CN1896268A - Extraction of activated micromolecular sheept germine from sheep embryo and its use - Google Patents
Extraction of activated micromolecular sheept germine from sheep embryo and its use Download PDFInfo
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Abstract
The extraction of active small molecular placentin from lamb embryo and its purpose that belongs to the bioengineering field includes the following steps: removing stomach and intestine from fresh lamb embryo of 40-90 days and cutting up, homogenizing with distilled water, frost thawing repeatedly at -20DEG C, filtrating, heating the filtrate and obtaining Filtrate A and Sediment A; ultra-filtrating Filtrate A and obtaining the transparent primrose yellow Filtrate B and Sediment B; mingling Sediment A and Sediment B, modifying pH to 2.0, adding pepsin, then modifying pH to 7.0, incubating at 100 +-2DEG C and preserving Filtrate C; active small molecular placentin is obtained by mingling Filtrate B and C that can be used in the preparation of skin care product. The placentin prepared in this invention has the molecular weight of 3000-6000 and contains mainly cytokines and small peptides, has high activity, low side-effect such as anaphylactic response, and increased skin absorption ratio.
Description
Technical field
The invention belongs to technical field of bioengineering, this technology is applied to preparation and produces the activated micromolecular sheep placental extract, but as raw material production external application cosmetics.
Background technology
Sheep placental extract is by biotechnology, extracts viable cell MIENS, hundred-percent maintenance embryo's active substance from Placenta caprae seu ovis.The constant, trace element, panimmunity sphaeroprotein, multiple antibody, Interferon, rabbit, Zadaxin (keeping cellular immune function, anti-ageing), bioactive peptide, the Urogastron (keeping skin elasticity) that contain abundant Yelkin TTS, kephalin (brain cell nutrient substance), lipopolysaccharides, multivitamin and needed by human in the sheep placental extract, insulin-like growth factor, platelet derived growth factor (elimination hematopoietic disorders), conversion growth factor, white corpuscle element and cancer-resisting substance.And contain humoral factor (regulate human body endocrine), the various active enzyme (improves digestive system function, promote conversion, decomposition and the absorption of nutrition), (elimination makes the oxyradical of cell aging to dismutase SOD, with the superoxide reduction, prevent melanin deposition, alleviate the damage of pair cell).The bile of lamb also has extremely strong antioxidant property in addition, can eliminate chloasma and prevent pachylosis.
Late nineteen eighties, on the Switzerland achievement basis of Carlow woods professor Japanese scientist, take the lead in developing the sheep placental extract injection, its appearance, caused global sensation in medical cosmetology nutrition circle, showed that the differentiation anti-ageing, that promote skin cells of sheep placental extract mystery upgrades, improves the microcirculation of skin cells, the wrinkle of releiving, endocrine regulation, the various color spots that go down, improves sleep quality and immunomodulatory, have comprehensive recovery health flesh can function.Sheep placental extract is by the consistent health care superfine product that is known as epoch-making beauty treatment and keeps fit of international medical community.
But, these effects competence exertion effect under certain conditions of sheep placental extract.The first fully is absorbed by the body, and does not destroy its composition; Its two be sheep placental extract must obtain the activation.Therefore external effect sheep placental extract product preferably all is an injection.But the risk of injection injection is bigger, and is also very high to the requirement of product innovation.
Key of the present invention is a method of having created the extraction of activated micromolecular sheep placental extract, and with this activated micromolecular sheep placental extract external application, can be absorbed by human skin, plays beautification function.Thereby both reduced the application risk of sheep placental extract, obtained the effect of anti-wrinkle, beautify people skin again.
Summary of the invention
But the object of the invention is to invent the method for extraction of activated micromolecular sheep placental extract from the sheep embryo a kind of external application, that have beauty functions.
The present invention includes following steps:
1) will remove the fresh sheep embryo chopping of 40～90 ages in days of stomach intestines after, adding distil water is made homogenate;
2) with homogenate multigelation under-20 ℃ of conditions, adjust pH is 3.0～3.5, filters, and transferring filtrate pH value again is 7.0;
3) filtrate being heated to 70 ± 1 ℃, being incubated after 30 ± 2 minutes, is under the condition of 8000g centrifugal 30 ± 2 minutes at centrifugal force, obtains filtered solution A and throw out A respectively;
4) with filtered solution A ultrafiltration, described ultrafiltration condition is: the pH value is 7.0, flow velocity 20～40ml/min, gets transparent faint yellow filtered solution B and sediment B;
5) throw out A and sediment B are merged, transfer pH to 2.0, add stomach en-, making pepsin concn is 5g/L, is incubated 4～6 hours under 37 ± 2 ℃ of conditions;
6) transfer pH to 7.0 again, under 100 ± 2 ℃ of conditions, be incubated 2～3 minutes, filter, get filtered solution C;
7) filtered solution B and filtered solution C are merged, be the activated micromolecular sheep placental extract.
Novelty of the present invention and superiority show:
1, the extraction of small molecules sheep placental extract: adopt low temperature multigelation and ultrafiltration to extract, obtain filtered solution B, i.e. the sheep placental extract of molecular weight control between 3000～6000, at this molecular weight ranges, major part is small-molecular peptides and cytokine composition.Be characterized in having reduced the irritated side effect of Denging, and improved the skin absorption rate.
2,70 ± 1 ℃ of processing of heating have been played better action to the activation sheep placental extract.Determine that through animal experiment and detection its activation is effective.
3, small molecules activation sheep placental extract can directly act on human skin, and plays the effect of beauty treatment anti-wrinkle.
4, macromolecular substance among the gastric pepsin digestion sheep embryo has not only increased small-molecular peptides, and has improved the content that comprises biologically active substances such as amino acid.
Second purpose of the present invention is to provide practical purposes for the activated micromolecular sheep placental extract.
The activated micromolecular sheep placental extract that above method is made is used to prepare external application to be dispeled wrinkle, beautifies the skin articles for use.
Be used to prepare lyophilized powder.Can the activated micromolecular sheep placental extract is half congealed with extra large bath, N.F,USP MANNITOL mixes, and is sub-packed in the container freeze-drying; Wherein, activated micromolecular sheep placental extract, extra large bath mass ratio half congealed, N.F,USP MANNITOL are 1～2: 4: 4.
Be used to prepare face paper.Can add perfume compound with after the activated micromolecular sheep placental extract sterilising treatment, be adsorbed in face paper.
Being used for the preparation eye pastes.Can add perfume compound with after the sterilization of activated micromolecular sheep placental extract, be adsorbed in a paster.
Be used to prepare beauty liquid.Can be with packing after the activated micromolecular sheep placental extract sterilising treatment.
Pass through mouse experiment, SOD detection, interleukin II (IL-2), Telomerase activity, the test of T cell subsets and beauty salon are on probation, the activated micromolecular sheep placental extract that proof is extracted by present method is used for skins such as human face, have the tiny wrinkle of the skin of dispeling, the effect of increase skin glossy, the freeze-dried sheep placenta extract powder of raw material making, facial mask, eye card, essence can be used for daily beautifying skin thus.
Embodiment
One, prepare the activated micromolecular sheep placental extract according to the following steps:
1, gets the fresh goat embryo of 40～90 ages in days, remove the stomach intestines, clean.
2, cut or shred.
3, adding distil water stirs, and makes homogenate.
4, with homogenate-20 ℃ of multigelations 3 times under-20 ℃ of conditions, adjust pH is 3.0-3.5, filters, and transferring pH value of filtrate again is 7.0.
5, filtrate being heated to 70 ± 1 ℃, being incubated after 30 ± 2 minutes, is under the condition of 8000g centrifugal 30 ± 2 minutes at centrifugal force, obtains filtered solution A and throw out A respectively;
6, with filtered solution A ultrafiltration, described ultrafiltration condition is: pH 7.0, flow velocity 20-40ml/min.Supernatant liquor should be transparent faint yellow after the ultrafiltration, gets filtered solution B and sediment B.
7, throw out A and sediment B are merged, transfer pH to 2.0, add stomach en-, under 37 ± 2 ℃ of conditions, be incubated 4～6 hours; The pepsin concn that adds is 5g/L.
8, transfer pH to 7.0 again, under 100 ± 2 ℃ of conditions, be incubated 2～3 minutes, filter, get filtered solution C.
9, filtered solution B and filtered solution C are merged, be the activated micromolecular sheep placental extract.
Two, analysis of components and active the detection: the activated micromolecular sheep placental extract carries out CurrentChromatogram mensuration, (T cellular immunization, humoral immunization, thymus gland weightening finish, free radical SOD, Telomerase Content detect the active detection of animal experiment.
1, analysis of components: Current Chromatogram measurement result is: the major ingredient molecular weight is 3500, and total molecular weight is between 3000～6000; Total protein content 1 grams per liter; As seen 7 peaks wherein contain multiple amino acids and other biological activity, comprise interleukin-, tethelin, SOD, EGF.
2, mouse live test sheep placental extract biological activity:
Activated micromolecular sheep placental extract experimentation on animals result
80 of BALB/c mouse are divided into 8 groups, and sample is not enough because of concentration, not freeze-drying.
Sheep placenta 0.8mg/ml numbering A1 0.8mg/ml
0.4mg/ml A2 0.4mg/ml
0.2mg/ml A3 0.3mg/ml
Placenta 0.8mg/ml B1 0.8mg/ml
0.4mg/ml B2 0.4mg/ml
0.2mg/ml B3 0.2mg/ml
Newborn sheep 0.4mg/ml C 0.4mg/ml
Contrast physiological saline 0.1mg/ml
Above solution is by body weight, and every 10G injects 0.1mg/ml
Continuously abdominal injection is 10 days, and the experimental group animal lacks appetite, weight loss, and drug withdrawal is after 4 days, the injection next day of changing into, it is normal that mouse recovers, and got blood on the 16th day to carry out every inspection, the results are shown in nextpage.
Table 1: free radical detected result
| SOD | EGF | |
| A1 | 184.28↑↑ | 124.24↑ |
| A2 | 122.12↑ | 126.38↑ |
| A3 | 94.44↑ | 113.04 |
| B1 | 103.66↑ | 69.5 |
| B2 | 95.66↑ | 90.18 |
| B3 | 109.27↑ | 84.3 |
| C | 118.8↑ | 97.68 |
| D | 87.18 | 118.92 |
The free radical detected result shows: the activated micromolecular sheep placental extract can obviously improve SOD, EGF, and showing has the promotion epidermal growth; Antioxygenation.
Table 2: antibody and interleukin detect
| IgG | IgA | IgM | IL-2 | |
| A1 | 2.58 | 0.454 | 0.464 | / |
| A2 | 2.66 | 0.462 | 0.4 | 204.82↑↑ |
| A3 | 3.04 | 0.446 | 0.292 | 180.24↑ |
| B1 | 4.12 | 0.418 | 0.594 | 122.8 |
| B2 | 2.6 | 0.312 | 0.478 | 158.78 |
| B3 | 4.04 | 0.444 | 0.288 | 123.32 |
| C | 2.92 | 0.32 | 0.352 | 197.60↑↑ |
| D | 2.76 | 0.584 | 0.566 | 152.2 |
Antibody and interleukin detected result show: the activated micromolecular sheep placental extract has the immunity function of adjusting and the effect of building up resistance.
Table 3:T cell subsets
| CD3+ | CD4 | CD8 | |
| 8mg/kg/d | 73.6 | 50.2 | 23.4 |
| 4mg/kg/d | 70.8 | 49 | 21.8 |
| 2mg/kg/d | 74 | 49 | 27.2 |
| control | 70 | 45.4 | 24.6 |
T cell subsets test-results shows: the activated micromolecular sheep placenta have the raising immunity.
Table 4: the body weight change of mouse
| 18/ III | 19 | 21 | 23 | 25 | 27 | 28-31 | 1/IV | 2-3/ IV | 7/IV | 9 | 11 | ||
| A | 1 | 20.9 | 21.6 | 22 | 23.3 | 23.96 | 21.9 | Stop injection | 24.6 | Detect every index | 24.2 | 24.4 | 24.6 |
| 2 | 20.5 | 20.9 | 21.6 | 23 | 23.3 | 23.2 | 24.2 | 24.2 | 24.8 | 25.0 | |||
| 3 | 20.7 | 21.3 | 22.2 | 23 | 20.8 | 24.0 | 24.9 | 25.6 | 26.4 | 26.8 | |||
| B | 1 | 18.4 | 19.6 | 20.7 | 22.2 | 21.9 | 22.8 | 24.5 | 24.6 | 24.8 | 25.4 | ||
| 2 | 19.2 | 18.7 | 19.4 | 21.1 | 22.6 | 21.1 | 23.9 | 25.2 | 25.8 | 26.0 | |||
| 3 | 18.8 | 18.4 | 19.9 | 21.3 | 21.9 | 20.6 | 24.0 | 24.6 | 24.8 | 25.8 | |||
| C | 20.8 | 21.7 | 22.9 | 22.8 | 21.4 | 21.0 | 24.8 | 24.8 | 25.2 | 24.8 | |||
| D | 19.9 | 19.9 | 20.2 | 22.4 | 22.8 | 23.2 | 25.25 | 24.6 | 25.8 | 25.7 | |||
Mouse body weight test-results shows: the activated micromolecular sheep placental extract does not have considerable change to the mouse body weight.
Three, use:
1, preparation lyophilized powder.
The activated micromolecular sheep placental extract is half congealed with extra large bath, N.F,USP MANNITOL mixes, and is sub-packed in the container freeze-drying; Wherein, activated micromolecular sheep placental extract, extra large bath mass ratio half congealed, N.F,USP MANNITOL are 1～2: 4: 4.
Use after cleaning face, lyophilized powder is spread on face has wrinkle, color spot on the face, observes after 7～10 days every day twice, and the phenomenon that obvious wrinkle reduces, skin is bright and clean is arranged.
2, preparation face paper or eye paste.
After activated micromolecular sheep placental extract sterilising treatment, add perfume compound, be adsorbed in face paper or eye paster.
Face paper is attached to once in a week on the face clean, waits to do the back and remove.Through four uses, there is obvious wrinkle to alleviate the effect bright and clean with skin.
The eye paster is attached to clean eye once a day, waits to do the back and remove.In a continuous week, the effect that obviously alleviates the tiny wrinkle of eye is arranged.
3, preparation beauty liquid.
With packing after the activated micromolecular sheep placental extract sterilising treatment.
Use after cleaning face, beauty liquid is spread on face has wrinkle, color spot on the face, observes after 7～10 days every day twice, obvious desalination wrinkle is arranged, increase the glossy effect.
Claims (10)
1, the method for extraction of activated micromolecular sheep placental extract from the sheep embryo is characterized in that may further comprise the steps:
1) will remove the fresh sheep embryo chopping of 40～90 ages in days of stomach intestines after, adding distil water is made homogenate;
2) with homogenate multigelation under-20 ℃ of conditions, adjust pH is 3.0～3.5, filters, and transferring filtrate pH value again is 7.0;
3) filtrate being heated to 70 ± 1 ℃, being incubated after 30 ± 2 minutes, is under the condition of 8000g centrifugal 30 ± 2 minutes at centrifugal force, obtains filtered solution A and throw out A respectively;
4) with filtered solution A ultrafiltration, described ultrafiltration condition is: the pH value is 7.0, flow velocity 20～40ml/min, gets transparent faint yellow filtered solution B and sediment B;
5) throw out A and sediment B are merged, transfer pH to 2.0, add stomach en-, making pepsin concn is 5g/L, is incubated 4～6 hours under 37 ± 2 ℃ of conditions;
6) transfer pH to 7.0 again, under 100 ± 2 ℃ of conditions, be incubated 2～3 minutes, filter, get filtered solution C;
7) filtered solution B and filtered solution C are merged, be the activated micromolecular sheep placental extract.
2, the purposes of the activated micromolecular sheep placental extract that makes of a kind of method according to claim 1 is characterized in that being used to prepare external application dispels wrinkle, beautifies the skin articles for use.
3,, it is characterized in that being used to prepare lyophilized powder according to the purposes of the described activated micromolecular sheep placental extract of claim 2.
4,, it is characterized in that the activated micromolecular sheep placental extract is half congealed with extra large bath, N.F,USP MANNITOL mixes, and is sub-packed in the container freeze-drying according to the purposes of the described activated micromolecular sheep placental extract of claim 3; Wherein, activated micromolecular sheep placental extract: extra large bath is half congealed: the mass ratio of N.F,USP MANNITOL is 1～2: 4: 4.
5,, it is characterized in that being used to prepare face paper according to the purposes of the described activated micromolecular sheep placental extract of claim 2.
6, according to the purposes of the described activated micromolecular sheep placental extract of claim 5, it is characterized in that adding perfume compound with after the sterilization of activated micromolecular sheep placental extract, be adsorbed in face paper.
7,, it is characterized in that being used for the preparation eye and paste according to the purposes of the described activated micromolecular sheep placental extract of claim 2.
8, according to the purposes of the described activated micromolecular sheep placental extract of claim 7, it is characterized in that adding perfume compound with after the sterilization of activated micromolecular sheep placental extract, be adsorbed in a paster.
9,, it is characterized in that being used to prepare beauty liquid according to the purposes of the described activated micromolecular sheep placental extract of claim 2.
10,, it is characterized in that packing after the activated micromolecular sheep placental extract sterilising treatment according to the purposes of the described activated micromolecular sheep placental extract of claim 2.
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Effective date of registration: 20120717 Address after: 225009 No. 15, Weiyang Road, Hanjiang District, Jiangsu, Yangzhou Patentee after: Yangzhou Yangda Lianhuan Pharmaceutical Gene Engineering Co.,Ltd. Address before: 225009 Yangzhou University, Jiangsu, South Road, No. 88 Patentee before: Yangzhou University |