CN1660125A - New capability and application of soybean isoflavone for enhancing and improving memory - Google Patents

Abstract

An application of soybean isoflavone in proparing the medicine or health-care food for improving memory power and treating the dysmnesia is disclosed.

Description

Soybean isoflavone strengthens and improves the new capability and the application thereof of memory

Technical field

The present invention relates to the new capability of crude soya bean isoflavonoid, particularly the application in strengthening and improve memory and preparation improvement memory and treatment dysmnesia disease medicament preparation belongs to the estrogenic application of natural plants.

Background technology

Phytoestrogen (Phytotoestrogen, be called for short PE) be meant that a class is present in the plant, can in conjunction with and activate mammal and the mankind estrogen receptor (Estrogen receptor ER), thereby has estrogen-like or the active nonsteroidal compound of estrogen antagonist sample.It extensively is present in as in 300 various plants such as beans, frumentum, fruit, vegetable, because the kind of plant, the factor differences such as growing environment in the place of production, so the content of contained phytoestrogen and kind difference are very big in the different plant.At present, to its definition and classification, scholars' view is different, and some scholar thinks and should be divided into four classes to it: isoflavone (Isoflavones), flavonoid, lignan (Lignans) and Coumarins (Coumestans).Wherein isoflavone mainly is distributed in the pulse family, and content is abundant especially in Semen sojae atricolor, so claim ' soybean isoflavone '.The lignan class comprises enterolactone and enterodiol, mainly is present in Semen Lini, corn, Semen Viciae fabae, the fruits and vegetables; Coumarins mainly is present in the plant of germination stage.

1, the research brief history of soybean isoflavone.

Soybean isoflavone is the phytoestrogen that research is more and effect is comparatively clear and definite at present, has plurality kinds of health care and pharmacological effect, and the mankind are had obvious health care effect, is subjected to many scientists' attention in the world.Soybean isoflavone is called as ' Semen sojae atricolor miracle ' (Soy Miracle) in the U.S..So understand the research brief history of soybean isoflavone, can better help to understand the biological effect of soybean isoflavone to human body, as shown in table 1:

The research brief history of table 1 soybean isoflavone

Age	Study brief history
		1931	Find to contain in the Semen sojae atricolor isoflavone (Walz, 1931) of high level;
Nineteen forty-one	The effective ingredient genistein (genistein) of soybean isoflavone be separated for the first time (Walter, 1941);
		?1940s	1 finds to contain the herbage relevant with the infertility of sheep (Bennetts etc., 1946) of high-load isoflavone; 2 Isoflavone and estrogen receptor (ER) have certain affinity;
1984	1 finds that the interior intestinal bacterium of human body plays an important role for absorption and the metabolism of isoflavone; Disease very beneficial (Axelson etc., 1984 that the biological activity of 2 Soy isoflavone causes for estrogen deficiency; Setchell etc., 1984);
		1993	The proof soybean isoflavone is (Coward etc., 1993) that the form with β-glucosides exists at nature;
Nineteen ninety-five	After the β of soybean isoflavone-glucosides form is absorbed by body, in the effect of jejunum via beta-glycosidase, make it to change into bioactive aglycon composition is arranged, and further produce its metabolite (Joannou etc., 1995) in distal small intestine;
		1998	King and Bursill have studied the pharmacokinetics (King ﹠ Bursill, 1998) of soybean isoflavone;

2, the classification of soybean isoflavone and chemical constitution.

Soybean isoflavone (Soy isoflavones is called for short SI) is the typical phytoestrogen that a class is extracted from Semen sojae atricolor, is the heterocycle phenolic compound.It mainly is distributed in the cotyledon and plumular axis of soybean seed, and kind skin content is less.Find that at present soybean isoflavone has 12 kinds, is divided into the aglycon (Aglycon) of free type and glucosides (Glucoside) two classes of conjunction type.Aglycon accounts for 2%～3% of total amount, comprises genistein or claims that (structure is 5 to genistein for Genistein, G, 7,4-tri hydroxy isoflavone), daidzein (Daidzein, D, structure is 7,4-dihydroxy isoflavone) and Glycitein (Glycetein, Gly).Glucosides accounts for 97%～98% of total amount, mainly with the malonyl genistin (6 "-O-malonylgenistin), the malonyl daiazi (6 "-O-malonyldaidzin), the form of genistin (Genistin) and daiazi (Daidzin) exists.Because the content of Glycitein (Glycetein) is very little in the Semen sojae atricolor, so what play biological effect in the soybean isoflavone is Genistein, Daidzein and glucosides thereof.The structure of glycosides and aglycon is as follows in the soybean isoflavone: wherein, G represents glucose.

The estrogenic chemical constitution of soybean isoflavone and mammal has surprising similarity, and the phenol ring in the structure is and the bonded key structure of estrogen receptor position.If metabolite equol (equol) and estrogenic certain monomer of soybean isoflavone are put together, can see that these two kinds of structures can be superimposed, and terminal hydroxyl almost can be overlapping.

3, the physiological function of soybean isoflavone.

(1) soybean isoflavone and growth of animal.

There are some researches show, gave rat skin lower injection soybean isoflavone 30mg/kg in continuous 16 days, the male rat daily gain improves 14%, the material anharmonic ratio descends 12%, back leg flesh weighs and the total rna content of back leg flesh significantly increases, the serum urea nitrogen level significantly reduces, and shows that muscle protein gathers increase in the body, and the myocyte increases.

Finding also that in addition the isoflavone phytoestrogen has facilitation to the growth of pig, but have sex difference, generally is to be better than female to male facilitation effect; Can promote proteinosis and influence the growth of broiler; Can influence GH gene transcription process and regulate the duckling growth; Also can pass through the elevating blood testosterone levels, the microbial metabolism that improves cud promotes the growth of ruminant.

The growth promoting function of isoflavone phytoestrogen also depends on the physiological status of its using dosage and animal.Heavy dose of and the long-time isoflavone plant estrogen that uses may suppress to grow.Zhou Yuchuan etc. (2002) discover that to Shaoxing duck after soybean isoflavone used a period of time, its growth promoting function disappeared, T in the serum ₄, the horizontal significance of IGF-1 descends, T3, GH and hypophysis GH mRNA do not have significant difference statistically, the hormonal readiness when being the soybean isoflavone growth promotion with short term tests is opposite tendency substantially.Its reason may be to occur the desensitization phenomenon behind the soybean isoflavone long duration of action, and promptly body descends to the sensitivity of soybean isoflavone.

The soybean isoflavone phytoestrogen promotes the mechanism of action of growth of animal: the one, directly act on the estrogen receptor of muscle cell.Studies show that the soybean isoflavone phytoestrogen can significantly promote the accumulation of rat and cock muscle protein, the myocyte is increased, total rna content raises; The 2nd, play a role indirectly by the concentration that influences endogenous hormone relevant in the body, enzymatic activity, metabolite with growth.A large amount of experiments show that the soybean isoflavone phytoestrogen can change the endocrinosity of body, improve Serum GH, prolactin antagonist, T ₃, testosterone and beta-endorphin the level affects growth; It reduces the somatostatin level by combining with estradiol receptor such as hypothalamus, hypophysis, thereby improves the GH level, promotes growth of animal.On the other hand, isoflavone phytoestrogen with estrogen activity combines estradiol receptor at hypothalamus with estradiol competitiveness, the performance estrogenic antagonist, the negative feedback that has weakened estradiol is regulated, improve interstitialcellstimulating hormone (ICSH) and short follicle estrogen concentrations in the blood plasma, cause the increase of endogenous testosterone, promote the buck protein synthesis, and then promote growth of animal by testosterone.

(2) soybean isoflavone and antioxidation, lipid metabolism and cardiovascular disease.

Human experimentation shows the edible beans material that contains high concentration isoflavone (about 132mg/d) of postmenopausal women, and visible LDLc significantly reduces.Scheiber etc. (2001) are 12 weeks of 42 postmenopausal women's continuous supplementation isoflavone (60mg/d), find that the LDLc oxidization time postpones 9.3%, shows that it has antioxidation, thereby can play a protective role to cardiovascular system.

Zooperal result of study has shown that also soybean isoflavone has antioxidation in body.Mary etc. (1999) make in the Semen sojae atricolor soybean isoflavone reduce to 0.97mg/kg by 9.41mg/kg with the ethanol elution method, and with this soybean protein with without the soybean protein that eluting the is handled adolescence monkey of feeding, the result confirms that actuating the arteries and veins hardener without T-CHOL (TC), low density lipoprotein, LDL (LDL), very low density lipoprotein (VLDL) (VLDL) and the apolipoprotein B (apoB) etc. of eluting treated animal all significantly descends, and high density lipoprotein (HDL), ApoA arteriosclerosis compositions such as (apoA) then obviously increase; Coronary atherosclerosis damage, peripheral blood vessel tremulous pulse lipidization and damage thereof are obviously reduced.Crouse etc. (1999) once estimated the influence of peas protein to blood fat in the blood plasma and lipoprotein concentration.The amount of institute's isoflavone-containing is respectively 3,27,37 and 62mg in the peas protein, and with casein in contrast, the peas protein that the result contains the 62mg isoflavone can significantly reduce TC, and increases HDLc, is the concentration dependence.Effect for reducing fat for soybean isoflavone, domestic also have relevant research, Yan Xianghua etc. (2000) observe the influence of soybean isoflavone to rat fat level and body endoperoxides state, the result shows: the plasma triglyceride level that soybean isoflavone significantly suppresses due to the high lipid food raises, and the body endoperoxide level rising that the feed high lipid food causes is had remarkable antagonism.Mainly show the free radical level that reduces in liver and the cardiac muscle, activity of glutathione peroxidase in rising liver superoxide dismutase and liver and the cardiac muscle reduces total antioxidation product (LPO) content in serum and liver, cardiac muscle and the aorta.This explanation soybean isoflavone has the reduction effect to plasma triglyceride, and can improve body endoperoxides abnormal state due to the high fat, alleviates the peroxide injury to body.

Experiment in vitro research further confirms ANTIOXIDATIVE EFFECT OF SOYBEAN ISOFLAVONES.With the non-oxidizability of external LDL model of oxidation research soybean isoflavone, find that genistein suppresses by Cu by prolonging connection diene formation time ²⁺The LDL oxidation that participates in.The genistein using dosage is 5 μ mol/L, and be (54.1 ± 5.1) minute action time; Matched group is (107.1 ± 1.6) minute, and this inhibitory action is certain dosage-reaction relation, and dosage is the peroxidization that the genistein of 200 μ mol/L can significantly suppress LDL.Daidezin has inhibitory action external to the HMG-CoA reductase.Infer that Daidezin also has similar effect in vivo, can reduce the formation of body inner cholesterol, blood fat reducing concentration.Kerry etc. (1998) also draw similar conclusion by in vitro study, and genistein can prolong by Cu ²⁺Inductive conjugated diene formation time and be dose dependent; By prolonging the formation time of malonaldehyde (MDA), reduce the concentration that MDA forms, the relative electrophoretic mobility (REM) that reduces LDL suppresses by the inductive oxidation of superoxide radical; Mix back LDL antioxidant activity strengthens with genistein.A large amount of experiment in vitro also show the atherosclerosis correlation factor of soybean isoflavone to other, as platelet, lymphocyte, mononuclear cell etc. benign influence are arranged all.50～300 μ mol/L genisteins energy Trombin inhibiting and platelet activating factor induced platelet aggregation, suppressing platelet activating factor stimulates thromboxane A ₂(TXA ₂) and the release of 5-HT.Genistein and Daidezin all can suppress TXA ₂With the platelet activation that collagen protein causes, reduce deposition and the gathering of platelet at the arterial injury position.Soybean isoflavone is more responsive to the endotheliocyte effect, 5 μ mol/L just can suppress the propagation of Medulla Bovis seu Bubali capillary endothelial cell, 10 μ mol/L can suppress the diastole of the inductive Canis familiaris L. coronary artery of the vascular permeability factor, can also suppress the generation of aortic smooth muscle cell nitric oxide (NO) during high concentration, suppress the motion of mononuclear phagocyte.These factor combineds effect are oozed out the arterial wall of damage and the inflammation degree alleviates, and suppress atherosclerosis (AS) and form.

Mechanism about the effect of soybean isoflavone angiocardiopathy preventing, Anthony etc. (1998) think have following several: 1. ldl receptor raises: soybean isoflavone can make ldl receptor generation forward regulate, make the active increase of ldl receptor, thereby promote the removing of cholesterol; 2. anti-oxidation characteristics: in vitro study shows that soybean protein has the LDL of reduction particle volume and prevents the effect of LDL over oxidation, and isoflavone can reduce the deposition of LDL granule on coronary arterial wall by this effect, thereby reduces atherosis incidence rate.There are some researches show that the easier LDL that incorporates into of the soybean isoflavone of esterification is to bring into play its antioxidation; 3. the propagation that suppresses vascular smooth muscle cell: cell culture finds that the genistein of 5 μ mol/L can reduce the activity of basilar fibers somatomedin (BFGF) and plasminogen activator, thereby the inhibition smooth muscle cell proliferation, and the propagation of this cell has important effect in atherosis the development; 4. thrombosis effect: active oxygen plays an important role in blood vessel injury and arterial thrombus.Cellular oxidation thing H ₂O ₂Participate in platelet activation, this process need tyrosine kinase participates in, genistein generates by the inhibitory action blocking platelet source property somatomedin (PDGF) to tyrosine kinase, and then cause biologically active pdgf to reduce, its accumulation on blood vessel is reduced, stop the generation of atherosclerosis (AS).

(3) soybean isoflavone and osteoporosis.

Confirmed that now the particularly contained isoflavone of soybean protein can increase bone formation, reduced bone and heavily absorb.For inquiring into the mechanism of action that phytoestrogen reduces bone loss, improves bone density; Fanti etc. (1998) are to SD rats underwent removal ovary postoperative genistein supplementation (GST); the result shows; compare with matched group; GST can obviously protect the removal ovary Os Mus to lose, and tectology shows, remarkable bone density improving; increase the osteoblast number, improve the bone formation rate.Biochemical measurement in serum and urine shows that Bone Gla protein and total deoxypyridinoline (Dpd) content all are significantly higher than matched group in the additional GST serum, and Bone Gla protein and Dpd are a little more than matched group in the urine.This result shows that phytoestrogen is different with estrogen to the protective effect of bone, and it is by stimulating bone formation but not the absorption again of inhibition bone.Find that phytoestrogen can make lumbar spine bmd that the rising of significance is arranged in the research of Potter etc. (1998) to 66 menopausal women bone densities variations.The women take respectively contain 56, peas protein and the casein in contrast and the defatted milk powder of 90mg isoflavone, took for 24 weeks continuously, adopt dual energy X-ray absorptiometry to measure spinal bone density, the result compares with matched group, and the peas protein group women who takes isoflavone-containing 90mg is at vertebra L ₁～L ₄Joint bone density and bone mineral content have significance rising (2%).Isoflavone mainly is incorporated into estrogen beta receptor in the osseous tissue, by suppressing osteoclast activity and bone resorption process, is maintained in the dynamic equilibrium between osteocyte and osteoclast, thereby effectively prevents bone loss.Ishida etc. (1998) report rat oral gavage daidzein and after genistein 50mg/kg/d4 week, can effectively prevent femur density, intensity, the grey decline that heavily reaches the calcium phosphorus content that removal ovary causes.

4, the metabolism of soybean isoflavone phytoestrogen and mechanism of action.

Soybean isoflavone in Semen sojae atricolor usually there not to be active glucosides combining form to exist, have only after the glucosidase of intestinal microbial population decomposes, form non-glucosides type isoflavone, through further metabolism, isoflavone can be converted into ethyl phenol and active stronger equol (equol) and oxygen demethyl angolensin (O-demethylangolesin, ODMA), lignan is converted into enteral fat (enterolactone) and enterodiol (enterodiol), these metabolite structures all to estradiol (17 β-estradiol) similar.The conjunction type isoflavone that is decomposed by intestinal microbial population is not difficult for being absorbed, and directly goes into intestinal from bile secretion and excretes; The isoflavone of desaccharide glycosides type can combine again with glucosides and form the active chemical compound of inanimate object through enterohepatic circulation.

Phytoestrogen all has the phenol ring, in human body, can be incorporated into two kinds of estrogen receptor (estrogen receptor, ER) ER α and ER β, but with the affinity of receptor far below estrogen, bring into play faint estrogen effect.Data shows, the part phytoestrogen is higher than ER α to the affinity of ER β, its estrogen-like effects may be beta mediated by ER-, because ER α is different in the expression of different tissues and cell with ER β, thereby phytoestrogen has different tissue specificities and brings into play different effects with the endogenous estrogen estradiol.Phytoestrogen also can combine ER with estradiol competitiveness and produce the estrogen antagonism simultaneously, so have the amphicheirality, bring into play which kind of effect in vivo actually, then depend primarily on the quantity and the type of the endogenous estrogen state and the estrogen receptor of its dosage and body.Generally, combine ER with the estrogen competition during low dosage and show as estrogenic antagonist; In produce certain estrogen activity during dosage; Can activate during high dose because of oestrogen deficiencies and fail activatory ER, produce the estrogen enhancement effect.

Soybean isoflavone is the phytoestrogen of a quasi-representative, also shows two kinds of important biologic activity from function: estrogen activity and estrogen antagonist activity.As for showing as the estrogen receptor level which kind of activity depends primarily on its local concentration, endogenous estrogen content and histoorgan.The estrogen activity of isoflavone is very weak, generally only is estradiol (E ₂) one thousandth to ten thousand/, the estrogen antagonist activity then mainly shows under the paranormal morbid state of body estrogen level.

Although the physiological action to soybean isoflavone has had extensive studies, and it is many similar to find that its physiological action and estrogenic effect have.And relevant soybean isoflavone is not seen document announcement as yet to the influence of learning and memory.But relevant estrogen influences the more existing document announcements of the research of cognitive function.Recent study shows, has estrogen receptor widely in the brain, and as on the male neuron of brain basal forebrain trk C estradiol receptor being arranged, hippocampal pyramidal cell has found that also estradiol receptor (ER) exists.In the central nervous system, estrogen not only acts on the interior neural circuit relevant with reproduction of brain influences reproductive process, but also acts on the neural circuit relevant with cognitive function, influences learning and memory.The neuron dendron plays an important role in the information processing of brain processing and memory form, and the Hippocampus dendritic spine induce and formation and estrogen closely related.The In vitro culture hippocampal cell, the more untreated remarkable increase of dendritic spine density through the estrogen processing, behind the adult female Mus removal ovary, the tree-shaped spinous process quantity of Hippocampus CA1 district pyramidal cell and density descend about 30%, after giving the estrogen processing, make this minimizing be blocked very soon even be reversed, shown that estrogen plays the formation of tree-shaped spinous process to induce and keep effect.Up-to-date research further is deep into the relevant district of brain function of estrogen action: Hippocampus is the brain district relevant with memory that generally acknowledges, estrogen can act on this zone, at the M of oophorectomize Mus by acetylcholine ₂Increase the adhesion of CA1 district nmda receptor, improve working memory power, the estrogen scalable hippocampus of low dosage improves the spatial memory ability.And find in the test of the out-phase in rat, the estrogen of low and high dose is scalable striatum district also, improve the non-space memory ability, Keenan is in the random experiment that the postmenopausal women carries out, adopt the meterological method to carry out the cognition assessment, find that prefrontal cortex and neural loop thereof play a major role in the cognition of estrogen regulating improves.In addition, postmenopausal women's decrease in estrogen may increase the susceptibility of basal forebrain and hippocampus cholinergic neuron; Simultaneously, apo E in the blood plasma (APOE) level raises, and APOE and amyloid beta combined effect have directly promoted the formation of amyloid fibrils, causes the density of synapse to reduce and the neurotoxicity increase.Because low estrogen, the neurotransmitter secretion of hypothalamus, limbic system also changes, and dopamine tension force descends, the epinephrine increased activity, the 5-hydroxy tryptamine secretion is impaired, these change except that causing the early stage symptom of menopause, and can cause nervous symptoms and hypomnesis late period, causes AD (Zhao Yuhong, Huang Ren, the Zhuqi cutting edge of a knife or a sword. estrogen is to the effect of brain. foreign medical science molecular biology fascicle, 2003,25 (2): 112～115).Estrin treatment can increase spay rat cerebral tissue choline transferring enzyme (ChAT) activity and K ⁺The release of the acetylcholine that stimulates, prolong the survival of cholinergic neuron, improve learning and memory function (Gibbs RB, Hashash A, Johnson DA.Effects of estrogen onpotassium-stimulated acetylcholine release in the hippocampus and overlyingcontex of adult rats[J] .Brain Res, 1997,749:143 ~ 146).Old women is after giving estrin treatment, and word memory aspect is apparently higher than non-estrin treatment matched group.Estrogen promotes the mechanism of memory to be owing to increase the result (Zhao Gang of Hippocampus, parahippocampal gyrus, temporal lobe and frontal lobe neuronal activity, Cai Dingfang. estrogen and brain function. foreign medical science, physiology, pathology science and clinical fascicle, 2001,21 (6): 424～426).Soybean isoflavone is structurally similar to estrogen, and has estrogenic many identity functions.But whether soybean isoflavone has the function of improving memory, and still need further research and discussion also are the purposes that the present invention researchs and solves.

Summary of the invention

The objective of the invention is to solve the deficiency that prior art exists, and provide a kind of soybean isoflavone to strengthen and improve the purposes of memory, have chemical constitution similar and physiological function according to soybean isoflavone to estrogen, and the closely-related mechanism of estrogen and memory function, research by experiment, the proof soybean isoflavone has the usefulness of improving learning and memory, makes soybean isoflavone increase a kind of new purposes, and has industrial utilization.

The present invention is achieved by the following technical solutions.

Soybean isoflavone is the phytoestrogen that a class is extracted from Semen sojae atricolor, be the heterocycle phenolic compound, what play biological effect mainly is genistein, daidzein and glucosides thereof, and the structure of glucosides and aglycon is as follows in the soybean isoflavone: wherein, G represents glucose

Soybean isoflavone has the performance that strengthens and improve memory; Activity or content to the higher mammal neurotransmitter are influential, make the active reduction of acetylcholinesterase in hippocampal tissue and the blood (AChE); Excitatory amino acid class neurotransmitter glutamate (Glu) in cerebral cortex and the hippocampal tissue is raise with aspartic acid (Asp) content, inhibitory aminoacid class neurotransmitter glycine (Gly) content is reduced.

Soybean isoflavone has the performance of memory reinforcing, is with soybean isoflavone normal mouse to be tested; Soybean isoflavone has the performance of improving memory, is with soybean isoflavone hyperlipidemia model mice and Chronic Aluminum poisoning model mice to be tested, and the dosage of soybean isoflavone is 30mg/kgbw-120mg/kgbw, wherein dose effect the best of 60mg/kgbw.

Soybean isoflavone is active influential to the higher mammal neurotransmitter, makes the active reduction of acetylcholinesterase in hippocampal tissue and the blood (AChE), and the dosage of its soybean isoflavone is 30-120mg/kgbw.

Soybean isoflavone can make the excitatory amino acid class neurotransmitter glutamate (Glu) in mouse brain cortex and the hippocampal tissue raise with aspartic acid (Asp) content, inhibitory aminoacid class neurotransmitter glycine (Gly) content is reduced, and the dosage of its soybean isoflavone is 30-120mg/kgbw.

The present invention also comprises application and soybean isoflavone compounds the application in preparation improvement memory and treatment dysmnesia disease medicament preparation of soybean isoflavone compounds in preparation hypermnesis health product.Described dysmnesia disease comprises organism metabolism obstacle, the dysmnesia that chemical compound is poisoned and year factor of waiting for a long time causes.In useful in preparing drug formulations or health product, the soybean isoflavone compounds uses separately and with a kind of as in the complex of soybean isoflavone compounds.

Beneficial effect of the present invention: the inventor is through continuous 3 years research, find that soybean isoflavone all is improved to some extent to normal, hyperlipidemia dysmnesia model and poisoning by aluminum obstacle mouse memory ability, can think that soybean isoflavone has the function that strengthens and improve memory.This result can be applicable to improve the clinical medicine practice of memory, is used to develop the medicine and the functional health care product of hypermnesis, has potential economic and social benefit.

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The specific embodiment

The present invention is by influence and the mechanism thereof of three groups of different experimental mouse proof soybean isoflavone to learning and memory, and first group is the normal growth mice; Second group is the hyperlipidemia model mice; The 3rd group is Chronic Aluminum poisoning model mice.

The learning and memory index adopts x ²Check; Other data experiment result and experimental data adopt means standard deviation, and (x ± s) expression handles with SAS 6.12 statistical analysis softwares.With P＜0.05 is the difference significance.

Embodiment 1: soybean isoflavone is to the experiment of normal growth mice

1. experimental arrangement

A. experimental animal feeding and packet transaction: experimental session is respectively organized mice and is put quiet environment, and sub-cage rearing is raised with normal diet, and the drinking-water of freely ingesting, drinking water are tap water.Change drinking-water and feedstuff every day, keep animal living environment ventilation and sanitation and hygiene.(12 ± 2) hour illumination on daytime, indoor temperature (20 ± 2) ℃, relative humidity 40%～60%.Mice is divided into 4 groups at random by sex after adapt to feeding a week: blank group (Control group), the basic, normal, high dosage group of soybean isoflavone (SI amount be respectively 30,60,120mg/kgbw).Every group 15.The soybean isoflavone powder is dissolved in 0.5% sodium carboxymethyl cellulose (CMC-Na) is mixed with desired concn, irritate stomach, 0.3ml/ days/only.Matched group is irritated stomach equivalent 0.5%CMC-Na solution.Continuous irrigation stomach one month.

B. detect index: memory ability; Acetylcholinesterase in Hippocampus and the blood (AChE) activity; Amino acid neurotransmitter (Asp, Glu and Gly) content in the cerebral tissue (Hippocampus and cerebral cortex).

The mensuration of memory ability adopts darkness avoidance test: test set is for keeping away camera bellows, finish preceding 7 days at raising experiment, train, mice is put into bright chamber, record mice extremity all enter before the darkroom in the time of bright chamber stop and are called into hole incubation period, after entering the darkroom, energising (voltage 50v) at once, the mice of electric shock flees back bright chamber, after 30 seconds, mice is taken out, promptly carry out repetition measurement in the 7th day after the training, and again mice was put into bright chamber, and observe hole incubation period.Memory ability is mainly according to repetition measurement and the preclinical variation of when training, when repetition measurement, observed 5 minutes, the mice that does not enter in 5 minutes during repetition measurement is " memory is good ", advance during repetition measurement and advance the preclinical difference in hole when the hole deducts training incubation period, greater than 30 seconds persons was " remembering medium ", and difference is " poor memory " less than 30 seconds persons.

2. experimental result

A. soybean isoflavone is to the influence of experiment mice ability of learning and memory:

The trend that soybean isoflavone processed group experiment mice ability of learning and memory is improved; High, in and low dosage soybean isoflavone group compare middle dosage group effect apparent in view (seeing Table 2) with matched group.

Table 2 soybean isoflavone is to the influence of experiment mice ability of learning and memory

Group	Number of animals	Memory is good			Remember medium			Poor memory
										Number of elements	????％		Number of elements	?????％		Number of elements	????％
		Matched group	???14	???5	??35.71		??7	???50.00										??2	??14.29
Low dosage SI group	???15									???7	??46.67		??7	???46.67		??1	??6.67
		Middle dosage SI group	???15	???8	??53.33		??7	???46.67										??0	??0.00
High dose SI group	???14									???5	??35.71		??8	???57.14		??1	??7.14

B. soybean isoflavone is to the active influence of acetylcholinesterase (AChE) in experiment mice blood and the hippocampal tissue:

Consult the result who respectively organizes experiment mice hippocampal tissue and blood cholinesterase vigor shown in the table 3, as can be seen from the table, compare with matched group, the soybean isoflavone group hippocampal tissue cholinesterase activity of 3 dosage all reduces, significant difference (P＜0.05).Wherein, low, middle dosage group is compared with high dose group, and difference reaches significant level (P＜0.05), and difference not significantly (P＞0.05) between middle dosage group and the low dose group.In addition, compare with matched group, the soybean isoflavone group blood cholinesterase vigor of 3 dosage all reduces, and difference reaches significant level (P＜0.05).High, middle dosage group and low dose group differences reach significant level (P＜0.05), and difference not significantly (P＞0.05) on the statistics between middle dosage group and the high dose group.

Table 3 soybean isoflavone is to the influence of experiment mice hippocampal tissue and whole blood cholinesterase activity

Group	Number of animals	Cholinesterase activity
				Blood	Hippocampus
		Matched group	???14			????14.16±1.45a	????321.55±15.12a
Low dosage SI group	???15			????10.97±1.14c	????273.68±12.65c
		Middle dosage SI group	???15			????12.16±0.84b	????263.72±16.20c
High dose SI group	???14			????12.18±0.82b	????292.09±7.76b

Annotate: with alphabetical different persons behind the column of figure, represent significant difference (P＜0.05) in the table, alphabetical identical person, expression difference is remarkable (p＞0.05) not

C. soybean isoflavone is to the influence of content of amino acids neurotransmitter in the experiment mice cerebral tissue:

Consult shown in the table 4, as can be seen, in the experiment mice cerebral cortex, soybean isoflavone experimental mice Asp content all is higher than matched group, significant difference (P＜0.05); Glu content also all is higher than matched group, and significant difference (P＜0.05) is learned check difference not significantly (P＞0.05) by statistics between low, high dose group; Gly content all is lower than matched group, and each group difference is (P＜0.05) significantly.

In the experiment mice hippocampal tissue, soybean isoflavone experimental mice Asp content all is higher than matched group, significant difference (P＜0.05), and middle and high dosage group Asp content difference not significantly (P＞0.05); Glu content all is higher than matched group, and each group difference all reaches significant level (P＜0.05); Gly content all is lower than matched group, significant difference (P＜0.05).Wherein difference does not reach significant level (P＞0.05) between low dose group and the high dose group.

Content of amino acids neurotransmitter (μ molg in the table 4 soybean isoflavone mouse brain tissue ^-1Wet tissue) influence

Group	Number of animals	Cortex				Hippocampus
									????Asp	?????Glu	????Gly	????Asp	????Glu	????Gly
		Matched group	???14	???0.501 ???± ???0.017d		???1.055 ???± ???0.041c	??0.568 ??± ??0.010a	??0.482 ??± ??0.017c							??1.076 ??± ??0.041d	??0.837 ??± ??0.024a
Low dosage SI group	???15								???0.565 ???± ???0.010c	???1.286 ???± ???0.023b	??0.517 ??± ??0.014c	??0.562 ??± ??0.022b	??1.488 ??± ??0.033b	??0.635 ??± ??0.030b
		Middle dosage SI group	???15	???0.623 ???± ???0.016a		???1.353 ???± ???0.021a	??0.440 ??± ??0.005d	??0.712 ??± ??0.028a							??1.545 ??± ??0.043a	??0.559 ??± ??0.029c
High dose SI group	???14								???0.580 ???± ???0.011b	???1.275 ???± ???0.120b	??0.537 ??± ??0.010b	??0.633 ??± ??0.050a	??1.401 ??± ??0.083c	??0.625 ??± ??0.016b

Annotate: with alphabetical different persons behind the column of figure, represent significant difference (P＜0.05) in the table, alphabetical identical person, expression difference is remarkable (p＞0.05) not

3. to the explanation and the explanation of experimental result

The physiologically active ingredient of Semen sojae atricolor research has in recent years become the focus that common people gaze at.Wherein, soybean isoflavone is one of functional components of gazing in the Semen sojae atricolor.Many domestic and international researchs have shown the effect of soybean isoflavone at aspects such as anti-curing oncoma, cardiovascular disease and osteoporosises, but the relevant soybean isoflavone of Shang Weijian influences the data of aspect to learning and memory function.Soybean isoflavone group in the present invention's experiment and matched group relatively show and improve trend preferably.This experimental result also shows, soybean isoflavone makes the active reduction of acetylcholinesterase in mice hippocampal tissue and the blood (AChE), and excitatory amino acid glutamic acid (Glu) raises with aspartic acid (Asp) content in cerebral cortex and the hippocampal tissue, and inhibitory aminoacid glycine (Gly) content then reduces; Prompting, soybean isoflavone may be relevant with the metabolism of blood and hippocampal tissue acetylcholine esterase and amino acid neurotransmitter to the improvement of memory function.

Learning and memory is the Premium Features of brain, and cerebral cortex is the important structure very relevant with learning and memory with Hippocampus.In the learning and memory forming process, often be accompanied by the biochemical variation of complicated nervous physiology, relate to multiple material.Think that at present learning and memory and cholinergic nerve of centrum phylogenetic relationship are very close, the central cholinergic system path is one of major avenues of approach that constitutes the conduction of study memory signal.Interrelated data is thought, acetylcholine (ACh) is the essential neurotransmitter of memory formation and the physiological foundation of longterm memory, and it can make neuron excited lastingly, the bunchiness discharge, the storage that helps remembering, the finishing of learning and memory task often are accompanied by the variation of acetyl choline content in the brain.Increase the decomposition of the synthetic of ACh or minimizing ACh in right amount, all can improve ability of learning and memory.And acetylcholine is synthetic by choline acetylase, and acetylcholinesterase (AChE) decomposes, thus in the brain choline acetylase and acetylcholine esterase (AChE) but vigor indirect reaction brain in the metabolic situation of acetylcholine.Existing discovering because hippocampal tissue cholinesterase activity rising in the brain is decomposed the interior acetylcholine of brain and quickened, and then reduces ability of learning and memory; And the inhibition cholinesterase activity can reduce dementia incidence rate, improves ability of learning and memory.In the present invention's experiment, soybean isoflavone makes acetylcholine esterase active reduction in the mice Hippocampus, and the prompting soybean isoflavone can be regulated the metabolism of Hippocampus acetylcholine, and in fact, the formation of Hippocampus and learning and memory is closely related.Therefore can think that soybean isoflavone can to keep the content of acetylcholine in these Hippocampus, guarantee that laboratory animal has ability of learning and memory preferably by suppressing the Hippocampus acetylcholine esterase active.In addition, in the present invention's experiment, soybean isoflavone also can suppress the activity of acetylcholine esterase in the mice peripheral blood, and the prompting soybean isoflavone can influence the metabolism of acetylcholine in the peripheral blood equally.Acetylcholine is as a kind of distribution peripheral neurotransmitter very widely, and the activity of internal organs and skeletal muscle is had regulating action very widely.And the method for testing of laboratory animal study memory is mainly evaluated with the avoidance pattern that connects with motion, and it is relevant to illustrate that therefore soybean isoflavone influence the change of the same learning and memory function with laboratory animal of the cholinesterase activity of peripheral blood.

Aminoacid such as Asp, Glu and Gly are central neurotransmitters.Asp and Glu are the higher excitatory amino acid class neurotransmitteies of mammal brain intensive amount; Gly then mainly plays the inhibition effect the central nervous system.Think that now it is the objective indicator that memory formed and consolidated neuron physiological activity in the process that cerebral hippocampus long time journey strengthens.Studies show that, the formation of rat LTP behind the learning training is relevant with the rising of the outer Glu concentration of brain inner cell, after discharging, the Glu mediator depends on the activation of N-methyl D-aspartic acid (NMDA) receptor, the formation of inducing LTP, thus behavior is regulated to learning and memory.And discover that in addition in the cerebral tissue such as the striatum of naturally-aged animal, cortical area and hippocampus, exicitatory/amino acid neurotransmitter Asp and Glu level obviously reduce, and the inhibitory aminoacid neurotransmitter levels obviously raises.In brain districts such as cerebral cortex and Hippocampus, Gly is the allosteric instrumentality of nmda receptor/passage, and the effect of excitatory amino acid is had regulating action.In this research, after soybean isoflavone is handled, excitatory amino acid Asp and Glu content raise and the reduction of inhibitory aminoacid Gly content in mouse brain cortex and the Hippocampus, the memory ability of mice improves simultaneously, and the prompting soybean isoflavone is relevant with its raising memory ability to the metabolic influence of amino acid neurotransmitter.

Embodiment 2: soybean isoflavone is to the experiment of hyperlipidemia model mice

1. experimental arrangement

A. prepare high lipid food:

Mice high lipid food prescription (Qi Chen chief editor): 10% Adeps Sus domestica, 10% yolk powder, 0.5% sodium cholate, 1% cholesterol, 78.5% normal feedstuff, normal feedstuff is provided by Test Animal Centre, Academy of Military Medical Sciences, P.L.A, is full price solid particle feedstuff.

B. experimental animal feeding and packet transaction:

Experiment mice is divided into 4 groups at random after adaptability is fed a week: 1 normal feedstuff matched group; 2 high lipid food model control group; 3 low dosage soybean isoflavone group: high lipid food+SI (50mg/kgbw); 4 high dose soybean isoflavone group: high lipid food+SI (100mg/kgbw).Every group 16, male and female half and half.The soybean isoflavone powder is dissolved in 0.5% sodium carboxymethyl cellulose (CMC-Na) is mixed with desired concn, irritate stomach, 0.3ml/ days/only.Normal feedstuff matched group and hyperlipidemia model matched group are all irritated stomach equivalent 0.5%CMC-Na solution.Continuous irrigation stomach one month.Experimental session is respectively organized mice and is put quiet environment, sub-cage rearing, and the drinking-water of freely ingesting, drinking water are tap water.Change drinking-water and feedstuff every day, keep animal living environment ventilation and sanitation and hygiene.(12 ± 2) hour illumination on daytime, indoor temperature (20 ± 2) ℃, relative humidity 40%～60%.Observe ordinary circumstances such as animal appearance (hair luster, brightness, sparse degree), expression (mental status, state of consciousness, reaction sensitivity), activity, appetite every day.Per 15 days weighing the weight of animals of experimental session also calculate the weight increase amount.

C. detect index:

Memory ability; Serum total cholesterol (TC), triglyceride (TG) and beta lipoprotein content; Superoxide dismutase in the serum (SOD) vigor; Acetylcholinesterase (AChE) activity in blood and the cerebral tissue (Hippocampus and cerebral cortex); Amino acid neurotransmitter (Asp, Glu and Gly) content in the cerebral tissue (Hippocampus and cerebral cortex).

2. experimental result

A. soybean isoflavone is to the influence of experiment mice memory ability

The assay method of memory ability is with embodiment 1

Consult table 5 as can be seen, hyperlipidemia model group experiment mice memory ability significantly is lower than matched group (P＜0.05); Low, high dose soybean isoflavone processed group all is significantly higher than hyperlipidemia model group (P＜0.05), but difference not significantly (P＞0.05) between low, the high dose soybean isoflavone processed group.

Table 5 soybean isoflavone is to the influence of experiment mice ability of learning and memory

Group	Number of animals	Memory is good			Remember medium			Poor memory
										Number of elements	?????％		Number of elements	?????％		Number of elements	?????％
		Matched group b	???16	???11	????68.75		??4	????25.00										???1	????6.25
The hyperlipidemia model group	???16									???3	????18.75		??3	????18.75		???10	????62.50
		High fat+low dosage SI group b	???16	???12	????75.00		??4	????25.00										???0	????0.00
High fat+high dose SI group b	???16									???10	????62.50		??5	????31.25		???1	????6.25

Annotate: with alphabetical different persons behind the column of figure, represent significant difference (P＜0.05) in the table; The identical person of letter, expression difference is remarkable (P＞0.05) not

B. soybean isoflavone is to the influence of experiment mice blood fat situation

Consult shown in the table 6, the comparison of hyperlipidemia model group and matched group blood fat situation, serum total cholesterol (TC), triglyceride (TG) and beta lipoprotein content significantly raise, and difference all reaches significant level (P＜0.05); Compare with the hyperlipidemia model group, soybean isoflavone is low, high dose processed group mice serum T-CHOL, triglyceride and beta lipoprotein content all significantly reduce (P＜0.05).

Table 6 soybean isoflavone is to the influence of experiment mice blood fat situation

Group	Number of animals	T-CHOL (TC)	Triglyceride (TG)	Beta lipoprotein
					Matched group	???16	??122.500±18.797b	??147.143±16.799b	??1.494±0.173d
The hyperlipidemia model group	???16	??171.711±25.559a	??171.429±12.218a	??2.711±0.235a
					High fat+low dosage SI group	???16	??97.500±22.061c	??121.429±18.263c	??2.032±0.130b
High fat+high dose SI group	???16	??107.738±24.335bc	??143.929±11.112b	??1.851±0.266c

Annotate: with alphabetical different persons behind the column of figure, represent significant difference (P＜0.05) in the table; The identical person of letter, expression difference is remarkable (P＞0.05) not

C. soybean isoflavone is to the active influence of superoxide dismutase (SOD) in the experiment mice serum:

Consult shown in the table 7, compare, active significantly descend (P＜0.05) of SOD in the hyperlipidemia model group mice serum with matched group; Behind the feed soybean isoflavone, activity of SOD in serum all significantly raises (P＜0.05), but difference not significantly (P＞0.05) between the soybean isoflavone processed group of low, high dose.

Table 7 soybean isoflavone is to the active influence of SOD in the experiment mice serum

Group	Number of animals	SOD vigor (U/ml)
			Matched group	?????16	?????1.322±0.261b
The hyperlipidemia model group	?????16	?????0.981±0.288c
			High fat+low dosage SI group	?????16	?????1.679±0.233a
High fat+high dose SI group	?????16	?????1.650±0.333a

Annotate: with alphabetical different persons behind the column of figure, represent that each group difference has significance (P＜0.05) in the table; Otherwise, represent that then difference do not have significance (P＞0.05)

D. soybean isoflavone is to the active influence of acetylcholinesterase (AChE) in experiment mice cerebral tissue and the blood:

Consult the active result of acetylcholinesterase (AChE) in experiment mice cerebral tissue shown in the table 8 and the blood.In the mice hippocampal tissue, hyperlipidemia model group cholinesterase activity is significantly higher than matched group (P＜0.05); The activity of soybean isoflavone processed group significantly is lower than hyperlipidemia model group (P＜0.05), but difference not significantly (P＞0.05) between low, the high dose soybean isoflavone processed group.In mouse brain cortex, cholinesterase activity relatively raises to some extent, but does not reach significant difference (P＞0.05) on the statistics in hyperlipidemia model group and the matched group blood; Soybean isoflavone processed group and model group differences be (P＜0.05) significantly; Difference not significantly (P＞0.05) between low, the high dose soybean isoflavone processed group.With matched group relatively, cholinesterase activity significantly raise (P＜0.05) in the hyperlipidemia model group mouse blood; But behind the soybean isoflavone of feeding, cholinesterase activity significantly reduces (P＜0.05), and wherein the effect of low dosage soybean isoflavone group is the most obvious.

Table 8 soybean isoflavone is to the influence of acetylcholine esterase active in experiment mice cerebral tissue and the blood

Group	Number of animals	Cholinesterase activity
					Hippocampus	Cortex	Blood
		Matched group	???16	??489.43±73.765b				??510.222±87.289ba	??5.521±0.715b
The hyperlipidemia model group	???16				??614.63±83.641a	??603.556±98.540a	??6.544±0.812a
		High fat+low dosage SI group	???16	??352.84±95.772c				??423.111±84.035c	??3.532±0.606c
High fat+high dose SI group	???16				??404.065±84.795cb	??360.889±96.394bc	??5.128±0.528b

Annotate: with alphabetical different persons behind the column of figure, represent significant difference (P＜0.05) in the table; Otherwise, then represent difference not remarkable (P＞0.05)

E. soybean isoflavone is to the influence of amino acid neurotransmitter in the experiment mice cerebral tissue (Asp, Glu and Gly) content:

Consult the situation of change of cortex content of amino acids neurotransmitter in experiment mice Hippocampus shown in the table 9 and the tissue.In Hippocampus, to compare with matched group, hyperlipidemia model group excitatory amino acid Asp and Glu content all reduce, significant difference (P＜0.05); Soybean isoflavone is low, high dose group all is significantly higher than the hyperlipidemia model group (P＜0.05).The Gly content difference of each group all reaches significance level (P＜0.05), and model group and soybean isoflavone group all are higher than matched group.In cortex, for Asp, each group difference all reaches significant difference (P＜0.05); The hyperlipidemia model group is lower than matched group; Low, high dose soybean isoflavone group all is higher than the hyperlipidemia model group.For Glu, hyperlipidemia model group content significantly is lower than soybean isoflavone processed group (P＜0.05), and all the other each group differences are remarkable (P＞0.05) not.For Gly, hyperlipidemia model group content significantly is lower than matched group (P＜0.05), and the soybean isoflavone processed group is significantly higher than hyperlipidemia model group (P＜0.05); Difference not significantly (P＞0.05) between low, the high dose soybean isoflavone group.

Table 9 soybean isoflavone is to content of amino acids neurotransmitter (μ molg in the mouse brain tissue ^-1Wet tissue) influence

Group	Number of animals	Cortex				Hippocampus
									????Asp	???Glu	??Gly	??Asp	????Glu	?????Gly
		Matched group	???16	???0.518 ???± ???0.031c		??1.263 ??± ??0.142a	??0.853 ??± ??0.038a	??0.536 ??± ??0.031a							???1.000 ???± ???0.081a	???0.417 ???± ???0.020d
The hyperlipidemia model group	???16								???0.434 ???± ???0.012d	??1.099 ??± ??0.038b	??0.836 ??± ??0.023b	??0.436 ??± ??0.016c	???0.845 ???± ???0.025c	???0.500 ???± ???0.015c
		High fat+low dosage SI group	???16	???0.631 ???± ???0.019a		??1.265 ??± ??0.055a	??0.985 ??± ??0.047c	??0.484 ??± ??0.012b							???0.907 ???± ???0.029b	???0.556 ???± ???0.025a
High fat+high dose SI group	???16								???0.544 ???± ???0.024b	??1.202 ??± ??0.091a	??0.942 ??± ??0.019c	??0.530 ??± ??0.022a	???1.032 ???± ???0.033a	???0.524 ???± ???0.017b

Annotate: with alphabetical different persons behind the column of figure, represent significant difference (P＜0.05) in the table; Otherwise, then represent difference not remarkable (P＞0.05)

3. to the explanation and the explanation of experimental result

SI can improve the memory function that causes because of hyperlipidemia and reduce.Blood lipid level raises and can cause cerebral arteries and the big cerebral blood flow of the impaired thin reduction of capillary endothelial cell function, makes the brain metabolism impaired, thereby has reduced arteriocerebral nutrition and the oxygen supply should increase cognitive dysfunction and dull-witted danger; Also can directly influence the neuronal degeneration relevant simultaneously with cognitive dysfunction.From medical science clinically, the hyperlipemic patients among the old people has higher ratio, and the senile dementia patient also has higher ratio in the old people simultaneously, prompting hyperlipidemia and dull-witted close the getting in touch that have.Studies show that of Kivipelto etc. (2002) study carefully middle age's serum total cholesterol raise (〉=6.5mmol/L) be the independent hazard factor that AD takes place.Its possible biological mechanism is serum cholesterol amyloid precursor protein (the amyloid precursor protein of cell of can affecting the nerves that raises, APP) metabolism, quicken the generation of A β and the deposition of beta amyloid speckle, and then cause the generation of cognitive dysfunction.There are some researches show that higher fatty acid or high-cholesterol diet improves cholesterol levels among transgenic mouse blood plasma and the central nervous system, promotes the deposition of beta amyloid speckle, form dull-witted; And cholesterol acyltransferase (ACAT) plays a significant role in the cholesterol regulating metabolism, has been used as the valuable pharmacological target of prevention and treatment AD.Experiment of the present invention shows SI functions for the treatment of hyperlipidemia mice TC and TG content, improves its memory function simultaneously, and it is relevant with its effect for reducing blood fat that prompting SI improves memory function.

Have data to show, senile dementia patient brain top layer and temporo layer cortex lipid peroxidation increase; Antioxidase SOD and GSH-Px are active in the cerebral tissue reduces, and lipid peroxidation product MDA level raises and causes motor behavior ability and short term memory capacity to descend; The ability of learning and memory in mice decline raises with brain lipofuscin content corresponding relation.These show that memory is closely related with the peroxidating and the antioxidant status of brain.In the research of the present invention, hyperlipidemia mouse memory ability obviously reduces, and its activity of SOD in serum obviously reduces, and SI significantly improves laboratory animal antioxidase SOD vigor, and memory ability obviously improves simultaneously.This further specifies, the relation of body antioxidant status and brain function, and it is relevant with its SOD activity improving to point out SI to improve high fat animal memory.

Acetylcholine (ACh) is central nervous system's an important mediator, also is the essential neurotransmitter that memory forms, the learning and memory task finish the variation that often is accompanied by the interior ACh content of brain.Interrelated data points out that the active rising of Hippocampus AChE can reduce laboratory animal study memory ability; And suppress the AChE vigor, and can reduce dementia incidence rate, improve ability of learning and memory.The activity of AChE is also relevant with people's cognitive state in the peripheral blood in addition.In the experiment of the present invention, SI significantly reduces mouse brain cortex and Hippocampus AChE activity, thereby regulates the metabolism of ACh in the cerebral tissue, and the formation of cerebral cortex and Hippocampus and memory is closely related.Therefore we think that SI keeps ACh content in the cerebral tissue by AChE activity in inhibition cerebral cortex and the Hippocampus, has ability of learning and memory preferably to guarantee laboratory animal.

Central nervous system's amino acid neurotransmitter has important function aspect the regulation and control learning and memory function.Keeping of brain function needs enough and equilibrated neurotransmitteies, and the relative equilibrium of irritability and inhibitory transmitter is most important to keeping the brain physiological function.In brain districts such as cerebral cortex and Hippocampus, glycine (Gly) is the allosteric instrumentality of N-methyl D-aspartic acid (NMDA) receptor/passage, and the effect of excitatory amino acid is had regulating action, rises in the central nervous system and suppresses and neuroprotective.In the experimentation of the present invention, Glu and Asp content significantly reduce in high fat mouse brain cortex and the Hippocampus, but the variation of Gly does not have clear regularity, and after replenishing SI, Asp, Glu and Gly content all significantly raise; In addition, high fat mouse memory ability significantly reduces, and behind the additional SI, memory ability significantly strengthens.Infer that thus SI causes that the mechanism that amino acid neurotransmitter content in the cerebral tissue raises with its promotion memory function has certain relation.

Embodiment 3: soybean isoflavone is to the experiment of Chronic Aluminum poisoning model mice

1. experimental arrangement

A. the foundation of Chronic Aluminum poisoning Model of Dementia:

Experiment mice is with aluminum chloride 100mg/kg (crystal aluminum chloride is mixed with the aqueous solution of 10g/L, autoclaving with distilled water), lumbar injection, 0.2ml/ only, the next day once, continuous 50 days.Matched group injecting normal saline (each group of volume and all the other is quite) lumbar injection, the next day once, continuous 50 days.

B. experimental animal feeding and packet transaction:

Experiment mice is divided into 3 groups at random by sex after adapting to two weeks of nursing: matched group, poisoning by aluminum model group and soybean isoflavone processed group (the soybean isoflavone amount is 60mg/kgbw, is called for short the SI group), 16 every group, male and female half and half.The soybean isoflavone powder is dissolved in 0.5% sodium carboxymethyl cellulose (CMC-Na) is mixed with desired concn, irritate stomach, 0.3ml/d/ only.Matched group is irritated stomach equivalent 0.5%CMC-Na solution.Continuous irrigation stomach two months.Experimental session is respectively organized mice and is put quiet environment, and sub-cage rearing is raised with normal diet (provided by Test Animal Centre, Academy of Military Medical Sciences, P.L.A, be full price solid normal diet), and the drinking-water of freely ingesting, drinking water are tap water.Change drinking-water and feedstuff every day, keep animal living environment ventilation and sanitation and hygiene.(12 ± 2) hour illumination on daytime, indoor temperature (20 ± 2) ℃, relative humidity 40%～60%.Observe ordinary circumstances such as animal appearance (hair luster, brightness, sparse degree), expression (mental status, state of consciousness, reaction sensitivity), activity, appetite every day, per 15 days weighing the weight of animals of experimental session.

C. detect index:

Memory ability; Superoxide dismutase in the serum (SOD) activity; Acetylcholinesterase (AChE) activity in cerebral tissue (Hippocampus and cerebral cortex) and the blood; Amino acid neurotransmitter (Asp, Glu and Gly) content in the cerebral tissue (Hippocampus and cerebral cortex).

2. experimental result

A. soybean isoflavone is to the influence of experiment mice memory ability:

The assay method of memory ability is with embodiment 1

Consult table 10 as can be seen, the memory ability of poisoning by aluminum group mice significantly is lower than matched group (P＜0.05), and the soybean isoflavone group is significantly higher than poisoning by aluminum group (P＜0.05), with the matched group basically identical.

Table 10 soybean isoflavone is to the influence of experiment mice memory ability

Group	Number of animals	Memory is good			Remember medium			Poor memory
										Number of elements	????％		Number of elements	?????％		Number of elements	????％
		Matched group b	???16	????8	???50.00		????7	???42.50										????1	???7.50
Model group a	???14									????2	???14.29		????3	???21.43		????9	???64.28
		The SI group b	???16	????9	???56.25		????5	???31.25										????2	???13.50

Annotate: different alphabetical footmarks are represented significant difference (P＜0.05) in the table

B. soybean isoflavone is to the active influence of superoxide dismutase (SOD) in the experiment mice serum:

Consult shown in the table 11, compare with matched group, poisoning by aluminum group mice serum SOD is active significantly to reduce (P＜0.05), and soybean isoflavone significantly improves poisoning by aluminum SOD in Mice vigor, and each group difference all reaches significant level (P＜0.05).

Table 11 soybean isoflavone is to the influence of experiment mice activity of SOD in serum (U/ml)

Group	Number of animals	The SOD vigor
			Matched group	????16	????1.386±0.236 a
Model group	????14	????0.586±0.232 c
			The SI group	????16	????1.014±0.177 b

Annotate: different alphabetical footmarks are represented significant difference (P＜0.05) in the table

C. soybean isoflavone is to the active influence of acetylcholine esterase (AChE) in experiment mice blood and the cerebral tissue:

Consult shown in the table 12, as can be seen, compare, cholinesterase activity significantly raise (P＜0.05) in poisoning by aluminum group mouse blood, cerebral cortex and the Hippocampus with matched group; And compare with the poisoning by aluminum group, the soybean isoflavone group is active obviously to be reduced, significant difference (P＜0.05); Difference not significantly (P＞0.05) between soybean isoflavone group and the matched group.

Table 12 soybean isoflavone is to the influence of cholinesterase activity (U) in experiment mice blood and the cerebral tissue

Group	Number of animals	Cholinesterase activity AChE activity
					Hippocampus	Cerebral cortex	Blood
		Matched group	????16	??309.167±81.081 b				??478.189±91.240 b	??4.483±0.675 b
Model group	????14				??589.167±92.381 a	??656.790±81.803 a	??5.111±0.544 a
		The SI group	????16	??297.500±100.834 b				??368.724±89.254 b	??4.372±0.564 b

Annotate: different alphabetical footmarks are represented significant difference (P＜0.05) in the table

D. soybean isoflavone is to the influence of amino acid neurotransmitter in the experiment mice cerebral tissue (Asp, Glu and Gly) content:

Consult shown in the table 13, situation of change for content of amino acids neurotransmitter in experiment mice cerebral cortex and the Hippocampus, as can be seen from the table, in cerebral cortex, compare with matched group, the Asp of poisoning by aluminum group and Glu content all significantly reduce (P＜0.05), and soybean isoflavone processing back content raises to some extent; The difference of Gly content between each group is not significantly (P＞0.05) all.In Hippocampus, the Asp of poisoning by aluminum group and Glu content are all than the remarkable reduction of matched group (P＜0.05), but after the soybean isoflavone processing, content raises, and is approaching with matched group; The Gly content of poisoning by aluminum group and soybean isoflavone group all is significantly higher than matched group (P＜0.05), and the soybean isoflavone group significantly is lower than poisoning by aluminum group (P＜0.05).

Table 13 soybean isoflavone is to content of amino acids neurotransmitter (μ molg in the mouse brain tissue ^-1Wet tissue) influence

Group	Number of animals	Cerebral cortex				Hippocampus
									????Asp	????Glu	????Gly	???Asp	???Glu	???Gly
		Matched group	???16	??0.635± ??0.031 a		???1.039± ???0.032 a	???0.493 ???± ???0.056 a	??0.646± ??0.008 a							??1.079± ??0.052 a	??0.504± ??0.016 a
Model group	???14								??0.499± ??0.048 b	???0.966± ???0.037 b	???0.501 ???0.028 a	??0.571± ??0.011 c	??0.965± ??0.020 b	??0.624± ??0.029 a
		The SI group	???16	??0.534± ??0.024 b		???0.975± ???0.104 ab	???0.495 ???± ???0.016 a	??0.611± ??0.009 b							??1.050± ??0.011 a	??0.523± ??0.015 b

Annotate: different alphabetical footmarks are represented significant difference (P＜0.05) in the table

3. to the explanation and the explanation of experimental result

Aluminum is a kind of neurotoxin, and aluminum enters and replaces Ca in the brain ²⁺And Mg ²⁺, be deposited in the brain in conjunction with forming stabilized complex with glutamic acid on the amino acid chain or arginic carboxyl.Be rich in the glutamic acid serotonergic neuron in brain in cerebral cortex, Hippocampus and the corpus amygdaloideum, al deposition causes the fibrinous unusual phosphorylation of Double helix in these positions, forms neural the entanglement, causes the formation of amyloid protein precursor, brings out cognitive disorder.It is a kind of dementia animal model commonly used that the Chronic Aluminum poisoning causes cognitive disorder.Aluminum is accumulated in cerebral tissue and also can be promoted the ferritin release Fe3+ ferrum latter can promote the generation of high toxicity free radical to give birth to, cause that also lipid peroxidation strengthens, and but lipid peroxidation strengthens and the various enzymes of increase coup injury, carrier, receptor, passage and the protein of free radical, also influence the formation of LTP when making the body Total antioxidant capacity reduce and keep, and then cause learning and memory to reduce.Jia Yichang (2001) studies show that, takes the photograph aluminum for a long time and can cause antioxidase SOD and the active reduction of GSH-Px in the cerebral tissue, and lipid peroxidation product MDA level raises and also causes motor behavior ability and short term memory capacity to descend.Superoxide dismutase (SOD) is an important antioxidase in the body, and its vigor height can reflect the ability of body removing oxygen-derived free radicals indirectly.Result of experiment of the present invention shows that poisoning by aluminum group mouse memory ability obviously reduces, and shows the Model of Dementia that has caused poisoning by aluminum in experiment periods.

The generation and the removing of normal physiological situation lower body free radical are in poised state.But when endogenous antioxidation mechanism changes, accumulate, can promote ferritin to discharge Fe as aluminum in the cerebral tissue ³⁺, Fe ³⁺Can promote the generation of high toxicity free radical, free radical accumulates in a large number in the body, too much oxygen-derived free radicals can be attacked the infringement neuron membrane, the normal function of interference cell endomembrane system, cause the aging and degeneration of neurocyte, participate in many rheological properties degenerative disease that increase, occur that ability of learning and memory goes down and cognitive dysfunction; But free radical increases the various enzymes of coup injury, carrier, receptor, passage and protein in addition, also influences the formation of LTP when the body Total antioxidant capacity is reduced and keeps, and then cause ability of learning and memory to reduce.Discover total SOD, Mn-SOD and the active obviously reduction of Cu/Zn-SOD in the dementia patients cerebral tissue, SOD content obviously reduces (Fu Hong in the serum, Wang Xuemei, Liu Gengxin, Deng. the research of mild cognitive impairment and radical metabolism and acetylcholine esterase dependency. Chinese gerontology magazine, 2004,24:17-18); Take the photograph aluminum for a long time and cause antioxidase SOD and the active reduction of GSH-Px in the cerebral tissue, lipid peroxidation product MDA level raises and also causes motor behavior ability and short term memory capacity to descend.Dull-witted generation and the free radical of this prompting has confidential relation.Superoxide dismutase (SOD) is an important antioxidase in the body, and its vigor height can reflect the ability of body removing oxygen-derived free radicals indirectly.Poisoning by aluminum group mice serum SOD vigor significantly descends in the experimentation of the present invention, the ability that body removing free radical is described reduces, and after replenishing soybean isoflavone, SOD is active significantly to raise, the laboratory animal memory ability improves simultaneously, the prompting soybean isoflavone improves memory ability and its raising body SOD vigor of poisoning by aluminum mice, thereby it is relevant to improve oxidation resistance.

Acetylcholine (ACh) is central nervous system's an important neurotransmitter, also is the essential neurotransmitter of memory formation and the physiological foundation of longterm memory.It is synthetic by choline acetylation transferring enzyme (ChAT), and acetylcholinesterase (AChE) decomposes, the learning and memory task finish the variation that often is accompanied by ACh content in the brain.Active (the Fu Hong that significantly raises of AChE among the mild cognitive impairment patients serum, Wang Xuemei, Liu Gengxin, Deng. the research of mild cognitive impairment and radical metabolism and acetylcholine esterase dependency. Chinese gerontology magazine, 2004,24:17-18), cause ACh to contain quantity not sufficient, cause central nervous system dysfunction, ability of learning and memory reduces; And the inhibition cholinesterase activity can reduce dementia incidence rate, improves ability of learning and memory.The primary structure basis that cerebral cortex and Hippocampus are considered to remember, abundant cholinergic neuron is all contained in these brain districts.Cerebral cortex and the active rising of Hippocampus AChE behind the mice poisoning by aluminum in this experiment, and soybean isoflavone can resist this variation, makes AChE active approaching with matched group.This is accompanied by the active variation of AChE after showing poisoning by aluminum, and soybean isoflavone can improve cholinergic neurotransmitter ACh content, thereby improve the central cholinergic system function indirectly by regulating the activity of AChE in the brain, strengthens learning and memory.In addition, soybean isoflavone also can suppress mice peripheral blood cholinesterase activity in the present invention's experiment, and the prompting soybean isoflavone can influence the metabolism of acetylcholine in the peripheral blood equally.Acetylcholine is as a kind of distribution peripheral neurotransmitter very widely, and the activity of internal organs and skeletal muscle is also had regulating action very widely.And the method for testing of laboratory animal study memory is mainly evaluated with the avoidance pattern that connects with motion, and therefore, to learn the change of memory function relevant with laboratory animal equally can to think cholinesterase activity that soybean isoflavone influence peripheral blood.

Central nervous system's amino acid neurotransmitter also has important function aspect the regulation and control learning and memory function.Excitatory amino acid glutamic acid (Glu) that presynaptic membrane discharges and aspartic acid (Asp) can cause Ca by combining with protein mediated glutamate receptor and N-methyl D-aspartic acid (NMDA) receptor-selective of G ²⁺Channel opener makes postsynaptic membrane Ca ²⁺In stream increase, strengthen the generation of (LTP) and keep thereby participate in long time journey that synapse transmits.And LTP is generally acknowledged it is the objective indicator that memory formed and consolidated neuron physiological activity in the process.The formation of rat LTP behind the learning training is relevant with the rising of the outer Glu concentration of brain inner cell, depends on the activation of NMDA-R after Glu discharges, the formation of inducing LTP, thus behavior is regulated to learning and memory.Aluminum can disturb the homergy of glutamic acid neurotransmitter and participate in the excititoxic of glutamic acid mediation and influence the formation of LTP, also can suppress Na in the cerebral tissue ⁺, K ⁺-ATP enzyme, Mg ²⁺The activity of-ATP enzyme influences the picked-up of synapse to glycine, glutamic acid.In the cerebral tissue such as the striatum of naturally-aged animal, cortical area and hippocampus, exicitatory/amino acid neurotransmitter Asp and Glu level obviously reduce, and in the aged mouse central nervous system, when Glu content reduces, the NMDA-R number also significantly reduces, and these change and the size of LTP is proportionate.In brain districts such as cerebral cortex and Hippocampus, glycine (Gly) is the allosteric instrumentality of nmda receptor/passage, and the effect of excitatory amino acid is had regulating action, rises in the central nervous system and suppresses and neuroprotective.This experimental result shows that excitatory amino acid Asp and Glu content all significantly reduce in poisoning by aluminum group mouse brain cortex and the Hippocampus; And inhibitory aminoacid Gly content in Hippocampus raises, so the ratio of excitatory amino acid/inhibitory aminoacid reduces, the excitement of cerebral tissue is destroyed with inhibition.And after the soybean isoflavone processing, excitatory amino acids content increases, and this may be closely related with its promotion learning and memory ability.

Claims (9)

1, soybean isoflavone strengthens and improves the new capability of memory, soybean isoflavone is the phytoestrogen that a class is extracted from Semen sojae atricolor, be the heterocycle phenolic compound, what play biological effect mainly is genistein, daidzein and glucosides thereof, the structure of glucosides and aglycon is as follows in the soybean isoflavone: wherein, G represents glucose

It is characterized in that soybean isoflavone has the performance that strengthens and improve memory; Activity or content to the higher mammal neurotransmitter are influential, make the active reduction of acetylcholinesterase in hippocampal tissue and the blood (AChE); Excitatory amino acid class neurotransmitter glutamate (Glu) in cerebral cortex and the hippocampal tissue is raise with aspartic acid (Asp) content, inhibitory aminoacid class neurotransmitter glycine (Gly) content is reduced.

2, soybean isoflavone according to claim 1 strengthens and improves the new capability of memory, it is characterized in that described soybean isoflavone has the performance of memory reinforcing, is with soybean isoflavone normal mouse to be tested; Soybean isoflavone has the performance of improving memory, is with soybean isoflavone hyperlipidemia model mice and Chronic Aluminum poisoning model mice to be tested, and the dosage of soybean isoflavone is 30mg/kgbw-120mg/kgbw.

3, soybean isoflavone according to claim 2 strengthens and improves the new capability of memory, it is characterized in that the dosage of described soybean isoflavone is 60mg/kgbw.

4, soybean isoflavone according to claim 1 strengthens and improves the new capability of memory, it is characterized in that, active influential to the higher mammal neurotransmitter makes that acetylcholinesterase in hippocampal tissue and the blood (AChE) is active to be reduced, and the dosage of its soybean isoflavone is 30-120mg/kgbw.

5, soybean isoflavone according to claim 1 strengthens and improves the new capability of memory, it is characterized in that, excitatory amino acid class neurotransmitter glutamate (Glu) in cerebral cortex and the hippocampal tissue is raise with aspartic acid (Asp) content, inhibitory aminoacid class neurotransmitter glycine (Gly) content is reduced, and the dosage of its soybean isoflavone is 30-120mg/kgbw.

6, the new application of soybean isoflavone compounds is characterized in that, the application of soybean isoflavone compounds in preparation hypermnesis health product.

7, the new application of soybean isoflavone compounds according to claim 6 is characterized in that, the application of soybean isoflavone compounds in preparation improvement memory and treatment dysmnesia disease medicament preparation.

8, the new application of soybean isoflavone compounds according to claim 7 is characterized in that, described dysmnesia disease comprises organism metabolism obstacle, the dysmnesia that chemical compound is poisoned and year factor of waiting for a long time causes.

9, according to the new application of claim 6 or 7 described soybean isoflavone compounds, it is characterized in that, described pharmaceutical preparation or health product comprise with the soybean isoflavone compounds and using separately and with a kind of as in the complex of soybean isoflavone compounds.