CN107179381A - The external gradient degradation method of multiple cross-linked chitosan or derivatives thereof gel - Google Patents
The external gradient degradation method of multiple cross-linked chitosan or derivatives thereof gel Download PDFInfo
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Abstract
The present invention relates to a kind of external gradient degradation method of multiple cross-linked chitosan gel rubber.Multiple cross-linked chitosan or derivatives thereof gel is degraded using free radical, and catabolite quantified using kjeldahl apparatus;Or multiple cross-linked chitosan or derivatives thereof gel is degraded using lysozyme, and use GB/T 5009.7 2016《The measure of reduced sugar in national food safety standard food》In the 3rd method potassium ferricyanide reducing sugar test method catabolite is quantified.Enzyme environment or free radical environment in this method analogue body, multiple cross-linked chitosan gel rubber is quantitatively degraded by enzyme or free radical, by the measure for determining the nitrogen content in catabolite in the content or catabolite of reduced sugar, determine the palliating degradation degree of chitosan gel rubber, so as to obtain the degradation curve of multiple cross-linked chitosan gel rubber, the degradation cycle of chitosan gel rubber is determined.
Description
Technical field
The present invention relates to the external gradient degradation method of multiple cross-linked chitosan or derivatives thereof gel.This method analogue body
Interior enzyme environment or free radical environment, are quantitatively dropped by enzyme or free radical to multiple cross-linked chitosan or derivatives thereof gel
Solution, by determining the measure of the nitrogen content in catabolite in the content or catabolite of reduced sugar, determines the degraded journey of gel
Degree, so as to obtain the degradation curve of multiple cross-linked chitosan or derivatives thereof gel, determines the degradation cycle of gel.
Background technology
Chitosan（Also known as glucosaminoglycan, English name and abbreviation：Chitosan, CS）It is that chitin passes through deacetylated processing
The product obtained afterwards, so being otherwise known as：" chitosan ".Chitosan is led to by 2-acetylamino-2-deoxy-D-glucose monomer
β-l are crossed, the straight-chain high-molecular compound that 4- glycosidic bonds are connected is insoluble in water, is but soluble in acid.Chitosan has good
Biocompatibility and biodegradability, nontoxic, physics and chemical property are stablized relatively, and cohesive is good, into fiber and film forming
Function admirable, is a kind of natural macromolecular material absorbed that can be degraded by organism.Chitosan has very high medical value：
Chitosan can reduce internal cholesterol；Also there is good fungistatic effect, the environment of bacterial growth can be unfavorable for；Chitosan
It can adsorb and drain internal heavy metal；Also can preventing hypertension, the effects such as improving body immunity.Research shows lysozyme
（A kind of content in human body fluid in the higher antibacterial material of content, tear at most, constitutes about human body tear protein content
40%）, Glucosamine can be turned into β-Isosorbide-5-Nitrae-Glucosamine glycosidic bond of catalyzing hydrolysis chitosan, therefore chitosan is in vivo
It can be degraded and absorbed.In addition, internal various active material, such as reactive oxygen species free radical and chitinase, glucolase, egg
More than the 30 kinds of enzyme preparations such as white enzyme, lipase, cellulase, hemicellulase and pectase also promote the katabolism of chitosan.Enzyme
To selectivity of the hydrolysis with height of polysaccharide, and without other side reactions.Therefore, chitosan is as a kind of very safe natural
Macromolecule polysaccharide, has been widely applied to biological medicine, the field such as organizational project.
As the material that can be used in biological medicine and field of tissue engineering technology, the degradation process of research chitosan very must
Will.Currently, for chitosan degradation process and degraded reagent has had many reports.The chitosan being currently known is in vivo
Can be by lysozyme (Lysozyme) or free radical cracking（Deng Qianying etc., chemistry world, 2005,6,338-340）, lysozyme can enter
Acetylglucosamine in chitosan is attacked, Isosorbide-5-Nitrae-glycosidic bond is occurred fracture and is degraded；Free radical directly can promote glycosidic bond to send out
Life is broken and degraded.Although cross-linked chitosan is using chitosan as main body, the Study on degradation for cross-linked chitosan rarely has
Report.Therefore, a kind of biodegrading process for being suitable for cross-linked chitosan is found, determines that the degraded trend of cross-linked chitosan also very must
Will.
The content of the invention
It is an object of the invention to provide a kind of external gradient degradation method of multiple cross-linked chitosan gel rubber.This method mould
Intend internal enzyme environment or free radical environment, multiple cross-linked chitosan gel rubber is quantitatively degraded by enzyme or free radical, passed through
The measure of the nitrogen content in catabolite in the content or catabolite of reduced sugar is determined, the degraded journey of chitosan gel rubber is determined
Degree, so as to obtain the degradation curve of multiple cross-linked chitosan gel rubber, determines the degradation cycle of chitosan gel rubber.
The external gradient degradation method for the multiple cross-linked chitosan gel rubber that the present invention is provided is to be handed over using free radical multiple
Connection chitosan or derivatives thereof gel is degraded, and catabolite is quantified using kjeldahl apparatus;Or use bacteriolyze
Enzyme is degraded to multiple cross-linked chitosan or derivatives thereof gel, and using GB/T 5009.7-2016《Food security country
The measure of reduced sugar in standard food》In the 3rd method potassium ferricyanide reducing sugar test method catabolite is quantified.
The step of external gradient degradation method for the multiple cross-linked chitosan gel rubber that the present invention is provided includes:
1）Configuration concentration is 0.001 ~ 0.1 mol/L, preferably 0.01 ~ 0.05 mol/L ascorbic acid solution, and with 1 ~ 6
Mol/L salt acid for adjusting pH is to 3 ~ 3.5；
2）Take the mL of cross-linked chitosan or derivatives thereof gel 1 ~ 10（Mass concentration scope is 10 ~ 500mg/mL）It is placed in 10 ~ 50
ML centrifuge tubes, add step 1）In the mL of ascorbic acid solution 1 ~ 10, and be cooled to 0 ~ 5 DEG C, the concentration of wherein ascorbic acid is
0.001 ~ 0.1 mol/L, preferably 0.01 ~ 0.05 mol/L；
2）The environment of 0 ~ 5 DEG C of holding, adds the mL of hydrogen peroxide solution 1 ~ 10, wherein hydrogen peroxide concentration must be with resisting with adding volume
The concentration of bad hematic acid is equal with volume, and the concentration of hydrogen peroxide is 0.001 ~ 0.1 mol/L, preferably 0.01 ~ 0.05 mol/L；
3）System is placed in thermostatic control oscillator vibration, concussion temperature sets 37 DEG C, is sampled every 10 ~ 20 min, every time
Take out the μ L of degradation solution 100 ~ 500, while supplement step 1 in the μ L of ascorbic acid solution 100 ~ 500, the total degradation time be 120 ~
1440min；
4）With concentration it is that 1mol/L sodium hydroxides are neutralized to behind pH=7.0 by the degradation solution after taking-up, is sealed in molecular cut off 10
In ~ 14 KDa bag filter, quantitatively dialysed using 5 mL pure water, dialysis time is 2 ~ 4 h；
5）The mL of dialyzate 4.0 is taken, the nitrogen content in dialyzate is detected using kjeldahl apparatus, kjeldahl apparatus, which can select, to be included
But the abundant Europe SKD-2000 full-automatic Kjeldahl determination devices in Shanghai are not limited to, detection process can refer to the kelvin carried during instrument purchase and determine
Requirement in nitrogen instrument operational manual, using the process of instrument not within protection scope of the present invention, the present invention will not be right
The concrete operations flow of instrument（Including parameter）Elaborate；
6）The degradation rate of gel will be calculated according to following three formula：
（Formula 1）
（Formula 2）
（Formula 3）
cN：The measuring samples total nitrogen content detected using kjeldahl apparatus（mg/g）；
m：Measuring samples quality（g）；
F：Moisture in measuring samples（%）；
V1：Sample cell quota of expenditure sulfuric acid solution volume（mL）；
V0：Blank tube quota of expenditure sulfuric acid solution volume（mL）；
c：The concentration of standard sulfuric acid solution（mol/L）；
c1：Carboxymethyl chitosan sugared content（mg/g）；
14：Nitrogen-atoms quality（Da）；
w：The degradation rate of carboxymethyl glucosaminoglycan in product（%）；
The ratio of X test fluid volumes for needed for the cumulative volume and kjeldahl apparatus of sample solution.
The external gradient degradation method for the multiple cross-linked chitosan gel rubber that the present invention is provided comprises the following steps:
1）Take the mL of cross-linked chitosan or derivatives thereof gel 1 ~ 10（Mass concentration scope is 10 ~ 500mg/mL）It is placed in 10 ~ 50
ML centrifuge tubes, add the phosphate buffer that 1 ~ 10 mL concentration is 0.01 ~ 0.05 mol/L（pH 7.0~7.4）, addition 400 ~
2000 u lysozymes, and it is cooled to 0 ~ 5 DEG C；
2）System is placed in thermostatic control oscillator vibration, concussion temperature sets 37 DEG C, is sampled every 1 ~ 2 h, takes out every time
The μ L of degradation solution 100 ~ 500, while supplementing the μ L of phosphate buffer 1 00 ~ 500 in step 1, the total degradation time is 24 ~ 72 h；
3）In the bag filter that degradation solution after taking-up is sealed in the KDa of molecular cut off 10 ~ 14, determined using 5 mL pure water
Amount dialysis, dialysis time is 2 ~ 4 h；
4）Take the mL of dialyzate 4.0, add the hydrochloric acid solution that 1 mL concentration is 12 mol/L, hydrolyze 1 under the conditions of 115 ~ 121 DEG C ~
5 h.Hydrolyzate is cooled to after 20 ~ 25 DEG C, using GB/T 5009.7-2016《Reduced sugar in national food safety standard food
Determine》In the 3rd method potassium ferricyanide reducing sugar test method detection hydrolyzate in reduced sugar content, detection process is not in this hair
Within bright protection domain, the present invention will not elaborate to detection process；
5）The degradation rate of gel will be calculated according to following formula：
m2= m1 × M / M1（Formula 4）
w=m2/ (c×m3/ρ)×100%（Formula 5）
M：Chitosan or derivatives thereof monosaccharide unit molecular weight（Da）；
m2：The gel quality of degraded（mg）；
m1：The reduced sugar detected according to potassium ferricyanide reducing sugar test method（That is Glucosamine）Quality（mg）；
M1：The molecular weight of Glucosamine（Da）.
w：The degradation rate of gel（%）.
c：Carboxymethyl chitosan concentration (mg/mL) in sample;
m3：Sample gross mass（g）；
ρ：The density of sample（g/mL）.
In the present invention selected chitosan or derivatives thereof molecular weight ranges be 20 KDa to 2 000 KDa, preferably divide
Son amount scope is 100 KDa to 800 KDa；Deacetylation scope is 60% to 99%, preferably 80% to 95%；It is selected in the present invention
Water-solubility chitosan derivative be mainly the derivative that side base modification is carried out to chitosan, sugared ring hexa-atomic to chitosan main chain enters
The modified derivative of row destruction is not suitable for the application then.The water-soluble side base of chitosan, which is modified, to be included：Pendant carboxyl groupsization are modified,
Such as carboxymethyl chitosan；Pendant aliphatic is chain modified, such as hydroxyl butyl chitosan；The sulfonated modification of side base, such as sulfonated shell gather
Sugar；And retain primaquine group or secondary amino group side base it is quaternised modified.The side base substitution of above-mentioned various side base modification of chitosan
It is 0.5 to 2.0 to spend scope.
Crosslinking agent selected in cross-linked chitosan or cross-linked chitosan derivative gel, gel is needed to use in the present invention
Including but not limited to polynary epoxy compounds, polyaldehyde base class compound, water-soluble polyhalohydrocarbon, and simultaneously containing above-mentioned
The compound of two or more functional groups（For example, epoxychloropropane etc.）.In above-mentioned crosslinking agent, polynary epoxide includes
Binary, ternary and quaternary epoxide, wherein, binary epoxide includes but is not limited to BDO two and shrinks sweet
Oily ether（BDDE）, 1,2,3,4- butane diepoxide and 1,2,7,8- diepoxyoctanes（DEO）Etc. compound；Ternary epoxy compound
Thing includes but is not limited to the compounds such as trimethyl propane triglycidyl ether；Quaternary epoxy compounds include but is not limited to season penta
The compounds such as the glycidol ether of tetrol four.Polyaldehyde base class compound includes but is not limited to glutaraldehyde, Geniposide etc..It is polynary water-soluble
Property halogenated hydrocarbons includes but is not limited to chloro- 2,3- butanediols of 1,4- bis- etc..
The multiple cross-linked chitosan mentioned in the present invention or derivatives thereof gel, can be obtained by two ways, and one is
Chitosan being repeatedly crosslinked or derivatives thereof gel, crosslinking numbers range is 2 ~ 5 times, the degree of cross linking（The quality degree of cross linking, is handed over to add
Join the ratio of agent quality and chitosan or derivatives thereof quality）Scope is 1 ~ 20%（Particular content is shown in what the applicant applied on the same day
Another patent of invention:The multiple cross-linked gel of chitosan or derivatives thereof and preparation method）；Secondly being a variety of different degrees of cross linking
Chitosan or derivatives thereof gel pass through the mixed product of homogeneous, degree of cross linking scope is 1 ~ 20%, can select 3 ~ 8 kinds of differences
The gel of the degree of cross linking is mixed（Particular content is shown in another patent of invention that the applicant applies on the same day:Bionic joint synovia
And preparation method thereof）.
The invention provides the external gradient degradation method of multiple cross-linked chitosan gel rubber, enzyme environment or freedom in analogue body
Basic ring border, is quantitatively degraded by enzyme or free radical to multiple cross-linked chitosan gel rubber, by determining to reduce in catabolite
The content of sugar or the measure of the nitrogen content in catabolite, determine the palliating degradation degree of chitosan gel rubber, so as to obtain multiple cross-linked
The degradation curve of chitosan gel rubber, precisely determines the degradation cycle of chitosan gel rubber.
The biodegrading process that the present invention is provided by it is quantitative detection gel catabolite to determine the degradation process of gel, can
It is simple to operate and result is accurate with the gradient degradation feature of prominent multiple cross-linked gel.The gradient degradation of gel, it was demonstrated that gel
The packing action for playing gel that in vivo can be stage by stage, gel enters internal early stage, and the gel on low crosslinking degree or top layer is first
It is degraded, the catabolite of gel（Glucosamine）It can play a part of stimulating regeneration with repairing, prolong over time
Long, the gel of high-crosslinking-degree or internal layer is slowly degraded, and filling effect can be both provided for a long time, can also be ensured the long period
Catabolite（Glucosamine）Release, prolonged histio-irritative regeneration completes with repairing until repairing.
Brief description of the drawings
Fig. 1：The free radical cracking curve of cross-linked chitosan gel.
Fig. 2：The lysozyme degradation curve of cross-linked chitosan gel.
Fig. 3：The free radical cracking curve of crosslinked carboxymethyl chitosan gel.
Fig. 4：The lysozyme degradation curve of crosslinked carboxymethyl chitosan gel.
Embodiment
The present invention is described in further detail by the following examples, and this is not intended to limit protection scope of the present invention.
The experimental method of unreceipted actual conditions in embodiment, generally according to the bar described in normal condition and handbook
Part, or according to the condition proposed by manufacturer；Material, reagent used etc., unless otherwise specified, can be obtained by commercial sources
.
The multiple cross-linked chitosan used in special instruction, the present invention or derivatives thereof gel is needed to use known
Method prepare, the preparation technology of cross-linked gel is not within protection scope of the present invention.Will not be right in the present invention
The preparation process of multiple cross-linked chitosan or derivatives thereof gel is illustrated.
In the kjeldahl apparatus operational manual that the detection process of kjeldahl apparatus will be carried with reference to instrument in embodiment
It is required that, using the process of instrument not within protection scope of the present invention.The present invention is by concrete operations flow not to instrument
（Including parameter）Elaborate.
The detection process of potassium ferricyanide reducing sugar test method will be with reference to GB/T 5009.7-2016 in embodiment《Food security
The measure of reduced sugar in national standard food》In the 3rd method potassium ferricyanide reducing sugar test method concrete operations flow, detected
Journey is not within protection scope of the present invention, and the present invention will not elaborate to detection process.
Embodiment 1：The free radical cracking of triple cross-linked chitosan gels
Configuration concentration is 0.01mol/L ascorbic acid solution, and is adjusted with 3 mol/L hydrochloric acid to pH=3；Take cross-linked chitosan
（Deacetylation 90%, molecular weight 20KDa, three crosslinkings, mass concentration 30mg/mL, the degree of cross linking 2%）The mL of gel 2 is placed in 50 mL
In centrifuge tube, the above-mentioned mL of ascorbic acid solution 2 being added, and being cooled to 0 DEG C, the wherein concentration of ascorbic acid is 0.01 mol/L；
The environment of 0 DEG C of holding, adds the mL of hydrogen peroxide solution 2, the concentration of hydrogen peroxide is 0.01 mol/L；System is placed in water bath with thermostatic control
In oscillator, concussion temperature sets 37 DEG C, and preceding 100min is sampled every 10min, is hereafter sampled every 20min, often
The secondary taking-up μ L of degradation solution 100, while the μ L of above-mentioned ascorbic acid solution 100, the total degradation time is 140min, total sampling amount 12
It is individual；It is respectively that 1mol/L sodium hydroxides are neutralized to behind pH=7.0 with concentration by the degradation solution after taking-up, is sealed in molecular cut off
In bag filter for 14 KDa, quantitatively dialysed using 5 mL pure water, dialysis time is 2 h；The mL of dialyzate 4.0 is taken, is used
Nitrogen content in the abundant Europe SKD-2000 full-automatic Kjeldahl determination devices detection dialyzate in Shanghai, and be scaled according to formula 1 to formula 3
The degradation rate of cross-linked chitosan gel, is as a result shown in patent of invention accompanying drawing 1.It can be seen that the chitosan of three crosslinkings coagulates from accompanying drawing
Glue shows the degraded trend of three gradients.
Embodiment 2：The lysozyme degraded of triple cross-linked chitosan gels
Take cross-linked chitosan（Deacetylation 90%, molecular weight 20KDa, three crosslinkings, mass concentration 30mg/mL, the degree of cross linking 2%）It is solidifying
The mL of glue 2 is placed in 50 mL centrifuge tubes, adds the phosphate buffer that 2 mL concentration are 0.01 mol/L（pH 7.2）, add
800 u lysozymes, and it is cooled to 0 DEG C；System is placed in thermostatic control oscillator vibration, concussion temperature set 37 DEG C, preceding 12h every
1 h is sampled, and is hereafter sampled every 2h, the μ L of degradation solution 100 is taken out every time, while it is molten to supplement above-mentioned phosphate-buffered
The μ L of liquid 100, the total degradation time is 14h；Degradation solution after taking-up is sealed in the bag filter that molecular cut off is 14 KDa,
Quantitatively dialysed using 5 mL pure water, dialysis time is 2 h；The mL of dialyzate 4.0 is taken, it is 12 mol/ to adopt 1 mL concentration of addition
L hydrochloric acid solution, hydrolyzes 2 h under the conditions of 115 DEG C.Hydrolyzate is cooled to after 25 DEG C, using GB/T 5009.7-2016《Food
The measure of reduced sugar in safe national standard food》In the 3rd method potassium ferricyanide reducing sugar test method detection hydrolyzate in reduced sugar
Content, and be scaled according to formula (4) and formula (6) degradation rate of cross-linked chitosan gel, as a result see patent of invention accompanying drawing
2.It can be seen that the chitosan gel rubber of three crosslinkings shows the degraded trend of three gradients from accompanying drawing.
Embodiment 3：The free radical cracking of sixfold crosslinked carboxymethyl chitosan gel
Configuration concentration is 0.01mol/L ascorbic acid solution, and is adjusted with 3 mol/L hydrochloric acid to pH=3；Take cross-linked carboxymethyl
Chitosan（Deacetylation 90%, molecular weight 50KDa, substitution value 1.2, mass concentration 30mg/mL, the degree of cross linking is mixed in equal volume is
2%th, 4%, 8%, 12%, 16% 5 kind of gel and non-crosslinked carboxymethyl chitosan）The mL of gel 2 is placed in 50 mL centrifuge tubes, in addition
State the mL of ascorbic acid solution 2, and be cooled to 0 DEG C, the wherein concentration of ascorbic acid is 0.01 mol/L；The environment of 0 DEG C of holding,
The mL of hydrogen peroxide solution 2 is added, the concentration of hydrogen peroxide is 0.01 mol/L；System is placed in thermostatic control oscillator vibration, shaken
Temperature sets 37 DEG C, and preceding 100min is sampled every 10min, is hereafter sampled every 20min, and degradation solution is taken out every time
100 μ L, while the μ L of above-mentioned ascorbic acid solution 100, the total degradation time is 250min；Respectively by the degradation solution after taking-up with dense
Spend and be neutralized to for 1mol/L sodium hydroxides behind pH=7.0, is sealed in the bag filter that molecular cut off is 14 KDa, using 5 mL
Pure water is quantitatively dialysed, and dialysis time is 2 h；The mL of dialyzate 4.0 is taken, using the abundant full-automatic kelvin of Europe SKD-2000 in Shanghai
Nitrogen content in azotometer detection dialyzate, and crosslinked carboxymethyl chitosan gel is scaled according to formula (1) to formula (3)
Degradation rate, is as a result shown in Fig. 3.It can be seen that the degraded that the chitosan gel rubber of sixfold crosslinking shows six gradients becomes from accompanying drawing
Gesture.
Embodiment 4：The lysozyme degraded of sixfold crosslinked carboxymethyl chitosan gel
Take crosslinked carboxymethyl chitosan（Deacetylation 90%, molecular weight 50KDa, substitution value 1.2, mass concentration 30mg/mL waits body
It is 2%, 4%, 8%, 12%, 16% 5 kind of gel and non-crosslinked carboxymethyl chitosan that product, which mixes the degree of cross linking,）Gel 2mL be placed in 50 mL from
In heart pipe, the phosphate buffer that 2 mL concentration are 0.01 mol/L is added（pH 7.2）, 800 u lysozymes are added, and cool down
To 0 DEG C；System is placed in thermostatic control oscillator vibration, concussion temperature sets 37 DEG C, and preceding 12h is sampled every 1 h, hereafter often
It is sampled every 2h, the μ L of degradation solution 100 is taken out every time, while supplementing the above-mentioned μ L of phosphate buffer solution 100, total degradation time
For 25h；Degradation solution after taking-up is sealed in the bag filter that molecular cut off is 14 KDa, quantified using 5 mL pure water
Dialysis, dialysis time is 2 h；The mL of dialyzate 4.0 is taken, the hydrochloric acid solution that 1 mL concentration is 12 mol/L is added, in 115 DEG C of bars
2 h are hydrolyzed under part.Hydrolyzate is cooled to after 20 DEG C, using GB/T 5009.7-2016《In national food safety standard food also
The measure of raw sugar》In in the 3rd method potassium ferricyanide reducing sugar test method detection hydrolyzate reduced sugar content, and according to formula (4)
The degradation rate of crosslinked carboxymethyl chitosan gel is scaled with formula (5), Fig. 4 is as a result seen.It can be seen that sixfold is handed over from accompanying drawing
Connection carboxymethyl chitosan gel shows the degraded trend of six gradients.
Claims (9)
1. a kind of external gradient degradation method of multiple cross-linked chitosan gel rubber, it is characterised in that：It is to many using free radical
Weight cross-linked chitosan or derivatives thereof gel is degraded, and catabolite is quantified using kjeldahl apparatus;Or use
Lysozyme is degraded to multiple cross-linked chitosan or derivatives thereof gel, and using potassium ferricyanide reducing sugar test method to degraded
Product is quantified.
2. in accordance with the method for claim 1, it is characterised in that described multiple cross-linked chitosan or derivatives thereof gel, lead to
Two ways acquisition is crossed, one is the chitosan that is repeatedly crosslinked or derivatives thereof gel, and crosslinking numbers range is 2 ~ 5 times, crosslinking
It is 1 ~ 20% to spend scope；Secondly chitosan for a variety of different degrees of cross linking or derivatives thereof gel passes through the mixed product of homogeneous,
Degree of cross linking scope is 1 ~ 20%, is mixed from the gel of 3 ~ 8 kinds of different degrees of cross linking.
3. a kind of external gradient degradation method of multiple cross-linked chitosan gel rubber, it is characterised in that including the step of:
1）Configuration concentration is 0.001 ~ 0.1 mol/L ascorbic acid solution, and with 1 ~ 6 mol/L salt acid for adjusting pH to 3 ~ 3.5；
2）The mL of cross-linked chitosan or derivatives thereof gel 1 ~ 10 is taken, mass concentration scope is 10 ~ 500mg/mL, is placed in 10 ~ 50
ML centrifuge tubes, add step 1）In the mL of ascorbic acid solution 1 ~ 10, and be cooled to 0 ~ 5 DEG C；
2）The environment of 0 ~ 5 DEG C of holding, adds the mL of hydrogen peroxide solution 1 ~ 10, wherein hydrogen peroxide concentration is with adding volume and Vitamin C
The concentration of acid is equal with volume, and the concentration of hydrogen peroxide is 0.001 ~ 0.1 mol/L；
3）System is placed in thermostatic control oscillator vibration, concussion temperature sets 37 DEG C, is sampled every 10 ~ 20 min, every time
The μ L of degradation solution 100 ~ 500 are taken out, while supplementing step 1）The middle μ L of ascorbic acid solution 100 ~ 500, the total degradation time be 120 ~
1440min；
4）With concentration it is that 1mol/L sodium hydroxides are neutralized to behind pH=7.0 by the degradation solution after taking-up, is sealed in molecular cut off 10
In ~ 14 KDa bag filter, quantitatively dialysed using 5 mL pure water, dialysis time is 2 ~ 4 h；
5）The mL of dialyzate 4.0 is taken, the nitrogen content in dialyzate is detected using kjeldahl apparatus；
6）The degradation rate of gel will be calculated according to following three formula：
（Formula 1）
（Formula 2）
（Formula 3）
cN：The measuring samples total nitrogen content detected using kjeldahl apparatus；
m：Measuring samples quality；
F：Moisture in measuring samples；
V1：Sample cell quota of expenditure sulfuric acid solution volume；
V0：Blank tube quota of expenditure sulfuric acid solution volume；
c：The concentration of standard sulfuric acid solution；
c1：Carboxymethyl chitosan sugared content；
14：Nitrogen-atoms quality；
w：The degradation rate of carboxymethyl glucosaminoglycan in product；
The ratio of X test fluid volumes for needed for the cumulative volume and kjeldahl apparatus of sample solution.
4. a kind of external gradient degradation method of multiple cross-linked chitosan gel rubber, it is characterised in that comprise the following steps:
1）The mL of cross-linked chitosan or derivatives thereof gel 1 ~ 10 is taken, mass concentration scope is 10 ~ 500mg/mL, is placed in 10 ~ 50
ML centrifuge tubes, the phosphate buffer that 1 ~ 10 mL concentration of addition is 0.01 ~ 0.05 mol/L, pH 7.0 ~ 7.4, addition 400 ~
2000 u lysozymes, and it is cooled to 0 ~ 5 DEG C；
2）System is placed in thermostatic control oscillator vibration, concussion temperature sets 37 DEG C, is sampled every 1 ~ 2 h, takes out every time
The μ L of degradation solution 100 ~ 500, while supplementing step 1）The middle μ L of phosphate buffer 1 00 ~ 500, the total degradation time is 24 ~ 72 h；
3）In the bag filter that degradation solution after taking-up is sealed in the KDa of molecular cut off 10 ~ 14, determined using 5 mL pure water
Amount dialysis, dialysis time is 2 ~ 4 h；
4）Take the mL of dialyzate 4.0, add the hydrochloric acid solution that 1 mL concentration is 12 mol/L, hydrolyze 1 under the conditions of 115 ~ 121 DEG C ~
5 h;Hydrolyzate is cooled to after 20 ~ 25 DEG C, and the content of reduced sugar in hydrolyzate is detected using potassium ferricyanide reducing sugar test method；
5）The degradation rate of gel will be calculated according to following formula：
m2= m1 × M / M1 （Formula 4）
w=m2/ (c×m3/ρ)×100%（Formula 5）
M：Chitosan or derivatives thereof monosaccharide unit molecular weight；
m2：The gel quality of degraded；
m1：The quality of the reduced sugar, i.e. Glucosamine that are detected according to potassium ferricyanide reducing sugar test method；
M1：The molecular weight of Glucosamine；
w：The degradation rate of gel（%）；
c：Carboxymethyl chitosan concentration in sample；
m3：Sample gross mass；
ρ：The density of sample.
5. according to the method described in claim 3,4, it is characterised in that described chitosan or derivatives thereof molecular weight ranges are
20 KDa to 2 000 KDa, deacetylation scope is 60% to 99%.
6. according to the method described in claim 3,4, it is characterised in that described water-solubility chitosan derivative is to chitosan
The derivative of side base modification is carried out, sugared ring hexa-atomic to chitosan main chain carries out the modified derivative of destruction and is not suitable for this Shen then
Please；Including：Pendant carboxyl groupsization are modified, and pendant aliphatic is chain modified, the sulfonated modification of side base, and retain primaquine group or secondary amino group
Group's side base is quaternised modified；The side base substitution value scope of above-mentioned various side base modification of chitosan is 0.5 to 2.0.
7. according to the method described in claim 3,4, it is characterised in that described water-solubility chitosan derivative is carboxymethyl shell
Glycan, hydroxyl butyl chitosan, sulfonated chitosan.
8. according to the method described in claim 3,4, it is characterised in that described cross-linked chitosan or cross-linked chitosan derivative
Selected crosslinking agent includes but is not limited to polynary epoxy compounds in gel, gel, and polyaldehyde base class compound is water-soluble
Polyhalohydrocarbon, and the compound containing above two or a variety of functional groups simultaneously；In above-mentioned crosslinking agent, polynary epoxy compound
Thing includes binary, ternary and quaternary epoxide, wherein, binary epoxide includes but is not limited to BDO two
Glycidol ether（BDDE）, 1,2,3,4- butane diepoxide and 1,2,7,8- diepoxyoctanes（DEO）；Ternary epoxide
Including but not limited to trimethyl propane triglycidyl ether；Quaternary epoxy compounds include but is not limited to the shrink of pentaerythrite four
Glycerin ether；Polyaldehyde base class compound includes but is not limited to glutaraldehyde, Geniposide；Polynary water-soluble halogenated hydrocarbons includes but is not limited to
The chloro- 2,3- butanediols of 1,4- bis-.
9. according to the method described in claim 3,4, it is characterised in that described multiple cross-linked chitosan or derivatives thereof gel,
It can be obtained by two ways, one is the chitosan that is repeatedly crosslinked or derivatives thereof gel, crosslinking numbers range is 2 ~ 5 times,
Degree of cross linking scope is 1 ~ 20%；Secondly chitosan for a variety of different degrees of cross linking or derivatives thereof gel is mixed by homogeneous
Product, degree of cross linking scope is 1 ~ 20%, is mixed from the gel of 3 ~ 8 kinds of different degrees of cross linking.
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| CN114280193A (en) * | 2021-12-28 | 2022-04-05 | 青岛琛蓝海洋生物工程有限公司 | Method for detecting gel crosslinking degree |
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| CN114280193A (en) * | 2021-12-28 | 2022-04-05 | 青岛琛蓝海洋生物工程有限公司 | Method for detecting gel crosslinking degree |
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