codonopsis polysaccharide selenizing of claim 1～7, it is characterized in that: the selenium content that adopts said method to obtain product is 500～5000 μ g/g.
9. according to the method for the described Radix Codonopsis polysaccharide selenizing of claim 1～7, it is characterized in that: the application of product in the preparation antitumor drug of adopting said method to obtain.
10. according to the method for the described Radix Codonopsis polysaccharide selenizing of claim 1～7, it is characterized in that: the product that adopts said method to obtain improves the medicine of immunizing power or the application in the healthcare products in preparation.
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| CN110105460A (en) * | 2019-05-24 | 2019-08-09 | 武汉轻工大学 | Selenizing carboxymethylpachymaran and preparation method and application |
| CN110526996A (en) * | 2018-05-24 | 2019-12-03 | 中国科学院大连化学物理研究所 | A kind of selenizing oligo-konjac glucomannan and its preparation and application |
| CN110679996A (en) * | 2019-10-31 | 2020-01-14 | 湖北中烟工业有限责任公司 | Tobacco leaf containing selenium-enriched polysaccharide |
| CN116211910A (en) * | 2022-12-15 | 2023-06-06 | 安徽济生元药业有限公司 | Preparation method of codonopsis pilosula nano-selenium capsule |
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| CN103720646A (en) * | 2013-12-06 | 2014-04-16 | 鼎正动物药业（天津）有限公司 | Enriched-Se isatis root polysaccharide oral liquid and preparation method thereof |
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| CN103739729B (en) * | 2013-12-06 | 2016-06-15 | 鼎正动物药业（天津）有限公司 | A kind of Indigowoad root selenium polysaccharide and preparation method thereof |
| CN104004107A (en) * | 2014-05-19 | 2014-08-27 | 兰州大学 | Selenide of radix codonopsis polysaccharide CPP1b, preparation method and application thereof |
| CN106267186A (en) * | 2016-09-21 | 2017-01-04 | 南京农业大学 | A kind of have garlic polysaccharide and the compositions of selenizing Radix Codonopsis polysaccharide strengthening immunization |
| CN110526996A (en) * | 2018-05-24 | 2019-12-03 | 中国科学院大连化学物理研究所 | A kind of selenizing oligo-konjac glucomannan and its preparation and application |
| CN110105460A (en) * | 2019-05-24 | 2019-08-09 | 武汉轻工大学 | Selenizing carboxymethylpachymaran and preparation method and application |
| CN110105460B (en) * | 2019-05-24 | 2021-08-27 | 武汉轻工大学 | Selenylation carboxymethyl pachyman and preparation method and application thereof |
| CN110679996A (en) * | 2019-10-31 | 2020-01-14 | 湖北中烟工业有限责任公司 | Tobacco leaf containing selenium-enriched polysaccharide |
| CN110679996B (en) * | 2019-10-31 | 2022-02-08 | 湖北中烟工业有限责任公司 | Tobacco leaf containing selenium-enriched polysaccharide |
| CN116211910A (en) * | 2022-12-15 | 2023-06-06 | 安徽济生元药业有限公司 | Preparation method of codonopsis pilosula nano-selenium capsule |
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