CN105085711B - A kind of preparation method and applications of chitosan oligosaccharide - Google Patents
A kind of preparation method and applications of chitosan oligosaccharide Download PDFInfo
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Abstract
A kind of preparation method and applications of chitosan oligosaccharide, the present invention relates to a kind of preparation method and applications of chitosan oligosaccharide.The present invention provides a kind of preparation method and applications of chitosan oligosaccharide.Method：Dissolve the chitosan in glacial acetic acid solution, then add hydrogen peroxide, heating stirring to 40~90 DEG C of constant temperature, reaction is used ethanol precipitation after terminating, filtered, and filter residue passes through drying, obtains chitosan oligosaccharide；Chitosan oligosaccharide prepared by the present invention is used for the medicine for preparing 10 plants of tumour cells for suppressing 5 kinds of people source tumours.It is effectively utilized its biocompatibility and tumour antagonistic activity.By analyzing the antitumor activity of chitosan oligosaccharide, help to promote anti-tumor agents development.The present invention is used for field of antineoplastic medicaments.
Description
Technical field
The present invention relates to a kind of preparation method and applications of chitosan oligosaccharide.
Background technology
Chitin (chitin) is widely present with the cell wall of the shell of shellfish, the crust of insect and fungi, being
Natural polymer the abundantest, content are only second to cellulose in nature.Chitin has a multiple functions characteristic, but its pole
The water soluble characteristic and biodegradability of difference significantly limit its extensive use.Relative to chitin is not soluble in water, diluted acid, dilute
The characteristic of alkali, ethanol or other organic solvents, chitosan (chitosan) is water-soluble and is showed in dilute acid soln higher
Viscosity.This dissolution characteristics also limit application of the chitosan in biological field.It is same to be used as chitin degrading product, shell widow
The dissolubility of sugared (chitosan oligosaccharides, COS) is more preferable, and viscosity is relatively low in physiological conditions, and this is due to
The sugar chain of chitosan oligosaccharide is shorter and free amine group.Contain an amino group and two in the glucoside residue repeat unit of chitosan oligosaccharide
Individual oh group, it is unique positively charged cation basic amine group oligosaccharide in nature.These characteristics of chitosan oligosaccharide cause
The concern of more and more researchers.There is the bioactivity of many document reports chitosan oligosaccharide, anti-oxidant, anti-inflammatory, antagonism are micro-
Biology, reduce cholesterol and enhancing immunocompetence, antitumor activity etc..Application of the chitosan oligosaccharide in many fields also has accordingly
Report, such as food, medicine, agricultural and environmental area.In addition, the good biocompatibility of chitosan oligosaccharide, nontoxic, to organs of living beings
Not sensitizing property, it is also set to develop into a kind of potential pharmaceutical carrier and tissue engineering bracket as nanometer -/micron system.
Tumour is to endanger a kind of common disease, the frequently-occurring disease of human health most serious.Tumour turns into the incidence of disease all over the world
With a major reason of the death rate, in the last few years with people life style change and environmental pollution aggravation, its send out
Sick rate is in ascendant trend, particularly developing country year by year.At present, the incidence of disease of China's tumour is 285.91/10 ten thousand, average every
It is per minute just to have 6 people to be diagnosed as malignant tumour.WHO predicts there are 20,140,000 newly-increased cases of cancers to the year two thousand thirty estimation whole world,
There to be 13,200,000 patients to die from cancer every year.There is document report, chitosan oligosaccharide has antitumor bioactivity, and its mechanism is direct
Suppress the growth of tumour cell, and have been reported that meronecrosis and apoptosis, reduce Tumor Angiongesis so as to realize suppression tumour
Cell shifts, it is also possible to is the effect for realizing antagonism tumor development indirectly by strengthening immunity of organisms.
Outer antitumor activity is many inside chitosan oligosaccharide, but its molecule mechanism is still to be unknown.Chitosan oligosaccharide has very
Good adhesion characteristics, can be combined with the surface glycoprotein of mammalian cell.Therefore, the characteristic changing of chitosan oligosaccharide positively charged
Cell membrane charging property, so as to destroy cell integrity, influence cell growth.Suzuki et al. has found that antineoplastic and tumour are thin
The electrostatic interaction of born of the same parents is the major reason that chitosan oligosaccharide has antitumor activity.Huang et al., which is used, has different charge numbers
Antagonism of the chitosan oligosaccharide to three plants of tumor cell lines, the results showed that the high chitosan oligosaccharide derivative of electrically charged amount can significantly reduce
The survival rate of tumour cell.
Except the influence, Huang et al. of electric charge finds that the molecular weight of chitosan oligosaccharide is also played emphatically its antitumor activity again
Act on.Salah et al. determines chitin, chitosan, small molecule chitin and people source tumour cell THP-1 antagonism is made
With, it is found that the antitumous effect of small molecule chitin is preferable, and as molecular weight reduces antitumous effect increase.
Angiogenesis provides metabolite, oxygen and nutriment, therefore quilt for the generation, development and transfer of tumor tissues
It is considered new action target spot in antitumor research.Chitosan oligosaccharide can suppress people source fibrosarcoma tissue matrix metalloproteinase
The expression of (matrix metalloproteinase-9, MMP-9), this occurs have important influence in metastases.MMP-9
Up-regulation matrix VEGF (Vascular Endothelial Growth Factor, VEGF) can be promoted to release
Put and angiogenesis.VEGF stimulates epithelial cell to expand and migrate, and this has regulating and controlling effect to vascular progenitor and angiogenesis.
Wu et al. determines inhibitory action of some chitosan oligosaccharide components to angiogenesis using several different biological methods, finds to suppress
Effect depends on the deacetylation and the degree of polymerization of component.
In addition, the immune-enhancing activity of chitosan oligosaccharide, which is also it, has one of major reason of antitumor activity.Suzuki etc.
People enhances immunocompetence using water-soluble preferably chitosan, and proves that chitosan oligosaccharide is swollen as immunocompetence enhancing inhibits
Knurl increases.Somebody thinks that chitosan oligosaccharide is unable to direct killing tumour, but by promoting the release of lymphokine to promote T- lymphs
Cell amplification plays tumour antagonistic activity.Suzuki et al. also confirm that chitosan oligosaccharide by activated lymphocyte discharge lymphokine come
Suppress tumour growth.In general, immunocompetence performance of the immunopotentiator by strengthening macrophage or neutrophil leucocyte is non-
Characteristic is immunocompetent.Most of immunopotentiators stimulate such as interferon, interleukin and ligandin immune response compound
Secretion, these compounds with will activating immune system after macrophage or lymphocyte surface receptors protein binding.One
A little immunopotentiators can be with specific receptors molecule competitive binding in infected tissue target cell.Chitosan oligomer can effectively promote
Enter the migratory activity of macrophage, because macrophage has chemotaxis.Ma et al. thinks chitosan oligomer under
Adjust the activation of MAPK and PI3K/Akt signal paths phosphorylation and NF- κ B and AP-1 and then suppress the secretion hair of IL-6 and TNF-α
Anti-inflammatory activity is waved.Chitosan oligosaccharide also plays immune-enhancing activity by strengthening antibody response.Chang et al. have studied shell widow
Sugar is to a kind of action effect for the influenza vaccines not inactivated.As a result show that chitosan oligosaccharide significantly improves the antibody content in serum,
Enhance the antiviral defense system of mouse.Chitosan oligosaccharide can also stimulating cytokine release, as interleukin I L-1 β and tumour cause
Necrosis factor TNF-α etc..
But existing chitosan oligosaccharide activity on tumour is suppressed is not good enough, and according to existing both at home and abroad from the point of view of bibliography,
The research of the administering mode and dosage of chitosan oligosaccharide in-vivo tumour antagonism is still blank, and chitosan oligosaccharide is to more tumor cell lines
The work for suppressing screening is rarely reported.Therefore the good chitosan oligosaccharide of activity is prepared, suppresses to have well on tumour medicine preparing
Application prospect.
The content of the invention
The present invention provides a kind of preparation method and applications of chitosan oligosaccharide.
A kind of preparation method of chitosan oligosaccharide, specifically prepared according to following steps：
Dissolve the chitosan in the glacial acetic acid solution that mass concentration is 0.1~5.0%, then add chitosan 5~25
The concentration of times weight is 10%~40% hydrogen peroxide, heating stirring to 40~90 DEG C of constant temperature, reacts 0.5~10h, then dense through quality
The ethanol precipitation for 60~100% is spent, is filtered, filter residue passes through drying, obtains the chitosan oligosaccharide of the degree of polymerization 2~30；Wherein chitosan oligosaccharide
The content of the middle degree of polymerization 2~10 is not less than 80%, the chitosan oligosaccharide purity not comprising moisture>98%；Chitosan is dense with quality
The mass volume ratio for spending the glacial acetic acid solution for 0.1~5.0% is 1g:(10~30) mL.
A kind of application of chitosan oligosaccharide refers to the medicine for preparing 10 plants of tumour cells for suppressing 5 kinds of people source tumours.
The present invention can not only effectively improve the utilization rate of abundant natural resources, be also effectively utilized its biocompatibility and
Tumour antagonistic activity.By analyzing the antitumor activity of chitosan oligosaccharide, help to drag anti-tumor agents development.It is dense in same test
The killing ability of chitosan oligosaccharide of the invention to HCT-116 and A549 is above commercially available chitosan oligosaccharide in the range of degree；It is dense in same test
Half-inhibition concentration excursion of the positive drug 5 FU 5 fluorouracil to the people source tumor cell line of ten kinds of separate sources in the range of degree
For 0.04 ± 0.01~12.1 ± 3.0 μ g/mL, chitosan oligosaccharide prepared by the present invention changes to the half-inhibition concentration of tumor cell line
Scope is 48.6 ± 7.0~1329.9 ± 93.4 μ g/mL.Chitosan oligosaccharide for ten kinds of separate sources people source tumor cell line most
Big inhibiting rate can reach more than 90%, even as high as 95%.The effect of killing tumour in chitosan oligosaccharide inside and outside prepared by the present invention
It is good.
Brief description of the drawings
Fig. 1 is that chitosan oligosaccharide prepared by embodiment 1 and three kinds of commercial chitosan oligosaccharides grow suppression to people source lung cancer tumor cell A549
Design sketch (n=4, ± sd) processed；Wherein a is COS, b COS-a, c COS-b, d COS-c；
Fig. 2 is that chitosan oligosaccharide prepared by embodiment 2 and three kinds of commercial chitosan oligosaccharides grow suppression to people source colon cancer cell HCT-116
Design sketch (n=4, ± sd) processed；Wherein a is COS, b COS-a, c COS-b, d COS-c；
Fig. 3 is inhibition figure (tumour of the chitosan oligosaccharide gastric infusion to S180 sarcoma transplantable tumor of the preparation of embodiment 4
Volume) (n=10, ± sd)；Wherein a is blank control group, and b is low dose group, and c is high dose group；
Fig. 4 is influence of the chitosan oligosaccharide gastric infusion of the preparation of embodiment 4 to tumor-bearing mice relative body weight；Wherein a is blank
Control group, b are low dose group, and c is high dose group；
Fig. 5 is inhibition figure (tumour body of the chitosan oligosaccharide gastric infusion to the residual knurl of S180 sarcoma of the preparation of embodiment 5
Product) (n=7, ± sd)；Wherein a is blank control group, and b is low dose group, and c is high dose group；
Fig. 6 is influence of the chitosan oligosaccharide gastric infusion of the preparation of embodiment 5 to tumor-bearing mice relative body weight；Wherein a is blank
Control group, b are low dose group, and c is high dose group；
Fig. 7 is that chitosan oligosaccharide intraperitoneal injection prepared by embodiment 6 is (swollen to the inhibition figure of the residual knurl of S180 sarcoma
Knurl volume) (n=10, ± sd)；Wherein a is blank control group, and b is Radix Astragali control group, and c is low dose group, and d is high dose group；
Fig. 8 is influence of the chitosan oligosaccharide intraperitoneal injection of the preparation of embodiment 6 to residual knurl mouse relative body weight；Wherein a is
Blank control group, b are Radix Astragali control group, and c is low dose group, and d is high dose group.
Embodiment
Technical solution of the present invention is not limited to the embodiment of act set forth below, in addition to each embodiment it
Between any combination.
Embodiment one：A kind of preparation method of chitosan oligosaccharide of present embodiment, is specifically prepared according to following steps
's：
Dissolve the chitosan in the glacial acetic acid solution that mass concentration is 0.1~5.0%, then add chitosan 5~25
The concentration of times weight is 10%~40% hydrogen peroxide, heating stirring to 40~90 DEG C of constant temperature, reacts 0.5~10h, then dense through quality
The ethanol precipitation for 60~100% is spent, is filtered, filter residue passes through drying, obtains the chitosan oligosaccharide of the degree of polymerization 2~30；Wherein chitosan oligosaccharide
The content of the middle degree of polymerization 2~10 is not less than 80%, the chitosan oligosaccharide purity not comprising moisture>98%；Chitosan is dense with quality
The mass volume ratio for spending the glacial acetic acid solution for 0.1~5.0% is 1g:(10~30) mL.
Present embodiment can not only effectively improve the utilization rate of abundant natural resources, also be effectively utilized its bio-compatible
Property and tumour antagonistic activity.By analyzing the antitumor activity of chitosan oligosaccharide, help to promote anti-tumor agents development.In identical survey
The killing ability of chitosan oligosaccharide of the invention to HCT-116 and A549 is above commercially available chitosan oligosaccharide in examination concentration range；In identical survey
Positive drug 5 FU 5 fluorouracil in concentration range is tried to change the half-inhibition concentration of the people source tumor cell line of ten kinds of separate sources
Scope is 0.04 ± 0.01~12.1 ± 3.0 μ g/mL, and chitosan oligosaccharide prepared by present embodiment suppresses to the half of tumor cell line
Change in concentration scope is 48.6 ± 7.0~1329.9 ± 93.4 μ g/mL.Chitosan oligosaccharide is thin for the people source tumour of ten kinds of separate sources
The maximal percentage inhibition of born of the same parents system can reach more than 90%, even as high as 95%.Kill chitosan oligosaccharide inside and outside prepared by present embodiment
The effect for hindering tumour is good.
Embodiment two：Present embodiment is unlike embodiment one：Dissolve the chitosan in quality
Concentration is in 3.5% glacial acetic acid solution, and then the concentration of addition 20 times of weights of chitosan is 30% hydrogen peroxide.It is other with it is specific
Embodiment one is identical.
Embodiment three：Present embodiment is unlike embodiment one or two：Described heating stirring
To 70 DEG C of constant temperature, 8h is reacted.It is other identical with embodiment one or two.
Embodiment four：Unlike one of present embodiment and embodiment one to three：Described mistake matter
Measure the ethanol precipitation that concentration is 95%.It is other identical with one of embodiment one to three.
Embodiment five：Unlike one of present embodiment and embodiment one to four：Described drying
Method for freeze-drying.It is other identical with one of embodiment one to four.
Embodiment six：A kind of application of chitosan oligosaccharide of present embodiment refers to be used to prepare to suppress 5 kinds of people source tumours
10 plants of tumour cells medicine.
Embodiment seven：Present embodiment is unlike embodiment six：5 kinds of described people source tumours are
Stomach cancer, kidney, lung cancer, breast cancer and colon cancer.It is other identical with embodiment six.
Embodiment eight：Present embodiment is unlike embodiment six or seven：10 plants of described tumours
Cell be gastric carcinoma cell lines BGC-823 and SGC-7901, renal carcinoma cell line KCC-853 and 786-O, lung cancer cell line A549 and
NCI-H460, breast cancer cell line MCF-7 and Bcap-37, colon carcinoma cell line HCT-116 and HT-29.Other and specific implementation
Mode six or seven is identical.
Embodiment nine：Unlike one of present embodiment and embodiment six to eight：Described medicine
Administering mode be daily gastric infusion, dosage 32mg/kg.It is other identical with one of embodiment six to eight.
Embodiment ten：Unlike one of present embodiment and embodiment six to nine：Described medicine
Administering mode be intraperitoneal injection every other day, dosage 4mg/kg.Other phases one of with embodiment six to nine
Together.
Beneficial effects of the present invention are verified using following examples：
Embodiment 1：The preparation method of the present embodiment chitosan oligosaccharide, specifically prepared according to following steps：
Dissolve the chitosan in the glacial acetic acid solution that mass concentration is 3.5%, then add heavy dense of 20 times of chitosan
Spend for 30% hydrogen peroxide, heating stirring to 70 DEG C of constant temperature, react 8h, then through the ethanol precipitation that mass concentration is 95%, filtering,
Filter residue passes through drying, obtains the chitosan oligosaccharide of the degree of polymerization 2~30；The content of the degree of polymerization 2~10 is not less than 80% wherein in chitosan oligosaccharide,
Chitosan oligosaccharide purity not comprising moisture>98%；Matter of the chitosan with mass concentration for 0.1~5.0% glacial acetic acid solution
Amount volume ratio is 1g:2mL.
The inhibitory activity that the chitosan oligosaccharide of the present embodiment and three kinds of commercially available chitosan oligosaccharides grow to people source lung cancer tumor cell A549
Compare；Commercially available pharmaceutical-gradechitosan chitosan oligosaccharide, be designated as COS-a；Commercially available food-grade chitosan oligosaccharide, is designated as COS-b；Commercially available shell is few
Sugar, it is designated as COS-c；Chitosan oligosaccharide manufactured in the present embodiment is designated as COS；Described experimental method is mtt assay.
First, cell culture
A, the preparation of nutrient solution：RPMI1640 culture mediums, Gibco companies of the U.S. are purchased from, it is prosperous that hyclone is purchased from Beijing member
Holy horse biotechnology research institute.After 56 DEG C of hyclone, 30min inactivations, added in 10% ratio in RPMI1640 culture mediums, 4
DEG C preserve.
B, sterilizing and sterile working：Before operation ultra violet lamp sterilize table top 30min, into super-clean bench before 75% ethanol it is net
Hand simultaneously wipes table top, and all operations should be carried out by alcolhol burner, and property must sterilize through flame, pay attention to sterile principle.
C, cell count：After alcohol washes tally and cover glass, dried with lens paper, cover glass is covered in counting
On plate.The tumour cell of exponential phase after trypsin solution is abandoned in centrifugation, is added appropriate nutrient solution by tryptic digestive juice,
After mixing, a small amount of cell suspension is drawn after diluting corresponding multiple with nutrient solution, is gently dropped in from edge on tally, makes cell suspension
Full of the space between tally and cover glass.Micro- Microscopic observation simultaneously counts corner block plaid inner cell number, if any flanging only
The cell on the wherein fixed both sides of pressure is calculated, calculation formula is as follows：
Cell number/mL=(4 big lattice TCS/4) × 104× extension rate
D, sample preparation：Certain chitosan oligosaccharide or commercial chitosan oligosaccharide are weighed, is dissolved in culture medium, bacteriological filtration membrane filtration is standby
With.
E, the preparation of MTT solution：Nutrient solution dissolves MTT powder, the 0.5mg/mL of system MTT solution for standby.
2nd, MTT is detected
It is 3000 cells per hole, by culture plate as CO by 100 μ L cell suspension inoculations in 96 orifice plates2Incubator
In, 37 DEG C, 5%CO2, culture 24h grows cell attachment under the conditions of saturated humidity.Add the chitosan oligosaccharide of 100 μ L various concentrations
Solution and 5 FU 5 fluorouracil solution, after control group adds 100 μ L blank cultures, continue to cultivate 72h.Abandoning supernatant, per hole
Add 100 μ L MTT solution, after lucifuge is incubated 4h, after abandoning supernatant, 200 μ L DMSO are added per hole, shake after being surveyed on ELIASA
It is scheduled on 570nm light absorption value.Inhibitory rate of cell growth is calculated according to following company：
Growth inhibition ratio (IR, %)=(1- experimental groups OD values/control group OD values) × 100
Experimental result：The suppression that the chitosan oligosaccharide of the present embodiment grows with commercially available chitosan oligosaccharide to people source lung cancer tumor cell A549
Design sketch is shown in accompanying drawing 1, compared with commercial chitosan oligosaccharide, chitosan oligosaccharide COS prepared by applicant to the fragmentation effects of A549 cells more
Substantially.
In the suppression that the chitosan oligosaccharide of the present embodiment grows with commercially available chitosan oligosaccharide to people source lung cancer tumor cell A549, this implementation
Inhibiting rate scope of the chitosan oligosaccharide of example in the range of institute's test concentrations is 82.0%-88.8%, and the inhibiting rate of commercially available chitosan oligosaccharide
In dose dependent, or even inhibiting rate is negative value in 1.25mg/mL.To the half of the commercially available chitosan oligosaccharide in measure concentration range
Inhibition concentration IC50 is fitted, and wherein COS-a and COS-c IC50 values are respectively 6.6582 ± 0.9814 and 5.3249 ±
0.8913mg/mL.In the range of institute's test concentrations, COS-b inhibiting rate is negative value, can not be fitted；The present embodiment
The inhibiting rate of chitosan oligosaccharide is relatively stable, can not be fitted, and the IC50 of the chitosan oligosaccharide of the present embodiment is fitted according to follow-up MTT experiment result
It is worth for 279.1 ± 25.4 μ g/mL, well below three kinds commercially available chitosan oligosaccharides.
People source lung cancer tumor cell A549 used in the present embodiment is commercially available prod.
Embodiment 2：The preparation method of the present embodiment chitosan oligosaccharide, specifically prepared according to following steps：
Dissolve the chitosan in the glacial acetic acid solution that mass concentration is 3.5%, then add heavy dense of 20 times of chitosan
Spend for 30% hydrogen peroxide, heating stirring to 70 DEG C of constant temperature, react 8h, then through the ethanol precipitation that mass concentration is 95%, filtering,
Filter residue passes through drying, obtains the chitosan oligosaccharide of the degree of polymerization 2~30；The content of the degree of polymerization 2~10 is not less than 80% wherein in chitosan oligosaccharide,
Chitosan oligosaccharide purity not comprising moisture>98%；Matter of the chitosan with mass concentration for 0.1~5.0% glacial acetic acid solution
Amount volume ratio is 1g:20mL.
The suppression that the chitosan oligosaccharide of the present embodiment and three kinds of commercially available chitosan oligosaccharides grow to people source colon cancer tumours cell HCT-116
Expression activitiy；Commercially available pharmaceutical-gradechitosan chitosan oligosaccharide, is designated as COS-a；Commercially available food-grade chitosan oligosaccharide, is designated as COS-b；Commercially available shell is few
Sugar, it is designated as COS-c；Chitosan oligosaccharide manufactured in the present embodiment is designated as COS；Described experimental method is mtt assay.
First, cell culture
A, the preparation of nutrient solution：RPMI1640 culture mediums, Gibco companies of the U.S. are purchased from, it is prosperous that hyclone is purchased from Beijing member
Holy horse biotechnology research institute.After 56 DEG C of hyclone, 30min inactivations, added in 10% ratio in RPMI1640 culture mediums, 4
DEG C preserve.
B, sterilizing and sterile working：Before operation ultra violet lamp sterilize table top 30min, into super-clean bench before 75% ethanol it is net
Hand simultaneously wipes table top, and all operations should be carried out by alcolhol burner, and property must sterilize through flame, pay attention to sterile principle.
C, cell count：After alcohol washes tally and cover glass, dried with lens paper, cover glass is covered in counting
On plate.The tumour cell of exponential phase after trypsin solution is abandoned in centrifugation, is added appropriate nutrient solution by tryptic digestive juice,
After mixing, a small amount of cell suspension is drawn after diluting corresponding multiple with nutrient solution, is gently dropped in from edge on tally, makes cell suspension
Full of the space between tally and cover glass.Micro- Microscopic observation simultaneously counts corner block plaid inner cell number, if any flanging only
The cell on the wherein fixed both sides of pressure is calculated, calculation formula is as follows：
Cell number/mL=(4 big lattice TCS/4) × 104× extension rate
D, sample preparation：Certain chitosan oligosaccharide or commercial chitosan oligosaccharide are weighed, is dissolved in culture medium, bacteriological filtration membrane filtration is standby
With.
E, the preparation of MTT solution：Nutrient solution dissolves MTT powder, the 0.5mg/mL of system MTT solution for standby.
2nd, MTT is detected
It is 3000 cells per hole, by culture plate as CO by 100 μ L cell suspension inoculations in 96 orifice plates2Incubator
In, 37 DEG C, 5%CO2, culture 24h grows cell attachment under the conditions of saturated humidity.Add the chitosan oligosaccharide of 100 μ L various concentrations
Solution and 5 FU 5 fluorouracil solution, after control group adds 100 μ L blank cultures, continue to cultivate 72h.Abandoning supernatant, per hole
Add 100 μ L MTT solution, after lucifuge is incubated 4h, after abandoning supernatant, 200 μ L DMSO are added per hole, shake after being surveyed on ELIASA
It is scheduled on 570nm light absorption value.Inhibitory rate of cell growth is calculated according to following company：
Growth inhibition ratio (IR, %)=(1- experimental groups OD values/control group OD values) × 100
Experimental result：Chitosan oligosaccharide manufactured in the present embodiment is with commercially available chitosan oligosaccharide to people source lung cancer tumor cell HCT-116
The inhibition figure of growth is shown in accompanying drawing 2, and compared with commercially available chitosan oligosaccharide, chitosan oligosaccharide COS prepared by applicant is to HCT-116 cells
Fragmentation effect becomes apparent.The chitosan oligosaccharide of the present embodiment grows with commercially available chitosan oligosaccharide to people source colon cancer tumours cell HCT-116
Suppression in, the inhibiting rate of the chitosan oligosaccharide of the present embodiment is -12.9~90.3%；And the highest inhibiting rate of three kinds of commercially available chitosan oligosaccharides
Respectively 88.9%, 88.4% and 87.2%, it is below the chitosan oligosaccharide highest inhibiting rate of the present embodiment.Half is carried out to histamine result
The half-inhibition concentration point of number inhibition concentration fitting, the chitosan oligosaccharide of the present embodiment and three kinds of commercially available chitosan oligosaccharides to HCT-116 cells
Wei not 2.5396 ± 0.2372,9.5104 ± 1.4619,9.6845 ± 0.2495 and 4.3789 ± 1.1299mg/mL.Thus may be used
See, the killing ability of the chitosan oligosaccharide of the present embodiment to HCT-116 is above commercially available chitosan oligosaccharide.
People source colon cancer tumours cell HCT-116 used is commercially available prod in the present embodiment.
Embodiment 3：The preparation method of the present embodiment chitosan oligosaccharide, specifically prepared according to following steps：
Dissolve the chitosan in the glacial acetic acid solution that mass concentration is 3.5%, then add heavy dense of 20 times of chitosan
Spend for 30% hydrogen peroxide, heating stirring to 70 DEG C of constant temperature, react 8h, then through the ethanol precipitation that mass concentration is 95%, filtering,
Filter residue passes through drying, obtains the chitosan oligosaccharide of the degree of polymerization 2~30；The content of the degree of polymerization 2~10 is not less than 80% wherein in chitosan oligosaccharide,
Chitosan oligosaccharide purity not comprising moisture>98%；Matter of the chitosan with mass concentration for 0.1~5.0% glacial acetic acid solution
Amount volume ratio is 1g:20mL.
The inhibitory activity grown by chitosan oligosaccharide to the people source tumor cell line of 10 kinds of separate sources, evaluates the present embodiment system
Standby chitosan oligosaccharide anti tumor activity in vitro；
First, cell culture
A, the preparation of nutrient solution：RPMI1640 culture mediums, Gibco companies of the U.S. are purchased from, it is prosperous that hyclone is purchased from Beijing member
Holy horse biotechnology research institute.After 56 DEG C of hyclone, 30min inactivations, added in 10% ratio in RPMI1640 culture mediums, 4
DEG C preserve.
B, sterilizing and sterile working：Before operation ultra violet lamp sterilize table top 30min, into super-clean bench before 75% ethanol it is net
Hand simultaneously wipes table top, and all operations should be carried out by alcolhol burner, and property must sterilize through flame, pay attention to sterile principle.
C, cell count：After alcohol washes tally and cover glass, dried with lens paper, cover glass is covered in counting
On plate.The tumour cell of exponential phase after trypsin solution is abandoned in centrifugation, is added appropriate nutrient solution by tryptic digestive juice,
After mixing, a small amount of cell suspension is drawn after diluting corresponding multiple with nutrient solution, is gently dropped in from edge on tally, makes cell suspension
Full of the space between tally and cover glass.Micro- Microscopic observation simultaneously counts corner block plaid inner cell number, if any flanging only
The cell on the wherein fixed both sides of pressure is calculated, calculation formula is as follows：
Cell number/mL=(4 big lattice TCS/4) × 104× extension rate
D, sample preparation：Certain chitosan oligosaccharide or commercial chitosan oligosaccharide are weighed, is dissolved in culture medium, bacteriological filtration membrane filtration is standby
With.
E, the preparation of MTT solution：Nutrient solution dissolves MTT powder, the 0.5mg/mL of system MTT solution for standby.
2nd, MTT is detected
It is 3000 cells per hole, by culture plate as CO by 100 μ L cell suspension inoculations in 96 orifice plates2Incubator
In, 37 DEG C, 5%CO2, culture 24h grows cell attachment under the conditions of saturated humidity.Add the chitosan oligosaccharide of 100 μ L various concentrations
Solution and 5 FU 5 fluorouracil solution, after control group adds 100 μ L blank cultures, continue to cultivate 72h.Abandoning supernatant, per hole
Add 100 μ L MTT solution, after lucifuge is incubated 4h, after abandoning supernatant, 200 μ L DMSO are added per hole, shake after being surveyed on ELIASA
It is scheduled on 570nm light absorption value.Inhibitory rate of cell growth is calculated according to following company：
Growth inhibition ratio (IR, %)=(1- experimental groups OD values/control group OD values) × 100
Experimental result：Inhibition of the chitosan oligosaccharide manufactured in the present embodiment to the people source tumor cell line of 10 kinds of separate sources
It is shown in Table 1.Positive drug 5 FU 5 fluorouracil to the half-inhibition concentration excursions of more plants of tumor cell lines for 0.04 ± 0.01~
12.1 ± 3.0 μ g/mL, chitosan oligosaccharide manufactured in the present embodiment to the half-inhibition concentration excursion of tumor cell line for 48.6 ±
7.0~1329.9 ± 93.4 μ g/mL.Chitosan oligosaccharide can reach more than 90% for the maximal percentage inhibition of tumor cell line, even
Up to 95%.
The people source tumor cell line of 10 kinds of separate sources used in the present embodiment is commercially available prod.
Fragmentation effect of the chitosan oligosaccharide of table 1 to the people source tumor cell line of 10 kinds of separate sources
Embodiment 4：The preparation method of the present embodiment chitosan oligosaccharide, specifically prepared according to following steps：
Dissolve the chitosan in the glacial acetic acid solution that mass concentration is 3.5%, then add heavy dense of 20 times of chitosan
Spend for 30% hydrogen peroxide, heating stirring to 70 DEG C of constant temperature, react 8h, then through the ethanol precipitation that mass concentration is 95%, filtering,
Filter residue passes through drying, obtains the chitosan oligosaccharide of the degree of polymerization 2~30；The content of the degree of polymerization 2~10 is not less than 80% wherein in chitosan oligosaccharide,
Chitosan oligosaccharide purity not comprising moisture>98%；Matter of the chitosan with mass concentration for 0.1~5.0% glacial acetic acid solution
Amount volume ratio is 1g:20mL.
Inhibition of the chitosan oligosaccharide gastric infusion to S180 sarcoma transplantable tumor is determined, it is evaluated and kills tumour in vivo
Effect and toxicity.
Experimental method：
First, the foundation of S180 sarcoma Transplanted tumor model
Normal saline dilution S180 cell suspensions, concentration are 2 × 1010Individual/L.Choose body weight 18-22g kunming mices 30.
Cell suspension, every 0.2mL is subcutaneously injected in the right shoulder of mouse.Mouse is randomly divided into 3 groups, every group 10.
1. medicine and dosage
It is divided into negative control group (gavage gives physiological saline), (gavage gives chitosan oligosaccharide to low dose group：400mg/kg) and
(gavage gives chitosan oligosaccharide to high dose group：800mg/kg).Chitosan oligosaccharide is dissolved in sterile saline, is diluted to required concentration.
2. medication
It is administered after inoculated tumour cell 96h, 1 time a day, successive administration 12 days, 0.2mL/20g gastric infusions (i.g.).
3. computational methods
S180 sarcoma Transplanted tumor model uses the diameter of vernier caliper measurement tumour, and the measurement of diameter of tumor is every 2
Once, the calculation formula of gross tumor volume is for its measurement：
V(mm3)=1/2 × a × b2(in formula：A and b is respectively the major diameter and minor axis of tumour)
Antitumor activity evaluation index is tumor-like hyperplasia (WIR%) and knurl volume inhibiting rate (VIR%), using following public affairs
Formula calculates：
WIR%=(1-WT/WC) × 100% is (in formula：WTFor treatment group's knurl weight, WCFor control group knurl weight)
VIR%=(1-VT/VC) × 100% is (in formula：VTFor treatment group's knurl volume, VCFor control group knurl volume)
Experimental result：As shown in accompanying drawing 3,4, compared with blank control group, the knurl volume of chitosan oligosaccharide treatment group mouse obtains
Effective suppression, the wherein knurl weight of high dose group and knurl volume have a significant difference (knurl weight P with control group<0.01；Knurl volume P<
0.05), the variation tendency of relative body weight and blank control group no significant difference.Specific experiment the results are shown in Table 2.
Inhibition of the chitosan oligosaccharide gastric infusion of table 2 to S180 sarcoma growth of transplanted human
Embodiment 5：The preparation method of the present embodiment chitosan oligosaccharide, specifically prepared according to following steps：
Dissolve the chitosan in the glacial acetic acid solution that mass concentration is 3.5%, then add heavy dense of 20 times of chitosan
Spend for 30% hydrogen peroxide, heating stirring to 70 DEG C of constant temperature, react 8h, then through the ethanol precipitation that mass concentration is 95%, filtering,
Filter residue passes through drying, obtains the chitosan oligosaccharide of the degree of polymerization 2~30；The content of the degree of polymerization 2~10 is not less than 80% wherein in chitosan oligosaccharide,
Chitosan oligosaccharide purity not comprising moisture>98%；Matter of the chitosan with mass concentration for 0.1~5.0% glacial acetic acid solution
Amount volume ratio is 1g:20mL.
Using chitosan oligosaccharide after the residual knurl modeling body operation tumor resection of mouse to tumour growth measurement chitosan oligosaccharide gavage
The inhibition to the residual knurl of S180 sarcoma is administered, evaluates it and kills the effect and toxicity of tumour in vivo.
Experimental method：
1st, the foundation of the residual knurl model of S180 sarcoma
Normal saline dilution S180 cell suspensions, concentration are 2 × 1010Individual/L.Choose body weight 18-22g kunming mices 21.
Cell suspension, every 0.2mL is subcutaneously injected in the right shoulder of mouse.Treat within 1 week or so gross tumor volume length to 200~300mm3Afterwards, perform the operation
Solid tumor is cut off, about remaining knurl volume is 40mm3, random packet on the day of operation, every group 7 and same day administration.
2nd, medicine and dosage
It is divided into negative control group (gavage gives physiological saline), (gavage gives chitosan oligosaccharide to low dose group：200mg/kg) and
(gavage gives chitosan oligosaccharide to high dose group：400mg/kg).Chitosan oligosaccharide is dissolved in sterile saline, is diluted to required concentration.
3rd, medication
The random packet same day is administered, 1 time a day, successive administration 12 days, 0.2mL/20g gastric infusions (i.g.).
4th, computational methods
The residual knurl model of S180 sarcoma uses the diameter of vernier caliper measurement tumour, and the measurement of diameter of tumor was every 2 days
Once, the calculation formula of gross tumor volume is for measurement：
V(mm3)=1/2 × a × b2(in formula：A and b is respectively the major diameter and minor axis of tumour)
Antitumor activity evaluation index is tumor-like hyperplasia (WIR%) and knurl volume inhibiting rate (VIR%), using following public affairs
Formula calculates：
WIR%=(1-WT/WC) × 100% is (in formula：WTFor treatment group's knurl weight, WCFor control group knurl weight)
VIR%=(1-VT/VC) × 100% is (in formula：VTFor treatment group's knurl volume, VCFor control group knurl volume)
Experimental result：As shown in accompanying drawing 5,6, compared with blank control group, the knurl volume of chitosan oligosaccharide treatment group mouse obtains
Effective suppression, the wherein knurl weight of high dose group have a significant difference (P with control group<0.05), and the change of relative body weight becomes
Gesture and blank control group no significant difference.Specific experiment the results are shown in Table 3.
Inhibition of the chitosan oligosaccharide gastric infusion of table 3 to the residual knurl of S180 sarcoma
Embodiment 6：The preparation method of the present embodiment chitosan oligosaccharide, specifically prepared according to following steps：
Dissolve the chitosan in the glacial acetic acid solution that mass concentration is 3.5%, then add heavy dense of 20 times of chitosan
Spend for 30% hydrogen peroxide, heating stirring to 70 DEG C of constant temperature, react 8h, then through the ethanol precipitation that mass concentration is 95%, filtering,
Filter residue passes through drying, obtains the chitosan oligosaccharide of the degree of polymerization 2~30；The content of the degree of polymerization 2~10 is not less than 80% wherein in chitosan oligosaccharide,
Chitosan oligosaccharide purity not comprising moisture>98%；Matter of the chitosan with mass concentration for 0.1~5.0% glacial acetic acid solution
Amount volume ratio is 1g:20mL.
Using chitosan oligosaccharide after the residual knurl modeling body operation tumor resection of mouse to tumour growth measurement chitosan oligosaccharide abdominal cavity
Drug administration by injection evaluates it and kills the effect and toxicity of tumour in vivo to the inhibition of the residual knurl of S180 sarcoma.
Experimental method：
1st, the foundation of the residual knurl model of S180 sarcoma
Normal saline dilution S180 cell suspensions, concentration are 2 × 1010Individual/L.Choose body weight 18-22g kunming mices 30.
Cell suspension, every 0.2mL is subcutaneously injected in the right shoulder of mouse.Treat within 1 week or so gross tumor volume length to 200~300mm3Afterwards, perform the operation
Solid tumor is cut off, about remaining knurl volume is 40mm3, random packet on the day of operation, every group 10 and same day administration.
2nd, medicine and dosage
It is divided into negative control group (physiological saline is given in intraperitoneal injection), (astragalus polyose is given in intraperitoneal injection to positive controls：
250mg/kg), (chitosan oligosaccharide is given in intraperitoneal injection to low dose group：50mg/kg) and high dose group (chitosan oligosaccharide is given in intraperitoneal injection：
250mg/kg).Astragalus polyose or chitosan oligosaccharide are dissolved in sterile saline, are diluted to required concentration.
3rd, medication
The random packet same day is administered, 1 time every other day, successive administration 11 days, 0.2mL/20g intraperitoneal injections (i.p.)
4th, computational methods
The residual knurl model of S180 sarcoma uses the diameter of vernier caliper measurement tumour, and the measurement of diameter of tumor was every 2 days
Once, the calculation formula of gross tumor volume is for measurement：
V(mm3)=1/2 × a × b2(in formula：A and b is respectively the major diameter and minor axis of tumour)
Antitumor activity evaluation index is tumor-like hyperplasia (WIR%) and knurl volume inhibiting rate (VIR%), using following public affairs
Formula calculates：
WIR%=(1-WT/WC) × 100% is (in formula：WTFor treatment group's knurl weight, WCFor control group knurl weight)
VIR%=(1-VT/VC) × 100% is (in formula：VTFor treatment group's knurl volume, VCFor control group knurl volume)
Inhibition of the chitosan oligosaccharide gastric infusion of table 4 to the residual knurl of S180 sarcoma
Experimental result：As shown in accompanying drawing 7,8, compared with blank control group, the knurl of chitosan oligosaccharide intraperitoneal injection treatment group mouse
Volume has obtained effective suppression, and the wherein knurl weight of low dose group, knurl volume and control group has significant difference (knurl weight P<0.05；
Knurl volume P<0.01), the variation tendency of relative body weight and blank control group no significant difference.Specific experiment the results are shown in Table 4.
In summary, the killing ability of chitosan oligosaccharide of the invention in same test concentration range to HCT-116 and A549
It is above commercially available chitosan oligosaccharide；People source tumour of the positive drug 5 FU 5 fluorouracil to ten kinds of separate sources in same test concentration range
The half-inhibition concentration excursion of cell line is 0.04 ± 0.01~12.1 ± 3.0 μ g/mL, chitosan oligosaccharide pair prepared by the present invention
The half-inhibition concentration excursion of tumor cell line is 48.6 ± 7.0~1329.9 ± 93.4 μ g/mL.Chitosan oligosaccharide is for ten kinds
The maximal percentage inhibition of the people source tumor cell line of separate sources can reach more than 90%, even as high as 95%.It is prepared by the present invention
Chitosan oligosaccharide inside and outside killing tumour effect it is good.
Claims (4)
1. a kind of purposes of chitosan oligosaccharide, it is characterised in that it is used for the medicine for preparing 10 plants of tumour cells for suppressing 5 kinds of people source tumours
Thing；
5 kinds of described people source tumours are stomach cancer, kidney, lung cancer, breast cancer and colon cancer；
10 plants of described tumour cells be gastric carcinoma cell lines BGC-823 and SGC-7901, renal carcinoma cell line KCC-853 and 786-O,
Lung cancer cell line A549 and NCI-H460, breast cancer cell line MCF-7 and Bcap-37, colon carcinoma cell line HCT-116 and HT-
29；
The preparation method of the chitosan oligosaccharide follows the steps below：
Dissolve the chitosan in the glacial acetic acid solution that mass concentration is 3.5%, then adding 20 times of heavy concentration of chitosan is
30% hydrogen peroxide, heating stirring to 40 ~ 90 DEG C of constant temperature, 0.5 ~ 10 h is reacted, then sunk through the ethanol that mass concentration is 60 ~ 100%
Form sediment, filtering, filter residue passes through drying, obtains the chitosan oligosaccharide of the degree of polymerization 2 ~ 30；Wherein the content of the degree of polymerization 2 ~ 10 is not low in chitosan oligosaccharide
In 80%, the chitosan oligosaccharide purity not comprising moisture>98%；Quality of the chitosan with mass concentration for 3.5% glacial acetic acid solution
Volume ratio is 1 g:(10~30) mL；The administering mode of described medicine is intraperitoneal injection every other day, dosage 4mg/
kg。
A kind of 2. purposes of chitosan oligosaccharide according to claim 1, it is characterised in that after adding hydrogen peroxide, heating stirring
To 70 DEG C of constant temperature, 8 h are reacted.
3. the purposes of a kind of chitosan oligosaccharide according to claim 1, it is characterised in that the mass concentration of described ethanol is
95%。
4. the purposes of a kind of chitosan oligosaccharide according to claim 1, it is characterised in that the method for described drying is dry for freezing
It is dry.
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