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
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
Fold 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
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
Fold changes of fluorescence intensity
Fold changes of fluorescence intensity
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)
Fold changes of Fluorescence
Fold changes of Fluorescence
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)
Fold changes of Fluorescence
Fold changes of Fluorescence
intensity against
intensity against
Sham
Sham--irradiated cells
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
Fold changes of Fluorescence
Fold changes of Fluorescence
intensity against
intensity against
irradiated cells
irradiated cells
Calpain activationCalpain activation
∗∗∗∗∗∗∗∗
60.060.0
80.080.0
100.0100.0
Survival Ratio (%)
Survival Ratio (%)
Cell damageCell damage
∗∗∗∗∗∗∗∗
1.41.4
1.61.6
1.81.8
Fold changes of Fluorescence
Fold changes of Fluorescence
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))))))))
Fold changes of Fluorescence
Fold changes of Fluorescence
intensity against
intensity against
Sham
Sham--irradiated cells
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
Fold changes of Fluorescence
Fold changes of Fluorescence
intensity against
intensity against
Sham
Sham--irradiated cells
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
Cell damageCell damage
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