CN116903705A - Method for purifying posterior pituitary element - Google Patents
Method for purifying posterior pituitary element Download PDFInfo
- Publication number
- CN116903705A CN116903705A CN202310738748.0A CN202310738748A CN116903705A CN 116903705 A CN116903705 A CN 116903705A CN 202310738748 A CN202310738748 A CN 202310738748A CN 116903705 A CN116903705 A CN 116903705A
- Authority
- CN
- China
- Prior art keywords
- posterior
- pituitary
- purifying
- centrifugate
- extraction
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 230000001817 pituitary effect Effects 0.000 title claims abstract description 42
- 238000000034 method Methods 0.000 title claims abstract description 34
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims abstract description 51
- 239000012528 membrane Substances 0.000 claims abstract description 34
- 238000000605 extraction Methods 0.000 claims abstract description 25
- 238000001914 filtration Methods 0.000 claims abstract description 19
- 238000005119 centrifugation Methods 0.000 claims abstract description 17
- 230000008014 freezing Effects 0.000 claims abstract description 12
- 238000007710 freezing Methods 0.000 claims abstract description 12
- 239000011259 mixed solution Substances 0.000 claims abstract description 7
- 239000004576 sand Substances 0.000 claims abstract description 7
- 239000007788 liquid Substances 0.000 claims description 26
- 238000009210 therapy by ultrasound Methods 0.000 claims description 14
- 238000000108 ultra-filtration Methods 0.000 claims description 13
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 12
- JLTCWSBVQSZVLT-CDIPANDDSA-N (2s)-n-[(2s)-6-amino-1-[(2-amino-2-oxoethyl)amino]-1-oxohexan-2-yl]-1-[(4r,7s,10s,13s,16s,19r)-19-amino-7-(2-amino-2-oxoethyl)-10-(3-amino-3-oxopropyl)-13-benzyl-16-[(4-hydroxyphenyl)methyl]-6,9,12,15,18-pentaoxo-1,2-dithia-5,8,11,14,17-pentazacycloicosan Chemical compound NCCCC[C@@H](C(=O)NCC(N)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@H]1NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC=2C=CC=CC=2)NC(=O)[C@H](CC=2C=CC(O)=CC=2)NC(=O)[C@@H](N)CSSC1.C([C@H]1C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CSSC[C@@H](C(N[C@@H](CC=2C=CC(O)=CC=2)C(=O)N1)=O)N)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCN=C(N)N)C(=O)NCC(N)=O)C1=CC=CC=C1 JLTCWSBVQSZVLT-CDIPANDDSA-N 0.000 claims description 11
- 108010070873 Posterior Pituitary Hormones Proteins 0.000 claims description 11
- 102000005320 Posterior Pituitary Hormones Human genes 0.000 claims description 11
- 239000012466 permeate Substances 0.000 claims description 7
- 230000001105 regulatory effect Effects 0.000 claims description 7
- 238000003756 stirring Methods 0.000 claims description 7
- 229960000583 acetic acid Drugs 0.000 claims description 6
- 239000012362 glacial acetic acid Substances 0.000 claims description 6
- 239000000047 product Substances 0.000 claims description 6
- 239000002033 PVDF binder Substances 0.000 claims description 3
- 229920002981 polyvinylidene fluoride Polymers 0.000 claims description 3
- 239000000126 substance Substances 0.000 abstract description 18
- 239000004480 active ingredient Substances 0.000 abstract description 17
- 206010062767 Hypophysitis Diseases 0.000 abstract description 8
- 210000003635 pituitary gland Anatomy 0.000 abstract description 8
- 230000002349 favourable effect Effects 0.000 abstract description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 22
- 239000000243 solution Substances 0.000 description 16
- 230000000052 comparative effect Effects 0.000 description 10
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 8
- 238000002360 preparation method Methods 0.000 description 8
- 239000000706 filtrate Substances 0.000 description 5
- 238000001471 micro-filtration Methods 0.000 description 5
- 239000012465 retentate Substances 0.000 description 5
- 235000013570 smoothie Nutrition 0.000 description 5
- 239000002244 precipitate Substances 0.000 description 4
- 235000018102 proteins Nutrition 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 239000007853 buffer solution Substances 0.000 description 3
- 239000011550 stock solution Substances 0.000 description 3
- GXBMIBRIOWHPDT-UHFFFAOYSA-N Vasopressin Natural products N1C(=O)C(CC=2C=C(O)C=CC=2)NC(=O)C(N)CSSCC(C(=O)N2C(CCC2)C(=O)NC(CCCN=C(N)N)C(=O)NCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(CCC(N)=O)NC(=O)C1CC1=CC=CC=C1 GXBMIBRIOWHPDT-UHFFFAOYSA-N 0.000 description 2
- 108010004977 Vasopressins Proteins 0.000 description 2
- 102000002852 Vasopressins Human genes 0.000 description 2
- 238000005054 agglomeration Methods 0.000 description 2
- 230000002776 aggregation Effects 0.000 description 2
- KBZOIRJILGZLEJ-LGYYRGKSSA-N argipressin Chemical compound C([C@H]1C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CSSC[C@@H](C(N[C@@H](CC=2C=CC(O)=CC=2)C(=O)N1)=O)N)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCN=C(N)N)C(=O)NCC(N)=O)C1=CC=CC=C1 KBZOIRJILGZLEJ-LGYYRGKSSA-N 0.000 description 2
- 238000005238 degreasing Methods 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000000149 penetrating effect Effects 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 229910021642 ultra pure water Inorganic materials 0.000 description 2
- 239000012498 ultrapure water Substances 0.000 description 2
- 229960003726 vasopressin Drugs 0.000 description 2
- 241000124008 Mammalia Species 0.000 description 1
- 101800000989 Oxytocin Proteins 0.000 description 1
- XNOPRXBHLZRZKH-UHFFFAOYSA-N Oxytocin Natural products N1C(=O)C(N)CSSCC(C(=O)N2C(CCC2)C(=O)NC(CC(C)C)C(=O)NCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(CCC(N)=O)NC(=O)C(C(C)CC)NC(=O)C1CC1=CC=C(O)C=C1 XNOPRXBHLZRZKH-UHFFFAOYSA-N 0.000 description 1
- 102100031951 Oxytocin-neurophysin 1 Human genes 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- 150000001945 cysteines Chemical class 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005265 energy consumption Methods 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 238000007654 immersion Methods 0.000 description 1
- 210000002418 meninge Anatomy 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- XNOPRXBHLZRZKH-DSZYJQQASA-N oxytocin Chemical compound C([C@H]1C(=O)N[C@H](C(N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CSSC[C@H](N)C(=O)N1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC(C)C)C(=O)NCC(N)=O)=O)[C@@H](C)CC)C1=CC=C(O)C=C1 XNOPRXBHLZRZKH-DSZYJQQASA-N 0.000 description 1
- 229960001723 oxytocin Drugs 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- BHZOKUMUHVTPBX-UHFFFAOYSA-M sodium acetic acid acetate Chemical compound [Na+].CC(O)=O.CC([O-])=O BHZOKUMUHVTPBX-UHFFFAOYSA-M 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/16—Oxytocins; Vasopressins; Related peptides
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Life Sciences & Earth Sciences (AREA)
- Peptides Or Proteins (AREA)
- Separation Using Semi-Permeable Membranes (AREA)
Abstract
The invention discloses a method for purifying posterior pituitary essence, which comprises the following specific steps: (1) Taking out the separated pituitary gland rear leaves, freezing, and crushing the pituitary gland rear leaves and a proper amount of ice cubes into ice sand to obtain mixed solution; (2) ultrasonically treating the ice sand; (3) Adding ethyl acetate into the mixed solution for extraction, and taking an extract; centrifuging the extract of (4) to obtain a centrifugate; (5) Filtering the centrifugate with a first-stage ultra-micro filter membrane and a second-stage ultra-micro filter membrane to obtain the finished product of pituitary posterior phylline. The method has simple process and high feasibility, combines the processes of freezing, ultrasonic, extraction and centrifugation, is favorable for removing high molecular weight substances, and ensures the yield and purity of active ingredients.
Description
Technical Field
The invention relates to a method for purifying posterior pituitary essence, belonging to the technical field of medicines.
Background
The main active ingredients of the posterior pituitary are posterior pituitary, mainly comprising vasopressin and oxytocin, both of which consist of nine amino acids, wherein vasopressin contains two cysteines, also known as octapeptides.
At present, the domestic extraction and purification of pituitary posterior phylline have the problems of complex extraction process, long time consumption, high cost and the like, and when the active ingredients of the pituitary posterior phylline are extracted, related high molecular weight substances cannot be thoroughly separated from the active ingredients, so that the purity and the yield are affected.
Disclosure of Invention
Aiming at the problems existing in the prior art, the invention provides a method for purifying pituitary posterior phyllin, which has simple process and high feasibility, combines freezing, ultrasonic, extraction and centrifugation processes, is favorable for removing high molecular weight substances, ensures the yield and purity of active ingredients, and has high preparation safety.
In order to achieve the above purpose, the present invention adopts the following technical scheme: a method for purifying posterior pituitary essence comprises the following specific steps:
(1) Taking out the separated pituitary posterior leaves, freezing for 2-3 hours at the temperature of minus 18-minus 15 ℃, and crushing the pituitary posterior leaves and a proper amount of ice cubes into ice;
(2) Placing the smoothie in the step (1) at 2-6 ℃ for ultrasonic treatment, so that high molecular weight substances in posterior pituitary are thinned, and active components are easily separated from the high molecular weight substances to obtain mixed liquor;
(3) Adding ethyl acetate into the mixed liquid obtained after ultrasonic treatment in the step (2) for extraction, fully stirring at 15-20 ℃, standing for layering, removing an upper layer liquid which is a high molecular weight substance extracted by ethyl acetate, and repeatedly extracting a lower layer liquid to obtain an extract;
(4) Centrifuging the extracted liquid obtained in the step (3) at 15-20 ℃, removing sediment, and repeatedly centrifuging the centrifugate to obtain centrifugate;
(5) Filtering the centrifugate in the step (4) through a first-stage ultra-micro filter membrane, wherein the molecular weight cut-off of the first-stage ultra-micro filter membrane is 10kD, removing cut-off solution, and collecting permeate;
(6) And (3) regulating the pH value of the centrifugate in the step (4) to 3-4 through glacial acetic acid, filtering through a secondary ultra-micro filtration membrane, removing filtrate, and collecting the retentate to obtain a finished product of pituitrin.
Preferably, the ice cubes are added in an amount 7 to 9 times the mass of the posterior pituitary, wherein the ice cubes are prepared by using ultrapure water.
Preferably, the conditions of the sonication: the ultrasonic power is 350-400W, and the ultrasonic time is 3-5 min.
Preferably, the extraction is repeated 1 to 4 times, preferably 2 to 3 times.
Preferably, the addition mass of the ethyl acetate is 1.5-2 times of the mass of the ice cubes after each extraction.
Preferably, the conditions of the centrifugation process: the centrifugal speed is 5500-7500 rpm, and the centrifugal time is 15-25 min.
Preferably, the centrifugation is repeated for 1 to 3 times, and the rotation speed of each centrifugation is not less than that of the last centrifugation, so that the removal of high molecular weight substances and residual ethyl acetate is facilitated, and the purity and yield of the active ingredients are improved.
Preferably, the primary ultrafiltration membrane and the secondary ultrafiltration membrane are made of polyvinylidene fluoride.
The invention has the beneficial effects that:
the invention takes the dried posterior pituitary as the raw material, the posterior pituitary is frozen and crushed with ice, and then ultrasonic treatment is utilized to refine the high molecular weight protein substances in the posterior pituitary, so as to destroy the agglomeration between the high molecular weight protein substances and the active ingredients, and the active ingredients are easy to separate from the high molecular weight protein substances; the ethyl acetate is adopted to extract the high molecular weight protein substances, so that the trouble that the rear leaf powder in a viscous state is not easy to separate when being adsorbed on filter paper in the traditional Buchner funnel filtration is avoided, the separation speed in the extraction process is increased, and the high molecular weight substances can be removed efficiently; further removing residual turbid substances in the solution by adopting centrifugal separation, and greatly improving the clarity of the solution; filtering with first-stage and second-stage filter membranes to improve purity and yield of active components; the preparation method has the advantages of simple process, high feasibility and high preparation safety, combines freezing, ultrasonic, extraction and centrifugation processes, is favorable for removing high molecular weight substances, and ensures the yield and purity of active ingredients; meanwhile, the number of filtering operation times in the prior art is effectively reduced, and the cost and the extraction period of the filtering membrane are reduced.
Detailed Description
The invention will now be more clearly and more fully described by way of the following specific examples, which are not intended to be limiting.
Obtaining posterior pituitary of the invention; extracting pituitary gland from mammal, placing into anhydrous acetone for fixation, dewatering and degreasing, removing meninges, immersing the stripped pituitary gland into proper amount of fresh anhydrous acetone, replacing fresh anhydrous acetone for 3-4 times to complete dewatering and degreasing, filtering the pituitary gland from the acetone immersion liquid, placing into a vacuum drier for drying at 30-50 ℃, separating front and rear pituitary gland leaves, and reserving rear pituitary gland leaves for standby.
The invention uses the material of the first-stage ultra-micro filter membrane and the second-stage ultra-filter membrane as polyvinylidene fluoride.
The ice blocks are prepared by adopting ultrapure water freezing.
Example 1
In this embodiment, a method for purifying posterior pituitary essence specifically includes the following steps:
(1) Taking out the separated pituitary back leaves, freezing at-15 ℃ for 3 hours, adding ice cubes which are 9 times of the mass of the pituitary back leaves, and crushing into smoothies;
(2) Placing the ice sand in the step (1) at 6 ℃ for ultrasonic treatment, wherein the ultrasonic power is 400W, and the ultrasonic time is 3min, so as to obtain a mixed solution;
(3) Adding ethyl acetate into the mixed liquid obtained after ultrasonic treatment in the step (2) for extraction, wherein the added mass of the ethyl acetate is 1.5 times of the mass of ice cubes, fully stirring at 15-20 ℃, standing for layering, removing the upper layer liquid, taking the lower layer liquid for repeated extraction, and repeating the extraction for 2 times to obtain an extract;
(4) Centrifuging the extracted liquid obtained in the step (3) at 15-20 ℃ for 25min at 5500rpm, removing precipitate, centrifuging the centrifugate repeatedly for 2 times at 6100rpm for 25min at 7000rpm for 20min, and obtaining centrifugate;
(5) Filtering the centrifugate in the step (4) through a first-stage ultra-micro filter membrane, wherein the molecular weight cut-off of the first-stage ultra-micro filter membrane is 10kD, removing cut-off solution, and collecting permeate;
(6) And (3) regulating the pH value of the centrifugate in the step (4) to 3-4 through glacial acetic acid, filtering through a secondary ultra-micro filtration membrane, removing filtrate, and collecting the retentate to obtain a finished product of pituitrin.
Example 2
In this embodiment, a method for purifying posterior pituitary essence specifically includes the following steps:
(1) Taking out the separated pituitary back leaves, freezing at-16deg.C for 2.5 hr, adding ice blocks with mass 8 times of that of pituitary back leaves, and crushing into smoothie;
(2) Placing the ice sand in the step (1) at 4 ℃ for ultrasonic treatment, wherein the ultrasonic power is 380W, and the ultrasonic time is 4min, so as to obtain a mixed solution;
(3) Adding ethyl acetate into the mixed liquid obtained after ultrasonic treatment in the step (2) for extraction, wherein the added mass of the ethyl acetate is 1.8 times of the mass of ice cubes, fully stirring at 15-20 ℃, standing for layering, removing the upper layer liquid, taking the lower layer liquid for repeated extraction, and repeating the extraction for 1 to obtain an extract;
(4) Centrifuging the extracted liquid obtained in the step (3) at 15-20 ℃ for 20min at 6000rpm, removing the precipitate, repeatedly centrifuging the centrifugate for 3 times, and repeatedly centrifuging at 7000rpm for 15min for the first, second and third times to obtain centrifugate;
(5) Filtering the centrifugate in the step (4) through a first-stage ultra-micro filter membrane, wherein the molecular weight cut-off of the first-stage ultra-micro filter membrane is 10kD, removing cut-off solution, and collecting permeate;
(6) And (3) regulating the pH value of the centrifugate in the step (4) to 3-4 through glacial acetic acid, filtering through a secondary ultra-micro filtration membrane, removing filtrate, and collecting the retentate to obtain a finished product of pituitrin.
Example 3
In this embodiment, a method for purifying posterior pituitary essence specifically includes the following steps:
(1) Taking out the separated pituitary back leaves, freezing at-18 ℃ for 2 hours, adding ice cubes which are 7 times the mass of the pituitary back leaves, and crushing into smoothies;
(2) Placing the ice sand in the step (1) at 2 ℃ for ultrasonic treatment, wherein the ultrasonic power is 350W, and the ultrasonic time is 5min, so as to obtain a mixed solution;
(3) Adding ethyl acetate into the mixed liquid obtained after ultrasonic treatment in the step (2) for extraction, wherein the added mass of the ethyl acetate is 2 times of the mass of ice cubes, fully stirring at 15-20 ℃, standing for layering, removing the upper layer liquid, taking the lower layer liquid for repeated extraction for 4 times, and obtaining an extract;
(4) Centrifuging the extracted liquid obtained in the step (3) at 15-20 ℃ for 25min at 7500rpm, removing the precipitate, and repeatedly centrifuging the centrifugate for 1 time at 7500rpm for 20min to obtain centrifugate;
(5) Filtering the centrifugate in the step (4) through a first-stage ultra-micro filter membrane, wherein the molecular weight cut-off of the first-stage ultra-micro filter membrane is 10kD, removing cut-off solution, and collecting permeate;
(6) And (3) regulating the pH value of the centrifugate in the step (4) to 3-4 through glacial acetic acid, filtering through a secondary ultra-micro filtration membrane, removing filtrate, and collecting the retentate to obtain a finished product of pituitrin.
Example 4
In this embodiment, a method for purifying posterior pituitary essence specifically includes the following steps:
(1) Taking out the separated pituitary back leaves, freezing at-18 ℃ for 2 hours, adding ice cubes which are 8 times the mass of the pituitary back leaves, and crushing into smoothies;
(2) Placing the ice sand in the step (1) at 4 ℃ for ultrasonic treatment, wherein the ultrasonic power is 360W, and the ultrasonic time is 4min, so as to obtain a mixed solution;
(3) Adding ethyl acetate into the mixed liquid obtained after ultrasonic treatment in the step (2) for extraction, wherein the added mass of the ethyl acetate is 2 times of the mass of ice cubes, fully stirring at 15-20 ℃, standing for layering, removing the upper layer liquid, taking the lower layer liquid for repeated extraction for 3 times, and obtaining an extract;
(4) Centrifuging the extracted liquid obtained in the step (3) at 15-20 ℃ for 20min at a centrifugation speed of 7000rpm, removing the precipitate, repeatedly centrifuging the centrifugate for 2 times, and obtaining centrifugate at a rotation speed of 7500rpm for 25min after the first and second repeated centrifugation;
(5) Filtering the centrifugate in the step (4) through a first-stage ultra-micro filter membrane, wherein the molecular weight cut-off of the first-stage ultra-micro filter membrane is 10kD, removing cut-off solution, and collecting permeate;
(6) And (3) regulating the pH value of the centrifugate in the step (4) to 3-4 through glacial acetic acid, filtering through a secondary ultra-micro filtration membrane, removing filtrate, and collecting the retentate to obtain a finished product of pituitrin.
Comparative example 1
The preparation procedure was the same as in example 4, except that: step (1) the separated pituitary gland She Zhijie is taken out and crushed into powder.
Comparative example 2
The preparation procedure was the same as in example 4, except that: step 2 is removed.
Comparative example 3
The preparation procedure was the same as in example 4, except that: removing the step (3) and the step (4), changing the precipitation of the extractant into filtration by adopting an ultrafiltration membrane with the molecular weight cut-off of 10-150 kD, and repeating the steps for 8 times.
Comparative example 4
The same as in example 1, except that: the ultrasonic power is 420W and the ultrasonic time is 2min.
Comparative example 5
The same as in example 3, except that: the ultrasonic power is 320W and the ultrasonic time is 6min.
Comparative example 6
The same as in example 1, except that: the centrifugal speed was 5000rpm and the centrifugal time was 25min.
Comparative example 7
The same as in example 3, except that: the centrifugal speed is 8000rpm, and the ultrasonic time is 25min.
Comparative example 8
The preparation procedure was the same as in example 4, except that: when the centrifugation is repeated, the rotation speed of the first and second centrifugation is 6500rpm, and the ultrasonic time is 25min.
Comparative example 9
A method for purifying posterior pituitary essence specifically comprises the following steps:
(1) Buffer solution preparation: preparing a CH3COONa-CH3COOH buffer solution with the concentration of 100mmol/L, and regulating the pH value to 5.0;
(2) Sample pretreatment: 2.0L of pituitary posterior leaflet hormone stock solution is measured, and the stock solution is prepared by the following steps: buffer 1:1 (volume ratio), adding buffer solution, stirring and mixing uniformly;
(3) Ultrafiltration: ultrafiltration is carried out by adopting an ultrafiltration membrane of 2kDa, the trapped solution is collected, and the ultrafiltration is stopped when the volume of the permeate end liquid reaches 90% of the initial volume of She Chaolv after pituitary;
(4) Dilution: accurately measuring the solution at the interception end, adding purified water according to 5 times of the volume, and adjusting the pH of a sample to 1.5 by adopting 2MH2SO 4;
(5) Reverse ultrafiltration: and (3) ultrafiltering the diluted intercepting end solution by adopting a 5kDa ultrafiltration membrane, collecting the penetrating end solution, and stopping ultrafiltering when the penetrating end solution volume reaches 90% of the initial volume of She Chaolv after pituitary.
The clarity, high molecular weight material impurity level and active ingredient yield for the above examples and comparative examples are shown in the following table:
from the above table, the process parameters of each step of the process effectively ensure the yield and purity of the active ingredient, the ultrasonic power is too high, the yield of the active ingredient can be influenced, the ultrasonic power is too low, and the yield and purity of the active ingredient are reduced; the centrifugal speed is too low, the yield of active ingredients is affected when the centrifugal speed is reduced in each repeated centrifugation, and the centrifugal speed is too high, so that the energy consumption is increased; the process of the invention is cooperated with each step of procedure, ice cubes and pituitary posterior leaves are mixed and crushed to form water extract, freezing and ultrasound are used for destroying the agglomeration between active ingredients and high molecular weight substances, ethyl acetate is used for extracting the high molecular weight substances, centrifugation is used for clarifying the solution, and a primary ultrafiltration membrane and a secondary ultrafiltration membrane are matched, so that the impurity removing effect and clarity of the solution are improved, the related substances of the stock solution are not introduced, and the yield and purity of the active ingredients are also effectively ensured.
In conclusion, the process provided by the invention has the advantages that the sequence of each step and the process parameters are matched in a synergistic way, the removal of high molecular weight substances is facilitated, and the yield and purity of active ingredients are ensured.
Finally, it should be noted that the above-mentioned embodiments are only for illustrating the technical solution of the present invention and not for limiting the technical solution of the present invention, and although the present invention has been described in detail with reference to the above-mentioned embodiments, it should be understood by those skilled in the art that the present invention may be modified or equivalently replaced without departing from the spirit and scope of the present invention, and any modification or partial replacement thereof should be included in the scope of the claims of the present invention.
Claims (8)
1. The method for purifying posterior pituitary essence is characterized by comprising the following specific steps:
(1) Taking out the separated pituitary posterior leaves, freezing for 2-3 hours at the temperature of minus 18-minus 15 ℃, and crushing the pituitary posterior leaves and a proper amount of ice cubes into ice;
(2) Placing the ice sand in the step (1) at 2-6 ℃ for ultrasonic treatment to obtain mixed solution;
(3) Adding ethyl acetate into the mixed liquid obtained after ultrasonic treatment in the step (2) for extraction, fully stirring at 15-20 ℃, standing for layering, and taking the lower layer liquid for repeated extraction to obtain an extract;
(4) Centrifuging the extracted liquid obtained in the step (3) at 15-20 ℃, and repeatedly centrifuging the centrifugate to obtain centrifugate;
(5) Filtering the centrifugate in the step (4) through a first-stage ultra-micro filter membrane, wherein the molecular weight cut-off of the first-stage ultra-micro filter membrane is 10kD, and collecting the permeate;
(6) And (3) regulating the pH value of the centrifugate in the step (4) to 3-4 through glacial acetic acid, filtering through a secondary ultra-micro filter membrane, retaining the molecular weight of the secondary ultra-micro filter membrane to be 1kD, and collecting the retaining liquid to obtain a finished product of pituitary posterior phyllin.
2. The method for purifying posterior pituitary according to claim 1, wherein the ice is added in an amount of 7 to 9 times the mass of posterior pituitary.
3. The method for purifying posterior pituitrin according to claim 1, wherein the condition of ultrasonic treatment: the ultrasonic power is 350-400W, and the ultrasonic time is 3-5 min.
4. The method for purifying posterior pituitrin according to claim 1, wherein the added mass of ethyl acetate is 1.5 to 2 times the mass of ice cubes per extraction.
5. The method for purifying posterior pituitrin according to claim 1, wherein the extraction is repeated 1 to 4 times.
6. The method for purifying posterior pituitrin according to claim 1, wherein the centrifugation conditions are as follows: the centrifugal speed is 5500-7500 rpm, and the centrifugal time is 15-25 min.
7. The method for purifying posterior pituitrin according to claim 1, wherein the centrifugation is repeated 1 to 3 times, and the rotational speed of each centrifugation is not less than the rotational speed of the last centrifugation.
8. The method for purifying posterior pituitrin according to claim 1, wherein the primary ultrafiltration membrane and the secondary ultrafiltration membrane are made of polyvinylidene fluoride.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2023104405082 | 2023-04-23 | ||
| CN202310440508 | 2023-04-23 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116903705A true CN116903705A (en) | 2023-10-20 |
Family
ID=88361045
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310738748.0A Pending CN116903705A (en) | 2023-04-23 | 2023-06-21 | Method for purifying posterior pituitary element |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116903705A (en) |
-
2023
- 2023-06-21 CN CN202310738748.0A patent/CN116903705A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP4680989B2 (en) | Reduction of phytic acid in protein isolation process | |
| CN105331665B (en) | A kind of method that pig brain proteolysis prepares brain polypeptide and brain small-molecular peptides | |
| US9179692B2 (en) | Production of protein isolates | |
| CN102488713B (en) | Method for preparing sheep placenta extract and sheep placenta hydrolyzed collagen concentrated solution | |
| US20070166798A1 (en) | Isolating chondroitin sulfate | |
| CN105925368B (en) | A method of walnut oil is prepared by water law demulsification walnut oil body | |
| CN107847891B (en) | Method for extracting saponin from agricultural products | |
| US20190359650A1 (en) | Methods for extracting keratin proteins | |
| JPH06116258A (en) | Production of low-caffeine green tea leaf catechins with low-caffeine content | |
| CN108977487A (en) | A kind of method and reaction system of subcritical fluid extraction and enzyme membrane coupling reaction preparation abalone activity glycopeptide | |
| CN101333245B (en) | Method for separating human serum albumin | |
| CN1152052C (en) | Process for preparing thymic peptide | |
| CN116903705A (en) | Method for purifying posterior pituitary element | |
| JP3988348B2 (en) | Method for producing water-soluble polysaccharide and clarification method for water-soluble polysaccharide aqueous solution | |
| JP2000506389A (en) | Method for producing fructose syrup from leuzelan plant | |
| US4128637A (en) | Process for producing thymosin | |
| KR101724118B1 (en) | Method for preparing high purity protein concentrate from rice bran | |
| CN109337952A (en) | A kind of extracting method of snake slough polypeptide | |
| CN105859827A (en) | Oligopeptides-1 active constituent extracting method | |
| EP4240838A1 (en) | Method of obtaining hyaluronidase from bovine testes | |
| Dendukuri et al. | Oil-free protein isolates from full-fat, dehulled mustard flour by microfiltration | |
| CN110642938A (en) | Collagen draws purification system | |
| JP2001072693A (en) | Production of purified yolk lecithin | |
| CN114634960B (en) | Preparation method of edible thymus peptide | |
| RU2648462C1 (en) | Membrane method for preparation of a drug based on prostatic peptides |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |