new antinew anti--aging concept: aging concept: uji tea...

New AntiNew Anti--Aging Concept: Aging Concept:

UJI TEA EXTRACT (B) BG inhibits UJI TEA EXTRACT (B) BG inhibits ccalpainalpain and blocks extremely and blocks extremely harmful “late phase” oxidative harmful “late phase” oxidative shock in skin shock in skin

Inflammation and skin aging

protein carbonylation protein carbonylation

Fine wrinkleFine wrinkleROSROS

DrynessDryness

UVUVprotein carbonylation protein carbonylation in SC and dermal matrixin SC and dermal matrix

ProPro--MMPMMP--11TIMPTIMP--11

LeucocytesLeucocytes

ElastaseElastase

Collagen, Elastin Collagen, Elastin

inflammationinflammation Deep wrinkleDeep wrinkle

Time course of ROS generation after UVB irradiation

1 h after1 h after

3 h after3 h after

25mJ/cm25mJ/cm22-- UVBUVB--RR

1.01.0

1.11.1

1.21.2

1.31.3

Fold changes of Fluorescence


intensity against

intensity against

irradiated cells

irradiated cells

∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗

5 h after5 h after

7 h after7 h after

After UVBAfter UVB--R (h)R (h)

:Sham:Sham--UVBUVB--RR :UVB:UVB--R 25mJ/cmR 25mJ/cm22

0.00.0

0.90.9

00 11 33 55 77Fold changes of Fluorescence


intensity against

intensity against

Sham

Sham--irradiated cells

irradiated cells

J Invest Dermatol Symp. Proceedings (2009) 14, 50J Invest Dermatol Symp. Proceedings (2009) 14, 50––5252

UVBUVB--irradiatedirradiated cellscells werewere loadedloaded withwith 2020 µµµµµµµµMM HH22DCFDADCFDA (cell(cell--

permeantpermeant 22',',77''--dichlorodihydrofluoresceindichlorodihydrofluorescein diacetate)diacetate) forfor 3030 minmin andandincubatedincubated furtherfurther 3030 minmin atat eacheach timetime.. IntracellularIntracellular ROSROS waswasmeasuredmeasured byby thethe fluorescencefluorescence intensityintensity (Ex(Ex:: 485485 nm/Emnm/Em:: 530530 nm)nm)ofof thethe cellcell lysateslysates.. SignificanceSignificance ∗∗∗∗∗∗∗∗<<00..0505..

ShamSham--UVBRUVBR

UVBUVB--R 25 mJ/cmR 25 mJ/cm22

88

1010

1212

1414

1616

1818∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗

ng/

ng/

µµ µµ µµ µµg protein

g protein

））））））））

∗∗∗∗∗∗∗∗

Time course profile of ILTime course profile of IL--11αααααααα secretion secretion after UVB irradiationafter UVB irradiation

Time (h)Time (h)

00

22

44

66

88

00 11 33 55 77

ILIL--11

αα αα αα αα （（（（（（（（ng/

ng/

∗∗∗∗∗∗∗∗

CellsCells werewere exposedexposed toto UVBUVB ((2525 mJ/cmmJ/cm22)).. ILIL--11αααααααα secretedsecreted intointo thethe mediummediumwaswas quantifiedquantified usingusing commercialcommercial assayassay ELISAELISA kits,kits, HumanHuman ILIL--11ααααααααImmunoassayImmunoassay (Quantikine)(Quantikine).. SignificanceSignificance *<*<00..0505,, **<**<00..0101..

ROS generation induced by IL-1αααα exposure

0 sec0 sec

60 sec60 sec

Fold changes of fluorescence intensity


--irradiated cells

irradiated cells

1.11.1

1.21.2

1.31.3

∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗

∗∗∗∗∗∗∗∗ILIL--11ααααααααControlControl

120 sec120 sec

180 sec180 sec



against sham

against sham--

0.00.0

0.90.9

1.01.0

00 55 1010 1515 2020 2525 3030

Time (min)Time (min)

CellsCells werewere loadedloaded withwith 2020 µµµµµµµµMM HH22DCFDADCFDA forfor 3030 minmin andand werewere thenthenincubatedincubated withwith 1010 ng/mLng/mL ILIL--11αααααααα.. IntracellularIntracellular ROSROS waswas measuredmeasured bybythethe fluorescencefluorescence intensityintensity (Ex(Ex:: 485485 nm/Emnm/Em:: 530530 nm)nm) ofof thethe cellcell lysateslysates..SignificanceSignificance *<*<00..0505,, **<**<00..0101..

Inte

nsit

yIn

tens

ity

ROSROSαααα

Cell Cell damage damage

propro--ILIL--11αααααααα

Profiles of biphasic ROS generation Profiles of biphasic ROS generation after UVB irradiationafter UVB irradiation

Time (h)Time (h)11 33 55 77

Inte

nsit

yIn

tens

ity

ROSROS

ROSROSCalpain Calpain

ILIL--11αααααααα

CaCa2+2+

Inactive calpain Inactive calpain ILIL--11αααααααα

propro--ILIL--11αααααααα

Calpain: Activation of ILCalpain: Activation of IL--11αααααααα

activationactivation

active calpain active calpain Calpain is a calcium-

dependent protein found in many parts of human

organism. Several medical reports on the role of calpain

in aging processes have been published.

NADPH oxidase NADPH oxidase (nicotinamide adenine (nicotinamide adenine

dinucleotide dinucleotide

Proposed scheme: Proposed scheme: CaCa2+2+--triggering triggering cascadecascade in ROS generationin ROS generation

ROSROS

CalpainCalpain

ProPro--ILIL--11αααααααα

CalpainCalpain

ILIL--11ααααααααCalpainCalpain

CaCa2+2+

CalpainCalpain

InflammationInflammationPigmentationPigmentationWrinklingWrinkling

Cell damageCell damage

CaCa2+2+

dinucleotide dinucleotide phosphatephosphate--oxidase)oxidase)



intensity against

intensity against

irradiated cells

irradiated cells

1.51.5

2.02.0

2.52.5

∗∗∗∗∗∗∗∗

∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗

∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗

Calpain activity after UVBCalpain activity after UVB--irradiationirradiation

Time (h)Time (h)



intensity against

intensity against

Sham


irradiated cells

0.00.0

0.50.5

1.01.0

00 11 22 33 44 55 66

∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗

AfterAfter UVBUVB irradiationirradiation ((2525 mJmJ/cm/cm22),), cellscells whichwhich hadhad beenbeen loadedloaded withwith 1010 µµµµµµµµMM SucSuc--LeuLeu--LeuLeu--ValVal--TyrTyr--AMCAMC forfor 11 hh atat eacheach timetime ((11,, 22,, 33,, 44 andand 66 h),h), werewere lysedlysed andand thethe fluorescencefluorescence intensityintensity (Ex(Ex:: 380380nm/nm/EmEm:: 460460nn m)m) waswas measuredmeasured.. SignificanceSignificance againstagainst shamsham--irradiationirradiation;; *<*<00..0505,, **<**<00..0101..

1.001.00

1.101.10

1.201.20



intensity against

intensity against

irradiated cells

irradiated cells

Calpain activationCalpain activation

∗∗∗∗∗∗∗∗

60.060.0

80.080.0

100.0100.0

Survival Ratio (%)

Survival Ratio (%)


∗∗∗∗∗∗∗∗

1.41.4

1.61.6

1.81.8



intensity against

intensity against

irradiated cells

irradiated cells

ROS generationROS generation

∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗∗

Effects of a Calpain inhibitor (ALLN) on Effects of a Calpain inhibitor (ALLN) on UVBUVB--induced reactionsinduced reactions

0.800.80

0.900.90

1.001.00

00 8080ALLNALLN（（（（（（（（µµµµµµµµMM））））））））



intensity against

intensity against

Sham


irradiated cells

0.00.0

20.020.0

40.040.0

00 8080ALLNALLN（（（（（（（（µµµµµµµµMM））））））））

Survival Ratio (%)

Survival Ratio (%)

0.80.8

11

1.21.2

ALLNALLN（（（（（（（（µµµµµµµµMM））））））））

00 8080



intensity against

intensity against

Sham


irradiated cells

After preloading with the calpain inhibitor, ALLN for 1 h, cells were exposed to UVB (25 mJ/cm2). Proteaseactivation was measured at 2 h after UVB-R, Survival ratio was assessed at 24 h after UVB-R by neutral redincorporation, and ROS generation was measured at 7 h after UVB-R. Significance; *<0.05, **<0.01.ALLN: N-Acetyl-L-leucyl-L-leucyl-L-norleucinal

UVBUVB--irradiatedirradiated cellscells secretedsecreted ILIL--11αααααααα fromfrom 55 hrhr afterafterUVBUVB--irradiationirradiation..

ILIL--11αααααααα significantlysignificantly stimulatedstimulated ROSROS generationgeneration..

Short summaryShort summary--11

TheThe latelate phasephase ROSROS causedcaused thethe UVBUVB--inducedinduced cellcelldamagedamage..

UVBUVB irradiationirradiation inducedinduced rapidrapid activationactivation ofof calpaincalpain..

TheseThese findingsfindings stronglystrongly indicateindicate thethe involvementinvolvement ofofthethe calpaincalpain--ILIL--11αααααααα cascadecascade onon thethe latelate phasephase generationgenerationofof ROSROS inin UVBUVB--irradiatedirradiated HaCaTHaCaT keratinocyteskeratinocytes..

AA calpaincalpain inhibitorinhibitor reducedreduced ROSROS generationgeneration bybyUVBUVB--irradiationirradiation..

AppliedApplied researchresearch::ToTo identifyidentify aa naturalnatural ingredientingredientthatthat couldcould preventprevent photoagingphotoaging

InvestigationsInvestigations

thatthat couldcould preventprevent photoagingphotoagingaccordingaccording toto thethe mechanismmechanismfoundfound inin thethe basicbasic researchresearch

CalpainCalpain

With CaWith Ca2+2+plant extractplant extract

fluorescence fluorescence (Ex=495 nm, Em=520 nm)(Ex=495 nm, Em=520 nm)

37 °°°°°°°°CC30min

Screening on calpain inhibition

FITC FITC (fluorescein (fluorescein

isothiocyanate)isothiocyanate)--caseincasein

(substrate)(substrate)

Ocimum sanctumOcimum sanctum

Grifola frondosaGrifola frondosa

Acanthopanax senticosusAcanthopanax senticosus

>100>100

>100>100

61.9261.92

Botanical nameBotanical name ICIC5050 value (value (µµµµµµµµg/mL)g/mL)

UJI TEA EXTRACTUJI TEA EXTRACT 13.8013.80

Summary 2Summary 2

ThisThis studystudy identifiedidentified aa novelnovel cascadecascade involvinginvolvingcalpaincalpain--ILIL--11αααααααα onon thethe UVBUVB--inducedinduced generationgeneration ofofROSROS inin keratinocyteskeratinocytes..

ToTo blockblock thatthat cascade,cascade, wewe screenedscreened forfor aa naturalnatural

11

22 ToTo blockblock thatthat cascade,cascade, wewe screenedscreened forfor aa naturalnaturalinhibitorinhibitor ofof calpaincalpain activityactivity..

AsAs anan effectiveeffective candidatecandidate ofof inhibitor,inhibitor, weweidentifiedidentified UjiUji TeaTea extractextract.. WeWe expectexpect UjiUji TeaTeaextractextract toto suppresssuppress thethe cellcell damagedamage inducedinduced bybyUVBUVB irradiationirradiation throughthrough thethe calpaincalpain--ILIL--11aacascadecascade ..

22

33

NADPH oxidaseNADPH oxidase

CaCa2+2+--triggering cascadetriggering cascadein ROS productionin ROS production

ROSROS

CalpainCalpain

ProPro--ILIL--1a1a

CalpainCalpain

ILIL--1a1a

CalpainCalpain

CaCa2+2+

CalpainCalpain

InflammationInflammationPigmentationPigmentationWrinklingWrinkling


CaCa2+2+

UJI TEA EXTRACT UJI TEA EXTRACT (B) BG(B) BG

In vivo test 1: reduction of UV induced In vivo test 1: reduction of UV induced inflammationinflammation

Test sample: UJI TEA EXTRACT(B)-BGDosage: Placebo, 1%, 2% and 3%Panel: 8 human subjects

Method: sites on the forearms of the panelists were Method: sites on the forearms of the panelists were treated with the test formulation (cream) and then irradiated with 1.5 MED (UVB+UVA) using Multiport Solar UV Simulator Model 601 (made by Solar Light).Test creams were applied immediately after application again, and the erythemas were evaluated 24 hours after irradiation.

Test cream formulationTest cream formulation

Formula Placebo 1% 2% 3%

Ingredients wt% wt% wt% wt%

A Phenoxyethanol 0.40 0.40 0.40 0.40

Water 89.00 83.00 82.00 81.00

B Carbomer 0.30 0.30 0.30 0.30

C Arginine 0.30 0.30 0.30 0.30 C Arginine 0.30 0.30 0.30 0.30

Water 10.00 10.00 10.00 10.00

DUJI TEA

EXTRACT(B)-BG1.00 2.00 3.00

E Water 5.00 5.00 5.00

Total 100.00 100.00 100.00 100.00

In vivo test results: reduction of UV In vivo test results: reduction of UV induced inflammationinduced inflammation

Erythema condition 24 hours after UV irradiation

Placebo 1% 2% 3%

UJI TEA EXTRACT(B)-BG

In vivo test results: reduction of UV In vivo test results: reduction of UV induced inflammationinduced inflammation

Redness of treated sites 24 hours after UV irradiation

4.0

5.0

6.0

7.0

4.3

0.0

1.0

2.0

3.0

4.0

Placebo 1% 2% 3%

a*

2.8

UJI TEA EXTRACT(B)-BG

In vivo test: use of Uji Tea Extract (B) BG helps to increase MED in humans

Uji Tea Extract (B) BG shows significant effect of suppression of UV induced erythema in vivo. In a sunscreen formulation, Uji Tea Extract (B) BG at 1% will provide SPF boosting effect of approximately 21% and at 3% use level the SPF will increase by 87%.

Addition of Uji Tea Extract (B) BG (3%) will provide the following

Suppression of UV-induced erythema

Change in MED against placebo (%) (*Placebo = 100%)

187200 provide the following SPF boosting effect:

Note: the SPF boosting effect is calculated on basis of UV-induced erythema suppression test results. The actual SPF increase may vary depending on the components of the actual formulation.

100121

155

187

020406080100120140160180200

Placebo UJI(1%)

UJI (2%)

UJI(3%)

Change inMED againstplacebo (%)(*Placebo =100%)

SPF 10 → 18.718.7

SPF 20 → 37.437.4

SPF 30 → 56.156.1

SPF 40 → 74.874.8

Conclusion: Conclusion: effects of Uji Tea Extract (B)effects of Uji Tea Extract (B)--BGBG

InhibitionInhibition ofof calpaincalpain expressionexpression..11

SuppressionSuppression ofof thethe cellcell damagedamage andandreductionreduction ofof erythemaerythema inducedinduced byby UVBUVBirradiationirradiation throughthrough thethe calpaincalpain--ILIL--11aacascadecascade ..

22

UJI

Definition of Uji Tea・・・・ Tea ｌｌｌｌeaves are specifically cultivated in 4 prefectures, Kyoto, Nara,

Shiga, and Mie prefectures

・・・・ Processed in Kyoto prefecture* by hand in a traditional way

*Uji city belongs to Kyoto pref.

Mie

Kyoto

Nara

Shiga

Ref: http://kyocha.or.jp/

Botanical InformationNameFamily namePart used

Tea classification

Product Information

Product name: UJI TEA EXTRACT (B)-BG

: Camellia sinensis: Theaceae: Leaf

: Green tea (meets UJI TEA definition)

UJI TEA EXTRACT (B)-BG

INCI NAME Composition

CAMELLIA SINENSIS LEAF EXTRACTBUTYLENE GLYCOL WATER

0.2%49.9%49.9%

Product name: UJI TEA EXTRACT (B)-BG

・ Japanese QD status・ Chinese INCI List 2007・ IECSC・ Origin of 1,3-butylene glycol・ ECOCERT

: Listed as an additive: 茶（CAMELLIA SINENSIS）叶提取物: Listed: Plant derived: Certified

new antinew anti--aging concept: aging concept: uji tea...

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