CN103937860A - Preparation method for sea cucumber polypeptide for promoting postoperation wound healing - Google Patents
Preparation method for sea cucumber polypeptide for promoting postoperation wound healing Download PDFInfo
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Abstract
A disclosed preparation method for sea cucumber polypeptide for promoting postoperation wound healing is characterized by comprising technological steps of pretreatment, heating denaturation, cooling fragmentation, heating enzyme-killing, coarse filtration and alcohol precipitation, vacuum condensation, standing precipitation, filtering and freeze drying. The prepared sea cucumber has good immunoloregulation effect on body, also has extremely substantial wound healing promotion effect, and is capable of promoting wound to heal 4-5 days early, and the healed wound is flat and no scar is left.
Description
Technical field:
The present invention relates to wound healing technical field, is a kind of preparation method who promotes the sea cucumber peptide of postoperative wound healing specifically.
Background technology:
In real life, some patient, such as diabetic subject waits after catcher surgical operation, wound healing is very slow, and this not only brings a lot of miseries to patient, and because healing is slow, the probability infecting increases greatly, therefore, how to take measures, promoting that postoperative wound heals as early as possible, is an important topic in surgical medicine.
Postoperative wound healing refers to that the tissues such as the skin at body post-operative wound position occur from the process more again after disconnected or damaged, is to comprise the regeneration of various tissues and the complex combination that granulation tissue hyperplasia, scar tissue form, and shows the synergy of various processes.The degree of damage and the regenerative power of tissue determine the mode of repairing, the time of healing and scar very much not.Therefore, principle of reatment should be to dwindle the surface of a wound (as involutory wound), prevent from damaging and promoting tissue regeneration.Although the regenerative power of tissue obtains during evolution, but still is subject to the impact of whole body and local condition.Therefore, should avoid some unfavorable factors, promotion regeneration and restoration creates favorable conditions.
At present, the wound healing expedite of medical circle widespread use, is mainly external application measure, and, outside wound, wound healing is protected from infection, promoted simultaneously to topical application of drug thing, by taking to promote the product of wound healing orally, almost do not have, and the research of this respect is also very rare.
As everyone knows, after sea cucumber live body is injured, self there is extremely strong reparation and regenerative power.Investigation is found, the 30 feet of wild sea cucumbers of Iceland that left and right is marine under water that grow from marine site, the North Atlantic, and the geographical position of its life is near the north polar circle, and seawater temperature remains on below 4 ℃ throughout the year, and winter, surface froze, uninhabited all the year round.In addition glacier snow mountain overburden soil ground, around without the pollution of any industry and mankind's activity.Live in the wild sea cucumber of Iceland in ice-cold waters, poor growth, finished product ginseng is all to have 10 years above wild sea cucumbers of joining age, is genuine old sea cucumber.It contains rich in protein, mineral substance and multiple sea cucumber active ingredient, and cholesterol level is almost nil.The wild sea cucumber of Iceland wild sea cucumber Iceland contains the high protein that enriches high-quality, sea cucumber protein becomes deal to account for 20.3%, cholesterol level is very low, every hectogram sea cucumber cholesterol is 0.02 milligram of content only, low-fat content is also very low, and fatty ingredient amount accounts for 0.06%, and acidic mucopolysaccharide is moderate, healthy nutritive value is relatively good, is the superfine product in sea cucumber.
Summary of the invention:
The object of the invention is to overcome the deficiency of above-mentioned prior art, and a kind of preparation method who promotes the sea cucumber peptide of postoperative wound healing is provided; Mainly solving the healing of existing promotion postoperative wound can only external application and without the problem of product for oral administration.
Technical scheme of the present invention is: promote the preparation method of the sea cucumber peptide of postoperative wound healing, its special character is, comprises the steps:
A pre-treatment, selecting fresh sea cucumber is raw material, cuts open, removes after internal organ, obtains fresh ginseng body;
B heat denatured, fresh ginseng body adds pure water, heated and boiled, and keep boiling 5-10 minute, make the sex change of albumen appropriateness;
The cooling fragmentation of c, add heat kill enzyme, be cooled to after room temperature, ginseng body is broken into fritter, and is again heated to 40-50 ℃, to temperature, add neutral protease, temperature 40-70 ℃, enzymolysis time 8-12h, then heated and boiled is killed enzyme;
D coarse filtration alcohol precipitation, kill after enzyme, is cooled to room temperature, with 30-50 object tiffany, carries out coarse filtration, adds 95%(V/V) alcohol, make ethanol content in system reach 50-70%(V/V), standing 5-10 hour, with 100 object filter-cloth filterings, precipitation separation, collects filtrate; Gained precipitation is for subsequent extracted sea cucumber polysaccharide;
E vacuum concentration, vacuum concentration gained filtrate, concentrated condition is: temperature 30-50 ℃, absolute pressure 1000-5000Pa, when volume reaches 1/10 left and right of greenery volume;
F staticly settles, and in concentrated solution, adding concentration is the tanning solution of 18-20%, and add-on is the 2-5% of concentrated solution volume, staticly settles 3-5 hour, and 100 order filter-cloth filterings, remove precipitation, collects filtrate;
G filters, and adds powdered active carbon, the 1-3%(w/V that add-on is filtrate volume in filtrate), system is heated to 50-80 ℃, maintain 1-2 hour; With 100 order filter-cloth filterings, and collect filtrate, the 1/3-1/5 by filtrate vacuum concentration to original volume;
H lyophilize, concentrated solution vacuum lyophilization, obtains pale yellow powder, is molecular weight and is no more than 2500 sea cucumber peptide product.
Further, the add-on of the neutral protease described in c step is the fresh ginseng body of 1000-5000U/kg.
Further, the vacuum lyophilization condition described in h step is: absolute pressure 50-100Pa, temperature 20-30 ℃, time 8-12h.
Compared with the prior art the preparation method of the sea cucumber peptide of promotion postoperative wound healing of the present invention has outstanding substantive distinguishing features and marked improvement, take fresh sea cucumber as raw material, the molecular weight that biological enzymolysis is produced is lower than 2500 sea cucumber bioactive peptide, not only body is had to good immunoregulation effect, and there is the wound healing promoter action of highly significant, research is found, this bioactive peptide can promote the formation of postoperative mouse wound circumference capillary vessel, compare with control group decimal, (control group mice generally needed about 13 days can to promote to shift to an earlier date healing in 4-5 days by wound, and experiment organization need 8-9 days), and the wound after healing is smooth, be difficult for scar.
Embodiment:
For Comprehension and Implementation better, below in conjunction with embodiment, describe the present invention in detail; Illustrated embodiment is only for explaining the present invention, not for limiting the scope of the invention.
Embodiment 1, and selecting the wild sea cucumber 100kg of fresh Iceland is raw material, cuts open, removes after the internal organ such as intestines, obtains fresh ginseng body 51.2kg; Then add 100kg pure water, heated and boiled, and keep boiling 8 minutes, make the sex change of albumen appropriateness; Be cooled to after room temperature, ginseng body is broken into fritter, and be again heated to 45 ℃, be beneficial to enzymolysis, to temperature, add neutral protease, add enzyme amount by the fresh ginseng body of 3000U/kg, enzymatic hydrolysis condition is: 55 ℃ of temperature, enzymolysis time 8-12h, to time, heated and boiled is killed enzyme; Kill after enzyme, be cooled to room temperature, and carry out coarse filtration with 40 object tiffanys, remove after oarse-grained impurity, add 95%(V/V) alcohol, be that in system, ethanol content reaches 60%(V/V), standing 8 hours, with 100 object filter-cloth filterings, precipitation separation, collect filtrate; Gained precipitation is for subsequent extracted sea cucumber polysaccharide; Vacuum concentrated filtrate gained filtrate, concentrated condition is: 40 ℃ of temperature, absolute pressure 3000Pa, when volume reaches 1/10 left and right of greenery volume; In concentrated solution, adding concentration is 19% tanning solution, and dosage is 3.5% of concentrated solution volume, staticly settles 4 hours, and 100 order filter-cloth filterings, remove precipitation, collects filtrate; In filtrate, add 3% powdered active carbon, the 2%(w/V that add-on is filtrate volume); System is heated to 65 ℃ of left and right, maintains 1.5 hours; With 100 order filter-cloth filterings, and collect filtrate.By filtrate vacuum concentration to 1/4 of original volume; Concentrated solution vacuum lyophilization, condition is: absolute pressure 75Pa, 25 ℃ of temperature, time 10h; Gained pale yellow powder, is molecular weight and is no more than 2500 sea cucumber peptide product, and this molecular weight product is no more than 2500 sea cucumber peptide content and is not less than 80%.
The sea cucumber peptide of above-described embodiment 1 is tested as follows:
1, laboratory animal:
Kunming kind small white mouse, body weight 18-22g, is buied by Chinese Institute of Preventive Medicine.
2, reagent:
Ether AR Tianjin Jin Dong Tian Zheng fine chemistry chemical reagent work
Dehydrated alcohol AR Laiyang City Kant Chemical Co., Ltd.
Double source chemical plant, formaldehyde AR Jiangsu
NaH
2pO
42H
2o AR Tianjin Jin Bei Fine Chemical Co., Ltd
Na
2hPO
412H
2o AR Tianjin Jin Bei Fine Chemical Co., Ltd
Dimethylbenzene AR Laiyang City Kant Chemical Co., Ltd.
1% hydrochloride alcohol, 1:1 glycerine albumen, hematoxylin solution, 0.5% Yihong solution, paraffin, physiological saline.
3, instrument:
Electronic horizon AEV-210 Xiang Yi balance equipment factory
The microscope E-200 south of the River Xin Feida of photoelectricity group opticinstrument company
Digital camera S-4500 Li Da digital technology company limited
Thermostatic drying chamber YB-1 Dongtai electrical apparatus factory
Electric-heated thermostatic water bath Longkou City instrument company of SAST
Slicing machine, crucible, beaker, triangular flask, wide-necked bottle, embedded box, spirit lamp, tweezers, scissors, knife blade.
4, experimental technique:
The anesthesia of 4.1 small white mouses:
In 250ml triangular flask, put into a small amount of absorbent cotton, pour 10ml ether into, left hand band rubber gloves is clamped mouse tail root with little finger of toe with nameless, middle finger, forefinger are picked up mouse skin of neck with thumb together with two ears, then mouse head is filled in to triangular flask, put in about 3cm, head around can not be attached on triangular flask wall, manual time-keeping, surveys best recovery time.
4.2.1 the microscler wound manufacture of small white mouse:
With knife blade, at the standardized microscler wound of mouse back, deeply reach subcutaneous muscle.
4.2.2 the manufacture of circular wound
The circular plastic collar that is 0.5cm by self-control diameter is caught color, being imprinted on mouse back positions, with aseptic nipper, from circle center, mouse skin is picked up, then by sterile scissors, along trace, wipe out skin of back, just can obtain size, the basically identical circular wound of the depth.
4.3 grouping experiments:
Get 20 body weight and be the small white mouse of 20 ± 2 grams, be divided at random 2 groups, the circular wound that to manufacture diameter be 0.5cm.Use tap water gavage 0.5ml, i.e. control group first group of every day; Use the sea cucumber peptide liquid gavage 0.5ml of embodiment 1, i.e. sea cucumber experimental group second group of every day.
4.4 wound healings are observed:
4.4.1 visual inspection:
Every other day with set square, measure wound size, and observe wound changing conditions.
4.4.2 under microscope, wound healing is observed:
Main angiogenesis distribution situation and the granulation tissue growing state observed under light microscopic.
Small white mouse is 1,3,5,7,11,13 natural gift 6 times after surgery, each each group is got 1 and is drawn neck to put to death, then with tweezers, pick up the peripheral skin of circular wound, with scissors, cut and be placed on (the inside upward) on the slide glass that scribbles 1:1 glycerine albumin glue, directly put micro-Microscopic observation, digital camera photography.
5, result and discussion:
The anaesthetic effect of 5.1 ether to small white mouse:
Can find out by experiment, the effect of anesthesia duration 60s is better, both there is no dead mouse, and recovery time is also comparatively suitable.
5.2 wound healing visual inspection results:
Result is observed and is found, the wound of gavage embodiment 1 sea cucumber peptide experimental group (2 groups) mouse, than control group (1 group) mouse wound, all can shift to an earlier date healing in 2 ~ 3 days, and after the wound healing of sea cucumber experimental group, smooth surface is without incrustation, and there is incrustation control group wound healing rear surface, therefore think that sea cucumber peptide plays significant promoter action to wound healing.
Measure rear 1,3,5,7,9,11,13, the 15 day wound area of small white mouse operation, be denoted as respectively S
1, S
3, S
5, S
7, S
9, S
11, S
13, S
15, result is as table 2.
Note: 1 ☆ has significant difference compared with the control, p < 0.05;
2 ☆ ☆ have utmost point significant difference compared with the control, p < 0.01.
From table 2 result, from the 7th day, the wound healing of gavage sea cucumber peptide group mouse was obvious, compares and has significant difference, p < 0.05 with control group; From the 7th day, compare with control group and there is utmost point significant difference, p < 0.01; From the 9th day, compare and there is significant difference with control group, p < 0.05; From the 11st day, compare with control group and there is utmost point significant difference, p < 0.01.
5.3 wound healing microscopic examination comparative results:
Postoperative 1 day, microscopic examination discovery, experimental group and control group wound circumference are all shown in a small amount of capillary vessel, periphery subcutis congestion and edema.
Postoperative 3 days, the visible a lot of new capillary vessels of experimental group wound circumference, and control group is wanted much less.
5 days after operation, experimental group wound circumference forms dense capillary vessel network, and control group does not have this phenomenon, but also many new lives' capillary vessel occurs.
Postoperative 7 days, experimental group wound circumference capillary vessel number started to reduce, have newborn thick blood vessel to occur, and control group wound circumference was still shown in there is more capillary vessel, there is no newborn thick blood vessel.
Postoperative 11 days, experimental group wound circumference capillary vessel number obviously reduced, and had obvious newborn extra heavy pipe to occur, control group wound circumference capillary vessel also reduces to some extent.
Postoperative 13 days, experimental group wound healed substantially, and wound circumference is recovered standard state substantially, and control group wound has seen have newborn thick blood vessel to occur, and wound does not also recover completely.
Embodiment 2, and selecting the wild fresh sea cucumber of Iceland is raw material, cuts open, removes after internal organ, obtains fresh ginseng body; Fresh ginseng body adds pure water, heated and boiled, and keep boiling 5 minutes, make the sex change of albumen appropriateness; Be cooled to after room temperature, ginseng body is broken into fritter, and be again heated to 40 ℃, to temperature, add neutral protease, the add-on of neutral protease is the fresh ginseng body of 1000U/kg, 40 ℃ of temperature, and enzymolysis time 12h, then heated and boiled is killed enzyme; Kill after enzyme, be cooled to room temperature, with 30 object tiffanys, carry out coarse filtration, add 95%(V/V) alcohol, make ethanol content in system reach 50%(V/V), standing 5 hours, with 100 object filter-cloth filterings, precipitation separation, collect filtrate; Gained precipitation is for subsequent extracted sea cucumber polysaccharide; Vacuum concentration gained filtrate, concentrated condition is: 30 ℃ of temperature, absolute pressure 5000Pa, when volume reaches 1/10 left and right of filtrate volume; In concentrated solution, adding concentration is 18% tanning solution, and add-on is 2% of concentrated solution volume, staticly settles 3 hours, and 100 order filter-cloth filterings, remove precipitation, collects filtrate; In filtrate, add 2% powdered active carbon, the 1%(w/V that add-on is filtrate volume), system is heated to 50 ℃, maintain 2 hours; With 100 order filter-cloth filterings, and collect filtrate, by filtrate vacuum concentration to 1/3 of original volume; Concentrated solution vacuum lyophilization, vacuum lyophilization condition is: absolute pressure 50Pa, 30 ℃ of temperature, time 12h.Obtain pale yellow powder, be molecular weight and be no more than 2500 sea cucumber peptide product.
Embodiment 3, and selecting the wild fresh sea cucumber of Iceland is raw material, cuts open, removes after internal organ, obtains fresh ginseng body; Fresh ginseng body adds pure water, heated and boiled, and keep boiling 10 minutes, make the sex change of albumen appropriateness; Be cooled to after room temperature, ginseng body is broken into fritter, and be again heated to 50 ℃, to temperature, add neutral protease, the add-on of neutral protease is the fresh ginseng body of 5000U/kg, temperature 70 C, and enzymolysis time 8h, then heated and boiled is killed enzyme; Kill after enzyme, be cooled to room temperature, with 50 object tiffanys, carry out coarse filtration, add 95%(V/V) alcohol, make ethanol content in system reach 70%(V/V), standing 10 hours, with 100 object filter-cloth filterings, precipitation separation, collect filtrate; Gained precipitation is for subsequent extracted sea cucumber polysaccharide; Vacuum concentration gained filtrate, concentrated condition is: temperature 50 C, absolute pressure 1000Pa, when volume reaches 1/10 left and right of filtrate volume; In concentrated solution, adding concentration is 20% tanning solution, and add-on is 5% of concentrated solution volume, staticly settles 5 hours, and 100 order filter-cloth filterings, remove precipitation, collects filtrate; In filtrate, add 4% powdered active carbon, the 3%(w/V that add-on is filtrate volume), system is heated to 80 ℃, maintain 1 hour; With 100 order filter-cloth filterings, and collect filtrate, by filtrate vacuum concentration to 1/5 of original volume, concentrated solution vacuum lyophilization, vacuum lyophilization condition is: absolute pressure 100Pa, 20 ℃ of temperature, time 8h, obtains pale yellow powder, is molecular weight and is no more than 2500 sea cucumber peptide product.
Claims (3)
1. the preparation method who promotes the sea cucumber peptide of postoperative wound healing, is characterized in that, comprises the steps:
A pre-treatment: selecting fresh sea cucumber is raw material, cuts open, removes after internal organ, obtains fresh ginseng body;
B heat denatured: fresh ginseng body adds pure water, heated and boiled, and keep boiling 5-10 minute, make protein denaturation;
The cooling fragmentation of c, add heat kill enzyme: be cooled to after room temperature, ginseng body be broken into fritter, and be again heated to 40-50 ℃, to temperature, add neutral protease, temperature 40-70 ℃, enzymolysis time 8-12h, then heated and boiled is killed enzyme;
D coarse filtration alcohol precipitation: kill after enzyme, be cooled to room temperature, with 30-50 object tiffany, carry out coarse filtration, add 95%(V/V) alcohol, make ethanol content in system reach 50-70%(V/V), standing 5-10 hour, with 100 object filter-cloth filterings, precipitation separation, collect filtrate; Gained precipitation is for subsequent extracted sea cucumber polysaccharide;
E vacuum concentration: vacuum concentration gained filtrate, concentrated condition is: temperature 30-50 ℃, absolute pressure 1000-5000Pa, when volume reaches 1/10 of filtrate volume;
F staticly settles: in concentrated solution, adding concentration is the tanning solution of 18-20%, and add-on is the 2-5% of concentrated solution volume, staticly settles 3-5 hour, and 100 order filter-cloth filterings, remove precipitation, collects filtrate;
G filters: in filtrate, add powdered active carbon, the 1-3%(w/V that add-on is filtrate volume), system is heated to 50-80 ℃, maintain 1-2 hour; With 100 order filter-cloth filterings, and collect filtrate, the 1/3-1/5 by filtrate vacuum concentration to original volume;
H lyophilize: concentrated solution vacuum lyophilization, obtain pale yellow powder, be molecular weight and be no more than 2500 sea cucumber peptide product.
2. the preparation method of the sea cucumber peptide of promotion postoperative wound according to claim 1 healing, is characterized in that, the add-on of the neutral protease described in c step is the fresh ginseng body of 1000-5000U/kg.
3. the preparation method of the sea cucumber peptide of promotion postoperative wound according to claim 1 healing, is characterized in that, the vacuum lyophilization condition described in h step is: absolute pressure 50-100Pa, temperature 20-30 ℃, time 8-12h.
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| CN104585798A (en) * | 2015-01-15 | 2015-05-06 | 大连卓尔高科技有限公司 | Method for preparing nutrients from sea cucumber water decoction |
| CN106581654A (en) * | 2016-12-27 | 2017-04-26 | 青岛琛蓝海洋生物工程有限公司 | Composite skin wound disinfectant and preparation method thereof |
| CN107397226A (en) * | 2017-06-30 | 2017-11-28 | 宁德师范学院 | A kind of reducing blood lipid sea cucumber polypeptide microcapsules and preparation method thereof |
| CN107549792A (en) * | 2017-09-13 | 2018-01-09 | 山东圣洲海洋生物科技股份有限公司 | A kind of sea cucumber peptide composite nutrition powder and its preparation technology |
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| CN112274536A (en) * | 2020-11-05 | 2021-01-29 | 江南大学 | Application of sea cucumber powder in preparation of medicine for promoting skin wound healing |
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| CN104585798A (en) * | 2015-01-15 | 2015-05-06 | 大连卓尔高科技有限公司 | Method for preparing nutrients from sea cucumber water decoction |
| CN106581654A (en) * | 2016-12-27 | 2017-04-26 | 青岛琛蓝海洋生物工程有限公司 | Composite skin wound disinfectant and preparation method thereof |
| CN107397226A (en) * | 2017-06-30 | 2017-11-28 | 宁德师范学院 | A kind of reducing blood lipid sea cucumber polypeptide microcapsules and preparation method thereof |
| CN107549792A (en) * | 2017-09-13 | 2018-01-09 | 山东圣洲海洋生物科技股份有限公司 | A kind of sea cucumber peptide composite nutrition powder and its preparation technology |
| CN107670097A (en) * | 2017-10-19 | 2018-02-09 | 北京九鼎君健医药科技项城有限公司 | A kind of oligopeptide Wound dressing for promoting diabetes wound healing and preparation method thereof |
| CN107670097B (en) * | 2017-10-19 | 2021-02-23 | 北京九鼎君健东肽生物科技项城有限公司 | Oligopeptide wound dressing for promoting diabetic wound healing and preparation method thereof |
| CN110613832A (en) * | 2019-10-31 | 2019-12-27 | 西安惠普生物科技有限公司 | Application and preparation method of sea cucumber polypeptide in medical apparatus |
| CN112274536A (en) * | 2020-11-05 | 2021-01-29 | 江南大学 | Application of sea cucumber powder in preparation of medicine for promoting skin wound healing |
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