CN106692192A

CN106692192A - Memory-enhancing mixture separated from blood plasma and preparation method and application of memory-enhancing mixture

Info

Publication number: CN106692192A
Application number: CN201611249420.9A
Authority: CN
Inventors: 崔文宏; 张丽丽; 孔毅荣; 郝敬雨; 石浩威; 任园园
Original assignee: Individual
Current assignee: Beijing Haosi Mass Spectrometry Technology Co ltd; Hunan Haosi Biotechnology Co ltd
Priority date: 2016-12-29
Filing date: 2016-12-29
Publication date: 2017-05-24
Anticipated expiration: 2036-12-29
Also published as: CN106692192B

Abstract

The invention belongs to the field of molecular biology, and provides a memory-enhancing mixture separated from blood plasma and a preparation method and application of the memory-enhancing mixture. During SDS-PAGE denaturing gel electrophoresis on the mixture, 11 clear stripes visible to naked eyes appear, and the stripes have the molecular weights of 25kD, 37kD, 41kD, 51kD, 66kD, 69kD, 88kD, 105kD, 145kD, 170kD and 200kD. Compared with the blood plasma and the current medicines, the mixture provided by the invention can more effectively improve the cognitive level of patients suffering from senile dementia; meanwhile, after long-time use, the mixture can greatly reduce side effects and medicine dependence caused by long-term use of Western medicines.

Description

It is a kind of to improve mixture for from blood plasma separate of memory and its preparation method and application

Technical field

The invention belongs to biology field, it is related to a kind of blood plasma separator, in particular it relates to one kind is divided from blood plasma From, mixture for strengthening memory and preparation method thereof, the mixture can be used to preventing, improve and treating senile dementia Disease, and with the effect of enhancing memory.

Background technology

Senile dementia is also called Alzheimer syndrome, is a kind of fatal disease of gradual cerebral function decline. Clinical manifestation constantly deteriorates for cognitive and memory function, and activity of daily living goes down, and has each neuropsychic symptom and behavior to hinder Hinder.Senile dementia is lethal dangerous suitable with tumour, cardiovascular and cerebrovascular disease etc., serious to threaten the health of the elderly and to whole Social development brings white elephant.This cause of disease is complicated, and pathogenesis is not yet illustrated completely, and characteristic pathological changes into β shallow lakes Nerve fibril is twined in the nerve cell that the extracellular senile plaque expelling and Protein tau Hyperphosphorylationof that powder sample proteinosis is formed are formed Knot, and neuron loss is with glial cells hyperplasia.

The drug therapy of current senile dementia is divided into symptomatic treatment and the major class of disease adjustment for the treatment of two.Wherein only four kinds The clinical efficacy of medicine (donepezil, Rivastigmine, galanthamine and Memantine) is more affirmed, has obtained U.S. FDA and ratified For clinical treatment.However, these medicines belong to symptomatic treatment medicine, it is only capable of improving clinical symptoms, disease can not be changed Sick process, and these medicines principally fall into chemical synthetic drug, there is different degrees of toxic and side effect.Cause

This, the newtype drug that research and development can adjust senile dementia process is particularly important at present.

In recent years result of study is proved：The cognitive water of aged rats can be lifted to the blood plasma of aged rats continuous injection youth mouse Flat, spatial memory, the ability that shakes down (Villeda S.A.Young blood reverses age-related impairments in cognitive function and synaptic plasticity in mice.Nature Medicine 2014；20:659-663).Its possible mechanism of action is containing thousands of kinds of albumen and various thin in blood plasma Intracellular cytokine, young blood plasma comprising some compositions of organized renewing vigor can be made, with age, these materials can disappear or It is destroyed.With modern separation technology, concentration is separated to the medicable plasma fraction of old dementia patients, for senile dementia Prevention and treatment, be a kind of developing direction of new regulation senile dementia process medicine.

The content of the invention

For the defect of prior art, an object of the present invention is to provide a kind of being separated from blood plasma for raising memory Mixture.

The second object of the present invention is the preparation method for providing said mixture.

The third object of the present invention is to provide the application of said mixture.

To achieve these goals, present invention employs following technical scheme：

In a first aspect, the present invention provides a kind of mixture for from blood plasma separate for improving memory, the SDS- of the mixture PAGE denaturation gel electrophoresis at least include the high-visible band of 11 naked eyes, and molecular size range is respectively：25kD、37kD、41kD、 51kD、66kD、69kD、88kD、105kD、145kD、170kD、200kD。

In the first aspect of the present invention, as a kind of preferred embodiment, analyzed by protein spectrum, in the mixture At least contain 106 kinds of albumen as shown in table 1 below；Preferably, the mixture also includes and the albumen shown in table 1 below Middle any small molecule for combining.

In the first aspect of the present invention, used as a kind of preferred embodiment, the blood plasma derives from mammal；It is preferred that Ground, the blood plasma derives from the mankind.

Second aspect, the present invention provides said mixture preparation method, comprises the following steps successively：Plasma collection, low temperature Filtering, low temperature ultrafiltration, cold ethanol precipitation, SD inactivations, centrifugation, anion exchange, for the first time concentration, molecular sieve, dialysis, for the second time Concentration step；Preferably, also include between the plasma collection and cold ethanol precipitation step：Filter at low temperature and low temperature ultrafiltration are walked Suddenly.

In the second aspect of the present invention, as a kind of preferred embodiment, in the plasma collection procedure, using anti-freezing Pipe collects blood, then by the way that supernatant is collected by centrifugation, obtains blood plasma；Preferably, in the centrifugation, centrifugal force is 500-1000g, Time is 10-30 minutes, and centrifuging temperature is 0-10 DEG C.

In the second aspect of the present invention, as a kind of preferred embodiment, in the filter at low temperature step, in pressure strip The blood plasma is filtered under part, is obtained filtrate；Preferably, the aperture of filter membrane be not less than 0.22 micron, it is more preferably described The aperture of filter membrane is 0.22 micron and 0.45 micron；Preferably, the temperature of the filtering is 0-5 DEG C, and pressure is 1-20MPa.

In the second aspect of the present invention, as a kind of preferred embodiment, in the low temperature ultrafiltration step, by the filter Liquid carries out film bag ultrafiltration by applying pressure, obtains low temperature ultrafiltration product；Preferably, the film bag molecular cut off is 3kD- 10kD；Preferably, the temperature of the ultrafiltration is 0-5 DEG C, and pressure is 1-20MPa；Preferably, the buffer solution that the ultrafiltration is used is One kind in phosphate buffer, Tris- hydrochloride buffers, HBS buffer solutions；It is highly preferred that the Tris- hydrochloride buffers Concentration is that 0.2-250mM, pH value are 7.0-9.2, and the concentration of the phosphate buffer is 1.0-450mM, pH value is 6.0-9.2, The concentration of the HBS buffer solutions is 0.5-600mM, pH value is 6.4-8.4；It is further preferred that the buffer solution is concentration 180mM, pH value are 8.0 Tris- hydrochloride buffers.

In the second aspect of the present invention, as a kind of preferred embodiment, in the cold ethanol precipitation step, will be described Low temperature ultrafiltration product or blood plasma stand after mixing with cold ethanol, supernatant are taken, as cold ethanol precipitation product；Preferably, it is described The temperature of cold ethanol is 0-10 DEG C；Preferably, the volume ratio of the cold ethanol and the low temperature ultrafiltration product or blood plasma is 1：(4- 8)；Preferably, the dwell temperature is 0-6 DEG C.

In the second aspect of the present invention, as a kind of preferred embodiment, in the SD inactivation steps, by the cold second Alcohol precipitated product stands after being suspended again with dense SD inactivators, obtains inactivating product；Preferably, the dwell temperature is 4~37 DEG C, the time is 6~18 hours；More preferably described dwell temperature is 20 DEG C, and the time is 12 hours；Preferably, the dense SD goes out Agent living contains N- butyl triphosphate, the tween of 1-4% of 0.6-4%.

In the second aspect of the present invention, as a kind of preferred embodiment, in the step with centrifugal separation, gone out described Retain supernatant after the centrifugation of life birth thing, obtain centrifugation product；Preferably, in the centrifugation, centrifugal force is 2000- 10000g, the time is 10-20 minutes, and temperature is 0-8 DEG C.

In the second aspect of the present invention, as a kind of preferred embodiment, in the anion exchange step, will be described Centrifugation product carries out step level wash-out in adding anion-exchange column, collects the eluent for obtaining every time, and the moon is obtained after mixing Ion exchange product；Preferably, in the step level wash-out, first eluted with first time elution buffer, obtain first time eluent； Second elution is used again, obtains waste liquid；Third time elution is used again, obtains third time eluent；Merge described First time eluent and third time eluent, obtain anion exchanged product；It is highly preferred that the first time eluent buffer solution In the sodium chloride containing 50-100mM, second eluent buffer solution contains the sodium chloride of 250-300mM, the third time Eluent buffer solution contains the sodium chloride of 450-500mM；It is highly preferred that the first time elution buffer, second wash-out are slow Fliud flushing, third time elution buffer are the Tris- hydrochloride buffers of concentration 0.2-200mM, pH value 7.0-9.2.

In the second aspect of the present invention, as a kind of preferred embodiment, in the first time concentration step, by described the moon Ion exchange product is concentrated, and obtains first time enriched product；Preferably, the volume of the dialysis product is that the concentration is produced Thing more than or equal to 5 times, more preferably 5-500 times；It is highly preferred that the volume of the first time enriched product is 500 microlitre -10 Milliliter；Preferably, the concentration is centrifuged using concentration tube, and the concentration tube allows the material of 1.0-2.5KD to pass through, it is described from In the heart, centrifugal force is 1000-3000g, and temperature is 2-8 DEG C；Preferably, the concentration is using concentration cup pressurization, the concentration Cup allows the material of 1.0-2.5KD to pass through, and the pressure of the pressurization is 1-15MPa.

In the second aspect of the present invention, as a kind of preferred embodiment, in the molecular sieve step, by the first time Enriched product is eluted in molecular sieve, obtains molecular sieve product；Preferably, 20- is contained in the elution buffer of the wash-out The sodium chloride of 500mM；Preferably, the elution buffer is Tris- hydrochloride buffers, and its concentration is 0.2-200mM, its pH value It is 7.0-9.2；It is highly preferred that the elution buffer is Tris- hydrochloride buffers, its concentration is 20mM, and its pH value is 8；It is excellent Selection of land, elution volume when starting to collect eluent is 15-35ml, and elution volume when stopping collecting eluent is 40-85ml.

In the second aspect of the present invention, as a kind of preferred embodiment, in the dialysis step, by the molecular sieve Product is dialysed, and is collected the product in Dialysis container and is obtained product of dialysing；Preferably, the elution buffer for using of dialysing It is the one kind in Tris- hydrochloride buffers, phosphate buffer, HBS buffer solutions；It is highly preferred that the Tris- hydrochloride buffers Concentration is that 0.2-250mM, pH value are 7.0-9.2, and the concentration of the phosphate buffer is 1.0-450mM, pH value is 6.0-9.2, The concentration of the HBS buffer solutions is 0.5-600mM, pH value is 6.4-8.4；It is further preferred that the elution buffer is 1mM, pH value are 8 Tris hydrochloride buffers；Preferably, the time of the dialysis is 20-72h；Preferably, the body of the dialysis Product ratio is 1:(100-10000).

In the second aspect of the present invention, as a kind of preferred embodiment, in the concentration step, the dialysis is produced Thing carries out second concentration, obtains second enriched product, as described mixture；Preferably, the volume of the dialysis product It is the enriched product more than or equal to 20 times, more preferably 20-100 times；Preferably, it is described concentration be using concentration tube from The heart, the concentration tube allows the material of 1.0-2.5KD to pass through, and in the centrifugation, centrifugal force is 1000-3000g；Preferably, institute It is that, using concentration cup pressurization, the concentration cup allows the material of 1.0-2.5KD to pass through, and the pressure of the pressurization is 1- to state concentration 20MPa。

The third aspect, the present invention provides a kind of pharmaceutical composition, comprising above-mentioned mixture and pharmaceutically acceptable load Body.

In the third aspect of the present invention, used as a kind of preferred embodiment, the pharmaceutically acceptable carrier is：Pharmacy Upper acceptable buffer solution, protein, gelatin, monose, polysaccharide, amino acid, chelating agent, sugar alcohol, polyethylene glycol and surface are lived One or more in property agent.

In the third aspect of the present invention, used as a kind of preferred embodiment, described pharmaceutical composition includes following component：1 The said mixture of times volume, the 8.5wt%NaCl of 9 times of volumes or the PBS of 1.5M, pH7.0；Preferably, the drug regimen Also include albumin, glucose and glutamine in thing；It is highly preferred that matter of the albumin in described pharmaceutical composition Amount percent by volume is 2%, and quality percent by volume of the glucose in described pharmaceutical composition is 1%, the paddy ammonia Quality percent by volume of the acid amides in described pharmaceutical composition is 3%.

Fourth aspect, the present invention provides a kind of sustained release preparation, comprising said mixture or aforementioned pharmaceutical compositions and medicine Acceptable biocompatible substance on；Preferably, the formulation of the sustained release preparation is liposome, microballoon, hydrogel, miniature oozes Saturating pump or microcapsules.

5th aspect, the present invention provides a kind of kit, comprising said mixture, aforementioned pharmaceutical compositions, above-mentioned sustained release Preparation.

6th aspect, the present invention provides said mixture, aforementioned pharmaceutical compositions, above-mentioned sustained release preparation, mentioned reagent box, Application in prevention, improvement or/and treatment senile dementia.

7th aspect, the present invention provides said mixture, aforementioned pharmaceutical compositions, above-mentioned sustained release preparation, mentioned reagent box, Application in memory medicine is strengthened.

The mixture that the present invention is provided can more effectively lift recognizing for patients of senile dementia than blood plasma and current medicine Know level.At the same time, this mixture of long-term taking can substantially reduce side effect and the medicine that long-term taking Western medicine brings Rely on.Preparation method of the invention may be directly applied to amplify metaplasia product, directly batch production can prepare substantial amounts of mixture. Each step of preparation method of the invention, parameter setting rationally, between act synergistically, the product of final products are improve jointly Matter.Mixture prepared by the present invention can be used for preventing, improve and treating senile dementia, and it may also operate as enhancing note in addition Recall the effect of power.

Brief description of the drawings

Fig. 1：In test case, the HC4201615 mixtures SDS- denaturation gel electrophoresis qualification result electrophoretograms of embodiment 1.Swimming Road M：Protein Marker；Swimming lane 1-4：HC4201615 mixtures.

Fig. 2：In test case, the microphoto of the primary hippocampal cells after different disposal.There is primary sea toward different cultures HC4201615 mixtures, and untreated human plasma are separately added into the DMEM culture mediums of horse cell, as experimental group；Control Physiological saline is added in group hippocampal cell.Hippocampal cell is imaged under the microscope after one day；(a), (b), (c) in figure It is respectively the primary hippocampal cells after HC4201615 mixtures, untreated human plasma, physiological saline (i.e. control group) treatment.

Fig. 3：In test case, the HC4201615 mixtures of embodiment 1 suppress the active column of primary hippocampal cells apoptosis Figure.HC4201615 mixtures are separately added into the DMEM culture mediums for there are primary hippocampal cells toward different cultures, and it is untreated Human plasma, as experimental group；Only add physiological saline in control group hippocampal cell；Hippocampal cell is carried out under the microscope after one day Counting, experiment with computing group cell number and cellular control unit number.

Fig. 4：In test case, the HC4201615 mixtures of embodiment 1 promote the work of Synaptic formation between primary hippocampal cells The block diagram of property；HC4201615 mixtures are separately added into the DMEM culture mediums for there are primary hippocampal cells toward different cultures, and Untreated human plasma, as experimental group；Only add physiological saline in control group hippocampal cell；Under the microscope to cynapse after one day Number is counted, experiment with computing group cynapse number and control group cynapse number.

Fig. 5：In test case, HC4201615 mixtures lifting alzheimer's disease (senile dementia) mouse of embodiment 1 Memory animal model data scatter diagram.HC4201615 mixture systems are expelled to alzheimer's disease mouse mould In type；By water maze laboratory, castering action of the administration to alzheimer's disease mouse memory power is evaluated.

Specific embodiment

In order to preferably be illustrated to technical characteristic of the invention and effect, below using specific embodiment to this hair It is bright to be described in detail, but the present invention is not limited thereto.

The present invention provides a kind of mixture for strengthening memory, and it derives from blood plasma, i.e., is isolated from blood plasma Mixture HC4201615, the mixture includes multiple proteins and various small molecules, and the SDS-PAGE of the mixture is denatured Gel electrophoresis have the high-visible band of 11 naked eyes, and the molecular weight of the band is：25kD、37kD、41kD、51kD、66kD、 69kD、88kD、105kD、145kD、170kD、200kD。

Preferably, the mixture at least contain 106 kinds of albumen, also containing with above-mentioned 160 kinds of protein bound small molecules. Analyzed and identified by protein spectrum (such as MS/MS), albumen such as table 1 below contained by the mixture.

Table 1

Preferably, the blood plasma derives from mammal, such as mankind, muroid etc. are preferably derived from the mankind.

The present invention also provides the preparation method of said mixture HC4201615, the mixture extracted from blood plasma prepare and Into the preparation method is comprised the following steps successively：Plasma collection, cold ethanol precipitation, SD inactivations, centrifugation, anion are handed over Change, for the first time concentration, molecular sieve, dialysis, second concentration step；As preferred embodiment, plasma collection procedure it Afterwards, before cold ethanol precipitation step, the preparation method also includes filter at low temperature step and low temperature ultrafiltration step.It is specific as follows：

S1 plasma collections：

Blood is collected using anticoagulant tube, then by the way that supernatant is collected by centrifugation, obtains blood plasma；Preferably, the centrifugal force is 500-1000g (can be the middle arbitrary values such as 500g, 750g, 800g, 1000g or any number range between the two), centrifugation Time be 10-30min (can be the middle arbitrary value such as 10min, 15min, 20min, 25min, 30min or it is any between the two Number range), centrifuging temperature be 0-10 DEG C (can for 0 DEG C, 2.5 DEG C, 5 DEG C, 7 DEG C, the middle arbitrary values such as 10 DEG C or it is any both it Between number range).

Further to control the quality of the final mixture for preparing, it is preferable that after the plasma collection procedure also Need to carry out filter at low temperature step and low temperature ultrafiltration step, carry out the steps such as cold ethanol precipitation again afterwards.

S2 filter at low temperature, the step is mainly the haemocyte that may be remained in removal blood plasma：

The blood plasma that the plasma collection procedure is obtained is added in filter, and applying pressure using peristaltic pump is filtered (pressure Power scope is 1-20MPa), obtain filtrate；The filter sizes of the filter are not less than 0.22 micron, and preferably 0.22 or 0.45 is micro- Rice, the temperature control of whole filter at 0-10 DEG C, preferably 0-5 DEG C.Exemplarily, above-mentioned pressure can for 1MPa, The middle arbitrary value such as 5MPa, 10MPa, 15MPa, 20MPa or any number range between the two, said temperature can for 0 DEG C, 2 DEG C, 4.5 DEG C, the middle arbitrary values such as 5 DEG C or any number range between the two.

Above-mentioned filter sizes can not be less than 0.22 micron, and otherwise haemocyte can not be removed by filtration；The temperature for filtering simultaneously Degree not above 5 DEG C, preferably 0-4 DEG C, not so the albumen inside final product can variability so as to lose activity.

The ultrafiltration of S3 low temperature is (for the material inside blood plasma to be replaced in the buffer solution determined in pH, so that convenient control The pH of solution system)：

The filtrate that the filter at low temperature is obtained carries out film bag ultrafiltration, using peristaltic pump apply pressure (scope be 1~ 20MPa), low temperature ultrafiltration product is obtained；In film bag ultra-filtration process, active ingredient can enter in buffer solution；The film bag retention Molecular weight be 3kD-10kD, the buffer solution that ultrafiltration is used be phosphate buffer, Tris- hydrochloride buffers and HBS buffer solutions Etc. conventional buffer solution, the temperature control of whole ultrafiltration apparatus is at 0-5 DEG C, it is preferable that the concentration of the Tris- hydrochloride buffers is 0.2-250mM, pH value are 7.0-9.2, and the concentration of the phosphate buffer is 1-450mM, pH value is 6.0-9.2, and the HBS delays The concentration of fliud flushing is 0.5-600mM, pH value is 6.4-8.4；It is highly preferred that it is 8.0 that the buffer solution is 180mM, pH value Tris- hydrochloride buffers.

The data area of parameter in the step is illustrated below by example, above-mentioned pressure can for 1MPa, 5MPa, The middle arbitrary value such as 10MPa, 15MPa, 20MPa or any number range between the two；Above-mentioned film bag molecular cut off can be The middle arbitrary value such as 3kD, 5kD, 7kD, 10kD or any number range between the two；The concentration of above-mentioned Tris- hydrochloride buffers Can for the middle arbitrary value such as 0.2mM, 1mM, 5mM, 10mM, 50mM, 100,125mM, 150mM, 200mM, 250mM or it is any both Between number range, pH value can be the middle arbitrary values such as 7.0,8.0,8.5,8.8,9.0 or any numerical value model between the two Enclose；The concentration of above-mentioned phosphate buffer can be in 1mM, 10mM, 50mM, 100mM, 200mM, 350mM, 400mM, 430mM etc. Arbitrary value or any number range between the two, pH value can be any in 6.0,7.0,8.0,8.5,8.8,9.0,9.2 etc. Value or any number range between the two；The concentration of above-mentioned HBS buffer solutions can for 0.5mM, 1mM, 10mM, 50mM, The middle arbitrary value such as 100mM, 200mM, 300mM, 350mM, 400mM, 500mM, 600mM or any number range between the two, PH value can be the middle arbitrary values such as 6.4,7.0,7.5,8.0,8.4 or any number range between the two.

Above-mentioned film bag aperture can not be more than 10kD, and not so some effective albumen are lost from ultra-filtration process；On Stating the temperature of ultrafiltration should control at 0-5 DEG C, not so the albumen inside final product can variability so as to lose activity.

(this step removes no effect, even virose egg to S4 cold ethanol precipitations by the method for cold ethanol precipitation Bai Zufen and these protein bound small molecules)：

The low temperature ultrafiltration product (if step 2, three being omitted, by blood plasma described in step one) is carried out into cold ethanol to sink Form sediment, obtain cold ethanol precipitation product；Wherein, ethanol is after 0-10 DEG C of precooling, and low temperature ultrafiltration product (or blood plasma) is with 1：(4-8) Volume ratio is mixed, and mixing is after 0-6 DEG C of placement, until precipitation automatic sedimentation takes the supernatant obtained after precipitation to becoming real, As cold ethanol precipitation product；It is preferred that the standing time is 8-16 hours, more preferably 12 hours.Described change refers in fact supernatant There is obvious line of demarcation with precipitation, supernatant becomes bright, precipitation quantity no longer increases.

The data area of parameter in the step is illustrated below by example, the temperature of above-mentioned precooling can for 0 DEG C, 2 DEG C, 5 DEG C, 8 DEG C, the middle arbitrary values such as 10 DEG C or any number range between the two, the temperature of placement can be after above-mentioned mixing 0 DEG C, 1 DEG C, 4 DEG C, 5 DEG C, the middle arbitrary values such as 6 DEG C or any number range between the two, above-mentioned ethanol and low temperature ultrafiltration product Volume ratio be 1：4、1：5、1：6、1:7、1:Arbitrary value or any number range between the two in 8 grades.

Above-mentioned ethanol and low temperature ultrafiltration product use different volumes ratio, and the albumen being precipitated out is different；While temperature Preferably must be held in the range of 0-6 DEG C, the albumen being not so settled out may be denatured.

(potential virus that this step is so that in initial feed human blood lose activity and by centrifugation for S5SD inactivations and centrifugation The foreign protein removal of aggregate and precipitate will be denatured, so as to improve the security of final products)：

The cold ethanol precipitation product and dense SD inactivators are mixed in equal volume, 4~37 DEG C of 6~18h of standing is put in, preferably 20 DEG C of standing 12h, are then centrifuged, and retain supernatant, as centrifugation product；The temperature of above-mentioned standing can not be low In 4 DEG C, the time can not be shorter than 6 hours, and not so the effect of inactivation of virus can be relatively more general, and the inactivation time of different virus is not yet Together.Preferably, the centrifugal force is 2000-10000g, and centrifugation time is 10-20min, and centrifuging temperature is 0-8 DEG C, above-mentioned centrifugation Power can not be less than 2000g, and centrifugation time can not be less than 10min, and the foreign protein for being not so denatured aggregate and precipitate occurs that sedimentation is unreal Situation.

SD inactivators contain the TnBP (N- butyl triphosphate) and 1.0~4% that percent by volume is 0.6~4% Tween80, preferably contains 2%TnBP and 2%Tween80.SD inactivators are formulated as follows：Gone out with the lane SD for preparing 100mL As a example by agent living, 0.6-4ml TnBP are dissolved in suitable quantity of water, are subsequently adding 1.0~4mLTween80, mended with water after being well mixed To 100mL.

The data area of parameter in the step is illustrated below by example, time of above-mentioned standing can for 6h, The middle arbitrary value such as 8h, 10h, 12h, 15h, 18h or any number range between the two, temperature can for 4 DEG C, 10 DEG C, 15 DEG C, 20 DEG C, 25 DEG C, 28 DEG C, 30 DEG C, 32 DEG C, the middle arbitrary values such as 35 DEG C or any number range between the two, the hundred of above-mentioned TnBP Point specific concentration can be the middle arbitrary value such as 0.3%, 0.5%, 1%, 1.5%, 2% or any number range between the two, on The percent concentration for stating Tween80 can be the middle arbitrary value such as 0.5%, 1%, 1.5%, 2% or any numerical value between the two Scope.Above-mentioned centrifugal force can for the middle arbitrary value such as 2000g, 2500g, 5000g, 7500g, 8000g, 10000g or it is any both Between number range, the time can be the middle arbitrary value such as 10min, 12min, 15min, 20min or any number between the two Value scope, temperature can be 0 DEG C, 1 DEG C, 2 DEG C, 3 DEG C, 4 DEG C, 6 DEG C, the middle arbitrary values such as 7 DEG C or any numerical value model between the two Enclose.

S7 anion exchanges (step prepare the albumen containing active ingredient and with these protein bound small molecules, Largely improve the curative effect of final product HC4201615)：

Step level wash-out in centrifugation product addition anion-exchange column (such as SourceQ), will be carried out, collects every The secondary eluent for obtaining, obtains anion exchanged product after mixing；

Wherein, in the step level wash-out, first eluted with first time elution buffer, obtain first time eluent；Again with Secondary elution buffer wash-out, obtains waste liquid；Eluted with third time elution buffer again, obtain third time eluent；Merge institute First time eluent and third time eluent are stated, anion exchanged product is obtained；Contain 50- in the first time elution buffer The sodium chloride of 100mM, second elution buffer contains the sodium chloride of 250-300mM, the third time elution buffer Sodium chloride containing 450-500mM；It is that 0.2-200mM, pH value are 7.0-9.2 that the elution buffer of three wash-outs is concentration Tris- hydrochloride buffers；Each elution speed is controlled in the range of instrument permission, such as 2-5ml/min.

The data area of parameter in the step is illustrated below by example, chlorine in above-mentioned first time elution buffer The concentration for changing sodium can be the middle arbitrary values such as 50mM, 60mM, 70mM, 80mM, 90mM, 100mM or any numerical value between the two Scope, in above-mentioned second elution buffer the concentration of sodium chloride can for 250mM, 260mM, 270mM, 280mM, 290mM, The middle arbitrary value such as 300mM or any number range between the two, the concentration of sodium chloride can in above-mentioned third time elution buffer Think the middle arbitrary value such as 450mM, 460mM, 470mM, 480mM, 490mM, 500mM or any number range between the two, on The concentration for stating Tris- hydrochloride buffers can be in 0.2mM, 0.5mM, 1mM, 10mM, 50mM, 100mM, 150mM, 200mM etc. Arbitrary value or any number range between the two, pH value can for the middle arbitrary values such as 7.0,8.0,8.5,9.2 or it is any both Between number range.

S8 concentrations for the first time (purpose of this step is to reduce the volume of sample, facilitates follow-up molecular sieve loading to operate)：

The anion exchanged product is carried out into first time concentration, first time enriched product is obtained；Wherein, the anion Exchange product volume be the enriched product more than or equal to 5 times, preferably 5-500 times, more preferably 50-500 times；It is preferred that Ground, the volume after concentration is 500 μ L-10mL；Preferably, the concentration is carried out by the way of concentration tube centrifugation, described Concentration tube allows the material of 1.0-2.5KD to pass through, and the centrifugal force is 1000-3000g, and temperature is 2-8 DEG C, and cycles of concentration reaches It is required that after stop concentration；Preferably, the concentration is carried out by the way of concentration cup pressurization, and the concentration cup is allowed The material of 1.0-2.5KD passes through, and the pressure limit of the pressurization is 1~15MPa.

When concentration tube used or concentration cup size are more than 2.5kD, inside the final mixture for preparing some effectively into Branch is lost in, but when concentration tube or concentration cup size are less than 1.0kD, concentration speed also can be very slow, takes long；Concentration When the pressure for using is less than 1MPa or centrifugal force less than 1000g, concentration speed can be very slow, takes long；Thickening temperature During higher than 8 DEG C, some active ingredients may be denatured so as to lose activity because of temperature drift in concentration process, thickening temperature During less than 2 DEG C, the price of temperature control system again can be of a relatively high, while there is enriched product assembling because temperature is too low The risk of precipitation.

The data area of parameter in the step is illustrated below by example, above-mentioned multiple can for 5,10,50, 100th, the middle arbitrary values such as 200,250,300,400,500 or any number range between the two, the concentration tube, concentration cup are permitted The size of the material for passing through perhaps be the middle arbitrary value such as 1.0KD, 1.25KD, 1.5KD, 1.75KD, 2KD, 2.25KD, 2.5KD or Any number range between the two, the centrifugal force be the middle arbitrary values such as 1000g, 1500g, 2000g, 2500g, 3000g or Any number range between the two, the temperature can for 2 DEG C, 4 DEG C, 5 DEG C, the middle arbitrary values such as 8 DEG C or it is any between the two Number range, the pressure is the middle arbitrary value such as 1MPa, 5MPa, 7.5MPa, 10MPa, 15MPa or any number between the two Value scope.

S9 molecular sieves (further sieve effective albumen and small molecule)：

By in first time enriched product addition molecular sieve, collection eluent obtains molecular sieve product after wash-out；Its In, contain 20-500mM sodium chloride in wash-out buffer solution used, only elute once；Preferably, when elution volume is 15- Start to collect eluent during 35ml, stop collecting eluent when elution volume is 40-85ml, the eluent that collection is obtained is HC4201615 stostes, as molecular sieve product；Preferably, described buffer solution is Tris- hydrochloride buffers, and concentration is 0.2- 200mM, pH value are 7.0-9.2；It is highly preferred that described buffer solution is Tris- hydrochloride buffers, concentration is 20mM, pH value is 8。

The data area of parameter in the step is illustrated below by example, sodium chloride in above-mentioned elution buffer Concentration can for the middle arbitrary value such as 20mM, 50mM, 100mM, 200mM, 250mM, 300mM, 400mM, 500mM or it is any both it Between number range, above-mentioned elution volume when starting to collect eluent can be in 15ml, 16ml, 17ml, 18ml, 20ml etc. Arbitrary value or any number range between the two, elution volume when eluent is collected in above-mentioned stopping can for 60ml, 65ml, The middle arbitrary value such as 70ml, 75ml, 80ml, 85ml or any number range between the two, above-mentioned Tris- hydrochloride buffers it is dense Degree can be the middle arbitrary value such as 0.2mM, 0.5mM, 1mM, 10mM, 50mM, 100mM, 150mM, 200mM or it is any between the two Number range, pH value can be the middle arbitrary values such as 7.0,8.0,8.5,9.2 or any number range between the two.

This step defines the concentration of sodium chloride in elution buffer, and start to collect and stop to collect eluent when Between point；When sodium chloride concentration is less than 20mM in elution buffer, Partial Protein can occur aggregate and precipitate because solubility is low, Pillar is caused to block the loss with active ingredient；When sodium chloride concentration inside elution buffer is higher than 500mM, this step Product can produce very big osmotic pressure because sodium chloride concentration is too high, so that following dialysis step needs is longer Time and the more dialyzates of consuming；Within the above-mentioned time period for starting and collecting and stop collection eluent, the wash-out being collected into Liquid is only active ingredient, otherwise the composition being not so collected into is containing many invalid or even poisonous compositions, otherwise it is exactly that some have Effect composition generation loss is reduced so as to cause curative effect.

S10 dialyses：

Dialysed during the molecular sieve product (HC4201615 stostes) is put into bag filter or pipe, collected saturating after dialysis Product in analysis bag or pipe, as dialysis product；Wherein, the elution buffer for being used during the dialysis is Tris- hydrochloride buffers Liquid, phosphate buffer or HBS buffer solutions；Preferably, the concentration of the Tris- hydrochloride buffers is that 0.2-250mM, pH value are 7.0-9.2, the concentration of the phosphate buffer is 1.0-450mM, pH value is 6.0-9.2, and the concentration of the HBS buffer solutions is 0.5-600mM, pH value are 6.4-8.4；It is highly preferred that the elution buffer is 20mM, the Tris hydrochloride buffers that pH value is 8 Liquid；Dialysis time is 20-72h, and dialysis volume ratio (volume ratio of dialyse product and dialyzate) is 1:(100-10000).This Dialysis operation in step, the impurity before can removing in step, such as inactivator, to prevent the medium from being prepared to downstream product Produce harmful effect.

Dialysis time is defined in this step；When dialysis time is less than 20 hours, it may occur that magazine removes incomplete feelings Condition；When dialysis time was more than 72 hours, the time cost of production can be dramatically increased again.

The data area of parameter in the step is illustrated below by example, above-mentioned Tris- hydrochloride buffers it is dense Degree can be the middle arbitrary values such as 0.2mM, 1mM, 5mM, 10mM, 50mM, 100mM, 125mM, 150mM, 200mM, 230mM or any Number range between the two, pH value can for the middle arbitrary values such as 7.0,7.5,8.0,8.5,8.8,9.0 or it is any between the two Number range；The concentration of above-mentioned phosphate buffer can for 1mM, 10mM, 50mM, 100mM, 200mM, 350mM, 400mM, The middle arbitrary value such as 450mM or any number range between the two, pH value can be 6.4,7.0,7.5,8.0,8.5,9,9.2 etc. Middle arbitrary value or any number range between the two；The concentration of above-mentioned HBS buffer solutions can for 0.5mM, 1mM, 10mM, The middle arbitrary value such as 50mM, 100mM, 200mM, 300mM, 350mM, 400mM, 500mM, 600mM or any numerical value between the two Scope, pH value can be the middle arbitrary values such as 6.4,7.0,8.0,8.4 or any number range between the two；Above-mentioned dialysis time Can be the middle arbitrary values such as 20h, 25h, 50h, 60h, 72h or any number range between the two；Above-mentioned dialysis volume ratio can Think 1:100、1:500、1:1000、1:2500、1:5000、1:Arbitrary value or any numerical value model between the two in 10000 grades Enclose.

S11 is concentrated for second：

The dialysis product is carried out into second concentration, second enriched product, as mixture HC4201615 is obtained； Wherein, condensate precursor product be concentration after volume more than or equal to 20 times, preferably 20-100 times；Preferably, the concentration is to adopt What the mode being centrifuged with concentration tube was carried out, the concentration tube allows the material of 1.0-2.5KD to pass through, and the centrifugal force is 1000- 3000g, temperature is 2-8 DEG C, and cycles of concentration stops concentration after reaching requirement；Preferably, the concentration is using concentration cup What the mode of pressurization was carried out, the concentration cup allows the material of 1.0-2.5KD to pass through, the pressure limit of the pressurization for 1~ 20MPa.The first purpose of this step is to improve the concentration of active principle in the mixture HC4201615, so as to improve unit The curative effect of composition；The second purpose is the osmotic pressure that solution is adjusted by concentration.Preparation method of the invention needs to concentrate twice Because：Concentration for the first time is the implementation for facilitating following molecular sieve step in order to reduce volume；Second concentration is in order to effective Ground controls the osmotic pressure of final product HC4201615 to meet the requirement of clinic physiologic infusion agent.

Define that concentration tube or concentration cup allow the magnitude range of the molecular weight of the material for passing through, use concentration in this step Centrifugal force scope during pipe, using concentration cup when pressure limit, concentration when temperature range；Concentration tube or concentration cup used When size is more than 2.5kD, some active ingredients inside the final mixture for preparing can be lost in, but when concentration tube or concentration cup are big During less than 1.0kD, concentration speed also can be very slow, takes long；The pressure that concentration is used is less than 1MPa or centrifugal force During less than 1000g, concentration speed can be very slow, takes long；When thickening temperature is higher than 8 DEG C, some are effective in concentration process Composition may be denatured so as to lose activity because of temperature drift, when thickening temperature is less than 2 DEG C, the price of temperature control system Again can be of a relatively high, while there is enriched product because the too low risk that aggregate and precipitate occurs of temperature.

The data area of parameter in the step is illustrated below by example, above-mentioned multiple can for 20 times, 25 times, 30 times, 50 times, 80 times, the middle arbitrary values such as 100 times or any number range between the two, the concentration tube, concentration cup are allowed The size of the material for passing through is the middle arbitrary value such as 1.0KD, 1.25KD, 1.5KD, 1.75KD, 2KD, 2.25KD, 2.5KD or appoints Meaning number range between the two, the centrifugal force is the middle arbitrary values such as 1000g, 1500g, 2000g, 2500g, 3000g or appoints Meaning number range between the two, the temperature can be 2 DEG C, 4 DEG C, 5 DEG C, the middle arbitrary values such as 8 DEG C or it is any between the two Number range, the pressure is the middle arbitrary values such as 1MPa, 5MPa, 7.5MPa, 10MPa, 15MPa or any numerical value between the two Scope.

The various common agents used in above-mentioned preparation method are prepared using conventional method.

The present invention further provides a kind of pharmaceutical composition, comprising the mixture HC4201615 and pharmaceutically acceptable Carrier.The pharmaceutically acceptable carrier includes：Pharmaceutically acceptable buffer solution, protein, gelatin, monose, polysaccharide, One or more in amino acid, chelating agent, sugar alcohol, polyethylene glycol and surfactant.

Used as preferred embodiment, described pharmaceutical composition includes following component：1 times of mixture of volume HC4201615, the 8.5wt%NaCl of 9 times of volumes or the PBS of 1.5M, pH7.0；Preferably, also include in described pharmaceutical composition Albumin, glucose and glutamine, wherein, quality percent by volume of the albumin in described pharmaceutical composition is 2%, quality percent by volume of the glucose in described pharmaceutical composition is 1%, and the glutamine is in the medicine Quality percent by volume in composition is 3%.

The present invention further provides a kind of sustained release preparation, comprising the mixture HC4201615 and pharmaceutically acceptable Biocompatible substance (also referred to as：Pharmaceutically acceptable carrier)；Preferably, the formulation of the sustained release preparation be liposome, Microballoon, hydrogel, Osmotic minipumps or microcapsules；Preferably, the pharmaceutically acceptable biocompatible substance is aqueous PH buffer solutions, including the buffer solution of phosphate, citrate or other organic acids, ascorbic acid or other antioxidants, low point Son amount (being no more than 10 residues) polypeptide, seralbumin, gelatin, immunoglobulin or other protein, polyvinylpyrrolidine Ketone or other hydrophilic polymers, glycine, glutamine, asparagine, arginine, lysine or other amino acid, monose, Disaccharides, glucose, mannose, dextrin or other carbohydrates, EDTA or other chelating agents, mannitol, sorbierite or other sugar alcohols, sodium Ion or other into salt counter ion,Polyethylene glycol (PEG),Or other non-ionic surfaces are lived Property agent.)

The present invention further provides a kind of kit, include：The mixture HC4201615 or/and comprising described mixed The aforementioned pharmaceutical compositions of compound HC4201615 or/and the above-mentioned sustained release agent comprising the mixture HC4201615.

The present invention further provides the mixture HC4201615 or/and comprising the upper of the mixture HC4201615 State pharmaceutical composition or/and the above-mentioned sustained release agent comprising the mixture HC4201615 or/and comprising the mixture The mentioned reagent box of HC4201615, in prevention, the medicine for improving or/and treating senile dementia or in enhancing memory Application in medicine.

Preparation, identification and application below by embodiment to inventive mixture are illustrated.Related in following examples And for example unreceipted specific experimental condition of molecular biology manipulations and method, refer to SambrookJ etc. chief editor, scientific publication Society, 2002, the specification of Molecular Cloning:A Laboratory guide (third edition) or corresponding product.

Preparation, identification and application below by embodiment to inventive mixture are illustrated.Make in following examples Primary hippocampal cells are according to following bibliography culture：Guo,W.,Y.Ji,et al.(2014)."Neuronal activity alters BDNF-TrkB signaling kinetics and downstream functions."J Cell Sci 127(Pt 10):2249-2260。)

Embodiment 1

The present embodiment is the preparation method of mixture HC4201615, is comprised the following steps：

S1：Collect blood plasma

Obtained by hospital donates blood, heparin tube is anticoagulant tube to the blood of Healthy People, rocks blood sampling in blood collection procedure in bloodletting Pipe, prevents blood clotting；Heparin tube containing blood is put into centrifuge, centrifugal force as 800g is set, is centrifuged 15 minutes, centrifugation Temperature is 4 degree；Then supernatant is carefully drawn with pipettor, the human plasma being as collected into.

S2：Filter at low temperature

During the blood plasma that will be collected into adds filter sizes to be 0.22 micron of filter, peristaltic pump is used to apply pressure 10MPa Filtered, temperature control of whole filter obtains filtrate at 0-4 DEG C during it.

S3：Low temperature ultrafiltration

By the film bag ultrafiltration that the molecular weight that the filtrate retains is 8kD, pressure 10MPa, its process are applied using peristaltic pump The middle buffer solution for using is the Tris hydrochloride buffers of concentration 180mM, pH8.0, and the temperature control of whole ultrafiltration apparatus is in 0-4 DEG C, collect the buffer solution after ultrafiltration and obtain low temperature ultrafiltration product.

S4：Cold ethanol precipitation

By the 0-4 DEG C of ethanol of precooling and the low temperature ultrafiltration product according to volume ratio 1:6 mixing, then at 0 DEG C of placement 8h, extremely sink Shallow lake automatic sedimentation becomes real, the supernatant after cold ethanol precipitation is obtained, as cold ethanol precipitation product.

S5：SD inactivation centrifugations

The cold ethanol precipitation product liquid and dense SD inactivators (containing 2%TnBP, 2%Tween80) are mixed in equal volume, is put 12 hours are stood in 20 DEG C, is then centrifuged 15 minutes in 5000g, 0-4 DEG C, retain supernatant, as centrifugation product.

S6：Anion exchange

The centrifugation product is added into anion-exchange column (such as SourceQ, purchased from GE Healthcare, average grain It is 30 microns to spend, and anion exchange column volume is 25 milliliters) in, first with the Tris- hydrochloride buffers containing 75mM sodium chloride (100mM, pH8.0) carries out first time wash-out, retains eluent；Then with the Tris- hydrochloride buffers containing 275mM sodium chloride (100mM, pH8.0) carries out second wash-out, discards eluent；Finally with the Tris- hydrochloride buffers containing 475mM sodium chloride (100mM, pH8.0) carries out third time wash-out, retains eluent；Merge above-mentioned first time and third time afford eluent, As anion exchanged product；Wherein, each elution speed is 4ml/min, and loading speed is 3ml/ml.

In above-mentioned each wash-out, start to collect when waiting UV-detector to show and albumen occur, albumen goes out to be over, and stops Collect, i.e., the eluent for being eluted each time.

S7：Concentrate for the first time

Take a small amount of Anion separation product to be added in the concentration tube that volume is 2mL, it is 2.5kD that concentration tube allows size Material pass through；Concentration tube is put into centrifuge again, sets centrifugal force as 1500g, design temperature is 4 DEG C, starts centrifuge, Start concentration, until final volume is 500 microlitres；Afterwards, by the addition again of the remaining dialysis product centrifuge tube, with Volume is reached 2mL, be centrifuged with identical parameter, be again concentrated to final volume for 500 microlitres；So circulation concentration, Plasma component after by dialysis is finally concentrated to volume for 500 microlitres, used as first time enriched product.

S8：Molecular sieve

By first time enriched product add molecular sieve in, with the Tris- hydrochloride buffer (concentration containing 100mM sodium chloride Be 1mM, pH value for 8) elute, when elution volume be 20ml when start collect eluent, when elution volume be 85ml when stop receive Collection eluent, the eluent that collection is obtained is HC4201615 stostes, as molecular sieve product.

S9：Dialysis

The molecular sieve product is added into aperture to be about in 0.25 nanometer of bag filter, then the bag filter is put into the burning of 1L In cup, then to 20mM Tris hydrochloride buffers (pH8.0) of 1L are added outside bag filter in the beaker, dialyse while stirring, dialyse Volume ratio is 1:5000；Dialysis temperature is 4 DEG C, and dialysis time is 24 hours, collects the product in bag filter as dialysis product.

S10：Concentrate for second

Take a small amount of dialysis product to be added in the concentration tube that volume is 2mL, it is the material of 2.5kD that concentration tube allows size Pass through；Concentration tube is put into centrifuge again, sets centrifugal force as 1500g, design temperature is 4 DEG C, starts centrifuge, is started dense Contracting, until final volume is 500 microlitres；Afterwards, by the addition again of the remaining dialysis product centrifuge tube, so that volume 2mL is reached, is centrifuged with identical parameter, be again concentrated to final volume for 500 microlitres；So circulation concentration, until inciting somebody to action Plasma component after dialysis is finally concentrated to volume for 500 microlitres, used as enriched product, as said mixture HC4201615.

Embodiment 2

In the present embodiment, in addition to S7 is different from embodiment 1, other preparation processes are same as Example 1, in this implementation In example, step S7 is specific as follows：

Anion exchanged product prepared by S7 in embodiment 1 is added in the concentration cup for allowing 2.5kD materials to pass through, and is covered Concentration cup lid, will concentrate the chromatography cabinet that cup is put into, and concentration cup then is connected into liquid nitrogen bottle, open liquid nitrogen bottle air valve, setting pressure Power is 10MPa；Under this air pressure, concentration cup starts product after concentration dialysis, and the volume for observing product after concentrating is before concentrating 1/50 when, stop concentration, obtain first time enriched product.

Embodiment 3

In the present embodiment, in addition to S10 is different from embodiment 1, other preparation processes are same as Example 1, in this reality Apply in example, S10 is specific as follows：

Dialysis product prepared by S10 in embodiment 1 is added in the concentration cup for allowing 2.5kD materials to pass through, and covers concentration Cup lid, will concentrate the chromatography cabinet that is put into of cup, and concentration cup then is connected into liquid nitrogen bottle, open liquid nitrogen bottle air valve, set pressure as 10MPa；Under this air pressure, concentration cup starts product after concentration dialysis, and the volume for observing product after concentrating is 1/ before concentrating When 50, stop concentration, obtain second enriched product, as mixture HC4201615.

Embodiment 4

The present embodiment is the preparation method of mixture HC4201615, and the method is comprised the following steps：

S1：Blood plasma is collected, the S1 operating methods with embodiment 1 are identical.

S2：Filter at low temperature, during the blood plasma that will be collected into adds filter sizes to be 0.45 micron of filter, is applied using peristaltic pump Plus-pressure 15MPa is filtered, and temperature control of whole filter obtains filtrate at 0-4 DEG C during it.

S3：Low temperature ultrafiltration

By the film bag ultrafiltration that the molecular weight that the filtrate retains is 3kD, pressure 20MPa, its process are applied using peristaltic pump The middle buffer solution for using is the Tris hydrochloride buffers of concentration 180mM, pH8.0, and the temperature control of whole ultrafiltration apparatus is in 0-4 DEG C, collect the buffer solution after ultrafiltration and obtain low temperature ultrafiltration product.

S4：Cold ethanol precipitation

By the 0-4 DEG C of ethanol of precooling and the low temperature ultrafiltration product according to volume ratio 1:4 mixing, place 16h, extremely then at 0 DEG C Precipitation automatic sedimentation becomes real, supernatant is obtained, as cold ethanol precipitation product.

S5：SD inactivation centrifugations

The cold ethanol precipitation product and dense SD inactivators (containing 2%TnBP, 2%Tween80) are mixed in equal volume, is put in After 4 DEG C stand 18 hours, it is centrifuged 20 minutes in 2000g, 0 DEG C, retains supernatant, as centrifugation product.

S6：Anion exchange

The centrifugation product is added into anion-exchange column (such as SourceQ, purchased from GE Healthcare, average grain It is 30 microns to spend, and anion exchange column volume is 25 milliliters) in, first with the Tris- hydrochloride buffers containing 80mM sodium chloride (100mM, pH8.0) carries out first time wash-out, retains eluent；Then with the Tris- hydrochloride buffers containing 275mM sodium chloride (100mM, pH8.0) carries out second wash-out, discards eluent；Finally with the Tris- hydrochloride buffers containing 475mM sodium chloride (100mM, pH8.0) carries out third time wash-out, retains eluent；Merge above-mentioned first time and third time afford eluent, As anion exchanged product；Wherein, each elution speed is 4ml/min, and loading speed is 3ml/ml.

S7：Concentrate for the first time

Take a small amount of Anion separation product to be added in the concentration tube that volume is 2mL, it is 1kD's that concentration tube allows size Material passes through；Concentration tube is put into centrifuge again, sets centrifugal force as 1000g, design temperature is 4 DEG C, starts centrifuge, is opened Begin to concentrate, until final volume is 500 microlitres；Afterwards, by the addition again of the remaining dialysis product centrifuge tube, so that Volume reaches 2mL, is centrifuged with identical parameter, is again concentrated to final volume for 500 microlitres；So circulation concentration, directly Plasma component to after by dialysis is finally concentrated to volume for 500 microlitres, used as first time enriched product.

S8：Molecular sieve

During first time enriched product added into molecular sieve, with the Tris- hydrochloride buffers containing 20mM sodium chloride, (concentration is 1mM, pH value start to collect eluent 8) to elute when elution volume is 20ml, stop collecting when elution volume is 85ml Eluent, the eluent that collection is obtained is HC4201615 stostes, as molecular sieve product.

S9：Dialysis

The molecular sieve product is added into aperture to be about in 0.25 nanometer of bag filter, then the bag filter is put into the burning of 1L In cup, then to 20mM Tris hydrochloride buffers (pH8.0) of 1L are added outside bag filter in the beaker, dialyse while stirring, dialyse Volume ratio is 1:100；Dialysis temperature is 4 DEG C, and dialysis time is 40 hours, obtains product of dialysing.

S10：Concentrate for second

Take a small amount of dialysis product to be added in the concentration tube that volume is 2mL, it is the material of 1.5kD that concentration tube allows size Pass through；Concentration tube is put into centrifuge again, sets centrifugal force as 1000g, design temperature is 4 DEG C, starts centrifuge, is started dense Contracting, until final volume is 500 microlitres；Afterwards, by the addition again of the remaining dialysis product centrifuge tube, so that volume 2mL is reached, is centrifuged with identical parameter, be again concentrated to final volume for 500 microlitres；So circulation concentration, until inciting somebody to action Plasma component after dialysis is finally concentrated to volume for 500 microlitres, used as enriched product, as said mixture HC4201615.

Embodiment 5

S2：Filter at low temperature

During the blood plasma that will be collected into adds filter sizes to be 0.45 micron of filter, peristaltic pump is used to apply pressure 20MPa Filtered, temperature control of whole filter obtains filtrate at 0-4 DEG C during it.

S3：Low temperature ultrafiltration

By the film bag ultrafiltration that the molecular weight that the filtrate retains is 10kD, pressure 1MPa, its process are applied using peristaltic pump The middle buffer solution for using is the Tris hydrochloride buffers of concentration 180mM, pH8.0, and the temperature control of whole ultrafiltration apparatus is in 0-4 DEG C, collect the buffer solution after ultrafiltration and obtain low temperature ultrafiltration product.

S4：Cold ethanol precipitation

By the 0-4 DEG C of ethanol of precooling and the low temperature ultrafiltration product according to volume ratio 1:8 mixing, place 10h, extremely then at 0 DEG C Precipitation automatic sedimentation becomes real, supernatant is taken, as cold ethanol precipitation product.

S5：SD is inactivated

The cold ethanol precipitation product is suspended again with SD inactivators (containing 1%TnBP, 1%Tween80), 37 DEG C are put in 6 hours are stood, the product after standing is inactivation product.

S6：Centrifugation

The inactivation product is centrifuged 10 minutes in 10000g, 4 DEG C, retains supernatant, as centrifugation product.

S7：Anion exchange

The centrifugation product is added into anion-exchange column (such as SourceQ, purchased from GE Healthcare, average grain It is 30 microns to spend, and anion exchange column volume is 25 milliliters) in, first with the Tris- hydrochloride buffers containing 50mM sodium chloride (100mM, pH8.0) carries out first time wash-out, retains eluent；Then with the Tris- hydrochloride buffers containing 250mM sodium chloride (100mM, pH8.0) carries out second wash-out, discards eluent；Finally with the Tris- hydrochloride buffers containing 450mM sodium chloride (100mM, pH8.0) carries out third time wash-out, retains eluent；Merge above-mentioned first time and third time afford eluent, As anion exchanged product；Wherein, each elution speed is 4ml/min, and loading speed is 3ml/ml.

S8：Concentrate for the first time

Take a small amount of Anion separation product to be added in the concentration tube that volume is 2mL, it is 2.5kD that concentration tube allows size Material pass through；Concentration tube is put into centrifuge again, sets centrifugal force as 3000g, design temperature is 4 DEG C, starts centrifuge, Start concentration, until final volume is 500 microlitres；Afterwards, by the addition again of the remaining dialysis product centrifuge tube, with Volume is reached 2mL, be centrifuged with identical parameter, be again concentrated to final volume for 500 microlitres；So circulation concentration, Plasma component after by dialysis is finally concentrated to volume for 500 microlitres, used as first time enriched product.

S9：Molecular sieve

By first time enriched product add molecular sieve in, with the Tris- hydrochloride buffer (concentration containing 500mM sodium chloride Be 1mM, pH value for 8) elute, when elution volume be 20ml when start collect eluent, when elution volume be 85ml when stop receive Collection eluent, the eluent that collection is obtained is HC4201615 stostes, as molecular sieve product.

S10：Dialysis

The molecular sieve product is added into aperture to be about in 0.25 nanometer of bag filter, then the bag filter is put into the burning of 1L In cup, then to 1mM Tris hydrochloride buffers (pH8.0) of 1L are added outside bag filter in the beaker, dialyse while stirring, dialyse Volume ratio is 1:10000；Dialysis temperature is 4 DEG C, and dialysis time is 20 hours, obtains product of dialysing.

S11：Concentrate for second

Take a small amount of dialysis product to be added in the concentration tube that volume is 2mL, concentration tube allows size to lead to for the material of 2kD Cross；Concentration tube is put into centrifuge again, sets centrifugal force as 3000g, design temperature is 4 DEG C, starts centrifuge, is started dense Contracting, until final volume is 500 microlitres；Afterwards, by the addition again of the remaining dialysis product centrifuge tube, so that volume 2mL is reached, is centrifuged with identical parameter, be again concentrated to final volume for 500 microlitres；So circulation concentration, until inciting somebody to action Plasma component after dialysis is finally concentrated to volume for 500 microlitres, used as enriched product, as said mixture HC4201615.

Embodiment 6

S2：Filter at low temperature

During the blood plasma that will be collected into adds filter sizes to be 0.22 micron of filter, apply pressure 1MPa using peristaltic pump and enter Row filtering, temperature control of whole filter obtains filtrate at 0-4 DEG C during it.

S3：Low temperature ultrafiltration

By the film bag ultrafiltration that the molecular weight that the filtrate retains is 5kD, pressure 15MPa, its process are applied using peristaltic pump The middle buffer solution for using for concentration 1mM, pH8.0 Tris hydrochloride buffers, the temperature control of whole ultrafiltration apparatus at 0-4 DEG C, Collect the buffer solution after ultrafiltration and obtain low temperature ultrafiltration product.

S4：Cold ethanol precipitation

By the 0-4 DEG C of ethanol of precooling and the low temperature ultrafiltration product according to volume ratio 1:5 mixing, place 12h, extremely then at 0 DEG C Precipitation automatic sedimentation becomes real, supernatant is taken, as cold ethanol precipitation product.

S5：SD inactivation centrifugations

The cold ethanol precipitation product is suspended again with SD inactivators (containing 1%TnBP, 1%Tween80), 20 DEG C are put in After standing 15 hours, it is centrifuged 15 minutes in 6000g, 6 DEG C, retains supernatant, as centrifugation product.

S6：Anion exchange

The centrifugation product is added into anion-exchange column (such as SourceQ, purchased from GE Healthcare, average grain It is 3 microns to spend, and anion exchange column volume is 25 milliliters) in, first with the Tris- hydrochloride buffers containing 100mM sodium chloride (100mM, pH8.0) carries out first time wash-out, retains eluent；Then with the Tris- hydrochloride buffers containing 300mM sodium chloride (100mM, pH8.0) carries out second wash-out, discards eluent；Finally with the Tris- hydrochloride buffers containing 500mM sodium chloride (100mM, pH8.0) carries out third time wash-out, retains eluent；Merge above-mentioned first time and third time afford eluent, As anion exchanged product；Wherein, each elution speed is 4ml/min, and loading speed is 3ml/ml.

In above-mentioned each wash-out, start to collect when waiting UV-detector to show and albumen occur, albumen goes out to be over, and stops Collect, i.e., the eluent for being eluted each time..

S7：Once concentrate

Take a small amount of Anion separation product to be added in the concentration tube that volume is 2mL, it is 2kD's that concentration tube allows size Material passes through；Concentration tube is put into centrifuge again, sets centrifugal force as 2000g, design temperature is 4 DEG C, starts centrifuge, is opened Begin to concentrate, until final volume is 500 microlitres；Afterwards, by the addition again of the remaining dialysis product centrifuge tube, so that Volume reaches 2mL, is centrifuged with identical parameter, is again concentrated to final volume for 500 microlitres；So circulation concentration, directly Plasma component to after by dialysis is finally concentrated to volume for 500 microlitres, used as first time enriched product.

S8：Molecular sieve

By first time enriched product add molecular sieve in, with the Tris- hydrochloride buffer (concentration containing 300mM sodium chloride Be 1mM, pH value for 8) elute, when elution volume be 20ml when start collect eluent, when elution volume be 85ml when stop receive Collection eluent, the eluent that collection is obtained is HC4201615 stostes, as molecular sieve product.

S9：Dialysis

The molecular sieve product is added into aperture to be about in 0.25 nanometer of bag filter, then the bag filter is put into the burning of 1L In cup, then to 1mM Tris hydrochloride buffers (pH8.0) of 1L are added outside bag filter in the beaker, dialyse while stirring, dialyse Volume ratio is 1:2500；Dialysis temperature is 4 DEG C, and dialysis time is 72 hours, obtains product of dialysing.

S10：Concentrate for second

Take a small amount of dialysis product to be added in the concentration tube that volume is 2mL, concentration tube allows size to lead to for the material of 1kD Cross；Concentration tube is put into centrifuge again, sets centrifugal force as 2000g, design temperature is 4 DEG C, starts centrifuge, is started dense Contracting, until final volume is 500 microlitres；Afterwards, by the addition again of the remaining dialysis product centrifuge tube, so that volume 2mL is reached, is centrifuged with identical parameter, be again concentrated to final volume for 500 microlitres；So circulation concentration, until inciting somebody to action Plasma component after dialysis is finally concentrated to volume for 500 microlitres, used as enriched product, as said mixture HC4201615.

Test case：

This test case is to be detected mixture HC4201615 prepared by embodiment 1-6 respectively.

First, SDS-PAGE denaturation gel electrophoresis identification, the authentication method is comprised the following steps：

(1) 2 microlitres are taken out from the mixture HC4201615 for finally preparing, its extinction is determined under ultraviolet 280nm Degree, so as to calculate the protein concentration of mixture HC4201615.

(2) the mixture HC4201615 of certain volume is taken, it is (blue rich purchased from Beijing with 1 microlitre of 5 × albumen sample-loading buffer Li De Bioisystech Co., Ltd, article No. D621) mixing, as need to carry out the sample of electrophoresis, there are 10 micrograms sample the inside Protein.

(3) electrophoresis Sample is warming up to 100 DEG C, heating makes protein denaturation in 20 minutes, and sample is put into ice immediately after On, start to run SDS-PAGE denaturation after waiting 5 minutes and go back virgin rubber (compound method that virgin rubber is gone back in SDS denaturation is as follows：30(w/ V) the acrylamide Acr-Bis (being purchased from GE Healthcare) of % takes 1.3ml, 1.5M Tris- hydrochloride buffers (pH8.8, purchase From GE Healthcare) take 1.3ml, the SDS of 10 (w/v) % and take 0.05ml, 10% (w/v) ammonium persulfate (purchased from GE Healthcare) take that 0.05ml, TEMED (purchased from GE Healthcare) take 0.003ml and water takes 2.3ml, altogether 5ml, Plastic can be solidified in room temperature) after mixing.When running glue, the albumen applied sample amount of each swimming lane is 10 micrograms, set run glue voltage as 100V, starts to run glue, runs the glue time for 1 hour.

(4) after running through glue, with coomassie brilliant blue staining liquid, (preparation method of the dyeing liquor is：By 2.5g Coomassie brilliant blues R-250 is dissolved in the ethanol solutions of 500ml 95%, adds the acetic acid solution of 100ml 85%, then, is supplemented to distilled water 1000ml, this dye liquor puts 4 DEG C and keeps stabilization at least 6 months) glue is dyeed.

The testing result of mixture HC4201615 prepared by embodiment 1 is referring to Fig. 1：Wherein：Swimming lane M：Molecular weight of albumen mark It is accurate；Swimming lane 1-4：HC4201615 mixtures；At least contain 11 visible polypeptides of naked eyes, its molecule in mixture HC4201615 Amount be respectively be successively from small to large 25kD, 37kD, 41kD, 51kD, 66kD, 69kD, 88kD, 105kD, 145kD, 170kD, 200kD.The testing result of embodiment 2-6 is same as Example 1.

2nd, proteomic image identification, the authentication method comprises the following steps：

(1) the mixture HC4201615 that will finally prepare is transferred in FALCON pipes, adds the sample of two volumes (formula of buffer solution is buffer solution：7.5M urea UREA, 1.5M thiocarbamide THIOUREA, 4 (w/v) %3- [3- (courage amido propyl) Dimethylamino] propane sulfonic acid inner salt CHAPS, 0.05 (w/v) % lauryl sodium sulfate SDS, 100mM dithiothreitol (DTT) DDT, each group Shown concentration before point is concentration of its respective components in buffer solution), and by 3kDa molecular weight cut- Off spin columns (Pall GmbH, Austria) centrifuge tube centrifugal concentrating, obtains concentrate.

(2) concentrate carries out reduction reaction with dithiothreitol (DTT), obtains reduzate；Wherein, concentrate and two sulphur threoses The volume ratio of alcohol is 1：3, the reaction time is 15 minutes, and temperature is room temperature.

(3) reduzate and iodoacetamide are reacted again, is obtained alkylate；Wherein, the reduzate with The volume ratio 1 of iodoacetamide：1, the reaction time is 15 minutes, and temperature is room temperature；

(4) then digestion reaction is carried out overnight at 37 DEG C with trypsase, wherein the body of alkylate and trypsase Product compares 1:1, obtain postdigestive peptide fragment.

(5) Trypsin Induced gained peptide fragment obtains sample by C18 chromatographies.

(6) gained sample traditional vacuum is dried and is then stored in -20 DEG C of refrigerators and analyzed for MS/MS.MS/MS is analyzed It is specific as follows：HPLC's is the systems of Ultimate 3000, wherein being equipped with (300 μm of PepMap100C-18trap column × 5mm) and two pillars of PepMap100C-18analytical column (75 μ m 250mm).Mass spectrograph uses Amazon Speed ETD, MS data acquisition range are 400-1400m/z, and the peptide fragment process range of MS/MS is 100-2800m/z.Then, Each MS data can automatically search for the top-quality CID MS/MS peaks spectrum of matched three.Jet hole voltage is set to 1400 volts.The temperature of nitrogen protection is 150 DEG C, and flow velocity is 3 liters/min.The protein identification of MS and unmarked quantitative (LFQ) Data analysis uses open-source software MaxQuant 1.3.0.5.By searching for SwissProt databases (version 10/ 2003 20354) protein to be identified, the qualification result of mixture HC4201615 prepared by embodiment 1 is referring to table 1. The testing result of embodiment 2-6 is same as Example 1.

3rd, mixture HC4201615 prepared by detection embodiment 1-6 has the external work for suppressing primary hippocampal cells apoptosis Property, promote hippocampal cell between Synaptic formation activity.The detection method is comprised the following steps：

(1) 2 microlitres are taken out from the mixture HC4201615 for finally preparing, its extinction is determined under ultraviolet 280nm Degree, so as to calculate the protein concentration of said mixture HC4201615.

(2) with 24 orifice plate culture primary hippocampal cells, culture used medium is DMEM, and condition of culture is 37 DEG C, 5vol% carbon dioxide.

(3) when cell density reaches every hole 4 × 10⁵During individual cell, toward culture medium in add mixture HC4201615, The mixture HC4201615 micrograms containing protein 10 00 added in each hole, as experimental group 1- mixtures HC4201615.Simultaneously Also include in experimental group：I.e. toward being separately added into the human plasma (embodiment 1 that protein content is 1000 micrograms in the culture medium in different holes The step of preparation method (1), prepares), it is named as experimental group 2- human plasmas.Control group is set simultaneously, in control group, etc. Every hole 4 × 10 is reached to cell density⁵During individual cell, 100 microlitres of physiological saline is added toward the culture medium in hole.

(4) cultured cells is continued 24 hours under conditions of original, then under an optical microscope to each experimental group and right The counting of cell quantity and cynapse quantity is carried out according to group.

The result of mixture HC4201615 prepared by embodiment 1, as in Figure 2-4, wherein Fig. 2 (a), (b), (c) difference It is according to the primary hippocampal cells obtained after experimental group 1-HC4201614, experimental group 2- human plasmas, control group treatment.Observable To the cynapse quantity that either hippocampal cell quantity is still formed, experimental group 1-HC4201615 mixtures are far longer than control Group, more than experimental group 2- human plasmas；In Fig. 3, the quantity of the primary hippocampal cells of experimental group 1 reaches about 1500-1800/square Centimetre, much larger than about 650-900/square centimeter of experimental group 2, and control group about 200-600/square centimeter, it was demonstrated that Mixture HC4201615 has the activity for suppressing primary hippocampal cells apoptosis.In Fig. 4, the Synaptic formation quantity of experimental group 1 reaches About 165/square centimeter, much larger than about 75/square centimeter of experimental group 2, and control group about 60/square centimeter, card Bright separator HC4201615 has the activity of Synaptic formation between promotion hippocampal cell.

The result of mixture HC4201615 prepared by embodiment 2 is as follows：The quantity of the primary hippocampal cells of experimental group 1 reaches To about 1350-1800/square centimeter, the about 200- of/square centimeter individual much larger than the about 650-900 of experimental group 2, and control group 600/square centimeter, it was demonstrated that mixture HC4201615 has the activity for suppressing primary hippocampal cells apoptosis.Experimental group 1 it is prominent Tactile quantity of formation reaches about 150/square centimeter, much larger than about 70/square centimeter of experimental group 2, and control group about 60 Individual/square centimeter, it was demonstrated that separator HC4201615 has the activity of Synaptic formation between promotion hippocampal cell.Prove embodiment 2 The HC4201615 mixtures of preparation have the activity of Synaptic formation between promotion hippocampal cell.

The result of mixture HC4201615 prepared by embodiment 3 is as follows：The quantity of the primary hippocampal cells of experimental group 1 reaches To about 1000-1600/square centimeter, the about 400- of/square centimeter individual much larger than the about 500-750 of experimental group 2, and control group 700/square centimeter, it was demonstrated that mixture HC4201615 has the activity for suppressing primary hippocampal cells apoptosis.Experimental group 1 it is prominent Tactile quantity of formation reaches about 150/square centimeter, much larger than about 70/square centimeter of experimental group 2, and control group about 50 Individual/square centimeter, it was demonstrated that separator HC4201615 has the activity of Synaptic formation between promotion hippocampal cell.Prove embodiment 3 The HC4201615 mixtures of preparation have the activity of Synaptic formation between promotion hippocampal cell.

The result of mixture HC4201615 prepared by embodiment 4 is as follows：The quantity of the primary hippocampal cells of experimental group 1 reaches To about 1200-1800/square centimeter, the about 200- of/square centimeter individual much larger than the about 650-900 of experimental group 2, and control group 600/square centimeter, it was demonstrated that mixture HC4201615 has the activity for suppressing primary hippocampal cells apoptosis.Experimental group 1 it is prominent Tactile quantity of formation reaches about 120/square centimeter, much larger than about 70/square centimeter of experimental group 2, and control group about 60 Individual/square centimeter, it was demonstrated that separator HC4201615 has the activity of Synaptic formation between promotion hippocampal cell.Prove embodiment 4 The HC4201615 mixtures of preparation have the activity of Synaptic formation between promotion hippocampal cell.

The result of mixture HC4201615 prepared by embodiment 5 is as follows：The quantity of the primary hippocampal cells of experimental group 1 reaches To about 1200-1900/square centimeter, the about 200- of/square centimeter individual much larger than the about 650-900 of experimental group 2, and control group 600/square centimeter, it was demonstrated that mixture HC4201615 has the activity for suppressing primary hippocampal cells apoptosis.Experimental group 1 it is prominent Tactile quantity of formation reaches about 100/square centimeter, much larger than about 70/square centimeter of experimental group 2, and control group about 60 Individual/square centimeter, it was demonstrated that separator HC4201615 has the activity of Synaptic formation between promotion hippocampal cell.Prove embodiment 5 The HC4201615 mixtures of preparation have the activity of Synaptic formation between promotion hippocampal cell.

The result of mixture HC4201615 prepared by embodiment 6 is as follows：The quantity of the primary hippocampal cells of experimental group 1 reaches To about 1200-1900/square centimeter, the about 200- of/square centimeter individual much larger than the about 650-900 of experimental group 2, and control group 600/square centimeter, it was demonstrated that mixture HC4201615 has the activity for suppressing primary hippocampal cells apoptosis.Experimental group 1 it is prominent Tactile quantity of formation reaches about 100/square centimeter, much larger than about 70/square centimeter of experimental group 2, and control group about 60 Individual/square centimeter, it was demonstrated that separator HC4201615 has the activity of Synaptic formation between promotion hippocampal cell.Prove embodiment 6 The HC4201615 mixtures of preparation have the activity of Synaptic formation between promotion hippocampal cell.

4th, mixture HC4201615 prepared by detection embodiment 1-6 can effectively lift alzheimer's disease (old age is silly Slow-witted disease) mouse memory, the detection method comprises the following steps：

(1) the present embodiment uses 5XFAD Elderly dementia patients models, orders in U.S. jackson laboratory, and Bred and raised according to zoopery standard；Each of which experimental model mouse all carries out identified for genes by rat-tail, Ensure the stabilization mutation of app gene and PS1 genes.

Mouse is grouped into：Experimental group 1- mixtures HC4201615：Using 20 14 week old 5XFAD male mices, injection Mixture HC4201615 prepared by embodiment 1；Experimental group 2- human plasmas：Using 20 14 week old 5XFAD male mices, injection The blood plasma that the step of 1 preparation method of embodiment (1) prepares；Control group：Using 20 14 week old 5XFAD male mices, note Penetrate physiological saline.

Various administrations are entered in Mice Body by tail vein injection, experimental group ensure 30 micrograms of protein/time dosage, Behaviors survey (specially water maze laboratory, learning ability and memory for detecting mouse) in first 24 days every three days to Medicine 1 time, is administered 8 times altogether.

(2) water maze laboratory：Carried out between 8 points of every morning at 1 point in afternoon.Water maze spatial memory training period is 4 days, Four times a day, training interval time is 10 minutes every time；In an experiment, every four mouse are randomly divided into a training group.For Each training group, the position of platform of water maze is probabilistically assigned, and keeps constant in whole training.In training, mouse from appoint Meaning position is released into water maze, and allows it that hiding platform was searched in 120 seconds.If mouse was not looked in 120 seconds To platform, it will be directed into platform.The time used by platform is found in training every time with the distance passed through by intelligent camera Head automatically records.

Water maze test is carried out after last time is trained 48 hours.Every time in test, every mouse is released into not to be had In the water maze of placement platform, and allow its freely activity 60 second.Its travelling route is automatically recorded by intelligent video camera head.Survey The examination phase is shorter than time training period one times, to avoid mouse from producing Depressive behavior.Time that mouse is spent in target quadrant and its The time that his three quadrants are spent is recorded, for the assessment to mouse memory power.

The result of mixture HC4201615 prepared by embodiment 1 is as shown in figure 5, the target quadrant of experimental group 1 stops memory Time is about 100 seconds, considerably longer than about 10 seconds of about the 25 of experimental group 2 second and control group.

The result of mixture HC4201615 prepared by embodiment 2 is as follows：The target quadrant of experimental group 1 stops memory time About 100 seconds, considerably longer than about 10 seconds of about the 25 of experimental group 2 second and control group.

The result of mixture HC4201615 prepared by embodiment 3 is as follows：The target quadrant of experimental group 1 stops memory time About 90 seconds, considerably longer than about 5 seconds of about the 10 of experimental group 2 second and control group.

The result of mixture HC4201615 prepared by embodiment 4 is as follows：The target quadrant of experimental group 1 stops memory time About 90 seconds, considerably longer than about 10 seconds of about the 25 of experimental group 2 second and control group.

The result of mixture HC4201615 prepared by embodiment 5 is as follows：The target quadrant of experimental group 1 stops memory time About 95 seconds, considerably longer than about 10 seconds of about the 25 of experimental group 2 second and control group.

The result of mixture HC4201615 prepared by embodiment 6 is as follows：The target quadrant of experimental group 1 stops memory time About 100 seconds, considerably longer than about 10 seconds of about the 25 of experimental group 2 second and control group.

Comparative example 1

In addition to the molecular weight of the film bag retention used in step (3) low temperature ultrafiltration step is for 13kD, other operating procedures It is same as Example 1.

The final product that the comparative example is obtained is named as C1, and C1 electrophoresis results are as follows：25kD、37kD、41kD、51kD、 66kD、69kD、88kD、105kD、145kD、170kD、200kD；Suppressed using product C1 is tested with test case identical method The activity of primary hippocampal cells apoptosis, the activity for promoting Synaptic formation between hippocampal cell, its result are as follows：

The quantity of the primary hippocampal cells of experimental group 1 reaches about 850-980/square centimeter, the about 640- of experimental group 2 850/square centimeter, and control group about 350-550/square centimeter.The Synaptic formation quantity of experimental group 1 reaches about 75 Individual/square centimeter, about 65/square centimeter of experimental group 2, about 55/square centimeter of control group.

Comparative example 2

In addition to step (4) cold ethanol precipitation step is eliminated, other operating procedures are same as Example 1.

The final product that the comparative example is obtained is named as C2, and C2 electrophoresis results are as follows：12kD、25kD、37kD、41kD、 51kD、66kD、69kD、88kD、105kD、145kD、170kD、200kD；The product is tested using with test case identical method C2 suppresses the activity of primary hippocampal cells apoptosis, promotes the activity of Synaptic formation between hippocampal cell, and its result is as follows：

The quantity of the primary hippocampal cells of experimental group 1 reaches about individual/square centimeter, about 630-840 of experimental group 2/flat Square centimetre, and control group about 650-840/square centimeter.The Synaptic formation quantity of experimental group 1 reaches about 79/square li Rice, about 70/square centimeter of experimental group 2, about 58/square centimeter of control group.

Comparative example 3

In addition to step (5) and (6) are eliminated, other operating procedures are same as Example 1.

The final product that the comparative example is obtained is named as C3, and C3 electrophoresis results are as follows：25kD、37kD、41kD、51kD、 66kD、69kD、88kD、105kD、145kD、170kD、200kD；Suppressed using product C3 is tested with test case identical method The activity of primary hippocampal cells apoptosis, the activity for promoting Synaptic formation between hippocampal cell, its result are as follows：

The quantity of the primary hippocampal cells of experimental group 1 reaches about 840-970/square centimeter, the about 650- of experimental group 2 840/square centimeter, and control group about 360-540/square centimeter.The Synaptic formation quantity of experimental group 1 reaches about 78 Individual/square centimeter, about 66/square centimeter of experimental group 2, about 58/square centimeter of control group.

Comparative example 4

In addition to step (8) and (9) are eliminated, other operating procedures are same as Example 1.

The final product that the comparative example is obtained is named as C4, and C4 electrophoresis results are as follows：15kD、20kD、25kD、37kD、 41kD、51kD、66kD、69kD、88kD、105kD、145kD、170kD、200kD；It is somebody's turn to do using with the test of test case identical method Product C4 suppresses the activity of primary hippocampal cells apoptosis, promotes the activity of Synaptic formation between hippocampal cell, and its result is as follows：

The quantity of the primary hippocampal cells of experimental group 1 reaches about 830-950/square centimeter, the about 650- of experimental group 2 840/square centimeter, and control group about 380-600/square centimeter.The Synaptic formation quantity of experimental group 1 reaches about 78 Individual/square centimeter, about 66/square centimeter of experimental group 2, about 58/square centimeter of control group.

Comparative example 5

In addition to the anion exchange step and the difference of embodiment 1 of step (7), other operating procedures are same as Example 1. The type of elution of step (7) is that gradient elution is eluted rather than step level, only have collected an eluent without repeatedly being divided Stage collects eluent.Anion exchange step is as follows in this comparative example：By centrifugation product, 200 microlitres are added to anion In exchange column Source Q, gradient elution is carried out with the Tris hydrochloride buffers (200mM, pH8.0) containing 500mM sodium chloride, Wherein the filler of anion-exchange column is Q Sepharose (being purchased from GE Healthcare), and particle mean size is 3 microns, anion It is 25 milliliters to exchange column volume, and elution speed is 4ml/min, and loading speed is 3ml/ml；Opened when elution volume is 35 milliliters Begin to collect eluent, stop collecting eluent when elution volume is 45 milliliters；The elution fractions being collected into, as the moon from Son exchanges product.

The final product that the comparative example is obtained is named as C5, and C5 electrophoresis results are as follows：41kD、51kD、66kD、69kD、 88kD、105kD、145kD、170kD；Suppress primary hippocampal cells apoptosis using product C5 is tested with test case identical method Activity, promote the activity of Synaptic formation between hippocampal cell, its result is as follows：

The quantity of the primary hippocampal cells of experimental group 1 reaches about 840-940/square centimeter, the about 650- of experimental group 2 840/square centimeter, and control group about 360-580/square centimeter.The Synaptic formation quantity of experimental group 1 reaches about 78 Individual/square centimeter, about 66/square centimeter of experimental group 2, about 56/square centimeter of control group.

Claims

It is 1. a kind of to improve the mixture for from blood plasma separate remembered, it is characterised in that：The SDS-PAGE denaturation glue of the mixture Electrophoresis at least includes the high-visible band of 11 naked eyes, and molecular size range is respectively：25kD、37kD、41kD、51kD、66kD、 69kD、88kD、105kD、145kD、170kD、200kD。
2. mixture according to claim 1, it is characterised in that：Analyzed by protein spectrum, at least contained in the mixture There are 106 kinds of albumen；Preferably, the mixture also includes and any protein bound small molecule in 106 kinds of albumen； It is highly preferred that 106 kinds of albumen includes：

What 14-3-3 albumen ζ/δ, 14-3-3 protein ε, 40S ribosomal protein S1 5, AP-4 complex subunits β -1, BPI were folded Family B member 1, BPI folds family B member 2, DNA recovery supports albumen, the isotype 2 of Hemicentin-1, HORMA structures Domain albumen 2, Ig λ chains, LVV- angiogenic peptides -7, LDH heartunit, MOB kinase activation agent 1B, NAD (P) transhydrogenase, Rho GDP- dissociation inhibitor 2, SLIT-NTRK samples albumen 4, S-adenosylmethionine synthase, TNFSF4 albumen, α -2-HS- glycoprotein, β -2- glycoprotein 1, γ-enolase, δ Lf Lactotransferrins, vitronectin, BLMH, C1Q subfraction are sub- Base C, Complement C_3, wing bud startup factor homologue, haptoglobin, prolactin inducible protein, the isotype of albumen argonaute-3 2nd, Protein S 100-A9, dimethylaniline monooxygenase 2, ubiquitin -60S ribosomal protein Ls 40, the α -2- sugar eggs rich in leucine In vain, Statherin, rich saliva acidity proline phosphoprotein 1/2, hepatocyte growth factor-like protein, glutamine gamma-glutamyl Transferase E, glutathione S-transferase P, glutathione peroxidase 3, lumican, fructosediphosphate aldolase A, mistake Site albumen 2, peroxisomal membrane protein PMP34, peroxide thing toxin 2, nuclear receptor conactivator 1, succinyl-CoA connections Enzyme subunit α, actin -1, basic saliva Pro-rich albumen 2, TBG, potassium voltage-gated ion Passage, basic peptide IB-6, with reference to ferrihemoglobin, argininosuccinate synthetase, polymerization immunoglobulin receptor, lysine- TRNA ligases, phosphotriose isomerase, ribose phosphate formoxyl glycinamidine cyclo-ligase, phosphoric acid cluster sequence albumen 2, chlorine from It is sub- anionite, zymogen granule protein 16 homologue B, mycin, annexin, olic acid soluble protein 1, fibrin ferment 2, solidifying Blood factor XIII B chains, malic dehydrogenase, G-6-P 1- dehydrogenases, factor Ⅰ isotype CRA_b, desmosome Spot albumen, desmoglein -1, humanized's kallikrein associated proteins, lysozyme C, lactoperoxidase, mammary gland pearl egg In vain-B, pigment epidermal derived factors, upper storage reservoir albumen, the scrotum element -3, retinol-binding proteins, difunctional 3'- AMPs 5'- Phosphosulfate synthase 1, serpin B12, silk polyprotein, the isoform H14 of myeloperoxidase, myosin B, tubulin α -1A chains, DBP, the isotype 2, extracellular CaM of enolase-phosphatase E1, It is plasminogen, zinc-α -2- glycoprotein, the isotype 2 of zinc finger protein DZIP1L, zinc transporter, sex hormone binding globulin, blood red Albumen, plasma kallikrein, ceruloplasmin, serum ferritin, blood platelet rabphilin Rab, blood platelet rabphilin Rab 2 Isotype 1, former albumen fat 3, aPoA, mucoprotein -5B, the sample albumen 1 of NGAL 1, neutrophil leucocyte alexin 1, turn Change grouth factor beta receptor-related proteins 1, cathepsin D.
3. mixture according to claim 1, it is characterised in that：The blood plasma derives from mammal；Preferably, the blood Slurry derives from the mankind.
4. the preparation method of any mixtures of a kind of claim 1-3, it is characterised in that：The preparation method successively include with Lower step：Plasma collection, filter at low temperature, low temperature ultrafiltration, cold ethanol precipitation, SD inactivations, centrifugation, anion exchange, first time are dense Contracting, molecular sieve, dialysis, second concentration step；Preferably, also include between the plasma collection and cold ethanol precipitation step： Filter at low temperature and low temperature ultrafiltration step.
5. preparation method according to claim 4, it is characterised in that：In the plasma collection procedure, using anticoagulant tube Blood is collected, then by the way that supernatant is collected by centrifugation, obtains blood plasma；

Preferably, in the centrifugation, centrifugal force is 500-1000g, and the time is 10-30 minutes, and centrifuging temperature is 0-10 DEG C.
6. the preparation method according to claim 4 or 5, it is characterised in that：In the filter at low temperature step, in pressure strip The blood plasma is filtered under part, is obtained filtrate；

Preferably, the aperture of filter membrane is that, not less than 0.22 micron, the aperture of more preferably described filter membrane is that 0.22 micron and 0.45 are micro- Rice；

Preferably, the temperature of the filtering is 0-5 DEG C, and pressure is 1-20MPa.
7. according to any described preparation methods of claim 4-6, it is characterised in that：In the low temperature ultrafiltration step, by institute State filtrate carries out film bag ultrafiltration by applying pressure, obtains low temperature ultrafiltration product；

Preferably, the film bag molecular cut off is 3kD-10kD；

Preferably, the temperature of the ultrafiltration is 0-5 DEG C, and pressure is 1-20MPa；

Preferably, the buffer solution that the ultrafiltration is used is in phosphate buffer, Tris- hydrochloride buffers, HBS buffer solutions Kind；It is highly preferred that the concentration of the Tris- hydrochloride buffers be 0.2-250mM, pH value be 7.0-9.2, the phosphate buffer Concentration be 1.0-450mM, pH value be 6.0-9.2, the concentration of the HBS buffer solutions is 0.5-600mM, pH value is 6.4-8.4； It is further preferred that the buffer solution is concentration 180mM, the Tris- hydrochloride buffers that pH value is 8.0.
8. according to any described preparation methods of claim 4-7, it is characterised in that：In the cold ethanol precipitation step, will The low temperature ultrafiltration product or blood plasma stand after mixing with cold ethanol, supernatant are taken, as cold ethanol precipitation product；

Preferably, the temperature of the cold ethanol is 0-10 DEG C；

Preferably, the volume ratio of the cold ethanol and the low temperature ultrafiltration product or blood plasma is 1：(4-8)；

Preferably, the dwell temperature is 0-6 DEG C.
9. according to any described preparation methods of claim 4-8, it is characterised in that：

In the SD inactivation steps, stood after the cold ethanol precipitation product is suspended again with dense SD inactivators, gone out Life birth thing；Preferably, the dwell temperature is 4~37 DEG C, and the time is 6~18 hours；More preferably described dwell temperature is 20 DEG C, the time is 12 hours；Preferably, the dense SD inactivators contain N- butyl triphosphate, the tween of 1-4% of 0.6-4%.

In the step with centrifugal separation, supernatant will be retained after the inactivation product centrifugation, obtain centrifugation product；It is preferred that Ground, in the centrifugation, centrifugal force is 2000-10000g, and the time is 10-20 minutes, and temperature is 0-8 DEG C.
10. according to any described preparation methods of claim 4-9, it is characterised in that：In the anion exchange step, will The centrifugation product carries out step level wash-out in adding anion-exchange column, collects the eluent for obtaining every time, after mixing To anion exchanged product；

Preferably, in the step level wash-out, first eluted with first time elution buffer, obtain first time eluent；Again with second Secondary elution, obtains waste liquid；Third time elution is used again, obtains third time eluent；Merging the first time washes De- liquid and third time eluent, obtain anion exchanged product；

It is highly preferred that the sodium chloride containing 50-100mM in the first time eluent buffer solution, second eluent delays Fliud flushing contains the sodium chloride of 250-300mM, and the third time eluent buffer solution contains the sodium chloride of 450-500mM；More preferably Ground, the first time elution buffer, second elution buffer, third time elution buffer are concentration 0.2-200mM, pH The Tris- hydrochloride buffers of value 7.0-9.2.
11. according to any described preparation methods of claim 4-10, it is characterised in that：In the first time concentration step, will The anion exchanged product is concentrated, and obtains first time enriched product；

Preferably, the volume of the anion exchanged product is the enriched product more than or equal to 5 times, more preferably 5-500 Times；It is highly preferred that the volume of the first time enriched product is 500 microlitres -10 milliliters；

Preferably, the concentration is centrifuged using concentration tube, and the concentration tube allows the material of 1.0-2.5KD to pass through, it is described from In the heart, centrifugal force is 1000-3000g, and temperature is 2-8 DEG C；

Preferably, the concentration is to use concentration cup pressurization, and the concentration cup allows the material of 1.0-2.5KD to pass through, described to add The pressure of pressure is 1-15MPa.
12. according to any described preparation methods of claim 4-11, it is characterised in that：In the molecular sieve step, will be described First time enriched product is eluted in molecular sieve, obtains molecular sieve product；

Preferably, the sodium chloride containing 20-500mM in the elution buffer of the wash-out；

Preferably, the elution buffer is Tris- hydrochloride buffers, and its concentration is 0.2-200mM, and its pH value is 7.0-9.2； It is highly preferred that the elution buffer is Tris- hydrochloride buffers, its concentration is 20mM, and its pH value is 8；

Preferably, elution volume when starting to collect eluent is 15-35ml, and elution volume when stopping collecting eluent is 40-85ml。
13. according to any described preparation methods of claim 4-12, it is characterised in that：In the dialysis step, will be described Molecular sieve product is dialysed, and is collected the product in Dialysis container and is obtained product of dialysing；

Preferably, the elution buffer for using of dialysing is in Tris- hydrochloride buffers, phosphate buffer, HBS buffer solutions It is a kind of；It is highly preferred that the concentration of the Tris- hydrochloride buffers be 0.2-250mM, pH value be 7.0-9.2, the phosphoric acid buffer The concentration of liquid is that 1.0-450mM, pH value are 6.0-9.2, and the concentration of the HBS buffer solutions is 0.5-600mM, pH value is 6.4- 8.4；It is further preferred that the elution buffer is 1mM, the Tris hydrochloride buffers that pH value is 8；

Preferably, the time of the dialysis is 20-72h；

Preferably, the volume ratio of the dialysis is 1:(100-10000).
14. according to any described preparation methods of claim 4-13, it is characterised in that：In the concentration step, will be described Dialysis product carries out second concentration, obtains second enriched product, as described mixture；

Preferably, the volume of the dialysis product is the enriched product more than or equal to 20 times, more preferably 20-100 times；

Preferably, the concentration is centrifuged using concentration tube, and the concentration tube allows the material of 1.0-2.5KD to pass through, it is described from In the heart, centrifugal force is 1000-3000g；

Preferably, the concentration is to use concentration cup pressurization, and the concentration cup allows the material of 1.0-2.5KD to pass through, described to add The pressure of pressure is 1-20MPa.
15. a kind of pharmaceutical compositions, comprising any described mixtures of claim 1-3 and pharmaceutically acceptable carrier.
16. pharmaceutical compositions according to claim 15, it is characterised in that the pharmaceutically acceptable carrier is：Medicine Acceptable buffer solution, protein, gelatin, monose, polysaccharide, amino acid, chelating agent, sugar alcohol, polyethylene glycol and surface on One or more in activating agent.
17. pharmaceutical compositions according to claim 16, it is characterised in that described pharmaceutical composition includes following component：1 Any described mixtures of claim 1-3 of times volume, the 8.5wt%NaCl of 9 times of volumes or the PBS of 1.5M, pH7.0；

Preferably, albumin, glucose and glutamine are also included in described pharmaceutical composition；It is highly preferred that the white egg The white quality percent by volume in described pharmaceutical composition is 2%, quality of the glucose in described pharmaceutical composition Percent by volume is 1%, and quality percent by volume of the glutamine in described pharmaceutical composition is 3%.
A kind of 18. sustained release preparations, it is any described comprising any described mixtures of claim 1-3 or claim 15-17 Pharmaceutical composition and pharmaceutically acceptable biocompatible substance；Preferably, the formulation of the sustained release preparation is lipid Body, microballoon, hydrogel, Osmotic minipumps or microcapsules.
19. a kind of kits, comprising any described mixtures of claim 1-3, any described medicines of claim 15-17 Composition, or sustained release preparation described in claim 18.
Any described mixtures of 20. claim 1-3, claim 15-17 any described pharmaceutical composition, claim Kit described in sustained release preparation, claim 19 described in 18, prevention, improve or/and treatment senile dementia in or Application in enhancing memory medicine.