research article dandelion extracts protect human skin...

Research ArticleDandelion Extracts Protect Human Skin Fibroblasts fromUVB Damage and Cellular Senescence

Yafan Yang1 and Shuangshuang Li2

1Sir Winston Churchill Collegiate and Vocational Institute Thunder Bay ON Canada P7C 1V52Cardiovascular and Metabolic Research Unit Lakehead University Thunder Bay ON Canada P7A 7T1

Correspondence should be addressed to Shuangshuang Li shu-li66hotmailcom

Received 2 December 2014 Accepted 17 February 2015

Academic Editor Felipe Simon

Copyright copy 2015 Y Yang and S Li This 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

Ultraviolet (UV) irradiation causes damage in skin by generating excessive reactive oxygen species (ROS) and induction of matrixmetalloproteinases (MMPs) leading to skin photoageing Dandelion extracts have long been used for traditional Chinese medicineand native American medicine to treat cancers hepatitis and digestive diseases however less is known on the effects of dandelionextracts in skin photoageing Here we found that dandelion leaf and flower extracts significantly protect UVB irradiation-inhibitedcell viability when added before UVB irradiation or promptly after irradiation Dandelion leaf and flower extracts inhibitedUVB irradiation-stimulated MMP activity and ROS generation Dandelion root extracts showed less action on protecting HDFsfrom UVB irradiation-induced MMP activity ROS generation and cell death Furthermore dandelion leaf and flower but notroot extracts stimulated glutathione generation and glutathione reductase mRNA expression in the presence or absence of UVBirradiation We also found that dandelion leaf and flower extracts help absorb UVB irradiation In addition dandelion extractssignificantly protected HDFs from H

2O2-induced cellular senescence In conclusion dandelion extracts especially leaf and flower

extracts are potent protective agents against UVB damage and H2O2-induced cellular senescence in HDFs by suppressing ROS

generation and MMP activities and helping UVB absorption

1 Introduction

When skin is aged skin is more transparent loose and frag-ile One of the most damaging actions on skin is from solarradiation especially from its ultraviolet (UV) componentleading to both clinical and histologic damage on human skin[1 2] UV irradiation causes distinct alterations of the connec-tive tissues by generating excessive reactive oxygen species(ROS) and induction of matrix metalloproteinases (MMPs)MMPs-mediated degradation of the collagenous extracellularmatrix (ECM) accounts for most of the connective tissuedamage that occurs in photodamaged skin [3 4] Dermalfibroblasts are responsible for generating ECM and allowingthe skin to recover from injury [5]

UV irradiation consists of three components UVA UVBand UVC Whereas UVA and UVB reach the earth in suffi-cient amounts to damage the skin UVC is almost completelyabsorbed by the ozone layer [2 6] UVB is particularlydamaging as it penetrates the epidermis and the upper part

of the dermis where it damages fibroblast cells and leadsto sunburn photoageing and skin cancer [7 8] In Canadasunlight is strong enough to cause skin cancer and prematureageing of the skin People often use sunscreen to protectthe skin from UV damaging from the sun and sunscreenabsorbs UV rays and prevents them from penetrating theskin However some sunscreen ingredients including oxy-benzone benzophenone and octocrylene have been shownto be potentially skin carcinogenic or penetrate into ourbody and have other health risks [9] It is necessary to findeffective safer and environmentally friendly nature productsfor antiageing and UV protection

Taraxacum officinale commonly known as dandelionis widely distributed in the warmer temperate zone of theNorthern Hemisphere [10] Dandelion extracts have beenused for centuries for traditionalChinesemedicine andnativeAmerican medicine to treat cancers hepatitis and digestivediseases [11ndash14] Dandelion extracts are shown to have anti-inflammatory antioxidant and anticarcinogenic activities

Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2015 Article ID 619560 10 pageshttpdxdoiorg1011552015619560

2 Oxidative Medicine and Cellular Longevity

however there are few scientific studies to investigate theeffects of dandelion extracts in the treatment of skin diseasesespecially photoageing [10 15]

The purpose of this study was to determine whetherdandelion leaf flower or root extracts can protect humandermal fibroblasts (HDFs) from UVB damage and cellularsenescence and the underlying mechanisms We found thatdandelion leaf and flower extracts but not root extracts arepotent protective agent against UVB damage and H

2O2-

induced cellular senescence in HDFs by suppressing ROSgeneration and MMP activities

2 Materials and Methods

21 Preparation of Dandelion Extract Dandelions werecollected at flowering stage in June-July 2013 from thehouse yards in Thunder Bay (latitude 48∘2210158405610158401015840N longitude89∘1410158404610158401015840W) Ontario Canada Leaf flower and root wereseparated cleaned and air-dried before extraction through4 hours of boiling in distilled water [13 16] The waterextracts were filtered through grade 1 Whatman filter paperunder vacuum concentrated and then dissolved in distilledwater following filtration through a 02 120583m filter to avoidcell contamination and 300mgmL stock aqueous extractwas prepared and stored at 4∘C for experimental use Theidentity of plant was confirmed by Dr Wei Cao (LakeheadUniversity) and the herbarium specimens were depositedin Cardiovascular and Metabolic Research Unit LakeheadUniversity for future reference

22 Cell Culture HDFs were obtained from American TypeCulture Collection (Manassas VA) and cultured with Dul-beccorsquos modified Eaglersquos medium supplemented with 10fetal bovine serum 100UmL penicillin and 100 120583gmLstreptomycin and grown in a CO

2incubator at 37∘C in 5

CO2[17 18]The experiments were performed when the cells

reached 70ndash80 confluence between passages 4 and 8 Inall studies cells were incubated in the serum-free mediumfor 12 h and then 10 serum added together with differenttreatments The media were changed every three days

23 Cellular Viability Assays Cell viabilities were measuredbased on conversion of yellow tetrazolium salt 3-(45-dim-ethylthiazol-2-yl)-25-diphenyltetrazolium bromide (MTT)to dark blue formazan by viable cells [19 20] Briefly cells atequal number were plated onto each well of 96-well plates for24 h After treatment 100 120583L medium containing 5mgmLMTTwas added to each well and the cells were then culturedat 37∘C for 4 h The medium was then discarded and thecrystals were dissolved in dimethyl sulfoxide and absorbanceof formazan products at 570 nm was measured in a Multi-skan spectrum microplate spectrophotometer (Thermo Lab-systems Franklin MA) The cells incubated with controlmedium were considered 100 viable

24 UVB Irradiation HDFs were rinsed with phosphatebuffered saline (PBS) and irradiated using a UVB lamp(UVB-18 Claremont CA) with a wavelength range of 280ndash315 nm [3 21] UVB was administered for 60 seconds

(200mJcm2) and the cells were cultured for another 24 hoursfollowing detection of cell viability Dandelion extracts wereadded 30minutes before or immediately after UVB radiationThe control cells were untreated with UVB irradiation

25 Measurement of ROS and Glutathione (GSH) Contents2101584071015840-Dichlorodihydrofluorescein diacetate (H

2DCFDA) (In-

vitrogen Carlsbad CA) was used to detect ROS [13 22]After treatments HDFs cultured in 35 mm plates werewashed with PBS once and then incubated with 1 120583gmLH2DCFDA in medium for 30 minutes at 37∘CThen the cells

were rinsed twice in PBS and the fluorescence signals weredetected using a fluorescentmicroscope ImageJ software wasused to quantify fluorescent intensity Total GSH contentswere measured using a commercial GSH assay kit (CaymanChemical Ann Arbor MI) as previously described

26 UV Absorption UV absorption ability of dandelionextracts in medium (300 120583gmL) under 280 290 300 and310 nm was measured with a UV-visible spectrophotometer[23]

27 Cell Staining for MMPs After treatments HDFs culturedin 35 mm plates were incubated with 1120583gmL DQ gelatin(Invitrogen) for 30 minutes to analyze MMP activity under afluorescentmicroscope ImageJ software was used to quantifyfluorescent intensity DQ gelatin is a fluorogenic substrateused to detectMMP activity [4 20] Upon digestion its greenfluorescence is revealed to be used to measure MMP activity

28 Determination of mRNA Level Total RNA of HDFswas isolated using TriReagent (Invitrogen) [24] First strandcDNA was prepared by reverse transcription using M-MuLV reverse transcriptase and random hexamer primersaccording to manufacturerrsquos protocol (New England Bio-labs Pickering ON) Real-time PCR was performed in aniCycler iQ5 apparatus (Bio-Rad Mississauga ON) associ-ated with the iCycler optical system software (version 31)using SYBR Green PCR Master Mix as described pre-viously The primers of glutathione reductase (GR) were51015840-AGGGCCGCCGTGGTGGAGAGC-31015840 (forward position265ndash285) and 51015840-ATGGGACTTGGTGAGATTGTTTTG-31015840(reverse position 475ndash498) These primers produced a prod-uct of 234 bp The primers of 120573-actin were purchased fromAmbion (Streetsville ON) which produce a product of295 bp A standard curve was constructed using a series ofdilution of total RNA (Ambion) transcribed to cDNA usingthe same protocol outlined above to confirm the sameamplifying efficiency in the PCR A standard melting curveanalysis was performed using the following thermal cyclingprofile 95∘C for 10 s 55∘C for 15 s and ramping to 95∘C at 1∘increments to confirm the absence of primer dimers RelativemRNA quantification was calculated by using the arithmeticformula 2minusΔΔCT where ΔCT is the difference between thethreshold cycle of a given target cDNA and an endogenousreference 120573-actin cDNA

29 Cell Ageing Cell ageing was evaluated with senescence-associated 120573-galactosidase (SA-120573-gal) staining [18 24] HDFs

Oxidative Medicine and Cellular Longevity 3

0 10 30 100 300 1000 3000

Cel

l via

bilit

y (

)

20406080

100120140

0

Leaf extract (120583gmL)

(a)

Cel

l via

bilit

y (

)

0 10 30 100 300 1000 30000

20406080

100120140

Flower extract (120583gmL)

(b)

0 10 30 100 300 1000 30000

20406080

100120140

Cel

l via

bilit

y (

)

Root extract (120583gmL)

(c)

Figure 1 Dandelion extracts have no effect on HDFs cell viability HDFs were treated with the extracts of dandelion leaf (a) flower (b) androot (c) at 10ndash3000 120583gmL for 24 hours and cell viability was then measured with MTT method 119899 = 4

cultured in 35 mm plates were treated with H2O2(100 120583M)

with or without dandelion extracts (300120583gmL) for 72 hoursAfter that the cells were fixed and stained with X-gal at pH60 overnight Nuclei were stained with 410158406-diamidino-2-phenylindole (DAPI 1120583gmL) for cell countingThe percent-age of ageing cells was calculated as the ratio of blue-stainedcells to total cells counted More than 600 cells were countedfrom each group

210 Materials and Data Analysis All chemicals were pur-chased from Sigma (St Louis MO) unless stated otherwiseStudentrsquos 119905-test in Microsoft excel was used to analyze databetween control and treatment group and the data werepresented asmeanplusmn standard error of themean Experimentswere repeated a minimum of 3 times Statistical significancewas set at 119875 lt 005

3 Results

31 Dandelion Leaf and Extracts Protect UVB Irradiation-Induced Cell Death The dandelion extracts were preparedfrom dandelion leaves flowers and root separately andthe extract rate was 106 112 and 127 respectivelyDandelion extracts alone at 10ndash3000120583gmL had no effect onHDFs cell viability (Figure 1) When dandelion extracts (30100 and 300 120583gmL) were added into HDFs for 30 minutesbefore UVB irradiation (Figures 2(a) and 2(b)) or whenadded promptly after irradiation (Figures 2(a) and 2(c)) leafand flower extracts significantly protected UVB irradiation-inhibited cell viability Dandelion root extracts had less

effect on UVB-induced cell death when added into HDFs30 minutes before UVB irradiation Although supplementof dandelion root extract at 300120583gmL immediately afterirradiation significantly protected HDFs from UVB-inducedcell damage it was quite inefficient compared with the samedose of leaf and flower extracts (Figure 2(b))

32 Dandelion Leaf and Flower Extracts Inhibit UVBIrradiation-Stimulated MMP Activity and Oxidative StressUVB irradiation strengthened the fluorescent intensity ofDQgelatin (Figure 3) and H

2-DCFDA (Figure 4) reflecting the

higher activities of MMPs and higher level of ROS whichleads to degradation of ECM and induction of cell death[4 13] Dandelion leaf and flower extracts (300120583gmL) sig-nificantly reversed UVB irradiation-induced MMP activityand ROS generation when added either before or after UVBirradiation (Figures 3 and 4) Dandelion root extracts showedless action on protecting HDFs from UVB irradiation-induced MMP activity and ROS generation

33 Dandelion Leaf and Flower Extracts Stimulate GSHGeneration andGRmRNAExpression GSH ismaster antiox-idant in our body [24] We observed that GSH level issignificantly lower in UVB-irradiated HDFs (0075mgmgprotein) which is completely reversed by dandelion leaf andflower extracts but not dandelion root extracts Dandelionleaf and flower extracts alone also significantly increasedGSH level (045mgmg protein and 046mgmg proteinresp) compared with the control cells (03mgmg protein)(Figure 5(a)) GR reduces glutathione disulfide to form GSH


Before UV

After UV

Control UV

Leaf Flower Root

(a)

0

20

40

60

80

100

120 Before UV

30 100 300Leaf

30 100 300 30 100 300Flower

Cel

l via

bilit

y (

)

Root

Con

trol (120583gmL)

lowastlowastlowast

lowastlowast

lowast

(b)

Root

0

20

40

60

80

100

120 After UV

30 100 300Leaf

30 100 300 30 100 300Flower

Cel

l via

bilit

y (

)

Con

trol

(120583gmL)

lowast

lowast

lowastlowast

lowastlowastlowast

(c)

Figure 2 Dandelion leaf and flower extracts protect HDFs from UVB irradiation-induced damage Dandelion extracts at the indicatedconcentrations were added 30 minutes before UVB (200mJcm2) (b) or promptly after UVB radiation (c) Cell viability was measured byMTT 24 hours after culture 119875 lt 005 versus control lowast119875 lt 005 versus UVB only Images in (a) were taken under microscope showingUVB irradiation damages cells but dandelion leaf and flower extracts (300120583gmL) protected the cells added from either before or after UVBradiation Under UVB irradiation the cells became smaller and less in number Scale bar 20 120583m 119899 = 3

and is an important GSH-maintaining gene [25] The mRNAexpression of GR was decreased by 620 in UVB-irradiatedHDFs in comparison with the control cells (Figure 5(b))Consistent with the GSH data incubation of HDFs withdandelion leaf and flower extracts significantly induced GRmRNA expression by 291 and 372 compared with the

control HDFs even in the presence of UVB irradiation Inaddition dandelion leaf and flower extracts alone stimu-lated GR mRNA expression by 894 and 1162 respec-tively Dandelion root extract had no effect on GR mRNAexpression in the presence or absence of UVB irradiation(Figure 5(b))


After UV

Control UV

Leaf Flower Root

Before UV

(a)

Relat

ive fl

uore

scen

t int

ensit

y

0

5

10

15

20

25

30

35

40UV

Before After Before After Before After

Con

trol

Leaf Flower Root

lowastlowast

lowastlowast

(b)

Figure 3 Dandelion leaf and flower extracts inhibit UVB irradiation-stimulatedMMP activity UVB irradiation strengthened the fluorescentintensity ofDQgelatin reflecting higherMMPactivity however dandelion leaf and flower extracts (300 120583gmL) significantly decreasedMMPactivity when added both before and after UVB irradiation Scale bar 20120583m (b) was the statistical analysis from (a) by imageJ software119875 lt 005 versus control lowast119875 lt 005 versus UVB only 119899 = 3 Scale bar 20120583m

34 Dandelion Leaf and Flower Extracts Help UV Absorp-tion As shown in Figure 6 dandelion extracts could helpabsorb UV irradiation At the wavelengths of 300 nm and310 nm flower and leaf extracts had the highest UV absorp-tion capacity At 290 nm dandelion root extracts displayedhigher UV absorption ability compared with dandelionleaf extract but significantly lower than dandelion flower

extract These data suggest the protective role of dandelionextracts against UV irradiation is partially through UVabsorption

35 Dandelion Leaf and Flower Extracts Protect H2O2-

Induced Cell Ageing Oxidative stress has been consideredone of crucial factors associated with cellular senescence To


Control UV

Leaf Flower Root

After UV

Before UV

(a)

Relat

ive fl

uore

scen

t int

ensit

y

UV

Before After Before After Before After0

100200300400500600700800900

1000

Con

trol

Leaf Flower Root

lowastlowast

lowastlowast

(b)

Figure 4 Dandelion leaf and flower extracts inhibit UVB irradiation-induced ROS production UVB irradiation caused significant increasesin the fluorescent intensity of H

2-DCFDA reflecting the higher level of ROS however dandelion leaf and flower extracts (300 120583gmL)

significantly decreased UVB irradiation-induced ROS generation when added both before and after UVB irradiation (b) was the statisticalanalysis from (a) by imageJ software 119875 lt 005 versus control lowast119875 lt 005 versus UVB only 119899 = 3 Scale bar 20 120583m

study the protective roles of dandelion extracts on oxidativestress-induced premature senescence we treated HDFswith H

2O2 one main source of oxidative stress HDFs

became enlarged and flattened with a decreased nucleus-to-cytoplasm ratio after H

2O2(100 120583M) treatment for 72 hours

(Figure 7(a)) showing the typical characteristics of senescent

cells [18 21]The cells were then stained for SA-120573-gal and thedata showed that 786 of cells in the H

2O2-treated group

become aged (stained with blue) however dandelion leafflower and root extracts (300 120583gmL) significantly protectH2O2-induced cell ageing by 618 733 and 400

respectively (Figure 7(b))


GSH

cont

ent (

mg

mg

prot

ein)

UV


01

02

03

04

05

06

Con

trol

Leaf Leaf

Flower Flow

er

Root Root

lowastlowastlowastlowast

(a)

GR

mRN

A ex

pres

sion

()

UV

0

50

100

150

200

250

Control Leaf LeafFlower FlowerRoot Root

lowastlowast

(b)

Figure 5 Dandelion leaf and flower extracts stimulate GSH generation and the expression of GR (a) Dandelion leaf and flower extractsprotected UVB irradiation-decreased GSH level Dandelion extracts (300120583gmL) were added 30 minutes before UVB or promptly afterUVB radiation and GSH was measured after additional 24-hour culture 119875 lt 005 versus control lowast119875 lt 005 versus UVB only 119899 = 3 (b)Dandelion leaf and flower extracts stimulated GR mRNA expression Dandelion extracts (300120583gmL) were added 30 minutes before UVBand GR mRNA was measured after additional 24-hour culture with real-time PCR 119875 lt 005 versus control lowast119875 lt 005 versus UVB only119899 = 3

002040608

11214

280 290 300 310

Dandelion flower

Dandelion leaf

Dandelion root

UVB wavelength (nm)

UV

B ab

sorp

tion

Figure 6 Dandelion extracts help absorb UV irradiation At wave-lengths of 300 nm and 310 nm flower and leaf extracts had the high-est UV absorption capacity UV absorption of dandelion extracts(300 120583gmL)wasmeasuredwith aUV spectrometer at 280 290 300and 310 nm respectively 119899 = 3

4 Discussion

In recent years interest on the research of herb medicinehas increased all over the world Many herb extracts haveshown therapeutic properties as reported elsewhere [2 619 20 23] Dandelion is one of the most common andrecognizable herbs and is found in almost every part ofthe world Many studies have shown that dandelion extractshave a wide range of pharmacological activities includinganticarcinogenic antioxidant anti-inflammatory and anti-heart burn activities [10 11 16] however there are limitedscientific studies investigating the antiageing and anti-UVactivity of dandelion extracts and very little is known aboutthe mechanisms of action

Skin ageing is mainly attributed to extrinsic (photoage-ing) and intrinsic (chronological ageing) processes that areoften manifested by increased wrinkles loss of tensionsand elasticity and altered pigmentation and so forth [1 8]Dermal fibroblasts are within the dermis layer of skin andare responsible for generating connective tissues includ-ing laminin and fibronectin which comprise the ECM [5]

In this study we found that exposure of HDFs to UVBleads to reduced cell viability Without dermal fibroblaststhe skin cannot properly recover from injury Recognitionof supplementing natural antioxidant components in skincare is growing important among dermatologists and othermedical professionals Sunscreens are considered to be thegold standard for protecting skin from UV damage It isrecently noted that sunscreen ingredients may become freeradicals themselves when activated by UV radiation Inaddition sunscreen chemicals may be absorbed into theskin causing harmful effect [9] Here we demonstratedthat supplement of dandelion leaf and flower extracts at aslower as 30 120583gmL rescued the cells from UVB-induced celldeath suggesting dandelion may be useful for preventingand treating skin photoageing More importantly dandelionextracts produce protective action when added at both beforeand after UVB irradiation pointing to the convenient use ofthis new productThe dandelion extracts at the concentrationas higher as 3mgmL did not affect cell growth so dandelionleaf and flower water extracts would be a green choice fordevelopment of novel sunscreens

It is well established that cutaneous exposure to UVirradiation causes higher activities of MMPs and excessiveproduction of ROS [4 8 26] Our studies further validatedthat UVB irradiation in HDFs induces significantly higherMMP activities and ROS level Increased activation of MMPscauses the degradation or disorganization of skin connectivetissues which would lead to increased skin wrinkles andloss of skin tone [5] Oxidative stress is one of the primarycauses of skin ageing and it is believed that oxidative stress iscaused by an imbalance between the production of ROS andskin cellrsquos ability to clean up the reactive intermediates [24]This is really the case and the present study found that UVBirradiation markedly decreased the content of GSH a masterantioxidant inside the cells The reduced expression of GR aGSH producing gene would be attributed to the lower levelof GSH by UVB irradiation [25] With less GSH generationthe cells will have less ability to remove overproducedROS Excitedly dandelion leaf and flower extracts attenuated


Control Leaf Flower Root

Cell senescence staining

Nucleusstaining

H2O2 (100120583M)

(a)

Control Leave Flower Root

Agi

ng ce

lls (

)

0102030405060708090

100lowast

H2O2 (100120583M)

(b)Figure 7 Dandelion leaf and flower extracts protect H

2O2-induced cell ageing (a)The images showed that H

2O2provokes cell ageing which

is protected by dandelion extracts The ageing cells had higher gal activity and stained with blue color HDFs were also incubated with DAPIfor nucleus staining for cell counting Scale bar 20120583m (b) was the statistical analysis from (a) HDFs were treated with H

2O2(100120583M) with

or without dandelion extracts (300 120583gmL) for 72 hours The percentage of aged cells was calculated from the number of blue-stained cells tototal cells counted lowast119875 lt 005 compared all other groups

UVB-inducedMMPactivation andROS generation with leafextracts having higher efficiency Incubation of HDFs witheither dandelion leaf or flower extracts alone significantlyincreased GR mRNA expression and GSH generation Evenin the presence of UVB irradiation dandelion leaf and flowerextracts maintained intracellular GSH level unchanged Allthese data indicate that dandelion leaf and flower extractscan protectHDFs fromUVB-induced cell death by increasingGSH generation inhibiting MMP activities and ROS genera-tion

Ageing at the cellular level is known as cellular senescence[18 24] Oxidative stress has been considered one of crucialfactors associated with cellular senescence Most of thesenescent cells have flattened cell morphology promiscuousgene expression a proinflammatory secretory response andpositive SA-120573-gal staining at pH 60 [18] Exposure of HDFsto lower doses ofH

2O2(100 120583M) for 72 hours induced prema-

ture senescence as evidenced by enlarged and flattened cellmorphology with a decreased nucleus-to-cytoplasm ratio

and most of the cells were stained blue for SA-120573-gal thetypical characteristics of senescent cells All these changesby H2O2in HDFs were significantly reversed by dandelion

extracts Dandelion extracts improve cellular ageing possiblyby directly altering the expressions of senescence-relatedgenes such as p53 or indirectly lowering oxidative stress

Unlike dandelion leaf and flower extracts dandelion rootextracts had less effect on UV protection Compared to dan-delion roots dandelion leaves and flowers are characterizedby higher polyphenol contents [10] So it is highly possiblethat polyphenols but not other components contribute to theprotective role of dandelion leaf and flower extracts againstUV damage Most of the components of dandelion leaf andflower extract have been isolated and identified and some ofthe important components of the extract including sesquiter-pene lactones and phenylpropanoids are believed to haveanti-inflammatory antioxidative and anticancer propertiesWhether these components are also playing the critical rolesin protecting HDFs from UVB damage needs to be tested


Other components which have not been fully characterizedalso need to be explored

In conclusion dandelionwater extracts are able to protectHDFs against UVB damage before irradiation and also whenadded promptly after irradiation via increased UV absorp-tion and reduced MMP activity and oxidative stress Theextracts are also able to prevent oxidative stress-induced pre-mature senescence however with both UV protection andantiageing the root extracts have the smallest effect com-pared with leaf and flower extractsThese findings can lead tomore effective safer and environmentally friendly antiageingand UV protection Future studies both preclinical andclinical will be necessary to test the efficacy of dandelionextracts for photoageing

Abbreviations

ECM Extracellular matrixHDF Human dermal fibroblastsGR Glutathione reductaseGSH GlutathioneMMP Matrix metalloproteinaseMTT 3-(45-Dimethylthiazol-2-yl)-25-

diphenyltetrazolium bromidePBS Phosphate buffered salineROS Reactive oxygen speciesSA-120573-gal Senescence-associated 120573-galactosidaseUV Ultraviolet

Conflict of Interests

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

Acknowledgments

The authors would like to thank Drs Yaoge Huang and WeiCao in Lakehead University for suggestion and comments onthis project as well as the identification of the plants and theyare also very grateful for the generous support and guidancefrom all the members in the Cardiovascular and MetabolicResearch Unit Lakehead University Thunder Bay Ontario

References

[1] J E Sanches Silveira and D M Myaki Pedroso ldquoUV light andskin agingrdquo Reviews on Environmental Health vol 29 no 3 pp243ndash254 2012

[2] B R Zhou H B Yin Y Xu et al ldquoBaicalin protects humanskin fibroblasts from ultraviolet A radiation-induced oxidativedamage and apoptosisrdquo Free Radical Research vol 46 pp 1458ndash1471 2012

[3] J Y Cherng L Y Chen andM F Shih ldquoPreventive effects of 120573-thujaplicin against UVB-induced MMP-1 and MMP-3 mRNAexpressions in skin fibroblastsrdquo American Journal of ChineseMedicine vol 40 no 2 pp 387ndash398 2012

[4] F Liebel S Kaur E Ruvolo N Kollias and M D SouthallldquoIrradiation of skin with visible light induces reactive oxygenspecies and matrix-degrading enzymesrdquo Journal of InvestigativeDermatology vol 132 no 7 pp 1901ndash1907 2012

[5] T Quan Z Qin Y Xu et al ldquoUltraviolet irradiation inducesCYR61CCN1 a mediator of collagen homeostasis throughactivation of transcription factor AP-1 in human skin fibrob-lastsrdquo Journal of Investigative Dermatology vol 130 no 6 pp1697ndash1706 2010

[6] Y P Hwang J H Choi H G Kim et al ldquoCultivated ginsengsuppresses ultraviolet B-induced collagenase activation viamitogen-activated protein kinases and nuclear factor 120581Bactiva-tor protein-1-dependent signaling in humandermal fibroblastsrdquoNutrition Research vol 32 no 6 pp 428ndash438 2012

[7] M Ichihashi andHAndo ldquoThemaximal cumulative solarUVBdose allowed to maintain healthy and young skin and preventpremature photoagingrdquo Experimental Dermatology vol 23 pp43ndash46 2014

[8] B Poljsak and R Dahmane ldquoFree radicals and extrinsic skinagingrdquoDermatology Research and Practice vol 2012 Article ID135206 4 pages 2012

[9] G Bens ldquoSunscreensrdquo Advances in Experimental Medicine andBiology vol 810 pp 429ndash463 2014

[10] K Schutz R Carle and A Schieber ldquoTaraxacummdasha reviewon its phytochemical and pharmacological profilerdquo Journal ofEthnopharmacology vol 107 no 3 pp 313ndash323 2006

[11] S J Chatterjee P Ovadje M Mousa C Hamm and S PandeyldquoThe efficacy of dandelion root extract in inducing apopto-sis in drug-resistant human melanoma cellsrdquo Evidence-BasedComplementary and Alternative Medicine vol 2011 Article ID129045 11 pages 2011

[12] M Hfaiedh D Brahmi and L Zourgui ldquoHepatoprotectiveeffect of Taraxacum officinale leaf extract on sodium dichro-mate-induced liver injury in ratsrdquo Environmental Toxicology2014

[13] C M Park J Y Park K H Noh J H Shin and Y S SongldquoTaraxacum officinale Weber extracts inhibit LPS-inducedoxidative stress and nitric oxide production via the NF-120581Bmodulation in RAW2647 cellsrdquo Journal of Ethnopharmacologyvol 133 no 2 pp 834ndash842 2011

[14] B-R Lee J-H Lee and H-J An ldquoEffects of taraxacumofficinale on fatigue and immunological parameters in micerdquoMolecules vol 17 no 11 pp 13253ndash13265 2012

[15] U-K Choi O-H Lee J H Yim et al ldquoHypolipidemic andantioxidant effects of dandelion (Taraxacum officinale) rootand leaf on cholesterol-fed rabbitsrdquo International Journal ofMolecular Sciences vol 11 no 1 pp 67ndash78 2010

[16] P Ovadje S Chatterjee C Griffin C Tran C Hamm and SPandey ldquoSelective induction of apoptosis through activation ofcaspase-8 in human leukemia cells (Jurkat) by dandelion rootextractrdquo Journal of Ethnopharmacology vol 133 no 1 pp 86ndash91 2011

[17] K Phetdee R Rakchai K Rattanamanee T Teaktong and JViyoch ldquoPreventive effects of tamarind seed coat extract onUVA-induced alterations in human skin fibroblastsrdquo Journal ofCosmetic Science vol 65 no 1 pp 11ndash24 2014

[18] X Shen Y Du W Shen B Xue and Y Zhao ldquoAdipose-derivedstem cells promote human dermal fibroblast function andincrease senescence-associated 120573-galactosidase mRNA expres-sion through paracrine effectsrdquoMolecularMedicine Reports vol10 no 6 pp 3068ndash3072 2014

[19] A Martınez E Conde A Moure H Domnguez and RJ Estevez ldquoProtective effect against oxygen reactive speciesand skin fibroblast stimulation of Couroupita guianensis leafextractsrdquo Natural Product Research vol 26 no 4 pp 314ndash3222012


[20] B-M Hwang E-M Noh J-S Kim et al ldquoCurcumin inhibitsUVB-induced matrix metalloproteinase-13 expression by sup-pressing the MAPK-p38JNK pathways in human dermalfibroblastsrdquo Experimental Dermatology vol 22 no 5 pp 371ndash374 2013

[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] M Majeed B Bhat S Anand A Sivakumar P Paliwal andK G Geetha ldquoInhibition of UV-induced ROS and collagendamage by Phyllanthus emblica extract in normal humandermal fibroblastsrdquo Journal of Cosmetic Science vol 62 no 1pp 49ndash56 2011

[23] A R Silva C Seidl A S Furusho M M S Boeno G CDieamant and A M Weffort-Santos ldquoIn vitro evaluation ofthe efficacy of commercial green tea extracts in UV protectionrdquoInternational Journal of Cosmetic Science vol 35 no 1 pp 69ndash77 2013

[24] G Yang K Zhao Y Ju et al ldquoHydrogen sulfide protects againstcellular senescence via s-sulfhydration of keap1 and activationof Nrf2rdquo Antioxidants and Redox Signaling vol 18 no 15 pp1906ndash1919 2013

[25] H-S Wong J-H Chen P-K Leong H-Y Leung W-MChan and K-M Ko ldquo120573-sitosterol protects against carbontetrachloride hepatotoxicity but not gentamicin nephrotoxicityin rats via the induction of mitochondrial glutathione redoxcyclingrdquoMolecules vol 19 no 11 pp 17649ndash17662 2014

[26] D M Palmer and J S Kitchin ldquoOxidative damage skin agingantioxidants and a novel antioxidant rating systemrdquo Journal ofDrugs in Dermatology vol 9 no 1 pp 11ndash15 2010

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


however there are few scientific studies to investigate theeffects of dandelion extracts in the treatment of skin diseasesespecially photoageing [10 15]

The purpose of this study was to determine whetherdandelion leaf flower or root extracts can protect humandermal fibroblasts (HDFs) from UVB damage and cellularsenescence and the underlying mechanisms We found thatdandelion leaf and flower extracts but not root extracts arepotent protective agent against UVB damage and H

2O2-

induced cellular senescence in HDFs by suppressing ROSgeneration and MMP activities

2 Materials and Methods

21 Preparation of Dandelion Extract Dandelions werecollected at flowering stage in June-July 2013 from thehouse yards in Thunder Bay (latitude 48∘2210158405610158401015840N longitude89∘1410158404610158401015840W) Ontario Canada Leaf flower and root wereseparated cleaned and air-dried before extraction through4 hours of boiling in distilled water [13 16] The waterextracts were filtered through grade 1 Whatman filter paperunder vacuum concentrated and then dissolved in distilledwater following filtration through a 02 120583m filter to avoidcell contamination and 300mgmL stock aqueous extractwas prepared and stored at 4∘C for experimental use Theidentity of plant was confirmed by Dr Wei Cao (LakeheadUniversity) and the herbarium specimens were depositedin Cardiovascular and Metabolic Research Unit LakeheadUniversity for future reference

22 Cell Culture HDFs were obtained from American TypeCulture Collection (Manassas VA) and cultured with Dul-beccorsquos modified Eaglersquos medium supplemented with 10fetal bovine serum 100UmL penicillin and 100 120583gmLstreptomycin and grown in a CO

2incubator at 37∘C in 5

CO2[17 18]The experiments were performed when the cells

reached 70ndash80 confluence between passages 4 and 8 Inall studies cells were incubated in the serum-free mediumfor 12 h and then 10 serum added together with differenttreatments The media were changed every three days

23 Cellular Viability Assays Cell viabilities were measuredbased on conversion of yellow tetrazolium salt 3-(45-dim-ethylthiazol-2-yl)-25-diphenyltetrazolium bromide (MTT)to dark blue formazan by viable cells [19 20] Briefly cells atequal number were plated onto each well of 96-well plates for24 h After treatment 100 120583L medium containing 5mgmLMTTwas added to each well and the cells were then culturedat 37∘C for 4 h The medium was then discarded and thecrystals were dissolved in dimethyl sulfoxide and absorbanceof formazan products at 570 nm was measured in a Multi-skan spectrum microplate spectrophotometer (Thermo Lab-systems Franklin MA) The cells incubated with controlmedium were considered 100 viable

24 UVB Irradiation HDFs were rinsed with phosphatebuffered saline (PBS) and irradiated using a UVB lamp(UVB-18 Claremont CA) with a wavelength range of 280ndash315 nm [3 21] UVB was administered for 60 seconds

(200mJcm2) and the cells were cultured for another 24 hoursfollowing detection of cell viability Dandelion extracts wereadded 30minutes before or immediately after UVB radiationThe control cells were untreated with UVB irradiation

25 Measurement of ROS and Glutathione (GSH) Contents2101584071015840-Dichlorodihydrofluorescein diacetate (H

2DCFDA) (In-

vitrogen Carlsbad CA) was used to detect ROS [13 22]After treatments HDFs cultured in 35 mm plates werewashed with PBS once and then incubated with 1 120583gmLH2DCFDA in medium for 30 minutes at 37∘CThen the cells

were rinsed twice in PBS and the fluorescence signals weredetected using a fluorescentmicroscope ImageJ software wasused to quantify fluorescent intensity Total GSH contentswere measured using a commercial GSH assay kit (CaymanChemical Ann Arbor MI) as previously described

26 UV Absorption UV absorption ability of dandelionextracts in medium (300 120583gmL) under 280 290 300 and310 nm was measured with a UV-visible spectrophotometer[23]

27 Cell Staining for MMPs After treatments HDFs culturedin 35 mm plates were incubated with 1120583gmL DQ gelatin(Invitrogen) for 30 minutes to analyze MMP activity under afluorescentmicroscope ImageJ software was used to quantifyfluorescent intensity DQ gelatin is a fluorogenic substrateused to detectMMP activity [4 20] Upon digestion its greenfluorescence is revealed to be used to measure MMP activity

28 Determination of mRNA Level Total RNA of HDFswas isolated using TriReagent (Invitrogen) [24] First strandcDNA was prepared by reverse transcription using M-MuLV reverse transcriptase and random hexamer primersaccording to manufacturerrsquos protocol (New England Bio-labs Pickering ON) Real-time PCR was performed in aniCycler iQ5 apparatus (Bio-Rad Mississauga ON) associ-ated with the iCycler optical system software (version 31)using SYBR Green PCR Master Mix as described pre-viously The primers of glutathione reductase (GR) were51015840-AGGGCCGCCGTGGTGGAGAGC-31015840 (forward position265ndash285) and 51015840-ATGGGACTTGGTGAGATTGTTTTG-31015840(reverse position 475ndash498) These primers produced a prod-uct of 234 bp The primers of 120573-actin were purchased fromAmbion (Streetsville ON) which produce a product of295 bp A standard curve was constructed using a series ofdilution of total RNA (Ambion) transcribed to cDNA usingthe same protocol outlined above to confirm the sameamplifying efficiency in the PCR A standard melting curveanalysis was performed using the following thermal cyclingprofile 95∘C for 10 s 55∘C for 15 s and ramping to 95∘C at 1∘increments to confirm the absence of primer dimers RelativemRNA quantification was calculated by using the arithmeticformula 2minusΔΔCT where ΔCT is the difference between thethreshold cycle of a given target cDNA and an endogenousreference 120573-actin cDNA

29 Cell Ageing Cell ageing was evaluated with senescence-associated 120573-galactosidase (SA-120573-gal) staining [18 24] HDFs


0 10 30 100 300 1000 3000

Cel

l via

bilit

y (

)

20406080

100120140

0


(a)

Cel

l via

bilit

y (

)

0 10 30 100 300 1000 30000

20406080

100120140


(b)

0 10 30 100 300 1000 30000

20406080

100120140

Cel

l via

bilit

y (

)


(c)





3 Results








Before UV

After UV

Control UV

Leaf Flower Root

(a)

0

20

40

60

80

100

120 Before UV

30 100 300Leaf

30 100 300 30 100 300Flower

Cel

l via

bilit

y (

)

Root

Con

trol (120583gmL)

lowastlowastlowast

lowastlowast

lowast

(b)

Root

0

20

40

60

80

100

120 After UV

30 100 300Leaf

30 100 300 30 100 300Flower

Cel

l via

bilit

y (

)

Con

trol

(120583gmL)

lowast

lowast

lowastlowast

lowastlowastlowast

(c)





After UV

Control UV

Leaf Flower Root

Before UV

(a)

Relat

ive fl

uore

scen

t int

ensit

y

0

5

10

15

20

25

30

35

40UV


Con

trol

Leaf Flower Root

lowastlowast

lowastlowast

(b)







Control UV

Leaf Flower Root

After UV

Before UV

(a)

Relat

ive fl

uore

scen

t int

ensit

y

UV


100200300400500600700800900

1000

Con

trol

Leaf Flower Root

lowastlowast

lowastlowast

(b)










2O2-treated group




GSH

cont

ent (

mg

mg

prot

ein)

UV


01

02

03

04

05

06

Con

trol

Leaf Leaf

Flower Flow

er

Root Root


(a)

GR

mRN

A ex

pres

sion

()

UV

0

50

100

150

200

250


lowastlowast

(b)


002040608

11214

280 290 300 310

Dandelion flower

Dandelion leaf

Dandelion root

UVB wavelength (nm)

UV

B ab

sorp

tion


4 Discussion








Nucleusstaining

H2O2 (100120583M)

(a)


Agi

ng ce

lls (

)

0102030405060708090

100lowast

H2O2 (100120583M)





2O2(100120583M) with












Abbreviations





Acknowledgments


References

































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0 10 30 100 300 1000 3000

Cel

l via

bilit

y (

)

20406080

100120140

0


(a)

Cel

l via

bilit

y (

)

0 10 30 100 300 1000 30000

20406080

100120140


(b)

0 10 30 100 300 1000 30000

20406080

100120140

Cel

l via

bilit

y (

)


(c)





3 Results








Before UV

After UV

Control UV

Leaf Flower Root

(a)

0

20

40

60

80

100

120 Before UV

30 100 300Leaf

30 100 300 30 100 300Flower

Cel

l via

bilit

y (

)

Root

Con

trol (120583gmL)

lowastlowastlowast

lowastlowast

lowast

(b)

Root

0

20

40

60

80

100

120 After UV

30 100 300Leaf

30 100 300 30 100 300Flower

Cel

l via

bilit

y (

)

Con

trol

(120583gmL)

lowast

lowast

lowastlowast

lowastlowastlowast

(c)





After UV

Control UV

Leaf Flower Root

Before UV

(a)

Relat

ive fl

uore

scen

t int

ensit

y

0

5

10

15

20

25

30

35

40UV


Con

trol

Leaf Flower Root

lowastlowast

lowastlowast

(b)







Control UV

Leaf Flower Root

After UV

Before UV

(a)

Relat

ive fl

uore

scen

t int

ensit

y

UV


100200300400500600700800900

1000

Con

trol

Leaf Flower Root

lowastlowast

lowastlowast

(b)










2O2-treated group




GSH

cont

ent (

mg

mg

prot

ein)

UV


01

02

03

04

05

06

Con

trol

Leaf Leaf

Flower Flow

er

Root Root


(a)

GR

mRN

A ex

pres

sion

()

UV

0

50

100

150

200

250


lowastlowast

(b)


002040608

11214

280 290 300 310

Dandelion flower

Dandelion leaf

Dandelion root

UVB wavelength (nm)

UV

B ab

sorp

tion


4 Discussion








Nucleusstaining

H2O2 (100120583M)

(a)


Agi

ng ce

lls (

)

0102030405060708090

100lowast

H2O2 (100120583M)





2O2(100120583M) with












Abbreviations





Acknowledgments


References

































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Before UV

After UV

Control UV

Leaf Flower Root

(a)

0

20

40

60

80

100

120 Before UV

30 100 300Leaf

30 100 300 30 100 300Flower

Cel

l via

bilit

y (

)

Root

Con

trol (120583gmL)

lowastlowastlowast

lowastlowast

lowast

(b)

Root

0

20

40

60

80

100

120 After UV

30 100 300Leaf

30 100 300 30 100 300Flower

Cel

l via

bilit

y (

)

Con

trol

(120583gmL)

lowast

lowast

lowastlowast

lowastlowastlowast

(c)





After UV

Control UV

Leaf Flower Root

Before UV

(a)

Relat

ive fl

uore

scen

t int

ensit

y

0

5

10

15

20

25

30

35

40UV


Con

trol

Leaf Flower Root

lowastlowast

lowastlowast

(b)







Control UV

Leaf Flower Root

After UV

Before UV

(a)

Relat

ive fl

uore

scen

t int

ensit

y

UV


100200300400500600700800900

1000

Con

trol

Leaf Flower Root

lowastlowast

lowastlowast

(b)










2O2-treated group




GSH

cont

ent (

mg

mg

prot

ein)

UV


01

02

03

04

05

06

Con

trol

Leaf Leaf

Flower Flow

er

Root Root


(a)

GR

mRN

A ex

pres

sion

()

UV

0

50

100

150

200

250


lowastlowast

(b)


002040608

11214

280 290 300 310

Dandelion flower

Dandelion leaf

Dandelion root

UVB wavelength (nm)

UV

B ab

sorp

tion


4 Discussion








Nucleusstaining

H2O2 (100120583M)

(a)


Agi

ng ce

lls (

)

0102030405060708090

100lowast

H2O2 (100120583M)





2O2(100120583M) with












Abbreviations





Acknowledgments


References

































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















After UV

Control UV

Leaf Flower Root

Before UV

(a)

Relat

ive fl

uore

scen

t int

ensit

y

0

5

10

15

20

25

30

35

40UV


Con

trol

Leaf Flower Root

lowastlowast

lowastlowast

(b)







Control UV

Leaf Flower Root

After UV

Before UV

(a)

Relat

ive fl

uore

scen

t int

ensit

y

UV


100200300400500600700800900

1000

Con

trol

Leaf Flower Root

lowastlowast

lowastlowast

(b)










2O2-treated group




GSH

cont

ent (

mg

mg

prot

ein)

UV


01

02

03

04

05

06

Con

trol

Leaf Leaf

Flower Flow

er

Root Root


(a)

GR

mRN

A ex

pres

sion

()

UV

0

50

100

150

200

250


lowastlowast

(b)


002040608

11214

280 290 300 310

Dandelion flower

Dandelion leaf

Dandelion root

UVB wavelength (nm)

UV

B ab

sorp

tion


4 Discussion








Nucleusstaining

H2O2 (100120583M)

(a)


Agi

ng ce

lls (

)

0102030405060708090

100lowast

H2O2 (100120583M)





2O2(100120583M) with












Abbreviations





Acknowledgments


References

































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Control UV

Leaf Flower Root

After UV

Before UV

(a)

Relat

ive fl

uore

scen

t int

ensit

y

UV


100200300400500600700800900

1000

Con

trol

Leaf Flower Root

lowastlowast

lowastlowast

(b)










2O2-treated group




GSH

cont

ent (

mg

mg

prot

ein)

UV


01

02

03

04

05

06

Con

trol

Leaf Leaf

Flower Flow

er

Root Root


(a)

GR

mRN

A ex

pres

sion

()

UV

0

50

100

150

200

250


lowastlowast

(b)


002040608

11214

280 290 300 310

Dandelion flower

Dandelion leaf

Dandelion root

UVB wavelength (nm)

UV

B ab

sorp

tion


4 Discussion








Nucleusstaining

H2O2 (100120583M)

(a)


Agi

ng ce

lls (

)

0102030405060708090

100lowast

H2O2 (100120583M)





2O2(100120583M) with












Abbreviations





Acknowledgments


References

































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















GSH

cont

ent (

mg

mg

prot

ein)

UV


01

02

03

04

05

06

Con

trol

Leaf Leaf

Flower Flow

er

Root Root


(a)

GR

mRN

A ex

pres

sion

()

UV

0

50

100

150

200

250


lowastlowast

(b)


002040608

11214

280 290 300 310

Dandelion flower

Dandelion leaf

Dandelion root

UVB wavelength (nm)

UV

B ab

sorp

tion


4 Discussion








Nucleusstaining

H2O2 (100120583M)

(a)


Agi

ng ce

lls (

)

0102030405060708090

100lowast

H2O2 (100120583M)





2O2(100120583M) with












Abbreviations





Acknowledgments


References

































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















Nucleusstaining

H2O2 (100120583M)

(a)


Agi

ng ce

lls (

)

0102030405060708090

100lowast

H2O2 (100120583M)





2O2(100120583M) with












Abbreviations





Acknowledgments


References

































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















Abbreviations





Acknowledgments


References

































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article dandelion extracts protect human skin...

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