JP2020003352A - Method of screening for skin aging improving agent - Google Patents
Method of screening for skin aging improving agent Download PDFInfo
- Publication number
- JP2020003352A JP2020003352A JP2018123293A JP2018123293A JP2020003352A JP 2020003352 A JP2020003352 A JP 2020003352A JP 2018123293 A JP2018123293 A JP 2018123293A JP 2018123293 A JP2018123293 A JP 2018123293A JP 2020003352 A JP2020003352 A JP 2020003352A
- Authority
- JP
- Japan
- Prior art keywords
- skin aging
- chemerin
- expression level
- screening
- skin
- 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.)
- Granted
Links
- 230000009759 skin aging Effects 0.000 title claims abstract description 51
- 238000000034 method Methods 0.000 title claims abstract description 36
- 238000012216 screening Methods 0.000 title claims abstract description 25
- 101710170513 Retinoic acid receptor responder protein 2 Proteins 0.000 claims abstract description 41
- 102100033914 Retinoic acid receptor responder protein 2 Human genes 0.000 claims abstract description 40
- 230000000694 effects Effects 0.000 claims abstract description 24
- 230000014509 gene expression Effects 0.000 claims description 53
- 210000004027 cell Anatomy 0.000 claims description 39
- 239000000203 mixture Substances 0.000 claims description 28
- 108090000623 proteins and genes Proteins 0.000 claims description 27
- 239000000126 substance Substances 0.000 claims description 27
- 235000013305 food Nutrition 0.000 claims description 25
- 210000004003 subcutaneous fat Anatomy 0.000 claims description 16
- 238000012360 testing method Methods 0.000 claims description 16
- 102000004169 proteins and genes Human genes 0.000 claims description 9
- 238000002360 preparation method Methods 0.000 claims description 6
- 238000013461 design Methods 0.000 claims description 5
- 230000009471 action Effects 0.000 claims description 4
- 208000024891 symptom Diseases 0.000 abstract description 12
- 210000003207 subcutaneous adipocyte Anatomy 0.000 abstract description 4
- 238000013459 approach Methods 0.000 abstract description 3
- 239000004129 EU approved improving agent Substances 0.000 abstract 2
- 210000003491 skin Anatomy 0.000 description 20
- 239000002609 medium Substances 0.000 description 18
- 239000003795 chemical substances by application Substances 0.000 description 17
- 108020004999 messenger RNA Proteins 0.000 description 17
- 239000002904 solvent Substances 0.000 description 15
- 239000000284 extract Substances 0.000 description 11
- 239000000047 product Substances 0.000 description 10
- 241000196324 Embryophyta Species 0.000 description 9
- 101000756632 Homo sapiens Actin, cytoplasmic 1 Proteins 0.000 description 8
- 230000007423 decrease Effects 0.000 description 8
- -1 polyoxyethylene Polymers 0.000 description 8
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 description 7
- 230000003712 anti-aging effect Effects 0.000 description 7
- 235000009508 confectionery Nutrition 0.000 description 7
- 210000002950 fibroblast Anatomy 0.000 description 7
- 229920002674 hyaluronan Polymers 0.000 description 7
- 229960003160 hyaluronic acid Drugs 0.000 description 7
- 235000018102 proteins Nutrition 0.000 description 7
- 238000012258 culturing Methods 0.000 description 6
- 238000004519 manufacturing process Methods 0.000 description 6
- 239000004278 EU approved seasoning Substances 0.000 description 5
- 101710128038 Hyaluronan synthase Proteins 0.000 description 5
- 238000000605 extraction Methods 0.000 description 5
- 235000011194 food seasoning agent Nutrition 0.000 description 5
- 235000013611 frozen food Nutrition 0.000 description 5
- 238000005259 measurement Methods 0.000 description 5
- PUPZLCDOIYMWBV-UHFFFAOYSA-N (+/-)-1,3-Butanediol Chemical compound CC(O)CCO PUPZLCDOIYMWBV-UHFFFAOYSA-N 0.000 description 4
- 230000006820 DNA synthesis Effects 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 108091013859 Mimecan Proteins 0.000 description 4
- 238000009004 PCR Kit Methods 0.000 description 4
- 238000011529 RT qPCR Methods 0.000 description 4
- 206010040954 Skin wrinkling Diseases 0.000 description 4
- 235000014113 dietary fatty acids Nutrition 0.000 description 4
- 238000011156 evaluation Methods 0.000 description 4
- 229930195729 fatty acid Natural products 0.000 description 4
- 239000000194 fatty acid Substances 0.000 description 4
- 239000000796 flavoring agent Substances 0.000 description 4
- 235000019634 flavors Nutrition 0.000 description 4
- 210000002435 tendon Anatomy 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 230000037303 wrinkles Effects 0.000 description 4
- ZWEHNKRNPOVVGH-UHFFFAOYSA-N 2-Butanone Chemical compound CCC(C)=O ZWEHNKRNPOVVGH-UHFFFAOYSA-N 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 3
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 102000027307 Mimecan Human genes 0.000 description 3
- 229920003171 Poly (ethylene oxide) Polymers 0.000 description 3
- 238000011530 RNeasy Mini Kit Methods 0.000 description 3
- 239000002299 complementary DNA Substances 0.000 description 3
- 238000012790 confirmation Methods 0.000 description 3
- 230000002500 effect on skin Effects 0.000 description 3
- 210000005175 epidermal keratinocyte Anatomy 0.000 description 3
- 230000036541 health Effects 0.000 description 3
- 235000012149 noodles Nutrition 0.000 description 3
- 235000014347 soups Nutrition 0.000 description 3
- 150000005846 sugar alcohols Polymers 0.000 description 3
- 241000251468 Actinopterygii Species 0.000 description 2
- 102000014777 Adipokines Human genes 0.000 description 2
- 108010078606 Adipokines Proteins 0.000 description 2
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- 240000007049 Juglans regia Species 0.000 description 2
- 235000009496 Juglans regia Nutrition 0.000 description 2
- 244000294411 Mirabilis expansa Species 0.000 description 2
- 235000015429 Mirabilis expansa Nutrition 0.000 description 2
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 2
- 235000010627 Phaseolus vulgaris Nutrition 0.000 description 2
- 244000046052 Phaseolus vulgaris Species 0.000 description 2
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 230000032683 aging Effects 0.000 description 2
- 150000001298 alcohols Chemical class 0.000 description 2
- 235000013361 beverage Nutrition 0.000 description 2
- 235000008429 bread Nutrition 0.000 description 2
- 150000001720 carbohydrates Chemical class 0.000 description 2
- 235000014633 carbohydrates Nutrition 0.000 description 2
- 235000013339 cereals Nutrition 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 238000010411 cooking Methods 0.000 description 2
- 235000013365 dairy product Nutrition 0.000 description 2
- 210000004207 dermis Anatomy 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 235000013399 edible fruits Nutrition 0.000 description 2
- 239000003925 fat Substances 0.000 description 2
- 235000019197 fats Nutrition 0.000 description 2
- 235000013332 fish product Nutrition 0.000 description 2
- 235000013312 flour Nutrition 0.000 description 2
- 239000003205 fragrance Substances 0.000 description 2
- 235000013376 functional food Nutrition 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 244000144972 livestock Species 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 235000013336 milk Nutrition 0.000 description 2
- 239000008267 milk Substances 0.000 description 2
- 210000004080 milk Anatomy 0.000 description 2
- 235000013536 miso Nutrition 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- GLDOVTGHNKAZLK-UHFFFAOYSA-N octadecan-1-ol Chemical compound CCCCCCCCCCCCCCCCCCO GLDOVTGHNKAZLK-UHFFFAOYSA-N 0.000 description 2
- 239000003921 oil Substances 0.000 description 2
- 235000019198 oils Nutrition 0.000 description 2
- 206010033675 panniculitis Diseases 0.000 description 2
- 150000008442 polyphenolic compounds Chemical class 0.000 description 2
- 235000013824 polyphenols Nutrition 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 235000015067 sauces Nutrition 0.000 description 2
- 235000013580 sausages Nutrition 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 235000013322 soy milk Nutrition 0.000 description 2
- PRAKJMSDJKAYCZ-UHFFFAOYSA-N squalane Chemical compound CC(C)CCCC(C)CCCC(C)CCCCC(C)CCCC(C)CCCC(C)C PRAKJMSDJKAYCZ-UHFFFAOYSA-N 0.000 description 2
- 238000007920 subcutaneous administration Methods 0.000 description 2
- 210000004304 subcutaneous tissue Anatomy 0.000 description 2
- 230000014616 translation Effects 0.000 description 2
- 235000020234 walnut Nutrition 0.000 description 2
- ALSTYHKOOCGGFT-KTKRTIGZSA-N (9Z)-octadecen-1-ol Chemical compound CCCCCCCC\C=C/CCCCCCCCO ALSTYHKOOCGGFT-KTKRTIGZSA-N 0.000 description 1
- WRIDQFICGBMAFQ-UHFFFAOYSA-N (E)-8-Octadecenoic acid Natural products CCCCCCCCCC=CCCCCCCC(O)=O WRIDQFICGBMAFQ-UHFFFAOYSA-N 0.000 description 1
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 1
- LEACJMVNYZDSKR-UHFFFAOYSA-N 2-octyldodecan-1-ol Chemical compound CCCCCCCCCCC(CO)CCCCCCCC LEACJMVNYZDSKR-UHFFFAOYSA-N 0.000 description 1
- GECRRQVLQHRVNH-MRCUWXFGSA-N 2-octyldodecyl (z)-octadec-9-enoate Chemical compound CCCCCCCCCCC(CCCCCCCC)COC(=O)CCCCCCC\C=C/CCCCCCCC GECRRQVLQHRVNH-MRCUWXFGSA-N 0.000 description 1
- LQJBNNIYVWPHFW-UHFFFAOYSA-N 20:1omega9c fatty acid Natural products CCCCCCCCCCC=CCCCCCCCC(O)=O LQJBNNIYVWPHFW-UHFFFAOYSA-N 0.000 description 1
- QSBYPNXLFMSGKH-UHFFFAOYSA-N 9-Heptadecensaeure Natural products CCCCCCCC=CCCCCCCCC(O)=O QSBYPNXLFMSGKH-UHFFFAOYSA-N 0.000 description 1
- 108010011485 Aspartame Proteins 0.000 description 1
- 102000008186 Collagen Human genes 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- 102000012422 Collagen Type I Human genes 0.000 description 1
- 108010022452 Collagen Type I Proteins 0.000 description 1
- 241001137251 Corvidae Species 0.000 description 1
- 229920000858 Cyclodextrin Polymers 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 1
- 229920001353 Dextrin Polymers 0.000 description 1
- 239000004375 Dextrin Substances 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 102000016942 Elastin Human genes 0.000 description 1
- 108010014258 Elastin Proteins 0.000 description 1
- 239000004386 Erythritol Substances 0.000 description 1
- UNXHWFMMPAWVPI-UHFFFAOYSA-N Erythritol Natural products OCC(O)C(O)CO UNXHWFMMPAWVPI-UHFFFAOYSA-N 0.000 description 1
- 229930091371 Fructose Natural products 0.000 description 1
- 239000005715 Fructose Substances 0.000 description 1
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 102000003918 Hyaluronan Synthases Human genes 0.000 description 1
- 108090000320 Hyaluronan Synthases Proteins 0.000 description 1
- 208000031226 Hyperlipidaemia Diseases 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 235000007688 Lycopersicon esculentum Nutrition 0.000 description 1
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 1
- YBHQCJILTOVLHD-YVMONPNESA-N Mirin Chemical compound S1C(N)=NC(=O)\C1=C\C1=CC=C(O)C=C1 YBHQCJILTOVLHD-YVMONPNESA-N 0.000 description 1
- 208000008589 Obesity Diseases 0.000 description 1
- 239000005642 Oleic acid Substances 0.000 description 1
- ZQPPMHVWECSIRJ-UHFFFAOYSA-N Oleic acid Natural products CCCCCCCCC=CCCCCCCCC(O)=O ZQPPMHVWECSIRJ-UHFFFAOYSA-N 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 239000004264 Petrolatum Substances 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 240000003768 Solanum lycopersicum Species 0.000 description 1
- 235000021355 Stearic acid Nutrition 0.000 description 1
- 244000228451 Stevia rebaudiana Species 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 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 1
- ULUAUXLGCMPNKK-UHFFFAOYSA-N Sulfobutanedioic acid Chemical class OC(=O)CC(C(O)=O)S(O)(=O)=O ULUAUXLGCMPNKK-UHFFFAOYSA-N 0.000 description 1
- 244000269722 Thea sinensis Species 0.000 description 1
- TVXBFESIOXBWNM-UHFFFAOYSA-N Xylitol Natural products OCCC(O)C(O)C(O)CCO TVXBFESIOXBWNM-UHFFFAOYSA-N 0.000 description 1
- 239000006096 absorbing agent Substances 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 239000000478 adipokine Substances 0.000 description 1
- 235000013334 alcoholic beverage Nutrition 0.000 description 1
- 150000005215 alkyl ethers Chemical class 0.000 description 1
- 150000008051 alkyl sulfates Chemical class 0.000 description 1
- SHGAZHPCJJPHSC-YCNIQYBTSA-N all-trans-retinoic acid Chemical compound OC(=O)\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C SHGAZHPCJJPHSC-YCNIQYBTSA-N 0.000 description 1
- 239000002280 amphoteric surfactant Substances 0.000 description 1
- 239000003945 anionic surfactant Substances 0.000 description 1
- 230000001153 anti-wrinkle effect Effects 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- BTFJIXJJCSYFAL-UHFFFAOYSA-N arachidyl alcohol Natural products CCCCCCCCCCCCCCCCCCCCO BTFJIXJJCSYFAL-UHFFFAOYSA-N 0.000 description 1
- 239000000605 aspartame Substances 0.000 description 1
- 235000010357 aspartame Nutrition 0.000 description 1
- IAOZJIPTCAWIRG-QWRGUYRKSA-N aspartame Chemical compound OC(=O)C[C@H](N)C(=O)N[C@H](C(=O)OC)CC1=CC=CC=C1 IAOZJIPTCAWIRG-QWRGUYRKSA-N 0.000 description 1
- 229960003438 aspartame Drugs 0.000 description 1
- 210000000270 basal cell Anatomy 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 description 1
- 235000015895 biscuits Nutrition 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 235000014121 butter Nutrition 0.000 description 1
- 238000010804 cDNA synthesis Methods 0.000 description 1
- 235000012970 cakes Nutrition 0.000 description 1
- 235000013574 canned fruits Nutrition 0.000 description 1
- 235000014613 canned/preserved soup Nutrition 0.000 description 1
- 235000013736 caramel Nutrition 0.000 description 1
- 239000004203 carnauba wax Substances 0.000 description 1
- 235000013869 carnauba wax Nutrition 0.000 description 1
- 229940082483 carnauba wax Drugs 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 235000013351 cheese Nutrition 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 235000015218 chewing gum Nutrition 0.000 description 1
- 229940112822 chewing gum Drugs 0.000 description 1
- 235000019219 chocolate Nutrition 0.000 description 1
- 238000003501 co-culture Methods 0.000 description 1
- 239000003240 coconut oil Substances 0.000 description 1
- 235000019864 coconut oil Nutrition 0.000 description 1
- 235000013353 coffee beverage Nutrition 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 235000014510 cooky Nutrition 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 235000012495 crackers Nutrition 0.000 description 1
- 239000006071 cream Substances 0.000 description 1
- 235000021438 curry Nutrition 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 235000021185 dessert Nutrition 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 235000019425 dextrin Nutrition 0.000 description 1
- 125000005131 dialkylammonium group Chemical group 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 150000002016 disaccharides Chemical class 0.000 description 1
- 235000015071 dressings Nutrition 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 229920002549 elastin Polymers 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 235000019414 erythritol Nutrition 0.000 description 1
- UNXHWFMMPAWVPI-ZXZARUISSA-N erythritol Chemical compound OC[C@H](O)[C@H](O)CO UNXHWFMMPAWVPI-ZXZARUISSA-N 0.000 description 1
- 229940009714 erythritol Drugs 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 150000002170 ethers Chemical class 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 230000037406 food intake Effects 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 239000003349 gelling agent Substances 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 241000411851 herbal medicine Species 0.000 description 1
- 229930195733 hydrocarbon Natural products 0.000 description 1
- 150000002430 hydrocarbons Chemical class 0.000 description 1
- 235000015243 ice cream Nutrition 0.000 description 1
- 238000010191 image analysis Methods 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000000415 inactivating effect Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 235000008446 instant noodles Nutrition 0.000 description 1
- 235000014109 instant soup Nutrition 0.000 description 1
- QXJSBBXBKPUZAA-UHFFFAOYSA-N isooleic acid Natural products CCCCCCCC=CCCCCCCCCC(O)=O QXJSBBXBKPUZAA-UHFFFAOYSA-N 0.000 description 1
- 229940119170 jojoba wax Drugs 0.000 description 1
- 210000002510 keratinocyte Anatomy 0.000 description 1
- 150000002576 ketones Chemical class 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 235000021056 liquid food Nutrition 0.000 description 1
- 239000006210 lotion Substances 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 235000013310 margarine Nutrition 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- HEBKCHPVOIAQTA-UHFFFAOYSA-N meso ribitol Natural products OCC(O)C(O)C(O)CO HEBKCHPVOIAQTA-UHFFFAOYSA-N 0.000 description 1
- 239000004200 microcrystalline wax Substances 0.000 description 1
- 235000019808 microcrystalline wax Nutrition 0.000 description 1
- 229940114937 microcrystalline wax Drugs 0.000 description 1
- 235000020124 milk-based beverage Nutrition 0.000 description 1
- 230000003020 moisturizing effect Effects 0.000 description 1
- 150000002772 monosaccharides Chemical class 0.000 description 1
- 210000001074 muscle attachment cell Anatomy 0.000 description 1
- JXTPJDDICSTXJX-UHFFFAOYSA-N n-Triacontane Natural products CCCCCCCCCCCCCCCCCCCCCCCCCCCCCC JXTPJDDICSTXJX-UHFFFAOYSA-N 0.000 description 1
- GOQYKNQRPGWPLP-UHFFFAOYSA-N n-heptadecyl alcohol Natural products CCCCCCCCCCCCCCCCCO GOQYKNQRPGWPLP-UHFFFAOYSA-N 0.000 description 1
- 239000002736 nonionic surfactant Substances 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 235000020824 obesity Nutrition 0.000 description 1
- QIQXTHQIDYTFRH-UHFFFAOYSA-N octadecanoic acid Chemical compound CCCCCCCCCCCCCCCCCC(O)=O QIQXTHQIDYTFRH-UHFFFAOYSA-N 0.000 description 1
- OQCDKBAXFALNLD-UHFFFAOYSA-N octadecanoic acid Natural products CCCCCCCC(C)CCCCCCCCC(O)=O OQCDKBAXFALNLD-UHFFFAOYSA-N 0.000 description 1
- ZQPPMHVWECSIRJ-KTKRTIGZSA-N oleic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(O)=O ZQPPMHVWECSIRJ-KTKRTIGZSA-N 0.000 description 1
- 229940055577 oleyl alcohol Drugs 0.000 description 1
- XMLQWXUVTXCDDL-UHFFFAOYSA-N oleyl alcohol Natural products CCCCCCC=CCCCCCCCCCCO XMLQWXUVTXCDDL-UHFFFAOYSA-N 0.000 description 1
- 229920001542 oligosaccharide Polymers 0.000 description 1
- 150000002482 oligosaccharides Chemical class 0.000 description 1
- 239000004006 olive oil Substances 0.000 description 1
- 235000008390 olive oil Nutrition 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 239000006072 paste Substances 0.000 description 1
- 239000002957 persistent organic pollutant Substances 0.000 description 1
- 235000019271 petrolatum Nutrition 0.000 description 1
- 229940066842 petrolatum Drugs 0.000 description 1
- 239000008194 pharmaceutical composition Substances 0.000 description 1
- 235000021110 pickles Nutrition 0.000 description 1
- 235000015108 pies Nutrition 0.000 description 1
- 239000002798 polar solvent Substances 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 229920001451 polypropylene glycol Polymers 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 235000008476 powdered milk Nutrition 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 235000013324 preserved food Nutrition 0.000 description 1
- 239000012264 purified product Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- HELXLJCILKEWJH-NCGAPWICSA-N rebaudioside A Chemical compound O([C@H]1[C@H](O)[C@@H](CO)O[C@H]([C@@H]1O[C@H]1[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O1)O)O[C@]12C(=C)C[C@@]3(C1)CC[C@@H]1[C@@](C)(CCC[C@]1([C@@H]3CC2)C)C(=O)O[C@H]1[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O1)O)[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O HELXLJCILKEWJH-NCGAPWICSA-N 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 229930002330 retinoic acid Natural products 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 235000019204 saccharin Nutrition 0.000 description 1
- CVHZOJJKTDOEJC-UHFFFAOYSA-N saccharin Chemical compound C1=CC=C2C(=O)NS(=O)(=O)C2=C1 CVHZOJJKTDOEJC-UHFFFAOYSA-N 0.000 description 1
- 229940081974 saccharin Drugs 0.000 description 1
- 239000000901 saccharin and its Na,K and Ca salt Substances 0.000 description 1
- HFHDHCJBZVLPGP-UHFFFAOYSA-N schardinger α-dextrin Chemical compound O1C(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(O)C2O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC2C(O)C(O)C1OC2CO HFHDHCJBZVLPGP-UHFFFAOYSA-N 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 230000037394 skin elasticity Effects 0.000 description 1
- 230000037393 skin firmness Effects 0.000 description 1
- 235000011888 snacks Nutrition 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 235000010356 sorbitol Nutrition 0.000 description 1
- 235000013555 soy sauce Nutrition 0.000 description 1
- 235000013599 spices Nutrition 0.000 description 1
- 235000011496 sports drink Nutrition 0.000 description 1
- 229940032094 squalane Drugs 0.000 description 1
- 239000008117 stearic acid Substances 0.000 description 1
- 235000013547 stew Nutrition 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 239000003760 tallow Substances 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 1
- 239000000892 thaumatin Substances 0.000 description 1
- 235000010436 thaumatin Nutrition 0.000 description 1
- 230000008719 thickening Effects 0.000 description 1
- 239000002562 thickening agent Substances 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 229960001727 tretinoin Drugs 0.000 description 1
- 150000003626 triacylglycerols Chemical class 0.000 description 1
- 235000015112 vegetable and seed oil Nutrition 0.000 description 1
- 239000008158 vegetable oil Substances 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- 239000000052 vinegar Substances 0.000 description 1
- 235000021419 vinegar Nutrition 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
- 239000000811 xylitol Substances 0.000 description 1
- 235000010447 xylitol Nutrition 0.000 description 1
- HEBKCHPVOIAQTA-SCDXWVJYSA-N xylitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)CO HEBKCHPVOIAQTA-SCDXWVJYSA-N 0.000 description 1
- 229960002675 xylitol Drugs 0.000 description 1
- 235000013618 yogurt Nutrition 0.000 description 1
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Cosmetics (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicines Containing Plant Substances (AREA)
- Peptides Or Proteins (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
Abstract
Description
本発明は、皮膚老化改善剤をスクリーニングする方法に関する。 The present invention relates to a method for screening a skin aging improving agent.
シワやタルミ等の皮膚老化症状は、見た目の印象に大きな影響を与えるものであるため、その改善への関心は高い。近年、皮膚老化症状の改善を目的とする、抗老化剤、抗シワ剤、保湿剤等の開発が盛んに行われている。それらは主に、コラーゲンやエラスチンといった肌の張りや弾力を保つ働きをする物質を産生する真皮の線維芽細胞をターゲットとするものや（特許文献１〜２等）、表皮基底細胞をターゲットとするものであった（例えば特許文献３等）。
Since skin aging symptoms such as wrinkles and tarmi have a great influence on the visual impression, there is a high interest in improving them. BACKGROUND ART In recent years, anti-aging agents, anti-wrinkle agents, moisturizing agents, and the like for the purpose of improving skin aging have been actively developed. They mainly target dermal fibroblasts that produce substances such as collagen and elastin that maintain skin firmness and elasticity (
従来の皮膚の抗老化剤では、ある程度の抗老化効果は認められるものの、十分に満足のいく効果が得られているとは言い難い。また、皮膚老化症状の生じるメカニズムのさらなる解明も求められている。
本発明は、かかる状況に鑑み、皮膚老化症状に対する新たなアプローチによる皮膚老化改善剤として有効な成分を探索することを目的とし、そのための新たなスクリーニング方法を確立することを課題とする。
Conventional anti-aging agents for skin have some anti-aging effects, but it is hard to say that satisfactory effects have been obtained. There is also a need for further elucidation of the mechanism by which skin aging occurs.
In view of such circumstances, an object of the present invention is to search for a component effective as a skin aging improving agent by a new approach to skin aging symptoms, and an object of the present invention is to establish a new screening method therefor.
本発明者は上記課題を解決するために鋭意研究を行った結果、アディポサイトカインのひとつであるケメリンに着目し、皮下脂肪細胞から分泌されるケメリンが真皮線維芽細胞等に影響を与えることを見出した。そして、ケメリンを不活性化することにより皮膚老化症状を改善することができるという知見を得て、本発明を完成するに至った。 The present inventors have conducted intensive studies to solve the above problems, and as a result, focused on chemerin, which is one of adipocytokines, and found that chemerin secreted from subcutaneous fat cells affects dermal fibroblasts and the like. Was. The inventors have found that inactivating chemerin can improve skin aging symptoms, and have completed the present invention.
すなわち、本発明は以下の通りである。
［１］皮下脂肪細胞中のケメリンの活性を指標として、皮膚老化改善剤をスクリーニングする方法。
［２］前記ケメリンの活性が、前記ケメリンを構成するタンパク質をコードする遺伝子又は前記タンパク質の発現量であり、被験物質を添加した皮下脂肪細胞における前記発現量が、被験物質を添加しなかった細胞における発現量と比較して小さい場合に、前記被験物質は皮膚老化改善作用を有すると判定する、［１］に記載の方法。
［３］［１］又は［２］に記載のスクリーニング方法を行う工程、及び前記工程により選択された物質を含有させる工程を含む、組成物の設計方法。
［４］前記組成物が皮膚老化改善用である、［３］に記載の設計方法。
［５］前記組成物が飲食品又は皮膚外用剤である、［３］又は［４］に記載の設計方法。
That is, the present invention is as follows.
[1] A method for screening a skin aging improving agent using the activity of chemerin in subcutaneous fat cells as an index.
[2] The activity of the chemerin is a gene encoding the protein constituting the chemerin or the expression level of the protein, and the expression level in subcutaneous adipocytes to which a test substance is added is a cell to which the test substance is not added. The method according to [1], wherein the test substance is determined to have a skin aging-improving activity when the expression level is smaller than the expression level in the above.
[3] A method for designing a composition, comprising a step of performing the screening method according to [1] or [2] and a step of including a substance selected by the step.
[4] The design method according to [3], wherein the composition is for improving skin aging.
[5] The design method according to [3] or [4], wherein the composition is a food or drink or an external preparation for skin.
本発明により、飲食品や皮膚外用剤に含有させるのに好適な、皮膚老化改善剤として有効な成分を探索できるスクリーニング方法が提供される。 According to the present invention, there is provided a screening method capable of searching for a component effective as a skin aging-improving agent, which is suitable for being contained in a food or drink or an external preparation for skin.
本発明のスクリーニングする方法は、皮下脂肪細胞中のケメリンの活性を指標とすることを特徴とする。
ケメリンは、皮下脂肪細胞から分泌されるアディポサイトカインの一種であり、加齢や肥満、高脂血症等により分泌が増えることが知られている。本発明者らは、ケメリンの発現量が増加するとその活性が増強して、皮膚老化症状の発生に関与する現象、例えば、ヒアルロン酸量、バーシカン量、皮膚支持構造（ＲＣ構造）等の減少が引き起こされることを見出した。より具体的には、ケメリンの活性化は、ヒアルロン酸又はバーシカンを合成する酵素が減少してヒアルロン酸量又はバーシカン量を減少させたり、皮下組織下部の皮下支持帯の網目構造を構成するタンパク質の発現量が減少して該網目構造が疎になったりすることを引き起こす。それらによって、真皮においてシワやタルミ等の皮膚老化症状が発現する。かかる皮膚老化フローにおいて、皮下脂肪細胞におけるケメリンの活性を減弱化することによって、皮膚老化症状が引き起こされるのを抑制し改善することができる。言い換えると、皮下脂肪細胞におけるケメリンの活性を抑制させるような物質は、皮膚老化改善剤となり得る。
The screening method of the present invention is characterized by using the activity of chemerin in subcutaneous fat cells as an index.
Chemerin is a kind of adipocytokine secreted from subcutaneous fat cells, and it is known that secretion increases due to aging, obesity, hyperlipidemia and the like. The present inventors have found that when the expression level of chemerin increases, its activity is enhanced, and phenomena related to the occurrence of skin aging symptoms, such as reduction in the amount of hyaluronic acid, versican, and the skin supporting structure (RC structure), are reduced. Found to be caused. More specifically, activation of chemerin decreases the amount of hyaluronic acid or versican by reducing the enzyme that synthesizes hyaluronic acid or versican, or reduces the amount of protein that constitutes the network structure of the subcutaneous support zone below the subcutaneous tissue. The amount of expression is reduced, and the network structure becomes sparse. By these, skin aging symptoms such as wrinkles and tarmi appear in the dermis. In such a skin aging flow, the occurrence of skin aging symptoms can be suppressed and improved by attenuating the activity of chemerin in subcutaneous fat cells. In other words, a substance that suppresses the activity of chemerin in subcutaneous fat cells can be an agent for improving skin aging.
ヒアルロン酸は、皮膚内部で貯水の役割を担っており、一般に加齢によってヒアルロン酸合成酵素が減少したり、ヒアルロン酸分解酵素が増加したりして、ヒアルロン酸量の減少が引き起こされる。ヒアルロン酸量が減少すると、肌の保水力が乏しくなり、乾燥やシワ、肌理の粗さなどが生じ、肌は老化した状態に至る。
バーシカンは、真皮が作られるときに線維芽細胞が働くための足場となるプロテオグリカンで、加齢に伴い減少し、皮膚老化や弾性力低下を招くことが知られている（フレグランスジャーナル44(1): 88 -88, 2016参照）。後述の実施例に示されるように、ケメリン
が活性化すると、バーシカン量の減少が引き起こされ、これはバーシカン産生に関与する酵素が減少したりバーシカン分解酵素が増加したりすることに因ると考えられる。
皮膚支持構造（ＲＣ構造）は、皮下組織下部の皮下支持帯（retinacula cutis；ＲＣ）と呼ばれる網目構造をいい、主にＩ型コラーゲンとミメカンという糖タンパク質とで構成される。該構造が疎になると皮膚深部の弾力性が低下し、タルミが引き起こされる（フレグランスジャーナル 44(2), 23-27, 2016参照）。後述の実施例に示されるように、ケメ
リンが活性化すると、ＲＣ構造を構成するタンパク質の発現量が減少し、ＲＣ構造の弱化（網目構造の疎化）が生じる。
Hyaluronic acid plays a role of storing water inside the skin, and generally, hyaluronic acid synthase decreases or hyaluronic acid degrading enzyme increases with aging, and the amount of hyaluronic acid decreases. When the amount of hyaluronic acid decreases, the water retaining ability of the skin becomes poor, and dryness, wrinkles, roughness of the skin and the like occur, and the skin is aged.
Versican is a proteoglycan that serves as a scaffold for fibroblasts to work when the dermis is made, and is known to decrease with age, leading to skin aging and decreased elasticity (Fragrance Journal 44 (1) : 88 -88, 2016). As shown in the Examples below, activation of chemerin causes a decrease in the amount of versican, which is thought to be due to a decrease in enzymes involved in versican production or an increase in versican-degrading enzymes. Can be
The skin support structure (RC structure) refers to a network structure called retinacula cutis (RC) below the subcutaneous tissue, and is mainly composed of type I collagen and a glycoprotein called mimecan. If the structure is sparse, the elasticity of the deep part of the skin decreases, causing tallness (see Fragrance Journal 44 (2), 23-27, 2016). As shown in the examples described below, when chemerin is activated, the expression level of the protein constituting the RC structure is reduced, and the RC structure is weakened (the network structure is loosened).
本発明のスクリーニング方法において指標となるケメリンの活性とは、通常はケメリンの発現量である。ここで、発現量とは、ケメリンを構成するタンパク質をコードする遺伝子のｍＲＮＡの転写量と、該タンパク質の翻訳量との何れかを指すものとする。 In the screening method of the present invention, the activity of chemerin, which is an indicator, is usually the expression level of chemerin. Here, the expression level refers to either the transcription level of the mRNA of the gene encoding the protein constituting chemerin or the translation level of the protein.
本発明のスクリーニング方法の好ましい態様においては、被験物質を添加した皮下脂肪細胞におけるケメリンの発現量が、被験物質を添加しなかった皮下脂肪細胞におけるケメリンの発現量（コントロール）と比較して小さい場合に、前記被験物質は皮膚老化改善作
用を有すると判定される。
In a preferred embodiment of the screening method of the present invention, the expression level of chemerin in subcutaneous fat cells to which the test substance is added is smaller than the expression level of chemerin in subcutaneous fat cells to which no test substance is added (control). Then, the test substance is determined to have a skin aging improving effect.
ケメリンをコードする遺伝子の発現量は、任意の方法を用いて測定することができる。例えば、当該遺伝子の配列に特異的に結合する配列を有するＤＮＡ断片をプライマーとして用いてＰＣＲを行い、定量的な検出を行う。なお、ケメリンをコードするヒトの遺伝子配列は公開されており、当業者は適宜プライマーを設計してＰＣＲに供することができる。
また、例えば、ケメリンタンパク質の細胞膜上の量を、常法で、例えば抗体を用いる免疫学的手法等で測定して、遺伝子の発現量としてもよい。
The expression level of the gene encoding chemerin can be measured using any method. For example, PCR is performed using a DNA fragment having a sequence that specifically binds to the sequence of the gene as a primer to perform quantitative detection. The human gene sequence encoding chemerin is publicly available, and those skilled in the art can appropriately design primers and use them for PCR.
In addition, for example, the amount of the chemerin protein on the cell membrane may be measured by a conventional method, for example, by an immunological technique using an antibody or the like, and used as the gene expression level.
本発明のスクリーニング方法に用いる細胞としては、皮下脂肪細胞を用いる。
細胞の培養の条件としては、通常の培養条件の他、本発明のスクリーニング方法の実行を妨げない、具体的にケメリンの発現量の測定を妨げない培養条件であれば、特段の限定なく適用することができる。
Subcutaneous fat cells are used as cells used in the screening method of the present invention.
As the conditions for culturing the cells, other than normal culture conditions, any culture conditions that do not hinder the execution of the screening method of the present invention and specifically do not hinder the measurement of the expression level of chemerin may be used without particular limitation. be able to.
本発明のスクリーニング方法が対象とする被験物質は、純物質、動植物由来の抽出物、またはそれらの混合物等のいずれであってもよい。
動植物由来の抽出物は、動物又は植物由来の抽出物自体のみならず、抽出物の画分、精製した画分、抽出物又は画分、精製物の溶媒除去物の総称を意味するものとし、植物由来の抽出物は、自生若しくは生育された植物、漢方生薬原料等として販売されるものを用いた抽出物、市販されている抽出物等が挙げられる。
抽出操作は、植物部位の全草を用いるほか、植物体、地上部、根茎部、木幹部、葉部、茎部、花、花蕾、果実等の部位を使用することできるが、予めこれらを粉砕あるいは細切して抽出効率を向上させることが好ましい。抽出溶媒としては、水、エタノール、イソプロピルアルコール、ブタノールなどのアルコール類、１，３−ブタンジオール、ポリプロピレングリコールなどの多価アルコール類、アセトン、メチルエチルケトンなどのケトン類、ジエチルエーテル、テトラヒドロフランなどのエーテル類等の極性溶媒から選択される一種又は二種以上が好適なものとして例示することができる。具体的な抽出方法としては、例えば、植物体等の抽出に用いる部位又はその乾燥物１質量部に対して、溶媒を１〜３０質量部加え、室温であれば数日間、沸点付近の温度であれば数時間浸漬し、室温まで冷却した後、所望により不溶物及び／又は溶媒除去し、カラムクロマトグラフィー等で分画精製する方法が挙げられる。
The test substance targeted by the screening method of the present invention may be any of a pure substance, an extract derived from animals and plants, or a mixture thereof.
An extract derived from animals and plants means not only the extract itself derived from animals or plants, but also a general term for the extract fraction, the purified fraction, the extract or fraction, and the solvent-removed product of the purified product, Examples of plant-derived extracts include native or grown plants, extracts using those sold as raw materials for Chinese herbal medicines, and commercially available extracts.
For the extraction operation, in addition to using the whole plant of the plant part, parts of the plant body, the above-ground part, the rhizome, the trunk, the leaf, the stem, the flower, the flower bud, the fruit, etc. can be used. Alternatively, it is preferable to improve the extraction efficiency by shredding. Examples of the extraction solvent include water, alcohols such as ethanol, isopropyl alcohol and butanol, polyhydric alcohols such as 1,3-butanediol and polypropylene glycol, ketones such as acetone and methyl ethyl ketone, and ethers such as diethyl ether and tetrahydrofuran. One or two or more selected from polar solvents such as the above can be exemplified as suitable ones. As a specific extraction method, for example, 1 to 30 parts by mass of a solvent is added to 1 part by mass of a site used for extraction of a plant or the like or a dried product thereof, and at room temperature for several days, at a temperature near the boiling point. If so, a method of immersing for several hours, cooling to room temperature, removing insolubles and / or solvent if desired, and fractionating and purifying by column chromatography or the like can be mentioned.
本発明のスクリーニング方法における手順の一例を以下に挙げるが、本発明の趣旨を逸脱しない限り以下の内容に限定されるものではなく、適宜変更して実施することができる。
まず、皮下脂肪細胞に被験物質を添加し、２４〜４８時間インキュベーションする。その後、該細胞からｍＲＮＡを抽出し、ケメリンをコードする遺伝子の発現量を、該遺伝子を特異的に検出するプライマーを用いてＲＴ−ＰＣＲを行い、定量的に測定する。コントロールとして被験物質を添加しなかった皮下脂肪細胞においても該遺伝子の発現量を測定する。被験物質を添加した細胞における該遺伝子の発現量が、被験物質を添加しなかった細胞における該遺伝子の発現量（コントロール）に対して小さい場合、前記被験物質は皮膚老化改善作用を有すると判定する。該判定された物質は、皮膚老化改善用の組成物に好適に含有し得る皮膚老化改善剤となり得る。
なお、発現量の変動の程度としては、コントロールに対して８０％以下が好ましく、６５％以下がより好ましく、５０％以下がさらに好ましい。
An example of the procedure in the screening method of the present invention will be described below, but the present invention is not limited to the following contents without departing from the gist of the present invention, and can be appropriately modified and carried out.
First, a test substance is added to subcutaneous fat cells, and incubated for 24 to 48 hours. Thereafter, mRNA is extracted from the cells, and the expression level of the gene encoding chemerin is quantitatively measured by RT-PCR using primers that specifically detect the gene. As a control, the expression level of the gene is also measured in subcutaneous fat cells to which no test substance is added. When the expression level of the gene in cells to which the test substance has been added is smaller than the expression level of the gene in cells to which the test substance has not been added (control), the test substance is determined to have a skin aging-improving effect. . The determined substance can be a skin aging improving agent that can be suitably contained in a composition for improving skin aging.
The degree of variation in the expression level is preferably 80% or less, more preferably 65% or less, and still more preferably 50% or less, relative to the control.
スクリーニングにより選択された被験物質が皮膚老化改善作用を有することは、例えば、該物質を含有する組成物を適用した被験者の自覚による評価、画像解析によるシワやタルミの評価、皮膚の粘弾物性の評価、皮膚の水分保持量の評価など、周知の方法によって
確認することができる。
That the test substance selected by screening has a skin aging improving effect is, for example, the subjective evaluation of the subject to whom the composition containing the substance is applied, the evaluation of wrinkles and tarmi by image analysis, the viscoelasticity of the skin It can be confirmed by a well-known method such as evaluation and evaluation of the amount of water retained in the skin.
本発明のスクリーニング方法により皮膚老化症状を改善する作用を有すると判定された物質（皮膚老化改善剤）は、任意の調製方法により組成物に含有させることができる。すなわち、本発明のスクリーニング方法は、組成物の設計に好適に用いることができる。かかる組成物としては、例えば飲食品組成物や、皮膚外用組成物等を好適に挙げられ、またこれらは皮膚老化改善用途や抗老化用途に好ましく適用できる。
本発明のスクリーニングにより皮膚老化症状を改善する作用を有すると判定された物質（皮膚老化改善剤）は、皮下脂肪細胞が分泌するケメリンに起因する皮膚老化の発生をターゲットとする新たな機序で皮膚老化症状を改善する点で、新たなアプローチによる有効な抗老化用組成物の配合成分となり得る。
The substance (skin aging improving agent) determined to have the effect of improving skin aging symptoms by the screening method of the present invention can be contained in the composition by any preparation method. That is, the screening method of the present invention can be suitably used for designing a composition. As such a composition, for example, a food and drink composition, a composition for external use on the skin and the like are preferably mentioned, and these can be preferably applied to a use for improving skin aging and an anti-aging use.
The substance (skin aging ameliorating agent) determined to have an effect of improving skin aging symptoms by the screening of the present invention is a novel mechanism targeting the occurrence of skin aging caused by chemerin secreted by subcutaneous fat cells. In terms of improving skin aging symptoms, it can be an effective anti-aging composition component by a new approach.
本発明のスクリーニングにより皮膚老化症状を改善する作用を有すると判定された物質（皮膚老化改善剤）を組成物に含有させる場合、その含有量（配合量）は、通常、０．００００１質量％以上、好ましくは０．０００１質量％以上、より好ましくは０．００１質量％以上であり、通常１５質量％以下、好ましくは１０質量％以下、より好ましくは５質量％以下である。皮膚老化改善剤の含有量（配合量）が少なすぎると所望の効果が得られにくい場合があり、多すぎると効果が頭打ちになるばかりか組成物の処方の自由度を損なう場合があるからである。また、組成物に含有させる皮膚老化改善剤の種類は、１種類のみでなく２種類以上であってもよい。
また、該組成物には、本発明のスクリーニングにより皮膚老化症状を改善する作用を有すると判定された物質以外の皮膚老化改善用成分もともに配合してもよい。
When a substance (skin aging improver) determined to have an effect of improving skin aging symptoms by the screening of the present invention is contained in the composition, the content (combination amount) is usually 0.00001% by mass or more. , Preferably 0.0001% by mass or more, more preferably 0.001% by mass or more, usually 15% by mass or less, preferably 10% by mass or less, more preferably 5% by mass or less. If the content (blending amount) of the skin aging improver is too small, it may be difficult to obtain the desired effect, while if it is too large, the effect may not only reach a plateau but also impair the flexibility of formulation of the composition. is there. The type of the skin aging improver to be contained in the composition is not limited to one type, and may be two or more types.
The composition may also contain a skin aging-improving component other than a substance determined to have an action of improving skin aging symptoms by the screening of the present invention.
本発明に係る皮膚老化改善剤を皮膚外用組成物に含有させる場合、その製造に際しては、化粧料、医薬部外品、医薬品などの製剤化で通常使用される成分を任意に配合することができる。
かかる任意成分としては例えば、スクワラン、ワセリン、マイクロクリスタリンワックスなどの炭化水素類、ホホバ油、カルナウバワックス、オレイン酸オクチルドデシルなどのエステル類、オリーブ油、牛脂、椰子油などのトリグリセライド類、ステアリン酸、オレイン酸、レチノイン酸などの脂肪酸、オレイルアルコール、ステアリルアルコール、オクチルドデカノール等の高級アルコール、スルホコハク酸エステルやポリオキシエチレンアルキル硫酸ナトリウム等のアニオン界面活性剤類、アルキルベタイン塩等の両性界面活性剤類、ジアルキルアンモニウム塩等のカチオン界面活性剤類、ソルビタン脂肪酸エステル、脂肪酸モノグリセライド、これらのポリオキシエチレン付加物、ポリオキシエチレンアルキルエーテル、ポリオキシエチレン脂肪酸エステル等の非イオン界面活性剤類、ポリエチレングリコール、グリセリン、１，３−ブタンジオール等の多価アルコール類、増粘・ゲル化剤、酸化防止剤、紫外線吸収剤、色剤、防腐剤、粉体等を任意に配合することができる。
該皮膚外用組成物は、常法に従って前述の成分を処理・配合することにより製造することができる。また、その形態は、例えば、ローション剤型、乳化剤型、オイル剤型など任意の剤型とすることができる。
When the skin aging improver according to the present invention is contained in a composition for external use on the skin, at the time of its production, ingredients commonly used in the preparation of cosmetics, quasi-drugs, pharmaceuticals, etc. can be arbitrarily blended. .
Such optional components include, for example, hydrocarbons such as squalane, petrolatum, microcrystalline wax, jojoba oil, carnauba wax, esters such as octyldodecyl oleate, olive oil, tallow, triglycerides such as coconut oil, stearic acid, Fatty acids such as oleic acid and retinoic acid, higher alcohols such as oleyl alcohol, stearyl alcohol and octyldodecanol, anionic surfactants such as sulfosuccinates and sodium polyoxyethylene alkyl sulfate, and amphoteric surfactants such as alkyl betaine salts , Surfactants such as dialkylammonium salts, sorbitan fatty acid esters, fatty acid monoglycerides, their polyoxyethylene adducts, polyoxyethylene alkyl ethers, polyoxy Nonionic surfactants such as tylene fatty acid esters, polyhydric alcohols such as polyethylene glycol, glycerin and 1,3-butanediol, thickening / gelling agents, antioxidants, ultraviolet absorbers, coloring agents, preservatives , Powder and the like can be arbitrarily compounded.
The composition for external use on the skin can be produced by treating and blending the above-mentioned components according to a conventional method. In addition, the form can be an arbitrary form such as a lotion form, an emulsifier form, and an oil form.
本発明に係る皮膚老化改善剤を飲食品に含有させる場合、その製造に際しては、食品製造において通常使用される成分を任意に配合することができる。
かかる任意成分としては例えば、タンパク質、炭水化物、脂肪、栄養素、調味料及び香味料等を用いることができる。炭水化物としては、単糖類、例えば、ブドウ糖、果糖など；二糖類、例えば、マルトース、スクロース、オリゴ糖など；及び多糖類、例えば、デキストリン、シクロデキストリンなどのような通常の糖及び、キシリトール、ソルビトール、エリトリトールなどの糖アルコールが挙げられる。香味料としては、天然香味料（タウマチン、ステビア抽出物等）及び合成香味料（サッカリン、アスパルテーム等）を使用す
ることができる。その他に、前述の医薬組成物で用いられる添加物であって通常食品にも添加されるものを同様に用いてもよい。
When the skin aging improver according to the present invention is contained in foods and drinks, components commonly used in the production of foods can be arbitrarily mixed during the production.
As such optional components, for example, proteins, carbohydrates, fats, nutrients, seasonings, flavors and the like can be used. Carbohydrates include monosaccharides such as glucose, fructose and the like; disaccharides such as maltose, sucrose, oligosaccharides and the like; and polysaccharides such as ordinary sugars such as dextrin, cyclodextrin and the like, and xylitol, sorbitol, Sugar alcohols such as erythritol; As the flavor, natural flavors (eg, thaumatin, stevia extract) and synthetic flavors (eg, saccharin, aspartame) can be used. In addition, additives that are used in the above-mentioned pharmaceutical compositions and that are usually added to foods may also be used.
飲食品の形態としては、液状、ペースト状、固体、粉末、顆粒等の形態を問わない。また、錠菓、流動食、飼料等も飲食品の態様に含まれる。 The form of the food or drink may be any of liquid, paste, solid, powder, granules and the like. Tablet confectionery, liquid food, feed, and the like are also included in the form of food and drink.
また、他に一般の飲食品に含有させる態様であってもよく、例えば、パン、マカロニ、スパゲッティ、めん類、ケーキミックス、から揚げ粉、パン粉の小麦粉製品；、即席めん、カップめん、レトルト・調理食品、調理缶詰め、電子レンジ食品、即席スープ・シチュー、即席みそ汁・吸い物、スープ缶詰め、フリーズ・ドライ食品等の即席食品；農産缶詰め、果実缶詰め、ジャム・マーマレード類、漬物、煮豆類、農産乾物類、シリアル（穀物加工品）等の農産加工品；水産缶詰め、魚肉ハム・ソーセージ、水産練り製品、水産珍味類、つくだ煮類等の水産加工品；畜産缶詰め・ペースト類、畜肉ハム・ソーセージ等の畜産加工品；加工乳、乳飲料、ヨーグルト類、乳酸菌飲料類、チーズ、アイスクリーム類、調製粉乳類、クリーム、その他の乳製品等の乳・乳製品；バター、マーガリン類、植物油等の油脂類；しょうゆ、みそ、ソース類、トマト加工調味料、みりん類、食酢類等の基礎調味料；調理ミックス、カレーの素類、たれ類、ドレッシング類、めんつゆ類、スパイス類、その他の複合調味料等の複合調味料・食品類；素材冷凍食品、半調理冷凍食品、調理済冷凍食品等の冷凍食品；キャラメル、キャンディー、チューインガム、チョコレート、クッキー、ビスケット、ケーキ、パイ、スナック、クラッカー、和菓子、米菓子、豆菓子、デザート菓子等の菓子類；炭酸飲料、天然果汁、果汁飲料、果汁入り清涼飲料、果肉飲料、果粒入り果実飲料、野菜系飲料、豆乳、豆乳飲料、コーヒー飲料、お茶飲料、粉末飲料、濃縮飲料、スポーツ飲料、栄養飲料、アルコール飲料等の飲料類；これら以外の食品等に、本発明に係る皮膚老化改善剤を添加してもよい。 In addition, other forms may be included in general foods and drinks, for example, bread, macaroni, spaghetti, noodles, cake mix, fried flour, bread flour products; instant noodles, cup noodles, retort / cooked foods , Cooking cans, microwave foods, instant soups and stews, instant miso soups and soups, canned soups, frozen and dried foods, and other instant foods; canned agricultural products, canned fruits, jams and marmalades, pickles, boiled beans, dried agricultural products, Agricultural products such as cereals (processed cereals); Canned fish products, fish ham / sausage, fish paste products, fishery delicacies, processed fish products such as tsukudani, livestock canned products / pastes, livestock processed products such as meat ham / sausage Processed milk, milk drinks, yogurt, lactic acid drinks, cheese, ice cream, powdered milk, cream, etc. Milk and dairy products such as dairy products; fats and oils such as butter, margarines and vegetable oils; basic seasonings such as soy sauce, miso, sauces, tomato seasonings, mirin and vinegar; cooking mixes and curry bases , Seasonings and foods such as sauces, dressings, noodle soups, spices, and other compound seasonings; frozen foods such as frozen foods, semi-cooked frozen foods, and cooked frozen foods; caramels, candies, and chewing gum , Chocolate, cookies, biscuits, cakes, pies, snacks, crackers, sweets such as Japanese confectionery, rice confectionery, bean confectionery, dessert confectionery; Fruit drinks, vegetable drinks, soy milk, soy milk drinks, coffee drinks, tea drinks, powdered drinks, concentrated drinks, sports drinks, nutritional drinks, alcoholic drinks, etc. Beverages; to foods other than the above, the skin aging improving agent according to the present invention may be added.
本発明に係る皮膚老化改善剤を含有する食品組成物の態様としては、通常の食品、飲料、機能性表示食品、特定保健用食品等の保健機能食品、サプリメント等が挙げられ、特に機能性表示食品が好ましい。
本発明に係る食品組成物を皮膚老化改善用途や抗老化用途とする場合、製品化の際にその有する有用性や機能性に関する表示を付してもよい。
かかる「表示」行為には、需要者に対して前記用途を知らしめるための全ての行為が含まれ、「皮膚老化の改善用」「抗老化用」といった用途を想起・類推させうるような表現であれば、表示の目的、表示の内容、表示する対象物・媒体等の如何に拘わらず、全て本発明の「表示」行為に該当する。
また、「表示」は、需要者が上記用途を直接的に認識できるような表現により行われることが好ましい。具体的には、飲食品に係る商品又は商品の包装、容器等に前記用途を記載したものを譲渡し、引き渡し、譲渡若しくは引き渡しのために展示し、輸入する行為、商品に関する広告、価格表、カタログ、パンフレット、ＰＯＰ等の販売現場における宣伝材等、若しくは取引書類に上記用途を記載して展示し、若しくは頒布し、又はこれらを内容とする情報に上記用途を記載して電磁気的（インターネット等）方法により提供する行為等が挙げられる。なお、本発明の食品組成物が保健機能食品等の行政が定める各種制度に基づいて認可を受けその認可のもとで実施される場合は、該認可に基づく態様で表示することが好ましい。
Examples of the food composition containing the skin aging improver according to the present invention include ordinary foods, beverages, functionally labeled foods, health functional foods such as foods for specified health use, supplements, and the like. Food is preferred.
When the food composition according to the present invention is used for improving skin aging or for anti-aging, the product composition may be labeled with its usefulness and functionality at the time of commercialization.
Such “display” actions include all actions for informing consumers of the use, and expressions that can remind / analyze uses such as “for improving skin aging” and “for anti-aging”. If so, all correspond to the "display" act of the present invention, regardless of the purpose of display, the content of the display, the object / medium to be displayed, and the like.
In addition, “display” is preferably performed in an expression that allows the consumer to directly recognize the use. To be specific, the goods related to food and drink or the packaging, containers, etc. of the goods are transferred, delivered, displayed for the purpose of transfer or delivery and imported, the advertisement of the goods, the price list, Catalogs, pamphlets, POPs and other advertising materials at the sales floor, or in business documents, exhibiting or distributing the above-mentioned applications, or by describing the above-mentioned applications in information containing these, and electromagnetically (such as the Internet) ) Act provided by the method. In addition, when the food composition of the present invention is approved based on various systems determined by the administration of health functional foods and the like, and is implemented under the approval, it is preferable to display in a form based on the approval.
本発明に係る皮膚老化改善剤を含有する食品組成物の好ましい摂取量は、成人が摂取する場合、該皮膚老化改善剤の固形物換算で１日当たり０．００１〜１０００ｍｇ／ｋｇとすることが、十分に効果が発揮される観点から好ましく、更に０．０１〜１００ｍｇ／ｋｇ、特に０．１〜１０ｍｇ／ｋｇとすることが好ましい。この１日分の量を一度に又は数回に分けて摂取することができる。また、単回摂取する他に、連続的に又は断続的に数週間〜数か月の間摂取することが好ましい。 The preferred intake of the food composition containing the skin aging improver according to the present invention, when taken by an adult, may be 0.001 to 1000 mg / kg per day in terms of solids of the skin aging improver. It is preferable from the viewpoint of sufficiently exerting the effect, and more preferably 0.01 to 100 mg / kg, particularly preferably 0.1 to 10 mg / kg. This daily dose can be taken at once or divided into several portions. In addition to the single ingestion, it is preferable to ingest it continuously or intermittently for several weeks to several months.
以下、本発明を実施例により更に詳細に説明するが、本発明は、その要旨を超えない限り、以下の実施例に限定されるものではない。 EXAMPLES Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples unless it exceeds the gist.
＜参考例１＞線維芽細胞におけるバーシカン産生に対するケメリンの影響の確認
DMEM培地（SIGMA社製）を用いて正常ヒト真皮線維芽細胞を２４ウェルプレートに２．
０×１０４ｃｅｌｌｓ／ウェル播種し、３７℃・５％ＣＯ２環境下で２日間培養した。培養後、培地を除去し、PBSにて細胞を洗浄後、ケメリン（Ｒ＆Ｄ ＳＹＳＴＥＭＳ製）（PBS溶液、１．５μＬ／ｍＬ培地、終濃度７．５ｎＭ）又は溶媒対照（PBS、１．５μＬ／ｍＬ培地）を含む、DMEM培地を加え、３７℃・５％ＣＯ２環境下にて、２４時間培養した。
RNeasy Mini Kit（QIAGEN製）を用いて上記線維芽細胞のｍＲＮＡを抽出し、Superscript VILO DNA synthesis Kit（Lifetechnologies製）を用いてｃＤＮＡを合成後、プライ
マー（QT00064064、ＱＩＡＧＥＮ製）を用いてバーシカンのｍＲＮＡ発現量をリアルタイムｑＰＣＲ法にて測定した。また、内在性コントロール遺伝子であるＡＣＴＢのｍＲＮＡ発現量を同様に測定した。測定には、QuantiFact SYBR GREEN PCR kit（QIAGEN製）を用
いた。
ケメリン添加群及び溶媒対照群におけるバーシカンｍＲＮＡ発現量について、ＡＣＴＢの発現量による補正を行い、溶媒対照群の発現量を１とした場合の、ケメリン添加群の相対発現量を算出した。結果を図１に示す。
<Reference Example 1> Confirmation of the effect of chemerin on versican production in fibroblasts
1. Using a DMEM medium (manufactured by SIGMA), normal human dermal fibroblasts were placed in a 24-well plate.
The cells were seeded at 0 × 10 4 cells / well and cultured at 37 ° C. in a 5% CO 2 environment for 2 days. After culturing, the medium was removed, and the cells were washed with PBS. Then, Chemerin (manufactured by R & D SYSTEMS) (PBS solution, 1.5 μL / mL medium, final concentration 7.5 nM) or a solvent control (PBS, 1.5 μL / mL) Medium), and cultured at 37 ° C. in a 5% CO 2 environment for 24 hours.
The mRNA of the above-mentioned fibroblasts is extracted using the RNeasy Mini Kit (manufactured by QIAGEN), and cDNA is synthesized using the Superscript VILO DNA synthesis Kit (manufactured by Lifetechnologies). The expression level was measured by the real-time qPCR method. In addition, the mRNA expression level of ACTB which is an endogenous control gene was measured in the same manner. For the measurement, QuantiFact SYBR GREEN PCR kit (manufactured by QIAGEN) was used.
The expression level of versican mRNA in the chemerin-added group and the solvent control group was corrected by the expression level of ACTB, and the relative expression level of the chemerin-added group was calculated when the expression level of the solvent control group was set to 1. The results are shown in FIG.
＜参考例２＞皮下腱細胞におけるＲＣ構成タンパク質産生に対するケメリンの影響の確認
Tenocyte Culture Medium（Dsファーマバイオメディカル社製）を用いて正常ヒト腱細
胞を２４ウェルプレートに２．０×１０４ｃｅｌｌｓ／ウェル播種し、３７℃・５％ＣＯ２環境下で１日間培養した。培養後、培地を除去し、ＰＢＳにて細胞を洗浄後、ケメリン（Ｒ＆Ｄ ＳＹＳＴＥＭＳ製）（PBS溶液、１．５μＬ／ｍＬ培地、終濃度７．５ｎＭ）又は溶媒対照（PBS、１．５μＬ／ｍＬ培地）を含む培地を加え、３７℃・５％ＣＯ２環境
下にて、４８時間培養した。
RNeasy Mini Kit（QIAGEN製）を用いて上記腱細胞のｍＲＮＡを抽出し、Superscript VILO DNA synthesis Kit（Lifetechnologies製）を用いてｃＤＮＡを合成後、プライマー
（QT00000483、ＱＩＡＧＥＮ製）を用いてミメカンのｍＲＮＡ発現量をリアルタイムｑＰＣＲ法にて測定した。また、内在性コントロール遺伝子であるＡＣＴＢのｍＲＮＡ発現量を同様に測定した。測定には、QuantiFact SYBR GREEN PCR kit（QIAGEN製）を用いた。
ケメリン添加群及び溶媒対照群におけるミメカンｍＲＮＡ発現量について、ＡＣＴＢの発現量による補正を行い、溶媒対照群の発現量を１とした場合の、ケメリン添加群の相対発現量を算出した。結果を図２に示す。
<Reference Example 2> Confirmation of the effect of chemerin on RC component protein production in subcutaneous tendon cells
Using a Tenocyte Culture Medium (manufactured by Ds Pharma Biomedical), normal human tendon cells were seeded at 2.0 × 10 4 cells / well in a 24-well plate and cultured at 37 ° C. in a 5% CO 2 environment for 1 day. After culturing, the medium was removed, and the cells were washed with PBS. Then, Chemerin (manufactured by R & D SYSTEMS) (PBS solution, 1.5 μL / mL medium, final concentration 7.5 nM) or a solvent control (PBS, 1.5 μL / mL) Medium), and cultured at 37 ° C. in a 5% CO 2 environment for 48 hours.
The mRNA of the above tendon cells was extracted using the RNeasy Mini Kit (QIAGEN), cDNA was synthesized using the Superscript VILO DNA synthesis Kit (Lifetechnologies), and mimecan mRNA expression was performed using primers (QT00000483, QIAGEN). The amount was measured by the real-time qPCR method. In addition, the mRNA expression level of ACTB which is an endogenous control gene was measured in the same manner. For the measurement, QuantiFact SYBR GREEN PCR kit (manufactured by QIAGEN) was used.
The expression level of mimecan mRNA in the chemerin-added group and the solvent control group was corrected by the expression level of ACTB, and the relative expression level of the chemerin-added group was calculated when the expression level of the solvent control group was set to 1. FIG. 2 shows the results.
＜参考例３＞表皮角化細胞におけるヒアルロン酸合成酵素産生に対するケメリンの影響の確認
Humedia-KG2培地（クラボウ製）を用いて培養ヒト成人表皮角化細胞（ＮＨＥＫ、クラ
ボウ製）を２４ウェルプレートに７．０×１０４ｃｅｌｌｓ／ウェル播種し、３７℃・５％ＣＯ２環境下で１日間培養した。培養後、培地を除去し、ＰＢＳにて細胞を洗浄後、ケメリン（Ｒ＆Ｄ ＳＹＳＴＥＭＳ製）（PBS溶液、１．５μＬ／ｍＬ培地、終濃度７．５ｎＭ）又は溶媒対照（PBS、１．５μＬ／ｍＬ培地）を含む培地を加え、３７℃・５％ＣＯ
２環境下にて、４８時間培養した。
RNeasy Mini Kit（QIAGEN製）を用いて上記角化細胞のｍＲＮＡを抽出し、Superscript
VILO DNA synthesis Kit（Lifetechnologies製）を用いてｃＤＮＡを合成後、プライマ
ー（QT00014903、ＱＩＡＧＥＮ製）を用いてＨＡＳ３のｍＲＮＡ発現量をリアルタイムｑＰＣＲ法にて測定した。また、内在性コントロール遺伝子であるＡＣＴＢのｍＲＮＡ発現量を同様に測定した。測定には、QuantiFact SYBR GREEN PCR kit（ＱＩＡＧＥＮ製）を
用いた。
ケメリン添加群及び溶媒対照群におけるＨＡＳ３ｍＲＮＡ発現量について、ＡＣＴＢの発現量による補正を行い、溶媒対照群の発現量を１とした場合の、ケメリン添加群の相対発現量を算出した。結果を図３に示す。
<Reference Example 3> Confirmation of the effect of chemerin on the production of hyaluronan synthase in epidermal keratinocytes
Using a Humedia-KG2 medium (manufactured by Kurabo Industries), cultured human adult epidermal keratinocytes (NHEK, manufactured by Kurabo Industries) were seeded at 7.0 × 10 4 cells / well in a 24-well plate, at 37 ° C. and 5% CO 2 environment. For 1 day. After culturing, the medium was removed, and the cells were washed with PBS. Then, Chemerin (manufactured by R & D SYSTEMS) (PBS solution, 1.5 μL / mL medium, final concentration 7.5 nM) or a solvent control (PBS, 1.5 μL / mL) Medium) at 37 ° C and 5% CO
Culture was performed for 48 hours under two environments.
The keratinocyte mRNA was extracted using RNeasy Mini Kit (QIAGEN)
After synthesizing cDNA using VILO DNA synthesis Kit (manufactured by Lifetechnologies), the expression level of HAS3 mRNA was measured by real-time qPCR using primers (QT00014903, manufactured by QIAGEN). In addition, the mRNA expression level of ACTB which is an endogenous control gene was measured in the same manner. For the measurement, QuantiFact SYBR GREEN PCR kit (manufactured by QIAGEN) was used.
The expression level of HAS3 mRNA in the chemerin-added group and the solvent control group was corrected by the ACTB expression level, and the relative expression level of the chemerin-added group was calculated when the expression level of the solvent control group was set to 1. The results are shown in FIG.
＜実施例１＞皮膚老化改善剤のスクリーニング
以下の手順で、ケメリンの遺伝子発現を指標に、皮膚老化改善剤のスクリーニングを行った。
増殖培地（CELL社製）を用い、正常ヒト皮下脂肪細胞を２４ウェルプレートに１．０×１０５ｃｅｌｌｓ／ウェルで播種し、３７℃・５％ＣＯ２環境下で２日間培養した。培養後、培地を除去し、ＰＢＳにて細胞を洗浄後、分化培地（CELL社製）に交換し、３７℃・５％ＣＯ２環境下にて、１０日間培養した。培養後、培地を除去し、ＰＢＳにて細胞を洗浄後、クルミポリフェノールエキス（Ｒ＆Ｄ ＳＹＳＴＥＭＳ製）１０μｇ／ｍＬ（終濃
度）又は溶媒対照（ＰＢＳ、１．５μＬ／ｍＬ培地）を含む維持培地を加え、３７℃・５％ＣＯ２環境下にて、４８時間培養した。培養後、培地を除去し、ＰＢＳにて細胞を洗浄後、QIAzol Lysis Reagent（ＱＩＡＧＥＮ社製）を用いて上記正常ヒト皮下脂肪細胞のｍＲＮＡを抽出し、Superscript VILO DNA synthesis Kit（Lifetechnologies社製）を用いてｃＤＮＡを合成後、プライマー（QT00091945、ＱＩＡＧＥＮ製）を用いてケメリンのｍＲＮＡ発現量をリアルタイムｑＰＣＲ法にて測定した。また、内在性コントロール遺伝子であるＡＣＴＢのｍＲＮＡ発現量を同様に測定した。測定には、QuantiFact SYBR GREEN PCR kit（ＱＩＡＧＥＮ社製）を用いた。エキス添加群及び溶媒対照群におけるケメリン
ｍＲＮＡ発現量について、ＡＣＴＢの発現量による補正を行い、溶媒対照群の発現量を１とした場合の、エキス添加群の相対発現量を算出した。結果を図４に示す。
<Example 1> Screening of skin aging improving agent A skin aging improving agent was screened by the following procedure using the expression of the gene for chemerin as an index.
Using a growth medium (manufactured by CELL), normal human subcutaneous adipocytes were seeded at 1.0 × 10 5 cells / well in a 24-well plate and cultured at 37 ° C. in a 5% CO 2 environment for 2 days. After culturing, the medium was removed, the cells were washed with PBS, replaced with a differentiation medium (manufactured by CELL), and cultured at 37 ° C. in a 5% CO 2 environment for 10 days. After the culture, the medium was removed, the cells were washed with PBS, and a maintenance medium containing 10 μg / mL (final concentration) of walnut polyphenol extract (manufactured by R & D SYSTEMS) or a solvent control (PBS, 1.5 μL / mL medium) was added. And cultured at 37 ° C. in a 5% CO 2 environment for 48 hours. After culturing, the medium was removed, the cells were washed with PBS, and the mRNA of the above normal human subcutaneous adipocytes was extracted using QIAzol Lysis Reagent (manufactured by QIAGEN), and Superscript VILO DNA synthesis Kit (manufactured by Lifetechnologies) was extracted. After cDNA synthesis using the primers, the expression level of chemerin mRNA was measured by real-time qPCR using a primer (QT00091945, manufactured by QIAGEN). In addition, the mRNA expression level of ACTB which is an endogenous control gene was measured in the same manner. For the measurement, QuantiFact SYBR GREEN PCR kit (manufactured by QIAGEN) was used. The expression level of chemerin mRNA in the extract-added group and the solvent control group was corrected by the expression level of ACTB, and the relative expression level of the extract-added group was calculated when the expression level of the solvent control group was set to 1. FIG. 4 shows the results.
本発明のスクリーニング方法により、皮膚老化改善剤として有効な成分を探索することができる。かかる皮膚老化改善剤は、皮膚老化改善用途の飲食品や皮膚外用剤に好適に含有させることができるため、産業上非常に有用である。 According to the screening method of the present invention, a component effective as a skin aging improving agent can be searched for. Such an agent for improving skin aging can be suitably contained in foods and drinks and external preparations for skin for improving skin aging, and is therefore extremely useful in industry.
Claims (5)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2018123293A JP7251036B2 (en) | 2018-06-28 | 2018-06-28 | Screening method for skin aging improving agent |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2018123293A JP7251036B2 (en) | 2018-06-28 | 2018-06-28 | Screening method for skin aging improving agent |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2020003352A true JP2020003352A (en) | 2020-01-09 |
| JP7251036B2 JP7251036B2 (en) | 2023-04-04 |
Family
ID=69099771
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2018123293A Active JP7251036B2 (en) | 2018-06-28 | 2018-06-28 | Screening method for skin aging improving agent |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP7251036B2 (en) |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100092974A1 (en) * | 2008-08-15 | 2010-04-15 | The Board Of Trustees Of The Leland Stanford Junior University | Methods for screening for modulators of ccrl2 |
| JP2010115131A (en) * | 2008-11-12 | 2010-05-27 | Kao Corp | Evaluation method and method for screening skin ageing improving agent and skin aging preventive |
| JP2013257347A (en) * | 2013-09-27 | 2013-12-26 | Pola Chem Ind Inc | Screening method and antiaging composition |
| JP2014528472A (en) * | 2011-10-11 | 2014-10-27 | 株式会社アモーレパシフィックＡｍｏｒｅｐａｃｉｆｉｃ Ｃｏｒｐｏｒａｔｉｏｎ | A composition for anti-aging through activation of a retinoic acid receptor containing a mixed extract of eggplant and pine needles as an active ingredient |
| JP2015003873A (en) * | 2013-06-20 | 2015-01-08 | 東洋精糖株式会社 | Adiponectin production promoter |
| JP2015193582A (en) * | 2014-03-28 | 2015-11-05 | 国立大学法人 東京大学 | Agent containing imidazoledipeptide |
| JP2017112918A (en) * | 2015-12-24 | 2017-06-29 | ポーラ化成工業株式会社 | Screening method for subcutaneous tissue structure improvement component |
| WO2017127827A1 (en) * | 2016-01-22 | 2017-07-27 | Tufts Medical Center | Compounds and methods for treating inflammation |
-
2018
- 2018-06-28 JP JP2018123293A patent/JP7251036B2/en active Active
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100092974A1 (en) * | 2008-08-15 | 2010-04-15 | The Board Of Trustees Of The Leland Stanford Junior University | Methods for screening for modulators of ccrl2 |
| JP2010115131A (en) * | 2008-11-12 | 2010-05-27 | Kao Corp | Evaluation method and method for screening skin ageing improving agent and skin aging preventive |
| JP2014528472A (en) * | 2011-10-11 | 2014-10-27 | 株式会社アモーレパシフィックＡｍｏｒｅｐａｃｉｆｉｃ Ｃｏｒｐｏｒａｔｉｏｎ | A composition for anti-aging through activation of a retinoic acid receptor containing a mixed extract of eggplant and pine needles as an active ingredient |
| JP2015003873A (en) * | 2013-06-20 | 2015-01-08 | 東洋精糖株式会社 | Adiponectin production promoter |
| JP2013257347A (en) * | 2013-09-27 | 2013-12-26 | Pola Chem Ind Inc | Screening method and antiaging composition |
| JP2015193582A (en) * | 2014-03-28 | 2015-11-05 | 国立大学法人 東京大学 | Agent containing imidazoledipeptide |
| JP2017112918A (en) * | 2015-12-24 | 2017-06-29 | ポーラ化成工業株式会社 | Screening method for subcutaneous tissue structure improvement component |
| WO2017127827A1 (en) * | 2016-01-22 | 2017-07-27 | Tufts Medical Center | Compounds and methods for treating inflammation |
Non-Patent Citations (1)
| Title |
|---|
| FARSAM, VIDA、外１２名: "Senescent fibroblast-derived Chemerin promotes squamous cell carcinoma migration.", ONCOTARGET, vol. 7, no. 50, JPN7022000794, 2016, pages 83554 - 83569, ISSN: 0004886579 * |
Also Published As
| Publication number | Publication date |
|---|---|
| JP7251036B2 (en) | 2023-04-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP6745250B2 (en) | Moringa extract | |
| JP6682073B2 (en) | Liver function improver | |
| JP4878771B2 (en) | Epidermal keratinocyte proliferating agent and use thereof | |
| JP6714928B2 (en) | Collagenase inhibitor | |
| JP7226843B2 (en) | Liver function improver | |
| JP2017141201A (en) | Glycosylation inhibitor | |
| JP2010095529A (en) | Composition for improving lipid metabolism | |
| JP2009155293A (en) | Fermented licorice extract and production method thereof, and external medicine for skin and food and drink for beautification containing the same | |
| JP5027361B2 (en) | Hyaluronic acid production promoter | |
| JP2020014413A (en) | Skin aging improver screening method | |
| JP7251036B2 (en) | Screening method for skin aging improving agent | |
| JP5832714B2 (en) | Tyrosinase activity inhibitor and melanin production inhibitor | |
| JP6718158B2 (en) | Liver function improver | |
| JP2010120946A (en) | Antiinflammatory composition | |
| JP4224593B2 (en) | Composition for suppressing fat accumulation comprising wasabi as an active ingredient | |
| JP7206725B2 (en) | Screening method for skin condition improving agent | |
| JP2010013416A (en) | Algae extract having hair-growing effect | |
| JP5422137B2 (en) | Whitening agent, tyrosinase activity inhibitor, melanin production inhibitor, and skin external preparation for whitening | |
| JP7565717B2 (en) | Composition | |
| JP7320270B2 (en) | Liver function improver | |
| JP2007300862A (en) | Collagen producing food | |
| JP2019013208A (en) | Method for producing oligosaccharide and action of the composition | |
| JP2007112721A (en) | Melanogenesis inhibitory composition, and beverage and food containing the same | |
| JP2023104573A (en) | Pharmaceutical composition for preventing or treating influenza and food composition for preventing or treating influenza | |
| JP2013082738A (en) | Platelet aggregation inhibitor |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20210407 |
|
| A977 | Report on retrieval |
Free format text: JAPANESE INTERMEDIATE CODE: A971007 Effective date: 20220222 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20220301 |
|
| A601 | Written request for extension of time |
Free format text: JAPANESE INTERMEDIATE CODE: A601 Effective date: 20220428 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20221004 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20221129 |
|
| TRDD | Decision of grant or rejection written | ||
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20230221 |
|
| A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20230303 |
|
| R150 | Certificate of patent or registration of utility model |
Ref document number: 7251036 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |