CN109553700A - Acylated chitosan oligosaccharide derivative of a kind of no cytotoxicity N- biguanides O- and preparation method thereof - Google Patents
Acylated chitosan oligosaccharide derivative of a kind of no cytotoxicity N- biguanides O- and preparation method thereof Download PDFInfo
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- C08B37/003—Chitin, i.e. 2-acetamido-2-deoxy-(beta-1,4)-D-glucan or N-acetyl-beta-1,4-D-glucosamine; Chitosan, i.e. deacetylated product of chitin or (beta-1,4)-D-glucosamine; Derivatives thereof
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Abstract
The present invention discloses acylated chitosan oligosaccharide derivative of a kind of no cytotoxicity N- biguanides O- and preparation method thereof; using chitosan oligosaccharide and dicyandiamide as reaction substrate; using microwave liquid phase synthesizing method; prepare raw material of the chitosan oligosaccharide biguanide hydrochloride as subsequent reactions; then select hydroxy cinnamic acid derivative as substrate; it under the effect of the mixed liquor of 7.5 phosphate buffer of pH and laccase solution, is reacted with chitosan oligosaccharide biguanide hydrochloride, obtains the acylated chitosan oligosaccharide derivative of N- biguanides O-.Biguanides and lipophilic group are introduced into the chitosan oligosaccharide that dissolubility is good, antibiotic property is good, bio-compatibility is good by the acylated chitosan oligosaccharide derivative of N- biguanides O- prepared by the present invention; product can be made to have both amphipathic property while realizing product low cytotoxicity.
Description
Technical field
The present invention relates to polymer modification fields, more particularly, and in particular to a kind of low cytotoxicity N- is bis-
Acylated chitosan oligosaccharide derivative of guanidine O- and preparation method thereof.
Background technique
Chitosan oligosaccharide is one kind of oligosaccharide, and scientific name oligosaccharides β-(Isosorbide-5-Nitrae) -2-amino-2-deoxy-D-Glucose is amino Portugal
The substance that grape sugar is got up by 2~20 β-(Isosorbide-5-Nitrae)-glucosides key connections, is gathered by the shell that chitin is formed after highly basic is handled
Sugar degradation generates, and unique a large amount of existing basic amine group oligosaccharides, degradable in natural sugar, easily absorbs, good water solubility can reduce blood
Blood lipid is pressed, it is a kind of bioactive materials of highly effective and safe that bioactivity is high.Chitosan oligosaccharide has very solely as chitosan
Special chemical structure contains largely free amino (- NH in strand2) or hydroxyl (- OH), both groups all have very strong
Reactivity, can with multiple compounds occur chemical reaction obtain the derivative of chitosan oligosaccharide.Currently, changing with chitosan oligosaccharide chemistry
The relevant document report of property is fewer, but due to the chemical similarity of the two, the chemical modification method suitable for chitosan is also same
Sample is applicable to chitosan oligosaccharide, and main method of modifying has: (1) be acylated: can by with organic acid and its derivative (acid anhydrides, acyl
Halogen etc.) reaction, it realizes that acylation modification, including the acylated generation ester of O- and N- are acylated and generate amide, imports aliphatic or aromatic series
Carboxyl groups；(2) be alkylated: the amino group on strand carries a pair of of lone pair electrons, can react with alkyl halide, can obtain
To corresponding N- alkylate；(3) it is etherified: passing through the examination of C-3 position hydroxyl and C-6 hydroxyls and hydrocarbylation on molecular skeleton
Agent reaction prepares ether；(4) sulfonates: to be prepared by being reacted with sulfonated reagent (sulfur dioxide, sulfur trioxide, concentrated sulfuric acid etc.), one
As carry out at low temperature；(5) quaternized: one is C-2 bit amino is alkylated completely, to generate quaternary ammonium salt, another kind is C-2
Amino, C-3 hydroxyl and C-6 hydroxyls and the epoxidation containing quaternary ammonium salt；(6) guanidinated: with contain NH2C (=NH) NH base
The compound and its derivatives reaction of group, guanidine saltization only carry out on amino, generally comprise single guanidine salt and Guanoctine.
Wherein, acylation modification is most common one kind, chitosan or chitosan oligosaccharide strand in its numerous chemical modification mode
On amino and hydroxyl, can chemically react with a variety of organic acids and its derivatives reaction, such as acid anhydrides or acyl chlorides, give birth to respectively
At the compound of esters and amides.Under normal circumstances, wherein the reactivity of each reactive group is followed successively by, amino > C6
Hydroxyl on the hydroxyl set > position C3.Peng Zheng etc. carries out N- acylation to chitosan as acylating reagent using maleic anhydride and changes
Property, the maleic anhydride acidation chitosan salt/nature rubber graft copolymer prepared has good antibiotic property, hydrophily, life
Reason activity and heat resistance, have further widened natural rubber in application range (Peng Zheng, Li Yongzhen, sieve in the fields such as health care
Bravely please a kind of maleic anhydride acidation chitosan salt/nature rubber graft copolymer of and preparation method thereof the Guangdong [P]:
CN101864051A,2010-10-20.).Zhou Xiangchis etc. are reacted by succinic acid with the O- acyl group of chitosan, and a kind of shell is prepared for
Glycan succinate, can be used as freshener, i.e., with the chitosan derivatives of delicate flavour, no chlorine, without sodium is solved existing
Technical problem in freshening seasoning containing a large amount of inorganic sodium ions, chloride ion.(Zhou Xiangchi, Xu Junyi, Lin Rongye, Liu Biqian,
The Zhejiang Liu Ling a kind of Chitosan succinate flavor enhancer [P]: CN102775518A, 2012-11-14.).Wang Jiangtao etc. is utilized
The reaction of O- acyl group, introduces p-benzoyl based structures on chitosan molecule skeleton, be prepared for a kind of molecular weight be 70~
The chitosan ester p-aminobenzoate of 80kDa, minimum inhibitory concentration point of the compound to staphylococcus aureus and aspergillus niger
Do not reach 0.1% and 0.25%, can be used as food preservation additive (Wang Jiangtao, Wang Hedong chitosan ester p-aminobenzoate and
The Shandong preparation method [P]: CN102153674A, 2011-08-17.).However, active in chitosan oligosaccharide or chitosan molecule chain
So that it is not readily dissolved in ordinary organic solvents, it is lipophilic that acylation modification before all passes through introducing for interaction between group hydrogen bond
Group (such as alkyl, naphthenic base, phenyl etc.) reduces active group number using acylation reaction, and then it is intermolecular to weaken chitosan oligosaccharide
And the Hyarogen-bonding of intramolecular, to improve its oil-soluble.
Guanidinated modification is the hot spot of chitosan or chitosan oligosaccharide study on the modification in recent years, refers in acid condition, makes
A series of reaction for being replaced the free amine group on chitosan oligosaccharide glycan with cyanoguanidines is different from above-mentioned acylation modification, guanidine radicals
Change the modified water solubility for tending to improve product, while promoting the stability and bioactivity of guanidine salt dissolving.Currently, both domestic and external grind
Study carefully in the guanidinated modification for focusing primarily upon chitosan: Du Yumin etc. is reacted using the chitosan of different molecular weight with formamidinesulfinic acid
Chitosan guanidine bisulfite salt derivative is obtained, then is reacted with hydrochloric acid, chitosan guanidine salt hydrochlorate is obtained, products therefrom has more
Wide bacteriostatic activity and broader antibacterial range (Du Yumin, Hu Ying, Hu Zhen chitosan guanidine salt derivative and preparation method thereof
The Hubei [P]: CN1944467,2007-04-11.)；Once culvert etc. is chemically modified glucocyamine, in one chlorine of its α substitution
Atom generates 1- chloroguanide acetic acid, then makes itself and chitosan reaction, prepares Chitosan, it is made to obtain excellent moisture absorption
Increase physiological activity (synthesis of Zeng Han, Zhang Lei Chitosan and its survey of moisture-absorbing moisture-keeping performance while performance of keeping humidity again
Fixed [J] Xinjiang Normal University journal, 2008, (01): 83-86+89. [2017-09-14])；The p-aminophenyl first such as Jin Shaodi
Acid is reacted with dicyandiamide, has synthesized intermediate to diguanidino benzoic acid hydrochloride, then biguanides Ji Benjia is reacted to obtain with thionyl chloride
Acyl chlorides, the latter carry out acylation reaction with chitosan in methylsulphur acid system and are made to biguanides base benzoyl chitosan hydrochloride, resist
Bacterium experiment shows the product antibacterial effect better than chitosan and to diguanidino benzoic acid hydrochloride (Jin Shaodi, Gao Zhaochang, Liu De
Coltfoal studies [J] daily chemical industry to the preparation of biguanides base benzoylation chitosan hydrochloride and its antibacterial activity, and 2011,41
(03): 176-179+207. [2017-09-14] .DOI:10.13218/j.cnki.csdc.2011.03.004).Based on this, by
There is similar structure in chitosan oligosaccharide and chitosan, and there is preferably water-soluble (chitosan cannot be directly dissolved in water), passes through use
Guanidine radicals is modified chitosan oligosaccharide, can not only improve the hydrophily of chitosan oligosaccharide and the stability of guanidine radicals, can also assign the two higher biology
Activity.
Summary of the invention
It is an object of the invention to overcome the deficiencies of the prior art and provide a kind of acylated shell of no cytotoxicity N- biguanides O- is few
Sugar derivatives and preparation method thereof utilizes the work of amino in the chitosan oligosaccharide that dissolubility is good, antibiotic property is good, bio-compatibility is good
Property, guanidinated structure is introduced, the hydrophily of chitosan oligosaccharide is improved；Then it is substituted by hydroxyl on chitosan oligosaccharide, is re-introduced into acylated base
Group, improves the lipophilicity of chitosan oligosaccharide, to prepare the acylated chitosan oligosaccharide derivative of amphipathic N- biguanides O-.
Technical purpose of the invention is achieved by following technical proposals:
No cytotoxicity N- biguanides O- is acylated chitosan oligosaccharide derivative, with chitosan oligosaccharide and dicyandiamide reaction preparation, with chitosan oligosaccharide
Side group NH2It is reacted under hydrochloric acid and microwave condition with dicyandiamide and forms the biguanidino groups bonded with chitosan oligosaccharide, then with shell
Hydroxyl is reacted with the carboxyl of hydroxy cinnamic acid derivative on oligosaccharides, so that hydroxy cinnamic acid derivative is keyed to by ester group
On chitosan oligosaccharide, it is shown below:
In above-mentioned chemical formula, m and n are respectively the degree of polymerization of acetylated unit and deacetylated unit in chitosan oligosaccharide.Shell is few
Sugar is the basic amine group oligosaccharides that positive charge is uniquely had in nature, its molecular weight is small, and dissolubility is fine, is easy by organism
It is absorbed and utilized, weight average molecular weight is 5000 hereinafter, it is preferred that 1500-3000 (KDa), deacetylation is 97% or more, preferably 98-
99%.
In the acylated chitosan oligosaccharide derivative of no cytotoxicity N- biguanides O-, biguanides degree of substitution is 40-80%, preferably 50-
70%；Acylation degree is between 0.20~1.45, preferably 0.5-1.2；In formula, R1:-CH=CH-CO-；R2:-OH or-H；R3:-OH ,-
H or-OCH3, i.e., when reactant hydroxy cinnamic acid derivative is cinnamic acid, R in product1For-CH=CH-CO-, R2For-H；R3
For-H；When hydroxy cinnamic acid derivative is caffeic acid, R in product1For-CH=CH-CO-, R2For-OH；R3For-OH；Hydroxy cinnamate
When acid derivative is ferulic acid, R in product1For-CH=CH-CO-, R2For-OH；R3For-OCH3；Hydroxy cinnamic acid derivative is
When p-Coumaric Acid, R in product1For-CH=CH-CO-, R2For-OH；R3For-H.
The preparation method of the acylated chitosan oligosaccharide derivative of no cytotoxicity N- biguanides O-, according to shown in following reaction equations
Step carries out:
During the preparation process, hydroxyl can be reacted with hydroxy cinnamic acid derivative in chitosan oligosaccharide, it is contemplated that reaction is lived
Property and steric hindrance influence, a possibility that hydroxyl participates in reaction on the position as shown in b, is greater than in b below chitosan oligosaccharide main chain
Hydroxyl.
Step 1, chitosan oligosaccharide is dispersed in hydrochloric acid and is stirred under the conditions of microwave 160W~560W, carry out first step microwave
Reaction；Later be added dicyandiamide aqueous solution and using hydrochloric acid adjustment pH value of reaction system be 1-2, continue microwave 100W~
It is stirred under the conditions of 600W, second step microwave reaction is carried out, so that chitosan oligosaccharide and dicyandiamide reaction prepare chitosan oligosaccharide Biguanide derivative；
Wherein:
In first step microwave reaction, microwave power be 300-400w, the reaction time be 3-7min, preferably 5-7min,
Mixing speed is 100-200 turns per minute.
In second step microwave reaction, microwave power be 300-400w, the reaction time be 5-30min, preferably 10-
20min, mixing speed are 100-400 turns per minute, and reaction temperature is 80~100 DEG C.
After reaction, reaction solution is cooled to room temperature, alcohol precipitation is carried out to mixed solution, is dried, grinding obtains chitosan oligosaccharide
Biguanide derivative powder.
The molar ratio of amino is (0.1-5): 1, preferably (1-3): 1, more preferred 1:1 in dicyandiamide and chitosan oligosaccharide.
Hydrochloric acid is the hydrochloride aqueous solution of 0.2~0.8mol/L, the use of hydrochloric acid adjustment pH value of reaction system is 1.
After reaction, reaction solution standing is cooled to room temperature, decompression suction filtration is carried out to mixed solution, dialyses (retention
Number-average molecular weight is 500Da), then repeatedly washed with dehydrated alcohol, it precipitates, vacuum drying is under the conditions of 50~80 DEG C to get arriving
Chitosan oligosaccharide biguanide hydrochloride powder.
Step 2, chitosan oligosaccharide biguanide hydrochloride powder and laccase prepared by step 1 are dispersed in phosphate buffer
In, and hydroxy cinnamic acid derivative is added and is reacted, to obtain the acylated chitosan oligosaccharide derivative of N- biguanides O-, in which:
The pH of phosphate buffer is 7-8.
Laccase is added as catalyst using laccase aqueous solution, and concentration is 1-5mg/mL (laccase quality/water body
Product), the volume ratio of laccase aqueous solution and phosphate buffer is (0.1-0.5): (40-50).
4~8h of reaction time, reaction temperature are 20~40 DEG C, 300~500r/min of mixing speed；Preferred reaction time 6
~8h, reaction temperature are 30~40 DEG C.
After reaction, reaction solution standing is cooled to room temperature, then with a large amount of phosphate buffers, ethyl alcohol and methanol rinse
Precipitating, is dried under vacuum to material moisture less than 0.5%~10% under the conditions of 50~80 DEG C, obtains target product
The molar ratio of hydroxyl is (0.1~5) in hydroxy cinnamic acid derivative and chitosan oligosaccharide biguanide hydrochloride: 1, preferably (1-
3): 1.
Preparation method of the present invention is simple and easy to do, and production cost is low, is suitable for industrialization；Gained N- biguanides O- of the invention is acylated
Chitosan oligosaccharide derivative combines the bioactivity of chitosan and guanidine compound, shows stronger bioactivity, guanidino group
On the one hand the use limitation for helping product tradition chitosan oligosaccharide improves its dissolubility in water, on the other hand can also improve
The poor disadvantage of the stability of traditional guanidine salt has important economic significance；The acylated chitosan oligosaccharide of gained N- biguanides O- of the invention spreads out
Biology introduces ester group structure, on the one hand, by the reduction of amino or hydroxyl quantity reduce chitosan oligosaccharide biguanides it is intermolecular and
The Hyarogen-bonding of intramolecular, to improve the oil-soluble of chitosan oligosaccharide biguanide hydrochloride；On the other hand, chitosan oligosaccharide biguanides hydrochloric acid
After acylation reaction, structural chain is changed salt, this has further changed the bioactivity of chitosan oligosaccharide biguanide hydrochloride,
It improves the lipophilicity and hydrophily of its oligosaccharide as a kind of acyl group and the dual modified material of guanidine radicals, is expected to be used as one
Kind novel biomaterial.
Detailed description of the invention
Fig. 1 is raw material chitosan oligosaccharide (COS), intermediate product chitosan oligosaccharide biguanide hydrochloride (COSG) and products therefrom N- of the present invention
Biguanides O- is acylated chitosan oligosaccharide derivative (O-COSG) infrared spectrum.
Fig. 2 is the uv atlas of products therefrom N- biguanides O- of the present invention acylated chitosan oligosaccharide derivative (O-COSG).
Fig. 3 is the acylated shell of chitosan oligosaccharide (COS), chitosan oligosaccharide biguanide hydrochloride (COSG) and products therefrom N- biguanides O- of the present invention
UV absorption spectrogram of the oligosaccharide derivative (O-COSG) in soybean oil butanol solution.
Fig. 4 is chitosan oligosaccharide (COS), chitosan oligosaccharide biguanide hydrochloride (COSG) and different esterification degrees under different dosing concentration
N- biguanides O- acylated chitosan oligosaccharide derivative (O-COSG) on the active influence of human hepatoma cell HepG2.
Specific embodiment
With specific implementation, the present invention will be described in further detail below, however, the present invention is not limited thereto.It is purchased using laccase
From Kmart (Tianjin) Chemical Industry Science Co., Ltd, the preparation for carrying out aqueous solution is used.
Embodiment 1
A. chitosan oligosaccharide biguanide hydrochloride is synthesized:
(1) 10g deacetylation 98% is weighed, molecular weight 3kDa chitosan oligosaccharide is dissolved in compound concentration in 0.8mol/L hydrochloric acid solution
For the chitosan oligosaccharide hydrochloric acid solution of 0.25g/mL, with the hydrochloric acid conditioning solution pH value of 1mol/L to 1, then chitosan oligosaccharide amino molal quantity is pressed
0.5 times, weigh the dicyandiamide of corrresponding quality at 60 DEG C stirring and dissolving in distilled water；
(2) aqueous solution of dicyandiamide is mixed with the hydrochloric acid solution of chitosan oligosaccharide, after adjusting pH value to 1, carries out microwave liquid phase
Synthesis controls reaction time 20min, and microwave reaction power is in 400w, and reaction temperature is 100 DEG C, mixing speed 400r/min；
(3) after reaction, reaction solution standing is cooled to room temperature, decompression suction filtration, dialysis (retention is carried out to mixed solution
Molecular weight is 500Da), then repeatedly washed, precipitated with dehydrated alcohol, under the conditions of 80 DEG C vacuum drying be to get to degree of substitution
55% chitosan oligosaccharide biguanide hydrochloride powder, structural formula are as follows:
B. synthesis N- biguanides O- is acylated chitosan oligosaccharide derivative:
(1) take the above-mentioned chitosan oligosaccharide biguanide hydrochloride powder of 2g, with 45mL phosphate buffer (50mmol/L, pH 7.5),
The 3mg/mL laccase solution of 0.20mL is sufficiently mixed, further according to 0.5 times of addition of hydroxyl moles on chitosan oligosaccharide biguanide hydrochloride
Hydroxy cinnamic acid derivative (i.e. cinnamic acid) controls reaction time 6h, and reaction temperature is 40 DEG C, mixing speed 500r/min；
(2) after reaction, reaction solution standing is cooled to room temperature, then with a large amount of phosphate buffers, ethyl alcohol and methanol
Rinsing precipitating, is dried under vacuum to material moisture less than 0.5%~10% under the conditions of 50~80 DEG C, pulverizes, obtain acyl
The acylated chitosan oligosaccharide derivative of the N- biguanides O- that change degree is 0.7438.
Embodiment 2
A. chitosan oligosaccharide biguanide hydrochloride is synthesized:
(1) 10g deacetylation 97% is weighed, the chitosan oligosaccharide of molecular weight 1.5kDa, which is dissolved in 0.2mol/L hydrochloric acid solution, to be prepared
Concentration is the chitosan oligosaccharide hydrochloric acid solution of 0.25g/mL, is rubbed with the hydrochloric acid conditioning solution pH value of 1mol/L to 1, then by chitosan oligosaccharide amino
0.5 times of your number, weigh the dicyandiamide of corrresponding quality at 45~60 DEG C stirring and dissolving in distilled water；
(2) aqueous solution of dicyandiamide is mixed with the hydrochloric acid solution of chitosan oligosaccharide, after adjusting pH value to 1, carries out microwave liquid phase
Synthesis controls reaction time 20min, and microwave reaction power is in 400w, and reaction temperature is 100 DEG C, mixing speed 100r/min；
(3) after reaction, reaction solution standing is cooled to room temperature, decompression suction filtration, dialysis (retention is carried out to mixed solution
Molecular weight is 500Da), then repeatedly washed, precipitated with dehydrated alcohol, under the conditions of 50 DEG C vacuum drying be to get to degree of substitution
55% chitosan oligosaccharide biguanide hydrochloride powder,
B. synthesis N- biguanides O- is acylated chitosan oligosaccharide derivative:
(1) take the above-mentioned chitosan oligosaccharide biguanide hydrochloride powder of 2g, with 45mL phosphate buffer (50mmol/L, pH 7.5),
The 3mg/mL laccase solution of 0.20mL is sufficiently mixed, further according to hydroxyl amino molal quantity on chitosan oligosaccharide biguanide hydrochloride 1 extraordinarily
Enter hydroxy cinnamic acid derivative (i.e. caffeic acid), control reaction time 8h, reaction temperature is 30 DEG C, mixing speed 300r/min；
(2) after reaction, reaction solution standing is cooled to room temperature, then with a large amount of phosphate buffers, ethyl alcohol and methanol
Rinsing precipitating, is dried under vacuum to material moisture less than 0.5%~10% under the conditions of 80 DEG C, pulverizes, obtain acylation degree
For the 0.5709 acylated chitosan oligosaccharide derivative of N- biguanides O-.
Embodiment 3
A. chitosan oligosaccharide biguanide hydrochloride is synthesized:
(1) 10g deacetylation 99% is weighed, the chitosan oligosaccharide of molecular weight 5kDa is dissolved in 0.2~0.8mol/L hydrochloric acid solution
Compound concentration is the chitosan oligosaccharide hydrochloric acid solution of 0.25g/mL, with the hydrochloric acid conditioning solution pH value of 1mol/L to 1, then presses chitosan oligosaccharide ammonia
0.5 times of base molal quantity, weigh the dicyandiamide of corrresponding quality at 50 DEG C stirring and dissolving in distilled water；
(2) aqueous solution of dicyandiamide is mixed with the hydrochloric acid solution of chitosan oligosaccharide, after adjusting pH value to 1, carries out microwave liquid phase
Synthesis controls reaction time 20min, and microwave reaction power is in 500w, and reaction temperature is 100 DEG C, mixing speed 300r/min；
(3) after reaction, reaction solution standing is cooled to room temperature, decompression suction filtration, dialysis (retention is carried out to mixed solution
Molecular weight is 500Da), then repeatedly washed, precipitated with dehydrated alcohol, under the conditions of 70 DEG C vacuum drying be to get to degree of substitution
55% chitosan oligosaccharide biguanide hydrochloride powder,
B. synthesis N- biguanides O- is acylated chitosan oligosaccharide derivative:
(1) take the above-mentioned chitosan oligosaccharide biguanide hydrochloride powder of 2g, with 45mL phosphate buffer (50mmol/L, pH 7.5),
The 3mg/mL laccase solution of 0.20mL is sufficiently mixed, further according to 0.10 times of hydroxyl amino molal quantity on chitosan oligosaccharide biguanide hydrochloride
It is added hydroxy cinnamic acid derivative (i.e. ferulic acid), controls reaction time 4h, reaction temperature is 40 DEG C, mixing speed 400r/
min；
(2) after reaction, reaction solution standing is cooled to room temperature, then with a large amount of phosphate buffers, ethyl alcohol and methanol
Rinsing precipitating, is dried under vacuum to material moisture less than 0.5%~10% under the conditions of 50~80 DEG C, pulverizes, obtain acyl
The acylated chitosan oligosaccharide derivative of the N- biguanides O- that change degree is 0.3306.
Embodiment 5
A. chitosan oligosaccharide biguanide hydrochloride is synthesized:
(1) 10g deacetylation 97% is weighed, the chitosan oligosaccharide of molecular weight 1.5kDa, which is dissolved in 0.2mol/L hydrochloric acid solution, to be prepared
Concentration is the chitosan oligosaccharide hydrochloric acid solution of 0.25g/mL, is rubbed with the hydrochloric acid conditioning solution pH value of 1mol/L to 1, then by chitosan oligosaccharide amino
0.5 times of your number, weigh the dicyandiamide of corrresponding quality at 45~60 DEG C stirring and dissolving in distilled water；
(2) aqueous solution of dicyandiamide is mixed with the hydrochloric acid solution of chitosan oligosaccharide, after adjusting pH value to 1, carries out microwave liquid phase
Synthesis controls reaction time 20min, and microwave reaction power is in 450w, and reaction temperature is 100 DEG C, mixing speed 100r/min；
(3) after reaction, reaction solution standing is cooled to room temperature, decompression suction filtration, dialysis (retention is carried out to mixed solution
Molecular weight is 500Da), then repeatedly washed, precipitated with dehydrated alcohol, under the conditions of 50 DEG C vacuum drying be to get to degree of substitution
55% chitosan oligosaccharide biguanide hydrochloride powder,
B. synthesis N- biguanides O- is acylated chitosan oligosaccharide derivative:
(1) take the above-mentioned chitosan oligosaccharide biguanide hydrochloride powder of 2g, with 45mL phosphate buffer (50mmol/L, pH 7.5),
The 3mg/mL laccase solution of 0.20mL is sufficiently mixed, further according to hydroxyl amino molal quantity on chitosan oligosaccharide biguanide hydrochloride 1 extraordinarily
Enter hydroxy cinnamic acid derivative (i.e. p-Coumaric Acid), control reaction time 8h, reaction temperature is 30 DEG C, mixing speed 300r/
min；
(2) after reaction, reaction solution standing is cooled to room temperature, then with a large amount of phosphate buffers, ethyl alcohol and methanol
Rinsing precipitating, is dried under vacuum to material moisture less than 0.5%~10% under the conditions of 80 DEG C, pulverizes, obtain acylation degree
For the 0.7905 acylated chitosan oligosaccharide derivative of N- biguanides O-.
In order to detect to the synthesis efficiency in the present invention, the present invention is using measurement esterification degree (DS) as judgement
Standard, esterification degree (DS) are commented according to the acyl group mass fraction contained in the acylated each unit of chitosan oligosaccharide derivative of N- biguanides O-
Estimate, (the measurement of Wu Weidou, Shi Wenrong, Ben Dongxu, Zhu Hui Acetylation starch esterification degree of specific method reference literature
The processing of [J] grain and oil, 2010, (05): 58-59. [2017-09-17]), calculation formula is as follows:
It arranges:
In formula, M is acyl group relative molecular mass (value of M is determined according to the type of hydroxy cinnamic acid derivative), and 318 be every
A guanidinated chitosan oligosaccharide unit relative molecular mass, 161 be each chitosan oligosaccharide unit relative molecular mass, and 1 is the opposite of H atom
Atomic mass, selecting the degree of substitution of chitosan oligosaccharide biguanide hydrochloride is 55%.
In formula, C is hydrochloric acid standard solution concentration (mol/L), V0It is respectively blank quota of expenditure liquor capacity and sample with V
It titrates quota of expenditure liquor capacity (mL), m is sample quality (g).
It is characterized using infrared and ultraviolet spectra, specific as follows:
| Fourier infrared spectrograph | Spectrum 100 | Perkin-Elmer company, the U.S. |
| UV, visible light is divided luminance meter | Shimadzu Uvmini-1240 | Japanese Shimadzu Corporation |
(1) the acylated chitosan oligosaccharide derivative infrared spectrum characterization of N- biguanides O-
The acylated chitosan oligosaccharide derivative about 2mg of the N- biguanides O- prepared in the embodiment of the present invention 1 is taken, dry KBr is about
200mg is placed in agate mortar, is uniformly mixed, is ground into a powder.It takes appropriate mixture to be transferred in compression mold, pressurizes at saturating
Bright or translucent sheet, is placed in infrared spectrophotometer and detects, 4000~400cm of test scope-1.Fig. 1 is raw material chitosan oligosaccharide
(COS), the acylated chitosan oligosaccharide derivative of intermediate product chitosan oligosaccharide biguanide hydrochloride (COSG), products therefrom N- biguanides O- of the present invention
(O-COSG) infrared spectrum, in figure: the variation tendency of the infrared spectroscopy figure line of COS infrared spectroscopy figure line and COSG, O-COSG is big
Cause identical, comparison can be seen that, O-COSG is in 1620~1640cm-1Place, 1060cm-1Vicinity absorption peak is remarkably reinforced, the two
Peak is all the characteristic peak of secondary amine group, illustrates that intramolecular secondary amine class group increases, and shows the acylated chitosan oligosaccharide derivative of N- biguanides O-
Guanidinated product is remained on amino.3400cm-1Vicinity absorption peak is that O-H the and N-H stretching vibration absworption peak of hydrogen bond is portion
Divide and be overlapped and broadening multi-absorption peak, compared with chitosan oligosaccharide, which is remarkably reinforced chitosan oligosaccharide biguanide hydrochloride, same explanation point
N-H content increases in son, in addition, the acylated chitosan oligosaccharide derivative of N- biguanides O- is in 2890~2920cm-1The absorption of methylmethylene
The increase at peak, 1740cm-1It is nearby O- acylated stretching vibration absworption peak and 1160cm-1Carbonyl absorption peak is all confirmed not
Hydroxy cinnamic acid derivative is successfully grafted on chitosan oligosaccharide strand while destroying guanidine radicals amino.
(2) the acylated chitosan oligosaccharide derivative biguanides ultraviolet spectra characterization of N- biguanides O-
The acylated chitosan oligosaccharide derivative of the N- biguanides O- prepared in the embodiment of the present invention 1 is taken to carry out uv scan, detection
Chromophore therein.The condition of scanning are as follows: 200~330nm of wavelength, sampling interval 0.5nm, scanning speed 400nm/min, slit
Width 4.0nm.Fig. 2 is the uv-spectrogram of N- biguanides O- acylated chitosan oligosaccharide derivative (O-COSG), is analyzed the spectrogram: N-
There is absorption peak in the vicinity 270nm in the acylated chitosan oligosaccharide derivative of biguanides O-, this is the characteristic absorption peak of guanidine radicals and carbonyl, is represented
Be C=N and C=O group.And due to being connected with-Cl ,-NH on guanidine radicals in derivative2Two auxochromes, so that acylated chitosan oligosaccharide
Two peaks cannot be distinguished to herein, because of position similar in the absorption peak of itself and carbonyl is in the red shift of biguanides ultraviolet absorption peak.
Investigate the solubility property of modified chitosan oligosaccharide.It is acylated precisely to weigh the N- biguanides O- prepared in the embodiment of the present invention 1
Chitosan oligosaccharide derivative 0.05g is dissolved into 5mL dimethyl sulfoxide, dimethylformamide, methylene chloride, methanol, tetrahydro furan respectively
It mutters, in hexamethylene, ethyl acetate, chloroform, peanut oil, soybean oil, is put into ultrasonator and vibrates one hour, stand number later
Hour, observation dissolution situation.Table 1 is the test result of the solubility property of the acylated chitosan oligosaccharide derivative of N- biguanides O-, and show: N- is bis-
The acylated chitosan oligosaccharide derivative of guanidine O- dissolve in dimethyl sulfoxide, dimethylformamide, methylene chloride, methanol, chloroform, peanut oil,
Soybean oil can be swollen in tetrahydrofuran, hexamethylene, ethyl acetate.Obviously, the introducing of acyl group largely reduced chitosan point
The effect of hydrogen bond between son improves the dissolubility of product in organic solvent.
N- biguanides O- is acylated chitosan oligosaccharide derivative
Note: √ represent dissolution, zero represent swelling, × represent it is insoluble
The soybean oil butanol solution 100mL for preparing 1g/L, takes solution 18mL to be placed in five 25mL conical flasks respectively,
Remaining liq is blank sample, and five conical flasks are separately added into 0.02g raw material chitosan oligosaccharide, chitosan oligosaccharide biguanide hydrochloride and embodiment 1
The acylated chitosan oligosaccharide derivative of resulting N- biguanides O-, in order to eliminate error, ultrasonator shakes at room temperature before experiment
Swing 2h.Baseline is done as reference liquid using the supernatant of soybean oil butanol solution, carries out ultraviolet whole process with ultraviolet spectrometry luminance meter
Scanning.Ultra-violet analysis condition: 200~330nm of wavelength, sampling interval 0.5nm scan 400nm/min, slit width 4.0nm.Figure
3 be chitosan oligosaccharide (COS), chitosan oligosaccharide biguanide hydrochloride (COSG) and N- biguanides O- acylated chitosan oligosaccharide derivative (O-COSG) in soybean
UV absorption spectrogram in oily butanol solution, table 2 are chitosan oligosaccharide (COS), chitosan oligosaccharide biguanides (COSG) and N- biguanides O- acylated
UV scanning data of the chitosan oligosaccharide derivative (O-COSG) in soybean oil butanol solution.
The ultraviolet whole scanning result of 2 COS, COSG, O-COSG of table
The acylated chitosan oligosaccharide derivative of chitosan oligosaccharide, chitosan oligosaccharide biguanide hydrochloride and N- biguanides O- is to unsaturated fat in grease
Acid shows certain adsorption capacity.Although they are all in 210cm-1Nearby there are absorption peaks, but COS, COSG, O- is added
Absorption peak area is respectively 22.213,19.609,21.624Abs*nm after COSG, in contrast to the absorption peak area of pure peanut oil
The equal decrease to some degree of 23.407Abs*nm shows that it has certain adsorption capacity to unsaturated fatty acid in grease.
And its adsorption rate that COS, COSG, O-COSG is added is respectively 5.329%, 16.226%, 7.617%, the reason is that chitosan oligosaccharide
In be rich in amino, have powerful Anion-adsorption ability, easily and grease in unsaturated fatty acid association reaction, reach absorption work
With not only containing amino in chitosan oligosaccharide guanidine, there are also guanidine radicals, thus suction-operated is more preferable.N- biguanides O- is acylated chitosan oligosaccharide derivative
Adsorption rate be greater than chitosan oligosaccharide, but be not so good as chitosan oligosaccharide guanidine, the introducing of phenyl ring, increases steric hindrance on hydroxy cinnamic acid derivative,
And then affect the combination of amino and unsaturated fatty acid.
Using mtt assay test cell activity, to judge the cell viability of experimental product in embodiment 1,2 and 3.Concrete operations
Steps are as follows: human hepatoma cell HepG2 is placed in the DMEM high glucose medium containing 15% fetal calf serum and is cultivated, condition
For 37 DEG C, 5%CO2, a culture solution is replaced for 24 hours.Cellular morphology is polygonal adherent growth.It takes and is in the thin of logarithmic growth phase
Born of the same parents' suspension is 1.5 × 10 by cell concentration4A/mL is inoculated in 96 porocyte culture plates, every 150 μ L of hole, 37 DEG C, 5%CO2Item
It is cultivated under part for 24 hours, after cell monolayer adherent growth, it is (dry using the culture solution containing drug to be administered processing respectively to cell
In advance), after continuing culture for 24 hours, the MTT solution that 15 μ L 5mg/mL now match is added in every hole, cultivates 4h, culture solution in orifice plate is sucked out, often
Hole adds 150 μ L dimethyl sulfoxide DMSO, and after vibrating a few minutes, object to be crystallized after completely dissolution, measures under 570nm wavelength
The light absorption value (OD value) of sample.16 groups are measured altogether, and 8 kinds of drugs, every kind of concentration is grouped into 200 μ g/mL of high dose group, middle dosage
Group 100 μ g/mL, 50 μ g/mL of low dose group.Concrete condition see the table below.
The reflection of Fig. 4 histogram is chitosan oligosaccharide, chitosan oligosaccharide biguanide hydrochloride and embodiment 1,2 under different dosing concentration
With 3 in the acylated chitosan oligosaccharide derivative of the N- biguanides O- for preparing on the active influence of human hepatoma cell HepG2, ordinate is inhaled bright
The size of angle value (OD value) is able to reflect out cell viability, and wherein the OD value of blanc cell experiment is 1.875, addition chitosan oligosaccharide,
The N- biguanides O- of chitosan oligosaccharide biguanide hydrochloride and different esterification degrees is acylated chitosan oligosaccharide derivative, and cell viability is significantly increased,
And improvement effect is different with the difference of dosage.Analysis: comparing each group of data can be seen that, chitosan oligosaccharide, chitosan oligosaccharide biguanides hydrochloric acid
The acylated chitosan oligosaccharide derivative administration group of the N- biguanides O- of salt and different esterification degrees inhales brightness value (OD value) number with respect to blank control group
According to being obviously improved, show that the N- for chitosan oligosaccharide, prepared chitosan oligosaccharide guanidine hydrochloride and the different esterification degrees that this experiment is selected is bis-
The acylated chitosan oligosaccharide derivative of guanidine O- is to the cell viability effect of being improved, no cytotoxicity effect.Especially in high dose intervention
Under, the cell viability value of COS, COSG, O-COSG-MDS, O-COSG-HDS group, which has, generally to be improved, and OD you can well imagine compared with blank elements
It is high by 33.51%, 44.85%, 42.78%, 43.81%.And the acylated chitosan oligosaccharide of N- biguanides O- of three kinds of different esterification degrees spreads out
Biology has a certain upgrade to cell activity, and correlation data can be found, under Isodose, the acylated chitosan oligosaccharide derivative of N- biguanides O-
The group OD value size of intervention sorts are as follows: O-COSG-LDS < O-COSG-MDS < O-COSG-HDS shows that esterification degree is higher, right
The castering action of HepG2 cell viability is more obvious.It observes simultaneously, the acylated chitosan oligosaccharide derivative of N- biguanides O- is to cell viability
There are apparent dose dependents for influence, and dosage is bigger, better to the promotion effect of cell viability, and equal no cytotoxicity.
The N- biguanides O- that remaining embodiment of the invention and according to the present invention content carry out technical arrangement plan preparation is acylated
Chitosan oligosaccharide derivative shows the performance being basically consistent with embodiment 1 above, and illustrates that technical solution of the present invention is realized for shell
The growth of oligosaccharides amphipathic (hydrophily and lipophilicity), increases the dissolubility of chitosan oligosaccharide and helps to improve cell activity not
Cellulotoxic side effect improves the application in cell active substance in preparation.Illustrative description has been done to the present invention above, has been answered
The explanation, in the case where not departing from core of the invention, any simple deformation, modification or other art technologies
Personnel can not spend the equivalent replacement of creative work to each fall within protection scope of the present invention.
Claims (10)
1. no cytotoxicity N- biguanides O- is acylated chitosan oligosaccharide derivative, which is characterized in that it is prepared with chitosan oligosaccharide and dicyandiamide reaction,
With chitosan oligosaccharide side group NH2It is reacted under hydrochloric acid and microwave condition with dicyandiamide and forms the biguanides base bonded with chitosan oligosaccharide
Group, then reacted with hydroxyl on chitosan oligosaccharide with the carboxyl of hydroxy cinnamic acid derivative, so that hydroxy cinnamic acid derivative passes through
Ester group is keyed on chitosan oligosaccharide, is shown below:
In above-mentioned chemical formula, m and n are respectively the degree of polymerization of acetylated unit and deacetylated unit in chitosan oligosaccharide, R1:-CH=
CH-CO-；R2:-OH or-H；R3:-OH ,-H or-OCH3。
2. the acylated chitosan oligosaccharide derivative of no cytotoxicity N- biguanides O- according to claim 1, which is characterized in that chitosan oligosaccharide
Weight average molecular weight is 5000 hereinafter, it is preferred that 1500-3000 (KDa), deacetylation is 97% or more, preferably 98-99%.
3. the acylated chitosan oligosaccharide derivative of no cytotoxicity N- biguanides O- according to claim 1, which is characterized in that cell-free
In the acylated chitosan oligosaccharide derivative of toxicity N- biguanides O-, biguanides degree of substitution is 40-80%, preferably 50-70%；Acylation degree between
0.20~1.45, preferably 0.5-1.2.
4. the acylated chitosan oligosaccharide derivative of no cytotoxicity N- biguanides O- according to claim 1, which is characterized in that work as reaction
When object hydroxy cinnamic acid derivative is cinnamic acid, R in product1For-CH=CH-CO-, R2For-H；R3For-H；Hydroxycinnamic acid spreads out
When biology is caffeic acid, R in product1For-CH=CH-CO-, R2For-OH；R3For-OH；Hydroxy cinnamic acid derivative is ferulic acid
When, R in product1For-CH=CH-CO-, R2For-OH；R3For-OCH3；When hydroxy cinnamic acid derivative is p-Coumaric Acid, in product
R1For-CH=CH-CO-, R2For-OH；R3For-H.
5. the preparation method of the acylated chitosan oligosaccharide derivative of no cytotoxicity N- biguanides O-, which is characterized in that as steps described below into
Row:
Step 1, chitosan oligosaccharide is dispersed in hydrochloric acid and is stirred under the conditions of microwave 160W~560W, it is anti-to carry out first step microwave
It answers；Dicyandiamide aqueous solution is added later and the use of hydrochloric acid adjustment pH value of reaction system is 1-2, continues in microwave 100W~600W
Under the conditions of stir, carry out second step microwave reaction so that chitosan oligosaccharide and dicyandiamide reaction prepare chitosan oligosaccharide Biguanide derivative；Its
In: in first step microwave reaction, microwave power is 300-400w, and the reaction time is 3-7min, and mixing speed is per minute
100-200 turns；In second step microwave reaction, microwave power be 300-400w, the reaction time be 5-30min, preferably 10-
20min, mixing speed are 100-400 turns per minute, and reaction temperature is 80~100 DEG C；Dicyandiamide rubs with amino in chitosan oligosaccharide
You are than being (0.1-5): 1；
Step 2, chitosan oligosaccharide biguanide hydrochloride powder and laccase prepared by step 1 are dispersed in phosphate buffer, and
Hydroxy cinnamic acid derivative is added to be reacted, to obtain the acylated chitosan oligosaccharide derivative of N- biguanides O-, in which: phosphate buffer
PH be 7-8,4~8h of reaction time, reaction temperature be 20~40 DEG C, 300~500r/min of mixing speed；Hydroxycinnamic acid
The molar ratio of hydroxyl is (0.1~5) in derivative and chitosan oligosaccharide biguanide hydrochloride: 1.
6. the preparation method of the acylated chitosan oligosaccharide derivative of no cytotoxicity N- biguanides O- according to claim 5, feature
It is, in step 1, in first step microwave reaction, microwave power is 300-400w, 5-7min of reaction time；Second
It walks in microwave reaction, microwave power is 300-400w, and the reaction time is 10-20min.
7. the preparation method of the acylated chitosan oligosaccharide derivative of no cytotoxicity N- biguanides O- according to claim 5, feature
It is, in step 1, the molar ratio of amino is (1-3) in dicyandiamide and chitosan oligosaccharide: 1；Hydrochloric acid is the chlorine of 0.2~0.8mol/L
Change aqueous solution of hydrogen, the use of hydrochloric acid adjustment pH value of reaction system is 1.
8. the preparation method of the acylated chitosan oligosaccharide derivative of no cytotoxicity N- biguanides O- according to claim 5, feature
It is, laccase is added as catalyst using laccase aqueous solution, and concentration is 1-5mg/mL (laccase quality/water body
Product), the volume ratio of laccase aqueous solution and phosphate buffer is (0.1-0.5): (40-50).
9. the preparation method of the acylated chitosan oligosaccharide derivative of no cytotoxicity N- biguanides O- according to claim 5, feature
It is, the molar ratio of hydroxyl is (1-3) in hydroxy cinnamic acid derivative and chitosan oligosaccharide biguanide hydrochloride: 1, the reaction time 6~
8h, reaction temperature are 30~40 DEG C.
10. the acylated chitosan oligosaccharide derivative of no cytotoxicity N- biguanides O- as described in one of claim 1-4 improves thin in preparation
Application in cytoactive substance, which is characterized in that improve that chitosan oligosaccharide is amphipathic and dissolubility, without cellulotoxic side effect.
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| CN115785291A (en) * | 2023-01-19 | 2023-03-14 | 常熟威怡科技有限公司 | Preparation method and application of biguanide crosslinked carboxyl cellulose material |
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