CN106110290A - A kind of preparation method of animal testis extract - Google Patents
A kind of preparation method of animal testis extract Download PDFInfo
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- CN106110290A CN106110290A CN201610739410.7A CN201610739410A CN106110290A CN 106110290 A CN106110290 A CN 106110290A CN 201610739410 A CN201610739410 A CN 201610739410A CN 106110290 A CN106110290 A CN 106110290A
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/01—Hydrolysed proteins; Derivatives thereof
- A61K38/012—Hydrolysed proteins; Derivatives thereof from animals
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
- A23J1/02—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from meat
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J3/00—Working-up of proteins for foodstuffs
- A23J3/04—Animal proteins
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
- A61K31/565—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol
- A61K31/568—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol substituted in positions 10 and 13 by a chain having at least one carbon atom, e.g. androstanes, e.g. testosterone
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
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Abstract
The present invention relates to polypeptide extractive technique field, particularly to the preparation method of a kind of animal testis extract.The preparation method of this animal testis extract includes: is homogenized by animal testis, enzymolysis and extraction, obtains enzymolysis crude extract and filtering residue；Enzymolysis crude extract obtains ultrafiltrate through ultrafiltration, and ultrafiltrate removes macromolecule impurity through agarose gel adsorption by hydrogen bond chromatographic process again, obtains enzymolysis and extraction refined liquid；Filtering residue uses ethanol extraction to concentrate, and obtains concentrating alcohol extract；Enzymolysis and extraction refined liquid is mixed with concentrating alcohol extract, lyophilization, obtain animal testis extract.Testis extraction and preparation technique of the present invention uses the method that toolenzyme extraction, ultrafiltration, agarose adsorption by hydrogen bond chromatographic separation and purification combine, micromolecule polypeptide class active substance is made to be enriched with, the water-fast testosterone of filtering residue alcohol extraction concentration and recovery, reduces the loss of active component.Its method is easy, and safely and effectively, product biological activity is good.
Description
Technical field
The present invention relates to polypeptide extractive technique field, particularly to the preparation method of a kind of animal testis extract.
Background technology
Along with the quickening of Modern Live rhythm, social competition is growing more intense, and inter personal contact complexity is nervous, the psychology mistake of people
The situation of the caused sub-health state of weighing apparatus is on the rise.Sub-health state refers to feel slight without clinical symptoms or symptom, but existing
The fuselage state of potential pathological information, this is an inferior health status of class, between boundary's health and disease.World Health Organization (WHO) will
Body is without organic disease, but has the state of some changing functions to be referred to as " third state ", is sub-health state.Subhealth state
Status and appearance is vigor decline, function and the symptom of adaptation decline of certain time.It is in the people of sub-health state, if
Dredging in time, subhealth state shade can be walked out, if developed as one pleases, then can change into disease.In subhealth state high-risk group, glycosuria
Disease, hypertension, tumor be again high-risk in high-risk, if intervened not in time, the life of people can be threatened, causing 40 years old with regard to premature death
Situation.
According to the order of severity of the state of an illness, sub-health state comprises successive several stages: wherein, with health tightly
Adjacent is referred to as " slight psychosoma imbalance ", and it is often with fatigue, insomnia, poor appetite, emotional instability etc. as primary symptom, but these
Imbalance easily recovers, and has recovered then to there is no from Healthy People different, slight psychosoma de-synchronization state crowd account for total crowd 25%～
28%.If this imbalance sustainable development, " latent clinic " state can be entered, the most present and developed into the high-risk of some disease and incline
To, lying dormant possible to the height of certain sick development, latent clinical state crowd exceedes the 1/3 of total crowd, and the people more than 40 years old
In Qun, ratio increases suddenly.Visible, the subhealth state problem with confirmed fatigue as cardinal symptom is 21 century to threaten the great of human health
Problem, and incidence rate is in the trend increased year by year.Therefore, how to reduce sub-health state crowd, increase health status crowd and become
Focus for medical research the most both at home and abroad.
Animal testis includes abundant testosterone, animal proteinum and polypeptide, has kidney invigorating and YANG supporting, YIN nourishing benefit essence, resisting fatigue, rush
Enter the effect of man's gonad development etc..Testosterone is also known as Testosterone or testosterone, by testis or the ovarian secretion of women of male, on kidney
Gland also secretes a small amount of testosterone, has maintenance muscle strength and quality, maintenance bone density and intensity, refreshes oneself and promotes physical ability etc. and makees
With.Testosterone can affect many body systems and function, including: hemopoiesis, internal calcium balance, bone mineralization, lipid metabolism, sugar generation
Thank and increase with prostate.It is main male sex hormone and anabolic hormone.Whether sex, health is had emphatically by it
The impact wanted, including gaining in strength, the effect such as immunologic function, antagonism osteoporosis.
Cultivated animals testis have food materials Representative properties, compared with other the kidney invigorating integration of edible and medicinal herbs raw material, security feature
Higher.Presently disclosed testis extractive technique, many employing water carry or enzyme assisted extraction method realizes, and extract product without refined.
Being additionally, since testosterone water insoluble, existing extracting method have lost the testosterone of more than 90%, causes the very big damage of active component
Losing, extraction efficiency and its lytic activity all ratios are relatively low.It is thus desirable to provide the testis that a kind of yield is high and active component content is high to carry
Take thing preparation method.
Summary of the invention
In view of this, the invention provides the preparation method of a kind of animal testis extract.This preparation method obtains activity
The content of polypeptide is high, and productivity is higher.
In order to realize foregoing invention purpose, the present invention provides techniques below scheme:
The invention provides the preparation method of a kind of animal testis extract, comprise the following steps:
Step 1: animal testis is homogenized, enzymolysis and extraction, obtains enzymolysis crude extract and filtering residue；
Step 2: enzymolysis crude extract obtains ultrafiltrate through ultrafiltration, ultrafiltrate is again through agarose gel adsorption by hydrogen bond chromatographic process
Remove macromolecule impurity, obtain enzymolysis and extraction refined liquid；
Filtering residue uses ethanol extraction to concentrate, and obtains concentrating alcohol extract；
Step 3: enzymolysis and extraction refined liquid is mixed with concentrating alcohol extract, lyophilization, obtain animal testis extract.
In some embodiments that the present invention provides, animal testis is selected from pig, cattle, horse, sheep, rabbit, fox any one
Or the testis of several animal.
As preferably, in step 1, enzymolysis and extraction comprises the following steps:
Transplanting even slurry mixes with water, regulates pH to 7.0～9.0；
Add protease, enzymolysis 2～8 hours under the conditions of 37～50 DEG C, obtain enzymolysis solution；
Enzymolysis solution is warming up to 80～95 DEG C, is incubated 5～20 minutes, makes enzyme-deactivating；
Enzymolysis solution after enzyme deactivation, through filtering, obtains enzymolysis crude extract and filtering residue.
As preferably, protease is one or both of trypsin or alkaline protease.
As preferably, the addition of protease is the 0.1%～0.5% of transplanting even slurry weight.
In some embodiments that the present invention provides, in step 1, enzymolysis and extraction comprises the following steps:
Step 1: transplanting even slurry mixes with water 1:1, regulates pH to 7.0～9.0.
Step 2: add protease, enzymolysis 2～8 hours under the conditions of 37～50 DEG C.Protease, for trypsin or alkalescence egg
One or both of white enzyme, addition is the 0.1%-0.5% of transplanting even slurry weight.
Step 3: enzymolysis solution is warming up to 80～95 DEG C, is incubated 5～20 minutes, makes enzyme-deactivating.
Step 4: enzymolysis solution after enzyme deactivation, through 300 mesh filtered through gauze, obtains enzymolysis crude extract and filtering residue.
As preferably, in step 2, the molecular cut off of the ultrafilter membrane that ultrafiltration uses is 10KD.
As preferably, agarose gel adsorption by hydrogen bond chromatograph is: using the agarose gel with affinity ligand is filler
Affinity chromatograph chromatograph；Affinity ligand is Quercetin aglucon, gallic acid aglucon, the one of beta-schardinger dextrin-aglucon.
The present invention provide some embodiments in, agarose gel adsorption by hydrogen bond chromatographic process particularly as follows:
The content of peptides of water use regulation ultrafiltrate is 15～45mg/mL, uses the flow velocity loading absorption of 5～8mL/min, on
Sample amount is the 10～20% of column volume；
Successively with 2 times of bed volumes of initial flow phase eluting, 2 times of bed volumes of pure water eluting, 2%～9% acetic acid is linear
Gradient elution 6～10 times of bed volumes, finally by 2 times of bed volumes of 9% acetic acid eluting, elution flow rate 10～30mL/min；
Collection absorbance is 280nm, the activity eluting peak more than 10%, merges into enzymolysis and extraction refined liquid.
As preferably, initial flow is ethanol, acetic acid and the solution of water composition mutually, ethanol initial flow mutually in volume
Percentage concentration is 20%-30%, acetic acid initial flow mutually in concentration expressed in percentage by volume be 2～5%.
As preferably, ethanol extraction concentrates and comprises the following steps:
(1) filtering residue and ethanol water volume ratio 1:(3 by weight～8) mix, 50～100 revs/min stirring extract 1～
3 hours, gained extracting solution by centrifugation, collected supernatant；After repeating to extract 2～3 times, merge supernatant；
(2) gained supernatant is through being evaporated to the 1/10～1/8 of original volume, obtains concentrating alcohol extract, reclaims ethanol；Subtract
The concentration pressure that pressure concentrates is 0.05～0.15MP, controls the temperature of supernatant less than 65 DEG C during concentrating under reduced pressure.
In some embodiments that the present invention provides, ethanol extraction concentrates and comprises the following steps:
(1) filtering residue and 95% ethanol volume ratio 1:3 by weight～8 mix, and 50～100 revs/min of stirring extractions 1～3 are little
Time.Extract is centrifuged 10～20 minutes through 3000 revs/min, collects supernatant.After repeating to extract 2～3 times, merge centrifugal supernatant
Liquid.
(2) centrifuged supernatant is through concentrating under reduced pressure, is original 1/10～1/8 to volume, obtains concentrating alcohol extract, reclaims second
Alcohol.
In the embodiment that the present invention provides, the preparation of animal testis extract includes step in detail below:
(1) animal testis peels off epididymal adipose tissues, cleans after removing a small amount of blood stains in surface, and colloid mill grinds, and obtains testis even
Slurry.
(2) transplanting even slurry mixes with water volume ratio 1:1 by weight, and the pH value of regulation mixed liquor is to 7.0～9.0.
(3) 0.1%～0.5% addition protease of transplanting even slurry weight, 37～50 DEG C of enzymolysis 2～8 hours are pressed in addition.
(4) enzymolysis solution is warming up to 80～95 DEG C, is incubated 5～20 minutes, makes enzyme-deactivating.
(5) enzymolysis solution after enzyme deactivation is by 300 mesh filtered through gauze, obtains enzymolysis crude extract and filtering residue.
(6) step (5) gained enzymolysis crude extract is through 10KD ultrafilter membrane ultrafiltration, obtains ultrafiltrate.
(7) step (6) gained ultrafiltrate is through agarose gel affinity chromatograph, collects and merges, obtains enzymolysis refined liquid.
(8) step (5) gained filtering residue and 95% ethanol volume ratio 1:3 by weight～8 mix, 50～100 revs/min of stirrings
Extract 1～3 hour.Extract is centrifuged 10～20 minutes through 3000 revs/min, collects supernatant.After repeating to extract 2～3 times, close
And centrifuged supernatant.
(9) alcohol extraction supernatant is 0.05～0.15MP, and temperature, less than concentrating under reduced pressure under the conditions of 65 DEG C, reclaims ethanol, makes
Alcohol extraction supernatant volume is concentrated into original 1/10～1/8, obtains concentrating alcohol extract.
(10) the enzymolysis and extraction refined liquid that step (7) obtains mixes with concentrating alcohol extract, and mixing is pulverized in lyophilization,
To the animal testis extract rich in testosterone and bioactive peptide.
In the embodiment that the present invention provides, for improving the biological activity of extract, use the agarose containing affinity ligand
Bioactive peptide in gel chromatography chromatograph enrichment enzymolysis and extraction ultrafiltrate.Agarose gel affinity chromatograph chromatograph is in the steps below
Realize:
(1) agarose gel is swelling: the w/v of 1:10 pressed by the agarose gel containing affinity ligand and distilled water
Mixing, after 60 DEG C of heating 2h, the static 22h of room temperature；Affinity ligand is Quercetin aglucon, gallic acid aglucon, beta-schardinger dextrin-aglucon
One；
(2) dress post: the agarose gel after swelling loads double glazing tubing string, by distilled water flushing 5～10 times of post bed body
Long-pending, irrigation flow rate is 50～80mL/min；
(3) balance: rinsing chromatographic column mutually by the initial flow of 2～5 times of bed volumes, flow velocity is 15～30mL/min.Just
Beginning to flow is ethanol, acetic acid and the solution of water composition mutually, and concentration of alcohol is 20%-30%, and acetic acid concentration is 2%～5%；
(4) loading: the content of peptides of water use regulation testis enzymolysis and extraction ultrafiltrate is 15～45mg/mL, uses 5～8mL/
The continuous loading of min flow velocity, loading volume is the 10%～20% of bed volume；
(5) eluting: successively with 2 times of bed volumes of initial flow phase eluting, 2 times of bed volumes of pure water eluting, 2%～9%
Acetic acid linear gradient elution 6～10 times of bed volumes, finally with 2 times of bed volumes of 9% acetic acid eluting, elution flow rate 10～
30mL/min；
(6) collect: collection absorbance is 280nm, the activity de-peak more than 10%, merges into enzymolysis and extraction refined liquid.
The most used agarose gel is affine absorption chromatograph column, can after initial flow the most again balance,
Can reuse.
The invention provides the preparation method of a kind of animal testis extract.The preparation method bag of this animal testis extract
Include:
Step 1: animal testis is homogenized, enzymolysis and extraction, obtains enzymolysis crude extract and filtering residue；
Step 2: enzymolysis crude extract obtains ultrafiltrate through ultrafiltration, ultrafiltrate is again through agarose gel adsorption by hydrogen bond chromatographic process
Remove macromolecule impurity, obtain enzymolysis and extraction refined liquid；
Filtering residue uses ethanol extraction to concentrate, and obtains concentrating alcohol extract；
Step 3: enzymolysis and extraction refined liquid is mixed with concentrating alcohol extract, lyophilization, obtain animal testis extract.
The device have the advantages that for:
1, testis extraction and preparation technique use toolenzyme extraction, filtration, ultrafiltration, agarose adsorption by hydrogen bond chromatographic isolation pure
Change the method combined, make extract small molecular polypeptide active substance be enriched with；
2, by filtering residue alcohol extraction, reclaimed water-fast testosterone in filtering residue, not only reduced active component in testis
Loss, also improves the active component content of extract；
3, the extract product purity that this preparation technology obtains is high, and impurity is few, and safety is good, steady quality；
4, the preparation technology of testis extract is not related to too much chemical reagent, and solvent is easily recycled safely and effectively, economical real
Good by property.
Detailed description of the invention
The invention discloses the preparation method of a kind of animal testis extract, in those skilled in the art can use for reference herein
Hold, be suitably modified technological parameter and realize.Special needs to be pointed out is, all similar replacements and change are to those skilled in the art
For be apparent from, they are considered as being included in the present invention.Preferably enforcement has been passed through in the method for the present invention and application
Example is described, related personnel substantially can in without departing from present invention, spirit and scope to method described herein and
Application is modified or suitably changes and combine, and realizes and applies the technology of the present invention.
The concrete detection method of indices:
A. content of peptides: use forint phenol algoscopy detection content of peptides.
Agents useful for same and concrete operations method are as follows:
Reagent: alkaline copper test solution takes sodium hydroxide 10g, sodium carbonate 50g, and the 400mL that adds water makes dissolving, as solution A；Take wine
Stone acid potassium 0.5g, the 50mL that adds water makes dissolving, separately takes blue vitriol 0.25g, and the 30mL that adds water makes dissolving, by two liquid mixing, as second liquid.
Before use, merge two liquid, and add water to 500mL.
Maneuver:
(1) preparation of reference substance solution takes bovine serum albumin reference substance, and add water the solution making every 1mL containing 0.3mg.
(2) preparation of need testing solution is prepared according to the method under each medicine item.
(3) the preparation precision of standard curve measure reference substance solution 0.0,0.1,0.3,0.5,0.7,0.9mL, put tool respectively
In plug test tube, respectively add water to 1.0mL, then be separately added into alkaline copper test solution 1.0mL, shake up, each addition forint phenol test solution (1 → 16)
4.0mL, mixes immediately, puts accurate response 5 minutes in 55 DEG C of water-baths, puts in psychrolusia 10 minutes, measures at the wavelength of 650nm
Trap；Simultaneously using No. 0 pipe as blank.Regression equation is calculated with corresponding reference substance solution concentration with trap.
(4) algoscopy precision measures need testing solution 1.0mL, and the method under the preparation of sighting target directrix curve, from " adding alkali
Property copper test solution " rise, measure in accordance with the law, by the concentration of regression equation calculation need testing solution, and be multiplied by extension rate, be polypeptide and contain
Amount.
B. testosterone: the testosterone concentration (reference food standard GB/T/T in LC-MS/MS sample
20758 " the mensuration LC-MS/MSs of testosterone, epitestosterone, progesterone residual quantity in Hepar Bovis seu Bubali and beef ")
C. total nitrogen: meso method measures the total nitrogen (with reference to 2010 editions two annex VII D of Chinese Pharmacopoeia) in sample.
D. fat content: utilize acid-hydrolysis method to measure fat content.Accurately weigh sample about 1.0g, put into the big examination of 50ml
Guan Zhong, the water Glass rod adding 8ml fully stirs evenly, and adds 10ml hydrochloric acid, in 70～80 DEG C of water-baths after mixing, left every 10min
The right side stirs evenly once till fat is free, about needs 40～50min.After taking out non-shock chilling, add 10ml ethanol, mix, cold
Being moved into by mixture in 100ml tool plug tube the most afterwards, rinse test tube with 25ml ether by several times, washing liquid is poured in the lump in tool plug tube, is added
Plug shakes 1min, is slowly turned by stopper and is stoppered after unclasping gas again, and static 15min carefully opens plug, mixes by petroleum ether-ether equivalent
Closing the fat of attachment, static 10～20min in liquid washout plug and cylinder, treat that supernatant fluid is clear, sucking-off supernatant is in the cone of constant weight
In shape bottle, then add 5ml ether in tool plug tube in shake, upper strata ether sucking-off will put into conical flask after standing.By conical flask water
Bath is evaporated, and puts 105 DEG C of oven drying 2h, weighs.
E. activity: use T cell Activity determination (de-E receptor method).1. the preparation of E receptor thymus T cells suspension is taken off: take new
Fresh porcine thymus, removes fat and shreds, and adds appropriate Hank ' s liquid and makes into cell suspension, and through 100 mesh screen, per minute 1500 leave
The heart 3～5 minutes, abandoning supernatant, add a small amount of Hank ' s liquid and beat, this solution is added the separation with 1/3 amount of filtrate
In the centrifuge tube of liquid, leave the heart 20 minutes with per minute 2000, the thymocyte cell in careful sucking-off intermediate layer, put into another centrifuge tube
In, add the washing of appropriate Hank ' s liquid, shake up, leave the heart 3～5 minutes with per minute 1500, abandoning supernatant, after washed once,
Adding appropriate Hank ' s liquid in precipitate, mixing, 45 DEG C of waters bath with thermostatic control are incubated 30 minutes (every shaking in 5 minutes once).With
Per minute 1500 leave the heart 3～5 minutes, abandoning supernatant, add appropriate Hank ' s liquid, after mixing, put 45 DEG C of waters bath with thermostatic control
It is incubated 30 minutes, after taking-up, leaves the heart 3～5 minutes with per minute 1500, supernatant discarded night.Three (operations are washed with Hank ' s
Ditto), finally suitably diluting with Hank ' s liquid and count, making ultimate density is that in every 1ml, (3 × 106)～(5 × 106) are individual carefully
Born of the same parents.2. the preparation of sheep red blood suspension: take appropriate Sanguis caprae seu ovis, washes three times (ditto) with Hank ' s liquid.Abandoning supernatant, adds appropriate
Hank ' s liquid dilutes and counts, make ultimate density be de-E receptor thymus T cells suspension concentration 8～10 times.3. need testing solution
Preparation: take test sample, be configured in every 1ml the solution containing 1mg with Hank ' s liquid.4. measure: take small test tube 6, wherein 3
Respectively adding Hank ' s liquid 0.1ml to oppose and look after, another 3 respectively add need testing solution 0.1ml and make to measure pipe, often add de-E receptor in pipe
Thymus T cells suspension 0.2ml, after 37 DEG C are incubated 1 hour, add sheep red blood suspension 0.2ml, shakes up, with 500 turns per minute
Centrifugal 3 minutes, putting into 4 DEG C of refrigerator overnight, next day takes out, supernatant discarded night, often each in pipe adds fixative one, shakes gently
Even, stand 10 minutes, add dyeing liquor 2 and also shake up, start counting up after standing 15 minutes, in field of microscope nattier blue relatively
Big cell is lymphocyte, altogether the number (no less than 200) of all lymphocytes, statistics in 16 block plaid of counting number plate
The cell number (in conjunction with the lymphocyte of more than 3 sheep red blood cell (SRBC)s) of E red rose pigment therein, tries to achieve knot flower percentage rate,
Average.It is the meansigma methods of test sample pipe or control tube.
Sample vigor=test sample measures pipe E rosette percentage rate control tube E rosette percentage rate.
In the preparation method of the animal testis extract that the present invention provides, material therefor, reagent or instrument all can be purchased by market
?.
Below in conjunction with embodiment, the present invention it is expanded on further:
The different genin and agarose gel adsorption chromatographic isolation effectiveness comparison of embodiment 1
1, the preparation of Testis Caprae seu Ovis enzymolysis and extraction refined liquid
(1) take FF rabbit testis 5kg, remove fat and rinse well, obtaining net weight 3.86Kg.The weight such as addition are pure
Change water, be homogenized 2 times under room temperature.
(2) homogenate regulating pH to 9.0, add 3.8g (0.1%) alkaline protease, 50 DEG C to carry out hydrolysis temperature 3 little
Time.After having reacted, 95 DEG C of degeneration 5min are with enzymolysis reaction, are cooled to room temperature.
(3) regulation enzymolysis solution pH to 7.5, adds 7.6g (0.2%) trypsin, and 37 DEG C of enzymolysis, after 2 hours, are warming up to 85
DEG C, keeping 15 minutes, 300 mesh non-woven fabrics filter, and obtain enzymolysis crude extract and filtering residue 1.6kg.
(4) above-mentioned Testis Caprae seu Ovis enzymolysis crude extract 10KD ultrafilter membrane ultra filtering clarifying, obtains slightly yellow clear transparent solutions
5.66 liters, content of peptides is 46.95mg/L.
2, chromatographic column prepares
1) gel swelling containing Quercetin aglucon, gallic acid aglucon and beta-schardinger dextrin-aglucon agarose gel respectively with
The w/v mixing of 1:10 pressed by distilled water, after 60 DEG C of heating 2h, and the static 22h of room temperature.
2) agarose gel after dress post is swelling loads double glazing tubing string, with 10 times of bed volumes of distilled water flushing, punching
Wash speed is 50mL/min.
3) balance rinses chromatographic column mutually by the initial flow of 5 times of bed volumes, and irrigation flow rate is 25mL/min.Begin to flow
Being Ethanol-Acetic Acid aqueous solution mutually, concentration of alcohol is 25%, and acetic acid concentration is 3%.
As stated above, test-type Quercetin genin and agarose gel affinity column, gallic acid aglucon fine jade are got out
Sepharose affinity column and each 1 of beta-schardinger dextrin-genin and agarose gel affinity column.Chromatographic column diameter 1.5cm is high
60cm。
3, loading
The content of peptides of water use regulation Testis Caprae seu Ovis enzymolysis ultrafiltrate is 15mg/mL, with the continuous loading of peristaltic pump, loading flow velocity
For 8mL/min.Above-mentioned three chromatographic column loading volume are 20mL, for the 18.8% of bed volume.
4, eluting
First use initial flow phase, 2 times of bed volumes of 15mL/min eluting；2 times of post bed body of redistilled water 10mL/min eluting
Long-pending；Linear gradient elution is carried out again with the acetic acid solution of 8 times of bed volumes.During linear gradient elution, acetic acid concentration progressively from
2% raises 9%, and acetic acid linear gradient flow velocity is progressively increased to 30mL/min from 15mL/min.Finally use 9% acetic acid solution,
2 times of bed volumes of 30mL/min eluting.
5, collect
Collection absorbance is 280nm, the activity eluting peak more than 10%, merges into Testis Caprae seu Ovis enzymolysis and extraction refined liquid.
6, result detection
Calculating the volume of detection enzymolysis and extraction refined liquid, detect its activity index, result see table 1.
The Activity Results of table 1 distinct methods detection enzymolysis and extraction refined liquid
| Method | Refined liquid volume (mL) | Activity (%) |
| Quercetin genin and agarose gel adsorption by hydrogen bond chromatographs | 15.5 | 20.2 |
| Gallic acid genin and agarose gel adsorption by hydrogen bond chromatographs | 16.3 | 15.4 |
| B-cyclodextrin genin and agarose gel adsorption by hydrogen bond chromatographs | 12.9 | 12.8 |
After find, the separating power of three kinds of genin and agarose gels is followed successively by: Quercetin aglucon ＞ gallic acid is joined
Base ＞ beta cyclodextrin aglucon, it is therefore preferable that Quercetin genin and agarose gel adsorption by hydrogen bond chromatograph carries out bioactive peptide separation.
Embodiment 2 piglets testis enzymolysis and extraction
1, the preparation of piglets testis enzymolysis and extraction refined liquid
(1) FF piglets testis 4.2kg is taken, the weights such as removal is fatty and rinses well, obtains net weight 3.1Kg, addition
Amount distilled water, is homogenized 2 times under room temperature.
(2) after homogenate being regulated pH to 7.5, add 9g (0.3%) trypsin, after 37 DEG C of enzymolysis time 3h, heat up
To 90 DEG C, insulation 10min makes enzyme denaturation, is cooled to room temperature, and 300 mesh non-woven fabrics filter, and obtain piglets testis enzymolysis crude extract and filter
Slag 1.3kg.
(3) above-mentioned enzymolysis crude extract 10KD ultrafilter membrane ultra filtering clarifying, obtains slightly yellow clear transparent solutions 4.83 liters, many
Peptide content is 36.35mg/mL.
(4) above-mentioned ultrafiltrate is isolated and purified through Quercetin genin and agarose gel adsorption by hydrogen bond thin layer chromatography, obtains enzymolysis
Hydrolysis kinetics liquid 3.6L, content of peptides is 9.63mg/mL, and activity is 25.8%.
Quercetin genin and agarose gel adsorption by hydrogen bond chromatographic purification piglets testis enzymolysis solution test tool in step (4)
Body is:
Chromatographic column prepares
(1) gel swelling agarose gel containing Quercetin aglucon is mixed by the w/v of 1:10 with distilled water, and 60
DEG C heating 2h after, the static 22h of room temperature.
(2) agarose gel after dress post is swelling loads rustless steel chromatographic column, with 5 times of bed volumes of distilled water flushing, punching
Wash speed is 75mL/min.
(3) balance rinses chromatographic column mutually by the initial flow of 2 times of bed volumes, and irrigation flow rate is 15mL/min.Begin to flow
Being Ethanol-Acetic Acid aqueous solution mutually, concentration of alcohol is 20%, and acetic acid concentration is 2%.
With reference to said method, getting out diameter 20cm, the chromatographic column of high 145cm, bed volume is 45.53L.
The content of peptides of loading water use regulation piglets testis enzymolysis ultrafiltrate is 15mg/mL, by the continuous loading of peristaltic pump,
Loading flow velocity is 8mL/min.
Eluting first uses initial flow phase, 2 times of bed volumes of 15mL/min eluting；2 times of posts of redistilled water 10mL/min eluting
Bed volume；Linear gradient elution is carried out again with the acetic acid solution of 8 times of bed volumes.During linear gradient elution, acetic acid concentration by
Walking and raise 9% from 2%, acetic acid linear gradient flow velocity is progressively increased to 30mL/min from 15mL/min.Finally molten with 9% acetic acid
Liquid, 2 times of bed volumes of 30mL/min eluting.
Collection absorbance is 280nm, the activity eluting peak more than 10%, merges into enzymolysis and extraction refined liquid, volume
For 3.6L.
After testing, the activity of piglets testis enzymolysis and extraction refined liquid is 25.8%, and content of peptides is 9.63mg/mL.
2, piglets testriol proposes the preparation of concentrated solution
(1) piglets testis enzymolysis filtering residue 1.3kg with 6.5L 95% ethanol mixes, after 2h is extracted in 75 revs/min of stirrings, and 3000
Rev/min centrifugal 15min, obtains supernatant 5.9L；
(2) the 1st centrifugation mixes with 4L 95% alcohol, after 2h is extracted in 100 revs/min of stirrings, and 3000 revs/min
Centrifugal 15min, obtains supernatant 3.6L；
(3) merging 2 centrifugal supernatant obtained, in 0.1MPa, 60 DEG C are evaporated to the suspension that volume is about 1L,
Testosterone concentration is 86.15mg/L.
3, the preparation of piglets testis extract
3.6L enzymolysis and extraction refined liquid mixes with 1L, lyophilization, obtains piglets testis extract 138.65g.
The enzymolysis and extraction of the big pig testis of embodiment 3
1, the preparation of big pig testis enzymolysis and extraction refined liquid
(1) take FF big pig testis 10kg, remove fat and rinse well, obtaining net weight 7.86Kg.The weights such as addition
Amount distilled water, is homogenized 2 times under room temperature.
(2), after homogenate being regulated pH to 8.5, add 31.4g (0.4%) alkaline protease and react, hydrolysis temperature
Being 50 DEG C, enzymolysis time is 5h.After having reacted, 85 DEG C of degeneration 15min are with enzymolysis reaction.Room temperature it is cooled to after degeneration,
300 mesh non-woven fabrics filter, and obtain enzymolysis crude extract and 3.2kg filtering residue.
(3) above-mentioned big pig testis enzymolysis crude extract 10KD ultrafilter membrane ultra filtering clarifying, obtains slightly yellow clear transparent solutions
11.26 liters, content of peptides is 48.09mg/L.
(4) above-mentioned ultrafiltrate is isolated and purified through Quercetin genin and agarose gel adsorption by hydrogen bond chromatography, obtains big pig
Enzymolysis and extraction refined liquid 7.3L, content of peptides is 11.63mg/mL, and activity is 21.6%.
Quercetin genin and agarose gel adsorption by hydrogen bond chromatographic purification big pig testis enzymolysis solution test tool in step (4)
Body is:
Chromatographic column prepares
The Quercetin genin and agarose gel adsorption by hydrogen bond chromatographic column of piglets testis enzymolysis refined liquid was prepared in employing, with 5
The initial flow of times bed volume rinses chromatographic column mutually, and irrigation flow rate is 30mL/min.Beginning to flow is that Ethanol-Acetic Acid is water-soluble mutually
Liquid, concentration of alcohol is 30%, and acetic acid concentration is 5%.
The content of peptides of loading water use regulation big pig testis enzymolysis ultrafiltrate is 45mg/mL, by the continuous loading of peristaltic pump,
Loading flow velocity is 5mL/min.
Eluting first uses initial flow phase, 2 times of bed volumes of 15mL/min eluting；2 times of posts of redistilled water 10mL/min eluting
Bed volume；Linear gradient elution is carried out again with the acetic acid solution of 6 times of bed volumes.During linear gradient elution, acetic acid concentration by
Walking and raise 9% from 2%, acetic acid linear gradient flow velocity is progressively increased to 30mL/min from 15mL/min.Finally molten with 9% acetic acid
Liquid, 2 times of bed volumes of 30mL/min eluting.
Collection absorbance is 280nm, the activity eluting peak more than 10%, merges into enzymolysis and extraction refined liquid, volume
For 7.3L.
2, the preparation of big pig testis alcohol extraction concentrated solution
(1) above-mentioned 3.2kg filtering residue mixes with 16L 95% ethanol, and 75 revs/min of stirrings are extracted 3 hours, and 3000 revs/min are centrifuged
15min, obtains supernatant 14.8L；
(2) the 1st centrifugation mixes with 10L 95% ethanol, and 50 revs/min of stirrings are extracted 3 hours, and 3000 revs/min are centrifuged
20min, obtains supernatant 9.1L；
(3) two times centrifugal gained supernatant merges, 0.05MPa, and 60 DEG C are evaporated to volume and are about 2.5L suspension, inspection
Survey testosterone concentration is 199.37mg/L.
3, the preparation of big pig testis extract
Above-mentioned 7.3L big pig enzymolysis and extraction refined liquid mixes with 2.5L alcohol extraction concentrated solution, and lyophilization obtains big pig testis and carries
Take thing 265.29g.
The enzymolysis and extraction of embodiment 4 bull testis
1, the preparation of bull testis enzymolysis and extraction refined liquid
(1) take the FF bull testis of 10kg, remove fat and rinse well, obtaining net weight 8.16Kg.The weight such as addition
Distilled water, is homogenized 2 times under room temperature.
(2), after homogenate being regulated pH to 9.0,50 DEG C of enzymolysis of 24.5g (0.3%) alkaline protease after 3 hours are added, 80
DEG C enzyme denaturing 20 minutes, is cooled to room temperature.
(3) enzymolysis solution after cooling adjusts pH to 7.5, adds trypsin 16.3g (0.2%) and continues enzymolysis 5 hours, afterwards
90 DEG C of degeneration 10min, with enzymolysis reaction, are cooled to room temperature after degeneration, 300 mesh non-woven fabrics filter, obtain enzymolysis crude extract and filter
Slag 3.6kg.
(4) above-mentioned bull testis enzymolysis crude extract 10KD ultrafilter membrane ultra filtering clarifying, obtains slightly yellow clear transparent solutions
12.10 liters, content of peptides is 54.78mg/L.
(5) above-mentioned ultrafiltrate is isolated and purified through Quercetin genin and agarose gel adsorption by hydrogen bond chromatography, obtains enzymolysis
Hydrolysis kinetics liquid 8.2L, content of peptides is 12.56mg/mL, and activity is 18.2%.
In step (5), the test of Quercetin genin and agarose gel adsorption by hydrogen bond chromatographic purification bull testis enzymolysis solution is concrete
For:
Chromatographic column prepares
Use the Quercetin genin and agarose gel adsorption by hydrogen bond chromatographic column preparing excessive pig testis enzymolysis refined liquid, with 4
The initial flow of times bed volume rinses chromatographic column mutually, and irrigation flow rate is 25mL/min.Beginning to flow is that Ethanol-Acetic Acid is water-soluble mutually
Liquid, concentration of alcohol is 25%, and acetic acid concentration is 3.5%.
The content of peptides of loading water use regulation bull testis enzymolysis ultrafiltrate is 40mg/mL, by the continuous loading of peristaltic pump, on
Sample flow velocity is 6mL/min.
Eluting first uses initial flow phase, 2 times of bed volumes of 15mL/min eluting；2 times of posts of redistilled water 10mL/min eluting
Bed volume；Linear gradient elution is carried out again with the acetic acid solution of 6 times of bed volumes.During linear gradient elution, acetic acid concentration by
Walking and raise 9% from 2%, acetic acid linear gradient flow velocity is progressively increased to 30mL/min from 15mL/min.Finally molten with 9% acetic acid
Liquid, 2 times of bed volumes of 30mL/min eluting.
Collection absorbance is 280nm, the activity eluting peak more than 10%, merges into enzymolysis and extraction refined liquid.
Owing to cattle enzymolysis ultrafiltrate volume is relatively big, above-mentioned loading, eluting divide 2 times and complete, and loading volume is 10L for the first time,
After eluting completes, collect 5.1L enzymolysis and extraction refined liquid.Again by flowing phase (Ethanol-Acetic Acid aqueous solution, the second of 4 times of bed volumes
Determining alcohol is 25%, and acetic acid concentration is 5%), after 30mL/min rebalancing chromatographic column, loading 6.6L again, after eluting, collect
To 3.1 liters of enzymolysis and extraction refined liquid, twice refined liquid merges use.
After testing, the activity of bull testis enzymolysis and extraction refined liquid is 18.2%, and content of peptides is 12.56mg/mL.
2, the preparation of bull testis alcohol extraction concentrated solution
(1) bull testis enzymolysis filtering residue 3.6kg with 18L 95% ethanol mixes, after 3h is extracted in 100 revs/min of stirrings, 3000 turns/
Separate heart 20min, obtain supernatant 16.7L；
(2) the 1st centrifugation mixes with 12L 95% ethanol, and after 2h is extracted in 75 revs/min of stirrings, 3000 revs/min are centrifuged
15min, obtains supernatant 11.2L；
(3) the 2nd centrifugation mixes with 12L 95% ethanol, and after 1h is extracted in 50 revs/min of stirrings, 3000 revs/min are centrifuged
10min, obtains supernatant 10.5L；
(4) merging 3 centrifugal supernatant obtained, in 0.05MPa, 65 DEG C are evaporated to volume and are about 3.9L suspendible
Liquid, testosterone concentration is 251.60mg/L.
3, the preparation of bull testis extract
8.2L enzymolysis and extraction refined liquid mixes with 3.9L alcohol extraction concentrated solution, and lyophilization obtains bull testis extract
484.55g。
The enzymolysis and extraction of embodiment 5 rabbit testis
1, the preparation of rabbit testis enzymolysis and extraction refined liquid
(1) take FF rabbit testis 3kg, remove fat and rinse well, obtaining net weight 1.95Kg.The weight such as addition are steamed
Distilled water, is homogenized 2 times under room temperature.
(2) homogenate regulation pH to 7.5, adds 1.95g (0.1%) trypsin, 40 DEG C of enzymolysis 2 hours, heats up 80 DEG C
Rear holding 20min, with enzymolysis reaction, is cooled to room temperature, and 300 mesh non-woven fabrics filter, and obtain enzymolysis crude extract and filtering residue 0.8kg.
(3) above-mentioned rabbit testis enzymolysis crude extract 10KD ultrafilter membrane ultra filtering clarifying, obtains slightly yellow clear transparent solutions 2.6
Rising, content of peptides is 29.75mg/L.
(4) above-mentioned ultrafiltrate Quercetin genin and agarose gel adsorption by hydrogen bond chromatography is isolated and purified, obtains enzymolysis
Hydrolysis kinetics liquid 1.9L, content of peptides is 7.13mg/mL, and activity is 22.5%.
In step (4), the test of Quercetin genin and agarose gel adsorption by hydrogen bond chromatographic purification rabbit testis enzymolysis solution is concrete
For:
Chromatographic column prepares
The Quercetin genin and agarose gel adsorption by hydrogen bond chromatographic column of bull testis enzymolysis refined liquid was prepared in employing, with 3 times
The initial flow of bed volume rinses chromatographic column mutually, and irrigation flow rate is 20mL/min.Beginning to flow is Ethanol-Acetic Acid aqueous solution mutually,
Concentration of alcohol is 25%, and acetic acid concentration is 2.5%.
The content of peptides of loading water use regulation rabbit testis enzymolysis ultrafiltrate is 25mg/mL, by the continuous loading of peristaltic pump, on
Sample flow velocity is 7.5mL/min.
Eluting first uses initial flow phase, 2 times of bed volumes of 15mL/min eluting；2 times of posts of redistilled water 10mL/min eluting
Bed volume；Linear gradient elution is carried out again with the acetic acid solution of 8 times of bed volumes.During linear gradient elution, acetic acid concentration by
Walking and raise 9% from 2%, acetic acid linear gradient flow velocity is progressively increased to 30mL/min from 15mL/min.Finally molten with 9% acetic acid
Liquid, 2 times of bed volumes of 30mL/min eluting.
Collection absorbance is 280nm, the activity eluting peak more than 10%, merges into enzymolysis and extraction refined liquid.
2, rabbit testriol proposes the preparation of concentrated solution
(1) rabbit testis enzymolysis filtering residue 0.8kg with 6.4L 95% ethanol mixes, after 1h is extracted in 50 revs/min of stirrings, 3000 turns/
Separate heart 10min, obtain supernatant 5.1L；
(2) the 1st centrifugation mixes with 4L 95% ethanol, and after 1h is extracted in 75 revs/min of stirrings, 3000 revs/min are centrifuged
20min, obtains supernatant 3.3L；
(3) merging 2 centrifugal supernatant obtained, in 0.1MPa, 65 DEG C are evaporated to the suspension that volume is about 1L,
Testosterone concentration is 148.38mg/L.
3, the preparation of rabbit testis extract
1.9L enzymolysis and extraction refined liquid mixes with 1L alcohol extraction concentrated solution, lyophilization, obtains rabbit testis extract 102.57g.
The enzymolysis and extraction of embodiment 6 Testis Caprae seu Ovis
1, the preparation of Testis Caprae seu Ovis enzymolysis and extraction refined liquid
(1) take FF rabbit testis 5kg, remove fat and rinse well, obtaining net weight 3.86Kg.The weight such as addition are pure
Change water, be homogenized 2 times under room temperature.
(2) homogenate regulating pH to 9.0, add 3.8g (0.1%) alkaline protease, 50 DEG C to carry out hydrolysis temperature 3 little
Time.After having reacted, 95 DEG C of degeneration 5min are with enzymolysis reaction, are cooled to room temperature.
(3) regulation enzymolysis solution pH to 7.5, adds 7.6g (0.2%) trypsin, and 37 DEG C of enzymolysis, after 2 hours, are warming up to 85
DEG C, keeping 15 minutes, 300 mesh non-woven fabrics filter, and obtain enzymolysis crude extract and filtering residue 1.6kg.
(4) above-mentioned Testis Caprae seu Ovis enzymolysis crude extract 10KD ultrafilter membrane ultra filtering clarifying, obtains slightly yellow clear transparent solutions
5.66 liters, content of peptides is 46.95mg/L.
(5) above-mentioned ultrafiltrate Quercetin genin and agarose gel adsorption by hydrogen bond chromatography is isolated and purified, obtains enzymolysis
Hydrolysis kinetics liquid 4.5L, content of peptides is 10.63mg/mL, and activity is 22.3%.
In step (5), the test of Quercetin genin and agarose gel adsorption by hydrogen bond chromatographic purification rabbit testis enzymolysis solution is concrete
For:
The Quercetin genin and agarose gel adsorption by hydrogen bond chromatographic column of rabbit testis enzymolysis refined liquid was prepared in employing, with 3.5
The initial flow of times bed volume rinses chromatographic column mutually, and irrigation flow rate is 20mL/min.Beginning to flow is that Ethanol-Acetic Acid is water-soluble mutually
Liquid, concentration of alcohol is 25%, and acetic acid concentration is 3%.
The content of peptides of loading water use regulation Testis Caprae seu Ovis enzymolysis ultrafiltrate is 30mg/mL, by the continuous loading of peristaltic pump, on
Sample flow velocity is 6.5mL/min.
Eluting first uses initial flow phase, 2 times of bed volumes of 15mL/min eluting；2 times of posts of redistilled water 10mL/min eluting
Bed volume；Linear gradient elution is carried out again with the acetic acid solution of 10 times of bed volumes.During linear gradient elution, acetic acid concentration by
Walking and raise 9% from 2%, acetic acid linear gradient flow velocity is progressively increased to 30mL/min from 15mL/min.Finally molten with 9% acetic acid
Liquid, 2 times of bed volumes of 30mL/min eluting.
Collection absorbance is 280nm, the activity eluting peak more than 10%, merges into enzymolysis and extraction refined liquid, volume
For 4.5L.
2, the preparation of Testis Caprae seu Ovis alcohol extraction concentrated solution
(1) Testis Caprae seu Ovis enzymolysis filtering residue 1.6kg with 8L 95% ethanol mixes, after 3h is extracted in 100 revs/min of stirrings, 3000 turns/
Separate heart 20min, obtain supernatant 6.9L；
(2) the 1st centrifugation mixes with 5L 95% ethanol, and after 2h is extracted in 75 revs/min of stirrings, 3000 revs/min are centrifuged
15min, obtains supernatant 4.2L；
(3) the 2nd centrifugation mixes with 5L 95% ethanol, and after 12h is extracted in 50 revs/min of stirrings, 3000 revs/min are centrifuged
10min, obtains supernatant 4.1L；
(4) merging 3 centrifugal supernatant obtained, in 0.1MPa, 60 DEG C are evaporated to volume and are about 1.9L suspension.
Detection testosterone concentration is 203.91mg/L.
3, the preparation of Testis Caprae seu Ovis extract
Above-mentioned 4.5L enzymolysis and extraction refined liquid mixes with 1.8L alcohol extraction concentrated solution, and lyophilization obtains Testis Caprae seu Ovis extract
201.73g。
Comparative example 1 Testis Caprae seu Ovis extracting solution preparation method
Take fresh Testis Caprae seu Ovis, smash to pieces through tissue mashing machine, add multigelation after tri-distilled water, with low temperature supercentrifuge from
The heart, supernatant is crossed post through hollow-fibre ultrafiltration device, is aseptically filtered, and is packaged into water white transparency Testis Caprae seu Ovis extracting solution.
Comparative example 2 bull testis particulate matter preparation method
Choosing the bull testis of fresh and healthy, purification is washed more than 3 times, to clean；
The bull testis just cleaned is positioned in the buffer saline that pH is 7.0 immersions, quickly removal blood and slime and other
Attachment；
The bull testis removing blood and slime is chopped into the fritter of 8cm, in bull testis fritter, is incorporated as bull testis weight 2 times
Distilled water, and add the papain of bull testis weight 2%, regulation pH is 3 at 15 DEG C of enzymolysis 4h；Remove supernatant, then will
The little particulate mass of bull testis after enzymolysis crosses 100 mesh screen, leaves and takes the upper solid matter of sieve, standby.
Test example contrast test
Being detected by obtained to embodiment 2-6 and comparative example 1-2 product, testing result is shown in Table 2:
Table 2 indices testing result
In contrast, watch test data understand, and the fat content of comparative example 1 and comparative example 2 is apparently higher than embodiment 2～6, testis
Ketone content (comparative example 1 does not detects testosterone concentration), content of peptides, Product Activity are significantly lower than embodiment 2～6.
It can thus be appreciated that this patent preparation method can make that testis decomposes more thorough, produce the little molecule of more high value,
In product after simultaneously processing, impurity reduces, and product purity improves, and Product Activity also significantly improves.
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For Yuan, under the premise without departing from the principles of the invention, it is also possible to make some improvements and modifications, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (10)
1. the preparation method of an animal testis extract, it is characterised in that comprise the following steps:
Step 1: animal testis is homogenized, enzymolysis and extraction, obtains enzymolysis crude extract and filtering residue；
Step 2: described enzymolysis crude extract obtains ultrafiltrate through ultrafiltration, described ultrafiltrate is again through agarose gel adsorption by hydrogen bond chromatograph
Method removes macromolecule impurity, obtains enzymolysis and extraction refined liquid；
Described filtering residue uses ethanol extraction to concentrate, and obtains concentrating alcohol extract；
Step 3: described enzymolysis and extraction refined liquid mixed with described concentration alcohol extract, lyophilization, obtains animal testis and extracts
Thing.
Preparation method the most according to claim 1, it is characterised in that described animal testis selected from pig, cattle, horse, sheep, rabbit,
The testis of any one or a few animal in fox.
Preparation method the most according to claim 1 and 2, it is characterised in that enzymolysis and extraction described in step 1 includes following step
Rapid:
Transplanting even slurry mixes with water, regulates pH to 7.0～9.0；
Add protease, enzymolysis 2～8 hours under the conditions of 37～50 DEG C, obtain enzymolysis solution；
Described enzymolysis solution is warming up to 80～95 DEG C, is incubated 5～20 minutes, makes enzyme-deactivating；
Enzymolysis solution after enzyme deactivation, through filtering, obtains enzymolysis crude extract and filtering residue.
Preparation method the most according to claim 3, it is characterised in that described protease is trypsin or alkaline protease
One or both.
5. according to the preparation method described in claim 3 or 4, it is characterised in that the addition of described protease is transplanting even slurry
The 0.1%～0.5% of weight.
Preparation method the most according to any one of claim 1 to 5, it is characterised in that ultrafiltration described in step 2 uses
The molecular cut off of ultrafilter membrane is 10KD.
Preparation method the most according to any one of claim 1 to 6, it is characterised in that described agarose gel hydrogen bond is inhaled
Attached chromatograph is: use the affinity chromatograph chromatograph that agarose gel is filler with affinity ligand；Described affinity ligand is Cortex querci dentatae
Element aglucon, gallic acid aglucon, the one of beta-schardinger dextrin-aglucon.
Preparation method the most according to any one of claim 1 to 7, it is characterised in that described agarose gel hydrogen bond is inhaled
Attached chromatographic process particularly as follows:
The content of peptides of water use regulation ultrafiltrate is 15～45mg/mL, uses the flow velocity loading absorption of 5～8mL/min, applied sample amount
For column volume 10～20%；
Successively with 2 times of bed volumes of initial flow phase eluting, 2 times of bed volumes of pure water eluting, 2%～9% acetic acid linear gradient
Eluting 6～10 times of bed volumes, finally by 2 times of bed volumes of 9% acetic acid eluting, elution flow rate 10～30mL/min；
Collection absorbance is 280nm, the activity eluting peak more than 10%, merges into enzymolysis and extraction refined liquid.
Preparation method the most according to claim 8, it is characterised in that described initial flow is ethanol, acetic acid and water group mutually
Become solution, described ethanol initial flow mutually in concentration expressed in percentage by volume be 20%-30%, described acetic acid is in initial flow phase
In concentration expressed in percentage by volume be 2～5%.
Preparation method the most according to any one of claim 1 to 9, it is characterised in that described ethanol extraction concentrates and includes
Following steps:
(1) filtering residue and ethanol water volume ratio 1:(3 by weight～8) mix, 50～100 revs/min of stirring extractions 1～3 are little
Time, gained extracting solution by centrifugation, collects supernatant；After repeating to extract 2～3 times, merge supernatant；
(2) gained supernatant is through being evaporated to the 1/10～1/8 of original volume, obtains concentrating alcohol extract, reclaims ethanol；Described subtract
The concentration pressure that pressure concentrates is 0.05～0.15MP, controls the temperature of supernatant less than 65 DEG C during described concentrating under reduced pressure.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110317847A (en) * | 2019-05-24 | 2019-10-11 | 科索瑞生物科技(天津)有限公司 | A kind of extract and its preparation method and application in animal tissue source |
| CN113827619A (en) * | 2021-09-07 | 2021-12-24 | 北京第一生物化学药业有限公司 | Animal testis extract and its preparation method and application |
| CN114632092A (en) * | 2022-05-17 | 2022-06-17 | 北京第一生物化学药业有限公司 | Application of testis tablet in preparing medicine with hypoglycemic activity |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1965868A (en) * | 2006-11-08 | 2007-05-23 | 甘肃省农业科学院 | An extraction preparation method of effective ingredients of Chinese medicine |
| US20130018022A1 (en) * | 2009-12-30 | 2013-01-17 | Industry-Academic Cooperation Foundation, Yeungnam University | Pharmaceutical composition including a testis extract as an active ingredient for treating and preventing anemia |
| CN103800617A (en) * | 2014-01-21 | 2014-05-21 | 马鞍山汉思生物科技有限公司 | Active bovine testis enteric capsule and preparation method thereof |
| CN104745664A (en) * | 2015-04-17 | 2015-07-01 | 浙江华尔成生物药业股份有限公司 | Preparation process of animal placenta extract |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1965868A (en) * | 2006-11-08 | 2007-05-23 | 甘肃省农业科学院 | An extraction preparation method of effective ingredients of Chinese medicine |
| US20130018022A1 (en) * | 2009-12-30 | 2013-01-17 | Industry-Academic Cooperation Foundation, Yeungnam University | Pharmaceutical composition including a testis extract as an active ingredient for treating and preventing anemia |
| CN103800617A (en) * | 2014-01-21 | 2014-05-21 | 马鞍山汉思生物科技有限公司 | Active bovine testis enteric capsule and preparation method thereof |
| CN104745664A (en) * | 2015-04-17 | 2015-07-01 | 浙江华尔成生物药业股份有限公司 | Preparation process of animal placenta extract |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110317847A (en) * | 2019-05-24 | 2019-10-11 | 科索瑞生物科技(天津)有限公司 | A kind of extract and its preparation method and application in animal tissue source |
| CN110317847B (en) * | 2019-05-24 | 2021-11-26 | 科索瑞生物科技(天津)有限公司 | Extract from animal tissue, and preparation method and application thereof |
| CN113827619A (en) * | 2021-09-07 | 2021-12-24 | 北京第一生物化学药业有限公司 | Animal testis extract and its preparation method and application |
| CN114632092A (en) * | 2022-05-17 | 2022-06-17 | 北京第一生物化学药业有限公司 | Application of testis tablet in preparing medicine with hypoglycemic activity |
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