CN117815148B - Peony callus extract and preparation method and application thereof - Google Patents
Peony callus extract and preparation method and application thereof Download PDFInfo
- Publication number
- CN117815148B CN117815148B CN202410238819.5A CN202410238819A CN117815148B CN 117815148 B CN117815148 B CN 117815148B CN 202410238819 A CN202410238819 A CN 202410238819A CN 117815148 B CN117815148 B CN 117815148B
- Authority
- CN
- China
- Prior art keywords
- peony
- peony callus
- callus extract
- extract
- callus
- 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.)
- Active
Links
- 235000006484 Paeonia officinalis Nutrition 0.000 title claims abstract description 221
- 206010020649 Hyperkeratosis Diseases 0.000 title claims abstract description 181
- 239000000284 extract Substances 0.000 title claims abstract description 172
- 238000002360 preparation method Methods 0.000 title claims abstract description 34
- 238000000605 extraction Methods 0.000 title abstract description 16
- 244000170916 Paeonia officinalis Species 0.000 title 1
- 241000736199 Paeonia Species 0.000 claims abstract description 225
- 238000004108 freeze drying Methods 0.000 claims abstract description 9
- 230000003712 anti-aging effect Effects 0.000 claims abstract description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 36
- 238000010025 steaming Methods 0.000 claims description 35
- 230000000694 effects Effects 0.000 claims description 33
- 238000002390 rotary evaporation Methods 0.000 claims description 20
- 239000000843 powder Substances 0.000 claims description 19
- 238000000034 method Methods 0.000 claims description 17
- 239000007788 liquid Substances 0.000 claims description 16
- 238000001914 filtration Methods 0.000 claims description 13
- 238000007710 freezing Methods 0.000 claims description 13
- 230000008014 freezing Effects 0.000 claims description 13
- 239000002904 solvent Substances 0.000 claims description 12
- 238000002137 ultrasound extraction Methods 0.000 claims description 10
- 238000002156 mixing Methods 0.000 claims description 8
- 239000006071 cream Substances 0.000 claims description 6
- 238000000227 grinding Methods 0.000 claims description 6
- 230000008439 repair process Effects 0.000 claims description 3
- 239000002994 raw material Substances 0.000 abstract description 14
- 241000196324 Embryophyta Species 0.000 abstract description 12
- 230000012010 growth Effects 0.000 abstract description 9
- 238000002474 experimental method Methods 0.000 abstract description 7
- 230000008635 plant growth Effects 0.000 abstract description 5
- 230000008901 benefit Effects 0.000 abstract description 4
- 231100000344 non-irritating Toxicity 0.000 abstract description 4
- 238000004519 manufacturing process Methods 0.000 abstract description 3
- 210000004027 cell Anatomy 0.000 description 76
- 239000000523 sample Substances 0.000 description 51
- 239000000243 solution Substances 0.000 description 38
- 230000014509 gene expression Effects 0.000 description 36
- 230000000052 comparative effect Effects 0.000 description 33
- NTYJJOPFIAHURM-UHFFFAOYSA-N Histamine Chemical compound NCCC1=CN=CN1 NTYJJOPFIAHURM-UHFFFAOYSA-N 0.000 description 32
- 210000003491 skin Anatomy 0.000 description 31
- 238000012360 testing method Methods 0.000 description 30
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 27
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 description 27
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 23
- BAWFJGJZGIEFAR-NNYOXOHSSA-O NAD(+) Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 BAWFJGJZGIEFAR-NNYOXOHSSA-O 0.000 description 20
- 108090000623 proteins and genes Proteins 0.000 description 19
- 239000000047 product Substances 0.000 description 18
- 238000011282 treatment Methods 0.000 description 18
- 229960001340 histamine Drugs 0.000 description 16
- 108020004414 DNA Proteins 0.000 description 15
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 15
- 239000000306 component Substances 0.000 description 13
- 238000003756 stirring Methods 0.000 description 13
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 12
- 108010005774 beta-Galactosidase Proteins 0.000 description 12
- 238000001514 detection method Methods 0.000 description 12
- 102000005936 beta-Galactosidase Human genes 0.000 description 11
- 239000001963 growth medium Substances 0.000 description 11
- 230000009758 senescence Effects 0.000 description 11
- 210000001519 tissue Anatomy 0.000 description 11
- 230000005778 DNA damage Effects 0.000 description 10
- 231100000277 DNA damage Toxicity 0.000 description 10
- 238000001816 cooling Methods 0.000 description 10
- 239000000126 substance Substances 0.000 description 10
- 102000008186 Collagen Human genes 0.000 description 8
- 108010035532 Collagen Proteins 0.000 description 8
- 230000032683 aging Effects 0.000 description 8
- 229920001436 collagen Polymers 0.000 description 8
- 101710088194 Dehydrogenase Proteins 0.000 description 7
- 239000004480 active ingredient Substances 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 7
- 238000010438 heat treatment Methods 0.000 description 7
- 230000006698 induction Effects 0.000 description 7
- 239000000463 material Substances 0.000 description 7
- 239000006228 supernatant Substances 0.000 description 7
- 238000005303 weighing Methods 0.000 description 7
- 239000012224 working solution Substances 0.000 description 7
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 6
- 108010000684 Matrix Metalloproteinases Proteins 0.000 description 6
- 239000002537 cosmetic Substances 0.000 description 6
- 238000005516 engineering process Methods 0.000 description 6
- 239000002609 medium Substances 0.000 description 6
- 229910052757 nitrogen Inorganic materials 0.000 description 6
- 230000035755 proliferation Effects 0.000 description 6
- 108020003175 receptors Proteins 0.000 description 6
- 238000002835 absorbance Methods 0.000 description 5
- 238000004113 cell culture Methods 0.000 description 5
- 239000012634 fragment Substances 0.000 description 5
- 239000003550 marker Substances 0.000 description 5
- 239000012528 membrane Substances 0.000 description 5
- 102000005962 receptors Human genes 0.000 description 5
- 239000002699 waste material Substances 0.000 description 5
- PRPINYUDVPFIRX-UHFFFAOYSA-N 1-naphthaleneacetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CC=CC2=C1 PRPINYUDVPFIRX-UHFFFAOYSA-N 0.000 description 4
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 4
- 206010015150 Erythema Diseases 0.000 description 4
- 102100028999 High mobility group protein HMGI-C Human genes 0.000 description 4
- 206010030113 Oedema Diseases 0.000 description 4
- UILPJVPSNHJFIK-UHFFFAOYSA-N Paeonol Chemical group COC1=CC=C(C(C)=O)C(O)=C1 UILPJVPSNHJFIK-UHFFFAOYSA-N 0.000 description 4
- HFCYZXMHUIHAQI-UHFFFAOYSA-N Thidiazuron Chemical compound C=1C=CC=CC=1NC(=O)NC1=CN=NS1 HFCYZXMHUIHAQI-UHFFFAOYSA-N 0.000 description 4
- 230000001154 acute effect Effects 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 238000004364 calculation method Methods 0.000 description 4
- 230000011382 collagen catabolic process Effects 0.000 description 4
- 231100000321 erythema Toxicity 0.000 description 4
- 210000003630 histaminocyte Anatomy 0.000 description 4
- 230000002401 inhibitory effect Effects 0.000 description 4
- 230000003834 intracellular effect Effects 0.000 description 4
- 239000004570 mortar (masonry) Substances 0.000 description 4
- 239000000419 plant extract Substances 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 108010016788 Cyclin-Dependent Kinase Inhibitor p21 Proteins 0.000 description 3
- 101001013159 Homo sapiens Myeloid leukemia factor 2 Proteins 0.000 description 3
- 101000995164 Homo sapiens Netrin-4 Proteins 0.000 description 3
- 102000002274 Matrix Metalloproteinases Human genes 0.000 description 3
- 102100034388 Netrin-4 Human genes 0.000 description 3
- 206010040914 Skin reaction Diseases 0.000 description 3
- 230000009471 action Effects 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 230000032677 cell aging Effects 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 238000011156 evaluation Methods 0.000 description 3
- 238000001704 evaporation Methods 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 230000008595 infiltration Effects 0.000 description 3
- 238000001764 infiltration Methods 0.000 description 3
- 230000014759 maintenance of location Effects 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 230000002829 reductive effect Effects 0.000 description 3
- 230000001105 regulatory effect Effects 0.000 description 3
- 230000004044 response Effects 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 230000035483 skin reaction Effects 0.000 description 3
- 231100000430 skin reaction Toxicity 0.000 description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 2
- 241001116389 Aloe Species 0.000 description 2
- 102100033270 Cyclin-dependent kinase inhibitor 1 Human genes 0.000 description 2
- 201000004624 Dermatitis Diseases 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 241000628997 Flos Species 0.000 description 2
- 101000669513 Homo sapiens Metalloproteinase inhibitor 1 Proteins 0.000 description 2
- 206010061218 Inflammation Diseases 0.000 description 2
- NWBJYWHLCVSVIJ-UHFFFAOYSA-N N-benzyladenine Chemical compound N=1C=NC=2NC=NC=2C=1NCC1=CC=CC=C1 NWBJYWHLCVSVIJ-UHFFFAOYSA-N 0.000 description 2
- YKRGDOXKVOZESV-WRJNSLSBSA-N Paeoniflorin Chemical group C([C@]12[C@H]3O[C@]4(O)C[C@](O3)([C@]1(C[C@@H]42)O[C@H]1[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O1)O)C)OC(=O)C1=CC=CC=C1 YKRGDOXKVOZESV-WRJNSLSBSA-N 0.000 description 2
- 206010033733 Papule Diseases 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 206010034972 Photosensitivity reaction Diseases 0.000 description 2
- 101710142276 Protein P21 Proteins 0.000 description 2
- 238000003559 RNA-seq method Methods 0.000 description 2
- 206010040880 Skin irritation Diseases 0.000 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
- 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 2
- 208000027418 Wounds and injury Diseases 0.000 description 2
- 239000013543 active substance Substances 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 239000003513 alkali Substances 0.000 description 2
- FPIPGXGPPPQFEQ-OVSJKPMPSA-N all-trans-retinol Chemical compound OC\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C FPIPGXGPPPQFEQ-OVSJKPMPSA-N 0.000 description 2
- 235000011399 aloe vera Nutrition 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 239000013592 cell lysate Substances 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 238000007599 discharging Methods 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 239000002532 enzyme inhibitor Substances 0.000 description 2
- 239000004744 fabric Substances 0.000 description 2
- 239000000835 fiber Substances 0.000 description 2
- 210000002950 fibroblast Anatomy 0.000 description 2
- 239000000834 fixative Substances 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 230000002757 inflammatory effect Effects 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- YLTGFGDODHXMFB-UHFFFAOYSA-N isoacetovanillon Natural products COC1=CC=C(C(C)=O)C=C1O YLTGFGDODHXMFB-UHFFFAOYSA-N 0.000 description 2
- 239000006166 lysate Substances 0.000 description 2
- 230000002132 lysosomal effect Effects 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 239000006870 ms-medium Substances 0.000 description 2
- 231100000286 mucous membrane, eye irritation or corrosion testing Toxicity 0.000 description 2
- 210000000633 nuclear envelope Anatomy 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- YKRGDOXKVOZESV-UHFFFAOYSA-N paeoniflorin Natural products O1C(C)(C2(CC34)OC5C(C(O)C(O)C(CO)O5)O)CC3(O)OC1C24COC(=O)C1=CC=CC=C1 YKRGDOXKVOZESV-UHFFFAOYSA-N 0.000 description 2
- MLIBGOFSXXWRIY-UHFFFAOYSA-N paeonol Natural products COC1=CC=C(O)C(C(C)=O)=C1 MLIBGOFSXXWRIY-UHFFFAOYSA-N 0.000 description 2
- 208000007578 phototoxic dermatitis Diseases 0.000 description 2
- 231100000018 phototoxicity Toxicity 0.000 description 2
- 150000008442 polyphenolic compounds Chemical class 0.000 description 2
- 235000013824 polyphenols Nutrition 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 239000011535 reaction buffer Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 239000002453 shampoo Substances 0.000 description 2
- 231100000475 skin irritation Toxicity 0.000 description 2
- 230000036556 skin irritation Effects 0.000 description 2
- 238000010181 skin prick test Methods 0.000 description 2
- 239000012192 staining solution Substances 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- 231100000820 toxicity test Toxicity 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- AJBZENLMTKDAEK-UHFFFAOYSA-N 3a,5a,5b,8,8,11a-hexamethyl-1-prop-1-en-2-yl-1,2,3,4,5,6,7,7a,9,10,11,11b,12,13,13a,13b-hexadecahydrocyclopenta[a]chrysene-4,9-diol Chemical compound CC12CCC(O)C(C)(C)C1CCC(C1(C)CC3O)(C)C2CCC1C1C3(C)CCC1C(=C)C AJBZENLMTKDAEK-UHFFFAOYSA-N 0.000 description 1
- ZKRFOXLVOKTUTA-KQYNXXCUSA-N 9-(5-phosphoribofuranosyl)-6-mercaptopurine Chemical compound O[C@@H]1[C@H](O)[C@@H](COP(O)(O)=O)O[C@H]1N1C(NC=NC2=S)=C2N=C1 ZKRFOXLVOKTUTA-KQYNXXCUSA-N 0.000 description 1
- 208000002874 Acne Vulgaris Diseases 0.000 description 1
- 101100298998 Caenorhabditis elegans pbs-3 gene Proteins 0.000 description 1
- 235000003880 Calendula Nutrition 0.000 description 1
- 240000001432 Calendula officinalis Species 0.000 description 1
- 108010077544 Chromatin Proteins 0.000 description 1
- 102000029816 Collagenase Human genes 0.000 description 1
- 108060005980 Collagenase Proteins 0.000 description 1
- 230000033616 DNA repair Effects 0.000 description 1
- 240000004638 Dendrobium nobile Species 0.000 description 1
- 238000008157 ELISA kit Methods 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 108091029865 Exogenous DNA Proteins 0.000 description 1
- 208000031448 Genomic Instability Diseases 0.000 description 1
- 102000018802 High Mobility Group Proteins Human genes 0.000 description 1
- 101710176246 High mobility group protein Proteins 0.000 description 1
- 101000986379 Homo sapiens High mobility group protein HMGI-C Proteins 0.000 description 1
- 101001013150 Homo sapiens Interstitial collagenase Proteins 0.000 description 1
- 101000845170 Homo sapiens Thymic stromal lymphopoietin Proteins 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- 102000013691 Interleukin-17 Human genes 0.000 description 1
- 108050003558 Interleukin-17 Proteins 0.000 description 1
- 229940124761 MMP inhibitor Drugs 0.000 description 1
- 102000000380 Matrix Metalloproteinase 1 Human genes 0.000 description 1
- 102000005741 Metalloproteases Human genes 0.000 description 1
- 108010006035 Metalloproteases Proteins 0.000 description 1
- 102100039364 Metalloproteinase inhibitor 1 Human genes 0.000 description 1
- 102000010750 Metalloproteins Human genes 0.000 description 1
- 108010063312 Metalloproteins Proteins 0.000 description 1
- 101710121532 Netrin-4 Proteins 0.000 description 1
- 108091093105 Nuclear DNA Proteins 0.000 description 1
- 244000236658 Paeonia lactiflora Species 0.000 description 1
- 235000008598 Paeonia lactiflora Nutrition 0.000 description 1
- 240000005001 Paeonia suffruticosa Species 0.000 description 1
- 235000003889 Paeonia suffruticosa Nutrition 0.000 description 1
- 241001106477 Paeoniaceae Species 0.000 description 1
- 206010057249 Phagocytosis Diseases 0.000 description 1
- 208000003251 Pruritus Diseases 0.000 description 1
- 241000220317 Rosa Species 0.000 description 1
- 102100031294 Thymic stromal lymphopoietin Human genes 0.000 description 1
- 101150030413 Timp gene Proteins 0.000 description 1
- 101150079992 Timp3 gene Proteins 0.000 description 1
- 108010037150 Transient Receptor Potential Channels Proteins 0.000 description 1
- 102000011753 Transient Receptor Potential Channels Human genes 0.000 description 1
- 101150072278 Tslp gene Proteins 0.000 description 1
- 206010000496 acne Diseases 0.000 description 1
- 239000011149 active material Substances 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 239000002390 adhesive tape Substances 0.000 description 1
- 235000019169 all-trans-retinol Nutrition 0.000 description 1
- 239000011717 all-trans-retinol Substances 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 230000000202 analgesic effect Effects 0.000 description 1
- 230000003266 anti-allergic effect Effects 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
- 239000000427 antigen Substances 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108010030694 avidin-horseradish peroxidase complex Proteins 0.000 description 1
- WQZGKKKJIJFFOK-FPRJBGLDSA-N beta-D-galactose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-FPRJBGLDSA-N 0.000 description 1
- 235000013361 beverage Nutrition 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 108091092356 cellular DNA Proteins 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000010094 cellular senescence Effects 0.000 description 1
- 210000003483 chromatin Anatomy 0.000 description 1
- 238000003927 comet assay Methods 0.000 description 1
- 231100000170 comet assay Toxicity 0.000 description 1
- 239000008358 core component Substances 0.000 description 1
- 239000007933 dermal patch Substances 0.000 description 1
- 210000004207 dermis Anatomy 0.000 description 1
- 230000006866 deterioration Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 230000002222 downregulating effect Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 239000003792 electrolyte Substances 0.000 description 1
- 230000001804 emulsifying effect Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 229940125532 enzyme inhibitor Drugs 0.000 description 1
- 210000004920 epithelial cell of skin Anatomy 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- 238000000799 fluorescence microscopy Methods 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 210000000245 forearm Anatomy 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 238000010413 gardening Methods 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000002439 hemostatic effect Effects 0.000 description 1
- 230000013632 homeostatic process Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000007373 indentation Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 230000007794 irritation Effects 0.000 description 1
- 230000007803 itching Effects 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 210000004373 mandible Anatomy 0.000 description 1
- 229940121386 matrix metalloproteinase inhibitor Drugs 0.000 description 1
- 239000003771 matrix metalloproteinase inhibitor Substances 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000003475 metalloproteinase inhibitor Substances 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000001616 monocyte Anatomy 0.000 description 1
- VMGAPWLDMVPYIA-HIDZBRGKSA-N n'-amino-n-iminomethanimidamide Chemical compound N\N=C\N=N VMGAPWLDMVPYIA-HIDZBRGKSA-N 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 210000004940 nucleus Anatomy 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000001151 other effect Effects 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 230000008782 phagocytosis Effects 0.000 description 1
- 238000004161 plant tissue culture Methods 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000005086 pumping Methods 0.000 description 1
- BOLDJAUMGUJJKM-LSDHHAIUSA-N renifolin D Natural products CC(=C)[C@@H]1Cc2c(O)c(O)ccc2[C@H]1CC(=O)c3ccc(O)cc3O BOLDJAUMGUJJKM-LSDHHAIUSA-N 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 230000037307 sensitive skin Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 230000001953 sensory effect Effects 0.000 description 1
- 230000009759 skin aging Effects 0.000 description 1
- 210000004927 skin cell Anatomy 0.000 description 1
- 230000037394 skin elasticity Effects 0.000 description 1
- 230000007480 spreading Effects 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 239000012089 stop solution Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 230000002936 tranquilizing effect Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 210000003934 vacuole Anatomy 0.000 description 1
- 230000024883 vasodilation Effects 0.000 description 1
- 230000017260 vegetative to reproductive phase transition of meristem Effects 0.000 description 1
Classifications
-
- 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
- A61K8/9783—Angiosperms [Magnoliophyta]
- A61K8/9789—Magnoliopsida [dicotyledons]
-
- 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
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
-
- 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
- A61K2800/74—Biological properties of particular ingredients
- A61K2800/78—Enzyme modulators, e.g. Enzyme agonists
- A61K2800/782—Enzyme inhibitors; Enzyme antagonists
-
- 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
- A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
- A61K2800/82—Preparation or application process involves sonication or ultrasonication
-
- 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
- A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
- A61K2800/84—Products or compounds obtained by lyophilisation, freeze-drying
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Dermatology (AREA)
- Engineering & Computer Science (AREA)
- Gerontology & Geriatric Medicine (AREA)
- Biotechnology (AREA)
- Botany (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Birds (AREA)
- Epidemiology (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention relates to a peony callus extract and a preparation method and application thereof, belonging to the technical field of plant extraction. According to the invention, the effective components of the peony callus are extracted by freeze drying and organic extracting solution, and experiments prove that the peony callus extract contains more active functional components and has better soothing and anti-aging effects; the peony callus extract is safe and non-irritating, and the raw material production process is more convenient for tracing the source; the peony callus extract provided by the invention has the advantages of simple preparation method and high extraction efficiency, and is not limited by plant growth cycle and growth season.
Description
Technical Field
The invention relates to a peony callus extract and a preparation method and application thereof, belonging to the technical field of plant extraction.
Background
The plant skin care raw material is an essence component extracted from plants, is developed by a modern process technology, and is added into a skin care product. Compared with the traditional skin care products with chemical components, the skin care products prepared by taking the plant extracts as the core components have a plurality of unique advantages, such as green and environment-friendly, the plant extracts are all derived from plants in the nature, and the sources are traceable, safe and green; the aloe beverage has complete functions and wide sources, such as aloe components with the effects of resisting acne, reducing swelling and inhibiting inflammation; the calendula component capable of resisting oxidation and relieving sensitive skin, the rose component capable of effectively removing free radicals and promoting blood circulation, the dendrobium nobile component capable of efficiently supplementing and locking water and regulating water-oil balance and the like. However, at present, plant extracts are mainly extracted from fresh or dried plants, so that the active ingredients, yield and the like of plant extracts are easily affected by weather factors, environment, regions and the like.
Peony (school name: paeonia suffruticosa Andr.) is a plant of Paeoniaceae, paeonia, and is a perennial deciduous shrub. Flowering period is 5 months, and fruit period is 6 months. China is the world's peony's auspicious place and the world's peony kingdom. The Chinese peony gardening varieties can be divided into 4 peony product populations, namely an original product population, a northwest product population, a south-river product population and a southwest product population according to the difference of cultivation areas and wild original seeds. Root bark is used as a medicine, and is called moutan bark, also called cortex moutan, scraped cortex moutan, etc. Modern researches have shown that cortex moutan has antibacterial, antiinflammatory, antiallergic, antitumor, hemostatic, blood stasis dispelling, heat and toxic materials clearing away, tranquilizing, analgesic, and other effects, and can promote phagocytosis of monocytes and enhance specific immunity. After the plant cells are stimulated by the wound, thin-walled cells with increased volume and dedifferentiated are formed on the wound surface, called callus, and can originate from living cells of various tissues in any organ of the plant body. Therefore, plant callus is obtained by tissue culture technology, the limitation of seasons or plant growth period can be ignored, and fresh materials can be obtained at any time. In addition, the growth conditions of plant callus culture are easily standardized, there is no risk of pathogenic or environmental pollution, and production can be expanded to industrial scale. The application of plant tissue culture in the field of cosmetics is not popularized yet, and the plant callus culture extract can provide more abundant and more stable raw material selection which accords with sustainable development for the field of cosmetics, and expands the application direction of skin care raw materials.
Disclosure of Invention
The invention aims to provide a peony callus extract, a preparation method and application thereof, and the preparation of the peony callus extract is not limited by plant growth period and growth season, has high content of active ingredients, has better effects of relieving, resisting aging and repairing, and is more suitable for being applied to the field of cosmetics.
In order to achieve the above purpose, a preparation method of peony callus extract comprises the following steps:
(1) Freeze-drying fresh peony callus to obtain freeze-dried peony callus;
(2) Grinding the freeze-dried peony callus to powder to obtain peony callus powder;
(3) Mixing peony callus powder and an organic extracting solution, performing ultrasonic extraction, and filtering to obtain peony callus extracting solution;
(4) And removing the solvent from the peony callus extract to obtain the peony callus extract.
In the prior art, peony is used as a raw material of a skin care product, flowers or root barks of peony plants are generally used as raw materials for extracting active ingredients, however, the extraction process for extracting the active ingredients by using the flowers or root barks of the peony plants is complex, a large amount of peony plants are required for extracting enough active ingredients, and the extraction efficiency is low. The peony plants have long growth cycle, mainly grow in spring, and the obtained raw materials are limited by time, season and climate environment. The invention adopts the peony callus as the extraction raw material for the first time, and the peony callus cultivated by the callus cultivation technology is not limited by the plant growth cycle and the growth season.
As a preferred embodiment of the method for preparing peony callus extract of the present invention, the organic extract comprises ethanol, methanol or acetone.
More preferably, the organic extract is ethanol.
As a preferred embodiment of the preparation method of the peony callus extract, the concentration of the ethanol is 30% -70%.
More preferably, the concentration of ethanol is 50%.
As a preferred embodiment of the preparation method of the peony callus extract of the present invention, the feed liquid ratio of the peony callus powder and the organic extract in the step (3) is 1:10-20.
More preferably, the feed liquid ratio of the peony callus powder and the organic extract in the step (3) is 1:10.
As a preferred implementation mode of the preparation method of the peony callus extract, the condition parameters of freeze drying are that the cold trap temperature is-80 ℃, the freezing chamber temperature is-45-35 ℃, the vacuum degree is 0-10 pa, and the vacuum freezing time is 36-60 h.
Preferably, the specific procedure of freeze-drying comprises: program 1:1 h-45 ℃ and vacuum degree 0; program 2:0.5 h, the temperature is minus 45 ℃ and the vacuum degree is 0; program 3:0.5 h, the temperature is-45 ℃ and the vacuum degree is 0-10 pa; program 4:0.5 h, at the temperature of minus 30 ℃ and the vacuum degree of 0-10 pa; program 5:1 h-10 ℃ and vacuum degree of 0-10 pa; program 6: 5h, 0 ℃, and the vacuum degree is 0-10 pa; program 7: 4h, 5 ℃, and the vacuum degree is 0-10 pa; program 8:3 h,10 ℃ and vacuum degree of 0-10 pa; program 9: 5h, 15 ℃ and vacuum degree of 0-10 pa; program 10: 5h, 20 ℃ and vacuum degree of 0-10 pa; program 11: 5h, 25 ℃, and the vacuum degree is 0-10 pa; program 12: 5h, 30 ℃ and vacuum degree of 0-10 pa; program 13: 5h, 30 ℃ and vacuum degree of 0-10 pa; program 14:7.5 h,35 ℃ and the vacuum degree is 0-10 pa.
As a preferred embodiment of the preparation method of peony callus extract of the present invention, the condition parameters of the ultrasonic extraction in the step (3) are as follows: ultrasonic power 2000W, ultrasonic temperature 25-45 deg.C, ultrasonic time 20-40min, ultrasonic extraction 3 times.
As a preferred embodiment of the method for preparing peony callus extract of the present invention, the solvent is removed in the step (4) by rotary evaporation.
As a preferred embodiment of the preparation method of the peony callus extract, the rotary evaporation comprises three rotary evaporation links, and the condition parameters of the first rotary evaporation link are as follows: steaming at 35-42deg.C at 80-120 rpm for 40-50min; the condition parameters of the second rotary evaporation link are as follows: steaming at 48 deg.C at 80-120 rpm for 30-45min; the condition parameters of the third rotary evaporation link are 48 ℃, the rotating speed is 80-120 rpm, and the rotary evaporation is 30-45 min.
The invention also provides a peony callus extract, which is prepared by adopting the preparation method of the peony callus extract.
The invention also provides application of the peony callus extract in preparing skin care products with anti-aging effect.
Collagen loss is considered one of the important "murders" of aging. Because collagen can penetrate through the horny layer to combine with skin epithelial cells, participate in and improve metabolism of skin cells, keep the integrity of moisture and fiber structures of the horny layer, and enable skin to absorb moisture and nutrient substances more easily. The loss of skin collagen is mainly regulated and controlled by MMPs genes, when the skin is stimulated by the outside to activate the MMPs gene expression, the collagen structure is destroyed to decompose the collagen in the skin, and the dermis of the skin is easy to collapse, so that the mandible is fuzzy and the face is loose. The research of the inventor discovers that the peony callus extract can inhibit the expression of the gene of the senescence protein P21 of the HaCaT cells, is beneficial to inhibiting the generation of senescence marker beta-galactosidase and slowing down the cell senescence; and can promote the expression of collagen degrading enzyme inhibitor genes in HaCaT cells, inhibit the expression of collagen degradation related metalloproteases, reduce collagen degradation, reduce collagen loss caused by aging, reduce skin elasticity and delay skin aging process.
The invention also provides application of the peony callus extract in preparing skin care products with relieving effect.
The invention also provides application of the peony callus extract in preparing skin care products with skin repair effect.
The invention also provides a skin care product, which comprises the peony callus extract.
As a preferred embodiment of the skin care product, the addition amount of the peony callus extract in the skin care product is 0.1% -5%.
As a preferred embodiment of the skin care product of the present invention, the skin care product includes a face cream, an eye cream, a toner or an essence.
Compared with the prior art, the invention has the beneficial effects that: the invention provides a peony callus extract, which is prepared by extracting effective components from peony callus by freeze drying and organic extracting solution, and experiments prove that the peony callus extract contains more active functional components and has better effects of relieving and resisting aging; the peony callus extract is safe and non-irritating, and the raw material production process is more convenient for tracing the source; the peony callus extract provided by the invention has the advantages of simple preparation method and high extraction efficiency, and is not limited by plant growth cycle and growth season.
Drawings
FIG. 1 is a chromatogram of peony callus extract prepared in example 1.
FIG. 2 is a chromatogram of the peony extract prepared in comparative example 1.
FIG. 3 is a statistical chart showing the effect of peony callus extract prepared in example 1 on the expression of senescence-associated marker genes.
FIG. 4 is a statistical chart showing the effect of peony callus extract prepared in example 1 on cell collagen-related gene expression.
FIG. 5 is a graph showing the comparison of tail DNA percentages of the peony callus extract prepared in example 1 and the peony extract prepared in comparative example 4.
FIG. 6 is a graph showing the results of comparative tail length tests of the peony callus extract prepared in example 1 and the peony extract prepared in comparative example 4.
FIG. 7 is a graph showing the results of caudal distance test of the peony callus extract prepared in example 1 and the peony extract prepared in comparative example 4.
FIG. 8 is a graph showing the results of measuring the beta-galactosidase activity of the peony callus extract prepared in example 1 and the peony extract prepared in comparative example 4.
FIG. 9 is a statistical chart of the number of senescent cells under treatment using the peony callus extract prepared in example 1 and the peony extract prepared in comparative example 4.
FIG. 10 is a statistical chart showing the effect of peony callus extract prepared in example 1 on the expression of genes related to the receptor of the cell receptor.
FIG. 11 is a graph showing the results of histamine release inhibition test for the peony callus extract prepared in example 1 and the peony extract prepared in comparative example 4.
Detailed Description
For a better description of the objects, technical solutions and advantages of the present invention, the present invention will be further described with reference to the following specific examples.
The peony callus used in the following examples was prepared by the following method:
(1) Peony callus induction: the flowers of the fresh peony are cut into tissue blocks with the thickness of about 10mm by a surgical knife and inoculated on an induction culture medium. The pH value of the callus induction culture medium is 5.8, and after the induction culture medium is inoculated with peony tissue blocks, dark reaction is carried out for 20-30 days at 25 ℃.
The peony callus induction culture medium comprises the following formula: 2g/L MS medium, 30g/L sucrose, 6-8 g/L agar, 0.5 mg/L6-BA (6-Benzylaminopurine ), 2mg/L TDZ (Thidiazuron, thidiazuron), 0.5 mg/L NAA (NAPHTHYLACETIC ACID ).
(2) Propagation of peony callus: and (3) picking peony callus in the induction culture medium, spreading the peony callus in the proliferation culture medium, and placing 2g of peony callus in each bottle of proliferation culture medium. The pH value of the callus proliferation culture medium is 5.8, the proliferation culture process needs to carry out dark reaction for 30-40 days at 25 ℃ and the proliferation peony callus is obtained after 40 days. The formula of the callus proliferation culture medium is as follows: 2g/L MS medium, 30g/L sucrose, 6-8 g/L agar, 0.5 mg/L6-BA (6-Benzylaminopurine ), 1mg/L TDZ (Thidiazuron, thidiazuron), 0.5 mg/L NAA (NAPHTHYLACETIC ACID ).
The peony used in the following comparative examples is commercially available.
Example 1
The embodiment of the invention provides a preparation method of peony callus extract, which comprises the following steps:
(1) Freeze-drying fresh peony callus at-80deg.C, freezing warehouse temperature of-45deg.C to-35deg.C, vacuum degree of 0-10 pa, and vacuum freezing of 48-h; in the 48 h drying process, the temperature and the vacuum degree of the freezing bin gradually change along with the freezing time, and the following trends are sequentially presented: program 1 (1 h, -45 ℃, vacuum 0), program 2 (0.5 h, -45 ℃, vacuum 0), program 3 (0.5 h, -45 ℃, vacuum 0-10 pa), program 4 (0.5 h, -30 ℃, vacuum 0-10 pa), program 5 (1 h, -10 ℃, vacuum 0-10 pa), program 6 (5 h,0 ℃, vacuum 0-10 pa), program 7 (4 h,5 ℃, vacuum 0-10 pa), program 8 (3 h,10 ℃, vacuum 0-10 pa), program 9 (5 h,15 ℃, vacuum 0-10 pa), program 10 (5 h,20 ℃, vacuum 0-10 pa), program 11 (5 h,25 ℃, vacuum 0-10 pa), program 12 (5 h,30 ℃, vacuum 0-10 pa), program 13 (5 h,30 ℃, vacuum 0-10 pa), program 14 (7.5 h,35 ℃, vacuum 0-10 calli pa) to obtain the peony tissue;
(2) Pre-cooling a mortar and a grinding pestle in advance (adding liquid nitrogen into the mortar for pre-cooling, repeating the operation for 2 times after the liquid nitrogen volatilizes), taking freeze-dried peony callus, and grinding the freeze-dried peony callus into powder in the liquid nitrogen to obtain peony callus powder;
(3) According to the feed liquid ratio of 1:10 weighing peony callus powder and 50% ethanol, mixing, performing ultrasonic extraction with ultrasonic power of 2000W and ultrasonic temperature of 45 ℃ and ultrasonic time of 30 min, repeating the extraction for 3 times, filtering and merging by using a Buchner funnel, and then filtering with a filter membrane of 0.22 mu m to obtain peony callus extract;
(4) Removing the solvent from the peony callus extract by a rotary evaporator, wherein the rotary evaporation step is carried out in three steps: the first step is rotary steaming condition: the temperature is 38 ℃, the rotating speed is 100rpm, and the rotary steaming is 45 min; second step rotary steaming condition: 48 ℃, rotating speed of 100rpm, and rotary steaming of 40 min; the third step of rotary steaming conditions are as follows: and (3) steaming at 48 ℃ at 100rpm for 40min to obtain the peony callus extract.
Examples 2 to 16
Examples 2 to 16 of the present invention explored the effects of the types and concentrations of the organic extracts, the feed-liquid ratios of peony callus powder and the organic extracts, the ultrasonic condition parameters, and the rotary evaporation condition parameters, and examples 2 to 16 of the present invention were identical to example 1 except for the parameters listed in table 1.
TABLE 1
Example 17
The difference between this embodiment and embodiment 1 is only that the step (4) is different, and the step (4) of this embodiment specifically is: removing the solvent from the peony callus extract by a rotary evaporator, wherein the rotary evaporation step is carried out in three steps: the first step is rotary steaming condition: rotary steaming at 35 deg.c and rotation speed of 80 rpm and rotary steaming at 50 min; second step rotary steaming condition: steaming at rpm and 48 deg.C for 45min; the third step of rotary steaming conditions are as follows: and (3) performing rotary steaming at 48 ℃ at the rotation speed of 80 rpm and the rotation speed of 45min to obtain the peony callus extract.
Example 18
The difference between this embodiment and embodiment 1 is only that the step (4) is different, and the step (4) of this embodiment specifically is: removing the solvent from the peony callus extract by a rotary evaporator, wherein the rotary evaporation step is carried out in three steps: the first step is rotary steaming condition: steaming at 42deg.C and rotation speed of 120 rpm for 40min; second step rotary steaming condition: steaming at rpm and 48 deg.C for 30min; the third step of rotary steaming conditions are as follows: and (3) performing rotary steaming at 48 ℃ at a rotation speed of 120 rpm and 30: 30min to obtain the peony callus extract.
Comparative example 1
The present comparative example provides a method for preparing peony callus extract, which only differs from example 1 in step (3), and the comparative example step (3) specifically comprises: according to the feed liquid ratio of 1:10, weighing peony callus powder and pure water, mixing, performing ultrasonic extraction, wherein the ultrasonic power is 2000W, the ultrasonic temperature is 45 ℃, the ultrasonic time is 30min, repeating the extraction for 3 times, filtering and merging by using a Buchner funnel, and then filtering with a filter membrane of 0.22 mu m to obtain peony callus extract.
Comparative example 2
The comparative example provides a preparation method of peony callus extract, comprising the following steps:
(1) Crushing fresh peony callus to obtain peony callus powder;
(2) According to the feed liquid ratio of 1:10 weighing peony callus powder and 50% ethanol, mixing, performing ultrasonic extraction with ultrasonic power of 2000W and ultrasonic temperature of 45 ℃ and ultrasonic time of 30 min, repeating the extraction for 3 times, filtering and merging by using a Buchner funnel, and then filtering with a filter membrane of 0.22 mu m to obtain peony callus extract;
(3) Removing the solvent from the peony callus extract by a rotary evaporator, wherein the rotary evaporation step is carried out in three steps: the first step is rotary steaming condition: the temperature is 38 ℃, the rotating speed is 100 rpm, and the rotary steaming is 45 min; second step rotary steaming condition: 48 ℃, the rotating speed is 100 rpm, and the rotary steaming is 40 min; the third step of rotary steaming conditions are as follows: and (3) performing rotary steaming for 40min at 48 ℃ at the rotating speed of 100 rpm to obtain the peony callus extract.
Comparative example 3
The present comparative example differs from example 1 only in the step (4), and the step (4) of the present example is specifically: removing the solvent from the peony callus extract by a rotary evaporator under the following conditions: and the rotating speed is 100 rpm at 48 ℃ and the rotary steaming is carried out for 120min.
Comparative example 4
The comparative example provides a preparation method of a peony extract, comprising the following steps:
(1) Taking fresh peony flowers for freeze drying, wherein the cold trap temperature is-80 ℃, the freezing bin temperature is-45 ℃ to-35 ℃, the vacuum degree is 0-10 pa, the vacuum freezing is 48: 48 h, and in the 48: 48 h drying process, the freezing bin temperature and the vacuum degree gradually change along with the freezing time, and the following trends are sequentially presented: program 1 (1 h, -45 ℃, vacuum 0), program 2 (0.5 h, -45 ℃, vacuum 0), program 3 (0.5 h, -45 ℃, vacuum 0-10 pa), program 4 (0.5 h, -30 ℃, vacuum 0-10 pa), program 5 (1 h, -10 ℃, vacuum 0-10 pa), program 6 (5 h,0 ℃, vacuum 0-10 pa), program 7 (4 h,5 ℃, vacuum 0-10 pa), program 8 (3 h,10 ℃, vacuum 0-10 pa), program 9 (5 h,15 ℃, vacuum 0-10 pa), program 10 (5 h,20 ℃, vacuum 0-10 pa), program 11 (5 h,25 ℃, vacuum 0-10 pa), program 12 (5 h,30 ℃, vacuum 0-10 pa), program 13 (5 h,30 ℃, vacuum 0-10 pa), program 14 (7.5 h,35 ℃, vacuum 0-10 pa), and freeze-dried peony is obtained;
(2) Pre-cooling a mortar and a grinding pestle in advance (adding liquid nitrogen into the mortar for pre-cooling, repeating the operation for 2 times after the liquid nitrogen volatilizes), taking freeze-dried peony flowers, and grinding the freeze-dried peony flowers into powder in the liquid nitrogen to obtain peony flower powder;
(3) According to the feed liquid ratio of 1:10 weighing flos moutan powder and 50% ethanol, mixing, performing ultrasonic extraction with ultrasonic power of 2000W, ultrasonic temperature of 25-45deg.C and ultrasonic time of 30: 30min, repeating extraction for 3 times, filtering with Buchner funnel, mixing, and filtering with 0.22 μm filter membrane to obtain flos moutan extractive solution;
(4) Removing the solvent from the peony extract by a rotary evaporator, wherein the rotary evaporation step is carried out in three steps: the first step is rotary steaming condition: the temperature is 35-42 ℃ and the rotating speed is 80-120 rpm; second step rotary steaming condition: spin steaming at 48deg.C for 30 min; the third step of rotary steaming conditions are as follows: and (3) performing rotary steaming at 48 ℃ for 30-45 min to obtain the peony extract.
Effect example 1
The detection of the active ingredients of the peony callus extract prepared in example 1 and the peony flower extract prepared in comparative example 4 is carried out by the following specific steps:
a. Test sample preparation: the peony callus extract prepared in example 1 and the peony flower extract prepared in comparative example 4 are dissolved in methanol solution respectively, and ultrasonic vibration is used for 20min to promote dissolution. And then oscillating and centrifuging, and repeating the ultrasonic oscillating and centrifuging step of the solution for 3 times. Collecting supernatant obtained in 3 times, evaporating to dryness, and evaporating to dryness to obtain a sample. The control group was a pure methanol solution without any sample.
B. HPLC determination of the active ingredients of the extract: the above test samples were quantitatively analyzed by HPLC. Adding methanol into the evaporated sample obtained in the step (1) for redissolving, centrifuging 10 min at the speed of 10 000 Xg, and taking supernatant to pass through an organic film; adopts binary gradient elution, the mobile phase A is acetonitrile, the mobile phase B is double distilled water (0.2% phosphoric acid), the column temperature is 25 ℃, the sample injection amount is 20 mu L, the flow rate is 1 mL/min, and the detection wavelength is 360 nm. Elution procedure: 0-4 min, 80-76% B; 4-20 min, 76-69% of B; 20-25 min, 69-60% B. The sample was repeated 3 times to obtain chromatograms of the respective effective substances in the peony callus extract prepared in example 1 and the peony extract prepared in comparative example 4, respectively.
The results are shown in FIGS. 1 and 2. The peaks in the test chromatograms are subjected to matching analysis according to the known peaks of the active substance standard substances, and the analysis results are as follows: the peony callus extract has a characteristic peak at 230 nm, wherein the component is paeoniflorin when the chromatographic retention time is 20.896 min, the peak area is 325.895 mAU min, and the content is about 0.29 mg/mL; the component is paeonol with the chromatographic retention time of 42.871 min, the peak area of 480.950 mAU min and the content of about 0.22 mg/mL; the peony extract did not match the corresponding peak for the corresponding retention time. As shown by the chromatographic results, the types of active substances in the peony callus extract are more than those of the peony extract, and the content of paeoniflorin and paeonol which are main components in the peony callus extract is higher than that of the peony extract.
Effect example 2 detection of Total polyphenol content of extract
The total polyphenol contents of the peony callus extracts prepared in examples 1 to 18 and comparative examples 1 to 4 were examined and analyzed, and the results are shown in Table 2.
TABLE 2
The results are shown in Table 2. According to the indexes of peony active material standard, solvent residue and the like, analyzing each extraction combination, and analyzing and extracting the combination result as follows: the peony callus can use organic extraction solution such as ethanol, methanol, acetone and the like as a solvent, wherein on the basis of a single factor test result, design-Expert software is used for analyzing the combined result, and the effect of the feed liquid ratio on the extraction effect of the peony callus extract is the greatest. From the results, the optimal combination of extracts from peony callus is: 50% ethanol and feed liquid ratio is 1: 10. the ultrasonic time was 30min.
Effect example 3 safety evaluation of peony callus extract
3.1 Cosmetic irritation evaluation was performed on the peony callus extracts prepared in examples 1 to 10 above.
The tested group is female, 400 persons are aged between 16 and 65 years, the skin of the tested person is healthy, the skin allergy history does not exist, the tested person meets the volunteer selection standard of the tested person, the tested person is randomly divided into 10 groups of 40 persons, and the peony callus extracts are prepared by respectively testing the examples 1-10.
The test method is a human body patch test. Selecting qualified patch tester, placing about 0.020-0.025g of test object into the patch tester, applying the patch tester with test object on the back or forearm flexor side of the subject with non-irritating adhesive tape, and applying on skin with palm light pressure for 24 hr. After removing the spot tester for 30min, the skin reaction is observed after the indentation disappears. If the result is negative, the spot test is observed again 24h and 48h after the spot test. The results were recorded according to 2007 "cosmetic health Specification" and 2015 "cosmetic safety Specification" as reference standards.
Evaluation criteria: level 0: a negative reaction; stage 1: suspicious reactions, only weak erythema; 2 stages: weak positive response, erythema, infiltration, edema, and possibly papules; 3 stages: strong positive reaction, erythema, infiltration, edema, papule, and reaction beyond the test area; 4 stages: very strong positive response, marked erythema, severe infiltration, edema, blepharospermia, and response beyond the test area.
The human skin patch experiment result shows that: the patch experiments of the peony callus extracts prepared in the examples 1-10 are carried out on each group of subjects, skin reactions of the subjects are observed 24h and 48h after the experiments, and the skin reactions of all the subjects are negative, so that the peony callus extracts prepared in the examples 1-10 are safe to use.
3.2 The peony callus extract prepared in example 1 was sent to Shanghai micro-spectral detection technology group Co., ltd for skin phototoxicity test, multiple skin irritation test, acute transdermal toxicity test, acute eye irritation test and skin allergy test.
The detection result shows that the peony callus extract prepared in the embodiment 1 has no skin phototoxicity; the results of the multiple skin irritation tests are non-irritating; the acute percutaneous toxicity test result belongs to a slightly toxic; the acute eye irritation test results were microstimulatory under no-rinse conditions; skin allergy test results showed no skin allergy.
Effect example 4 anti-aging efficacy test of peony callus extract
4.1 Effect of peony callus extract on the expression of anti-aging marker genes
The specific experimental steps are as follows:
(1) Preparing a culture solution containing peony callus extracts: diluting the peony callus extract prepared in the embodiment 1 into different concentration gradients by adopting a DMEM minimal medium, and screening to obtain a DMEM culture solution containing 1% of peony callus as a test sample.
(2) Preparation of HaCaT cells: haCaT cells with good growth state were inoculated into 6-well culture plates at a density of 5×10 3 cells per well, and cultured overnight at 37℃in a 5% CO 2 environment.
(3) Adding culture solution containing peony callus extract: the waste culture solution in the cell culture plate cultured overnight was removed, and corresponding treatments were performed according to the control group and the sample group (set sample group: DMEM culture solution 2 mL containing 1% callus of 1% peony; control group: DMEM culture solution 2 mL), 3 replicates were set for each treatment, and incubation was performed 24 h after addition.
(4) Whole genome assay excavated cell skin care target gene expression: removing the culture medium of the cells, collecting the cells, adding a lysate, extracting total RNA of the cells, detecting the concentration and purity of the RNA, measuring the gene expression of the whole genome of the cells after the peony callus extract is treated by adopting an RNA-Seq sequencing technology, and excavating the genes of skin care targets of the cells.
(5) Analyzing the relative expression quantity of the cell senescence-associated characteristic genes: the obtained whole gene sequence file was mass analyzed using Nanoplot and low quality sequences were filtered out. Then carrying out reference genome expression quantity comparison and calculation on a cell gene sample by using hisat software and DESeq2 software, and finally carrying out gene expression quantity difference analysis by using DESeq2 to obtain the gene relative expression quantity of a cell senescence protein related gene CDKN1A (CYCLIN DEPENDENT KINASE inhibitor 1A, p21 protein), a cell senescence marker beta-galactosidase related gene NTN4 (Netrin 4) and HMGA2 (High Mobility Group AT-Hook 2, high mobility group protein A2); the relative gene expression levels of the cell collagen related genes MMPs (matrix metalloprotein, matrix metalloproteinase) and TIMP (metallopeptidase inhibitor, matrix metalloproteinase inhibitor) were obtained.
The results are shown in FIGS. 3-4. As can be seen from FIG. 3, the peony callus extract prepared in example 1 can inhibit the expression of senescence protein P21 protein gene CDKN1A, which is one of senescence markers, so that the expression level is reduced by 11.9%; the peony callus extract prepared in the example 1 can promote the expression of NTN4 and HMGA2 genes, increase the expression level of NTN4 by 23.53% and the expression level of HMGA2 by 19.25%, thereby inhibiting the activity of beta-galactosidase. As can be seen from fig. 4, the peony callus extract prepared in example 1 can down-regulate the expression of the gene MMP related to collagen degradation of HaCaT cells, up-regulate the expression of the gene TIMP, which is a collagen degradation inhibitor; the peony callus extract prepared in the embodiment 1 can down-regulate the expression of 5 matrix metalloproteinases MMP, wherein the most typical collagen degrading enzyme MMP1 has the largest down-regulating amplitude, and the expression level is down-regulated by 45.07%; the TIMP gene is a natural MMP inhibitor in cells, and the peony callus extract can improve the expression of two metal matrix enzyme inhibitors, and can improve the expression level of the TIMP3 gene by 17.79% at most.
4.2 DNA repair action of peony callus extract
Anti-aging cell test model UVB irradiation damage model was generally selected, and DNA damage was used to represent cell aging. DNA damage is one of the leading causes of aging. Physiological changes caused by DNA damage in turn increase genomic instability, thereby amplifying the deterioration of homeostasis during aging. When various exogenous and exogenous DNA damage factors induce the breaking of cell DNA chains, the supercoiled structure of DNA is destroyed, and under the action of cell lysate, the membrane structures of cell membrane, nuclear membrane and the like are destroyed, and the proteins, RNA and other components in the cell are diffused into the cell lysate, while the nuclear DNA can only stay in place due to the too large molecular weight. Under neutral conditions, DNA fragments can enter gel to migrate, under the action of alkali treatment and alkali electrolyte, DNA is uncoiled, damaged DNA is broken and fragments are released, and due to the small molecular weight of the DNA, the DNA fragments can leave the core DNA and move to the anode in the electrophoresis process to form comet-like images, and undamaged DNA parts keep a sphere shape. The more serious the damage of DNA, the more fragments and the smaller the fragments are, the larger the amount of DNA transferred during electrophoresis, the longer the transfer distance, the longer the tail length and the enhanced tail fluorescence intensity can be observed under a fluorescence microscope. Under certain conditions, the distribution of the DNA migration distance (comet tail length) and the DNA content (fluorescence intensity) are linearly related to the DNA damage degree, so that the tail moment (the product of the tail DNA content and the tail length) becomes a main basis for quantitatively measuring the cell DNA damage degree.
The specific experimental steps are as follows:
(1) Preparing a test sample: the peony callus extracts prepared in example 1 and the peony extract prepared in comparative example 4 are respectively diluted into different concentration gradients by adopting a DMEM minimal medium, and a DMEM culture solution (MDC) containing 0.1% of the peony callus extracts and a DMEM culture solution (MD) containing 0.1% of the peony extracts are obtained by screening.
(2) Preparation of HaCaT cells: haCaT cells with good growth state were inoculated into 6-well culture plates at a density of 5×10 3 cells per well, and cultured overnight at 37℃in a 5% CO 2 environment.
(3) Adding a culture solution containing a peony raw material of super A: the waste culture solution in the cell culture plate cultured overnight is removed, after being stimulated by adding hydrogen peroxide, the DMEM culture solution containing 0.1% of peony callus and the DMEM culture solution containing 0.1% of peony extract are respectively added, 3 repetitions are set for each treatment, and after the addition, 24h is incubated.
(4) Comet assay to determine cellular DNA damage: cells were collected by centrifuging cells 2 min at a relative centrifugal force of 700 Xg and the supernatant was discarded. Precipitation was performed once with pre-chilled PBS (without Mg 2+ and Ca 2+), centrifuged and the supernatant discarded. Finally, cells were resuspended at 1x10 5 cells/mL in pre-chilled PBS (without Mg 2+ and Ca 2+); washing and decoloring according to the steps of the specification; slides were observed by inverted fluorescence microscopy using FITC filters.
The experimental results are shown in fig. 5-7. Experimental results show that the peony callus extract and the peony extract have good effects of repairing DNA damage to moderately severe DNA damage; at the same concentration, the repair effect of the peony callus extract on the DNA damage caused by ultraviolet is better than that of the peony extract.
4.3 Detection of beta-galactosidase Activity of peony callus extract
Cell senescence is a phenomenon in which cells undergo hypofunction and gradually tend to die under normal environmental conditions. Aged cells appear as enlarged cell volumes, flattened; the nucleus becomes large, the nuclear membrane is sunken, and chromatin is gathered, condensed and cracked; the intracytoplasmic granules increase and vacuoles are formed; increased lysosomal content results in increased lysosomal enzyme β -galactosidase activity. The above characteristics of aging cells are the basis for various cell aging detection means. Among them, detection of senescence-associated beta-galactosidase is a classical method for effectively detecting cellular senescence. The SA-beta-gal is used as a substrate to react with beta-gal in senescent cells to convert the substrate to blue-green, so that the senescent cells can be observed under a common optical microscope.
The specific experimental steps are as follows:
(1) Preparing a test sample: the peony callus extracts prepared in example 1 and the peony extract prepared in comparative example 4 were diluted to different concentration gradients respectively using DMEM minimal medium, and were grouped as follows:
Test example sample 1 (0.1% mdc): DMEM broth containing 0.1% of peony callus extract prepared in example 1;
test example sample 2 (0.5% mdc): DMEM broth containing 0.5% of peony callus extract prepared in example 1;
test example sample 3 (1% mdc): DMEM broth containing 1% of peony callus extract prepared in example 1;
Comparative example sample 1 (0.1% md): DMEM broth containing 0.1% of the peony extract prepared in comparative example 4;
comparative sample 2 (0.5% md): DMEM broth containing 0.5% of the peony extract prepared in comparative example 4;
Comparative example sample 3 (1% md): DMEM broth containing 1% of the peony extract prepared in comparative example 4.
(2) Preparation of fibroblasts: fibroblasts well grown were inoculated into 6-well plates at a density of 5X 10 3 per well and cultured overnight at 37℃in a 5% CO 2 environment.
(3) Additive sample processing
The waste culture solution in the cell culture plate cultured overnight is removed, corresponding sample treatments are respectively added to the cells according to the group in (1) after the hydrogen peroxide is added for stimulation, 3 replicates are arranged for each treatment, and the cells are incubated for 24 h after the addition.
(4) Fluorescent staining assay to determine beta-galactosidase activity: the cell-processing culture solution in (3) was removed, washed 1 time with PBS, and 1mL of the staining fixative solution was added thereto, and the cells were fixed at room temperature for 15 min hours. The fixative was aspirated and the cells were washed 3 times with PBS 3 min times. PBS was removed in its entirety, and 1ml SA-. Beta. -gal staining solution was added to each well. Incubation at 37 ℃ was carried out overnight, and the staining solution was prevented from evaporating by sealing with a preservative film, and then observed under an optical microscope and photographed.
The results are shown in fig. 8 and 9. As can be seen from fig. 8, the increase of the blue-green substance (β -galactosidase) in the model composition fiber cells treated with hydrogen peroxide represents that the cells are in an aging state. After MDC (peony callus extract) and MD (peony extract) were added to cells in a senescent state, both were seen to alleviate the senescent condition in the model group. As the MDC, MD concentration of the added sample increased, the cellular β -galactosidase activity decreased, and the green material in the figure decreased. As can be seen from fig. 9, the MDC-treated groups had lower percentages of senescent cells than the MD-treated groups at different concentrations, and the experimental groups after 0.5% MDC treatment had lower percentages of senescent cells than the control groups, with significant differences; the percentage of senescent cells in the experimental group after 1% MDC treatment is obviously lower than that in the control group, and the differences are extremely obvious; in contrast, the MD treated group showed a decrease in the percentage of senescent cells only in the 1% concentration treated group. Compared with the peony extract, the peony callus extract can reduce the activity of beta-galactosidase in cells and the percentage of cells aged in vivo better, i.e. the effect of delaying cell aging is better than that of the peony extract.
4.4 Determination of NAD + content in peony callus extract
(1) Preparation of samples
Preparation of tissue samples: after washing the tissue with ice-pre-chilled PBS, about 30mg of peony callus from example 1 was weighed, placed in a homogenizer, and homogenized at room temperature or on ice with 400. Mu.l of NAD +/NADH extract. Then, the mixture was centrifuged at 12,000g at 4℃for 5-10 minutes, and the supernatant was taken as a sample to be tested for use.
(2) Preparation of the kit
A. Preparation of NADH standard: after 655. Mu.l of NADH preparation is sucked up and 5mg of NADH provided by the kit is fully dissolved, the 10mM NADH standard substance is obtained. 10mM NADH standard was stored in the dark at-80℃after appropriate packaging.
Setting NADH standard curve: the 10mM NADH standard is diluted with NAD +/NADH extract to a proper concentration gradient, for example, several concentrations of 0, 0.25, 0.5, 1,2, 4, 6, 8 and 10. Mu.M can be set for initial detection, and 20. Mu.l of standard is added to each well of a 96-well plate during detection, which corresponds to 0, 5, 10, 20, 40, 80, 120, 160 and 200pmol of NADH per well. If necessary, the concentration range of the standard can be appropriately adjusted in the subsequent experiments according to the NADH content in the sample. Wherein the spot with the concentration of 0. Mu.M is a blank spot and contains only NAD +/NADH extract.
C. Preparing ethanol dehydrogenase working solution: the ethanol dehydrogenase is diluted 45-fold with the reaction buffer, for example, 2. Mu.l of ethanol dehydrogenase is added to 88. Mu.l of the reaction buffer, and 90. Mu.l of ethanol dehydrogenase working solution is obtained. For each standard or sample, 90. Mu.l of ethanol dehydrogenase working solution is required, and an appropriate amount of ethanol dehydrogenase working solution is prepared according to the number of the standard or sample to be detected.
(3) Sample measurement
A. Determination of the total amount of NAD + and NADH in the sample: 20 μl of the test sample was pipetted into a 96-well plate, and duplicate wells of the sample were recommended for reduced experimental error. If the total amount of NAD + and NADH in the sample is found to be too high and exceeds the range of the standard curve, the sample needs to be properly diluted by using NAD +/NADH extracting solution and then detected; if the total amount is too low, the amount of the cell or tissue sample to be used should be increased.
B. Determination of NAD +, NADH content or NAD+/NADH ratio in the sample: sucking 50-100 μl of the sample to be tested into a centrifuge tube, and heating on a water bath or a PCR instrument at 60 ℃ for 30 minutes to decompose the NAD + and NADH content or the determination of NAD +/NADH ratio in the NAD + sample: 50-100 μl of the sample to be tested is pipetted into a centrifuge tube and heated on a 60℃water bath or PCR instrument for 30 minutes to decompose NAD +.
C. please refer to table 3 below, blank wells, standard wells and sample wells were set using 96-well plates. Adding ethanol dehydrogenase working solution, and mixing.
TABLE 3 Table 3
D. Incubate at 37℃for 10 min in the dark.
E. The color-developing solution was mixed appropriately, then 10. Mu.l of the color-developing solution was added to each well, and the mixture was incubated at 37℃for 30 minutes in the dark, at which time orange-yellow formazan was formed. The absorbance at 450nm was measured.
(4) Calculation of the amount of NAD +/NADH in the sample
A. And calculating the average absorbance of each point in the standard substance group, and subtracting the absorbance of the blank control group to obtain the absorbance of each standard substance. And drawing a standard curve by taking the concentration of NADH as an abscissa and the absorbance as an ordinate.
B. The total concentration of NAD + and NADH or the concentration of NADH in the samples of cells, tissues, etc. is calculated according to the standard curve. The concentration of NAD + and the total amount of NADH in the sample was detected without heat treatment at 60 ℃ (NADtotal); after heat treatment at 60 ℃, the concentration of NADH in the sample was detected.
C. The amount of NAD + in the sample was calculated according to the following calculation formula
[NAD+] = [NADtotal]- [NADH]
Conclusion: the concentration of NADtotal in peony callus was: 1.795 Mu M; the concentration of NADH is: 1.7722. Mu.M, calculated to: the concentration of NAD + is: 15.9216mg/g, approximately 1000 times the NAD + content in human blood.
Effect example 5 detection of soothing efficacy of peony callus extract
5.1 Effect of peony callus extract on expression of soothing marker Gene
The specific experimental steps are as follows:
(1) Preparing a culture solution containing peony callus extracts: diluting the peony callus extract prepared in the embodiment 1 into different concentration gradients by adopting a DMEM minimal medium, and screening to obtain a DMEM culture solution containing 1% of peony callus as a test sample.
(2) Preparation of HaCaT cells: haCaT cells with good growth state were inoculated into 6-well culture plates at a density of 5×10 3 cells per well, and cultured overnight at 37℃in a 5% CO 2 environment.
(3) Adding culture solution containing peony callus extract: the waste culture solution in the cell culture plate cultured overnight was removed, and corresponding treatments were performed according to the control group and the sample group (set sample group: DMEM culture solution 2 mL containing 1% callus of 1% peony; control group: DMEM culture solution 2 mL), 3 replicates were set for each treatment, and incubation was performed 24 h after addition.
(4) Whole genome assay excavated cell skin care target gene expression: removing the culture medium of the cells, collecting the cells, adding a lysate, extracting total RNA of the cells, detecting the concentration and purity of the RNA, measuring the gene expression of the whole genome of the cells after the peony callus extract is treated by adopting an RNA-Seq sequencing technology, and excavating the genes of skin care targets of the cells.
(5) Analyzing the relative expression level of the receptor genes of the cell receptor: the obtained whole gene sequence file was mass analyzed using Nanoplot and low quality sequences were filtered out. And then carrying out reference genome expression quantity comparison and calculation on the cell gene sample by using hisat software and DESeq2 software, and finally carrying out gene expression quantity difference analysis by using DESeq2 to obtain a cell receptor related gene TPRV (tansient receptor potential vanilloid, transient receptor potential channel).
As shown in the figure 10, the peony callus extract can inhibit the expression of the gene TPRV4 related to the sensory nerves of the HaCaT cells, and the expression is reduced by 21.98%; inhibiting TSLP gene expression (TSLP is one of induction factors of interleukin 17), reducing its expression level by 76.31%, and reducing the generation of lymphoimmune factor.
5.2 Detection of inhibition of histamine Release by peony callus extract
There are many histamine substances in the skin, which are released when the skin is damaged or inflammatory and allergic reactions occur. Histamine has strong vasodilation effect, and can increase permeability of capillary vessel and vena cava, and plasma leaks into tissue, resulting in local tissue edema, and further inducing skin pain and itching feeling. The method of enzyme linked immunosorbent assay can sensitively detect the change of the histamine content in the cell sample.
The specific experimental method is as follows:
(1) Preparing a test sample: the peony callus extracts prepared in example 1 and the peony extract prepared in comparative example 4 were diluted to different concentration gradients respectively using DMEM minimal medium, and were grouped as follows:
Test example sample (0.5% mdc): DMEM broth containing 0.5% of peony callus extract prepared in example 1;
Comparative sample (0.5% MD): DMEM broth containing 0.5% of the peony extract prepared in comparative example 4.
(2) Preparation of mast cells: mast cells were centrifuged and resuspended in PBS to a cell concentration of about 1X 10 6/mL, and then added to complete medium containing C48/80 (5. Mu.g/mL), incubated at 37℃for 1h and centrifuged.
(3) Sample solution adding treatment: the waste culture solution in the cell culture plate cultured overnight was removed, and corresponding treatments were performed according to the sample in (1), 3 replicates were set for each treatment, and 24h was cultured in a 5% CO 2 incubator at 37℃after addition.
(4) Measurement of mast cell histamine content by enzyme-linked immunosorbent assay: centrifuging the cultured mast cells to obtain a supernatant, repeatedly freezing and thawing (so as to destroy cells and release intracellular components), and detecting by using a human Histamine (HIS) enzyme-linked immunosorbent assay kit: 50 mu L of each of the sample supernatant and the standard substance was added to the enzyme-labeled coated plate, 50 mu L of biotin antigen working solution was added, and the reaction was carried out at 37℃for 30 min. Plates were washed 5 times, 50. Mu.L of avidin-HRP working solution was added and reacted at 37℃for 30 min times. The plate was washed 5 times, 50. Mu.L of each of the color-developing solutions A, B was added, and reacted at 37℃for 10: 10 min. 50. Mu.L of stop solution was added. The OD values within 10 min at 450 nm were read using a microplate reader.
The results are shown in figure 11, where an increase in cell histamine levels after the model group addition stimulus, compared to the control group, indicates that the cells were now in a sensitive inflammatory state, followed by the addition of different concentrations of MDC and MD treatments. The decrease in histamine levels occurred in both experimental and model groups, indicating that both MDC and MD were effective in reducing intracellular histamine levels and reducing the state of sensitivity. Wherein the histamine content after MDC treatment is obviously lower than MD, the intracellular histamine content after 0.5% peony callus extract treatment is 0.42 ng/mL, and the intracellular histamine content after 0.5% peony extract treatment is 1.40 ng/mL. From the above, the peony callus extract has better effect of relieving histamine release than the peony extract, i.e. the peony callus extract has better effect of relieving histamine release.
Application example 1
1. The application example provides a face cream, which comprises the peony callus extract prepared in the example 1, and the specific formula is shown in table 4.
TABLE 4 Table 4
The preparation method of the face cream comprises the following steps:
(1) Sequentially weighing the phase A raw materials, stirring and dispersing uniformly, heating to 80-85 ℃, and preserving heat for 30 minutes; (2) Sequentially weighing the phase B raw materials, putting into an oil pan, heating to 80-85 ℃, and completely dissolving; (3) Pumping the B-phase raw material into the A-phase emulsifying pot, homogenizing for 5-7 minutes, and uniformly stirring; (4) Cooling to 50-60deg.C, adding C phase material, and stirring; (5) Cooling to 40-45deg.C, adding phase D material, and stirring; (6) And filtering and discharging by using 200-mesh filter cloth after each physicochemical index is qualified.
Application example 2
The application example provides a shampoo, which comprises the peony callus extract prepared in the example 1, and the specific formula is shown in table 5.
TABLE 5
The preparation method of the shampoo comprises the following steps:
(1) Stirring and dissolving the phase A uniformly, and heating to 75-80 ℃ while stirring; (2) adding the phase B and stirring uniformly; (3) adding the phase C and stirring uniformly; (4) cooling to 55-60 ℃, adding phase D, and stirring uniformly; (5) Cooling to 40-45deg.C, adding phase E and phase F, and stirring; (6) Detecting physicochemical indexes, cooling to 30-35deg.C after being qualified, and filtering with 400 mesh sieve.
Application example 3
The application example provides an essence, which comprises the peony callus extract prepared in the example 1, and the specific formula is shown in table 6.
TABLE 6
The preparation method of the essence comprises the following steps:
(1) Sequentially weighing the phase A raw materials, stirring and dispersing uniformly, heating to 80-85 ℃, and preserving heat for 30 minutes; (2) Cooling to 40-45deg.C, adding phase B material, stirring, adding phase C material, and stirring; (3) And filtering and discharging by using 200-mesh filter cloth after each physicochemical index is qualified.
Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention.
Claims (13)
1. The preparation method of the peony callus extract is characterized by comprising the following steps:
(1) Freeze-drying fresh peony callus to obtain freeze-dried peony callus;
(2) Grinding the freeze-dried peony callus to powder to obtain peony callus powder;
(3) Mixing peony callus powder and an organic extracting solution, performing ultrasonic extraction, and filtering to obtain peony callus extracting solution;
(4) Removing the solvent from the peony callus extract to obtain the peony callus extract;
The organic extracting solution is ethanol;
the feed liquid ratio of the peony callus powder to the organic extracting solution in the step (3) is 1:10-20.
2. The method for preparing peony callus extract according to claim 1, wherein the concentration of ethanol is 30% -70%.
3. The method for preparing peony callus extract according to claim 1, wherein the condition parameters of freeze drying are cold trap temperature of-80 ℃, freezing chamber temperature of-45 ℃ to-35 ℃, vacuum degree of 0-10 pa and vacuum freezing time of 36-60 h.
4. The method for preparing peony callus extract according to claim 1, wherein the condition parameters of the ultrasonic extraction in the step (3) are as follows: ultrasonic power 2000W, ultrasonic temperature 25-45 deg.C, ultrasonic time 20-40min, ultrasonic extraction 3 times.
5. The method of preparing peony callus extract according to claim 1, wherein the solvent is removed in step (4) by rotary evaporation.
6. The method for preparing peony callus extract according to claim 5, wherein the rotary evaporation comprises three rotary evaporation links, and the condition parameters of the first rotary evaporation link are as follows: steaming at 35-42deg.C at 80-120 rpm for 40-50min; the condition parameters of the second rotary evaporation link are as follows: rotary steaming at 48 deg.C at 80-120 rpm for 30-45min; the condition parameters of the third rotary evaporation link are 48 ℃, the rotating speed is 80-120 rpm, and the rotary evaporation is 30-45 min.
7. A peony callus extract, characterized in that the peony callus extract is prepared by the preparation method of the peony callus extract according to any one of claims 1-6.
8. The use of peony callus extract as defined in claim 7 for preparing skin care products with anti-aging effect.
9. Use of the peony callus extract as defined in claim 7 for preparing skin care products with soothing effect.
10. The use of peony callus extract as defined in claim 7 for preparing skin care product with skin repair effect.
11. A skin care product, characterized in that it comprises the peony callus extract as claimed in claim 7.
12. The skin care product according to claim 11, wherein the addition amount of peony callus extract in the skin care product is 0.1% -5%.
13. The skin care product according to claim 11, wherein the skin care product comprises a face cream, eye cream, toner or essence.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410238819.5A CN117815148B (en) | 2024-03-04 | 2024-03-04 | Peony callus extract and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410238819.5A CN117815148B (en) | 2024-03-04 | 2024-03-04 | Peony callus extract and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN117815148A CN117815148A (en) | 2024-04-05 |
| CN117815148B true CN117815148B (en) | 2024-06-04 |
Family
ID=90504389
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202410238819.5A Active CN117815148B (en) | 2024-03-04 | 2024-03-04 | Peony callus extract and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117815148B (en) |
Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20050041259A (en) * | 2003-10-30 | 2005-05-04 | 윤성용 | Use of a callus for development of natural cosmetics |
| CN108721203A (en) * | 2018-08-09 | 2018-11-02 | 广州暨南生物医药研究开发基地有限公司 | Skin care item and preparation method thereof are repaired in a kind of whitening of the extract of stem cell containing tree peony |
| CN109602006A (en) * | 2019-01-09 | 2019-04-12 | 山东贝世康生物科技有限公司 | A kind of culture method of purification and application containing tree peony Stem Cell Activity object |
| KR101981016B1 (en) * | 2018-11-02 | 2019-05-21 | 남연희 | Functional moisturizing, antioxidant and wrinkle-improving functional cosmetic composition and preparing method thereof |
| KR102119511B1 (en) * | 2019-12-30 | 2020-06-05 | 주식회사 다이아린 | Functional cosmetic composition |
| CN111494262A (en) * | 2020-04-27 | 2020-08-07 | 渠志灿 | Peony extract and preparation method thereof |
| CN111528096A (en) * | 2020-06-03 | 2020-08-14 | 西藏农牧学院 | Callus induction method for peony embryos |
| CN113855599A (en) * | 2021-10-28 | 2021-12-31 | 济南泽润生物科技有限公司 | Whitening anti-inflammatory peony extract, preparation method thereof and application thereof in cosmetics |
| CN114209624A (en) * | 2021-12-24 | 2022-03-22 | 美出莱(杭州)化妆品有限责任公司 | Peony fermentation stock solution and preparation method and application thereof |
| CN115252530A (en) * | 2022-05-09 | 2022-11-01 | 蓝宇创科(广州)生物科技有限公司 | Peony root-bark cell sap, skin external preparation containing peony root-bark cell sap, and preparation method and application of peony root-bark cell sap |
| CN116492261A (en) * | 2023-05-26 | 2023-07-28 | 美出莱(杭州)化妆品有限责任公司 | Supermolecule peony peptide composition and preparation method thereof |
| CN117442650A (en) * | 2023-08-28 | 2024-01-26 | 信康美(福建)化妆品有限公司 | Application of alcohol extract of paeonia suffruticosa root in preparation of anti-inflammatory composition |
-
2024
- 2024-03-04 CN CN202410238819.5A patent/CN117815148B/en active Active
Patent Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20050041259A (en) * | 2003-10-30 | 2005-05-04 | 윤성용 | Use of a callus for development of natural cosmetics |
| CN108721203A (en) * | 2018-08-09 | 2018-11-02 | 广州暨南生物医药研究开发基地有限公司 | Skin care item and preparation method thereof are repaired in a kind of whitening of the extract of stem cell containing tree peony |
| KR101981016B1 (en) * | 2018-11-02 | 2019-05-21 | 남연희 | Functional moisturizing, antioxidant and wrinkle-improving functional cosmetic composition and preparing method thereof |
| CN109602006A (en) * | 2019-01-09 | 2019-04-12 | 山东贝世康生物科技有限公司 | A kind of culture method of purification and application containing tree peony Stem Cell Activity object |
| KR102119511B1 (en) * | 2019-12-30 | 2020-06-05 | 주식회사 다이아린 | Functional cosmetic composition |
| CN111494262A (en) * | 2020-04-27 | 2020-08-07 | 渠志灿 | Peony extract and preparation method thereof |
| CN111528096A (en) * | 2020-06-03 | 2020-08-14 | 西藏农牧学院 | Callus induction method for peony embryos |
| CN113855599A (en) * | 2021-10-28 | 2021-12-31 | 济南泽润生物科技有限公司 | Whitening anti-inflammatory peony extract, preparation method thereof and application thereof in cosmetics |
| CN114209624A (en) * | 2021-12-24 | 2022-03-22 | 美出莱(杭州)化妆品有限责任公司 | Peony fermentation stock solution and preparation method and application thereof |
| CN115252530A (en) * | 2022-05-09 | 2022-11-01 | 蓝宇创科(广州)生物科技有限公司 | Peony root-bark cell sap, skin external preparation containing peony root-bark cell sap, and preparation method and application of peony root-bark cell sap |
| CN116492261A (en) * | 2023-05-26 | 2023-07-28 | 美出莱(杭州)化妆品有限责任公司 | Supermolecule peony peptide composition and preparation method thereof |
| CN117442650A (en) * | 2023-08-28 | 2024-01-26 | 信康美(福建)化妆品有限公司 | Application of alcohol extract of paeonia suffruticosa root in preparation of anti-inflammatory composition |
Non-Patent Citations (3)
| Title |
|---|
| Xiangtao,et.al..Callus induction and transcriptomic analysis of in vitro embryos at different developmental stages of peony.《Frontiers in Plant Science》.2023,第第13卷卷第1-14页. * |
| 王吉凤等.5 个芍药品种愈伤组织诱导及分化研究.《北京林业大学学报》.2010,第第32卷卷(第第3期期),第213-216页. * |
| 肖培根.《中华医学百科全书》.中国协和医科大学出版社,2022,第139页. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN117815148A (en) | 2024-04-05 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Dhindsa et al. | Water stress and protein synthesis: V. protein synthesis, protein stability, and membrane permeability in a drought-sensitive and a drought-tolerant Moss | |
| CN110538112A (en) | Glycyrrhiza glabra extract composition with function of enhancing skin immune barrier and preparation method and application thereof | |
| Sanguigno et al. | Oligosaccharidic fractions derived from Triticum vulgare extract accelerate tissutal repairing processes in in vitro and in vivo models of skin lesions | |
| CN116120420A (en) | Bird's nest polypeptide composition, preparation method thereof and application thereof in anti-aging and whitening | |
| CN117815148B (en) | Peony callus extract and preparation method and application thereof | |
| TW202106329A (en) | Viola mandshuricaextract, extraction method thereof, and use thereof for enhancing fibroblast cell proliferation, for enhancing mitochondrial activity, and for increasing skin luster | |
| CN109414600A (en) | The extract of sensitive plant neoblast and its purposes in beautifying skin | |
| DMT et al. | Antioxidant activities of crude extracts from medicinal plants used by diabetic patients in Eastern Botswana | |
| WO2021196079A1 (en) | Energy-activating, anti-wrinkle, anti-inflammatory, and whitening orchid embryo tissue extract | |
| Danial et al. | Seed histology of recalcitrant Eurycoma longifolia plants during germination and its beneficial attribute for hairy roots production | |
| CN113855597A (en) | Antioxidant, anti-inflammatory and soothing repair composition, preparation method and application thereof | |
| Abdel-Aziz et al. | In vitro Cytotoxicity, Antimicrobial, Antioxidant Activities and HPLC Finger Print Analyses of the Extracts of Ceiba insignis Leaves Growing in Egypt | |
| US20180303749A1 (en) | Cosmetic use of extracts derived from somatic embryo enriched plant cell cultures and cosmetic compositions containing those extracts | |
| Nurviani et al. | The inhibition of Tobamovirus by using the extract of banana flower | |
| TWI793940B (en) | Method for preparing ferment of saussurea involucrata and skin complexion improved composition | |
| CN118121514B (en) | Whitening plant composition and application thereof | |
| CN104069156A (en) | Medicine composition for treating rhinitis as well as preparation method and application thereof | |
| Bagam et al. | A NEW REPORT ON CORDYCEPS CICADAE FROM WESTERN GHATS, INDIA | |
| Grochowska et al. | Changes in the leucoanthocyanidin content in the leaves of biennially bearing apple trees | |
| CN114848566B (en) | Preparation method and application of dendrobium officinale extract for resisting skin photoaging | |
| CN112174848B (en) | Oleoylethanolamide compound with antibacterial activity in parasitic loranthus, preparation method and application thereof | |
| Rizka | Development of a gel spray formulation based on banana peel (Musa paradisiaca L.) as an approach to support environmental sustainability | |
| CN118319803A (en) | Deep repair composition and application thereof | |
| Reddy et al. | Optimization of biomass yield and asiaticoside accumulation in the callus cultures from the leaves of Centella asiatica (L). urban | |
| Grilli et al. | Influence of age and storage temperature on RNA metabolism in durum wheat seeds |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |