CN1240381C - Use of 5-hydroxymethyl furfural in preparation of medicine and healthcare product for prevention and treatment of degenerative disease of nervous system and cognize hurt - Google Patents
Use of 5-hydroxymethyl furfural in preparation of medicine and healthcare product for prevention and treatment of degenerative disease of nervous system and cognize hurt Download PDFInfo
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- RJGBSYZFOCAGQY-UHFFFAOYSA-N hydroxymethylfurfural Natural products COC1=CC=C(C=O)O1 RJGBSYZFOCAGQY-UHFFFAOYSA-N 0.000 title description 6
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- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The present invention relates to the application of 5-hydroxymethyl-2-furfural or a derivative thereof in preparing pharmaceuticals and health-care products for preventing / treating nerve retrogression diseases or cognitive imparement.
Description
Invention field
The present invention relates to 5 hydroxymethyl furfural and derivant thereof in preparation control neurodegenerative diseases and the medicine of cognitive impairment and the purposes in the health product.
Background technology
Neurodegenerative diseases (claiming neurodegenerative disease again) is meant owing to a series of diseases that the damage of decay property causes disordered brain function appear in central nervous system too early, comprises senile dementia, parkinson disease and Huntington Chorea etc.These diseases are feature with memory, language, the dyskinesia of chronic progressivity, have brought white elephant for society and family, and its prevalence increases with age year by year.China has entered aging society, and prevention and treatment to these diseases reduce its harm, have crucial meaning.
Summary of the invention
The purpose of this invention is to provide 5 hydroxymethyl furfural and derivant thereof in preparation control neurodegenerative diseases and the medicine of cognitive impairment and the purposes in the health product.
5 hydroxymethyl furfural, full name: 5 hydroxymethyl 2 furaldehyde, English name: 5-hydroxymethyl-2-furaldehyde (5-HMF) or 5-(hydroxy methyl) furfural.
Structural formula:
This chemical compound can extraction from Fructus Corni, separation, purification and get.Also can chemosynthesis.Can also buy from the market.
The inventor finds that through cell and animal experiment this chemical compound has good improvement effect for pathology, the pathophysiological change of senile dementia, parkinson disease and Huntington Chorea.Thereby finished the present invention.
5-HMF of the present invention can adopt oral medication, can accept the form use of compositions with various pharmaceutical preparatioies such as tablet, capsule and pharmacy.
5-HMF of the present invention also can make ejection preparation.
According to pharmaceutical composition of the present invention, can contain a certain amount of 5-HMF as active component, also can contain pharmaceutically acceptable carrier, these pharmaceutically acceptable carriers can be the various carriers that are widely used in the prior art in the medicine, as excipient, water for example.Pharmaceutical composition of the present invention can prepare by method well known in the prior art such as mixing, granulation, tabletting.Pharmaceutical composition of the present invention can also comprise the optional components that various pharmacy are used, as flavouring agent, coloring agent, sweeting agent etc.
The oral consumption of 5-HMF is generally the 0.5-10mg/kg body weight/day, the general 0.1-5mg/kg body weight/day of injection consumption.
Description of drawings
Fig. 1 is that 5-HMF causes the influence that neurocyte produces A β to sodium azide.
Embodiment
Below in conjunction with embodiment the present invention is described.
Embodiment 1
5-HMF 10mg, carboxymethyl cellulose 650mg, lactose 320mg and magnesium stearate 20mg pulverize said components, mix, and tabletting is made tablet.
Embodiment 2
5-HMF 100mg and normal saline 100mL mix, and make injection.
Pharmacodynamics test
(1) 5 hydroxymethyl furfural (5-HMF) is to the preventive and therapeutic effect of senile dementia
Alzheimer disease (Alzheimer ' s disease, AD) be a kind of chronic brain neurodegenerative diseases, clinical is first symptom with hypomnesis, develops into gradually that memory completely loses, aphasis, dyskinesia.Recent study think this sick pathomechanism main with brain in amyloid beta (A β) too much the neurofibrillary tangles, mitochondrial defects, the free radical that cause of the excessive phosphorylation of inflammatory reaction, microtubule-associated protein tau of deposition generation increases, calcium overload etc. is closely related.Our following series of studies finds that 5-HMF all has good improvement effect to these pathology damage.
1,5-HMF is to the influence of AD patient's fused cell
Mitochondrial defects is the characteristic pathological change that is prevalent in tissues such as alzheimer disease patient's brain and platelet.We adopt the ρ of AD patient's platelet mitochondrial DNA and no mitochondrial DNA
0Cell fusion has been set up AD patient's transfer mitochondrial DNA cell line model (AD patient's fused cell).This model can be simulated the multiple senile dementia pathology factor that is caused by mitochondrial injury, and carries out pharmaceutical intervention research.
(1) 5-HMF is to the influence of AD patient's fused cell reactive oxygen species
1) experiment purpose
Active oxygen (ROS) major part produces in the mitochondrion electron transfer process, is normal physiological processes.When having defective, can produce the ROS above normal level at mitochondrion electron transport chain (ETC), pair cell causes damage.The influence of 5-HMF to AD fused cell intracellular reactive oxygen species generation level observed in this research.
2) experimental technique
5-HMF 100 μ g/ml and AD fused cell preincubate 24 hours, with 2 ', ROS in 7 '-dichlorofluorescein (DCF-DA) fluorescent probe labeled cell with the level that laser confocal microscope (LSCM) is observed ROS in the individual cells, monitors the ROS dynamic change in real time; Producing work one with stream Schwann Cells instrument (FCM) pair cell integral level ROS estimates.Statistical method is 1 with DCF-DA fluorescent value in the normal 143B cell, represents ROS level in the fused cell with fused cell DCF-DA fluorescent value with the ratio of DCF-DA fluorescent value in the normal 143B cell.
3) experimental result
The result shows that 5-HMF can make active oxygen (ROS) level in AD patient's fused cell reduce by 24% (table 1).
Table 1. 5-HMF is to the influence of active oxygen (ROS) level in the AD fused cell
| Group | N | ROS (ratio) |
| Normal young group AD model group AD+5-HMF | 5 5 5 | 0.92±0.44* 1.50±0.29 1.14±0.33 |
M ± SD; * compare with the AD model group P＜0.05.
(2) 5-HMF is to the influence of AD patient's fused cell mitochondrial membrane potential
1) experiment purpose
Studies confirm that mitochondrial membrane potential (MMP) reduces about 30% than normal old contrast in the senile dementia patient cell line.This laboratory observation 5-HMF is to the influence of AD patient's fused cell MMP.
2) experimental technique
AD patient's fused cell routine is cultured to second day, and selecting concentration is 5-HMF and the fused cell preincubate 24 hours of 100 μ g/ml, with LSCM, FCM mensuration MMP.With Rh123 fluorescent value in the normal cell 143B cell is 1, with fused cell Rh123 fluorescent value with the ratio of Rh123 fluorescent value in the normal 143B cell represent MMP level in the fused cell organize between relatively.
3) experimental result
The result shows that 5-HMF can make AD patient's fused cell MMP level improve 80.4%, reaches near normal level (table 2).
Table 2. 5-HMF is to the influence of AD patient's fused cell mitochondrial membrane potential (MMP)
| Group | N | MMP |
| Young contrast AD model AD+5-HMF | 6 5 5 | 1.00±0.18** 0.51±0.20 0.92±0.65 |
M ± SD; Compare with the AD model group * P＜0.01.
(3) 5-HMF is to the influence of AD patient's fused cell endochylema calcium
1) experiment purpose
In the mitochondrion deficiency disorders, often unbalance with the adjusting of endochylema calcium, have and studies show that in AD patient's cell line have the calcium of existence unbalance.This experiment is further inquired into 5-HMF and whether the regulating power of AD patient's fused cell mitochondrial storage calcium ability and endochylema calcium is produced certain influence.
2) experimental technique
With 5-HMF100 μ g/ml preincubate 24 hours, with Fluo-3/AM is the cytoplasm calcium ion fluorescent probe, measure endochylema basis calcium and the post-stimulatory rate of change of mitochondrion oxidative phosphorylation uncoupling agent (CCCP) with LSCM and FCM, represent the basic calcium ion concentration of fused cell and be subjected to the post-stimulatory Fluo-3 change in fluorescence of CCCP rate with Fluo-3 fluorescent value relative value in fused cell and the 143B cell.
3) experimental result
The result shows that 5-HMF is hatched and can be reduced Ca in AD patient's fused cell endochylema
2+Concentration (table 3); And improve AD patient's fused cell endochylema calcium at the post-stimulatory rate of change 24.5% of CCCP (table 4), show that 5-HMF can improve the storage calcium ability of intracellular calcium store and the ability of regulating calcium homeostasis.
Table 3. 5-HMF is to the influence of AD patient's fused cell basis concentration of cytoplasm calcium ion
| Group | N | Ca 2+(ratio) |
| Young contrast AD model AD+5-HMF | 6 5 5 | 1.21±0.35 1.63±0.95 1.23±0.59 |
M±SD
Table 4. 5-HMF is subjected to the influence of the post-stimulatory endochylema calcium of CCCP rate of change to AD patient's fused cell
| Group | N | Ca 2+(ratio) |
| Young contrast AD model AD+5-HMF | 6 5 5 | 0.98±0.58 0.35±0.18* 0.60±0.10 |
M ± SD; * compare with the AD model group P＜0.05.
2,5-HMF causes the influence of AD cell model to mitochondrial injury
The defective of mitochondrial respiratory chain complex IV function is a key factor of distributing type and delayed AD morbidity, at patient's AD brain cell the active decline of mitochondrion complex IV is arranged all.We have adopted mitochondrial respiratory chain complex IV sodium azide (NaN3) to induce the variation relevant with the AD pathogeny, and have observed the pharmacodynamic action of 5-HMF.
(1) 5-HMF suppresses NaN3 and causes senile dementia cell model A β and express and increase
1) experiment purpose
A β over-deposit in brain is to cause the major reason of cranial nerve cell inflammatory reaction, necrosis.Whether this experiment discussion 5-HMF expresses to increase to A β in the senile dementia cell model born of the same parents inhibitory action.
2) experimental technique
Variable concentrations 5-HMF (50 μ g/ml, 100 μ g/ml) discards culture fluid after hatching 24 hours altogether with the SH-SY5Y neurocyte.Model group and 5-HMF protection group all add 50mmol/L NaN3, hatch altogether 4 hours with cell, and matched group adds isopyknic culture fluid.The method of using immunocytochemistry detects, with A β in the A β 1-16 antibody recognition born of the same parents.
3) experimental result
Microscopically is observed (300 *): normal cell A β mainly is distributed in the cell cytosol, and it is more shallow to dye; Model group part cell rounding, A β dyeing is deepened in the endochylema; 5-HMF protection cellular morphology is recovered, and A β dyeing shoals than model group in the endochylema, and dose-dependence is arranged.
The result shows that NaN3 can cause neurocyte A β to express enhancing; 5-HMF has the effect (accompanying drawing) that model cell A β expresses that suppresses.
(2) 5-HMF improves the survival rate of sodium azide damage AD patient fused cell
1) experiment purpose
This laboratory observation 5-HMF causes the protective effect of AD patient's fused cell damage to NaN3.
2) experimental technique
AD patient's fused cell routine is cultured to second day, and the 5-HMF of selection variable concentrations (12.5--200 μ g/ml) and fused cell preincubate 24 hours are abandoned supernatant; NaN3 and cell with 50mmol/L were hatched 2 hours, measured the survival rate of cell.
3) experimental result
The result shows that 5-HMF can improve the survival rate (table 5) that NaN3 causes AD patient's fused cell damage model.
Table 5. 5-HMF causes the influence of AD patient's fused cell damage to sodium azide (NaN3)
| Normal group | Model group (NaN3) | 5-HMF(μg/ml) | |||||
| 12.5 | 25 | 50 | 100 | 200 | |||
| MTT value (O.D. 550nm) | 0.58± 0.01 ** | 0.26± 0.02 | 0.33± 0.03 * | 0.39± 0.02 ** | 0.43± 0.02 ** | 0.44± 0.02 ** | 0.41± 0.03 ** |
M ± SD;
*P＜0.05;
*Compare with model group p＜0.01.
(3) 5-HMF improves NaN3 damage SH-SY5Y neuron survival rate and protection cell membrane integrity
1) experiment purpose
SH-SY5Y cell behaviour neuroblastoma cell system, its form is similar to neuron to physiologically active.This experiment adopts NaN3 to cause SH-SY5Y cell injury model, observes the pharmacological action of 5-HMF to this model.
2) experimental technique
The 5-HMF of various dose and SH-SY5Y cell preincubate 24 hours discard culture fluid, and the NaN3 damage that adds 50mmol/L was measured MTT value and lactic acid dehydrogenase (LDH) and spilt rate after 4 hours.The result is expressed as mean ± standard deviation, and each dosage is established three parallel holes, the experiment triplicate, and trend is identical as a result.
3) experimental result
The result shows that 5-HMF can increase the survival rate that NaN3 causes the neural cell injury model, reduces the interior LDH of cell and spills rate, the integrity (table 6) of protection cell membrane.
Table 6. 5-HMF causes SH-SY5Y neurocyte survival rate to NaN3 and descends and the destructive influence of cell membrane
| Group | N | MTT (550nm) | LDH spills rate (%) |
| Normal NaN3 model model+5-HMF 6.25 μ g/ml 5-HMF 12.5 μ g/ml 5-HMF 25 μ g/ml 5-HMF 50 μ g/ml 5-HMF 100 μ g/ml 5-HMF 200 μ g/ml | 3 3 3 3 3 3 3 3 | 0.251± 0.034** 0.050±0.011 0.072±0.024 0.082± 0.085* 0.084± 0.032* 0.062± 0.017* 0.080± 0.019* 0.088±0.005 | 9.155± 3.700** 28.05±6.746 23.24±1.992 19.01±3.659 16.20±3.659* 11.27±2.542* 13.62±3.614* 13.03±4.482* |
M ± SD; * P＜0.05;
*P＜0.01 is compared with the NaN3 model group.
(4) 5-HMF antagonism NaN3 causes the decline of SH-SY5Y neurocyte mitochondrion transmembrane potential
1) experiment purpose
Studies show that mitochondrial respiratory chain complex IV inhibitor NaN3 can make neurocyte mitochondrion transmembrane potential descend.This research is observed 5-HMF NaN3 is caused the influence that SH-SY5Y neurocyte mitochondrion transmembrane potential descends.
2) experimental technique
5-HMF (50 μ g/ml, 100 μ g/ml) was hatched 24 hours with the SH-SY5Y neurocyte, added 50mmol/L NaN3 damage 4 hours, and the normal control group adds isopyknic serum-free medium, observes the protective effect that medicine descends to the mitochondrion transmembrane potential.Adopt Rhodamine 123 labeled mitochondria transmembrane potentials, PI labelling dead cell utilizes biological microscope and laser confocal microscope to gather fluorescence distribution and change that image is observed transmembrane potential, and with image analysis calculation fluorescence area and fluorescence intensity.
3) experimental result
Normal SH-SY5Y cell is shown in morphologic observation, and cell is sprawled well, and aixs cylinder is long; Rhodamine123 fluorescence mainly is distributed in the endochylema, and a small amount of distribution is also arranged in the aixs cylinder, and fluorescence is strong, does not see dead cell.NaN3 damage group cell, it is round that cellular morphology partly becomes, the aixs cylinder retraction; The fluorescence that is distributed in the Rhodamine 123 of endochylema obviously weakens, and the fluorescence in the aixs cylinder then disappears, and the visible part cell death.5-HMF protection group cell, cellular morphology is kept well, and the part cell axon is longer; The fluorescence that is distributed in the Rhondamine 123 of endochylema strengthens, and the fluorescence in the aixs cylinder occurs, and dose-dependence is arranged, and does not see dead cell.
But the minimizing of mitochondrial membrane potential fluorescence area due to the 5-HMF antagonism sodium azide and the reduction of fluorescence intensity are shown in graphical analysis, and dose-dependence (table 7) is arranged.
Table 7. 5-HMF causes the influence that SH-SY5Y neurocyte mitochondrial membrane potential descends to NaN3
| Group | GTG | Area |
| Normal NaN3 model model+5-HMF 50 μ g/ml 5-HMF 100 μ g/ml | 61.65±11.91* 42.52±20.85 54.21±15.10* 60.40±19.69* | 630.70±288.72** 279.94±97.88 394.77±125.28* 583.92±250.15* |
M ± SD; * P＜0.05; * P＜0.01 is compared with the NaN3 model group.
(5) NaN3 is caused the protective effect that SH-SY5Y neurocyte skeleton damages
1) experiment purpose
The neurofibrillary tangles that the excessive phosphorylation of Tau albumen causes in the senile dementia patient cranial nerve cell is typical case's performance of cytoskeleton damage.Cytoskeleton mainly is made up of microtubule, microfilament.This experiment causes at NaN3 whether observation 5-HMF has protective effect to its skeleton damage on the senile dementia cell model.
2) experimental technique
The 5-HMF of variable concentrations (50 μ g/ml, 100 μ g/ml) was hatched 24 hours altogether with the SH-SY5Y cell, the NaN3 of adding 50mmol/L and SH-SY5Y cytosis 4 hours, and the normal control group adds isopyknic serum-free medium.Utilize DM1 Alpha antibodies identification α-tubulin; two anti-combinations with DM1 α with the TRITC labelling of red fluorescent material labelling to discern microtubule; utilize the Phalloidin labelling actin filament of green fluorescence material FITC labelling; utilize the laser confocal microscope images acquired; observe the distribution of microtubule and microfilament, and 5-HMF causes the protective effect of microtubule, microfilament damage to NaN3 at the SH-SY5Y cell.With amplification be 200 * image carry out graphical analysis with image analyzer.
3) experimental result
Morphologic observation (1000 *): normal group SH-SY5Y cell, cellular morphology is good, microtubule is widely distributed, and distribution is all arranged in kytoplasm, aixs cylinder and dendron, is netted, fine and close rule, fibre bundle is thick, and microfilament mainly is distributed in kytoplasm, and a small amount of distribution is also arranged in the aixs cylinder, then do not see the distribution of microfilament fluorescence in the dendron, fluorescence is strong.NaN3 damage group cell, part cell cell space become circle, and aixs cylinder shortens, the microtubule disorder of caving in, and microfilament shrinks, and the normal cell of structure is loose, and fluorescence distribution reduces, and fluorescence dies down.It is normal that 5-HMF protection group cell, cellular morphology recover, and aixs cylinder occurs, the fine and close rule of microtubule, and fibre bundle is thicker, the microfilament densification, distribution increases, and fluorescence is stronger, and fluorescence intensity, fluorescence distribution area all have dose-dependence.
Graphical analysis is the result show, 5-HMF causes the minimizing of fluorescence area of microtubule and microfilament to NaN3 and the reduction of fluorescence intensity all has protective effect, and dose-dependence (table 8,9) is arranged.
Table 8. 5-HMF causes the protective effect of SH-SY5Y neurocyte micro-duct injury to NaN3
| Group | GTG | Area |
| Normal NaN3 model model+5-HMF 50 μ g/ml 5-HMF 100 μ g/ml | 89.05±28.72** 66.97±37.65 78.59±27.64* 83.58±27.38* | 775.47±425.34** 389.03±93.62 451.80±139.21* 539.02±245.02** |
M ± SD; * P＜0.05; * P＜0.01 is compared with the NaN3 model group.
Table 9. 5-HMF causes the protective effect of SH-SY5Y neurocyte microfilament damage to NaN3
| Group | GTG | Area |
| Normal NaN3 model model+5-HMF 50 μ g/ml 5-HMF 100 μ g/ml | 76.47±26.67** 50.81±19.08 60.09±18.37* 72.73±31.72* | 544.58±287.56** 341.78±133.54 392.22±182.38* 411.24±132.49* |
M ± SD; * P＜0.05, * * P＜0.01 is compared with the NaN3 matched group.
3,5-HMF causes the influence of senile dementia cell model to inhibitors of phosphatases
Studies show that the main cause of neurofibrillary tangles is that the excessive phosphorylation of microtubule-associated protein Tau albumen causes cytoskeleton generation pathologic to change in the AD brain.The reduction of protein phosphatase enzymatic activity is the major reason of the excessive phosphorylation of Tau albumen in patient's AD brain.Sloping field acid (Okadaic acid, OA) be the inhibitor of phosphoprotein phosphatase-1 (PP-1), PP-2A, can induce SH-SY5Y cell line the excessive phosphorylation of Tau albumen to occur, and with the disappearance of synapse, aixs cylinder retraction, cell death, the factor that can be used for studying the nerve fiber degeneration of AD and regulate Tau protein phosphorylation level.SK-N-SH cell behaviour neuroblastoma cell system, form is similar to neuron to physiologically active.We use OA and the SK-N-SH cell has been set up the senile dementia cell model, and use this model and carried out drug research.
(1) 5-HMF antagonism OA causes the decline of SK-N-SH neurocyte survival rate
1) experiment purpose
This experiment adopts OA to cause SK-N-SH cell injury model, observes the pharmacological action of 5-HMF to this model.
2) experimental technique
5-HMF and SK-N-SH cell with various dose were hatched 24 hours, discarded culture fluid, and the OA that adds 150nmol/L damaged 24 hours, measured cell MTT value.The result is expressed as mean ± standard deviation, three parallel holes of each dosage, and the experiment triplicate, trend is identical as a result, with phase contrast microscope observation of cell form.
3) experimental result:
The result shows that 5-HMF can improve the survival rate (table 10) that OA causes the neural cell injury model.
Table 10. 5-HMF causes the influence that SK-N-SH neurocyte survival rate descends to OA
| Group | N | MTT (550nm) |
| Normal OA model model+5-HMF 12.5 μ g/ml 5-HMF 25 μ g/ml 5-HMF 50 μ g/ml 5-HMF 100 μ g/ml 5-HMF 200 μ g/ml | 4 4 4 4 4 4 4 | 0.129±0.008** 0.103±0.007 0.127±0.009* 0.130±0.006* 0.128±0.011* 0.142±0.004* 0.141±0.014* |
Mean ± SD; * P＜0.05;
*P＜0.01 is compared with the OA model group.
Phase contrast microscope observation of cell form: normal cell is adherent good, and aixs cylinder is sturdy longer, and dendron branch is arranged, and synapse is abundant, and cell space and projection differ respond well.OA damage group cell attachment is bad, endochylema aixs cylinder retraction, and dendron and synapse disappear, and it is shinny that cell space becomes circle.It is more sturdy that most of cell axon is organized in the 5-HMF protection, and dendron recovers, and it is better to differ effect, and good dose-dependent relationship is arranged.
(2) 5-HMF causes the protective effect of SK-N-SH neurocyte skeleton damage to OA
1) experiment purpose
The neurofibrillary tangles that the excessive phosphorylation of Tau albumen causes in the senile dementia patient cranial nerve cell is typical case's performance of cytoskeleton damage.Cytoskeleton mainly is made up of microtubule, microfilament.This experiment causes at OA whether observation 5-HMF has protective effect to its skeleton damage on the senile dementia cell model.
2) experimental technique
The 5-HMF of variable concentrations (50 μ g/ml, 100 μ g/ml) was hatched 24 hours altogether with the SH-SY5Y cell, the OA of adding 100nmol/L and SH-SY5Y cytosis 4 hours, and the normal control group adds isopyknic serum-free medium.Utilize DM1 Alpha antibodies identification α-tubulin; anti-ly combine to discern microtubule with two of the TRITC labelling of red fluorescent material labelling with DM1 α; utilize the Phalloidin labelling actin filament of green fluorescence material FITC labelling; utilize the laser confocal microscope images acquired; observe the distribution of microtubule and microfilament, and 5-HMF causes the protective effect of microtubule, microfilament damage to NaN3 at the SH-SY5Y cell.With amplification be 200 * image carry out graphical analysis with image analyzer.
3) experimental result
Show with microscope (100 *) observation: normal cell is sprawled well, the fine and close rule of kytoplasm microtubule, and the microtubule fibre bundle is thick, and fluorescence is strong; OA damage group cellular morphology becomes circle, and the kytoplasm microtubule subsides loose, and the microtubule fibre bundle attenuates, and content obviously reduces, and fluorescence dies down; The fine and close rule of protection group part cell cytoplasm microtubule, aixs cylinder microtubule fibre bundle is thicker, and fluorescence is stronger, and good dose-dependent relationship is arranged.
Show with image analysis system (1000 *) observed result: normal cell is sprawled well, the densification of kytoplasm microfilament, and fluorescence is strong; OA damage group cell cytoplasm microfilament is loose disorderly, and fluorescence dies down; Protection group part cell cytoplasm microfilament is fine and close, and fluorescence is stronger, and is dose-dependence.
The result shows, but the minimizing (table 11,12) of the reduction of the microtubule microfilament fluorescence intensity due to the 5-HMF antagonism OA and fluorescence area.
Table 11. 5-HMF causes the protective effect of SK-N-SH neurocyte micro-duct injury to OA
| Group | GTG | Area |
| Normal OA model OA+5-HMF 50 μ g/ml 5-HMF 100 μ g/ml | 110.19±23.65** 73.73±39.85 85.51±18.83* 103.72±25.14* | 624.11±112.34* 475.86±94.36 537.31±142.36* 592.43±177.42* |
M ± SD; * P＜0.05, * * P＜0.01 is compared with the OA model group.
Table 12. 5-HMF causes the protective effect of SK-N-SH neurocyte microfilament damage to OA
| Group | GTG | Area |
| Normal OA model OA+5-HMF 50 μ g/ml 5-HMF 100 μ g/ml | 160.03±46.39** 57.20±29.48 69.04±14.54* 82.76±21.50* | 592.75±199.26* 409.03±274.96 559.09±243.49* 583.28±271.14* |
M ± SD; * P＜0.05, * * P＜0.01 is compared with the OA model group.
4,5-HMF causes the protective effect of AD patient's fused cell damage to hydrogen peroxide
1) experiment purpose
Free radical increases and can promote A β deposition and inflammatory cytokine to increase, and damages biomembrane, DNA and protein etc., is one of important pathogeny of AD.This experimental observation 5-HMF is to active oxygen hydrogen peroxide (H
2O
2) due to cell injury whether have antagonism.
2) experimental technique
AD patient's fused cell model routine is cultured to second day, and selecting concentration is the 5-HMF of 12.5--200 μ g/ml and fused cell preincubate 24 hours, abandons culture fluid; H with 500 μ mol/L
2O
2Damaged 2 hours, and measured the survival rate of cell with mtt assay.
3) experimental result
The result shows that 5-HMF can significantly improve H
2O
2Cause the cell survival rate of AD patient's fused cell damage, have antioxidation (table 13).
Table 13. 5-HMF is to H
2O
2Cause the influence (mtt assay) that AD patient's fused cell survival rate descends
| Normal group | Model group (H2O2) | Model+5-HMF (μ g/ml) | |||||
| 12.5 | 25 | 50 | 100 | 200 | |||
| MTT value (O.D. 550nm) | 0.58± 0.01** | 0.27± 0.01 | 0.35± 0.02* | 0.37± 0.01** | 0.39± 0.02* | 0.38± 0.02** | 0.31± 0.03** |
M ± SD;
*P＜0.05;
*Compare with model group p＜0.01.
(2) 5-HMF is to Parkinsonian preventive and therapeutic effect
Parkinson disease (Parkinson ' s disease, PD) claim Parkinsonism again, be in, old normal carrying out property of the brain neurodegenerative disease that takes place.Its pathomechanism is because the dopaminergic neuron of substantia nigra of midbrain is degenerated, degeneration, make by nigrostriatal bundle, act on striatal mediator dopamine minimizing, cause in the striatum dopamine and acetylcholine dysequilibrium and fall ill, wherein nerve degeneration, degeneration relate to mechanism such as oxidative stress damage, mitochondria dysfunction, intracellular Ca2+ imbalance.Below research is studied the pharmacological action of 5-HMF around these mechanism.
1,5-HMF is to the influence of Parkinsonian's fused cell model
Discover the active reduction of mitochondrial respiratory chain complex I in the parkinson disease brain, the mitochondrial function defective.We adopt the ρ of PD patient's platelet mitochondrial DNA and no mitochondrial DNA
0Cell fusion has been set up PD patient's transfer mitochondrial DNA cell line model (PD patient's fused cell).And on this model, carried out pharmaceutical research.
(1) 5-HMF is to the influence of reactive oxygen species in PD patient's fused cell
1) experiment purpose
Active oxygen (ROS) major part produces in the mitochondrion electron transfer process, is normal physiological processes.When having defective, can produce ROS at mitochondrion electron transport chain (ETC) above normal level.The influence of 5-HMF to PD fused cell intracellular reactive oxygen species generation level observed in this research.
2) experimental technique
5-HMF 100 μ g/ml and PD patient's fused cell preincubate 24 hours are measured ROS with laser confocal microscope (LSCM), stream Schwann Cells instrument (FCM).With 2 ', ROS in 7 '-dichlorofluorescein (DCF-DA) fluorescent probe labeled cell is with LSCM) level of observing ROS in the individual cells, monitor the ROS dynamic change in real time; Producing work one with FCM pair cell integral level ROS estimates.Statistical method is 1 with DCF-DA fluorescent value in the normal cell 143B cell, represents ROS level in the fused cell with fused cell DCF-DA fluorescent value with the ratio of DCF-DA fluorescent value in the normal 143B cell.
3) experimental result
The result shows that 5-HMF can make the ROS level in PD patient's fused cell reduce by 56% (table 14).
Table 14. 5-HMF is to the influence of reactive oxygen species in PD patient's fused cell
| Group | N | ROS (ratio) |
| Normal young PD model PD+5-HMF | 5 6 6 | 0.92±0.44* 1.78±0.67 1.22±0.40 |
M ± SD; * compare with the PD model group P＜0.05.
(2) 5-HMF is to the influence of PD patient's fused cell mitochondrial membrane potential
1) experiment purpose
Studies confirm that PD patient's cell exists mitochondrial membrane potential (MMP) to reduce.This laboratory observation 5-HMF is to the influence of PD fused cell MMP.
2) experimental technique
PD patient's fused cell routine is cultured to second day, and selecting concentration is 5-HMF and the fused cell preincubate 24 hours of 100 μ g/ml, with LSCM, FCM mensuration MMP.With Rh123 fluorescent value in the normal cell 143B cell is 1, with fused cell Rh123 fluorescent value with the ratio of Rh123 fluorescent value in the normal 143B cell represent MMP level in the fused cell organize between relatively.
3) experimental result
The result shows that 5-HMF can make PD fused cell MMP level improve 56%, reaches near normal level (table 15).
Table 15. 5-HMF is to the influence of PD fused cell mitochondrial membrane potential (MMP)
| Group | N | MMP |
| Normal young PD model PD+5-HMF | 6 6 6 | 1.00±0.18** 0.50±0.21 0.78±0.18* |
M ± SD;
*P＜0.05;
*Compare with the PD model group p＜0.01.
(3) 5-HMF is to the influence of PD patient's fused cell endochylema calcium
1) experiment purpose
In the mitochondrion deficiency disorders, often unbalance with the adjusting of endochylema calcium, have and studies show that in PD patient's cell line have the calcium of existence unbalance.This experiment is further inquired into 5-HMF and whether the regulating power of PD patient's fused cell mitochondrial storage calcium ability and endochylema calcium is produced certain influence.
2) experimental technique
With 5-HMF100 μ g/ml and PD patient's fused cell preincubate 24 hours, with Fluo-3/AM is the cytoplasm calcium ion fluorescent probe, measure endochylema basis calcium and the post-stimulatory rate of change of mitochondrion oxidative phosphorylation uncoupling agent (CCCP) with LSCM and FCM, represent the basic calcium ion concentration of fused cell and be subjected to the post-stimulatory Fluo-3 change in fluorescence of CCCP rate with Fluo-3 fluorescent value relative value in fused cell and the 143B cell.
3) experimental result
The result shows that 5-HMF is hatched and can be reduced Ca in PD patient's fused cell endochylema
2+Concentration (table 16), and can improve PD patient's fused cell endochylema calcium at the post-stimulatory rate of change 24.5% of CCCP (table 17) shows that 5-HMF can improve the ability of the stable state of the storage calcium ability of intracellular calcium store and endochylema calcium.
Table 16. 5-HMF is to the influence of endochylema basis, PD patient's fused cell basis calcium ion concentration
| Group | N | Ca 2+(ratio) |
| Normal young PD model PD+5-HMF | 6 6 6 | 1.21±0.35 1.54±0.79 0.94±0.79 |
M±SD
Table 17. 5-HMF is subjected to the influence of the post-stimulatory endochylema calcium of CCCP rate of change to PD patient's fused cell
| Group | N | Ca 2+(ratio) |
| Normal young PD model PD+5-HMF | 6 6 6 | 0.98±0.58 0.38±0.26 0.66±0.12 |
M±SD
2,5-HMF is to H
2O
2Cause the protective effect of PD patient's fused cell damage
1) experiment purpose
Free radical increases in the pathogenic process of PD and has played important function.5-HMF is to H for this laboratory observation various dose
2O
2The protective effect of damage PD patient fused cell.
2) experimental technique
This model fused cell routine is cultured to second day, and selecting concentration is the 5-HMF of 12.5,25,50,100,200 μ g/ml and fused cell preincubate 24 hours, H
2O
2500 μ mol/L damage 2 hours is with the survival rate of mtt assay mensuration cell.
3) experimental result
The result shows, except that 12.5 μ g/ml dosage groups, other each dosage 5-HMF are obvious antagonism H all
2O
2Damage PD patient fused cell improves cell survival rate (table 18).
Table 18. 5-HMF is to H
2O
2The influence of damage PD patient fused cell survival rate
| Normal group | Model group (H2O2) | Model+5-HMF (μ g/ml) | |||||
| 12.5 | 25 | 50 | 100 | 200 | |||
| MTT value (O.D. 550nm) | 0.63± 0.01 ** | 0.52± 0.02 | 0.58± 0.01 | 0.63± 0.01 ** | 0.62± 0.01 * | 0.64± 0.02 ** | 0.64± 0.03 ** |
M ± SD;
*P＜0.05;
*Compare with model group p＜0.01.
3,5-HMF causes the protective effect of PD patient's cell injury to NaN3
1) experiment purpose
Intend observing the protective effect of 5-HMF to mitochondrial respiratory chain inhibitor NaN3 damage PD patient fused cell.
2) experimental technique
PD patient's fused cell routine is cultured to second day, and selecting concentration is 5-HMF and the fused cell preincubate 24 hours of 12.5,25,50,100,200 μ g/ml, NaN350mmol/L damage 2 hours, the survival rate of mensuration cell.
3) experimental result
The result shows, but PD patient's fused cell survival rate descends due to the 5-HMF antagonism NaN3, has cytoprotection (table 19).
Table 19. 5-HMF causes the protective effect of PD patient's fused cell damage to NaN3
| Normal group | Model group NaN3 | Model+5-HMF (μ g/ml) | |||||
| 12.5 | 25 | 50 | 100 | 200 | |||
| MTT value (O.D. 550nm) | 0.63± 0.01 ** | 0.43± 0.02 | 0.48± 0.02 * | 0.50± 0.01 ** | 0.52± 0.01 ** | 0.53± 0.02 ** | 0.54± 0.03 ** |
M ± SD;
*P＜0.05,
*Compare with model group p＜0.01.
4,5-HMF causes the influence of parkinsonian mouse model to MPTP
1) experiment purpose
Parkinsonian pathomechanism is because the dopaminergic neuron of substantia nigra of midbrain is degenerated, degeneration, makes by nigrostriatal bundle, acts on striatal mediator dopamine and reduce, and causes dopamine and acetylcholine dysequilibrium in the striatum and falls ill.MPTP can pass through specific inhibition mitochondrion complex I, produces dopamine neuron in a kind of selectivity neurotoxin infringement black substance, produces and quite similar pathology, the physiological change of clinical parkinson patient.This experiment is intended to observe 5-HMF causes the damage of mice substantia nigra dopaminergic neuron to MPTP behavioristics's improvement effect.
2) experimental technique
Male C57B1 mice gave 5-HMF continuous irrigation stomach after 7 days, and lumbar injection MPTP 30mg/kg modeling gavages 5-HMF14 days more continuously, during carry out following pole-climbing training and timing, and set up normal group and model group to compare.
The experiment apparatus is long 0.5 meter of self-control stainless steel, and diameter 1cm (twining with adhesive plaster on it) top is the wooden ball of a diameter 2.5cm.Before animal gives MPTP, carry out pole-climbing training earlier, climb at the bottom of the bar by masthead in the guiding animal 15 seconds, every training 4 times, training finishes and promptly it is carried out the pole-climbing timing, gives behind the MPTP in 1-2 hour animal to be carried out the mensuration of pole-climbing time for the second time.Concrete assay method is as follows: hold mouse tail, its head is placed masthead portion (place ball on be as the criterion with the two hind legs of mice) downwards, allow it climb down naturally, the record mice is the platform required time at the bottom of standing in masthead to two forelimb contact levers, gives behind the MPTP to repeat as above to measure in 1-2 hour.(measure 120 seconds time limits, mice can not be held bar, complete natural downslide person, writing down its pole-climbing time is 120 seconds).Calculate and respectively organize mice processing front and back pole-climbing time difference (the pole-climbing time is shorter than when being untreated after handling, and its data record is 0).Respectively organize mice during the METHOD FOR CONTINUOUS DETERMINATION modeling and handle front and back pole-climbing time difference.The harmony of pole-climbing time reflection animal well then the pole-climbing time short, inaccurate coordination then the pole-climbing time long.
3) experimental result:
The 5-HMF administration group mice pole-climbing time is compared remarkable shortening with model group, show that this medicine can significantly improve the sports coordination ability (table 20) of parkinson model mice.
Table 20. 5-HMF causes the influence of pole-climbing time of Parkinson disease model mice to MPTP
| Group | N | The pole-climbing time (second) |
| Normal control MPTP model model+5-HMF (10mg/kg) model+5-HMF (100mg/kg) | 6 6 5 6 | 10.47±2.31* 92.28±23.97 67.93±27.05 39.00±21.63* |
M ± SD; * compare with the MPTP model group P＜0.05
(3) 5-HMF is to the preventive and therapeutic effect of Huntington Chorea
1) experiment purpose
(Huntington Disease HD) claims the Huntington chorea again to Huntington Chorea, is a kind of common Basal ganglia and cerebral cortex neurodegenerative disease, and Clinical symptoms is the action of dancing sample and the dementia of chronic progressive external.This experiment intends utilizing the Huntington Chorea rat model of mitochondrion complex II/III inhibitor 3-nitropropionic acid (3-NPA) foundation, observes 5-HMF to the ethological improvement effect of this rat model.
2) experimental technique
The 3-NPA of lumbar injection 10mg/kg next day of the SD rat, modeling is 20 days altogether, the 5-HMF of the preceding 1 week beginning of modeling subcutaneous injection 30mg/kg every day, 6 weeks of administration altogether.
Morris water maze test: detect the space learning memory ability.Mainly form by round pool and automatic record system two parts.Every animal is trained 2 times every day, trains 4 days.The person that do not find the platform in 120 seconds causes platform with it, places 30 seconds, guides its Learning and Memory.Repeated above operation in 2-4 days.Data acquisition detects automatically by image with processing and analytical system is finished.Draw the time of swimming out of (promptly going into the pond) and swimming distance to the time of finding platform.
Keep away dark reaction: detect the passive avoidance ability of learning and memory.Trained in the 1st day, and the rat head was outwards put into kept away the bright chamber of camera bellows, animal generally can enter the darkroom and be subjected to electric shock 1-2 time, does not enter as animal, then drives in the darkroom, makes it to produce memory, totally 5 minutes.Tested in the 2nd day, the time that the record extremity all enter the number of times in darkroom and enter the darkroom was incubation period, as does not enter, by record in 300 seconds.
Spacious field analysis: detect motor function.Rat is put into the spacious field analysis case that divides 25 grids at the bottom of the case into, observe animal in 5 minutes stride the lattice number of times, stand number of times and Li Mao number of times.Times of exercise reduces, and shows that motor function is poor more.
3) experimental result
Compare with the 3-NPA model group, the 5-HMF administration group water maze time of swimming out of all has to a certain degree shortening with the swimming distance in each schedule, at mean difference bigger (table 21,22); Keep away dark experiment prolongation of latency, errors number minimizing (table 23), show that this medicine improves to the memory ability of Huntington Chorea rat model.The experiment of spacious field analysis shows that 5-HMF can make Huntington Chorea rat motor number of times increase, and makes it motor function improve (table 24).
Table 21. 5-HMF swims out of the time influence of (second) to Huntington Chorea rat model Morris water maze
| Group | N | First day | Second day | The 3rd day | The 4th day | On average |
| Normal control 3-NPA model model+5-HMF | 10 8 11 | 93.1± 24.9 89.9± 40.2 80.5± 48.7 | 61.5± 36.5 86.9± 41.2 62.6± 47.8 | 51.5± 51.8 84.5± 38.4 61.1± 39.4 | 19.3± 16.7 29.1± 33.0 26.4± 25.9 | 52.6± 16.1 89.5± 43.1 56.5± 33.7 |
M±SD
Table 22. 5-HMF to Huntington Chorea rat Morris water maze swim out of the distance (centimetre) influence
| Group | N | First day | Second day | The 3rd day | The 4th day | On average |
| Normal control 3-NPA model model+5-HMF | 10 8 11 | 1928.0±7 24.1 2220.1±9 52.3 1534.7±9 21.6 | 1700.5± 711.5 1846.3± 848.0 1148.42± 797.8 | 1007.5± 906.3 2005.9± 1036.3 1245.8± 726.5 | 460.8± 500.5 797.3± 875.9 527.5± 484.8 | 1140.8± 464.0 1636.9± 737.9 1100.5± 617.9 |
M±SD
Table 23. 5-HMF keeps away the influence of dark experiment to the Huntington Chorea rat
| Group | N | Incubation period (second) | Errors number |
| Normal control group 3-NPA model model+5-HMF | 10 8 11 | 173.2±15.5 105.0±82.3 173.3±22.3 | 0.22±0.44* 1.62±1.40 0.09±0.30* |
M ± SD; * compare with model group P＜0.01
Table 24. 5-HMF is to the influence of the spacious field analysis experiment of Huntington Chorea rat
| Group | N | The number of times of standing | Stride the lattice number of times | Reason hair number of times |
| Normal control group NPA model model+5-HMF | 10 8 11 | 7.11±8.43* 1.38±2.77 1.45±2.34 | 27.67±33.40 11.25±9.94 17.09±24.76 | 1.89±1.90 0.87±1.12 1.72±2.49 |
M ± SD; * compare with model group P＜0.05
Conclusion
By above series of studies, we find that 5-HMF can have good improvement effect in the many-sides such as survival rate, stabilizing cell membrane, adjusting calcium ion balance, the antagonism excessive phosphorylation of microtubule-associated protein and A β deposition of pathology damage to senile dementia from antioxidation, raising neurocyte; Can effectively improve the anti-damage ability of parkinson disease cell model and the behavioral competence of animal model; And can improve the memory ability and the motor function of Huntington Chorea rat model.Therefore, the 5-HMF compounds can be as the medicine and the health product of control cranial nerve degenerative disease and cognitive impairment.
Claims (16)
1, the purposes of 5 hydroxymethyl 2 furaldehyde in the medicine of preparation preventing/treating neurodegenerative diseases or cognitive impairment.
2, according to the purposes of claim 1, wherein neurodegenerative diseases is a senile dementia.
3, according to the purposes of claim 1, wherein neurodegenerative diseases is parkinson disease.
4, according to the purposes of claim 1, wherein neurodegenerative diseases is a Huntington Chorea.
5, the purposes of 5 hydroxymethyl 2 furaldehyde in the health product of preparation preventing/treating neurodegenerative diseases or cognitive impairment.
6, according to the purposes of claim 5, wherein neurodegenerative diseases is a senile dementia.
7, according to the purposes of claim 5, wherein neurodegenerative diseases is parkinson disease.
8, according to the purposes of claim 5, wherein neurodegenerative diseases is a Huntington Chorea.
9, the purposes of 5 hydroxymethyl 2 furaldehyde in the pharmaceutical composition of preparation prevention neurodegenerative diseases and cognitive impairment.
10, according to the purposes of claim 9, wherein neurodegenerative diseases is a senile dementia.
11, according to the purposes of claim 9, wherein neurodegenerative diseases is parkinson disease.
12, according to the purposes of claim 9, wherein neurodegenerative diseases is a Huntington Chorea.
13, the purposes of 5 hydroxymethyl 2 furaldehyde in the pharmaceutical composition of preparation treatment neurodegenerative diseases and cognitive impairment.
14, according to the purposes of claim 13, wherein neurodegenerative diseases is a senile dementia.
15, according to the purposes of claim 13, wherein neurodegenerative diseases is parkinson disease.
16, according to the purposes of claim 13, wherein neurodegenerative diseases is a Huntington Chorea.
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| TWI607004B (en) * | 2011-12-20 | 2017-12-01 | 日本阿明諾化學有限公司 | Method for producing hydroxymethyl furfural derivative, and use of treated products for process of hot water heating stems of the asparagus plant, and use of treated products for process of heating stems of the asparagus plant |
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