CN1333294A - Novel process for extracting purified sheed fetal agent and method for testing biological activity thereof - Google Patents
Novel process for extracting purified sheed fetal agent and method for testing biological activity thereof Download PDFInfo
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Abstract
The present invention discloses a process for extracting and purifying sheep placenta extract and a method for detecting its biological activity. It said invention modern biological technology is adopted to extract and purify sheep placental protein component, and mainly includes the following steps: homogenizing, salt precipitation (ammonium sulfate salt or sodium chloride salt), low-temp. centrifugation, low-temp. ultrafiltration, dextran chromatography, nucleoprotein detection and amino acid composition analysis, etc. The sheep placenta extract obtained by said method possesses the advantages of high purity, good, homogeneity, strong biological activity, high yield and low cost, so that it has high practical value.
Description
The present invention relates to medicine biological technique, relate in particular to a kind of new extraction purifying sheep placental extract technology and its bioactive method of detection.
Contain nearly hundred kinds of effective ingredients that promote body tissue cellular metabolism and reparation in the Placenta caprae seu ovis, along with the progress of medicine biological technique in recent years, the albumen and the polypeptide fraction that obtain in the purer Placenta caprae seu ovis have become possibility, yet the method for currently used traditional extraction process for from food technology, deriving, (as described in Chinese patent application numbers 98101188 and 98115761), not only too coarse, the so-called sheep placental extract that extracts is actually the structural proteins all in the Placenta caprae seu ovis and the mixture of functional protein and polysaccharide, has immunogenicity, at all can not be medicinal, simultaneously because enzyme liberating, protein denaturation, the influence of factors such as temperature variation, the substance bioactivity that is extracted is poor, only be used for industries such as beauty and make-up a little less than the specificity, we can say with existing method and extract sheep placental extract, not only extract less than sheep placental extract but also wasted the Placenta caprae seu ovis resource of a large amount of preciousnesses.
Purpose of the present invention is exactly in order to overcome the defective of existing sheep placental extract extracting method, and provide a kind ofly have a kind of new extraction purifying sheep placental extract technology of characteristics such as purity height, an even property is good, biological activity is strong and yield is many, cost is low and detect its bioactive method.
The technical scheme that realizes the object of the invention is:
A kind of new extraction purifying sheep placental extract technology is characterized in, comprising: extracting and saltout, ultrafiltration and desalination, dialysis and concentrate and carboxymethyl cellulose dextrane gel cation-exchange chromatography (hereinafter to be referred as CM-SephadexC
50Column chromatography) four steps;
Described extracting and the step of saltouing comprise:
Fresh or refrigerated sheep placenta are wiped out fetal membrane and great vessels, clean remaining clot, chopping is smashed to pieces in tissue mashing machine more repeatedly, makes homogenate; This homogenate is stirred in ice bath, abandon precipitation, get the supernatant liquor restir, salt precipitation promptly obtains containing the crude extract of sheep placental extract;
The step of described ultrafiltration and desalination comprises: be filter just in the daltonian filter in 30000 kilodaltons～4000 in cutoff value earlier, adopting cutoff value again under the cold condition of 0-15 degree Celsius is that the daltonian filter in 100 dalton～800 carries out classification ultrafiltration and desalination;
Described dialysis and spissated step comprise: with crude extract with a small amount of cold deionized water dissolving after, more thoroughly the dialysis; Collect dialyzate and concentrated, calculated population is long-pending, measures protein concn;
Described CM-SephadexC
50The column chromatography step comprises: with the dialysis of sheep placental extract crude extract concentrated solution, be splined on the CM-SephedexC that crosses with same damping fluid balance in advance then
50(2.5 * 50cm) posts carry out wash-out, obtain first protein peak; When treating that this protein peak drops to baseline position, successive can obtain second protein peak and the 3rd protein peak respectively with above-mentioned phosphate buffered saline buffer wash-out again; Collect above-mentioned CMF respectively
1, CMF
2And CMF
3Component, difference called after sheep placental extract 1, sheep placental extract 2 and sheep placental extract 3 its protein concn to be measured and biological activitys.
Above-mentioned a kind of new extraction purifying sheep placental extract technology, wherein, the described requirement that Placenta caprae seu ovis is made homogenate is: under 1 ℃～6 ℃ condition, the per kilogram tissue block adds the 0.15mol/L ammoniumsulphate soln 2L that contains 0.5mmol/L phenmethyl semi-annular jade pendant acyl fluorides (hereinafter to be referred as PMSF), in tissue mashing machine, smashed to pieces repeatedly 3 minutes, and made homogenate.
Above-mentioned a kind of new extraction purifying sheep placental extract technology, wherein, described dialysis be with crude extract with a small amount of cold deionized water dissolving after, place in the dialysis tubing of cutoff value＜4000, stir down constantly at 1 ℃-4 ℃, water is thoroughly dialysed.
A kind of method that detects the sheep placental extract biologic activity is characterized in, comprises molecular weight determination and amino acid composition analysis two big steps;
Described molecular weight determination is to adopt sodium lauryl sulphate-Polyacrylamide gel electrophoresis, the steps include: standard protein and testing protein sample are added in isopyknic sample buffer behind the mixing, and the predefined procedure electrophoresis is pressed in heating then; When sample begins to enter in the glue of upper strata, be concentrated gradually; When the dyestuff forward position enters separation gel, and reach separation gel when bottom, stripping glue and mark; The decolouring back is measured from the separation gel section start to labeling dye district band and the distance of zone of protein central position; Be ordinate zou with standard protein molecular weight logarithm then, (Rm) is X-coordinate with relative mobility, makes typical curve on semi-logarithmic coordinate paper; Rm value according to testing protein is calculated its corresponding molecular weight on typical curve.
Described amino acid composition analysis method is carried out on the sour automatic analyser of base.
The bioactive mensuration employing of described sheep placental extract component tritiated thymidine (hereinafter to be referred as
3H-TdR) method of mixing is carried out, and the steps include:
Get inoblast and behind 0.25% tryptic digestion, make cell suspension, be inoculated on the culture plate, change nutrient solution after 24 hours, place 37 ℃, 5%CO
2Continue under the saturated humidity condition to cultivate; The different components of sheep placental extract that add doubling dilution next day are with continuing cultivation under the condition.Finish after 48 hours to cultivate, the nutrient solution that inclines adds 0.25% tryptic digestion 3min, and the bull cell is collected the device collecting cell on the glass fibre filter disc, and the washing back is fixing, and oven dry is put in the scintillation solution again, surveys the cpm value of each sample hose by instrument; Being X-coordinate with the protein concn that contains the sheep placental extract component again, is ordinate zou with the cpm number in every hole, traces
3H-TdR mixes curve, one of them (ED of activity unit
50) be defined as the content of the required sheep placental extract component of the active half of maximal stimulation.
Aforesaid method, wherein, described damping fluid is made of 10% policapram 12-alkyl sulfonyl sodium (hereinafter to be referred as SDS), 1% thioglycol, 0.01mol/LPH7.8 sodium phosphate.
Aforesaid method, wherein, described electrophoretic electrophoresis system is: concentrated gum concentration is 3%, PH:6.8, resolving gel concentration are 15%, PH:8.8.
Aforesaid method, wherein, described concentration time was about 40 minutes-50 minutes.
Aforesaid method, wherein
Aforesaid method, wherein, described scintillation solution is the toluene solution of 0.3%PPO and 0.03%POPOP.
Because the present invention has adopted above technical scheme, adopt modern biotechnology to extract purifying Placenta caprae seu ovis protein ingredient, significantly be better than disclosing at present material institute reported method, have characteristics such as purity height, an even property is good, biological activity is strong and yield is many, cost is low with the sheep placental extract that this method obtained, great practical value is arranged.
The present invention extracts purifying Placenta caprae seu ovis component 1 by using modern medicine biological technique, component 2 and component 3, difference called after sheep placental extract 1, sheep placental extract 2, sheep placental extract 3.And further foundation detects its bioactive method.For further development and utilization sheep placental extract provides a feasible approach.
Further specify the features and advantages of the present invention below in conjunction with embodiment and accompanying drawing.
Fig. 1 is the schema of the separation and purification of sheep placental extract of the present invention.
The present invention mainly comprises the extracting of use sulfuric acid amine salt, salt precipitation, and low-temperature centrifugation, the low temperature ultrafiltration, methods such as cation-exchange chromatography are carried out the separation and purification sheep placental extract and are set up the content of sheep placental extract activity test method two aspects.
The flow process of the separation and purification of sheep placental extract is seen Fig. 1.
The step of the separation and purification of sheep placental extract is:
(1) extracting and saltouing
Fresh or refrigerated sheep placenta are wiped out fetal membrane and great vessels, clean remaining clot, rub with physiological saline, claim weight in wet base, under 4 ℃ of conditions, every kg tissue block adds the 0.15mol/L ammoniumsulphate soln 2L that contains 0.5mmol/LPMSF, smashed to pieces repeatedly 3 minutes with DS-l type tissue mashing machine, make homogenate.With 6mol/LHCL the PH of homogenate is transferred to 4.5, mechanical stirring is 2 hours in the ice bath, and 7500rpm/min is centrifugal 60 minutes then, abandons precipitation, and supernatant liquor is regulated PH to 6.0 with lmol/LNaOH, and this supernatant liquor slowly adds solid (NH under constantly stirring
4)
2SO
4Powder 230g/L, salt precipitation.Next day 7500rpm/min, centrifugal 60 minutes, abandon precipitation, the gained supernatant liquor adds solid ammonium sulfate powder 300g/L again, salt precipitation, 7500rpm/min, centrifugal 60 minutes, abandon supernatant liquor, resulting precipitation is the crude extract that contains sheep placental extract.
[2] ultrafiltration:
The selection of ultra-filtration equipment: can select for use shallow road system's ultra-filtration equipment or closed system that whipping appts or tubular fibre system ultra-filtration equipment are arranged according to condition, wherein with hollow-fibre ultrafiltration device efficient the best.Selected the plate and frame ultrafiltration apparatus of the SINR of Shanghai Atomic Nucleus Inst., Chinese Academy of Sciences's production for use for reducing cost of investment present method.
The selection of ultra-filtration membrane: the selection of film mainly comprises two aspects, be the selection of film cutoff value molecular weight on the one hand, mainly reach to such an extent that separate effect and come selective membrane according to the molecular weight size of processed material and requirement, molecular weight as separated material is 25000KD, reach 90% separating effect, then but the selective membrane cutoff value is 20000KD, and can to select cutoff value be 10000KD or littler film as reaching 100% separating effect.Select the relatively good speed that leaches of separating effect again than film faster on the other hand.The ultra-filtration membrane that present method has selected Chinese Academy of Sciences's Shanghai nucleus to be developed, selecting cutoff value earlier for use is 4000 dalton, the pure water flow is the aseptic ultra-filtration membrane of SPK high polymer alloy of 30-40 liter/square metre/hour, and then to select cutoff value for use be the aseptic ultra-filtration membranes of 600 daltonian SPK high polymer alloys, under the cold condition of 0-10 degree, carry out classification ultrafiltration and desalination, obtained splendid separating effect (available red, orange, green, blue, yellow (ROGBY) is measured the purity of isolate).
[3] dialysis and concentrated
With crude extract with a small amount of cold deionized water dissolving after, place in the dialysis tubing of cutoff value＜4000, stir down constantly at 4 ℃, water is thoroughly dialysed; Collect dialyzate and concentrated, calculated population is long-pending, measures protein concn.
[4] CM-SephadexC
50Column chromatography
With sheep placental extract crude extract concentrated solution 20-40ml,, be splined on the CM-SephedexC that crosses with same damping fluid balance in advance then to the 0.1mol/LPH6.0PBS dialysis
50(2.5 * 50cm) posts carry out wash-out under the monitoring of nucleic acid-protein detector and registering instrument, flow velocity is 50ml/hr, can obtain first protein peak (CMF
1), when treating that protein peak drops to baseline position, successive can obtain second protein peak (CMF respectively with the above-mentioned phosphate buffered saline buffer wash-out that contains 0.15mol/LNaCL and 0.6mol/LNaCL again
2) and the 3rd protein peak (CMF
3).Collect above-mentioned CMF respectively
1, CMF
2And CMF
3Component, difference called after sheep placental extract 1, sheep placental extract 2 and sheep placental extract 3 its protein concn to be measured and biological activitys.
Above-mentioned 3 components find that through biological activity assay the 3T3 cell is had very strong short cell fission activity, and the biological activity of buffer solution elution thing CM3d that wherein contains 0.6mol/1NaCL is the highest.The 1kg Placenta caprae seu ovis is available sheep placental extract, sheep placental extract, sheep placental extract 3 behind the above-mentioned steps purifying, and productive rate is respectively 400～450mg, 200～250mg and 40～60mg.
The method of sheep placental extract Biological Detection of the present invention comprises that molecular weight determination, amino acid composition analysis, the bioactive mensuration of sheep placental extract component adopt
3H-TdR mixes method:
1. molecular weight determination:
Adopt sodium lauryl sulphate-Polyacrylamide gel electrophoresis (SodiumDodeySulfate-PolyacrylamideGelElectrophoresis, SDS-PAGE) method determining molecular weight: standard protein and testing protein sample are added in isopyknic sample buffer behind (10%SDS-1% thioglycol-0.01mol/LPH7.8 sodium phosphate buffer) mixing, heating is 3 minutes in boiling water, (concentrating gum concentration is 3% to add same electrophoresis system by predefined procedure then, PH:6.8, resolving gel concentration is 15%, PH:8.8) electrophoresis in, last groove positive pole, following groove negative pole.Voltage transfers to 50V (about 8V/CM), and sample begins to enter in the glue of upper strata, is concentrated (about 40-50 minute) gradually.When the dyestuff forward position entered separation gel (lower floor), voltage transferred to 120V, continued electrophoresis, reached the separation gel bottom until bromjophenol blue, transferred voltage to zero, cut off the electricity supply.Stripping glue and mark.The monoblock separation gel is immersed in the 0.25% coomassie brilliant blue R250 solution dyeing 5 hours.Take out gel, put into destainer with water rinse for several times and decolour.The decolouring back is measured from the separation gel section start to the dye area band and the distance of zone of protein central position.Be ordinate zou with standard protein molecular weight logarithm then, (Rm) is X-coordinate with relative mobility, makes typical curve on semi-logarithmic coordinate paper.Rm value according to testing protein is calculated its corresponding molecular weight on typical curve.
2. amino acid composition analysis:
On the 835-50 of Hitachi type automatic analyzer for amino acids, carry out (analyze pre-treatment: 6NHCL, 110 ℃, hydrolysis in 20 hours, sample concentration 0.6ml/ml), assist to finish by the Chinese Academy of Sciences is biochemical.
The bioactive mensuration of sheep placental extract component (
3H-TdR mixes method):
Get the 3T3 inoblast, make cell suspension behind 0.25% tryptic digestion, with containing the nutrient solution of 10% calf serum by 2 * 10
4/ mm
3Density is inoculated on 96 well culture plates, changes the DMEM nutrient solution that contains 0.5% calf serum after 24 hours, places 37 ℃, 5%CO
2Continue under the saturated humidity condition to cultivate.The different components of sheep placental extract that add doubling dilution next day are with continuing cultivation under the condition.After 48 hours, every Kong Jiahan
3The DMEM liquid 50 μ l (1 μ ci) of H-TdR, continue to cultivate and finish after 4 hours to cultivate, nutrient solution inclines, add 0.25% tryptic digestion 3min, the bull cell is collected the device collecting cell on the glass fibre filter disc, after distillation is washed 6 times, 5% Tricholroacetic Acid is fixed, dried 1 hour for 60-80 ℃, put in the scintillation solution (toluene solution of 0.3%PPO and 0.03%POPOP), liquid scintillation counter is surveyed the cpm value of each sample hose automatically.With the protein concn that contains the sheep placental extract component is X-coordinate, is ordinate zou with the cpm number in every hole, traces
3H-TdR mixes curve, one of them (ED of activity unit
50) be defined as the content of the required sheep placental extract component of the active half of maximal stimulation.
The extract that contains different sheep placental extract is seen Fig. 5 to the influence of 3T3 cytoactive.
(see figure 9) behind the sheep placental extract 3 that adds 10ng/ml in the nutrient solution at serum-free, the CPM value of capillary endothelium CMEC and arteries smooth cell ASMC is increased to 4807.0 ± 326.72 and 3260.4 ± 268.67 from 2459 ± 268.90 and 1699.4 ± 203.96 respectively, the per-cent that increases is respectively: 95.47% and 91.90%, and difference is (P＜0.01) extremely significantly.
Sheep placental extract of the present invention contains more immunoglobulin (Ig), active polysaccharide, tethelin, cell active factor, is a kind of biologically active substance that has highly bioactive promotion cellular metabolism and prevent aging.Studies have shown that it makes it have high potential using value to promotion epidermal cell proliferation, the biology characteristics such as apoptosis, increase passage that delay, and can cell cultured supernatant regenerate, have resistibility and the beauty treatment and the function in delaying senility of raise immunity, raising body, the composition of sheep placental extract has reached 500 Hongkong dollars/ml in the price of Hongkong market at present.Sheep placental extract of the present invention is except that having above-mentioned effect, also may all have short propagation and splitting action to the cell in all mesoderm sources, therefore, it can produce wide influence to reproductive system, hemopoietic system, cardiovascular systems, nerve and endocrine system, can further develop as increasing brain cell activity, memory, improve the biotechnological formulation of the elderly's thinking and strengthen hematopoietic function, promoting the new bio medicine that myocardial cell and liver cell are repaired.
Claims (9)
1, a kind of new extraction purifying sheep placental extract technology is characterized in that, comprising: extracting and saltout, ultrafiltration and desalination, dialysis and concentrate and four steps of carboxymethyl cellulose dextrane gel cation-exchange chromatography;
Described extracting and the step of saltouing comprise:
Fresh or refrigerated sheep placenta are wiped out fetal membrane and great vessels, clean remaining clot, chopping is smashed to pieces in tissue mashing machine more repeatedly, makes homogenate; This homogenate is stirred in ice bath, abandon precipitation, get the supernatant liquor restir, salt precipitation promptly obtains containing the crude extract of sheep placental extract;
The step of described ultrafiltration and desalination comprises: be filter just in the daltonian filter in 30000 kilodaltons～4000 in cutoff value earlier, adopting cutoff value again under the cold condition of 0-15 degree Celsius is that the daltonian ultra-fine filter in 100 dalton～800 carries out classification ultrafiltration and desalination;
Described dialysis and spissated step comprise: with crude extract with a small amount of cold deionized water dissolving after, more thoroughly the dialysis; Collect dialyzate and concentrated, calculated population is long-pending, measures protein concn;
Described carboxymethyl cellulose dextrane gel cation-exchange chromatography step comprises: sheep placental extract crude extract concentrated solution is dialysed, be splined on the carboxymethyl cellulose dextrane gel cation-exchange chromatography post of crossing with same damping fluid balance in advance then and carry out wash-out, obtain first protein peak; When treating that this protein peak drops to baseline position, successive can obtain second protein peak and the 3rd protein peak respectively with above-mentioned phosphate buffered saline buffer wash-out again; Collect above-mentioned CMF respectively
1, CMF
2And CMF
3Component, difference called after sheep placental extract 1, sheep placental extract 2 and sheep placental extract 3 its protein concn to be measured and biological activitys.
2, a kind of new extraction purifying sheep placental extract technology according to claim 1, it is characterized in that, the described requirement that Placenta caprae seu ovis is made homogenate is: under 1 ℃～6 ℃ condition, the per kilogram tissue block adds the 0.15mol/L ammoniumsulphate soln 2L that contains 0.5mmol/L phenmethyl semi-annular jade pendant acyl fluorides, in tissue mashing machine, smashed to pieces repeatedly 3 minutes, and made homogenate.
3, a kind of new extraction purifying sheep placental extract technology according to claim 1, it is characterized in that, described dialysis be with crude extract with a small amount of cold deionized water dissolving after, place in the dialysis tubing of cutoff value＜4000, constantly stir down at 1 ℃-4 ℃, water is thoroughly dialysed.
4, a kind of method that detects the sheep placental extract biologic activity is characterized in that, comprises molecular weight determination and amino acid composition analysis two big steps;
Described molecular weight determination is to adopt sodium lauryl sulphate-Polyacrylamide gel electrophoresis, the steps include: standard protein and testing protein sample are added in isopyknic sample buffer behind the mixing, and the predefined procedure electrophoresis is pressed in heating then; When sample begins to enter in the glue of upper strata, be concentrated gradually; When the dyestuff forward position enters separation gel, and reach separation gel when bottom, stripping glue and mark; The decolouring back is measured from the separation gel section start to labeling dye district band and the distance of zone of protein central position; Be ordinate zou with standard protein molecular weight logarithm then, (Rm) is X-coordinate with relative mobility, makes typical curve on semi-logarithmic coordinate paper; Rm value according to testing protein is calculated its corresponding molecular weight on typical curve.
Described amino acid composition analysis method is carried out on the sour automatic analyser of base.
The bioactive mensuration of described sheep placental extract component adopts the tritiated thymidine method of mixing to carry out, and the steps include:
Get inoblast and behind 0.25% tryptic digestion, make cell suspension, be inoculated on the culture plate, change nutrient solution after 24 hours, place 37 ℃, 5%CO
2Continue under the saturated humidity condition to cultivate; The different components of sheep placental extract that add doubling dilution next day are with continuing cultivation under the condition.Finish after 48 hours to cultivate, the nutrient solution that inclines adds 0.25% tryptic digestion 3min, and the bull cell is collected the device collecting cell on the glass fibre filter disc, and the washing back is fixing, and oven dry is put in the scintillation solution again, surveys the cpm value of each sample hose by instrument; Being X-coordinate with the protein concn that contains the sheep placental extract component again, is ordinate zou with the cpm number in every hole, traces tritiated thymidine and mixes curve, one of them (ED of activity unit
50) be defined as the content of the required sheep placental extract component of the active half of maximal stimulation.
5, method according to claim 4 is characterized in that, described damping fluid is made of 10% policapram 12-alkyl sulfonyl sodium, 1% thioglycol, 0.01mol/LPH7.8 sodium phosphate.
6, method according to claim 4 is characterized in that, described electrophoretic electrophoresis system is: concentrated gum concentration is 3%, and PH:6.8, resolving gel concentration are 15%, PH:8.8.
7, method according to claim 4 is characterized in that, described concentration time was about 40 minutes-50 minutes.
8, method according to claim 4 is characterized in that, and is described
9, method according to claim 4 is characterized in that, described scintillation solution is the toluene solution of 0.3%PPO and 0.03%POPOP.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101231403B (en) * | 2007-01-10 | 2011-10-05 | Nec液晶技术株式会社 | Reflective LCD equipment having excellent image quality |
| CN102274241A (en) * | 2011-08-10 | 2011-12-14 | 周伟 | Method for extracting sheep embryo extract and medicinal composition comprising same |
| CN102329827A (en) * | 2011-07-12 | 2012-01-25 | 湖州康海斯生物科技有限公司 | Sheep placenta amino acid and low molecular weight polypeptide, and preparation method for sheep placenta amino acid and low molecular weight polypeptide |
| CN102329842A (en) * | 2011-07-12 | 2012-01-25 | 湖州康海斯生物科技有限公司 | Lamb placenta polypeptide and preparation method thereof |
| CN102488713A (en) * | 2011-12-26 | 2012-06-13 | 重庆大学 | Method for preparing sheep placenta extract and sheep placenta hydrolyzed collagen concentrated solution |
| CN108323750A (en) * | 2017-01-20 | 2018-07-27 | 内蒙古草原鑫河食品有限公司 | A kind of active constituent and its extracting method and application with liver protection |
| CN109350627A (en) * | 2018-11-28 | 2019-02-19 | 广东冠龙生物科技有限公司 | A kind of freeze-dried sheep placenta extract powder and preparation method thereof |
| CN111647039A (en) * | 2020-07-07 | 2020-09-11 | 肖启森 | Method for extracting sheep embryo element active peptide |
| CN113577005A (en) * | 2021-08-02 | 2021-11-02 | 京美瑞禾健康科技(辽宁)有限公司 | Sheep placenta ultramicro factor mask liquid and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101231403B (en) * | 2007-01-10 | 2011-10-05 | Nec液晶技术株式会社 | Reflective LCD equipment having excellent image quality |
| CN102329842B (en) * | 2011-07-12 | 2014-10-29 | 湖州康海斯生物科技有限公司 | Lamb placenta polypeptide and preparation method thereof |
| CN102329827A (en) * | 2011-07-12 | 2012-01-25 | 湖州康海斯生物科技有限公司 | Sheep placenta amino acid and low molecular weight polypeptide, and preparation method for sheep placenta amino acid and low molecular weight polypeptide |
| CN102329842A (en) * | 2011-07-12 | 2012-01-25 | 湖州康海斯生物科技有限公司 | Lamb placenta polypeptide and preparation method thereof |
| CN102329827B (en) * | 2011-07-12 | 2014-11-26 | 湖州康海斯生物科技有限公司 | Sheep placenta amino acid and low molecular weight polypeptide, and preparation method for sheep placenta amino acid and low molecular weight polypeptide |
| CN102274241A (en) * | 2011-08-10 | 2011-12-14 | 周伟 | Method for extracting sheep embryo extract and medicinal composition comprising same |
| CN102274241B (en) * | 2011-08-10 | 2014-11-05 | 周伟 | Method for extracting sheep embryo extract and medicinal composition comprising same |
| CN102488713A (en) * | 2011-12-26 | 2012-06-13 | 重庆大学 | Method for preparing sheep placenta extract and sheep placenta hydrolyzed collagen concentrated solution |
| CN102488713B (en) * | 2011-12-26 | 2013-04-03 | 重庆大学 | Method for preparing sheep placenta extract and sheep placenta hydrolyzed collagen concentrated solution |
| CN108323750A (en) * | 2017-01-20 | 2018-07-27 | 内蒙古草原鑫河食品有限公司 | A kind of active constituent and its extracting method and application with liver protection |
| CN109350627A (en) * | 2018-11-28 | 2019-02-19 | 广东冠龙生物科技有限公司 | A kind of freeze-dried sheep placenta extract powder and preparation method thereof |
| CN111647039A (en) * | 2020-07-07 | 2020-09-11 | 肖启森 | Method for extracting sheep embryo element active peptide |
| CN113577005A (en) * | 2021-08-02 | 2021-11-02 | 京美瑞禾健康科技(辽宁)有限公司 | Sheep placenta ultramicro factor mask liquid and preparation method thereof |
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