CN108175770A

CN108175770A - It is a kind of to treat the reagent of kidney failure by acting on adenosine receptor

Info

Publication number: CN108175770A
Application number: CN201610586709.3A
Authority: CN
Inventors: 柴秋; 柴一秋; 朱碧纯; 彭秀秀; 金轶伟
Original assignee: ZHEJIANG PROV INST OF SUBTROPICAL CROPS
Current assignee: ZHEJIANG PROV INST OF SUBTROPICAL CROPS
Priority date: 2014-07-27
Filing date: 2016-07-22
Publication date: 2018-06-19
Also published as: CN104173365A; CN104173365B

Abstract

The present invention relates to nitrogen 6 (2 ethoxy) adenosine [N (6) (2 hydroxyethyl) adenosine, HEA] and its derivative as adenosine A₁Purposes of the receptor stimulating agent in prepared by drug or food：HEA and its derivative are for the treatment disease related with adenosine receptor modulators, such as tranquilizing soporific, analgesia, anticonvulsion, resistance apoplexy, Parkinson's disease, opioid addiction and renal ischemia/reperfusion injury.The present invention provides a kind of new method for nervous system and the related disease of kidney.

Description

It is a kind of to treat the reagent of kidney failure by acting on adenosine receptor

This application claims part U. S. application, application numbers：14/805,742, the applying date：On July 22nd, 2015 it is preferential Power.

Technical field

The present invention relates to a kind of new adenosine a1 receptor agonists, particularly, are related to nitrogen 6- (2- ethoxys) adenosine [N (6)-(2-hydroxyethyl)-adenosine, HEA] and its derivative as adenosine a1 receptor agonists；With it as gland Glycosides A1 receptor stimulating agents have related disorders available for preventing, treating in prepared by medicine preparation or food with adenosine receptor adjusting material New application.

Background technology

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.In these cordyceps sinensis, Paecilomyces cicadae (Paecilomyces Cicadae) is one of which important role.Traditional Chinese Medicine is remembered It is carried with tranquilizing soporific, anti-frightened epilepsy, Chi Zong, the morbid night crying of babies and other effects, but simultaneously no evidence be shown to be that specific substance rise Effect, how is the mechanism of effect.(the Advance on Pharmacological Activities China National folk medicine 2009,9 of Qiu Jie Song Jie people cicada fungus： 4-6；Thunder helps influence fungus journal 15January of the star difference condition of culture to Cordyceps pruinosa production N6- (2- ethoxys) adenosine 2014,33(1):103‐113)。

The close phase of physiological effects such as numerous result of study prompting adenosine receptors and neuronal excitation, locomitivity adjusting It closes.Have an impact to the mechanism of action of the active drug for the treatment of schizophrenia, depression, epilepsy and anxiety (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 The receptor immune molecule important for body, it is closely related with inflammatory reaction.A₁、A_2aReceptor generally takes part in adenosine to sleep, feelings The adjusting of many physiology such as thread and pathologic process.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 find that A2BR can be with Increase astroglia release IL-6, imply that A2b may participate in inflammatory process.A3 receptors are in the horizontal of intracerebral and it is to gland Glycosides affinity it is horizontal all far below A1 and A2A receptors, physiological action is currently not that fully aware of (king appoints firelight or sunlight Pan to build spring gland Glycosides 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.)。

Numerous studies show selective 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 intracellular second messenger cyclic adenosine monophosphate (cAMP) concentration, 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 receptors and A2A receptors；Millan The experiment of MJ shows that cause anxiety and angst resistance effect are related with the blocking of A1 receptors and excitement respectively, the mouse of A1R gene delections 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, and molecular weight is 36 600.Activation A1 receptors can play Protect the effect of neuron.It is now recognized that its possible mechanism is：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 is reduced so as to protect neuron.(in the Effect study progress of ancestor's pattern river in Henan Province adenosine A 1 receptor State's Pharmacological Bulletin 2,008 24 (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 ' positions repair Decorations.It is documented that N⁶The neplanocin of substitution 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 analog NECA has been widely used for explaining by A₂Biological effect caused by receptor activation.In the structural modification effect of other ribose, The metalepsis of 2 '-position can lose compatibility completely, and the unsubstituted hydroxyl in 3 '-position is efficiently necessary.(kenneth The pharmacology and structure-activity relationship pharmacy of the general receptor of A.Jacobson glands are in progress the phase of volume 1,992 16 the 4th).

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

Invention content

The present invention has surprisingly found that, nitrogen 6- (2- ethoxys) adenosine (N6- (2-hydroxyethyl)-adenosine, HEA) and derivative is a kind of new adenosine receptor excitement reagent.They are to adenosine A₁Receptor all has stronger selectivity. By specific bond A1 receptors and a series of physiological and biochemical activity is generated, so as to adjust the work(in terms of neural and protection kidney Energy.

On the one hand, the present invention relates to a kind of exciting reagent of new adenosine receptor, which includes HEA and derivative.

In some specific embodiments, the adenosine receptor is adenosine A₁、A_2A、A_2B、A₃One kind in receptor or It is several.

In some preferred embodiments, adenosine receptor A₁Receptor.

In some specific embodiments, HEA and derivative are that a kind of the exciting of new specific bond A1 receptors tries Agent.

In some modes, the nitrogen 6- (2- ethoxys) adenosine [N6- (2-hydroxyethyl)-adenosine, HEA] such as lower structure

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

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

In other specific embodiment, the HEA of artificial synthesized HEA and natural product extraction, which equally has, adjusts nerve And the function of protection kidney etc..

On the other hand, the present invention provide a kind of HEA and derivative be used to prepare prevention or treatment faint from fear, analgesia, Purposes in the nervous systems such as apoplexy, Parkinson or opioid addiction, sleep-disorder or protection renal reagent.

A further object for invention be to provide a kind of prevention and treatment convulsions, analgesia, headstroke, Parkinson, drug habit, Pharmaceutical agent in the diseases such as the nervous systems such as sleep-disorder or protection renal, the wherein reagent include HEA and its derivative.

The another object of invention is provided with a kind of prevention and treatment convulsions, headstroke, Parkinson, drug habit, sleep barrier The food in the diseases such as nervous systems or protection renal such as hinder, the wherein food includes HEA and its derivative.

In another aspect, the present invention is directed to addition adenosine receptor antagonists experiment is tested and combined by adenosine receptor affinity Proof HEA is adenosine a1 receptor agonists, plays prevention and treatment convulsions, analgesia, headstroke, Parkinson, drug habit, sleeps The nervous system diseases such as dormancy obstacle or protection renal are worked by adenosine A 1 receptor.

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

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

Preferably, according to the purposes or preparation, the preparation can be used for ischemia-reperfusion in treatment kidney transplant to damage The purposes such as wound, the inhibition inflammation in anti-kidney failure and Apoptosis.

In other preferred modes, extraction or culture of microorganism of the HEA from cordyceps sinensis or microorganism or microorganism Extract.Preferably, HEA is from Periostracum cicadae, cordyceps sinensis [Ophiocordyceps sinensis (Berk.) G.H.Sung, J.M.Sung, Hywel-Jones＆Spatafora], Cordyceps militaris [Cordyceps militaris (L.) Link], And its acquisition can be detached in artificial culture, acquisition can also be synthesized.Preferably, HEA is from Paecilomyces cicadae The culture of (Paecilomyces Cicadae) extracts acquisition from entity or coremium, cultural hypha object.

Analgesic effect can be found, such as Chinese patent application in Chinese invention disclosed patent about HEA Specific descriptions in 200410094511.0, reference of the patent application as all present invention；Kidney is protected about HEA Effect can be found in Chinese invention disclosed patent, such as specifically retouching in Chinese patent application 201280049909.5 It states, reference of the patent application as all present invention.The method of drug combination is increased for the first time in this patent by adding Add the supposition of selective A1 receptor antagonists to the progress of HEA mechanism of action further, tentatively judge it for A1 receptor stimulating agents.

Periostracum cicadae have the function of it is similar to cordyceps sinensis, can alternatively product, there is multiple adjustment effect, but The neuroprotections such as Periostracum cicadae calmness, hypnosis, anticonvulsion and the chemical nature and the mechanism of action of protection renal are had not been reported.This Outer Periostracum cicadae, Cordyceps militaris apply in preventing or treating the diseases such as headstroke, Parkinson and drug habit and refer to for the first time, It has new value when containing HEA.

So another aspect of the present invention, provides a kind of examination prevented or treat headstroke, Parkinson and drug habit Agent, the culture of the reagent including Paecilomyces cicadae (Paecilomyces Cicadae), from entity or coremium, cultural hypha object Middle extraction of substance.

Preferably, which includes HEA.

This research finds that HEA and its derivative have the prevention and treatment disease related with adenosine A 1 receptor based on zoopery Disease effect.Further investigation revealed that the protective effect of HEA can be by A₁Receptor antagonist prompts HEA and its derivative It can be as a kind of novel A₁Adenosine receptor agonist is for treating calmness, hypnosis, analgesia, anticonvulsion, apoplexy, Parkinson, right A variety of diseases such as acute renal failure caused by anti-habituation or renal ischemia/reperfusion injury.

The present invention also provides a kind for the treatment of or prevention mammal or mankind's convulsions, pain, insomnia, apoplexy, Parkinsons Or the method for opioid addiction, wherein applying HEA and derivative to mammal or the mankind.

Preferably, the HEA of application and derivative exist in the form of tablet, aqua, mixture, dry mixture etc..

Preferably, these drugs application includes other auxiliary reagents, such as stable reagent etc..

Preferably, the HEA intends celebrating mould fungi or the extract of the fungal cultures from cordyceps sinensis, Cordyceps militaris, cicada.

Therefore the effective active composition HEA that is extracted in Periostracum cicadae of the present invention and carry out range of animal experiment, probe into Effects of the HEA in the nervous system related with adenosine A 1 receptor and renal-related conditions are treated, it is found that it can serve as adenosine The agonist of A1 receptors plays rational medical usage；Tested by adenosine receptor affinity and combine addition adenosine receptor it is short of money Anti-agent is tested, and has probed into HEA and adenosine A 1 and the relationship of A2a receptor affinities, as a result HEA is prompted to have and adenosine A 1 receptor knot Height parent's energy property of conjunction, and prove that HEA is a kind of new selective adenosine A₁Receptor stimulating agent.Clinically available safe selection Property adenosine A₁Receptor stimulating agent type is limited, and therefore, the cordyceps sinensis such as Periostracum cicadae, cordyceps sinensis, Cordyceps militaris can contain this because of it Active constituent and be worth with high medical, edible.The present invention, which is expected to be developed into prevention the nervous system disease and protects kidney, to be had The potential drugs of pass, and correlation function food, health products performance are can be made into disease beneficial use.

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

Naturally it extracts

The present invention extracts a kind of natural active compound HEA of separation using chemical method from Periostracum cicadae, by pharmacology Experiment finds HEA as a kind of adenosine class A₁Receptor 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, and for raw material, 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 purifications.

HEA structural formulas are as follows：

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

HEA of the present invention can be derived from from Periostracum cicadae (O.sinensis), Cordyceps militaris (Cordyceps Militaris), Cordyceps pruinosa (Cordyceps pruinosa), cordyceps sinensis (Ophiocordyceps sinensis) etc. It generates and separation is extracted in the cordyceps sinensis class fungi of HEA.HEA can be obtained by cultivating the above-mentioned cordyceps sinensis class fungi such as Paecilomyces cicadae bacterium The analysis product obtained, this product can be detached from fructification, mycelia, spore, cultural hypha base and be obtained.HEA can also come It synthesizes, can also directly be bought by commercially produced product derived from artificial chemistry.

It is artificial synthesized

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

Definition：

It faints from fear, is the brain dysfunction 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.At present, it is more Medicine combination therapy convulsions is very universal, but major part can cause toxic side effect and adverse reaction.

Cerebral ischemia (cerebral infarction or headstroke) is a kind of very common nervous centralis damage, has serious disability rate and higher Lethality.It is investigated according to Chinese Medical Association：Headstroke has become first of China city and people in the countryside and has disabled and extremely at present Die reason.The diagnosis and treatment field of ischemic stroke is underestimated, misjudgment phenomenon is serious；Admission rate is only about 6%, far below developed country 30% or so ratio, and real definite effective drug there is no Patients with Cerebral Infarction to 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,>65 years old crowds about 1%；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-disorder.

Sleep-disorder means the various functions obstacle showed during Sleep-Wake.According to incompletely statistics, China is each Class sleep-disorder 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 that can influence spirit Except state, the immunity of human body can be also reduced, thus various diseases can be caused to occur.Research shows that sleep-disorder 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, and form tolerance, Showing as morphine usage amount, gradually increase and administration time interval are shortened.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, shed tears, perspire, 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.

Renal ischemic reperfusion injury is the main reason for leading to acute renal failure, it is also possible to some chronic kidney diseases The occurrence and development of disease are related, and acute renal failure is the rapid decline that kidney removes noxious material ability in blood, and leads to blood The accumulation of metabolic waste in liquid, such as urea.One investigation result shows, China 40 years old or more possesses kidney trouble illness rate about 8- 9%, the patient in kidney failure latter stage can only finally select kidney transplant or the purification of lifelong blood, blood, and larger warp is brought to family Ji and psychological burden.

" food ", which is here meant that, refers to any substance that can be eaten for the mankind or mammal.This food also wraps Include any health food, functional food or the food commonly understood, existing form can be with beverage, tablet, molten The forms such as liquid exist.These food can be solid, semisolid, fluid form presence.

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

Advantageous effect

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

Description of the drawings：

Fig. 1 .1 [³H] saturation curves (Fig. 1 .1A) that are combined with rat cortex membrane receptor of-DPCPX and Scatchard (figures 1.1B) map, note:B. radionuclide binding protein；F. floating preteins (Kd=0.12nmol/L, Bmax=2140fmol/mg Albumen).

Fig. 1 .2 [³H]-MSX-2 full (Fig. 1 .2A) that is combined with rat cortex membrane receptor and curve and Scatchard ((scheme 1.2B)) map, note:B. radionuclide binding protein；F. floating preteins (Kd=10.90nmol/L, Bmax=5235fmol/ mg protein)。

Fig. 2 .1 HEA (15mg/kg, 40mg/kg, 60mg/kg, ip) cause pentylenetetrazole the influence of convulsions incidence, as a result Display HEA (40mg/kg, ip) can significantly extend life span, embody anticonvulsant effect (n=8；*P<0.05, * * P< 0.01 with control group ratio).

Fig. 2 .2 adenosine As₁Influence of the receptor selective antagonists to HEA anticonvulsant actions, DPCPX can make that HEA's is anticonvulsion Falling (n=8 with obvious effects；*P<0.05, * * P<0.01 with control group ratio, #P<0.05, ##P<0.01 and administration group HEA40mg/ Kg ratios).

Fig. 2 .3 adenosine As_2AInfluence of the receptor selective antagonists to HEA anticonvulsant actions, Zm241385 is to the anti-frightened of HEA The effect of fainting has not significant impact (n=8；*P<0.05, * * P<0.01 with control group ratio, #P<0.05, ##P<0.01 and administration group HEA40mg/kg ratios).

Fig. 3 .1 Periostracum cicadaes extracts (1500mg/kg), HEA (5mg/kg, 7.5mg/kg, 12mg/kg, ip) are to dMCAO The rat nerve functional status scoring of cortex, as a result shows compared with model group, it is bright that HEA can obtain the nervous function of cerebral injury It is aobvious to improve.(n=8；*P<0.05, * * P<0.01 with to model group ratio).

Fig. 3 .2 Periostracum cicadaes extracts (1500mg/kg), HEA (5mg/kg, 7.5mg/kg, 12mg/kg, ip) are to dMCAO The TTC dyeing of cortex measures infarct size, as a result shows compared with model group, HEA (7.5mg/kg, ip) can make the stalk of cerebral injury Unleavened dough, which accumulates, to be obviously reduced.(n=8；*P<0.05, * * P<0.01 with model group ratio).

Fig. 3 .3 HEA (7.5mg/kg, ip) groups are to the protective effect of the cerebral ischemia of dMCAO cortexes and joint DPCPX (1mg/ Kg, ip) the rat brain tectology of HAE cerebral protections is blocked to dye HE (400X), it as a result shows compared with model group, HEA groups can make cellular swelling degree and cell nuclear alteration make moderate progress, and cell number increases.

Fig. 3 .4 HEA (7.5mg/kg, ip) groups are to the protective effect of the cerebral ischemia of dMCAO cortexes and joint DPCPX (1mg/ Kg, ip) block HAE cerebral protections tunel detection rat cerebral cortex Apoptosis situation (400X), as a result display and mould Type group is compared, and rat cerebral cortex infarct Penumbra zone area apoptosis rate can be used to be substantially reduced for HEA groups.

Fig. 4 .1. Periostracum cicadaes extracts (1500mg/kg, ip), HEA (5mg/kg, 10mg/kg, 15mg/kg, ip) are right MPTP causes the influence of mouse movement dysfunction.(n=8；*P<0.05, * * P<0.01 with control group ratio；#P<0.05, ##P< 0.01 with model group ratio)

Fig. 4 .2 Periostracum cicadaes extracts (1500mg/kg, ip), HEA (5mg/kg, 10mg/kg, 15mg/kg, ip) are right The influence of dopaminergic neuron reduction caused by MPTP.(n=8；*P<0.05, * * P<0.01 with control group ratio；#P< 0.05, ##P<0.01 with model group ratio)

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

Have during Fig. 6 .1.HEA (25mg/kg, sc) independent role to the 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<0.05, * * P<0.01 with solvent group ratio；#P<0.05, ##P<0.01 with administration group ratio).

Fig. 7 .1.HEA combine influence of the adenosine receptor to reduction pain mouse writhing number, as a result show HEA (15mg/ Kg the writhing number of mouse) can be substantially reduced, under the action of DPCPX (1mg/kg), the analgesic activity of HEA is by apparent short of money Anti- (n=8；*P<0.05, * * P<0.01 with solvent group ratio；#P<0.05, ##P<0.01 with administration group HEA15mg/kg ratios).

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

Fig. 8 .2 are 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<0.05, * * P<0.01 compared with the control group；#P<0.05, ##P<0.01 compared with morphine group).

Fig. 9 .1 HEA (5mg/kg, 7.5mg/kg, 10mg/kg, ip), selective A₁AR antagonist DPCPX 1mg/kg, connection Administration group DPCPX (1mg/kg)+HEA (5mg/kg) and DPCPX (1mg/kg)+HEA (7.5mg/kg) is closed to mouse Ischemia Reperfusion Blood serum Bun (A) and the influence (n=5 of BUN (B) level when after note for 24 hours；^*P<0.05,^**P<0.01 compared with sham-operation group；^#P< 0.05,^##P<0.01 compared with IR groups).Compared with sham-operation group：IR group mouse significantly increase Scr, and BUN is horizontal.With IR groups ratio Compared with：It is horizontal that mouse pretreatment HEA (5mg/kg, 7.5mg/kg, 10mg/kg) is substantially reduced Scr and BUN；Pretreatment selectivity Apparent increase Scr, BUN are horizontal respectively by A1AR antagonists DPCPX (1mg/kg) and DPCPX (1mg/kg)+HEA (7.5mg/kg), And DPCPX (1mg/kg), DPCPX (1mg/kg)+HEA (5mg/kg) and three groups of DPCPX (1mg/kg)+HEA (7.5mg/kg) it Between Scr and BUN levels there is no notable difference (P>0.05).

Fig. 9 .2 HEA (5mg/kg, 7.5mg/kg, 10mg/kg, ip), selective A₁AR antagonist DPCPX 1mg/kg, connection Administration group DPCPX (1mg/kg)+HEA (5mg/kg) and DPCPX (1mg/kg)+HEA (7.5mg/kg) is closed to mouse Ischemia Reperfusion The influence (HE × 400) that renal pathology changes after note.Note：A：Sham-operation group；B：IR groups；C：HEA 2.5mg/kg groups；D：HEA 5mg/kg groups；E：HEA 7.5mg/kg groups；F：DPCPX (1mg/kg) group；G：DPCPX(1mg/kg)+HEA(5mg/kg)；H： DPCPX(1mg/kg)+HEA(7.5mg/kg).Sham-operation group (A) renal tissue structure is normal, on rarely seen part renal tubule Chrotoplast is denaturalized, and local renal tubule intracavitary has the non-viable non-apoptotic cell to come off and vacuolar degeneration.Compared with the kidney of sham-operation group mouse, IR groups (B) nephridial tissue is shown in a large amount of small official's epithelial cell swelling, vacuolar degenerations, and serious position sees the necrosis of rill sheet, comes off, kidney is small Pipe official jargon is expanded, and partly sees that epithelial cells fragments and brush border come off.HEA2.5mg/kg groups (C), HEA 5mg/kg groups (D) and The change of HEA 7.5mg/kg groups (E) specimens pathological is substantially reduced compared with IR groups, and only swelling occurs in part cell, comes off and vacuole becomes Property, non-viable non-apoptotic cell is less, and renal tubule shape keeps good.On the contrary, the pretreatment selectivity A1AR antagonists before IR is damaged DPCPX can aggravate histology necrosis, and cell comes off completely substantially, the expansion of renal tubule official jargon, seldom see the kidney of structural integrity Tubular epithelial cell.DPCPX+HEA similarly aggravates renal histology necrosis.

Fig. 9 .3 Pathologicals scoring (n=5；^*P<0.05,^**P<0.01 compared with sham-operation group；^#P<0.05,^##P<0.01 Compared with IR groups) renal tubular-interstitial injuries of IR groups scores than sham-operation group apparent increase (P<0.01).HEA 5mg/kg groups, HEA7.5mg/kg groups and HEA 10mg/kg groups and the obvious reduction renal tubular-interstitial injury scoring (P of IR groups<0.01, P< 0.01, P<0.01).On the contrary, pretreatment DPCPX 1mg/kg, DPCPX (1mg/kg)+HEA (5mg/kg) and DPCPX (1mg/ Kg)+HEA (7.5mg/kg) significantly increases renal tubular-interstitial injury scoring (P<0.01, P<0.01, P<0.01) compared with IR groups Compared with, and renal tubular-interstitial injury scoring no significant difference (P between three groups of groups>0.05).

Fig. 9 .4 are from qualitative upper electricity consumption sem observation HEA (5mg/kg, 7.5mg/kg, 10mg/kg, ip), selective A₁AR antagonisms Agent DPCPX 1mg/kg, administering drug combinations group DPCPX (1mg/kg)+HEA (5mg/kg) and DPCPX (1mg/kg)+HEA (7.5mg/ Kg) to mouse

The influence of apoptosis in renal tubular epithelial cells situation.Note：A：Sham-operation group；B：IR groups；C：HEA 2.5mg/kg groups；D： HEA 5mg/kg groups；E：HEA 7.5mg/kg groups；F：DPCPX (1mg/kg) group；G：DPCPX(1mg/kg)+HEA(5mg/kg)； H：DPCPX(1mg/kg)+HEA(7.5mg/kg).Sham-operation group (A) nucleus is entirely complete.The structure of nucleus and organelle It clearly easily debates, nuclear membrane is double-deck, and mitochondria is complete, and mitochondrial cristae is apparent.IR groups (B) karyopyknosis, swelling have denaturation； The mitochondria of neither one structural integrity；Nuclear fractions dissolve；Chromatin condensation is blocking and side is gathered；Mitochondria vacuole；Cell Edge is less smooth；Cell membrane obscures, fold；Chromatin is sparse in fine particulate, irregular distribution, obscure boundary, cell Swelling is starched, organelle structure destroys.HEA 5mg/kg groups (C) integrally make moderate progress compared with ischemia-reperfusion group, partial mitochondrial knot Structure is complete, and mitochondrial cristae is apparent.But with larger lipid vacuole.HEA 7.5mg/kg groups (D) nuclear membrane compared with IR groups and HEA 5mg/kg groups (C) are double-deck apparent, and most of mitochondria is normal, and mitochondrial cristae is more apparent, but has part lysosome.HEA 10mg/kg groups (E) are apparent compared with IR group chondriosome protectives, and most of structure of mitochondria is complete, but has partial mitochondrial swelling occur. The basic mitochondrias without complete structure structure of selectivity A1AR antagonists DPCPX (F) are pre-processed before ischemic, there are a large amount of lyases Body and lipid vacuole generate.Drug combination DPCPX+HEA (G, H) karyopyknosis, swelling, chromatin side are pre-processed before ischemic Collection, the mitochondria of basic neither one complete structure, the apoptosis degree than IR groups are more serious.

Fig. 9 .5 are from quantitatively with HEA from TUNEL (5mg/kg, 7.5mg/kg, 10mg/kg, ip), selective A₁AR is short of money Anti-agent DPCPX 1mg/kg, administering drug combinations group DPCPX (1mg/kg)+HEA (5mg/kg) and DPCPX (1mg/kg)+HEA The influence (TUNEL × 400) of (7.5mg/kg) to Mouse Renal Tubular Epithelial Cells apoptosis situation.Note：A：Sham-operation group；B：IR Group；C：HEA 2.5mg/kg groups；D：HEA 5mg/kg groups；E：HEA 7.5mg/kg groups；F：DPCPX (1mg/kg) group；G：DPCPX (1mg/kg)+HEA(5mg/kg)；H：DPCPX (1mg/kg)+HEA (7.5mg/kg) sham-operation group renal tissue only has only a few Apoptosis, and IR group Apoptosis showed increaseds, compared with IR groups, Apoptosis number is reduced, but still more for HEA treatment groups In sham-operation group.And selectivity A1AR antagonists DPCPX and drug combination DPCPX+HEA apoptotic cells are pre-processed significantly than IR group Increase.

Fig. 9 .6 TUNEL apoptotic indexes (n=5；^*P<0.05,^**P<0.01 compared with sham-operation group；^#P<0.05,^##P< 0.01 compared with IR groups) IR groups apoptotic index is than sham-operation group showed increased (P<0.05), HEA treatment groups (5mg/kg, 7.5mg/ Kg, 10mg/kg) apoptotic index is considerably less than IR groups (P<0.01).And pre-process selectivity A1AR antagonists DPCPX (1mg/kg) Apoptotic index and drug combination [DPCPX (1mg/kg)+HEA (5mg/kg)；DPCPX (1mg/kg)+HEA (7.5mg/kg)] apoptosis Index is significantly bigger (P than IR groups<0.01).

Fig. 9 .7 HEA (5mg/kg, 7.5mg/kg, 10mg/kg, ip), selective A₁AR antagonist DPCPX 1mg/kg, connection Administration group DPCPX (1mg/kg)+HEA (5mg/kg) and DPCPX (1mg/kg)+HEA (7.5mg/kg) is closed to Murine Bone Marrow peroxidating Influence (the n=5 of object enzyme (MPO) activity；^*P<0.05,^**P<0.01 compared with sham-operation group；^#P<0.05,^##P<0.01 with IR groups Compared to).Compared with sham-operation group：MPO activity dramatically increases (P in IR group mouse kidney cortex<0.01).Compared with IR groups：C57 Mouse pretreatment HEA 5mg/kg and 7.5mg/kg before IR is damaged significantly reduce MPO activity (P<0.05)；On the contrary, pretreatment Selective A1AR antagonists DPCPX (1mg/kg) and DPCPX (1mg/kg)+HEA (5mg/kg) significantly increase MPO activity (P< 0.05).And DPCPX (1mg/kg) group, DPCPX (1mg/kg)+HEA (5mg/kg) group and DPCPX (1mg/kg)+HEA (7.5mg/ Kg MPO activity is statistically no significant difference (P between) organizing three groups of groups>0.05).

Fig. 9 .8 HEA (5mg/kg, 7.5mg/kg, 10mg/kg, ip), selective A₁AR antagonist DPCPX 1mg/kg, connection Administration group DPCPX (1mg/kg)+HEA (5mg/kg) and DPCPX (1mg/kg)+HEA (7.5mg/kg) is closed to mouse cortex renis ICAM-1, IL-1 β, TNF-α mRNA gene expressions influence (n=5；^*P<0.05,^**P<0.01 compared with sham-operation group；^#P< 0.05,^##P<0.01 compared with IR groups).The mRNA of ICAM-1 in IR group mouse cortex renis, TNF-α and IL-1 β are expressed with sham-operation Obvious increase (the P of group<0.01).Compared with IR groups：Pretreatment HEA 5mg/kg, 7.5mg/kg and 10mg/kg are significantly reduced The high expression of the mRNA of ICAM-11, TNF-α and IL-1 β.On the contrary, pretreatment selectivity A1AR antagonist DPCPX (1mg/ Kg), DPCPX (1mg/kg)+HEA (5mg/kg) and DPCPX (1mg/kg)+HEA (5mg/kg) respectively increase proinflammatory cytokines The mrna expression amount of ICAM-1, TNF-α and IL-1 β, wherein TNF-α and the mrna expression amount of IL-1 β significantly increase, and ICAM-1 Increase be not statistically significant.In addition, between three groups of DPCPX groups and administering drug combinations DPCPX+HEA groups proinflammatory cytokines base Because expression is not statistically significant (P>0.05).

Specific embodiment

Embodiment 1.HEA is A₁The selective agonist of receptor

The preparation of 1.1 membrane receptor proteins

Brain is taken using Wistar rats broken end, cerebral cortex and corpus straitum is isolated, weighs respectively, by 1:10 add in 10 times The Tris-HCL buffer solutions (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 precipitation is mixed in 50mM Tris-HCL buffer solutions again, uses Coomassie Brilliant Blue (Bradford methods) determines rat cerebral cortex protein concentration as 0.8mg/ml, rat striatum brain tissue homogenate protein content 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 experiment

Memebrane protein and corresponding ligand (adenosine al receptor ligands Binding experiment are added in reaction tube：Rat cortex brain tissue Homogenate and the combination that corresponding ligand is 0.2nM [3H] DPCPX；Adenosine A 2 A receptor ligand binding assay：Rat striatum brain group Knit the combination of homogenate and corresponding ligand for 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) the saturation curve combined, with Scatchard linear transformation methods, acquires balance (the Kd values that A1 is combined are 0.14nmol/L, Bmax 2290fmol/mg to dissociation constant；A_2AWith reference to Kd values be 11.48nmol/ L, Bmax 5657fmol/mg).

Measure the HEA (10 that pipe adds in various concentration^-9、10^-8、10^-7、10^-6、10^-5Mol/L), mixing is after shaking bath 25 DEG C of incubation 30min, aspirate reaction solution using cell harvestor, pass it through GF/B glass filters (Watman), terminate reaction, With Tris-HCl wash buffers 3 times, 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 measure 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 result of the test

Experiment measures HEA and adenosine A₁The Ki values of competitive binding are 89.5nmol/L, with adenosine A_2AThe Ki values 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 A₁With higher selectivity (Fig. 1 .1-1.2).

The affinity experiment of artificial synthesized HEA and its derivative

Wherein, HEA (artificial synthesized), R1 are C (CH3) 2CH2OH (derivative 1), CH (CH3) CH2OH (derivative 2) or C (CH3) four kinds of substances such as 3 (derivatives 3) have carried out the sample similar with this experiment, measure artificial synthesized HEA and its derivative 1-3 (summary of specific experiment data) similar with the result of HEA, as a result also prompts artificial synthesized HEA and its derivative 1-3 to A₁Tool There is higher selectivity.

Applications 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 experiment 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 groups (0.1mgkg^-1, ip), HEA groups (15mg/kg, 40mg/kg, 60mg/kg), DPCPX groups (2mg kg^-1, ip), ZM241385 groups (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 antagonists 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 antagonist 5min is given Pentylenetetrazole (100mgkg^-1, ip), observe mouse eclamptogenic reaction to PTZ.

2.2 Testing index

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

2.3 result of the test

The Behavior Test result of animal prompts us HEA 40mgkg^-1Group, can pole significantly reduce it is small caused by pentylenetetrazole The death rate that mouse is fainted from fear.In addition, specificity A₁Receptor antagonist DPCPX (2mgkg^-1, ip) and it can significantly 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, available for fainting from fear Clinical treatment and prevention (Fig. 2).

Applications of the embodiment 3.HEA in cerebral ischemia

The preparation of 3.1 test samples

It is raw material that precision, which weighs the Periostracum cicadae after drying, using 50% ethyl alcohol as solvent extraction, 2h/ times, filters, merges Filtrate is configured to the solution for later use that effective quantity containing sample is 1500mg/kg.

3.2 animal models 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 skins are opened at the corner of the eyes and external auditory canal line within the eye Notch, separation fascia, musculature, exposes skull；3. inhaling a small amount of physiological saline with cotton ball wipes skull to getting a clear view；④ Fascia, exposure skull are detached under surgical operation microscope, a diameter of 2mm circular holes are bored at 1/3 on bone ridge；5. with a small amount of physiology The extra bone bits of normal saline washing, push meninx aside, expose arteria cerebri media MCA；6. rat dorsal position is fixed, hit exactly along throat portion Notch detaches bilateral carotid CCA, is passed through with surgical thread, temporarily not ligatures；7. finding MCA under surgical operation microscope, use Unipolar electrocoagulator is cooled down after burning with normal saline flushing again；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 rats are randomly divided into 7 groups：Sham-operation group (1%DMSO, ip), model group, Periostracum cicadae extract group 1500mg/ Kg, HEA 5mg/kg groups, HEA 7.5mg/kg groups, HEA12mg/kg groups, HEA7.5mg/kg+DPCPX1mg/kg groups.Each group medicine Object in the preoperative 30min of dMCAO is given be injected intraperitoneally once respectively respectively, and sham-operation group and model group give 1% dimethyl sulfoxide (DMSO), Rats in sham-operated group only opens cranium exposure arteria cerebri media, not solidifying to close arteria cerebri media and abdominal cavity injection, A₁Selective adenosine receptor Antagonist DPCPX gives before 10min is administered and is injected intraperitoneally.

3.3 index determining

3.3.1 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 apparent neurologic impairment；1 point:Not tensible offside forelimb；2 points:To right during walking Side rotates；3 points:Topple over during walking to offside；4 points:It spontaneous cannot walk, the loss of consciousness.

3.3.2TTC the measure to brain infarction area is dyed：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 put 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 positioned over tweezers and is equipped with In the avoid light box of 2%TTC, room temperature is incubated 15min, and digital camera is fixed at brain piece vertical direction 30cm and is taken pictures.Normal brain activity It is in cerise that succinate dehydrogenase in tissue Mitochondria is reacted with TTC, and infarcted region is not colored due to lacking mitochondria.Stalk Unleavened dough product accounts for the percentage of full brain to represent with infarcted region.

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

3.3.4tunel cerebral cortex cells apoptosis situation is detected

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

3.4 experimental result

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

Applications of the embodiment 4.HEA in Parkinson

The preparation of 4.1 test samples

4.2 animal models and medication

24 ± 1g male C57BL/6 mouse are randomly divided into 6 groups, control group, model group, Periostracum cicadae extract group (1500mg/kg) (extracting method is with reference to examples of implementation 3), HEA (5mg/kg, 10mg/kg, 15mg/kg) group.Using 1- methyl- 4 phenyl -1,2,3,6- tetrahydropyridines (1-methyl-4-phenyl-1,2,6-tetrahydropyridine, MPTP) induction Mouse PD models, Periostracum cicadae crude extract group and various concentration HEA groups respectively continuous intraperitoneal injection 14 days and in the 11st day to MPTP30mg/kg is injected intraperitoneally in 1h before medicine, and continuous 4 days, 1 day after last time administration, the behaviouristics for carrying out pole-climbing ability was surveyed Examination, post-tensioning neck execution in 4 days take corpus straitum.Control group gives normal saline, and model group is before the 11st day gives physiological saline 1h, be injected intraperitoneally MPTP30mg/kg, continuous 4 days.

4.3 index determining

4.3.1 pole-climbing method measures mouse movement ability

By mouse upside down, long 50cm is placed in, 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.

4.3.2 immunohistochemical method measures the quantity of TH positive cells 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-mouses (1 of Alexa fluor；1000, Molecular Probes) fluorescence microscopes are taken pictures, to black substance TH Positive Cell Counts.

4.4 result of the test

Result of the test shows that Periostracum cicadae extract and HEA (10mg/kg, ip) can improve the limb for improving mouse in various degree The body coordination ability increases TH positive cell quantities, prompts clinical treatment and prevention that HEA can be used for Parkinson's disease, Periostracum cicadae Extract has the function of when containing HEA identical.

Applications of the embodiment 5.HEA in downern is prepared

5.1 animal packets and medication

Male ICR mouse is randomly divided into 4 groups, selects adenosine A₁R antagonists 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 groups are by small After mouse intraperitoneal injection antagonist 10min, then HEA is injected intraperitoneally.

5.2 detection method

It completes to be administered after 15min and mouse is put into single opaque rectangular autonomic activities case (50cm × 50cm × 40cm) It is interior, step on lattice number in camera record 5min.Same lattice are entered into mouse four limbs to calculate, as animal activity index.

5.3 experimental result

HEA (15mg/kg, ip) is notable to the inhibition of mouse autonomic activities.Prompting HEA can be played in nervous system Sedation.

Applications of the embodiment 6.HEA in drug of sleeping is prepared

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 intragastric administration on mice administration 30min, threshold agent is injected intraperitoneally in each group animal 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 the sleeping time caused by mouse threshold dose yellow Jackets, But no difference of science of statistics, prompting cicada extract HEA have certain collaboration amobarbital to Sleep latency compared with the control group The effect of sodium.

Embodiment 7：Purposes of the HEA in analgesia

7.1 animal packets and medication

Cleaning grade male mouse of kunming, 18~22g of weight select adenosine A₁R antagonists DPCPX.Mouse be divided into solvent group, HEA (15mg/kg) group, DPCPX (1mg/kg) and DPCPX+HEA (1mg/kg+15mg/kg) group；Wherein DPCPX+HEA groups by After mouse peritoneal injection antagonist 10min, then HEA is injected intraperitoneally.

7.2 detection method

Using writhing method, treatment dosage is administered 30~40min pneumoretroperitoneums and injects 0.7% acetic acid, record small white mouse 15 minutes Interior writhing number calculates the number of pain writhing response that HEA reductions generate 0.7% acetic acid.

7.3 experimental result

HEA (15mg/kg, ip) significantly reduces the writhing number of pain mouse, illustrates that the analgesic effect of HEA is notable, passes through Adenosine A 1 receptor antagonists are added, analgesic effect is fallen after rise, and the analgesic effect of HEA is prompted to may be by excited adenosine A 1 receptor and is risen Effect.

Embodiment 8.HEA or medicinal fungus containing HEA and extract are treated and prevented on addictive drug preparing Using

The preparation of 8.1 test samples

It is raw material that precision, which weighs the Periostracum cicadae after drying, 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 purifications, and step by step arithmetic divides Sample B, C, D are not obtained.The effective quantity of sample containing each component (HEA) is each configured to as 1500mg/kg, 750mg/kg, 100mg/ The solution for later use of kg, 20mg/kg.

8.2 animal packets and medication

Cleaning grade male mouse of kunming, 18~22g of weight are 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)：It tests first three day and allows it in case Middle free shuttling 15min naturally has a preference for black box according to rat, therefore selects white box (d-1, d0) records every big as with medicine-chest 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 d1, d3, d5, d7 intraperitoneal injection hydrochloric acid (10mg/kg, ip), whitepack training 50min, d2, d4, d6, d8 After isometric physiological saline is injected intraperitoneally, black box training 50min, administration group 15min administrations physiology before injection morphine daily Brine group all gives physiological saline Cpp expression in 8 days：Rat is not made any processing head and places two box intersections towards whitepack by d9, Its free activity 15min in two boxes is allowed to record the residence time situation of rat in white box；Cpp subsides：Start within d10 days, it is real It tests group and control group gives training 50min, d11, d13, d15, d17 training of physiological saline d10, d12, d14, d16 whitepack 50min；Rat is not made any processing head and places two box intersections towards whitepack by d18, allows its free activity 15min in two boxes, Record residence time situation of the rat in white box；Cpp resume combustion：D19 morphine groups give 5mg/kg, and administration group is before morphine 15min is administered, and each group mouse head softly places two box intersections freely activity 15min towards whitepack, records activity condition.

8.3 Testing index

Place Preference index：Mouse is recorded in the whitepack residence time of different times, analyzes the different habituation stages, HEA or Person contain HEA medicinal fungus and extract to the mouse CPP effects of morphine induction.

8.4 result of the test

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 Periostracum cicadae sample A is given to During The Withdrawal Period or its substep carries Object sample (B, C) is taken to be substantially less than morphine group, particularly sample D extremely significantly low with the medicine-chest time with the medicine-chest time after handling In morphine group, HEA is prompted to can be used for the drug-seeking behavior after giving up, had certain anti-additive.

Examples of implementation 9：Purposes of the HEA in anti-kidney failure

9.1 animal packets, modeling and medication

Male 20-25g of C57BL/6 mouse, animal are fed after a week through adaptability, are randomly divided into 8 groups：Sham-operation group, IR Group, HEA 5mg/kg groups, HEA 7mg/kg groups, HEA 10mg/kg groups, DPCPX (1mg/kg) group, DPCPX (1mg/kg)+HEA (5mg/kg) is organized and DPCPX (1mg/kg)+HEA (7.5mg/kg) group.

Mouse is preoperative to weigh, yellow Jackets 50mg/kg intraperitoneal injection of anesthesia mouse, and median abdominal incision opens abdomen, passivity point From and fully expose the kidney base of a fruit, close the Bilateral Renal base of a fruit with noninvasive artery clamp folder and cause renal ischaemia, kidney color is become at once from cerise Atropurpureus, the method for the whether successful determination method reference literature 7,8 of experimental model.Artery clamp is unclamped during ischemic 30min, is restored Perfusion, kidney gradually become scarlet by kermesinus, show Reperfu- sion success.Suture abdominal wound.Animal freely takes the photograph water and food.

Sham-operation group mouse only receives out abdomen, the free kidney base of a fruit and suture abdomen operation.Remaining group mouse establishes kidney IR Model.Respective concentration drug is injected intraperitoneally in each administration group 15min before ischemic, and then 15min is simultaneously before ischemic for administering drug combinations Administration.

9.2 Testing index

9.2.1 renal function detects

Reperfu- sion for 24 hours after anesthetized mice again, obtain venous blood.With biochemistry analyzer (Dimension Xpand plus are purchased from Siemens Company), picric acid rate method and zymetology performance rate method detection Scr (serum is respectively adopted Creatinine) and BUN (blood urea nitrogen) is horizontal.

9.2.2 renal tissues pathology inspection

Reperfu- sion for 24 hours after anesthetized mice again, obtain kidney sample.Kidney sample is after the fixation of 10% formalin, stone Wax embeds, slice, row hematoxylin-eosin (hematoxylin-eosin staining, HE) dyeing, optical microphotograph Microscopic observation Renal histopathology changes.

9.2.3 prepared by electron microscopic section and subcellular situation is observed

Piece is taken the photograph using transmission electron microscope (H7500 is purchased from Hitachi Ltd) observation.It is completed in Wenzhou Medical University's Electron Microscopy Room.

9.2.4 situ end labeling analyzes Apoptosis

The renal tissue that part is taken to preserve, routine paraffin wax embed 4 μm of slices.It is grasped by the method for kit specification Make.Positive staining result judges：Positive cell shows as nucleus in brown color or sepia.10 400 times are randomly selected to regard Open country calculates apoptotic index AI%=positive cell numbers/all total number of cells × 100% in the visual field.

9.2.5MPO analysis

MPO analyses are measured according to the operation of real kit specification.

9.2.6 the gene expression of Real-time PCR Analysis ICAM-1, TNF-α and IL-1 β；Used in RT-PCR reaction systems Primer sequence is as follows:

GAPDH：Upstream 5-GAGACCTTCAACACCCCAGC-3；Downstream

5-ATGTCACGCACGATTTCCC-3；

ICAM-1：Upstream 5-TCTTCTGAGCGGCGTCG-3.Downstream

5-TTGCCAGGTCCAGTTCCC-3；IL-1β：Upstream 5-TGGGAAACAACAGTGGTCAGG-3.Downstream 5- CATCAGAGGCAAGGAGGAAAAC-3；

TNF-α：Upstream 5-AACTTAGAAAGGGGATTATGGCT-3.Downstream 5-TCAGGGAAGAATCTGGAAAGGT-3.

Pcr amplification reaction system：12.5 μ L, Forward primer (10 μm of ol/ of IQ SYBR Green Super mix L) 1 μ L, Reverse primer (10 μm of ol/L) 1 μ L, cDNA (being diluted with water into consistent level) 10.5 μ L.Reaction condition： 50.0℃3min、95.0℃3min；95.0 DEG C of 10s, 60.0 DEG C of 10s, 72.0 DEG C of 20s, totally 45 recycle.Experimental result is by fluorescence Quantitative PCR analysis software BIO-RAD CFX Manager are counted and are calculated automatically.Target gene mRNA expressions use 2- Δ Δ Ct methods calculate, and to be standardized on the basis of GAPDH mRNA in corresponding sample.

9.3 result of the test

Set about in terms of by detecting blood serum Bun, BUN levels and observation renal pathology morphological change, determine cicada fungus worm Careless extract N6- (2- ethoxys) adenosines are used to having protective effect to mouse kidney ischemical reperfusion injury, then by combining The method addition selectivity A1AR antagonists DPCPX of medicine speculates N6- (2- ethoxys) gland glycoside action mechanism, thus it is speculated that N6- (2- ethoxys) adenosine may be by exciting adenosine A 1AR, and scavenging activated oxygen mitigates peroxidatic reaction of lipid, mitigates cell Apoptosis reduces the release of inflammatory factor, protects the kidney (Fig. 9 .1- Fig. 9 .8) of ischemia-reperfusion.

Claims

1. a kind of preparation treated in the horizontal preparation that inosine, urea nitrogen are reduced in kidney failure or renal ischemic reperfusion injury, Middle said preparation is included using N6- (2- ethoxys) adenosine.

2. a kind of treat kidney failure or renal ischemic reperfusion injury mesonephric tubule Epithelial Cell Apoptosis preparation, wherein, said preparation packet Include N6- (2- ethoxys) adenosine.

3. a kind of treat renal inflammation preparation in kidney failure or renal ischemic reperfusion injury, wherein, said preparation includes N6- (2- hydroxyls Ethyl) adenosine.

4. according to the preparation described in one of claim 1-3, wherein, the N6- (2- ethoxys) adenosine includes being used for reducing The content of inosine (Scr) or/and urea nitrogen (Bun) in blood.

5. preparation according to claim 4, wherein, the inflammation is by inflammatory factor TNF-a, IL-I β or ICAM-1 One of or the common high expression of their threes caused by.

6. preparation according to claim 5, wherein, the N6- (2- ethoxys) adenosine inhibit TNF-a, IL-I β, One in ICAM-1, the expression of two or three.

7. according to the reagent described in one of claim 1-6, wherein, the N6- (2- ethoxys) adenosine a concentration of 0.1~ 20mg/kg, preferably 5~7.5mg/kg.

8. according to the reagent described in one of claim 1-6, wherein, N6- (2- ethoxys) adenosine is worked by adenosine receptor.

9. according to the reagent described in one of claim 1-8, wherein the HEA is from Periostracum cicadae, Cordyceps militaris, cordyceps sinensis And its extract of artificial culture.

10.N6- (2- ethoxys) adenosines and derivative reduce flesh in treatment kidney failure or renal ischemic reperfusion injury is prepared Purposes on glycosides, the level of urea nitrogen, apoptosis in renal tubular epithelial cells or renal inflammation reagent, wherein N6- (2- ethoxys) Adenosine and derivative act on adenosine receptor and work.

11. purposes according to claim 10, wherein, the adenosine receptor is A₁、A_2A、A_2B、A₃In one kind or several Kind.

12. purposes according to claim 11, wherein, wherein, adenosine receptor A₁Receptor.