erjingwan extracts exert antiaging effects of skin through...

Research ArticleErjingwan Extracts Exert Antiaging Effects of Skin throughActivating Nrf2 and Inhibiting NF-120581B

Hairong Zhong1 Choyoung Hong2 Zhouxin Han1 Seung Jin Hwang2 Byunghyun Kim2

Zihang Xu1 Junmun Lee2 Ki Hoon Kim3 Mu Hyun Jin 2 and Chunpu Zou 1

1School of Basic Medical Science Shanghai University of Traditional Chinese Medicine Shanghai 201203 China2Oriental Herbal Medicine Research Team LG Household amp Health Care Co Seoul 07795 Republic of Korea3Safety Researcher Lab LG Household amp Health Care Co Seoul 07795 Republic of Korea

Correspondence should be addressed to Mu Hyun Jin mhjinlghnhcom and Chunpu Zou chunpuzoushutcmeducn

Received 4 November 2018 Revised 26 January 2019 Accepted 24 February 2019 Published 2 May 2019

Academic Editor Yong C Boo

Copyright copy 2019 Hairong Zhong et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

In oriental medicine mixtures of medical plants are always used as prescriptions for diseases Natural products extracted fromherbs have great potential antiaging effects Previous studies and clinical trials have shown several critical functions of Erjingwan(EJW) such as nourishing Yin kidney tonifying and aging-resistance We assumed that EJW extracts exerted the antiaging effectsthrough nourishing Yin We examined the antiaging effects of EJW extracts on healthy human skin by noninvasive measurementsThen we estimated the cell proliferation and DPPH radical scavenging rate Western blotting analysis was used to determine theexpressions of matrix metalloproteinase-1 (MMP-1) type I collagen (COL1A2) p-NF-120581B NF-120581B p-I120581B120572 I120581B120572 p-Nrf2 and HO-1EJW extracts did not affect moisture content TEWL and skin chroma while it significantly improved skin glossiness and skinelasticity Moreover EJW extracts could downregulate theMMP1 expression and upregulate the COL1A2 expression In addition itpromoted the Nrf2 pathway while it inhibited the NF-120581B pathway With the application of cream containing EJW extracts the skinaging state was significantly improved Furthermore in vitro studies showed that EJW extracts contributed to the repair of skinafter injury Taken together the antiaging effects of EJW extracts were related to its antioxidant and anti-inflammatory abilities

1 Introduction

Skin is the largest organ of the human body and thefunctions of skin include protection sensation regulationof body temperature secretion and excretion absorptionand metabolism [1] Skin aging often results in inordinatefunctions and destroyed structures which are both affectedby endogenous and exogenous factors Aging-induced struc-tural changes include flattened dermoepidermal connectionsaltered epidermal thickness cell size and cell shape dermalatrophy reduced fibroblasts mast cells and blood vessels hairloss and decreased hair pigmentation Functional changesinclude cell turnover wound repair barrier function secre-tion of sweat and sebum and reduced synthesis of vitaminD Endogenous factors include cellular metabolism geneticshormones and metabolic processes and exogenous factorsinclude chronic exposures to light pollution ionizing radia-tion chemicals and toxins No matter what kind of reasons

the representative externalmanifestations are wrinkles spotsskin laxity and roughness

Themechanisms underlying the skin aging remain largelyunexplored It is well accepted that the main cause ofskin aging and wrinkle formation is changes in collagenAbout 90 of the proteins in dermis are collagen Matrixmetalloproteinases (MMPs) and MMP-1 subfamily may leadto collagen degradation in the skin [2] Oxidative stress playsan important role in the progress of aging Oxidative stress isthe most harmful contributor to skin aging both internallyand externally and over production of reactive oxygenspecies (ROS) can induce the imbalance between free radicalproduction and antioxidant defense Previous studies haveshown that the increased generation of ROS is significantlycorrelated with aging process [3] Skin inflammation playsan important role in accelerated aging [4] Two importanttranscription factors nuclear factor-120581B (NF-120581B) and nuclearfactor (erythroid-derived 2)-like 2 (Nrf2) play fundamental

HindawiEvidence-Based Complementary and Alternative MedicineVolume 2019 Article ID 5976749 15 pageshttpsdoiorg10115520195976749

2 Evidence-Based Complementary and Alternative Medicine

roles in regulating cellular response to inflammation andoxidant stress Nrf2 is considered as an auxiliary protectivefactor in the regulation of antioxidation anti-inflammationand expressions of detoxification proteins [5] IKKNF-120581Binhibitors can decreaseDNAdamage that induces senescenceand aging [6]

People always prefer healthy and younger-looking skinwhich represents youth and beauty There are many anti-aging strategies including preventive measures cosmeticstrategies topical and systemic medications and invasiveprocedures (such as chemical peeling vitamin A fluorouracilointment laser intense pulsed light skin filling operationdermabrasion cryotherapy and photodynamic therapy) [78] Many antiaging skin care products have been developedin history and over the years advanced technologies andnew theories have greatly contributed to the convenient andeffective use of the skin care products Many studies haveshown that topical application of antiaging products canimprove the appearance of skin and topical application ofvitamins minerals and botanical ingredients can improvefine wrinkles freckles and pigmentation More and moreattention has been paid to botanical ingredients due to theirless toxicity and side effects [9]

Erjingwan (EJW) decoction is composed of Lyciumbarbarum and Polygonatum sibiricum which is recordedin General Medical Collection of Royal Benevolence EJWis a classic tonic formula which can maintain youth andprevent decrepitude It has been investigated that Lyciumbarbarumpolysaccharide candecreaseDNAdamage throughreducing oxidative stress [10] Moreover it can inhibit cellaging through the p53-mediated pathway [11] Polygona-tum sibiricum is usually applied for Qi- or Yin-deficiencysyndrome and it has kidney-tonifying ability Polygonatumsibiricum has been validated to have antioxidant effects andit can enhance telomerase activity [12 13] Therefore thecompatibility of these two medicinal herbs contributes to theantiaging process

Based on previous studies we hypothesized that EJWextracts could be used in antiaging products In the presentstudy we aimed to explore the mechanisms underlying theantiaging effects of EJW extracts and assess its antioxidantand anti-inflammatory abilities We first confirmed the effec-tiveness of EJW extracts Human foreskin fibroblasts (HFFs)were treated with H

2O2to induce the damage Then some

targets were determined to evaluate if EJW extracts couldreverse such H

2O2-induced damage The DPPH free radical

scavenging assay was conducted to validate the antioxidantability of EJW extracts Western blotting analysis was usedto examine the expressions of MMP1 and COL1A2 as well asNF-120581B and Nrf2 pathways

2 Materials and Methods

21 Clinical Trial

211 Preparation of EJWCream The samples were producedby LG Household amp Health Care Co including creamcontaining EJW extracts with traditional Chinese medicine

(TCM) nourishing Yin therapy (sample A) and cream with-out active ingredient (sample B)

Mix 100ml solvent (Water 88 Butylene Glycol 10 12-Hexanediol 2) with 10g Lycium Chinense Fruit Extract10g Polygonatum Sibiricum Extract 10g Poria Cocos Extract10g Polygonum multiflorum respectively Then extract themat normal temperature for 3 days and filter Then mix theextractions together and filer again

The components of the cream are listed in Table 5

212 Main Instruments Following instruments were usedin the present study including human skin rapid opticalimaging system PRIMOS Pico (GFMesstechnik GmbH Ger-many) stratum corneumwater component tester Corneome-ter (CourageampKhazakaGermany) epidermalwater loss ratetester Tewameter TM300 (Courage amp Khazaka Germany)CR-400 MINOLTA (KONICA Japan) skin gloss testerGL200 (Courage amp Khazaka Germany) skin elasticity testerDual Cutometer MPA580 (Courage amp Khazaka Germany)and balance sense quantity 001 g (METTLER Switzerland)

213 Participants The study was reviewed and approved bythe National Quality Supervision and Inspection Center forPerfume and Perfume Cosmetics Each participant providedwritten informed consent before any study-related examina-tion or procedure was performed and the study adhered tothe tenets of the Declaration of Helsinki The participantswere selected according to inclusion and exclusion criteriaThe participants were required to voluntarily participate inthe test and sign the informed consent be healthy Chinesewomen be between 30 to 45 years old have obvious wrinklesor fine lines in the eye test area (grade 2-3 of crows feet)keep the skin of arms dry (the base value of skin moisturein the forearm test area was between 15-45 au) and refrainto use any cosmetics on the face from the night before thetest On the day of the test participants should clean theface and forearms before the test have no severe systemicdiseases no immunodeficiency or autoimmune diseases andreceive no skin cosmetology treatment and other tests benot highly sensitive to body constitution not sensitive tocommon cosmetics and not suffering from pollen allergy orother allergic reactions ensure that no steroid drugs andimmunosuppressive agents were used in the last monthensure not to participate in other clinical studies in the last 3months agree to use photos of local skin and self-assessmentresults in the data have no plan for outdoor activities ortravel to the south and have no excessive sun exposureduring the test period ensure that the facial products ofthe same function other than the test products were notused during the test ensure that the face was not treatedby antiaging whitening grinding or other cosmetic projectsduring the test period and ensure that no drugs of antiagingand whitening effects were administered orally well use theproducts understand and fill in the project questionnaire andcooperate with the staffs and have the chance to participatein further tests in the future The exclusion criteria were asfollows people with allergic dermatitis or having a history ofskin diseases (severe freckles atopic skin diseases psoriasis

Evidence-Based Complementary and Alternative Medicine 3

severe acne dermatitis and so on) or people not suitable toparticipate in the project according to the clinical evaluationpeople receiving treatment in dermatology or people usingskin whitening and antiaging drugs within 1 month thetest area in the arms had a large area of birthmark scratchwhite spot pigmented nevus and keloid the face had alarge area of birthmark scratch white spot pigmented nevusand keloid woman who was pregnant breast-breeding andmenopause or who intended to conceive during the testand woman who was not suitable as the object of thistest

A total of 22 healthy Chinese women (30-45 years old)were enrolled in this randomized double-blind study and21 of them were recruited as participants while one wasexcluded from subsequent analysis due to the agreement ofwithdrawal

214 Treatment andTest Requirements Thetest environmentrequired a temperature of 22plusmn 2∘C and a humidity of40-60

Cream containing effective components (EJW extracts)was applied on the skin of the left face and one forearm for4 weeks while cream without such components was appliedon the other side as a control

During 4 weeks participants were requested to cleanthe face and arms every morning and evening and then05 g of each cream was carefully spread to the face andforearm followed by gentle massage until the cream was fullyabsorbed

215 Noninvasive measurements of the skin (Table 1)

216 Test Schedule Tests were carried out according toa predesigned schedule as follows test 0W (before use)20161129 Tuesday test 2W 20161213 Tuesday and test4W 20161227 Tuesday) Specific implementation process isshown in Figure 1

217 Allergy Test (24 h Patch Test) In order to examinewhether the skin was allergic to the cream containing EJWextracts 24 h patch test was performed A total of 20 maleswithout dermatosis were selected in the test Briefly 15 120583L testsamples were spread on their backs for 24 h and the degree ofallergic reaction was observed after 2 h Criteria of judgmentwere set as follows (Table 2)

22 In Vitro Study

221 Cell Culture HFF cells (Cellular Bank of ChineseAcademy of Sciences) were maintained in Dulbeccorsquos Mod-ified Eaglersquos medium (DMEM Gibco USA) supplementedwith 10 heat inactivated fetal bovine serum (Gibco USA)and 1 penicillin streptomycin under standard conditions(37∘C 5 CO

2)

222 Cell Viability Cell viability was assessed byCell Count-ing Kit-8 (CCK-8 Byotime China) Briefly HFF cells wereseeded into 96-well plates at a density of 4times103well andincubated under normal conditions for 24 h The cells were

divided into three groups including control group modelgroup and drug group Moreover the drug group wasfurther divided into three subgroups with three different finalconcentrations (10 100 and 200 120583gmL) After 6 h 10 120583LCCK-8 reagent was added to each well and the mixturewas incubated at 37∘C for additional 2 h Subsequently theabsorbance of each well was determined at a wavelength of450 nm using the Synergy 2 Multi-Mode Microplate Reader(BioTek Instrument Int Winooski VT USA) and the cellviability was calculated with the formula as follows cellviability () = average value of drug groupaverage value ofcontrol group

223 DPPH Free Radical Scavenging Assay Stock solution ofDPPH (D9132 Sigma Aldrich USA) was prepared by adding9464 mg DPPH into 3 mL 80 absolute ethanol yielding aconcentration of 8 mM and such stock solution was furtherdiluted to 200 120583Mwhen the test was conducted Four groupswere designed in this assay and reagents in each group arelisted in Table 3 Vitamin C (A610021 Sangon China) wasused as the positive control Five concentrations (006250125 025 05 and 1 mgmL) of EJW extracts were carriedout in the test and each concentration was conducted intriplicate

After reagents were mixed well in 96-well plates themixturewas incubated at 37∘C for 30min and the absorbancewas determined at a wavelength of 517 nm using Synergy2 Multi-Mode Microplate Reader Independent experimentswere performed in triplicate The inhibition rates were cal-culated with the formula as follows Inhibition rate = (1-(B-C)(A-D)) lowast 100 A represents the absorbance of totalDPPH B represents the absorbance of sample C representsthe absorbance of sample control and D represents theabsorbance of blank control The graph was plotted byGraphPad prism 60 and then the IC50 value was calculatedfrom the graph

224 Western Blotting Analysis HFF cells in logarithmicgrowth were seeded into 6-well plates at a density of 1times105cellswell and incubated in the incubator for 24 h Except forthe control group cells were incubated with 2 mL of 0015 H2O2for 15 min Culture medium in the model group

was replaced by 2 mL fresh culture medium while 2 mL of250 120583gmL EJW extracts was added to the drug group andincubated for 24 h and 48 h Next cells were lysed by 40 120583LRIPA buffer (Beyotime China) on ice for 15 min and thecell lysates were centrifuged at 12000 rpm for 15 min at 4∘CProtein concentrations were determined by BCA method(Sangon China) Equal amounts of proteins (15 120583g) weresubjected to 10 sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) and then transferred onto nitro-cellulose membranes (GE) The membranes were blockedby Quick Block (Beyotime China) for 10 min and thenincubated with primary antibodies against COL1A2 (CY-5120 Abways China) MMP1 (D220093 Sangon China) p-NF-120581B (ser536) (11014 SAB USA) NF-120581B (D220135 SangonChina) p-I120581B120572 (ser32ser36) (D155066 Sangon China)I120581B120572 (D120138 Sangon China) p-Nrf2 (S40) (CY-6573


Declined to participate(n=1)

Participate in the Tests(n=21)

Left side for test sampleright side for control sample

Test again after using the creamfor two weeks and four weeks

Confirm the amount of product each time test

Collect the questionnaires andanalyse

Assessed for eligibility(n=22)

Adverse Reaction Questionnaireneed being filled in when it occurs

will be tested stay in constant temperatureand humidity)

Test the initial value(cleaning the area

Figure 1 Study flowchart of the participants describing trial progress

Table 1 Observe projects and test site

Observe and test projects Test area and control area1 Moisture content (MPA10 Comeometer CM825) Inside of forearms2 Trans epidermal water loss rate (MPA10 Tewameter TM300) Inside of forearms3 Skin chroma (CR-400) Joint of the eye midline and nose wing4 Skin glossiness (MPA10 Skin-Glossmeter GL200) Cheekbones5 Skin elasticity (Dual CutometerMPA580) Corner of the eyes

Table 2 CTFAICDRG guideline textbook of contact dermatitis

Reaction Score Symptoms and criteria of judgment- 0 Adiaphoria+- 05 Light erythema+ 10 Obvious erythema++ 20 Severe erythema with swelling+++ 30 Severe erythema with vesicles and swelling


Table 3 Reagents in different groups

Groups Sample (120583L) 200 120583MDPPH (120583L) 80 absolute ethanol (120583L)A - 200 50B 50 200 -C 50 - 200D - - 250

Table 4 The primer sequences for target genes

MMP1 forward 51015840-GAGCAGATGTG-GACCATGCC-31015840

MMP1 reverse 51015840-TGGGCCTGGTTGAAAAGCAT-31015840

COL1A2 forward 51015840-G-TGCTACTGGTGCTGCCG-31015840

COL1A2 reverse 51015840-CACACCCTGGGGACCTTCAG-31015840

HO-1 forward 51015840-GGCCATGAACTTTGTCCGGT-31015840

HO-1 reverse 51015840-TCAGTGCCCACGGTAAGGAA-31015840

GAPDH forward 51015840-GGCACAGTCAAGGCTGAGAATG-31015840

GAPDH reverse 51015840-ATGGTG-GTGAAGACGCCAGTA-31015840

Abways China) or HO-1 (sc-136960 Santa Cruz USA) at4∘C overnight Subsequently the blots were washed withTBST for three times and then incubated with correspondingHRP-conjugated secondary antibodies (15000 dilution) at37∘C for 30 min Immunoreactive bands were visualized withECL reagents (Millipore USA) and imaging densitometrywas quantified using ImageJ (NIH USA)

225 qRT-PCR HFF cells were seeded into 6-well plates at adensity of 7times104well 24 h prior to different treatments More-over 0015 H

2O2was used to treat the model group and

EJWgroup for 15minThenH2O2solution was removed and

then HFF cells in the EJW group were further incubated with200 120583gmL EJW extracts for 48 h Total RNA was extractedand then reversely transcribed into cDNA with PrimeScriptRT MasterMix kit (Takara) qRT-PCR was performed usingSYBR Premix Ex Taq kit (Takara) on LightCycler 96 SW 11The relative expressions of target genes at the mRNA levelwere determined using the 2-ΔΔCT method and GAPDH wasselected as the housekeeping gene The primer sequences fortarget genes are listed in Table 4

23 Statistical Analysis All data were expressed as meanplusmn SEM and analyzed using SPSS190 (IBM) Comparisonsbetween experimental groups were conducted using one-wayANOVA while multiple comparisons were performed usingthe LSD method Statistical significance was defined as P lt005 or P lt 001

3 Results

31 Antiaging Effects of EJW-Containing Cream Moisturecontent was determined using MPA10 Comeometer CM825The higher value indicates the higher moisture contentMoisture content in the area applied with cream containingEJW extracts for 2 weeks and 4 weeks was significantlyimproved compared with their initial values as well as thecontrol area (Figure 2(a)) No significant difference in the

moisture increment was observed between the two groupssuggesting that the cream containing EJW extracts couldnot effectively increase the moisture of stratum corneumcompared with the control sample (Figure 2(b))

As an important indicator of skin function transepider-mal water loss rate (TEWL) is widely accepted as the mostreliable method to examine the skin barrier function [14]MPA 10 Tewameter TM300 was used to determine TEWLinside of forearms (left and right) Values of TEWL in boththe control area and test area after 2 weeks and 4 weeks oftreatment were significantly decreased compared with theirinitial values (Figure 2(c)) However there was no significantdifference in the reduction of TEWLbetween the two areas offorearms indicating that the cream containing EJW extractscould not effectively decrease the TEWL of the forearm skincompared with the control sample (Figure 2(d))

The measurements of skin chroma included Llowast (bright-ness of color) and ITA∘ (individual topology angle) whichwere evaluated by CR-400 The most widely used standardcolor system is Commission Internationale de lrsquoEclairage(CIE) Lab system including Llowast (lightness value) alowast (theamount of green or red) and blowast (the amount of yellowor blue) The three axes record color in a 3-dimensionalspace [15] The calculation formula of ITA∘ is tan-1[(Llowast minus50)blowast] times 180120587 ITA∘ is the angle between Llowast and blowast inthe 3-dimensional space which has a positive correlationwith Llowast and a negative correlation with blowast [16] The greaterthe values of Llowast and ITA∘ are the brighter the skins areFigures 3(a) 3(b) 3(c) and 3(d) show that skin brightnessparameters Llowast and ITA∘ in both the test area and controlarea after 2 weeks and 4 weeks of treatment were significantlyimproved compared with their initial values Meanwhile nosignificant difference of skin chroma between the two areaswas observedThis result suggested that the cream containingEJW extracts could not effectively improve the skin chromacompared with the control sample

Though there was no significant difference in moisturecontent TEWL and skin chroma between the test area and


Week

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sture

(au

)

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lowastlowastlowastlowastlowastlowast


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(f)

Figure 2 EJW extracts significantly improve skin aging (a b) Moisture content was measured by MPA10 Comeometer CM825 (c d) MPA10 Tewameter TM300 was used to measure the TEWL (e f) Skin glossiness was measured by MPA10 Skin-Glossmeter GL200 lowast Plt005 lowastlowastPlt001 lowast lowast lowast Plt0001 lowast lowast lowastlowast Plt00001 Data were shown as mean plusmn SD

control area the skin glossiness in the area applied withcream containing EJW extracts for 2 weeks and 4 weekswas significantly improved compared with initial value whileit in the control area showed no difference (Figure 2(e))

Meanwhile the skin glossiness in the test area was obviouslyhigher than that in the control area (Figure 2(f)) This resultindicated that EJW extracts played an important role inimproving the skin glossiness compared with the control


Week

25

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Figure 3 The skin chroma was evaluated by CR-400 lowast Plt005 lowastlowast Plt001 lowastlowastlowast Plt0001 lowastlowastlowastlowast Plt00001 Data were shown as mean plusmn SD

sample Moreover the skin elasticity test showed that theR1 value was significantly decreased and the R2 value wassignificantly increased only in the test area (Figures 4(a) 4(b)4(c) and 4(d)) R1 value (skin firmness parameter) and R2value (skin elasticity parameter) are two parameters of skinelasticity The lower the R1 value is the firmer the skin is Thehigher the R2 value is the resilient the skin is In a word thecream containing EJW extracts could strongly improve theskin elasticity compared with the control sample

32 Allergy Test The sample groups included SLS (sodiumlauryl sulfate) MPO (2-methyl- 13-propanediol) EJW (rawmaterial) control 02 EJW (sample) 05 EJW (sample)and 1 EJW (sample) SLS (Sodium lauryl sulfate or sodiumdodecyl sulfate SDS) is an anionic surfactant with thesample CH3(CH2)11SO4Na It is a common component ofmany domestic cleansing personal hygiene and cosmeticpharmaceutical and food products And SLS is used as a

positive control in human skin primary irritation test All thesample groups showed no reaction on the skin (Figure 5)

33 EJW Extracts Reverse the Damage of H2O2 to HFFCells In Vitro H

2O2was used as a stressor in the test

Briefly 0015 H2O2was used to treat the HFF cells in

vitro for different durations The results showed that thecell viability was significantly decreased in a time-dependentmanner (Figure 6(a)) Then we examined the expressions ofMMP1 and COL1A2 at the protein level in HFF cells treatedwith 0015 H

2O2for different durations and our data

showed that the expression of MMP1 was increased whilethe expression of COL1A 2 was decreased (Figure 6(b))ThenHFF cells were incubated with 100 120583gmL or 200 120583gmL EJWextracts for 24 h and 48 h after H

2O2exposure and we found

that the cell viability of EJW-treated cells was significantlyincreased after 48 h (Figure 6(c)) Western blotting analysisalso showed that the MMP1 expression was increased the


Week

Skin

com

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minus04

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lowastlowastlowast

lowastlowastlowast

(d)

Figure 4 The skin elasticity was measured by Dual Cutometer MPA580 lowast Plt005 lowastlowast Plt001 lowast lowast lowast Plt0001 lowast lowast lowastlowast Plt00001 Data wereshown as mean plusmn SD

0

02

04

SLS MPO EJW1 (raw material)

Control EJW02 (sample)

EJW05 (sample)

EJW1 (sample)

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

Figure 5The formula of primary irritation index (PII) is [sum(score timesNo of responses)]20 (No of total subjects)times1 (total stimulus evaluationtimes) Criteria of judgment were set according to CTFAICDRG guideline text book of contact dermatitis (Table 2) lowast P lt 005 lowastlowast P lt 001Data were shown as mean plusmn SD


Cel

l via

bilit

y (

)

CON 3 h 4 h 6 h

20

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4 h6 h

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(b)

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24 h 48 h

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CONMODEL10 gmL

100 gmL200 gmL

(c)

Figure 6 EJW extracts can improve the cell viability of H2O2-treatedHFF cells (a) Cell viability was significantly decreased by the treatment

of 0015 H2O2 (b) HFF cells were treated with 0015 H

2O2for 5 10 15 20 30 min (c) After exposure to 0015 H

2O2for 15 min HFF

cells were incubated with EJW extracts (10 100 200 120583gmL) for 24 h and 48 h indicates the significant difference between control groupand model group and lowast indicates the significant difference between model group and EJW group lowast and Plt005lowastlowast and Plt001 lowast lowast lowastand Plt0001 lowast lowast lowastlowast and Plt00001 Data were shown as mean plusmn SD

COL1A2 expression was decreased after H2O2exposure and

48 h exposure to EJW extracts could reverse such expressionpattern (Figures 8(b) and 8(c))

34 EJW Extracts Restore H2O2-Induced Damage throughModulating the Antioxidant and Anti-Inflammatory Path-ways We showed that EJW extracts could significantlyimprove the skin glossiness and skin elasticity in healthyhuman skin and reverse the H

2O2-induced damage of HFF

cells in vitro We next explored why EJW extracts had sucheffects We first tested the DPPH inhibition ability of EJWextracts in vitro using Synergy 2 Multi-Mode MicroplateReader (BioTek Instrument Int Winooski VT USA) Fig-ure 7(a) shows that the EJW extracts significantly inhibitedthe DPPHactivity and the IC50was 0531mgmLThe results

indicated that the antiaging effects of EJW extracts wererelated to the antioxidant ability Therefore we next assessedthe Nrf2 pathway an antioxidation pathway As expected theNrf2 pathway was activated by the exposure to EJW extractsfor 24 h The expressions of p-Nrf2 and HO-1 were bothsignificantly increased Moreover the H

2O2-activated NF-120581B

pathway was also downregulated by EJW extracts Westernblotting analysis showed that the expressions of p-NF-120581b andp-I120581B120572 were both significantly decreased (Figures 9(a) 9(b)and 9(c))

4 Discussion

Herbal medicine has long been used in Asian countries andherbal extracts can effectively reverse aging signs Therefore


Concentration (mgmL)

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Figure 7 EJW extracts significantly inhibit the activity of DPPH (a) DPPH inhibition rates of EJW extracts (00625 0125 025 05 and 1mgmL) were examined Different concentrations of EJW extracts were incubated with DPPH for 30 min at 37∘C (b) Vitamin C (125 25 510 20 40 and 80 120583gmL) was used as the positive control (Table 3) Data were shown as mean plusmn SD

herbal ingredients have become a good choice for antiagingtherapies [17] Nowadays a great deal of attention has beenpaid to herbal medicine worldwide Topical application ofherb extracts has attracted more andmore attention due to itsgood skin care effect and low cost as well as noninvasive andless side effects Hundreds of plant extracts from plant seedsstems skins and fruits have been used in antiaging cosmetics[18] Green tea extracts have shown antiphotoaging effectin clinical trial [19] Algal extracts can resist UVB-inducedskin lesion in BALBC mice due to anti-inflammatory andantioxidative effects [20]

Herbal medicine has also long been used in China andmany herbal medicines are recorded to have antiaging effectsand used as tonic formula in TCM therapies EJW decoctionis one of the classic decoctions Firstly our clinical trialvalidated the antiaging effects of EJW Clinical trials includedthe evaluations of moisture content TEWL skin chromaskin glossiness and skin elasticity They were all noninvasivedetections of skin changes which could be easily accepted byparticipants The results indicated that EJW extracts couldnot increase the moisture content and keep water fromrunning away (TEWL) Besides it had no obvious whiteningeffects (Llowast and ITA∘) On the contrary skin glossiness andskin elasticity (R1 and R2) were both significantly improvedTherefore dull and loose skin caused by aging could beimproved by EJW extracts

To explore the mechanism underlying antiaging effects ofEJW extracts H2O2 was used to induce the senescent stateas an in vitro model of oxidative stress Most contributingfactors of skin aging such as mitochondrial dysfunctionintracellular communication changes genomic instabilitycell senescence and extracellular matrix (ECM) decompo-sition are the consequences of oxidative processes H

2O2is

an ROS signaling agent and generation of free radicals canlead to cell death protein degradation and inflammatoryreaction [21] To confirm the senescent state of HFF cells weexamined the cell viability by CCK-8 assay The cell viability

was significantly decreased upon the exposure to H2O2

Besides the increased MMP1 expression and decreasedCOL1A2 expression also indicated the cell injury Furtherstudies revealed that EJW extracts could reverse the H

2O2-

induced injury EJW extracts could promote the proliferationof HFF cells after H2O2 treatment upregulate the expressionof COL1A2 and downregulate the expression of MMP1leading to reduced oxidative stress Based on our findingswe deduced that the antiaging effects of EJW extracts wererelated to the antioxidative effects Therefore we conductedthe DPPH free radical scavenging assay to validate suchassumption

Extensive studies have been focused on NF-120581B and Nrf2pathways Simultaneous regulation of NF-120581B and Nrf2 path-ways plays an important role in many diseases For exampleNrf2 improves lupus nephritis by inhibiting oxidative injuryand negatively regulating the NF-120581B pathway [22] Theclassical NF-120581B pathway is activated by stimuli such as TNF-120572 IL-1 H

2O2 LPS or microbial infection further triggering

the expressions of downstream genes including variousinflammatory cytokines and chemokines after inducing theproteasome-mediated degradation of I120581B proteins [23] NF-120581B pathway plays an important role in the occurrence andregulation of aging It is not only activated in aging butalso directly involved in the development of aging-relateddiseases For example inhibition ofNF-120581B can reduce cellularsenescence and oxidative damage in mice or even extend thehealthspan in elderly patients [24]

Nrf2 has detoxification and antioxidant effects and thebeneficial effects of Nrf2 have been previously reported inmany diseases UVB can induce more inflammation in theNrf2 KO mice compared with the WT mice while Nrf2 canprotect the skin from UVB-induced inflammation and sun-burn reaction [25] Keap1 is an important negative regulatorof Nrf2 in vivo Under normal redox conditions Nrf2 binds totypicalNrf2 repressor proteinKeap1 in cytoplasm and is easilydegraded by ubiquitin-proteasome pathway However Nrf2


MMP1

COLA1

GAPDH

HFFCON Model EJW

002040608

11214

CON Model EJW

COL1

A2

prot

ein

leve

l(n

orm

aliz

ed C

ON

= 1

)

08509

0951

10511

115

CON Model EJW

MM

P1 p

rote

in le

vel

(nor

mal

ized

CO

N =

1)

(a)

002040608

11214

CON Model EJW

Relat

ive m

RNA

expr

essio

n

005

115

225

3

CON Model EJWRe

lativ

e mRN

Aex

pres

sion


(b)

HFF

MMP1

GAPDH

COLA1

CON Model EJW

002040608

11214

CON Model EJW

COL1

A2

prot

ein

leve

l(n

orm

aliz

ed C

ON

= 1

)

002040608

11214

CON Model EJW

MM

P1 p

rote

in le

vel

(nor

mal

ized

CO

N =

1)

lowastlowast

lowastlowastlowast

(c)

Figure 8 EJW extracts restore the expression of COL1A2 by reducing the MMP1 expression (a) HFF cells were seeded into the 6-well platesand exposed to 0015 H

2O2for 15 min The expressions of COL1A2 and MMP1 at 24 h were examined by Western blotting analysis (b c)

At 48 h the expressions of COL1A2 and MMP1 were assessed by RT-qPCR and Western blotting analysis The concentration of EJW was200120583gmL indicates the significant difference between control group and model group and lowast indicates the significant difference betweenmodel group and EJW group lowast and Plt005 lowastlowast and Plt001 lowast lowast lowast and Plt0001 lowast lowast lowastlowast and Plt00001 Data were shown asmean plusmn SD

dissociates fromKeap1when exposed to stressors or inducersmoves to the nucleus binds to the ARE and triggers theexpressions of Nrf2-regulated genes Therefore the expres-sions of multiple antioxidant enzymes such as superoxidedismutase (SOD) and biphase detoxifying enzymes such asheme oxygenase 1 (HO-1) glutathione S transferase (GSTs)andNAD(P)Hquinone oxidoreductase 1 (NQO1) begin to beupregulated [26ndash28] HO-1 acts as a rate-limiting enzyme forheme degradation in all Nrf2-regulated enzymes and it hasantioxidant and anti-inflammatory immunomodulatory andantiapoptotic effects Once Nrf2 binds to ARE on the HO-1promoter HO-1 can be induced in many cells to exert its owndefensive effect [5]

Moreover there is an interaction between Nrf2 NF-120581Band inflammation [29] NF-120581B can downregulate the Nrf2pathway while the Nrf2-regulated gene HO-1 can inhibit NF-120581B activation NF-120581B blocks the binding of CREB-binding

protein (CBP) to Nrf2 or promotes the interaction of HDAC3with CBP or MafK [30 31] Our results confirmed that EJWextracts might promote the Nrf2 pathway and inhibit the NF-120581B pathway contributing to restoration of the redox home-ostasis EJW extracts possessed anti-inflammatory activitiesand excellent ROS-scavenging abilities This could explainwhy EJW exhibited great antiaging effects on skin Howeverthere was a complex link between ROS aging and lifespan[26] On one hand low levels of oxidative stress are foundto be good for longevity and health [32] On the other handit is difficult to quantify the experimental ROS level Gen-erally the results of the experiments may be easy to deviateand produce unexpected results [21] Therefore completelyscavenging free radicals may not be a good choice In thepresent study the successful establishment of H

2O2-treated

model was confirmed by examining cell viability and theexpressions ofMMP1 andCOL1A2 EJWextractsmight lower


0

05

1

15

2

25

3

35

CON Model EJW

Rela

tive m

RNA

expr

essio

n

lowastlowast

(a)

p-IB (ser32 ser36)

IB

p-NFB (ser536)

NFB

GAPDH

HO-1

p-Nrf2 (S40)

HFFCON Model EJW

(b)

0

02

04

06

08

1

12

CON Model EJW

p-N

rf2 (S

40) p

rote

in le

vel

(nor

mal

ized

CO

N =

1)

0

05

1

15

2

25

CON Model EJW

HO

-1 p

rote

in le

vel

(nor

mal

ized

CO

N =

1)

0

02

04

06

08

1

12

14

CON Model EJW

p-N

FkB

NFk

B pr

otei

n le

vel

(nor

mal

ized

CO

N =

1)

002040608

112141618

CON Model EJW

p-Ik

BaIk

Ba p

rote

in le

vel

(nor

mal

ized

CO

N =

1)

lowast


lowastlowast

(c)

Figure 9 EJW extracts activate Nrf2 and inhibit NF-120581B to improve the aging HFF cells (a b) HFF cells were seeded into the 6-well platesand then exposed to 0015 H

2O2for 15 min The expression of HO-1 was detected by RT-qPCR and the expressions of p-NF-120581B NF-120581B

p-I120581B120572 I120581B120572 and p-Nrf2 were examined byWestern blotting analysis (c) Data were pooled from three independent experiments indicatesthe significant difference between control group and model group and lowast indicates the significant difference between model group and EJWgroup lowast and Plt005 lowastlowast and Plt001 lowast lowast lowast and Plt0001 lowast lowast lowastlowast and Plt00001


Table 5 The components of the cream

ComponentsWATERCYCLOHEXASILOXANEHYDROGENATED POLYDECENEDIPROPYLENE GLYCOLGLYCERINMACADAMIA TERNIFOLIA SEED OILTRIETHYLHEXANOINBIS-PEG-18 METHYL ETHER DIMETHYL SILANE12-HEXANEDIOLSTEARYL ALCOHOLGLYCERYL STEARATEPEG-40 STEARATEPENTAERYTHRITYL TETRAETHYLHEXANOATECETEARYL ALCOHOLSTEARIC ACIDPEG-100 STEARATESORBITAN STEARATEHYDROGENATED LECITHINCETEARYL OLIVATESORBITAN OLIVATEDIMETHICONEDIMETHICONEVINYL DIMETHICONE CROSSPOLYMERLACTOBACILLUSSOYBEAN FERMENT EXTRACTSACCHAROMYCESPOTATO EXTRACT FERMENT FILTRATESACCHAROMYCESBARLEY SEED FERMENT FILTRATEPANTHENOLXANTHAN GUMACRYLATESC10-30 ALKYL ACRYLATE CROSSPOLYMERCARBOMERTROMETHAMINETRISODIUM EDTAFRAGRANCELYCIUM CHINENSE FRUIT EXTRACTPOLYGONATUM SIBIRICUM EXTRACTPOLYGONUMMULTIFLORUM ROOT EXTRACTPORIA COCOS EXTRACTSOPHOCARPINE

ROS to standard levels through the regulation of oxidation-reduction balance and the specific mechanism needs to befurther explored

According to the aging theory of TCM aging is a resultof decline in vital energy Therefore maintaining the vitalenergy is the most important aspect in antiaging strategiesYin essence is the most important basic substance of bodywhich is important for the body to maintain the balance ofYin and Yang As two sides of a coin a balance between Yinand Yang is similar to the balance between antioxidant andoxidant while Yin represents antioxidant andYang representsoxidant Over oxidative stress is the imbalance of Yin andYang In TCM theory inflammation is caused by the excessive

Yang which can be released by Yin-tonifying or cleared up bycold-cool drug [33]The antiaging herbs have some commonproperties For example they can supplement the vital energyto the body intervene with disease and have multitargets toregulate the biological process [34] One important futuredirection of the antiaging effects of EJW extracts is to findthe target groups for Yin or Yang

5 Conclusions

Taken together our results indicated that EJW extracts couldsignificantly improve the skin aging state including skinglossiness and skin elasticity EJW extracts might be involvedin redox regulation by activating Nrf2 pathway and inhibitingNF-120581B pathway leading to scavenging of free radicals as wellas MMP1 downregulation and COL1A2 upregulation Theantiaging effect was related to the balance between Yin andYang where redox homeostasis was one of the aspects Inconclusion like many other herbal medicines EJW extractscould be applied in the antiaging products

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon requset

Disclosure

Hairong Zhong and Choyoung Hong are both the first co-authors

Conflicts of Interest

The authors declare that there are no conflicts of interest

Authorsrsquo Contributions

Choyoung Hong Byunghyun Kim Mu Hyun Jin andChunpu Zou conceived the study Hairong Zhong ZhouxinHan ZihangXuChoyoungHong andChunpuZou searchedthe literature selected the prescription extracted the mate-rials performed the experiments and analyzed the dataJunmun Lee made cream with Erjingwan extracts and KiHoon Kim conducted the skin allergy test for human ofthe cream Hairong Zhong wrote the manuscript ChoyoungHong Byunghyun Kim Seung Jin Hwang Junmun LeeKi Hoon Kim and Mu Hyun Jin helped with the studydesign and the revision of the manuscript Dr Ki Hoon Kimwas added after acceptance All authors read and approvedthe final version of the manuscript Hairong Zhong andChoyoung Hong contributed equally to this work

Acknowledgments

This work was supported by LG Household amp Health CareCo from the Republic of Korea The authors are grateful fortheir support


References

[1] T Kanaki E Makrantonaki and C C Zouboulis ldquoBiomarkersof skin agingrdquo Reviews in Endocrine and Metabolic Disordersvol 17 no 3 pp 433ndash442 2016

[2] J Varani M K Dame L Rittie et al ldquoDecreased collagenproduction in chronologically aged skin roles of age-dependentalteration in fibroblast function and defective mechanical stim-ulationrdquo 13e American Journal of Pathology vol 168 no 6 pp1861ndash1868 2006

[3] J R Speakman andC Selman ldquoThe free-radical damage theoryaccumulating evidence against a simple link of oxidative stressto ageing and lifespanrdquo BioEssays vol 33 no 4 pp 255ndash2592011

[4] A Freund A V Orjalo P Desprez and J Campisi ldquoInflam-matory networks during cellular senescence causes and conse-quencesrdquo Trends in Molecular Medicine vol 16 no 5 pp 238ndash246 2010

[5] A Loboda M Damulewicz E Pyza A Jozkowicz and JDulak ldquoRole of Nrf2HO-1 system in development oxidativestress response and diseases an evolutionarily conservedmech-anismrdquo Cellular and Molecular Life Sciences vol 73 no 17 pp3221ndash3247 2016

[6] J S Tilstra A R Robinson J Wang et al ldquoNF-120581B inhibitiondelays DNA damagendashinduced senescence and aging in micerdquo13e Journal of Clinical Investigation vol 122 no 7 pp 2601ndash2612 2012

[7] R GancevicieneA I Liakou ATheodoridis EMakrantonakiand C C Zouboulis ldquoSkin anti-aging strategiesrdquo Dermatoen-docrinol vol 4 no 3 pp 308ndash319 2012

[8] L He Dermatology and Cosmetology Peoplersquos Health Publish-ing House Beijing China 2011

[9] F Afaq and H Mukhtar ldquoPhotochemoprevention by botanicalantioxidantsrdquo Skin Pharmacology and Physiology vol 15 no 5pp 297ndash306 2002

[10] H Wu H Guo and R Zhao ldquoEffect of Lycium barbarumpolysaccharide on the improvement of antioxidant ability andDNA damage in NIDDM ratsrdquo Yakugaku Zasshi vol 126 no 5pp 365ndash371 2006

[11] G Xia N Xin W Liu H Yao Y Hou and J Qi ldquoInhibitoryeffect of Lycium barbarum polysaccharides on cell apoptosisand senescence is potentially mediated by the p53 signalingpathwayrdquo Molecular Medicine Reports vol 9 no 4 pp 1237ndash1241 2014

[12] Y Wang X Wu and G Zhang ldquoExperimental study onanti-oxidative effect of Polygonatum Polysaccharides in ratsrdquoChinese Modern Doctor vol 49 no 6 2011

[13] Y LiWChengHDeng andP Zhang ldquoEffects of polygonatumpolysaccharide on telomerase activity in brain andgonadofApptransgenic micerdquoChinese NewDrugs and Clinical Remedies vol27 pp 844ndash846 2008

[14] D Antonov S Schliemann and P Elsner ldquoMethods for theassessment of barrier functionrdquo in Skin Barrier Function vol49 ofCurrent Problems in Dermatology pp 61ndash70 S Karger AG2016

[15] S Taylor W Westerhof S Im and J Lim ldquoNoninvasivetechniques for the evaluation of skin colorrdquo Journal of theAmerican Academy of Dermatology vol 54 no 5 pp S282ndashS290 2006

[16] A Chardon I Cretois and C Hourseau ldquoSkin colour typologyand suntanning pathwaysrdquo International Journal of CosmeticScience vol 13 no 4 pp 191ndash208 1991

[17] C RThornfeldt and R L Rizer ldquoSuperior efficacy of an herbal-based cosmeceutical compared with common prescription andcosmetic antiaging therapiesrdquo Journal of Drugs in Dermatology(JDD) vol 15 no 2 pp 218ndash223 2016

[18] Z D Draelos ldquoCosmeceuticals Procedures in Cosmetic Der-matology Seriesrdquo Australas J Dermatol vol 46 p 287 2015

[19] A E Chiu J L Chan D G Kern S Kohler W E Rehmusand A B Kimball ldquoDouble-blinded placebo-controlled trial ofgreen tea extracts in the clinical and histologic appearance ofphotoaging skinrdquo Dermatologic Surgery vol 31 supplement 1pp 855ndash860 2005

[20] A Al-fawwaz M Abu-Al-Basal and H A Tawil ldquoEvaluatingthe Effect of Algal Extracts against Ultraviolet B-Induced SkinDamage in BALBcMicerdquo Journal of Environmental Science andEngineering B vol 4 no 11 pp 583ndash590 2015

[21] A Kammeyer and R M Luiten ldquoOxidation events and skinagingrdquo Ageing Research Reviews vol 21 pp 16ndash29 2015

[22] T Jiang F Tian H-T Zheng et al ldquoNrf2 suppresses lupusnephritis through inhibition of oxidative injury and theNF-120581B-mediated inflammatory responserdquoKidney International vol 85no 2 pp 333ndash343 2014

[23] K Taniguchi and M Karin ldquoNF-120581B inflammation immunityand cancer coming of agerdquo Nature Reviews Immunology vol18 no 5 pp 309ndash324 2018

[24] J S Tilstra A R Robinson J Wang et al ldquoNF-120581B inhibitiondelays DNA damagemdashinduced senescence and aging in micerdquo13e Journal of Clinical Investigation vol 122 no 7 pp 2601ndash2612 2012

[25] C L Saw M-T Huang Y Liu T O Khor A H Conneyand A-N Kong ldquoImpact of Nrf2 on UVB-induced skininflammationphotoprotection and photoprotective effect ofsulforaphanerdquoMolecular Carcinogenesis vol 50 no 6 pp 479ndash486 2011

[26] Y Huang W Li Z-Y Su and A-N T Kong ldquoThe complexityof the Nrf2 pathway beyond the antioxidant responserdquo 13eJournal of Nutritional Biochemistry vol 26 no 12 pp 1401ndash14132015

[27] T Suzuki andMYamamoto ldquoMolecular basis of theKeap1-Nrf2systemrdquo Free Radical Biology amp Medicine B vol 88 pp 93ndash1002015

[28] E Kansanen S M Kuosmanen H Leinonen and A-LLevonenn ldquoTheKeap1-Nrf2 pathwaymechanisms of activationand dysregulation in cancerrdquoRedox Biology vol 1 no 1 pp 45ndash49 2013

[29] S Reuter S C Gupta M M Chaturvedi and B B AggarwalldquoOxidative stress inflammation and cancer how are theylinkedrdquo Free Radical Biology amp Medicine vol 49 no 11 pp1603ndash1616 2010

[30] G-H Liu J Qu and X Shen ldquoNF-120581Bp65 antagonizes Nrf2-ARE pathway by depriving CBP from Nrf2 and facilitatingrecruitment ofHDAC3 toMafKrdquoBiochimica et Biophysica Actavol 1783 no 5 pp 713ndash727 2008

[31] M P Seldon G Silva N Pejanovic et al ldquoHeme oxygenase-1 inhibits the expression of adhesion molecules associatedwith endothelial cell activation via inhibition of NF-120581B RelAphosphorylation at serine 276rdquo13e Journal of Immunology vol179 no 11 pp 7840ndash7851 2007

[32] M Ristow and S Schmeisser ldquoExtending life span by increasingoxidative stressrdquo Free Radical Biology amp Medicine vol 51 no 2pp 327ndash336 2011


[33] B Ou D Huang M Hampsch-Woodill and J A FlanaganldquoWhen east meets west The relationship between yin-yang andantioxidation-oxidationrdquo 13e FASEB Journal vol 17 no 2 pp127ndash129 2003

[34] Y-S Ho K-F So and R C-C Chang ldquoAnti-aging herbalmedicinemdashhow and why can they be used in aging-associatedneurodegenerative diseasesrdquo Ageing Research Reviews vol 9no 3 pp 354ndash362 2010

Stem Cells International

Hindawiwwwhindawicom Volume 2018


MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine


Behavioural Neurology

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom


roles in regulating cellular response to inflammation andoxidant stress Nrf2 is considered as an auxiliary protectivefactor in the regulation of antioxidation anti-inflammationand expressions of detoxification proteins [5] IKKNF-120581Binhibitors can decreaseDNAdamage that induces senescenceand aging [6]

People always prefer healthy and younger-looking skinwhich represents youth and beauty There are many anti-aging strategies including preventive measures cosmeticstrategies topical and systemic medications and invasiveprocedures (such as chemical peeling vitamin A fluorouracilointment laser intense pulsed light skin filling operationdermabrasion cryotherapy and photodynamic therapy) [78] Many antiaging skin care products have been developedin history and over the years advanced technologies andnew theories have greatly contributed to the convenient andeffective use of the skin care products Many studies haveshown that topical application of antiaging products canimprove the appearance of skin and topical application ofvitamins minerals and botanical ingredients can improvefine wrinkles freckles and pigmentation More and moreattention has been paid to botanical ingredients due to theirless toxicity and side effects [9]

Erjingwan (EJW) decoction is composed of Lyciumbarbarum and Polygonatum sibiricum which is recordedin General Medical Collection of Royal Benevolence EJWis a classic tonic formula which can maintain youth andprevent decrepitude It has been investigated that Lyciumbarbarumpolysaccharide candecreaseDNAdamage throughreducing oxidative stress [10] Moreover it can inhibit cellaging through the p53-mediated pathway [11] Polygona-tum sibiricum is usually applied for Qi- or Yin-deficiencysyndrome and it has kidney-tonifying ability Polygonatumsibiricum has been validated to have antioxidant effects andit can enhance telomerase activity [12 13] Therefore thecompatibility of these two medicinal herbs contributes to theantiaging process

Based on previous studies we hypothesized that EJWextracts could be used in antiaging products In the presentstudy we aimed to explore the mechanisms underlying theantiaging effects of EJW extracts and assess its antioxidantand anti-inflammatory abilities We first confirmed the effec-tiveness of EJW extracts Human foreskin fibroblasts (HFFs)were treated with H

2O2to induce the damage Then some

targets were determined to evaluate if EJW extracts couldreverse such H

2O2-induced damage The DPPH free radical

scavenging assay was conducted to validate the antioxidantability of EJW extracts Western blotting analysis was usedto examine the expressions of MMP1 and COL1A2 as well asNF-120581B and Nrf2 pathways

2 Materials and Methods

21 Clinical Trial

211 Preparation of EJWCream The samples were producedby LG Household amp Health Care Co including creamcontaining EJW extracts with traditional Chinese medicine

(TCM) nourishing Yin therapy (sample A) and cream with-out active ingredient (sample B)

Mix 100ml solvent (Water 88 Butylene Glycol 10 12-Hexanediol 2) with 10g Lycium Chinense Fruit Extract10g Polygonatum Sibiricum Extract 10g Poria Cocos Extract10g Polygonum multiflorum respectively Then extract themat normal temperature for 3 days and filter Then mix theextractions together and filer again

The components of the cream are listed in Table 5

212 Main Instruments Following instruments were usedin the present study including human skin rapid opticalimaging system PRIMOS Pico (GFMesstechnik GmbH Ger-many) stratum corneumwater component tester Corneome-ter (CourageampKhazakaGermany) epidermalwater loss ratetester Tewameter TM300 (Courage amp Khazaka Germany)CR-400 MINOLTA (KONICA Japan) skin gloss testerGL200 (Courage amp Khazaka Germany) skin elasticity testerDual Cutometer MPA580 (Courage amp Khazaka Germany)and balance sense quantity 001 g (METTLER Switzerland)

213 Participants The study was reviewed and approved bythe National Quality Supervision and Inspection Center forPerfume and Perfume Cosmetics Each participant providedwritten informed consent before any study-related examina-tion or procedure was performed and the study adhered tothe tenets of the Declaration of Helsinki The participantswere selected according to inclusion and exclusion criteriaThe participants were required to voluntarily participate inthe test and sign the informed consent be healthy Chinesewomen be between 30 to 45 years old have obvious wrinklesor fine lines in the eye test area (grade 2-3 of crows feet)keep the skin of arms dry (the base value of skin moisturein the forearm test area was between 15-45 au) and refrainto use any cosmetics on the face from the night before thetest On the day of the test participants should clean theface and forearms before the test have no severe systemicdiseases no immunodeficiency or autoimmune diseases andreceive no skin cosmetology treatment and other tests benot highly sensitive to body constitution not sensitive tocommon cosmetics and not suffering from pollen allergy orother allergic reactions ensure that no steroid drugs andimmunosuppressive agents were used in the last monthensure not to participate in other clinical studies in the last 3months agree to use photos of local skin and self-assessmentresults in the data have no plan for outdoor activities ortravel to the south and have no excessive sun exposureduring the test period ensure that the facial products ofthe same function other than the test products were notused during the test ensure that the face was not treatedby antiaging whitening grinding or other cosmetic projectsduring the test period and ensure that no drugs of antiagingand whitening effects were administered orally well use theproducts understand and fill in the project questionnaire andcooperate with the staffs and have the chance to participatein further tests in the future The exclusion criteria were asfollows people with allergic dermatitis or having a history ofskin diseases (severe freckles atopic skin diseases psoriasis










22 In Vitro Study


2)









































3 Results







Week

Moi

sture

(au

)

10

20

30

40

50




20 420 4

(a)

Week

Moi

sture

(au

)

5

10

15

20

_

_

2 4


(b)

Week

TEW

L (g

hm

2)

5

10

15

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

20 420 4


(c)

Week

TEW

L (g

hm

2)

2 4

minus8

minus6

minus4

minus2

_

_


(d)

Week

Skin

glo

ssin

ess (

au)

5

6

7

8

9

_

_lowastlowast

lowastlowastlowast

20 420 4


(e)

Week

Skin

glo

ssin

ess (

au)

minus1

0

1

2

3


2 4


(f)





Week

25

30

35

40

Test area

Control area

0 2 40 2 4

Skin

brig

htne

ss p

aram

eter

IT

∘


lowastlowastlowast

(a)

Week

minus3

0

3

6

_ _

Skin

brig

htne

ss p

aram

eter

IT

∘

2 4

Test area

Control area(b)

Week

Skin

brig

htne

ss p

aram

eter

s Llowast

55

60

65

Test area

Control area

0 2 40 2 4


lowast

(c)

Week

minus3

0

3

Skin

brig

htne

ss p

aram

eter

s Llowast

__

Test area

Control area

2 4

(d)














Week

Skin

com

pact

ness

par

amet

er R

1 va

lue

005

010

015

020

__

Test area

Control area

0 2 40 2 4

lowastlowastlowast

lowastlowast

(a)

Week

Skin

com

pact

ness

par

amet

er R

1 va

lue

minus01

00

01

Test area

Control area

2 4

lowast

lowast

(b)

Week

Skin

elas

tic p

aram

eter

R2

valu

e

02

04

06

08

10_

_

Test area

Control area

0 2 40 2 4


(c)

Week

Skin

elas

tic p

aram

eter

R2

valu

e

minus04

minus02

00

02

04

Test area

Control area

2 4

lowastlowastlowast

lowastlowastlowast

(d)


0

02

04



EJW05 (sample)

EJW1 (sample)

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast



Cel

l via

bilit

y (

)

CON 3 h 4 h 6 h

20

40

60

80

100

120

CON3 h

4 h6 h

(a)

GAPDH

MMP1

COL1A2


(b)

Cel

l via

bilit

y (

)

24 h 48 h

40

80

120


CONMODEL10 gmL

100 gmL200 gmL

(c)




2O2for 15 min HFF










4 Discussion




Inhi

bitio

n ra

te (

)

20

40

60

80

IC50 0531mgmL

00625 0125 025 05 1

(a)

Concentration (gmL)

Inhi

bitio

n ra

te (

)

10 100

20

40

60

80

100

IC502427gmL

(b)





2O2is




2O2-







MMP1

COLA1

GAPDH

HFFCON Model EJW

002040608

11214

CON Model EJW

COL1

A2

prot

ein

leve

l(n

orm

aliz

ed C

ON

= 1

)

08509

0951

10511

115

CON Model EJW

MM

P1 p

rote

in le

vel

(nor

mal

ized

CO

N =

1)

(a)

002040608

11214

CON Model EJW

Relat

ive m

RNA

expr

essio

n

005

115

225

3

CON Model EJWRe

lativ

e mRN

Aex

pres

sion


(b)

HFF

MMP1

GAPDH

COLA1

CON Model EJW

002040608

11214

CON Model EJW

COL1

A2

prot

ein

leve

l(n

orm

aliz

ed C

ON

= 1

)

002040608

11214

CON Model EJW

MM

P1 p

rote

in le

vel

(nor

mal

ized

CO

N =

1)

lowastlowast

lowastlowastlowast

(c)







2O2-treated



0

05

1

15

2

25

3

35

CON Model EJW

Rela

tive m

RNA

expr

essio

n

lowastlowast

(a)

p-IB (ser32 ser36)

IB

p-NFB (ser536)

NFB

GAPDH

HO-1

p-Nrf2 (S40)

HFFCON Model EJW

(b)

0

02

04

06

08

1

12

CON Model EJW

p-N

rf2 (S

40) p

rote

in le

vel

(nor

mal

ized

CO

N =

1)

0

05

1

15

2

25

CON Model EJW

HO

-1 p

rote

in le

vel

(nor

mal

ized

CO

N =

1)

0

02

04

06

08

1

12

14

CON Model EJW

p-N

FkB

NFk

B pr

otei

n le

vel

(nor

mal

ized

CO

N =

1)

002040608

112141618

CON Model EJW

p-Ik

BaIk

Ba p

rote

in le

vel

(nor

mal

ized

CO

N =

1)

lowast


lowastlowast

(c)










5 Conclusions


Data Availability


Disclosure






Acknowledgments



References








































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




























22 In Vitro Study


2)









































3 Results







Week

Moi

sture

(au

)

10

20

30

40

50




20 420 4

(a)

Week

Moi

sture

(au

)

5

10

15

20

_

_

2 4


(b)

Week

TEW

L (g

hm

2)

5

10

15

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

20 420 4


(c)

Week

TEW

L (g

hm

2)

2 4

minus8

minus6

minus4

minus2

_

_


(d)

Week

Skin

glo

ssin

ess (

au)

5

6

7

8

9

_

_lowastlowast

lowastlowastlowast

20 420 4


(e)

Week

Skin

glo

ssin

ess (

au)

minus1

0

1

2

3


2 4


(f)





Week

25

30

35

40

Test area

Control area

0 2 40 2 4

Skin

brig

htne

ss p

aram

eter

IT

∘


lowastlowastlowast

(a)

Week

minus3

0

3

6

_ _

Skin

brig

htne

ss p

aram

eter

IT

∘

2 4

Test area

Control area(b)

Week

Skin

brig

htne

ss p

aram

eter

s Llowast

55

60

65

Test area

Control area

0 2 40 2 4


lowast

(c)

Week

minus3

0

3

Skin

brig

htne

ss p

aram

eter

s Llowast

__

Test area

Control area

2 4

(d)














Week

Skin

com

pact

ness

par

amet

er R

1 va

lue

005

010

015

020

__

Test area

Control area

0 2 40 2 4

lowastlowastlowast

lowastlowast

(a)

Week

Skin

com

pact

ness

par

amet

er R

1 va

lue

minus01

00

01

Test area

Control area

2 4

lowast

lowast

(b)

Week

Skin

elas

tic p

aram

eter

R2

valu

e

02

04

06

08

10_

_

Test area

Control area

0 2 40 2 4


(c)

Week

Skin

elas

tic p

aram

eter

R2

valu

e

minus04

minus02

00

02

04

Test area

Control area

2 4

lowastlowastlowast

lowastlowastlowast

(d)


0

02

04



EJW05 (sample)

EJW1 (sample)

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast



Cel

l via

bilit

y (

)

CON 3 h 4 h 6 h

20

40

60

80

100

120

CON3 h

4 h6 h

(a)

GAPDH

MMP1

COL1A2


(b)

Cel

l via

bilit

y (

)

24 h 48 h

40

80

120


CONMODEL10 gmL

100 gmL200 gmL

(c)




2O2for 15 min HFF










4 Discussion




Inhi

bitio

n ra

te (

)

20

40

60

80

IC50 0531mgmL

00625 0125 025 05 1

(a)

Concentration (gmL)

Inhi

bitio

n ra

te (

)

10 100

20

40

60

80

100

IC502427gmL

(b)





2O2is




2O2-







MMP1

COLA1

GAPDH

HFFCON Model EJW

002040608

11214

CON Model EJW

COL1

A2

prot

ein

leve

l(n

orm

aliz

ed C

ON

= 1

)

08509

0951

10511

115

CON Model EJW

MM

P1 p

rote

in le

vel

(nor

mal

ized

CO

N =

1)

(a)

002040608

11214

CON Model EJW

Relat

ive m

RNA

expr

essio

n

005

115

225

3

CON Model EJWRe

lativ

e mRN

Aex

pres

sion


(b)

HFF

MMP1

GAPDH

COLA1

CON Model EJW

002040608

11214

CON Model EJW

COL1

A2

prot

ein

leve

l(n

orm

aliz

ed C

ON

= 1

)

002040608

11214

CON Model EJW

MM

P1 p

rote

in le

vel

(nor

mal

ized

CO

N =

1)

lowastlowast

lowastlowastlowast

(c)







2O2-treated



0

05

1

15

2

25

3

35

CON Model EJW

Rela

tive m

RNA

expr

essio

n

lowastlowast

(a)

p-IB (ser32 ser36)

IB

p-NFB (ser536)

NFB

GAPDH

HO-1

p-Nrf2 (S40)

HFFCON Model EJW

(b)

0

02

04

06

08

1

12

CON Model EJW

p-N

rf2 (S

40) p

rote

in le

vel

(nor

mal

ized

CO

N =

1)

0

05

1

15

2

25

CON Model EJW

HO

-1 p

rote

in le

vel

(nor

mal

ized

CO

N =

1)

0

02

04

06

08

1

12

14

CON Model EJW

p-N

FkB

NFk

B pr

otei

n le

vel

(nor

mal

ized

CO

N =

1)

002040608

112141618

CON Model EJW

p-Ik

BaIk

Ba p

rote

in le

vel

(nor

mal

ized

CO

N =

1)

lowast


lowastlowast

(c)










5 Conclusions


Data Availability


Disclosure






Acknowledgments



References








































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of





















































3 Results







Week

Moi

sture

(au

)

10

20

30

40

50




20 420 4

(a)

Week

Moi

sture

(au

)

5

10

15

20

_

_

2 4


(b)

Week

TEW

L (g

hm

2)

5

10

15

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

20 420 4


(c)

Week

TEW

L (g

hm

2)

2 4

minus8

minus6

minus4

minus2

_

_


(d)

Week

Skin

glo

ssin

ess (

au)

5

6

7

8

9

_

_lowastlowast

lowastlowastlowast

20 420 4


(e)

Week

Skin

glo

ssin

ess (

au)

minus1

0

1

2

3


2 4


(f)





Week

25

30

35

40

Test area

Control area

0 2 40 2 4

Skin

brig

htne

ss p

aram

eter

IT

∘


lowastlowastlowast

(a)

Week

minus3

0

3

6

_ _

Skin

brig

htne

ss p

aram

eter

IT

∘

2 4

Test area

Control area(b)

Week

Skin

brig

htne

ss p

aram

eter

s Llowast

55

60

65

Test area

Control area

0 2 40 2 4


lowast

(c)

Week

minus3

0

3

Skin

brig

htne

ss p

aram

eter

s Llowast

__

Test area

Control area

2 4

(d)














Week

Skin

com

pact

ness

par

amet

er R

1 va

lue

005

010

015

020

__

Test area

Control area

0 2 40 2 4

lowastlowastlowast

lowastlowast

(a)

Week

Skin

com

pact

ness

par

amet

er R

1 va

lue

minus01

00

01

Test area

Control area

2 4

lowast

lowast

(b)

Week

Skin

elas

tic p

aram

eter

R2

valu

e

02

04

06

08

10_

_

Test area

Control area

0 2 40 2 4


(c)

Week

Skin

elas

tic p

aram

eter

R2

valu

e

minus04

minus02

00

02

04

Test area

Control area

2 4

lowastlowastlowast

lowastlowastlowast

(d)


0

02

04



EJW05 (sample)

EJW1 (sample)

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast



Cel

l via

bilit

y (

)

CON 3 h 4 h 6 h

20

40

60

80

100

120

CON3 h

4 h6 h

(a)

GAPDH

MMP1

COL1A2


(b)

Cel

l via

bilit

y (

)

24 h 48 h

40

80

120


CONMODEL10 gmL

100 gmL200 gmL

(c)




2O2for 15 min HFF










4 Discussion




Inhi

bitio

n ra

te (

)

20

40

60

80

IC50 0531mgmL

00625 0125 025 05 1

(a)

Concentration (gmL)

Inhi

bitio

n ra

te (

)

10 100

20

40

60

80

100

IC502427gmL

(b)





2O2is




2O2-







MMP1

COLA1

GAPDH

HFFCON Model EJW

002040608

11214

CON Model EJW

COL1

A2

prot

ein

leve

l(n

orm

aliz

ed C

ON

= 1

)

08509

0951

10511

115

CON Model EJW

MM

P1 p

rote

in le

vel

(nor

mal

ized

CO

N =

1)

(a)

002040608

11214

CON Model EJW

Relat

ive m

RNA

expr

essio

n

005

115

225

3

CON Model EJWRe

lativ

e mRN

Aex

pres

sion


(b)

HFF

MMP1

GAPDH

COLA1

CON Model EJW

002040608

11214

CON Model EJW

COL1

A2

prot

ein

leve

l(n

orm

aliz

ed C

ON

= 1

)

002040608

11214

CON Model EJW

MM

P1 p

rote

in le

vel

(nor

mal

ized

CO

N =

1)

lowastlowast

lowastlowastlowast

(c)







2O2-treated



0

05

1

15

2

25

3

35

CON Model EJW

Rela

tive m

RNA

expr

essio

n

lowastlowast

(a)

p-IB (ser32 ser36)

IB

p-NFB (ser536)

NFB

GAPDH

HO-1

p-Nrf2 (S40)

HFFCON Model EJW

(b)

0

02

04

06

08

1

12

CON Model EJW

p-N

rf2 (S

40) p

rote

in le

vel

(nor

mal

ized

CO

N =

1)

0

05

1

15

2

25

CON Model EJW

HO

-1 p

rote

in le

vel

(nor

mal

ized

CO

N =

1)

0

02

04

06

08

1

12

14

CON Model EJW

p-N

FkB

NFk

B pr

otei

n le

vel

(nor

mal

ized

CO

N =

1)

002040608

112141618

CON Model EJW

p-Ik

BaIk

Ba p

rote

in le

vel

(nor

mal

ized

CO

N =

1)

lowast


lowastlowast

(c)










5 Conclusions


Data Availability


Disclosure






Acknowledgments



References








































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















Week

Moi

sture

(au

)

10

20

30

40

50




20 420 4

(a)

Week

Moi

sture

(au

)

5

10

15

20

_

_

2 4


(b)

Week

TEW

L (g

hm

2)

5

10

15

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

20 420 4


(c)

Week

TEW

L (g

hm

2)

2 4

minus8

minus6

minus4

minus2

_

_


(d)

Week

Skin

glo

ssin

ess (

au)

5

6

7

8

9

_

_lowastlowast

lowastlowastlowast

20 420 4


(e)

Week

Skin

glo

ssin

ess (

au)

minus1

0

1

2

3


2 4


(f)





Week

25

30

35

40

Test area

Control area

0 2 40 2 4

Skin

brig

htne

ss p

aram

eter

IT

∘


lowastlowastlowast

(a)

Week

minus3

0

3

6

_ _

Skin

brig

htne

ss p

aram

eter

IT

∘

2 4

Test area

Control area(b)

Week

Skin

brig

htne

ss p

aram

eter

s Llowast

55

60

65

Test area

Control area

0 2 40 2 4


lowast

(c)

Week

minus3

0

3

Skin

brig

htne

ss p

aram

eter

s Llowast

__

Test area

Control area

2 4

(d)














Week

Skin

com

pact

ness

par

amet

er R

1 va

lue

005

010

015

020

__

Test area

Control area

0 2 40 2 4

lowastlowastlowast

lowastlowast

(a)

Week

Skin

com

pact

ness

par

amet

er R

1 va

lue

minus01

00

01

Test area

Control area

2 4

lowast

lowast

(b)

Week

Skin

elas

tic p

aram

eter

R2

valu

e

02

04

06

08

10_

_

Test area

Control area

0 2 40 2 4


(c)

Week

Skin

elas

tic p

aram

eter

R2

valu

e

minus04

minus02

00

02

04

Test area

Control area

2 4

lowastlowastlowast

lowastlowastlowast

(d)


0

02

04



EJW05 (sample)

EJW1 (sample)

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast



Cel

l via

bilit

y (

)

CON 3 h 4 h 6 h

20

40

60

80

100

120

CON3 h

4 h6 h

(a)

GAPDH

MMP1

COL1A2


(b)

Cel

l via

bilit

y (

)

24 h 48 h

40

80

120


CONMODEL10 gmL

100 gmL200 gmL

(c)




2O2for 15 min HFF










4 Discussion




Inhi

bitio

n ra

te (

)

20

40

60

80

IC50 0531mgmL

00625 0125 025 05 1

(a)

Concentration (gmL)

Inhi

bitio

n ra

te (

)

10 100

20

40

60

80

100

IC502427gmL

(b)





2O2is




2O2-







MMP1

COLA1

GAPDH

HFFCON Model EJW

002040608

11214

CON Model EJW

COL1

A2

prot

ein

leve

l(n

orm

aliz

ed C

ON

= 1

)

08509

0951

10511

115

CON Model EJW

MM

P1 p

rote

in le

vel

(nor

mal

ized

CO

N =

1)

(a)

002040608

11214

CON Model EJW

Relat

ive m

RNA

expr

essio

n

005

115

225

3

CON Model EJWRe

lativ

e mRN

Aex

pres

sion


(b)

HFF

MMP1

GAPDH

COLA1

CON Model EJW

002040608

11214

CON Model EJW

COL1

A2

prot

ein

leve

l(n

orm

aliz

ed C

ON

= 1

)

002040608

11214

CON Model EJW

MM

P1 p

rote

in le

vel

(nor

mal

ized

CO

N =

1)

lowastlowast

lowastlowastlowast

(c)







2O2-treated



0

05

1

15

2

25

3

35

CON Model EJW

Rela

tive m

RNA

expr

essio

n

lowastlowast

(a)

p-IB (ser32 ser36)

IB

p-NFB (ser536)

NFB

GAPDH

HO-1

p-Nrf2 (S40)

HFFCON Model EJW

(b)

0

02

04

06

08

1

12

CON Model EJW

p-N

rf2 (S

40) p

rote

in le

vel

(nor

mal

ized

CO

N =

1)

0

05

1

15

2

25

CON Model EJW

HO

-1 p

rote

in le

vel

(nor

mal

ized

CO

N =

1)

0

02

04

06

08

1

12

14

CON Model EJW

p-N

FkB

NFk

B pr

otei

n le

vel

(nor

mal

ized

CO

N =

1)

002040608

112141618

CON Model EJW

p-Ik

BaIk

Ba p

rote

in le

vel

(nor

mal

ized

CO

N =

1)

lowast


lowastlowast

(c)










5 Conclusions


Data Availability


Disclosure






Acknowledgments



References








































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















Week

25

30

35

40

Test area

Control area

0 2 40 2 4

Skin

brig

htne

ss p

aram

eter

IT

∘


lowastlowastlowast

(a)

Week

minus3

0

3

6

_ _

Skin

brig

htne

ss p

aram

eter

IT

∘

2 4

Test area

Control area(b)

Week

Skin

brig

htne

ss p

aram

eter

s Llowast

55

60

65

Test area

Control area

0 2 40 2 4


lowast

(c)

Week

minus3

0

3

Skin

brig

htne

ss p

aram

eter

s Llowast

__

Test area

Control area

2 4

(d)














Week

Skin

com

pact

ness

par

amet

er R

1 va

lue

005

010

015

020

__

Test area

Control area

0 2 40 2 4

lowastlowastlowast

lowastlowast

(a)

Week

Skin

com

pact

ness

par

amet

er R

1 va

lue

minus01

00

01

Test area

Control area

2 4

lowast

lowast

(b)

Week

Skin

elas

tic p

aram

eter

R2

valu

e

02

04

06

08

10_

_

Test area

Control area

0 2 40 2 4


(c)

Week

Skin

elas

tic p

aram

eter

R2

valu

e

minus04

minus02

00

02

04

Test area

Control area

2 4

lowastlowastlowast

lowastlowastlowast

(d)


0

02

04



EJW05 (sample)

EJW1 (sample)

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast



Cel

l via

bilit

y (

)

CON 3 h 4 h 6 h

20

40

60

80

100

120

CON3 h

4 h6 h

(a)

GAPDH

MMP1

COL1A2


(b)

Cel

l via

bilit

y (

)

24 h 48 h

40

80

120


CONMODEL10 gmL

100 gmL200 gmL

(c)




2O2for 15 min HFF










4 Discussion




Inhi

bitio

n ra

te (

)

20

40

60

80

IC50 0531mgmL

00625 0125 025 05 1

(a)

Concentration (gmL)

Inhi

bitio

n ra

te (

)

10 100

20

40

60

80

100

IC502427gmL

(b)





2O2is




2O2-







MMP1

COLA1

GAPDH

HFFCON Model EJW

002040608

11214

CON Model EJW

COL1

A2

prot

ein

leve

l(n

orm

aliz

ed C

ON

= 1

)

08509

0951

10511

115

CON Model EJW

MM

P1 p

rote

in le

vel

(nor

mal

ized

CO

N =

1)

(a)

002040608

11214

CON Model EJW

Relat

ive m

RNA

expr

essio

n

005

115

225

3

CON Model EJWRe

lativ

e mRN

Aex

pres

sion


(b)

HFF

MMP1

GAPDH

COLA1

CON Model EJW

002040608

11214

CON Model EJW

COL1

A2

prot

ein

leve

l(n

orm

aliz

ed C

ON

= 1

)

002040608

11214

CON Model EJW

MM

P1 p

rote

in le

vel

(nor

mal

ized

CO

N =

1)

lowastlowast

lowastlowastlowast

(c)







2O2-treated



0

05

1

15

2

25

3

35

CON Model EJW

Rela

tive m

RNA

expr

essio

n

lowastlowast

(a)

p-IB (ser32 ser36)

IB

p-NFB (ser536)

NFB

GAPDH

HO-1

p-Nrf2 (S40)

HFFCON Model EJW

(b)

0

02

04

06

08

1

12

CON Model EJW

p-N

rf2 (S

40) p

rote

in le

vel

(nor

mal

ized

CO

N =

1)

0

05

1

15

2

25

CON Model EJW

HO

-1 p

rote

in le

vel

(nor

mal

ized

CO

N =

1)

0

02

04

06

08

1

12

14

CON Model EJW

p-N

FkB

NFk

B pr

otei

n le

vel

(nor

mal

ized

CO

N =

1)

002040608

112141618

CON Model EJW

p-Ik

BaIk

Ba p

rote

in le

vel

(nor

mal

ized

CO

N =

1)

lowast


lowastlowast

(c)










5 Conclusions


Data Availability


Disclosure






Acknowledgments



References








































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















Week

Skin

com

pact

ness

par

amet

er R

1 va

lue

005

010

015

020

__

Test area

Control area

0 2 40 2 4

lowastlowastlowast

lowastlowast

(a)

Week

Skin

com

pact

ness

par

amet

er R

1 va

lue

minus01

00

01

Test area

Control area

2 4

lowast

lowast

(b)

Week

Skin

elas

tic p

aram

eter

R2

valu

e

02

04

06

08

10_

_

Test area

Control area

0 2 40 2 4


(c)

Week

Skin

elas

tic p

aram

eter

R2

valu

e

minus04

minus02

00

02

04

Test area

Control area

2 4

lowastlowastlowast

lowastlowastlowast

(d)


0

02

04



EJW05 (sample)

EJW1 (sample)

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast



Cel

l via

bilit

y (

)

CON 3 h 4 h 6 h

20

40

60

80

100

120

CON3 h

4 h6 h

(a)

GAPDH

MMP1

COL1A2


(b)

Cel

l via

bilit

y (

)

24 h 48 h

40

80

120


CONMODEL10 gmL

100 gmL200 gmL

(c)




2O2for 15 min HFF










4 Discussion




Inhi

bitio

n ra

te (

)

20

40

60

80

IC50 0531mgmL

00625 0125 025 05 1

(a)

Concentration (gmL)

Inhi

bitio

n ra

te (

)

10 100

20

40

60

80

100

IC502427gmL

(b)





2O2is




2O2-







MMP1

COLA1

GAPDH

HFFCON Model EJW

002040608

11214

CON Model EJW

COL1

A2

prot

ein

leve

l(n

orm

aliz

ed C

ON

= 1

)

08509

0951

10511

115

CON Model EJW

MM

P1 p

rote

in le

vel

(nor

mal

ized

CO

N =

1)

(a)

002040608

11214

CON Model EJW

Relat

ive m

RNA

expr

essio

n

005

115

225

3

CON Model EJWRe

lativ

e mRN

Aex

pres

sion


(b)

HFF

MMP1

GAPDH

COLA1

CON Model EJW

002040608

11214

CON Model EJW

COL1

A2

prot

ein

leve

l(n

orm

aliz

ed C

ON

= 1

)

002040608

11214

CON Model EJW

MM

P1 p

rote

in le

vel

(nor

mal

ized

CO

N =

1)

lowastlowast

lowastlowastlowast

(c)







2O2-treated



0

05

1

15

2

25

3

35

CON Model EJW

Rela

tive m

RNA

expr

essio

n

lowastlowast

(a)

p-IB (ser32 ser36)

IB

p-NFB (ser536)

NFB

GAPDH

HO-1

p-Nrf2 (S40)

HFFCON Model EJW

(b)

0

02

04

06

08

1

12

CON Model EJW

p-N

rf2 (S

40) p

rote

in le

vel

(nor

mal

ized

CO

N =

1)

0

05

1

15

2

25

CON Model EJW

HO

-1 p

rote

in le

vel

(nor

mal

ized

CO

N =

1)

0

02

04

06

08

1

12

14

CON Model EJW

p-N

FkB

NFk

B pr

otei

n le

vel

(nor

mal

ized

CO

N =

1)

002040608

112141618

CON Model EJW

p-Ik

BaIk

Ba p

rote

in le

vel

(nor

mal

ized

CO

N =

1)

lowast


lowastlowast

(c)










5 Conclusions


Data Availability


Disclosure






Acknowledgments



References








































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















Cel

l via

bilit

y (

)

CON 3 h 4 h 6 h

20

40

60

80

100

120

CON3 h

4 h6 h

(a)

GAPDH

MMP1

COL1A2


(b)

Cel

l via

bilit

y (

)

24 h 48 h

40

80

120


CONMODEL10 gmL

100 gmL200 gmL

(c)




2O2for 15 min HFF










4 Discussion




Inhi

bitio

n ra

te (

)

20

40

60

80

IC50 0531mgmL

00625 0125 025 05 1

(a)

Concentration (gmL)

Inhi

bitio

n ra

te (

)

10 100

20

40

60

80

100

IC502427gmL

(b)





2O2is




2O2-







MMP1

COLA1

GAPDH

HFFCON Model EJW

002040608

11214

CON Model EJW

COL1

A2

prot

ein

leve

l(n

orm

aliz

ed C

ON

= 1

)

08509

0951

10511

115

CON Model EJW

MM

P1 p

rote

in le

vel

(nor

mal

ized

CO

N =

1)

(a)

002040608

11214

CON Model EJW

Relat

ive m

RNA

expr

essio

n

005

115

225

3

CON Model EJWRe

lativ

e mRN

Aex

pres

sion


(b)

HFF

MMP1

GAPDH

COLA1

CON Model EJW

002040608

11214

CON Model EJW

COL1

A2

prot

ein

leve

l(n

orm

aliz

ed C

ON

= 1

)

002040608

11214

CON Model EJW

MM

P1 p

rote

in le

vel

(nor

mal

ized

CO

N =

1)

lowastlowast

lowastlowastlowast

(c)







2O2-treated



0

05

1

15

2

25

3

35

CON Model EJW

Rela

tive m

RNA

expr

essio

n

lowastlowast

(a)

p-IB (ser32 ser36)

IB

p-NFB (ser536)

NFB

GAPDH

HO-1

p-Nrf2 (S40)

HFFCON Model EJW

(b)

0

02

04

06

08

1

12

CON Model EJW

p-N

rf2 (S

40) p

rote

in le

vel

(nor

mal

ized

CO

N =

1)

0

05

1

15

2

25

CON Model EJW

HO

-1 p

rote

in le

vel

(nor

mal

ized

CO

N =

1)

0

02

04

06

08

1

12

14

CON Model EJW

p-N

FkB

NFk

B pr

otei

n le

vel

(nor

mal

ized

CO

N =

1)

002040608

112141618

CON Model EJW

p-Ik

BaIk

Ba p

rote

in le

vel

(nor

mal

ized

CO

N =

1)

lowast


lowastlowast

(c)










5 Conclusions


Data Availability


Disclosure






Acknowledgments



References








































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of





















Inhi

bitio

n ra

te (

)

20

40

60

80

IC50 0531mgmL

00625 0125 025 05 1

(a)

Concentration (gmL)

Inhi

bitio

n ra

te (

)

10 100

20

40

60

80

100

IC502427gmL

(b)





2O2is




2O2-







MMP1

COLA1

GAPDH

HFFCON Model EJW

002040608

11214

CON Model EJW

COL1

A2

prot

ein

leve

l(n

orm

aliz

ed C

ON

= 1

)

08509

0951

10511

115

CON Model EJW

MM

P1 p

rote

in le

vel

(nor

mal

ized

CO

N =

1)

(a)

002040608

11214

CON Model EJW

Relat

ive m

RNA

expr

essio

n

005

115

225

3

CON Model EJWRe

lativ

e mRN

Aex

pres

sion


(b)

HFF

MMP1

GAPDH

COLA1

CON Model EJW

002040608

11214

CON Model EJW

COL1

A2

prot

ein

leve

l(n

orm

aliz

ed C

ON

= 1

)

002040608

11214

CON Model EJW

MM

P1 p

rote

in le

vel

(nor

mal

ized

CO

N =

1)

lowastlowast

lowastlowastlowast

(c)







2O2-treated



0

05

1

15

2

25

3

35

CON Model EJW

Rela

tive m

RNA

expr

essio

n

lowastlowast

(a)

p-IB (ser32 ser36)

IB

p-NFB (ser536)

NFB

GAPDH

HO-1

p-Nrf2 (S40)

HFFCON Model EJW

(b)

0

02

04

06

08

1

12

CON Model EJW

p-N

rf2 (S

40) p

rote

in le

vel

(nor

mal

ized

CO

N =

1)

0

05

1

15

2

25

CON Model EJW

HO

-1 p

rote

in le

vel

(nor

mal

ized

CO

N =

1)

0

02

04

06

08

1

12

14

CON Model EJW

p-N

FkB

NFk

B pr

otei

n le

vel

(nor

mal

ized

CO

N =

1)

002040608

112141618

CON Model EJW

p-Ik

BaIk

Ba p

rote

in le

vel

(nor

mal

ized

CO

N =

1)

lowast


lowastlowast

(c)










5 Conclusions


Data Availability


Disclosure






Acknowledgments



References








































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















MMP1

COLA1

GAPDH

HFFCON Model EJW

002040608

11214

CON Model EJW

COL1

A2

prot

ein

leve

l(n

orm

aliz

ed C

ON

= 1

)

08509

0951

10511

115

CON Model EJW

MM

P1 p

rote

in le

vel

(nor

mal

ized

CO

N =

1)

(a)

002040608

11214

CON Model EJW

Relat

ive m

RNA

expr

essio

n

005

115

225

3

CON Model EJWRe

lativ

e mRN

Aex

pres

sion


(b)

HFF

MMP1

GAPDH

COLA1

CON Model EJW

002040608

11214

CON Model EJW

COL1

A2

prot

ein

leve

l(n

orm

aliz

ed C

ON

= 1

)

002040608

11214

CON Model EJW

MM

P1 p

rote

in le

vel

(nor

mal

ized

CO

N =

1)

lowastlowast

lowastlowastlowast

(c)







2O2-treated



0

05

1

15

2

25

3

35

CON Model EJW

Rela

tive m

RNA

expr

essio

n

lowastlowast

(a)

p-IB (ser32 ser36)

IB

p-NFB (ser536)

NFB

GAPDH

HO-1

p-Nrf2 (S40)

HFFCON Model EJW

(b)

0

02

04

06

08

1

12

CON Model EJW

p-N

rf2 (S

40) p

rote

in le

vel

(nor

mal

ized

CO

N =

1)

0

05

1

15

2

25

CON Model EJW

HO

-1 p

rote

in le

vel

(nor

mal

ized

CO

N =

1)

0

02

04

06

08

1

12

14

CON Model EJW

p-N

FkB

NFk

B pr

otei

n le

vel

(nor

mal

ized

CO

N =

1)

002040608

112141618

CON Model EJW

p-Ik

BaIk

Ba p

rote

in le

vel

(nor

mal

ized

CO

N =

1)

lowast


lowastlowast

(c)










5 Conclusions


Data Availability


Disclosure






Acknowledgments



References








































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















0

05

1

15

2

25

3

35

CON Model EJW

Rela

tive m

RNA

expr

essio

n

lowastlowast

(a)

p-IB (ser32 ser36)

IB

p-NFB (ser536)

NFB

GAPDH

HO-1

p-Nrf2 (S40)

HFFCON Model EJW

(b)

0

02

04

06

08

1

12

CON Model EJW

p-N

rf2 (S

40) p

rote

in le

vel

(nor

mal

ized

CO

N =

1)

0

05

1

15

2

25

CON Model EJW

HO

-1 p

rote

in le

vel

(nor

mal

ized

CO

N =

1)

0

02

04

06

08

1

12

14

CON Model EJW

p-N

FkB

NFk

B pr

otei

n le

vel

(nor

mal

ized

CO

N =

1)

002040608

112141618

CON Model EJW

p-Ik

BaIk

Ba p

rote

in le

vel

(nor

mal

ized

CO

N =

1)

lowast


lowastlowast

(c)










5 Conclusions


Data Availability


Disclosure






Acknowledgments



References








































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of

























5 Conclusions


Data Availability


Disclosure






Acknowledgments



References








































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















References








































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of


























of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



















erjingwan extracts exert antiaging effects of skin through...

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