CN105085711A - Preparation method and application of chitobiose - Google Patents
Preparation method and application of chitobiose Download PDFInfo
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- CN105085711A CN105085711A CN201510519424.3A CN201510519424A CN105085711A CN 105085711 A CN105085711 A CN 105085711A CN 201510519424 A CN201510519424 A CN 201510519424A CN 105085711 A CN105085711 A CN 105085711A
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Abstract
The invention relates to a preparation method and application of chitobiose. The preparation method comprises the following steps of dissolving chitobiose in glacial acetic acid solution; adding hydrogen peroxide; heating and stirring until the temperature is 40-90 DEG C and incubating; precipitating with ethanol after reaction is finished; filtering; and drying filter residues to obtain the chitobiose. The prepared chitobiose is used for preparing medicines for suppressing 10 strains of tumor cells of 5 kinds of humanized tumors. Biological compatibility and tumor atagonistics activity of the chitobiose are utilized effectively. By analyzing antitumor activity of the chitobiose, development of anti-tumor medicaments is promoted. The invention is used for the field of anti-tumor medicines.
Description
Technical field
The present invention relates to a kind of preparation method and application thereof of oligochitosan.
Background technology
Chitin (chitin) extensively exists with the cell wall of the shell of Crustacean, the crust of insect and fungi, and be the natural polymer that occurring in nature enriches the most, content is only second to Mierocrystalline cellulose.Chitin has several functions characteristic, but the water soluble characteristic of its extreme difference and biodegradability significantly limit its widespread use.The characteristic of, diluted acid, diluted alkaline, ethanol or other organic solvents water insoluble relative to chitin, chitosan (chitosan) water soluble also presents higher viscosity in dilute acid soln.This dissolution characteristics also limit the application of chitosan at biological field.Same as chitin degrading product, the solvability of oligochitosan (chitosanoligosaccharides, COS) is better, and viscosity is lower in physiological conditions, and this is because the sugar chain of oligochitosan is shorter and free amine group.Containing an amino group and two oh groups in the glucoside residue repeating unit of oligochitosan, it is the unique positively charged positively charged ion basic amine group oligose of occurring in nature.These characteristics of oligochitosan cause the concern of more and more investigator.There is a lot of bibliographical information biological activity of oligochitosan, anti-oxidant, anti-inflammatory, antagonistic microbe, reduction cholesterol and strengthen immunocompetence, antineoplastic activity etc.The application of oligochitosan in a lot of field also has corresponding report, as food, medicine, agricultural and environmental area.In addition, the biocompatibility that oligochitosan is good, nontoxic, not sensitizing property to organs of living beings, also make it develop into a kind of potential pharmaceutical carrier and tissue engineering bracket as nanometer-/micron system.
Tumour is harm humans healthy the most serious a class common disease, frequently-occurring disease.Tumour has become a major reason of M & M all over the world, and in the last few years along with the change of people life style and the aggravation of environmental pollution, its sickness rate was in ascendant trend, particularly developing country year by year.At present, the sickness rate of China's tumour is 285.91/10 ten thousand, and average every day, per minute just had 6 people to be diagnosed as malignant tumour.WHO predicts, estimates that there are 2,014 ten thousand newly-increased cases of cancers in the whole world, 1,320 ten thousand patients will be had to die from cancer every year to the year two thousand thirty.There is bibliographical information, oligochitosan has antineoplastic biological activity, its mechanism is the growth of direct inhibition tumor cell, and there are report necrocytosis and apoptosis, reduce tumor-blood-vessel growth thus realize inhibition tumor cell transfer, also be likely by enhancing body immunizing power, indirectly realize the effect that development occurs antagonize Tumor.
The inside and outside antitumor activity of oligochitosan is a lot, but its molecule mechanism is still unknown.Oligochitosan has good adhesion characteristics, can be combined with the surface glycoprotein of mammalian cell.Therefore, the characteristic changing of oligochitosan positively charged cytolemma charging property, thus destroy cell integrity, affect Growth of Cells.The people such as Suzuki find that the electrostatic interaction of antitumor drug and tumour cell is the major reason that oligochitosan has anti-tumor activity.The people such as Huang uses has the antagonistic action of different charge number oligochitosan to three strain tumor cell lines, and result shows that oligochitosan derivative that electrically charged amount is high significantly can reduce the survival rate of tumour cell.
Except the impact of electric charge, the people such as Huang find that again the molecular weight of oligochitosan also plays an important role its anti-tumor activity.The people such as Salah determine chitin, chitosan, small molecules chitin to the antagonistic action of people source tumour cell THP-1, find that the chitinous antitumous effect of small molecules is better, and increase along with molecular weight reduces antitumous effect.
Vasculogenesis is the generation of tumor tissues, development and transfer provide meta-bolites, oxygen and nutritive substance, is therefore considered to novel action target spot in antitumor research.Oligochitosan can suppress people source fibrosarcoma to organize the expression of matrix metalloproteinase (matrixmetalloproteinase-9, MMP-9), and this has important impact in metastases.The rise of MMP-9 can promote that matrix VEGF (VascularEndothelialGrowthFactor, VEGF) discharges and vasculogenesis.VEGF stimulates epithelial cell amplification and migration, and this has regulating and controlling effect to vascular progenitor and angiogenesis.The people such as Wu adopt several different biological method to determine some oligochitosan components to the restraining effect of vasculogenesis, find that inhibition depends on deacetylation and the polymerization degree of component.
In addition, the immune-enhancing activity of oligochitosan is also one of its major reason with anti-tumor activity.The people such as Suzuki utilize water-soluble good chitosan to enhance immunological competence, and prove that oligochitosan inhibits tumor propagation along with immunocompetence strengthens.Somebody thinks that oligochitosan can not direct killing tumour, but by promoting that the release of lymphokine promotes that T-Lymphocyte expansion plays tumour antagonistic activity.The people such as Suzuki also confirm that oligochitosan carrys out Tumor suppression growth by activated lymphocyte release lymphokine.In general, to play non-characteristic by the immunocompetence strengthening scavenger cell or neutrophil leucocyte immunocompetent for immunostimulant.Most of immunostimulant stimulates the secretion as immune response compounds such as Interferon, rabbit, interleukin and ligandins, these compounds with scavenger cell or lymphocyte surface receptors protein binding after just can activating immune system.Some immunostimulants can be combined with specific receptors molecule competes in infected tissue target cell.Chitosan oligopolymer effectively can promote the migratory activity of scavenger cell, this is because scavenger cell has chemotaxis.The people such as Ma think that chitosan oligopolymer is by lowering the activation of MAPK and PI3K/Akt signal path phosphorylation and NF-κ B and AP-1 and then suppressing the secretion of IL-6 and TNF-α to play anti-inflammatory activity.Oligochitosan also passes through enhancing antibody response performance immune-enhancing activity.The people such as Chang have studied the action effect of oligochitosan to a kind of influenza vaccines of non-deactivation.Result shows that oligochitosan significantly improves the anti-body contg in serum, enhances the antiviral defense system of mouse.Oligochitosan can also the release of stimulating cytokine, as interleukin I L-1 β and tumor lethal factor TNF-α etc.
But existing oligochitosan is active not good enough on Tumor suppression, and according to existing domestic and international reference, the administering mode of oligochitosan in-vivo tumour antagonistic action and the research of dosage are still blank, and oligochitosan suppresses the work of screening to rarely have report to many tumor cell lines.Therefore prepare active good oligochitosan, have good application prospect preparing on Tumor suppression medicine.
Summary of the invention
The invention provides a kind of preparation method and application thereof of oligochitosan.
A preparation method for oligochitosan, specifically prepares according to following steps:
Chitosan being dissolved in mass concentration is in the glacial acetic acid solution of 0.1 ~ 5.0%, then adding the heavy concentration of chitosan 5 ~ 25 times is 10% ~ 40% hydrogen peroxide, intensification is stirred to 40 ~ 90 DEG C of constant temperature, reaction 0.5 ~ 10h, again through mass concentration be the alcohol settling of 60 ~ 100%, filter, filter residue, through super-dry, obtains the oligochitosan of the polymerization degree 2 ~ 30; Wherein in oligochitosan, the content of the polymerization degree 2 ~ 10 is not less than 80%, does not comprise the oligochitosan purity >98% of moisture content; Chitosan and mass concentration are the mass volume ratio of the glacial acetic acid solution of 0.1 ~ 5.0% is 1g:(10 ~ 30) mL.
A kind of application of oligochitosan refers to the medicine of the 10 strain tumour cells for the preparation of suppression 5 kinds of people source tumours.
The present invention not only effectively can improve the utilization ratio of abundant natural resources, also effectively make use of its biocompatibility and tumour antagonistic activity.By analyzing the anti-tumor activity of oligochitosan, contribute to dragging anti-tumor agents development.In same test concentration range oligochitosan of the present invention to the kill capability of HCT-116 and A549 all higher than commercially available oligochitosan; In same test concentration range, the half-inhibition concentration variation range of positive drug 5 FU 5 fluorouracil to the people source tumor cell line of ten kinds of different sourcess is 0.04 ± 0.01 ~ 12.1 ± 3.0 μ g/mL, and oligochitosan prepared by the present invention half-inhibition concentration variation range to tumor cell line is 48.6 ± 7.0 ~ 1329.9 ± 93.4 μ g/mL.Oligochitosan all can reach more than 90%, even up to 95% for the maximal percentage inhibition of the people source tumor cell line of ten kinds of different sourcess.Oligochitosan inside and outside killing tumor cells prepared by the present invention effective.
Accompanying drawing explanation
Fig. 1 be embodiment 1 prepare oligochitosan and three kinds of commercial oligochitosans to people source lung cancer tumor cell A549 growth inhibitory effect figure (n=4, ± sd); Wherein a is COS, b be COS-a, c be COS-b, d is COS-c;
Fig. 2 be embodiment 2 prepare oligochitosan and three kinds of commercial oligochitosans to people source colon cancer cell HCT-116 growth inhibitory effect figure (n=4, ± sd); Wherein a is COS, b be COS-a, c be COS-b, d is COS-c;
Fig. 3 is that the oligochitosan gastric infusion of embodiment 4 preparation is to the inhibition figure (gross tumor volume) (n=10, ± sd) of S180 sarcoma transplanted tumor; Wherein a is blank group, and b is low dose group, and c is high dose group;
Fig. 4 is that the oligochitosan gastric infusion of embodiment 4 preparation is on the impact of tumor-bearing mice relative body weight; Wherein a is blank group, and b is low dose group, and c is high dose group;
Fig. 5 is that the oligochitosan gastric infusion of embodiment 5 preparation is to the inhibition figure (gross tumor volume) (n=7, ± sd) of the residual knurl of S180 sarcoma; Wherein a is blank group, and b is low dose group, and c is high dose group;
Fig. 6 is that the oligochitosan gastric infusion of embodiment 5 preparation is on the impact of tumor-bearing mice relative body weight; Wherein a is blank group, and b is low dose group, and c is high dose group;
Fig. 7 is that the oligochitosan intraperitoneal injection of embodiment 6 preparation is to the inhibition figure (gross tumor volume) (n=10, ± sd) of the residual knurl of S180 sarcoma; Wherein a is blank group, and b is Radix Astragali control group, and c is low dose group, and d is high dose group;
Fig. 8 is that the oligochitosan intraperitoneal injection of embodiment 6 preparation is on the impact of residual knurl mouse relative body weight; Wherein a is blank group, and b is 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 following cited embodiment, also comprises the arbitrary combination between each embodiment.
Embodiment one: the preparation method of a kind of oligochitosan of present embodiment, specifically prepare according to following steps:
Chitosan being dissolved in mass concentration is in the glacial acetic acid solution of 0.1 ~ 5.0%, then adding the heavy concentration of chitosan 5 ~ 25 times is 10% ~ 40% hydrogen peroxide, intensification is stirred to 40 ~ 90 DEG C of constant temperature, reaction 0.5 ~ 10h, again through mass concentration be the alcohol settling of 60 ~ 100%, filter, filter residue, through super-dry, obtains the oligochitosan of the polymerization degree 2 ~ 30; Wherein in oligochitosan, the content of the polymerization degree 2 ~ 10 is not less than 80%, does not comprise the oligochitosan purity >98% of moisture content; Chitosan and mass concentration are the mass volume ratio of the glacial acetic acid solution of 0.1 ~ 5.0% is 1g:(10 ~ 30) mL.
Present embodiment not only effectively can improve the utilization ratio of abundant natural resources, also effectively make use of its biocompatibility and tumour antagonistic activity.By analyzing the anti-tumor activity of oligochitosan, contribute to promoting anti-tumor agents development.In same test concentration range oligochitosan of the present invention to the kill capability of HCT-116 and A549 all higher than commercially available oligochitosan; In same test concentration range, the half-inhibition concentration variation range of positive drug 5 FU 5 fluorouracil to the people source tumor cell line of ten kinds of different sourcess is 0.04 ± 0.01 ~ 12.1 ± 3.0 μ g/mL, and the half-inhibition concentration variation range of oligochitosan to tumor cell line prepared by present embodiment is 48.6 ± 7.0 ~ 1329.9 ± 93.4 μ g/mL.Oligochitosan all can reach more than 90%, even up to 95% for the maximal percentage inhibition of the people source tumor cell line of ten kinds of different sourcess.Oligochitosan inside and outside killing tumor cells prepared by present embodiment effective.
Embodiment two: present embodiment and embodiment one unlike: chitosan being dissolved in mass concentration is in the glacial acetic acid solution of 3.5%, and then adding the heavy concentration of chitosan 20 times is 30% hydrogen peroxide.Other is identical with embodiment one.
Embodiment three: present embodiment and embodiment one or two unlike: described intensification is stirred to 70 DEG C of constant temperature, reaction 8h.Other is identical with embodiment one or two.
Embodiment four: one of present embodiment and embodiment one to three unlike: described mistake mass concentration is the alcohol settling of 95%.Other is identical with one of embodiment one to three.
Embodiment five: one of present embodiment and embodiment one to four unlike: the method for described drying is lyophilize.Other is identical with one of embodiment one to four.
Embodiment six: the application of a kind of oligochitosan of present embodiment refers to the medicine of the 10 strain tumour cells for the preparation of suppression 5 kinds of people source tumours.
Embodiment seven: present embodiment and embodiment six unlike: 5 kinds of described people source tumours are cancer of the stomach, kidney, lung cancer, mammary cancer and colorectal carcinoma.Other is identical with embodiment six.
Embodiment eight: present embodiment and embodiment six or seven unlike: 10 described strain tumour cells are 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 is identical with embodiment six or seven.
Embodiment nine: one of present embodiment and embodiment six to eight unlike: the administering mode of described medicine is gastric infusion every day, and dosage is 32mg/kg.Other is identical with one of embodiment six to eight.
Embodiment ten: one of present embodiment and embodiment six to nine are intraperitoneal injection every other day unlike the administering mode of: described medicine, and dosage is 4mg/kg.Other is identical with one of embodiment six to nine.
Following examples are adopted to verify beneficial effect of the present invention:
Embodiment 1: the preparation method of the present embodiment oligochitosan, specifically prepare according to following steps:
Chitosan being dissolved in mass concentration is in the glacial acetic acid solution of 3.5%, then adding the heavy concentration of chitosan 20 times is 30% hydrogen peroxide, intensification is stirred to 70 DEG C of constant temperature, reaction 8h, again through mass concentration be the alcohol settling of 95%, filter, filter residue, through super-dry, obtains the oligochitosan of the polymerization degree 2 ~ 30; Wherein in oligochitosan, the content of the polymerization degree 2 ~ 10 is not less than 80%, does not comprise the oligochitosan purity >98% of moisture content; Chitosan and mass concentration are the mass volume ratio of the glacial acetic acid solution of 0.1 ~ 5.0% is 1g:2mL.
The oligochitosan of the present embodiment compares the inhibit activities that people source lung cancer tumor cell A549 grows with three kinds of commercially available oligochitosans; Commercially available pharmaceutical-gradechitosan chitosan oligosaccharide, be designated as COS-a; Commercially available food grade oligochitosan, is designated as COS-b; Commercially available oligochitosan, is designated as COS-c; Oligochitosan prepared by the present embodiment is designated as COS; Described experimental technique is mtt assay.
One, cell cultures
The preparation of a, nutrient solution: RPMI1640 substratum, is purchased from Gibco company of the U.S., and foetal calf serum is purchased from Beijing unit Heng Shengma biotechnology research institute.After foetal calf serum 56 DEG C, 30min deactivation, add in RPMI1640 substratum in 10% ratio, 4 DEG C of preservations.
B, sterilizing and aseptic technique: ultra violet lamp sterilizing table top 30min before operation, before entering super clean bench, 75% ethanol is washed one's hands and wiping table top, and all operations should carry out on spirit lamp side, and belongings through flame sterilizing, must note aseptic principle.
C, cell counting: after alcohol washes tally and cover glass, dry with lens paper, cover glass is covered on tally.Tryptic digestive juice by the tumour cell of logarithmic phase, centrifugal abandon trypsin solution after, add appropriate nutrient solution, after mixing, draw a small amount of cell suspension after diluting corresponding multiple with nutrient solution, gently drop in tally from edge, make cell suspension be full of space between tally and cover glass.Basis of microscopic observation also counts corner block plaid inner cell number, and only calculate if any flanging the cell that pressure wherein fixes both sides, calculation formula is as follows:
Cell count/mL=(4 large lattice total cellular score/4) × 10
4× extension rate
D, sample preparation: take certain oligochitosan or commercial oligochitosan, be dissolved in substratum, bacteriological filtration membrane filtration is for subsequent use.
The preparation of e, MTT solution: nutrient solution dissolves MTT powder, the MTT solution for standby of the 0.5mg/mL of system.
Two, MTT detects
By 100 μ L cell suspension inoculations in 96 orifice plates, every hole is 3000 cells, by culture plate as CO
2in incubator, 37 DEG C, 5%CO
2, cultivate 24h under saturated humidity condition cell attachment grown.Add oligochitosan solution and the 5 FU 5 fluorouracil solution of 100 μ L different concns, after control group adds 100 μ L blank cultures, continue to cultivate 72h.Abandoning supernatant, every hole adds 100 μ LMTT solution, and after lucifuge hatches 4h, after abandoning supernatant, every hole adds 200 μ LDMSO, is determined at the light absorption value of 570nm after concussion in microplate reader.Inhibitory rate of cell growth is calculated according to following company:
Growth inhibition ratio (IR, %)=(1-experimental group OD value/control group OD value) × 100
Experimental result: the oligochitosan of the present embodiment and commercially available oligochitosan are shown in accompanying drawing 1 to the inhibition figure that people source lung cancer tumor cell A549 grows, and compared with commercial oligochitosan, oligochitosan COS prepared by applicant is more obvious to the fragmentation effect of A549 cell.
In the suppression that the oligochitosan of the present embodiment and commercially available oligochitosan grow people source lung cancer tumor cell A549, the inhibiting rate scope of oligochitosan within the scope of institute's test concentrations of the present embodiment is 82.0%-88.8%, and the inhibiting rate of commercially available oligochitosan is dose-dependently, even when 1.25mg/mL, inhibiting rate is negative value.Carried out matching to the half-inhibition concentration IC50 of the commercially available oligochitosan measured in concentration range, wherein the IC50 value of COS-a and COS-c is respectively 6.6582 ± 0.9814 and 5.3249 ± 0.8913mg/mL.Within the scope of institute's test concentrations, the inhibiting rate of COS-b is negative value, cannot carry out matching; The inhibiting rate of the oligochitosan of the present embodiment is more stable, cannot matching, and is 279.1 ± 25.4 μ g/mL according to the IC50 value of the oligochitosan of follow-up MTT experiment result matching the present embodiment, well below three kinds of commercially available oligochitosans.
The present embodiment people source lung cancer tumor cell A549 used is commercially available prod.
Embodiment 2: the preparation method of the present embodiment oligochitosan, specifically prepare according to following steps:
Chitosan being dissolved in mass concentration is in the glacial acetic acid solution of 3.5%, then adding the heavy concentration of chitosan 20 times is 30% hydrogen peroxide, intensification is stirred to 70 DEG C of constant temperature, reaction 8h, again through mass concentration be the alcohol settling of 95%, filter, filter residue, through super-dry, obtains the oligochitosan of the polymerization degree 2 ~ 30; Wherein in oligochitosan, the content of the polymerization degree 2 ~ 10 is not less than 80%, does not comprise the oligochitosan purity >98% of moisture content; Chitosan and mass concentration are the mass volume ratio of the glacial acetic acid solution of 0.1 ~ 5.0% is 1g:20mL.
The oligochitosan of the present embodiment compares the inhibit activities that people source colon cancer tumours cell HCT-116 grows with three kinds of commercially available oligochitosans; Commercially available pharmaceutical-gradechitosan chitosan oligosaccharide, is designated as COS-a; Commercially available food grade oligochitosan, is designated as COS-b; Commercially available oligochitosan, is designated as COS-c; Oligochitosan prepared by the present embodiment is designated as COS; Described experimental technique is mtt assay.
One, cell cultures
The preparation of a, nutrient solution: RPMI1640 substratum, is purchased from Gibco company of the U.S., and foetal calf serum is purchased from Beijing unit Heng Shengma biotechnology research institute.After foetal calf serum 56 DEG C, 30min deactivation, add in RPMI1640 substratum in 10% ratio, 4 DEG C of preservations.
B, sterilizing and aseptic technique: ultra violet lamp sterilizing table top 30min before operation, before entering super clean bench, 75% ethanol is washed one's hands and wiping table top, and all operations should carry out on spirit lamp side, and belongings through flame sterilizing, must note aseptic principle.
C, cell counting: after alcohol washes tally and cover glass, dry with lens paper, cover glass is covered on tally.Tryptic digestive juice by the tumour cell of logarithmic phase, centrifugal abandon trypsin solution after, add appropriate nutrient solution, after mixing, draw a small amount of cell suspension after diluting corresponding multiple with nutrient solution, gently drop in tally from edge, make cell suspension be full of space between tally and cover glass.Basis of microscopic observation also counts corner block plaid inner cell number, and only calculate if any flanging the cell that pressure wherein fixes both sides, calculation formula is as follows:
Cell count/mL=(4 large lattice total cellular score/4) × 10
4× extension rate
D, sample preparation: take certain oligochitosan or commercial oligochitosan, be dissolved in substratum, bacteriological filtration membrane filtration is for subsequent use.
The preparation of e, MTT solution: nutrient solution dissolves MTT powder, the MTT solution for standby of the 0.5mg/mL of system.
Two, MTT detects
By 100 μ L cell suspension inoculations in 96 orifice plates, every hole is 3000 cells, by culture plate as CO
2in incubator, 37 DEG C, 5%CO
2, cultivate 24h under saturated humidity condition cell attachment grown.Add oligochitosan solution and the 5 FU 5 fluorouracil solution of 100 μ L different concns, after control group adds 100 μ L blank cultures, continue to cultivate 72h.Abandoning supernatant, every hole adds 100 μ LMTT solution, and after lucifuge hatches 4h, after abandoning supernatant, every hole adds 200 μ LDMSO, is determined at the light absorption value of 570nm after concussion in microplate reader.Inhibitory rate of cell growth is calculated according to following company:
Growth inhibition ratio (IR, %)=(1-experimental group OD value/control group OD value) × 100
Experimental result: oligochitosan prepared by the present embodiment and commercially available oligochitosan are shown in accompanying drawing 2 to the inhibition figure that people source lung cancer tumor cell HCT-116 grows, compared with commercially available oligochitosan, oligochitosan COS prepared by applicant is more obvious to the fragmentation effect of HCT-116 cell.In the suppression that the oligochitosan of the present embodiment and commercially available oligochitosan grow people source colon cancer tumours cell HCT-116, the inhibiting rate of the oligochitosan of the present embodiment is-12.9 ~ 90.3%; And the highest inhibiting rate of three kinds of commercially available oligochitosans is respectively 88.9%, 88.4% and 87.2%, all lower than the highest inhibiting rate of oligochitosan of the present embodiment.Carry out half-inhibition concentration matching to suppression result, the oligochitosan of the present embodiment and the half-inhibition concentration of three kinds of commercially available oligochitosans to HCT-116 cell are respectively 2.5396 ± 0.2372,9.5104 ± 1.4619,9.6845 ± 0.2495 and 4.3789 ± 1.1299mg/mL.Can see thus, the oligochitosan of the present embodiment to the kill capability of HCT-116 all higher than commercially available oligochitosan.
People source colon cancer tumours cell HCT-116 used in the present embodiment is commercially available prod.
Embodiment 3: the preparation method of the present embodiment oligochitosan, specifically prepare according to following steps:
Chitosan being dissolved in mass concentration is in the glacial acetic acid solution of 3.5%, then adding the heavy concentration of chitosan 20 times is 30% hydrogen peroxide, intensification is stirred to 70 DEG C of constant temperature, reaction 8h, again through mass concentration be the alcohol settling of 95%, filter, filter residue, through super-dry, obtains the oligochitosan of the polymerization degree 2 ~ 30; Wherein in oligochitosan, the content of the polymerization degree 2 ~ 10 is not less than 80%, does not comprise the oligochitosan purity >98% of moisture content; Chitosan and mass concentration are the mass volume ratio of the glacial acetic acid solution of 0.1 ~ 5.0% is 1g:20mL.
By the inhibit activities of oligochitosan to the people source tumor cell line growth of 10 kinds of different sourcess, evaluate oligochitosan anti tumor activity in vitro prepared by the present embodiment;
One, cell cultures
The preparation of a, nutrient solution: RPMI1640 substratum, is purchased from Gibco company of the U.S., and foetal calf serum is purchased from Beijing unit Heng Shengma biotechnology research institute.After foetal calf serum 56 DEG C, 30min deactivation, add in RPMI1640 substratum in 10% ratio, 4 DEG C of preservations.
B, sterilizing and aseptic technique: ultra violet lamp sterilizing table top 30min before operation, before entering super clean bench, 75% ethanol is washed one's hands and wiping table top, and all operations should carry out on spirit lamp side, and belongings through flame sterilizing, must note aseptic principle.
C, cell counting: after alcohol washes tally and cover glass, dry with lens paper, cover glass is covered on tally.Tryptic digestive juice by the tumour cell of logarithmic phase, centrifugal abandon trypsin solution after, add appropriate nutrient solution, after mixing, draw a small amount of cell suspension after diluting corresponding multiple with nutrient solution, gently drop in tally from edge, make cell suspension be full of space between tally and cover glass.Basis of microscopic observation also counts corner block plaid inner cell number, and only calculate if any flanging the cell that pressure wherein fixes both sides, calculation formula is as follows:
Cell count/mL=(4 large lattice total cellular score/4) × 10
4× extension rate
D, sample preparation: take certain oligochitosan or commercial oligochitosan, be dissolved in substratum, bacteriological filtration membrane filtration is for subsequent use.
The preparation of e, MTT solution: nutrient solution dissolves MTT powder, the MTT solution for standby of the 0.5mg/mL of system.
Two, MTT detects
By 100 μ L cell suspension inoculations in 96 orifice plates, every hole is 3000 cells, by culture plate as CO
2in incubator, 37 DEG C, 5%CO
2, cultivate 24h under saturated humidity condition cell attachment grown.Add oligochitosan solution and the 5 FU 5 fluorouracil solution of 100 μ L different concns, after control group adds 100 μ L blank cultures, continue to cultivate 72h.Abandoning supernatant, every hole adds 100 μ LMTT solution, and after lucifuge hatches 4h, after abandoning supernatant, every hole adds 200 μ LDMSO, is determined at the light absorption value of 570nm after concussion in microplate reader.Inhibitory rate of cell growth is calculated according to following company:
Growth inhibition ratio (IR, %)=(1-experimental group OD value/control group OD value) × 100
Experimental result: oligochitosan prepared by the present embodiment to the inhibition of the people source tumor cell line of 10 kinds of different sourcess in table 1.The half-inhibition concentration variation range of positive drug 5 FU 5 fluorouracil to many strains tumor cell line is 0.04 ± 0.01 ~ 12.1 ± 3.0 μ g/mL, and the half-inhibition concentration variation range of oligochitosan to tumor cell line prepared by the present embodiment is 48.6 ± 7.0 ~ 1329.9 ± 93.4 μ g/mL.Oligochitosan all can reach more than 90%, even up to 95% for the maximal percentage inhibition of tumor cell line.
The people source tumor cell line of 10 kinds of different sourcess that the present embodiment is used is commercially available prod.
Table 1 oligochitosan is to the fragmentation effect of the people source tumor cell line of 10 kinds of different sourcess
Embodiment 4: the preparation method of the present embodiment oligochitosan, specifically prepare according to following steps:
Chitosan being dissolved in mass concentration is in the glacial acetic acid solution of 3.5%, then adding the heavy concentration of chitosan 20 times is 30% hydrogen peroxide, intensification is stirred to 70 DEG C of constant temperature, reaction 8h, again through mass concentration be the alcohol settling of 95%, filter, filter residue, through super-dry, obtains the oligochitosan of the polymerization degree 2 ~ 30; Wherein in oligochitosan, the content of the polymerization degree 2 ~ 10 is not less than 80%, does not comprise the oligochitosan purity >98% of moisture content; Chitosan and mass concentration are the mass volume ratio of the glacial acetic acid solution of 0.1 ~ 5.0% is 1g:20mL.
Measure oligochitosan gastric infusion to the inhibition of S180 sarcoma transplanted tumor, evaluate effect and the toxicity of killing tumor cells in its body.
Experimental technique:
One, the foundation of S180 sarcoma Transplanted tumor model
Normal saline dilution S180 cell suspension, concentration is 2 × 10
10individual/L.Choose body weight 18-22g kunming mice 30.In mouse right shoulder subcutaneous injection cell suspension, every 0.2mL.Mouse is divided into 3 groups at random, often organizes 10.
1. medicine and dosage
Be divided into negative control group (gavage gives physiological saline), low dose group (gavage gives oligochitosan: 400mg/kg) and high dose group (gavage gives oligochitosan: 800mg/kg).Oligochitosan is dissolved in stroke-physiological saline solution, is diluted to desired concn.
2. medication
Administration after inoculated tumour cell 96h, every day 1 time, successive administration 12 days, 0.2mL/20g gastric infusion (i.g.).
3. method of calculation
S180 sarcoma Transplanted tumor model uses the diameter of vernier caliper measurement tumour, and the measurement of diameter of tumor was measured once every 2 days, and the calculation formula of gross tumor volume is:
V (mm
3)=1/2 × a × b
2(in formula: a and b is respectively major diameter and the minor axis of tumour)
Antitumor activity evaluation index is tumor-like hyperplasia (WIR%) and knurl volume inhibiting rate (VIR%), adopts following formulae discovery:
WIR%=(1-W
t/ W
c) × 100% is (in formula: W
tfor treatment group knurl weight, W
cfor control group knurl weight)
VIR%=(1-V
t/ V
c) × 100% is (in formula: V
tfor treatment group knurl volume, V
cfor control group knurl volume)
Experimental result: as shown in accompanying drawing 3,4, compared with blank group, the knurl volume of oligochitosan treatment group mouse obtains effective suppression, and wherein heavy the and knurl volume of the knurl of high dose group and control group have significant difference (knurl weight P<0.01; Knurl volume P<0.05), and the variation tendency of relative body weight and blank group no significant difference.Specific experiment the results are shown in Table 2.
Table 2 oligochitosan gastric infusion is to the inhibition of S180 sarcoma growth of xenografted
Embodiment 5: the preparation method of the present embodiment oligochitosan, specifically prepare according to following steps:
Chitosan being dissolved in mass concentration is in the glacial acetic acid solution of 3.5%, then adding the heavy concentration of chitosan 20 times is 30% hydrogen peroxide, intensification is stirred to 70 DEG C of constant temperature, reaction 8h, again through mass concentration be the alcohol settling of 95%, filter, filter residue, through super-dry, obtains the oligochitosan of the polymerization degree 2 ~ 30; Wherein in oligochitosan, the content of the polymerization degree 2 ~ 10 is not less than 80%, does not comprise the oligochitosan purity >98% of moisture content; Chitosan and mass concentration are the mass volume ratio of the glacial acetic acid solution of 0.1 ~ 5.0% is 1g:20mL.
After adopting mouse residual knurl modeling body operation tumor resection, oligochitosan measures oligochitosan gastric infusion to the inhibition of the residual knurl of S180 sarcoma to tumor growth, evaluates effect and the toxicity of killing tumor cells in its body.
Experimental technique:
1, the foundation of the residual knurl model of S180 sarcoma
Normal saline dilution S180 cell suspension, concentration is 2 × 10
10individual/L.Choose body weight 18-22g kunming mice 21.In mouse right shoulder subcutaneous injection cell suspension, every 0.2mL.Within about 1 week, treat that gross tumor volume grows to 200 ~ 300mm
3after, excision solid tumor, about remaining knurl volume is 40mm
3, operation random packet on the same day, often organizes 7 and administration on the same day.
2, medicine and dosage
Be divided into negative control group (gavage gives physiological saline), low dose group (gavage gives oligochitosan: 200mg/kg) and high dose group (gavage gives oligochitosan: 400mg/kg).Oligochitosan is dissolved in stroke-physiological saline solution, is diluted to desired concn.
3, medication
Random packet administration on the same day, every day 1 time, successive administration 12 days, 0.2mL/20g gastric infusion (i.g.).
4, method of calculation
The residual knurl model of S180 sarcoma uses the diameter of vernier caliper measurement tumour, and the measurement of diameter of tumor was measured once every 2 days, and the calculation formula of gross tumor volume is:
V (mm
3)=1/2 × a × b
2(in formula: a and b is respectively major diameter and the minor axis of tumour)
Antitumor activity evaluation index is tumor-like hyperplasia (WIR%) and knurl volume inhibiting rate (VIR%), adopts following formulae discovery:
WIR%=(1-W
t/ W
c) × 100% is (in formula: W
tfor treatment group knurl weight, W
cfor control group knurl weight)
VIR%=(1-V
t/ V
c) × 100% is (in formula: V
tfor treatment group knurl volume, V
cfor control group knurl volume)
Experimental result: as shown in accompanying drawing 5,6, compared with blank group, the knurl volume of oligochitosan treatment group mouse obtains effective suppression, wherein the knurl of high dose group is heavy has significant difference (P<0.05) with control group, and the variation tendency of relative body weight and blank group no significant difference.Specific experiment the results are shown in Table 3.
Table 3 oligochitosan gastric infusion is to the inhibition of the residual knurl of S180 sarcoma
Embodiment 6: the preparation method of the present embodiment oligochitosan, specifically prepare according to following steps:
Chitosan being dissolved in mass concentration is in the glacial acetic acid solution of 3.5%, then adding the heavy concentration of chitosan 20 times is 30% hydrogen peroxide, intensification is stirred to 70 DEG C of constant temperature, reaction 8h, again through mass concentration be the alcohol settling of 95%, filter, filter residue, through super-dry, obtains the oligochitosan of the polymerization degree 2 ~ 30; Wherein in oligochitosan, the content of the polymerization degree 2 ~ 10 is not less than 80%, does not comprise the oligochitosan purity >98% of moisture content; Chitosan and mass concentration are the mass volume ratio of the glacial acetic acid solution of 0.1 ~ 5.0% is 1g:20mL.
After adopting mouse residual knurl modeling body operation tumor resection, oligochitosan measures oligochitosan intraperitoneal injection to the inhibition of the residual knurl of S180 sarcoma to tumor growth, evaluates effect and the toxicity of killing tumor cells in its body.
Experimental technique:
1, the foundation of the residual knurl model of S180 sarcoma
Normal saline dilution S180 cell suspension, concentration is 2 × 10
10individual/L.Choose body weight 18-22g kunming mice 30.In mouse right shoulder subcutaneous injection cell suspension, every 0.2mL.Within about 1 week, treat that gross tumor volume grows to 200 ~ 300mm
3after, excision solid tumor, about remaining knurl volume is 40mm
3, operation random packet on the same day, often organizes 10 and administration on the same day.
2, medicine and dosage
Be divided into negative control group (abdominal injection gives physiological saline), positive controls (abdominal injection gives astragalus polysaccharides: 250mg/kg), low dose group (abdominal injection gives oligochitosan: 50mg/kg) and high dose group (abdominal injection gives oligochitosan: 250mg/kg).Astragalus polysaccharides or oligochitosan are dissolved in stroke-physiological saline solution, are diluted to desired concn.
3, medication
Random packet administration on the same day, 1 time every other day, successive administration 11 days, 0.2mL/20g intraperitoneal injection (i.p.)
4, method of calculation
The residual knurl model of S180 sarcoma uses the diameter of vernier caliper measurement tumour, and the measurement of diameter of tumor was measured once every 2 days, and the calculation formula of gross tumor volume is:
V (mm
3)=1/2 × a × b
2(in formula: a and b is respectively major diameter and the minor axis of tumour)
Antitumor activity evaluation index is tumor-like hyperplasia (WIR%) and knurl volume inhibiting rate (VIR%), adopts following formulae discovery:
WIR%=(1-W
t/ W
c) × 100% is (in formula: W
tfor treatment group knurl weight, W
cfor control group knurl weight)
VIR%=(1-V
t/ V
c) × 100% is (in formula: V
tfor treatment group knurl volume, V
cfor control group knurl volume)
Table 4 oligochitosan gastric infusion is to the inhibition of the residual knurl of S180 sarcoma
Experimental result: as shown in accompanying drawing 7,8, compared with blank group, the knurl volume of oligochitosan abdominal injection treatment group mouse obtains effective suppression, and wherein heavy, the knurl volume of the knurl of low dose group has significant difference (knurl weight P<0.05 with control group; Knurl volume P<0.01), and the variation tendency of relative body weight and blank group no significant difference.Specific experiment the results are shown in Table 4.
In sum, in same test concentration range oligochitosan of the present invention to the kill capability of HCT-116 and A549 all higher than commercially available oligochitosan; In same test concentration range, the half-inhibition concentration variation range of positive drug 5 FU 5 fluorouracil to the people source tumor cell line of ten kinds of different sourcess is 0.04 ± 0.01 ~ 12.1 ± 3.0 μ g/mL, and oligochitosan prepared by the present invention half-inhibition concentration variation range to tumor cell line is 48.6 ± 7.0 ~ 1329.9 ± 93.4 μ g/mL.Oligochitosan all can reach more than 90%, even up to 95% for the maximal percentage inhibition of the people source tumor cell line of ten kinds of different sourcess.Oligochitosan inside and outside killing tumor cells prepared by the present invention effective.
Claims (10)
1. a preparation method for oligochitosan, is characterized in that the method is prepared according to following steps:
Chitosan being dissolved in mass concentration is in the glacial acetic acid solution of 0.1 ~ 5.0%, then adding the heavy concentration of chitosan 5 ~ 25 times is 10% ~ 40% hydrogen peroxide, intensification is stirred to 40 ~ 90 DEG C of constant temperature, reaction 0.5 ~ 10h, again through mass concentration be the alcohol settling of 60 ~ 100%, filter, filter residue, through super-dry, obtains the oligochitosan of the polymerization degree 2 ~ 30; Wherein in oligochitosan, the content of the polymerization degree 2 ~ 10 is not less than 80%, does not comprise the oligochitosan purity >98% of moisture content; Chitosan and mass concentration are the mass volume ratio of the glacial acetic acid solution of 0.1 ~ 5.0% is 1g:(10 ~ 30) mL.
2. the preparation method of a kind of oligochitosan according to claim 1, it is characterized in that the described mass concentration that is dissolved in by chitosan is in the glacial acetic acid solution of 3.5%, then adding the heavy concentration of chitosan 20 times is 30% hydrogen peroxide.
3. the preparation method of a kind of oligochitosan according to claim 1, is characterized in that described intensification is stirred to 70 DEG C of constant temperature, reaction 8h.
4. the preparation method of a kind of oligochitosan according to claim 1, is characterized in that described mistake mass concentration is the alcohol settling of 95%.
5. the preparation method of a kind of oligochitosan according to claim 1, is characterized in that the method for described drying is lyophilize.
6. the application of oligochitosan that obtains of preparation method as claimed in claim 1, is characterized in that the medicine of this oligochitosan for the preparation of 10 strain tumour cells of suppression 5 kinds of people source tumours.
7. the application of a kind of oligochitosan according to claim 6, is characterized in that 5 kinds of described people source tumours are cancer of the stomach, kidney, lung cancer, mammary cancer and colorectal carcinoma.
8. the application of a kind of oligochitosan according to claim 6, is characterized in that 10 described strain tumour cells are 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.
9. the application of a kind of oligochitosan according to claim 6, it is characterized in that the administering mode of described medicine is gastric infusion every day, dosage is 32mg/kg.
10. the application of a kind of oligochitosan according to claim 6, the administering mode that it is characterized in that described medicine is intraperitoneal injection every other day, and dosage is 4mg/kg.
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| CN106943424A (en) * | 2017-05-23 | 2017-07-14 | 哈尔滨工业大学 | A kind of application of chitosan oligosaccharide |
| CN107158024A (en) * | 2017-06-21 | 2017-09-15 | 哈尔滨工业大学 | A kind of application of chitosan oligosaccharide |
| CN110179810A (en) * | 2019-06-24 | 2019-08-30 | 江南大学 | A kind of pharmaceutical composition with antitumor action |
| CN110279862A (en) * | 2019-07-09 | 2019-09-27 | 上海市第六人民医院 | A kind of anti-cancer composition and its application in the drug of preparation treatment osteosarcoma |
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| CN105968147A (en) * | 2016-05-13 | 2016-09-28 | 上海艾苛密进出口有限公司 | Preparation method of oligochitosan |
| CN105968147B (en) * | 2016-05-13 | 2018-12-14 | 上海艾苛密进出口有限公司 | The preparation method of chitosan oligosaccharide |
| CN105949350A (en) * | 2016-06-14 | 2016-09-21 | 王斐芬 | Chitosan oligosaccharide preparation method |
| CN106943424A (en) * | 2017-05-23 | 2017-07-14 | 哈尔滨工业大学 | A kind of application of chitosan oligosaccharide |
| CN107158024B (en) * | 2017-06-21 | 2021-04-02 | 哈尔滨工业大学 | Application of chitosan oligosaccharide |
| CN107158024A (en) * | 2017-06-21 | 2017-09-15 | 哈尔滨工业大学 | A kind of application of chitosan oligosaccharide |
| CN110179810A (en) * | 2019-06-24 | 2019-08-30 | 江南大学 | A kind of pharmaceutical composition with antitumor action |
| CN110179810B (en) * | 2019-06-24 | 2022-03-25 | 江南大学 | A pharmaceutical composition with anti-tumor effect |
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