JP5958983B2 - Skin external preparation for hair growth and hair growth using lactic acid bacteria - Google Patents
Skin external preparation for hair growth and hair growth using lactic acid bacteria Download PDFInfo
- Publication number
- JP5958983B2 JP5958983B2 JP2015142170A JP2015142170A JP5958983B2 JP 5958983 B2 JP5958983 B2 JP 5958983B2 JP 2015142170 A JP2015142170 A JP 2015142170A JP 2015142170 A JP2015142170 A JP 2015142170A JP 5958983 B2 JP5958983 B2 JP 5958983B2
- Authority
- JP
- Japan
- Prior art keywords
- hair
- lactic acid
- hair growth
- cells
- acid bacteria
- 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
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 title description 106
- 239000004310 lactic acid Substances 0.000 title description 53
- 235000014655 lactic acid Nutrition 0.000 title description 53
- 230000003779 hair growth Effects 0.000 title description 51
- 241000894006 Bacteria Species 0.000 title description 49
- 238000002360 preparation method Methods 0.000 title description 30
- 239000000284 extract Substances 0.000 claims description 25
- 241000194035 Lactococcus lactis Species 0.000 claims description 13
- 235000014897 Streptococcus lactis Nutrition 0.000 claims description 13
- 239000004480 active ingredient Substances 0.000 claims description 10
- 210000004027 cell Anatomy 0.000 description 70
- 238000012360 testing method Methods 0.000 description 30
- 230000008099 melanin synthesis Effects 0.000 description 28
- 210000003491 skin Anatomy 0.000 description 28
- 108050007372 Fibroblast Growth Factor Proteins 0.000 description 27
- 102000018233 Fibroblast Growth Factor Human genes 0.000 description 27
- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 description 26
- 229940126864 fibroblast growth factor Drugs 0.000 description 26
- 238000011282 treatment Methods 0.000 description 24
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 22
- 210000004209 hair Anatomy 0.000 description 22
- 201000004384 Alopecia Diseases 0.000 description 20
- 108010073929 Vascular Endothelial Growth Factor A Proteins 0.000 description 20
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 description 20
- 230000003676 hair loss Effects 0.000 description 20
- 208000024963 hair loss Diseases 0.000 description 20
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 description 19
- 230000000694 effects Effects 0.000 description 19
- 239000003112 inhibitor Substances 0.000 description 19
- 239000000243 solution Substances 0.000 description 17
- 239000012228 culture supernatant Substances 0.000 description 15
- 239000013641 positive control Substances 0.000 description 15
- 230000002401 inhibitory effect Effects 0.000 description 14
- 239000002126 C01EB10 - Adenosine Substances 0.000 description 13
- 229960005305 adenosine Drugs 0.000 description 13
- 238000004519 manufacturing process Methods 0.000 description 12
- 241000194036 Lactococcus Species 0.000 description 11
- 239000003795 chemical substances by application Substances 0.000 description 11
- XUMBMVFBXHLACL-UHFFFAOYSA-N Melanin Chemical compound O=C1C(=O)C(C2=CNC3=C(C(C(=O)C4=C32)=O)C)=C2C4=CNC2=C1C XUMBMVFBXHLACL-UHFFFAOYSA-N 0.000 description 10
- 238000002835 absorbance Methods 0.000 description 10
- BJRNKVDFDLYUGJ-RMPHRYRLSA-N hydroquinone O-beta-D-glucopyranoside Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=CC=C(O)C=C1 BJRNKVDFDLYUGJ-RMPHRYRLSA-N 0.000 description 10
- 239000002609 medium Substances 0.000 description 10
- 239000000126 substance Substances 0.000 description 10
- 230000020411 cell activation Effects 0.000 description 9
- 238000005259 measurement Methods 0.000 description 9
- 230000001737 promoting effect Effects 0.000 description 9
- 239000012085 test solution Substances 0.000 description 8
- 230000003778 catagen phase Effects 0.000 description 7
- 230000002500 effect on skin Effects 0.000 description 7
- 230000031774 hair cycle Effects 0.000 description 7
- 230000006711 vascular endothelial growth factor production Effects 0.000 description 7
- 230000003698 anagen phase Effects 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 6
- 239000011159 matrix material Substances 0.000 description 6
- WHNFPRLDDSXQCL-UAZQEYIDSA-N α-msh Chemical compound C([C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](C(C)C)C(N)=O)NC(=O)[C@H](CO)NC(C)=O)C1=CC=C(O)C=C1 WHNFPRLDDSXQCL-UAZQEYIDSA-N 0.000 description 6
- 102400000740 Melanocyte-stimulating hormone alpha Human genes 0.000 description 5
- 101710200814 Melanotropin alpha Proteins 0.000 description 5
- 230000009471 action Effects 0.000 description 5
- 229960000271 arbutin Drugs 0.000 description 5
- 230000032823 cell division Effects 0.000 description 5
- 230000012010 growth Effects 0.000 description 5
- 239000004615 ingredient Substances 0.000 description 5
- BJRNKVDFDLYUGJ-UHFFFAOYSA-N p-hydroxyphenyl beta-D-alloside Natural products OC1C(O)C(O)C(CO)OC1OC1=CC=C(O)C=C1 BJRNKVDFDLYUGJ-UHFFFAOYSA-N 0.000 description 5
- 238000005406 washing Methods 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- QIGBRXMKCJKVMJ-UHFFFAOYSA-N Hydroquinone Chemical compound OC1=CC=C(O)C=C1 QIGBRXMKCJKVMJ-UHFFFAOYSA-N 0.000 description 4
- 239000000499 gel Substances 0.000 description 4
- 238000000034 method Methods 0.000 description 4
- 230000000813 microbial effect Effects 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 239000002504 physiological saline solution Substances 0.000 description 4
- 239000000843 powder Substances 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 239000012086 standard solution Substances 0.000 description 4
- 230000001954 sterilising effect Effects 0.000 description 4
- 238000004659 sterilization and disinfection Methods 0.000 description 4
- 239000011550 stock solution Substances 0.000 description 4
- 230000001629 suppression Effects 0.000 description 4
- 238000002965 ELISA Methods 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 230000004913 activation Effects 0.000 description 3
- 230000003712 anti-aging effect Effects 0.000 description 3
- 239000006071 cream Substances 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 239000002552 dosage form Substances 0.000 description 3
- 239000000839 emulsion Substances 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 239000006210 lotion Substances 0.000 description 3
- 239000002674 ointment Substances 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- 230000000638 stimulation Effects 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
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 241000192132 Leuconostoc Species 0.000 description 2
- 239000004909 Moisturizer Substances 0.000 description 2
- 206010042496 Sunburn Diseases 0.000 description 2
- 239000006096 absorbing agent Substances 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 230000003213 activating effect Effects 0.000 description 2
- 239000002870 angiogenesis inducing agent Substances 0.000 description 2
- 239000003963 antioxidant agent Substances 0.000 description 2
- 235000006708 antioxidants Nutrition 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 239000002537 cosmetic Substances 0.000 description 2
- 238000012136 culture method Methods 0.000 description 2
- 235000005911 diet Nutrition 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 210000002615 epidermis Anatomy 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 239000003205 fragrance Substances 0.000 description 2
- 238000004108 freeze drying Methods 0.000 description 2
- 239000008187 granular material Substances 0.000 description 2
- 239000003102 growth factor Substances 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 230000003308 immunostimulating effect Effects 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 238000012423 maintenance Methods 0.000 description 2
- 230000001333 moisturizer Effects 0.000 description 2
- 235000016709 nutrition Nutrition 0.000 description 2
- 230000001766 physiological effect Effects 0.000 description 2
- 239000000049 pigment Substances 0.000 description 2
- 239000003755 preservative agent Substances 0.000 description 2
- 239000011535 reaction buffer Substances 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- GHMLBKRAJCXXBS-UHFFFAOYSA-N resorcinol Chemical compound OC1=CC=CC(O)=C1 GHMLBKRAJCXXBS-UHFFFAOYSA-N 0.000 description 2
- 238000010517 secondary reaction Methods 0.000 description 2
- 239000002453 shampoo Substances 0.000 description 2
- -1 softeners Substances 0.000 description 2
- 239000012089 stop solution Substances 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 239000004094 surface-active agent Substances 0.000 description 2
- 239000002562 thickening agent Substances 0.000 description 2
- 102000012422 Collagen Type I Human genes 0.000 description 1
- 108010022452 Collagen Type I Proteins 0.000 description 1
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 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
- 208000001382 Experimental Melanoma Diseases 0.000 description 1
- 102100028071 Fibroblast growth factor 7 Human genes 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 101001060261 Homo sapiens Fibroblast growth factor 7 Proteins 0.000 description 1
- 101000962469 Homo sapiens Transcription factor MafF Proteins 0.000 description 1
- 101000808011 Homo sapiens Vascular endothelial growth factor A Proteins 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- 241000186660 Lactobacillus Species 0.000 description 1
- 101800001751 Melanocyte-stimulating hormone alpha Proteins 0.000 description 1
- 241000192001 Pediococcus Species 0.000 description 1
- 102100027467 Pro-opiomelanocortin Human genes 0.000 description 1
- 108010090804 Streptavidin Proteins 0.000 description 1
- 102100039187 Transcription factor MafF Human genes 0.000 description 1
- 229930003268 Vitamin C Natural products 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000033115 angiogenesis Effects 0.000 description 1
- 230000003796 beauty Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 235000013361 beverage Nutrition 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 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
- 230000036770 blood supply Effects 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 239000003183 carcinogenic agent Substances 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000004040 coloring Methods 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 235000013365 dairy product Nutrition 0.000 description 1
- 238000004042 decolorization Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000004332 deodorization Methods 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 230000000378 dietary effect Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000002900 effect on cell Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000018661 fibroblast growth factor production Effects 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 239000006260 foam Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 229930182470 glycoside Natural products 0.000 description 1
- 150000002338 glycosides Chemical class 0.000 description 1
- 239000003966 growth inhibitor Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 210000003780 hair follicle Anatomy 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 102000058223 human VEGFA Human genes 0.000 description 1
- 230000016507 interphase Effects 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 229940039696 lactobacillus Drugs 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000010899 nucleation Methods 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 238000010979 pH adjustment Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 230000008470 skin growth Effects 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- 235000019154 vitamin C Nutrition 0.000 description 1
- 239000011718 vitamin C Substances 0.000 description 1
- 230000002087 whitening effect Effects 0.000 description 1
Images
Landscapes
- Cosmetics (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Description
本発明は、特定の乳酸菌を含有する皮膚外用剤に関し、詳しくは、ラクトコッカス属乳酸菌を有効成分とする育毛・発毛剤に関する。 The present invention relates to an external preparation for skin containing a specific lactic acid bacterium, and more particularly to a hair growth / hair growth agent containing a lactic acid bacterium of the genus Lactococcus as an active ingredient.
乳酸菌によるヒトの健康や美容に対する効果は広く知られ、食品や飲料として食生活の中に取り込まれて活用されてきている。ラクトコッカス属乳酸菌は、乳製品製造において古くから利用されており、乳酸菌の中でも高い安全性を有するグループとして広く関心を集めている。ラクトコッカス属乳酸菌の生理的効用としては、経口投与による整腸効果、免疫賦活効果、老化抑制効果等が知られている（例えば特許文献１、２参照）。
The effects of lactic acid bacteria on human health and beauty are widely known and have been incorporated into the diet as food and beverages. Lactococcus lactic acid bacteria have been used for a long time in the production of dairy products, and have attracted widespread interest as a highly safe group among lactic acid bacteria. Known physiological effects of Lactococcus lactic acid bacteria include intestinal regulation effects, immunostimulatory effects, antiaging effects, and the like by oral administration (see, for example,
外用での乳酸菌の効用としては、例えばロイコノストック属乳酸菌がメラニン生成抑制作用を有すること（例えば特許文献３参照）や、ラクトバチルス属、ペディオコッカス属、ロイコノストック属の乳酸菌が養毛・育毛作用を有すること（例えば特許文献４参照）が知られている。また、乳酸菌成分を使用した化粧品が複数上市されている。
しかしながら、ラクトコッカス属乳酸菌の外用における生理的効用はこれまで報告されていない。
As for the utility of lactic acid bacteria for external use, for example, the lactic acid bacteria of the genus Leuconostoc have an inhibitory action on melanin production (see, for example, Patent Document 3), and the lactic acid bacteria of the genus Lactobacillus, Pediococcus and Leuconostoc -It has been known that it has a hair-growth action (for example, see Patent Document 4). In addition, a plurality of cosmetics using lactic acid bacteria components are marketed.
However, the physiological utility of Lactococcus lactic acid bacteria in the external application has not been reported so far.
本発明の課題は、乳酸菌の中でも安全性が高いラクトコッカス属乳酸菌を利用した皮膚外用剤を初めて提供することであり、特にメラニン産生抑制剤および育毛・発毛剤を提供することである。 The subject of this invention is providing the skin external preparation using the Lactococcus lactic acid bacteria with high safety | security among lactic acid bacteria for the first time, and is providing the melanin production inhibitor and the hair growth and hair growth agent especially.
本発明者は、ラクトコッカス属乳酸菌の中から外用での優れた生理活性を有する菌株を鋭意探索した結果、ラクトコッカス・ラクティス・サブスピーシーズ クレモリス（Lactococcus lactis subsp. cremoris） H61株およびラクトコッカス・ラクティス・サブスピーシーズ ラクティス（Lactococcus lactis subsp. lactis） G50株が特に優れた美白作用および育毛・発毛作用を有することを見出し、本発明に到達した。 As a result of earnest search for strains having excellent physiological activity for external use from among lactic acid bacteria belonging to the genus Lactococcus, the present inventors have found that Lactococcus lactis subsp. Cremoris H61 strain and Lactococcus lactis -Subspecies Lactococcus lactis subsp. Lactis G50 strain was found to have a particularly excellent whitening action and hair growth / hair growth action, and reached the present invention.
すなわち、請求項１に係る本発明は、ラクトコッカス・ラクティス・サブスピーシーズ クレモリス（Lactococcus lactis subsp. cremoris） Ｈ６１株（ＮＩＴＥ Ｐ−９２）またはその抽出物のみを有効成分として含有することを特徴とする育毛・発毛・脱毛抑制用皮膚外用剤である。
請求項２に係る本発明は、ラクトコッカス・ラクティス・サブスピーシーズ ラクティス（Lactococcus lactis subsp. lactis） Ｇ５０株（ＦＥＲＭ Ｐ−１８４１５）またはその抽出物のみを有効成分として含有することを特徴とする育毛・発毛・脱毛抑制用皮膚外用剤である。
That is, the present invention according to
The present invention according to claim 2 is characterized in that it contains only Lactococcus lactis subsp. Lactis G50 strain (FERM P-18415) or an extract thereof as an active ingredient. It is a skin external preparation for suppressing hair growth and hair loss.
本発明によれば、乳酸菌の中でも安全性が高いラクトコッカス属乳酸菌を利用した皮膚外用剤が初めて提供される。特に、ラクトコッカス属の特定の乳酸菌株またはその抽出物を有効成分とするメラニン産生抑制剤および育毛・発毛剤が初めて提供される。育毛・発毛・脱毛抑制作用については、H61株とG50株がヘアサイクルの中の異なる時期にそれぞれ顕著な効果を発揮することから、両菌株を併用することによって、より効果の高い育毛・発毛剤を提供できる。また、当該乳酸菌株の機能性は死菌体またはその抽出物でも見られることから、様々な形態での利用が可能である。 ADVANTAGE OF THE INVENTION According to this invention, the skin external preparation using Lactococcus lactic acid bacteria with high safety | security among lactic acid bacteria is provided for the first time. In particular, a melanin production inhibitor and a hair growth / hair growth agent comprising a specific lactic acid strain of the genus Lactococcus or an extract thereof as an active ingredient are provided for the first time. With regard to hair growth, hair growth, and hair loss inhibiting effects, the H61 strain and G50 strain exhibit remarkable effects at different times in the hair cycle. A hair agent can be provided. Further, since the functionality of the lactic acid strain is also observed in dead cells or extracts thereof, it can be used in various forms.
以下、本発明を詳細に説明する。
本発明は特定の乳酸菌またはその抽出物を有効成分として含有する皮膚外用剤に関するものである。
Hereinafter, the present invention will be described in detail.
The present invention relates to a skin external preparation containing a specific lactic acid bacterium or an extract thereof as an active ingredient.
〔乳酸菌について〕
本発明の乳酸菌としては、ラクトコッカス・ラクティス・サブスピーシーズ クレモリス H61株（以下、「H61」と略記することもある。）及び／又はラクトコッカス・ラクティス・サブスピーシーズ ラクティス G50株（以下、「G50」と略記することもある。）を用いる。本発明において両菌株は単独で用いても、混合して用いても良い。
[About lactic acid bacteria]
Examples of the lactic acid bacteria of the present invention include Lactococcus lactis subspecies Cremolis strain H61 (hereinafter also abbreviated as “H61”) and / or Lactococcus lactis subspecies lactis G50 strain (hereinafter “G50”). May be abbreviated as). In the present invention, both strains may be used alone or in combination.
H61は、本発明者が経口投与による老化抑制剤として既に特許（特許第4604207号）を取得している乳酸菌株であり、独立行政法人製品評価技術基盤機構 特許微生物寄託センターに受託番号NITE P-92として寄託されている。また、本菌株は独立行政法人農業生物資源研究所の農業生物資源ジーンバンクにもMAFF No.400007として寄託されている。しかし、H61が皮膚外用剤として有用な機能性を有することは、これまで知られていない。
本発明において、H61の培養は一般的な乳酸菌の培養法に従って行うことができる。
H61 is a lactic acid strain that the inventor has already obtained a patent (Patent No. 4604207) as an anti-aging agent by oral administration, and has received the accession number NITE P- Deposited as 92. This strain is also deposited as MAFF No. 400007 in the Genebank of Agricultural Bioresources, an independent administrative agency. However, it has not been known so far that H61 has a useful functionality as an external preparation for skin.
In the present invention, H61 can be cultured according to a general lactic acid bacteria culture method.
G50は、本発明者が経口投与により免疫賦活作用を有する乳酸菌株として既に特許（特許第3671212号）を取得している乳酸菌株であり、独立行政法人製品評価技術基盤機構 特許微生物寄託センターに受託番号FERM P-18415として寄託されている。しかし、G50が皮膚外用剤として有用な機能性を有することは、これまで知られていない。
本発明において、G50の培養は一般的な乳酸菌の培養法に従って行うことができる。
G50 is a lactic acid strain that the present inventor has already obtained a patent (patent No. 3671212) as a lactic acid strain having an immunostimulatory effect by oral administration, and is entrusted to the National Institute for Product Evaluation Technology Patent Microorganism Depositary Center. Deposited under the number FERM P-18415. However, it has not been known so far that G50 has a useful functionality as a skin external preparation.
In the present invention, G50 can be cultured according to a general culture method for lactic acid bacteria.
〔皮膚外用剤〕
本発明に係る皮膚外用剤は、上記乳酸菌またはその抽出物を含有することを特徴とする。
本発明において「皮膚外用剤」とは、皮膚上に投与する形態の組成物の総称であって、皮膚外用医薬組成物、医薬部外品、化粧料、皮膚洗浄料、浴用剤などを総括して意味する言葉である。当該皮膚外用剤の形態は特に限定されるものではなく、使用方法に応じて、例えばローション、乳液、ゲル、クリーム、軟膏剤、粉末、顆粒、経皮吸収剤等の種々の剤型の製剤とすることができる。
[Skin external preparation]
The external preparation for skin according to the present invention contains the lactic acid bacterium or an extract thereof.
In the present invention, the term “external skin preparation” is a general term for a composition in a form to be administered onto the skin. It is a word that means. The form of the external preparation for skin is not particularly limited. Depending on the method of use, for example, preparations of various dosage forms such as lotions, emulsions, gels, creams, ointments, powders, granules, transdermal absorption agents and the like can do.
本発明の皮膚外用剤は上記乳酸菌またはその抽出物を有効成分として含み、当該皮膚外用剤の調製は公知の方法に従って行うことができる。本発明の皮膚外用剤には、上記乳酸菌またはその抽出物以外にも、本発明の効果を奏する範囲内で、各種担体や添加剤、他の薬効成分などが含まれていても良い。 The skin external preparation of the present invention contains the lactic acid bacterium or an extract thereof as an active ingredient, and the preparation of the skin external preparation can be performed according to a known method. The external preparation for skin of the present invention may contain various carriers, additives, other medicinal ingredients, and the like within the scope of the effects of the present invention, in addition to the lactic acid bacteria or the extract thereof.
本発明においては、上記乳酸菌の培養物をそのまま皮膚外用剤として使用しても良いが、当該培養物について常法により濃縮、洗浄、精製、滅菌、pH調整、脱臭、脱色等の処理を行ったものを用いても良い。上記乳酸菌の培養物は、例えばTYG培地などを用いて、通性嫌気の条件下で1〜2日間程度培養することにより得ることができる。また上記培養物から遠心分離により菌体を集菌することができ、さらに生理食塩水や滅菌水などで1〜2回洗浄して用いることもできる。 In the present invention, the culture of lactic acid bacteria may be used as it is as an external preparation for skin. However, the culture was subjected to treatments such as concentration, washing, purification, sterilization, pH adjustment, deodorization, decolorization and the like by conventional methods. A thing may be used. The culture of the lactic acid bacteria can be obtained by culturing for about 1 to 2 days under facultative anaerobic conditions using, for example, a TYG medium. In addition, cells can be collected from the culture by centrifugation, and further washed once or twice with physiological saline or sterilized water.
また、本発明の乳酸菌は生菌体であっても死菌体であっても同等の機能性を発揮することから、本発明の皮膚外用剤において上記乳酸菌は特に生菌として含まれる必要はない。一般的な加熱滅菌操作による殺菌・滅菌処理物や、破砕物、凍結乾燥物等として含まれていても良い。 In addition, since the lactic acid bacterium of the present invention exhibits equivalent functionality regardless of whether it is a live cell or a dead cell, the lactic acid bacterium need not be included as a live cell in the external preparation for skin of the present invention. . It may be contained as a sterilized / sterilized product by a general heat sterilization operation, a crushed product, a freeze-dried product, or the like.
本発明において「抽出物」とは、上記乳酸菌の生菌体または死菌体を加熱処理した後に通常の方法で抽出するか、加熱処理しない場合には熱水（85〜100℃）を用いて通常の方法で抽出することにより得ることができる。例えば代表的には、上記乳酸菌培養物から集菌して得た菌体を生理食塩水で洗浄し、加熱処理をしてから凍結乾燥し、それを抽出溶媒に懸濁し、遠心分離して菌体を除去することによって得ることができる。
抽出溶媒としては、生理食塩水、滅菌水、培地などが挙げられる。
上記抽出物はそのまま用いても良く、濃縮乾固等して用いても良い。
In the present invention, the term “extract” means that the lactic acid bacteria live cells or dead cells are extracted by a normal method after heat treatment, or hot water (85 to 100 ° C.) is used when heat treatment is not performed. It can be obtained by extraction by a usual method. For example, typically, the cells obtained from the above lactic acid bacteria culture are washed with physiological saline, heat-treated, freeze-dried, suspended in an extraction solvent, centrifuged and centrifuged. It can be obtained by removing the body.
Examples of the extraction solvent include physiological saline, sterilized water, a culture medium, and the like.
The above extract may be used as it is, or may be used after concentration to dryness.
〔メラニン産生抑制用皮膚外用剤〕
本発明の乳酸菌は、後述の実施例に示すように優れたメラニン産生抑制作用を有しているため、本発明の皮膚外用剤はメラニン産生抑制剤として用いることができる。
本発明のメラニン産生抑制剤の剤型としては、例えば、ローション、乳液、ゲル、クリーム、軟膏剤、粉末、顆粒、経皮吸収剤等が例示される。
[External skin preparation for inhibiting melanin production]
Since the lactic acid bacteria of this invention have the outstanding melanin production inhibitory effect as shown in the below-mentioned Example, the skin external preparation of this invention can be used as a melanin production inhibitor.
Examples of the dosage form of the melanin production inhibitor of the present invention include lotions, emulsions, gels, creams, ointments, powders, granules, and transdermal absorption agents.
本発明のメラニン産生抑制剤は、皮膚に塗布することによりメラニン産生抑制作用を発揮することができる。本発明のメラニン産生抑制剤における上記乳酸菌またはその抽出物の含有量は、メラニン産生抑制効果を奏する限り特に制限されないが、例えば、乾燥菌体として50μg〜10mg/mLとすることができ、好ましくは250μg〜1mg/mL、より好ましくは500μg〜1mg/mLとすることができる。乳酸菌抽出物の場合は、上記の菌体濃度に相当する菌体抽出物を含有することが好ましい。 The melanin production inhibitor of this invention can exhibit a melanin production inhibitory effect by apply | coating to skin. The content of the lactic acid bacterium or the extract thereof in the melanin production inhibitor of the present invention is not particularly limited as long as it exerts a melanin production inhibitory effect, but can be, for example, 50 μg to 10 mg / mL as a dry cell, preferably It can be 250 μg to 1 mg / mL, more preferably 500 μg to 1 mg / mL. In the case of a lactic acid bacterium extract, it is preferable to contain a microbial cell extract corresponding to the above microbial cell concentration.
本発明のメラニン産生抑制剤には、上記乳酸菌またはその抽出物以外に、通常皮膚外用剤で使用される任意成分を含有することができる。かかる任意成分としては、水、アルコール、油性成分、保湿剤、柔軟剤、界面活性剤、増粘剤、防腐剤、紫外線吸収剤、抗酸化剤、緩衝剤、色素、香料、粉体類、皮膚栄養剤等が例示できる。また、ビタミンC、ハイドロキノン、レゾルシノール、アルブチンおよびこれらの誘導体等、他のメラニン産生抑制剤を含有することもできる。 The melanin production inhibitor of the present invention may contain optional components usually used in a skin external preparation, in addition to the lactic acid bacteria or the extract thereof. Such optional ingredients include water, alcohol, oily ingredients, moisturizers, softeners, surfactants, thickeners, preservatives, UV absorbers, antioxidants, buffers, pigments, fragrances, powders, skin A nutrient etc. can be illustrated. In addition, other melanin production inhibitors such as vitamin C, hydroquinone, resorcinol, arbutin, and derivatives thereof may be contained.
〔育毛・発毛用および／または脱毛抑制用皮膚外用剤〕
毛周期（ヘアサイクル）は成長期、退行期、休止期に分けられる。そのうち成長期では、毛乳頭に分布している毛細血管から栄養を受けて、毛母細胞が細胞分裂を行い、角化しながら次々に分裂してできる新しい細胞によって表皮側に押し上げられ、毛を形成していく。退行期では、毛細血管からの血液供給を受けられなくなった毛母細胞は分裂を停止し、毛が表皮側に押し上げられていく。休止期になると、毛乳頭と毛が完全に離れ、毛包との固着がなくなるため、シャンプーや摩擦などによって毛は自然と抜け落ち、やがて再び成長期の準備に入る。
[Skin preparation for hair growth / hair growth and / or hair loss suppression]
The hair cycle is divided into a growth phase, a regression phase, and a resting phase. During the growth period, the hair matrix cells receive nutrition from the capillaries distributed in the hair papilla, and the hair matrix cells undergo cell division and are pushed up to the epidermis side by new cells that divide one after another and form hair. I will do it. In the regression phase, hair matrix cells that cannot receive blood supply from capillaries stop dividing, and the hair is pushed up to the epidermis side. In the rest period, the hair papilla is completely separated from the hair, and the hair follicle is not fixed. Therefore, the hair naturally falls off due to shampoo, friction, and the like, and then begins preparation for the growth period again.
繊維芽細胞増殖因子（FGF-7）及び血管内皮増殖因子（VEGF）は、いずれも毛乳頭細胞から分泌される増殖因子である。FGF-7は、毛母細胞に作用してその増殖を促進することにより毛髪成長を促進し、ヘアサイクルでの成長期に有効であると考えられている。VEGFは、血管新生を促進し、それにより毛母細胞が活性化することで、発毛が促進されると考えられている。VEGFは血管新生因子であることから、成長期だけでなく、毛細血管の減少が始まる退行期にも有効であると考えられている。 Fibroblast growth factor (FGF-7) and vascular endothelial growth factor (VEGF) are both growth factors secreted from hair papilla cells. FGF-7 acts on hair matrix cells and promotes their proliferation to promote hair growth and is considered to be effective during the growth phase of the hair cycle. VEGF is believed to promote hair growth by promoting angiogenesis and thereby activating hair matrix cells. Since VEGF is an angiogenic factor, it is considered to be effective not only in the growth phase but also in the regression phase where the reduction of capillaries begins.
本発明の乳酸菌またはその抽出物は、後述の実施例に示すように優れた毛乳頭細胞賦活作用、繊維芽細胞増殖因子産生促進作用、血管内皮増殖因子産生促進作用を有しているため、本発明の皮膚外用剤は育毛・発毛剤および／または脱毛抑制剤として用いることができる。 Since the lactic acid bacterium of the present invention or an extract thereof has excellent dermal papilla cell activating effect, fibroblast growth factor production promoting effect, and vascular endothelial growth factor production promoting effect as shown in the examples described later, The skin external preparation of the invention can be used as a hair growth / hair growth agent and / or hair loss inhibitor.
具体的には、毛乳頭細胞賦活化試験において、H61は細胞分裂を促進する効果が顕著であり、G50は細胞を維持する効果が顕著であると認められた。また、H61では高いFGF-7産生促進作用が、G50では高いVEGF産生促進作用が認められた。したがって、H61は成長期において育毛作用を発揮し、G50は成長期〜退行期において発毛・脱毛抑制作用を発揮するものと期待される。このように、H61とG50はヘアサイクルの中の異なる時期においてそれぞれ顕著な効果を発揮することから、両菌株を併用することによって、より効果の高い育毛・発毛・脱毛抑制剤を提供できる。 Specifically, in the dermal papilla cell activation test, it was recognized that H61 had a remarkable effect of promoting cell division and G50 had a remarkable effect of maintaining cells. Further, H61 showed a high FGF-7 production promoting action, and G50 showed a high VEGF production promoting action. Therefore, it is expected that H61 exhibits a hair growth effect during the growth period, and G50 exhibits a hair growth / hair loss inhibiting effect during the growth period to the regression phase. Thus, since H61 and G50 exhibit remarkable effects at different times in the hair cycle, a combination of both strains can provide a more effective hair growth / hair growth / hair loss inhibitor.
本発明の育毛・発毛・脱毛抑制剤の剤型としては、例えば、ローション、乳液、ゲル、クリーム、軟膏剤、ヘアートニック、リンス、シャンプー、ヘアーフォーム、ヘアーゲル等が例示される。 Examples of the dosage form of the hair growth / hair growth / hair loss inhibitor of the present invention include lotions, emulsions, gels, creams, ointments, hair arts, rinses, shampoos, hair foams, hair gels and the like.
本発明の育毛・発毛・脱毛抑制剤は、皮膚に塗布することにより育毛・発毛・脱毛抑制作用を発揮することができる。本発明の育毛・発毛・脱毛抑制剤における上記乳酸菌またはその抽出物の含有量は、育毛・発毛・脱毛抑制効果を奏する限り特に制限されないが、例えば、乾燥菌体として0.1μg〜10mg/mLとすることができ、好ましくは1μg〜10mg/mL、より好ましくは1〜100μg/mLとすることができる。乳酸菌抽出物の場合は、上記の菌体濃度に相当する菌体抽出物を含有することが好ましい。 The hair growth / hair growth / hair loss inhibitor of the present invention can exert a hair growth / hair growth / hair loss inhibitory effect when applied to the skin. The content of the lactic acid bacterium or the extract thereof in the hair growth / hair growth / hair loss inhibitor of the present invention is not particularly limited as long as it exhibits the effect of hair growth / hair growth / hair loss suppression, for example, 0.1 μg to 10 mg / kg as dry cells. mL, preferably 1 μg to 10 mg / mL, more preferably 1 to 100 μg / mL. In the case of a lactic acid bacterium extract, it is preferable to contain a microbial cell extract corresponding to the above microbial cell concentration.
本発明の育毛・発毛・脱毛抑制剤には、上記乳酸菌またはその抽出物以外に、通常皮膚外用剤で使用される任意成分を含有することができる。かかる任意成分としては、水、アルコール、油性成分、保湿剤、柔軟剤、界面活性剤、増粘剤、防腐剤、紫外線吸収剤、抗酸化剤、緩衝剤、色素、香料、粉体類、皮膚栄養剤等が好ましく例示できる。また、アデノシン等、他の育毛・発毛剤または脱毛抑制剤を含有することもできる。 The hair growth / hair growth / hair loss inhibitor of the present invention can contain, in addition to the lactic acid bacteria or the extract thereof, optional components that are usually used in external preparations for skin. Such optional ingredients include water, alcohol, oily ingredients, moisturizers, softeners, surfactants, thickeners, preservatives, UV absorbers, antioxidants, buffers, pigments, fragrances, powders, skin A nutritional agent etc. can illustrate preferably. Further, other hair growth / hair growth agents or hair loss inhibitors such as adenosine can also be contained.
以下に実施例、および比較例を挙げて本発明を具体的に説明するが、本発明はこれらに限定されるものではない。 Hereinafter, the present invention will be specifically described with reference to Examples and Comparative Examples, but the present invention is not limited thereto.
実施例１（ヒト毛乳頭細胞賦活効果）
毛乳頭細胞を被験物質（H61又はG50）添加下で培養後、生細胞数を測定し、また繊維芽細胞増殖因子（FGF-7）、血管内皮増殖因子（VEGF）産生量をELISA法にて測定した。
Example 1 (human hair papilla cell activation effect)
After cultivating dermal papilla cells with the addition of a test substance (H61 or G50), the number of viable cells is measured, and fibroblast growth factor (FGF-7) and vascular endothelial growth factor (VEGF) production are determined by ELISA. It was measured.
（１）細胞
ヒト毛乳頭細胞（TOYOBO）は、白色人種、51歳女性由来のヒト頭髪毛乳頭培養細胞を購入して使用した。
(1) Cells Human dermal papilla cells (TOYOBO) were purchased and used from human race, 51-year-old female human dermal papilla cultured cells.
（２）ヒト頭髪毛乳頭細胞の培養条件
毛乳頭細胞は、付属の毛乳頭細胞専用培地（TOYOBO）を用い、CO２インキュベーター（5％CO２、37℃）内で培養した。培養に使用したフラスコはＩ型コラーゲンコート（Cellmatrix Type I-C、新田ゼラチン）して使用した。細胞の継代には付属の毛乳頭細胞専用サブカルチャーセット（TOYOBO）を用いて細胞をフラスコより剥離し、使用した。
(2) Culture conditions for human hair hair papilla cells Hair papilla cells were cultured in a CO 2 incubator (5% CO 2 , 37 ° C.) using the attached hair papilla cell dedicated medium (TOYOBO). The flask used for the culture was a type I collagen coat (Cellmatrix Type IC, Nitta Gelatin). For cell passage, cells were detached from the flask using the attached subculture set (TOYOBO) exclusively for dermal papilla cells and used.
（３）被験物質調整
ラクトコッカス・ラクティス・サブスピーシーズ クレモリス H61およびラクトコッカス・ラクティス・サブスピーシーズ ラクティス G50は、予めそれぞれTYG培地で常法に従って一昼夜培養し、洗浄、加熱処理（121℃、15分間）を行った後、凍結乾燥して得られた乾燥菌体を被検物質とした。なお、当該乾燥菌体0.001mgあたりに含まれる菌数は×10５レベルであった。
(3) Preparation of test substance Lactococcus lactis sub-species Cremolis H61 and Lactococcus lactis sub-species lactis G50 were each pre-cultured in TYG medium overnight according to a conventional method, washed and heat-treated (121 ° C., 15 minutes) Then, dried cells obtained by freeze-drying were used as test substances. In addition, the number of bacteria contained per 0.001 mg of the dry cells was at a level of × 10 5 .
各被験物質を100mg/mL濃度となるよう毛乳頭細胞専用培地によく懸濁し、10倍濃縮溶液として使用した。また、濾過滅菌処理において前述懸濁液を遠心分離（12000×g、5分間）し、上清を低吸着タイプの0.45μm濾過滅菌フィルター（Millex-HV、Millex）にて滅菌処理を行った。濾過液を10倍濃縮溶液として使用した。 Each test substance was well suspended in a medium exclusively for hair papilla cells to a concentration of 100 mg / mL, and used as a 10-fold concentrated solution. Further, in the filtration sterilization treatment, the suspension was centrifuged (12000 × g, 5 minutes), and the supernatant was sterilized with a low adsorption type 0.45 μm filtration sterilization filter (Millex-HV, Millex). The filtrate was used as a 10-fold concentrated solution.
陽性対照のアデノシン（Wako）は25mg/mL濃度となるようジメチルスルホキシド（DMSO）（nacalai tesque）に溶解した。本試験ではアデノシン終濃度25μg/mLとし、このときの溶媒（DMSO）濃度は0.1％になる。 Positive control adenosine (Wako) was dissolved in dimethyl sulfoxide (DMSO) (nacalai tesque) to a concentration of 25 mg / mL. In this test, the final concentration of adenosine is 25 μg / mL, and the solvent (DMSO) concentration at this time is 0.1%.
（４）細胞賦活試験
細胞を1.2×10４cells/0.3ｍｌ/ウェルで48ウェルプレートに播種した。CO２インキュベーター内（5％CO２、37℃）で、1日間培養後、被験物質（0.001mg/mL, 0.1mg/mL, 10mg/mL）を含む培地に置換した。その後、1日間及び3日間培養し、それぞれの細胞の増殖性（viability）を生細胞数測定試薬SF（nacalai tesque）で比較検討した。n＝3にて行った。その際、培養上清を回収し、後述のように培養上清中のFGF-7,VEGF産生量をELISA法で測定した。
(4) Cell activation test Cells were seeded in a 48-well plate at 1.2 × 10 4 cells / 0.3 ml / well. After culturing for 1 day in a CO 2 incubator (5% CO 2 , 37 ° C.), the medium was replaced with a medium containing a test substance (0.001 mg / mL, 0.1 mg / mL, 10 mg / mL). Thereafter, the cells were cultured for 1 day and 3 days, and the viability of each cell was compared with a living cell number measuring reagent SF (nacalai tesque). n = 3. At that time, the culture supernatant was collected, and the production amounts of FGF-7 and VEGF in the culture supernatant were measured by ELISA as described later.
生細胞数測定において、培養上清を除去した細胞に生細胞数測定試薬SFを10％含む培地を添加（300μL/ウェル）した。添加後、30分、90分後に培養上清（100μL/ウェル）を別の96ウェルプレートに移し、マイクロプレートリーダーにて吸光度測定した。90分、30分の値から1時間あたりの吸光度変化量を算出した。 In the measurement of the number of viable cells, a medium containing 10% of the viable cell count measuring reagent SF was added to the cells from which the culture supernatant was removed (300 μL / well). 30 minutes and 90 minutes after the addition, the culture supernatant (100 μL / well) was transferred to another 96-well plate, and the absorbance was measured with a microplate reader. The amount of change in absorbance per hour was calculated from the values for 90 minutes and 30 minutes.
培養1日後の結果を図１に、培養3日後の結果を図２に示す。図中、‘Non’は未処理対照、‘25μg/mL Ade’はアデノシン25μg/mLを添加した陽性対照（DMSO濃度 0.1％）、‘0.1％DMSO’は前記陽性対照からアデノシンを除いたもの、縦軸は生細胞数と比例関係にある1時間当りの吸光度ABSの変化量（単位：ABS/hr）、をそれぞれ示す。
被験物質処理1日間ではH61株、G50株ともに低濃度域に細胞賦活性が認められた（図１）。H61株は0.001mg/mLで対照（Non）の1.28倍、G50株は0.1mg/mLで1.24倍であった。一方、被験物質処理3日間ではH61株、G50株ともに0.1mg/mL濃度区で最大の賦活性を示したが、G50株の方がより賦活性を示した（H61株は対照（Non）の1.47倍、G50株は2.21倍）（図２）。一方、陽性対照であるアデノシンは、1日間の処理で0.1％DMSO区の1.29倍に生細胞数が上昇したが、3日間の処理では細胞の賦活性が認められなかった。
The results after 1 day of culture are shown in FIG. 1, and the results after 3 days of culture are shown in FIG. In the figure, “Non” is an untreated control, “25 μg / mL Ade” is a positive control to which 25 μg / mL of adenosine is added (DMSO concentration 0.1%), “0.1% DMSO” is a positive control in which adenosine is excluded, The vertical axis represents the amount of change in absorbance ABS per hour (unit: ABS / hr) that is proportional to the number of living cells.
In one day of treatment with the test substance, cell activation was observed in the low concentration range for both the H61 strain and the G50 strain (FIG. 1). The H61 strain was 0.001 mg / mL, 1.28 times the control (Non), and the G50 strain was 0.1 mg / mL, 1.24 times. On the other hand, in the test substance treatment for 3 days, the H61 strain and the G50 strain showed the maximum activation in the 0.1 mg / mL concentration group, but the G50 strain showed the more activation (the H61 strain was the control (Non) 1.47 times, G50 strain 2.21 times) (Figure 2). On the other hand, the positive control, adenosine, increased the number of living cells to 1.29 times that of 0.1% DMSO in the treatment for 1 day, but the activation of the cells was not observed in the treatment for 3 days.
なお、培養上清回収前に細胞形態を顕微鏡観察したところ、H61、G50ともに細胞形態の異常は認められないことから大きな細胞毒性はないと判断できる。 When the cell morphology was observed with a microscope before collecting the culture supernatant, no abnormal cell morphology was observed for both H61 and G50, so it can be determined that there was no significant cytotoxicity.
（５）FGF-7産生試験
細胞を5×10３cells/0.1ｍｌ/ウェルで96ウェルプレートに播種したこと以外は、上述（４）の試験と同様の条件で培養した細胞から回収した培養上清を、測定まで-80℃で保存した。測定時に氷上で解凍し、FGF7 Human ELISA kit（abcam）にて培養上清中のFGF-7量を測定した。予備検討により培養上清を希釈せず、原液を測定することとした。詳細なプロトロールを以下に示す。
(5) FGF-7 production test The culture recovered from the cells cultured under the same conditions as in the above test (4) except that the cells were seeded in a 96-well plate at 5 × 10 3 cells / 0.1 ml / well. Kiyo was stored at -80 ° C until measurement. At the time of measurement, the sample was thawed on ice, and the amount of FGF-7 in the culture supernatant was measured with FGF7 Human ELISA kit (abcam). In the preliminary study, it was decided to measure the stock solution without diluting the culture supernatant. Detailed protocol is shown below.
〔FGF-7測定プロトコール〕
(1)FGF-7標準原液（50ng/ml）をもとに、反応緩衝液を用いて400、133.3、44.44、14.81、4.94、1.65、0.55pg/mlのFGF-7標準溶液を調製する。
(2)FGF-7標準溶液および検体（培養上清）100μLをFGF-7固相化マイクロプレートの各ウェルに添加し、室温にて2時間30分反応する（一次反応）。
(3)ウェル内の溶液を除去し、洗浄液で4回洗浄する。
(4)ビオチン標識抗FGF-7抗体溶液を各ウェルに100μLずつ添加し、室温で60分間反応させる（二次反応）。
(5)ウェル内の溶液を除去し、洗浄液で4回洗浄する。
(6)ストレプトアビジン溶液100μLを各ウェルに添加し、室温で45分反応する。
(7)酵素基質溶液100μLを各ウェルに添加し、遮光・室温下で30分間静置する（発色反応）。
(8)反応停止溶液を各ウェルに50μlずつ添加し、プレートミキサーで1分間混和後、マイクロプレートリーダーで各ウェルの吸光度を測定する（測定波長450nm）。
(9)標準曲線から、検体中のFGF-7濃度を算出する。
[FGF-7 measurement protocol]
(1) Prepare 400, 133.3, 44.44, 14.81, 4.94, 1.65, 0.55 pg / ml FGF-7 standard solution from the FGF-7 standard stock solution (50 ng / ml) using the reaction buffer.
(2) Add 100 μL of FGF-7 standard solution and specimen (culture supernatant) to each well of the FGF-7 solid-phased microplate and react at room temperature for 2 hours 30 minutes (primary reaction).
(3) Remove the solution in the well and wash 4 times with the washing solution.
(4) Add 100 μL of biotin-labeled anti-FGF-7 antibody solution to each well and allow to react at room temperature for 60 minutes (secondary reaction).
(5) Remove the solution in the well and wash 4 times with the washing solution.
(6) Add 100 μL of streptavidin solution to each well and react at room temperature for 45 minutes.
(7) Add 100 μL of the enzyme substrate solution to each well and let stand for 30 minutes at room temperature in the dark (coloring reaction).
(8) Add 50 μl of the reaction stop solution to each well, mix with a plate mixer for 1 minute, and then measure the absorbance of each well with a microplate reader (measurement wavelength: 450 nm).
(9) Calculate the FGF-7 concentration in the specimen from the standard curve.
培養1日後のFGF-7産生量を図３に、培養3日後のFGF-7産生量を図４に、それぞれ示す。図中、‘Non’は未処理対照、‘25μg/mL Ade’はアデノシン25μg/mLを添加した陽性対照（DMSO濃度 0.1％）、‘0.1％DMSO’は前記陽性対照からアデノシンを除いたもの、をそれぞれ示す。図３、４の縦軸は培養上清中の繊維芽細胞増殖因子FGF-7の量（単位：pg/mL）を示す。
処理１日間ではH61株、G50株ともに最高濃度である10mg/mLで顕著にFGF-7の産生を促進していた（H61株は対照（Non）の3.85倍、G50株は7.10倍）が、陽性対照であるアデノシンでは、0.1％DMSO区と比べてFGF-7の産生促進効果は見られなかった（図３）。処理3日間では、H61株において0.001mg/mL（1μg/mL）、0.1mg/mL（100μg/mL）もFGF-7の蓄積が認められた（それぞれ対照（Non）の1.57倍、1.48倍）（図４）。しかし、G50株は処理3日間で1μg/mL、100μg/mL濃度において10mg/mL区と同程度まで増加しなかった。陽性対照であるアデノシンは、処理期間3日間で0.1％DMSO区の1.66倍の増加を示した。
FIG. 3 shows the amount of FGF-7 produced after 1 day of culture, and FIG. 4 shows the amount of FGF-7 produced after 3 days of culture. In the figure, “Non” is an untreated control, “25 μg / mL Ade” is a positive control to which 25 μg / mL of adenosine is added (DMSO concentration 0.1%), “0.1% DMSO” is a positive control in which adenosine is excluded, Respectively. 3 and 4 indicate the amount (unit: pg / mL) of fibroblast growth factor FGF-7 in the culture supernatant.
In one day of treatment, both H61 and G50 strains significantly promoted the production of FGF-7 at the highest concentration of 10 mg / mL (H61 strain was 3.85 times that of control (Non) and G50 strain was 7.10 times). The positive control adenosine did not show the FGF-7 production-promoting effect compared to the 0.1% DMSO group (FIG. 3). In 3 days of treatment, accumulation of FGF-7 was also observed in the H61 strain at 0.001 mg / mL (1 μg / mL) and 0.1 mg / mL (100 μg / mL) (1.57 times and 1.48 times of the control (Non), respectively) (FIG. 4). However, the G50 strain did not increase to the same level as the 10 mg / mL group at 1 μg / mL and 100 μg / mL at 3 days after treatment. The positive control, adenosine, showed a 1.66-fold increase over the 0.1% DMSO group over the 3-day treatment period.
（６）VEGF産生試験
上述（４）の試験の条件で培養した細胞から回収した培養上清を、測定まで-80℃で保存した。測定時に氷上で解凍し、Human VEGF Quantikine ELISA（R & D Systems）にて培養上清中のVEGF量を測定した。予備検討により培養上清を３倍希釈し測定することとした。詳細なプロトロールを以下に示す。
(6) VEGF production test Culture supernatants collected from cells cultured under the test conditions described in (4) above were stored at -80 ° C until measurement. At the time of measurement, the sample was thawed on ice, and the amount of VEGF in the culture supernatant was measured by Human VEGF Quantikine ELISA (R & D Systems). In the preliminary study, the culture supernatant was diluted 3 times and measured. Detailed protocol is shown below.
〔VEGF測定プロトコール〕
(1)VEGF標準原液（2000pg/ml）をもとに、反応緩衝液を用いて1000、500、250、125、62.5、31.2、15.6pg/mlのVEGF標準溶液を調製する。
(2)VEGF標準溶液および検体（培養上清）200μLを、50μLの希釈溶液を各ウェルに添加したVEGF固相化マイクロプレートに添加し、室温にて2時間反応する（一次反応）。
(3)ウェル内の溶液を除去し、洗浄液で3回洗浄する。
(4)西洋ワサビペルオキシダーゼ結合VEGF抗体溶液を各ウェルに200μLずつ添加し、室温で2時間反応させる（二次反応）。
(5)ウェル内の溶液を除去し、洗浄液で3回洗浄する。
(6)基質溶液200μLを各ウェルに添加し、室温で20分反応する。
(7)反応停止溶液を各ウェルに50μlずつ添加し、プレートミキサーで1分間混和後、マイクロプレートリーダーで各ウェルの吸光度を測定する（測定波長450nm）。
(8)標準曲線から、検体中のVEGF濃度を算出する。
[VEGF measurement protocol]
(1) Based on the VEGF standard stock solution (2000 pg / ml), prepare 1000, 500, 250, 125, 62.5, 31.2, 15.6 pg / ml VEGF standard solutions using the reaction buffer.
(2) VEGF standard solution and 200 μL of specimen (culture supernatant) are added to a VEGF-immobilized microplate to which 50 μL of diluted solution has been added to each well, and reacted at room temperature for 2 hours (primary reaction).
(3) Remove the solution in the well and wash 3 times with the washing solution.
(4) 200 μL of horseradish peroxidase-conjugated VEGF antibody solution is added to each well and allowed to react at room temperature for 2 hours (secondary reaction).
(5) Remove the solution in the well and wash 3 times with the washing solution.
(6) Add 200 μL of substrate solution to each well and react for 20 minutes at room temperature.
(7) Add 50 μl of the reaction stop solution to each well, mix with a plate mixer for 1 minute, and then measure the absorbance of each well with a microplate reader (measurement wavelength: 450 nm).
(8) Calculate the VEGF concentration in the specimen from the standard curve.
培養1日後のVEGF産生量を図５に、培養3日後のVEGF産生量を図６に、それぞれ示す。図中、‘Non’は未処理対照、‘Ade’はアデノシン25μg/mLを添加した陽性対照（DMSO濃度 0.1％）、‘0.1％DMSO’は前記陽性対照からアデノシンを除いたもの、をそれぞれ示す。図５、６の縦軸は培養上清中の血管内皮増殖因子VEGFの量（単位：pg/mL）を示す。
処理期間1日においてH61、G50ともに産生促進効果を示したが（0.001mg/mL、0.1mg/mL、10mg/mLでH61株は、それぞれ対照（Non）の3.54倍、3.29倍、2.51倍であり、G50株は2.76倍、3.44倍、5.77倍）、なかでもG50の10mg/mLは顕著に増加していた（図５）。一方、処理期間3日においてもH61、G50ともに0.001mg/mL（1μg/mL）、0.1mg/mL（100μg/mL）濃度で増加を示したが（H61株で対照（Non）の1.93倍、2.01倍、G50株で1.81倍、1.78倍）、10mg/mL濃度は無添加区（Non）と同程度まで減少していた（図６）。一般に過剰に合成された成分がフィードバック効果により合成が抑制される場合がある。1日目において過剰に分泌されたことで3日目において抑制作用が働いた可能性が考えられる。陽性対照であるアデノシンは、1日処理ではVEGFの産生促進効果は見られなかったが、3日間処理で1.23倍の促進作用を示した。
FIG. 5 shows the amount of VEGF produced after 1 day of culture, and FIG. 6 shows the amount of VEGF produced after 3 days of culture. In the figure, 'Non' is an untreated control, 'Ade' is a positive control (DMSO concentration 0.1%) added with adenosine 25 µg / mL, and '0.1% DMSO' is a positive control excluding adenosine. . 5 and 6 indicate the amount of vascular endothelial growth factor VEGF (unit: pg / mL) in the culture supernatant.
Although H61 and G50 showed production-promoting effects in the treatment period of 1 day (0.001 mg / mL, 0.1 mg / mL and 10 mg / mL, the H61 strain was 3.54 times, 3.29 times and 2.51 times the control (Non), respectively. The G50 strain was 2.76 times, 3.44 times, 5.77 times), and in particular, 10 mg / mL of G50 was remarkably increased (FIG. 5). On the other hand, even in the treatment period of 3 days, both H61 and G50 showed increases in concentrations of 0.001 mg / mL (1 μg / mL) and 0.1 mg / mL (100 μg / mL) (H93 strain 1.93 times the control (Non), 2.01 times, 1.81 times and 1.78 times in the G50 strain), and the 10 mg / mL concentration decreased to the same level as in the non-added group (Non) (FIG. 6). In general, synthesis of an excessively synthesized component may be suppressed due to a feedback effect. It may be possible that the inhibitory action worked on the third day due to excessive secretion on the first day. The positive control, adenosine, showed no VEGF production promoting effect on 1-day treatment, but showed a 1.23-fold promoting effect on 3-day treatment.
（７）考察
細胞賦活試験においてH61及びG50の毛乳頭細胞への有効性が認められた。しかし、その効果・性質は異なっていた。H61は、細胞賦活試験では処理期間1日で低濃度域で比較的作用を示すことから、細胞分裂を促進する効果があることが考えられる（培養1日は細胞増殖曲線のうち増殖期、培養3日は維持期を検証することになる）。一方、G50は、処理期間3日で顕著に細胞賦活性が現れたことから、細胞の抗老化など細胞維持に関与するものと考えられる。ヘアサイクルにおいて細胞分裂の盛んな成長期にはH61が、分裂を停止し細胞を維持する段階の退行期にはG50が、有効に作用する可能性がある。
(7) Discussion The effectiveness of H61 and G50 on dermal papilla cells was confirmed in the cell activation test. However, the effects and properties were different. In the cell activation test, H61 is relatively effective in a low concentration range in a treatment period of 1 day, so it is considered that H61 has an effect of promoting cell division. 3 days will verify the maintenance period). On the other hand, G50 is thought to be involved in cell maintenance such as anti-aging of cells because cell activation appeared remarkably in the treatment period of 3 days. There is a possibility that H61 works effectively in the growth phase where cell division is active in the hair cycle, and G50 works in the regression phase of stopping the division and maintaining the cells.
細胞増殖因子産生に及ぼす効果として、G50は処理期間1日でVEGFの大部分を促進することから、短期的な作用効果があると考えられる。VEGFは血管新生因子であり、成長期だけでなく毛細血管の減少が始まる退行期にも有効であることから、G50は退行期において短期間使用で作用（主に発毛・脱毛抑制作用）を発揮する可能性がある。一方、H61は低濃度であっても処理期間3日でゆるやかにFGF-7の産生を促進することから、長期的効果があると考えられる。FGF-7は毛母細胞分裂を促進することから、ヘアサイクルでの成長期に有効であると考えられる。したがって、H61は成長期において長期間使用で作用（主に育毛作用）を発揮する可能性がある。 As an effect on cell growth factor production, G50 promotes most of VEGF in a treatment period of 1 day, so it is considered to have a short-term effect. Since VEGF is an angiogenic factor and is effective not only in the growth phase but also in the regression phase where the reduction of capillaries begins, G50 acts in the regression phase with a short period of use (mainly hair growth / hair loss inhibition) There is a possibility of exerting. On the other hand, H61 is considered to have a long-term effect because it gradually promotes the production of FGF-7 in a treatment period of 3 days even at a low concentration. Since FGF-7 promotes hair matrix cell division, it is considered to be effective during the growth phase of the hair cycle. Therefore, H61 may exert an action (mainly hair growth action) when used for a long time in the growth period.
以上に述べたように、本実施例により、ラクトコッカス属乳酸菌H61、G50はいずれも高い育毛・発毛効果および脱毛抑制効果を奏することが示された。育毛・発毛剤および／または脱毛抑制剤における乳酸菌抽出物の添加濃度は0.001〜10mg/mLとすることが好ましく、0.001〜0.1mg/mL（いずれも乾燥菌体換算）とすることがより好ましいことがわかった。H61とG50はヘアサイクルの中の異なる時期においてそれぞれ顕著な効果を発揮することから、両菌株を併用することによって、より効果の高い育毛・発毛剤および／または脱毛抑制剤を提供できる。また、両菌株の育毛・発毛作用および／または脱毛抑制作用は死菌体（乾燥菌体）の抽出物でも見られることから、幅広い加工処理に対応できると考えられる。 As described above, according to the present Example, it was shown that both Lactococcus lactic acid bacteria H61 and G50 have high hair growth / hair growth effect and hair loss inhibiting effect. The concentration of the lactic acid bacteria extract in the hair growth / hair growth agent and / or hair loss inhibitor is preferably 0.001 to 10 mg / mL, more preferably 0.001 to 0.1 mg / mL (all in terms of dry cells). I understood it. Since H61 and G50 exhibit remarkable effects at different times in the hair cycle, a combination of both strains can provide a more effective hair growth / hair growth inhibitor and / or hair loss inhibitor. Moreover, since the hair-growth / hair growth action and / or hair loss-inhibiting action of both strains is also observed in the extract of dead cells (dry cells), it is considered that it can cope with a wide range of processing.
参考例（メラニン産生抑制効果）
マウスB16メラノーマ細胞(以下「B16細胞」と略す。)は、日焼けやシミの原因となるメラニンを産生する特徴を有する。また,α-メラノサイト刺激ホルモン(以下「α-MSH」と略す。)により、メラニン産生が促進されることが知られている。このため、日焼けやシミをターゲットとしたメラニン産生の細胞モデル系として研究などに広く用いられている。本試験では、B16細胞がα-MSH刺激または未刺激条件下で産生するメラニンを測定し、未処置対照のメラニン産生量に対する試験液（H61又はG50を含有）添加時のメラニン産生量からメラニン産生率を求め、メラニン産生抑制作用を評価した。
Reference example (melanin production inhibitory effect)
Mouse B16 melanoma cells (hereinafter abbreviated as “B16 cells”) have the characteristic of producing melanin that causes sunburn and spots. It is also known that melanin production is promoted by α-melanocyte stimulating hormone (hereinafter abbreviated as “α-MSH”). For this reason, it is widely used in research as a cell model system for melanin production targeting sunburn and spots. In this test, melanin produced by B16 cells under α-MSH stimulation or unstimulated conditions was measured, and melanin production was calculated from the amount of melanin produced when the test solution (containing H61 or G50) was added to the melanin production of the untreated control. The rate was obtained and the melanin production inhibitory action was evaluated.
（１）試験液の調製
ラクトコッカス・ラクティス・サブスピーシーズ クレモリス H61およびラクトコッカス・ラクティス・サブスピーシーズ ラクティス G50は、予めそれぞれTYG培地で常法に従って一昼夜培養し、洗浄、加熱処理（121℃、15分間）を行った後、凍結乾燥して得られた乾燥菌体を検体とした。なお、当該乾燥菌体500μgあたりに含まれる菌数は10８レベルであった。
(1) Preparation of test solution Lactococcus lactis subspecies Cremolis H61 and Lactococcus lactis subspecies lactis G50 were each preliminarily cultured in TYG medium overnight for a day, washed, and heated (121 ° C., 15 minutes) The dried cells obtained by freeze-drying were used as specimens. The number of bacteria contained per 500 μg of the dried cells was 10 8 levels.
検体に生理食塩液を加え30秒間かくはんした後、遠心分離(18000g、5分間)し、上清を分取した。これを50mg/mL試験液原液とし、培地で希釈して検体濃度4000、2000及び1000μg/mLの試験液を調製した。 A physiological saline solution was added to the sample and stirred for 30 seconds, followed by centrifugation (18000 g, 5 minutes), and the supernatant was collected. This was used as a 50 mg / mL test solution stock solution and diluted with a medium to prepare test solutions having specimen concentrations of 4000, 2000, and 1000 μg / mL.
（２）試験操作
B16細胞を24ウェルプレートに播種後1日間培養した。4000、2000及び1000μg/mLの各試験液を添加した（検体の終濃度は1000、500及び250μg/mL）。この際、α-MSH刺激条件では、α-MSH（和光純薬工業株式会社）を終濃度5μmol/Lとなるように添加した。培地のみを加えたものを未処置対照、アルブチン（和光純薬工業株式会社）を終濃度1mmol/Lとなるように加えたものを陽性対照として同様に試験を行った。4日間培養後、倒立型位相差顕微鏡にて観察及び撮影した。24ウェルプレートの培養上清を除去し，1mol/L水酸化ナトリウム溶液を加え、80℃、1時間加温し、B16細胞に蓄積したメラニンを抽出した。主な試験条件を表１に示した。
(2) Test operation
B16 cells were cultured for 1 day after seeding in 24-well plates. 4000, 2000, and 1000 μg / mL of each test solution was added (final concentrations of specimens were 1000, 500, and 250 μg / mL). At this time, α-MSH (Wako Pure Chemical Industries, Ltd.) was added at a final concentration of 5 μmol / L under α-MSH stimulation conditions. The same test was performed using a medium alone as an untreated control and an arbutin (Wako Pure Chemical Industries, Ltd.) added to a final concentration of 1 mmol / L as a positive control. After culturing for 4 days, it was observed and photographed with an inverted phase contrast microscope. The culture supernatant of the 24-well plate was removed, 1 mol / L sodium hydroxide solution was added, and the mixture was heated at 80 ° C. for 1 hour to extract melanin accumulated in B16 cells. The main test conditions are shown in Table 1.
（３）測定方法
マイクロプレートリーダー（SpectraMax M2e ，Molecular Devices Corporation）を用い、抽出したメラニンの吸光度を405nmにて測定した。
(3) Measurement method The absorbance of the extracted melanin was measured at 405 nm using a microplate reader (SpectraMax M2e, Molecular Devices Corporation).
（４）算出方法
未処置対照の吸光度に対する各試験液の吸光度から、次式によりメラニン産生率を算出した。陽性対照は各試験液と同様に算出した。
(4) Calculation method From the absorbance of each test solution relative to the absorbance of the untreated control, the melanin production rate was calculated by the following formula. The positive control was calculated in the same manner as each test solution.
メラニン産生率（％）＝Sa／CN ×100
Sa：各試験液の吸光度
CN：未処置対照の吸光度の平均値（n＝3）
Melanin production rate (%) = Sa /
Sa: Absorbance of each test solution
CN: Average absorbance of untreated controls (n = 3)
（５）結果と考察
メラニン産生抑制試験の結果を表２、３および図７、８に示す。なお、図７、８の棒グラフのうち、黒はコントロール（未処置対照）を、ドットはアルブチン1mM添加区（陽性対照）を、斜線は被検物質（H61又はG50）500μg/mL添加区を、それぞれ示す。
H61、G50とも、陽性対照であるアルブチンよりはやや劣るものの、メラニン産生抑制効果を示した。特にα-MSH刺激条件では、乳酸菌無添加の未処置対照に比べて、H61はメラニン産生を68％に抑制し、G50は81％に抑制することができた（いずれも500μg/mL添加時）。このことから、メラニン産生に及ぼす影響は乳酸菌の種類により異なると考えられた。
また、乳酸菌抽出物の添加濃度は250〜1000μg/mLとすることが好ましく、500〜1000μg/mL（いずれも乾燥菌体換算）とすることがより好ましいことがわかった。
なお、アルブチンは発ガン性物質であるハイドロキノンの配糖体であることから、使用濃度によっては安全性に問題がある場合がある。一方、本発明の乳酸菌はいずれも長い食経験を持つラクトコッカス属乳酸菌であり、安全性が高いと思われる。
(5) Results and Discussion The results of the melanin production inhibition test are shown in Tables 2 and 3 and FIGS. In the bar graphs of FIGS. 7 and 8, black represents a control (untreated control), a dot represents an
Although both H61 and G50 were slightly inferior to the positive control arbutin, they showed an inhibitory effect on melanin production. In particular, under α-MSH stimulation conditions, H61 was able to suppress melanin production to 68% and G50 to 81% compared to the untreated control without addition of lactic acid bacteria (both at 500 μg / mL). . From this, it was considered that the effect on melanin production varies depending on the type of lactic acid bacteria.
Moreover, it turned out that it is preferable to set the addition density | concentration of a lactic-acid-bacteria extract to 250-1000 microgram / mL, and it is more preferable to set it as 500-1000 microgram / mL (all are dry cell conversion).
Arbutin is a glycoside of hydroquinone, which is a carcinogenic substance, and may have a safety problem depending on the concentration used. On the other hand, the lactic acid bacteria of the present invention are all Lactococcus lactic acid bacteria having a long dietary experience and are considered to be highly safe.
本発明によれば、乳酸菌の中でも安全性が高いラクトコッカス属乳酸菌の特定菌株を利用した皮膚外用剤（メラニン産生抑制剤および育毛・発毛・脱毛抑制剤）が初めて提供される。また、当該乳酸菌株の機能性は死菌体や抽出物でも見られることから、様々な形態での利用が可能である。
したがって、本発明は化粧品、医薬部外品、医薬品等の分野に好適に利用できる。
According to the present invention, a skin external preparation (melanin production inhibitor and hair growth / hair growth / hair loss inhibitor) using a specific strain of Lactococcus lactic acid bacteria, which is highly safe among lactic acid bacteria, is provided for the first time. Moreover, since the functionality of the lactic acid strain is also observed in dead cells and extracts, it can be used in various forms.
Therefore, the present invention can be suitably used in the fields of cosmetics, quasi drugs, pharmaceuticals and the like.
Claims (2)
Lactococcus lactis subsp. Cremoris H61 strain (NITE P-92) or an extract thereof, containing only an active ingredient as an active ingredient .
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2015142170A JP5958983B2 (en) | 2015-07-16 | 2015-07-16 | Skin external preparation for hair growth and hair growth using lactic acid bacteria |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2015142170A JP5958983B2 (en) | 2015-07-16 | 2015-07-16 | Skin external preparation for hair growth and hair growth using lactic acid bacteria |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2013237797A Division JP5783648B2 (en) | 2013-11-18 | 2013-11-18 | Skin external preparation for suppressing melanin production or for hair growth / hair growth using lactic acid bacteria |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2015187171A JP2015187171A (en) | 2015-10-29 |
| JP5958983B2 true JP5958983B2 (en) | 2016-08-02 |
Family
ID=54429589
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2015142170A Active JP5958983B2 (en) | 2015-07-16 | 2015-07-16 | Skin external preparation for hair growth and hair growth using lactic acid bacteria |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP5958983B2 (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101801764B1 (en) * | 2017-06-01 | 2017-11-27 | 한국식품연구원 | Lactobacillus curvatus WIKIM55 having promoting the hair growth |
| KR102429031B1 (en) * | 2017-09-11 | 2022-08-04 | (주)아모레퍼시픽 | Novel Lactococcus lactis W-camellia derived from white camellia and a composition for preventing hair loss or promoting hair growth containing the same |
| KR102429032B1 (en) * | 2017-09-11 | 2022-08-04 | (주)아모레퍼시픽 | Novel Lactococcus lactis R-camellia derived from red camellia and a composition for preventing hair loss or promoting hair growth containing the same |
| KR20230088343A (en) | 2020-10-21 | 2023-06-19 | 닛신 쇼꾸힝 홀딩스 가부시끼가이샤 | Foods, cosmetics, quasi-drugs, or pharmaceuticals containing lactic acid bacteria and having hair growth/hair growth effects |
Family Cites Families (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH04264016A (en) * | 1991-02-18 | 1992-09-18 | Yakult Honsha Co Ltd | Melanin production inhibitor |
| WO2000069399A1 (en) * | 1999-05-14 | 2000-11-23 | Japan Applied Microbiology Research Institute Co., Ltd. | Hair growth stimulants |
| JP3671212B2 (en) * | 2001-09-17 | 2005-07-13 | 独立行政法人農業・生物系特定産業技術研究機構 | Lactococcus lactic acid bacteria useful for immunostimulation |
| JP4604207B2 (en) * | 2005-03-16 | 2011-01-05 | 独立行政法人農業・食品産業技術総合研究機構 | Lactic acid bacteria having anti-aging action and uses thereof |
| JP4547320B2 (en) * | 2005-09-08 | 2010-09-22 | 花王株式会社 | Hair nourishing agent |
| JP2011168520A (en) * | 2010-02-17 | 2011-09-01 | Kikumasamune Shuzo Kk | Lactic acid bacterium having melanogenesis inhibitory action and use thereof |
| JP5783648B2 (en) * | 2013-11-18 | 2015-09-24 | 国立研究開発法人農業・食品産業技術総合研究機構 | Skin external preparation for suppressing melanin production or for hair growth / hair growth using lactic acid bacteria |
-
2015
- 2015-07-16 JP JP2015142170A patent/JP5958983B2/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| JP2015187171A (en) | 2015-10-29 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP6670166B2 (en) | Cosmetics | |
| JP5588474B2 (en) | Composition for promoting collagen and / or hyaluronic acid production | |
| KR101908679B1 (en) | A fermented composition for stimulating growth of hairs contaning natural extract | |
| JP5958983B2 (en) | Skin external preparation for hair growth and hair growth using lactic acid bacteria | |
| JP5972667B2 (en) | Hyaluronic acid synthesis promoter and topical skin preparation | |
| JP5783648B2 (en) | Skin external preparation for suppressing melanin production or for hair growth / hair growth using lactic acid bacteria | |
| JP2020090544A (en) | Cosmetics and food and drink composition | |
| JP2017114814A (en) | Cosmetics | |
| KR20150013994A (en) | Composition for hair or scalp treatment comprising fermented black garlic residue and method of preparing the same | |
| TWI492768B (en) | Cell protective agent | |
| JP6234465B2 (en) | Collagen fiber bundling ability enhancer | |
| KR101627242B1 (en) | Skin composition comprising the purified extract of fermented chestnut bur for anti-wrinkle or anti-bacterial activity | |
| KR101685121B1 (en) | Skin composition comprising the purified extract of fermented chestnut bur showing anti-aging or anti-inflammatory activity | |
| KR101541843B1 (en) | Skin composition comprising the extract of fermented chestnut bur for anti-wrinkle or anti-bacterial activity | |
| JP6090854B2 (en) | Composition for promoting collagen production | |
| JP6861134B2 (en) | Hyaluronic acid production promoter and hyaluronic acid production promoter food | |
| EP2902022B1 (en) | Vegfc production promoter | |
| JP4499371B2 (en) | Method for producing proanthocyanidins derived from peanuts | |
| KR101716777B1 (en) | Composition for antiaging and antiwrinkle which comprises extracts of fermented persimmon using Pediococcus pentosaceus DK1 | |
| JP6805399B2 (en) | Skin care agent containing plant-derived lactic acid bacteria | |
| KR102284168B1 (en) | A novel bacterium from marine resources, Lentisphaera araneosa, cosmetic compositions using the same | |
| JP2007131602A (en) | Bleaching external preparation | |
| KR101541842B1 (en) | Skin composition comprising the extract of fermented chestnut bur showing skin whitening and anti-aging or anti-inflammatory activity | |
| KR20220096261A (en) | Micrococcus luteus strain and its use for improving hair or scalp condition | |
| JP5462491B2 (en) | Proanthocyanidin trimer |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20150722 |
|
| A977 | Report on retrieval |
Free format text: JAPANESE INTERMEDIATE CODE: A971007 Effective date: 20160426 |
|
| 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: 20160607 |
|
| A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20160615 |
|
| R150 | Certificate of patent or registration of utility model |
Ref document number: 5958983 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |