CN109529012A - A kind of metal oxide nano bletilla striata pheretima albumen composition preparation method and application - Google Patents
A kind of metal oxide nano bletilla striata pheretima albumen composition preparation method and application Download PDFInfo
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Abstract
The invention discloses a kind of metal oxide nano bletilla striata pheretima albumen composition preparation method and applications, prepare pheretima stress protein, layered metal oxide nano-carrier is prepared, metal oxide nano bletilla striata pheretima albumen composition is prepared, the compound is applied in rat brain injury reparation.Compared with prior art, the present invention enters the effective component self assembly of pheretima stress protein and bletilla in stratified nano materials, it is a kind of with sustained release, economic medical preparation to be prepared for, the degradation of albumen and the oxidational losses of traditional Chinese medicine ingredients are also effectively reduced while saving drug effect, have been finally reached the purpose for promoting craniocerebral injury rehabilitation.
Description
Technical field
The present invention relates to animal protein and vegetable drug nano traditional Chinese medicine compound preparation technical field, especially a kind of metal
Oxidate nano bletilla striata pheretima albumen composition preparation method and application.
Background technique
Craniocerebral injury is a kind of common damage of modern society, especially in traffic accident case.Patient is due to external object
Shock of the body to brain, seriously destroys the normal function of brain, so as to cause the temporarily or permanently cognition of property, physiology and the heart
In function forfeiture.Craniocerebral injury at present has become the primary cause of disease dead and disabled in 45 years old or less crowd.Chinese traditional medicine sleeper
Albumen is it is verified that the proliferation and differentiation of wound tissue's cell can be effectively facilitated, the ability of acceleration of wound healing.But because
It is ground dragon protein as a kind of large biological molecule, is very easy to that protein degradation occurs, there is consumption in actual use
The problems such as amount is big, and economic cost is high.The bletilla striata has astringing to arrest bleeding on herbal pharmacology, and the effect of detumescence and promoting granulation, but it is in reality
It is excessive that there is also consumptions in the use of border, it is difficult to the problems such as merchandized handling.
It would therefore be highly desirable to develop a kind of metal oxide nano bletilla striata pheretima albumen composition.
Summary of the invention
The invention aims to solve the deficiencies in the prior art, a kind of metal oxide nano bletilla striata is provided
Dragon protein compound preparation method and application.
In order to achieve the above objectives, the present invention is implemented according to following technical scheme:
A kind of metal oxide nano bletilla striata pheretima albumen composition preparation method, comprising the following steps:
S1, preparation pheretima stress protein:
Fresh and alive pheretima is cleaned repeatedly with 0.9% NaCl solution, removes body surface dirt, the pheretima after cleaning is at 25 DEG C
Under be placed in and allow within 4 hours in the normal saline solution of the continuous oxygen system of sky air pump its internal silt of spuing；With water suction after pulling out
Paper removes surface moisture and is transferred in beaker；It is quickly stirred with glass bar rapidly after the sucrose of pheretima total weight 10% is added
Even, waiting the yellow-green liquid collected in beaker after 10min is pheretima stress protein, the anti-protein degradation enzyme of 1X is added, so
The NaN that concentration is 0.02% is added afterwards3Pheretima stress protein liquid is obtained as preservative, is stored spare；
S2, layered metal oxide nano-carrier is prepared
Weigh the Ni (NO of 6mmol3)2It is added in 300ml water after mixing constantly with the dodecyl sodium sulfate of 30mmol
It is stirred overnight, is then transferred in hydrothermal synthesis reaction kettle, natural cooling after reaction kettle is heated to 110 DEG C of reactions 16 hours；It crosses
The mixed liquor of the product obtained after filter ethyl alcohol and water is rinsed to the colourless postposition of washing lotion in a vacuum drying oven, dry at 70 DEG C
12 hours, resulting powder and 10g Na2HPO4Porcelain boat is put into tube furnace after being put into porcelain boat two sides, wherein NaH2PO4One end
It is placed on air inlet side, powder sample one end is placed on tube furnace gas outlet side, N2It is natural after being heated to 350 DEG C under environment
Cooling obtains NiP layered metal oxide nano-carrier；
S3, metal oxide nano bletilla striata pheretima albumen composition is prepared
The 10g bletilla striata is weighed, is filtered after decocting half an hour with deionized water, utilizes Rotary Evaporators removing more after collecting solution
Gained pasty mass is freeze-dried by remaining moisture, collects bletilla striata extract powder；By gained bletilla striata extract powder and S1
In the pheretima stress protein liquid that is prepared be mixed to get pheretima stress protein mixed liquor, it is rear 2 times of pheretimas then are added stress
The concentration of mixed liquid of protein volume is that the Tris-HCl buffer that 20mM, pH are 7.2 obtains mixed liquor；Measure mixed liquor 20ml with
5.0gNiP layered metal oxide nano-carrier mixes, and stirs 8 hours at 4 DEG C；It is centrifuged mixed liquor and collects precipitating, freeze-drying
Collect powder afterwards to get metal oxide nano bletilla striata pheretima albumen composition is arrived.
Further, pheretima selects Eisenia Foetida in the S1.
Further, the volume ratio of ethyl alcohol and water is 1:1 in the S2.
Further, bletilla striata extract powder carries out mixing system according to mass ratio 1:1 with pheretima stress protein liquid in the S3
Obtain pheretima stress protein mixed liquor.
In addition, the present invention also provides a kind of application of metal oxide nano bletilla striata pheretima albumen composition, use is above-mentioned
Metal oxide nano bletilla striata pheretima albumen composition made from preparation method is used as rat brain injury repair medicine.
The principle of the present invention are as follows: the slow of ground dragon protein effective component is controlled by NiP layered metal oxide nano-carrier
On The Drug Release achievees the purpose that medicament-saving consumes, and NiP layered metal oxide nano-carrier has blocked ground dragon protein effective component
With extraneous contact, to effectively inhibit the degradation of protein ingredient and the oxidation of effective ingredient, metal oxygen produced by the present invention
Compound nanometer bletilla striata pheretima albumen composition can promote the proliferation of cell, nerve cell is promoted to restore regeneration and restore, therefore, can
As rat brain injury repair medicine.
Compared with prior art, the effective component self assembly of pheretima stress protein and bletilla is entered stratiform and received by the present invention
In rice material, to be prepared for a kind of with sustained release, economic medical preparation, also effective drop while saving drug effect
The low degradation of albumen and the oxidational losses of traditional Chinese medicine ingredients have been finally reached the purpose for promoting craniocerebral injury rehabilitation.
Detailed description of the invention
Fig. 1 is the characterization of NiP layered metal oxide nano-carrier made from the embodiment of the present invention；A is 2000 times of amplification
NiP layered metal oxide nano-carrier electronics surface sweeping MIcrosope image；B is the laminated metal the NiP oxygen for amplifying 10000 times
The electronics surface sweeping MIcrosope image of compound nano-carrier；C is the X-ray powder diffraction of NiP layered metal oxide nano-carrier
Figure；D is the medicament slow release experimental result of NiP layered metal oxide nano-carrier.
Fig. 2 is the H&E coloration result of the Damage of Rats position Cerebral cortex in application example；(a) it is blank group, is (b) multiple
Close object group.
Fig. 3 is the Nissl's staining result of the Damage of Rats position Cerebral cortex in application example；(a) it is blank group, is (b) multiple
Close object group.
Fig. 4 is the Apoptosis result of the Damage of Rats position Cerebral cortex in application example；(a) it is blank group, is (b) multiple
Close object group.
Specific embodiment
To make the objectives, technical solutions, and advantages of the present invention more comprehensible, with reference to embodiments, to the present invention into
Row is further to be described in detail.Described herein the specific embodiments are only for explaining the present invention, is not used to limit invention.
A kind of metal oxide nano bletilla striata pheretima albumen composition preparation method of the embodiment of the present embodiment, including it is following
Step:
S1, preparation pheretima stress protein:
Fresh and alive pheretima (Eisenia Foetida) is cleaned repeatedly with 0.9% NaCl solution, body surface dirt is removed, after cleaning
Pheretima be placed at 25 DEG C and allow within 4 hours in the normal saline solution of the continuous oxygen system of sky air pump its internal silt of spuing；
Surface moisture is removed with blotting paper after pulling out and is transferred in beaker；Glass is used rapidly after the sucrose of pheretima total weight 10% is added
Stick quickly stirs evenly, and waiting the yellow-green liquid collected in beaker after 10min is pheretima stress protein, and the anti-egg of 1X is added
Then the NaN that concentration is 0.02% is added in white degrading enzyme3Pheretima stress protein liquid is obtained as preservative, is stored spare；
S2, layered metal oxide nano-carrier is prepared
Weigh the Ni (NO of 6mmol3)2It is added in 300ml water after mixing constantly with the dodecyl sodium sulfate of 30mmol
It is stirred overnight, is then transferred in hydrothermal synthesis reaction kettle, natural cooling after reaction kettle is heated to 110 DEG C of reactions 16 hours；It crosses
The mixed liquor of ethyl alcohol and water that the product obtained after filter is 1:1 with volume ratio rinses to washing lotion is colourless and is placed on vacuum oven
In, 12 hours dry at 70 DEG C, resulting powder and 10g Na2HPO4Porcelain boat is put into tube furnace after being put into porcelain boat two sides,
Wherein NaH2PO4One end is placed on air inlet side, and powder sample one end is placed on tube furnace gas outlet side, N2Add under environment
Heat to natural cooling after 350 DEG C obtains NiP layered metal oxide nano-carrier；
S3, metal oxide nano bletilla striata pheretima albumen composition is prepared
The 10g bletilla striata is weighed, is filtered after decocting half an hour with deionized water, utilizes Rotary Evaporators removing more after collecting solution
Gained pasty mass is freeze-dried by remaining moisture, collects bletilla striata extract powder；By gained bletilla striata extract powder and S1
In the pheretima stress protein liquid that is prepared be mixed to get pheretima stress protein mixed liquor according to mass ratio 1:1, it is rear to be then added 2
The concentration of times pheretima stress protein mixeding liquid volume is that the Tris-HCl buffer that 20mM, pH are 7.2 obtains mixed liquor；It measures
Mixed liquor 20ml is mixed with 5.0gNiP layered metal oxide nano-carrier, is stirred 8 hours at 4 DEG C；It is centrifuged mixed liquor and collects and sink
It forms sediment, powder is collected after freeze-drying to get metal oxide nano bletilla striata pheretima albumen composition is arrived.
The sustained release performance of NiP layered metal oxide nano-carrier is tested: compound concentration 0.4mg/ml,
BSA Tris-HCl (pH=7.2) buffer of 0.8mg/ml, 1.2mg/ml, 1.6mg/ml and 2.0mg/ml utilize
Nanodrop (2000C) measures its light absorption value OD280nm at 20 DEG C at 280nm respectively, is calculated by linear regression
It is A=0.065C+0.001 to the linear relationship of light absorption value and protein concentration, wherein A is light absorption value, and C is protein concentration,
Coefficient R 2=0.9991.Weigh the Tris-HCl of 2.0g metal oxide nano bletilla striata pheretima albumen composition mixing 4ml
(pH=7.2) it is placed in shaking table at 4 DEG C after buffer, every 30min with pipetting after hand held centrifuge rapid centrifugation 60s
200ul supernatant measures its OD280nm, and it is slow that fresh 200ul Tris-HCl (pH=7.2) is supplemented into system after measurement every time
Fliud flushing.
Fig. 1 a and Fig. 1 b are scanning electron microscope (SEM) figure of prepared NiP layered metal oxide nano-carrier
Picture, wherein the amplification factor of Fig. 1 a is 2000 times, and the amplification factor of Fig. 1 b is 10000 times.Morphology observation is it can be found that prepared
NiP layered metal oxide nano-carrier accumulated by sheet, constructed stephanoporate framework in space, entire material is in space point
Cloth is substantially uniform.Through measuring, these channel diameters are 800 ± 400nm, and the ground dragon protein that exists for further loading in duct provides
Space.SEM Surface energy spectrum analysis confirms that the atomic percent of Ni atom and P atom is about 1:1 in material.Fig. 1 c is NiP layers
There is apparent diffraction at 20 °, 26 °, 33 °, 59 ° and 73 ° in the X-ray powder diffraction figure of shape metal oxide nano carrier
Peak thinks that the atomic percent of Ni atom and P atom in 1:1 or so, illustrates to have had successfully been obtained NiP layers afterwards compared with document
Shape metal oxide nano carrier.
Dotted line describes the variation feelings in NiP nanometer earthworm albumen composition protein concentration in buffer solution system in Fig. 1 d
Condition, and block curve is then that protein concentration is managed in buffer when not having pharmaceutical carrier to participate in situation under identical initial protein concentration
By situation of change.If apparent as can be seen that being participated in without NiP layered metal oxide nano-carrier, theoretically on 9 hours left sides
Concentration in right system is lower than 0.02mg/ml, and this time can be extended to 14 small for NiP nanometer earthworm albumen composition
When or so, increase 55%.Illustrate that the compound has preferable albumen slow-release capability.
Application Example
S1,60 (220 ± 10g) SD rats are taken to be randomly divided into compound rat group (30) and blank using the same manner
Groups of control (30)；
Before S2, modeling to SD rat carry out 6 hours fasting for solids and liquids, later to rat using intraperitoneal injection yellow Jackets into
Row anesthesia, shave rat crown hair, hit using cranium brain striking device rat brain: blow speed 5.30m/s is beaten
Depth 4.5mm is hit, residence time 400ms is hit.Gentamicin sulphate 6 is instilled to wound with 2ml injection to drip, and utilizes hand
Art needle sutures rat scalp.All experimental mouses carry out single cage raising, and feeding environment temperature is 25 DEG C；
S3, the nanometer earthworm albumen composition 5.0g being prepared is uniformly mixed with the 0.9% of 20ml NaCl solution
Afterwards respectively at wound the 1st day, stomach-filling is carried out to rat oral chamber within the 4th day, the 7th day and 15 days, each stomach-filling 2ml, one time a day；
Physiological saline is perfused in blank group；Rat is put to death with carbon dioxide smother play after 15 days, and takes damage location Cerebral cortex according to reagent
Box operation instructions carry out H&E dyeing, Nissl's staining and Apoptosis test result respectively such as Fig. 2, Fig. 3, Fig. 4.
In Fig. 2, spot indicates nucleus, and the number of cell in tissue is determined using the distribution situation of observation nucleus
Amount.By observation, it can be seen that, in compound group, spot is more, illustrates that the cell quantity in compound group is greater than blank group,
To prove that compound group can promote the proliferation of cell.
In Fig. 3, each circle is a neuronal cell, and the quantity in compound group is higher than blank group, is illustrated logical
The treatment of compound is crossed, the nerve cell recovery effects in the Cerebral cortex of rat are preferable.Compound, which has, promotes nerve cell to restore
Regenerated ability.
In Fig. 4, light tone spot represents the cell of apoptosis.Light tone spot in blank group is significantly more than compound
Group illustrates the treatment by compound, and the apoptosis situation in tissue is reduced, and compound can promote cell to restore.
The limitation that technical solution of the present invention is not limited to the above specific embodiments, it is all to do according to the technique and scheme of the present invention
Technology deformation out, falls within the scope of protection of the present invention.
Claims (5)
1. a kind of metal oxide nano bletilla striata pheretima albumen composition preparation method, which comprises the following steps:
S1, preparation pheretima stress protein:
Fresh and alive pheretima is cleaned repeatedly with 0.9% NaCl solution, removes body surface dirt, the pheretima after cleaning is underlying at 25 DEG C
Allow within 4 hours in the normal saline solution for having the continuous oxygen system of air pump its internal silt of spuing；It is removed after pulling out with blotting paper
It removes surface moisture and is transferred in beaker；It is quickly stirred evenly with glass bar rapidly after the sucrose of pheretima total weight 10% is added,
Wait 10min after collect beaker in yellow-green liquid be pheretima stress protein, the anti-protein degradation enzyme of 1X is added, then plus
Enter the NaN that concentration is 0.02%3Pheretima stress protein liquid is obtained as preservative, is stored spare；
S2, layered metal oxide nano-carrier is prepared
Weigh the Ni (NO of 6mmol3)2It is added in 300ml water and is stirred continuously after mixing with the dodecyl sodium sulfate of 30mmol
Overnight, it is then transferred in hydrothermal synthesis reaction kettle, natural cooling after reaction kettle is heated to 110 DEG C of reactions 16 hours；After filtering
The mixed liquor of obtained product ethyl alcohol and water is rinsed to the colourless postposition of washing lotion in a vacuum drying oven, and dry 12 is small at 70 DEG C
When, resulting powder and 10g Na2HPO4Porcelain boat is put into tube furnace after being put into porcelain boat two sides, wherein NaH2PO4It places one end
In air inlet side, powder sample one end is placed on tube furnace gas outlet side, N2Natural cooling after being heated to 350 DEG C under environment
Obtain NiP layered metal oxide nano-carrier；
S3, metal oxide nano bletilla striata pheretima albumen composition is prepared
The 10g bletilla striata is weighed, is filtered after decocting half an hour with deionized water, utilizes Rotary Evaporators removing superfluous water after collecting solution
Point, gained pasty mass is freeze-dried, bletilla striata extract powder is collected；It will be made in gained bletilla striata extract powder and S1
Standby obtained pheretima stress protein liquid is mixed to get pheretima stress protein mixed liquor, rear that 2 times of pheretima stress proteins are then added
The concentration of mixeding liquid volume is that the Tris-HCl buffer that 20mM, pH are 7.2 obtains mixed liquor；Measure mixed liquor 20ml with
5.0gNiP layered metal oxide nano-carrier mixes, and stirs 8 hours at 4 DEG C；It is centrifuged mixed liquor and collects precipitating, freeze-drying
Collect powder afterwards to get metal oxide nano bletilla striata pheretima albumen composition is arrived.
2. metal oxide nano bletilla striata pheretima albumen composition preparation method according to claim 1, it is characterised in that:
Pheretima selects Eisenia Foetida in the S1.
3. metal oxide nano bletilla striata pheretima albumen composition preparation method according to claim 1, it is characterised in that:
The volume ratio of ethyl alcohol and water is 1:1 in the S2.
4. metal oxide nano bletilla striata pheretima albumen composition preparation method according to claim 1, it is characterised in that:
Bletilla striata extract powder and pheretima stress protein liquid carry out being mixed to prepare pheretima stress protein mixed according to mass ratio 1:1 in the S3
Close liquid.
5. a kind of application of metal oxide nano bletilla striata pheretima albumen composition, it is characterised in that: using such as claim 1-4
Metal oxide nano made from a kind of any metal oxide nano bletilla striata pheretima albumen composition preparation method is white
Splendid achnatherum pheretima albumen composition is used as rat brain injury repair medicine.
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| CN111743997A (en) * | 2020-07-08 | 2020-10-09 | 陕西中医药大学 | Paste traditional Chinese medicine composition based on earthworm protein paste and preparation method thereof |
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| CN111743997A (en) * | 2020-07-08 | 2020-10-09 | 陕西中医药大学 | Paste traditional Chinese medicine composition based on earthworm protein paste and preparation method thereof |
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Application publication date: 20190329 |