CN106589157A

CN106589157A - Selenium modification method for improving immunological competence of radix pseudostellariae polysaccharides

Info

Publication number: CN106589157A
Application number: CN201611189833.2A
Authority: CN
Inventors: 秦韬; 黄帆; 黄一帆; 李健; 马玉芳; 黄小红; 俞道进; 任喆
Original assignee: Fujian Agriculture and Forestry University
Current assignee: Fujian Agriculture and Forestry University
Priority date: 2016-12-21
Filing date: 2016-12-21
Publication date: 2017-04-26

Abstract

The invention provides a selenium modification method for improving the immunological competence of radix pseudostellariae polysaccharides. The selenium modification method comprises the following steps: taking the radix pseudostellariae polysaccharides, reacting with Na2SeO3 under certain conditions, cooling to a room temperature after the end of reaction, adjusting a pH value to 5-6 with anhydrous sodium carbonate, centrifuging, filtering and putting a solution into a 1000-D dialysis bag for flowing water dialysis; taking a dialysate sample once every 6 hours, detecting whether the dialysate sample contains sodium selenite or not by using an ascorbic acid method, and stopping dialysis when no sodium selenite residue exists or the red is not displayed; and performing Sephadex G-100 column chromatography on the solution to obtain the radix pseudostellariae polysaccharides. When nitric acid is used for reaction, the cleavage of a polysaccharide chain can be caused, so that oligosaccharides are formed, and the low yield of selenium-containing polysaccharides is caused. By using glacial acetic acid, the cleavage of the polysaccharide chain is difficultly caused, so that the yield of selenium-containing polysaccharides is improved.

Description

One kind improves the immunocompetent selenizing method of modifying of Pseudostellaria Polysaccharide

Technical field

The invention belongs to veterinary field, and in particular to one kind improves the immunocompetent selenizing modification side of Pseudostellaria Polysaccharide Method.

Background technology

Radix Pseudostellariae is《Chinese Pharmacopoeia》The conventional Chinese medicine for recording, the effects such as with replenishing QI to invigorate the spleen, promoting the production of body fluid for nourishing the lung, is clinically used for The symptoms such as insufficiency of the spleen body is tired, inappetence, eak after being ill.Pharmacological testing shows that Pseudostellaria Polysaccharide has enhancing human body immunity function Effect, Pseudostellaria Polysaccharide is neutral glucan polymers.

Selenium is the necessary element of vital movement, mankind's cancer-resisting, slow down aging, improve immunity of organisms, prevention and The treatment aspect such as cardiovascular disease and Keshan disease is all played an important role.The existence form of selenium has inorganic selenium and organic selenium two Kind, comparatively organic selenium toxicity is low, Small side effects, can preferably beam back the physiologically active of selenium.

The method of the polysaccharide of selenizing at present includes nitric acid-sodium selenite method, and the method has three：1. bag filter is used Some polysaccharide are had during dialysis to flow out, and cause the extraction ratio of selenizing polysaccharide not high.Although 2. using the barium chloride can as catalyst To improve the preparation speed of selenizing polysaccharide, but while the residual of barium chloride can be caused, make selenizing polysaccharide impure.3. check selenizing to repair The success or not of decorations generally uses Hydride Generation-atomic Fluorescence Spectrometry, infrared spectrometry, but to the molecule before and after its selenizing Amount is unclear.

The content of the invention

It is an object of the invention to provide a kind of improve the immunocompetent selenizing method of modifying of Pseudostellaria Polysaccharide.

For achieving the above object, the present invention is adopted the following technical scheme that：

One kind improves the immunocompetent selenizing method of modifying of Pseudostellaria Polysaccharide, and described method includes as follows：

（1）The mg of Pseudostellaria Polysaccharide 500 is taken, in being placed in 50 mL water, after dissolving under magnetic stirring, is put respectively in three-necked bottle, delayed The glacial acetic acid and the g of magnesium ferrite 0.6 of the slow mL of Deca 2；Addition Na₂SeO₃200-400mg, at 50-90 DEG C 6-10h is reacted；

（2）Reaction is cooled to room temperature after terminating, and with natrium carbonicum calcinatum pH value 5～6 is adjusted, and centrifugation is filtered, and solution loads 1000 D Bag filter flowing water is dialysed；A dialysate samples were taken per 6 hours, is detected whether containing sodium selenite with Vitamin C acid system, to without Asia Sodium selenate stops dialysis when remaining or do not show redness；

（3）Solution Jing Sephadex G-100 column chromatographies, set flow velocity as 12 mLh^-1, collect eluent；Eluent Jing subtracts Pressure concentration, lyophilization；Obtain selenizing Pseudostellaria Polysaccharide；

Determined the Se content of selenizing Pseudostellaria Polysaccharide, divided with Hydride Generation-atomic Fluorescence Spectrometry, infrared spectrometer, liquid matter Son amount etc..Determine the immune-enhancing activity of selenizing Pseudostellaria Polysaccharide：Promote macrophage centering including selenizing Pseudostellaria Polysaccharide Red phagocytic activity, NO yields and IL-6, TNF-α secretion.

Table

It is an advantage of the current invention that：

Compared with prior art, 1. carrying out reaction with nitric acid can cause polysaccharide chain break, form oligosaccharide, cause the product of selenium polysaccharide Rate is not high.By the present invention in that being difficult to make sugar chain rupture with glacial acetic acid, the yield of selenizing polysaccharide is improve.2. made using barium chloride Although the preparation speed of selenizing polysaccharide can be improved for catalyst, while the residual of barium chloride can be caused, selenizing polysaccharide is made not It is pure.By adding magnesium ferrite, one side magnesium ferrite is a kind of solid catalyst to the present invention, is not readily dissolved in water, it is ensured that selenizing is more The purity of sugar；Another aspect magnesium ferrite is more beneficial for forming five-membered ring monohydrate selenium dioxide ester.3. inevitably there be selenium polysaccharide after reacting The presence of some oligosaccharide, so by Sephadex G-100 column chromatographies, further purification selenium polysaccharide.

Description of the drawings

Fig. 1 selenizings Pseudostellaria Polysaccharide and Pseudostellaria Polysaccharide to mouse macrophage TNF-α and IL-6 secretory volumes, in pillar A-e difference letter representation significant differences (P<0.05)。

Specific embodiment

Embodiment 1

（1）9 parts of 500 mg of Pseudostellaria Polysaccharide is taken, in being placed in 50 mL water, after dissolving under magnetic stirring, three-necked bottle is put respectively In, it is slowly added dropwise the glacial acetic acid and the g of magnesium ferrite 0.6 of 2 mL；Addition Na₂SeO₃200-400mg, at 50-90 DEG C 6- is reacted 10h；

（2）Reaction is cooled to room temperature after terminating, and with natrium carbonicum calcinatum pH value 6 is adjusted, and centrifugation is filtered, and it is saturating that solution loads 1000 D The flowing water dialysis of analysis bag；A dialysate samples were taken per 6 hours, is detected whether containing sodium selenite with Vitamin C acid system, to without sub- selenium Sour sodium stops dialysis when remaining or do not show redness；

（3）Solution Jing Sephadex G-100 column chromatographies, set flow velocity as 12 mLh^-1, collect eluent；Eluent Jing subtracts Pressure concentration, lyophilization；9 kinds of selenizing Pseudostellaria Polysaccharides are respectively obtained, sRPP is labeled as successively₁-sRPP₉；

After using the method, the yield of selenium polysaccharide is improve.With selenium polysaccharide yield obtained in traditional method typically in 15%- 30%, using selenium polysaccharide yield after the method in 35%-67%.

Embodiment 2

Mice free choice feeding and drinking-water, temperature control is shone at 22 ± 1 DEG C using 12 h circulation lights.Mice is randomly divided into 11 groups, It is divided into healthy control group, administration group, and cyclophosphamide (CY) model group, 20 per group, all Animal adaptabilities raise 7 d. Healthy control group and cyclophosphamide (CY) model group subcutaneous injection normal saline, continue 14 d.Administration group sRPP₂, sRPP₃, RPP Respectively it is divided into 3 groups of three concentration（1 mg/mL, 2 mg/mL, 3 mg/mL）, totally 9 groups, the mL of subcutaneous administrations 0.2 continuously gives The d of medicine 14.15th day starts, except healthy control group, 9 administration groups and cyclophosphamide (CY) model group lumbar injection ring phosphinylidyne Amine, 80 mg/kgd, 1 day 1 time, continuous injection 3 days.

24 h after last time injection CY, pluck eyeball and take blood, separate serum, enter according to the description of ELISA kit The strict operation of row, determines TNF-α in serum, the measure of IL-6 contents.

TNF-α, IL-6 assay results

The TNF-α of each group, the OD values of IL-6 are shown in Fig. 1.The TNF-α of healthy control group and the OD values of IL-6 are noticeably greater than CY models Group (P<0.05)；The TNF-α of tri- dosage groups of RPP be noticeably greater than CY model group (P<0.05), in 0.2 mg and 0.4 mg agent IL-6 during amount group be noticeably greater than CY model group (P<0.05)。 sPRR₂And sPRR₃The TNF-α and IL-6 of three dosage groups shows Write more than CY model group (P<0.05)。

sPRR₂In 0.2 mg and 0.6 mg dosage groups, sPRR₃In three dosage groups, TNF-α be noticeably greater than RPP groups (P <0.05)；sPRR₃In three dosage groups, TNF-α is noticeably greater than sRPP₂Group (P<0.05)。

sPRR₂And sPRR₃In 0.2 mg and 0.4 mg dosage groups, IL-6 be noticeably greater than RPP groups (P<0.05)；sPRR₂With sPRR₃The content difference of IL-6 is not notable in each dosage group（P>0.05）.

The foregoing is only presently preferred embodiments of the present invention, all impartial changes done according to scope of the present invention patent with Modification, should all belong to the covering scope of the present invention.

Claims

It is 1. a kind of to improve the immunocompetent selenizing method of modifying of Pseudostellaria Polysaccharide, it is characterised in that：Described method includes as follows：

（1）The mg of Pseudostellaria Polysaccharide 500 is taken, in being placed in 50 mL water, after dissolving under magnetic stirring, is put respectively in three-necked bottle, delayed The glacial acetic acid and the g of magnesium ferrite 0.6 of the slow mL of Deca 2；Addition Na₂SeO₃200-400mg, at 50-90 DEG C 6-10h is reacted；

（2）Reaction is cooled to room temperature after terminating, and with natrium carbonicum calcinatum pH value 5～6 is adjusted, and centrifugation is filtered, and solution loads 1000 D Bag filter flowing water is dialysed；A dialysate samples were taken per 6 hours, is detected whether containing sodium selenite with Vitamin C acid system, to without Asia Sodium selenate stops dialysis when remaining or do not show redness；

（3）Solution Jing Sephadex G-100 column chromatographies, set flow velocity as 12 mLh^-1, collect eluent；Eluent Jing reduces pressure Concentration, lyophilization；Obtain selenizing Pseudostellaria Polysaccharide.