CN107929321A - A kind of immunocyte gel preparation with long preservation period and preparation method - Google Patents
A kind of immunocyte gel preparation with long preservation period and preparation method Download PDFInfo
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- CN107929321A CN107929321A CN201711387344.2A CN201711387344A CN107929321A CN 107929321 A CN107929321 A CN 107929321A CN 201711387344 A CN201711387344 A CN 201711387344A CN 107929321 A CN107929321 A CN 107929321A
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- 238000002360 preparation method Methods 0.000 title claims abstract description 115
- 238000004321 preservation Methods 0.000 title claims abstract description 34
- 235000014066 European mistletoe Nutrition 0.000 claims abstract description 32
- 235000012300 Rhipsalis cassutha Nutrition 0.000 claims abstract description 32
- 241000221012 Viscum Species 0.000 claims abstract description 32
- 238000003756 stirring Methods 0.000 claims abstract description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 11
- 239000008367 deionised water Substances 0.000 claims abstract description 10
- 229910021641 deionized water Inorganic materials 0.000 claims abstract description 10
- ALSTYHKOOCGGFT-KTKRTIGZSA-N (9Z)-octadecen-1-ol Chemical compound CCCCCCCC\C=C/CCCCCCCCO ALSTYHKOOCGGFT-KTKRTIGZSA-N 0.000 claims abstract description 8
- HDSBZMRLPLPFLQ-UHFFFAOYSA-N Propylene glycol alginate Chemical compound OC1C(O)C(OC)OC(C(O)=O)C1OC1C(O)C(O)C(C)C(C(=O)OCC(C)O)O1 HDSBZMRLPLPFLQ-UHFFFAOYSA-N 0.000 claims abstract description 8
- 229940055577 oleyl alcohol Drugs 0.000 claims abstract description 8
- XMLQWXUVTXCDDL-UHFFFAOYSA-N oleyl alcohol Natural products CCCCCCC=CCCCCCCCCCCO XMLQWXUVTXCDDL-UHFFFAOYSA-N 0.000 claims abstract description 8
- 239000000770 propane-1,2-diol alginate Substances 0.000 claims abstract description 8
- 235000010409 propane-1,2-diol alginate Nutrition 0.000 claims abstract description 8
- 229920002307 Dextran Polymers 0.000 claims abstract description 7
- 238000000034 method Methods 0.000 claims abstract description 7
- 210000004698 lymphocyte Anatomy 0.000 claims description 18
- 239000006228 supernatant Substances 0.000 claims description 16
- 239000000843 powder Substances 0.000 claims description 15
- 238000012360 testing method Methods 0.000 claims description 14
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 12
- 239000012141 concentrate Substances 0.000 claims description 10
- 210000001744 T-lymphocyte Anatomy 0.000 claims description 8
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 claims description 7
- 229920002134 Carboxymethyl cellulose Polymers 0.000 claims description 7
- 229920002472 Starch Polymers 0.000 claims description 7
- 239000011575 calcium Substances 0.000 claims description 7
- 229910052791 calcium Inorganic materials 0.000 claims description 7
- 239000001768 carboxy methyl cellulose Substances 0.000 claims description 7
- 235000010948 carboxy methyl cellulose Nutrition 0.000 claims description 7
- 239000008112 carboxymethyl-cellulose Substances 0.000 claims description 7
- 238000000605 extraction Methods 0.000 claims description 7
- 239000008107 starch Substances 0.000 claims description 7
- 235000019698 starch Nutrition 0.000 claims description 7
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 claims description 7
- UREZNYTWGJKWBI-UHFFFAOYSA-M benzethonium chloride Chemical compound [Cl-].C1=CC(C(C)(C)CC(C)(C)C)=CC=C1OCCOCC[N+](C)(C)CC1=CC=CC=C1 UREZNYTWGJKWBI-UHFFFAOYSA-M 0.000 claims description 6
- 239000011159 matrix material Substances 0.000 claims description 6
- CPKVUHPKYQGHMW-UHFFFAOYSA-N 1-ethenylpyrrolidin-2-one;molecular iodine Chemical compound II.C=CN1CCCC1=O CPKVUHPKYQGHMW-UHFFFAOYSA-N 0.000 claims description 5
- 229920001479 Hydroxyethyl methyl cellulose Polymers 0.000 claims description 5
- 229920000153 Povidone-iodine Polymers 0.000 claims description 5
- 210000001185 bone marrow Anatomy 0.000 claims description 5
- 238000005119 centrifugation Methods 0.000 claims description 5
- 230000001186 cumulative effect Effects 0.000 claims description 5
- 210000004405 cytokine-induced killer cell Anatomy 0.000 claims description 5
- UYAAVKFHBMJOJZ-UHFFFAOYSA-N diimidazo[1,3-b:1',3'-e]pyrazine-5,10-dione Chemical compound O=C1C2=CN=CN2C(=O)C2=CN=CN12 UYAAVKFHBMJOJZ-UHFFFAOYSA-N 0.000 claims description 5
- 235000019441 ethanol Nutrition 0.000 claims description 5
- 125000005909 ethyl alcohol group Chemical group 0.000 claims description 5
- 239000003292 glue Substances 0.000 claims description 5
- 229960001621 povidone-iodine Drugs 0.000 claims description 5
- 229940116423 propylene glycol diacetate Drugs 0.000 claims description 5
- 238000002137 ultrasound extraction Methods 0.000 claims description 5
- 239000003814 drug Substances 0.000 abstract description 11
- 229940079593 drug Drugs 0.000 abstract description 10
- 241000147041 Guaiacum officinale Species 0.000 abstract description 5
- 229940091561 guaiac Drugs 0.000 abstract description 5
- 230000007774 longterm Effects 0.000 abstract description 5
- 230000000857 drug effect Effects 0.000 abstract description 2
- 239000000499 gel Substances 0.000 description 87
- 230000004083 survival effect Effects 0.000 description 18
- 210000004027 cell Anatomy 0.000 description 13
- 239000000203 mixture Substances 0.000 description 13
- 230000000694 effects Effects 0.000 description 11
- 238000002474 experimental method Methods 0.000 description 10
- 210000003491 skin Anatomy 0.000 description 10
- 239000000463 material Substances 0.000 description 6
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 3
- 239000011248 coating agent Substances 0.000 description 3
- 238000000576 coating method Methods 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- -1 hydroxyethyl methyl Chemical group 0.000 description 3
- 230000035699 permeability Effects 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
- 241000700159 Rattus Species 0.000 description 2
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 238000005260 corrosion Methods 0.000 description 2
- 230000018044 dehydration Effects 0.000 description 2
- 238000006297 dehydration reaction Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 230000029142 excretion Effects 0.000 description 2
- 230000001408 fungistatic effect Effects 0.000 description 2
- 230000028993 immune response Effects 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 239000008194 pharmaceutical composition Substances 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- ULWHHBHJGPPBCO-UHFFFAOYSA-N propane-1,1-diol Chemical class CCC(O)O ULWHHBHJGPPBCO-UHFFFAOYSA-N 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 239000002023 wood Substances 0.000 description 2
- NIXOWILDQLNWCW-UHFFFAOYSA-N Acrylic acid Chemical compound OC(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 229920002125 Sokalan® Polymers 0.000 description 1
- 210000001015 abdomen Anatomy 0.000 description 1
- 230000001133 acceleration Effects 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 235000010443 alginic acid Nutrition 0.000 description 1
- 229920000615 alginic acid Polymers 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229960001631 carbomer Drugs 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 210000004443 dendritic cell Anatomy 0.000 description 1
- 230000035617 depilation Effects 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 125000004494 ethyl ester group Chemical group 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 239000003349 gelling agent Substances 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- 210000003714 granulocyte Anatomy 0.000 description 1
- 210000003630 histaminocyte Anatomy 0.000 description 1
- 239000000017 hydrogel Substances 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 229910052740 iodine Inorganic materials 0.000 description 1
- 239000011630 iodine Substances 0.000 description 1
- 230000007794 irritation Effects 0.000 description 1
- 235000015110 jellies Nutrition 0.000 description 1
- 239000008274 jelly Substances 0.000 description 1
- 229940070765 laurate Drugs 0.000 description 1
- 238000002386 leaching Methods 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 210000001616 monocyte Anatomy 0.000 description 1
- 230000004899 motility Effects 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000000149 penetrating effect Effects 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
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- 210000000130 stem cell Anatomy 0.000 description 1
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- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
Classifications
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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
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/14—Blood; Artificial blood
- A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
- A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
- A61K47/14—Esters of carboxylic acids, e.g. fatty acid monoglycerides, medium-chain triglycerides, parabens or PEG fatty acid esters
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
- A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
- A61K47/38—Cellulose; Derivatives thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/46—Ingredients of undetermined constitution or reaction products thereof, e.g. skin, bone, milk, cotton fibre, eggshell, oxgall or plant extracts
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
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Abstract
The present invention provides a kind of immunocyte gel preparation with long preservation period and preparation method, the gel preparation to be made of immunocyte and gel-type vehicle, and the gel-type vehicle is mainly made of following component：Deionized water, dextran, mistletoe leaf extract, propylene glycol alginate, oleyl alcohol, guaiac gum；This method includes：Prepare mistletoe leaf extract；Mistletoe leaf extract is dissolved in deionized water, is stood overnight, adds propylene glycol alginate, oleyl alcohol and guaiac gum, stirring, adds dextran, stirring, configures gel-type vehicle, immunocyte will be added in gel-type vehicle, stirred evenly.The present invention can ensure cytoactive of the immunocyte during long-term preserve, meanwhile, the present invention can not only improve the stability of immunocyte gel preparation, ensure the preparation drug effect stored for a long time, and can improve drug release efficiency, prevent from going mouldy, highly practical.
Description
Technical field
The invention belongs to gel preparation technical field, more particularly to a kind of immunocyte gel preparation with long preservation period and
Preparation method.
Background technology
At present, the diagnoses and treatment for disease comes into molecule and cell epoch, and stem cell, immunocyte have very big
Treatment potentiality, immunocyte refers to participate in immune response or generally comprising leaching with the relevant cell of immune response, immunocyte
Bar cell, Dendritic Cells, Monocytes/Macrophages, granulocyte, mast cell etc..Immunocyte can be divided into it is a variety of, in human body
In various immunocytes serve as important role.At this stage, the application of immunocyte is more limited to, both at home and abroad there is not yet having
Immunocyte preparation.
Gel preparation is that a kind of novel pharmaceutical formulation gel preparation of domestic rising in recent years refers to medicine and can form gel
Auxiliary material the glop or semisolid preparation of solution, suspension or emulsion type is made, it is often general with matrix to prepare gel preparation
It is made of high molecular materials such as water, glycerine or propane diols and cellulose derivative, carbomer and alginates, this gelling agent is
Hydrogel, although it is easy to coating and removes, drug release is slow, and easily dehydration seriously affects gel system with going mouldy without greasy feeling
The quality of agent and various performances.
The content of the invention
In order to solve the above problem existing for existing gel preparation, have the present invention provides one kind and stablize well
Property, drug release is fast and the immunocyte gel preparation with long preservation period that is not easy dehydration and goes mouldy.
Concrete technical scheme of the present invention is as follows：
The present invention provides a kind of immunocyte gel preparation with long preservation period, the gel preparation by immunocyte and
Gel-type vehicle forms, and 0.3 × 10 is contained in gel-type vehicle described in per g11-1.2×1011A immunocyte, the gel-type vehicle master
To be made of the component of following parts by weight：
The present invention serves not only as gel-type vehicle by adding dextran and mistletoe leaf extract in gel preparation, and
And it provides nutritional ingredient for the survival of immunocyte, the survival rate of immunocyte ensure that, in addition, propylene glycol alginate,
The addition of oleyl alcohol and guaiac gum can improve the stability of gel-type vehicle, preserve immunocyte available for long-term, add at the same time
Propylene glycol alginate, oleyl alcohol and guaiac gum can also improve the drug release efficiency of gel preparation, prevent from going mouldy, it is highly practical.
Further, the immunocyte is selected from T lymphocytes, bone-marrow-derived lymphocyte, K lymphocytes, NK lymphocytes, CIK
One kind in cell.
Preferably, the extracting method of the mistletoe leaf extract is as follows：
Mistletoe leaf is cleaned, is freeze-dried, and by dry mistletoe leaf grind into powder；
The powder is taken to be added in test tube, addition 10ml absolute ethyl alcohols in powder described in per g, ultrasonic extraction 30~
60min, centrifugation, supernatant is transferred in another test tube；Extraction 2 times is repeated to residue, and the supernatant extracted every time is closed
And；
It is concentrated under reduced pressure to the supernatant obtained in step S2, obtains the concentrate that cumulative volume is less than 5mL；
The concentrate is fully dissolved with deionized water, and is settled to 50mL, that is, obtains the mistletoe leaf extract.
The mistletoe leaf extract prepared by parameter setting special in the above method and method can improve cell
Resistance, reduces loss of activity of the immunocyte during preservation, is that the preservation of cell improves stable control environment.
Further, the gel-type vehicle further includes the component of following parts by weight：
Starch phosphate sodium 0.5-1
Calcium carboxymethylcellulose 0.1-1.4
Hydroxyethylmethylcellulose 0.1-0.6.
The present invention in gel-type vehicle by adding starch phosphate sodium, calcium carboxymethylcellulose, hydroxyethyl methyl fiber
The drug release rate of gel-type vehicle can be significantly improved in element, Transdermal absorption performance is more preferable, can also improve immunocyte in gel
Dispersiveness in matrix, and the starch phosphate sodium of addition, calcium carboxymethylcellulose, hydroxyethylmethylcellulose will not be to exempting from
Epidemic disease cell produces interference and absorption, does not interfere with the activity of immunocyte.
Further, the gel-type vehicle further includes the component of following parts by weight：
Propylene glycol diacetate 0.2-1.2
Lauric isopropropanolamide 0.1-1.5.
The present invention can be carried significantly by the propylene glycol diacetate and lauric isopropropanolamide that are added in gel-type vehicle
The moisture retention of high gel-type vehicle, so that gel preparation is more comfortable with skin contact, ensure that the viscous of gel preparation and skin
Knot property, can significantly reduce irritation of the gel preparation to skin.
Further, the gel-type vehicle further includes the component of following parts by weight：
Phemerol chloride 0.1-0.5
Isopropanol 0.1-1
Povidone-iodine 0.1-0.8.
The blending constituent of the phemerol chloride, isopropanol and the povidone-iodine that are added in gel preparation provided by the invention can have
Effect plays the role of anti-corrosion, sterilization, provides the foundation for long-term preserve of immunocyte, effectively reduces the survival of immunocyte
Rate, ensure that cytoactive, reduce loss of activity.
Preferably, the gel-type vehicle is mainly made of the component of following parts by weight：
Above-mentioned gel-type vehicle provided by the invention is optimum substrate component, by the mixing of mentioned component, can not only be prolonged
The storage life of long immunocyte, and can ensure activity of the immunocyte during long-term preserve, while effectively have and protect
Wet, anti-corrosion effect, while it is fast to release the drug, it is highly practical.
Present invention also offers a kind of preparation method of immunocyte gel preparation with long preservation period, this method include with
Lower step：
S1：Prepare mistletoe leaf extract；
Mistletoe leaf extract, be dissolved in deionized water by S2, stands overnight, and adds propylene glycol alginate, oleyl alcohol and more
Wood glue is created, stirs 10min, 3500 turns/min of rotating speed, adds dextran, stirs 13min, 500 turns/min of rotating speed, configures gel
Matrix, to add 0.3 × 10 in every g gel-type vehicles11-1.2×1011A immunocyte, stirs evenly.
Preferably, in step S1, the preparation method of the mistletoe leaf extract is as follows：
S1：Mistletoe leaf is cleaned, is freeze-dried, and by dry mistletoe leaf grind into powder；
S2：The powder is taken to be added in test tube, addition 10ml absolute ethyl alcohols in powder described in per g, ultrasonic extraction 30~
60min, centrifugation, supernatant is transferred in another test tube；Extraction 2 times is repeated to residue, and the supernatant extracted every time is closed
And；
S3：It is concentrated under reduced pressure to the supernatant obtained in step S2, obtains the concentrate that cumulative volume is less than 5mL；
S4：The concentrate is fully dissolved with deionized water, and is settled to 50mL, that is, obtains the mistletoe leaf extraction
Thing.
Further, the immunocyte is selected from T lymphocytes, bone-marrow-derived lymphocyte, K lymphocytes, NK lymphocytes, CIK
One kind in cell.
Beneficial effects of the present invention are as follows：Added in immunocyte gel preparation with long preservation period provided by the invention
Dextran, mistletoe leaf extract can ensure cytoactive of the immunocyte during long-term preserve, in addition, the gel
The propylene glycol alginate, oleyl alcohol and the guaiac gum that are added in matrix can not only improve the stability of immunocyte gel preparation,
Ensure the preparation drug effect stored for a long time, and drug release efficiency can be improved, prevent from going mouldy, it is highly practical.
Embodiment
The present invention is described in further detail with reference to following embodiments.
Embodiment 1
A kind of immunocyte gel preparation with long preservation period, the gel preparation is by immunocyte and gel-type vehicle group
Into, per gel-type vehicle described in g in contain 0.3 × 1011A immunocyte, the gel-type vehicle is mainly by the component of following weight number
Composition：
Wherein, immunocyte is T lymphocytes.
Embodiment 2
A kind of immunocyte gel preparation with long preservation period, the gel preparation is by immunocyte and gel-type vehicle group
Into, per gel-type vehicle described in g in contain 0.8 × 1011A immunocyte, the gel-type vehicle is mainly by the component of following weight number
Composition：
Wherein, immunocyte is bone-marrow-derived lymphocyte.
The preparation method of the gel preparation comprises the following steps：
S1：Prepare mistletoe leaf extract；
Mistletoe leaf extract, be dissolved in deionized water by S2, stands overnight, and adds propylene glycol alginate, oleyl alcohol and more
Wood glue is created, stirs 10min, 3500 turns/min of rotating speed, adds dextran, stirs 13min, 500 turns/min of rotating speed, configures gel
Matrix, to add 0.8 × 10 in every g gel-type vehicles11A immunocyte, stirs evenly.
In step S1, the preparation method of the mistletoe leaf extract is as follows：
S1：Mistletoe leaf is cleaned, is freeze-dried, and by dry mistletoe leaf grind into powder；
S2：Take the powder to be added in test tube, 10ml absolute ethyl alcohols, ultrasonic extraction are added in powder described in per g
50min, centrifugation, supernatant is transferred in another test tube；Extraction 2 times is repeated to residue, and the supernatant extracted every time is closed
And；
S3：It is concentrated under reduced pressure to the supernatant obtained in step S2, obtains the concentrate that cumulative volume is less than 5mL；
S4：The concentrate is fully dissolved with deionized water, and is settled to 50mL, that is, obtains the mistletoe leaf extraction
Thing.
Embodiment 3
A kind of immunocyte gel preparation with long preservation period, the gel preparation is by immunocyte and gel-type vehicle group
Into, per gel-type vehicle described in g in contain 1.2 × 1011A immunocyte, the gel-type vehicle is mainly by the component of following weight number
Composition：
Wherein, immunocyte is K lymphocytes.
Embodiment 4
A kind of immunocyte gel preparation with long preservation period, the gel preparation is by immunocyte and gel-type vehicle group
Into, per gel-type vehicle described in g in contain 0.5 × 1011A immunocyte, the gel-type vehicle is mainly by the component of following weight number
Composition：
Wherein, immunocyte is NK lymphocytes.
Embodiment 5
A kind of immunocyte gel preparation with long preservation period, the gel preparation is by immunocyte and gel-type vehicle group
Into, per gel-type vehicle described in g in contain 0.3 × 1011A immunocyte, the gel-type vehicle is mainly by the component of following weight number
Composition：
Wherein, immunocyte is CIK cell.
Embodiment 6
A kind of immunocyte gel preparation with long preservation period, the gel preparation is by immunocyte and gel-type vehicle group
Into, per gel-type vehicle described in g in contain 1.2 × 1011A immunocyte, the gel-type vehicle is mainly by the component of following weight number
Composition：
Wherein, immunocyte is CIK cell.
Embodiment 7
A kind of immunocyte gel preparation with long preservation period, the gel preparation is by immunocyte and gel-type vehicle group
Into, per gel-type vehicle described in g in contain 0.6 × 1011A immunocyte, the gel-type vehicle is mainly by the component of following weight number
Composition：
Wherein, immunocyte is T lymphocytes.
Embodiment 8
A kind of immunocyte gel preparation with long preservation period, the gel preparation is by immunocyte and gel-type vehicle group
Into, per gel-type vehicle described in g in contain 1.2 × 1011A immunocyte, the gel-type vehicle is mainly by the component of following weight number
Composition：
Wherein, immunocyte is K lymphocytes.
Embodiment 9
A kind of immunocyte gel preparation with long preservation period, the gel preparation is by immunocyte and gel-type vehicle group
Into, per gel-type vehicle described in g in contain 0.3 × 1011A immunocyte, the gel-type vehicle is mainly by the component of following weight number
Composition：
Wherein, immunocyte is NK lymphocytes.
Reference examples 1
A kind of immunocyte gel preparation with long preservation period, the gel preparation is by immunocyte and gel-type vehicle group
Into, per gel-type vehicle described in g in contain 0.3 × 1011A immunocyte, the gel-type vehicle is mainly by the component of following weight number
Composition：
Wherein, immunocyte is T lymphocytes.
Reference examples 2
A kind of immunocyte gel preparation with long preservation period, the gel preparation is by immunocyte and gel-type vehicle group
Into, per gel-type vehicle described in g in contain 1.2 × 1011A immunocyte, the gel-type vehicle is mainly by the component of following weight number
Composition：
Wherein, immunocyte is K lymphocytes.
Reference examples 3
A kind of immunocyte gel preparation with long preservation period, the gel preparation is by immunocyte and gel-type vehicle group
Into, per gel-type vehicle described in g in contain 0.3 × 1011A immunocyte, the gel-type vehicle is mainly by the component of following weight number
Composition：
Wherein, immunocyte is CIK cell.
Reference examples 4
A kind of immunocyte gel preparation with long preservation period, the gel preparation is by immunocyte and gel-type vehicle group
Into, per gel-type vehicle described in g in contain 0.6 × 1011A immunocyte, the gel-type vehicle is mainly by the component of following weight number
Composition：
Wherein, immunocyte is T lymphocytes.
Experiment 1, immunocyte gel preparation freeze the detection of rear immunocyte vigor
Take the embodiment of the present invention 1,2,3,5,7,9 and the gel preparation of reference examples 1-4, be placed in liquid nitrogen, respectively jelly
Deposit 1 month, 2 months, 3 months, 6 months, 9 months and 12 months and taken out from liquid nitrogen, is placed in 37 DEG C of water-bath, recovery 1-
2min, carries out cell count with trypan blue staining, calculates excretion body survival rate, testing result is shown in Table 1.
The testing result of immunocyte motility rate in 1 each embodiment of table and reference examples gel preparation
From table 1 it follows that the embodiment of the present invention 1,3,5,7 is for check experiment 1-4, it is provided by the invention
Gel preparation property is stablized, and can preserve immunocyte for a long time, and after preserving 12 months, immunocyte survival rate exceedes
80.6%, in addition, embodiment 9 is most preferred embodiment provided by the invention, drawn by above-mentioned experiment, embodiment 9 provides solidifying
For glue preparation after preserving 12 months, the survival rate of immunocyte can still reach 97.8%；
In addition, the phemerol chloride, isopropanol and the povidone-iodine that are added in the gel preparation that embodiment 7 provides, can make to coagulate
For glue preparation after preserving 12 months, the survival rate of immunocyte has reached 94.1%, is significantly higher than embodiment 5, embodiment 3 and reality
Example 1 is applied, for this, it may be said that phemerol chloride, isopropanol and the povidone-iodine of bright addition can improve the survival rate of immunocyte, with
Reference examples 4 are compared, and can be derived that, these three materials lack any one, it can result in the survival rate of immunocyte and declines.
The propylene glycol diacetate and lauric isopropropanolamide added in the gel preparation that embodiment 5 provides can make to be immunized
The survival rate of cell is higher than embodiment 3 and embodiment 1, for this, it may be said that the propylene glycol diacetate and laurate isopropyl of bright addition
Alkylolamides can significantly improve the survival rate of immunocyte, can show that both materials, arbitrarily lack one with the contrast of reference examples 3
Kind, the survival rate of cell is reduced.
Starch phosphate sodium, calcium carboxymethylcellulose, the hydroxyethyl methyl added in the gel preparation that embodiment 3 provides is fine
Dimension element the survival rate of immunocyte can be made to be higher than embodiment 1, for this reason, increased starch phosphate sodium, calcium carboxymethylcellulose,
Hydroxyethylmethylcellulose can improve the survival rate of immunocyte, compared with reference examples 2, it can be deduced that, these three materials lack
Lack any one, survival rate reduces.
Compared with Example 2, gel preparation prepared by the preparation method provided in example 2 preserves thin embodiment 1
Born of the same parents' survival rate is higher than embodiment 1, for this, it may be said that and it is bright, it can be used in preserving immunocyte for a long time in the preparation method.
For embodiment 1 compared with reference examples 1, the survival rate of cell is significantly higher than reference examples 1, can illustrate for this, the present invention
The gel preparation of offer can be used for preserving immunocyte for a long time, the cell survival rate preserved from the gel preparation of reference examples 1
As can be seen that when each component in gel preparation is replaced, delete can influence cell viability that gel preparation freezes and
The time frozen.
Experiment 2, in vitro release
The release detection of pharmaceutical composition:With reference to 2010 editions versions《Chinese Pharmacopoeia》Annex XIXD vitro drug releases degree is examined
Look into.
Experimental method：Adult male rats are taken, are put to death, skin of abdomen is peeled off, is carrying out depilation processing, be placed in physiological saline
In it is spare, using Franz diffusion cells, effective infiltrating area is 0.8cm2, reception medium is physiological saline, and acceptance pool volume is
5mL。
Rat skin after processing is uniformly divided into 4 pieces of homalographic, is divided into 1 group of experiment, 2 groups of experiment, control 1-2 groups,
The immunocyte gel preparation of 1 group of the skin chunk coating embodiment of the present invention 1 is tested, the skin chunk that 2 groups of test example coats this hair
The gel preparation of reference examples 1-2 is respectively coated in the immunocyte gel preparation of bright embodiment 3, the skin chunk of control 1-2 groups；Every piece
Skin respectively coats 0.2g and repairs gel, and the skin chunk for coating gel preparation is placed in supply pool in 37 DEG C, 500r/min progress
Whole acceptable solutions are removed (while supplementing normal saline) by penetrating absorption respectively at 0.5,1,2,4,6,8,10,12h,
8000r/min centrifuges 10min, takes supernatant, and the number of excretion body, the accumulation of unit of account area are calculated using trypan blue staining
Transdermal penetration amount Qn, with QnMap to time t, and the straight line portion linear regression in curve, gained straight slope, that is, stable state are oozed
Saturating speed Js (a/cm2H), 2 be the results are shown in Table.
2 in vitro release result of the test (n=3) of table
From Table 2, it can be seen that the permeability for the gel preparation that embodiment 1 provides is far above reference examples 1, so of the invention
The component provided in embodiment 1, it is any to delete one kind, drug release effect well will not reached, permeability will reduce, in addition, with
Embodiment 3 is compared, and the starch phosphate sodium that is added in embodiment 3, calcium carboxymethylcellulose, hydroxyethylmethylcellulose can be shown
The percutaneous permeability for improving gel preparation is write, drug release rate is fast, and compared with reference examples 2, these three materials arbitrarily lack or replace
One kind, will not reach good effect.
Experiment 3, the stability test of immunocyte gel preparation provided by the invention
The gel preparation that above example 1,3,5 provides and the gel preparation that reference examples 1 and 3 provide are taken respectively, in temperature
At 25 DEG C ± 2 DEG C of degree, relative humidity is placed 6 months under conditions of being 10% ± 5%, 1 month during experiment, 2 months, 3
The moon, 6 the end of month are separately sampled once, detect the character, principal component content (labelled amount %), moisture of gel preparation, it turns out that,
The Contents of Main Components of embodiment 1,3,5 and reference examples 3 has no significant change, but the gel system that embodiment 1 and embodiment 3 provide
Agent is slightly shrivelled, and viscosity reduces, and the moisture in embodiment 1 and the gel preparation of the offer of embodiment 3 is less than embodiment 5；And compare
Main component significantly reduces in the gel preparation that example 1 and 3 provides, and preparation is shrivelled, inviscid, and gel preparation substantially deepens, explanation
Gel preparation provided by the invention has certain moistening effect, compared with reference examples 3, the propane diols two that is added in embodiment 5
Ethyl ester and lauric isopropropanolamide can have moistening effect, and both components arbitrarily lack one kind, and effect will reduce.
Experiment 4, immunocyte gel preparation safety evaluatio with long preservation period
Example 1,3,5,7 and the gel preparation of reference examples 1 and reference examples 4, are both placed in closed container, in temperature
For 40 DEG C ± 2 DEG C, relative humidity is placed 30 days under conditions of being 60% ± 10%, and the bacterium in acceleration detection gel preparation is total
Number.
3 gel preparation safety evaluatio of table
From above-mentioned 3 result of table, compared with reference examples 1, the gel preparation provided in the embodiment of the present invention 1 has necessarily
Fungistatic effect, compared with embodiment 1,3,5, the phemerol chloride that is added in the gel preparation that embodiment 7 provides, isopropanol and poly-
Dimension iodine ketone can effectively inhibit bacteria growth, ensure that the safety storage of gel preparation, embodiment 7, can compared with reference examples 4
Learn, these three components arbitrarily lack or replace one kind, will not reach good fungistatic effect.
The present invention is not limited to above-mentioned preferred forms, anyone can show that other are various under the enlightenment of the present invention
The product of form, however, make any change in its shape or structure, it is every that there is skill identical or similar to the present application
Art scheme, is within the scope of the present invention.
Claims (10)
1. a kind of immunocyte gel preparation with long preservation period, it is characterised in that the gel preparation is by immunocyte and coagulates
Gel matrix forms, and 0.3 × 10 is contained in gel-type vehicle described in per g11-1.2×1011A immunocyte, the gel-type vehicle are main
It is made of the component of following parts by weight：
2. immunocyte gel preparation with long preservation period as claimed in claim 1, it is characterised in that the immunocyte choosing
One kind from T lymphocytes, bone-marrow-derived lymphocyte, K lymphocytes, NK lymphocytes, CIK cell.
3. immunocyte gel preparation with long preservation period as claimed in claim 1, it is characterised in that the mistletoe leaf carries
Take the extracting method of thing as follows：
Mistletoe leaf is cleaned, is freeze-dried, and by dry mistletoe leaf grind into powder；The powder is taken to be added to
In test tube, 10ml absolute ethyl alcohols are added in powder described in per g, 30~60min of ultrasonic extraction, centrifugation, supernatant is transferred to separately
In one test tube；Extraction 2 times is repeated to residue, and the supernatant extracted every time is merged；
It is concentrated under reduced pressure to the supernatant obtained in step S2, obtains the concentrate that cumulative volume is less than 5mL；
The concentrate is fully dissolved with deionized water, and is settled to 50mL, that is, obtains the mistletoe leaf extract.
4. immunocyte gel preparation with long preservation period as claimed in claim 1, it is characterised in that the gel-type vehicle is also
Include the component of following parts by weight：
Starch phosphate sodium 0.5-1
Calcium carboxymethylcellulose 0.1-1.4
Hydroxyethylmethylcellulose 0.1-0.6.
5. immunocyte gel preparation with long preservation period as claimed in claim 4, it is characterised in that the gel-type vehicle is also
Include the component of following parts by weight：
Propylene glycol diacetate 0.2-1.2
Lauric isopropropanolamide 0.1-1.5.
6. immunocyte gel preparation with long preservation period as claimed in claim 5, it is characterised in that the gel-type vehicle is also
Include the component of following parts by weight：
Phemerol chloride 0.1-0.5
Isopropanol 0.1-1
Povidone-iodine 0.1-0.8.
7. immunocyte gel preparation with long preservation period as claimed in claim 6, it is characterised in that the gel-type vehicle master
To be made of the component of following parts by weight：
8. a kind of preparation method of immunocyte gel preparation with long preservation period, it is characterised in that this method includes following step
Suddenly：
S1：Prepare mistletoe leaf extract；
Mistletoe leaf extract, be dissolved in deionized water by S2, stands overnight, and adds propylene glycol alginate, oleyl alcohol and guaiaci lignum
Glue, stirs 10min, 3500 turns/min of rotating speed, adds dextran, stirs 13min, 500 turns/min of rotating speed, configures gel base
Matter, to add 0.3 × 10 in every g gel-type vehicles11-1.2×1011A immunocyte, stirs evenly.
9. the preparation method of immunocyte gel preparation with long preservation period as claimed in claim 8, it is characterised in that step
In S1, the preparation method of the mistletoe leaf extract is as follows：
S1：Mistletoe leaf is cleaned, is freeze-dried, and by dry mistletoe leaf grind into powder；
S2：The powder is taken to be added in test tube, addition 10ml absolute ethyl alcohols in powder described in per g, ultrasonic extraction 30~
60min, centrifugation, supernatant is transferred in another test tube；Extraction 2 times is repeated to residue, and the supernatant extracted every time is closed
And；
S3：It is concentrated under reduced pressure to the supernatant obtained in step S2, obtains the concentrate that cumulative volume is less than 5mL；
S4：The concentrate is fully dissolved with deionized water, and is settled to 50mL, that is, obtains the mistletoe leaf extract.
10. the preparation method of immunocyte gel preparation with long preservation period as claimed in claim 8, it is characterised in that institute
State the one kind of immunocyte in T lymphocytes, bone-marrow-derived lymphocyte, K lymphocytes, NK lymphocytes, CIK cell.
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| CN114667997A (en) * | 2022-04-14 | 2022-06-28 | 杭州百凌生物科技有限公司 | Buffer solution for immunohistochemical detection of quality control product and preparation method and application thereof |
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| CN107929321B (en) | 2021-04-02 |
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Application publication date: 20180420 Assignee: Tang Yi Huikang (Shenzhen) Biomedical Technology Co.,Ltd. Assignor: Beijing tangyihuikang Biomedical Technology Co.,Ltd. Contract record no.: X2024980006121 Denomination of invention: A long-term preserved immune cell gel preparation and its preparation method Granted publication date: 20210402 License type: Common License Record date: 20240523 |