CN104873531B - A kind of adenosine receptor excitement reagent and application thereof - Google Patents

Abstract

The present invention relates to nitrogen 6- (2- ethoxy) adenosines [N (6)-(2-hydroxyethyl)-adenosine, HEA] and its derivative as adenosine A₁Purposes HEA and its derivative of the receptor stimulating agent in drug or food preparation are for treating the nervous system disease related with adenosine receptor adjusting material, such as tranquilizing soporific, anticonvulsion, brain protection, Parkinson's disease, drug habit.The present invention provides a kind of source new drugs for prevention the nervous system disease.

Description

A kind of adenosine receptor excitement reagent and application thereof

Technical field

The present invention relates to a kind of new adenosine a1 receptor agonists to be particularly related to nitrogen 6- (2- ethoxy) adenosine [N (6)-(2-hydroxyethyl)-adenosine, HEA] and its derivative as adenosine a1 receptor agonists；With it as gland Glycosides A1 receptor stimulating agent can be used for treating nervous system related with adenosine receptor adjusting material in medicine preparation or food preparation The new application of disease.

Background technique

Periostracum cicadae is more nematode grass [Ophiocordyceps sobolifera (Hill ex Watson) G.H.Sung, J.M.Sung, Hywel-Jones&Spatafora ,] parasitism mountain cicada (Cicada flammatus Distant), Bamboo cicada (Platylomia pieli koto) nymph and the worm for growing more root entities in the polypide that is formed full of mycelia head Grass.Traditional Chinese Medicine records it with tranquilizing soporific, anti-frightened epilepsy, Chi Zong, the morbid night crying of babies and other effects, but simultaneously no evidence is shown to be that tool In action, how is the mechanism of effect for the substance of body.(the Advance on Pharmacological Activities China National of Qiu Jie Song Jiemin cicada fungus is civil Medical 2009,9:4-6；Thunder helps star difference condition of culture to the influence fungus journal of Cordyceps pruinosa production N6- (2- ethoxy) adenosine 15January 2014,33(1):103‐113)。

The close phase of physiological effects such as numerous result of study prompt adenosine receptors and neuronal excitation, locomitivity adjusting It closes.To treatment schizophrenia, depression, epilepsy and anxiety active drug mechanism of action have an impact (Franklin PH, Zhang G,Trpp ED,Murray TF,1989.Adenosine A₁receptor activation mediates suppression of(-)-bicuculline methiodide-induced seizures in rat prepiriform cortex.The Journal of Pharmacology and Experimental Therapeutics.251(3):1229- 1236；Lai DM, Tu YK, Liu IM, Cheng JT 2005, Increase of adenosine A₁receptor gene expression in cerebral ischemia of Wistar rats Neuroscience Letters387:59–6； Dunwiddie TV,Worth T,1982.Sedative and anticonvulsant effects of adenosine analogs in mouse and rat.The Journal of Pharmacology and Experimental Therapeutics.,220(1):70-76；Ismayilova N,Crossman A,Verkhratsky A,et al.Ef fects of adenosine A₁,dopamine D1and metabotropic glutamate 5receptors- modulating agents on locomotion of the reserpinised rats[J].Eur J Pharmacol, 2004,497(2):187-195.)。

Adenosine receptor is distributed in each position of whole body, by A as a kind of excitatory neurotransmitter₁、A_2A、A_2B、A₃, 4 kinds of hypotypes Composition, and 4 kinds of hypotypes are all g protein coupled receptors.Wherein A₁Receptor acts on the most extensive the sensibility highest of adenosine.A_2A Receptor is the important immune molecule of body, closely related with inflammatory reaction.A₁、A_2aReceptor generally takes part in adenosine to sleep, feelings The adjusting of many physiology and pathologic process such as thread.Due to lacking A_2BLigands specific, be not at present very deep to the research of A2b Enter, but zhou and zhong et al. refer to that adenosine high aggregation can activate A2B under certain pathological states, and finds that A2BR can be with Increase astroglia and discharge IL-6, implies that A2b may participate in inflammatory process.A3 receptor is right with its in the level of intracerebral The horizontal of adenosine affinity is all far below A1 and A2A receptor, and physiological action is currently not (Wang Renye Pan Jianchun fully aware of Adenosine and its receptor in August, 2006 the 4th phase of volume 33 of the biological action foreign medical science pharmacy fascicle in nervous system；Zhou AM,Li WB,Li QJ,et al.A short cerebral ischemic preconditioning up-regulates adenosine receptors in the hippocampal CA1regionof rat s[J].Neurosci Res, 2004,48(4):397-404.Zhong H,Belardinelli L,Maa T,et al.Synergy between A2B adenosine receptors and hypoxia in activating human lung fibroblast s[J].AmJ Respir Cell Mol Biol,2005,32(1):2-8.)。

A large number of studies show that selectivity A₁Adenosine receptor agonist has a variety of nerves as endogenous neural Protective substances Defencive function: the research of such as Taiwo has shown that the A on peripheral sensory tip₁After receptor is activated, adenyl cyclase can inhibit (adenylyl cyclase, AC) declines second messenger's cyclic adenosine monophosphate (cAMP) concentration intracellular, and then generates analgesia and make With；Kaster etc. has found what the antidepression sample effect of adenosine was seemingly realized by the activation of A1 receptor and A2A receptor；Millan The experiment of MJ shows that cause anxiety and angst resistance effect are related with the blocking of A1 receptor and excitement respectively, the mouse of A1R gene delection There are more anxiety performance (Taiwo YO, Levine JD.Further confirmation of the role of adenyl Cyclase and of cAMP-dependent protein kinase in primary afferent Hyperalgesia [J] .Neuroscience, 1991,44 (1): 131~135；Kaster MP,Rosa AO,Rosso MM, et al.Adenosine administration produces an antidepressant like effect in mice:evidence for the involvement of A1and A2A receptors[J].Neurosci Lett, 2004,355(1):21-24.；Millan MJ.The neurobiology and control of anxious states [J].Prog Neurobiol,2003,70(2):83-244；).

Adenosine A 1 receptor is the glycoprotein containing 326 amino acid, molecular weight 36600.Activation A1 receptor can play Protect the effect of neuron.It is now recognized that its possible mechanism are as follows: on the one hand, A1 is inhibited excitatory neurotransmitter such as paddy by physical efficiency The release of propylhomoserin protects cell by reducing the excitability of cell.On the other hand, the A1 on postsynaptic membrane is activated to be made by physical efficiency Intracellular outward potassium flow increases, because excitability reduces to protect neuron.(in the Effect study progress of Zong Kaiqi adenosine A 1 receptor State's Pharmacological Bulletin 200824 (5): 573~6)

One's early years Jacobson KA et al. reports that adenosine interferes its use on clinical medicine because it is metabolized unstability. Hereafter, some relatively stable analogs have been synthesized in succession, these compounds are primarily directed to adenosine N⁶, 2- and 5 ' position repairs Decorations.It is documented that N⁶The neplanocin replaced is proven to have A₁Receptor-selective, as CPA and CHA has 400~800 A₁Selectivity.CCPA is 1500 times, the A of S-ENBA₁It is selective then stronger, up to 4700 times.And 5 '-substituted adenosines analogs NECA has been widely used for explaining by A₂Biological effect caused by receptor activation.In the structural modification effect of other ribose, 2 '- The metalepsis of position can lose compatibility completely, and the unsubstituted hydroxyl in 3 '-positions is efficiently necessary.(kenneth The pharmacology and structure-activity relationship pharmacy of the general receptor of A.Jacobson gland are in progress the phase of volume 199216 the 4th)

Present existing A₁Agonist is mostly N⁶Substituted adenosine derivative, including CCPA, CHA, CPA etc., they are to A₁ All there is stronger selectivity.

Summary of the invention

The present invention has surprisingly found that nitrogen 6- (2- ethoxy) adenosine and derivative are a kind of new adenosine receptor excitement examinations Agent.They are to A₁All there is stronger selectivity.It is this to pass through specific bond A1 receptor and generate a series of Physiology and biochemistry work It is dynamic, to adjust neurological function.

On the one hand, the present invention relates to a kind of exciting reagent of new adenosine receptor, which includes nitrogen 6- (2- ethoxy) Adenosine and derivative.

In some specific embodiments, the adenosine receptor is A₁、A_2A、A_2B、A₃One or more of.

In some preferred embodiments, adenosine receptor A₁Receptor.

In some specific embodiments, nitrogen 6- (2- ethoxy) adenosine and derivative are a kind of new specific bonds The exciting reagent of A1 receptor.

In some modes, the nitrogen 6- (2- ethoxy) adenosine [N (6)-(2-hydroxyethyl)- Adenosine, HEA] such as flowering structure

In other specific embodiments, the derivative of the HEA is following general formula (1)

Wherein, R1 is the alkyl or hydroxyl of branch or straight chain.In some preferred modes, R1 be C (CH3) 2CH2OH, CH (CH3) CH2OH or C (CH3) 3.

On the other hand, the present invention provide a kind of nitrogen 6- (2- ethoxy) adenosine and derivative be used to prepare treatment or Prevent the purposes in convulsions, apoplexy, Parkinson or opioid addiction reagent.

A further object for invention is to provide the nerves such as a kind of prevention and treatment convulsions, headstroke, Parkinson, drug habit Pharmaceutical agent in systemic disease, wherein the reagent includes HEA and its derivative.

The another object of invention is provided with the nerveous systems such as a kind of prevention and treatment convulsions, headstroke, Parkinson, drug habit Food in disease of uniting, wherein the food includes HEA and its derivative.

On the other hand, the present invention, which provides a kind of nitrogen 6- (2- ethoxy) adenosine and derivative, improves sleep being used to prepare Purposes in the reagent of obstacle or raising sleep quality.

On the one hand, the present invention provides a kind of for improving sleep, prevention convulsions, calm pharmaceutical agent, wherein the reagent Including HEA and its derivative.

Preferably, according to the purposes or reagent, the reagent is as pharmaceutical agent, food reagent.

Preferably, according to the purposes or reagent, its derivative of nitrogen 6- (2- ethoxy) adenosine is comprised the following structure General formula:

Wherein, R1 is the alkyl of branch or straight chain.

Preferably, R1 is C (CH3) 2CH2OH, CH (CH3) CH2OH or C (CH3) 3.

Preferably, according to the purposes or reagent, it is because of nitrogen 6- that playing, which improves sleep disturbance or raising sleep quality, (2- ethoxy) adenosine and its derivative are realized as adenosine receptor agonist.Preferably, adenosine receptor A₁、A_2A、 A_2B、A₃One or more of.Preferably, adenosine receptor A₁Receptor.

Preferably, in above-mentioned reagent or purposes, the nitrogen 6- (2- ethoxy) adenosine from animals and plants, microorganism, The extract of culture of microorganism.Preferably, the nitrogen 6- (2- ethoxy) adenosine came from from Periostracum cicadae, Cordyceps militaris, winter The extract of worm summer grass and its artificial culture.

Preferably, in above-mentioned reagent or purposes, the reagent is food.Preferably, the reagent is food packet Include health food.

In some specific embodiments, this new purposes, reagent, food or health food are by nitrogen 6- (2- ethoxy) adenosine and derivative act on adenosine receptor to realize.In some specific embodiments, the gland Glycosides receptor is A₁、A_2A、A_2B、A₃One or more of.In some preferred embodiments, adenosine receptor A₁Receptor.? In some specific embodiments, nitrogen 6- (2- ethoxy) adenosine and derivative are a kind of swashing for new specific bond A1 receptor Dynamic reagent, in some modes, the nitrogen 6- (2- ethoxy) adenosine [N (6)-(2-hydroxyethyl)-adenosine, HEA] such as flowering structure

In other specific embodiments, the derivative of the HEA is following general formula (1)

Wherein, R1 is the alkyl or hydroxyl of branch or straight chain.In some preferred modes, R1 be C (CH3) 2CH2OH, CH (CH3) CH2OH or C (CH3) 3.

Periostracum cicadae has function similar with cordyceps sinensis, can be used as its substitute, has multiple nervous system tune Section effect, but the chemical nature of the neuroprotections such as Periostracum cicadae calmness, hypnosis, anticonvulsion and the mechanism of action have not been reported.

In other preferred modes, nitrogen 6- (2- ethoxy) adenosine is mentioned from animals and plants or microorganism or microorganism It takes or the extract of culture of microorganism.Preferably, nitrogen 6- (2- ethoxy) adenosine is from Periostracum cicadae, Cordyceps militaris [Cordyceps militaris (L.) Link], cordyceps sinensis [Ophiocordyceps sinensis (Berk.) G.H.Sung, J.M.Sung, Hywel-Jones&Spatafora] and its artificial culture in can separate and lived.

This research finds that HEA and its derivative have the function of preventing and treating the nervous system disease based on zoopery. Further investigation revealed that the neuroprotection of HEA can be by A₁Receptor antagonist prompts HEA and its derivative that can make For a kind of novel A₁Adenosine receptor agonist is for treating calmness, hypnosis, anticonvulsion, apoplexy, Parkinson and confrontation habituation etc. A variety of the nervous system diseases.

HEA described in this patent is similarly N⁶Substituted adenosine derivative is on the amino of the adenine group of adenosine It is connected with " 2- ethoxy " group, to keeping bioactivity most important, N6 replaces β-D-RIBOSE of reservation 9 In base, OH base replaces with NH₂, COOH then loses activity, may acid, alkali substituent group it is unfavorable to activity, N6 substituent group is chain alkane Tranquilizing soporific activity can be improved in hydrocarbon, and also enhances as carbochain increases activity, this may be improved with the hydrophobicity of substituent group has It closes.

We have probed into the relationship of HEA and A1 and A2a receptor affinity and have carried out zoopery in the present invention, we It was found that HEA has the Gao Qinneng in conjunction with adenosine A 1 receptor and can serve as the agonist of adenosine A 1 receptor, its conjunction is played The medical usage of reason.

On the other hand, the present invention is extracted the effective active composition HEA in Periostracum cicadae, affine by joint adenosine receptor Power experiment proves that HEA is a kind of new selective adenosine A for the first time₁Receptor stimulating agent.Clinically available safe selection sexual gland Glycosides A₁Receptor stimulating agent is limited, therefore Periostracum cicadae can have high medical, edible valence because it contains this active constituent Value.The present invention is expected to be developed into the potential drugs of prevention the nervous system disease, and can be made into correlation function food, health care product hair It waves to disease beneficial use.

The source of HEA can be nature extraction or artificial synthesized.

Naturally it extracts

The present invention is extracted from Periostracum cicadae using chemical method and separates a kind of natural active compound HEA, by pharmacology HEA is as a kind of adenosine class A for test discovery₁Receptor stimulating agent can prepare and prevent to obtain in the drugs for nervous such as convulsions Using.

The present invention uses the Periostracum cicadae manually cultivated for raw material, and preparation process is as follows: 50% second of Periostracum cicadae fructification Alcohol reflux extract obtains single active ingredient HEA through UF membrane, macroreticular resin and Sephadex LH20 column separating purification.

HEA structural formula is as follows:

Its molecular formula are as follows: C₁₂H₁₇N₅O₅Molecular weight are as follows: 311.297 chemical names are as follows: N (6)-(2- Hydroxyethyl) the method for extraction and purification referenced patent of-adenosine HEA.

It is artificial synthesized

HEA or derivative of the invention can also be artificial synthesized.

Definition:

It faints from fear, is the brain dysfunction due to caused by many reasons, show as unexpected whole body or local muscle group is in Tatanic and clonic twitches, are often accompanied by the disturbance of consciousness.If see a doctor takes relieving convulsion measure not in time, can threat to life.And it faints from fear Easy recurrent exerbation, twitch behavior can be alleviated in a short time, but the long-term progress sexual development of pathological biochemistry variation.Currently, more Medicine combination therapy convulsions is very universal, but major part can cause toxic side effect and adverse reaction.

Cerebral ischemia (cerebral infarction) is a kind of very common nervous centralis damage, has serious disability rate and higher lethal Rate.Investigate according to Chinese Medical Association: headstroke have become China city and people in the countryside first is disabled and the cause of death at present. The diagnosis and treatment field of ischemic stroke is underestimated, misjudgment phenomenon is serious；Admission rate is only about 6%, left far below developed country 30% Right ratio, and there is no real definite effective drug that Patients with Cerebral Infarction can be allowed to be benefited.

Parkinson's disease (PD) also known as shaking plasy are one of most common neurodegenerative diseases.Epidemiology is shown, is suffered from Sick rate is 15~3,28/,100,000 populations, and > 65 years old crowd about 1%；Disease incidence is 10~21/,100,000 populations/year.The maximum danger of this disease Evil is patients ' life quality degradation, can't take care of oneself, and multiple complications often occur, such as sleep disturbance.

Sleep disturbance means the various dysfunctions showed during Sleep-Wake.According to incompletely statistics, China is each Class sleep disturbance person accounts for the 38% of crowd, higher than the ratio in the world 27%.The insufficient harm of extended sleep is in addition to will affect spirit Except state, the immunity of human body can be also reduced, thus will lead to various diseases and occur.Studies have shown that sleep disturbance and glycosuria A variety of diseases such as disease, cerebral apoplexy, epilepsy, dementia, children's intelligence development, renal impairment, sex dysfunction are associated.

Opioid drug includes natural opium alkaloid such as morphine or artificial synthesized antalgesic such as pethidine, they Habituation be due to some chronic pain patients, after continuous Reusability morphine, effect can gradually weaken, form tolerance, Morphine usage amount is shown as to be gradually increased and the shortening of administration time interval.Patient can occur ill sexual preference and generate dependence, Including psychic dependence and physical dependence.Withrawal symptom can be generated after 6~10 hours once being discontinued, appearance dysphoria, Insomnia pain, runny nose, sheds tears, perspires, trembling, vomitting, diarrhea, collapse or even threat to life.Such patient has strong thirsty The desire of medication is sought, can go to obtain drug by fair means or foul, not only seriously damage the health of drug user, can also cause serious society Problem.

" food " again here be meant that any substance that can be edible for the mankind or mammal.This food also wraps Any health food, functional food are included, or the food commonly understood, existing form can be with beverage, tablet, molten The forms such as liquid exist.These food, which can be solid, semisolid, fluid form, to be existed.

" pharmaceutical agent " is the reagent on the ordinary meaning of this field, can be tablet, solvent, semisolid is easy or injection Body etc..Pharmaceutical agent referred herein can be the medicament forms with treatment disease, be also possible to play health medicine It acts on and exists.

Beneficial effect

The present invention provides a kind of exciting reagent of new adenosine receptor, particularly, provides the new A1 adenosine receptor of one kind Exciting reagent.The new reagent provides new approach to treat or prevent such disease.

Detailed description of the invention:

Fig. 1 .1 [³H] saturation curve (Fig. 1 .1A) and Scatchard (figure of the-DPCPX in conjunction with rat cortex membrane receptor 1.1B) map, wherein, B. radionuclide binding protein；F. floating preteins (Kd=0.12nmol/L, Bmax=2140fmol/ Mg albumen).

Fig. 1 .2 [³H] full (Fig. 1 .2A) and curve and Scatchard (figure of the-MSX-2 in conjunction with rat cortex membrane receptor 1.2B) map, note: B. radionuclide binding protein；F. floating preteins (Kd=10.90nmol/L, Bmax=5235fmol/ Mg albumen (protein).

Fig. 2 .1HEA (15mg/kg, 40mg/kg, 60mg/kg, ip) causes the influence of convulsions incidence to pentylenetetrazole, as a result shows Show that HEA (40mg/kg, ip) can significantly extend life span, embodies anticonvulsant effect (n=8；* P < 0.01 P < 0.05, * * With control group ratio).

Fig. 2 .2 adenosine A₁Influence of the receptor selective antagonists to HEA anticonvulsant action, DPCPX can make that HEA's is anticonvulsion Effect obviously falls (n=8 after rise；P < 0.01 P < 0.05, b a and control group ratio, P < 0.01 P < 0.05, B A and administration group ratio).

Fig. 2 .3 adenosine A_2AInfluence of the receptor selective antagonists to HEA anticonvulsant action, Zm241385 is to the anti-frightened of HEA The effect of fainting has not significant impact (n=8；P < 0.01 P < 0.05, b a and control group ratio, P < 0.01 P < 0.05, B A and administration group Than).

Fig. 3 .1HEA (5mg/kg, 7.5mg/kg, 12mg/kg, ip) comments the rat nerve functional status of dMCAO cortex Point, as the result is shown compared with model group, HEA can make cerebral injury nervous function be improved significantly.(n=8；* P < 0.05, * * P < 0.01 with to model group ratio).

Fig. 3 .2HEA (5mg/kg, 7.5mg/kg, 12mg/kg, ip) measures infarct size to the TTC dyeing of dMCAO cortex, As the result is shown compared with model group, HEA (7.5mg/kg, ip) can be such that the infarct size of cerebral injury is obviously reduced.(n=8；* P < 0.01 P < 0.05, * * and model group ratio).

Fig. 3 .3HEA (7.5mg/kg, ip) group to the protective effect of the cerebral ischemia of dMCAO cortex and DPCPX (1mg/kg, Ip the rat brain tectology of HAE cerebral protection) is blocked to dye HE (400X), as the result is shown compared with model group, HEA Group can make cellular swelling degree and cell nuclear alteration make moderate progress, and cell number increases.

Fig. 3 .4HEA (7.5mg/kg, ip) group to the protective effect of the cerebral ischemia of dMCAO cortex and DPCPX (1mg/kg, Ip) block HAE cerebral protection tunel detect rat cerebral cortex Apoptosis situation (400X), as the result is shown with model Group is compared, and rat cerebral cortex infarct Penumbra zone area apoptosis rate can be used to be substantially reduced for HEA group.

Fig. 4 .1.HEA (5mg/kg, 10mg/kg, 15mg/kg, ip) causes the influence of mouse movement dysfunction to MPTP. (n=8；* P < 0.01 P < 0.05, * * and control group ratio；#P < 0.05, ##P < 0.01 and model group ratio)

Fig. 4 .2HEA (5mg/kg, 10mg/kg, 15mg/kg, ip) is to the shadow of dopaminergic neuron reduction caused by MPTP It rings.(n=8；* P < 0.01 P < 0.05, * * and control group ratio；#P < 0.05, ##P < 0.01 and model group ratio)

Fig. 5 .1.HEA combines influence of the adenosine receptor to mouse autonomic activities, and HEA (15mg/kg) can be significant as the result is shown Autonomic activities number in normal mouse 5min is reduced, under the action of DPCPX (4mg/kg), the sedation of HEA is by obvious Antagonism (n=8；* P < 0.01 P < 0.05, * * and solvent group ratio；#P < 0.05, ##P < 0.01 and administration group ratio).

Have when Fig. 6 .1.HEA (25mg/kg, sc) independent role to sleeping time caused by mouse threshold dose yellow Jackets A degree of extension, has not significant impact Sleep latency, and when combining adenosine receptor antagonists, the syngignoscism of HEA is bright Aobvious suppressed (n=8；* P < 0.01 P < 0.05, * * and solvent group ratio；#P < 0.05, ##P < 0.01 and administration group ratio).

Fig. 7 .1 is the shadow of HEA and Periostracum cicadae comprising HEA and its extract to CPP establishment stage morphine induction habituation Ring (n=8；* P < 0.01 P < 0.05, * * is compared with the control group)

Fig. 7 .2 is that HEA and Periostracum cicadae comprising HEA and its extract are lighted the stage morphine induction of relapsing to CPP and relapsed Influence (the n=8 of habituation；* P < 0.01 P < 0.05, * * is compared with the control group；#P < 0.05, ##P < 0.01 is compared with morphine group)

Specific embodiment

Embodiment 1

1.HEA is A₁The selective agonist of receptor

The preparation of 1.1 membrane receptor proteins

Brain is taken using Wistar rat broken end, cerebral cortex and corpus straitum is isolated, weighs respectively, is added 10 times by 1:10 The Tris-HCL buffer (50mM, PH7.5) of volumes ice cold, tissue homogenate abandon supernatant after suspension centrifugation, repeat above-mentioned solution After washing 3 times, supernatant is abandoned in centrifugation again, and precipitating is mixed in 50mM Tris-HCL buffer again, uses Coomassie Brilliant Blue It is 0.8mg/ml, rat striatum brain tissue homogenate protein content that (Bradford method), which determines rat cerebral cortex protein concentration, For 1.3mg/ml.Be stored in after packing -80 DEG C it is spare.(Li M,Kang RX,Shi JG,Liu GT,Zhang JJ, 2013.Anticonvulsant Activity of B2,an Adenosine Analog,on Chemical Convulsant-Induced Seizures,PLoS One Jun25；8(6):e67060)

1.2 adenosine receptor ligands combine test

Memebrane protein and corresponding ligand (adenosine al receptor ligands Binding experiment: rat cortex brain tissue are added in reaction tube The combination that homogenate and corresponding ligand are 0.2nM [3H] DPCPX；Adenosine A 2 A receptor ligand binding assay: rat striatum brain group Homogenate and corresponding ligand are knitted as the combination of 0.75nM [3H] MSX-2), measure respectively [³H]-DPCPX([³H]-MSX-2) and it is big Mouse cortex adenosine A₁(rat striatum adenosine A_2AR) saturation curve combined acquires balance with Scatchard linear transformation method (the Kd value that A1 is combined is 0.14nmol/L, Bmax 2290fmol/mg to dissociation constant；A_2AIn conjunction with Kd value be 11.48nmol/ L, Bmax 5657fmol/mg).

Measure the HEA (10 that various concentration is added in pipe^-9、10^-8、10^-7、10^-6、10^-5Mol/L), in shaking bath after mixing 25 DEG C of incubation 30min aspirate reaction solution using cell harvestor, pass it through GF/B glass filters (Watman), terminate reaction, It is rinsed 3 times with Tris-HCl buffer, filter membrane is removed to be fitted into the scintillation vial of the scintillation solution containing 4ml after drying and be measured by each 3ml Radioactivity.Radioactive counts are carried out to filter membrane by scintillation counter.It is corresponding in the presence of the compound of measurement various concentration 3 [³H]-DPCPX or [³H]-MSX-2 combine percentage.(Li W,Wang YF,Li M,YUE ZG,Shi JG,Zhang JJ, 2011,Sedative and hypnotic effects of a novel ligand YZG-404for adenosine A1receptor,J Int Pharm Res,Vol.38,No.3,June)

1.3 test result

Experiment measures HEA and adenosine A₁The Ki value of competitive binding is 89.5nmol/L, with adenosine A_2AThe Ki value of competitive binding About 8921.4nmol/L.HEA is to adenosine A₁Affinity be approximately it to adenosine A_2AAs a result 100 times of affinity prompt HEA pairs A₁Selectivity with higher.

The experiment of 2.HEA derivative

The derivative of institute's experiment is object derived from the 3 of following structural:

Wherein, R1 is 3 (derivative of C (CH3) 2CH2OH (derivative 1), CH (CH3) CH2OH (derivative 2) or C (CH3) Etc. 3) three kinds of substances have carried out the experiment similar with this experiment 1, and the derivative 1-3 for measuring HEA is similar with the result of HEA (specific Experimental data is omited), as a result also prompt HEA derivative 1-3 to A₁Selectivity with higher.

Application of the embodiment 2.HEA in anticonvulsion

2.1 animal models and medication

Male ICR mouse, 18~22g；Purchased from Wenzhou Medical College's animal experimental center.Animal adapts to environment at least before testing 5d.Room temperature is maintained at 25 DEG C, ad lib and water inlet.Healthy male ICR mouse by weight be randomly divided into control group (1%DMSO, Ip), model group, CCPA group (0.1mgkg^-1, ip), HEA group (15mg/kg, 40mg/kg, 60mg/kg), DPCPX group (2mg kg^-1, ip), ZM241385 group (1mgkg^-1、5mg·kg^-1, ip), DPCPX+HEA (2mgkg^-1+ 40mg/kg, ip) group and ZM241385+HEA(1mg·kg^-1+ 40mg/kg, 5mgkg^-1+ 40mg/kg ip) group.Wherein adenosine A₁R receptor antagonist DPCPX (or A₂R receptor antagonist ZM241385) it gives and is injected intraperitoneally before 10min is administered, administration is completed after 15min again Give pentylenetetrazole (100mgkg^-1, ip) and inducing mouse convulsions；Antagonist group is used alone, is given after giving antagonist 5min Pentylenetetrazole (100mgkg^-1, ip), mouse is observed to the eclamptogenic reaction of PTZ.

2.2 Testing index

Life span and the death rate after record each group convulsions occurs respectively.

2.3 test result

The Behavior Test result of animal prompts our HEA 40mgkg^-1Group, can be small caused by extremely significant reduction pentylenetetrazole The death rate that mouse is fainted from fear.In addition, specificity A₁Receptor antagonist DPCPX (2mgkg^-1, ip) and it can obviously inhibit that HEA's is anticonvulsion Effect, and specificity A_2AReceptor antagonist ZM241385 (1mgkg^-1、5mg·kg^-1, ip) do not have to the anticonvulsant action of HEA It significantly affects.As a result, it is presumed that HEA may be by exciting A₁Receptor takes part in the effect for adjusting and fainting from fear, and can be used for fainting from fear Clinical treatment and prevention.

Application of the embodiment 3.HEA in cerebral ischemia

3.1 animal model and medication

Using intraluminal middle cerebral artery occlusion in rats Distal occlusion cerebral ischemic model.1. according to 10% chloraldurate of rat body weight (3ml/kg) gives intraperitoneal injection of anesthesia.2. lying on one's side and fixing Rat Right, 1cm skin is opened at the corner of the eyes and external auditory canal line within the eye Notch separates fascia, musculature, exposes skull；3. inhaling a small amount of physiological saline with cotton ball wipes skull to getting a clear view； 4. separating fascia, exposure skull under surgical operation microscope, drill straight diameter is 2mm circular hole on the bone ridge 1/3；5. with a small amount of raw It manages salt water and rinses extra bone bits, push meninx aside, expose arteria cerebri media MCA；6. rat dorsal position is fixed, just along throat portion Middle notch separates bilateral carotid CCA, is passed through with surgical thread, temporarily not ligatures；7. MCA is found under surgical operation microscope, Cooled down again with normal saline flushing after being burnt with unipolar electrocoagulator；8. ligaturing bilateral common carotid arteries after coagulation is complete immediately, block 60min；9. suture head wound, bilateral carotid rope after 1h, skin suture modeling are completed.SD rat is randomly divided into 6 groups: Sham-operation group (1%DMSO, ip), model group, HEA 5mg/kg group, HEA 7.5mg/kg group, HEA12mg/kg group, HEA7.5mg/kg+DPCPX1mg/kg group.Each group drug is given respectively in the preoperative 30min of dMCAO primary, vacation is injected intraperitoneally respectively Operation group and model group give 1% dimethyl sulfoxide, and rats in sham-operated group only opens cranium exposure arteria cerebri media, not solidifying to close in brain Artery and abdominal cavity injection, A₁Selective adenosine receptor antagonist DPCPX gives before 10min is administered and is injected intraperitoneally.

3.2 index determining

3.2.1 according to Longa EZ etc. when Neuroscore animal is awake^[4]5 grade of 4 point-score standard of neurologically handicapped to big Mouse behaviouristics scores.0 point: without obvious neurologic impairment；1 point: not tensible opposite side forelimb；2 points: to right when walking Side rotation；3 points: toppling over when walking to opposite side；4 points: it spontaneous cannot walk, the loss of consciousness.

3.2.2TTC it dyes the measurement to brain infarction area: after modeling is completed for 24 hours, thoracic cavity exposure will be opened after rat anesthesia Heart, after 250mL saline infusions, the marrow that breaks takes out brain tissue, is placed in minus 80 DEG C of frosts.The rat brain of adfreezing is set and cuts brain mould In tool, from antinion to occipital pole (being free of cerebellum part), 5 are continuously cut, piece thickness 2mm.Carefully brain piece is placed in tweezers and is equipped with In the avoid light box of 2%TTC, room temperature is incubated for 15min, and digital camera is fixed at brain piece vertical direction 30cm and is taken pictures.Normal brain activity Succinate dehydrogenase in tissue Mitochondria is reacted with TTC in cerise, and infarcted region is not colored due to lacking mitochondria.Stalk Unleavened dough product accounts for the percentage of full brain with infarcted region to indicate.

32.3 cerebral tissue morphology dye HE: after rat modeling is completed 24 hours, anesthesia, cardiac perfusion takes brain tissue It is fixed in 4% paraformaldehyde；Conventional dehydration embeds, coronal section, is dyed after slice with Hematoxylin-eosin.

3.2.4tunel cerebral cortex cells apoptosis situation is detected

It takes brain tissue fixed in paraformaldehyde, carries out tunel method according to the requirement of kit after paraffin section completion Colour developing washing, dehydration, transparent, mounting.

3.3 experimental result

Experimental result shows that HEA (7.5mg/kg) is obviously improved the nervous symptoms of dMCAO rat, while being substantially reduced cerebral infarction Unleavened dough product reduces brain cortex tissue structure caused by ischemic and becomes loose, and cell number significantly reduces, rat cerebral cortex infarct Penumbra zone area apoptosis rate is substantially reduced.The above-mentioned protective effect of institute can be by selective adenosine A₁Receptor antagonist institute antagonism, mentions Show that HEA has certain cerebral protection, and this neuroprotection is likely to be and passes through adenosine A₁It mediates.

Application of the embodiment 4.HEA in Parkinson

4.1 animal models and medication

24 ± 1g male C57BL/6 mouse, is randomly divided into 5 groups, using -4 phenyl -1,2 of 1- methyl, 3,6- tetrahydropyridines The mouse PD model of (1-methyl-4-phenyl-1,2,6-tetrahydropyridine, MPTP) induction, HEA (5mg/kg, 10mg/kg, 15mg/kg) continuous intraperitoneal injection 14 days and the 1h before administration in the 11st day respectively, MPTP30mg/kg is injected intraperitoneally, Continuous 4 days, 1 day after last time administration, the Behavior test of pole-climbing ability is carried out, post-tensioning neck execution in 4 days takes corpus straitum.Control Group gives same amount of normal saline, and model group gave 1h before physiological saline at the 11st day, is injected intraperitoneally MPTP30mg/kg, and continuous 4 It.

4.2 index determining

4.2.1 pole-climbing method measures mouse movement ability

Upside down by mouse, it is placed in long 50cm, it is required from top to bottom along bar to record mouse for the rod top of thick 10mm Time calculates the time difference before and after modeling, and does statistical analysis.

4.2.2 immunohistochemical method measures the quantity of TH positive cell in substantia nigra of midbrain

After behaviouristics detects, heart is perfused with 4% paraformaldehyde and takes brain, fixed, is routinely dehydrated, embeds, is taken black substance Region different parts coronal section, using tyrosine hydroxylase as the specific marker of neuron.Primary antibody is Monoclonal mouse TH Antibody (1；1000, Sigma), secondary antibody is 488 fluorescent marker goat anti-mouse (1 of Alexa fluor；1000, Molecular Probes) fluorescence microscope is taken pictures, to black substance TH Positive Cell Counts.

4.3 test result

Test result shows that HEA (10mg/kg, ip) significantly improves the limbs coordination ability for improving mouse, and it is positive to increase TH Cell quantity prompts HEA to can be used for clinical treatment and the prevention of Parkinson's disease.

Embodiment 5.HEA is preparing the application in downern

5.1 animal packet and medication

Male ICR mouse is randomly divided into 4 groups, selects adenosine A₁R antagonist DPCPX.Mouse is divided into solvent group, HEA (15mg/kg) group, DPCPX (4mg/kg) and DPCPX+HEA (4mg/kg+15mg/kg) group；Wherein DPCPX+HEA group is by small After antagonist 10min is injected intraperitoneally in mouse, then HEA is injected intraperitoneally.

5.2 detection method

Mouse is put into individually opaque rectangular autonomic activities case (50cm × 50cm × 40cm) after completing administration 15min It is interior, lattice number is stepped in camera record 5min.It enters into same lattice with mouse four limbs to calculate, as animal activity index.

5.3 experimental result

HEA (15mg/kg, ip) is significant to the inhibitory effect of mouse autonomic activities.Prompt HEA can be played in nervous system Sedation.

Application of the embodiment 6.HEA in preparation sleep drug

6.1 animal packets and medication

Male ICR mouse is randomly divided into 4 groups, respectively control group, positive diazepam (1mg/kg) group, HEA (25mg/kg) Group and DPCPX+HEA (2mg/kg+25mg/kg) group.After 30min is administered in intragastric administration on mice, threshold agent is injected intraperitoneally in groups of animals Measure yellow Jackets 50mg/kg.

6.2 Testing index:

Using mouse righting reflex loss in 15min up to 1min as sleep standard, record each group mouse Sleep latency and Sleep time.

6.3 experimental result

HEA (25mg/kg, sc) has a degree of extension to sleeping time caused by mouse threshold dose yellow Jackets, But no difference of science of statistics, prompt cicada extract HEA have certain collaboration amobarbital to Sleep latency compared with the control group The effect of sodium.

Embodiment 7.HEA or medicinal fungus containing HEA and extract treat and prevent on addictive drug in preparation Using

The preparation of 7.1 test samples

It is raw material that precision, which weighs the Periostracum cicadae after drying, is labeled as sample A, using 50% ethyl alcohol as solvent extraction, 2h/ Secondary, filtering, merging filtrate passes sequentially through UF membrane, macroporous resin purification and Sephadex LH20 column purification, and step by step arithmetic divides Sample B, C, D are not obtained.Be each configured to the effective quantity of sample containing each component be 1500mg/kg, 750mg/kg, 100mg/kg, The solution for later use of 20mg/kg.

7.2 animal packet and medication

Cleaning grade male mouse of kunming, 18~22g of weight, be divided into control group, morphine group, sample sets (A:1500mg/kg, B:750mg/kg, C:100mg/kg, D:20mg/kg).Natural preference test (d-2, d-1, d0): experiment first three days allow it in case Middle free shuttling 15min naturally has a preference for black box according to rat, therefore selects white box as with medicine-chest, and (d-1, d0) records every big Residence time of the mouse in white box takes the average value of two days test results as rat in the basic value with the medicine-chest time.Cpp is built It is vertical: morphine processing group: after hydrochloric acid (10mg/kg, ip) is injected intraperitoneally in d1, d3, d5, d7, whitepack training 50min, d2, d4, d6, d8 After isometric physiological saline is injected intraperitoneally, physiology is administered in black box training 50min, administration group 15min before injection morphine daily All given in salt water group 8 days physiological saline Cpp expression: d9 by rat with no treatment head towards whitepack place two box intersections, It is allowed to move freely residence time situation of the 15min record rat in white box in two boxes；Cpp subsides: d10 days start, real It tests group and control group gives training 50min, d11, d13, d15, d17 training of physiological saline d10, d12, d14, d16 whitepack 50min；By rat, head allows it to move freely 15min in two boxes to d18 towards whitepack two box intersections of placement with no treatment, Record residence time situation of the rat in white box；Cpp re-ignition: d19 morphine group gives 5mg/kg, and administration group is before morphine 15min administration, each group mouse head softly place two box intersections towards whitepack and move freely 15min, record activity condition.7.3 inspection Survey index

Place Preference index: record mouse analyzes the different habituation stages in the whitepack residence time of different times, HEA or The medicinal fungus and extract that person contains HEA are to the mouse CPP effect of morphine induction.

7.4 test result

The experimental results showed that the phase that formed repeatedly gives Periostracum cicadae sample A or its step by step arithmetic object (B, C, D) lures morphine The mouse led is not significantly different with residence time of medicine-chest, but gives Periostracum cicadae sample A to During The Withdrawal Period or its substep mentions Morphine group, especially sample D are being substantially less than extremely significant low with the medicine-chest time with the medicine-chest time after taking object sample (B, C) to handle In morphine group, drug-seeking behavior after prompting HEA to can be used for giving up, have it is certain resist it is additive.

Claims (5)

1. a kind of nitrogen 6- (2- ethoxy) adenosine is being used to prepare treatment or prevention convulsions or pa gold as sole active agent Purposes in the drug of gloomy disease.

2. purposes according to claim 1, wherein nitrogen 6- (2- ethoxy) adenosine from animals and plants, microorganism, The extract of culture of microorganism.

3. purposes according to claim 1, wherein nitrogen 6- (2- ethoxy) adenosine comes from Periostracum cicadae, pupa worm The extract of grass, cordyceps sinensis and its artificial culture.

4. purposes according to claim 1, wherein play the role for the treatment of or prevention is fainted from fear or Parkinson is because of nitrogen 6- (2- ethoxy) adenosine is realized as adenosine receptor agonist.

5. purposes according to claim 4, wherein adenosine receptor A₁Receptor.