CN108003257B - A kind of chitosan oligosaccharide and its preparation method and application with specific structure - Google Patents
A kind of chitosan oligosaccharide and its preparation method and application with specific structure Download PDFInfo
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Abstract
The invention discloses a kind of chitosan oligosaccharide and its preparation method and application with specific structure.The reducing end and non-reducing end of all components of the chitosan oligosaccharide are aminoglucose sugar, deacetylation 50%-80%.The chitosan oligosaccharide can be obtained by enzyme hydrolysis chitosan substrate, used enzyme specific recognition and can hydrolyze the glycosidic bond that aminoglucose sugar and Glucosamine are formed, so that its reducing end of all components of chitosan oligosaccharide and non-reducing end that hydrolysis obtains are aminoglucose sugar.Compared to the chitosan oligosaccharide of ordinary construction, the chitosan oligosaccharide of specific structure of the present invention has higher inhibitory activity to liver cancer cells, can be applied to the fields such as prevention and the adjuvant treatment of liver cancer.
Description
Technical field
The invention belongs to chitosan oligosaccharide applied technical fields, and in particular to a kind of chitosan oligosaccharide and its preparation with specific structure
Methods and applications.
Background technique
Chitosan oligosaccharide (Chitooligosaccharides, COS) is Glucosamine and N-acetylglucosamine through β -1,
Oligomer of the degree of polymerization less than 20 that 4 glucosides key connections are formed has the bioactivity such as anti-inflammatory, antitumor and immunological regulation.It grinds
Study carefully display, the structure of chitosan oligosaccharide, including structure features such as acetyl group site distributions in the degree of polymerization, deacetylation and oligosaccharides at it
Bioactivity plays decisive role in playing.The high temperature highly basic that traditional handicraft prepares chitin involved in chitosan process takes off second
Acyl, product chitosan only just have preferable solubility in acid in deacetylation > 80%.Therefore, existing industrial chitosan
Deacetylation is usual > and 80%, the acetyl content in chitosan is less.The chitosan oligosaccharide prepared using above-mentioned chitosan as substrate it is de-
Acetyl degree > 80%, therefore the acetylation monosaccharide component contained on chitosan oligosaccharide chain is also accordingly reduced, corresponding difference chitosan oligosaccharide component
Limited amount.
Even mutually deacetylated (Kurita K, the Sannan T, Iwakura of chitin may be implemented using low temperature alkaline process
Y.Studies on chitin,.4.Evidence for formation of block and random copolymers
of N-acetyl-D-glucosamine and D-glucosamine by heterogeneous and homogeneous
hydrolyses[J].Makromolekulare Chemie-Macromolecular Chemistry and Physics,
1977,178(12):3197-3202.Liu great Sheng, Wei Yuanan, Jiang Linbin wait the deacetylated preparation water soluble shells of ultrafine chitin poly-
Research [J] food science and technology of sugar, 2007,32 (9): 108-110.), deacetylation > 50% can be obtained using the above method
Low deacetylation chitosan.Due to N-acetylglucosamine (hereinafter referred to as A) and amino on the sugar chain of the type chitosan
Glucose (hereinafter referred to as D) content is higher, therefore can recognize the hydrolase of A and D, such as chitinase and chitosan enzyme
The type chitosan is hydrolyzed.The study found that different chitosan hydrolyzate enzymes identify exist to the glycosidic bond at hydrolysis position
Larger difference, if glycoside hydrolase Families 18 (GH18) chitinase can only identify and hydrolyze A-A and A-D type glycosidic bond, and
GH19 chitinase can only then identify and hydrolyze A-A and D-A type glycosidic bond；For chitosan enzyme, according to what is identified to glycosidic bond
Difference, be substantially classified as three hypotypes (I, II and III): I type can recognize hydrolysis D-D and A-D, and II type only identifies D-D, and
Type III can recognize D-D and D-A glycosidic bond.Even if hydrolyzing substrate is identical low deacetylation chitosan, but not due to these
Same enzyme has differences the identification of substrate, therefore can obtain different types of structure using different chitosanases
Chitosan oligosaccharide, the distribution such as the degree of polymerization and acetyl group site is different, and the chitosan oligosaccharide of these architectural differences can in particular organisms activity
Can there can be larger difference.
Summary of the invention
It is an object of the present invention to provide a kind of novel, the chitosan oligosaccharides and preparation method thereof with specific structure.Except this it
Outside, the purposes of novel chitosan oligosaccharide in medicine, the especially application in anti-liver cancer and anti-field is also found in present inventor.
To achieve the above object, the technical solution adopted in the present invention is as follows:
A kind of chitosan oligosaccharide with specific structure, shown in the structure of the chitosan oligosaccharide such as formula (Ι),
Wherein, n 0-18；R is H or COCH3。
The deacetylation of chitosan oligosaccharide of the invention is 50%-80%.Preferably, the deacetylation of the chitosan oligosaccharide is
56%-78%, it is further preferred that the deacetylation of the chitosan oligosaccharide is 60%-70%, still more preferably de-
Acetyl degree is 60-65%, and most preferred deacetylation is 62%.The deacetylation refers to chitosan oligosaccharide structure shown in formula (Ι)
In, deacetylation chain link accounts for the score of total chain link.Assuming that the constitutional repeating unit of chitosan oligosaccharide is 100, wherein acetylamino Portugal
Sugared structural unit is 22, and Glucosamine structural unit is 78, then corresponding deacetylation is 78%.By controlling formula
R is COCH in (Ι)3Quantitative proportion, that is, can control corresponding deacetylation.
Low deacetylation chitosan described herein refers to chitosan of the deacetylation less than 80%.
The present invention also provides the preparation method of the chitosan oligosaccharide with specific structure, this method are as follows: pass through enzyme hydrolysis
Chitosan substrate obtains the chitosan oligosaccharide, and used enzyme specific recognition and can hydrolyze aminoglucose sugar and Glucosamine
The glycosidic bond of formation, its reducing end of all components of chitosan oligosaccharide and non-reducing end for obtaining hydrolysis are aminoglucose sugar.Institute
State the specific steps of chitosan oligosaccharide preparation method are as follows: (1) deacetylated acquisition chitosan substrate is carried out to chitin, it is de- by controlling
The acetylization reaction time obtains the chitosan of different deacetylations；(2) it utilizes specific recognition and hydrolyzes the enzyme of D-D type glycosidic bond
The chitosan substrate is hydrolyzed, the chitosan oligosaccharide product is obtained.
Preferably, the deacetylation of the chitosan be 50%-80%, further preferably 56%-78%, further
Preferably, the deacetylation of the chitosan oligosaccharide is 60%-70%, and deacetylation still more preferably is 60-65%, most
Preferred deacetylation is 62%.The deacetylation of chitosan can be controlled by the control de-acetyl chitin reaction time
System.
In the present invention, the deacetylation of chitin carries out under conditions of alkaline low-temperature.It can be used for deacetylated
Alkali include sodium hydroxide and potassium hydroxide, preferred alkali is sodium hydroxide.Deacetylated temperature range is 40 DEG C -90 DEG C, excellent
The temperature range of choosing is 50 DEG C -60 DEG C.The concentration range 40%-50% of aqueous slkali, preferably 45%.Deacetylation
Time range is 0.5h-24h, preferably 2h-6h.
In a specific embodiment of the invention, chitin in the sodium hydroxide solution of 45wt% at 60 DEG C into
Row deacetylation controlled the deacetylation time in the range of 0.5 to 24 hour, can get 50%-80% and takes off second
The chitosan of acyl degree.
The present invention is using specific recognition and capable of hydrolyzing the enzyme of D-D type glycosidic bond and carry out water to the chitosan substrate
Solution.Preferably, the enzyme is neutral proteinase.Neutral proteinase used in the embodiment of the present invention is from withered grass gemma
The natural protease thick enzyme (Neutral protease) of bacillus.
Different enzymes needs different hydrolysising conditions to the hydrolysis of chitosan substrate.According to the enzyme viability used, ability
The technical staff in domain can determine suitable hydrolysising condition by testing.In the present invention, it is preferred to use neutral protein water
Solving enzyme, the actual conditions of the neutral proteinase hydrolysis chitosan are as follows: neutral protein enzyme dosage is the 2wt%-25wt% of substrate,
Preferably 5wt%-20wt%, more preferably 5wt%-15wt%；Hydrolysis temperature is 25-55 DEG C, and preferred temperature is 30-
45 DEG C, more preferably 35-45 DEG C；Hydrolysis time is 30-60h, preferably 40-50h.
Purposes the present invention also provides the chitosan oligosaccharide with specific structure as drug.
Invention further provides the chitosan oligosaccharides with specific structure to prepare the application in medicines resistant to liver cancer.
The present invention also provides a kind of pharmaceutical composition, the pharmaceutical composition contain the chitosan oligosaccharide with specific structure or
The chitosan oligosaccharide pharmaceutically acceptable salt is as active component.
Pharmaceutical composition of the invention further contains and is pharmaceutically subjected to other than containing chitosan oligosaccharide of the invention
Carrier.Here " pharmaceutically acceptable carrier " refers to pharmaceutically acceptable material, composition or medium, as liquid or solid is filled out
Material, diluent, auxiliary material, solvent or encapsulating material, including from certain part of an organ or body to another organ or body
Certain part carry or transport main pharmaceutical reagent.Each carrier must be " can receive ", can be compatible with the drug of other forms
It is not damaged at and to patient.Some examples that can be used as pharmaceutically acceptable carrier include: sugar, such as lactose, grape
Sugar and sucrose sugar；Starch, such as wheaten starch and potato starch starch；Cellulose and its derivates, as sodium carboxymethyl cellulose,
Ethyl cellulose, cellulose acetate, powdered gum tragacanth, malt, gelatin, talcum powder；Auxiliary material, such as cocoa butter and suppository wax；
Oil, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soya-bean oil；Glycol, such as butanediol；Polyalcohol, such as
Glycerol, sorbierite, mannitol and polyethylene glycol；Ester, such as ethyl oleate and ethyl laurate；Agar；Buffer, such as magnesium hydroxide
And aluminium hydroxide；Alginic acid；Apirogen water；Physiological saline；Ringer's solution；Ethyl alcohol；Phosphate buffer and other are nontoxic
Apply the compatible substance in pharmaceutical preparation.
Compared with prior art, beneficial effects of the present invention are as follows:
1, it is poly- to the shell lower than 80% deacetylation by the chitosanase using different hydrolytic sites identification types
Sugar is hydrolyzed, and is identified on this basis the structure of product chitosan oligosaccharide and evaluates their differences in terms of anticancer activity
It is different.It is surprisingly found by the inventors that the chitosan oligosaccharide of one of specific structure type is substantially better than other in terms of resisting liver cancer activity
Chitosan oligosaccharide has biggish application potential in anti-liver cancer and anti-field.Its structure law of the chitosan oligosaccharide of the specific structure is: chitosan oligosaccharide
The reducing end and non-reducing end of all components are aminoglucose sugar.
2, the chitosan oligosaccharide for the specific structure that present invention research obtains has better pharmacological activity, can be used in medical neck
Domain.Specifically, the chitosan oligosaccharide of the specific structure of present disclosure has the activity for inhibiting liver cancer cell growth, can be applied to
Prepare medicines resistant to liver cancer or pharmaceutical composition.
Detailed description of the invention
Fig. 1 is 62% chitosan of deacetylation in the embodiment of the present invention 11H-NMR map.
Fig. 2 is the MALDI-TOF mass spectrogram of different type chitosan oligosaccharide in the embodiment of the present invention 2.
Fig. 3 A- Fig. 3 D is the 1H-NMR and 13C-NMR of chitosan oligosaccharide COS-62-PA and COS-62-NP in the embodiment of the present invention 2
Map.Wherein, Fig. 3 A and Fig. 3 B correspond to COS-62-PA's1H-NMR and13C-NMR spectrogram；Fig. 3 C and Fig. 3 D correspond to COS-62-NP
1H-NMR and 13C-NMR spectrogram.
Fig. 4 be in the embodiment of the present invention 3 different types of structure chitosan oligosaccharide to the graphic data of HepG2 cell growth effect
Figure.
Fig. 5 A- Fig. 5 B is that various concentration chitosan oligosaccharide COS-62-NP and positive drug 5Fu are thin to HepG2 in the embodiment of the present invention 4
The bar shaped datagram that intracellular growth influences；Wherein Fig. 5 A corresponds to the schematic diagram that COS-62-NP inhibits cells survival rate, the corresponding sun of Fig. 5 B
Property medicine 5Fu inhibit cells survival rate schematic diagram.
Fig. 6 A- Fig. 6 E is that different desacetylated degree chitosans are corresponding in the embodiment of the present invention 51H-NMR map；Wherein Fig. 6 A
It is the corresponding deacetylation of 66%, Fig. 6 C is 70%, Fig. 6 D couple that corresponding deacetylation, which is the corresponding deacetylation of 56%, Fig. 6 B,
The deacetylation answered is that the corresponding deacetylation of 74%, Fig. 6 E is 78%.
Fig. 7 be in the embodiment of the present invention 5 different desacetylated degree chitosans to the graphic data of HepG2 cell growth effect
Figure.
Specific embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.It is not specified in embodiment specific
Condition person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer is
It can be with conventional products that are commercially available.
Embodiment 1: different types of structure low deacetylation chitosan oligosaccharide preparation
With reference to Liu great Sheng etc., (Liu great Sheng, Wei Yuanan, Jiang Linbin wait preparation of water-soluble chitosan by deacetylation of ultrafine chitin
Research [J] food science and technology, 2007,32 (9): 108-110.) method prepare low deacetylation chitosan, specific method is such as
Under: the chitin powder 30g after pulverizing is weighed, is added in 10 times of 45% sodium hydroxide solution, stirs evenly, then
Be warming up to 60 DEG C of progress deacetylation 2h, be centrifuged after completion of the reaction, sediment with 60% alcohol-water mixed solution wash to
Without alkalinity, product is obtained after dry.It uses1H-NMR measures its deacetylation (referring to Fig. 1), according to Fig. 1's1H-NMR map determines
Its deacetylation is 62%.Two parts of chitosan of preparation are weighed, every part of 10g is added to water-soluble equipped with 1.5% acetic acid of 200mL respectively
In the isothermal reaction kettle of liquid, it is sufficiently stirred and makes it completely dissolved.Reaction temperature is adjusted to 40 DEG C, is separately added into 1 gram of neutral protein
Enzyme dry powder (be abbreviated as NP, enzyme activity: 50,000 U/g get profit bioengineering Co., Ltd purchased from Taian Shandong letter) and 1g papain
Powder (be abbreviated as PA, enzyme activity: 50,000 U/g are purchased from Pangbo Bioengineering Co Ltd, Nanning), isothermal reaction 48 hours.Reaction knot
Shu Hou, centrifugation removal insoluble substance, supernatant are concentrated into 50-100mL through Rotary Evaporators at 40 DEG C, then freeze-dried finished product
Chitosan oligosaccharide, is successively named as COS-62-NP (the chitosan oligosaccharide product after referring to neutral proteinase hydrolysis) and COS-62-PA (refers to pawpaw
Chitosan oligosaccharide product after protease hydrolytic).
Embodiment 2: the composition and Structural Identification of different type chitosan oligosaccharide
The chitosan oligosaccharide component in above-mentioned three kinds of chitosan oligosaccharides is identified using MALDI-TOF mass spectrometry method.Specific method
Are as follows: three kinds of chitosan oligosaccharide sample COS-62-NP, COS-62-PA and COS-MP-162 for preparing or buying are weighed, are configured with ultrapure water
The aqueous solution for being 2mg/mL at concentration, it is each to draw on 1 μ L point sample to sample panel, it is each that 1 μ L matrix 2 is added after it is spontaneously dried,
5- dihydroxy-benzoic acid (DHB) solution uses III smartbeam type MALDI-TOF mass spectrograph of autoflex after its drying
(Bruker company) is detected (cation reflective-mode).Mass Spectrometer Method result is as shown in Figure 2: respectively correspond COS-62-NP,
The mass spectrogram of COS-62-PA and COS-MP-162；To represent N-acetylglucosamine in Fig. 2 with A convenient for distinguishing, D is represented
Glucosamine, subsequent number represent the number containing the monosaccharide, and the two adduction is the degree of polymerization of oligosaccharides.From the mass spectrum of Fig. 2
As a result from the point of view of, commodity chitosan oligosaccharide COS-MP-162 is mainly grouped as by complete deacetylated chitosan oligosaccharide group, and low deacetylation chitosan oligosaccharide
(COS-62-NP and COS-62-PA) is then made of the oligosaccharide compositions containing more N-acetylglucosamine.In addition, according to fig. 2
Mass spectrogram known to: the degree of polymerization of low deacetylation chitosan oligosaccharide (COS-62-NP and COS-62-PA) is between 2-20.Not only such as
This, even identical substrate, using different enzyme hydrolysis, there is also apparent difference, neutral proteinase water for the structure of oligosaccharides
It is higher to solve deacetylation of the chitosan oligosaccharide of low deacetylation chitosan acquisition in detection range, the oligosaccharide compositions containing acetyl group
In there is only 1-3 acetyl group, and papain hydrolysis low deacetylation chitosan obtains the chitosan oligosaccharide in detection range
Deacetylation is lower, contains 3-6 acetyl group.
It is further since MALDI-TOF mass spectrometry method can only preferably detect the component below of molecular weight 2000
The structure feature of the different type chitosan oligosaccharide of preparation is further confirmed that, is used1H-NMR and13C-NMR is to COS-62-PA
And the reducing end and non-reducing end structure feature of COS-62-NP are analyzed, and as shown in Fig. 3 A- Fig. 3 D, wherein Fig. 3 A and Fig. 3 B
Corresponding COS-62-PA's1H-NMR and13C-NMR spectrogram；Fig. 3 C and Fig. 3 D correspond to 1H-NMR and the 13C-NMR spectrum of COS-62-NP
Figure.The spectrogram of Fig. 3 A- Fig. 3 D the results show that the reducing end of COS-62-PA includes monosaccharide unit A and D (as shown in Figure 3A) simultaneously,
And non-reducing end also includes A and D (as shown in Figure 3B) simultaneously.The result illustrates may be simultaneously containing there are many water in papain
The chitosan hydrolyzate enzyme of type is solved, to generate reducing end and the more complex chitosan oligosaccharide product of non-reducing end structure.It is opposite and
Speech, the structure of COS-62-NP is more regular to be followed: reducing end and non-reducing end are made of D sugar unit (such as Fig. 3 C and 3D institute
Show), there is the structure feature for being easy to differentiate.This may be since play chitosan hydrolysis in neutral proteinase is more single
One enzyme, according to the reducing end of product and non-reducing end characteristic, it should be a kind of II for being only capable of identifying and hydrolyzing D-D glycosidic bond
Shell dextranase.Oligosaccharide compositions at least contain that there are two D in this MALDI-TOF mass spectral results (as shown in Figure 2) with COS-62-NP
Monosaccharide is also very identical.
Embodiment 3: different types of structure chitosan oligosaccharide resisting liver cancer activity compares
MEM culture medium (10%FBS, S/P, 1% will be added after human hepatoma cell line HepG2's digestion of culture to logarithmic phase
NAEE) it is diluted to 1 × 104A/ml, 100 μ l/well are inoculated with 3 piece of 96 porocyte culture plates, 37 DEG C of 5%CO2In incubator overnight
It cultivates completely adherent to cell.Prepare three kinds of chitosan oligosaccharide (COS-62-NP, COS-62-PA and COS-MP-162) aqueous solutions
(10mg/ml), in 0.22 μm of membrane filtration degerming in iuntercellular super-clean bench.With MEM culture medium by three kinds of shell widows of above-mentioned preparation
Sugar aqueous solution is diluted to 200 μ g/ml and (takes the oligosaccharide solution of 60 μ l 10mg/ml that MEM culture medium 2940 μ l to 3ml), 100 μ is added
L/well is inoculated with 3 piece of 96 porocyte culture plates (the final concentration of 100 μ g/ml of chitosan oligosaccharide), 37 DEG C of 5%CO2Continue to train in incubator
It supports.Simultaneously using the 5 FU 5 fluorouracil (5Fu) of same concentrations as positive control, negative control is made with blank cultures MEM.Administration
It is added in 96 porocyte culture plates of 72h afterwards and continues to cultivate 4h at 37 DEG C in 20 μ l/well, 5%CO2 incubator of MTT, with row
The liquid that rifle is sucked out in culture plate in each hole discards, and 100 μ l/well of DMSO is added, measures the OD490 value in each hole, and use
The mapping of OriginPro8.5 software.With MEM culture medium group for 100% cells survival rate, three kinds of different chitosan oligosaccharides and sun are calculated
The cells survival rate of property comparison medicine group, concrete outcome are as shown in Figure 4.The results show that positive drug 5Fu grew HepG2 cell
Inhibiting effect is most obvious, cells survival rate be only 11.64%, COS-62-NP of control group also have to the growth of HepG2 cell it is bright
Aobvious inhibiting effect, cells survival rate is only 69.87% compareed, and chitosan oligosaccharide COS-62-PA and COS-MP-162 is thin to HepG2
The inhibition of born of the same parents is unobvious.
Embodiment 4: influence of the concentration to chitosan oligosaccharide COS-62-NP resisting liver cancer activity
MEM culture medium (10%FBS, S/P, 1%NAEE) dilution will be added after culture to the HepG2 cell dissociation of logarithmic phase
To 1 × 104A/ml, 100 μ l/well are inoculated with 3 piece of 96 porocyte culture plates, 37 DEG C of 5%CO2It is incubated overnight in incubator to cell
It is completely adherent.It prepares chitosan oligosaccharide COS-62-NP aqueous solution (10mg/ml), is removed in 0.22 μm of membrane filtration in iuntercellular super-clean bench
Bacterium.200 μ g/ml, 100 μ g/ml, 50 μ g/ml, 20 μ g/ml and 2 μ g/ml, 100 μ l/well are successively diluted to MEM culture medium
3 piece of 96 porocyte culture plates of inoculation (chitosan oligosaccharide final concentration correspondence halves), 37 DEG C of 5%CO2Continue to cultivate in incubator.While with
The 5 FU 5 fluorouracil (5Fu) of same concentrations is used as positive control, makees negative control with blank cultures MEM.The 96 of 72h after administration
MTT 20 μ l/well, 5%CO are added in porocyte culture plates2Continue to cultivate 4h at 37 DEG C in incubator, is sucked out and is cultivated with the volley of rifle fire
Liquid in plate in each hole discards, and 100 μ l/well of DMSO is added, measures the OD490 value in each hole, and use
The mapping of OriginPro8.5 software.With MEM culture medium group for 100% cells survival rate, various concentration chitosan oligosaccharide COS- is calculated
The cells survival rate of 62-NP and positive control drug group, as fig. 5 a and fig. 5b, wherein Fig. 5 A corresponds to COS-62- to concrete outcome
NP inhibits the schematic diagram of cells survival rate, and Fig. 5 B corresponds to the schematic diagram that positive drug 5Fu inhibits cells survival rate.The results show that shell
Oligosaccharides COS-62-NP also increase accordingly the inhibitory activity of HepG2 cell, positive drug 5Fu also obtains phase with the increasing of concentration
As a result, only inhibitory effect is more obvious.
Embodiment 5: influence of the deacetylation to chitosan oligosaccharide resisting liver cancer activity
To determine influence of the deacetylation to neutral proteinase hydrolysis low deacetylation chitosan product resisting liver cancer activity, into
For one step on the basis of 62% chitosan of deacetylation, with reference to Liu great Sheng etc., (Liu great Sheng, Wei Yuanan, Jiang Linbin wait the ultra-fine first of
Deacetylated research [J] the food science and technology for preparing water soluble chitosan of shell element, 2007,32 (9): 108-110.) method, 60
By adjusting deacetylation time (there are 1h, 3h, 6h, 9h, 12h in the reaction time respectively) at DEG C, prepare again different deacetylated
The chitosan of degree, passes through1H-NMR determines deacetylation, and as shown in Fig. 6 A- Fig. 6 E, wherein the corresponding deacetylation of Fig. 6 A is
The corresponding deacetylation of 56%, Fig. 6 B is that the corresponding deacetylation of 66%, Fig. 6 C is that the corresponding deacetylation of 70%, Fig. 6 D is
The corresponding deacetylation of 74%, Fig. 6 E is 78%.According to Fig. 6 A- Fig. 6 E's1H-NMR spectrum data determines that deacetylation is distinguished
For 56% (the deacetylation time is 1h), 66% (the deacetylation time is 3h), 70% (the deacetylation time is 6h),
74% (the deacetylation time is 9h) and 78% (the deacetylation time is 12h).In method use in reference implementation example 1
Above-mentioned chitosan substrate is hydrolyzed in property protease, and obtained product is successively denoted as DA56, DA66, DA70, DA74 and DA78,
Original COS-62-NP is also denoted as DA62 and is used for resisting liver cancer activity evaluation together.
The specific method reference implementation example 4 of above-mentioned difference deacetylation chitosan oligosaccharide resisting liver cancer activity evaluation, the shell used are few
Sugared final concentration is 100 μ g/mL.Simultaneously using the 5 FU 5 fluorouracil (5Fu) of same concentrations as positive control, with blank cultures
MEM makees negative control, as a result as shown in Figure 7.By the data result of Fig. 7 it is found that different deacetylation chitosan oligosaccharides all have one
Fixed resisting liver cancer activity, the resisting liver cancer activity of chitosan oligosaccharide is best when wherein deacetylation is 62%.
It above are only part preferred embodiment of the invention, the present invention is not limited in the content of embodiment.For ability
For technical staff in domain, can there are various change and change in the conception range of technical solution of the present invention, made
What changes and change, within that scope of the present invention.
Claims (5)
1. the application of the chitosan oligosaccharide with specific structure in medicine preparation, it is characterised in that: the drug is medicines resistant to liver cancer；
Shown in the structure of the chitosan oligosaccharide of the specific structure such as formula (Ι),
Wherein, n 0-18；R is H or COCH3；The deacetylation of the chitosan oligosaccharide is 50%-80%；
The chitosan oligosaccharide of the specific structure the preparation method comprises the following steps: obtain the chitosan oligosaccharide by enzyme hydrolysis chitosan substrate, adopted
Enzyme specific recognition and can hydrolyze the glycosidic bond that Glucosamine and Glucosamine are formed, and the shell for obtaining hydrolysis is few
Its reducing end of all components and non-reducing end of sugar are Glucosamine；The enzyme is neutral proteinase.
2. the application of the chitosan oligosaccharide according to claim 1 with specific structure in medicine preparation, it is characterised in that: institute
The deacetylation for stating chitosan oligosaccharide is 56%-78%.
3. a kind of anti-liver cancer drug compositions, the chitosan oligosaccharide or the chitosan oligosaccharide which contains specific structure are pharmaceutically
Acceptable salt is as active component；Shown in the structure of the chitosan oligosaccharide of the specific structure such as formula (Ι),
Wherein, n 0-18；R is H or COCH3；The deacetylation of the chitosan oligosaccharide is 50%-80%；
The chitosan oligosaccharide of the specific structure the preparation method comprises the following steps: obtain the chitosan oligosaccharide by enzyme hydrolysis chitosan substrate, adopted
Enzyme specific recognition and can hydrolyze the glycosidic bond that Glucosamine and Glucosamine are formed, and the shell for obtaining hydrolysis is few
Its reducing end of all components and non-reducing end of sugar are Glucosamine；The enzyme is neutral proteinase.
4. a kind of pharmaceutical composition according to claim 3, it is characterised in that: the deacetylation of the chitosan oligosaccharide is
56%-78%.
5. pharmaceutical composition according to claim 3, described pharmaceutical composition further contains pharmaceutically acceptable
Carrier.
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| AU2017101852A AU2017101852A4 (en) | 2017-10-11 | 2017-11-08 | Chitooligosaccharide with a specific structure and preparation method and application thereof |
| CA3041417A CA3041417C (en) | 2017-10-11 | 2017-11-08 | Chitooligosaccharide of specific structure, preparation method therefor and use thereof |
| PCT/CN2017/109990 WO2019071688A1 (en) | 2017-10-11 | 2017-11-08 | Chitooligosaccharide of specific structure, preparation method therefor and use thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1654483A (en) * | 2005-01-26 | 2005-08-17 | 哈尔滨工业大学 | Process for preparing water-soluble chitosan oligosaccharide |
| CN1746193A (en) * | 2005-09-15 | 2006-03-15 | 武汉大学 | Preparation of low-molecular weight chitoglycan or chitooligose |
| CN1803849A (en) * | 2005-12-16 | 2006-07-19 | 武汉大学 | Method for preparing complete water soluble low molecular weight chitosan/chitooligosaccharace |
| CN102653569A (en) * | 2012-05-11 | 2012-09-05 | 中国科学院海洋研究所 | Preparation method of chitosan oligosaccharide with single polymerization degree |
| CN107236721A (en) * | 2017-07-28 | 2017-10-10 | 中科荣信（苏州）生物科技有限公司 | A kind of bacillus subtilis chitosan enzyme and its preparation method and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1654483A (en) * | 2005-01-26 | 2005-08-17 | 哈尔滨工业大学 | Process for preparing water-soluble chitosan oligosaccharide |
| CN1746193A (en) * | 2005-09-15 | 2006-03-15 | 武汉大学 | Preparation of low-molecular weight chitoglycan or chitooligose |
| CN1803849A (en) * | 2005-12-16 | 2006-07-19 | 武汉大学 | Method for preparing complete water soluble low molecular weight chitosan/chitooligosaccharace |
| CN102653569A (en) * | 2012-05-11 | 2012-09-05 | 中国科学院海洋研究所 | Preparation method of chitosan oligosaccharide with single polymerization degree |
| CN107236721A (en) * | 2017-07-28 | 2017-10-10 | 中科荣信（苏州）生物科技有限公司 | A kind of bacillus subtilis chitosan enzyme and its preparation method and application |
Non-Patent Citations (3)
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| Action Pattern of Bacillus sp. No. 7-M Chitosanase on Partially N-Acetylated Chitosan;Masato Izume, et al.;《Biosci. Biotech. Biochem.》;19921231;第56卷(第3期);第448-453页 |
| 壳寡糖单体的制备及表征;杨国宁;《中国优秀硕士学位论文全文数据库-工程科技I辑》;20130215(第2期);第16-50页 |
| 壳聚糖酶的研究进展;张虎 等;《化学通报》;19991231(第5期);第32-35页 |
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| CA3041417C (en) | 2020-06-09 |
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