CN105852125A - 芦荟干细胞冻干粉及其制备方法和应用 - Google Patents
芦荟干细胞冻干粉及其制备方法和应用 Download PDFInfo
- Publication number
- CN105852125A CN105852125A CN201610268493.6A CN201610268493A CN105852125A CN 105852125 A CN105852125 A CN 105852125A CN 201610268493 A CN201610268493 A CN 201610268493A CN 105852125 A CN105852125 A CN 105852125A
- Authority
- CN
- China
- Prior art keywords
- stem cell
- aloe
- aloe stem
- lyophilized powder
- preparation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 241001116389 Aloe Species 0.000 title claims abstract description 122
- 235000011399 aloe vera Nutrition 0.000 title claims abstract description 122
- 210000000130 stem cell Anatomy 0.000 title claims abstract description 114
- 239000008176 lyophilized powder Substances 0.000 title claims abstract description 43
- 238000002360 preparation method Methods 0.000 title claims abstract description 30
- 241000196324 Embryophyta Species 0.000 claims abstract description 23
- 239000007787 solid Substances 0.000 claims abstract description 18
- 238000000034 method Methods 0.000 claims abstract description 16
- 239000006143 cell culture medium Substances 0.000 claims abstract description 12
- 238000000338 in vitro Methods 0.000 claims abstract description 9
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 claims abstract description 7
- 229930195725 Mannitol Natural products 0.000 claims abstract description 7
- 238000007710 freezing Methods 0.000 claims abstract description 7
- 230000008014 freezing Effects 0.000 claims abstract description 7
- 239000000594 mannitol Substances 0.000 claims abstract description 7
- 235000010355 mannitol Nutrition 0.000 claims abstract description 7
- 239000002609 medium Substances 0.000 claims description 17
- 230000002520 cambial effect Effects 0.000 claims description 14
- 210000004027 cell Anatomy 0.000 claims description 12
- 229930192334 Auxin Natural products 0.000 claims description 9
- 230000003321 amplification Effects 0.000 claims description 9
- 239000002363 auxin Substances 0.000 claims description 9
- 238000004108 freeze drying Methods 0.000 claims description 9
- SEOVTRFCIGRIMH-UHFFFAOYSA-N indole-3-acetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CNC2=C1 SEOVTRFCIGRIMH-UHFFFAOYSA-N 0.000 claims description 9
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 9
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 8
- 150000001720 carbohydrates Chemical class 0.000 claims description 8
- 229910017053 inorganic salt Inorganic materials 0.000 claims description 8
- 235000018102 proteins Nutrition 0.000 claims description 8
- 108090000623 proteins and genes Proteins 0.000 claims description 8
- 102000004169 proteins and genes Human genes 0.000 claims description 8
- 238000005286 illumination Methods 0.000 claims description 7
- 235000001014 amino acid Nutrition 0.000 claims description 6
- 150000001413 amino acids Chemical class 0.000 claims description 6
- 229930003231 vitamin Natural products 0.000 claims description 6
- 235000013343 vitamin Nutrition 0.000 claims description 6
- 239000011782 vitamin Substances 0.000 claims description 6
- 229940088594 vitamin Drugs 0.000 claims description 6
- 150000003722 vitamin derivatives Chemical class 0.000 claims description 6
- 229920001817 Agar Polymers 0.000 claims description 5
- 239000008272 agar Substances 0.000 claims description 5
- 239000012141 concentrate Substances 0.000 claims description 4
- 239000002537 cosmetic Substances 0.000 claims description 4
- 235000013305 food Nutrition 0.000 claims description 4
- 238000010257 thawing Methods 0.000 claims description 4
- 239000003814 drug Substances 0.000 claims description 3
- 238000012546 transfer Methods 0.000 claims description 2
- 239000001963 growth medium Substances 0.000 abstract description 8
- 238000000605 extraction Methods 0.000 abstract description 4
- 230000015572 biosynthetic process Effects 0.000 abstract 2
- 238000012258 culturing Methods 0.000 abstract 2
- 241000894006 Bacteria Species 0.000 abstract 1
- AFHJQYHRLPMKHU-XXWVOBANSA-N Aloin Natural products O=C1c2c(O)cc(CO)cc2[C@H]([C@H]2[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O2)c2c1c(O)ccc2 AFHJQYHRLPMKHU-XXWVOBANSA-N 0.000 description 6
- 229930006000 Sucrose Natural products 0.000 description 6
- AFHJQYHRLPMKHU-OSYMLPPYSA-N aloin A Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1[C@@H]1C2=CC(CO)=CC(O)=C2C(=O)C2=C(O)C=CC=C21 AFHJQYHRLPMKHU-OSYMLPPYSA-N 0.000 description 6
- AFHJQYHRLPMKHU-UHFFFAOYSA-N isobarbaloin Natural products OC1C(O)C(O)C(CO)OC1C1C2=CC(CO)=CC(O)=C2C(=O)C2=C(O)C=CC=C21 AFHJQYHRLPMKHU-UHFFFAOYSA-N 0.000 description 6
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 description 6
- 239000005720 sucrose Substances 0.000 description 6
- 239000006285 cell suspension Substances 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 4
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 4
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 4
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 4
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 4
- 239000008101 lactose Substances 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 230000001954 sterilising effect Effects 0.000 description 4
- PRPINYUDVPFIRX-UHFFFAOYSA-N 1-naphthaleneacetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CC=CC2=C1 PRPINYUDVPFIRX-UHFFFAOYSA-N 0.000 description 3
- AFWYNOWVYKOMGZ-UHFFFAOYSA-N 8-benzyl-7h-purine-2,6-diamine Chemical compound N=1C2=NC(N)=NC(N)=C2NC=1CC1=CC=CC=C1 AFWYNOWVYKOMGZ-UHFFFAOYSA-N 0.000 description 3
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 3
- 239000001110 calcium chloride Substances 0.000 description 3
- 229910001628 calcium chloride Inorganic materials 0.000 description 3
- 239000012930 cell culture fluid Substances 0.000 description 3
- 229960002523 mercuric chloride Drugs 0.000 description 3
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 description 3
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 description 2
- 239000004471 Glycine Substances 0.000 description 2
- 239000007836 KH2PO4 Substances 0.000 description 2
- 108010059820 Polygalacturonase Proteins 0.000 description 2
- 229930003268 Vitamin C Natural products 0.000 description 2
- 206010052428 Wound Diseases 0.000 description 2
- 208000027418 Wounds and injury Diseases 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 229910052927 chalcanthite Inorganic materials 0.000 description 2
- 239000000645 desinfectant Substances 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 108010093305 exopolygalacturonase Proteins 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 238000003306 harvesting Methods 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 2
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 2
- 230000008929 regeneration Effects 0.000 description 2
- 238000011069 regeneration method Methods 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 125000000185 sucrose group Chemical group 0.000 description 2
- 235000019154 vitamin C Nutrition 0.000 description 2
- 239000011718 vitamin C Substances 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 241001289435 Astragalus brachycalyx Species 0.000 description 1
- 108010059892 Cellulase Proteins 0.000 description 1
- 229920002558 Curdlan Polymers 0.000 description 1
- 239000001879 Curdlan Substances 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 235000002917 Fraxinus ornus Nutrition 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 241000234280 Liliaceae Species 0.000 description 1
- 235000014676 Phragmites communis Nutrition 0.000 description 1
- 229920001231 Polysaccharide peptide Polymers 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- CPUHNROBVJNNPW-UHFFFAOYSA-N aloin A Natural products OC1C(O)C(O)C(CO)OC1OC1C2=CC(CO)=CC(O)=C2C(=O)C2=C(O)C=CC=C21 CPUHNROBVJNNPW-UHFFFAOYSA-N 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 238000012832 cell culture technique Methods 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 229940106157 cellulase Drugs 0.000 description 1
- 230000006835 compression Effects 0.000 description 1
- 238000007906 compression Methods 0.000 description 1
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 1
- 229910000366 copper(II) sulfate Inorganic materials 0.000 description 1
- 238000000315 cryotherapy Methods 0.000 description 1
- 229940078035 curdlan Drugs 0.000 description 1
- 235000019316 curdlan Nutrition 0.000 description 1
- 230000000249 desinfective effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 238000002481 ethanol extraction Methods 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 239000003337 fertilizer Substances 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 230000035876 healing Effects 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 230000002519 immonomodulatory effect Effects 0.000 description 1
- 239000007943 implant Substances 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000001788 irregular Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
- 150000008442 polyphenolic compounds Chemical class 0.000 description 1
- 235000013824 polyphenols Nutrition 0.000 description 1
- 108010022457 polysaccharide peptide Proteins 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 229910001220 stainless steel Inorganic materials 0.000 description 1
- 239000010935 stainless steel Substances 0.000 description 1
- 238000013517 stratification Methods 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 230000029663 wound healing Effects 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/88—Liliopsida (monocotyledons)
- A61K36/886—Aloeaceae (Aloe family), e.g. aloe vera
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/88—Liliopsida (monocotyledons)
- A61K36/896—Liliaceae (Lily family), e.g. daylily, plantain lily, Hyacinth or narcissus
-
- 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
- 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
- A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
-
- 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
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/10—Preparation or pretreatment of starting material
- A61K2236/11—Preparation or pretreatment of starting material involving culturing conditions, e.g. cultivation in the dark or under defined water stress
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/10—Preparation or pretreatment of starting material
- A61K2236/17—Preparation or pretreatment of starting material involving drying, e.g. sun-drying or wilting
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/10—General cosmetic use
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Pharmacology & Pharmacy (AREA)
- Medicinal Chemistry (AREA)
- Mycology (AREA)
- Biotechnology (AREA)
- Botany (AREA)
- Microbiology (AREA)
- Alternative & Traditional Medicine (AREA)
- Medical Informatics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Birds (AREA)
- Dermatology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Cosmetics (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
本发明涉及一种芦荟干细胞冻干粉的制备方法,包括以下步骤:从芦荟中获取含有形成层的组织;将所述含有形成层的组织置于固体培养基中培养后,分离出芦荟干细胞;再将所述芦荟干细胞置于植物干细胞培养基中进行体外培养,获得扩增后的芦荟干细胞;所述扩增后的芦荟干细胞经冷冻破碎后浓缩,收获芦荟干细胞提取物;将所述芦荟干细胞提取物与甘露醇混合后,除菌、冻干后,收获芦荟干细胞冻干粉。通过从芦荟茎尖中提取芦荟干细胞,对芦荟干细胞进行放大培养后,提取芦荟干细胞提取物,之后进行冻干处理获得芦荟干细胞,通过该方法制备芦荟干细胞冻干粉周期较短,产率较高,冻干方法简单易行,提取效率较高,具有较高的使用价值。
Description
技术领域
本发明涉及生物技术领域,特别涉及一种芦荟干细胞冻干粉及其制备方法和应用。
背景技术
植物干细胞(plant stem cell)是未分化的细胞,具有很强的自我更新和持续分裂能力。植物干细胞培养技术,是在传统组培技术的基础上,以植物干细胞为目标,诱导、分离和培养外植体,建立相应的干细胞培养体系植物干细胞培养技术,不仅丰富了植物培养技术,而且为天然产物的商业化生产,乃至植物生物技术的发展,提供了新的研究方向和契机。
芦荟是一种多年生的肉质草本植物,属百合科芦荟属,传统上作为泻药使用,近年来,也在化妆品和食品行业中得到了广泛的应用。随着研究的深入,芦荟的抗氧化能力、抑菌、消炎、抗肿瘤、免疫调节和辅助治疗糖尿病的作用也相继被报道出来。芦荟中的芦荟素A、创伤激素和聚糖肽甘露等具有抗病毒感染、促进伤口愈合复原的作用,又可以消炎杀菌、吸热消肿、软化皮肤、保持细胞活力,凝胶多糖与愈伤酸联合还具有愈合创伤活性的功效。因此,提供一种能够快速制备活性成分含量较高且适合医用的芦荟干细胞制品十分必要。
发明内容
有鉴于此,本发明要解决的技术问题在于提供一种芦荟干细胞冻干粉及其制备方法。
本发明解决其技术问题所采用的技术方案是:一种芦荟干细胞冻干粉的制备方法,包括以下步骤:
从芦荟中获取含有形成层的组织;
将所述含有形成层的组织置于固体培养基中培养后,分离出芦荟干细胞;
再将所述芦荟干细胞置于植物干细胞培养基中进行体外培养,获得扩增后的芦荟干细胞;
所述扩增后的芦荟干细胞经冷冻破碎后浓缩,收获芦荟干细胞提取物;
将所述芦荟干细胞提取物与甘露醇混合后,除菌、冻干后,收获芦荟干细胞冻干粉。
在本发明提供的芦荟干细胞冻干粉的制备方法中,所述冻干步骤之前,
还需调整所述芦荟干细胞提取物中的蛋白质浓度至30~70μg/ml。
在本发明提供的芦荟干细胞冻干粉的制备方法中,所述冻干条件为:温度-20℃~-35℃,真空度50~200Pa,冻干时间为36~48小时。
在本发明提供的芦荟干细胞冻干粉的制备方法中,所述冷冻破碎过程为:将所述扩增后的芦荟干细胞放入-50~-100℃低温环境下冷冻2~3小时,然后取出解冻,重复3~5次。
在本发明提供的芦荟干细胞冻干粉的制备方法中,所述植物干细胞培养基包括无机盐、维生素、氨基酸、生长素、糖类和琼脂。
在本发明提供的芦荟干细胞冻干粉的制备方法中,所述固体培养基包括无机盐、维生素、氨基酸、生长素、糖类和活性炭。
在本发明提供的芦荟干细胞冻干粉的制备方法中,所述体外培养过程是在恒温培养摇床中培养7~14天后,再转移至生物反应器中进行大规模培养。
在本发明提供的芦荟干细胞冻干粉的制备方法中,所述体外培养的条件为温度为30℃~37℃,每小时光照5~10min。
本发明进一步保护上述芦荟干细胞冻干粉的制备方法所获得的芦荟干细胞冻干粉。
本发明进一步保护上述芦荟干细胞冻干粉在制备食品、药品、保健品或化妆品中的应用。
实施本发明提供的芦荟干细胞提取物及其的制备方法,可以达到以下有益效果:通过从芦荟茎尖中提取芦荟干细胞,对芦荟干细胞进行放大培养后,提取芦荟干细胞提取物,之后进行冻干处理获得芦荟干细胞,通过该方法制备芦荟干细胞冻干粉周期较短,产率较高,冻干方法简单易行,提取效率较高,且冻干粉中所含有的芦荟活性成分较高,特别是芦荟苷,具有较高的医学使用价值。
具体实施方式
本发明提供的芦荟干细胞冻干粉的制备方法,包括以下步骤:
S1、从芦荟中获取含有形成层的组织;
S2、将步骤S1中获得的含有形成层的组织置于固体培养基中进行培养后,分离出芦荟干细胞;
S3、将步骤S2中获得的芦荟干细胞置于植物干细胞培养基中进行体外培养,获得扩增后的芦荟干细胞;
其中,所述植物干细胞培养基包括无机盐、维生素、氨基酸、生长素、糖类和琼脂。其中,无机盐包括:CaCl2、KH2PO4、KNO3、MgSO4、NH4NO3、CuSO4·5H2O等;糖类为蔗糖或乳糖等;优选地,植物干细胞培养基中,生长素的浓度为0.5~1.5mg/L、蔗糖或乳糖的质量分数为2~5%,琼脂的浓度为5.5~7.5g/L;植物干细胞培养基为MS培养基。
S4、步骤S3获得的扩增后芦荟干细胞经冷冻破碎后浓缩,收获芦荟干细胞提取物;
S5、将步骤S4中获得的芦荟干细胞提取物与甘露醇混合后,除菌,冻干,即获得芦荟干细胞冻干粉。
步骤S1中,由于芦荟茎尖中含有大量的具有自我更新能力又能产生具有持续分裂能力的干细胞,因此,本发明中优先采用从芦荟茎尖中获取芦荟干细胞。含有形成层的组织的获取过程,包括以下步骤:
取芦荟茎尖,冲洗15~20分钟,酒精消毒,再用升汞液消毒10~20分钟后,无菌水冲洗数次,剥离获取形成层的组织。
步骤S2中,固体培养基包括无机盐、维生素、氨基酸、生长素、糖类和活性炭;其中,无机盐包括:CaCl2、KH2PO4、KNO3、MgSO4、NH4NO3、CuSO4·5H2O等;糖类为蔗糖或乳糖等。优选地,固体培养基为MS培养基;且生长素的浓度为0.5~1.5mg/L、蔗糖或乳糖的质量分数为2~5%;更为优选地,固体培养基中还包括萘乙酸和苄氨基腺嘌呤,萘乙酸的浓度为0.5~1.5mg/L,苄氨基腺嘌呤的浓度为0.5~1.5mg/L。
进一步地,该步骤中,将含有形成层的组织置于固体培养基中进行培养的条件为:培养温度为23~29℃,光照10~12小时,光照强度1500~2000lx,PH值为5.6~5.9,培养7~14天。
进一步地,含有形成层的组织经固体培养基培养培养后获得生长良好的形成层干细胞团,还需要采用酶解法分离出芦荟干细胞。本发明优先采用质量分数为0.2-0.3%纤维素酶溶液和质量分数为0.2-0.3%果胶酶溶液对形成层干细胞团进行酶解8~12小时。
步骤S3中,体外培养芦荟干细胞的过程是在恒温培养摇床中进行的,摇床转速为100转/分钟~120转/分钟,温度为30℃~37℃,每小时光照5~10min,培养时间为7天~14天后,得到单细胞悬液。然后将单细胞悬液转移至生物反应器进行大规模培养,在生物反应器中培养第4~7天时,加入新鲜的植物干细胞培养基,继续培养后收获扩增后的芦荟干细胞。
步骤S4中,将步骤S3中获得的扩增后的芦荟干细胞放入-50~-100℃低温环境下冷冻2~3小时,然后取出解冻,重复3~5次,最后一次解冻后用超声波破碎仪破碎细胞,收集滤液,真空蒸发浓度,即获得芦荟干细胞提取物。
步骤S5中,在对芦荟干细胞提取物进行冻干之前,还需要调整芦荟干细胞提取物中的蛋白质浓度,使得蛋白质浓度为30~70μg/ml,降低蛋白质浓度。
进一步地,甘露醇在芦荟干细胞提取物中的质量分数为4~6%,芦荟干细胞提取物的冻干条件为:温度-20℃~-35℃,真空度50~200Pa,冻干时间为36~48小时。
本发明进一步保护上述方法所获得的芦荟干细胞冻干粉在制备食品、药品、保健品或化妆品中的应用。
为了使本发明的目的、技术方案及优点更加清楚明白,以下结合实施例,对本发明进行进一步详细说明。应当理解,此处所描述的具体实施例仅用以解释本发明,并不用于限定本发明。
实施例1
本实施例中,芦荟干细胞冻干粉的制备方法,包括以下步骤:
S1、从芦荟中获取含有形成层的组织;
选择无化肥农药污染、绿色有机种植的芦荟,并选取生长状况较佳的芦荟茎尖,用无菌水冲洗芦荟茎尖表面附着物15~20分钟,再用75%的酒精进行表面消毒30秒,然后,用0.1%的升汞液消毒10~20分钟,最后用无菌水反复冲洗5次,洗去植物表面的升汞残液;将灭菌后的芦荟茎尖切成5mm×5mm大小,用接种刀剥离出含有形成层的组织。
S2、将含有形成层的组织置于固体培养基中进行培养,分离出芦荟干细胞;
将步骤S1中剥离出的含有形成层的组织接种至固体培养基中,以每1g组织接种5cm2固体培养基的密度进行接种,培养后,形成层组织为均一生长的平板状组织,而其他组织则为不规则的聚集生长物;将再生的形成层组织与其他组织分离开,以每1g组织接种5cm2固体培养基的密度,将再生的形成层组织接种至新鲜的固体培养基中进行培养。其中,固体培养基中还含有浓度为1.0mg/L的萘乙酸和浓度为1.0mg/L的苄氨基腺嘌呤,且固体培养基为MS培养基,含有浓度为232mg/L的CaCl2、140mg/L的KH2PO4、1642mg/L的KNO3、130.54mg/L的MgSO4、1560mg/L的NH4NO3、0.021mg/L的CuSO4·5H2O、60mg/L的维生素C、、2mg/L的甘氨酸、1.0mg/L的生长素、质量分数为3%的蔗糖和活性炭;培养条件为:温度26℃,光照11小时,光照强度为1800lx,PH值为5.6~5.9,培养14天;即可收获形成层组织来源的芦荟干细胞团;用质量分数为0.25%的果胶酶溶液和质量分数为0.25%的纤维素酶对芦荟干细胞团进行酶解12小时,分离出芦荟干细胞。
S3、将芦荟干细胞置于植物干细胞培养基中进行体外培养;
将步骤S2中酶解后的芦荟干细胞转移至植物干细胞培养液中,并置于恒温培养摇床上培养。
其中,植物干细胞培养液为MS培养基,其中含有浓度为372mg/L的CaCl2、181mg/L的KH2PO4、1790mg/L的KNO3、173.54mg/L的MgSO4、1840mg/L的NH4NO3、0.022mg/L的CuSO4·5H2O、60mg/L的维生素C、1.58mg/L的甘氨酸、1.5mg/L的生长素、质量分数为3%的蔗糖和浓度为6.0g/L的琼脂等添加物;摇床的转速为110转/分钟,培养温度为34℃,每小时光照8分钟,培养第7天,加入新鲜的植物干细胞培养液至细胞密度为1×107个/ml,然后继续培养,培养后用100目的不锈钢筛网过滤,除去细胞团、残渣等,得到芦荟干细胞悬液;
然后,将芦荟干细胞悬液转移至20L生物反应器中进行大规模培养,生物反应器中添加有5L的植物干细胞培养基,培养第7天时,加入10L的新鲜培养基,继续培养后收获细胞。对照组培养在生物反应器中持续培养20天,中间不添加培养基。培养后收获细胞,称重。
步骤S4、芦荟干细胞提取物的制备;
收集步骤S3中获得芦荟干细胞悬液,用1um过滤器过滤,去掉培养基,收获截留的细胞;并将细胞放入-80℃的超低温冷冻2小时,然后取出解冻30分钟,重复上述操作5次,最后一次室温解冻后,用超声波破碎仪破碎细胞,过滤器过滤后,搜集滤液,于旋转式真空蒸发器内浓缩,即可获得芦荟干细胞提取物。
S5、芦荟干细胞冻干粉的制备;
采用BCA法对步骤S4中获得的芦荟干细胞提取物进行蛋白质浓度测定,根据该蛋白质浓度,加入无菌蒸馏水调节至蛋白终浓度为50.0±0.5μg/ml;再加入甘露醇,使得甘露醇在芦荟干细胞提取物中的质量分数为5%,然后,进行灭菌和冻干处理,冻干的温度为-30℃,真空度为150Pa,冻干时间为48小时,即可得到芦荟干细胞冻干粉。
S6、芦荟干细胞冻干粉质量鉴定;
采用HPLC法测定芦荟干细胞冻干粉中芦荟苷的含量
色谱条件色谱柱:LichrosperC18(250mm×4.6mm,5μm)流动相:乙腈-水(25:75);流速:1.0mL·min-1;柱温:25℃;检测波长:355nm;进样量:10LL。
将芦荟冻干粉,以75%乙醇提取3次,合并提取液经旋转蒸发、冷冻干燥后,以蒸馏水溶解后,采用HPLC法测定多酚含量:
芦荟干细胞冻干粉中,芦荟苷的含量为150.80mg/g;而直接从芦荟萃取液中,芦荟苷的含量仅为32.35mg/g;由此可知,通过本发明提供的方法所获得的芦荟干细胞冻干粉中,芦荟苷的含量显著增加。
以上对本发明的实施例进行了描述,但是本发明并不局限于上述的具体实施方式,上述的具体实施方式仅仅是示意性的,而不是限制性的,本领域的普通技术人员在本发明的启示下,在不脱离本发明宗旨和权利要求所保护的范围情况下,还可做出很多形式,这些均属于本发明的保护之内。
Claims (10)
1.一种芦荟干细胞冻干粉的制备方法,其特征在于,包括以下步骤:
从芦荟中获取含有形成层的组织;
将所述含有形成层的组织置于固体培养基中培养后,分离出芦荟干细胞;
再将所述芦荟干细胞置于植物干细胞培养基中进行体外培养,获得扩增后的芦荟干细胞;
所述扩增后的芦荟干细胞经冷冻破碎后浓缩,收获芦荟干细胞提取物;
将所述芦荟干细胞提取物与甘露醇混合后,除菌、冻干后,收获芦荟干细胞冻干粉。
2.根据权利要求1所述的芦荟干细胞冻干粉的制备方法,其特征在于,所述冻干步骤之前,还需调整所述芦荟干细胞提取物中的蛋白质浓度至30~70μg/ml。
3.根据权利要求2所述的芦荟干细胞冻干粉的制备方法,其特征在于,所述冻干条件为:温度-20℃~-35℃,真空度50~200Pa,冻干时间为36~48小时。
4.根据权利要求3所述的芦荟干细胞冻干粉的制备方法,其特征在于,所述冷冻破碎过程为:将所述扩增后的芦荟干细胞放入-50~-100℃低温环境下冷冻2~3小时,然后取出解冻,重复3~5次。
5.根据权利要求1所述的芦荟干细胞冻干粉的制备方法,其特征在于,所述植物干细胞培养基包括无机盐、维生素、氨基酸、生长素、糖类和琼脂。
6.根据权利要求1所述的芦荟干细胞冻干粉的制备方法,其特征在于,所述固体培养基包括无机盐、维生素、氨基酸、生长素、糖类和活性炭。
7.根据权利要求1所述的芦荟干细胞冻干粉的制备方法,其特征在于,所述体外培养过程是在恒温培养摇床中培养7~14天后,再转移至生物反应器中进行大规模培养。
8.根据权利要求7所述的芦荟干细胞冻干粉的制备方法,其特征在于,所述体外培养的条件为温度为30℃~37℃,每小时光照5~10min。
9.一种根据权利要求1~8任一项所述的芦荟干细胞冻干粉的制备方法所获得的芦荟干细胞冻干粉。
10.根据权利要求9所述的芦荟干细胞冻干粉在制备食品、药品、保健品或化妆品中的应用。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201610268493.6A CN105852125A (zh) | 2016-04-27 | 2016-04-27 | 芦荟干细胞冻干粉及其制备方法和应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201610268493.6A CN105852125A (zh) | 2016-04-27 | 2016-04-27 | 芦荟干细胞冻干粉及其制备方法和应用 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN105852125A true CN105852125A (zh) | 2016-08-17 |
Family
ID=56629274
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201610268493.6A Pending CN105852125A (zh) | 2016-04-27 | 2016-04-27 | 芦荟干细胞冻干粉及其制备方法和应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN105852125A (zh) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN108721202A (zh) * | 2018-08-07 | 2018-11-02 | 广州暨南生物医药研究开发基地有限公司 | 一种玫瑰茄干细胞冻干粉眼霜及其制备方法 |
CN109568226A (zh) * | 2018-11-27 | 2019-04-05 | 山东奥博森生物药业股份有限公司 | 一种芦荟干细胞提取物 |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN104498425A (zh) * | 2014-11-28 | 2015-04-08 | 广州赛莱拉干细胞科技股份有限公司 | 雪莲干细胞的分离培养方法及在制备抗衰老产品中的应用 |
CN104711215A (zh) * | 2015-04-09 | 2015-06-17 | 广州赛莱拉干细胞科技股份有限公司 | 一种苹果干细胞的培养方法及培养的苹果干细胞 |
CN104928229A (zh) * | 2015-06-03 | 2015-09-23 | 广州暨南生物医药研究开发基地有限公司 | 一种铁皮石斛干细胞冻干粉的制备方法及其用途 |
CN105238736A (zh) * | 2015-11-11 | 2016-01-13 | 大连大学 | 一种铁皮石斛干细胞的培养方法及其应用 |
EP3010482A1 (en) * | 2013-06-19 | 2016-04-27 | Stemtech International, Inc. | Methods and compositions for enhancing stem cell mobilization |
-
2016
- 2016-04-27 CN CN201610268493.6A patent/CN105852125A/zh active Pending
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP3010482A1 (en) * | 2013-06-19 | 2016-04-27 | Stemtech International, Inc. | Methods and compositions for enhancing stem cell mobilization |
CN104498425A (zh) * | 2014-11-28 | 2015-04-08 | 广州赛莱拉干细胞科技股份有限公司 | 雪莲干细胞的分离培养方法及在制备抗衰老产品中的应用 |
CN104711215A (zh) * | 2015-04-09 | 2015-06-17 | 广州赛莱拉干细胞科技股份有限公司 | 一种苹果干细胞的培养方法及培养的苹果干细胞 |
CN104928229A (zh) * | 2015-06-03 | 2015-09-23 | 广州暨南生物医药研究开发基地有限公司 | 一种铁皮石斛干细胞冻干粉的制备方法及其用途 |
CN105238736A (zh) * | 2015-11-11 | 2016-01-13 | 大连大学 | 一种铁皮石斛干细胞的培养方法及其应用 |
Non-Patent Citations (2)
Title |
---|
孟广云等: "《野生蔬菜保鲜与加工技术》", 31 January 2007 * |
张尚民: "万州芦荟产业深加工发展的分析", 《农产品加工·学刊》 * |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN108721202A (zh) * | 2018-08-07 | 2018-11-02 | 广州暨南生物医药研究开发基地有限公司 | 一种玫瑰茄干细胞冻干粉眼霜及其制备方法 |
CN108721202B (zh) * | 2018-08-07 | 2021-06-29 | 广州暨南生物医药研究开发基地有限公司 | 一种玫瑰茄干细胞冻干粉眼霜及其制备方法 |
CN109568226A (zh) * | 2018-11-27 | 2019-04-05 | 山东奥博森生物药业股份有限公司 | 一种芦荟干细胞提取物 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP3593429B2 (ja) | 冬虫夏草の人工培養方法 | |
CN101215527A (zh) | 一种蚕蛹虫草的培育方法 | |
JP2007537737A (ja) | 中国冬虫夏草無性型菌(HirsutellahepialiChen&Shen)の工業発酵生産方法 | |
CN104381011B (zh) | 一种具有高侵染活性的冬虫夏草菌种材料的制备及侵染钩蝠蛾幼虫的方法 | |
CN105368720B (zh) | 棉花内生真菌cef-082及其在棉花黄萎病防治中的应用 | |
CN110495344A (zh) | 一种人工驯化野生羊肚菌的栽培种植方法 | |
CN107460151B (zh) | 一种球形赖氨酸芽孢杆菌及其防治南方根结线虫的应用 | |
CN103215311A (zh) | 一种利用黑曲霉菌转化生产优质沉香的方法 | |
CN108823140A (zh) | 一种防治黄龙病的桐枯生物转化微生物菌剂及其制备方法 | |
CN100407905C (zh) | 杜鹃兰人工种子制作方法 | |
CN105962367A (zh) | 芦荟干细胞提取物及其制备方法和应用 | |
CN105103948B (zh) | 一种蚕蛹虫草的培育方法 | |
CN1759665A (zh) | 组培诱导虎杖毛状根制备虎杖苷的方法 | |
CN105852125A (zh) | 芦荟干细胞冻干粉及其制备方法和应用 | |
CN110317735B (zh) | 生防寡雄腐霉菌及应用 | |
CN107858300A (zh) | 针对番茄的防病、促生、品质提升以及抗逆的解淀粉芽胞杆菌2yn11及其应用 | |
CN106010978A (zh) | 冬虫夏草无性型中国被毛孢纯菌种的培养方法 | |
CN110066199A (zh) | 一种优质赤芍配方肥及其制备方法和使用方法 | |
CN104628434B (zh) | 一种抗黑果枸杞病理真菌的生物肥的制备方法 | |
KR100459869B1 (ko) | 누에배지 또는 누에번데기 배지를 사용한 코디셉스시넨시스의 배양방법 | |
CN110218109A (zh) | 一种优质蔓越莓生长调控剂及其制备方法和使用方法 | |
CN106106151A (zh) | 苦参的良种培育方法 | |
CN106967633B (zh) | 一株抗坏血酸克吕沃尔菌及其应用 | |
CN101486991B (zh) | 一种用细胞工程培养繁殖泛生墙藓原丝体的方法 | |
CN109220794A (zh) | 一种荸荠种质的超低温脱毒保存方法 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
CB02 | Change of applicant information |
Address after: 518057, room 2, building 10, Shenzhen biological incubation center, No. 302, Nanshan District hi tech, Shenzhen, Guangdong Applicant after: ISTEM REGENERATIVE MEDICINE SCI-TECH CO., LTD. Address before: 518057, Guangdong Province, Nanshan District high tech Zone, South Ring Road, No. 29, No. 02 building, international students, building No. 15 Applicant before: ISTEM REGENERATIVE MEDICINE SCI-TECH CO., LTD. |
|
COR | Change of bibliographic data | ||
C10 | Entry into substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
WD01 | Invention patent application deemed withdrawn after publication | ||
WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20160817 |