Research Article Djulis ( Chenopodium formosanum Koidz...

Research ArticleDjulis (Chenopodium formosanum Koidz)Water Extract and Its Bioactive Components Ameliorate DermalDamage in UVB-Irradiated Skin Models

Yong-Han Hong1 Ya-Ling Huang2 Yao-Cheng Liu2 and Pi-Jen Tsai3

1Department of Nutrition I-Shou University Kaohsiung Taiwan2Department of Seafood Science National Kaohsiung Marine University Kaohsiung Taiwan3Department of Food Science National Pingtung University of Technology and Science Pingtung Taiwan

Correspondence should be addressed to Yong-Han Hong yonghanisuedutw

Received 12 July 2016 Revised 25 September 2016 Accepted 10 October 2016

Academic Editor Chia-Chien Hsieh

Copyright copy 2016 Yong-Han Hong et alThis is an open access article distributed under theCreativeCommonsAttributionLicensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Dermal photoaging is a condition of skin suffering inappropriate ultraviolet (UV) exposure and exerts inflammation tissuealterations redness swelling and uncomfortable feelings Djulis (Chenopodium formosanum Koidz) is a cereal food and itsantioxidant and pigment constituents may provide skin protection from photoaging but it still lacks proved experiments Inthis study protective effects of djulis extract (CFE) on UVB-irradiated skin were explored The results showed that HaCaT cellswith 150 120583gmL CFE treatment had higher survival and less production of interleukin- (IL-) 6 matrix metalloprotease- (MMP-) 1and reactive oxygen species (ROS) in UVB-irradiated conditions Subsequently in animal studies mice supplemented with CFE(100mgkg BW)were under UVB irradiation and had thinner epidermis and lower IL-6 levels in skin layerThese data demonstratethat bioactive compounds possessing the potency of antiphotoaging exist in CFE Following that we found rutin and chlorogenicacid (10ndash100120583M) could significantly increase cell viability and decrease the production of IL-6 in UVBmodels Additionally djulispigment-betanin has no effect of increasing cell viability in this studyOur findings suggest CFE can protect skin againstUV-induceddamage and this protection is mainly from contributions of rutin and chlorogenic acid

1 Introduction

Djulis (Chenopodium formosanum Koidz) is a native cerealplant in Taiwan and its nutritional values have been doc-umented with high levels of starch dietary fiber proteinsand grain-limited essential amino acids (eg lysine) Thisgets djulis viewed as a whole-nutritious grain food especiallyvaluable for vegetarians Apart from grain food use djulisalso gets more and more attention on the use of health foodand skincare based on antioxidant and pigment-containingproperties in recent years [1] However that still needs moreexperiments to clarify functional bioactivities and compo-nents of djulis

For now it has been known that exposure of the skinto solar UV radiation can result in reactive oxygen species-(ROS-) related oxidative stress DNA damage inflammatory

responses dysregulation of immune function and even theinitiation of carcinogenesis [2] InUV lights UVB is themaincaused factor of skin photodamage also called photoagingdue to its strong energy and permeability into skin layersWhile concerning molecular mechanisms ROS-mediatedmitogen-activated protein kinase (MAPK) signaling path-way is involved and related expressions of inflammatorymediators and matrix metalloproteinases (MMPs) are oftenhighlighted in the process In these mediators interleukin-(IL-) 6 TNF-120572 and TGF1205731 play crucial roles in skin abnor-malities including infiltration of inflammatory cells rednessswelling and uncomfortable feelings [3 4] As for MMPsmolecules they act as proteolytic substances which cancleavage dermal extracellular matrix such as fibrillar collagenand elastin thus resulting in the damage of skin integrity[5] It would be seen from this that ROS inflammatory

Hindawi Publishing CorporationBioMed Research InternationalVolume 2016 Article ID 7368797 8 pageshttpdxdoiorg10115520167368797

2 BioMed Research International

mediators andMMPs could be indicators to test whether thesample possesses the ability of skin protection For instancethrough reducing the production of inflammatory cytokinesphytochemicals from Scutellaria baicalensis and the seedsof quinoa (Chenopodium quinoa Willd) have been provedto have protective functions on skin in UVB models [6 7]In a classification of plant taxonomy djulis has the samegenus as quinoa Although previous studies have pointed outbioactive compounds including betanins epigallocatechinsferulic acids and certain flavonoids in djulis and proposedbeneficial functions [1 8] the skincare potency of djulis hasnever been tested under UVB models

The aim of this study is to examine protective effects ofdjulis water extracts on skin photoaging in in vitro and invivo UVB-irradiated models and confirm which bioactivecompounds are main contributors

2 Materials and Methods

21 Sample Preparation and Constituents Measurement Thedjulis (Chenopodium formosanum Koidz) seeds were har-vested from the National Pingtung University of Science andTechnology The extracts were prepared by soaking 1 g ofseeds grains in 5mL of distilled water in the dark at 4∘C for24 h After filtration the filtrate was then freeze-dried as sam-ples (the water extract of Chenopodium formosanum KoidzCFE) for further analysis The dry weight of this extractwas 32 g and the yield was 106 It was stored at minus20∘Cuntil using it To analyze components of CFE total pheno-lic contentwasmeasured spectrophotometrically by using themodified Folin-Ciocalteumethod and the samples were sub-jected to HPLC to identify betanin and phenolic compoundsaccording to the method described by the previous study [1]

22 Cell Culture The keratinocyte cells from human skin(HaCaT) were grown in Dulbeccorsquos modified Eagle medium(DMEM) supplemented with 10 (vv) heat-inactivated fetalbovine serum penicillin (100 unitsmL) and streptomycin(100 unitsmL) These culture reagents were commercialproducts (Gibco Grand Island NY USA) The cells wereincubated in an atmosphere of 5 CO2 at 37

∘C and wereregularly subcultured for experiment uses In samples testcells were seeded in a 96-well plate at a concentrationof 1 times 105 cellsmL Following one-day incubation cellsmedia were removed and replaced by phosphate buffersaline (PBS) for UV irradiation The plate was placed in anUV CROSSLINKER (XLE-1000B SPECTROLINE) whichset peak spectral emission electric tension frequency andthe distance from cells at 315 nm 120V 60Hz and 15 cmrespectively When cells were exposed to 30mJcm2UVBtime of irradiation was averagely 30 sec Then these cellswere treated with media containing various concentrationsof CFE pure compounds fromCFE or ascorbic acid for 24 hAfter that cell supernatants were collected for the analysisof inflammation-related mediators and cells in the bottom ofplate were for cell viability

23 Cell Viability The cell viability was assayed by MTT(3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide)

method After collecting cell supernatants an aliquot of55 120583Lwell of DMEM containing 05mgmLMTT was addedinto the plate for 3 h incubation Then the plate wasadded with an aliquot of 100 120583Lwell of isopropanol contain-ing 004NHCl and appropriately shaken for dissolving outcolored crystals Cell viability values at absorbance of 570 nmwere determined by an ELISA reader (Bio-Rad LaboratoriesHercules CA USA) In this study to confirm the data of cellviability we counted cell number by trypan blue stainingBriefly we seeded cells onto 6 cm dish (1 times 106 cells) for24 h incubation After washing twice by PBS cells were irra-diated with UVB at dose of 30mJcm2 Then these cells weretreated with CFE for 24 h Following that cells were har-vested for counting cell number

24 IL-6 and MMP-1 Measurements Production of IL-6(eBioscience Minneapolis MN USA) and MMP-1 (RampDMinneapolis MN USA) in cell supernatants was assayedusing commercial ELISA kits Briefly primary anti-IL-6 orMMP-1 antibodies were coated onto 96-well plates Afterovernight incubation plate wells were washed with washingbuffer and blocked with blocking solution for 1 h Afterwashingwells diluted supernatants or standard reagentswereadded into wells and the plate was incubated for 2 h Nextwells were washed and then added with biotin-conjugatedanti-IL-6 or MMP-1 antibodies for 1 h Following the washthe wells were added with horseradish peroxidase- (HRP-)conjugated streptavidin for 30min washed and incubatedwith tetramethylbenzidine Absorbance was measured by anELISA reader at 620 nm Data were calculated according tostandard curves

25 Intracellular Reactive Oxygen Species (ROS) Measure-ment Detecting levels of dichlorofluorescein (DCF) which isderived fromdichlorodihydrofluorescein-diacetate (DCFDA)under oxidative stress can represent the production of ROSIn this experiment cells were seeded at a concentration of 1times 106 cells in a 6 cm dish for 24 h and irradiated with UVB(30mJcm2) then they were treated with CFE or positivecontrols for 3 hNext cellswere collected in a tube and labeledwith 5 120583M DCFDA (Invitrogen Carlsbad CA USA) for30min After that cells were centrifuged and washed twicewith phosphate buffer saline (PBS) and intracellular DCFlevels were detected by flow cytometer (Accuri CytometersInc Ann Arbor MI USA)

26 Cell Cycle Analysis Cell cycle analysis was performed todetermine the proportion of apoptotic sub-G1 hypodiploidcells In brief cells were seeded at a concentration of 1 times106 cells in a 6 cm dish for 24 h Then cells were irradiatedwith UVB (30mJcm2) and treated with CFE or positivecontrols for 24 h Afterwards cells were harvested and fixedin 70 ethanol for 24 h at minus20∘C then they were washedtwice with PBS and introduced with PBS containing PIsolution at 4∘C Following overnight incubation cellularfluorescein levels were assayed by flow cytometer In the assayeach cycle phase was quantified from histograms by usingsoftware programs and changes of values in cycle phases werecalculated to evaluate CFErsquos effects

BioMed Research International 3

Table 1 The effect of CFE treatment on cell cycle in UVB-irradiated HaCaT cells

UVB 30mJcm2 of cellsSub-G1 G0G1 S G2M

Control minus 37 plusmn 18 320 plusmn 108 158 plusmn 53 288 plusmn 60

Vehicle + 297 plusmn 70 281 plusmn 55 155 plusmn 47 143 plusmn 52

CFE (150120583gmL) + 136 plusmn 59lowastlowast 313 plusmn 24 143 plusmn 26 229 plusmn 31lowastlowast

AA (20 120583M) + 55 plusmn 07lowastlowast 430 plusmn 55lowastlowast 226 plusmn 42lowast 207 plusmn 36lowast

Each value represents the mean plusmn SD of three independent experiments Compared with the vehicle the statistical difference was analyzed by Studentrsquos t-testand indicated a significance as lowast119901 lt 005 and lowastlowast119901 lt 001 Compared with the control the statistical difference was analyzed and indicated a significance as119901 lt 005

27 Animals Study Eight-week-old female BALBc micewere purchased from the National Animal Center (TaipeiTaiwan) Mice were maintained in an air-conditioned roomat 23 plusmn 2∘C on a regulated 12 h light-dark cycle and fed anonpurified diet (Lab Rodent Chow 5001 Ralston PurinaInc St Louis MO USA) for adaptation Animal care andhandling are conformed to the National Institutes of HealthrsquosGuide for the Care and Use of Laboratory Animals Inoral experiment mice at age of 10 wk were divided intofive groups control vehicle LCFE (40mgkg BW) HCFE(100mgkg BW) and AA (ascorbic acid 25mgkg BW) andeach group had 8ndash10 mice Water or samples were orallyadministered to these mice for 14 consecutive days Onday 8 all mice dorsal skin hairs were removed by medicalshaver and they received UVB irradiation (150mJcm2) for7 consecutive days except for the control mice Mice skinthickness was daily measured once by using a Quick Minicaliper (Verniercaliper number 1202-100 Beijing C amp CTrad Beijing China) until mice sacrifice Following thesacrifice mice dorsal and ear skin tissues were collectedfor histology observation In topical experiment forty micewere averagely assigned into four groups control (emulsionreagent) vehicle (emulsion reagent) CFE (8mgmouse inemulsion reagent) and AA (0392mgmouse in emulsionreagent) After all mice dorsal skin hairs were removed micereceived 150mJcm2 UVB irradiation for seven consecutivedays The control mice did not receive UVB irradiation asshame mice In each day mice received topical applicationwith respective samples at volume of 200120583L after UVBirradiationThen mice skin thicknesses were daily measureduntil mice sacrifice After sacrifice mice dorsal and ear skintissues were collected for epidermis histology observation

28 Tissue Histology Skin specimens were fixed in 10 buff-ered formalin Tissue sections measuring 3 120583m were createdfrom the paraffinized blocks and stained with hematoxylinand eosin (TA01HE kit BioTnA Inc Kaohsiung Taiwan)For further immunohistochemistry the paraffin sectionswere deparaffinized in xylene and rehydrated in a gradedalcohol series treated with 3 H2O2 for 10min and boiledwith the citrate buffer (pH 6) for 40min then blockingwith the immunoblock for 1 h at room temperature Thesections were incubated with rabbit anti-IL-6 antibody (bs-0379R Bioss IncWoburnMA USA) or rabbit anti-collagenI antibody (ab34710 Abcam Inc Cambridge MA USA) for2 h and then treated with mouserabbit probe HRP labeling

(BioTnA TAHC03D) for 30min at room temperature Per-oxidase activity was developed in diaminobenzidine-H2O2solution (BioTnA TAHC03D) for 5min at room temperatureand counterstain with hematoxylin (BioTnA TA01NB)

29 Statistical Analysis All data are presented as meansplusmn SD or SEM of at least three independent experimentsSignificant differences among the groups were determined byusing unpaired Studentrsquos t-test and Duncanrsquos multiple rangetests During UV irradiation period skin thicknesses amonggroups were statistically analyzed and compared by usingthe linear mixed model (LMM) 119901 value less than 005 wasconsidered statistically significant

3 Results and Discussions

31 CFE Protects from UVB-Induced Cell Deaths in Cell Mod-els This study used keratinocyte cell line exposed to UVBradiation to mimic the model of skin photoaging As cellswere under UVB irradiation at doses of 0ndash100mJcm2 wefoundUVB at dose of 20ndash30mJcm2 caused cells mortality of40ndash50 Previous studies have indicated that UVB at dose of20ndash40mJcm2 is close to the average minimal erythema dose(MED) for human populations [9 10] Thus UVB at dose of30mJcm2 was conducted in the following experiments InFigure 1(a) CFE at concentrations of 10ndash150 120583gmL did notcause HaCaT cells deaths (without UVB irradiation) Whilecells were under UVB exposure their viability decreasedby 60 however the decrease could be reversed by CFEtreatments (Figure 1(a) right) We confirmed the effect ofCFE on cell viability by using trypan blue staining The datashowed that CFE (150120583gmL) could have higher cell numbercompared to the vehicle in an UVB-irradiated model (CFE084 plusmn 009 times 106 cells versus vehicle 065 plusmn 010 times 106 cells)In addition the observation in Table 1 that 30 of UVB-irradiated cells (vehicle) were arrested in Sub-G1 phase (aDNA content less than 2 n) was given whereas cells with CFEtreatment at concentration of 150 120583gmL only has 135plusmn59arrest in Sub-G1 phase and higher S + G2M phase It isalso known that UVB-induced cell death is mainly throughapoptosis [11] Further it is essential to confirm if CFE mightalter DNA intactness and cause precancerous DNA-damagedcells to survive We detected the degree of DNA methylationby a commercial kit (ab117128 Abcam) as the assay ofDNA protection The data show there was no change ofpercentage of 5-methylcytosine (5-mC) in DNA in cells with


wo UVB UVB

Cel

l via

bilit

y (

of t

he co

ntro

l)

0

20

40

60

80

100

120

140

Control (0)Vehicle (0)

lowastlowast

10120583gmL CFE

50120583gmL CFE100120583gmL CFE150120583gmL CFE

(a)

Control Vehicle CFE AA

ROS

leve

ls (

of t

he co

ntro

l)

0

20

40

60

80

100

120

140

160

180

lowast

lowastlowast

(150120583gmL)

(b)

Control Vehicle 10 50 100 150 AA0

20

40

60

80

100

120

(120583gmL)

IL-6

prod

uctio

n (p

g104

cells

)

lowast

lowast lowast

(c)


20

40

60

80

100

120

(120583gmL)

MM

P-1

prod

uctio

n (p

g104

cells

)

lowastlowast

(d)

Figure 1 Effects of CFE treatments on cell viability (a) and the production of ROS (b) IL-6 (c) and MMP-1 (d) in UVB-irradiated HaCaTcells Ascorbic acid (AA) at concentration of 50120583Mwas as a positive control Comparedwith the vehicle the statistical differencewas analyzedby Studentrsquos t-test and indicated a significance as lowast119901 lt 005 or lowastlowast119901 lt 001 Compared with the control the statistical difference was analyzedindicated a significance as 119901 lt 005

UVB irradiation (30mJcm2) compared to the control (187versus 171) Meanwhile CFE had comparable percentageof 5mC in cells (179) showing CFE did not affect DNAintactness (unpublished data) The data indicate that CFEmight attenuate cell apoptosis and subsequently augment cellviability in UVB-irradiated keratinocytes

32 CFE Reverses UVB-Induced Cellular Mediators in CellModels Oxidative stress and mediators derived from UVBirradiation are the main cause of dermal harmful effectsincluding inflammation structural alteration and even morecells deaths Therefore we assayed the production of reactiveoxygen species (ROS) IL-6 and MMP-1 As shown inFigure 1(b) HaCaT cells under UVB radiation (vehicle)would have 16-fold ROS production compared to the controlwhereas CFE treatment at concentration of 150 120583gmL made

UVB-irradiated cells release less ROS production that iscomparable to that of the control It is well known thatUV radiation causes ROS-mediated oxidative stress whichtriggers cellular transcriptional activations including NF-120581BAP-1 and MAPK induction or STAT3 tyrosine phosphory-lation increasing the production of inflammatory mediatorsand progressive cell death [12 13] Accordingly scavengingROS is expected to regulate skin photodamage Our previousstudies show CFE is a strong antioxidant to remove freeradicals [1 14] hence we suggest that CFErsquos potency resultsfrom its antioxidant

For the change of cellular response UVB-induced pro-duction of IL-6 and MMP-1 was significantly reduced byCFE treatments dosedependently (Figures 2(c) and 2(d)) Inthis experiment 50 120583M ascorbic acid (AA) was used as thepositive control to confirm our data and AA did have the


0 1 2 3 4 5 6 7

Skin

thic

knes

s (120583

m)

0

200

400

600

800

1000

ControlVehicle AALCFE (40mgkg BW)

HCFE (100mgkg BW)

(a)

Vehicle LCFE HCFE AA

Epid

erm

is th

ickn

ess (

120583m

)

0

5

10

15

20

25

30

35

Control

lowast

lowastlowast

(b)

Duration (day)0 1 2 3 4 5 6 7

Skin

thic

knes

s (120583

m)

0

200

400

600

800

1000

ControlVehicle AA

CFE (8mgmouse)

(c)

Vehicle CFE AA

Epid

erm

is th

ickn

ess (

120583m

)

0

5

10

15

20

25

30

35

Control

lowast

(d)

Figure 2 Effects of CFE treatments on skin (a c) and epidermis (b d) thickness in UVB-irradiated mice (a) (b) and (c) (d) show the dataof oral and topical application experiments respectively Bar values are means plusmn SEM 119899 = 8ndash10 Compared with the vehicle the statisticaldifference was analyzed by Studentrsquos t-test and indicated a significance as lowast119901 lt 005 or lowastlowast119901 lt 001 Compared with the control the statisticaldifference was analyzed and indicated a significance as 119901 lt 005

dermal protectionThe same tendency is adherent to a Boyceet alrsquos study [15]

33 CFE Improves UVB-Induced Skin Alterations in AnimalModels Subsequently we conducted animal experiments toprove in vivo effects of CFE In Figure 2(a) the vehicle (onlyUVB-irradiated) micersquos skin thickness increased day by day(119901 lt 0001) and had a closely 700 120583m increase at day 3compared to the control But this increase was significantlyattenuated by CFE supplement at doses of 40 and 100mgkg BW For the measurement of epidermis changes collected

mice dorsal skins were stained with HampE The data inFigure 2(b) revealed that mean epidermis thickness in CFEsupplementmicewas significantly less than that in the vehiclemice Furthermore we explored whether skin protectioncould be displayed via topical application of CFE As shownin Figure 2(c) emulsion CFE could reduce the increased skinthickness compared to other groups during UVB radiationperiod (all 119901 values lt 005 by LMM model) The same eventwas represented in tissues staining (Figure 2(d)) and CFEtreatment (101 plusmn 096 120583m) had less epidermis thickness thanthe vehiclersquos (266 plusmn 402 120583m) This data shows that CFE


Con

trol

DAPI IL-6 Merge

Vehi

cleH

CFE

LCFE

AA

Figure 3 Effects of CFE treatments on skin IL-6 production in UVB-irradiated mice In experiments mice skin biopsies were processed forstaining A fluorescent microscopic image (100x) showing IL-6 stained mouse skin cells in green and 410158406-diamidino-2-phenylindole (DAPI)stained all nuclei in blue (100x)

intervention by oral or skin administration has the beneficialprotection fromUVB-induced skin alterations To investigatethe modulation of cellular mediators by CFE treatment inanimal models we used IHC assay to detect the expressionsof IL-6 and collagen in skin As shown in Figure 3 comparedto the vehicle group dermal IL-6 expression in the HCFEgroup was significantly decreased Dermal collagen type Iexpressions in CFE groups were slightly higher than that inthe vehicle group (data not shown)

When the skin is under UVB radiation proinflamma-tory cytokines would be strongly produced to cause cuta-neous inflammation and their outcomes including erythemaedema and epidermal hyperplasia are often recognized asmarkers of skin damage [16 17] In our test UVB did inducesignificantly the production of IL-6 that is close to 150ndash200 pg104 cells in cell culture tests and the same tendency isalso seen in animal models (Figure 3) Since IL-6 is involvedin the regulation of MAPK signaling cascade and stimulatesthe production of MMPs which can cause fibrillar collagen

breakdown and thus leads to premature skin damage [18 19]it is suggested that CFE can lower IL-6 and make less dermalstructural alterations in here

Besides improvement effects of CFE by oral route canwork inUVBmodels making us have an interest in exploringthe linkage between skin and systemic immuneWe analyzedsplenocytic cytokines profile of mice in this study and foundIL-6 production in the vehicle mice is 25-fold that inthe control whereas mice with CFE supplement had lowerproduction (the vehicle versusHCFE 198plusmn039 versus 074plusmn011 ngmL119901 = 002)Thus we suggest that CFE supplementpotentially modulates systemic inflammation and T cellfunction which might be correlated with the improvementof skin alterations This also demonstrates that IL-6 is a keyfactor in protection against UVB-induced skin alterations

34 Main Phytochemicals Contribute to CFErsquos BeneficialPotency in UVB-Irradiated Models In CFE this study iden-tified eight phytochemical compounds including betanin


Cel

l via

bilit

y (

of t

he co

ntro

l)

0102030405060708090

100110

Con

trol

Vehi

cle

Beta

nins CA FA

Rutin VA

EGCG A

A

1120583M10120583M100120583M

lowast lowast lowastlowastlowastlowast

(a)

Con

trol

Vehi

cle

Beta

nins CA FA

Rutin VA

EGCG A

A

0

50

100

150

200

250

300

IL-6

prod

uctio

n (p

g104

cells

)

1120583M10120583M100120583M

lowast

lowastlowast lowast lowastlowast lowast

lowastlowastlowast

(b)

Figure 4 Effects of identified compounds from CFE on cell viability (a) and IL-6 production (b) in UVB-irradiated HaCaT cells Betaninschlorogenic acid (CA) ferulic acid (FA) rutin vanillic acid (VA) EGCG (20 120583M) and ascorbic acid (AA 50 120583M) were used Bar valuesare means plusmn SEM 119899 = 5 Compared with the vehicle statistical differences were analyzed by Studentrsquos t-test and indicated a significance aslowast119901 lt 005 or lowastlowast119901 lt 001 The statistical difference between the control and vehicle represents a significance as 119901 lt 005

(1643mg100 g) rutin (330mg100 g) vanillic acid (85mg100 g) chlorogenic acid (65mg100 g) gallic acid (51mg100 g) epicatechin (43mg100 g) and ferulic acid (9mg100 g)The ration of compounds content showed betanin wasthe major pigment and rutin was the major constitute ofpolyphenols followed by chlorogenic acid and catechins Toanalyze the effectiveness of compounds (Figure 4) we foundrutin (10 120583M) and chlorogenic acid (100 120583M) could signifi-cantly increase cell viability but decrease the production of IL-6The data imply these two are possible active components ofCFE in UVBmodels For rutin its protection from cell deathof HaCaT cells under chronic UVC exposure (18 Jcm2) andinhibition in MMP-1 production from dermal fibroblastsunder UVB exposure had been reported [20 21] Basicallyour data is attached to these reports For chlorogenic acid nostudies clearly proved its effects in UVBmodels only a reportsupposed its antioxidant and photostability to UV light couldfight against UV irradiation [22] In here we experimentallyproved chlorogenic acid had the protective potency

Besides treatment of vanillic acid or ferulic acid (100120583M)did not increase cell viability but decreased the productionof IL-6 (Figure 4(b)) These two components have been indi-cated as major improved contributors of Chromolaena odor-ata extracts in H2O2- and xanthine oxidase-induced models[23]Thismight demonstrate that these two also provide a lit-tle part of anti-inflammation in CFE Out of our expectationreverse data had been seen djulis pigment-betanin (100120583M)in this model did not affect cell viability but increased IL-6production Although this dose is 20-fold the converted doseof betanin (sim45 120583M) driven from CFE (150 120583gmL) betaninat dose manner is notable to discuss its different effects infuture work In this experiment the positive control EGCGand ascorbic acid act as positive controls in reference toprevious studies [2 24]

4 Conclusion

Our findings show that CFE can provide protection fromUVB-induced skin damage and rutin and chlorogenic acidin CFE act as main bioactive compounds in skin protection

Competing Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors would like to thank the financial support of theMinistry of Science and Technology Taiwan (MOST 103-2313-B-214-001MY2)

References

[1] P-J Tsai Y-S Chen C-H Sheu and C-Y Chen ldquoEffect ofnanogrinding on the pigment and bioactivity of djulis (Cheno-podium formosanum Koidz)rdquo Journal of Agricultural and FoodChemistry vol 59 no 5 pp 1814ndash1820 2011

[2] F Afaq V M Adhami and H Mukhtar ldquoPhotochemopreven-tion of ultraviolet B signaling and photocarcinogenesisrdquoMuta-tion ResearchFundamental andMolecularMechanisms ofMuta-genesis vol 571 no 1-2 pp 153ndash173 2005

[3] S Pillai C Oresajo and J Hayward ldquoUltraviolet radiation andskin aging roles of reactive oxygen species inflammation andprotease activation and strategies for prevention of inflam-mation-induced matrix degradationmdasha reviewrdquo InternationalJournal of Cosmetic Science vol 27 no 1 pp 17ndash34 2005

[4] J Uitto ldquoThe role of elastin and collagen in cutaneous agingintrinsic aging versus photoexposurerdquo Journal of Drugs inDermatology vol 7 no 2 pp s12ndashs16 2008


[5] P Brenneisen H Sies and K Scharffetter-Kochanek ldquoUltra-violet-B irradiation andmatrixmetalloproteinases from induc-tion via signaling to initial eventsrdquo Annals of the New YorkAcademy of Sciences vol 973 pp 31ndash43 2002

[6] C Nishigori ldquoCellular aspects of photocarcinogenesisrdquo Photo-chemical and Photobiological Sciences vol 5 no 2 pp 208ndash2142006

[7] Y Kimura andM Sumiyoshi ldquoEffects of various flavonoids iso-lated from Scutellaria baicalensis roots on skin damage in acuteUVB-irradiated hairlessmicerdquo Journal of Pharmacy andPharm-acology vol 63 no 12 pp 1613ndash1623 2011

[8] C-C Chyau C-C Chu S-Y Chen and P-D Duh ldquoDjulis(Chenopodiun formosaneum) and its bioactive compoundsprotect against oxidative stress in human HepG2 cellsrdquo Journalof Functional Foods vol 18 pp 159ndash170 2015

[9] S Lisby R Gniadecki andHCWulf ldquoUV-inducedDNAdam-age in human keratinocytes quantitation and correlation withlong-term survivalrdquo Experimental Dermatology vol 14 no 5pp 349ndash355 2005

[10] D N Syed and H Mukhtar ldquoGender bias in skin cancer role ofcatalase revealedrdquo Journal of Investigative Dermatology vol 132no 3 pp 512ndash514 2012

[11] N-L Wu J-Y Fang M Chen C-J Wu C-C Huang and C-F Hung ldquoChrysin protects epidermal keratinocytes fromUVA-and UVB-induced damagerdquo Journal of Agricultural and FoodChemistry vol 59 no 15 pp 8391ndash8400 2011

[12] C S Sander H Chang F Hamm P Elsner and J JThiele ldquoRoleof oxidative stress and the antioxidant network in cutaneouscarcinogenesisrdquo International Journal of Dermatology vol 43no 5 pp 326ndash335 2004

[13] T Bito and C Nishigori ldquoImpact of reactive oxygen species onkeratinocyte signaling pathwaysrdquo Journal of Dermatological Sci-ence vol 68 no 1 pp 3ndash8 2012

[14] P-J Tsai C-H Sheu P-H Wu and Y-F Sun ldquoThermal andpH stability of betacyanin pigment of djulis (Chenopodium for-mosanum) in Taiwan and their relation to antioxidant activityrdquoJournal of Agricultural and Food Chemistry vol 58 no 2 pp1020ndash1025 2010

[15] S T Boyce A P Supp V B Swope andG DWarden ldquoVitaminC regulates keratinocyte viability epidermal barrier and base-ment membrane in vitro and reduces wound contraction aftergrafting of cultured skin substitutesrdquo Journal of InvestigativeDermatology vol 118 no 4 pp 565ndash572 2002

[16] H Mukhtar and C A Elmets ldquoPhotocarcinogenesis mecha-nisms models and human health implicationsrdquo Photochemistryand Photobiology vol 63 no 4 pp 356ndash357 1996

[17] S Lembo A Balato R Di Caprio et al ldquoThe modulatoryeffect of ellagic acid and rosmarinic acid on ultraviolet-B-induced cytokinechemokine gene expression in skin keratino-cyte (HaCaT) cellsrdquo BioMed Research International vol 2014Article ID 346793 8 pages 2014

[18] D N Syed F Afaq and H Mukhtar ldquoDifferential activationof signaling pathways by UVA and UVB radiation in normalhuman epidermal keratinocytesrdquo Photochemistry and Photobi-ology vol 88 no 5 pp 1184ndash1190 2012

[19] R-F Lin X-X Feng C-W Li et al ldquoPrevention of UV radia-tion-induced cutaneous photoaging inmice by topical adminis-tration of patchouli oilrdquo Journal of Ethnopharmacology vol 154no 2 pp 408ndash418 2014

[20] S Pastore A Potapovich V Kostyuk et al ldquoPlant polyphenolseffectively protect HaCaT cells from ultraviolet C-triggered

necrosis and suppress inflammatory chemokine expressionrdquoAnnals of the New York Academy of Sciences vol 1171 pp 305ndash313 2009

[21] E Hwang S-Y Park H J Lee et al ldquoVigna angularis waterextracts protect against ultraviolet B-exposed skin aging in vitroand in vivordquo Journal of Medicinal Food vol 17 no 12 pp 1339ndash1349 2014

[22] D P Rivelli C A H Filho R L Almeida C D Ropke T CH Sawada and S B M Barros ldquoChlorogenic acid UVA-UVBphotostabilityrdquo Photochemistry and Photobiology vol 86 no 5pp 1005ndash1007 2010

[23] T-T Phan LWang P See R J Grayer S-Y Chan and S T LeeldquoPhenolic compounds ofChromolaena odorata protect culturedskin cells from oxidative damage implication for cutaneouswound healingrdquo Biological and Pharmaceutical Bulletin vol 24no 12 pp 1373ndash1379 2001

[24] C-C Huang W-B Wu J-Y Fang et al ldquo(-)-Epicatechin-3-gallate a green tea polyphenol is a potent agent against UVB-induced damage inHaCaT keratinocytesrdquoMolecules vol 12 no8 pp 1845ndash1858 2007

Submit your manuscripts athttpwwwhindawicom

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ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


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mediators andMMPs could be indicators to test whether thesample possesses the ability of skin protection For instancethrough reducing the production of inflammatory cytokinesphytochemicals from Scutellaria baicalensis and the seedsof quinoa (Chenopodium quinoa Willd) have been provedto have protective functions on skin in UVB models [6 7]In a classification of plant taxonomy djulis has the samegenus as quinoa Although previous studies have pointed outbioactive compounds including betanins epigallocatechinsferulic acids and certain flavonoids in djulis and proposedbeneficial functions [1 8] the skincare potency of djulis hasnever been tested under UVB models

The aim of this study is to examine protective effects ofdjulis water extracts on skin photoaging in in vitro and invivo UVB-irradiated models and confirm which bioactivecompounds are main contributors

2 Materials and Methods

21 Sample Preparation and Constituents Measurement Thedjulis (Chenopodium formosanum Koidz) seeds were har-vested from the National Pingtung University of Science andTechnology The extracts were prepared by soaking 1 g ofseeds grains in 5mL of distilled water in the dark at 4∘C for24 h After filtration the filtrate was then freeze-dried as sam-ples (the water extract of Chenopodium formosanum KoidzCFE) for further analysis The dry weight of this extractwas 32 g and the yield was 106 It was stored at minus20∘Cuntil using it To analyze components of CFE total pheno-lic contentwasmeasured spectrophotometrically by using themodified Folin-Ciocalteumethod and the samples were sub-jected to HPLC to identify betanin and phenolic compoundsaccording to the method described by the previous study [1]

22 Cell Culture The keratinocyte cells from human skin(HaCaT) were grown in Dulbeccorsquos modified Eagle medium(DMEM) supplemented with 10 (vv) heat-inactivated fetalbovine serum penicillin (100 unitsmL) and streptomycin(100 unitsmL) These culture reagents were commercialproducts (Gibco Grand Island NY USA) The cells wereincubated in an atmosphere of 5 CO2 at 37

∘C and wereregularly subcultured for experiment uses In samples testcells were seeded in a 96-well plate at a concentrationof 1 times 105 cellsmL Following one-day incubation cellsmedia were removed and replaced by phosphate buffersaline (PBS) for UV irradiation The plate was placed in anUV CROSSLINKER (XLE-1000B SPECTROLINE) whichset peak spectral emission electric tension frequency andthe distance from cells at 315 nm 120V 60Hz and 15 cmrespectively When cells were exposed to 30mJcm2UVBtime of irradiation was averagely 30 sec Then these cellswere treated with media containing various concentrationsof CFE pure compounds fromCFE or ascorbic acid for 24 hAfter that cell supernatants were collected for the analysisof inflammation-related mediators and cells in the bottom ofplate were for cell viability

23 Cell Viability The cell viability was assayed by MTT(3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide)

method After collecting cell supernatants an aliquot of55 120583Lwell of DMEM containing 05mgmLMTT was addedinto the plate for 3 h incubation Then the plate wasadded with an aliquot of 100 120583Lwell of isopropanol contain-ing 004NHCl and appropriately shaken for dissolving outcolored crystals Cell viability values at absorbance of 570 nmwere determined by an ELISA reader (Bio-Rad LaboratoriesHercules CA USA) In this study to confirm the data of cellviability we counted cell number by trypan blue stainingBriefly we seeded cells onto 6 cm dish (1 times 106 cells) for24 h incubation After washing twice by PBS cells were irra-diated with UVB at dose of 30mJcm2 Then these cells weretreated with CFE for 24 h Following that cells were har-vested for counting cell number

24 IL-6 and MMP-1 Measurements Production of IL-6(eBioscience Minneapolis MN USA) and MMP-1 (RampDMinneapolis MN USA) in cell supernatants was assayedusing commercial ELISA kits Briefly primary anti-IL-6 orMMP-1 antibodies were coated onto 96-well plates Afterovernight incubation plate wells were washed with washingbuffer and blocked with blocking solution for 1 h Afterwashingwells diluted supernatants or standard reagentswereadded into wells and the plate was incubated for 2 h Nextwells were washed and then added with biotin-conjugatedanti-IL-6 or MMP-1 antibodies for 1 h Following the washthe wells were added with horseradish peroxidase- (HRP-)conjugated streptavidin for 30min washed and incubatedwith tetramethylbenzidine Absorbance was measured by anELISA reader at 620 nm Data were calculated according tostandard curves

25 Intracellular Reactive Oxygen Species (ROS) Measure-ment Detecting levels of dichlorofluorescein (DCF) which isderived fromdichlorodihydrofluorescein-diacetate (DCFDA)under oxidative stress can represent the production of ROSIn this experiment cells were seeded at a concentration of 1times 106 cells in a 6 cm dish for 24 h and irradiated with UVB(30mJcm2) then they were treated with CFE or positivecontrols for 3 hNext cellswere collected in a tube and labeledwith 5 120583M DCFDA (Invitrogen Carlsbad CA USA) for30min After that cells were centrifuged and washed twicewith phosphate buffer saline (PBS) and intracellular DCFlevels were detected by flow cytometer (Accuri CytometersInc Ann Arbor MI USA)

26 Cell Cycle Analysis Cell cycle analysis was performed todetermine the proportion of apoptotic sub-G1 hypodiploidcells In brief cells were seeded at a concentration of 1 times106 cells in a 6 cm dish for 24 h Then cells were irradiatedwith UVB (30mJcm2) and treated with CFE or positivecontrols for 24 h Afterwards cells were harvested and fixedin 70 ethanol for 24 h at minus20∘C then they were washedtwice with PBS and introduced with PBS containing PIsolution at 4∘C Following overnight incubation cellularfluorescein levels were assayed by flow cytometer In the assayeach cycle phase was quantified from histograms by usingsoftware programs and changes of values in cycle phases werecalculated to evaluate CFErsquos effects
















wo UVB UVB

Cel

l via

bilit

y (

of t

he co

ntro

l)

0

20

40

60

80

100

120

140


lowastlowast

10120583gmL CFE


(a)


ROS

leve

ls (

of t

he co

ntro

l)

0

20

40

60

80

100

120

140

160

180

lowast

lowastlowast

(150120583gmL)

(b)


20

40

60

80

100

120

(120583gmL)

IL-6

prod

uctio

n (p

g104

cells

)

lowast

lowast lowast

(c)


20

40

60

80

100

120

(120583gmL)

MM

P-1

prod

uctio

n (p

g104

cells

)

lowastlowast

(d)







0 1 2 3 4 5 6 7

Skin

thic

knes

s (120583

m)

0

200

400

600

800

1000


HCFE (100mgkg BW)

(a)


Epid

erm

is th

ickn

ess (

120583m

)

0

5

10

15

20

25

30

35

Control

lowast

lowastlowast

(b)

Duration (day)0 1 2 3 4 5 6 7

Skin

thic

knes

s (120583

m)

0

200

400

600

800

1000

ControlVehicle AA

CFE (8mgmouse)

(c)

Vehicle CFE AA

Epid

erm

is th

ickn

ess (

120583m

)

0

5

10

15

20

25

30

35

Control

lowast

(d)






Con

trol

DAPI IL-6 Merge

Vehi

cleH

CFE

LCFE

AA








Cel

l via

bilit

y (

of t

he co

ntro

l)

0102030405060708090

100110

Con

trol

Vehi

cle

Beta

nins CA FA

Rutin VA

EGCG A

A

1120583M10120583M100120583M


(a)

Con

trol

Vehi

cle

Beta

nins CA FA

Rutin VA

EGCG A

A

0

50

100

150

200

250

300

IL-6

prod

uctio

n (p

g104

cells

)

1120583M10120583M100120583M

lowast


lowastlowastlowast

(b)




4 Conclusion


Competing Interests


Acknowledgments


References


































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
















wo UVB UVB

Cel

l via

bilit

y (

of t

he co

ntro

l)

0

20

40

60

80

100

120

140


lowastlowast

10120583gmL CFE


(a)


ROS

leve

ls (

of t

he co

ntro

l)

0

20

40

60

80

100

120

140

160

180

lowast

lowastlowast

(150120583gmL)

(b)


20

40

60

80

100

120

(120583gmL)

IL-6

prod

uctio

n (p

g104

cells

)

lowast

lowast lowast

(c)


20

40

60

80

100

120

(120583gmL)

MM

P-1

prod

uctio

n (p

g104

cells

)

lowastlowast

(d)







0 1 2 3 4 5 6 7

Skin

thic

knes

s (120583

m)

0

200

400

600

800

1000


HCFE (100mgkg BW)

(a)


Epid

erm

is th

ickn

ess (

120583m

)

0

5

10

15

20

25

30

35

Control

lowast

lowastlowast

(b)

Duration (day)0 1 2 3 4 5 6 7

Skin

thic

knes

s (120583

m)

0

200

400

600

800

1000

ControlVehicle AA

CFE (8mgmouse)

(c)

Vehicle CFE AA

Epid

erm

is th

ickn

ess (

120583m

)

0

5

10

15

20

25

30

35

Control

lowast

(d)






Con

trol

DAPI IL-6 Merge

Vehi

cleH

CFE

LCFE

AA








Cel

l via

bilit

y (

of t

he co

ntro

l)

0102030405060708090

100110

Con

trol

Vehi

cle

Beta

nins CA FA

Rutin VA

EGCG A

A

1120583M10120583M100120583M


(a)

Con

trol

Vehi

cle

Beta

nins CA FA

Rutin VA

EGCG A

A

0

50

100

150

200

250

300

IL-6

prod

uctio

n (p

g104

cells

)

1120583M10120583M100120583M

lowast


lowastlowastlowast

(b)




4 Conclusion


Competing Interests


Acknowledgments


References


































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0 1 2 3 4 5 6 7

Skin

thic

knes

s (120583

m)

0

200

400

600

800

1000


HCFE (100mgkg BW)

(a)


Epid

erm

is th

ickn

ess (

120583m

)

0

5

10

15

20

25

30

35

Control

lowast

lowastlowast

(b)

Duration (day)0 1 2 3 4 5 6 7

Skin

thic

knes

s (120583

m)

0

200

400

600

800

1000

ControlVehicle AA

CFE (8mgmouse)

(c)

Vehicle CFE AA

Epid

erm

is th

ickn

ess (

120583m

)

0

5

10

15

20

25

30

35

Control

lowast

(d)






Con

trol

DAPI IL-6 Merge

Vehi

cleH

CFE

LCFE

AA








Cel

l via

bilit

y (

of t

he co

ntro

l)

0102030405060708090

100110

Con

trol

Vehi

cle

Beta

nins CA FA

Rutin VA

EGCG A

A

1120583M10120583M100120583M


(a)

Con

trol

Vehi

cle

Beta

nins CA FA

Rutin VA

EGCG A

A

0

50

100

150

200

250

300

IL-6

prod

uctio

n (p

g104

cells

)

1120583M10120583M100120583M

lowast


lowastlowastlowast

(b)




4 Conclusion


Competing Interests


Acknowledgments


References


































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Con

trol

DAPI IL-6 Merge

Vehi

cleH

CFE

LCFE

AA








Cel

l via

bilit

y (

of t

he co

ntro

l)

0102030405060708090

100110

Con

trol

Vehi

cle

Beta

nins CA FA

Rutin VA

EGCG A

A

1120583M10120583M100120583M


(a)

Con

trol

Vehi

cle

Beta

nins CA FA

Rutin VA

EGCG A

A

0

50

100

150

200

250

300

IL-6

prod

uctio

n (p

g104

cells

)

1120583M10120583M100120583M

lowast


lowastlowastlowast

(b)




4 Conclusion


Competing Interests


Acknowledgments


References


































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Cel

l via

bilit

y (

of t

he co

ntro

l)

0102030405060708090

100110

Con

trol

Vehi

cle

Beta

nins CA FA

Rutin VA

EGCG A

A

1120583M10120583M100120583M


(a)

Con

trol

Vehi

cle

Beta

nins CA FA

Rutin VA

EGCG A

A

0

50

100

150

200

250

300

IL-6

prod

uctio

n (p

g104

cells

)

1120583M10120583M100120583M

lowast


lowastlowastlowast

(b)




4 Conclusion


Competing Interests


Acknowledgments


References


































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Research Article Djulis ( Chenopodium formosanum Koidz...

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Transcript of Research Article Djulis ( Chenopodium formosanum Koidz...