化粧品における植物資源の付加価値向上に関する研究

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1 1 4 1. 5 2. 7 3. 10 2 32 1. 33 2. 35 3. 39 4. 50 3 56 1. 57 2. 58 3. 61 4. 65 4 74 1. 75 2. 77 3. 80 4. 85 5 89 1. 90 2. 92

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4. 103 6 108 1. 109 2. 112 3. 116 4. 119 7 128 1. 129 2. 131 3. 134 141 143 144 Sumary 159

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1 2 3 4 5 4 5 6 7 6

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1. 5000 [1,2] VPK VPK [1] VPK [3] [4,5] [1,2,3]

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2014

[6]

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2. 2.1. Table 1 2.2. Table1 2.3. (DPPH ) DPPH 1,1-diphenyl-2-picrylhydrazyl [23] 0.1M

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Ascorbic acid (1-10 µg/ml) 40µl

60µl ethanol 0.1mM DPPH

96-well plate 30 517 nm (A517)

3

Scavenging effect (%) = 100-(A-Ab) / (A0-A0b) ×100 A0: A517 of DPPH without sample A0b: A517 without sample and DPPH A: A517 of sample and DPPH Ab: A517 of sample without DPPH

2.4.

NB1RGB MEXT National Bio-Resource

Project RIKEN BRC 10% (FBS;

Biowest) 0.2% NaHCO3 Minimum Essential Media-alpha

(MEMα; Life Technologies Corp.) 37 °C 5% CO2

NB1RGB 96 (3×103 cells/well)-well 10% FBS

8 MEMα

16 [24]

2.5.

(HEKn; GIBCO)

keratinocyte growth factor (HKGS; Invitrogen) Medium 154 (Invitrogen) 5% CO2 37˚C

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Medium 154 16 [25]

2.6.

Normal human hair follicle dermal papilla cells,

hHFDPC APPLICATIONS 1.0 % FCS

0.5 % ITT insulin-transferrin-triiodothyronine 1.0 % BPE 0.5 % Cyp cyproterone acetate PCGM

TOYOBO 8 FCS, ITT, BPE, Cyp

PCGM

16 [26]

2.7.

3×103 cells/well 96-well

24 37˚C 30

10 mM calcein-AM (Dojindo) phosphate-buffered saline

(PBS) em/ex, 485/530 nm

SpectraMax® i3x fluorescence microplate reader (MOLECULAR DEVICES)

3

2.8. RNA

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0.001-0.01% 2,4,8,24 TRI reagent (Merck)

RNA TRI reagent Merck [24,25]

2.9. PCR

mRNA Primescript RT reagent kit (Takara bio inc.)

oligo dT primer cDNA

mRNA SYBR Premix Ex Taq II (Takara bio inc.)

LightCycler 96 (Roche) PCR

ct Cq GAPDH Cq

[27]

Table 2

IGF-1 AR 5αR2

TGF-β IL-1α IL-1β IL-6 COX2 iNOS [26]

3. 3.1.

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[46] DPPH

10 DPPH Fig. 1-1

Fig. 1-2 Fig. 1-2

10

[47,48] Fig. 1-3

malabar nut holy basil neem emblic myrobalan 4

4 Fig. 1-4

Neem 3

malabar nut holy basil emblic myrobalan NB1RGB

HEKn malabar

nut holy basil emblic myrobalan NB1RGB

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Table 3 4

17 18

malabar nut holy basil emblic myrobalan

3.2.

Fig. 1-11 1-12 1-13 (hHFDPC)

IGF-1 IGF-1

OFF

AR 5αR2 TGFβ

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Table 1 Plants and condition of extract

English name Local name in SriLanka Scientific name Part Solvent/Plant Solvent Referance

bougainvillea bouganvillea Bougainvillea grabla flower 7.5 70% EtOH

purple fruited pea eggplant welthibbatu Solanum trilobatum shoot 4.0 70% EtOH [7]

country borage kapparawalliya Plectranthus amboinicus leaf 3.0 70% EtOH [8]

malabar nut adhatoda Justicia adhatoda leaf 5.0 70% EtOH [9]

long pepper thippili Piper longum leaf 7.5 70% EtOH [10]

holy basil maduruthala Ocimum tenuiflorum shoot 3.0 70% EtOH [11]

air plant akkapana Kalanchoe pinnata leaf 2.0 70% EtOH [12]

plumed cockscomb kiri-henda Celosia argentea shoot 2.5 70% EtOH [13]

neem kohomba Azadirachta indica leaf 15.0 50% BG [14]

balipoovu polpala Aerva lanata shoot 7.5 50% EtOH [15]

balloon-vine wel penera Cardiospermum halicacabum shoot 7.5 50% EtOH [16]

emblic myrobalan nelli Phyllanthus emblica leaf 7.5 70% EtOH [17]

indian copperleaf kuppameniya Acalypha indica shoot 7.5 50% EtOH [18]

spreading hogweed pita sudu sarana Boerhavia diffusa shoot 7.5 70% EtOH [19]

curry leaf karapincha Murraya koenigii leaf 7.5 70% EtOH [20]

indian pennywort gotukola Centera asiatica shoot 7.5 70% EtOH [21]

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Table 2. Nucleotide sequences of primers used in this study.

Primers Sequences Direction Reference

Quantitative real time-PCR HAS1

HAS1-F 3’-CGCTAACTACGTCCCTCTGC-5’ Sense [28]

HAS1-R 3’-CCAGTACAGCGTCAACATGG-5’ Anti-sense

HAS2

HAS2-F 3’-GCCTCATCTGTGGAGATGGT-5’ Sense [29]

HAS2-R 3’-ATGCACTGAACACACCCAAA-5’ Anti-sense

HYAL1

HYAL1-F 3’-CCAAGGAATCATGTCAGGCCATCAA-5’ Sense [30]

HYAL1-R 3’-CCCACTGGTCACGTTCAGG-5’ Anti-sense

HYAL2

HYAL2-F 3’-GGCTTAGTGAGATGGACCTC-5’ Sense [30]

HYAL2-R 3’-CCGTGTCAGGTAATCTTTGAG-5’ Anti-sense

versican

VCAN 3-F 3’-TGAGAACCCTGTATCGTTTTGAGA-5’ Sense [31]

VCAN 3-R 3’-CGTTAAGGCACGGGTTCATT-5’ Anti-sense

aggrecan

ACAN-F 3’-TCGAGGACAGCGAGGCC-5’ Sense [32]

ACAN-R 3’-TCGAGGGTGTAGCGTGTAGAGA-5’ Anti-sense

CD44

CD44-F 3’-GCTATTGAAAGCCTTGCAGAG-5’ Sense [33]

CD44-R 3’-CGCAGATCGATTTGAATATAACC-5’ Anti-sense

COL1A1

COL1A1-F 3’-CACCAATCACCTGCGGTACAGAA-5’ Sense [34]

COL1A1-R 3’-CAGATCACGTCATCGCACAAC-5’ Anti-sense

COL3A1

COL3A1-F 3’-CCCACTATTATTTTGGCACAACAG-5’ Sense [35] COL3A1-R 3’-AACGGATCCTGAGTCACAGACA-5’ Anti-sense

COL7A1

COL7A1-F 3’-CTCAGCAGCTATCACCTGGAC-5’ Sense [36]

COL7A1-R 3’-TGTCCACCACACGTAGTTCAA-5’ Anti-sense

MMP1 MMP1-F 3’-TGTGGTGTCTCACAGCTTCC-5’ Sense [30] MMP1-R 3’-CTTGCCTCCCATCATTCTTC-5’ Anti-sense acid ceramidase acid ceramidase-F

3’-CGTACAGAGGTGCAGTTCCA-5’ Sense original

acid ceramidase-R 3’-GTAGGCCAGGCAATTTTTCA-5’ Anti-sense bFGF bFGF-F 3’-AGAGCGACCCTCACATCAAG-5’ Sense [37] bFGF-R 3’-ACTGCCCAGTTCGTTTCAGT-5’ Anti-sense FGF7 FGF7-F 3’-CATGAACACCCGGAGCACTAC-5’ Sense [38] FGF7-R 3’-CACTGTGTTCGACAGAAGAGTCTTC-5’ Anti-sense VEGF

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IL-1α

IL-1α-F 3’-TGGCTCATTTTCCCTCAAAAGTTG-5’ Sense [40]

IL-1α-R 3’-AGAAATCGTGAAATCCGAAGTCAAG-5’ Anti-sense

cox2

COX2-F 3’-TGAGCATCTACGGTTTGCTG-5’ Sense [41]

COX2-R 3’-TGCTTGTCTGGAACAACTGC-5’ Anti-sense

TGF-β

TGF-β-F 3’-GCCCTGGACACCAACTATTG-5’ Sense [42]

TGF-β-R 3’-GTCCAGGCTCCAAATGTAGG-5’ Anti-sense

AQP3

AQP3-F 3’-GTCACTCTGGGCATCCTCAT-5’ Sense [43]

AQP3-R 3’-TATTCCAGCACCCAAGAAGG-5’ Anti-sense

Laminin5

Laminin5-F 3’-GCCTGGAGTACAACGAGGTC-5’ Sense original

Laminin5-R 3’-AGTTGGCAAACTTGATGAGGAC-5’ Anti-sense

COL17A1

COL17A1-F 3’-CGAGACTTTCGACTACTCAGAGC-5’ Sense original COL17A1-R 3’-GAGGACGAGAACAAGCTGAC-5’ Anti-sense

ITGA6

ITGA6-F 3’-TCTCGCTGGGATCTTGATGC-5’ Sense original

ITGA6-R 3’-CCTAGAGCGTTTAAAGAATCCAC-5’ Anti-sense

CERS3

CERS3-F 3’-TCTCTGCTGACTGCATCTATTG-5’ Sense original

CERS3-R 3’-GAAGCCAGAATCTTTCCAACC-5’ Anti-sense

ELOVL1

ELOVL1-F 3’-GGACTTCTCTCTGGCCCTG-5’ Sense original

ELOVL1-R 3’-CGTGCTTCATCACCTCTTGG-5’ Anti-sense

ELOVL4

ELOVL4-F 3’-GATTCTCCCCCTGTTCACATC-5’ Sense original

ELOVL4-R 3’-TTCAGACCGAAGAATGAGTGAC-5’ Anti-sense

FLG

FLG-F 3’-GAAGGTGAAGGTCGGAGTC-5’ Sense original

FLG-R 3’-GAAGATGGTGATGGGATTTC-5’ Anti-sense

TGM1

TGM1-F 3’-CGAAGGCTCTGGGTTACAGA-5’ Sense original

TGM1-R 3’-TGTCACTGTTTCATTGCCTCC-5’ Anti-sense

KRT1

KRT1-F 3’-TGAGCTGAATCGTGTGATCC-5’ Sense original

KRT1-R 3’-CCAGGTCATTCAGCTTGTTC-5’ Anti-sense

GAPDH

GAPDH-F 3’-GAAGGTGAAGGTCGGAGTC-5’ Sense [44]

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Fig. 1-1

Free radical scavenging activity of several plants extracts harvested in Sri Lanka through 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. Results of plants judged as suitable for the subsequent screening are shown. The results are presented as a relative of the control (0%). The values are shown as the mean ± SE of three independent experiments

1(ƒ u[ƒ Qƒ “ƒ rƒ Šƒ Aj 0.0001 0.001 0.01 0.1 1 0 50 100 *** %(v/v) DP P H r ad ic al s ca ve ng in g ac tiv ity (% ) 0

Bougainvillea grabla 2(ƒ Eƒ Fƒ ‹ƒ eƒ Bƒ oƒ bƒ gƒ Dj

0.0001 0.001 0.01 0.1 1 0 50 100 *** %(v/v) DP P H r ad ic al s ca ve ng in g ac tiv ity (% ) 0

Solanum trilobatum Plectranthus amboinicus3(ƒ Jƒ bƒ pƒ ‰ƒƒ Šƒ „j

0.0001 0.001 0.01 0.1 1 0 50 100 ** %(v/v) DP P H r ad ic al s ca ve ng in g ac tiv ity (% ) 0 Justicia adhatoda4(ƒ Aƒ _ƒ gƒ _j 0.0001 0.001 0.01 0.1 1 0 50 100 %(v/v) DP P H r ad ic al s ca ve ng in g ac tiv ity (% ) 0

Piper longum5(ƒ eƒ Bƒ bƒ sƒ Šj

0.0001 0.001 0.01 0.1 1 0 50 100 * %(v/v) DP P H r ad ic al s ca ve ng in g ac tiv ity (% ) 0 Ocimum tenuiflorum6(ƒ }ƒ hƒ Dƒ ‹ƒ Tƒ ‰j 0.0001 0.001 0.01 0.1 1 0 50 100 *** %(v/v) DP P H r ad ic al s ca ve ng in g ac tiv ity (% ) 0

Kalanchoe pinnata7(ƒ Aƒ bƒ Jƒ o[ƒ ij

0.0001 0.001 0.01 0.1 1 0 50 100 *** %(v/v) DP P H r ad ic al s ca ve ng in g ac tiv ity (% ) 0 Celosia argentea8(ƒ Lƒ Šƒ wƒ “ƒ _j 0.0001 0.001 0.01 0.1 1 0 50 100 ** %(v/v) DP P H r ad ic al s ca ve ng in g ac tiv ity (% ) 0 Azadirachta indica 0 0.0001 0.001 0.01 0.1 1 0 50 100 _*** *** ** %(v/v) D P P H r a d ica l sca ve n g in g a ct ivi ty( % ) Phyllanthus emblicaS19 0.0001 0.001 0.01 0.1 1 0 50 100 _*** _*** ** %(v/v) DP P H r ad ic al s ca ve ng in g ac tiv ity (% )

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Fig. 1-2

Free radical scavenging activity of several plants extracts harvested in Sri Lanka through 1,1-diphenyl-2-picrylhydrazyl

Aerva lanata S1 0.0001 0.001 0.01 0.1 1 0 50 100 %(v/v) DP P H r ad ic al s ca ve ng in g ac tiv ity (% ) S13 0.0001 0.001 0.01 0.1 1 0 50 100 %(v/v) DP P H r ad ic al s ca ve ng in g ac tiv ity (% ) 0 Cardiospermum halicacabum S25 0.0001 0.001 0.01 0.1 1 0 50 100 * %(v/v) DP P H r ad ic al s ca ve ng in g ac tiv ity (% ) 0

Acalypha indica Boerhavia diffusaS33

0.0001 0.001 0.01 0.1 1 0 50 100 %(v/v) DP P H r ad ic al s ca ve ng in g ac tiv ity (% ) 0 Murraya koenigiiS73 0.0001 0.001 0.01 0.1 1 0 50 100 *** %(v/v) DP P H r ad ic al s ca ve ng in g ac tiv ity (% ) 0 Centella asiaticaS41 0.0001 0.001 0.01 0.1 1 0 50 100 _*** %(v/v) DP P H r ad ic al s ca ve ng in g ac tiv ity (% ) 0

Spondias dulcis (leaf)S49

0.0001 0.001 0.01 0.1 1 0 50 100 _*** *** %(v/v) DP P H r ad ic al s ca ve ng in g ac tiv ity (% ) 0

Spondias dulcis (fruit) S57

0.0001 0.001 0.01 0.1 1 0 50 100 %(v/v) DP P H r ad ic al s ca ve ng in g ac tiv ity (% ) 0

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Fig. 1-3

Cell viability of NB1RGB detected by calcein assay. Results of plant samples are shown. Cells were treated with various concentrations (0-0.3%) of each plant extracts for 24h, and percent cell viability was calculated relative to that of untreated controls. The values are shown as the mean ± SE of three independent experiments. (*P < 0.05, **P < 0.001 and ***P < 0.001 vs. control (0.0%)

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Fig. 1-4

Cell viability of HEKn detected by calcein assay. Results of plant samples judged as suitable for the subsequent screening are shown. Cells were treated with various concentrations (0-0.3%) of each plant extracts for 24h, and percent cell viability was calculated relative to that of untreated controls. The values are shown as the mean ± SE of three independent experiments.

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Fig.1-5 Gene expression levels in fibroblast cells after exposure to malabar nut extract. The mRNA expression levels were normalized to GAPDH expression, and the relative gene expression levels in the cells at 2, 4, 8, and 24 h after

HAS1 0 2-0.014-0.018-0.01₂₄-000 1 2-0. 1 4-0. 1 8-0. 1 24-0 .1 0.0 0.5 1.0 1.5 2.0 2.5 *** *** *** ** * hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) 0 2 4 8 24 2 4 8 24(hr) 0.01% 0.1% HAS1 R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) 0 2 4 8 24 2 4 8 24(hr) 0.01% 0.1% HAS2 0 2-0.014-0.018-0.01 24-0 001_2-0.1_4-0.1_8-0.1 24-0 .1 0 1 2 3 4 *** *** *** hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) HAS2 R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) 0 2 4 8 24 2 4 8 24(hr) 0.01% 0.1% HYAL1 0 2-0. 01 4-0. 01 8-0. 01 24-0 .01_2-0.1_4-0.1_8-0.1 24-0 .1 0.0 0.5 1.0 1.5 2.0 2.5 *** *** ** * *** * hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) HYAL1 R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) 0 2 4 8 24 2 4 8 24(hr) 0.01% 0.1% HYAL2 0 2-0. 01 4-0. 01 8-0. 01 24-0 001_2-0.1_4-0.1_8-0.1 24-0 .1 0.0 0.5 1.0 1.5 ** *** ** hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) HYAL2 R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) 0 2 4 8 24 2 4 8 24(hr) 0.01% 0.1% versican 0 2-0. 01 4-0. 01 8-0. 01 24-0 001_2-0.1_4-0.1_8-0.1 24-0 .1 0.0 0.5 1.0 1.5 ** ***_{*** ***}*** hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) versican R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) 0 2 4 8 24 2 4 8 24(hr) 0.01% 0.1% aggrecan 0 2-0. 01 4-0. 01 8-0. 01 24-0 001_2-0.1_4-0.1_8-0.1 24-0 .1 0.0 0.5 1.0 1.5 *** *** *** *** *** *** *** Re la tiv e m RNA e xp re ss io n le ve l (G A P D H ) aggrecan R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) 0 2 4 8 24 2 4 8 24(hr) 0.01% 0.1% CD44 0 2-0.014-0.018-0.01₂₄-000 1 2-0.14-0.18-0.1₂₄-0.1 0.0 0.5 1.0 1.5 ** * ** Re la tiv e m RNA e xp re ss io n le ve l (G A P D H ) CD44 R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) 0 2 4 8 24 2 4 8 24(hr) 0.01% 0.1% COL1A1 0 2-0. 01 4-0. 01 8-0. 01 24-0 001_2-0.1_4-0.1_8-0.1 24-0 .1 0.0 0.5 1.0 1.5 2.0 ** ****** *** *** *** *** hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) COL1A1 R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) 0 2 4 8 24 2 4 8 24(hr) 0.01% 0.1% COL3A1 0 2-0.014-0.018-0.01₂₄-000 1 2-0. 1 4-0. 1 8-0. 1 24-0 .1 0 1 2 3 _*** ** * ** ** hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) COL3A1 R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) 0 2 4 8 24 2 4 8 24(hr) 0.01% 0.1% COL7A1 0 2-0.014-0.018-0.01₂₄-000 1 2-0.14-0.18-0.1₂₄-0.1 0.0 0.5 1.0 1.5 *** *** ***_*** * *** *** hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) COL7A1 R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) 0 2 4 8 24 2 4 8 24(hr) 0.01% 0.1% MMP1 0 2-0. 01 4-0. 01 8-0. 01 24-0 001_2-0.1_4-0.1_8-0.1 24-0 .1 0.0 0.5 1.0 1.5 *** *** *** *** *** *** *** hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) MMP1 R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) 0 2 4 8 24 2 4 8 24(hr) 0.01% 0.1% acid ceramidase 0 2-0.014-0.018-0.01₂₄-000 1 2-0.14-0.18-0.1₂₄-0.1 0.0 0.5 1.0 1.5 2.0 *** * * ***** *** hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) acid ceramidase R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) 0 2 4 8 24 2 4 8 24(hr) 0.01% 0.1% bFGF 0 2-0.014-0.018-0.01₂₄-000 1 2-0. 1 4-0. 1 8-0. 1 24-0 .1 0.0 0.5 1.0 1.5 *** *** *** *** *** *** hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) bFGF R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) 0 2 4 8 24 2 4 8 24(hr) 0.01% 0.1% FGF7 0 2-0.014-0.018-0.01₂₄-000 1 2-0.14-0.18-0.1₂₄-0.1 0.0 0.5 1.0 1.5 2.0 2.5 ** *** ** ** ** hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) FGF7 R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) 0 2 4 8 24 2 4 8 24(hr) 0.01% 0.1% VEGF 0 2-0.0 1 4-0.0 1 8-0.0 1 24-0 001_2-0.1_4-0.1_8-0.1 24-0 .1 0 1 2 3 4 ** *** *** *** *** *** *** *** hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) VEGF R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) 0 2 4 8 24 2 4 8 24(hr) 0.01% 0.1% IL-1a 0 2-0.014-0.018-0.01₂₄-000 1 2-0. 1 4-0. 1 8-0. 1 24-0 .1 0 1 2 3 4 ** * ** ** ** Re la tiv e m RNA e xp re ss io n le ve l (G A P D H ) IL-1α R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) 0 2 4 8 24 2 4 8 24(hr) 0.01% 0.1% cox2 0 2-0. 01 4-0. 01 8-0. 01 24-0 001_2-0.1_4-0.1_8-0.1 24-0 .1 0.0 0.5 1.0 1.5 2.0 _*** * * ** ** ** hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) cox2 R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) 0 2 4 8 24 2 4 8 24(hr) 0.01% 0.1% TGF-b 0 2-0.014-0.018-0.01₂₄-000 1 2-0. 1 4-0. 1 8-0. 1 24-0 .1 0.0 0.5 1.0 1.5 * *** *** * *** *** hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) TGF-β R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) 0 2 4 8 24 2 4 8 24(hr) 0.01% 0.1% AQP3 0 2-0.014-0.018-0.01₂₄-000 1 2-0.14-0.18-0.1₂₄-0.1 0.0 0.5 1.0 1.5 2.0 *** * _*** *** ** hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) AQP3 R e la tive m R N A e xp re ssi o n le ve l (G A P D H )

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Fig.1-6 Gene expression levels in keratinocyte cells after exposure to malabar nut extract. The mRNA expression levels were normalized to GAPDH expression, and the relative gene expression levels in the cells at 2, 4, 8, and 24 h after

0 2 4 8 24 2 4 8 24(hr) 0.01% 0.1% HAS1 0 2-0. 01 4-0. 01 8-0. 01 24-0 .01_2-0.1_4-0.1_8-0.1 24-0 .1 0.0 0.5 1.0 1.5 2.0 2.5 * * * hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) HAS1 R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) 0 2 4 8 24 2 4 8 24(hr) 0.01% 0.1% HAS2 0 2-0.014-0.018-0.01₂₄-0.0 1 2-0. 1 4-0. 1 8-0. 1 24-0 .1 0.0 0.5 1.0 1.5 2.0 ** ****** *** *** hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) HAS2 R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) 0 2 4 8 24 2 4 8 24(hr) 0.01% 0.1% HYAL1 0 2-0. 01 4-0. 01 8-0. 01 24-0 .01_2-0.1_4-0.1_8-0.1 24-0 .1 0 1 2 3 *** ** * hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) HYAL1 R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) 0 2 4 8 24 2 4 8 24(hr) 0.01% 0.1% HYAL2 0 2-0. 01 4-0. 01 8-0. 01 24-0 .01_2-0.1_4-0.1_8-0.1 24-0 .1 0.0 0.5 1.0 1.5 2.0 _*** _*** ** hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) HYAL2 R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) 0 2 4 8 24 2 4 8 24(hr) 0.01% 0.1% versican 0 2-0. 01 4-0. 01 8-0. 01 24-0 .01 2-0. 1 4-0. 1 8-0. 1 24-0 .1 0.0 0.5 1.0 1.5 2.0 2.5 * *** hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) versican R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) 0 2 4 8 24 2 4 8 24(hr) 0.01% 0.1% CD44 0 2-0. 01 4-0. 01 8-0. 01 24-0 .01_2-0.1_4-0.1_8-0.1 24-0 .1 0.0 0.5 1.0 1.5 2.0 ** * ** Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) CD44 R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) 0 2 4 8 24 2 4 8 24(hr) 0.01% 0.1% IL-1a 0 2-0. 01 4-0. 01 8-0. 01 24-0 .01_2-0.1_4-0.1_8-0.1 24-0 .1 0 1 2 3 *** *** * ** Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) IL-1α R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) 0 2 4 8 24 2 4 8 24(hr) 0.01% 0.1% cox2 0 2-0.014-0.018-0.01₂₄-0.0 1 2-0.14-0.18-0.124-0 .1 0.0 0.5 1.0 1.5 ** *** * *** * * hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) cox2 R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) 0 2 4 8 24 2 4 8 24(hr) 0.01% 0.1% TGF-b 0 2-0.014-0.018-0.01₂₄-0.012-0.14-0.18-0.124-0 .1 0.0 0.5 1.0 1.5 2.0 2.5 *** *** hr Re la tiv e m RNA e xp re ss io n le ve l (G A P D H ) TGF-β R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) AQP3 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.01% AQP3 0 2-0. 01% 4-0. 01% 8-0. 01% 24-0 .01%_2-0.1%_4-0.1%_8-0.1% 24-0 .1% 0.0 0.5 1.0 1.5 2.0 *** *** *** *** *** *** *** *** hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) Laminin5 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.01% Lminin5 0 2-0. 01% 4-0. 01% 8-0. 01% 24-0 .01%_2-0.1%_4-0.1%_8-0.1% 24-0 .1% 0.0 0.5 1.0 1.5 2.0 2.5 ** *** *** *** *** * hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) COL17A1 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.01% COL17 0 2-0. 01% 4-0. 01% 8-0. 01% 24-0 .01%_2-0.1%_4-0.1%_8-0.1% 24-0 .1% 0.0 0.5 1.0 1.5 2.0 2.5 *** *** *** *** *** *** hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.01% ITGA6 0 2-0. 01% 4-0. 01% 8-0. 01% 24-0 .01% 2-0. 1% 4-0. 1% 8-0. 1% 24-0 .1% 0.0 0.5 1.0 1.5 2.0 *** *** *** *** hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) ITGA6 R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) CERS3 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.01% CERS3 0 2-0. 01% 4-0. 01% 8-0. 01% 24-0 .01%_2-0.1%_4-0.1%_8-0.1% 24-0 .1% 0.0 0.5 1.0 1.5 2.0 _*** * ** *** hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) ELOVL1 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.01% ELOVL1 0 2-0.01 % 4-0.01 % 8-0.01 % 24-0 .01% 2-0. 1% 4-0. 1% 8-0. 1% 24-0 .1% 0.0 0.5 1.0 1.5 2.0 2.5 *** * *** * ** hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) ELOVL4 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.01% ELOVL4 0 2-0. 01% 4-0. 01% 8-0. 01% 24-0 .01%_2-0.1%_4-0.1%_8-0.1% 24-0 .1% 0 1 2 3 4 *** * *** *** * *** * hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) FLG 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.01% FLG 0 2-0. 01% 4-0. 01% 8-0. 01% 24-0 .01%_2-0.1%_4-0.1%_8-0.1% 24-0 .1% 0 5 10 15 *** *** * ** *** *** hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) TGM1 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.01% TGM1 0 2-0. 01% 4-0. 01% 8-0. 01% 24-0 .01%_2-0.1%_4-0.1%_8-0.1% 24-0 .1% 0 2 4 6 8 *** * *** *** * *** *** hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.01% KRT1 0 2-0. 01% 4-0. 01% 8-0. 01% 24-0 .01%_2-0.1%_4-0.1%_8-0.1% 24-0 .1% 0 1 2 3 4 * *** _*** * ** hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) KRT1 R e la tive m R N A e xp re ssi o n le ve l (G A P D H )

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Fig.1-7 Gene expression levels in fibroblast cells after exposure to holy basil extract. The mRNA expression levels were normalized to GAPDH expression, and the relative gene expression levels in the cells at 2, 4, 8, and 24 h after initiation

HAS1 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.01% HAS1 0 2-0.014-0.018-0.01₂₄-0.0 1 2-0.14-0.18-0.1₂₄-0.1 0.0 0.5 1.0 1.5 2.0 2.5 *** *** *** ** ** * hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) HAS2 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.01% HAS2 0 2-0.014-0.018-0.01₂₄-0.0 1 2-0. 1 4-0. 1 8-0. 1 24-0 .1 0 1 2 3 *** *** ** * * ** ** hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) HYAL1 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.01% HYAL1 0 2-0. 01 4-0. 01 8-0. 01 24-0 .01_2-0.1_4-0.1_8-0.1 24-0 .1 0 2 4 6 *** ** ** ** ** ** hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) HYAL2 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.01% HYAL2 0 2-0.014-0.018-0.01₂₄-0.0 1 2-0. 1 4-0. 1 8-0. 1 24-0 .1 0.0 0.5 1.0 1.5 *** ** _** hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) versican 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.01% versican 0 2-0.014-0.018-0.01₂₄-0.0 1 2-0. 1 4-0. 1 8-0. 1 24-0 .1 0.0 0.5 1.0 1.5 *** ****** _*** * *** ** hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) aggrecan 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.01% aggrecan 0 2-0. 01 4-0. 01 8-0. 01 24-0 .01_2-0.1_4-0.1_8-0.1 24-0 .1 0.0 0.5 1.0 1.5 *** *** *** *** *** *** *** *** Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) CD44 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.01% CD44 0 2-0. 01 4-0. 01 8-0. 01 24-0 .01 2-0. 1 4-0. 1 8-0. 1 24-0 .1 0.0 0.5 1.0 1.5 * ** * Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) COL1A1 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.01% COL1A1 0 2-0.014-0.018-0.01₂₄-0.0 1 2-0. 1 4-0. 1 8-0. 1 24-0 .1 0.0 0.5 1.0 1.5 2.0 *** *** * ** *** *** hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) COL3A1 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.01% COL3A1 0 2-0.0 1 4-0.0 1 8-0.0 1 24-0 .01 2-0.14-0.18-0.124-0 .1 0 1 2 3 4 *** ** *** *** ** hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) COL7A1 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.01% COL7A1 0 2-0.014-0.018-0.01₂₄-0.0 1 2-0.14-0.18-0.1₂₄-0.1 0.0 0.5 1.0 1.5 *** _*** * _** *** *** *** * hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) MMP1 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.01% MMP1 0 2-0.0 1 4-0.0 1 8-0.0 1 24-0 .01_2-0.1_4-0.1_8-0.1 24-0 .1 0.0 0.5 1.0 1.5 2.0 *** *** ** *** *** *** ** *** hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) acid ceramidase 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.01% acid ceramidase 0 2-0. 01 4-0. 01 8-0. 01 24-0 .01_2-0.1_4-0.1_8-0.1 24-0 .1 0.0 0.5 1.0 1.5 ********* ** *** *** *** *** hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) bFGF 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.01% bFGF 0 2-0.014-0.018-0.01₂₄-0.0 1 2-0. 1 4-0. 1 8-0. 1 24-0 .1 0.0 0.5 1.0 1.5 ** *** ** *** *** *** *** hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) FGF7 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.01% FGF7 0 2-0. 01 4-0. 01 8-0. 01 24-0 .01_2-0.1_4-0.1_8-0.1 24-0 .1 0.0 0.5 1.0 1.5 2.0 * ** * hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) VEGF 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.01% VEGF 0 2-0. 01 4-0. 01 8-0. 01 24-0 .01_2-0.1_4-0.1_8-0.1 24-0 .1 0 1 2 3 4 *** *** * *** *** *** ** *** hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) IL-1α 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.01% IL-1a 0 2-0. 01 4-0. 01 8-0. 01 24-0 .01_2-0.1_4-0.1_8-0.1 24-0 .1 0 1 2 3 4 *** * *** * * * Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) cox2 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.01% cox2 0 2-0.014-0.018-0.01₂₄-0.0 1 2-0. 1 4-0. 1 8-0. 1 24-0 .1 0.0 0.5 1.0 1.5 2.0 2.5 *** *** * *** *** *** hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) TGF-β 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.01% TGF-b 0 2-0.014-0.018-0.01₂₄-0.0 1 2-0. 1 4-0. 1 8-0. 1 24-0 .1 0.0 0.5 1.0 1.5 *** * *** *** *** * hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) AQP3 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.01% AQP3 0 2-0. 01 4-0. 01 8-0. 01 24-0 .01_2-0.1_4-0.1_8-0.1 24-0 .1 0.0 0.5 1.0 1.5 *** * ** hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) R e la tive m R N A e xp re ssi o n le ve l (G A P D H )

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Fig.1-8 Gene expression levels in keratinocyte cells after exposure to holy basil extract. The mRNA expression levels were normalized to GAPDH expression, and the relative gene expression levels in the cells at 2, 4, 8, and 24 h after

HAS1 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.01% R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) HAS1-use 0 2-0.01 % 4-0.01 % 8-0.01 % 24-0 .01%_2-0.1%_4-0.1%_8-0.1% 24-0 .1% 0.0 0.5 1.0 1.5 hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) N.D. N.D. N.D. N.D. N.D. N.D.N.D. N.D. HAS2 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.01% HAS2 0 2-0. 01% 4-0. 01% 8-0. 01% 24-0 .01%_2-0.1%_4-0.1%_8-0.1% 24-0 .1% 0 1 2 3 4 ** ** ** *** *** hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) HYAL1 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.01% HYAL1 0 2-0. 01% 4-0. 01% 8-0. 01% 24-0 .01% 2-0. 1% 4-0. 1% 8-0. 1% 24-0 .1% 0 2 4 6 * ** *** *** *** hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) HYAL2 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.01% HYAL2 0 2-0. 01% 4-0. 01% 8-0. 01% 24-0 .01%_2-0.1%_4-0.1%_8-0.1% 24-0 .1% 0.0 0.5 1.0 1.5 2.0 * ** *** *** *** *** hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) versican 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.01% versican 0 2-0.01 % 4-0.01 % 8-0.01 % 24-0 .01%_2-0.1%_4-0.1%_8-0.1% 24-0 .1% 0.0 0.5 1.0 1.5 2.0 2.5 * ** hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) CD44 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.01% CD44 0 2-0.01 % 4-0.01 % 8-0.01 % 24-0 .01%_2-0.1%_4-0.1%_8-0.1% 24-0 .1% 0.0 0.5 1.0 1.5 ** ** *** *** *** *** Re la tiv e m RNA e xp re ss io n le ve l (G A P D H ) R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.01% IL-1a 0 2-0.01 % 4-0.01 % 8-0.01 % 24-0 .01% 2-0. 1% 4-0. 1% 8-0. 1% 24-0 .1% 0 1 2 3 ** *** *** *** *** Re la tiv e m RNA e xp re ss io n le ve l (G A P D H ) IL-1α R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) cox2 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.01% cox2 0 2-0.01 % 4-0.01 % 8-0.01 % 24-0 .01%_2-0.1%_4-0.1%_8-0.1% 24-0 .1% 0.0 0.5 1.0 1.5 2.0 *** *** * * * hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) TGF-β 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.01% TGF-b 0 2-0.01 % 4-0.01 % 8-0.01 % 24-0 .01%_2-0.1%_4-0.1%_8-0.1% 24-0 .1% 0.0 0.5 1.0 1.5 2.0 *** *** * _* * ** hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) AQP3 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.01% AQP3 0 2-0.01 % 4-0.01 % 8-0.01 % 24-0 .01%_2-0.1%_4-0.1%_8-0.1% 24-0 .1% 0.0 0.5 1.0 1.5 2.0 *** *** *** *** *** *** *** *** hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) Laminin5 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.01% Lminin5 0 2-0.01 % 4-0.01 % 8-0.01 % 24-0 .01% 2-0. 1% 4-0. 1% 8-0. 1% 24-0 .1% 0.0 0.5 1.0 1.5 2.0 2.5 ** *** *** *** *** _* hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) COL17A1 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.01% COL17 0 2-0.01 % 4-0.01 % 8-0.01 % 24-0 .01% 2-0. 1% 4-0. 1% 8-0. 1% 24-0 .1% 0.0 0.5 1.0 1.5 2.0 2.5 *** *** *** *** *** *** hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.01% ITGA6 0 2-0.01 % 4-0.01 % 8-0.01 % 24-0 .01%_2-0.1%_4-0.1%_8-0.1% 24-0 .1% 0.0 0.5 1.0 1.5 2.0 *** *** *** *** hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) ITGA6 R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) CERS3 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.01% CERS3 0 2-0. 01% 4-0. 01% 8-0. 01% 24-0 .01%_2-0.1%_4-0.1%_8-0.1% 24-0 .1% 0.0 0.5 1.0 1.5 2.0 _*** * ** *** hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) ELOVL1 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.01% ELOVL1 0 2-0. 01% 4-0. 01% 8-0. 01% 24-0 .01%_2-0.1%_4-0.1%_8-0.1% 24-0 .1% 0.0 0.5 1.0 1.5 2.0 2.5 *** * *** * ** hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) ELOVL4 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.01% ELOVL4 0 2-0.01 % 4-0.01 % 8-0.01 % 24-0 .01%_2-0.1%_4-0.1%_8-0.1% 24-0 .1% 0 1 2 3 4 *** * *** *** * *** * hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) FLG 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.01% FLG 0 2-0.01 % 4-0.01 % 8-0.01 % 24-0 .01%_2-0.1%_4-0.1%_8-0.1% 24-0 .1% 0 5 10 15 *** *** * ** *** *** hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) TGM1 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.01% TGM1 0 2-0.01 % 4-0.01 % 8-0.01 % 24-0 .01% 2-0. 1% 4-0. 1% 8-0. 1% 24-0 .1% 0 2 4 6 8 *** * *** *** * *** *** hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.01% KRT1 0 2-0.01 % 4-0.01 % 8-0.01 % 24-0 .01% 2-0. 1% 4-0. 1% 8-0. 1% 24-0 .1% 0 1 2 3 4 * *** _*** * ** hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) KRT1 R e la tive m R N A e xp re ssi o n le ve l (G A P D H )

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Fig.1-9 Gene expression levels in fibroblast cells after exposure to emblic myrobalan extract. The mRNA expression levels were normalized to GAPDH expression, and the relative gene expression levels in the cells at 2, 4, 8, and 24 h

HAS1 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.003% HAS1 0 2-0.00 1 4-0.00 1 8-0.00 1 24-0 .001 2-0.00 3 4-0.00 3 8-0.00 3 24-0 .003 0.0 0.5 1.0 1.5 *** *** *** *** *** ***_{*** ***} hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) HAS2 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.003% HAS2 0 2-0.00 1 4-0.00 1 8-0.00 1 24-0 .001 2-0.00 3 4-0.00 3 8-0.00 3 24-0 .003 0.0 0.5 1.0 1.5 2.0 ** ** *** ** *** *** hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) HYAL1 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.003% HYAL1 0 2-0.00 1 4-0.00 1 8-0.00 1 24-0 .001 2-0.00 3 4-0.00 3 8-0.00 3 24-0 .003 0.0 0.5 1.0 1.5 2.0 ** * _{*** *** ***} *** hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) HYAL2 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.003% HYAL2 0 2-0. 001 4-0. 001 8-0. 001 24-0 .001 2-0. 003 4-0. 003 8-0. 003 24-0 .003 0.0 0.5 1.0 1.5 *** *** ****** *** *** * * hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) versican 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.003% versican 0 2-0.00 1 4-0.00 1 8-0.00 1 24-0 .001 2-0.00 3 4-0.00 3 8-0.00 3 24-0 .003 0.0 0.5 1.0 1.5 ** *** *** ** *** *** * ** hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) aggrecan 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.003% aggrecan 0 2-0. 001 4-0. 001 8-0. 001 24-0 .001 2-0. 003 4-0. 003 8-0. 003 24-0 .003 0.0 0.5 1.0 1.5 2.0 _* ** Re la tiv e m RNA e xp re ss io n le ve l (G A P D H ) R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) CD44 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.003% CD44 0 2-0. 001 4-0. 001 8-0. 001 24-0 .001 2-0. 003 4-0. 003 8-0. 003 24-0 .003 0.0 0.5 1.0 1.5 *** *** ***_*** ** * Re la tiv e m RNA e xp re ss io n le ve l (G A P D H ) R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) COL1A1 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.003% COL1A1 0 2-0.0 01 4-0.0 01 8-0.0 01 24-0 .001 2-0.0 03 4-0.0 03 8-0.0 03 24-0 .003 0 1 2 3 *** *** ** hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) COL3A1 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.003% COL3A1 0 2-0.00 1 4-0.00 1 8-0.00 1 24-0 .001 2-0.00 3 4-0.00 3 8-0.00 3 24-0 .003 0 1 2 3 *** *** ** * * hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) COL7A1 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.003% COL7A1 0 2-0.00 1 4-0.00 1 8-0.00 1 24-0 .001 2-0.00 3 4-0.00 3 8-0.00 3 24-0 .003 0.0 0.5 1.0 1.5 * * * hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) MMP1 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.003% MMP1 0 2-0. 001 4-0. 001 8-0. 001 24-0 .001 2-0. 003 4-0. 003 8-0. 003 24-0 .003 0.0 0.5 1.0 1.5 *** ** * ** *** _*** *** hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) acid ceramidase 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.003% acid ceramidase 0 2-0.00 1 4-0.00 1 8-0.00 1 24-0 .001 2-0.00 3 4-0.00 3 8-0.00 3 24-0 .003 0.0 0.5 1.0 1.5 *** *** ** *** *** *** hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) bFGF 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.003% bFGF 0 2-0.00 1 4-0.00 1 8-0.00 1 24-0 .001 2-0.00 3 4-0.00 3 8-0.00 3 24-0 .003 0.0 0.5 1.0 1.5 ****** ** *** *** *** hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) FGF7 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.003% FGF7 0 2-0.00 1 4-0.00 1 8-0.00 1 24-0 .001 2-0.00 3 4-0.00 3 8-0.00 3 24-0 .003 0.0 0.5 1.0 1.5 *** ** * _** *** *** *** hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) VEGF 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.003% VEGF 0 2-0.00 1 4-0.00 1 8-0.00 1 24-0 .001 2-0.00 3 4-0.00 3 8-0.00 3 24-0 .003 0 1 2 3 ** *** *** *** *** *** *** hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) IL-1α 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.003% IL-1a 0 2-0.00 1 4-0.00 1 8-0.00 1 24-0 .001 2-0.00 3 4-0.00 3 8-0.00 3 24-0 .003 0.0 0.5 1.0 1.5 2.0 ** *** *** *** Re la tiv e m RNA e xp re ss io n le ve l (G A P D H ) R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) cox2 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.003% cox2 0 2-0.00 1 4-0.00 1 8-0.00 1 24-0 .001 2-0.00 3 4-0.00 3 8-0.00 3 24-0 .003 0.0 0.5 1.0 1.5 ** _*** *** ****** *** *** hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) TGF-β 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.003% TGF-b 0 2-0.00 1 4-0.00 1 8-0.00 1 24-0 .001 2-0.00 3 4-0.00 3 8-0.00 3 24-0 .003 0.0 0.5 1.0 1.5 2.0 ** * *** *** *** *** hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) AQP3 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.003% AQP3 0 2-0.00 1 4-0.00 1 8-0.00 1 24-0 .001 2-0.00 3 4-0.00 3 8-0.00 3 24-0 .003 0 1 2 3 4 5 ** *** *** *** *** *** *** ** hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) R e la tive m R N A e xp re ssi o n le ve l (G A P D H )

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Fig.1-10 Gene expression levels in keratinocyte cells after exposure to emblic myrobalan extract. The mRNA expression levels were normalized to GAPDH expression, and the relative gene expression levels in the cells at 2, 4, 8, and 24 h

HAS1 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.003% HAS1 0 2-0. 001% 4-0. 001% 8-0. 001% 24-0 .001 % 2-0. 003% 4-0. 003% 8-0. 003% 24-0 .003 % 0 1 2 3 4 *** *** ** * hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) HAS2 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.003% HAS2 0 2-0. 001% 4-0. 001% 8-0. 001% 24-0 .001 % 2-0. 003% 4-0. 003% 8-0. 003% 24-0 .003 % 0.0 0.5 1.0 1.5 *********_{*** *** *** ***} hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) HYAL1 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.003% HYAL1 0 2-0. 001% 4-0. 001% 8-0. 001% 24-0 .001 % 2-0. 003% 4-0. 003% 8-0. 003% 24-0 .003 % 0 1 2 3 4 *** *** ** * ** hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) HYAL2 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.003% HYAL2 0 2-0. 001% 4-0. 001% 8-0. 001% 24-0 .001 % 2-0. 003% 4-0. 003% 8-0. 003% 24-0 .003 % 0 1 2 3 *** *** *** hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) versican 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.003% versican 0 2-0. 001% 4-0. 001% 8-0. 001% 24-0 .001 % 2-0. 003% 4-0. 003% 8-0. 003% 24-0 .003 % 0 1 2 3 4 *** *** ***** * hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) CD44 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.003% CD44 0 2-0. 001% 4-0. 001% 8-0. 001% 24-0 .001 % 2-0. 003% 4-0. 003% 8-0. 003% 24-0 .003 % 0.0 0.5 1.0 1.5 2.0 2.5 *** ** * *** * Re la tiv e m RNA e xp re ss io n le ve l (G A P D H ) R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) IL-1α 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.003% IL-1a 0 2-0. 001% 4-0. 001% 8-0. 001% 24-0 .001 % 2-0. 003% 4-0. 003% 8-0. 003% 24-0 .003 % 0 2 4 6 8 *** ** * *** * *** *** *** Re la tiv e m RNA e xp re ss io n le ve l (G A P D H ) R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) cox2 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.003% cox2 0 2-0. 001% 4-0. 001% 8-0. 001% 24-0 .001 % 2-0. 003% 4-0. 003% 8-0. 003% 24-0 .003 % 0 1 2 3 *** ** * *** ** hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) TGF-β 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.003% TGF-b 0 2-0. 001% 4-0. 001% 8-0. 001% 24-0 .001 % 2-0. 003% 4-0. 003% 8-0. 003% 24-0 .003 % 0 1 2 3 _*** * *** * *** _*** *** *** hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) AQP3 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.003% AQP3 0 2-0. 001% 4-0. 001% 8-0. 001% 24-0 .001 % 2-0. 003% 4-0. 003% 8-0. 003% 24-0 .003 % 0 1 2 3 _*** * *** hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) Laminin5 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.003% Lminin5 0 2-0. 001% 4-0. 001% 8-0. 001% 24-0 .001 % 2-0. 003% 4-0. 003% 8-0. 003% 24-0 .003 % 0 1 2 3 4 *** * *** *** hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) COL17A1 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.003% COL17 0 2-0. 001% 4-0. 001% 8-0. 001% 24-0 .001 % 2-0. 003% 4-0. 003% 8-0. 003% 24-0 .003 % 0 1 2 3 4 *** *** *** *** ** *** hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) ITGA6 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.003% ITGA6 0 2-0. 001% 4-0. 001% 8-0. 001% 24-0 .001 % 2-0. 003% 4-0. 003% 8-0. 003% 24-0 .003 % 0.0 0.5 1.0 1.5 2.0 2.5 *** hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) CERS3 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.003% CERS3 0 2-0. 001% 4-0. 001% 8-0. 001% 24-0 .001 % 2-0. 003% 4-0. 003% 8-0. 003% 24-0 .003 % 0.0 0.5 1.0 1.5 *** *** *** hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) HAS2 0 2-0. 001% 4-0. 001% 8-0. 001% 24-0 .001 % 2-0. 003% 4-0. 003% 8-0. 003% 24-0 .003 % 0.0 0.5 1.0 1.5 *********_{*** *** *** ***} hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) ELOVL1 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.003% ELOVL1 0 2-0. 001% 4-0. 001% 8-0. 001% 24-0 .001 % 2-0. 003% 4-0. 003% 8-0. 003% 24-0 .003 % 0.0 0.5 1.0 1.5 2.0 2.5 *** * ** hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) ELOVL4 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.003% ELOVL4 0 2-0. 001% 4-0. 001% 8-0. 001% 24-0 .001 % 2-0. 003% 4-0. 003% 8-0. 003% 24-0 .003 % 0 1 2 3 4 *** * *** ** hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) FLG 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.003% FLG 0 2-0. 001% 4-0. 001% 8-0. 001% 24-0 .001 % 2-0. 003% 4-0. 003% 8-0. 003% 24-0 .003 % 0 1 2 3 *** * *** _*** *** *** *** hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) TGM1 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.003% TGM1 0 2-0. 001% 4-0. 001% 8-0. 001% 24-0 .001 % 2-0. 003% 4-0. 003% 8-0. 003% 24-0 .003 % 0 1 2 3 4 *** *** *** hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) R e la tive m R N A e xp re ssi o n le ve l (G A P D H ) KRT1 0 2 4 8 24 2 4 8 24(hr) 0.001% 0.003% KRT1 0 2-0.0 01% 4-0.0 01% 8-0.0 01% 24-0 .001 % 2-0.0 03% 4-0.0 03% 8-0.0 03% 24-0 .003 % 0 2 4 6 8 10 *** * *** *** *** *** *** hr Re la tiv e m RNA e xp re ss io n le ve l (G A P DH ) R e la tive m R N A e xp re ssi o n le ve l (G A P D H )

(30)

Table 3

1/2 positive

(31)

Table 4

1/2 positive

(32)

Fig. 1-11

Expression of IGF-1 in hHFDPC. Total RNA was extracted from hHFDPC treated with 0.03% NLE for 2-24h, and the expression level of IGF-1 was determined by qRT-PCR.

(33)

Fig. 1-12

Expression of AR, 5αR2, and TGFβ in hHFDPC. Total RNA was extracted from hHFDPC treated with 0.03% NLE for 2-24h, and the expression level of these factors were determined by qRT-PCR.

(34)

Fig. 1-13

Expression of pro-inflammatory cytokines in hHFDPC. Total RNA was extracted from hHFDPC treated with 0.03% NLE for 2-24h, and the expression level of pro-inflammatory cytokines were determined by qRT-PCR.

(35)

(36)

1.

2 3

inner cosmetics outer cosmetics

in&out

The First International Congress on the Menopause 1976 6

1976

(37)

[1] [2] [1] [3,4] [5,6] [7] [8] . [9] Rosa rugosa Rosa multiflora [7,10]

(38)

16 [11]

[12] Tarnichene

Rosa ×damascene Bulgarian Rose Damascene

Water Enio Bonchev Production Ltd., Bulgaria 3 5

Bulgarian Rose Damascene Water

[13]

2. 2.1.

2015 10 2

(39)

2.2. 50 ml/ 2.3. 2.3.1. 35 55 3 5 17 2.3.2. 2.4. 0 30 60 CRC GHQ30

(40)

2.5. 2.5.1. 17 0 30 60 2.5.2. GHQ30 17 GHQ30 0 60 2.5.3. 0 60 2.5.4. 17 Skin Checker 0 30 60

(41)

0 1 2 3 4 2.5.5. 17 SKICON-200EX 0 30 60 2.5.6. 17 0 30 60 2.5.7. 17 pH R3 0 30 60 2.5.8. 17

(42)

0 30 60

2.5.9.

17 ACTH ECLIA CLEIA

0 30 60 LH CLI FSH CLI HPLC HPLC 0 60 2.6. Microsoft Excel 2010 Stat Mate F 5 t 3. 3.1. 35 55

(43)

17 45.5 5.2 17 3.2. 17 0 30 60 0 15 30 60 5 60 33.3 0 15 30 4 60 7

(44)

60 46.7 0 15 60 2 60 13.3 0 17 30 5 60 12 60 70.6 0 16 30 8 60 11 60 68.8 0 16 30 3 60 6 60 37.5

(45)

0 17 30 3 60 5 60 29.4 0 17 30 60 3 60 17.6 0 10 30 2 60 4 60 40.0 0 14 30 3 60 6 60 42.9

(46)

0 7 30 2 60 3 60 42.9 0 16 30 60 2 60 13.3 0 10 30 4 60 3 60 30.0 0 11 30 3 60 6 60 54.5

(47)

0 8 30 2 60 3 60 37.5 0 13 30 2 60 3 60 23.1 0 7 30 60 1 60 14.3 0 16 30 60 2 60 12.5

(48)

0 15 30 4 60 6 60 37.5 0 8 30 3 60 1 60 12.5 0 8 30 1 60 4 60 50.0 0 10 30 4 60 5 60 50.0

(49)

0 16 30 2 60 6 60 37.5 0 17 30 3 60 9 60 52.9 0 16 60 6 60 37.5 0 17 60 2 60 11.8 0 16

(50)

60 43.8 0 16 30 2 60 9 60 56.3 0 1 5 1 1 2 5 30 2 60 1 60 20.0 0 1 10 1 5 9 1 1 4 8 30 60 3.3. GHQ30 17 GHQ30 0 60

(51)

2-1 0 60 0 9 F-02 F-03 F-04 F-05 F-06 F-09 F-11 F-13 F-14 60 60 3.4. 17 0 30 60 0 30 60 ***: p<0.001 (Fig. 2-2) 3.5. 17 0 30 60 0 30 60 ***: p<0.001 (Fig. 2-3) 3.6. 17 0

(52)

30 60 0 (Fig 2-4) 3.7. 17 pH 0 30 60 30 60 0 3.8. 17 MCV MCH MCHC 0 30 60 30 60 0 3.9. 17 ACTH 0 30 60 LH FSH 0 60 ACTH 0 30

(53)

60 *: p<0.05 (Fig. 2-5) 4. 35 55 17 60 1 0 30 60 GHQ30 F-01 F-02 F-06 F-07 F-09 F-12 F-15 7 ACTH 0 30 30 0 [14] ACTH 7 F-02 1 7 3 4 2 6 GHQ30 7 0 3 60

(54)

17 3 14 12 GHQ30 0 9 60 GHQ30 17 16 [15] 30 60

(55)

inner

cosmetics outer cosmetics

(56)

Fig. 2-1 0 60 GHQ30

(57)

Fig. 2-3

(58)

Fig. 2-5

(59)

(60)

1. 4 [1,2] [3,4] Artemisia 250 30 [5] A.princeps A.montana A.montana

(61)

[7]

[8]

[9,10] Nagata

[11]

Near-infrared spectroscopy : NIRS NIRS

[12-14] A.montana A.inceps [15] A.montana A.montana NIRS 2.

(62)

ethanol EtOH n-hexane triethyl citrate

1,8-Cineol camphor borneol bornyl acetate o-cymene caryophyllene

oxide β-thujone 2.2. Artemisia montana 2012 7 8 2.3. 50g L Dean-Stark 2 50.1mg 2.4. GC/MS

Agilent 7890A Agilent Technology

GC Agilent 5975 Agilent Technology MS

DB-wax 0.25mm I.D 30m 0.25µm

injection temp. 250°C column temp. 40°C (0.5 min) - 40-230°C (3°C/min) split ratio 10:1 He flow 3.0 ml/min.

2.5.

1,8-cineol borneol caryophyllen oxide thujone o-cymene bornyl

(63)

3 2.6. 55 19~24 33 22 10mg/mL 7 2.7. COCORO triethyl citrate (10 mg/ml) 10 19 24 5 5 5 2.8. NIRS ∆[oxy-Hb] DynaSense Pocket NIRS Duo

Ventrolateral Prefrontal : VLPFC

(64)

60 10mg/mL 60 60 18 19 24 8 10 2.9. Table 3-1 3. 3.1. A.montana A.montana 0.10 Artemisia 0.05 1.00 [15-19] A.princeps 0.0049 A.montana A.princeps GC/MS Fig.3-1

borneol 1,8-cineol camphor

16.3 15.4 13.7%

Piperitone caryophyllene oxide o-cymene β-thujone bornyl

(65)

GC/MS Table 3-2 1,8-cineol borneol camphor

840–900 µg/g caryophyllene oxide 256 µg/g

caryophyllene oxide

AEDA Aroma Extract Dilution Analysis 9

Table 3-3

FD flavor dilution

1,8-cineol FD=7 borneol FD=6

o-cymene β-thujone bornyl acetate

FD 5 1,8-cineol borneol

3.2.

Fig.3-2 9

(66)

3.3. [20] [21] Fig.3-3 3.4. NIRS NIRS oxy-Hb [22]

Ventrolateral Prefrontal : VLPFC oxy-Hb ∆[oxy-Hb]

NIRS

NIRS 18

[oxy-Hb] Fig.3-4 (A)

[oxy-Hb] Fig.3-4 (B)

(67)

[oxy-Hb] 60 [oxy-Hb] [oxy-Hb] [oxy-Hb] 3 [oxy-Hb] Table 3-4 (-3.0 × 10-4 µM/s) (-2.0 × 10-4 µM/s) 3.5. Fig.3-4 NIRS Table 3-3 1,8-cineol

borneol β-thujone bornyl acetate ο-cymene

Table 3-3

NIRS ∆[oxy-Hb]

2/5 Table 3-5

10 mg/mL EtOH solutions Δ[oxy-Hb]

(68)

Δ[oxy-Hb] 1,8-cineol -2.0 × 10-4 µM/s

o-cymene bornyl acetate β-thujone

borneol +1.0 × 10-4 µM/s 1,8-cineol 4. A.montana NIRS [23] NIRS NIRS 1,8-cineol A.montana

(69)

Table 3-1 Contents of model yamayomogi oil Compound Components (mg/g) 1,8-cineol 0.535 borneol 0.530 β-thujone 0.085 bornyl acetate 0.045 o-cymene 0.041 Triethyl citrate 8.764

(70)

Fig. 3-1 GC-MS chromatogram of yamayomogi oil.

Peak 1:1,8-cineol, peak 2: o-cymene, peak 3: β-Thujone, peak 4: camphor, peak 5: bornul acetate, peak 6: terpinene-4-ol, peak 7: borneol, peak 8: piperitone, peak 9: caryophyllene oxide

1 5 1 2 10. 20. 30.0 40. 50. Ab u n d an ce (× 10 5 ) Retention time 0 1 2 3 4 5 6 7 8 9

(71)

Table 3-2 Chemical composition of yamayomogi oil Compound RIa _Relative Content (%)b Identificationc α-pinene 948 0.89 MS, RI camphen 968 1.81 MS, RI β-pinene 990 0.39 MS, RI β-phellandrene 998 0.45 MS, RI α-phellandrene 1027 0.44 MS, RI α-terpinene 1038 0.41 MS, RI 1,8-cineol 1054 15.39 MS, RI, Co γ-terpinene 1095 0.55 MS, RI o-cymene 1116 3.21 MS, RI, Co 6-methyl-hepten-2-one 1189 0.92 MS, RI β-thujone 1263 2.77 MS, RI, Co α-thujone 1298 1.64 MS, RI 1-octen-3-ol 1317 1.62 MS, RI camphor 1352 13.67 MS, RI, Co pinocarvone 1408 0.45 MS, RI

bornyl acetate 1420 2.69 MS, RI, Co

caryophyllene 1429 0.52 MS, RI terpinen-4-ol 1443 1.61 MS, RI lavandulyl acatate 1459 0.51 MS, RI bornyl isobutyrate 1490 1.58 MS, RI borneol 1537 16.26 MS, RI, Co piperitone 1555 5.51 MS, RI

caryophyllene oxide 1800 3.91 MS, RI, Co

spathulenol 1956 0.94 MS, RI

thymol 2017 0.93 MS, RI

α-bisabolol 2049 0.94 MS, RI

Total 80.01

a_{Retention index (RI) relative to the homologous series of n-alkanes}

(C6-C26) on the DB-WAX capillary column.

b_{Relative area (peak area relative to total peak area)}

(72)

Table 3-3 Aroma impact compounds in yamayomogi oil

No. RI Compound Aroma

description

FD Content (µg/g dry weight) 1 1054 1,8-cineol Young leaf 7 897.5 ± 16.5

2 1116 o-cymene Cut grass 5 41.1 ± 14.3

3 1263 β-thujone Muscat 5 84.3 ± 13.0

4 1352 camphor Wet leaf 2 889.6 ± 25.4

5 1420 bornyl acetate Citrus 5 45.3 ± 16.0

6 1443 terpinen-4-ol Watermelon 5 7 1537 borneol Oriental medicine 6 839.8 ± 29.4 8 1555 piperitone Antiseptic 2

9 1800 caryophyllene oxide Mint 2 255.6 ± 9.2 Each compounds of FD value measured by AEDA method.

(73)

Fig. 3-2 Results of sensory tests of yamayomogi oil and lavender oil. The subject sniffed a mouillette dipped in 10 mg/ml EtOH solutions of yamayomogi oil or lavender oil, and answered in seven stage evaluation to 9 questions. Each value is the mean ± SE, n=55.

(74)

Fig. 3-3 Effect of sniffing yamayomogi oil on saliva α-amylase activities. Five minutes after sniffing the yamayomogi oil test solution, saliva

α-amylase activities were measured by a COCORO meter. Each value is the mean ± SE, n=10.

0 10 20 30 40 50 60 70 80

Triethyl citrate (Vehicle) Yomogi Oil

S al iva α -a m yl as e a ct ivi ti es (kIU /L )

(75)

Fig.3-4 Time courses of ∆[oxy-Hb] by sniffing yamayomogi oil and reference oils.

(76)

Table 3-4 Slopes of ∆[oxy-Hb] by sniffing yamayomogi oil and reference oils

Sample slope (µM/s)

Yomogi Oil -3.0 × 10-4

Ylang ylang oil +2.0 × 10-4 Lavernder oil -2.0× 10-4 Control (EtOH) -0.6 × 10-4

Each value was calculated by time courses of ∆[oxy-Hb] in Fig. 3.

Table 3-5 Slopes of NIRS test (Δ[oxy-Hb])

Sample Slope (µM/s)

Yomogi Oil -3.0 × 10-4

Model Yomogi Oil -1.3 × 10-4 1,8-cineol -2.0 × 10-4

borneol +1.0 × 10-4

β-thujone -0.1× 10-4

bornyl acetate -0.3× 10-4

(77)

(78)

1.

[1]

Bernd H.

Schmitt [2,3]

(79)

[4]

5

[5]

[2]

SENSE FEEL

THINK ACT RELATE

5 5 Table 4-1 [3] Bernd H.Schmitt 300

(80)

5 1705 300 Bernd H.Schmitt Fig. 4-1 2. 2.1. 2.1.1. Fig. 4-2 85 15

(81)

2.1.2. 1705 2 1989 listen 33 2004 300 2.1.3.

(82)

2

2.1.4.

30

MIRAGE

(83)

2.1.5. Table 4-2 3. Bernd H.Schmitt Fig. 4-3 [6] 3.1. SENSE

(84)

3.2.

FEEL

(85)

[7]

3.3.

THINK

(86)

3.4.

(87)

3.5.

RELATE

HP

(88)

3.6. SENSE FEEL 300 THINK ACT RELATE 4. Bernd

H.Schmitt SENSE, FEEL, THINK, ACT, RELATE

(89)

Table 4-1 Bernd H.Schmitt Fig 4-1 SENSE FEEL ( ) THINK ACT ( ) RELATE

(90)

Fig. 4-2 Table 4-2 4P Product Price Place Promotion

(91)

Table 4-3 B.H.Schmitt SENSE FEEL THINK ACT RELATE

(92)

(93)

1.

1.1.

100

(94)

100 [1]

[2] [3]

100

[4] [5]

(95)

2. 2.1. 2.1.1.

(96)

2.1.2.

(97)

(98)

3. 3.1. 480 Fig. 5-1 Fig. 5-2 3.2.

(99)

3.2.1. Table 5-1 3.2.2. SEM Table 5-2 [6] 4 Table 5-3 3.2.3.

(100)

3.3. MOT 3.3.1. 3 2039 9641 3.3.2. 3.3.3. 2003

(101)

3.3.4

2003 TORAYA CAFÉ

3.4. 3.4.1.

(102)

MOT [8] QCD Cultivate Cultivate Education Culture Cultivate

(103)

SENSE FEEL THINK ACT RELATE

(104)

[9]

( )

Fig. 5-3 4

(105)

3.4.3.

(106)

3.4.4. [10] AQUOS WACOAL DIA AQUOS WACOAL DIA

(107)

4.

(108)

Fig. 5-1

(109)

Table 5-1 4P Product Price 2,625 /740g Place Promotion Table 5-2 SENSE FEEL THINK ACT RELATE

(110)

Table 5-3

(111)

(112)

1. 1.1.

Bernd H.Schmitt

[1,2] [3-6]

(113)

[7] Bernd H.Schmitt 1.2. 1.2.1. Fig. 6-1 1990

(114)

1.2.2.

630 2017 1956

[9]

(115)

Fig. 6-2

2009

2011

2.

(116)

Bernd H.Schmitt 2011 2009 2.1. 2011 Table 6-1 2009 2.1.1. SENSE

(117)

SENSE

2.1.2.

(118)

2.1.3.

THINK

2.1.4.

ACT

(119)

RELATE 3. Table 6-2 2010 [11-14] Fig. 6-3 Fig. 6-4 3.1.

(120)

SENSE

3.2. FEEL

3.3.

(121)

3.4. ACT

3.5.

RELATE

(122)

Fig. 6-5 4. 4.1. 2009 2010 Bernd H.Schmitt 2011 2012 Fig. 6-6 2009 100 Bernd H.Schmitt 2011 4 [15] 4.2.

(123)

(124)

(125)

(126)

4.3.

(127)

Fig. 6-1

(128)

Table 6-1 Bernd.H.Schmitt 2009 Table 6-2 Bernd.H.Schmitt 2011 2009 SENSE FEEL THINK ACT RELATE 2011 SENSE FEEL THINK ACT RELATE

(129)

Fig. 6-3

(130)

Fig. 6-5

Fig. 6-6 2009 100

(131)

(132)

1. 1.1.

Bernd H.Schmitt

(133)

NPO 2011 Fig. 7-1 2011 1.3. NPO NPO 3

(134)

[1] [2-6] NPO 2010 1956 2009 2011 Fig. 7-2 2.

(135)

Bernd H.Schmitt [7] 2.1. SENSE Fig. 7-3 2.2. FEEL

(136)

2.3.

THINK( )

FEEL

2.4. ACT

(137)

2.5.

RELATE

3. 3.1.

Fig. 7-4 Bernd H.Schmitt

2011 2012

(138)

30%

3.2.

Table 7-2

[8]

3.3.

(139)

ACT RELATE

3.4.

(140)

(141)

Fig. 7-1

(142)

Table 7-1 Bernd H.Schmitt

(143)

Fig. 7-4

2010 100

(144)

malabar nut holy basil emblic myrobalan 3

(145)

(146)

(147)

1 [1] H (2014) [2] (2017) [3] (2009) [4] (2006) [5] (2007) [6] Spitravel (2013)

[7] Nature’s Beauty Creations Ltd., A COLLECTION OF MEDICINAL PLANTS IN SRI LANKA Revised Edition (2014) 165

(148)

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Sagane, Toshihiro Watanabe, R.G.S.Wijesekara, Antioxidant activities of traditional plants in Sri Lanka by DPPH free radical-scavenging assay, Data in Brief (In Press)

[24] Takao Someya, Katsura Sano, Kotaro Hara, Yoshimasa Sagane, Toshihiro Watanabe, R.G.S.Wijesekara, Fibroblast and keratinocyte gene expression following exposure to extracts of neem plant (Azadirachta indica), Data in Breif 16 (2017) 982-992

[25] Takao Someya, Katsura Sano, Kotaro Hara, Yoshimasa Sagane, Toshihiro Watanabe, R.G.S.Wijesekara, Fibroblast and keratinocyte gene expression following exposure to the extracts of holy basil plant (Ocimum tenuiflorum), malabar nut plant (Justicia adhatoda), and emblic myrobalan plant (Phyllanthus emblica), Data in Breif 17 (2018) 24-46

[26] Kyoko Dodo, Katsura Sano, Kotaro Hara, Takao Someya, Effects of neem (Melia azadirachta L.) leaf extract on human dermal papilla cells, Proceedings of 8th International Symposium on Agriculture and Environment (ISAE 2018) 60-62

[27] K. Maeda-Sano, M. Gotoh, T. Morohoshi, T. Someya, H. Murofushi, K. Murakami-Murofushi, Cyclic phosphatidic acid and lysophosphatidic acid induce hyaluronic acid synthesis via CREB transcription factor regulation in human skin fibroblasts, Biochim Biophys Acta.1841(9) (2014) 1256-1263

[28] K. Röck, M. Meusch, N. Fuchs, J. Tigges, P. Zipper, E. Fritsche, J. Krutmann, B. Homey, J. Reifenberger, J. W. Fischer, Estradiol protects