CN109512838A

CN109512838A - A kind of rabbit fur extractive and its preparation method and application

Info

Publication number: CN109512838A
Application number: CN201710829641.1A
Authority: CN
Inventors: 冯世庆; 孔晓红; 姚雪; 冯翊坤; 李波; 孙超
Original assignee: Tianjin Xiaoxi Bio Medical Science & Technology Co Ltd
Current assignee: Tianjin Xiaoxi Bio Medical Science & Technology Co Ltd
Priority date: 2017-09-15
Filing date: 2017-09-15
Publication date: 2019-03-26
Anticipated expiration: 2037-09-15
Also published as: CN109512838B

Abstract

This application involves a kind of rabbit fur extractives and its preparation method and application.Particularly, this application provides a kind of methods for being used to prepare rabbit fur extractive, and the rabbit fur extractive prepared by the method.In addition, present invention also provides the purposes of the composition comprising the rabbit fur extractive and the rabbit fur extractive.

Description

A kind of rabbit fur extractive and its preparation method and application

Technical field

This application involves field of immunology and biomedicine field.Particularly, this application involves a kind of rabbit fur extractive, with And its preparation method and application.More specifically, this application provides a kind of method for being used to prepare rabbit fur extractive, Yi Jitong Cross the rabbit fur extractive that the method prepares.In addition, present invention also provides the composition comprising the rabbit fur extractive, And the purposes of the rabbit fur extractive.

Background technique

Vaccination with vaccinia virus rabbit inflammation skin extract is containing the inflammation skin for sending out the rabbit of acne from inoculation vaccinia virus The extract of the active material of the non-protein of separation is extracted in tissue.Such extract is liquid in the state of after being extracted Body, but solid can also be made by drying.It has been reported that such extract can be used for treating Lumbago, neck shoulder wrist synthesis Sign, symptomatic neuralgia, the itching with skin disease (eczema, dermatitis, nettle rash), allergic rhinitis, subacute optic nerve The creeping chill of myelopathy (SMON) sequelae-abnormal perception-pain (see, for example, CN103110663A).Based on such extract Bioactivity, developed containing Vaccination with vaccinia virus rabbit inflammation skin extraction liquid preparation (Neurotropin/ NEUR0TR0PIN is also referred to as " NTP " or " NTP preparation " in this application).

However, it is time-consuming and laborious to prepare such rabbit fur extractive using traditional handicraft.For example, Chinese patent application Rabbit is disclosed in CN1493302A, CN103110663A, CN105163746A, CN106109498A, CN106573020A The extracting method of extract；However, only the time-consuming of preliminary extraction step has just reached 6-11 days, non-in these extracting methods It is often time-consuming.At the same time, in the extracting method of traditional rabbit fur extractive, it usually needs use organic solvent (such as benzene Phenol), this extract safety and reliability and in terms of result in potential risks.

Therefore, in the art, need to develop the new method for preparing rabbit fur extractive, to reduce the consumption of extraction step When, improve extraction efficiency.In addition, in the art, it is also necessary to the new method for preparing rabbit fur extractive is developed, to reduce or keep away Exempt from the use of organic solvent (such as phenol), to improve the safety and reliability of rabbit fur extractive obtained.

Summary of the invention

Present inventor develops a kind of new method for preparing rabbit fur extractive after largely studying.With Traditional method for preparing rabbit fur extractive is compared, and the preliminary extraction step (that is, step (B)) of preparation method of the present invention only needs 12-30 hours, the time-consuming of the step can reduce by more than 75%, to substantially increase extraction efficiency.At the same time, of the invention Preparation method can not use any organic solvent (such as phenol), so as to ensure the quality of rabbit fur extractive obtained More stable, safe and reliable (for example, any organic solvent is free of in rabbit fur extractive obtained).In addition, the hair of the application Bright people has surprisingly been found that the rabbit fur extractive prepared by the method for the invention contains the γ-aminobutyric acid of higher amount (γ-aminobutyric acid, GABA), citrulling, and/or carnosine, and therefore have superior technical effect (for example, With higher GABA activity).Therefore, the application refers at least to following summary of the invention.

In one aspect, the present invention provides a kind of method for being used to prepare rabbit fur extractive, the method includes following Step:

(A) skin of the hair acne from the rabbit for being vaccinated with vaccinia virus is provided；

(B) skin is mixed with water, and stirred 2-10 hours, obtain mixture；Then, by the pH of the mixture It is adjusted to 9.5-10.5, and continues stirring 10-20 hours；

(C) (such as filtering, filter and/or be centrifuged) is separated by solid-liquid separation to the product of step (B), to obtain the One extracting solution；With

(D) rabbit fur extractive is prepared using first extracting solution.

In certain preferred aspects, the vaccinia virus (vaccinia virus) can be the acne of any strain Seedling diseases poison, including but not limited to vaccinia virus Li Site (Lis ter) strain, vaccinia virus Dalian (Dairen) strain, vaccinia virus Ikeda (Ikeda) strain, EM-63 plants of vaccinia virus, vaccinia virus New York community health office (New York City Board of Heal th) strain, vaccinia virus ankara (Ankara) strain, vaccinia virus Copenhagen (Copenhagen) strain, vaccinia virus day Altar (Tian Tan) strain, WR plants of vaccinia virus, buffalo pox virus etc..

In certain preferred aspects, the rabbit can be the arbitrary rabbit for belonging to Lagomorpha, including but not limited to Cave rabbit (that is, rabbit that rabbit is tamed), hare (such as Japanese hare), pika, snow hare etc..In certain preferred implementations In scheme, the rabbit is rabbit.In certain preferred aspects, the rabbit can be the family of any kind (breed) Rabbit, including but not limited to, Japanese white kind rabbit and New Zealand White kind (New Zealand is white) rabbit.

The skin for being vaccinated with the hair acne of the rabbit of vaccinia virus can be obtained by various methods.In certain preferred embodiment party In case, by giving rabbit inoculation vaccinia virus, then the inflammation skin of acne is sent out in acquisition, to obtain the rabbit for being vaccinated with vaccinia virus Send out the skin of acne.In certain preferred aspects, it is commercially available for being vaccinated with the skin of the hair acne of the rabbit of vaccinia virus 's.

In certain preferred aspects, before carrying out step (B), the skin is pre-processed.Certain In preferred embodiment, the pretreatment includes freezing, and thaws, cleans, and disinfection, ultrasonication, is crushed or its is any Combination.In certain preferred aspects, before carrying out step (B), the skin is cleaned and/or is sterilized.? In certain preferred embodiments, before carrying out step (B), break process is carried out to the skin.In certain preferred realities It applies in scheme, before carrying out step (B), the skin is cleaned, is sterilized and break process.In certain preferred implementations In scheme, before carrying out step (B), the skin is freezed, is thawed, is cleaned, is sterilized and break process.Certain excellent In the embodiment of choosing, the break process includes that (a) is ground in liquid nitrogen, (b) is crushed in pulverizer, or (c) (a) and (b) combination.In certain preferred aspects, before carrying out step (B), break process is carried out to the skin, from And the skin is broken for particulate matter.Preferably, the partial size of the particulate matter is 50-500 μm, such as 50-75 μm, 75-100 μm, 100-150 μm, 150-200 μm, 200-500 μm or 75-200 μm.In certain preferred aspects, step is being carried out (B) before, break process is carried out to the skin, then the particulate matter exists so that the skin is broken for particulate matter Placed under 30-40 DEG C (such as 30-32 DEG C, 32-35 DEG C, 35-37 DEG C or 37-40 DEG C) 4-16 hours (for example, 4-8 hours, 8-12 Hour or 12-16 hours).Not by any theoretical contained, it is generally recognized that the break process of skin is advantageous, because it can promote Into the extraction of the active material in skin histology.

In certain preferred aspects, in step (B), the w/v (g/ml) of the skin and water is 1: (1-5), such as 1:(1-1.5), 1:(1.5-2), 1:(2-2.5) and, 1:(2.5-3), 1:(3-4) or 1:(4-5).Certain preferred Embodiment in, in step (B), the w/v of the skin and water is 1:2.5.In certain preferred embodiments In, it, at room temperature (such as 15-35 DEG C, 15-20 DEG C, 20-25 DEG C, 25-30 DEG C or 30-35 DEG C), will be described in step (B) Skin is mixed with water, and is stirred 2-10 hours (for example, 2-4 hours, 4-6 hours, 6-8 hours or 8-10 hours), to obtain Mixture.

In certain preferred aspects, in step (B), the pH of the mixture is adjusted to 9.5- using alkali 10.5, such as it is adjusted to pH10.In certain preferred aspects, the alkali is inorganic base, such as NaOH or KOH.At certain In a little preferred embodiments, in step (B), the pH of the mixture is adjusted to 9.5-10.5, and in raised temperature Under (such as 40-50 DEG C, 40-42 DEG C, 42-45 DEG C, 45-48 DEG C or 48-50 DEG C) continue stirring 10-20 hours (for example, 12-14 Hour, 14-16 hours, 16-18 hours or 18-20 hours).

In the method for the invention, in step (B), by using two stage extraction scheme, that is, first it is extracted with water, It is extracted again with alkaline solution (especially aqueous slkali), substantially reduces extraction time spent by the step (that is, can foreshorten to 12-30 hours).Compared to reported extracting method before, such as (it is preliminary for extracting method disclosed in CN103110663A Extraction step time-consuming reaches 6-11 days), the time-consuming of the preliminary extraction step (that is, step (B)) of the method for the present invention can reduce by more than 75%, to substantially increase extraction efficiency.

In certain preferred aspects, in step (C), by filtering or filtering the product progress to step (B) It is separated by solid-liquid separation；Preferably, with 50-500 μm (such as 50-75 μm, 75-100 μm, 100-150 μm, 150-200 μm, 200-500 μ M or 75-200 μm) pore size filter, the product of step (B) is filtered or is filtered.

In certain preferred aspects, the step of the method for the present invention (D) the following steps are included:

(D1) pH of first extracting solution is adjusted to 4.0-5.0, and is heated, then, carry out solid-liquid point From (such as being filtered, filter and/or be centrifuged), to obtain the second extracting solution；

(D2) pH of second extracting solution is adjusted to 9.0-10.0, and is heated, then, carry out solid-liquid point From (such as being filtered, filter and/or be centrifuged), to obtain third extracting solution；

(D3) pH of the third extracting solution is adjusted to 3.5-5.5, and is contacted with adsorbent, made in third extracting solution Active material is adsorbed on adsorbent；

(D4) product of step (D3) is separated by solid-liquid separation (such as filtering, filter and/or be centrifuged), collects absorption Agent；With

(D5) adsorbed active material on adsorbent is extracted with the Extraction solvent that pH is 9.0-11.0, to obtain the 4th Extracting solution, as rabbit fur extractive.

In certain preferred aspects, in step (D1), the pH of first extracting solution is adjusted to using acid 4.0-5.0, such as it is adjusted to pH4.5.In certain preferred aspects, the acid is inorganic acid, such as HCl.Certain In preferred embodiment, in step (D1), the pH of first extracting solution is adjusted to 4.0-5.0, and be heated to 85- 105 DEG C (such as 85-90 DEG C, 90-95 DEG C, 95-100 DEG C or 100-105 DEG C).In certain preferred aspects, in step (D1) in, the heat treatment is carried out 0.5-1.5 hours, such as 0.5-1 hours or 1-1.5 hours.It is not restrained by any theory, Generally, it is considered that the heat treatment under acid condition can help to remove the partial impurities albumen in extracting solution.In certain preferred implementations It in scheme, in step (D1), is separated by solid-liquid separation by centrifugation and subsequent filtering or suction filtration, is extracted to obtain second Liquid.

In certain preferred aspects, in step (D2), the pH of second extracting solution is adjusted to using alkali 9.0-10.0 such as being adjusted to pH9.5.In certain preferred aspects, the alkali is inorganic base, such as NaOH or KOH. In certain preferred aspects, in step (D2), the pH of second extracting solution is adjusted to 9.0-10.0, and heat To 85-105 DEG C (such as 85-90 DEG C, 90-95 DEG C, 95-100 DEG C or 100-105 DEG C).In certain preferred aspects, exist In step (D2), the heat treatment is carried out 0.25-1.5 hours, such as 0.25-0.5 hours, 0.5-1 hours or 1-1.5 small When.In certain preferred aspects, in step (D2), after heating to second extracting solution, by it 10-20 hours (for example, 10-12 hours, 12-14 hours, 14-16 hours, 16-18 hours or 18-20 hours) are stirred, then again It is separated by solid-liquid separation, to obtain third extracting solution.Not by any theoretical contained, it is generally accepted that at the heating under alkaline condition Reason and subsequent stir process can help to remove the partial impurities albumen in extracting solution.In certain preferred aspects, In step (D2), by filter or filter (for example, using aperture be 0.22-0.65 μm (such as 0.45 μm) filter membrane) come into Row is separated by solid-liquid separation, to obtain third extracting solution.

In certain preferred aspects, in step (D3), the pH of the third extracting solution is adjusted to using acid 3.5-5.5, such as 3.5-4.0,4.0-4.5,4.5-5.0 or 5.0-5.5.In certain preferred aspects, the acid is Inorganic acid, such as HCl.In certain preferred aspects, in step (D3), the adsorbent is selected from active carbon, kaolinite Soil and any combination thereof.In certain preferred aspects, in adsorbent used in step (D3) and step (B) The weight ratio (g/g) of used skin is 1:(10-50), such as 1:(10-15), 1:(15-20), 1:(20-25), 1:(25- 30), 1:(30-40) or 1:(40-50).In certain preferred aspects, it in step (D3), can be connect with adsorbent Prior to, concurrently with, or after touching, the pH of the third extracting solution is adjusted to 3.5-5.5.In certain preferred aspects, exist In step (D3), the pH of the third extracting solution is adjusted to 3.5-5.5, and contacts (such as mixing) with adsorbent, is then existed (such as 15-35 DEG C, 15-20 DEG C, 20-25 DEG C, 25-30 DEG C or 30-35 DEG C) stirs 1-4 hours at room temperature, such as 1-2 hours, 2-3 hours or 3-4 hours.In certain preferred aspects, in step (D3), the adsorbent is filled in absorption In column, then contacted with the adjusted third extracting solution of pH value, to adsorb the active material in third extracting solution.Not by any Theory is restrained, it is generally accepted that stirring and/or the use of packed column appropriate can promote the absorption of the active material in third extracting solution In on adsorbent.

In certain preferred aspects, in step (D4), by filter or filter (for example, using filter paper) come pair The product of step (D3) is separated by solid-liquid separation, to obtain the adsorbent for having adsorbed active material.

In certain preferred aspects, step (D5) includes the extraction for being 9.0-11.0 by the adsorbent and pH Solvent contact (such as mixing), is then separated by solid-liquid separation (such as being filtered, filter and/or be centrifuged), to obtain the 4th Extracting solution.In certain preferred aspects, in step (D5), the Extraction solvent is alkaline solution (for example, alkalinity Aqueous solution, methanol solution, ethanol solution, aqueous isopropanol, or any combination thereof；It is preferred that the solution of alkali).Certain preferred In embodiment, in step (D5), using pH be 9.0-11.0 (such as pH is 9.0-9.5,9.5-10.0,10.0-10.5 or 10.5-11.0) alkaline solution (for example, the aqueous solution of alkalinity, methanol solution, ethanol solution, aqueous isopropanol or its is any Combination；It is preferred that the solution of alkali) extract the active material adsorbed on adsorbent.In certain preferred aspects, the alkali Solution be inorganic base aqueous solution, such as the aqueous solution of NaOH or KOH.In certain preferred aspects, step (D3) Used in adsorbent and step (D5) used in Extraction solvent w/v (g/ml) be 1:(10-100), example Such as 1:(10-20), 1:(20-30), 1:(30-40) and, 1:(40-50), 1:(50-60) and, 1:(60-70), 1:(70-80), 1: (80-90) or 1:(90-100).In certain preferred aspects, in step (D5), in the condition for heating and/or stirring Under, the active material adsorbed on adsorbent is extracted with the Extraction solvent.Not by any theoretical contained, it is generally accepted that appropriate Stirring and/or heat treatment can promote active material and elute from adsorbent.In certain preferred aspects, in step (D5) in, 40-50 DEG C at a temperature of (such as 40-42 DEG C, 42-45 DEG C, 45-48 DEG C or 48-50 DEG C), and in the item of stirring Under part (such as stirring 1-4 hours, such as 1-2 hours, 2-3 hours or 3-4 hours), adsorbent is extracted with the Extraction solvent The active material of upper absorption.In certain preferred aspects, in step (D5), adsorbent is extracted with the Extraction solvent The active material of upper absorption is one or many, and for example, at least 2 times；It is then combined with the extracting solution for extracting acquisition each time, to obtain Obtain the 4th extracting solution.It can be readily appreciated that extracting used each time when extracting the active material adsorbed on adsorbent with Extraction solvent Extraction solvent can be it is same or different.For example, in certain preferred aspects, in step (D5), first First time extraction is carried out with the Extraction solvent that pH is 9.0-10.0, then carries out with the Extraction solvent that pH is 10.0-11.0 again Second extraction is then combined with the extracting solution for extracting acquisition twice, to obtain the 4th extracting solution.

In certain preferred aspects, the method further includes selected from following one or more after step (D) A step:

(E1) ultrafiltration is carried out to the 4th extracting solution；

(E2) the 4th extracting solution is heated；

(E3) pH of the 4th extracting solution is adjusted to 6.5-7.5；

(E4) the 4th extracting solution is diluted or is concentrated；

(E5) sterilization treatment is carried out to the 4th extracting solution；With

(E6) frozen dried is carried out to the 4th extracting solution.

In certain preferred aspects, in step (E1), molecular weight boundary used by ultrafiltration is 5-40KD, example Such as 5-10KD, 10-15KD, 15-20KD, 20-30KD or 30-40KD.In certain preferred aspects, in step (E2) In, the 4th extracting solution is heated to 60-100 DEG C, such as 60-70 DEG C, 70-80 DEG C, 80-90 DEG C or 90-100 DEG C, and appoint Selection of land stands 10-18 hours, such as 10-12 hours, 12-14 hours, 14-16 hours or 16-18 hours.Certain preferred In embodiment, in step (E3), the pH of the 4th extracting solution is adjusted to 6.5-7.5 using acid, for example, 6.5-7.0 or 7.0-7.5.In certain preferred aspects, the acid is inorganic acid, such as HCl.In certain preferred aspects, In step (E4), using water for injection, physiological saline or physiological buffer are diluted the 4th extracting solution.Certain In preferred embodiment, in step (E4), the 4th extracting solution is concentrated into desired concentration.In certain preferred realities It applies in scheme, in step (E5), using high pressure sterilization, ultra-high temperature sterilization (UHT), pasteurization, filtration sterilization or its is any Combination carries out sterilization treatment to the 4th extracting solution.

In certain preferred aspects, the method further includes 1 in step (E1)-(E6) after step (D) A, 2,3,4,5 or 6 steps.In certain preferred aspects, the method is also wrapped after step (D4) Include step (E1)-(E5).It in the method for the invention, can according to actual needs, with any desired after step (D) Sequence carries out 1,2,3,4,5 or 6 step in step (E1)-(E6).

In certain preferred aspects, the method for the present invention the step of in (B), phenol is not used.Certain preferred Embodiment in, the method for the present invention the step of in (B), do not use organic solvent.In certain preferred aspects, root Phenol is not used according to method of the invention.In certain preferred aspects, according to the method for the present invention without using organic molten Agent.It is generally believed that both sides technical effect can be achieved in the use of phenol: on the one hand, phenol can be used as fungicide and play sterilizing The effect of disinfection；On the other hand, phenol can promote the extraction of active material as organic solvent.However, in some cases, In the extraction of active material or the preparation process of drug, it is expected that being avoided as much as using any organic solvent (such as benzene Phenol), to avoid residual of the such organic solvent (such as phenol) in final product, influencing subsequent application, (such as clinic is answered With).In certain preferred embodiments of the invention, method of the invention (especially step (B)) does not need to have using any Solvent (such as phenol), to be free of any organic solvent in rabbit fur extractive obtained.Therefore, method of the invention with And thus rabbit fur extractive obtained is particularly advantageous.

On the other hand, the present invention provides a kind of rabbit fur extractives, are made by the above method.As retouched above It states, preferred preparation method of the invention can not use any organic solvent.Correspondingly, pass through rabbit obtained by such method Skin extraction will be entirely free of any organic solvent, this is particularly advantageous in some cases.

In addition, rabbit fur extractive is the extract that the inflammation skin histology of the rabbit of acne is sent out from inoculation vaccinia virus, In the substance containing pole multiple types.In other words, rabbit fur extractive and the preparation using rabbit fur extractive preparation are not with specific 1 Kind or number of substances cannot determine it simply by the content of measurement a certain kind or several effective components as effective component Activity/efficiency.In order to objectively evaluate activity/efficiency of rabbit fur extractive, it has been suggested that by measurement bioactivity (potency) come into The effective component of row rabbit fur extractive is quantitative.In short, according to " day pharmacology will " (volume 72 the 5th, 573-584 pages, 1976 Year) in the method recorded, using SART stress (repeatedly cold load) animal (it is chronic lower than intact animal of pain threshold Stressed animal), by inflammation, pressurization (Randal l-Sel itto) method carries out analgesic test enough, measures the town of rabbit fur extractive Pain coefficient.In inflammation foot pressurization, Pressure stimulation is applied to the tail portion of mouse, until mouse shows the pressurization of fugue reaction Weight is index, measures analgesic effect.So-called analgesia coefficient is the pressurization weight that will measure after giving drug divided by giving medicine The value that the pressurization weight measured before object obtains.About the activity of rabbit fur extractive, to show the analgesia coefficient of predetermined value or more Situation finds out the ratio for being judged as positive animal number of elements as the analgesic effect positive, as ease pain efficient (%).With Afterwards, according to the standard items measurement for being diluted to various concentration as a result, calculating ED₅₀Value.Definition such as get off as a result, for commenting The unit of the bioactivity (potency) of valence rabbit fur extractive: by ED₅₀Value is that the rabbit of dosage 100mg/kg (mouse weight) mentions Taking activity definition shown by object lmg is 1Neurotropin unit (that is, 1NU).In this application, when description rabbit fur extractive Or preparation active ingredient amount (potency) when, use unit as NU (sometimes also directly referred to as " unit ", U) Lai Jinhang table Show.In the art, NU unit has been used for the active ingredient amount (potency) for describing rabbit fur extractive or related preparations.For example, Presently commercially available NTP injection contains 1.2NU unit/ml effective component, NTP tablet contain 4NU unit/piece it is effective at Point.In this application, " Neurotropin unit ", " unit ", " NU " and " U " has the same meaning, and is used interchangeably.

It has further been found by the present inventors that the rabbit prepared with conventional method (such as method disclosed in CN103110663A) Bark extract is compared, and the every NU unit of rabbit fur extractive of the invention contains the γ-aminobutyric acid (γ-of higher amount Aminobutyric acid, GABA), citrulling, and/or carnosine.

γ-aminobutyric acid (abbreviation GABA) is a kind of naturally occurring nonprotein amino acid.Studies have shown that GABA is to feed Important inhibiting nerve conveys substance in newborn animal central nervous systems, in human brain cortex, hippocampus, thalamus, substrate It plays an important role in neuromere and cerebellum, and there is adjustment effect to the multiple functions of body.For example, GABA can reduce nerve First activity, prevents nerve cell from overheating (overfiring), realizes effect antianxity.In addition, GABA can also act on ridge The vasomotor center of marrow is effectively facilitated the expansion of blood vessel, realizes the effect to reduce blood pressure.Citrulling is a kind of alpha amino acid. Studies have shown that citrulling can make papaverine, can be used for enhancing male's sexual and the treatment of sexual dysfunction.In addition, melon Propylhomoserin can also safeguard the lung function in Jiankang, improve mental clarity, help improve cranial nerve cell storage and recall to message Ability.Carnosine, i.e. β-alanyl-L-histidin are a kind of dipeptides being made of two kinds of amino acid of Beta-alanine and L-Histidine. Studies have shown that carnosine has very strong oxidation resistance, and animal experimental data is shown, carnosine can delay the life of cancer cell It is long, oxidative pressure caused by alcohol is taken precautions against, reduces chronic liver injury caused by ethyl alcohol, and there is neuroprotection, it can Prevent permanent cerebral ischemic injury.Therefore, the rabbit of the present invention of the GABA containing higher amount, citrulling, and/or carnosine mentions Object is taken to be particularly advantageous.The rabbit fur extractive prepared by the method for the invention be it is unique, have preferably application before Scape.

Therefore, on the other hand, the present invention provides a kind of rabbit fur extractives, do not contain any organic solvent (example Such as phenol), and containing at least 0.8ng γ-aminobutyric acid in the rabbit fur extractive of every NU unit, such as containing at least 1ng, until Few 2ng, at least 5ng, at least 10ng, at least 20ng, at least 50ng, at least 70ng, at least 100ng, at least 120ng, or at least 150ng γ-aminobutyric acid.In certain preferred aspects, contain 0.8-150ng in the rabbit fur extractive of every NU unit γ-aminobutyric acid, such as contain 0.8-1ng, 1-2ng, 2-5ng, 5-10ng, 10-20ng, 20-50ng, 50-70ng, 70- 100ng, 100-120ng or 120-150ng γ-aminobutyric acid.

In certain preferred aspects, containing at least 4ng carnosine in the rabbit fur extractive of every NU unit, such as containing At least 5ng, at least 7ng, at least 10ng, at least 12ng, at least 15ng, at least 20ng, at least 22ng, at least 25ng, or at least 30ng carnosine.In certain preferred aspects, containing 4-30ng carnosine in the rabbit fur extractive of every NU unit, such as containing 4-5ng, 5-7ng, 7-10ng, 10-12ng, 12-15ng, 15-20ng, 20-22ng, 22-25ng or 25-30ng carnosine.

In certain preferred aspects, containing at least 60ng citrulling in the rabbit fur extractive of every NU unit, such as Containing at least 80ng, at least 100ng, at least 120ng, at least 150ng, at least 170ng, or at least 200ng citrulling.Certain In preferred embodiment, 60-200ng citrulling is contained in the rabbit fur extractive of every NU unit, such as contain 60-80ng, 80- 100ng, 100-120ng, 120-150ng, 150-170ng or 170-200ng citrulling.

In certain preferred aspects, the rabbit fur extractive prepares by means of the present invention.

On the other hand, the present invention also provides a kind of composition, it includes rabbit fur extractives as described above (especially It is the rabbit fur extractive prepared by the method for the invention).

In this application, inventor has found after numerous studies, utilizes the skin of the hair acne for the rabbit for being vaccinated with vaccinia virus Skin and the rabbit fur extractive prepared can promote various kinds of cell, and (such as oligodendroglia, mescenchymal stem cell are (such as between marrow Mesenchymal stem cells) and neural stem cell) proliferation；Also, the rabbit fur extractive prepared by the method for the invention has spy Not significant, the good effect for promoting cell Proliferation.Therefore, rabbit fur extractive of the invention can be added to cell culture In base, for promoting cell Proliferation.Therefore, in certain preferred aspects, the composition is cell culture medium, packet Containing the rabbit fur extractive prepared by the method for the invention.In certain preferred aspects, the cell culture medium is It is added to the DMEM culture medium, F12 culture medium or DMEM/F12 culture medium of rabbit fur extractive according to the present invention.DMEM culture Base, F12 culture medium or DMEM/F12 culture medium can prepare acquisition by conventional method, or can also be commercially available.Certain In preferred embodiment, the cell culture medium also includes additional reagent, such as serum (such as fetal calf serum), cell The factor, B27 cell culture additive (B27), or any combination thereof.In certain preferred aspects, the cell factor Including but not limited to, epithelical cell growth factor (EGF), LIF ELISA (LIF), fibroblast growth factor (FGF；Including but not limited to aFGF and bFGF), or any combination thereof.

In addition, before it has been reported that the rabbit fur extractive prepared using the skin of the hair acne for the rabbit for being vaccinated with vaccinia virus It can be used for the treatment of a variety of diseases or illness.Such disease or illness include but is not limited to Lumbago, neck shoulder wrist syndrome, disease Shape nerve pain, the itching with skin disease (such as eczema, dermatitis, nettle rash), allergic rhinitis, subacute optic nerve ridge Creeping chill-abnormal perception-pain, postherpetic neuralgia, scapulohumeral periarthritis, arthritis deformans of marrow disease sequelae etc. are (referring to example Such as CN103110663A).Moreover, it has been found that the rabbit fur extractive can be used for treating central nervous system disease (such as wound Property cerebral injury (TBI) and spinal cord injury (SCI)), it can be used for promoting the motor function recovery after spinal cord injury, or for inhibiting glue Matter cicatrization (glial scar especially after spinal cord injury is formed).Therefore, rabbit fur extractive of the invention can be used for The drug or pharmaceutical composition of the disease or illness are treated in preparation.Therefore, in certain preferred aspects, the combination Object is pharmaceutical composition, also includes pharmaceutically acceptable carrier or excipient.In certain preferred aspects, institute Stating pharmaceutical composition is the injection containing rabbit fur extractive according to the present invention.In certain preferred aspects, described Pharmaceutical composition is the tablet containing rabbit fur extractive according to the present invention.In certain preferred aspects, the drug Composition, for the pharmaceutical dosage form being administered suitable for systemic vein, such as injection or freeze dried powder.

As discussed above, rabbit fur extractive of the invention can promote the proliferation of various kinds of cell.Therefore, at another Aspect additionally provides rabbit fur extractive of the invention for cultured cell in vitro or the purposes for promoting cell Proliferation in vitro. In certain preferred aspects, the cell is selected from oligodendroglia, (such as medulla mesenchyma is dry for mescenchymal stem cell Cell) and neural stem cell.In certain preferred aspects, the purposes is used for non-diagnostic or therapeutic purposes.

On the other hand, the purposes that rabbit fur extractive of the invention is used to prepare composition, the combination are additionally provided Object is for cultivating cell or for promoting cell Proliferation.In certain preferred aspects, the cell is selected from oligodendroglia Cell, mescenchymal stem cell (such as mesenchymal stem cell) and neural stem cell.In certain preferred aspects, institute Stating composition is the cell culture medium comprising rabbit fur extractive of the present invention.In certain preferred aspects, the composition For the DMEM culture medium comprising rabbit fur extractive of the present invention, F12 culture medium or DMEM/F12 culture medium.In certain preferred implementations In scheme, the cell culture medium also includes additional reagent, such as serum (such as fetal calf serum), cell factor, B27 thin Born of the same parents cultivate additive (B27), or any combination thereof.In certain preferred aspects, the cell factor includes but unlimited In, epithelical cell growth factor (EGF), LIF ELISA (LIF), fibroblast growth factor (FGF；Including but not It is limited to aFGF and bFGF), or any combination thereof.

On the other hand, the present invention provides a kind of cultured cell in vitro or the external method for promoting cell Proliferation, Include the steps that using rabbit fur extractive of the invention.In certain preferred aspects, the cell is selected from oligodendroglia Cell, mescenchymal stem cell (such as mesenchymal stem cell) and neural stem cell.In certain preferred aspects, institute The step of method of stating includes, cultivates the cell in the cell culture medium containing rabbit fur extractive of the present invention.Certain preferred Embodiment in, the cell culture medium be the DMEM culture medium comprising rabbit fur extractive of the present invention, F12 culture medium or DMEM/F12 culture medium.In certain preferred aspects, the cell culture medium also includes additional reagent, such as blood (such as fetal calf serum) clearly, cell factor, B27 cell culture additive (B27), or any combination thereof.In certain preferred realities It applies in scheme, the cell factor includes but is not limited to epithelical cell growth factor (EGF), LIF ELISA (LIF), Fibroblast growth factor (FGF；Including but not limited to aFGF and bFGF), or any combination thereof.In certain preferred implementations In scheme, the method is used for non-diagnostic or therapeutic purposes.

As discussed above, rabbit fur extractive of the invention can be used in the treatment of a variety of diseases or illness.Another A aspect additionally provides the purposes of rabbit fur extractive of the invention in medicine preparation, and the drug is for treating Lumbago, neck Shoulder wrist syndrome, symptomatic neuralgia, with the itching of skin disease (such as eczema, dermatitis, nettle rash), allergic rhinitis, Creeping chill-abnormal perception-pain, postherpetic neuralgia, scapulohumeral periarthritis, the morphotropism of subacute optic neuropathy sequelae are closed Section is scorching or central nervous system disease, for promoting the motor function recovery after spinal cord injury, or for inhibiting glial scar shape At.In certain preferred aspects, the central nervous system disease is selected from traumatic brain injury (TBI) and spinal cord injury (SCI).In certain preferred aspects, the drug is for promoting the motor function recovery after spinal cord injury.Certain In preferred embodiment, the drug is for inhibiting glial scar to be formed, and especially the glial scar after spinal cord injury is formed. In certain preferred aspects, the spinal cord injury serve as reasons (such as caused by traffic accident or the wounds such as fall) spine fracture or Spinal cord injury caused by dislocating.In certain preferred aspects, the glial scar formation is by colloid after spinal cord injury Caused by hyperplasia.In certain preferred aspects, the drug also includes pharmaceutically acceptable carrier or figuration Agent.In certain preferred aspects, the drug is the pharmaceutical dosage form being administered suitable for systemic vein, for example, injection or Freeze dried powder.

Advantageous effect of the invention

Compared with prior art, the method for preparing rabbit fur extractive of the invention has the advantages that in the present invention In the preliminary extraction step (that is, step (B)) of preparation method, by using two stage extraction scheme, that is, first it is extracted with water, It is extracted again with alkaline solution (especially aqueous slkali), substantially reduces extraction time spent by the preliminary extraction step (that is, can It foreshortens to 12-30 hours).Compared to reported extracting method, such as extracting method disclosed in CN103110663A before (its preliminary extraction step time-consuming reaches 6-11 days), the time-consuming of the preliminary extraction step (that is, step (B)) of preparation method of the present invention can 75% is reduced by more than, to substantially increase extraction efficiency.

In addition, in some preferred cases, compared with prior art, preparation method (especially step of the invention (B)) it does not need using any organic solvent (such as phenol), so as to ensure the quality of rabbit fur extractive obtained more Stable, safe and reliable (for example, any organic solvent is free of in rabbit fur extractive obtained).Therefore, preparation side of the invention Method and thus rabbit fur extractive obtained is particularly advantageous.

In addition, present inventor also found, made with conventional method (such as method disclosed in CN103110663A) The standby rabbit fur extractive obtained is compared, and the rabbit fur extractive prepared by the method for the invention contains γ-ammonia of higher amount Base butyric acid (γ-aminobutyric acid, GABA), citrulling, and/or carnosine, and therefore there is superior technical effect (for example, there is higher GABA activity).Therefore, preparation method of the invention and thus rabbit fur extractive obtained is special It is not advantageous.Rabbit fur extractive of the invention has more excellent validity and safety, has better application prospect.

In addition, present inventor also found, rabbit fur extractive of the invention is able to suppress the colloid after spinal cord injury Cicatrization, and significantly improve the BBB scoring of spinal cord injury animal.Rabbit fur extractive of the invention is for the fortune after spinal cord injury Dynamic functional rehabilitation has significant facilitation.

Embodiment of the present invention is described in detail below in conjunction with drawings and examples, but those skilled in the art Member it will be understood that, following drawings and embodiment are merely to illustrate the present invention, rather than the restriction to the scope of the present invention.With reference to the accompanying drawings With the following detailed description of preferred embodiment, various purposes of the invention and advantageous aspect are to those skilled in the art It will be apparent.

Detailed description of the invention

Fig. 1 shows the HPLC testing result (abscissa: time/min of rabbit fur extractive obtained in embodiment 1；It is vertical to sit Mark: absorbance/mAU).

Fig. 2 show the LV absorption spectrum of rabbit fur extractive obtained in embodiment 1 testing result (abscissa: wavelength/ nm；Ordinate: absorbance).

Fig. 3 A shows the result (fluorescence for carrying out Immunofluorescence test in embodiment 2 with cell of the anti-O4 antibody to culture Microphoto).The results show that the cell cultivated is that anti-O4 antibody test is positive, it is oligodendroglia.

Fig. 3 B shows result (the fluorescence microscopy photograph detected in embodiment 2 with cell of the DAPI decoration method to culture Piece), wherein show the nucleus being colored.

Fig. 3 C is the merging image of Fig. 3 A and Fig. 3 B.The results show that the cell and DAPI of the anti-O4 antibody test positive dye Cell ratio be 1:1.

Fig. 4 A-4C respectively illustrates the 2nd day (Fig. 4 A), the 4th day (Fig. 4 B), the 6th day (Fig. 4 C) after starting culture, training Support the proliferative conditions of each group oligodendroglia in the cell culture medium of the rabbit fur extractive containing various concentration.

Fig. 5 A-5B respectively illustrates the 2nd day (Fig. 5 A) and the 4th day (Fig. 5 B) after starting culture, is incubated at containing not With the proliferative conditions of each group mesenchymal stem cell in the cell culture medium of the rabbit fur extractive of concentration.

Fig. 6 A shows the light micrograph for the cell cultivated in embodiment 4.The results show that the cell table cultivated Revealing the distinctive bright, three-dimensional sense of neural stem cell by force, around has the cell ball form of suspension of radial burr.

Fig. 6 B shows the result for carrying out Immunofluorescence test in embodiment 4 to the cell of culture with anti-Nestin antibody (fluorescence micrograph).The results show that the cell cultivated is that anti-Nest in antibody test is positive, it is neural stem cell.

Fig. 6 C shows result (the fluorescence microscopy photograph detected in embodiment 4 with cell of the DAPI decoration method to culture Piece), wherein show the nucleus being colored.

Fig. 6 D is the merging image of Fig. 6 B and Fig. 6 C.The results show that the cell and DAPI of the anti-Nestin antibody test positive The ratio of the cell of dyeing is 1:1.

Fig. 7 A-7C respectively illustrates the 1st day (Fig. 7 A), the 2nd day (Fig. 7 B), the 3rd day (Fig. 7 C) after starting culture, training Support the proliferative conditions of each group neural stem cell in the cell culture medium of the rabbit fur extractive containing various concentration.

Fig. 8 A-8D is respectively illustrated in GABA (10 μM), NTP injection, NTP injection+Bic (Bicuculline, 10 μM), rabbit fur extractive of the invention or rabbit fur extractive+Bic of the invention (10 μM) In the presence of, the GABA electric current of hippocampus of rats cell.Fig. 8 A-8B is respectively illustrated, cell sample 1 (Fig. 8 A) and cell sample Product 2 (Fig. 8 B) respond GABA, rabbit fur extractive of the invention or rabbit fur extractive+Bic GABA electricity of the invention Stream.Fig. 8 C-8D is respectively illustrated, and cell sample 1 (Fig. 8 C) and cell sample 2 (Fig. 8 D) respond GABA, NTP injection or NTP Injection+Bic GABA electric current.

Fig. 9 shows that rats with spinal cord injury receives the movement after the rabbit fur extractive treatment of physiological saline or prescribed dose Functional rehabilitation situation (that is, BBB scores).

Figure 10 shows that rats with spinal cord injury receives the colloid after the rabbit fur extractive treatment of physiological saline or prescribed dose Cicatrization situation (that is, GFAP immunostaining).

Specific embodiment

It is intended to illustrate the present invention embodiment (rather than limiting the invention) referring now to following and describes the present invention.Ability Field technique personnel know that embodiment describes the present invention by way of example, and is not intended to limit scope of the present invention.

Experimental method used in following embodiments is unless otherwise specified conventional method.Made in following embodiments Reagent and material etc. are unless otherwise specified the product of commercially available purchase.Wherein, used portion of reagent and reagent The source of box is as follows:

DMEM/F12 cell culture medium is purchased from Gibco company；

Cck-8 kit is purchased from Bei Bo company；

B27 is purchased from Invi trogen company；

Anti- O4 antibody, anti-Nestin antibody and anti-beta III-microtubulin antibody are purchased from Sigma-Aldrich company；

Anti- GFAP antibody and anti-CNPase antibody are purchased from Abcam company；With

BFGF, EGF and LIF are purchased from Peprotech company.

The preparation of 1. rabbit fur extractive of embodiment

(New Zealand White kind rabbit, weight are greater than the rabbit of acquisition intradermal vaccination vaccinia virus Li Site (Lister) strain It is frozen in -20 DEG C of refrigerator-freezer by the rabbit of hair acne 2.5kg) in advance, spare.

(A) rabbit freezed is taken out from -20 DEG C of refrigerator-freezers, weighs 80g, is first cut into 2-3 centimetres of fritter, and side trimming is placed In liquid nitrogen.After all cutting, rabbit is put in pulverizer, is crushed as particulate matter (75-200 μm).After crushing Rabbit is placed in beaker, and adds preservative film sealing, and waiting defrosting to room temperature, (every half an hour, measurement is primary；Ordinary circumstance Under, after liquid nitrogen processing, rabbit can reach room temperature at 2.5-3 hours).Then, rabbit is placed in constant incubator, in It is placed 12 hours at 37 ± 2 DEG C.

(B) ultrapure water of product obtained in (A) and 200ml is put into beaker, is stirred at room temperature 6 hours.

(C) mixture obtained in (B) is transferred in round-bottomed flask, while stirring extremely with 5mol/L NaOH adjustment pH 10.0 ± 0.5, then extracted 12-18 hours (overnight) at 45 DEG C.Then, it using the Suction filtration device of connection vacuum pump, uses 80 mesh filter clothes, filter the mixture in round-bottomed flask, and will filter out liquid (the first extracting solution) and be transferred to new round bottom In flask.

(D) liquid (the first extracting solution) being filtered out obtained in (C), pH is adjusted to 4.5 with 5mol/LHCl while stirring ± 0.5, and heated 60 minutes at 95 DEG C.After heating, entire mixture is cooled to 50 DEG C hereinafter, 10000rpm centrifugation 1 is small When, filtering leaves and takes supernatant (the second extracting solution) into new round-bottomed flask.

(E) pH is adjusted to 9.5 with 5mol/L NaOH while stirring by supernatant obtained in (D) (the second extracting solution) ± 0.5, and heated at 95 DEG C 30 minutes, it then cools down and is stirred overnight (12-18 hours).Then, with the pumping of connection vacuum pump Device is filtered, entire mixture is filtered using 0.45 μm of filter membrane, liquid (third extracting solution) will be filtered out and be transferred to new burning In cup.

(F) it filters out obtained in (E) and 4g active carbon is added in liquid (third extracting solution), use 5mol/L while stirring PH is adjusted to 4.5 ± 0.5 by HCl.After being stirred at room temperature 2 hours, with filter paper pressure filtration, leaching active carbon, filtrate is discarded. Then, ultrapure water 200ml is added in the active carbon for having adsorbed effective component, is while stirring adjusted pH with 5mol/L NaOH It is 10.0 ± 0.5.After being stirred 2 hours at 45 DEG C, pressure filtration (filter paper, 0.22 μm of filter membrane), by filtrate (the 4th extracting solution) It is transferred in blue lid bottle.The rabbit fur extractive of the immune rabbit of 4th extracting solution obtained, as vaccinia virus.

(G) filtrate obtained in (F) (the 4th extracting solution) is added in ultrafilter, with the filter membrane of molecular weight boundary 10KD Ultrafiltration is carried out, is then transferred to filtrate in new blue lid bottle, further to remove undesired impurity.Blue lid bottle is heated, is made It obtains filtrate and is heated to 80 DEG C, and save one late (10~18 hours), be later cooled to room temperature filtrate (the 5th extracting solution).Will The filtrate (the 5th extracting solution) arrived is in subsequent experiment.

(H) the UV absorption value of filtrate (the 5th extracting solution) obtained in (G) is measured, it, will then by addition water for injection Its absorbance at 270nm is adjusted to 0.4-0.5.In the case, the concentration of the effective component of rabbit fur extractive is about 1.2NU/ml.Then pH is adjusted to 7.0 ± 0.5 with 5mol/LHCl.

(I) product obtained in (H) is sampled detection, comprising: (1) carry out HPLC detection to it to confirm 5 types The content of the nucleic acid base of type；(2) feature of its LV absorption spectrum is measured；(3) content of various amino acid is measured；(4) into Row LC-MS/MS detection.

(J) product obtained in (H) is carried out at 121 DEG C with autoclave to 20-30 minutes moist heat sterilizations, is protected It deposits, it is spare.

(1) HPLC is detected

According to manufacturer specification, using highly effective liquid phase chromatographic system (Thermofisher Scientific, Ul Timate3000), HPLC detection is carried out to product obtained in (H) using the following conditions:

Sample volume: 10 μ g；Wavelength: 260.0nm；Flow velocity: 0.6ml/min；Runing time: 20min；Dilution gfactor: 1.0.

The testing result of HPLC is shown in table 1 and Fig. 1 in.

The HPLC testing result of the rabbit fur extractive of the immune rabbit of 1. vaccinia virus of table

The HPLC testing result of table 1 and Fig. 1 show that in prepared rabbit fur extractive, the nucleic acid base of 5 seed types contains Amount meets relevant criterion.

(2) detection of LV absorption spectrum

It is produced using ultraviolet specrophotometer (Shimadzu, UV1800) to obtained in (H) according to manufacturer specification The detection of object progress LV absorption spectrum.The testing result of LV absorption spectrum is shown in table 2 and Fig. 2.

The LV absorption spectrum testing result of the rabbit fur extractive of the immune rabbit of 2. vaccinia virus of table

Number	Wavelength (nm)	Absorbance
			1	339.60	0.062
2	269.80	0.484
			3	245.50	0.393

The HPLC testing result of table 2 and Fig. 2 show that the obtained the maximum absorption of prepared rabbit fur extractive is in 268-272nm In the range of.

(3) detection of amino acid content

According to manufacturer specification, using ion chromatograph (Dionex, ICS-3000), to product obtained in (H) into The detection of row amino acid content.The testing result of amino acid content is shown in Table 3.

The amino acid content testing result of the rabbit fur extractive of the immune rabbit of 3. vaccinia virus of table

Type	Content (mg/kg)
		Arginine	39.043
Lysine	0.375
		Alanine	7.393
Threonine	3.646
		Glycine	4.527
Valine	2.370
		Serine	7.586
Proline	3.472
		Isoleucine	11.723
Leucine	22.798
		Methionine	3.293
Histidine	1.323
		Phenylalanine	9.979
Glutamic acid	1.568
		Aspartic acid	1.950
Cystine	0.314
		Tyrosine	3.728

The testing result of table 3 is shown, at least 13 kinds of amino acid are contained in prepared rabbit fur extractive.

(4) LC-MS/MS is detected

In addition, detecting LC-MS/MS method to product obtained in (H).Before detection, by acquisition in (H) The concentration (potency) of rabbit fur extractive is adjusted to 1.2NU/ml, and by the NTP injection of commercially available acquisition (Neurotropin/ NEUR0TR0PIN, 1.2NU/ml) it is used as control.Used instrument and testing conditions are as follows:

Liquid chromatogram (LC)

Instrument: SHIMAZDU 20AXR；

Chromatographic column: Phexnomonex Bi-phenyl 2.6um, 3.0X 50mm；

Mobile phase A: water (contains 0.1% formic acid)；Mobile phase B: acetonitrile (contains 0.1% formic acid)；

Flow velocity: 0.5mL/min；Sampling volume: 3uL；Column temperature: 35 DEG C；

Elution program:

Second order ms (MS/MS)

Instrument: QTRAP5500 (AB SCIEX), the source ESI

MS/MS condition:

Source parameters	Numerical value
		CUR	30psi
IS	5500(+),-4500(-)
		TEM	500℃
GS1	50psi
		GS 2	50psi

Compound test condition:

Quantitative result is as follows:

Analyte	Prepared rabbit fur extractive, ng/mL	NTP injection, ng/mL
			Citrulling	204	39.5
Carnosine	31.1	2.54
			GABA	179	0.463
L-carnitine	111	126

Above-mentioned testing result shows that the every NU unit of rabbit fur extractive prepared by the present embodiment contains about 170ng citrulling, About 25.9ng carnosine, about 149ng GABA, significantly larger than commercially available NTP injection；Wherein, the content of GABA improves about 380 Times, the content of carnosine improves about 12 times, and the content of citrulling improves about 5 times.

2. rabbit fur extractive of embodiment promotes the assessment of the effect of oligodendrocyte proliferation

The present embodiment application oligodendroglia studies the work that rabbit fur extractive made from embodiment 1 promotes it proliferation With.

Oligodendroglia is extracted out of the SD female rats in newborn 48h cerebral cortex.After passage 5 times, aobvious The form of micro- microscopic observation oligodendroglia.Then, by the cell after passing on 5 times in the coated orifice plate middle berth of poly-D-lysine Plate.The 1st day after bed board, using oligodendroglia specific antibody (anti-O4 antibody) and DAPI decoration method to the cell of culture into Row identification and characterization.

Experimental result is as shown in figs. 3 a-3 c.Fig. 3 A, which is shown, carries out immunofluorescence inspection with cell of the anti-O4 antibody to culture The result (fluorescence micrograph) of survey.The results show that the cell cultivated is that anti-O4 antibody test is positive, it is that oligodendroglia is thin Born of the same parents.Fig. 3 B shows the result (fluorescence micrograph) detected with cell of the DAPI decoration method to culture, wherein shows The nucleus being colored.Fig. 3 C is the merging image of Fig. 3 A and Fig. 3 B.The results show that the cell of the anti-O4 antibody test positive and The ratio of the cell of DAPI dyeing is 1:1.This shows that cultivated cell is oligodendroglia, and cell purity is 100%.

Oligodendroglia is layered in 96 orifice plates according to the density of every 5000 cells in hole, 7 experimental groups and 1 are set Control group, in which:

The cell culture medium that 7 experimental groups use is DMEM/F12 culture medium, wherein the embodiment 1 for adding concentration respectively is made Standby rabbit fur extractive, make its final concentration in the medium be respectively 0.2NU/ml, 0.5NU/ml, 1NU/ml, 1.5NU/ml, 2NU/ml,2.5NU/ml,3NU/ml；

The cell culture medium that 1 control group uses is DMEM/F12 culture medium, wherein being added with PBS.

By 8 groups of cells in 37 DEG C, 5% CO₂Culture in incubator.The the 2nd, 4,6 day after starting culture, according to reagent Box specification carries out proliferation detection to the cell cultivated in 96 orifice plates under the conditions of 450nm using cck-8 kit.

Experimental result is as shown in figs. 4 a-4 c.Fig. 4 A-4C respectively illustrates the 2nd day (Fig. 4 A), the 4th after starting culture It its (Fig. 4 B), the 6th day (Fig. 4 C), is incubated at each group in the cell culture medium of the rabbit fur extractive containing various concentration and dashes forward less glue The proliferative conditions (absorbance measured at 450nm using cck-8 kit) of cell plastid.The results show that prepared rabbit Extract is in concentration and time dependence to the proliferation of oligodendroglia.In addition, result is also shown, prepared rabbit It can be for example, 0.5-3NU/ml in cell culture medium that bark extract, which promotes the concentration of oligodendrocyte proliferation,.These knots Fruit shows that prepared rabbit fur extractive can promote the proliferation of oligodendroglia.For example, can use comprising concentration for The cell culture medium culture oligodendroglia of the rabbit fur extractive of 1.5NU/ml, to promote the proliferation of the cell.

3. rabbit fur extractive of embodiment promotes the assessment of the effect of Proliferation of Bone Mesenchymal Stem Cells

Mesenchymal stem cell in the present embodiment application rat femur studies rabbit fur extractive pair made from embodiment 1 It promotes the effect of proliferation.

Mesenchymal stem cell is extracted out of wistar female rats femur in newborn 48h.It, will after passage 5 times Mesenchymal stem cell is layered in 96 orifice plates according to the density of every 5000 cells in hole, and 7 experimental groups and 1 control are arranged Group, in which:

By 8 groups of cells in 37 DEG C, 5% CO₂Culture in incubator.The the 2nd and 4 day after starting culture, according to reagent Box specification carries out proliferation detection to the cell cultivated in 96 orifice plates under the conditions of 450nm using cck-8 kit.

Experimental result is as indicated by figures 5 a-5b.Fig. 5 A-5B respectively illustrates the 2nd day (Fig. 5 A) and the 4th after starting culture Its (Fig. 5 B), is incubated at each group mesenchymal stem cell in the cell culture medium of the rabbit fur extractive containing various concentration Proliferative conditions (absorbance measured at 450nm using cck-8 kit).The results show that prepared rabbit fur extractive pair The proliferation of mesenchymal stem cell is in concentration and time dependence.In addition, result is also shown, prepared rabbit is mentioned The concentration for taking object to promote Proliferation of Bone Mesenchymal Stem Cells can be for example, 0.2-3NU/ml in cell culture medium.These knots Fruit shows that prepared rabbit fur extractive can promote the proliferation of mesenchymal stem cell.Include concentration for example, can use For the cell culture medium culture mesenchymal stem cell of the rabbit fur extractive of 2NU/ml, to promote the proliferation of the cell.

4. rabbit fur extractive of embodiment promotes the assessment of the effect of nerve stem cell proliferation

The present embodiment application neural stem cell studies the effect that rabbit fur extractive made from embodiment 1 promotes it proliferation.

Neural stem cell is extracted out of pregnant 14 days SD Fetal Rat mouse cerebral cortex, in 5%CO₂Under, DMEM/F12 it is thin Culture in born of the same parents' culture medium (being added with bFGF, EGF, LIF and B27).After passage 3 times, the form of cell is observed under the microscope. Then, by pass on 3 times after cell in the coated orifice plate of poly-D-lysine bed board.The 1st day after bed board, neural stem cell is used Specific antibody (anti-Nestin antibody) and DAPI decoration method are identified and are characterized to the cell of culture.

Experimental result is as illustrated in figs. 6 a-6d.Fig. 6 A shows the light micrograph of cultivated cell.The results show that The cells show cultivated goes out the cell that the distinctive bright, three-dimensional sense of neural stem cell by force, around has the suspension of radial burr Spherical state.Fig. 6 B shows result (the fluorescence microscopy photograph that Immunofluorescence test is carried out with cell of the anti-Nestin antibody to culture Piece).The results show that the cell cultivated is that anti-Nestin antibody test is positive, it is neural stem cell.Fig. 6 C shows use The result (fluorescence micrograph) that DAPI decoration method detects the cell of culture, wherein show the nucleus being colored. Fig. 6 D is the merging image of Fig. 6 B and Fig. 6 C.The results show that the cell and DAPI of the anti-Nest in antibody test positive dye it is thin The ratio of born of the same parents is 1:1.This shows that cultivated cell is neural stem cell, and cell purity is 100%.

Neural stem cell is layered in 96 orifice plates according to the density of every 20000 cells in hole, 3 experimental groups and 1 are set Control group, in which:

The cell culture medium that 3 experimental groups use is DMEM/F12 culture medium (having been added bFGF, EGF, LIF and B27), Rabbit fur extractive prepared by the embodiment 1 of concentration is wherein added respectively, and making its final concentration in the medium is respectively 0.5NU/ ml,1NU/ml,2NU/ml；

The cell culture medium that 1 control group uses is DMEM/F12 culture medium (having been added bFGF, EGF, LIF and B27), Wherein it is added with PBS.

By 4 groups of cells in 37 DEG C, 5% CO₂Culture in incubator.The the 1st, 2,3 day after starting culture, according to reagent Box specification carries out proliferation detection to the cell cultivated in 96 orifice plates under the conditions of 450nm using cck-8 kit.

Experimental result is as shown in Figure 7 A-7C.Fig. 7 A-7C respectively illustrates the 1st day (Fig. 7 A), the 2nd after starting culture Its (Fig. 7 B), the 3rd day (Fig. 7 C), each group nerve cord being incubated in the cell culture medium of the rabbit fur extractive containing various concentration The proliferative conditions of cell (cell is opposite to survive).The results show that prepared rabbit fur extractive is proliferated the rush of neural stem cell Effect is in concentration and time dependence.In addition, result is also shown, prepared rabbit fur extractive promotes nerve stem cell proliferation Concentration can be for example, 0.5-2NU/ml in cell culture medium.These results indicate that prepared rabbit fur extractive can promote Into the proliferation of neural stem cell.For example, the cell culture medium culture comprising concentration for the rabbit fur extractive of 2NU/ml can be used Neural stem cell, to promote the proliferation of the cell.In addition, result is also shown, in the presence of the rabbit fur extractive of 2NU/ml Under, the neural stem cell cultivated still shows that suspension, bright, three-dimensional sense by force, around have the thin of the suspension of radial burr Born of the same parents' spherical shape state, remains the feature of neural stem cell.

Assessment of 5. rabbit fur extractive of embodiment to the effect of SD rat neuronal cell

In the present embodiment, using patch clamp technique, the rabbit fur extractive of the preparation of embodiment 1 is had evaluated to SD rat primary The effect of neuronal cell, the especially influence to the GABA signal path of the cell (GABA receptor).Used instrument For EPC10 amplifier system (HEKA company)；The sample tested is rabbit fur extractive (1.2NU/ml) prepared by embodiment 1, And the NTP injection (1.2NU/ml) of commercially available acquisition.

1. material and method

1.1 experiment agents useful for same

PBS/HEPES/Glucose buffer: 6g glucose, 7.38g HEPES, addition PBS 1000ml (TaKaRa, T900), PH=7.38 is adjusted using NaOH.Filtration sterilization.

Cell adhere-wall culture base: 50ml FBS (gibco, 10099-141), 0.55ml glutamic acid (s igma, G1251- 100G), 5ml penicillin/streptomycin 100X (Hyclone, sv30010), be added in 500ml DMEM culture medium (Gibco, 10313021)。

Neurobasal culture medium: 10ml B27Supplement (gibco, 17504044), 1.25ml L-Glutamine 200mM (sigma, 59202C-100ML), 5ml penicillin/streptomycin 100X (Hyclone, sv30010), is added to 500ml In Neurobasal Medium (gibco, 21103049).

1.2 experiment instruments

The separation and culture of 1.3 rat hippocampus nerve cells

1) culture plate is handled with poly-D-lysine, is advisable with covering culture plate bottom, extra poly is drawn after 30min and relies ammonia Acid sets 37 degree of CO₂Incubator is stayed overnight.

2) culture plate is washed 2-3 times with PBS before experiment, dry spare.

3) PBS (containing sugar) each 6 pipe into 50ml and 15ml centrifuge tube is dispensed, -20 degree is placed in and freezes 30min, become ice Aqueous mixtures.

4) experiment is divided into two parts to be put into isopropanol with operating equipment and impregnates 1h.A portion: fine forceps two, rainbow One, film scissors, curved scissors and eye scissors each 1, spoon one, for removing brain tissue；Another part: nerve forceps 2, greatly 1, scissors, eye scissors one, for removing mouse embryo.

5) two box of trash ice (the underlying ice bag of ice chest) is taken, experimental bench is cleaned up, ice bag is set on experimental plate, spray alcohol is most Amount is accomplished sterile.

6) prepare 35mm ware (built-in filter membrane) 3-4 sterile, 1ml liquid-transfering gun and sterile pipette tips.

7) newborn SD rat suckling mouse is sterilized with 75% ethyl alcohol, is breaked end rapidly.

8) brain separated is put into the PBS of ice, waits 2min.

9) brain taking-up is put into the plate containing ice PBS, a filter paper is put in bottom, convenient for fixed brain tissue, makes it not It is slided in plate.

10) skull is peeled off, exposure simultaneously opens cerebral cortex, finds thalamus, the film on surface is cleaned out.

11) thalamus is taken out, is placed in the 10ml centrifuge tube of PBS containing ice.

12) free thalamus is put into the PBS of ice and is washed 1-2 times.

13) PBS is blotted, typsin is added, puts and incubates 20min in 37 degree of incubators, every 5min rocks.

14) tissue after enzymic digestion is taken out, enzyme solution is sucked, the PBS that ice is added terminates reaction, cleans 2-3 times (no with PBS Vibrate tissue).

15) 2min is stood, supernatant liquid is removed.

16) DMEM (for neuro) that 1ml is added is blown and beaten；After piping and druming 15 times, 2min is stood, supernatant is collected, puts Enter in centrifuge tube (centrifuge tube holding low temperature).

17) 1ml DMEM is added, continues piping and druming 10 times, regathers supernatant and enter in same ware.Third time adds 1ml again, After 3 times, indigested tissue is discarded for repetitive operation.

18) supernatant being collected into is put into the plate completed in advance (700000/ hole) (6 orifice plates), shaken up.

19) after 4 hours, under jog is several, dead cell and fragment are siphoned away, then with DMEM clean it is primary after, use Neurobasal culture medium is cultivated.

20) second day, half changed liquid, replaced Neurobasal culture medium.Hereafter, it was changed every two days primary.

21) hippocampal neuron epileptiform discharge model is established: the hippocampal neuron by vitro culture to the 12nd day changes liquid.Nothing Magnesium processing group is gently washed 3 times with no magnesium HBSS, is removed in culture medium and is retained magnesium ion, then uses no magnesium HBSS culture 3h instead, Then restore normal incubation medium or the culture medium containing 10 μM of SSA, incubated cell start to detect after 24 hours.

2. electrophysiological recording

According to the manufacturer's instructions, the acquisition of cellular electrophysiologicalsensor signal is carried out using EPC-10 amplifier (HEKA), and It stores data in PatchMaster (HEKA) software.

In short, drawing instrument (P97, Sutter Ins truments) for glass capillary (BF150-86- with microelectrode 10, Sut ter Ins truments) it is drawn into recording electrode.Electrode fills interior liquid: 130mM CsCl, 1.6mM MgCl₂, 10mM HEPES,5mM EGTA,2mM Na-ATP,pH7.3.It is grasped in inverted microscope (IX71, Olympus) lower-pilot microelectrode Vertical instrument (MP285, Sut ter Ins truments) touches recording electrode on cell, gives negative-pressure ward, forms G Ω envelope It connects.After forming G Ω sealing-in, flying capacitance compensation is carried out, then proceedes to give negative pressure, inhales broken cell film, form whole-cell recording technique Mode.Then it carries out the compensation of capacitor at a slow speed and records membrane capacitance and series resistance.

GABA electric current recording method:

Intracellular fluid: 130mM CsCl, 0.1mM CaCl₂,2mM MgCl₂,1.1mM EGTA,5mM Na₂ATP and 10mM HEPES, pH=7.15, are adjusted with CsOH；

Extracellular fluid: 140mM NaCl, 5mM CsCl, 2mM CaCl₂,1mM MgCl₂, 5mM HEPES and 10mM Glucose, pH=7.35 are adjusted with NaOH.

20 μM of-DNQX, 20 μM of-DAP5 and 300nM-TTX are added in external solution.

Under voltage-clamp mode, after -70mV stablizes five minutes, recorded at -70mV.

3. experimental result

Using above-mentioned experimental program, in GABA (10 μM), NTP injection, NTP injection+Bic (Bicucul Line, 10 μM), rabbit fur extractive+Bic (10 μM) for preparing of the rabbit fur extractive for preparing of embodiment 1 or embodiment 1 In the presence of, record the GABA electric current (being tested respectively with 2 parts of cell samples) of hippocampus of rats cell.It is used The concentration (potency) of NTP injection and rabbit fur extractive is 1.2*10^-2NU/ml.Bic is a kind of competitiveness GABAA Receptor antagonist inhibits GABA signal (electric current) for specificity.Experimental result is as shown in table 4-5 and Fig. 8 A-8D.

Table 4: the GABA electric current (pA) of rabbit fur extractive prepared by hippocampus of rats cellular response embodiment 1

Table 5: the GABA electric current (pA) of hippocampus of rats cellular response NTP

Fig. 8 A-8B is respectively illustrated, and cell sample 1 (Fig. 8 A) and cell sample 2 (Fig. 8 B) respond GABA, rabbit of the invention Bark extract or rabbit fur extractive+Bic GABA electric current of the invention.Fig. 8 C-8D is respectively illustrated, cell sample 1 (Fig. 8 C) and cell sample 2 (Fig. 8 D) respond GABA, NTP injection or NTP injection+Bic GABA electric current.Knot Fruit shows, compared with NTP injection, rabbit fur extractive of the invention can stimulate hippocampus of rats cell to generate significantly more Strong GABA electric current.The experiment conclusion verified in this experimental result and embodiment 1 in rabbit fur extractive of the invention (that is, contain Have the GABA of higher amount) it is consistent.Rabbit fur extractive of the invention has better application prospect.

Assessment of 6. rabbit fur extractive of embodiment to the therapeutic effect of spinal cord injury

In the present embodiment, rabbit fur extractive produced above is studied to spinal cord injury using the spinal cord injury model of rat Therapeutic effect.

In short, being hit using 8 week old female Wista rats using New York University (NYU) standard spinal cord injury (Impactor Model II) hits spinal cord in 10 segment of rat chest, constructs the animal model of spinal cord injury.24 is small after damage When, rabbit fur extractive prepared by embodiment 1 respectively with the dosage intraperitoneal injection of 25NU/kg, 50NU/kg, 100NU/kg, Once a day, 2 weeks after continuing to damage.In this experiment, while 2 groups of negative controls are set, one group is not to be subjected to spinal cord injury Rat, another group is the rats with spinal cord injury treated with physiological saline.Then, pass through BBB scoring (Basso Beat Tie Bresnahan motor function scores) and GFAP (glial fibril lary acidic protein) immunostaining The functional rehabilitation of experimental animal and the size of glial scar after observation treatment, wherein according to Basso DM, Beattie MS, Bresnahan JC.A sensi tive and rel iable locomotor rating scale for open field testing in rats.J Neurotrauma 1995；12 (1): method described in 1-21 scores to measure BBB, also, Method (Enrichment of differentiated, the stel late as trocytes described according to Levi G. et al. in cerebel lar interneuron cul tures as studied by GFAP immunof luorescence and autoradiographic uptake patterns with[³H]D-aspartate and[³H]GABA.Brain Res.1983Nov；312 (2): 227-41) Lai Jinhang GFAP immunostaining.Experimental result is as shown in figs. 9-10.

Fig. 9-10 shows that rabbit fur extractive is compared with physiological saline is to the therapeutic effect of rats with spinal cord injury.Wherein, Fig. 9 shows that rats with spinal cord injury receives the motor function recovery after the rabbit fur extractive treatment of physiological saline or prescribed dose Situation (that is, BBB scores).Figure 10 shows that rats with spinal cord injury receives physiological saline or the rabbit fur extractive of prescribed dose is controlled Glial scar formational situation (that is, GFAP immunostaining) after treatment.

From fig. 9, it can be seen that the BBB scoring of rabbit fur extractive treatment group rat is significantly higher than saline control group, this Show that rabbit fur extractive of the invention can remarkably promote the motor function recovery after Spinal Cord Injury in Rats, can be used for treating spinal cord Damage.From fig. 10 it can be seen that the GFAP expression of the rat of rabbit fur extractive treatment group is substantially less than the rat of control group GFAP expression, this shows that rabbit fur extractive of the invention is able to suppress the formation of glial scar, after promoting spinal cord injury Tissue repair and functional rehabilitation.

These the experimental results showed that, compared with the control group, rabbit fur extractive of the invention can substantially reduce glial scar Formation, promote Spinal Cord Injury in Rats after motor function recovery.It is indicated above that rabbit fur extractive of the invention is to inhibition spinal cord Damage and inhibition glial scar, which are formed, has determining curative effect.

Although a specific embodiment of the invention has obtained detailed description, those skilled in the art will appreciate that root According to all introductions having disclosed, details can be carry out various modifications and be changed, and these change in guarantor of the invention Within the scope of shield.Full scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. a kind of method for being used to prepare rabbit fur extractive, the described method comprises the following steps:

(B) skin is mixed with water, and stirred 2-10 hours, obtain mixture；Then, the pH of the mixture is adjusted To 9.5-10.5, and continue stirring 10-20 hours；

(C) (such as filtering, filter and/or be centrifuged) is separated by solid-liquid separation to the product of step (B), is mentioned to obtain first Take liquid；With

(D) rabbit fur extractive is prepared using first extracting solution；

Preferably, the vaccinia virus is selected from vaccinia virus Li Site (Lister) strain, vaccinia virus Dalian (Dairen) strain, acne Seedling diseases poison Ikeda (Ikeda) strain, EM-63 plants of vaccinia virus, vaccinia virus New York community health office (New York City Board of Health) strain, vaccinia virus ankara (Ankara) strain, vaccinia virus Copenhagen (Copenhagen) strain, acne The seedling diseases poison the Temple of Heaven (Tian Tan) strain, WR plants of vaccinia virus and buffalo pox virus；

Preferably, the rabbit is selected from cave rabbit (such as Japanese white kind rabbit and New Zealand White kind rabbit), hare (example Such as Japanese hare), pika, snow hare；

Preferably, before carrying out step (B), the skin is pre-processed；Preferably, the pretreatment includes freezing, It thaws, cleans, disinfection, ultrasonication is crushed, or any combination thereof；For example, before carrying out step (B), to the skin Break process is carried out, (partial size of the particulate matter is, for example, 50-75 μm, 75-100 μ so that the skin is broken for particulate matter M, 100-150 μm, 150-200 μm, 200-500 μm)；

Preferably, in step (B), the w/v (g/ml) of the skin and water is 1:(1-5), such as 1:(1-1.5), 1:(1.5-2), 1:(2-2.5), 1:(2.5-3), 1:(3-4) or 1:(4-5)；

It preferably, at room temperature (such as 15-20 DEG C, 20-25 DEG C, 25-30 DEG C or 30-35 DEG C), will be described in step (B) Skin is mixed with water, and is stirred 2-10 hours (for example, 2-4 hours, 4-6 hours, 6-8 hours or 8-10 hours), to obtain Mixture；

Preferably, in step (B), the pH of the mixture is adjusted to 9.5-10.5 using alkali, such as be adjusted to pH10；It is excellent Selection of land, the alkali are inorganic base, such as NaOH or KOH；

Preferably, in step (B), the pH of the mixture is adjusted to 9.5-10.5, and at elevated temperatures (such as 40-42 DEG C, 42-45 DEG C, 45-48 DEG C or 48-50 DEG C) continue stirring 10-20 hours (for example, 12-14 hours, 14-16 hours, 16-18 hours or 18-20 hours)；

Preferably, in step (C), the product of step (B) is separated by solid-liquid separation by filtering or filtering；Preferably, with 50- The filter hole of 500 μm (such as 50-75 μm, 75-100 μm, 100-150 μm, 150-200 μm, 200-500 μm or 75-200 μm) Diameter is filtered or is filtered to the product of step (B)；

Preferably, in step (B), phenol is not used；

Preferably, in step (B), organic solvent is not used；

Preferably, the method does not use phenol；

Preferably, the method does not use organic solvent.

2. the method for claim 1 wherein, the step (D) the following steps are included:

(D1) pH of first extracting solution is adjusted to 4.0-5.0, and is heated, then, be separated by solid-liquid separation (example Such as filtered, filter and/or be centrifuged), to obtain the second extracting solution；

(D2) pH of second extracting solution is adjusted to 9.0-10.0, and is heated, then, is separated by solid-liquid separation (such as being filtered, filter and/or be centrifuged), to obtain third extracting solution；

(D3) pH of the third extracting solution is adjusted to 3.5-5.5, and is contacted with adsorbent, make the activity in third extracting solution Substance is adsorbed on adsorbent；

(D4) product of step (D3) is separated by solid-liquid separation (such as filtering, filter and/or be centrifuged), collects adsorbent； With

(D5) adsorbed active material on adsorbent is extracted with the Extraction solvent that pH is 9.0-11.0, is extracted to obtain the 4th Liquid, as rabbit fur extractive.

Preferably, in step (D1), the pH of first extracting solution is adjusted to 4.0-5.0 using acid, such as be adjusted to pH4.5；Preferably, the acid is inorganic acid, such as HCl；

Preferably, in step (D1), the pH of first extracting solution is adjusted to 4.0-5.0, and be heated to 85-105 DEG C of (example Such as 85-90 DEG C, 90-95 DEG C, 95-100 DEG C or 100-105 DEG C)；Preferably, the heat treatment carries out 0.5-1.5 hours, example Such as 0.5-1 hours or 1-1.5 hours；

Preferably, in step (D2), the pH of second extracting solution is adjusted to 9.0-10.0 using alkali, such as be adjusted to pH9.5；Preferably, the alkali is inorganic base, such as NaOH or KOH；

Preferably, in step (D2), the pH of second extracting solution is adjusted to 9.0-10.0, and be heated to 85-105 DEG C (such as 85-90 DEG C, 90-95 DEG C, 95-100 DEG C or 100-105 DEG C)；Preferably, in step (D2), it is described heat into Row 0.25-1.5 hours, such as 0.25-0.5 hours, 0.5-1 hours or 1-1.5 hours；Preferably, in step (D2), right After second extracting solution is heated, be stirred for 10-20 hours (for example, 10-12 hours, 12-14 hours, 14-16 Hour, 16-18 hours or 18-20 hours), it is then separated by solid-liquid separation again, to obtain third extracting solution；

Preferably, in step (D2), by filtering or filtering (for example, the use of aperture being 0.22-0.65 μm (such as 0.45 μm) Filter membrane) be separated by solid-liquid separation, to obtain third extracting solution；

Preferably, in step (D3), the pH of the third extracting solution is adjusted to 3.5-5.5, such as 3.5-4.0 using acid, 4.0-4.5,4.5-5.0 or 5.0-5.5；Preferably, the acid is inorganic acid, such as HCl；

Preferably, in step (D3), the adsorbent is selected from active carbon, kaolin and any combination thereof；

Preferably, the weight ratio (g/g) of adsorbent used in step (D3) and skin used in step (B) is 1: (10-50), such as 1:(10-15), 1:(15-20), 1:(20-25) and, 1:(25-30), 1:(30-40) or 1:(40-50)；

Preferably, in step (D3), prior to, concurrently with, or after being contacted with adsorbent, by the pH tune of the third extracting solution It is whole to arrive 3.5-5.5；

Preferably, in step (D3), the pH of the third extracting solution is adjusted to 3.5-5.5, and contact with adsorbent, then (such as 15-35 DEG C, 15-20 DEG C, 20-25 DEG C, 25-30 DEG C or 30-35 DEG C) stirs 1-4 hours at room temperature, such as 1-2 small When, 2-3 hours or 3-4 hours；

Preferably, in step (D3), the adsorbent is filled in adsorption column, is then extracted with the adjusted third of pH value Liquid contact, to adsorb the active material in third extracting solution；

Preferably, in step (D4), the product of step (D3) is consolidated by filtering or filtering (for example, using filter paper) Liquid separation, to obtain the adsorbent for having adsorbed active material；

Preferably, step (D5) includes contacting the adsorbent with the Extraction solvent that pH is 9.0-11.0, then carrying out solid-liquid It separates (such as being filtered, filter and/or be centrifuged), to obtain the 4th extracting solution；

Preferably, in step (D5), the Extraction solvent is alkaline solution (for example, the aqueous solution of alkalinity, methanol solution, second Alcoholic solution, aqueous isopropanol, or any combination thereof；It is preferred that the solution of alkali)；Preferably, in step (D5), it is using pH The alkaline solution of 9.0-11.0 (such as pH is 9.0-9.5,9.5-10.0,10.0-10.5 or 10.5-11.0) is (for example, alkalinity Aqueous solution, methanol solution, ethanol solution, aqueous isopropanol, or any combination thereof；It is preferred that the solution of alkali) it extracts on adsorbent The active material of absorption；Preferably, the solution of the alkali is the aqueous solution of inorganic base, such as the aqueous solution of NaOH or KOH；

Preferably, the w/v of adsorbent used in step (D3) and Extraction solvent used in step (D5) (g/ml) it is 1:(10-100), such as 1:(10-20), 1:(20-30), 1:(30-40), 1:(40-50) and, 1:(50-60), 1: (60-70), 1:(70-80), 1:(80-90) or 1:(90-100)；

Preferably, it in step (D5), under conditions of heating and/or stirring, is extracted with the Extraction solvent and is inhaled on adsorbent Attached active material；Preferably, in step (D5), 40-50 DEG C at a temperature of (such as 40-42 DEG C, 42-45 DEG C, 45-48 DEG C or 48-50 DEG C), and under stirring conditions (such as stirring 1-4 hours, such as it is 1-2 hours, 2-3 hours or 3-4 small When), the active material adsorbed on adsorbent is extracted with the Extraction solvent；

Preferably, one or many with the active material adsorbed on Extraction solvent extraction adsorbent in step (D5), example Such as at least 2 times；It is then combined with the extracting solution for extracting acquisition each time, to obtain the 4th extracting solution；Preferably, in step (D5) In, first carry out first time extraction with the Extraction solvent that pH is 9.0-10.0, then again with pH be 10.0-11.0 Extraction solvent into Second of extraction of row, is then combined with the extracting solution for extracting acquisition twice, to obtain the 4th extracting solution.

3. the method for claims 1 or 2, wherein the method further includes selected from following one or more after step (D) Step:

(E1) ultrafiltration is carried out to the 4th extracting solution；

(E2) the 4th extracting solution is heated；

(E3) pH of the 4th extracting solution is adjusted to 6.5-7.5；

(E4) the 4th extracting solution is diluted or is concentrated；

(E6) frozen dried is carried out to the 4th extracting solution；

Preferably, in step (E1), molecular weight boundary used by ultrafiltration is 5-40KD, such as 5-10KD, 10-15KD, 15- 20KD, 20-30KD or 30-40KD；

Preferably, in step (E2), the 4th extracting solution is heated to 60-100 DEG C, such as 60-70 DEG C, 70-80 DEG C, 80-90 DEG C or 90-100 DEG C, and optionally, stand 10-18 hours, for example, 10-12 hours, 12-14 hours, 14-16 hours or 16-18 hours；

Preferably, in step (E3), the pH of the 4th extracting solution is adjusted to 6.5-7.5 using acid, for example, 6.5-7.0 or 7.0-7.5；Preferably, the acid is inorganic acid, such as HCl；

Preferably, in step (E4), using water for injection, physiological saline or physiological buffer carry out the 4th extracting solution Dilution；

Preferably, in step (E5), using high pressure sterilization, ultra-high temperature sterilization (UHT), pasteurization, filtration sterilization or its What is combined, and carries out sterilization treatment to the 4th extracting solution；

Preferably, the method further includes 1,2,3,4,5 or 6 in step (E1)-(E6) after step (D) A step.

4. a kind of rabbit fur extractive is made by the method for any one of claim 1-3.

5. a kind of rabbit fur extractive does not contain any organic solvent (such as phenol), and the rabbit fur extractive of every NU unit In containing containing 0.8-150ng gamma-amino fourth at least 0.8ng γ-aminobutyric acid, such as the rabbit fur extractive of every NU unit Acid, such as contain 0.8-1ng, 1-2ng, 2-5ng, 5-10ng, 10-20ng, 20-50ng, 50-70ng, 70-100ng, 100- 120ng or 120-150ng γ-aminobutyric acid；

Preferably, it is also extracted containing at least 4ng carnosine, such as the rabbit of every NU unit in the rabbit fur extractive of every NU unit Also contain 4-30ng carnosine in object, such as contains 4-5ng, 5-7ng, 7-10ng, 10-12ng, 12-15ng, 15-20ng, 20- 22ng, 22-25ng or 25-30ng carnosine；

Preferably, at least 60ng citrulling, such as the rabbit of every NU unit is also contained in the rabbit fur extractive of every NU unit Also contain 60-200ng citrulling in extract, such as contain 60-80ng, 80-100ng, 100-120ng, 120-150ng, 150-170ng or 170-200ng citrulling；

Preferably, the rabbit fur extractive is prepared by the method for any one of claim 1-3.

6. a kind of composition, it includes the rabbit fur extractives of claim 4 or 5；

Preferably, the composition is cell culture medium, such as DMEM culture medium, F12 culture medium or DMEM/F12 culture medium；It is excellent Selection of land, the cell culture medium also include additional reagent, such as serum (such as fetal calf serum), cell factor, B27 cell Additive is cultivated, or any combination thereof；Preferably, the cell factor is selected from epithelical cell growth factor (EGF), leukaemia suppression The factor (LIF) processed, fibroblast growth factor (FGF；Including but not limited to aFGF and bFGF), or any combination thereof；

Preferably, the composition is pharmaceutical composition, also includes pharmaceutically acceptable carrier or excipient；It is preferred that Ground, described pharmaceutical composition are injection, tablet or freeze dried powder.

7. the purposes of the rabbit fur extractive of claim 4 or 5 in medicine preparation, the drug is for treating Lumbago, neck shoulder Wrist syndrome, symptomatic neuralgia, the itching with skin disease (such as eczema, dermatitis, nettle rash), allergic rhinitis, Asia Creeping chill-abnormal perception-pain of acute optic nerve myelopathy sequelae, postherpetic neuralgia, scapulohumeral periarthritis, morphotropism joint Scorching or central nervous system disease, for promoting the motor function recovery after spinal cord injury, or for inhibiting glial scar to be formed, Glial scar especially after spinal cord injury is formed；

Preferably, the central nervous system disease is selected from traumatic brain injury (TBI) and spinal cord injury (SCI)；

Preferably, the spinal cord injury is by spine fracture or caused spinal cord injury of dislocating；

Preferably, the drug also includes pharmaceutically acceptable carrier or excipient；

Preferably, the drug is the pharmaceutical dosage form being administered suitable for systemic vein, such as injection or freeze dried powder.