DermaPep™ A350 - TRI-K...any side effects. Function Anti-wrinkle for face and body Increase of...

DermaPep™ A350 Retinoic acid- like peptide

ep Experience the magic of science DermaP

Experience The Magic of Science

rebecca

TRI-K Industries

Anti-aging DermaPep™ A350

DermaPep™ A420

DermaPep™ A440

DermaPep™ A530

DermaPep™ A350

DermaPep™ UL

DermaPep™ W220

DermaPep™ W411

DermaPep™ A350

DermaPep™ A440

DermaPep™ A530

DermaPep™ UL

DermaPep™ W220

DermaPep™ W411

Whitening

Anti-inflammatory

DermaPep™ A350 Retinoic Acid-like Peptide

Introduction

Aging of skin is an intricate biological process

composed of two types, intrinsic and extrinsic aging.

While intrinsic aging is an inevitable process, extrinsic

aging called photoaging occurs via the clinical and

histological consequences of chronic sun exposure, and

responsible for the majority of age-related changes,

including wrinkles, roughness and mottled

hyperpigmentation. These age-related changes are

known to be caused by collagen break-down, thus the

loss of structural support, by excessive expression and

production of the matrix metalloproteinases (MMPs).

Retinoic acid has been widely used both for topical

therapy of several skin diseases and to improve the

aspect of aging skin. However, topical retinoic acid

induces irritation of the skin, which precludes its use in

some skin diseases. In addition, retinoic acid has been

classified as prescription drug, which also precludes its

use as cosmetic active.

In vitro and in vivo study results show that Myristoyl

tripeptide-31, a multifunctional retinoic acid-like

tripeptide, is very effective in reversing age-related

changes, such as wrinkles, mottled hyperpigmentation,

and can be a powerful substitute for retinoids without

any side effects.

Function

Anti-wrinkle for face and body Increase of collagen synthesis Providing exceptional anti-aging benefits Anti-aging effect against environmental stimulus such as UV irradiation

Applications

DermaPep™ A350 can be incorporated in cosmetic

formulations such as emulsions, oily sera, gels and

creams for anti-aging and anti-wrinkle purpose.

Formulation Dermatological tolerance : Standard testing has been performed on DermaPep™ A350 which has showed neither cytotoxic effects nor any irritation or sensitization reaction in healthy volunteers with an occlusive single patch test. Recommended concentration : 2-4 %

Product Information Appearance : Transparent solution INCI/CTFA-Declaration : Myristoyl Tripeptide-31 (and) Butylene Glycol Active ingredient content : 0.1% Myristoyl Tripeptide-31 Powder purity : 95 % up Preservative : None

DermaPep™ A350 Safety

Cell Cytotoxicity Test

Hs68 cells were seeded into 96-well plates (2 x 103) and

treated with different concentrations of Myristoyl

tripeptide-31 for 24 hours. Cell viability was assessed by

MTT assay and was shown relative to untreated

control. Absorbance was measured by an ELISA reader

at 540 nm.

Skin Irritation Test

Evaluation and grading of skin irritation

Erythema (no redness = 0 to very strong redness=5)

After 24 hrs, mean score = 0.00 (max = 0)


Oedema (no oedema = 0 to very strong oedema =5)



Scaling (no scaling = 0 to very strong scaling = 5)



MTT Assay

Skin irritation test was performed on 31 healthy Asian

volunteers (Spincontrol Asia (study report number IR-

6Q01-MW-DE08), Bangkok, Thailand).

100 95

101 95

84

0

20

40

60

80

100

120

0 2 5 10 25

Concentration (ppm)

Cel

l vi

abili

ty (

% o

f co

ntr

ol)

Mutagenicity (Ames Test)

Myristoyl tripeptide-31 at 5 different concentrations

(20,40,80,160,320 ppm) were evaluated to predict its

potential to cause mutagenicity. Five different

auxotrophic mutants were used to assesse directly or in

the presence of liver S9 fractions.

No mutagenicity was observed.

In vitro Ocular Irritation Test

DermaPep™ A350 at 3 different concentrations (2, 4,

8 %) and butylene glycol were evaluated with the

ocular irritection assay system (CA, USA).

The ocular results demonstrated that the DermaPepTM

A350 was classified as mild irritants.

DermaPep™ A350 In vitro efficacy

Hs68 cells were seeded into 6-well plates and

pretreated with the retinoic acid or Myristoyl

tripeptide-31 followed by UV radiation or not for 48

hours. Equal amounts of media were analyzed by ELISA

assay for MMP-I. Cell viability was used for sample

standardization.

Myristoyl tripeptide-31 significantly decreased MMP-1

production.

Inhibition of MMP-1 production after UV irradiation

Inhibition of MMP-1 Production Inhibition of MMP-1 Production

Retinoic acid Myristoyl Tripeptide-31

0.8 1.5 2

(ppm) 1 - - - -

-

100

59

68 65

52

0

20

40

60

80

100

120

MM

P1

pro

du

ctio

n

(% o

f co

ntr

ol)

- 41 % - 48 %

100

147

82

106

84 77

0

50

100

150

200

MM

P1

pro

du

ctio

n

(% o

f co

ntr

ol)


0.8 1.5 2

(ppm) 1 - - - -

-

UVA 30J/cm2 + - + + + +

- 138 % - 149 %

100

242

41

212

55 44

0

50

100

150

200

250

300

MM

P1

pro

du

ctio

n

(% o

f co

ntr

ol)


0.8 1.5 2

(ppm) 1 - - - -

-

UVB 80mJ/cm2 + - + + + +

- 160 % - 149 %


Inhibition of MMP-1 expression (RT-PCR)

MMP-1

GAPDH

NT R.A 1.5 0.8 2

Myristoyl Tripeptide-31

(ppm)

Inhibition of MMP-1 mRNA expression

Hs68 cells were seeded into 6-well plates and treated

with retinoic acid or Myristoyl tripeptide-31 . After 48

hours, total cellular RNA was extracted and reverse-

transcription PCR was performed to determine MMP-1.

GAPDH mRNA level was used for sample

standardization.

Myristoyl tripeptide-31 significantly inhibited the MMP-

1 expression.

Inhibition of MMP-1 and MMP-3 mRNA expression after UVA irradiation


pretreated with retinoic acid or Myristoyl tripeptide-31.

followed by 30J/cm2 UVA. After 48 hours, total cellular

RNA was extracted and reverse-transcription PCR was

performed to determine MMP-1.and MMP-3. GAPDH

mRNA level was used for sample standardization.

Myristoyl tripeptide-31 considerably inhibited UVA

stimulated MMP-1 and MMP-3 expression.

100

41

70

56

44

0

20

40

60

80

100

120

MM

P1

exp

ress

ion

(%

of

con

tro

l)


0.8 1.5 2

(ppm) 1 - - - -

-

-59 % -56 %

100

152

102 102 98 100

131

92 100 97

0

20

40

60

80

100

120

140

160

MM

Ps

exp

ress

ion

(%

of

con

tro

l)


1

(ppm) 1 -

-

UVA 30J/cm2 + - + +

2

-

+

MMP-1

GAPDH

NT R.A 2


(ppm)

UVA 30J/cm2

1

MMP-3

-

MMP-3 MMP-1


Effect on Procollagen 1 production


pretreated with retinoic acid or Myristoyl tripeptide-31.

followed by 30J/cm2 UVA. After 48 hours equal

amounts of cell lysates were subjected to

electrophoresis and analyzed by western blot for

procollagen type I.

Myristoyl tripeptide-31 considerably increased the

production of type I procollagen.

Stimulation of Procollagen I Production

Procollagen I

-actin

NT R.A 0.8 1.5 2

UVA 30J/cm2



0.8 1.5 2

(ppm) 1 - - -

UVA 30J/cm2 + + + + +

1

3.15

2.2 2.3

3.35

0

1

2

3

4

Pro

colla

gen

I P

rod

uct

ion

(R

atio

)


Comparison with Retinoids


pretreated with retinyl palmitate/retinol only or retinyl

palmitate/retinol with Myristoyl TrIpeptide-31.

followed by 30J/cm2 UVA. Equal amounts of media

were analyzed by ELISA assay for MMP-I. Cell viability

was used for sample standardization

Myristoyl Tripeptide-31 showed much better MMP-1

reducing effect than retinyl palmitate or retinol alone.

Comparison with Retinyl Palmitate on MMP-1 Inhibition

Comparison with Retinol on MMP-1 Inhibition


Retinyl palmitate

UVA 30J/cm2

(ppm)

+ + + + + + + + + + - 1 3 3 10 1 10 20 20 -

2 - 2 - - 2 - 2 2

100

181 183

80

157

74

144

67

96

47 61

0

30

60

90

120

150

180

210

MM

P1

pro

du

ctio

n

(% o

f co

ntr

ol)


Retinol

UVA 30J/cm2

(ppm)

+ + + + + + + + + - 5 5 10 10 1 20 20 -

- 2 - 2 - - 2 2

100

229

60

236

54

113 91

114 108

53

0

50

100

150

200

250

300

MM

P1

pro

du

ctio

n

(% o

f co

ntr

ol)


Signaling of MMP-1 expression


pretreated with the retinoic acid or Myristoyl

Tripeptide-31 for 24 and then stimulated by UV

irradiation. After 30 minutes, total cellular RNA was

extracted and reverse-transcription PCR was performed

to determine c-jun and c-fos mRNA level. -actin was

used for sample standardization.

Myristoyl Tripeptide-31 significantly decreased UVB

induced c-fos mRNA expression.

AP-1 component gene expression(PCR)

c-jun/c-fos gene expression Mechanism of MMP-1 expression

Several MMPs are strongly regulated by AP-1.

100

129 128

121

111

100 102 99 92 92

60

80

100

120

140

mR

NA

exp

ress

ion

(%

of

con

tro

l)

c-jun c-fos

(ppm) 1 2 4

UV + + + +


-

c-fos

c-jun

GAPDH

(ppm) 1 2 4

UV + + + +


-


Inhibition of ROS Induced MMP-1 Production and AP-1 Signaling

100

153

95 91

0

50

100

150

MM

P1

pro

du

ctio

n

(% o

f co

ntr

ol)

- 117 %


1 2

(ppm)

-

H2O2 (500uM) + - + +

Inhibition of MMP-1 production against H2O2


with Myristoyl Tripeptide-31 followed by or H2O2.

Equal amounts of media were analyzed by ELISA assay

for MMP-I. Cell viability was used for sample

standardization. For AP-1 signaling, total cellular RNA

was extracted and reverse-transcription PCR was

performed to determine c-jun and c-fos mRNA level.

GAPDH was used for sample standardization.

Myristoyl Tripeptide-31 significantly decreased ROS

radical stimulated MMP-1 production and c-fos mRNA

expression.


c-fos

c-jun

GAPDH

100

208 193

180 167

100 122 119 113 119

0

50

100

150

200

mR

NA

exp

ress

ion

(%

of

con

tro

l)

c-jun c-fos

(ppm) 1 2 4

H2O2 + + + +


-


Inhibition of MMP-1 production against SNAP

100

185

144

114

0

50

100

150

MM

P1

pro

du

ctio

n

(% o

f co

ntr

ol)

- 84 %


1 2

(ppm)

-

SNAP(500uM) + - + +


with Myristoyl Tripeptide-31 followed by SNAP(S-

nitroso-N-acetylpenicillamine. Equal amounts of media

were analyzed by ELISA assay for MMP-I. Cell viability

was used for sample standardization. For AP-1

signaling, total cellular RNA was extracted and reverse-

transcription PCR was performed to determine c-jun

and c-fos mRNA level. GAPDH was used for sample

standardization.

Myristoyl Tripeptide-31 significantly decreased RNS

radical stimulated MMP-1 production and c-fos mRNA

expression.


100

127

109

101 101 100

119 118 120 120

60

80

100

120

140

mR

NA

exp

ress

ion

(%

of

con

tro

l)

c-jun c-fos

(ppm) 1 2 4

SNAP(100uM) + + + +


-

Inhibition of RNS Induced MMP-1 Production and AP-1 Signaling

c-fos

c-jun

GAPDH


Melanogenic Gene Expression (RT-PCR)

Tyrosinase

GAPDH

NT


0.1 0.5 1

MITF

POMC

(ppm)

TRP1

TRP2

2 5

Down-Regulation of Melanogenic Genes

Human primary melanocyte HEMn-LP cells were

treated with various concentrations of Myristoyl

tripeptide-31. After 48 hours, total cellular RNA was

extracted and reverse-transcription PCR was performed

to determine tyrosinase, POMC, MITF, TRP1 and TRP2.

-actin mRNA level was used for sample

standardization.

Myristoyl tripeptide-31 significantly down-regulated

the expression of melanogenic genes.

Myristoyl tripeptide-31, As an Anti-inflammatory Agent

Human keratinocyte HaCaT cells were treated with

Myristoyl tripeptide-31 and then stimulated with or

without UVB (100 mJ/cm2). After 48 hours, total cellular

RNA was extracted and reverse-transcription PCR was

performed to determine IL-1, IL-1β, IL-6. GAPDH

mRNA level was used for sample standardization.

Myristoyl tripeptide-31 significantly inhibited UVB-

induced expression of proinflammatory cytokines.

Proinflammatory cytokines Level (RT-PCR)

NT (ppm)

UVB 100mJ/cm2

IL-1

IL-1

IL-6

GAPDH

-38%

-132%

-60%

Myr-tripeptide-31

0.5 1 2

Myr-tripeptide-31

0.5 1 2


CHIP Assay

Hs68(human skin fibroblasts) cells were seeded into 6-

well plates and treated with Myristoyl Tripeptide-31.

After 48 hours, total cellular RNA was extracted. Each

total RNA samples (200ng) was labeled and amplified

using Low Input Quick Amp labeling kit (Agilent

Technologies, CA.). The Cy3-labeled aRNAs were

resuspended in 50 ul of hybridization solution (Agilent

Technologies, CA.). After labeled aRNAs were placed

on Agilent SurePrint G3 Human GE 8x60K array

(Agilent Technologies, CA.) and covered by a Gasket 8-

plex slide (Agilent Technologies, CA.). The slides were

hybridized for 17hr at 65°C oven. The hybridized slides

were washed in 2X SSC, 0.1% SDS for 2 min, 1 X SSC for

3min, and then 0.2 X SSC for 2 min at room

temperature. The slides were centrifuged at 30,000 rpm

for 20 sec to dry.

Data analysis

The array was analyzed using an Agilent scanner with

associated software. Gene expression levels were

calculated with Feature Extraction v10.7.3.1 (Agilent

Technologies, CA.) Relative signal intensities for each

gene were generated using the Robust Multi-Array

Average algorithm. The data were processed based on

quantile normalization method using the GeneSpring

GX 11.5.1 (Agilent Technologies, CA.).

CHIP data of ECM Protein Gene

Index ProbeName Description Gene

Symbol

Genbank

Accession UniGeneID

UV/NT

intensity

A350/UV

intensity

3181 A_23_P161698 matrix metallopeptidase 3 (stromelysin 1, progelatinase) (MMP3), m

RNA MMP3 NM_002422 Hs.375129 2.803817166 0.704587549

3547 A_23_P1691 matrix metallopeptidase 1 (interstitial collagenase) (MMP1), transcrip

t variant 1, mRNA MMP1 NM_002421 Hs.83169 1.564405238 0.599597805

16187 A_24_P82106 matrix metallopeptidase 14 (membrane-inserted) (MMP14), mRNA MMP14 NM_004995 Hs.2399 1.135234216 0.640397536

1031 A_23_P120472 transcription factor AP-2 gamma (activating enhancer binding protein

2 gamma) (TFAP2C), mRNA TFAP2C NM_003222 Hs.473152 1.331976314 0.597596818

25105 A_33_P3304668 collagen, type I, alpha 1 (COL1A1), mRNA COL1A1 NM_000088 Hs.172928 0.445770446 1.152406116

8628 A_23_P42322 collagen, type XI, alpha 2 (COL11A2), transcript variant 1, mRNA COL11A2 NM_080680 Hs.390171 1.001852164 1.742267974

31278 A_33_P3398236 collagen, type XXVIII, alpha 1 COL28A1 AJ890452 Hs.491104 0.934087883 1.856850378

20182 A_33_P3231953 collagen, type XII, alpha 1 (COL12A1), transcript variant long, mRNA COL12A1 NM_004370 Hs.101302 0.594571671 1.440269232

22078 A_33_P3259393 hyaluronan and proteoglycan link protein 3 (HAPLN3), mRNA [NM_1

78232] HAPLN3 NM_178232 Hs.447530 0.762367083 1.321780416

14650 A_24_P355720 fibroblast growth factor 4 (FGF4), mRNA FGF4 NM_002007 Hs.1755 0.705654698 1.5252397

15179 A_24_P402438 transforming growth factor, beta 2 (TGFB2), transcript variant 2, mR

NA TGFB2 NM_003238 Hs.133379 0.971181556 1.252144007

13766 A_24_P274270 signal transducer and activator of transcription 1, 91kDa (STAT1), tra

nscript variant beta, mRNA STAT1 NM_139266 Hs.642990 2.028586279 0.603417946


CHIP Assay

Hs68(human skin fibroblasts) cells were seeded into 6-

well plates and treated with Myristoyl Tripeptide-31.

After 48 hours, total cellular RNA was extracted. Each

total RNA samples (200ng) was labeled and amplified

using Low Input Quick Amp labeling kit (Agilent

Technologies, CA.). The Cy3-labeled aRNAs were

resuspended in 50 ul of hybridization solution (Agilent

Technologies, CA.). After labeled aRNAs were placed

on Agilent SurePrint G3 Human GE 8x60K array

(Agilent Technologies, CA.) and covered by a Gasket 8-

plex slide (Agilent Technologies, CA.). The slides were

hybridized for 17hr at 65°C oven. The hybridized slides

were washed in 2X SSC, 0.1% SDS for 2 min, 1 X SSC for

3min, and then 0.2 X SSC for 2 min at room

temperature. The slides were centrifuged at 30,000 rpm

for 20 sec to dry.

Data analysis

The array were analyzed using an Agilent scanner with

associated software. Gene expression levels were

calculated with Feature Extraction v10.7.3.1 (Agilent

Technologies, CA.) Relative signal intensities for each

gene were generated using the Robust Multi-Array

Average algorithm. The data were processed based on

quantile normalization method using the GeneSpring

GX 11.5.1 (Agilent Technologies, CA.).

CHIP data of Pro-inflammatory Related Gene

Index ProbeName Description Gene

Symbol

Genbank

Accession UniGeneID

A350/UV

intensity

21238 A_33_P3246833 interleukin 1 receptor antagonist (IL1RN), transcript variant 4, mRNA IL1RN NM_173843 Hs.81134 0.618890459

12985 A_24_P200023 interleukin 1 receptor, type I (IL1R1), mRNA IL1R1 NM_000877 Hs.701982 0.697941489

10576 A_23_P73780 interleukin 1 receptor-associated kinase 1 (IRAK1), transcript variant 1, mRNA IRAK1 NM_001569 Hs.522819 0.645198774

11156 A_23_P85209 interleukin 13 receptor, alpha 2 (IL13RA2), mRNA IL13RA2 NM_000640 Hs.336046 0.711123756

1964 A_23_P138680 interleukin 15 receptor, alpha (IL15RA), transcript variant 2, mRNA IL15RA NM_172200 Hs.524117 0.760231452

5072 A_23_P252062 peroxisome proliferator-activated receptor gamma (PPARG), transcript variant

3, mRNA PPARG NM_138711 Hs.162646 0.696303581

28110 A_33_P3350726 peroxisome proliferator-activated receptor gamma (PPARG), transcript variant

3, mRNA PPARG NM_138711 Hs.162646 0.655867065

1078 A_23_P121253 tumor necrosis factor (ligand) superfamily, member 10 (TNFSF10), transcript v

ariant 1, mRNA TNFSF10 NM_003810 Hs.478275 0.499606944

2139 A_23_P14174 tumor necrosis factor (ligand) superfamily, member 13b (TNFSF13B), transcrip

t variant 1, mRNA TNFSF13B NM_006573 Hs.525157 0.698981681

10479 A_23_P71530 tumor necrosis factor receptor superfamily, member 11b (TNFRSF11B), mRN

A

TNFRSF11

B NM_002546 Hs.81791 0.595563436

4776 A_23_P218646 tumor necrosis factor receptor superfamily, member 6b, decoy (TNFRSF6B),

mRNA TNFRSF6B NM_003823 Hs.434878 0.775638905

25168 A_33_P3305571 tumor necrosis factor receptor superfamily, member 6b, decoy (TNFRSF6B),

mRNA TNFRSF6B NM_003823 Hs.434878 0.755130528

3365 A_23_P165624 tumor necrosis factor, alpha-induced protein 6 (TNFAIP6), mRNA TNFAIP6 NM_007115 Hs.437322 0.813895078

DermaPep™ A350 In vivo efficacy

Anti-Aging Efficacy on Asian Skin Type Wrinkle Reduction on number, area and length

In a clinical study with 30 Asian healthy female

volunteers (Bangkok), aged 40 to 60, 2 % formula

containing DermaPep™ A350 had been applied twice

daily during 12 weeks on the face area. Evaluation was

performed by replica analysis (evaluation of the anti-

wrinkle effect) and digital photography.

DermaPep™ A350 showed significant anti-wrinkle

effect even after 4 weeks.

Illustrative Picture_Case No.F00667

-30

-35 -33

-40

-30

-20

-10

0

Wri

nkl

e re

du

ctio

n p

erce

nta

ge

(%)

30, 35, 33 %

Improvement

o 4 (week)

Wrinkle number

Wrinkle area

Wrinkle length

Improved in 85 % of the volunteers

2% DERMAPEP™ A350

Code F00667

T0 T+4 weeks T+8 weeks T+12 weeks

Number of wrinkles 38 16 2 3

Total wrinkle area (mm²) 11.11 2.77 0.46 1.04

Total wrinkle length (mm) 47.16 15.17 2.13 3.75



volunteers (Bangkok), aged 40 to 60, 2% formula

containing DermaPep™ A350 has been applied twice

daily during 12 weeks on the face area. Evaluation was

performed by chromameter (Chromameter CR-300® ,

Minolta, Japan) and digital photography.

The degree of whitening effect was estimated by

following parameters :

Whitening Efficacy on Asian Skin Type

Lightening Effect of skin tone

L* = Luminosity (Lightness)

ITA˚ = Arc tan ((L*-50)/b*) 180π

Increase of L* and ITA˚ indicates a decrease in skin

pigmentation.

L* is luminosity which represents relative brightness from total darkness (L*=0) to absolute white (L*=100)

0

0.8

1.1

1.8

0

0.5

1

1.5

2

D0

L* parameter evolution in comparison to D0

D28 D56

Increase of L* value in 80% of volunteers

D84

A whitening agent must increase Individual Typological Angle ITA˚ parameter.

-11.8

10.2

-15

-10

-5

0

5

10

15ITA˚ % variation in comparison to D0

Increase of ITA˚ value in 80% of volunteers

D0 D84 +22.0 %


Anti-Aging , Depigmentation and Skin Pore Contraction Efficacy on Human Skin


volunteers (Korea), aged 30 to 60, 20ppm of myristoyl

tripeptide-31 had been applied twice daily during 8

weeks on face area. Efficacy was evaluated by replica

analysis (evaluation of the anti-wrinkle effect) and

digital photography.

DermaPep™ A350 clearly demonstrated significant

anti-wrinkle, depigmentation and skin pore

contraction efficacy in 8 weeks.

Illustrative Pictures

DermaPepTM A350 showed significant improvement on skin pore size, mottled hyperpigmentation and wrinkle on human skin.

Case 1. Case 2.


Skin Pore Contraction Efficacy


volunteers (Korea), aged 30 to 50, 20ppm of Myristoyl

tripeptide-31 had been applied twice daily during 8

weeks on the face area. Skin pore reduction was

evaluated by DM-3.

DermaPep™ A350 significantly reduced skin pore size

in 8 weeks.

Facial Pore Contraction

32.4

30.6

28.8

27

28

29

30

31

32

33

D0 D28 D56

Fac

ial P

ore

s A

rea

(mm

2)

-11.3 %

DermaPepTM A350 significantly reduced skin pore size by 11.3% in 8 weeks.

MIWON COMMERCIAL CO., LTD. 405-3 MONGRAE-DONG, DANWON-GU, ANSAN-SHI, GYEONGGI, 425-100, KOREA PHONE +82-31-8084-8410 FAX +82-31-492-2061 www.dermapep.com, www.mwc.com

Personal Care Ingredients Business Unit http://www.dermapep.com E-mail : [email protected]

Last revised on Apr 17th, 2013

rebecca

TRI-K Industries

DermaPep™ A350 - TRI-K...any side effects. Function Anti-wrinkle for face and body Increase of...

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Transcript of DermaPep™ A350 - TRI-K...any side effects. Function Anti-wrinkle for face and body Increase of...