CN104788586B - Chondroitin sulfate strontium and preparation method thereof - Google Patents
Chondroitin sulfate strontium and preparation method thereof Download PDFInfo
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Abstract
The present inventor is related to a kind of chondroitin sulfate strontium and preparation method thereof.The molecular formula of the chondroitin sulfate strontium is：H2O(C14H19NSrO14S)x, wherein, x is 60~100 integer.The problem of above-mentioned chondroitin sulfate strontium can not only avoid causing blood vessel and the calcium at capillary position to stockpile and cause petechiae and hemotoncus, additionally it is possible to preferable auxiliary treatment osteoporosis and osteoarthritis.
Description
Technical field
The invention belongs to biological technical field, more particularly to a kind of chondroitin sulfate strontium and preparation method thereof.
Background technology
Chondroitin sulfate is a kind of glutinous polysaccharose substance extracted from animal cartilage, mainly by D-Glucose aldehydic acid and N-
Acetyl-D-amino galactolipin is formed.It has protective effect to bone, can prevent bone and is hardened to become fragile, prevent further calcium
Hardening, bone is kept certain pliability, contribute to the recovery of osteoproliferation, in Bone Defect Repari, the preventing and treating etc. of arthropathy
Have the function that important.
The method of tradition extraction chondroitin sulfate mainly has alkali carries enzymatic isolation method, alkaline extraction, ultrasonic assistant method and acetic acid to carry
Follow the example of, the chondroitin sulfate that these conventional methods are prepared mainly exists in the form of sodium chondroitin sulfate, and chondroitin sulfate
Element is better than sodium to the affinity of calcium, after use, easily causes blood vessel and the calcium at capillary position to stockpile and cause petechiae and blood
It is swollen, effect is poor to be prevented to osteoporosis and osteoarthritis.
The content of the invention
In consideration of it, being necessary to provide a kind of chondroitin sulfate strontium, the chondroitin sulfate strontium can avoid blood vessel and blood capillary
Petechiae and the problem of hemotoncus caused by the calcium of pipe portion position stockpiles, and being capable of preferably auxiliary treatment osteoporosis and Bones and joints
It is scorching.
In addition, also provide a kind of preparation method of chondroitin sulfate strontium.
A kind of chondroitin sulfate strontium, the molecular formula of the chondroitin sulfate strontium is H2O(C14H19NSrO14S)x, wherein, x is
60~100 integer.
A kind of preparation method of chondroitin sulfate strontium, comprises the following steps：
The aqueous solution of sodium chondroitin sulfate is crossed into cation exchange resin column, when the pH value of efflux is changed into 4.5 from neutrality
~5 start to collect efflux, until the pH value of efflux is changed into 6.5~7, stop collecting；
The pH value for the efflux collected is adjusted to 6~6.5, then adds soluble strontium salt, stirring and dissolving 20 minutes
~60 minutes, through standing reaction 3 hours~5 hours, reaction solution is obtained, wherein, the water-soluble strontium salt and the chondroitin sulfate
The mass ratio of plain sodium is 0.4:1~0.8:1；And
The aqueous solution that the ethanol that weight/mass percentage composition is 90%~95% is added in the reaction solution obtains mixed liquor, and
The weight/mass percentage composition of ethanol in the mixed liquor is 60%~65%, and the mixed liquor is stood, then filtered, is divided
Minor is H2O(C14H19NSrO14S)xChondroitin sulfate strontium, wherein, x be 60~100 integer.
In one of the embodiments, the cationic ion-exchange resin in the cation exchange resin column and the sulfuric acid are soft
The mass ratio of ossein sodium is 3:1~4:1.
In one of the embodiments, in the aqueous solution of the sodium chondroitin sulfate, the quality of the sodium chondroitin sulfate
Percentage composition is 5%~10%.
In one of the embodiments, the water-soluble strontium salt is strontium chloride or Strontium Ranelate.
In one of the embodiments, the time that the mixed liquor is stood is 12 hours~15 hours.
In one of the embodiments, in addition to suction filtration the step that the suction filtration thing obtained is cleaned and dried, it is described
Cleaning and the step dried are：The suction filtration thing is cleaned using absolute ethyl alcohol, is then dried in vacuo 2 in 60 DEG C~80 DEG C
Hour~4 hours.
In one of the embodiments, the preparation process of the sodium chondroitin sulfate is included：
It will be crushed after cartilage degreasing, obtain cartilage powder；
The cartilage powder is mixed with deionized water, then adds basifier, is stirred in 30 DEG C~40 DEG C to described soft
Bone powder dissolves, and obtains Cartilage solution, wherein, the mass ratio of the basifier and the cartilage is 0.01:1~0.5:1, the alkali
Agent is sodium hydroxide or sodium acid carbonate；
The pH value of the Cartilage solution is adjusted to 8~9, pancreatin is added, is digested 4 hours~5 hours in 40 DEG C~50 DEG C, then
It is warming up to 80 DEG C~100 DEG C insulation reactions 2 hours~4 hours, it is filtered, obtain settled solution, the pancreatin and the cartilage
Mass ratio be 0.0045:1~0.0055:1；And
The pH value of the settled solution is adjusted to 6~6.5, absolute ethyl alcohol is added and obtains mixture, and in the mixture
Ethanol weight/mass percentage composition be 70%~75%, then filter, obtain sodium chondroitin sulfate.
In one of the embodiments, in the step of cartilage powder being mixed with the deionized water, it is described go from
The quality of sub- water is 5 times~30 times of the quality of cartilage.
In one of the embodiments, it is by the step of cartilage degreasing：By the cartilage 80 in deionized water
DEG C~100 DEG C of boilings 2 hours~3 hours, after removing the grease, in deionized water 80 DEG C~100 DEG C boilings 2 hours~
4 hours, dried after cooling.
Above-mentioned chondroitin sulfate strontium is a kind of new chondroitin sulfate, and the strontium in the chondroitin sulfate strontium can pass through calcium channel
Into cell, combined in the cell with relevant calcium binding site, have and promote skeleton development and osteoid to form and adjust bone
The multiple action of metabolism, and strontium has the function that to promote Oesteoblast growth and suppresses osteoclast formation, on a cellular level,
Strontium can be reduced bone reabsorption by osteoclast and by Gegenbaur's cell promoting bone growing, and chondroitin sulfate is to the parent of strontium and calcium
It is suitable with ability, can avoid using it is traditional in the form of sodium chondroitin sulfate existing for chondroitin sulfate easily cause blood vessel
And the calcium at capillary position stockpiles, and the problem of cause petechiae and hemotoncus, additionally it is possible to preferable auxiliary treatment osteoporosis
And osteoarthritis.
Brief description of the drawings
Fig. 1 is the flow chart of the preparation method of the chondroitin sulfate strontium of an embodiment；
Fig. 2 represent be embodiment 1 chondroitin sulfate strontium concentration be respectively 0mmol/L, 1mmol/L, 3mmol/L and
The relative expression quantity of interleukin (IL-1 β) mRNA during 5mmol/L；
Fig. 3 represent be embodiment 1 chondroitin sulfate strontium concentration be respectively 0mmol/L, 1mmol/L, 3mmol/L and
The relative expression quantity of collagen (CollagenI) mRNA during 5mmol/L；
Fig. 4 represent be embodiment 1 chondroitin sulfate strontium concentration be respectively 0mmol/L, 1mmol/L, 3mmol/L and
The relative expression quantity of memebrane protein (RANKL) mRNA during 5mmol/L；
When what Fig. 5 was represented is that the concentration of sodium chondroitin sulfate is respectively 0mmol/L, 1mmol/L, 3mmol/L and 5mmol/L
Interleukin (IL-1 β) mRNA relative expression quantity；
When what Fig. 6 was represented is that the concentration of sodium chondroitin sulfate is respectively 0mmol/L, 1mmol/L, 3mmol/L and 5mmol/L
Collagen (CollagenI) mRNA relative expression quantity；
When what Fig. 7 was represented is that the concentration of sodium chondroitin sulfate is respectively 0mmol/L, 1mmol/L, 3mmol/L and 5mmol/L
Memebrane protein (RANKL) mRNA relative expression quantity.
Embodiment
Chondroitin sulfate strontium and preparation method thereof is made mainly in combination with drawings and the specific embodiments below further details of
Explanation.
The chondroitin sulfate strontium of one embodiment, its molecular formula are H2O(C14H19NSrO14S)x, wherein, x is 60~100
Integer.
The chondroitin sulfate strontium can apply to Bone Defect Repari, arthropathy prevents etc..
Above-mentioned chondroitin sulfate strontium is a kind of new chondroitin sulfate, and the strontium in the chondroitin sulfate strontium can pass through calcium channel
Into cell, combined in the cell with relevant calcium binding site, have and promote skeleton development and osteoid to form and adjust bone
The multiple action of metabolism, and chondroitin sulfate is suitable to the affinity of strontium and calcium, avoids using traditional with chondroitin sulfate
Chondroitin sulfate existing for the form of plain sodium easily causes blood vessel and the calcium at capillary position to stockpile, and causes petechiae and hemotoncus
Problem.
As shown in figure 1, the preparation method of the chondroitin sulfate strontium of an embodiment, available for preparing above-mentioned chondroitin sulfate
Strontium, the preparation method comprise the following steps：
Step S110：The aqueous solution of sodium chondroitin sulfate is crossed into cation exchange resin column, when efflux pH value therefrom
Property be changed into 4.5~5 and start to collect efflux, until the pH value of efflux is changed into 6.5~7, stop collecting.
Wherein, sodium chondroitin sulfate can be bought by market；Can also be by voluntarily preparing.
Wherein, the preparation process of sodium chondroitin sulfate is as follows：It will be crushed after cartilage degreasing, obtain cartilage powder；Will be soft
Bone powder mixes with deionized water, then adds basifier, is dissolved in 30 DEG C~40 DEG C stirrings to cartilage powder, it is molten to obtain cartilage
Liquid；Wherein, the mass ratio of basifier and cartilage is 0.01:1~0.5:1；Basifier is sodium hydroxide or sodium acid carbonate；Adjust soft
The pH value of bone solution adds pancreatin, digested 4 hours~5 hours in 40 DEG C~50 DEG C, then be warming up to 80 DEG C~100 DEG C to 8~9
Insulation reaction 2 hours~4 hours, it is filtered, obtain settled solution, the mass ratio of pancreatin and cartilage is 0.0045:1~
0.0055:1；The pH value of settled solution is adjusted to 6~6.5, absolute ethyl alcohol is added and obtains mixture, and the ethanol in mixture
Weight/mass percentage composition is 70%~75%, then filters, obtains sodium chondroitin sulfate.
Wherein, the pH value of Cartilage solution is adjusted into the step of 8~9, the use of reagent is the hydrochloric acid that concentration is 3~4mol/L
The aqueous solution.
Wherein, in the step of cartilage powder being mixed with deionized water, the quality of deionized water is 5 times of the quality of cartilage
~30 times.
Wherein, the pH value of settled solution is adjusted into the step of 6~6.5, and concentration is the water of 3~4mol/L hydrochloric acid
The pH value of solution regulation settled solution is to 6~6.5.
Wherein, it is by the step of cartilage degreasing：By cartilage, 80 DEG C~100 DEG C boilings 2 hours~3 are small in deionized water
When, after removing grease, 80 DEG C~100 DEG C boilings 2 hours~4 hours in deionized water, dried after cooling.To ensure cartilage
In grease be removed as far as possible.
In step S110, in the aqueous solution of sodium chondroitin sulfate, the weight/mass percentage composition of sodium chondroitin sulfate for 5%~
10%.
In step S110, the mass ratio of cationic ion-exchange resin and sodium chondroitin sulfate in cation exchange resin column is
3:1~4:1.So that sodium is by complete exchange in sodium chondroitin sulfate.
Wherein, cationic ion-exchange resin is with styrene and divinylbenzene polymerization, through polymer prepared by sulfuric acid sulfonation.
Cationic ion-exchange resin meets water and can be exchanged with each other certain electron ion in itself a certain active ion and water, that is, sends out
Raw displacement reaction, removes soluble ion in water removal.
Step S120：The pH value for the efflux collected is adjusted to 6~6.5, then adds water-soluble strontium salt, stirring and dissolving 20
Minute~60 minutes, through standing reaction 3 hours~5 hours, obtain reaction solution.Wherein, water-soluble strontium salt and sodium chondroitin sulfate
Mass ratio be 0.4:1~0.8:1.
Wherein, in step S120, the pH value of efflux of collection is adjusted to 6~6.5, the reagent used is that quality percentage contains
Measure the aqueous solution of the strontium hydroxide for 10%~20%.The pH value for the efflux collected using the aqueous solution regulation of strontium hydroxide can
The purity of product is influenceed to avoid introducing extraneous ion in preparation process.
Wherein, water-soluble strontium salt is strontium chloride or Strontium Ranelate.Strontium is as the important trace element in skeleton composition
One of, cell can be entered by calcium channel, be combined in the cell with relevant calcium binding site, had and promote skeleton development and class
The formation of sclerotin and the multiple action for adjusting Bone m etabolism.
Step S130：The aqueous solution for the ethanol that weight/mass percentage composition is 90%~95% is added in reaction solution, is mixed
Liquid is closed, and the weight/mass percentage composition of the ethanol in mixed liquor is 60%~65%, and mixed liquor is stood, then filtered, is divided
Minor is H2O(C14H19NSrO14S)xChondroitin sulfate strontium, wherein, x be 60~100 integer.
By making the weight/mass percentage composition of the ethanol in mixed liquor be 60%~65%, so that chondroitin sulfate strontium precipitates.
Wherein, in step S130, the time that mixed liquor is stood is 12 hours~15 hours.
Wherein, in addition to the step S130 suction filtration things for filtering to obtain the step cleaned and dried, clean and dry
The step of be：Cleaned to filtering thing using absolute ethyl alcohol, be then dried in vacuo 2 hours~4 hours in 60 DEG C~80 DEG C.Tool
Body, cleaned 3 times using absolute ethyl alcohol to filtering thing.
The preparation method of above-mentioned chondroitin sulfate strontium is simple, is raw material by using sodium chondroitin sulfate, and by using
Above-mentioned steps, chondroitin sulfate strontium can be prepared, be a kind of new chondroitin sulfate；Strontium energy in the chondroitin sulfate strontium
Cell is entered by calcium channel, combined in the cell with relevant calcium binding site, has and promotes skeleton development and osteoid shape
Into the multiple action with regulation Bone m etabolism, and strontium has the function that to promote Oesteoblast growth and suppresses osteoclast formation,
On cellular level, strontium can be reduced bone reabsorption by osteoclast and by Gegenbaur's cell promoting bone growing, and chondroitin sulfate
It is suitable to the affinity of strontium and calcium so that upper chondroitin sulfate strontium not only avoid using traditional with sodium chondroitin sulfate
Chondroitin sulfate existing for form easily causes blood vessel and the calcium at capillary position to stockpile, and the problem of cause petechiae and hemotoncus,
And being capable of preferably auxiliary treatment arthritis and osteoporosis etc..And the preparation method of above-mentioned chondroitin sulfate strontium is prepared
The high purity more than 95% of chondroitin sulfate strontium.And the raw material used in the preparation method of sulfuric acid ossein strontium is cartilage, source
Extensively.
It is specific embodiment part below：
Embodiment 1
The preparation process of the chondroitin sulfate strontium of the present embodiment is as follows：
(1) preparation of sodium chondroitin sulfate：By animal cartilage in deionized water 80 DEG C of boilings 3 hours, after removing grease,
80 DEG C of boilings 4 hours in deionized water, are dried after cooling.
Crushed after cartilage degreasing, obtain cartilage powder, cartilage powder is placed in retort, add going for 5 times of cartilage
Ionized water mixes, and is then 0.01 according to the mass ratio of sodium hydroxide and cartilage:1, sodium hydroxide is added, is stirred in 40 DEG C to soft
Bone powder dissolves, and obtains Cartilage solution；Concentration be 3mol/L hydrochloric acid the aqueous solution adjust Cartilage solution pH value to 8, press
It is 0.0045 according to the mass ratio of pancreatin and cartilage:1, pancreatin is added, is digested 4 hours in 40 DEG C, then be warming up to 80 DEG C of insulation reactions 4
Hour, it is filtered, obtain settled solution；Concentration is that the aqueous solution of 3mol/L hydrochloric acid adjusts the pH value of settled solution to 6,
Add absolute ethyl alcohol and obtain mixture, and the weight/mass percentage composition of the ethanol in mixture is 70%, then filters, obtains sulfuric acid
Chondroitin sodium.
(2) aqueous solution for the sodium chondroitin sulfate that weight/mass percentage composition is 8% is crossed into cation exchange resin column, works as outflow
The pH value of liquid collects efflux since neutrality is changed into 4.5, until the pH value of efflux is changed into 7, stops collecting.Wherein, sun from
The mass ratio of cationic ion-exchange resin and sodium chondroitin sulfate in sub-exchange resin post is 3:1.
(3) use quality percentage composition be 10% strontium hydroxide the pH value of efflux collected of aqueous solution regulation to 6,
Then strontium chloride is added, stirring and dissolving 20 minutes, through standing reaction 3 hours, obtains reaction solution, wherein, strontium chloride is soft with sulfuric acid
The mass ratio of ossein sodium is 0.4:1.
(4) aqueous solution that the ethanol that weight/mass percentage composition is 90% is added in reaction solution obtains mixed liquor, and mixed liquor
In ethanol weight/mass percentage composition be 60%, by mixed liquor stand 12 hours, then filter, using absolute ethyl alcohol to filter thing
Cleaning 3 times, then it is dried in vacuo 4 hours in 60 DEG C, it is H to obtain molecular formula2O(C14H19NSrO14S)60Chondroitin sulfate strontium.
The purity of the chondroitin sulfate strontium of the present embodiment is 95%.
Embodiment 2
The preparation process of the chondroitin sulfate strontium of the present embodiment is as follows：
(1) preparation of sodium chondroitin sulfate：By animal cartilage in deionized water 100 DEG C of boilings 2 hours, remove grease
Afterwards, 100 DEG C of boilings 2 hours in deionized water, are dried after cooling.
Crushed after cartilage degreasing, obtain cartilage powder, cartilage powder is placed in retort, add going for 30 times of cartilage
Ionized water mixes, and is then 0.5 according to the mass ratio of sodium acid carbonate and cartilage:1, sodium acid carbonate is added, is stirred in 30 DEG C to soft
Bone powder dissolves, and obtains Cartilage solution；Concentration be 4mol/L hydrochloric acid the aqueous solution adjust Cartilage solution pH value to 9, press
It is 0.0055 according to the mass ratio of pancreatin and cartilage:1, pancreatin is added, is digested 5 hours in 50 DEG C, then be warming up to 100 DEG C of insulation reactions
It is 2 hours, filtered, obtain settled solution；Concentration be 4mol/L hydrochloric acid the aqueous solution adjust settled solution pH value to
6.5, add absolute ethyl alcohol and obtain mixture, and the weight/mass percentage composition of the ethanol in mixture is 75%, then filters, obtains
Sodium chondroitin sulfate.
(2) aqueous solution for the sodium chondroitin sulfate that weight/mass percentage composition is 5% is crossed into cation exchange resin column, works as outflow
The pH value of liquid collects efflux since neutrality is changed into 5, until the pH value of efflux is changed into 6.5, stops collecting.Wherein, sun from
The mass ratio of cationic ion-exchange resin and sodium chondroitin sulfate in sub-exchange resin post is 4:1.
(3) use quality percentage composition be 20% strontium hydroxide the aqueous solution regulation collect efflux pH value to
6.5, strontium chloride is then added, stirring and dissolving 60 minutes, through standing reaction 5 hours, obtains reaction solution, wherein, strontium chloride and sulphur
The mass ratio of aching and limp ossein sodium is 0.8:1.
(4) aqueous solution that the ethanol that weight/mass percentage composition is 95% is added in reaction solution obtains mixed liquor, and mixed liquor
In ethanol weight/mass percentage composition be 65%, by mixed liquor stand 15 hours, then filter, using absolute ethyl alcohol to filter thing
Cleaning 3 times, then it is dried in vacuo 2 hours in 80 DEG C, it is H to obtain molecular formula2O(C14H19NSrO14S)80Chondroitin sulfate strontium.
The purity of the chondroitin sulfate strontium of the present embodiment is 98%.
Embodiment 3
The preparation process of the chondroitin sulfate strontium of the present embodiment is as follows：
(1) preparation of sodium chondroitin sulfate：By animal cartilage in deionized water 90 DEG C of boilings 2.5 hours, remove grease
Afterwards, 90 DEG C of boilings 3.5 hours in deionized water, are dried after cooling.
Crushed after cartilage degreasing, obtain cartilage powder, cartilage powder is placed in retort, add going for 20 times of cartilage
Ionized water mixes, and is then 0.2 according to the mass ratio of sodium hydroxide and cartilage:1, sodium hydroxide is added, is stirred in 35 DEG C to soft
Bone powder dissolves, and obtains Cartilage solution；Concentration be 4mol/L hydrochloric acid the aqueous solution adjust Cartilage solution pH value to 8, press
It is 0.0050 according to the mass ratio of pancreatin and cartilage:1, pancreatin is added, is digested 4 hours in 50 DEG C, then be warming up to 90 DEG C of insulation reactions 3
Hour, it is filtered, obtain settled solution；Concentration is that the aqueous solution of 4mol/L hydrochloric acid adjusts the pH value of settled solution to 6,
Add absolute ethyl alcohol and obtain mixture, and the weight/mass percentage composition of the ethanol in mixture is 72%, then filters, obtains sulfuric acid
Chondroitin sodium.
(2) aqueous solution for the sodium chondroitin sulfate that weight/mass percentage composition is 10% is crossed into cation exchange resin column, works as stream
The pH value for going out liquid collects efflux since neutrality is changed into 4.5, until the pH value of efflux is changed into 6.5, stops collecting.Wherein,
The mass ratio of cationic ion-exchange resin and sodium chondroitin sulfate in cation exchange resin column is 3.5:1.
(3) use quality percentage composition be 15% strontium hydroxide the pH value of efflux collected of aqueous solution regulation to 6,
Then Strontium Ranelate is added, stirring and dissolving 30 minutes, through standing reaction 4 hours, obtains reaction solution, wherein, Strontium Ranelate and sulphur
The mass ratio of aching and limp ossein sodium is 0.6:1.
(4) aqueous solution that the ethanol that weight/mass percentage composition is 95% is added in reaction solution obtains mixed liquor, and mixed liquor
In ethanol weight/mass percentage composition be 60%, by mixed liquor stand 14 hours, then filter, using absolute ethyl alcohol to filter thing
Cleaning 3 times, then it is dried in vacuo 3 hours in 70 DEG C, it is H to obtain molecular formula2O(C14H19NSrO14S)100Chondroitin sulfate strontium.
The purity of the chondroitin sulfate strontium of the present embodiment is 96%.
Embodiment 4
The preparation process of the chondroitin sulfate strontium of the present embodiment is as follows：
(1) preparation of sodium chondroitin sulfate：By animal cartilage in deionized water 80 DEG C of boilings 3 hours, after removing grease,
100 DEG C of boilings 2 hours in deionized water, are dried after cooling.
Crushed after cartilage degreasing, obtain cartilage powder, cartilage powder is placed in retort, add going for 5 times of cartilage
Ionized water mixes, and is then 0.4 according to the mass ratio of sodium acid carbonate and cartilage:1, sodium acid carbonate is added, is stirred in 40 DEG C to soft
Bone powder dissolves, and obtains Cartilage solution；The pH value of the aqueous solution regulation Cartilage solution for the hydrochloric acid that concentration is 3mol/L is 9, is pressed
It is 0.0050 according to the mass ratio of pancreatin and cartilage:1, pancreatin is added, is digested 4 hours in 40 DEG C, then be warming up to 80 DEG C of insulation reactions 4
Hour, it is filtered, obtain settled solution；Concentration is that the aqueous solution of 3mol/L hydrochloric acid adjusts the pH value of settled solution to 6,
Add absolute ethyl alcohol and obtain mixture, and the weight/mass percentage composition of the ethanol in mixture is 75%, then filters, obtains sulfuric acid
Chondroitin sodium.
(2) aqueous solution for the sodium chondroitin sulfate that weight/mass percentage composition is 6% is crossed into cation exchange resin column, works as outflow
The pH value of liquid collects efflux since neutrality is changed into 4.8, until the pH value of efflux is changed into 6.8, stops collecting.Wherein, it is positive
The mass ratio of cationic ion-exchange resin and sodium chondroitin sulfate in ion exchange resin column is 3:1.
(3) use quality percentage composition be 10% strontium hydroxide the aqueous solution regulation collect efflux pH value to
6.5, Strontium Ranelate is then added, stirring and dissolving 50 minutes, through standing reaction 2.5 hours, obtains reaction solution, wherein, thunder Buddhist nun acid
The mass ratio of strontium and sodium chondroitin sulfate is 0.7:1.
(4) aqueous solution that the ethanol that weight/mass percentage composition is 95% is added in reaction solution obtains mixed liquor, and mixed liquor
In ethanol weight/mass percentage composition be 65%, by mixed liquor stand 13 hours, then filter, using absolute ethyl alcohol to filter thing
Cleaning 3 times, then it is dried in vacuo 4 hours in 60 DEG C, it is H to obtain molecular formula2O(C14H19NSrO14S)90Chondroitin sulfate strontium.
The purity of the chondroitin sulfate strontium of the present embodiment is 95%.
As a result detect：
Sodium chondroitin sulfate and the chondroitin sulfate strontium of embodiment 1 are subjected to mRNA detection of expression.
Osteoarthritis is the chronic joint disease that articular cartilage degeneration is characterized, and Osteoarthritis cartilage cell epimatrix closes
Into with to degrade unbalance be one of major reason for causing cartilage degeneration.Wherein, matrix metalloproteinase is to cartilage cell epimatrix
There is degradation, make arthroncus, external force resistance effect declines, and ultimately results in articular cartilage damage.And due to interleukin (IL-1 β)
Can inducer substance metalloprotein expression of enzymes, and promote its activation, so as to cause cartilage matrix to be degraded.Memebrane protein (RANKL) is
Memebrane protein on Gegenbaur's cell film, osteoclast can be activated, prevents osteoclast apoptosis.And collagen (CollagenI)
It is the important component of articular cartilage.Therefore, by detecting interleukin (IL-1 β), collagen (CollagenI) and film egg
The mRNA of (RANKL) relative expression quantity obtains the effect of sodium chondroitin sulfate and chondroitin sulfate strontium in vain.
MRNA relative expression quantities are detected using quantitative real time PCR Instrument (ABI, Vii A7), detect the reagent used
For RNAiso Plus (TaKaRa, D9108A) and SYBR Green dye method mRNA detection kits (Geneup).Specific method
For：Material to be detected (chondroitin sulfate strontium or sodium chondroitin sulfate) is added in the DMEM culture mediums containing 10%FBS respectively
The culture solution for the material to be detected that concentration is 1mmol/L, 3mmol/L and 5mmol/L is configured to, without chondroitin sulfate strontium
Every group in triplicate as a control group for DMEM culture mediums (containing 10%FBS)；After cultivating 48 hours respectively, extracted with Trizol total
RNA, reverse transcription obtain 4 cDNA samples；Then interleukin (IL-1 β), the collagen in 4 cDNA are detected respectively
(CollagenI) and memebrane protein (RANKL) mRNA relative expression quantities.
Fig. 2 represent be embodiment 1 chondroitin sulfate strontium concentration be respectively 0mmol/L, 1mmol/L, 3mmol/L and
The relative expression quantity of interleukin (IL-1 β) mRNA during 5mmol/L；What Fig. 3 was represented is the dense of the chondroitin sulfate strontium of embodiment 1
The relative expression of collagen (CollagenI) mRNA when degree is respectively 0mmol/L, 1mmol/L, 3mmol/L and 5mmol/L
Amount；Fig. 4 represent be embodiment 1 chondroitin sulfate strontium concentration be respectively 0mmol/L, 1mmol/L, 3mmol/L and
The relative expression quantity of memebrane protein (RANKL) mRNA during 5mmol/L.What Fig. 5 was represented is that the concentration of sodium chondroitin sulfate is respectively
The relative expression quantity of interleukin (IL-1 β) mRNA when 0mmol/L, 1mmol/L, 3mmol/L and 5mmol/L；Fig. 6 represent be
The collagen when concentration of sodium chondroitin sulfate is respectively 0mmol/L, 1mmol/L, 3mmol/L and 5mmol/L
(CollagenI) mRNA relative expression quantity；What Fig. 7 was represented is that the concentration of sodium chondroitin sulfate is respectively 0mmol/L, 1mmol/
L, memebrane protein during 3mmol/L and 5mmol/L (RANKL) mRNA relative expression quantity.
From Fig. 2~7 as can be seen that with the increase of chondroitin sulfate strontium or the concentration of sodium chondroitin sulfate, interleukin
(IL-1 β) mRNA relative expression quantity gradually decreases, and collagen (CollagenI) mRNA relative expression quantity gradually increases,
Memebrane protein (RANKL) mRNA relative expression quantity gradually decreases.And compared with the sodium chondroitin sulfate of same concentrations, although sulfuric acid
The relative expression quantity of the interleukin (IL-1 β) of chondroitin strontium is more or less the same, but collagen (CollagenI) mRNA relative table
Significantly increased up to amount, and the relative expression quantity of memebrane protein (RANKL) also considerably reduces, it is clear that the property of chondroitin sulfate strontium
Sodium chondroitin sulfate can be better than, chondroitin sulfate strontium being capable of preferably auxiliary treatment arthritis and osteoporosis.
Embodiment described above only expresses the several embodiments of the present invention, and its description is more specific and detailed, but simultaneously
Therefore the limitation to the scope of the claims of the present invention can not be interpreted as.It should be pointed out that for one of ordinary skill in the art
For, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to the guarantor of the present invention
Protect scope.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.
Claims (1)
1. a kind of preparation method of chondroitin sulfate strontium, it is characterised in that comprise the following steps：
(1) preparation of sodium chondroitin sulfate：By animal cartilage in deionized water 100 DEG C of boilings 2 hours, after removing grease, then
100 DEG C of boilings 2 hours in deionized water, are dried after cooling；
Cartilage crushed after being dried, obtains cartilage powder, and cartilage powder is placed in retort, adds the deionized water of 30 times of cartilage
Mixing, it is then 0.5 according to the mass ratio of sodium acid carbonate and cartilage:1, sodium acid carbonate is added, in 30 DEG C of stirrings to cartilage powder
Dissolving, obtains Cartilage solution；Concentration be 4mol/L hydrochloric acid the aqueous solution adjust Cartilage solution pH value to 9, according to pancreatin
Mass ratio with cartilage is 0.0055:1, pancreatin is added, is digested 5 hours in 50 DEG C, then it is warming up to 100 DEG C of insulation reactions 2 hours,
It is filtered, obtain settled solution；Concentration be 4mol/L hydrochloric acid the aqueous solution adjust settled solution pH value to 6.5, add
Enter absolute ethyl alcohol and obtain mixture, and the weight/mass percentage composition of the ethanol in mixture is 75%, then filters, it is soft to obtain sulfuric acid
Ossein sodium；
(2) aqueous solution for the sodium chondroitin sulfate that weight/mass percentage composition is 5% is crossed into cation exchange resin column, when efflux
PH value collects efflux since neutrality is changed into 5, until the pH value of efflux is changed into 6.5, stops collecting, wherein, cation is handed over
The cationic ion-exchange resin and the mass ratio of sodium chondroitin sulfate changed in resin column is 4:1；
(3) use quality percentage composition be 20% strontium hydroxide the pH value of efflux collected of aqueous solution regulation to 6.5, so
After add strontium chloride, stirring and dissolving 60 minutes, through standing reaction 5 hours, obtain reaction solution, wherein, strontium chloride and chondroitin sulfate
The mass ratio of plain sodium is 0.8:1；
(4) aqueous solution that the ethanol that weight/mass percentage composition is 95% is added in reaction solution obtains mixed liquor, and in mixed liquor
The weight/mass percentage composition of ethanol is 65%, and mixed liquor is stood into 15 hours, then filtered, and is cleaned using absolute ethyl alcohol to filtering thing
3 times, then it is dried in vacuo 2 hours in 80 DEG C, it is H to obtain molecular formula2O(C14H19NSrO14S)80Chondroitin sulfate strontium.
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