CN103374079B - Methylate fucose or methylate sulphating fucose or its salt and Synthesis and applications - Google Patents
Methylate fucose or methylate sulphating fucose or its salt and Synthesis and applications Download PDFInfo
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Abstract
The present invention relates to series to methylate fucose or methylate sulphating fucose or its salt, and preparation method thereof and preparing the application in anti-parkinson drug.This patent, by preparing oligosaccharides to algal polysaccharide sulfate desulfurization is Esterification, is separated to serial high-purity methyl fucose and the sulphating fucose that methylates through column chromatography purification.Methylate fucose and the neural cell injury that sulphating fucose induces for neurotoxin that methylates has neuroprotective, may be used for medicine and the healthcare products of preparing prevention and therapy nerve degenerative diseases.
Description
Technical field
The present invention relates to methylate fucose or methylate sulphating fucose or its salt and its preparation method and application.
Background technology
Algal polysaccharide sulfate is a kind of sulfated polysaccharides from phaeophyta, has the multiple effects such as anticoagulation, anti-inflammatory, antitumor, antiviral, antiulcer agent.But due to the source difference (comprising cultivation ground, the time of harvesting, kind etc.) of brown alga, the structure obtained is also different, and known has, sulfation fucosan, sulfation rock algae glucuronic acid mannosans, sulfated fucogalactan etc.
Sulfated polysaccharides is developed as pharmaceuticals, is necessary to determine its structure.Enzyme is as a kind of important means of structural analysis, but there is no now the enzyme of commercially available decomposition algal polysaccharide sulfate, and the structure of algal polysaccharide sulfate is different because originating difference, therefore it is carried out to the desulfation of part, for structure elucidation be separated aspect and can bring huge facility.We find that this technology may be used for the preparation of fucose in the research of algal polysaccharide sulfate desulfation.
Unhomogeneity due to polysaccharose substance structure constrains the development and application of polyose medicament.And oligosaccharides is because have clear and definite constitutional features, and have many biological activitys, thus oligosaccharide kind medicine has developable prospect.The invention provides a class to methylate fucose and preparation method thereof.
Along with the increase year by year of average human life, the world is just stepping into the global aging epoch.The neurodegenerative disorders relevant with the age also increases thereupon, in central nervous system, main manifestations weakens with process of inhibition for excited, and brain function reduces, hypomnesis and forfeiture, occur that the Progressive symmetric erythrokeratodermia of recognition function goes down and the increasing the weight of of emotionally disturbed subsequently.Senile dementia become cardiovascular and cerebrovascular diseases and tumour after the 4th disease.At present, whole world over-65s crowd person in middle and old age property prevalence of dementia is about 5%.And senile dementia is because of the pathogeny of its complexity, can not make a breakthrough in control in the recent period, therefore west some experts even predict that, senile dementia will be the 21 century mankind first killer.Parkinson's disease are also known as Parkinsonism, middle-aged and old common central nervous system degenerative diseases, Parkinsonian major pathologic features is after the cell generation pathologic because being arranged in midbrain position " black substance " changes, the synthesis of Dopamine HCL reduces, suppress the function of vagusstoff to reduce, then the excitation of vagusstoff strengthens relatively.Unbalance result just there is " Parkinsonism " in both.Latest information shows, in state-owned 1,700,000 Parkinsonians, wherein in the crowd of more than 55 years old, every 100 people just have 1 patient Parkinson, estimate annual newly-increased patient Parkinson about 100,000 people.Senile dementia and Parkinson's disease all bring huge pressure to patient, family and society, the whole world is made to give numerous concerns to it, the important topic that medicine research of the nerve degenerative diseases such as senile dementia and Parkinson's disease becomes in geriatrics field.What the present invention obtained methylate fucose and the sulphating fucose that methylates have significant neuroprotective to the neural cell injury that neurotoxin causes, and the medicine for nerve degenerative diseases provides new approach.
Summary of the invention
The invention provides a class to methylate fucose and its preparation method and application.BROAD SUMMARY is as follows:
One class methylates fucose or methylate sulphating fucose or its salt,
(1) composition sugar: Fucose and derivative thereof;
(2) methylate the C4 position of position at non-reducing end Fucose;
(3) Fucose connects with α (1 → 4) glycosidic link;
(4) the sulphating fucose that methylates or methylate represented with following general expression (I) is sugar composed required composition;
In formula, R is H or SO
3h(or SO
3na), R1 is H or SO
3h(or SO
3na); The R2 integer that to be H, n be between 1-20.
One methylates fucose, and its structural formula is as following general expression (II), and in formula, n is the integer between 1-20;
One methylates sulphating fucose or its salt, and its structural formula is as following general expression (III), and in formula, n is the integer between 1-20;
Methylate the preparation method of fucose or methylate sulphating fucose or its salt:
(1) algal polysaccharide sulfate being dissolved in volume ratio is 5-20%DMSO/ methanol solution, the mass concentration of algal polysaccharide sulfate in DMSO/ methanol solution is 0.5%-5%, temperature of reaction 70-120 DEG C, reaction times is 3-10h, after completion of the reaction, reaction mixture adopts membrane separation technique purifying, lyophilize, thus obtains oligosaccharide mixture;
(2) oligosaccharide mixture obtained, adopt preparation HPLC purifying, chromatographic column adopts maltose post (" Click " Maltose post), moving phase is that acetonitrile/water/100mM ammonium formate-formic acid (pH=3.2) damping fluid carries out gradient elution, above-mentioned sample after chromatographic column is separated is collected respectively according to the timed interval, dry up with nitrogen, obtain the oligosaccharides of purifying.
Wherein algal polysaccharide sulfate derives from the Laminariales marine alga in brown alga.
Methylate fucose or methylate sulphating fucose or the application of its salt in the medicine preparing prevention and therapy nerve degenerative diseases and healthcare products.
Described nerve degenerative diseases comprises Parkinson's disease and alzheimer's disease.
Nerve cell apoptosis plays a part very important in neural cell injury, and medicine stops it to further develop by intervening apoptotic process, plays a protective role to neurocyte.SH-SY5Y cell derived is in the neuroblastoma of people, and its physiology and morphology function is similar to normal neurons, tapered, and has obvious aixs cylinder, is the current good a kind of cell of Studies On Neuronal function in the world.SY5Y cell and MES23.5 dopaminergic cell are the neural cell model that research alzheimer's disease and Parkinson's disease are commonly used respectively, methylate fucose and the sulphating fucose that methylates damages two kinds of clones to neurotoxin respectively and has obvious repair, shows that they may be used for nervous system disorders as Parkinson's disease and the medicine of senile dementia and the preparation of healthcare products.
Tool of the present invention has the following advantages:
1. the fucose that methylates of the present invention has unique methylate structure and glycosidic link structure with the sulphating fucose that methylates.
2. the fucose that methylates can adopt DMSO/ methyl alcohol devulcanization to react preparation.After further investigation, we can utilize usually chemical process to determine and the homogeneous sulfation fucose that methylates to obtain structure.
3. confirm that the fucose that methylates of desulfurization has the neurotoxicity to antineurotoxin induction first, for the preparation of neurodegenerative disease medicine and healthcare products.
Accompanying drawing explanation
Fig. 1 is that gained of the present invention methylates fucose and the ESI-MS analysis of spectra of sulfation fucose 1 of methylating.
Fig. 2 is that gained of the present invention methylates fucose and the half preparative HPLC spectrogram of sulfation fucose 1 of methylating.
Fig. 3 is that gained of the present invention methylates fucose 1-(1) mass analysis spectrogram (on) and second order ms spectrogram (under).
Fig. 4 is that gained of the present invention methylates fucose 1-(2) mass analysis spectrogram (on) and second order ms spectrogram (under).
Fig. 5 is that gained of the present invention methylates fucose 1-(3) mass analysis spectrogram (on) and second order ms spectrogram (under).
Fig. 6 is that gained of the present invention methylates fucose 1-(4) mass analysis spectrogram (on) and second order ms spectrogram (under).
Fig. 7 is that gained of the present invention methylates fucose 1-(5) mass analysis spectrogram (on) and second order ms spectrogram (under).
Fig. 8 is that gained of the present invention methylates fucose 1-(6) mass analysis spectrogram (on) and second order ms spectrogram (under).
Fig. 9 is that gained of the present invention methylates fucose 1-(7) mass analysis spectrogram (on) and second order ms spectrogram (under).
Figure 10 is that gained of the present invention methylates fucose 1-(8) mass analysis spectrogram (on) and second order ms spectrogram (under).
Figure 11 is that gained of the present invention methylates fucose 1-(9) mass analysis spectrogram (on) and second order ms spectrogram (under).
Figure 12 is that gained of the present invention methylates fucose 1-(10) mass analysis spectrogram (on) and second order ms spectrogram (under).
Figure 13 is that gained of the present invention methylates fucose 1-(11) mass analysis spectrogram (on) and second order ms spectrogram (under).
Figure 14 is that gained of the present invention methylates fucose 1-(12) mass analysis spectrogram (on) and second order ms spectrogram (under).
Figure 15 is that gained of the present invention methylates fucose 1-(13) mass analysis spectrogram (on) and second order ms spectrogram (under).
Figure 16 is that gained of the present invention methylates fucose 1-(14) mass analysis spectrogram (on) and second order ms spectrogram (under).
Figure 17 is that gained of the present invention methylates fucose and the mass analysis spectrogram of sulfation fucose 2 of methylating.
Figure 18 is that gained of the present invention methylates fucose and the mass analysis spectrogram of sulfation fucose 3 of methylating.
Figure 19 is that gained of the present invention methylates fucose and the mass analysis spectrogram of sulfation fucose 4 of methylating.
Embodiment
By embodiment, the present invention is specifically described below, but the present invention has more than and is limited to following case study on implementation scope.
Embodiment 1 methylates the preparation of fucose or its salt and methylate sulphating fucose or its salt, refining and structural analysis.
(1) methylate fucose or its salt and methylate sulphating fucose or its salt mixture is prepared
The preparation method of the sulfation that methylates fucose, it take fucoidan as raw material, can operate as follows, ratio algal polysaccharide sulfate being dissolved in volume is the DMSO/ methanol solution of 10%, the mass concentration of algal polysaccharide sulfate in DMSO/ methanol solution is 1.5%, temperature of reaction 90 DEG C, the reaction times is 10h, and the degradation of mixture obtained; After completion of the reaction, employing molecular weight cut-off is the nanofiltration membrane nanofiltration of 500, removes small-molecule substance, and trapped fluid concentrates, lyophilize, thus obtains oligosaccharide mixture A.By the oligosaccharide mixture A of gained by ESI-MS analyzing molecules quality, accompanying drawing 1 is the ESI-MS figure of oligosaccharide mixture.
(2) refining
Embodiment 1-(1) in the fucose and methylate after the lyophilize of sulphating fucose of methylating that obtains, adopt half preparative HPLC preparation, condition is as follows:
Chromatographic column: " Click " Maltose;
Flow velocity: 3mL/ divides
Column temperature: 30 DEG C;
Moving phase: acetonitrile/water/ammonium formate formic acid buffer.100mM ammonium formate formic acid buffer (pH=3.2) keeps 10% volumetric concentration, and acetonitrile is 30 minutes internal volume concentration is from 80% to 50% above, and 10 minutes volumetric concentrations are below from 50% to 15%.Minute internal volume concentration is from 10% to 40% in 30 above for water, and 10 minutes volumetric concentrations are below from 40% to 75%.
Detector: evaporat light scattering device
Sampling volume: 100 μ L
To above-mentioned sample after chromatographic column is separated, from the 4th minute, collected 1 pipe at interval of 1 minute.Obtain HPLC figure as indicated with 2.Sample nitrogen after collection dries up, and carries out mass analysis with UPLC-ESI-MS to its component.Because raw material adopts the component only containing Fucose and semi-lactosi, the quality that therefore each component is corresponding and molecular formula as shown in table 1.Its corresponding ESI-MS figure sees appendix.
The quality that each component of table 1 is corresponding and molecular formula
(3) structural analysis
ESI-CID-MS/MS is adopted to analyze the corresponding structure of each component.Name adopts below Domon-Costello. and shows each oligosaccharides 1-(1)-the physical property of (14), oligosaccharides 1-(1)-(14) corresponding with the order of above-mentioned pipe number respectively.
(a) oligosaccharides 1-(1)
Fig. 3 (under) shown in, the analytical results of second order ms is as follows, and quasi-molecular ions m/z225 illustrates the methyl alcohol losing a part, and fragments characteristic peak m/z139 illustrates that C2 position is by sulphating simultaneously, illustrates that methyl is not at reducing end simultaneously.Thus to obtain its structure of molecular ion peak m/z257 be MeFucSO
3h(Na).
(b) oligosaccharides 1-(2)
Fig. 4 (under) shown in, the analytical results of second order ms is as follows: C series: C1(177), C1-MeOH(145), C1-MeOH-H2O(127); B series: B2(305), B1(159); Y-series: Y1(163);
0,3a series:
0,3a
2(233) be characteristic ion, be inferred as 1-4 glycosidic link, and infer that methyl is at non-reducing end according to B/C series, and in C4 position.Thus to obtain its structure of molecular ion peak m/z323 be MeFuc(1-4) Fuc.
(c) oligosaccharides 1-(3)
Fig. 5 (under) shown in, the analytical results of second order ms is as follows: M-MeOH(371) illustrate that methyl exists.According to supposition above, illustrate that methyl is at non-reducing end.C-MeOH series: C1'(225); X series:
0,2x
1-F(139), be speculated as 2-SO3
-at non-reducing end.Thus to obtain its structure of molecular ion peak m/z403 be MeFuc [2SO
3h(Na)] (1-4) Fuc.
(d) oligosaccharides 1-(4)
Fig. 6 (under) shown in, the analytical results of second order ms is as follows: C series: C2(323), C1(177), C1-MeOH(145), C1-MeOH-H2O(127); B series: B2(305), B1(159); Z series: Z2(291), Z1(145); Y-series: Y1(163); X series:
0,2x
1(205)
0,3a series:
0,3a
2(233),
0,3a
3(379)
Characteristic ion, is inferred as 1-4 glycosidic link, and infers that methyl is at non-reducing end according to B/C series, and in C4 position.Thus to obtain its structure of molecular ion peak m/z469 be MeFuc(1-4) Fuc(1-4) Fuc.
(e) oligosaccharides 1-(5)
Fig. 7 (under) shown in, the analytical results of second order ms is as follows: M-MeOH(517) illustrate that methyl exists.According to supposition above, illustrate that methyl is at non-reducing end.C series: C2(403), C1 (257); C-MeOH series: C3'(517), C2'(371), C1'(225);
0,2x series:
0,2x
2-2F(139),
0,2x
1-1F(285) be speculated as 2-SO3
-at non-reducing end.Thus to obtain its structure of molecular ion peak m/z549 be MeFuc [2SO
3h(Na)] (1-4) Fuc(1-4) Fuc.
(f) oligosaccharides 1-(6)
Fig. 8 (under) shown in, the analytical results of second order ms is as follows: C series: C3(469), C2(323), C1(177), C1-MeOH(145) and, C1-MeOH-H2O(127); B series: B3(451), B3-H2O(433), B2(305) and, C1(159); Z series: Z2(291), Z1(145); Y-series: Y1(163); X series:
0,2x
1(205)
0,3a series:
0,3a
2(233),
0,3a
3(379),
0,3a
4(525) characteristic ion, is inferred as 1-4 glycosidic link, and infers that methyl is at non-reducing end according to B/C series, and in C4 position.Thus to obtain its structure of molecular ion peak m/z615 be MeFuc(1-4) Fuc(1-4) Fuc(1-4) Fuc.
(g) oligosaccharides 1-(7)
Fig. 9 (under) shown in, the analytical results of second order ms is as follows: M-MeOH(663) illustrate that methyl exists.According to supposition above, illustrate that methyl is at non-reducing end.C series: C3(549), C2(403), C1 (257); C-MeOH series: C3'(517), C2'(371), C1'(225);
0,2x series:
0,2x
2-2F(139),
0,2x
1-1F(285) be speculated as 2-SO3
-at non-reducing end.Thus to obtain its structure of molecular ion peak m/z695 be MeFuc [2SO
3h(Na)] (1-4) Fuc(1-4) Fuc(1-4) Fuc.
(h) oligosaccharides 1-(8)
Figure 10 (under) shown in, the analytical results of second order ms is as follows: C series: C4 (615), C3(469), C2(323), C1(177) and, C1-MeOH(145), C1-MeOH-H2O(127); B series: B4(597), B3(451), B2(305) and, C1(159); Z series: Z3(437), Z2(291), Z2-H2O(273) and, Z1(145); Y-series: Y1(163); X series:
0,2x
1(205);
0,3a series:
0,3a
2(233),
0,3a
3(379),
0,3a
4(525),
0,3a
5(671) characteristic ion, is inferred as 1-4 glycosidic link, and infers that methyl is at non-reducing end according to B/C series, and in C4 position.Thus to obtain its structure of molecular ion peak m/z761 be MeFuc(1-4) Fuc(1-4) Fuc(1-4) Fuc(1-4) Fuc.
(i) oligosaccharides 1-(9)
Figure 11 (under) shown in, the analytical results of second order ms is as follows: C-MeOH series: C4(663), C3'(517), C2'(371), C1'(225);
0,2x series:
0,2x
2-2F(139), be speculated as 2-SO3
-at non-reducing end.Thus to obtain its structure of molecular ion peak m/z841 be MeFuc [2SO
3h(Na)] (1-4) Fuc(1-4) Fuc(1-4) Fuc(1-4) Fuc.
(j) oligosaccharides 1-(10)
Figure 12 (under) shown in, the analytical results of second order ms is as follows: C series: C4 (615), C3(469), C2(323), C1(177) and, C1-MeOH(145), C1-MeOH-H2O(127); B series: B5 (743), B4(597), B3(451), B2(305) and, C1(159); Z series: Z3(437), Z2(291), Z2-H2O(273) and, Z1(145); Y-series: Y1(163); X series:
0,2x
1(205);
0,3a series:
0,3a
2(233),
0,3a
3(379),
0,3a
4(525),
0,3a
5(671) characteristic ion, is inferred as 1-4 glycosidic link, and infers that methyl is at non-reducing end according to B/C series, and in C4 position.Thus to obtain its structure of molecular ion peak m/z907 be MeFuc(1-4) Fuc(1-4) Fuc(1-4) Fuc(1-4) Fuc(1-4) Fuc.
(k) oligosaccharides 1-(11)
Figure 13 (under) shown in, the analytical results of second order ms is as follows: C-MeOH series: C5 (809), C4(663), C3'(517), C2'(371) and, C1'(225);
0,2x series:
0,2x
2-2F(139) be speculated as 2-SO3
-at non-reducing end.Thus to obtain its structure of molecular ion peak m/z987 be MeFuc [2SO
3h(Na)] (1-4) Fuc(1-4) Fuc(1-4) Fuc(1-4) Fuc(1-4) Fuc.
(l) oligosaccharides 1-(12)
Figure 14 (under) shown in, the analytical results of second order ms is as follows: C series: C6(907), C5(761), C4 (615), C3(469), C2(323) and, C1(177), C1-MeOH(145) and, C1-MeOH-H2O(127); B series: B6(889), B5 (743), B4(597), B3(451) and, B2(305), C1(159); Z series: Z3(437), Z2(291), Z2-H2O(273) and, Z1(145); Y-series: Y2(309), Y1(163); X series:
0,2x
1(205);
0,3a series:
0,3a
2(233),
0,3a
3(379),
0,3a
4(525),
0,3a
5(671) characteristic ion, is inferred as 1-4 glycosidic link, and infers that methyl is at non-reducing end according to B/C series, and in C4 position.Thus to obtain its structure of molecular ion peak m/z1053 be MeFuc(1-4) Fuc(1-4) Fuc(1-4) Fuc(1-4) Fuc(1-4) Fuc(1-4) Fuc.
(m) oligosaccharides 1-(13)
Figure 15 (under) shown in, the analytical results of second order ms is as follows: C-MeOH series: C6(955), C5 (809), C4(663), C3'(517), C2'(371) and, C1'(225);
0,2x series:
0,2x
2-2F(139) be speculated as 2-SO3
-at non-reducing end.Thus to obtain its structure of molecular ion peak m/z1133 be MeFuc [2SO
3h(Na)] (1-4) Fuc(1-4) Fuc(1-4) Fuc(1-4) Fuc(1-4) Fuc(1-4) Fuc.
(n) oligosaccharides 1-(14)
Figure 16 (under) shown in, the analytical results of second order ms is as follows: C series: C7(1053), (C6(907), C5(761), C4 (615), C3(469), C2(323), C1(177), C1-MeOH(145), C1-MeOH-H2O(127); B series: B7(1035), B6(889), B5 (743), B4(597) and, B3(451), B2(305) and, C1(159); Z series: Z3(437), Z2(291), Z2-H2O(273) and, Z1(145); Y-series: Y2(309), Y1(163); X series:
0,2x
1(205);
0,3a series:
0,3a
2(233),
0,3a
3(379),
0,3a
4(525),
0,3a
5(671),
0,3a
6(817),
0,3a
7(963) characteristic ion, is inferred as 1-4 glycosidic link, and infers that methyl is at non-reducing end according to B/C series, and in C4 position.Thus to obtain its structure of molecular ion peak m/z1199 be MeFuc(1-4) Fuc(1-4) Fuc(1-4) Fuc(1-4) Fuc(1-4) Fuc(1-4) Fuc(1-4) Fuc.
Embodiment 2 methylates the preparation of fucose or its salt and methylate sulphating fucose or its salt, refining and structural analysis.
(1) methylate fucose or its salt and methylate sulphating fucose or its salt mixture is prepared
The preparation method of the sulfation that methylates fucose, it take algal polysaccharide sulfate as raw material, can operate as follows, 1) taking the ratio that algal polysaccharide sulfate is dissolved in volume is 10%DMSO/ methanol solution, algal polysaccharide sulfate mass concentration is 0.5%, temperature of reaction 80 DEG C, the reaction times is 5h; 2) after completion of the reaction, tap water dialysis 2-3 days, distill water dialysis 1-2 days.Lyophilize.Thus obtaining oligosaccharide mixture, accompanying drawing 17 is schemed for its ESI-MS.
(2) refining and structural analysis
Refining and structural analysis adopts the method for embodiment 1 to process, and obtains result as described in Example 1.
Embodiment 3 methylates the preparation of fucose or its salt and methylate sulphating fucose or its salt, refining and structural analysis.
(1) methylate fucose or its salt and methylate sulphating fucose or its salt mixture is prepared
The preparation method of the sulfation that methylates fucose, it take algal polysaccharide sulfate as raw material, can operate as follows, 1) taking the ratio that algal polysaccharide sulfate is dissolved in volume is 10%DMSO/ methanol solution, algal polysaccharide sulfate mass concentration is 5%, temperature of reaction 80 DEG C, the reaction times is 5h; 2) after completion of the reaction, tap water dialysis 2-3 days, distill water dialysis 1-2 days.Lyophilize.Thus obtaining oligosaccharide mixture, accompanying drawing 18 is schemed for its ESI-MS.
(2) refining and structural analysis
Refining and structural analysis adopts the method for embodiment 1 to process, and obtains result as described in Example 1.
Embodiment 4 methylates the preparation of fucose or its salt and methylate sulphating fucose or its salt, refining and structural analysis.
(1) methylate fucose or its salt and methylate sulphating fucose or its salt mixture is prepared
The preparation method of the sulfation that methylates fucose, it take algal polysaccharide sulfate as raw material, can operate as follows, 1) taking the ratio that algal polysaccharide sulfate is dissolved in volume is 10%DMSO/ methanol solution, the mass concentration of algal polysaccharide sulfate is 2.5%, temperature of reaction 80 DEG C, the reaction times is 7h; 2) after completion of the reaction, nanofiltration desalination, lyophilize.Thus obtaining oligosaccharide mixture, accompanying drawing 19 is schemed for its ESI-MS.
(2) refining and structural analysis
Refining and structural analysis adopts the method for embodiment 1 to process, and obtains result as described in Example 1.
Test example 1 methylates fucose or methylate sulphating fucose or its salt can to the toxic action of antineurotoxin 6-OHDA
This test measures cell survival rate by mtt assay and reflects its toxic action size to antineurotoxin 6-OHDA, and its effect in neuroprotective is described.
The MES23.5 dopaminergic cell of taking the logarithm vegetative period, centrifugal after single cell suspension is made in piping and druming, be diluted to 2 × 10 with the DMEM/F12 substratum containing 10% new-born calf serum
5the cell suspension of individual cell/ml, every hole 200 μ l is inoculated in 96 orifice plates, is placed in 37 DEG C, 5%CO
2cultivate in incubator.Add after adherent with Dulbecco'smodificationofEagle'smedium/F12(DMEM/F12) sample (1mg/ml, 0.1mg/ml) that dissolves of nutrient solution (Gibco company) and 6-OHDA(100 μm of ol/L) Dual culture 24h.Test sample comprises: sample 1 is oligosaccharide mixture A in embodiment 1, and sample 2 is oligosaccharides 1-(5 in embodiment 1), sample 3 is oligosaccharides 1-(9 in embodiment 1); Sample 4 is oligosaccharides 1-(11 in embodiment 1).
Every hole adds the 3-(4 of 5mg/ml afterwards, 5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide bromine salt (MTT) 20 μ l, cultivate 4h in 37 DEG C of incubators.After abandoning supernatant, every hole adds dimethyl sulfoxide (DMSO) (DMSO) 150 μ l, and automatic elisa reading instrument (R-T-2100C, Shenzhen Lei Du company) colorimetric (wavelength 570nm), measures its absorbance to reflect the survival rate of MES23.5 cell.MT reconnaissance T measurement result is pointed out, when only adding 6-OHDA, the survival rate of cell obviously declines, and use sample 1-4(1mg/ml respectively) and (0.1mg/ml) and 6-OHDA are hatched altogether time, except sample 2 does not show obvious activity under 0.1mg/ml concentration, other each group can the decline of obvious T suppression cell survival rate, has statistical significance compared with 6-OHDA independent role group.Show to methylate fucose and the sulphating fucose that methylates can have significant protective effect to the MES23.5 cell of neurotoxin damage.
Table 2. oligosaccharide sample (1mg/ml and 0.1mg/ml) is on the impact of cell survival rate
* (P < 0.05), compared with control group; # (P < 0.05), compared with 6-OHDA group; ## (P < 0.01), compared with 6-OHDA
Test example 2 methylates the provide protection of fucose to neuroblastoma strain SH-SY5Y
The present invention take cell survival rate as index; have studied methylate fucose and the provide protection of sulphating fucose to beta amyloid peptide (β-amyloidpeptide, the A β) damage model of neuroblastoma strain SH-SY5Y cell that methylate.
Material: neurocyte SH-SY5Y cell strain, A β
25-35segment is synthesized by brain aging laboratory, Xuanwu Hospital of Capital University of Medical Science Beijing, high performance liquid chromatography purifying, purity more than 98%, serum-free MEM wiring solution-forming, considers film suction filtration ,-20 DEG C of preservations for 0.22 μm.
Test sample comprises: sample 1 is oligosaccharide mixture A in embodiment 1, and sample 2 is oligosaccharides 1-(5 in embodiment 1), sample 3 is oligosaccharides 1-(9 in embodiment 1); Sample 4 is oligosaccharides 1-(11 in embodiment 1).
Test method: cell culture condition is add 10% foetal calf serum (Hyclone, SH30070.03), 15mmol.L in MEM (GibcoBRL, 41500-067) substratum
-1hEPES (DNNcompany), penicillin 1x10
5iU.L
-1, Streptomycin sulphate 1x10
5iU.L
-1, 5%CO
2, 37 DEG C, change weekly liquid twice, go down to posterity once.By SY5Y cell with density for 1 × 10
3individual cell is inoculated in 96 orifice plates (Costar product), is divided into control group, A β after 3 days
25-35group and A β
25-35+ sample sets, the administration concentration of each test sample is respectively 1mg/mL and 0.1mg/mL, often organize 8 holes, add respectively with Dulbecco'smodificationofEagle'smedium/F12(DMEM/F12 in each sample group) sample (1mg/ml, 0.1mg/ml two concentration) that dissolves of nutrient solution (Gibco company) and A β
25-35dual culture 24h.Inoculate latter 24 hours every holes and add MTT (5mg/ml) 20 μ l, 37 ° of C hatch 4 hours, sucking-off nutrient solution, and every hole adds DMSO200 μ l, jolting 10 minutes, measures 550nm place optical density value (OD) by microplate reader (DiagnosticPasteurLP400).
Result: as shown in table 3, A β
25-35after adding, the survival rate of SH-SY5Y cell obviously declines, and A β is described
25-35major injury cell, and use sample 1-4(1mg/ml respectively) and (0.1mg/ml) and A β
25-35when hatching altogether, all samples all makes the Neuronal Survival rate of damaged significantly improve, and illustrates that methylate fucose or the sulphating fucose that methylates can repair the neurocyte of damage, has obvious neuroprotective.
Nerve cell apoptosis plays a part very important in neural cell injury, and medicine stops it to further develop by intervening apoptotic process, plays a protective role to neurocyte.SH-SY5Y cell derived is in the neuroblastoma of people, and its physiology and morphology function is similar to normal neurons, tapered, and has obvious aixs cylinder, is the current good a kind of cell of Studies On Neuronal function in the world.
SY5Y cell and MES23.5 dopaminergic cell are the neural cell model that research alzheimer's disease and Parkinson's disease are commonly used respectively, methylate fucose and the sulphating fucose that methylates damages two kinds of clones to neurotoxin respectively and has obvious repair, shows that they may be used for nervous system disorders as Parkinson's disease and the medicine of senile dementia and the preparation of healthcare products.
Table 3. oligosaccharide sample (1mg/ml and 0.1mg/ml) is on the impact of SY5Y cell survival rate
* (P < 0.05), compared with control group; # (P < 0.05), compared with damage group; ## (P < 0.01), compared with damage group.
Claims (7)
1. a class methylates fucose or methylate sulphating fucose or its salt, it is characterized in that:
(1) composition sugar: Fucose and derivative thereof;
(2) methylate the C4 position of position at non-reducing end Fucose;
(3) Fucose connects with α (1 → 4) glycosidic link;
(4) the sulphating fucose that methylates or methylate represented with following general expression (I) is sugar composed required composition;
In formula, R is H, R1 is H or SO
3h or SO
3na; The R2 integer that to be H, n be between 0-6.
2. methylate a fucose, and its structural formula is as following general expression (II), and in formula, n is the integer between 0-6;
3. methylate a sulphating fucose, and its structural formula is as following general expression (III), and in formula, n is the integer between 0-6;
4. methylate described in claim 1,2 or 3 preparation method of fucose or methylate sulphating fucose or its salt, it is characterized in that:
(1) algal polysaccharide sulfate being dissolved in volume ratio is 5-20%DMSO/ methanol solution, the mass concentration of algal polysaccharide sulfate in DMSO/ methanol solution is 0.5%-5%, temperature of reaction 70-120 DEG C, reaction times is 3-10h, after completion of the reaction, reaction mixture adopts membrane separation technique purifying, lyophilize, thus obtains oligosaccharide mixture;
(2) oligosaccharide mixture obtained, adopt preparation HPLC separation and purification, chromatographic column adopts maltose post, moving phase is that the damping fluid of acetonitrile/water/100mM ammonium formate-formic acid pH=3.2 carries out gradient elution, above-mentioned sample after chromatographic column is separated is collected respectively according to the timed interval, dry up with nitrogen, obtain the oligosaccharides of purifying.
5. method according to claim 4, is characterized in that: wherein algal polysaccharide sulfate derives from the Laminariales marine alga in brown alga.
6. methylate described in a claim 1,2 or 3 fucose or methylate sulphating fucose or the application of its salt in the medicine preparing prevention and therapy nerve degenerative diseases or healthcare products.
7. application according to claim 6, is characterized in that: described nerve degenerative diseases comprises Parkinson's disease and/or alzheimer's disease.
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| CN101152208A (en) * | 2006-09-27 | 2008-04-02 | 中国科学院海洋研究所 | Application of brown seaweed polyoses sulfate in preparing medicament for treating senile dementia |
| CN101301310A (en) * | 2007-05-08 | 2008-11-12 | 首都医科大学 | Use of brown alga polysaccharide sulfate in preventing and treating Parkinson's disease |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101152208A (en) * | 2006-09-27 | 2008-04-02 | 中国科学院海洋研究所 | Application of brown seaweed polyoses sulfate in preparing medicament for treating senile dementia |
| CN101301310A (en) * | 2007-05-08 | 2008-11-12 | 首都医科大学 | Use of brown alga polysaccharide sulfate in preventing and treating Parkinson's disease |
Non-Patent Citations (2)
| Title |
|---|
| Sulfated Polysaccharides from the Egg Jelly Layer Are Species- specific Inducers of Acrosomal Reaction in Sperms of Sea Urchins;Ana-Paula Alves etal.;《The Journal of Biological Chemistry》;19970314;第272卷(第11期);第6965-6971页 * |
| 海洋硫酸多糖不同脱硫方法的比较研究;刘鑫;《中国优秀硕士学位论文全文数据库工程科技I辑》;20120415(第4期);第17页 * |
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