CN107158038A - The preparation method and applications of bioactive substance - Google Patents
The preparation method and applications of bioactive substance Download PDFInfo
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- CN107158038A CN107158038A CN201710402954.9A CN201710402954A CN107158038A CN 107158038 A CN107158038 A CN 107158038A CN 201710402954 A CN201710402954 A CN 201710402954A CN 107158038 A CN107158038 A CN 107158038A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/56—Materials from animals other than mammals
- A61K35/63—Arthropods
- A61K35/64—Insects, e.g. bees, wasps or fleas
- A61K35/644—Beeswax; Propolis; Royal jelly; Honey
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/48—Reproductive organs
- A61K35/50—Placenta; Placental stem cells; Amniotic fluid; Amnion; Amniotic stem cells
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- A—HUMAN NECESSITIES
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- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
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- A61K8/98—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin
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- A61K8/982—Reproductive organs; Embryos, Eggs
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/98—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin
- A61K8/987—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin of species other than mammals or birds
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Abstract
The invention discloses the preparation method of bioactie agent, bioactie agent of the invention is honeybee royal embryo factor, placenta transcription factor and royal jelly transcription factor.The invention also discloses the application of honeybee royal embryo factor extract solution, placenta transcription factor extract solution, royal jelly transcription factor extract solution in terms of skin care item.The invention also discloses a kind of skin cream and preparation method thereof.The skin cream whitening of the present invention, anti-ageing, smoothing wrinkle composition function introduction：The transcription factor that queen bee fetus, royal jelly, Human plactnta are extracted, without allergy, is less than 3000 small component nanometer technologies.Skin-absorbable, go deep into epidermis, corium, hypodermis.Physical and chemical index, bioactivity, go out virus, bacterium（Using：wilfriedfranke（Germany）Produce human blood DLE in enormous quantities to go out the improvement of malicious method）Product safety, effectively.
Description
Technical field
The present invention relates to health care and beauty treatment fields, and in particular to the preparation method and applications of bioactive substance,
More particularly to honeybee royal embryo factor extract solution, placenta transcription factor extract solution, royal jelly transcription factor extract solution are preparing skin care item
The application of aspect, the application especially in terms of skin cream.
Background technology
Lawrence inventions transfer factor (Transfer factor, TF) in 1949, Levin in 1969 applies TF first
One immunologic deficiency disease for the treatment of succeeds.Placenta prepares transfer factor, experimental study and clinical practice have biologically active peptide,
Nucleic acid, 23 kinds of trace elements etc..Human plactnta contains 312 kinds of special transcription factor (23.P65)；Clinic trial is comprehensive to Behcet
Close disease, it is chorionitis, psoriasis, leukopenia, encephalitis B, chronic hepatitis B, hepatitis, recurrent herpes simplex, red
Yabbi sore is cured and coordinates Chemotherapy in Treating Malignant Tumor, there is certain effect.Can nutrition protection skin elasticity it is soft, to skin
There is beautification function.Acute and chronic dermatitis, skin splash (senile plaque expelling) pigmentation are significantly disappeared.
Human plactnta has 312 kinds of transcription factor (TF) this product contents：It is more than 2mg/ml in terms of biologically active polypeptide.
1. placenta TF is the holy product of beauty, can improve the spot on coarse aging skin, desalination skin, skin whitening.Tire
The effect of disk TF both unrestraint tyrosinases, does not destroy melanin yet or reduces the effect of melanin.Its lightening mechanism can
To be explained from the angle of " activating cell "：Cell is activated, and metabolic function is improved, the function enhancing of metabolism of pigment,
Cutin updates Metabolism of Normal, and skin is just naturally pale.
2. nucleic acid：(DNA RNA) this product content：E=7.8, A260/A280 ratio are more than more than 1.8.DNA
(deoxyribonucleic、DNA):Endonuclear function is：The synthesis of repetition DNA and protein.When DNA lacks, skin
Fed without appropriate neonatal cell, just easily form aging phenomenon.Clinical practice：Small molecule placenta dna can be absorbed by the skin, and be closed
Into the genetic constructions of neoblast, cell is set to be in the vigorous state of vitality, cell turnover speed is fast, so as to play anti crease and anti senile
Old effect.
3. it is micro-：This product placenta transcription factor (HP-TF) is containing 23 kinds of trace elements (being shown in Table 4).Trace element is anti-
Effect in aging is the focus that aging biology is studied in the world in recent years.Numerous studies show：Cellular replication, suppression are freely
Base, raising immunologic function and the close trace element of anti-aging mainly have zinc, selenium, placenta selenium (se) content 0.18-0.52mg/
Kg, copper, manganese.
4. collagen (collagen), HP-TF hydrolysising amino acid contents (being shown in Table 2).Soluble collagen hydrolysis contains
15 kinds of amino acid nutrient materials, are easily absorbed by the skin, and can promote epidermal cell vigor, have additional nutrients, and effectively eliminate the thin of skin
Small wrinkle.
Queen bee fetus, royal jelly function：Protein can be promoted to synthesize, accelerate the metabolism of body, enhancing histocyte is again
Raw ability.Honeybee royal embryo factor and the royal jelly factor are nano micromolecule bioactive substances, can infiltrate through skin histology, can make
Skin keeps tender and lovely, fine and smooth, glossy and high resilience.It is the various skin splash caused by endocrinopathy, pigmentation, pregnant
Be pregnent spot, senile plaque expelling disappears healing substantially.
Shoot up in proteomics nearly more than ten year and ripe subject.The viewpoint that proteomics is integrated：I.e. in order to build
The biosystem that vertical and maintenance one is normally run, protein is how to function and act synergistically.
The nucleic acid of extraction is tested with polypeptide through protein reversible：The change of variable residue (small peptide) does not influence protein
Conformation and function.This product can make amino acid sequence (small peptide) revert back to primary structure by Caloric test.It is denatured renaturation experiment
Prove amino acid, ribonucleic acid enzymatic reversion space structure and function.It is clinical cure autoimmune disorder, SLE, Behcet, yellowish-brown
The diseases such as spot.
Protein is biological in vivo functionality macromolecule (also referred to as functional macromolecule)：The master of protein in vivo
Function is wanted to include the reaction (enzyme) in catalysis biological body, Metabolism regulation (polypeptide hormone, such as insulin), body movement (muscle egg
In vain, dynein and flagellin), matter transportation (hemoglobin and film transport protein), immune (antibody), heredity control it is (multiple
Device-archaeal dna polymerase and spiral-processed, which goes to stablize albumen, to be collectively formed) [6].
The concept of current gene therapy has larger extension, the method and principle of every use molecular biology, in core
The flat upper methods for the treatment of diseases carried out of sour water all can be described as gene therapy.DNA segment with hereditary information is referred to as gene.
Gene therapy has two kinds of approach, and one is ex vivo methods (in vitro method), is to cultivate recipient cell in vitro, is transferred to
Foreign gene, by appropriate selection system, the recipient cell of restructuring is fed back in patient's body, allows exogenous gene expression to change
Kind patient symptom；Two be in vivo methods (in vivo method), and this method does not need cell transplantation, exogenous DNA directly is injected into human body
It is interior, it is expressed in vivo and play therapeutic action (suction, oral, intramuscular injection, intravenous injection).In vivo methods compare ex
Vivo methods are simpler, direct and economy, and treatment is also relatively more definite, the conventional direct transfering means of internal gene have it is virus-mediated,
Liposome-mediated and gene direct injection etc..We are invention human placental transfer factor (injection, oral, external application) in 1977.HP-TF
The interior mixture containing nucleic acid (DNA, RNA) and gal4 amino acid (micromolecule polypeptide), nucleic acid is with it in some cases
The complex compound constituted with protein, form performance bioactivity.Its in vivo method (in vivo) gene therapy is acted on；To patient
Gene substitution (gene replacement), gene amendment (gene correction), genetic modification (gene
Augmentation), gene activation (gene activation), gene inactivation (gene inactivation), immunological regulation
(immune adjuetment) clinical practices, with a variety of diseases are cured, are the applications of in vivo method (in vivo) gene therapy
[22]。
Gene and protein modification：DNA, also known as DNA, are mainization of chromosome also referred to as " hereditary particle "
Study point.DNA is a kind of molecule, can constitute genetic command, and to guide biological development and vital functions to operate, its major function is
The information storage of chronicity, can liken to " blueprint " or " recipe ", the instruction included in it, be other intracellular chemical combination of construction
Thing is as necessary to protein and RNA.DNA fragmentation with hereditary information is referred to as gene.Other DNA sequence dnas, some are direct
Played a role with construction itself, some then participate in the expression of hereditary information.
DNA chemical component is just had determined that early in 1930s, it is referred to as nucleotides by 4 kinds
(nucleotide) base unit composition, wherein base has 4 kinds, is adenine (adenine), guanine respectively
(guanine), thymidine (thymine) and cytimidine (cytosine) .DNA molecules be by many nucleotides be formed by connecting with
The phosphate of another nucleotides links together, and forms sugar-phosphate skeleton, constitutes DNA backbone.
Ao Keya it is entitled " polynucleotides and amino acid code V of synthesis " and paper, with nitrous acid treatment tobacco mosaic disease
The RNA of poison can cause single base oxidation deoxidation, and this deoxidation can occur two kinds of changes, uracil is instead of born of the same parents phonetic
Pyridine, that is, cause C-U displacement；Guanine is set to instead of adenine, i.e. A-G displacement.Due to the base replacement on nucleotide chain,
Their nucleotide sequence is changed, so as to cause in this protein and peptide an amino acid quilt on some ad-hoc location
Another amino acid is replaced.
The characteristic of nucleic acid is entirely to be represented by its base sequence, and this sequence is exactly specified protein amino acid sequence
The password of row.That is the nucleotide sequence in nucleic acid defines the amino acid sequence in protein.
Nucleic acid body is the organelle of protein synthesis, performs the function of protein synthesis.With complete by hereditary information from
Transcriptions of the mRNA to polypeptide chain.
Peptide chain is then to be contracted to go a hydrone to generate with the amino of another amino acid by the carbonyl of an amino acid, with
The amino acid chain that this mode is combined is called polypeptide.Because peptide bond is covalent, so relative is stable (COOH, NH2).
HP-TF is proteomics application：Prlmary structure of protein is the basis of its space conformation, participates in functional activity portion
The change of the amino acid residue of position or the amino acid residue in specific conformation key position can cause the change of function, the mankind
There are many diseases all extremely relevant with protein, such as sickle-cell anemia (Sickle-cell anemin) basic reason is
Because genetic gene mutation causes amino acid residue in haemoglobin molecule to be replaced.Referred to as molecular disease (molecular
disease)。
HP-TF nucleic acid can be treated with active peptide：Autoimmunity disease, lupus erythematosus (are cured patient working healthily 32
Year), LES it is viral, B-mode with hepatitis C, Behcet syndromes.
The primary structure of amino acid is determined that amino acid is connected by the order of genetic code by peptide bond by gene；Ammonia
Base acid primary structure be space structure basis, primary structure determine two grades, three-level, quaternary structure.Treating a variety of diseases has
Effect, is the complementation and mutual assistance of gene and protein (active peptide), corrects the group of human body cell metabolism, signal transduction and regulated and control network
Structure function is knitted, makes pathological tissues/cell rapid apoptosis, recovers normal function.
The content of the invention
Technical problem：In view of the shortcomings of the prior art, first technical problem to be solved by this invention is to provide biology
The preparation method of active material, the bioactive substance be queen bee fetus transcription factor, placenta transcription factor or royal jelly transcription because
One kind in son.
The invention solves the problems that second technical problem there is provided queen bee fetus transcription factor extract solution, placenta transcription factor
The application of extract solution, royal jelly transcription factor extract solution in terms of skin care item.
The invention solves the problems that the 3rd technical problem be to provide a kind of skin cream.
The invention solves the problems that the 4th technical problem be to provide a kind of preparation method of skin cream.
Honeybee royal embryo factor, the royal jelly factor, the combination of placenta transcription factor compound, are the holy product of beauty.
The skin cream of the present invention is made up of a variety of compounds such as nucleic acid, polypeptide transcription factors, into human body, replaces modification disease
Become gene cell and meet the application that proteomics is integrated theory with practice.
Technical scheme：In order to solve the above-mentioned technical problem, the invention provides the preparation method of bioactive substance, including
Following steps：
1) pre-process：Collect detection HIV, HBV, HCV, syphilis and remove debris for negative queen bee fetus, placenta or royal jelly,
Put less than -18 DEG C refrigerations；
2) by step 1) pretreated queen bee fetus, placenta add normal saline dilution, and royal jelly dilutes with 95% ethanol,
Then homogenate is worn into high-speed homogenization or colloid respectively put less than -18 DEG C freeze 24~48 hours；
3) by step 2) obtain three kinds homogenate are extracted after freeze-thaw respectively, with 3000 revs/min of temperature control 1~4 DEG C, centrifuge 15 points
Clock, stays supernatant to be placed in less than -18 DEG C freezings in 24~48 hours, take freezing liquid to put 4-10 DEG C of thawing to obtain lysate, take dissolving
Liquid fine filtering, the filtrate obtained using the NF membrane ultrafiltration of doughnut molecular weight 3000 i.e. respectively extract by queen bee fetus transcription factor
Liquid, placenta transcription factor extract solution, royal jelly transcription factor extract solution.
Wherein, above-mentioned steps 2) in physiological saline mass concentration be 0.9%.
Present invention also includes queen bee fetus transcription factor extract solution, the placenta transcription that above-mentioned preparation method is prepared
The application of factor extract solution, royal jelly transcription factor extract solution in terms of skin care item.
A kind of skin cream, it contains the queen bee fetus transcription factor extract solution prepared, placenta transcription factor extract solution, honeybee
Royal jelly transcription factor extract solution.
Wherein, above-mentioned skin cream includes following components：
Aqueous phase：Glycerine, potassium hydroxide, sodium hydroxide, Methyl p-hydroxybenzoate, deionized water；
Oil phase：Stearic acid, glycerin monostearate, 1618 alcohol, propyl p-hydroxybenzoate, goose oil；
Additive：VE, queen bee fetus transcription factor extract solution, placenta transcription factor extract solution, royal jelly transcription factor are extracted
Liquid, royal jelly essence, nipagin ethylester, essence.
Glycerin monostearate：It is that auxiliary emulsifying agent has moisturizing effect, lotion is fine and smooth, lubricate, stably；Moisture is not after frost
Easily isolate.
1618 alcohol：Assistant for emulsifying agent has moisturizing, sticky, promotion (Ab is W/O) skin hydration effect.Assign skin emolliency and
Smoothly the sensation of (A25 is O/W), can avoid cream from thinning, thicker phenomenon occur during long-time is placed and is stored.
The oily skin-moisturizing, skin effect of glycerine, goose；
KOH：With bases neutralization generation stearate soap occurs for stearic acid.For emulsifying agent.
Placenta TF, royal jelly essence, queen bee fetus TF etc.：' antioxygen is turned into antiultraviolet repairing skin tissue's ' keep the skin wet nutrition '
With '.
Wherein, above-mentioned skin cream includes following components by weight percentage：Stearic acid 8~10%, glycerin monostearate
0.5~1%, 1618 alcohol 3~7%, propyl p-hydroxybenzoate 0.1~0.3%, goose oil 1~5%, glycerine 5-12%, potassium hydroxide 0.1-
0.6%th, sodium hydroxide 0.1-0.6%, Methyl p-hydroxybenzoate 0.1-0.6%, surplus are deionized water；Additive adds water phase by oil phase
The percentage of total amount adds VE0.1%, royal jelly transcription factor extract solution 5-15%, queen bee fetus transcription factor extract solution after emulsification
5~15%, placenta transcription factor extract solution 5~15%, royal jelly essence 5%, nipagin ethylester 0.1%, essence 0.5%.
Oil phase：Stearic acid, glycerin monostearate, 1618 alcohol, propyl p-hydroxybenzoate, goose oil；
Aqueous phase：Glycerine, potassium hydroxide, sodium hydroxide, Methyl p-hydroxybenzoate, deionized water；
Additive：VE, royal jelly extract solution, honeybee royal embryo factor extract solution, placental transfer factor extract solution, nipalgin second
Fat, royal jelly essence, essence.
Present invention also includes the preparation method of above-mentioned skin cream, comprises the following steps：
1) by aqueous phase, oil phase difference water-bath heating, 75-95 DEG C of aqueous phase, 70-85 DEG C of oil phase, indwelling 20 minutes after dissolving；
2) after oil phase filtering, stirring is added in aqueous phase, and stirring same direction is mixed, middling speed emulsification；
3) 65 DEG C of addition additives stir evenly into cream after emulsifying；
4) still aging 24 hours, packing.
Beneficial effect：Relative to prior art, the present invention has advantages below：
1) transcription factor skin cream of the invention is gene therapy：Skin cream is to extract nucleic acid from embryonic stem cell (ES)
(DNA RNA) active peptides, various active material；Skin disease can be cured：Lupus erythematosus LES, youth acne, solar lentigines skin
Inflammation, allergic dermatitis, chloasma, senile plaque expelling regression effect are notable.With activating cell metabolic function, regeneration speed is improved
Rate, dies the fast velocity modulation of sick cell, neoplastic skin is naturally pale.
2) transcription factor of the invention has bioactive substance：Placenta and fetus come from same embryonated egg, hereditary thing
Matter is identical (30.p62).Human plactnta is containing 312 kinds of transcription factors.To human disease treatment's advantage：Convenient material drawing, product safety, should
With (external application, oral, injection) effectively.
3) skin cream whitening of the invention, anti-ageing, smoothing wrinkle composition function introduction：What queen bee fetus, royal jelly, Human plactnta were extracted
Transcription factor, without allergy, is less than 3000 small molecule nanometer technologies.Skin-absorbable, go deep into epidermis, corium, hypodermis.Reason
Change index, bioactivity, virus of going out, bacterium (use：Wilfriedfranke (Germany) produces human blood DLE in enormous quantities and gone out poison side
The improvement of method) product safety, effectively.
Embodiment
Embodiment 1
1st, materials and methods
1.1 material：Queen bee fetus, royal jelly are purchased from Jinhu County Lao Shi bee farms.Placenta is purchased from Jinhu County's county hospital, Yangzhou hospital.
Other reagents are conventional reagent.
1.2 key instrument：Ultraviolet specrophotometer, high speed low temperature centrifugal machine, filter (doughnut), Biochemical autoanalysis
Instrument, purifying working room, clean work station, colloid mill, microscope, refrigerator-freezer, refrigerator, vacuum drying chamber, constant water bath box, batch are thrown
Product then another oil (gas) filling device.Produce increase cleaning equipment in enormous quantities：Air purifying room, equipment of De-ionized Water, manufacturing machine equipment.
1.3 queen bee fetus factor extraction methods：
1. pre-process：
2. handle qualified queen bee fetus addition 0.9% and take queen bee fetus plus 0.9% physiological saline, 1.5:1 (3kg queen bee fetus larvas
Plus 2kg0.9% sodium chloride freezing solution) mix, high-speed homogenization.Less than -18 DEG C are put to freeze 24 hours.
3. centrifuge：With 3000 revs/min of temperature control 1 DEG C, centrifuge 15 minutes, stay supernatant to be placed in less than -18 DEG C freezings in 24 hours；
4. take freezing liquid to put 5 DEG C of refrigerators thawings and obtain lysate, take lysate fine filtering (100 mesh)；
5. sodium is filtered：Using the NF membrane of doughnut molecular weight 3000, the filtrate that ultrafiltration is obtained is queen bee fetus transcription factor.
1.4 placenta transcription factor extracting methods：
1. pre-process：2. qualified placenta plus 0.9 sodium chloride solution, 1 are taken:1 (5kg placentas add 5kg refrigerate 5 DEG C of 0.9% chlorination
Sodium) high-speed homogenization, put less than -18 DEG C and freeze 48 hours.
3. freeze thawing is centrifuged, and 15min is centrifuged with 1 DEG C of 3000r/min of temperature-controlled centrifuge.
4. supernatant is taken, less than -18 DEG C freezings in 48 hours are put.
5. freeze thawing：Freezing liquid is taken to put 10 DEG C of thawings.
6. centrifuge：3000 revs/min of lysate temperature control 4 DEG C is taken to centrifuge 15 minutes.
7. nanofiltration：Using the NF membrane of doughnut molecular weight 3000, what ultrafiltration was obtained is placenta transcription factor.
1.5 royal jelly transcription factor extracting methods：
1. pre-process：
2. qualified fresh royal jelly freeze thawing plus 95% ethanol 1 are handled:1 (10kg royal jelly add -18 DEG C freezing 95% ethanol
10kg) colloid mill is homogenized, and is put -18 DEG C of refrigerator-freezers and is freezed 24 hours.
3. centrifuge：Extract and 3000 revs/min of royal jelly liquid temperature control 1 DEG C 15 minutes is taken after freeze-thaw, stay supernatant to be placed in less than -18 DEG C
Freeze within 36 hours.
5. freeze thawing：Freezing liquid is taken to put 4 DEG C of refrigerator-freezers 12 hours, fine filtering, reserved filtrate.
6. nanofiltration：With the filter membrane of doughnut molecular weight 3000, ultrafiltration obtains royal jelly transcription factor.
1.6 Quality Control：
1), outward appearance：Above-mentioned honeybee royal embryo factor, royal jelly transcription factor, placenta transcription factor solution for clarification white or
Slightly yellow liquid.
2), protein：20% sulfosalicylic acid is used, little cloudy or precipitation must not occur.
3), pH value：Potentiometry surveys shallow lake PH and should be 6.5-7.5
4), HBSAg, HCV, HIV standard reagent box are less than 2ng/ml (concentrated 10 times), and finished product sterilizes for 10 hours through 60 DEG C
Testing result should be negative.
5), ultraviolet absorption value LOD value is determined.UV-absorbance maximum UV absorption light E=between 250-260nm
7.8.A260/A280nm more than or equal to 1.8, content of peptides is more than 2mg/ per unit ml, Ribose concentration 80ug/ml.
6), chromatographic analysis visible obvious 2 peaks of glucan G25 (Sephaclex).260nm/280nm has maximum suction
Receipts value.
7) Atomic Absorption Spectrometry resultant metal element, trace element.
Placenta transcription factor trace element analysis result：
HP-TF (Human plactnta transcription factor) HS-TF (people's spleen transcription factor) SS-TF (pig spleen transcription factor) are taken to make micro member
Plain analysis result：
Table 1HP-TF, HS-TF and SS-TF trace element analysis
Note：1. instrument.U.S. J-A I 100, the road plasma emission spectrometer of vacuum type 63；
2. concentration unit PPM.1PPM=1ug/ml；
3.Na overlength (TF 0.9%NaCl dissolve).
8), biological property：Sterility test, asepsis growth pyrogen testing is answered by the detection of biological products code：Rabbit is tested,
Cavy hypodermic injection, it is no to suppurate, be still living and in good health without downright bad animal.
9) bioactivity denaturation is tested with renaturation：
1. protein structure renaturation, I and II structure：
The linear order that amino acid is interconnected to constitute with peptide bond is referred to as the primary structure of protein.
The denaturation mechanism of protein：The essence of protein denaturation be exactly protein molecule time key destruction cause two grades, three
Level, the change of quaternary structure, lose original bioactivity, often weaken or lose etc. property change in a mild condition,
The space conformation of protein is relaxation rather than chaotic, hydrogen bond fracture, and the accumulation force between base is destroyed.When change sexual factor
Protein can return to native conformation after releasing, and this denaturation claims reversible denaturation.
This experiment placenta transcription factor polypeptide, after alternating temperature, transcription factor small molecule peptide fragment reinstatement primary structure,
With bioactivity.
2. placenta transcription factor is denatured and renaturation experimental procedure：
A, 10ml mls/branch of finished product placenta transcription factor (HP-TF) is taken, (white glass ampulla, transparent liquid, containing polypeptide
12mg/ branch) take 4.
B, put water bath and heat 20-37 DEG C, gently shake in 10-30 minute that naked eyes occur visible obvious cotton-shaped.
C, 0.05-0.1 milliliters of draw solution are put on slide, with low power and high power sem observation.
D, visible obvious micromolecule polypeptide renaturation primary structure long-chain, form, uniform in size, (low power is regarded quantity 10-20 bars
It is wild) the visible catenation primary structure of high power lens.Without other forms and debris.
E, placenta transcription factor (HP-TF) are unwind：It is another to take with a collection of HP-TF finished products 10 that (10 milliliters every contain polypeptide 12
Milligram), 25-37 DEG C of water bath is put, gently shakes and occurs naked eyes visible cotton-shaped (i.e. primary structure is formed) in 10-30 minutes, after
It is continuous to put 25-37 DEG C of water bath, 10-30 minutes, occur from cotton-shaped, transparency liquid (temperature and ribosomes connecting peptides) is reverted to again
Prove that HP-TF has bioactivity.
3. protein structure renaturation one-level secondary structure advantage：
Advantage of the present invention：
A, the true primary structure of peptide chain can be observed under the microscope recover.
B, the present invention are available for batch detection to inspect by random samples, convenient material drawing.
C, technological operation are simply not required to another reagent adding.
One of quick, true and reliable bioactivity detection method of D, result.
4. E Rosette Test activity ratios are used, higher than control 30%；E-RFC is tested, and (E Rosette Tests) refers to that placenta drenches
Bar cell linkages sheep red blood cell (SRBC), raw percentage.Lymphocyte links sheep red blood cell (SRBC), increases placenta transcription factor, is sharp
Increase percentage after work, activity ratio is higher than virgin control group 30% (1 two batches of sample results of table).
The activation method of biological activity test-lymphocyte E receptor.The cord blood of anticoagulant heparin, plus separation of lymphocytes
Liquid, suctions out buffy coat, is washed with Hank liquid after 2 times, then be made into 5 × 10 with Hank liquid3Lymphocyte suspension.Take
0.1ml lymphocyte suspensions add 0.2mlHPTF, and mixing is put 37 DEG C of water-baths and rushed 30 minutes, is allowed to activate, and then adds 1% sheep red
Cell 0.2ml, by E-RFC test method(s)s, observation counts 200 lymphocytes and obtains Activated T-lymphocytes number, as a result as table 2 illustrates
HP-TF has bioactivity.
Lymphocyte E receptor activation method (table 2)
Table 2HP-TF (placenta transcription factor) is to the activity ratio of T cell E acceptors
10) laboratory and start batch production, free amino acid and peptide mapping can be surveyed, dissociate base analysis.HP-TF
Contents of Amino Acids：
HP-TF adds upper machine 0.4mg albumen/milliliter after hydrochloric acid hydrolysis to be examined through Hitdch835-50 automatic amino acid analyzers
Survey, as a result see (table 3).Per unit HP-TF total amino acid contents containing hydrolysis are that 2.1946mg is slightly higher compared with document report.Amino acid contains
Amount, 1 milliliter of detection sample introduction contains 1.0973mg/1ml, 2 milliliters of submitted sample content.Quantity unit one at a time, 2 milliliters=
2.194mg/2ml (0.4mg detecting instrument drying processes method)
Table 3HP-TF hydrolysising amino acid contents
8) the Human plactnta factor of the invention, queen bee fetus transcription factor, the content of peptides testing result of royal jelly transcription factor；
Table 4
Embodiment 2
Substantially the same manner as in Example 1, different is, the colloid mill that placenta transcription factor extracting method step 2 is used
Homogenate；Step 2 in royal jelly transcription factor extracting method) use place's high-speed homogenization step；
The Human plactnta factor, queen bee fetus transcription factor, the content of peptides testing result of royal jelly transcription factor of the present invention；
Table 5
Embodiment 3
Substantially the same manner as in Example 1, different is, the extraction side of queen bee fetus transcription factor and royal jelly transcription factor
Method step 3) in centrifugation temperature control be 4 DEG C 3000 revs/min,
The Human plactnta factor, queen bee fetus transcription factor, the content of peptides testing result of royal jelly transcription factor of the present invention；
Table 6
Influence transcription factor content factor：
A, raw material quality：Cell content is big, and concentration is high, and the big concentration of water content is low.
B, high-speed homogenization, freeze thawing, centrifugation, operating process：Ensure that clasmatosis is good, yield is high.Freeze thawing time, temperature control
It is required that accurate.
Below C, 2-20 DEG C of room temperature of operation, to prevent product loss of bioactivity.
D, ultrafiltration：Filter membrane quality, is maintained, and is cleaned, operation, voltage, and flow velocity regulation, the too fast concentration of flow velocity is relatively low.
The skin cream preparation technology of embodiment 4
Raw material is constituted：Stearic acid, glycerin monostearate, 1618 alcohol, glycerine, KOH, TF honeybee royal embryo factor, placenta peptide turn
Move the factor (nucleic acid, polypeptide), royal jelly essence, preservative, deionized water, essence, VE, goose oil.
Formula：
Additive by oil phase add water mutually emulsification after total amount percentage add:
The preparation method of skin cream：
1) by aqueous phase, oil phase difference water-bath heating, 95 DEG C of aqueous phase, 85 DEG C of oil phase, indwelling 20 minutes after dissolving；
2) after oil phase filtering, stirring is added in aqueous phase, and stirring same direction is mixed, middling speed emulsification；
3) 65 DEG C of addition additives stir evenly into cream after emulsifying；
4) still aging 24 hours, packing.
Embodiment 5
It is essentially the same with embodiment 4, except that, stearic acid 8%, glycerin monostearate 0.5%, 1618 alcohol 7%,
Propyl p-hydroxybenzoate 0.3%, goose oil 1%, glycerine 5%, potassium hydroxide 0.1%, sodium hydroxide 0.6%, Methyl p-hydroxybenzoate 0.1%,
Surplus is deionized water；Additive by oil phase add water mutually emulsification after total amount percentage add VE0.1%, royal jelly extract solution
15%th, honeybee royal embryo factor extract solution 15%, placental transfer factor extract solution 15%, royal jelly essence 5%, nipagin ethylester 0.1%, perfume
Essence 0.5%.In preparation process in the present embodiment, when aqueous phase, oil phase difference water-bath heating, 75 DEG C of aqueous phase, 70 DEG C of oil phase.
Embodiment 6
It is essentially the same with embodiment 4, except that, stearic acid 9%, glycerin monostearate 0.8%, 1618 alcohol 3%,
Propyl p-hydroxybenzoate 0.1%, goose oil 5%, glycerine 12%, potassium hydroxide 0.6%, sodium hydroxide 0.1%, Methyl p-hydroxybenzoate 0.6%,
Surplus is deionized water；Additive by oil phase add water mutually emulsification after total amount percentage add VE0.1%, royal jelly extract solution
10%th, honeybee royal embryo factor extract solution 10%, placental transfer factor extract solution 10%, royal jelly essence 5%, nipagin ethylester 0.1%, perfume
Essence 0.5%.In preparation process in the present embodiment, when aqueous phase, oil phase difference water-bath heating, 85 DEG C of aqueous phase, 80 DEG C of oil phase.
The application of the skin cream of embodiment 7 safely and effectively has no toxic side effect
Clinical practice：The skin cream of embodiment 4~6 is applied through clinical people more than 5000, daily 2-3 times, human body skin checking,
It works well.Observation of curative effect is cured and efficient：Youth acne 75%, solar dermatitis 67%, allergic dermatitis 87%, butterfly
Butterfly spot 58%, chloasma 52%, small wrinkle regression 62%, senile plaque expelling regression 50%, acne freckle regression 75%, dermal melanin
Disappear 50%, Pi Lin coarse 65%.
Claims (7)
1. the preparation method of bioactive substance, it is characterised in that comprise the following steps：
1）Pretreatment：Collect detection HIV, HBV, HCV, syphilis and remove debris for negative queen bee fetus, placenta or royal jelly, put -18
Refrigerated below DEG C；
2）By step 1）Pretreated queen bee fetus, placenta add normal saline dilution, and royal jelly is diluted with 95% ethanol, then
Worn into respectively with high-speed homogenization or colloid homogenate put less than -18 DEG C freeze 24 ~ 48 hours；
3）By step 2）Three kinds of obtained homogenate are extracted after freeze-thaw respectively, with 3000 revs/min of temperature control 1 ~ 4 DEG C, are centrifuged 15 minutes, are stayed
Supernatant is placed in less than -18 DEG C freezings in 24 ~ 48 hours, takes freezing liquid to put 4 ~ 10 DEG C of thawings to obtain lysate, take lysate fine filtering,
I.e. respectively queen bee fetus transcription factor extract solution, placenta turn the filtrate obtained using the NF membrane ultrafiltration of doughnut molecular weight 3000
Record factor extract solution, royal jelly transcription factor extract solution.
2. the preparation method of bioactive substance according to claim 1, it is characterised in that the step 2）In physiology
The mass concentration of salt solution is 0.9%.
3. the queen bee fetus transcription factor extract solution that the preparation method described in claim 1 or 2 is prepared, placenta transcription factor are carried
Take the application of liquid, royal jelly transcription factor extract solution in terms of skin care item.
4. a kind of skin cream, it is characterised in that it contains honeybee royal embryo factor extract solution, the placenta that claim 1 or 2 is prepared
Transcription factor extract solution, royal jelly transcription factor extract solution.
5. skin cream according to claim 4, it is characterised in that the skin cream includes following components：
Aqueous phase：Glycerine, potassium hydroxide, sodium hydroxide, Methyl p-hydroxybenzoate, deionized water；
Oil phase：Stearic acid, glycerin monostearate, 1618 alcohol, propyl p-hydroxybenzoate, goose oil；
Additive：VE, queen bee fetus transcription factor extract solution, placenta transcription factor extract solution, royal jelly transcription factor extract solution, king
Starch essence, nipagin ethylester, essence.
6. skin cream according to claim 5, it is characterised in that the skin cream includes with the following group by weight percentage
Point：It is stearic acid 8 ~ 10%, glycerin monostearate 0.5 ~ 1%, 1618 alcohol 3 ~ 7%, propyl p-hydroxybenzoate 0.1 ~ 0.3%, goose oil 1 ~ 5%, sweet
Oily 5-12%, potassium hydroxide 0.1-0.6%, sodium hydroxide 0.1-0.6%, Methyl p-hydroxybenzoate 0.1-0.6%, surplus are deionized water；
The additive by oil phase add water mutually emulsification after total amount percentage add VE0.1%, royal jelly transcription factor extract solution 5-15%,
Queen bee fetus transcription factor extract solution 5 ~ 15%, placenta transcription factor extract solution 5 ~ 15%, royal jelly essence 5%, nipagin ethylester 0.1%, perfume
Essence 0.5%.
7. the preparation method of the skin cream described in claim 6, it is characterised in that comprise the following steps：
1）By aqueous phase：Glycerine, potassium hydroxide, sodium hydroxide, Methyl p-hydroxybenzoate, deionized water；Oil phase：Stearic acid, monostearate
The other water-bath heating of glyceride, 1618 alcohol, propyl p-hydroxybenzoate, goose oil, 75-95 DEG C of aqueous phase, 70-85 DEG C of oil phase, indwelling after dissolving
20 minutes；
2）After oil phase filtering, stirring is added in aqueous phase, and stirring same direction is mixed, middling speed emulsification；
3）65 DEG C of addition additives after emulsification：VE, royal jelly transcription factor extract solution, queen bee fetus transcription factor extract solution, placenta
Transcription factor extract solution, royal jelly essence, nipagin ethylester, essence stir evenly into cream；
4）Still aging 24 hours, packing.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1145196A (en) * | 1995-01-24 | 1997-03-19 | 郭金刚 | Placenta peptide oral liquid and its prodn. method |
| CN1562086A (en) * | 2004-03-25 | 2005-01-12 | 夏介青 | Method for extracting bee essence from bee embryo |
| WO2009136291A2 (en) * | 2008-05-09 | 2009-11-12 | Regenics As | Cellular extracts |
| CN104940100A (en) * | 2015-06-19 | 2015-09-30 | 成都清科生物科技有限公司 | Human placenta extract, method for preparing the same and application |
| CN105456294A (en) * | 2014-09-05 | 2016-04-06 | 中国辐射防护研究院 | Preparing method of placenta peptide liquor for livestock and placenta peptide liquor for livestock |
-
2017
- 2017-06-01 CN CN201710402954.9A patent/CN107158038A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1145196A (en) * | 1995-01-24 | 1997-03-19 | 郭金刚 | Placenta peptide oral liquid and its prodn. method |
| CN1562086A (en) * | 2004-03-25 | 2005-01-12 | 夏介青 | Method for extracting bee essence from bee embryo |
| WO2009136291A2 (en) * | 2008-05-09 | 2009-11-12 | Regenics As | Cellular extracts |
| CN105456294A (en) * | 2014-09-05 | 2016-04-06 | 中国辐射防护研究院 | Preparing method of placenta peptide liquor for livestock and placenta peptide liquor for livestock |
| CN104940100A (en) * | 2015-06-19 | 2015-09-30 | 成都清科生物科技有限公司 | Human placenta extract, method for preparing the same and application |
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Application publication date: 20170915 |