CN101513425A - Antiepileptic effect and application of fructose-1,6-diphosphate and dehydroascorbate - Google Patents
Antiepileptic effect and application of fructose-1,6-diphosphate and dehydroascorbate Download PDFInfo
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Abstract
The invention relates to antiepileptic effect and action mechanism of fructose-1,6-diphosphate (F1, 6BP) and dehydroascorbate (DHA) and application thereof in preventing and curing epilepsy. The F1, 6BP and the DHA can not only effectively control or inhibit epilepsy attack of animal with various acute and chronic epilepsy, but also effectively prevent generation and development of epilepsy, and even radically cure epilepsy. Antiepileptic effect mechanism of the F1, 6BP is to inhibit excess glycolysis controlled by the fructose-1,6-diphosphate and converse glycolysis of glucose to pentose phosphorylation metabolism so as to increase the level of reduction type glutathione (GSH) in brain. The DHA stimulates activity of enzyme in process of pentose phosphate metabolism to increase the level of the GSH in brain so as to perform antiepileptic effect. The special antiepileptic effect and action mechanism of the F1, 6BP and the DHA support new application thereof in preventing and curing epilepsy disease.
Description
Technical field
The present invention relates to 1,6-fructose diphosphate (fructose-1,6-bisphosphate, F1,6BP) with oxidized form vitamin C (dehydroascorbate, antiepileptic action DHA) and mechanism of action and their new application aspect control and treatment epileptic condition.F1,6BP and DHA can not only control or suppress the epilepsy of various acute and Chronic Epilepsy animal effectively, and can prevent the generation and the development of epilepsy effectively, even the radical cure epilepsy.F1, the antiepileptic action of 6BP and DHA all is to bring into play its effect by the metabolism of regulating sugar, promptly suppress 2, the too much glycolysis of 6-fructose diphosphate regulation and control and/or stimulation glucose utilization increase reduced glutathion in the brain (GSH) level by the pentose phosphate metabolic pathway.F1, the antiepileptic action of 6BP and DHA uniqueness and mechanism of action have supported their new application aspect control and treatment epilepsy disease.
Background technology
Epilepsy is a kind of common not normal chronic disease of central nervous system function of outbreak repeatedly.According to World Health Organization's recent statistics, nearly 50,000,000 artificial epileptics in the worldwide.Existing antiepileptic mainly is control or suppresses induced seizures, but can not effect a radical cure the cause of disease that causes epilepsy, in case be in particular in and stop using medicine, most epileptic's state of an illness are reaccessed.And existing antiepileptic has serious adverse effects and side effect more, such as cognitive impairment, and emotion changes and neuroendocrine disturbance etc.Therefore, seek and induced seizures can be controlled or suppress to exploitation, can effect a radical cure again cause epileptics thereby and the few new drug of toxic and side effects, significant for the treatment of epileptics.
Although multiple factor is proved to be relevant with the cause of disease of epilepsy with complicated gene reciprocal action, it is unusual that energy metabolism finally appears in epileptic's brain, particularly the Developmental and Metabolic Disorder of glucose.Glucose is the topmost energy matter of brain.Two important channels of glucose metabolism be glycolytic pathway (glycolysis pathway) and pentose phosphate approach (pentose phosphatepathway, PPP).
Glycolysis is mainly by phosphofructose enzyme-1 (phosphofructokinase-1, PFK1) regulation and control.Under normal circumstances, glucolytic end product is an acetone acid.But under neural fever and epilepsy state, glycolysis significantly increases, and a large amount of acetone acid is converted into lactic acid and causes lactic acid accumulation in the brain.Current research shows, the glycolysis of a large amount of lactic acid of this generation (being too much glycolysis, excessive glycolysis) mainly by 2,6-fructose diphosphate (fructose-2,6-bisphosphate, F2,6BP) regulation and control.F2, (phosphofructokinase-1 PFK2) produces and is the strong agonist of PFK1 6BP by phosphofructose enzyme-2.Because PFK2 only exists in the interior glial cell (astrocytes) of brain, therefore this too much glycolysis only limits in the glial cell.Numerous generations that studies show that too much glycolysis and epilepsy is closely related with development: (1) too much glycolysis is the universal phenomenon of epileptic patient and epilepsy animal; (2) glycolysis increases along with the increase of neural activity; (3) hyperglycemia is relevant with the epilepsy activity, and hypoglycemia tool antiepileptic action, the antiepileptic action of (4) Ketogenic diet (90% fat, 3% carbohydrate, 7% protein) depends on the hypoglycemia level of keeping.
Another metabolic pathway PPP of glucose is by discharging the glutathion (GS of DPNH I (NDAPH) with oxidized form
SG) be reduced into the glutathion (GSH) of reduced form.GSH is a most important free radical scavenger in the mammalian nervous system, and has very strong antiepileptic action.Numerous studies show that interior GSH of brain and epilepsy activities have substantial connection: (1) GSH and its precursor acetylcysteine all have very strong direct antiepileptic action; (2) GSH loss or low animal are easy to produce automatic epilepsy; (3) GSH loss or low and extracellular high-level excitatory amino acid (glutamic acid) is closely related, and glutamic acid and epilepsy are closely related; (4) the GSH loss or lowly be initiated oxidation stress, the key factor of mitochondrial function disorder and inflammation.
In a word, too much the generation of glycolysis and epilepsy is closely related, and the important cause of disease that interior GSH loss of brain or the outer high-level excitatory amino acid glutamic acid of oxidative stress, inflammation, mitochondrial function disorder and brain inner cell low and that cause are the epilepsy generations.Therefore suppress F2 in the glial cell, the too much glycolysis of 6BP regulation and control and stimulate glucose PPP metabolism and the chemical compound that increases GSH level in the brain can effectively prevent the generation and the development of epilepsy.Given this, we attempt researching and developing the novel antiepileptic that selectivity suppresses too much glycolysis and increases GSH level in the brain.In the present invention we concrete disclosed be 1, the 6-fructose diphosphate (F1,6BP) and oxidized form support one's family antiepileptic action and the mechanism of action of C (DHA).F1,6BP and DHA all can control or suppress induced seizures effectively to various acute and Chronic Epilepsy animal, and can prevent the generation and the development of epilepsy.The more important thing is stopping F1 in chronic rat epilepsy model, all animals epilepsy all do not occur and show effect once more after 6BP and the DHA administration, point out both to treat effectively and may effect a radical cure epilepsy.F1,6BP and DHA produce its antiepileptic action by regulating carbohydrate metabolism (suppress too much glycolysis and/or stimulate the PPP of glucose to increase GSH level in the brain).
Summary of the invention
Disclosed by the invention is F1, the antiepileptic action of 6BP and DHA and mechanism of action and their application aspect control and treatment epileptic condition.Its summary of the invention mainly comprises:
1, F1,6BP and DHA have tangible antiepileptic action to the acute epilepsy of rat that alginic acid, pilocarpine and pentetrazole bring out.
2, F1, the rat Chronic Epilepsy that 6BP and DHA bring out pilocarpine and pentetrazole has prevention and therapeutical effect.Stopping F1, all animals epilepsy all do not occur and show effect once more after 6BP and the DHA administration, point out both to treat effectively and may effect a radical cure epilepsy.
3, F1,6BP and DHA have significant protective effect to the nerve injury that pilocarpine and alginic acid bring out acute epilepsy animal.F1,6BP also have significant protective effect to the cerebral ischemia nerve injury.
4, F1, the antiepileptic action mechanism of 6BP is by suppressing 2, thus the too much glycolysis of 6-fructose diphosphate regulation and control and glucose is transformed into the pentose phosphate metabolism from glycolysis increases reduced glutathion (GSH) level in the brain.
5, DHA increases in the brain GSH level by the activity that stimulates phosphopentose metabolic pathway enzyme to bring into play its antiepileptic action.
6, suppress F2, the too much glucolytic chemical compound of 6BP regulation and control has antiepileptic action.
7, the chemical compound of inhibition phosphofructose enzyme-2 (PFK2) has antiepileptic action.
8, the chemical compound of inhibition lactic acid dehydrogenase 5 hypotypes (LDH5) has antiepileptic action.
9, the chemical compound that stimulates glucose PPP metabolic pathway to increase reduced glutathion (GSH) level in the brain has antiepileptic action.
10, increase glucose-6-phosphate dehydrogenase (6-GPD) and the active chemical compound of transldolase (transaldolase) and have antiepileptic action and antioxidation.
11, compare with existing antiepileptic, (be widely used in the medicine of treatment epilepsy in a kind of world wide such as valproic acid, also be research epilepsy new drug positive control drug commonly used), Ketogenic diet and 2-deoxyglucose (2-deoxyglucose, 2DG, a kind of glycolytic inhibitor also has antiepileptic action), F1,6BP and DHA control and treatment epileptic condition have following characteristics: (1) has the wide spectrum antiepileptic action.F1,6BP and DHA all have effect to various acute and Chronic Epilepsy animal, and stronger than the effect of existing antiepileptic.(2) F1,6BP and DHA can effectively treat and may effect a radical cure epileptic condition.Existing antiepileptic mainly is control or suppresses induced seizures, to the effect of the caused nerve injury unprotect of epilepsy, and can not effect a radical cure the cause of disease that causes epilepsy.F1,6BP and DHA can not only effectively control or suppress induced seizures, prevent the generation and the development of epilepsy, and caused nerve injury has very significant protective effect to epilepsy.The more important thing is stopping F1 in chronic rat epilepsy model, all animals epilepsy all do not occur and show effect once more after 6BP and the DHA administration, point out both to treat effectively and may effect a radical cure epilepsy.(3) F1,6BP and DHA have fewer toxic and side effects.Existing antiepileptic has serious adverse effects more, such as cognitive impairment, and emotion changes and neuroendocrine disturbance.The utilization that suppresses whole glucose is the reason that Ketogenic diet and 2-deoxyglucose produce untoward reaction such as cognitive impairment.And the utilization that suppresses whole glucose will cause in the brain GSH and γ-Ji butanoic acid (GABA) level low or damaged and increase the weight of epilepsy or produce other untoward reaction.And F1, the antiepileptic action mechanism of 6BP and DHA is that selectivity suppresses F2, and the too much glycolysis of 6BP regulation and control and stimulation glucose pentose phosphate metabolic pathway increase GSH level in the brain, and the utilization to whole glucose does not exert an influence.(4) F1,6BP are used for the report that toxic side effect is not seen in cardiopathic treatment clinically.
12, F1,6BP and DHA have prospect because the antiepileptic action and the mechanism of action of their uniquenesses aspect prevention and the treatment epileptic condition.
Description of drawings
Fig. 1 (Fig 1) is two kinds of antuepileptics 1 of the present invention, and the 6-fructose diphosphate (F1,6BP) and oxidized form C (DHA) schematic arrangement of supporting one's family;
Fig. 2 (Fig 2) is the present invention 1, and (F1 is 6BP) to the antiepileptic action of rat acute epilepsy due to the pilocarpine for the 6-fructose diphosphate;
Fig. 3 (Fig 3) is the present invention 1, and (F1 is 6BP) to the antiepileptic action of rat acute epilepsy due to the alginic acid for the 6-fructose diphosphate;
Fig. 4 (Fig 4) is the present invention 1, and (F1 is 6BP) to the antiepileptic action of rat acute epilepsy due to the pentetrazole for the 6-fructose diphosphate;
Fig. 5 (Fig 5) is the present invention 1, and (F1 is 6BP) to the antiepileptic action of rat chronic epilepsy due to the pentetrazole for the 6-fructose diphosphate;
Fig. 6 (Fig 6) is the present invention 1, and (F1 is 6BP) to the antiepileptic action of rat chronic epilepsy due to the pilocarpine for the 6-fructose diphosphate;
Fig. 7 (Fig 7) is fasting to the blood sugar level influence of epilepsy animal due to the pilocarpine;
Fig. 8 (Fig 8) be lactic acid and acetone acid to 1, the 6-fructose diphosphate (F1,6BP) and the antiepileptic action of 2-deoxyglucose;
Fig. 9 (Fig 9) is 1, and (F1 is 6BP) to the influence of reduced form glutathione for the 6-fructose diphosphate.
The concrete example of implementing
Provide the enforcement example below and further specify the present invention, but do not limit the invention.
(1): 1, the concrete enforcement example of 6-fructose diphosphate antiepileptic action
1.1, acute epilepsy model
Three kinds of different acute epilepsy models comprise pilocarpine (Pilocarpine, Pilo), alginic acid (Kainic acid, KA) and pentetrazole (Pentylenetetrazole PTZ) is used to test 1,6-fructose diphosphate (F1, antiepileptic action 6BP).Valproic acid (VPA, a kind of medicine that is widely used in the treatment epilepsy, also be research epilepsy new drug positive control drug commonly used), 2-deoxyglucose (2-deoxyglucose, 2-DG, a kind of glycolytic inhibitor also has antiepileptic action) and ketogenic diet (a kind of nutriment of treatment epilepsy) be used as contrast.Bull Sprague Dawley rat (200-230 gram) is at lumbar injection (ip) F1,6BP or VPA or 2-DG or normal saline (epilepsy group), or feed ketogenic diet after 1 hour, lumbar injection pilocarpine (300mg/kg) again, or pentetrazole (50mg/kg), or alginic acid (10mg/kg).The epilepsy behavior of observing 6 hours (Pilo and KA model) or 20 minutes (PTZ model) animals then continuously comprises epilepsy time, epilepsy grade, epilepsy persistent period, the weight of animals variation and death etc.
1.1A pilocarpine model
10 animals of epilepsy group all produce epilepsy, the epilepsy time, (latency to the first forelimb clonus afterpilocarpine) was 14 ± 1 minutes, and continue at least 5 hours, and the epilepsy grade is 5.5 ± 0.4 grades (Fig. 2 A), 4 animals are dead in 24 hours.1,6-fructose diphosphate antiepileptic action is relevant with dosage.Low dose group (0.25g/kg, n=5) not effect.In only have in 10 animals of dosage (0.5g/kg) 3 to have only grade more than or equal to 3 epilepsy.Only have 2 to have only grade in 10 animals of high dose (1.0g/kg) more than or equal to 3 epilepsy.1, in the 6-fructose diphosphate in the high dose group animal epilepsy time obviously prolong, epilepsy grade and epilepsy persistent period are all significantly reduced not animal dead.Compare with epilepsy treated animal electroencephalogram (EEG), 1,5 of 6-fructose diphosphate high dose group do not have the electroencephalogram of the animal of epilepsy row not show epilepsy activity (Fig. 2 B) yet.The acute epilepsy that 2-deoxyglucose (0,25g/kg) is led pilocarpine is also effective.6 animals of 2-deoxyglucose group only have one to have only epilepsy (grade is 3), and epilepsy grade and epilepsy persistent period all significantly reduce.(0.3g/kg n=9) can reduce epilepsy grade and epilepsy persistent period, but its effect does not have 1 to valproic acid yet, and 6-fructose diphosphate and 2-deoxyglucose are strong.Have 6 to have only epilepsy and 1 animal dead is arranged in 9 animals of valproic acid group.Ketogenic diet is to the not effect of the inductive acute epilepsy of pilocarpine.
1.1B alginic acid model
Have 9 to have only serious epilepsy (grade is more than or equal to 3) in 10 animals of the inductive epilepsy group of alginic acid, 1 animal has slight epilepsy (rhythmicity is nodded), its epilepsy time, (latency to the first wet dog shake after kainic acid) was 38 ± 4 minutes, and epilepsy grade and epilepsy persistent period are respectively 3.7 ± 0.3 and 3.7 ± 0.2 hours (Fig. 3).There is not animal dead.1,6 fructose diphosphate are also relevant with dosage to the effect of the inductive epilepsy of alginic acid.Low dose group (0.25g/kg, n=6) not effect.Middle dosage (0.5g/kg) and high dose (1.0g/kg) can obviously prolong the epilepsy time and obviously reduce epilepsy grade and epilepsy persistent period.In in 8 animals of dosage group 2 do not have epilepsy, 3 have only slight epilepsy (rhythmicity is nodded), remaining 3 have only serious epilepsy, the average rank of its epilepsy is 2.5 ± 0.5.3 do not have epilepsy in 8 animals of high dose group, and 3 have only slight epilepsy, and remaining 2 have only serious epilepsy, and the average rank of its epilepsy is 1.4 ± 0.5.2-deoxyglucose (0.25g/kg, n=6; 0.5g/kg, n=3) only can prolong the epilepsy time, other epilepsy behavior and epilepsy group relatively do not have statistical difference.Although valproic acid (0.3g/kg, n=6) treated animal has serious epilepsy, and valproic acid obviously prolongs the epilepsy time and reduces epilepsy grade and epilepsy persistent period.Ketogenic diet is to the not effect of the inductive acute epilepsy of alginic acid.
1.1C pentetrazole model:
All 9 animals of epilepsy group by pentylenetetrazole induction all have clonic epilepsy (tonic-clonic seizures), the epilepsy time, (latency to the first forelimb clonus after pentylenetetrazole) was 76 ± 6 seconds, and the epilepsy persistent period is 170 ± 54 seconds (Fig. 4).1, the low middle Senior Three dosage of 6-fructose diphosphate all can prolong the epilepsy time and reduce the epilepsy persistent period.8 animals of high dose group have 3 not have epilepsy.2-deoxyglucose (0.25g/kg, n=5; 0.5g/kg n=5) organizing all animals all has epilepsy 2-deoxyglucose can not prolong the epilepsy time.Have 2 not have epilepsy in 8 animals of valproic acid group (0.3g/kg), the epilepsy time of 6 animals obviously prolongs and the shortening of epilepsy persistent period.In addition, other establishes a treated animal (9) and drinks 30mL 0.5%1 every day, 6-fructose diphosphate (this dosage is equivalent to 0.25 gram/kilogram/every day), successive administration 7 days.At the 7th day, all animal lumbar injection pentetrazoles (50mg/kg).The result shows: 6 do not have epilepsy in 9 animals.This result not only illustrates 1, and the 6-fructose diphosphate can effectively suppress the acute epilepsy of pentylenetetrazole induction, and shows 1, and the 6-fructose diphosphate can be by the mode administration of drinking.Like this in the Chronic Epilepsy zoopery, 1, the 6-fructose diphosphate will adopt this route of administration.
1.2, the Chronic Epilepsy model
1.2A pentetrazole is lighted method model (PTZ kindling model)
Bull Sprague Dawley rat (180-200g) lumbar injection pentetrazole continuous 6 day every day (40mg/kg), the dosage that changes pentetrazole every day then into are that 35mg/kg has an epilepsy phase at least up to animal in for three days on end.Inject pentetrazole lumbar injection pentetrazole (30mg/kg) and write down the epilepsy grade of animal again after 24 hours the last time.The epilepsy animal gives normal saline (n=6) or 1,6-fructose diphosphate (0.5g/kg, n=6; 1.0g/kg, n=6).The epilepsy grade that writes down animal respectively is as the preceding contrast of treatment.At second day, all animal lumbar injection pentetrazoles (30mg/kg) gave normal saline or 1,6-fructose diphosphate again after one hour.In 60 minutes, observe the epilepsy behavior of animal and write down the epilepsy grade.The result shows: with comparing before the treatment, all animals of normal saline group have higher epilepsy grade, and 1, the epilepsy grade of all animals of two dosage groups of 6-fructose diphosphate obviously reduces (Fig. 5 A).
In order to prove that 1,6 two-phosphofructose is maybe can suppress the development that pentetrazole is lighted the inductive epilepsy of method, other 14 animals were first day lumbar injection pentetrazole (50mg/kg) relief time seven day their orthobiosis.At the 7th day, all epilepsy animals were distinguished intraperitoneal injection of saline (n=6) or 1 at lumbar injection pentetrazole (40mg/kg) after one hour, and the 6-fructose diphosphate (0.5g/kg, n=8).This medication repeats five days.Observe the epilepsy behavior of animal and write down the epilepsy grade after each administration.The result shows: behind the 6th injection pentetrazole, generalized tonic-clonic seizures all appears in all animals of normal saline group, and 1, the animal of 6-fructose diphosphate group does not see generalized tonic-clonic seizures (Fig. 5 B).
1.2B, pilocarpine model (The pilocarpine rat model of temporal lobe epilepsy)
Bull Sprague Dawley rat (180-200g) lumbar injection pilocarpine (300mg/kg).Forelimb clonic spasm (3 grades of epilepsies) occurs or stand and fall (4 grades of epilepsies) and the animal of keeping 2 hours is defined as this animal SE (statusepilepticus) is arranged.After 2 hours, SE animal lumbar injection stable (5mg/kg) is to improve the animal dis motility rate.Animal SE occurred after seven days, 8 epilepsy behaviors of observing animal to 6 pm in every day from morning.All animals several epilepsy behaviors occurred and comprise facial clonic spasm, muscle twitches, forelimb clonic spasm or stand and fall in 10 to 40 days.Occurring for the first time, forelimb clonic spasm is defined as automatic epilepsy for the first time.Occurring for the first time, the time of automatic epilepsy is 21 ± 2.7 days.Animal with automatic epilepsy be divided into the epilepsy group (normal saline, n=6) and the medicine group (0.5%1,6 two-phosphofructose, n=6) and continuous respectively two weeks drink normal saline and 0.5%1,6-fructose diphosphate (every animal 30mL every day).All animals of epilepsy group continue to occur the epilepsy behavior.1, the 6-fructose diphosphate in 7 days, stop the epilepsy behavior fully and discontinuing medication all animals of back in two weeks epilepsy not show effect (Fig. 6, F1,6BP/1).The epilepsy activity does not appear in the EEG(electroencephalography) of these animal brain cortexes.Bibliographical information is arranged, and the automatic epilepsy outbreak is after 60 days, and the epilepsy of this animal model has dependent form to medicine.
In order to prove 1, the 6-fructose diphosphate is or is effective to the epilepsy of this drug dependence that other 6 epilepsy animals drink 0.5%1,6-fructose diphosphate (every animal 30mL every day) in the automatic epilepsy outbreak after 60 days in continuous two weeks.The result shows 1, the 6-fructose diphosphate stoped the epilepsy behavior fully in 9 days and all animals of back epilepsy in 10 days of discontinuing medication is not reaccessed (Fig. 6, F1,6BP/2).These results show 1, and the 6-fructose diphosphate can effectively suppress Chronic Epilepsy and may effect a radical cure epileptic condition.
1.3, suppress carbohydrate metabolism and have antiepileptic action
1.3A: fasting is to the influence of epilepsy animal
Drink 16 bull animals of normal diet and measure its blood sugar concentration respectively, its meansigma methods is 123.9 ± 4.1mg/DL.Animal is divided into two groups then, and a treated animal is fed normal diet as epilepsy control group, another treated animal fasting 48 hours.The fasting the weight of animals alleviates 15.1 ± 0.4%, and blood sugar concentration is reduced to 79.3 ± 7.7mg/DL.The equal lumbar injection pilocarpines of two treated animals (300mg/kg) are observed the behavior of six hours animal epilepsies then continuously.Serious epilepsy (grade is greater than 3) all appears in all animals of epilepsy group.Have six dull behavior to occur in 30 minutes in seven animals of fasting group behind the injection pilocarpine, animal behavior is tending towards normal after 30 minutes.Clonic spasm (Fig. 7) appears in the another animal, and its blood sugar concentration is 97mg/DL, is the highest in seven animals of fasting group.Above result shows that blood sugar lowering concentration has antiepileptic action.Known, dietary restriction is relevant with blood sugar lowering concentration to epileptic's therapeutical effect with ketogenic diet to the antiepileptic action of genetic animal.
1.3B: the antiepileptic action of phosphofructose enzyme-2 (PFK2) inhibitor
Current research shows the too much glycolysis that produces a large amount of lactic acid mainly by 2, and (6BP is produced by phosphofructose enzyme-2 (PFK2) in the glial cell 6-fructose diphosphate for F2,6BP) regulation and control, and F2.Therefore, we infer that the inhibitor of PFK2 can reduce the PFK2 activity and reduce F2, thereby the generation of 6BP suppresses too much glycolysis and brings into play its antiepileptic action.Three kinds of different PFK2 chemical inhibitor phosphoenolpyruvate carboxylate (phosphoenolpyruvate for this reason, PEP), it is active and estimate their antiepileptic action (Fig. 7) that itaconate (itaconatey) and citrate (citrate) are used to suppress PFK2.Three groups of different animals respectively the lumbar injection sodium itaconate (250mg/kg, n=7), sodium citrate (500mg/kg, n=5) and normal saline lumbar injection pilocarpine (300mg/kg) again after a hour.Other establishes two groups of different animals brain side room injection PEP (n=5) or PBS (matched group) respectively.All animals of normal saline and PBS matched group forelimb clonic spasm all occurs and stand or fall.Sodium itaconate has antiepileptic action, has four not have epilepsy in seven animals.Same citric acid and PEP also have antiepileptic action.Three do not have epilepsy in five animals of sodium citrate group, and in five animals of PEP group four do not have epilepsy.These three all animals of administration group did not have animal dead in 24 hours.Also obtain similar result in the animal model of living alginic acid of these three kinds of PFK2 inhibitor and pentetrazole.Above result shows that suppressing the active chemical compound of PFK2 has antiepileptic action, just suppresses by 2 of PFK2 generation, and the too much glycolysis of 6-fructose diphosphate regulation and control has antiepileptic action.
Four kinds of PFK2 isomerase PFK2.1, PFK2.1, PFK2.3, PFK2.3 and PFK2.4 are arranged in the known glial cell.Wherein produce 2 with PFK2.3, (F2, ability 6BP) is the strongest for the 6-fructose diphosphate.Therefore, we infer suppress the PPFK2.3 activity can more effective blocking-up or reduce the too much glycolysis that causes epilepsy.In the acute animal epileptic model of pilocarpine, we have successfully used siRNA (small intering RNA) technology to suppress the activity of PFK2.3, its result causes the PFK2.3 protein level to reduce and produces 2, thereby the Disability of 6-fructose diphosphate suppresses too much glycolysis and produces the stronger antiepileptic activity of PFK2 inhibitor.Above result shows that PFK2.3 just plays an important role to the too much glycolysis that causes epilepsy, is that action target spot may be the new method of exploitation treatment epilepsy new drug with PFK2.3.
1.3C: the antiepileptic action of lactic acid dehydrogenase 5 hypotypes (LDH5) inhibitor
As previously mentioned, too much glycolysis to be converted into lactic acid with a large amount of acetone acid be feature.This conversion needs the participation of lactic acid dehydrogenase 5 hypotypes (LDH5).Suppress the LDH5 activity and can suppress too much glycolysis.Therefore the inhibitor of LDH5 enzyme may have antiepileptic action.The inhibitor dehydrocholic acid (deoxycholate) and the gossypol (gossypol) of two kinds of different LDH5 enzymes are used to assess its antiepileptic action.Three groups of different animals respectively brain side room injection Carachol (0.5mg in 10 μ L PBS, n=10), gossypol (1mg in 10 μ L PBS, n=6) and PBS lumbar injection pilocarpine (300mg/kg) again after a hour.All animals of PBS matched group forelimb clonic spasm all occurs and stand or fall.Carachol has significant antiepileptic action, has 6 not have epilepsy in 10 animals, and 4 animals have slight epilepsy.Same gossypol also has antiepileptic action.Have 3 not have epilepsy in 6 animals, 2 animals rhythmicity occurs and nod, and forelimb clonic spasm appears in 2 animals.These results show that the chemical compound that suppresses lactic acid dehydrogenase 5 hypotypes (LDH5) has antiepileptic action, are that action target spot also may be a kind of new method of exploitation treatment epilepsy new drug with the LDH5 enzyme.
1.4, lactic acid and acetone acid be to 1, the influence of 6-fructose diphosphate and 2-deoxyglucose antiepileptic action
Our experimental result shows 1, and (F1, antiepileptic action 6BP) is better than the 2-deoxyglucose to the 6-fructose diphosphate.This may be owing to F1, and 6BP not only suppresses too much glycolysis (promptly reducing the lactic acid accumulation) and stimulates the glucose pentose phosphateization to increase GSH level in the brain for separating.GSH has antiepileptic action.And 2-deoxyglucose antiepileptic action only is to bring into play its effect by suppressing the glucose glycolysis.Therefore, we infer that ectogenic lactic acid will abolish the antiepileptic action of 2-deoxyglucose, but only reduce by 1, the antiepileptic activity of 6-fructose diphosphate.In order to confirm this hypothesis, animal is distinguished lumbar injection 2-deoxyglucose (0.25g/kg) or 1,6-fructose diphosphate (1.0g/kg) at lumbar injection sodium lactate (0.5g/kg) after 30 minutes.Lumbar injection pilocarpine (300mg/kg) again after 30 minutes.48 hours pneumoretroperitoneums of another treated animal fasting are injected sodium lactate (0.5g/kg) after 30 minutes pneumoretroperitoneum injection pilocarpines (300mg/kg).The result shows: the 2-deoxyglucose adds the lactic acid group and fasting adds the lactic acid group and the pilocarpine group compares, and the epilepsy behavior of two treated animals is obviously difference not.The ectogenic lactic acid of this presentation of results can be abolished the antiepileptic action of 2-deoxyglucose and fasting.But lactic acid only reduces by 1, the antiepileptic activity of 6-fructose diphosphate (Fig. 8).Acetone acid is the precursor of lactic acid, under normal circumstances directly enters the Krebs circulation.If it is relevant with epilepsy to produce the too much glycolysis of a large amount of lactic acid, so ectogenic acetone acid has different influences with exogenous lactic acid to the epilepsy activity.Our experimental result shows that ectogenic acetone acid (500mg/kg) do not abolish the antiepileptic action of 2-deoxyglucose and fasting.These results have supported above hypothesis and have illustrated that the lactic acid accumulation that too much glycolysis causes is closely related with epilepsy.
1.5,1, the 6-fructose diphosphate is to the influence of reduced glutathion (GSH)
Known ectogenic 1, (F1 6BP) can not be as the carbohydrate metabolism material, but can stimulate the metabolism of glucose pentose phosphate and improve the production of NADPH and can improve the GSH level for the 6-fructose diphosphate.The tool antiepileptic action of GSH own.So F1,6BP may be by correcting GSH loss or low its antiepileptic action of bringing into play in the epilepsy brain.For this reason, we have studied F1, and 6BP is to the variation (Fig. 9) of GSH content in intact animal and three different time points brains of the inductive Chronic Epilepsy animal of pilocarpine.Animal is divided into intact animal's group (normal saline group), epilepsy animal groups and F1,6BP treatment group.F1,6BP treatment treated animal is drunk 30mL0.5%1,6-fructose diphosphate, successive administration 7 days every day.These three different time points are respectively: behind the injection pilocarpine two hours (Pilo 2h), take place after SE10 days (epilepsy does not also appear, 10d), and epilepsy initial stage (occurring epilepsy 10-15 days) and postepileptic stage (occurring epilepsy 50-60 days).The GSH level is by high-performance liquid chromatography method.Its result shows: two hours animal GSH level and intact animal's group (Normal) animal no significant difference behind the injection pilocarpine.After 10 days when epilepsy does not also occur the GSH level significantly raise, drop to normal level in epilepsy initial stage GSH level then, it is in all seriousness to continue to drop to about normal level in postepileptic stage GSH level at last.And F1, the epilepsy of the animal of 6BP treatment group stops fully and does not reaccess (Fig. 6) the horizontal intact animal's treated animal of the GSH of these animals no significant difference in all animals of back epilepsy in 10 days of discontinuing medication.Above result shows F1, and 6BP can correct the interior GSH loss of epilepsy brain or lowly bring into play its antiepileptic action.The front has proved F1, and the 6BP antiepileptic action is better than the 2-deoxyglucose.May be because F1,6BP can not only suppress glycolysis and can be by stimulating glucose pentose phosphate metabolism raising GSH level.And the antiepileptic action of 2-deoxyglucose is only brought into play by suppressing glycolysis.
The GSH level is measured by high performance liquid chromatography (HPLC) method.Instrument: HP-1100 type high-efficient phase chromatogram instrument.Chromatographic condition: chromatographic column Eclipse AAA 150 * 4.6mm, 3.5 μ m; Detect wavelength 338nm, reference wavelength 390nm; 40 ℃ of temperature; Flow velocity 1.5mL/min; Mobile phase-A 40mM Na
2HPO4, pH 7.8, mobile phase-B acetonitrile/methanol/water: 45/45/10.Reference substance: the glutathion of reduced form (GSH, purity is purchased the company in SIGMA greater than 98%), with reference to the standard fabrication method of amino acid derivativges, GSH and OPA reagent reacting are made the reference substance titer of 0.5mg/mL.The preparation of standard curve: the reference substance titer of getting concentration and be 0.5mg/mL is respectively with 0.1,0.2, and 0.5,5.0,10.0,20.0,30.0 μ L sample introductions are analyzed under above-mentioned chromatographic condition.With peak area (Y) be vertical coordinate and sample concentration (X, μ g) for abscissa map standard curve and to calculate regression equation be Y=1378.9397X+4.5802, its sample concentration is good line style relation between 0.2 to 6.0 μ g, correlation coefficient is 0.9999.Precision test: get concentration and be the GSH reference substance liquid continuous sample introduction 6 times of 0.5mg/mL, each 5 μ L measure the peak area of GSH under aforementioned chromatographic condition, and the RSD of its peak area is 2.14%.Stability test: the GSH reference substance liquid of getting concentration and be 0.5mg/mL is respectively at 0,1,6,8,12 hour sample introduction, and each 5 μ L measure the peak area of rosmarinic acid under aforementioned chromatographic condition, and the RSD of its peak area is 4.56% (n=5).The preparation of sample and analysis: adopt the reference substance same procedure to be prepared into that the sample test liquid is measured its trap under above-mentioned chromatographic condition the sample and GSH content in the calculation sample from regression equation is.
1.6,1, the neuroprotective of 6-fructose diphosphate
1.6A 1, the 6-fructose diphosphate is to the protective effect of the inductive nerve injury of pilocarpine
In experiment 1.1A, major injury (Nissl dyeing) appears in the brain cortex neurocyte of 6 animals of epilepsy group (not dead after 24 hours at the injection pilocarpine, its epilepsy grade is greater than 5), and degree of injury is 2.8 ± 0.2 grades.The neurocyte in Hippocampus CA4 of these animals and dentation gyrus district also has major injury (Fluoro-Jade B dyeing), and its average damaging cells number is 282 ± 33.1, the brain cortex neural cell injury degree of 5 animals in the 6-fructose diphosphate medicine group (in comprising and its epilepsy grade of high dose group animal more than or equal to 3) reduces greatly, and degree of injury is 0.9 ± 0.2 grade.The neural cell injury that Hippocampus CA4 of these animals and dentation brain district are returned also reduces greatly, and its average damaging cells number is 78 ± 23.1,5 of high dose group do not have the animal of epilepsy behavior not see brain cortex neural cell injury in the 6-fructose diphosphate.Damage does not appear in the neurocyte that Hippocampus CA4 of these animals and dentation brain district are returned yet.Above result shows: 1, and the 6-fructose diphosphate has significant protective effect to the inductive neural cell injury of pilocarpine.
1.6B 1 ,-fructose diphosphate is to the protective effect of the inductive nerve injury of alginic acid
In experiment 1.1B, major injury (50% animal appears at the CA1/2 district, and 100% animal appears at the CA3/4 district, Fluoro-Jade B dyeing) appears in the hippocampal neurons of 9 animals of epilepsy group (serious epilepsy is arranged, and grade is more than or equal to 3).Its average damaging cells number is 61 ± 13.By Nissl dyeing, the cell loss in CA3/4 district is high-visible.1, the 6-fructose diphosphate has significant protective effect to the inductive neural cell injury of alginic acid.1, there are not 5 animals of epilepsy neural cell injury not occur in the 6-fructose diphosphate in the high dose group.Though and neural cell injury also appears in 5 animals with serious epilepsy, its degree of injury reduces greatly, and its average damaging cells number is 15 ± 3.
1.6C 1, the 6-fructose diphosphate is to the protective effect of cerebral ischemia nerve injury
Cause former being commissioned to train to repose with excitatory toxicity material NMDA through cell injury, 1,6-fructose diphosphate (3.5mM) has obvious protective effect to this excitatory neuron cell injury.
(2): the concrete enforcement example of oxidized form vitamin C antiepileptic action
2.1, acute epilepsy model
Three kinds of different acute epilepsy models comprise that pilocarpine (Pilo), alginic acid (KA) and pentetrazole (PTZ) are used to assess oxidized form vitamin C (dehydroasxorbate, antiepileptic action DHA).Valproic acid is used as positive control.Bull Sprague Dawley rat (200-230 gram) from intracerebroventricular DHA 10 μ l (10mM) (ip), or lumbar injection valproic acid (0.3g/kg) or normal saline (epilepsy group), lumbar injection pilocarpine (300mg/kg) again, or pentetrazole (50mg/kg), or alginic acid (10mg/kg).The epilepsy behavior of observing six hours (Pilo and KA model) or 20 minutes (PTZ model) animals then continuously comprises epilepsy time, epilepsy grade, epilepsy persistent period, the weight of animals variation and death etc.
2.1A pilocarpine model
10 animals of epilepsy group all produce epilepsy, and the epilepsy time is 14 ± 1 minutes, and continues at least 5 hours, and the epilepsy grade is 5.5 ± 0.4 grades, and 4 animals are dead in 24 hours.Epilepsy did not appear in 98 of animals of DHA group in 48 hours, forelimb clonic spasm, no animal dead appearred in an animal at 54 minutes.4 of DHA groups do not have the EEG(electroencephalography) of the animal of epilepsy behavior not show the epilepsy activity.Valproic acid can obviously reduce epilepsy grade and epilepsy persistent period.3 do not have epilepsy 6 to have only epilepsy and have 1 animal dead in 24 hours in 9 animals.
2.1B, the alginic acid model
Have 9 to have only serious epilepsy (grade is more than or equal to 3) in 10 animals of the inductive epilepsy group of alginic acid, 1 animal has slight epilepsy (rhythmicity is nodded), its epilepsy time is 38 ± 4 minutes, and epilepsy grade and epilepsy persistent period were respectively 3.7 ± 0.3 and 3.7 ± 0.2 hours.DHA can prevent epilepsy.86 of animals do not have epilepsy, and other 2 slight epilepsy only occurs.Although 6 animals of valproic acid group all have serious epilepsy, valproic acid obviously prolongs the epilepsy time and reduces epilepsy grade and epilepsy persistent period.
2.1C pentetrazole model
All 9 animals of epilepsy group by pentylenetetrazole induction all have clonic epilepsy, and the epilepsy time is 76 ± 6 seconds, and the epilepsy persistent period is 170 ± 54 seconds.DHA can prevent epilepsy.8 animals have 6 not have epilepsy.There are 4 not have epilepsy in 8 animals of valproic acid group (0.3g/kg).
2.2, the Chronic Epilepsy model
The pilocarpine model: experimental technique and epilepsy group (normal saline) and epilepsy behavior and index observing are identical with previous experiments 1.2B.But medicine group every day from intracerebroventricular 10 μ l oxidized form vitamin Cs (DHA, 10mM).All animals of epilepsy group continue to occur the epilepsy behavior.The oxidized form vitamin C stoped the epilepsy behavior fully in 5 days and all animals of back epilepsy in two weeks of discontinuing medication is not reaccessed.The epilepsy activity does not appear in the EEG(electroencephalography) of these animal brain cortexes.In order to prove whether the oxidized form vitamin C has drug dependence to the inductive Chronic Epilepsy of pilocarpine, other 6 epilepsy animals are continuous two all intracerebroventricular 10 μ l oxidized form vitamin Cs after automatic epilepsy showed effect 60 days.The result shows that the oxidized form vitamin C stoped the epilepsy behavior fully in 8 days and all animals of back epilepsy in 15 days of discontinuing medication is not reaccessed.These results prove that the oxidized form vitamin C can effectively suppress Chronic Epilepsy and may effect a radical cure epileptic condition.
2.3, the ascorbic neuroprotective of oxidized form
2.3A the oxidized form vitamin C is to the protective effect of the inductive nerve injury of pilocarpine
In experiment 2.1A, major injury (Nissl dyeing) appears in the brain cortex neurocyte of 6 animals of epilepsy group (not dead after 24 hours at the injection pilocarpine, its epilepsy grade is greater than 5), and degree of injury is 2.8 ± 0.2 grades.The neurocyte in Hippocampus CA4 of these animals and dentation gyrus district also has major injury (Fluoro-Jade B dyeing), and its average damaging cells number is 282 ± 33.The brain cortex neurocyte of 8 animals of oxidized form vitamin C medicine group is not seen obvious damage.The neurocyte that Hippocampus CA4 of these animals and dentation brain district are returned is also for obviously damage occurring.Above result shows: the oxidized form vitamin C has significant protective effect to the inductive neural cell injury of pilocarpine.
2.3B the oxidized form vitamin C is being tested the protective effect of the inductive nerve injury of alginic acid
2.1B in, major injury (50% animal appears at the CA1/2 district, and 100% animal appears at the CA3/4 district, Fluoro-Jade B dyeing) appears in the hippocampal neurons of 9 animals of epilepsy group (serious epilepsy is arranged, and grade is more than or equal to 3).Its average damaging cells number is 61 ± 13.By Nissl dyeing, the cell loss in CA3/4 district is high-visible.The oxidized form vitamin C has significant protective effect to the inductive neural cell injury of alginic acid.There are not 6 animals of epilepsy not see neural cell injury in the oxidized form vitamin C medicine group.And other 2 have only the animal of slight epilepsy slight neural cell injury only to occur.
2.4, the oxidized form vitamin C is to the influence of reduced glutathion (GSH)
Experimental results show that: in the primitive cell culture experiment, the oxidized form vitamin C can by stimulate phosphopentose metabolic pathway enzyme comprise glucose-6-phosphate dehydrogenase (6-phosphogluconate dehydrogenase, 6-PDG) and the activity of transldolase (transaldolase) and significantly improve the GSH level.GSH loss or lowly be one of important cause of disease that causes epilepsy in the brain as previously mentioned, and the chemical compound that increases GSH level in the brain can effectively prevent the generation and the development of epilepsy.We infer that the ascorbic antiepileptic action of oxidized form is relevant with raising GSH level.For this reason, we have studied the variation of oxidized form vitamin C to GSH content in three different time points brains of the inductive Chronic Epilepsy animal of pilocarpine.Animal is divided into intact animal's group (normal saline group), epilepsy animal groups and oxidized form vitamin C treatment group.Oxidized form vitamin C treatment treated animal every day is from intracerebroventricular 10 μ l oxidized form vitamin Cs (10mM), successive administration 7 days.These three different time points are respectively: behind the injection pilocarpine two hours (Pilo 2h), take place after SE10 days (epilepsy does not also appear, 10d), and epilepsy initial stage (occurring epilepsy 10-15 days) and postepileptic stage (occurring epilepsy 50-60 days).The GSH level is by high-performance liquid chromatography method.Its result shows: two hours animal GSH level and intact animal's group (Normal) animal no significant difference behind the injection pilocarpine.After 10 days when epilepsy does not also occur the GSH level significantly raise, drop to normal level in epilepsy initial stage GSH level then, at last postepileptic stage GSH level continue to drop to about normal level half.And the epilepsy of the animal of oxidized form vitamin C treatment group stops fully and stopping not reaccess the horizontal no significant difference of GSH of the GSH level of these animals and intact animal's treated animal with about all animals of back epilepsy in 15 days.Above result shows that the oxidized form vitamin C may be by correcting low its antiepileptic action of bringing into play of epilepsy animal GSH level.
Claims (9)
1,1, (F1 is 6BP) with oxidized form vitamin C (dehydroascorbate, DHA) antiepileptic action and mechanism of action and their application aspect control and treatment epileptic condition for fructose-1,6-bisphosphate for the 6-fructose diphosphate.
2, F1 according to claim 1, the antiepileptic action of 6BP and DHA is characterized in that: F1, and 6BP and DHA can not only control or suppress various acute and Chronic Epilepsy animal induced seizures effectively, and can prevent the generation and the development of epilepsy effectively, even radical cure epilepsy.Various acute epilepsy animal comprise by pilocarpine (Piloacarpine, Pilo), alginic acid (Kainic acid, KA) and pentetrazole (Pentylenetetrazole, the acute epilepsy animal of the rat of PTZ) bringing out.The Chronic Epilepsy animal comprises the rat Chronic Epilepsy animal of being brought out by pilocarpine and pentetrazole.
3, F1 according to claim 1, the antiepileptic action of 6BP and DHA is characterized in that: F1,6BP and DHA have significant protective effect to the nerve injury of the acute epilepsy animal that pilocarpine and alginic acid bring out.F1,6BP also have significant protective effect to the cerebral ischemia nerve injury.
4, F1 according to claim 1, the antiepileptic action mechanism of 6BP and DHA, it is characterized in that: F1,6BP is by suppressing 2, thus the too much glycolysis of 6-fructose diphosphate regulation and control and glucose is transformed into the pentose phosphate metabolism from glycolysis increases glutathion (GSH) level of reduced form in the brain and bring into play its antiepileptic action; And DHA antiepileptic action mechanism is to increase GSH level in the brain by the activity that stimulates phosphopentose metabolic pathway enzyme.
5, suppress phosphofructose enzyme-2 (phosphofructokinase-2, PFK2) active chemical compound and their antiepileptic action.
6, suppress active chemical compound of lactic acid dehydrogenase 5 hypotypes (LDH5) and their antiepileptic action.
7, stimulate glucose pentose phosphate metabolic pathway (PPP) activity and increase the chemical compound of GSH level in the brain and their antiepileptic action.
8,1,6-fructose diphosphate and oxidized form vitamin C respectively separately or associating, and merge other drug or merge preparation and be used to prevent and treat the application of epileptic condition.
9, according to claim 1 and the described F1 of claim 8,6BP and DHA are used for control and the treatment epileptic condition can be used as medicine or functional health care product uses.Medicine or functional health care product, its preparation comprise oral administration route of administration preparation and non-oral administration route preparation.Described oral Preparation comprises tablet, oral liquid, pill, drop pill, capsule, soft capsule, effervescent, slow releasing agent, controlled release agent, targeting preparation; Described non-oral administration route preparation comprises injection, powder pin, infusion solutions, suppository.
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