CN101489543A - 3-hydroxy fatty acid and its derivatives for improving of learning and/or memory of subjects - Google Patents

Abstract

The pharmaceutical composition for improving of learning and/or memory of subjects comprising 3-hydroxy fatty acid and its derivatives as active components, and the use of the compounds in the manufacture of medicaments for improving of learning and/or memory of subjects. escape response latency.

Description

3-hydroxy fatty acid and its derivatives for improving of learning and/or memory of subjects

Improve study and/or the memory capability of subject³- hydroxy fatty acid and its derivative technical field

The present invention relates to the purposes of hydroxy fatty acid and its derivative in the study of subject and/or memory capability is improved.The invention further relates to include pharmaceutical composition of the be confused compound as active component.The relevant knowledge of background technology learning and memory

Learning and memory is that animal changes itself behavior or produces new behavior to adapt to the necessary process of environment, and the learning and memory of the mankind is the basis for understanding and changing the objective world and participating in social practice.Physiologists claim to learn to be primarily referred to as nervous system to receive external environment information and influence the process of itself behavior.Memory is then comprising note with recalling two aspects：Note refers to brain from the information of extraneous numerous and complicated, extract the useful information formation in part and experience the marking, selected section information forms short-term memory from the marking is experienced again, finally selected part information formation such a process of long-term memory from short-term memory；Recall, refer to situation of the brain according to current outside afferent message, the rapid related information experience for extracting brain storage, with reference to extraneous afferent message, generates the process of the reaction of certain Psychology and behavior or response.

The type of study can be divided into three kinds (Schunk, 1996).

The first study is non-Associative learning, also known as simple study, and it need not form certain between stimulation and reaction and clearly contact, and habituation and sensitization are to belong to such study.

Two kinds of study of the mat woven of fine bamboo strips are Associative learning, and it includes classical conditioned reflex and operant conditioned reflex.Classical conditioned reflex is the approach that animal learning judges environmental concerns, such as Pavlov (Pavlov, 1927) is first carnivorous to light and then to animal in zoopery, by several times repeatedly after, animal is to the reaction of light with being identical to the reaction of meat：Light can also cause salivary secretion.One exemplary of operant conditioned reflex：Hungry rat is put into the test box for the depression bar for having protrusion in a side walls by experimenter, rat has pressed bar because of accidental chance, then food is obtained soon, the probability of its following depression bar is higher than spontaneity, so illustrate the reflection that Zoological Society is rewarded with food, grasp after this information, animal is once be in starvation, and it, which will be arrived in this case, produces corresponding reaction and obtain food（ Ferster and Skinner, 1957 ) 。

Three kinds of study of the mat woven of fine bamboo strips are Hybrid Learning, and Hybrid Learning includes latent learning, vicarious learning etc. again.Latent learning refers to influence of the potential experience to results of learning, for example, once entered the animal of labyrinth environment, although and be not affected by certain training, be again introduced into when undergoing training always than being easier to learn from the animal for being introduced into the environment；Vicarious learning refers to the ability that some animals have the behaviors such as action, the sound of the other animals of imitation, and this study form is generally existing in higher mammal and the mankind（Ormrod, 1999 ) .

The formation of memory is constituted by obtaining, consolidating and reproduce three processes.Sensory system is obtained by the process of intracerebral input signal, this process is easily disturbed by extraneous factor；Consolidation be the information obtained in intracerebral code storage and the stage kept, the information of long storage always has great importance and frequent recurrent information to bion；Reproduction is that the information for storing intracerebral extracts the process for being allowed to be reproduced in consciousness（Han Taizhen, Wu Fumei, 1998).

Human mind can be divided into three phases by the length according to memory time-histories.The stage of the mat woven of fine bamboo strips one is sensory storage, after environmental stimuli occurs, and a number of information enters from sense organ stores referred to as sensory storage, also known as immediate memory in mutually multitudinous system, the duration hundreds of, Bo second was by one second or so（The warm rising sun is elementary, and 1995).

Second stage is short-term memory, also known as primary memory.Short-term memory is on the basis of sensory storage, to the result being processed further of word, numeral, word or other information.The duration of short-term memory is general at several seconds to one minute or so（ Miller, 19⁵6 ) 。

Phase III is long-term memory, also known as long-term memory.It is by the result of short-term memory reprocessabilty.Time from a few minutes, several days to several years that it keeps, or even throughout one's life.Long-term memory is generally further divided into two types, i.e. second memory and mat woven of fine bamboo strips three-level memory.Second memory is a kind of long-term memory with stored by weak or slightly strong traces of memory, is easily forgotten about.Tertiary memory is that one kind is deeply engraved in haunting memory, has very strong traces of memory, the information of storage is called at any time（ Schweickert, 1993 ) .Strengthen the development situation of memory medicine

On the basis of the Remarkable Progress On Electric Artificial of geneticist and neuroscientist to human gene, brain structure and neurochemical, artificially improving intelligence by a certain measure will be possibly realized.At present, many " improving memory medicine " are in clinical trial in the world, some cognitive enhancers that can improve memory commercially occur（Table 1), these cognitive enhancers are also referred to as " clever medicine ", and the CNS such as developed by Warner Lambert companies can Inverse AchE inhibitor Tacrine (Hakansson, 1993)；Some medicines for being used for memory disorders patient simultaneously, Clinical Practice proves that the intelligence of healthy population can also be strengthened, and it is forgetful because of caused by aging to improve that many people take this kind of medicine, such as galanthamine（Sanda, 1995) and U.S. bent phosphatide（Morris et al, 1998 ) ；The medicine of the class of ritalin one treatment mental illness can also be used for some regular cerebral functions of enhancing, and it can improve the active disease children's study of trouble into malen, also there is similar effect to normal child（Jiao Pengtao etc. is 2006).Table 1 developed or develop relevant enhancing memory medicine

Table is noted： AchE:(acetylcholinesterase) Columbia Univ USA professor's Eric's bank Dares（Eric Kandel) it is the forerunner that memory enhancers are studied, he finds the secret of memory by the research to sea slug, and obtains the Nobel Prize（Kandel, 2001 ) .He occurs in the cynapse between two neurons by some modes in discovery learning：When the CREB albumen in nerve cell（cAMP response Element binding protein, cAMP response element binding proteins）When being activated, the cynapse is just reinforced, and CREB albumen also plays a role in the memory of drosophila and mouse is formed.

Before a neuron increases CREB naturally, NMDA (N-methyl-D-aspartate, N- the methyl asparatates on neuron membrane）Passage is switched on, and allows cation to flow into the nerve cell by NMDA passages, then a succession of cascade reaction for causing CREB to activate of these cations triggering（Arumugam, et al 2005 ).Shimuzu etc. (2000) has found that the quantity of NMDA acceptors in increase hippocampus of mice area can cause mouse to obtain more preferable achievement in a kind of experiment of spatial memory.I oneself recall relevant disease and its treatment

There are many diseases to reduce the learning and memory ability of patient at present, so as to cause memory disorders or decline, strong influence is brought to normal live of EI of people, this kind of disease includes：The various degenerative diseases of brain（Such as alzheimer ', Mo's disease, Alzheimer's disease, abbreviation AD), brain trauma and boxer's dementia；The apoplexy sequelas such as binswanger disease, cerebral infarction and cerebral hemorrhage；Postencephalitis；The cerebral anoxia sequelae such as anthracemia；Deficiency disease encephalopathic（Such as Wernicke encephalopathics（ Reuler et al 1985 ) ) ；Alcoholism and biochemistry are for dyskinetic encephalopathic；Mental illness etc..This kind of disease may be collectively referred to as remembering relevant disease.

It is the most serious to betide the Alzheimer's of old and presenium in these diseases.The disease is found first by German neurosurgeon Alois Alzheimer, it is the central nervous system degenerative disease (Muhamamad et al 1999) based on a kind of progressive cognitive disorder and Memory impairment, increases with advancing age.The AD incidences of disease are 4 ~ 12% in the old man of 65 years old or so according to statistics, and the incidence of disease is 20 40% (applying Anguo etc., 2000) in the elderly of more than 85 years old.Memory disorders or decline be a complicated pathologic process, it is every cause the brains such as frontal lobe, temporal lobe, hippocampal gyrus, thalamus, cingulate gyrus, diencephalon and formatio reticularis mesencephali extremely can reduce the memory of patient（ Andrew et al 2005 ) .

Effective method treatment memory disorders are there is no at present.Although the medicines such as the expansion blood vessel medicine of the precursor of lecithin fat foods, neurotransmitter, ritalin, dihydroergotoxine class have certain curative effect, while the training to memory function also has certain help（Wilson et al l997), but the ideal effect that people expect can't be reached.With the increase of improvement and the average life span of people's living standard, elderly population are increasing in the world, and society also enters aging society therewith, therefore seems most important to the research of these diseases and its treatment.People are learning any section Xue Zhi simultaneously During knowledge, memory is all be unable to do without, and the biggest obstacle learnt is no more than poor memory.Strengthen memory can promptly, exactly, the knowledge and skills that enduringly mastery learning is crossed, can also understand better, with these knowledge and skills.

3- hydroxy fatty acids are briefly explained

Polyhydroxyalkanoate（Polyhydroxyalkanoates, abbreviation PHA) construction unit there is diversity, it has now been found that PHA has an at least 125 kinds different monomer structures, and new monomer is constantly found to come out；PHA construction unit of the carbon chain lengths between 4-14 carbon also can have been synthesized chemically, and the group that can be modified on side chain.Because PHA biodegrading process is different, a greater variety of monomer structures and derivative can be obtained, enrich research application of these products in terms of biology, wherein 3-hydroxybutyrate is as a part for biological organic metabolite ketoboidies, and its each side physiological function widely studied（Chen and Wu, 2005).

Ketoboidies is the product during fatty acid catabolism, is formed only in liver, and it includes 3-hydroxybutyric acid, acetoacetate and acetone, and wherein 3-hydroxybutyrate content is more.Liver contains the enzyme system of synthesis ketoboidies, therefore can generate ketoboidies, but lacks the enzyme using ketoboidies, therefore can not aoxidize ketoboidies, and the ketoboidies of generation need to be through blood transportation to the further oxidation Decomposition of extrahepatic tissue.Liver output ketoboidies provides the energy for extrahepatic tissue, ensures the energy supply of brain during to hypoglycemia, to maintain its normal physiological function aspect to play an important role（Amiel et al, l l).

Utilize 3-hydroxybutyrate（3-hydroxybutyric acid or 3HB) and other metabolic precursor thereofs as drug regimen, myocardial efficiency can be strengthened to improve the glucose utilization efficiency (Cross et al, 1995) of heart；The energy can also be provided for the patient of diabetes and anti-insulin disease（Rackley et al, 1981)；These composition of medicine can also prolong Slow or prevent the brain damage related to memory（Reger et al, 2004).

3- hydroxy fatty acids（3-hydroxyalkanoic acid, abbreviation 3HA) monomer and its derivative have very particular advantage：

3HA monomers and its derivative water white transparency, and with slight fragranced, the additive of most of food can be made, it also is adapted for oral（ Plecko et al, 2002 ) ；

There is no 3HA metabolic enzyme in liver, all additives as normal diet will not be by liver metabolism after being absorbed, but transporting directly to utilize in its extrahepatic tissue, so as to quickly act（ Rasmussen et al, 1998 ) ；

They can directly pass through blood-brain barrier, directly reach in cerebral nerve tissue that there is provided battalion Support and can protect and prolong the aging and death of Slow nerve cells（Tieu et al, 2003) content of the invention

The present invention is based in part on following discovery：The 3- hydroxy fatty acids and its derivative of the present invention can remarkably promote the propagation of nerve cell, prolong Slow or suppress apoptosis and the death of nerve cell, and improve the S phases in CDC and G2/M phase cell quantities；In mouse test, the compounds of this invention substantially shortens the incubation period in learning process, significantly enhances the learning ability and space exploration ability of mouse, and the memory capability of mouse is significantly strengthened.

Therefore, one aspect of the present invention provides a kind of pharmaceutical compositions for being used to improve the study and/or memory capability of subject of c, and it includes formula (I) compound as active component,

0

OH- CH_; C— OR,

(I)

Or its pharmaceutically acceptable salt, solvate, hydrate, enantiomter or pro-drug, wherein,

Selected from by H, C C₉Alkyl or cycloalkyl, the group that constitutes of aryl and nontoxic metal ion；

R₂Selected from by H, C₉Alkyl or cycloalkyl, alkoxy and aryl constitute group；And pharmaceutically acceptable carrier.

Inventionwithout being bound to any specific theory, it is believed that when the compounds of this invention is amphipathic molecule, for favourable across blood-brain barrier.When the compounds of this invention is salt or acid, the effect across blood-brain barrier may be slightly poor, but still has good effect.

Therefore, the substituent in the compounds of this invention！^ and R₂Comprising carbon number be usually no more than 9.In a preferred embodiment of the invention, selected from by H, C_rC₇Alkyl and nontoxic metal ion constitute group；It is highly preferred that selected from by H, C₅Alkyl and nontoxic metal ion constitute group；It is further preferred that selected from by H, C_rC₃Alkyl and nontoxic metal ion constitute group.

In another preferred embodiment of the present invention, R₂Selected from by H, C C₇Alkyl, C C₇Alkoxy and aryl constitute Group；It is highly preferred that R₂Selected from by H, C₅Alkane Base, C C₅Alkoxy constitute group；It is further preferred that R₂Selected from by H, C_rC₃Alkyl, C₃Alkoxy constitute group.

In the compounds of this invention, nontoxic metal ion preferably is Na+, K+ and Ca²⁺。

Particularly preferred specific the compounds of this invention is：3-hydroxybutyrate methyl esters；Ethyl 3-hydroxybutanoate；3-hydroxybutyrate；3-HBA sodium；DL-3- Sodium γ-Hydroxybutrates.

Another aspect of the present invention provides a kind of method for being used to improve the study and/or memory capability of subject, including the above-claimed cpd of effective dose is applied to the subject.

Further aspect of the present invention provides above-claimed cpd and is preparing the purposes in being used to improve the study of subject and/or the medicine of memory capability.

According to a preferred embodiment of the present invention, compound of the invention can be used for treating the memory relevant disease in subject, include but is not limited to：Brain degenerative disease（Such as Alzheimer's）, brain trauma and boxer's dementia；The apoplexy sequelas such as binswanger disease, cerebral infarction and cerebral hemorrhage；Postencephalitis；Cerebral anoxia sequelae；Deficiency disease encephalopathic（Such as Wernicke encephalopathics）；Alcoholism and the dyskinetic encephalopathic of biochemical metabolism；Mental illness.The brief description of accompanying drawing

Fig. 1 is ethyl 3-hydroxybutanoate infrared analysis figure：The type Fourier Transform Near Infrared instruments of Magna 750, KBr tablettings, scanning spectrum area is 4 000 ~ 500cm-¹, scanning times are 20 times, and resolution ratio is that 4 cm- Fig. 2 are ethyl 3-hydroxybutanoate GC-MS analysis charts：Shimadzu gas chromatograph-mass spectrometers (GC-MSQP5050A)；Temperature programming（60 keep 5 min, 60 ~ 240,2.C/ minutes, 240-290,23.C/ minutes, 290 " C is kept for 5 minutes）；The V of injector temperature 280, split ratio 20:1, carrier gas：He, 1 ml/ minutes；Ionization mode EI, the eV of ionization energy 70, ion source temperature：180, ion stream：20 A, full scan acquisition mode (scanning range (M/ Z) 33 ~ 300amu).

Fig. 3 is 3-hydroxybutyrate methyl esters infrared analysis figure：The type Fourier transformation near infrared light Pass instrument of Magna 750, KBr tablettings, scanning spectrum area is 4 000-500 cm¹, scanning times be 20 times, resolution ratio for (：！^¹。

Fig. 4 is block diagram, shows the value-added influence of 3-hydroxybutyrate, 3-hydroxybutyrate methyl esters and ethyl 3-hydroxybutanoate on rabbit brain cortex neural spongiocyte.（a):Each material is acted on 24 hours； (b):Each material is acted on 4S hours. Fig. 5 is fluorescent microscopy images, shows ethyl 3-hydroxybutanoate to the apoptosis of rabbit brain cortex neural spongiocyte and dead influence：Cell culture condition is 37,5% C0₂, 2 %FBS DMEM nutrient solutions；Annexin-V-FITC cell apoptosis detection kit staining cell creep plates（The thick cover glasses of 0.13-0.17 mm are placed in 12 orifice plates, Ι Ο Ο μ Ι 1x10 are added^sIndividual/ml cell suspension；After cell attachment culture 4 hours, add⁹The % FBS DMEM nutrient solutions of 00 μ 12 continue to cultivate²0 hour）, NIKON TE2000-E fluorescence microscopes take pictures.（_a):Control； (b): 0.004 g/1³- 3-hydroxyethyl butyrate function cells 48 hours.

Fig. 6 is Flow cytometry result, shows influence of the ethyl 3-hydroxybutanoate to the rabbit Deiter's cells cycle.（a):Control；（b): 0.005_g/ l ethyl 3-hydroxybutanoates handle cell.

Fig. 7 shows the change of mouse learning and memory performances in MWM experiments.（a):The ability that orientation navigation experiment measurement mouse learns to water maze；（b):The ability of space exploration experiment measurement mouse locus memory；（c):Detection mouse memory retains situation behind 48 hours of space exploration experiment；（d):Detection mouse memory retains situation behind 45 days of space exploration experiment.

Fig. 8 shows the measurement result of mouse liver crude fat content.

Fig. 9 shows the measurement result of mice serum cholesterol level.

Figure 10 shows mouse femur bonemarrow nucleated cells number.

Figure 11 shows that immunohistochemical method identifies primary mouse cranial glia cell.

Figure 12 is Flow Cytometry detection 3-HBA sodium, the influence of DL-3- Sodium γ-Hydroxybutrates and 3- beta-hydroxymethyl butyrates to mouse glial cell apoptosis, is respectively：Negative control group), 3-HBA sodium treatment group（B), DL-3- Sodium γ-Hydroxybutrates treatment group（C) with 3-hydroxybutyrate methyl esters treatment group（d ) .

Figure 13 is Flow Cytometry detection 3-HBA sodium, the influence of DL-3- Sodium γ-Hydroxybutrates and 3- beta-hydroxymethyl butyrates to mouse glial cell apoptosis.

Figure 14 is fluorescence microscope home position observation 3-HBA sodium, the influence of DL-3- Sodium γ-Hydroxybutrates and 3-hydroxybutyrate methyl esters to mouse neuroglia shield Apoptosis.（a)、 （b)、 （c)、 （D) it is respectively negative control group, 3-HBA sodium, DL-3- Sodium γ-Hydroxybutrates and 3-hydroxybutyrate methyl esters treatment group.

Figure 15 is calcium imaging method detection 3-HBA sodium, the influence of DL-3- Sodium γ-Hydroxybutrates and 3-hydroxybutyrate methyl esters to mouse Deiter's cells intracellular free calcium level.（A) it is laser co-focusing microphotograph result, respectively negative control group, 3-HBA sodium, DL-3- Sodium γ-Hydroxybutrates and 3-hydroxybutyrate methyl esters treatment group from left to right.（B) it is the statistical result of each control group intracellular free calcium level. Figure 16 is that calcium imaging method detects influence of the various concentrations 3-hydroxybutyrate methyl esters to Deiter's cells intracellular free calcium level.

Figure 17 is that calcium imaging method is detected at different conditions, the influence of various concentrations 3-HBA sodium and DL-3- Sodium γ-Hydroxybutrates to Deiter's cells intracellular free calcium level.（_a):Using containing Ca²⁺With M ^ NPM; (b):Using not containing Ca²⁺With Mg^ NPM.

Figure 18 is that calcium imaging method detects calcium ion analog nitredipine (nitrendipines）The influence produced to intracellular calcium ion change caused by D- 3-hydroxybutyrates sodium, DL-3- Sodium γ-Hydroxybutrates and 3-hydroxybutyrate methyl esters.Embodiment

One aspect of the present invention provides a kind of pharmaceutical composition for being used to improve the study and/or memory capability of subject, and it is included：

As formula (I) compound of active component,

0

(I)

Or its pharmaceutically acceptable salt, solvate, hydrate, enantiomter or pro-drug, wherein,

Selected from by H, C C₉Alkyl or cycloalkyl, the group that constitutes of aryl and nontoxic metal ion；

R₂Selected from by H, C_rC₉Alkyl or cycloalkyl, the group that constitutes of C alkoxy and aryl；And pharmaceutically acceptable carrier.

It is preferred that specific formula (I) compound include：3-hydroxybutyrate methyl esters, ethyl 3-hydroxybutanoate, 3-hydroxybutyrate (including its salt, such as sodium salt, calcium salt, sylvite）, 3- hydroxycaproic acids methyl esters, 3- hydroxy ethyl caproates, 3- hydroxycaproic acids (including its salt, such as sodium salt, calcium salt, sylvite）.

The inventors discovered that, formula (I) compound（Hereinafter referred to as " the compounds of this invention "）Easily it is absorbed by organisms and works, drug effect significantly has no side effect.Formula (I) compound shows that the propagation of nerve cell can be remarkably promoted in cytologic experiment, improves the S phases in CDC and G2 M phase cell quantities.In water maze laboratory（Morris Water Maze) experiment In, formula (I) compound substantially shortens the incubation period in learning process, significantly enhances the learning ability and space exploration ability of mouse, and the memory capability that mouse is then embodied in memory retains step is significantly strengthened.Mouse increased weight when taking such newtype drug is very fast, but its liver crude fat content and serum cholesterol content, all without significant change, the increase of body weight is then embodied in the increase of the bone density of mouse.

The compounds of this invention shows that these small molecules can significantly improve the propagation of nerve cell in low concentration during neuronal cell cultures by MTT analysis results（Fig. 4 a, 4b), while they can prolong Slow or the apoptosis for suppressing nerve cell and death（Fig. 5 a, 5b)；Flow cytometry analysis result then indicates these small molecules and promotes the propagation of nerve cell to be that they enhance the splitting ability of nerve cell, is in particular in S phases and G in the cell cycle₂The obvious increase of/M phase cells（Fig. 6 a, 6b).

The compounds of this invention feeding mouse passes through Morris Water Maze after one month

(Morris water mazes）To detect the ability of learning and memory of contrast mouse（Morris et al, 1984 ) .Orientation navigation experiment result shows the EL of each group as increasing for test number (TN) is gradually shortened, and the EL of each administration experimental mice is shorter than control group, and with the significance difference opposite sex

( P<0.05 ) ；In space exploration experiment, each concentration 3-hydroxybutyrate methyl esters group mouse cross-platform number of times in 60 seconds is more all than control group, with significant difference（P<0.05 ) .Space exploration experiment terminates to carry out memory retention test after 48 h, as a result shows and each concentration³The EL of-beta-hydroxymethyl butyrate group mouse has the significance difference opposite sex compared with control group（P<0.05), wherein 30 3-hydroxybutyrate methyl esters group mouse performances in mg/kg/ days are the most obvious（Fig. 7 a, 7b).

The mouse of feeding the compounds of this invention shows being remarkably reinforced for learning ability and space orientation ability in Morris Water Maze Behaviors surveys, it is more permanent that memory retains, and searches out rapidly and climbs up easily by enhanced memory and space orientation ability and hides in platform under water

(Fig. 7 c, 7d).

Mouse is during feeding the compounds of this invention, and body weight increases comparatively fast compared with control group, and is going strong than control group mice, and the increase of body weight is embodied in the increase of bone marrow cell and bone density（Figure 10).

Pass through the determination experiment of serum cholesterol content, cholesterol level in administration group mice serum is similar to Dui Zhao Group, and in the determination experiment of liver crude fat content, the content outline of administration group mouse liver crude fat is less than control group mice, but being not significantly different property（Fig. 8 and 9).Therefore take the compounds of this invention and will not increase the cholesterol level in blood, will not also increase the content of crude fat in liver, it is to avoid suffer from the danger of angiocardiopathy and fatty liver. Another aspect of the present invention provides a kind of method for being used to improve the study and/or memory capability of subject, including formula (I) compound or its pharmaceutically acceptable salt, solvate, hydrate, enantiomter or pro-drug of effective dose are applied to the subject.

Further aspect of the present invention provides formula (I) compound or its pharmaceutically acceptable salt, solvate, hydrate, enantiomter or pro-drug and is preparing the purposes in being used to improve the study of subject and/or the medicine of memory capability.

Term " 3- hydroxy fatty acids and its derivative " used herein has the general structure of formula (I), including³- hydroxy fatty acid and its ester, salt etc..It is not inconsistent unless otherwise stated or substantially with context, term " 3- hydroxy fatty acids and its derivative " used herein generally can be with term " formula (I) compound " or " the compounds of this invention " used interchangeably.

The compounds of this invention can be obtained by a variety of methods such as the hydrolysis and alcoholysis of all kinds of polyhydroxyalkanoates（Chen and Wu, 2005), by distillation purifying, pass through FOR and GC-

MS is analyzed to identify very high purity, and the accessory substance of cell growth is endangered without double bond etc..

Term " subject " used herein, refers to any mammal, for example, mouse, rat, rabbit, dog, ox, and primate, such as monkey and people." subject, the mammal of illness, such as mammal with failure of memory or Alzheimer's, particularly people can be referred to；Not ill healthy mammal can also be referred to.It will be understood by those skilled in the art that for certain purposes, such as improving and learning into malen or improve work performance, healthy subject, which may also need to improve, to learn and/or memory capability.

Unless otherwise stated, term " pharmaceutically acceptable salt " used herein includes the nontoxic bronsted lowry acids and bases bronsted lowry addition salts of compound.Acceptable nontoxic acid-addition salts include those salt derived from known in the art organic and inorganic acid or alkali.Acid includes：For example, hydrochloric acid, phosphoric acid, sulfuric acid, acetic acid, lactic acid, butanedioic acid, citric acid, malic acid, maleic acid etc..Substantially it is that acid compound can be with various pharmaceutically acceptable alkali forming salts.It is the alkali that those form nontoxic base addition salts, i.e., available for the alkali for preparing pharmaceutically acceptable base addition salts, described salt contains the acceptable cation of pharmacology, such as, but not limited to, alkali metal or alkali salt, especially calcium, magnesium, sodium or sylvite.Suitable organic base includes, but are not limited to,

N, N- dibenzyl-ethylenediamin, chloroprocanine, choline, diethanol amine, ethylenediamine, lysine and procaine.

Unless otherwise stated, term " hydrate " used herein refers to that the compounds of this invention is further combined by non-covalent intermolecular force with a certain amount of water.

Unless otherwise stated, term " pro-drug " used herein refers to：Can be in life Thing condition is (external or internal）The derivative of the lower compound that the present invention is provided by hydrolysis, oxidation or other reactions.The example of pro-drug includes, but are not limited to, the derivative of the compounds of this invention, and the derivative includes biological hydrolyzable part, such as biological hydrolyzable acid amides, biological hydrolyzable ester or biological hydrolyzable carbamate moiety.Other examples of pro-drug include：Include-NO ,-N0₂,-ONO or-ON0₂The derivative of partial formula (I) compound of the invention.Pro-drug can typically use well known method to prepare, for example in Burger's Medicinal Chemistry and Drug Discovery, 172-178, (Manfred E. Wolff are edited 949- 982, the mat woven of fine bamboo strips five editions, 1995 and Design of Prodrugs Bundgaard are edited, Elsdvier, New York 1985) described in.

Unless otherwise old fan, the acid amides, ester or carbamate that term " biological hydrolyzable acid amides " used herein, " biological hydrolyzable ester ", " biological hydrolyzable carbamate " refer to compound respectively can be with：1) bioactivity of compound is not hindered, but can give the internal property of compound advantageous, and such as intake, action time or effect start；Or be 2) that biology is inactive, but can be using conversion in the body as biologically active cpds.The example of biological hydrolyzable ester includes, but are not limited to, rudimentary protective embankment base ester, lower acyloxyalkyl esters (such as acetoxy-methyl, Acetoxvethyl, aminocarbonyloxymethyl, valeryl epoxide Yue bases and pivaloyloxyethyl esters）, cholinester, and amido alkyl base ester (such as acetamidomethyl ester）.The example of biological hydrolyzable acid amides includes, but are not limited to, lower alkyl, alpha-amino acid amides, alkoxy amide, and alkylaminoalkylcarbonyl acid amides.The example of biological hydrolyzable carbamate includes, but are not limited to, low-grade alkylamine, substituted ethylenediamine, amino acid, hydroxy alkyl amine, and polyetheramine.

" enantiomter " of the compounds of this invention includes racemic, enantiomeric enrichment or enantiomer-pure compound.Term " enantiomer-pure " used herein, unless otherwise stated, refers to a kind of enantiomter for including compound, and other enantiomters substantially free of the compound.The compound of typical enantiomer-pure includes a kind of enantiomter for being greater than about 95% compound by weight, such as D- types or L-type enantiomter.Term " enantiomeric enrichment " used herein, unless otherwise stated, refers to include to be greater than about 50%, preferably greater than about 70%, a kind of enantiomter of more preferably greater than about 80% compound by weight.Term " racemic " used herein or " racemic modification ", unless otherwise stated, refer to do not have active mixture by what the levo form and d-isomer of equivalent were constituted.For example, the racemic modification of 3-hydroxybutyrate can be expressed as DL-³- hydroxybutyric acid. The compound or its pharmaceutically acceptable salt, solvate, hydrate, enantiomter or pro-drug of formula (I)（Active component) it can be used in its own form, but will be generally administered in the form of pharmaceutical composition, in described composition, active component is combined with pharmaceutically useful carrier.According to administering mode, described pharmaceutical composition will preferably include 0.05 to 99 wt % (percentage by weight), more preferably 0.05 to 80 wt %, more preferably 0.10 to 70 wt %, and more preferably 0.10 to 50 wt % active component, all percentage by weights are all using the weight of total composition as base.

Term " alkyl " used herein refers to the side chain and the radical of saturated aliphatic alkyl of straight chain of the carbon atom with given quantity.For example, alkyl " be defined as straight or branched with 1, the group of 2,3,4,5,6,7,8 or 9 carbon.For example, " C alkyl " particularly including methyl, ethyl, n-propyl, i-propyl, n-butyl, tert-butyl, iso-butyl, amyl group, hexyl, heptyl, octyl group, nonyl etc..

Term " cycloalkyl " used herein refers to the monocyclic radical of saturated aliphatic alkyl with given number of carbon atoms.For example, " C₃-C₉Cycloalkyl " include cyclopropyl, methyl-cyclopropyl, 2,2- dimethyl-cyclobutyls, 2- ethyI-cyclopentyls, cyclohexyl etc..

Term " alkoxy " used herein represents the protective embankment base or cycloalkyl that indicate carbon number connected by oxygen bridge." alkoxy, the thus definition including abovementioned alkyl and cycloalkyl.

Term used herein " aryl, refer to the isocyclic aryl for including 5 to 10 annular atoms.Representational example includes but is not limited to phenyl, tolyl, anthryl, fluorenyl, indenyl, pyridine radicals and naphthyl and including 5, the benzo-fused isocyclic part of 6,7,8-tetrahydronaphthalene.Isocyclic aryl can be unsubstituted or substituted.In one embodiment, isocyclic aryl is phenyl.

Term " nontoxic metal ion " used herein refers to does not have the metal ion of overt toxicity, such as, but not limited to Na for subject⁺、 K U Ca²\ Mg² Zn²⁺、 Fe²⁺、 Fe³⁺Deng.

Term " effective dose " used herein refers to being enough the amount of the biology for causing animal doctor or clinician to be found in animal or human body or the reactive compound of medical response." effective dose " of the compounds of this invention can be determined by those skilled in the art according to factors such as method of administration, the body weight of subject, age, the state of an illness.

Present invention also offers the method for preparing Yao Wu Group compounds of the present invention, it includes being mixed the compound or its pharmaceutically acceptable salt, solvate, hydrate, enantiomter or pro-drug of formula defined above (I) with pharmaceutically useful carrier.

The pharmaceutical composition can be with such as emulsifiable paste, solution, suspension, aerosol and dry powder formulations Form locally administration (for example delivering medicine to skin or lung and/or air flue)；Such as can by oral in the form of tablet, glue Nang, syrup, powder or particle Formulations for systemic administration；Or can in the form of solution or suspension parenteral；Or can be with subcutaneous administration.

The composition of the present invention can be obtained with well-known conventional carrier in the prior art by conventional method.Therefore, the composition for oral application can include for example one or more colouring agents, sweetener, flavouring and/or preservative.

Suitable pharmaceutical acceptable carrier for tablet includes：For example, inert diluent such as lactose, sodium carbonate, calcium phosphate or calcium carbonate；Granulation agent and disintegrant such as cornstarch or alginic acid；Adhesive such as starch；Lubricant such as magnesium stearate, stearic acid or talcum powder；Preservative such as ethyl p-hydroxybenzoate or propyl ester；And antioxidant, such as ascorbic acid.Tablet can not be coated or be coated to change its disintegration and absorption of the subsequent active component in intestines and stomach, or improve its stability and/or outward appearance, in either case in, the well-known method all using conventional coating agents and in the prior art.

Composition for oral application can be glutoid glue Nang or soft gelatin glue Nang forms.In glutoid glue Nang, by active component and inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin are mixed；In soft gelatin glue Nang, active component is mixed with water or oil such as peanut oil, atoleine or olive oil.

Aqueous suspension generally comprises the active component and one or more supensoid agents of fine powder form, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methyl cellulose, mosanom, polyvinylpyrrolidone, bassora gum and Arabic gum；Condensation product (such as polyoxyethylene stearate of dispersant or wetting agent such as lecithin or oxyalkylene and aliphatic acid）.Described aqueous suspension can also include one or more preservatives（Such as ethyl p-hydroxybenzoate or propyl ester), antioxidant (such as ascorbic acid), colouring agent, flavouring, and/or sweetener（Such as sucrose, saccharin or aspartame）.

Oil-based suspension can be by being suspended in vegetable oil by active component（Such as peanut oil, olive oil, sesame oil or coconut oil）Or mineral oil (such as atoleine）In prepared.The oil-based suspension can also include thickener such as beeswax, solid paraffin or cetanol.Sweetener can be added, and these materials and flavouring provide a kind of agreeable to the taste oral formulations as described above.Preservative treatment can be carried out to these compositions by adding antioxidant such as ascorbic acid.

Active component and dispersant or wetting agent, supensoid agent and one or more preservatives are generally comprised suitable for preparing the dispersible powder and particle of aqueous suspension by adding water.With these above-mentioned materials to suitable dispersant or wetting agent and supensoid agent as described above.Can be with There is other excipient such as sweetener, flavouring and colouring agent.

The pharmaceutical composition of the present invention can also be the form of oil in water emulsion.Its oil phase can be vegetable oil such as olive oil or peanut oil, or mineral oil such as atoleine, or in these materials any material mixture.Suitable emulsifying agent can be for example naturally occurring natural gum such as Arabic gum or the condensation product such as polyoxyethylene sorbitan glycan monoleate of bassora gum, naturally occurring phosphatide such as soybean lecithin, lecithin, the ester derived from aliphatic acid and hexitan or partial ester (such as sorbitan monooleate) and described partial ester and ethylene oxide.The emulsion can also include sweetener, flavouring and preservative.

Syrup and elixir can be prepared with sweetener such as glycerine, propane diols, sorbierite, aspartame or sucrose, and can also include Slow and agent, preservative, flavouring and/or colouring agent.

The pharmaceutical composition can also be the sterile aqueous or oil-based suspension form of injectable, and can be prepared according to known method with one or more suitable dispersants that have already mentioned above or wetting agent and supensoid agent.The sterile preparation of injectable can also be the sterile solution or suspension of the injectable in parenteral acceptable non-toxic diluent or solvent, for example positioned at

Solution in 1,3-BDO.

Topical formulations, such as emulsifiable paste, ointment, gel and aqueous or oily solution or suspension generally can by using conventional method well-known in the art, with it is conventional can local application excipient or diluent active component is prepared and prepared.

It comprising average diameter be that such as 30 μ ι η or lower particle splits to obtain very thin powder type that the composition being administered by insufflation, which can be, and the powder has the one or more acceptable carrier of physiology such as lactose only comprising active component or also in itself.Then, the powder for insufflation can be easily maintained at comprising in such as glue Nang of 1 to 50 mg active components, and glue Nang is used with turboinhalator (turbo-inhaler) device.

Can be conventional pressurised aerosol form for the composition by inhalation, described aerosol is arranged to active component with comprising splitting to be allocated in the form of the aerosol of thin solid or liquid droplets very much.Conventional aerosol propellants such as volatile fluorinated hydrocarbons or hydro carbons can be used and the active component with aerosol device metered amounts to distribute can be easily arranged.

It should be recognized that dosage will change with compound used, administering mode, required treatment and the state of an illness.The typical daily dose scope for having mammal to be treated to receive is per the mg of kg body weight 0.05 to 75 mg active components.For people, daily dose preferably is, for example l-10mg/kg.If desired, the daily dose can be administered in the form of fractionated dose.According to principle well-known in the art, the exact magnitude and method of administration of the active component given depend on body weight, age, sex and the treated particular condition of treated subject.

In order to which the present invention is explained in greater detail, embodiments of the invention are provided below in conjunction with accompanying drawing.These embodiments shall not be construed as limitation of the scope of the invention merely for the sake of the purpose of explanation and illustration.Embodiment 1:The preparation of ethyl 3-hydroxybutanoate adds 5 g PHB and 50 ml chloroform in round-bottomed flask.Water-bath condenser pipe is installed, and is put into oil bath pan, the slow heating stirring dissolvings of Slow.Separately take 100 ml ethanol to be put into 150 ml conical flasks, add the mixing of 2 ml concentrated sulfuric acids stirring at normal temperature, above-mentioned ethanol-concentrated sulfuric acid solution is added in the round-bottomed flask of PHB chloroformic solutions, mix.Temperature control is 80 or so.48 hours reaction time.Terminate after reaction, suction filtration removal of impurities.Filtrate is transferred in 250 ml separatory funnels, 25 ml distilled water are added, stratification collects lower floor's organic phase, adds 25 ml NaHC0₃Neutralize once, 25 ml distillation washings are hasty and careless once to be separated with separatory funnel, and organic phase anhydrous sodium sulfate drying filters out sodium sulphate.Organic phase Rotary Evaporators evaporate chloroform and drying, and Jian Ya Zheng Evaporated (110 ± 10,2900 Pa) are obtained².8 g products, pass through Fourier infrared spectrum（FTIR) it is used in conjunction with gas phase color praseodymium-mass spectrum（GC-MS) analysis determines that product is high-purity ethyl 3-hydroxybutanoate（Fig. 1, Fig. 2).Embodiment 2:The preparation of 3-hydroxybutyrate methyl esters adds 5 g PHB and 50 ml chloroforms in round-bottomed flask.Water-bath condenser pipe is installed, and is put into oil bath pan, the slow heating stirring dissolvings of Slow.Separately take 100 ml methanol to be put into 150 ml conical flasks, add the mixing of 2 ml concentrated sulfuric acids stirring at normal temperature, above-mentioned methanol-concentrated sulfuric acid solution is added in the round-bottomed flask of PHB chloroformic solutions, mix.Temperature control is 80 or so.48 hours reaction time.Terminate after reaction, suction filtration removal of impurities.Filtrate is transferred in 250 ml separatory funnels, 25 ml distilled water are added, stratification collects lower floor's organic phase, adds 25 ml NaHC0₃Neutralize once, 25 ml distilled water washed once, and be separated with separatory funnel, and organic phase anhydrous sodium sulfate drying filters out sodium sulphate.Organic phase Rotary Evaporators evaporate chloroform and drying, and vacuum distillation (80 ± 10 °C, 4800 Pa) obtains 3.0 g products, analyze true by FTIR Fixed output quota thing is 3-hydroxybutyrate methyl esters（Fig. 3).Embodiment 3:The preparation of rabbit brain cortex neural glue shield cell is taken after white rabbit cerebral tissue with sterile scissors, carefully divests the fibre compositions such as meninx and blood vessel, Hanks liquid is put into after shredding（Containing the mg/L streptomysins of 100 U/mL penicillin+100）Middle to rinse 1-2 times, in the Hanks liquid for being placed in 30-50 times of volume, piping and druming repeatedly can be made into cell suspension（Brain tissue is soft）；In suspension injection centrifuge tube, it is stored at room temperature 5-10 minutes, cell or cell mass sink naturally, the debris such as fat floats on suspension top layer, absorbs supernatant, 2-3 times repeatedly, obtain more cell component；Added into final pellets thing and contain 20 % FBS (hyclones in right amount）DMEM nutrient solutions（Dulbecco's Minimum Eagle's Medium), by 300 mesh screen filtrations, count cell and adjust cell concentration, be inoculated with blake bottle, 37,5% C0₂Culture；After cell growth is converged, with 0.²5 % Trypsin Induceds passages are handled.Embodiment 4:MTT (Thiazolyl blues）What method detection ethyl 3-hydroxybutanoate was bred to rabbit neuroglia shield cell influences the good cell of upgrowth situation（The cell that embodiment 3 is obtained, by 3 Secondary Cultures）Handled 2 minutes with pancreatin digestive juice, absorb digestive juice, added fresh medium and single cell suspension is made.Blood counting chamber detects cell quantity.It is inoculated in 96 orifice plates（ 5xl0³Individual cells/well）, 37V, 5% C0₂Cell attachment culture 24 hours.Remove nutrient solution, PBS (phosphoric acid Slow fliud flushings pH 7.2) is cleaned 2 times.Add the DMEM nutrient solutions of filtration sterilization（Containing 10 % FBS), wherein containing a series of concentration gradients（0th, 0.00025,0.0005,0.001,0.005,0.01,0.05 g/1) ethyl 3-hydroxybutanoate, each concentration does 6 parallel laboratory tests.Continue after cultivating 24 hours and 48 hours, MTT detection cytoactives.MTT (5.0 is added in 96 orifice plates per hole_δ/ 1) 1,37 °C of 10 μ incubate 4 hours.Supernatant is abandoned, DMSO (dimethyl sulfoxide (DMSO)s are added）100 μ 1, vibrate in several minutes, 30 minutes and extinction at 570 nm are read on full-automatic ELIASA, are worth.

The 0.00025-0.01 g/1 refreshing colloid of ethyl 3-hydroxybutanoate processing rabbit is after cell 24 hours, and MTT light absorption values are respectively 0.11 ± 0.01,0.09 ± 001,0.12 ± 001,0.14 ± 0.02,0.12 ± 0.02, with control group（0.06 ± 0.01) compare with significant difference（Ρ<005) (Fig. 4 a)；The 0.00025-0.01 g/1 refreshing colloid of ethyl 3-hydroxybutanoate processing rabbit is small through cell 48 Shi Hou, MTT light absorption value are respectively 016 ± 001,019 ± 0.01,0.2 0.01 and control group（0·1²± 001) compare with significant difference（Ρ<0·0⁵) (Fig. 4 b)；The propagation of rabbit Deiter's cells can be remarkably promoted by illustrating the ethyl 3-hydroxybutanoate of low concentration.Embodiment 5:Mtt assay detects the good cell of influence upgrowth situation of the 3-hydroxybutyrate methyl esters to rabbit glial cell proliferation（The cell that embodiment 3 is obtained, by 3 Secondary Cultures）Pancreatin digestive juice is handled 2 minutes, absorbs digestive juice, adds fresh medium and single cell suspension is made.Blood counting chamber detects cell quantity.It is inoculated in 96 orifice plates（5xl0³Individual cells/well）, 37,5% C0₂Cell attachment culture 24 hours.Remove nutrient solution, PBS 2 times.Add the DMEM nutrient solutions of filtration sterilization（Containing 10%FBS), wherein the 3-hydroxybutyrate methyl esters containing a series of concentration gradients (0,0.00025,0.0005,0.001,0.005,0.01,0.05 g/1), each concentration does 6 parallel laboratory tests.Continue after cultivating 24 hours and 48 hours, MTT detection cytoactives.1,37 °C of MTT (5.0g/l) 10 μ is added in 96 orifice plates per hole to incubate 4 hours.Supernatant is abandoned, DMSO lOO l are added, vibrates in several minutes, 30 minutes and light absorption value at 570 nm is read on full-automatic ELIASA.The refreshing colloid of 3-hydroxybutyrate Yue esters processing rabbit of each concentration is after cell 24 hours, the MTT light absorption values of all concentration groups（0.1³

± 0.03,015 ± 002,0.12 ± 0.01,0,12 ± 001,0.11 ± 0.01,0.10 ± 0.02) and control group（0.06 ± 0.01) compared to all with significant difference（Ρ<0·0⁵) (figure；Equally, the refreshing colloid of 3-hydroxybutyrate methyl esters processing rabbit of each concentration is through cell⁴⁸After hour, the MTT light absorption values of all concentration groups（0.15 ± 0.01,0.17 ± 0.01,018 ± 0.01,0.16 ± 0.01,0.15 ± 0.01,0.15 ± 0.01) there is significant difference compared with control group (012 ± 001)

( Ρ<0.05) (Fig. 4 b).As a result all concentration in experiment are indicated³- beta-hydroxymethyl butyrate can remarkably promote the propagation of rabbit Deiter's cells.Embodiment 6:Mtt assay detects the good cell of influence upgrowth situation of the DL-3- hydroxybutyric acids to rabbit glial cell proliferation（The cell that embodiment 3 is obtained, by 3 Secondary Cultures）Pancreatin digestive juice is handled 2 minutes, absorbs digestive juice, adds fresh medium and single cell suspension is made.Blood counting chamber detects cell quantity.It is inoculated in 96 orifice plates（5xl0³Individual cells/well）, 37 °C, 5% C0₂Cell attachment culture 24 hours.Remove nutrient solution, PBS 2 times.Add the DMEM nutrient solutions of filtration sterilization（Containing 10%FBS), wherein containing a series of dense Spend gradient（0th, 0.00025,0.0005,0.001,0.005,0.01,0.05 g/1) racemic DL-3- hydroxybutyric acids, each concentration does 6 parallel laboratory tests.Continue after cultivating 24 hours and 48 hours, MTT detection cytoactives.MTT (5.0 g/l) 10 μ are added in 96 orifice plates per hole, 37 °C incubate 4 hours.Supernatant is abandoned, the μ of DMSO 100 are added, vibrates in several minutes, 30 minutes and light absorption value at 570 nm is read on full-automatic ELIASA.0.001st, the 0.005 g/1 refreshing colloid of DL-3- hydroxybutyric acids processing rabbit is after cell 24 hours, and MTT light absorption values are respectively 0.09 ± 0.01 0.12 ± 0.03, with control group（0.06 ± 0.01) compare with significant difference (P<0.05) (Fig. 4 a);DL-3- hydroxybutyric acids handle the refreshing colloid of rabbit after cell 48 hours, only the MTT light absorption values of 0.005 g/1 concentration groups（020 ± 003) and control group（0.12 ± 0.01) compare with significant difference（Ρ<0.05) (Fig. 4 b).As a result the DL- of show lower concentration³- hydroxybutyric acid can promote the propagation of nerve cell, but be not as obvious as 3-hydroxybutyrate methyl esters and ethyl 3-hydroxybutanoate effect.Embodiment 7:The preparation of influence cell print of the fluorescence microscope home position observation ethyl 3-hydroxybutanoate to rabbit neuroglia shield Apoptosis：The good cell of upgrowth situation（The cell that embodiment 3 is obtained, by 3 Secondary Cultures）Handled 1 minute by pancreatin digestive juice, absorb digestive juice, added fresh medium and single cell suspension is made.Blood counting chamber detects cell quantity.By 0.1；3-0.1⁷Cover glass thick mm is placed in 12 orifice plates, adds Ι Ο Ο μ Ι lxlO⁵Individual/ml cell suspension； 37Ό, 5% C0₂After cell attachment culture 4 hours, add DMEM nutrient solutions of 900 μ 1 containing 2 % FBS and continue to cultivate 20 hours.Discard nutrient solution, PBS²It is secondary.1 ml is added containing 0.004 g/1³The DMEM nutrient solutions of -3-hydroxyethyl butyrate, culture⁴⁸Hour.Discard nutrient solution, the PBS washings cell of 1 ml water coolings 2 times（It is gently adherent to add and pour out liquid）.Annexin-V-FITC cell apoptosis detection kits are dyed：500 μ Binding Buffer are added (with reference to Slow fliud flushings）, the Ρ Ι (propidium iodides of Ι Ο μ Ι Annexin-V-FITC, 5 μ 1）, gently shake up, reacted 15 minutes during room temperature is dark.Cover glass is taken out, upper machine is placed on wave carrier piece（NIKON inverted microscope TE2000-E) observation.Fluorescent microscopy images show, ethyl 3-hydroxybutanoate significantly prolongs Slow and the apoptosis for suppressing nerve cell and death, the cell of control treatment lies substantially in death phase, and more cell all dyes the fluorescence that takes on a red color by PI, and small part cell is in green fluorescence by annexin dyeing（Fig. 5 a).And the cell of 0.004 g/1 ethyl 3-hydroxybutanoates processing lies substantially in the apoptosis phase, most cells are in green fluorescence, few part cell all by aimexin dyeing The fluorescence that takes on a red color is dyed by PI（Fig. 5 b).The Flow Cytometry of embodiment 8 detects 3- hydroxyl target ethyl butyrates to the rabbit Deiter's cells cycle

The preparation and dyeing of flow cytometer cell sample：The good cell of upgrowth situation（The cell that embodiment 3 is obtained, by 3 Secondary Cultures）Handled 2 minutes with pancreatin digestive juice, absorb digestive juice, added fresh medium and single cell suspension is made.Blood counting chamber detects cell quantity.It is inoculated in glass Tissue Culture Flask（5xl0⁴Individual cell/ml), 37,5% C0₂Cell attachment culture 24 hours.Remove nutrient solution, PBS (phosphoric acid Slow fliud flushings pH 7.2) is cleaned 2 times.Add the DMEM nutrient solutions of filtration sterilization（Containing 10 % FBS), wherein the ethyl 3-hydroxybutanoate containing 0.005 g/1.Trypsin digestion cell, 1 ml nutrient solutions blow even, and 1000 rpm are centrifuged 10 minutes.Supernatant discarding, PBS 2 times, 0.5 ml PBS blow even.Cell is picked up and is blown into the ethanol of the % precoolings of 5 ml 70 by 5ml syringes, and 4 °C of fixations are stayed overnight.1000 rpm, which are centrifuged, collects fixed cell, PBS 2 times for 10 minutes.0.5 ml PBS are resuspended cell and gently blow even（Prevent clasmatosis).Add 1.5 μ RNase A to final concentration of 60 g/ml, 37 digestion 30 minutes.0.25 ml PI solution is added to final concentration of 20 g/ml, lucifuge is dyed 30 minutes in water-bath.Upper machine（BECKMAN Coulter Epics XL flow cytometers）Detection.

The cell colony being collected into by software analysis flow cytometer, shows that ethyl 3-hydroxybutanoate improves S phases and G₂The cell proportion of/M phases.S phases cell number and G₂The ratio that/M phase cell numbers account for whole cell colony number is capable of the splitting degrees of reacting cells.Deiter's cells is after 0.005 g/1 ethyl 3-hydroxybutanoate processing, S phases cell number and G₂/ M phase cell numbers add 9.1% and 9.6% respectively, illustrate that ethyl 3-hydroxybutanoate significantly promotes Deiter's cells division and enters S phases and G₂/ M the phases, the splitting degree of cell is improved, so as to promote the propagation of cell（Fig. 6 a, 6b).Embodiment 9:Morris water mazes（Morris Water Maze, MWM) experiment mice rearing conditions and its packet situation

KM mouse：The g of body weight 20 ± 2;Female and male half and half；Cleaning grade.Ancestral temple is supported：Cleaning grade mouse experiment receptacle；Temperature： 25±2 °C ;Humidity： 40-60 % ;Illumination：12 hours Light and shade replaces；Drinking-water：Ultra-pure water, freely drinks；Feed：Mouse full nutrition pellet；Feeding manner：Cage.

Experiment mice packet situation is as follows：

Negative control group：11 mouse, feed, continuous 30 days naturally.

Positive controls：9 mouse, feed the acetyl-L-carnitine aqueous solution（30 mg/ g/d), continuous 30 days.

Sample experiments group：30 mouse（Each concentration group 10, totally three groups）, feed the 3- beta-hydroxymethyl butyrate aqueous solution（20th, 30,40mg/Kg/d), continuous 30 days.Embodiment 10:The change orientation navigation experiment (Place navigation) of Morris water mazes observation mouse learning behavior is used to measure the ability that mouse learns water maze.Experiment lasts 5 days, daily training 4 times（Place of entry of 4 training mouse respectively from four different quadrants enters water；If mouse did not found platform in 60 seconds, platform need to be led to, at this moment incubation period be designated as 60 seconds, every time training interval 60 seconds）, a place of entry is randomly choosed during training, mouse is put into water towards pool wall, observes and records：Mouse finds and climbed up route map and the incubation period of platform（Escape Latency, EL ) .As a result the EL of each group in embodiment 9 is shown as increasing for test number (TN) is gradually shortened, and the EL of positive controls and sample experiments group mouse is shorter than negative control group.Carry out one-way analysis of variance respectively to each group result, 30mg/kg/ days acetyl-L-carnitine groups and low concentration during the mat woven of fine bamboo strips five days（20th, 30 mg/kg.d) 3-hydroxybutyrate methyl esters group EL values be respectively 12.63 ± 4.58 seconds, 11.24 ± 3.14 seconds and 8.45 ± 2.09 seconds, with negative control group（19.55 ± 494 seconds) compare with the significance difference opposite sex（Ρ<0.05), and climb up platform route it is also simpler than negative control group.The learning ability of this explanation mouse is significantly improved.The EL values of 40 mg/kg.d 3-hydroxybutyrate methyl esters groups are 12.21 ± 3.05 seconds no difference of science of statistics compared with negative control group（P>0.05) (Fig. 7 a).

Space exploration is tested（Spatial Probe Test) it is used to measure after mouse association searching platform, to the ability of platform space position memory.Orientation navigation removes horizontal stand after testing the 5th day, and then mouse is put into water by an optional place of entry towards pool wall, and data are measured in three times：The number of times of original platform position is passed through in 60 seconds；Record mouse route map result of search platform in 60 seconds shows each concentration（20th, 30,40 mg/kg.d) 3-hydroxybutyrate methyl esters group cross-platform number of times in 60 seconds is respectively 6.07 ± 2.22>8.13 ± 2.83,5.85 ± 2.19, all compare negative control group（4.20 ± 1.16) many, wherein 30 mg/kg.d 3-hydroxybutyrate methyl esters groups are the most Substantially, and all there is significant difference（Ρ<0·0⁵) .Cross-platform number of times is 5.52 ± 1.94, slightly above negative control group, but difference is not notable in acetyl-L-carnitine group 60 seconds（Ρ>0.05) (Fig. 7 b).

The mouse of all progress Morris water maze tests is in progress memory retention test behind 48 hours that space exploration is tested（Retention Test ) .Carry out, be spaced 5 minutes in three times.Mouse inserts water from the fixed bit back to platform, is quantum clock using 60 seconds from water is entered to climbing up the time of platform as EL.Mouse is climbed up and removed after platform into cage rapidly, until next experiment.As a result the EL values for showing left-handed acetyl Rou Du Jian Group and each concentration (20,30,40mg/kg.d) 3- beta-hydroxymethyl butyrate group mouse are respectively 20.05 ± 4.25 seconds, 19.01 ± 4.41 seconds, 15.51 ± 6.17 seconds and 19.45 ± 7.52 seconds, with negative control group（28.08 ± 10.84 seconds）Compared to the significance difference opposite sex（Ρ<0.05), wherein the EL of 30 mg/kg.d 3-hydroxybutyrate methyl esters groups has highly significant otherness compared with negative control group（Ρ<001) (Fig. 7 c).30 mg/kg.d 3-hydroxybutyrate methyl esters groups and negative control group mouse natural feeding proceed memory and retain experiment after 45 days, the EL values of this group of mouse are 8.78 ± 3.21 seconds, still than negative control group (18.98 ± 7.52 seconds）Much lower, memory retention is significantly better than negative control group（Difference extremely shows chopsticks Ρ<001) (Fig. 7 d).Embodiment 11:The clean small flask of apparatus,Soxhlet's is supplied dry 2 hours by the quantitative determination of mouse liver crude fat in 103-105, and drier cooling is weighed（The weight is designated as B).Weigh a certain amount of sample（The liver organization of each group mouse in Example 9,103-105 °C of high temperature is dried to constant weight）（The weight is designated as C), grind, wrapped with filter paper, is put into extraction pipe.1/2 volume petroleum ether is added into small flask, apparatus,Soxhlet's each several part is connected,⁸Heating distillation 14-20 hours in 0 °C of water-bath.Extraction is finished, and waits petroleum ether to take out filter paper bag when fully flowing into small flask, then is flowed back once, and washing extraction is managed and continues petroleum ether liquid level in heating, pipe to be leached and do not flowed into before small flask close to siphon pipe upper end, pours out extraction pipe petrochina ether.If leaving petroleum ether in small flask, continue heating evaporation and steamed to the greatest extent to solvent.Small flask is dried 0.5 hour in 103-105'C, taking-up is placed in drier being cooled to room temperature, weighs（The weight is designated as A ").Computational methods：Crude fat content %=(A-B)/Cxl00% (A:Small flask and crude fat are weighed altogether； B:Small flask weight； C:Example weight）.As a result the crude fat content for showing negative control group mouse is 9.71 ± 1.46 %, acetyl-L-carnitine group and each concentration (20,30,40 mg/kg.d) 3-hydroxybutyrate methyl esters group The crude fat content of mouse is respectively 10.24 ± 3.35 %, 9.85 ± 2.76 %, and 10.86 ± 0.79 %, 8.00 ± 1.28 %, the content of the mouse liver crude fat of experiment each group do not have significant difference（Fig. 8).Embodiment 12:The dosing solution reagent of serum cholesterol：10 % liquor ferri trichloridis： 10g FeCl₃.6H₂0 is dissolved in phosphoric acid, is settled to 100 ml, is stored in brown bottle；Phosphorus sulphur ferron：The ml of 10 % liquor ferri trichloridis 1.5 is taken in 100 ml brown volumetric flasks, enriching sulfuric acid to scale；Cholesterol standard stock solution：The mg of cholesterol 80, is dissolved in absolute ethyl alcohol, is settled to 100 ml;Cholesterol standard liquid：Liquid storage is diluted 10 times with absolute ethyl alcohol, this titer contains 0.08 mg cholesterol per ml.

Test Bu Sudden：The L of each group mice serum 75 is placed in centrifuge tube in Example 9, first plus after 0.3 ml absolute ethyl alcohols shake up, then adds the ml of absolute ethyl alcohol 1.5 to shake up, 3000 rpm centrifuge 5 min after 10 min, take supernatant standby（Ethanol extract）.Separately centrifuge tube is taken to number, blank tube：0.75 ml absolute ethyl alcohols；Standard pipe：0.75 ml cholesterol standard liquids；Sample cell：0.75 ml ethanol extracts.Each pipe all adds the ml of phosphorus sulphur ferron 0.75 and shakes hook, after 10 minutes, spectrophotometric measurement OD₅₆₍₎.As a result the serum cholesterol content for showing negative control group mouse is 0.154 ± 0.03 %, acetyl-L-carnitine group and each concentration（20th, 30,40 mg/kg.d) serum cholesterol content of 3-hydroxybutyrate methyl esters group mouse is respectively 0.154 ± 0.05 %, 0125 ± 003 %, 0158 ± 0.02 %, 0.102 ± 0.01 %, the content of the wherein mice serum cholesterol of 40mg/kg.d 3- beta-hydroxymethyl butyrates group is substantially less than negative control group, but other each experimental groups do not have significant difference then compared with control group（Fig. 9).The level of cholesterol in serum has obvious relation with coronary heart disease illness rate, if the content rise of cholesterol in serum, can undoubtedly increase the probability that mammal obtains coronary heart disease.What we used in testing³- beta-hydroxymethyl butyrate and acetyl-L-carnitine do not increase the serum cholesterol content of mouse, therefore animal will not be caused to obtain the danger of coronary heart disease.Embodiment 13:Each group mouse neck in embodiment 9 is evaded position and put to death by myeloid cell count, every mouse takes 2 femurs, every femur goes out bone marrow cell with the % acetums of 10 ml 3, the cell number of 4 block plaids is counted on hemacytometer, gained cell number is multiplied by 2.5x100 000, as 1 femur Bonemarrow nucleated cells number.Experimental result shows that the bonemarrow nucleated cells number of acetyl-L-carnitine group every femur of mouse is 8125 ± 1.24 χ 10⁶It is individual, 20 mg/kg.d, 30 mg/kg.d and 40 mg/kg.d³The bonemarrow nucleated cells number of-beta-hydroxymethyl butyrate group every femur of mouse is 85 ± 089,8.95 ± 122,87 ± 106 χ 10⁶It is individual, and the bonemarrow nucleated cells number of every femur of negative control group mouse is only⁵·⁹⁷⁵±1·³⁹ <10^δIndividual, the bonemarrow nucleated cells number of acetyl-L-carnitine group and each concentration 3-hydroxybutyrate methyl esters group every femur of mouse has notable difference compared with negative control group（Ρ<001) (Figure 10).Embodiment 14:The preparation of mouse cranial glia cell takes newborn 1 day Bal/c mouse, execution of beheading using etherization and aseptically.Take whole mouse brain and be stored under freezing conditions in sterile D-Hanks liquid（Containing the mg/L streptomysins of 50 U/mL penicillin+50）.The fibre compositions such as meninx and blood vessel are carefully divested with sterile scissors under anatomical lens, by brain tissue be transferred to aseptic plastic culture it is sub- in, add 6 ml and contain 20 % hyclones through 37 °C of preheatings（FBS DMEM nutrient solutions).It is 15 mm to be shredded brain tissue with aseptic operation knife³The fritter of left and right simultaneously passes through the membrane filtration in 0.05 mm apertures.The cell being filtrated to get is centrifuged 10 minutes under 1200 rpm.Abandon after supernatant and add the DMEM nutrient solutions resuspension containing 20 % FBS.Above-mentioned centrifugation step is repeated, the cell being finally collected into is suspended in the DMEM nutrient solutions containing 20 % FBS, cell count simultaneously adjusts cell concentration, is inoculated with blake bottle, 37O,⁵% C0₂Culture.After cell growth is converged, handled with 0.25 % Trypsin Induceds passage.Embodiment 15:Immunocytochemical method visitor determines the good cell of mouse cranial glia cell growth condition（The cell that embodiment 14 is obtained, by 3 Secondary Cultures）Handled 2 minutes with pancreatin digestive juice, absorb digestive juice, added fresh medium and single cell suspension is made.Blood counting chamber detects cell quantity.It is inoculated in 96 orifice plates（5xl0³Individual cells/well）, 37,5% C0₂Nutrient solution is changed in cell attachment culture after 24 hours, continue to cultivate 48 hours.Absorb after nutrient solution, with 0.1 M PBS (phosphoric acid Slow fliud flushing ρ Η 7²) cleaning culture plate 2 times, 4 % paraformaldehyde solutions fix cell 30 minutes.Fixer is removed, culture plate is cleaned 2 times with 0.1 M PBS solutions.By detecting whether cell contains GFAP（GFAP the identification of Deiter's cells) is carried out.Primary antibodies are anti-using rabbit-anti GFAP Body, with 1:100 dilution proportions are in the PBS solution containing 0.3 % Triton X-100 and 5 % sheep blood serums.Secondary antibody uses goat-anti rabbit Cy3- IgG antibodies, with 1:30 dilution proportions are in containing 0.³In % Triton X-100 PBS solution.Primary antibodies are 4.Reaction overnight under the conditions of C, with 0.1 M PBSs after reaction；Secondary antibody is reacted 2 hours at room temperature, with 0.1 M PBSs after reaction.Nucleus is redyed using DAPI, is dyed 30 minutes at room temperature；PBS three times.Under fluorescence inverted microscope（NIKON Elipse TE2000) observation Immuncytochemical detection result（Multiplication factor： 10 x 20 ) .The fluorescence micrograph result randomly selected shows that Primary antibodies are combined with GFAP, and the secondary antibody with Cy3 fluorescence radiation groups is combined with Primary antibodies, and red fluorescence is can observe under fluorescence inverted microscope（Cy3 sends fluorescence）, nucleus dyed by DAPI, sends green fluorescence（Figure 11).Immunocytochemistry result proves that the cell separated in embodiment 14 is mouse cranial glia cell.Embodiment 16:Flow Cytometry detection 3-HBA sodium, DL-3- Sodium γ-Hydroxybutrates and

What 3-hydroxybutyrate methyl esters suppressed to mouse glial cell apoptosis influences the good cell of upgrowth situation（The cell that embodiment 14 is obtained, by 3 Secondary Cultures）Handled 2 minutes with pancreatin digestive juice, absorb digestive juice, added fresh medium and single cell suspension is made.Blood counting chamber detects cell quantity, is inoculated in 6 orifice plates（1 x10^sIndividual cells/well）.Negative control group, and 3-HBA sodium, DL-3- Sodium γ-Hydroxybutrates and 3-hydroxybutyrate methyl esters treatment group are set respectively（Concentration is 10 mM).Use DMEM+20%FBS nutrient solutions, 37.(, 5% C0₂Cell attachment culture 24 hours, absorbs nutrient solution, and PBS (phosphoric acid Slow fliud flushings pH 7.2) is cleaned 2 times, after adding the culture of serum-free DMEM nutrient solutions 24 hours, respectively to D-³- Sodium γ-Hydroxybutrate, DL-³10 mM D-3- Qiang Ji Ding Suan Satisfied, 10 mM DL-3- Sodium γ-Hydroxybutrates and 10 mM 3-hydroxybutyrates methyl esters are added in-Sodium γ-Hydroxybutrate and 3-hydroxybutyrate methyl esters treatment group to continue to cultivate 48 hours.

Cell is dyed using Annexin-V-FITC cell apoptosis detection kits and Flow cytometry is carried out：Trypsin digestion cell, 1 ml nutrient solutions blow hook, and 1000 rpm are centrifuged 10 minutes.Supernatant discarding, PBS 2 times, 0.5 ml PBS blow even.Cell is picked up and is blown into the ethanol of the % precoolings of 5 ml 70 by 5ml syringes, and 4 fixations are stayed overnight.1000 rpm, which are centrifuged, collects fixed cell, PBS 2 times for 10 minutes.0.5 ml PBS are resuspended cell and gently blow even（Prevent clasmatosis).The RNase A of 1.5 μ 1 to final concentration of 60 g/ml are added, 37 °C digest 30 minutes；0.25 ml PI solution is added to final concentration of 20 g/inl, water-bath Middle lucifuge is dyed 30 minutes；Upper machine（BECKMAN Coulter Epics XL flow cytometers) detection.

Flow Cytometry testing result is shown：The Apoptosis ratio of negative control group is 94.3%, the Apoptosis ratio of 3-HBA sodium, DL-3- Sodium γ-Hydroxybutrates and 3-hydroxybutyrate methyl esters treatment group is respectively (Figure 12 a of 84.3 %, 86.1% and 87.9%, 12b, 12c, 12d), the Apoptosis ratio of 3-HBA sodium, DL-3- Sodium γ-Hydroxybutrates and 3-hydroxybutyrate methyl esters treatment group has reduction than negative control group（Figure 13).Embodiment 17:Influence cell culture condition and the control group setting of fluorescence microscope home position observation 3-HBA sodium, DL-3- Sodium γ-Hydroxybutrates and 3-hydroxybutyrate methyl esters to mouse neuroglia shield Apoptosis are identical with embodiment 16.6 orifice plates after culture are cleaned 3 times with PBS solution, according to the explanation of DAPI reaction kits, add 100 DAPI Binding Buffer into culture plate respectively (with reference to Slow fliud flushings）With 5 μ DAPI (2- (4-amidinophenyl) -6-indolecarbamidine dihydrochloride) dye liquor.Dyeing removes dye liquor after terminating, PBS solution is cleaned 3 times, Tissue Culture Plate is observed using fluorescence inverted microscope（NIKON Elipse fluorescence inverted microscope TE2000).The fluorescence micrograph result randomly selected is shown, the nucleus of apoptotic cell can be dyed by DAPI and send blue-fluorescence, 3-HBA sodium, DL-3- Sodium γ-Hydroxybutrates and 3-hydroxybutyrate methyl esters treatment group are compared with negative control group, apoptotic cell number has been reduced, and wherein 3-hydroxybutyrate methyl esters treatment group apoptotic cell number is considerably less than control group（Figure 14).Embodiment 18:The good cell of the influence upgrowth situation of calcium imaging method detection 3-HBA sodium, DL-3- Sodium γ-Hydroxybutrates and 3-hydroxybutyrate methyl esters to mouse Deiter's cells intracellular free calcium level（The cell that embodiment 14 is obtained, by 3 Secondary Cultures）Handled 1 minute by pancreatin digestive juice, absorb digestive juice, added fresh medium and single cell suspension is made；Blood counting chamber detects cell quantity；By 0.1：3-0.17 mm thickness passes through polylysine（Poly-L-lysine) coated cover glass is placed in 6 orifice plates, and 1x10 is added into culture plate^sIndividual/ml cell suspension and DMEM+20%FBS nutrient solutions, 5% C0₂Cell attachment culture 24 hours.Control group sets identical with embodiment 16.Remove after nutrient solution, add under the conditions of 2 mM calcium ions specific stain fura-4/AM, 37 °C and dye 20 Minute；Generic physiological solution（ΝΡΜ, 45 mM NaCl, 5 mM KC1, 1.8 mM CaCl₂, 0.8 mM MgCl₂, 10 mM glucose, and 10 mM Hepes pH 7.4) cleaning；3-HBA sodium, DL-3- Sodium γ-Hydroxybutrates and 3-hydroxybutyrate methyl ester solution are prepared with NPM；Three kinds of solution respectively with 1 ml/min VELOCITY DIFFUSION by cell, and by control solution flow rate control solution concentration (10 mM 3-HBAs sodium, 10 mM DL-3- Sodium γ-Hydroxybutrates and 10 mM 3-hydroxybutyrate methyl esters）.Calcium imaging results are recorded using laser confocal microscope (Zeiss LSM 510).

Calcium image checking result shows that 3-HBA sodium, DL-3- Sodium γ-Hydroxybutrates and 3- beta-hydroxymethyl butyrates are responsible for the unexpected rise of cell intracellular free calcium level, has statistical significant difference compared with negative control group（P<0.05), raising amount is respectively that Sodium γ-Hydroxybutrate treatment group improves 60 units, and Sodium γ-Hydroxybutrate treatment group improves 120 units, and 3-hydroxybutyrate methyl esters treatment group improves 160 units（Figure 15).In addition, compared with DL-3- Sodium γ-Hydroxybutrates and 3-HBA sodium, the activity of 3-hydroxybutyrate methyl esters is only the above two one thousandth, thus with more obvious action（Figure 15 b).In addition, the raising of intracellular calcium concentration has corresponding relation with 3-hydroxybutyrate methyl acetate concentrations, 10 mM 3-hydroxybutyrates methyl esters can cause intracellular free calcium level to improve 240 units, and 5 mM 3-hydroxybutyrates methyl esters can only cause intracellular free calcium level to improve about 100 units（Figure 16).It may be concluded that influence of the 3-hydroxybutyrate methyl esters to intracellular free calcium level has concentration dependent.

Cause the elevated mechanism of intracellular free calcium level for research 3-HBA sodium, DL-3- Sodium γ-Hydroxybutrates and 3-hydroxybutyrate methyl esters, study whether the increased calcium ion of intracellular derives from extracellular NPM solution, or source and the release in cellular calcium storehouse, respectively using containing Ca²⁺And Mg²⁺NPM and do not eat the NPM of both cations and tested.As a result show, using containing Ca²⁺And Mg²⁺NPM when, 5 mM, 10 mM 3-HBAs sodium and 5 mM, 10 mM DL-3- Sodium γ-Hydroxybutrates are responsible for intracellular free calcium level and raised suddenly：Compared with negative control, 5 mM 3-HBA sodium（Referred to as (D) -3-HBNa or D-3- HBNa), at 69.7 seconds, calcium ion concentration maximum incrementss are 111 units, 5 mM DL-3- Sodium γ-Hydroxybutrates（Referred to as（DL) -3-HBNa or DL-3-HBNa), at 59.2 seconds, calcium ion concentration maximum incrementss are 142.5 units, 10 mM 3-HBA sodium, at 57.4 seconds, calcium ion concentration maximum incrementss are 96 units, 10 mM 3-HBA sodium, 61.⁵During the second, calcium ion concentration maximum incrementss are 1⁵³.4 unit（Figure 17 a).It can draw, the effect of DL-3- Sodium γ-Hydroxybutrates is better than 3-HBA sodium, and with significant difference (P<0.05), the various concentrations of material of the same race, its action effect no significant difference（ P<0.05 ). Using not containing Ca²⁺And Mg²⁺NPM when, 5mM, 10 mM 3-HBAs sodium and 5 mM, 10 mM DL-3- Sodium γ-Hydroxybutrates are equally responsible for intracellular free calcium level and raised suddenly：Compared with negative control, 5 mM 3-HBA sodium, at 53.3 seconds, calcium ion concentration maximum incrementss are 6³.4 unit, 5 mM DL-3- Sodium γ-Hydroxybutrates, at 36 seconds, calcium ion concentration maximum incrementss are³6.⁵Unit, 10 mM 3-HBA sodium, at 73.8 seconds, calcium ion concentration maximum incrementss are 91 units, 10 mM DL-3- Sodium γ-Hydroxybutrates, 7⁵·⁹During the second, calcium ion concentration maximum incrementss are 54.1 units（Figure 17 b).Therefore it can draw, using not containing Ca²⁺And M_g ²⁺NPM when, two kinds of materials cause intracellular free calcium level it is elevated effect declined.

With reference to above-mentioned analysis, it is concluded that, 3-HBA sodium, DL-3- Sodium γ-Hydroxybutrates and 3-hydroxybutyrate methyl esters can not only be by promoting extracellular flow of calcium ions to cause intracellular calcium ion to raise, also can be by causing the release in cellular calcium storehouse to raise intracellular free calcium level.

For further research 3-HBA sodium, DL-3- Sodium γ-Hydroxybutrates and 3-hydroxybutyrate methyl esters（Referred to as M (D) -3-HB or M-3-HB) cause the elevated mechanism of intracellular free calcium level, from calcium ion analog Nitredipine, striving property restrains calcium channel unexpectedly.As a result show, add after 10 μ Μ Nitredipine, 3-HBA sodium, DL-3- Sodium γ-Hydroxybutrates and intracellular free calcium level caused by 3-hydroxybutyrate methyl esters raise phenomenon and are obviously reduced respectively, caused by 10 mM3- beta-hydroxymethyl butyrates calcium ion concentration rise maximum drop to 120 units by 250 units, caused by 3 mM 3-HBA sodium calcium ion concentration rise maximum by⁷0 unit drops to 42 units, 3 mM DL-³Calcium ion concentration rise maximum drops to 4 by 90 units caused by-Sodium γ-Hydroxybutrate⁵.²Unit（Fig. 1,18b, 18c).As a result show, striving property inhibits the valtage-gated type calcium channel of L-type to Nitredipine unexpectedly, cause the reduction of three kinds of material effects.It was therefore concluded that, three kinds of materials are by influenceing the valtage-gated type calcium channel of L-type so as to further cause intracellular calcium concentration to raise.

Bibliography involved by herein, including patent document, scientific paper, publication etc., entire contents are included herein by reference.

It should be appreciated that without departing from the spirit and scope of the present invention, one of ordinary skill in the art can make various changes and improvement to it in form and details, and these are all considered to fall into protection scope of the present invention. Bibliography：

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Claims (1)

1. ', ' ", "

   1. a kind of pharmaceutical composition for being used to improve the study and/or memory capability of subject, it is included：

   As formula (I) compound of active component,

   0

   0H— C —CH₂— C— 0R】

   (I)

   Or its pharmaceutically acceptable salt, solvate, hydrate, enantiomter or pro-drug, wherein,

   Selected from by H, C_rC₉Alkyl, C₃-C₉Cycloalkyl, the group that constitutes of aryl and nontoxic metal ion；

   R₂Selected from by H, C C₉Alkyl, C₃-C₉Cycloalkyl, C_rC₉Alkoxy and aryl constitute Group；And

   Pharmaceutically acceptable carrier.

   2. pharmaceutical composition according to claim 1, wherein selected from by H, C C₇Alkyl and nontoxic metal ion constitute group.

   3. Yao Wu Group compounds according to claim 2, wherein selected from by H, C C₅Alkyl and nontoxic metal ion constitute group.

   4. pharmaceutical composition according to claim 3, wherein selected from by H,（ C₃Alkyl and nontoxic metal ion constitute group.

   5. pharmaceutical composition according to claim 4, wherein the nontoxic metal ion is Na+, K+ and Ca²⁺。

   6. according to the pharmaceutical composition of any one of claim 1-5, wherein R₂Selected from by H, c₇Alkyl, c_rc₇Alkoxy and aryl constitute group.

   7. pharmaceutical composition according to claim 6, wherein R₂Selected from by H, pit foundation, c c₅Alkoxy constitute group.

   8. Yao Wu Group compounds according to claim 7, wherein R₂Selected from by H, C C₃Alkyl, c c₃Alkoxy constitute Group.

   9. pharmaceutical composition according to claim 1, wherein selected from by H, C₂Alkyl, Na⁺The group constituted with K+, and R₂The free C of ease₂Alkyl constitute group.

   10. according to the pharmaceutical composition of claim 1, wherein formula (I) compound is selected from the group being made up of following member：

   3-hydroxybutyrate Yue esters；

   Ethyl 3-hydroxybutanoate；

   3-hydroxybutyrate；

   3-HBA sodium；

   DL-3- Sodium γ-Hydroxybutrates.

   Π are according to the pharmaceutical composition of any one of claim 1-10, and wherein described pharmaceutical composition is for treating the memory relevant disease in subject.

   12.-kind be used for improve subject study and/or memory capability method, including to the subject apply effective dose formula (I) compound,

   OH— CH— CH₂— C— OR

   R₂

   (I)

   Or its pharmaceutically acceptable salt, solvate, hydrate, enantiomter or pro-drug, wherein,

   Selected from by H,（Wide ^ alkyl, C₃-C₉Cycloalkyl, the group that constitutes of aryl and nontoxic metal ion；

   R₂Selected from by H,（：Wide ^ alkyl, C C₉Cycloalkyl, C C₉Alkoxy and aryl constitute group.

   13. the method that 12 are required according to wooden fork profit, wherein selected from by H, C₇Alkyl and nontoxic metal ion constitute group.

   14. method according to claim 13, wherein R are selected from the group being made up of H, C Cs alkyl and nontoxic metal ion.

   15. method according to claim 14, wherein selected from by H,（^-(₃Alkyl and nontoxic metal ion constitute group.

   16. method according to claim 15, wherein the nontoxic metal ion is Na+, K⁺ And Ca²⁺。

   17. according to the method for any one of claim 12-16, wherein R₂Selected from by H, CVC₇Alkyl, c c₇Alkoxy and aryl constitute group.

   18. method according to claim 17, wherein R₂Selected from by H,（：Wide ^ alkyl, c_sProtective embankment epoxide constitute group.

   19. method according to claim 18, wherein R₂Selected from by H, alkyl, C C₃Alkoxy constitute group.

   20. method according to claim 12, wherein selected from by H,（^ alkyl, Na⁺And K⁺The group of composition, and R₂Selected from by C₂Alkyl constitute group.

   Method according to claim 12, wherein formula 21. (I) compound is selected from the group being made up of following member：

   3-hydroxybutyrate methyl esters；

   Ethyl 3-hydroxybutanoate；

   3-hydroxybutyrate；

   3-HBA sodium；

   DL-3- Sodium γ-Hydroxybutrates.

   22. according to the method for any one of claim 12-21, wherein methods described is used to treat the memory relevant disease in subject.

   Formula 23. (I) compound

   OH— CH— C¾— C— OR,

   R,

   (I)

   Or its pharmaceutically acceptable salt, solvate, hydrate, enantiomter or pro-drug are preparing the purposes in being used to improve the study of subject and/or the medicine of memory capability, wherein,

   Selected from by H,（Extensively (₉Alkyl, C₃-C₉Cycloalkyl, the group that constitutes of aryl and nontoxic metal ion；

   R₂Selected from by H, alkyl, C₃-C₉Ring protective embankment base, C₉Alkoxy and aryl constitute group；And Pharmaceutically acceptable carrier.

   24. purposes according to claim 23, wherein selected from by H, C_rC₇Alkyl and nontoxic metal ion constitute group.

   25. purposes according to claim 24, wherein selected from by H,（：The group that ^ alkyl and nontoxic metal ion is constituted.

   26. purposes according to claim 25, wherein selected from by H,（ C₃Alkyl and nontoxic metal ion constitute group.

   27. purposes according to claim 26, wherein the nontoxic metal ion is Na+, K⁺And Ca²⁺。

   28. according to the purposes of any one of claim 23-27, wherein R₂Selected from by H, C₇Alkyl, alkoxy and aryl constitute group.

   29. purposes according to claim 28, wherein R₂Selected from by H,（^- (^ alkyl, C₅Alkoxy constitute group.

   30. purposes according to claim 29, wherein selected from by H,（^-<₃Alkyl, c c₃Alkoxy constitute group.

   31. purposes according to claim 23, wherein selected from by H, C C₂Alkyl, the group that constitutes of Na+ and K+, and R₂Selected from by C C₂Alkyl constitute group.

   Purposes according to claim 23, wherein formula 32. (I) compound is selected from the group being made up of following member：

   3-hydroxybutyrate methyl esters；

   Ethyl 3-hydroxybutanoate；

   3-hydroxybutyrate；

   3-HBA sodium；

   DL-3- Sodium γ-Hydroxybutrates.

   33. according to the purposes of any one of claim 23-32, wherein the medicine is used to treat the memory relevant disease in subject.