Research ArticleComparative Hair Restorer Efficacy of Medicinal Herb onNude (Foxn1nu) Mice

Shahnaz Begum1 Mi Ra Lee1 Li Juan Gu1 Md Jamil Hossain2

Hyun Kyoung Kim1 and Chang Keun Sung1

1 Department of Food Science and Technology College of Agriculture and Biotechnology Chungnam National UniversityDaejeon 305-764 Republic of Korea

2Molecular Genetics and Genomics Lab Department of Horticulture Chungnam National UniversityDaejeon 305-764 Republic of Korea

Correspondence should be addressed to Chang Keun Sung sungck80gmailcom

Received 6 May 2014 Accepted 8 August 2014 Published 13 November 2014

Academic Editor Sanyog Jain

Copyright copy 2014 Shahnaz Begum et alThis is an open access article distributed under theCreativeCommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Eclipta alba (L) Hassk Asiasarum sieboldii (Miq) F Maek (Asiasari radix) and Panax ginseng C A Mey (red ginseng) aretraditionally acclaimed for therapeutic properties of various human ailments Synergistic effect of each standardized plant extractwas investigated for hair growth potential on nude mice as these mutant mice genetically lack hair due to abnormal keratinizationDried plant samples were ground and extracted by methanol Topical application was performed on the back of nude micedaily up to completion of two hair growth generations The hair density and length of Eclipta alba treated mice were increasedsignificantly (119875 gt 0001) than control mice Hair growth area was also distinctly visible in Eclipta alba treated mice On the otherhand Asiasari radix and Panax ginseng treated mice developing hair loss were recognized from the abortive boundaries of haircoverage Histomorphometric observation of nude mice skin samples revealed an increase in number of hair follicles (HFs) Thepresence of follicular keratinocytes was confirmed by BrdU labeling S-phase cells in HFs Therefore Eclipta alba extract andorphytochemicals strongly displayed incomparability of hair growth promotion activity than others Thus the standardized Ecliptaalba extract can be used as an effective alternative and complementary treatment against hair loss

1 Introduction

Hair is a dynamic mini-organ of mammalian skin thatoriginates from the hair follicle and serves multiple functionsthroughout adult life Inside the skin every HF is particularlyactive and is composed of fully keratinized epithelial cellsHair growth is coordinated by hormones and it commandsthe follicle to undergo appropriate changes during thisprocess This process is followed by specific cyclic order andcharacterized by anagen (growth phase) catagen (regres-sion) and telogen (resting phase) [1] Human hair folliclesalso pose a unique paradox as the hormones androgensgrowth factor may cause stimulation of hair growth Onthe other hand genetically acquired disorder inhibit togenerate well differentiated hair follicles or shorten anagenphase these phenomena may cause baldness pattern [2 3]

Although human hair loss is not life threatening alopecia(hair lossbalding) may cause serious psychological distress[4] These may reflect on some sociopsychological contextsNovel therapies are therefore required to prevent hair loss andpromote hair growth In this context developing alternativemedicine has increased interest in the recent years Untilnow complete lack of strong scientific evidence of effectivecomponent of natural plant extract and limited to their hairpromotion mechanisms has kept natural hair care away frommainstream research Although the search for the treatmenthair loss research resulted in some synthetic drugs associatedside effects are highly remarkableTherefore herbal productsnow emerged as an alternate to the synthetic drugs in thiscurrent state

Studies of various research groups reported that hundredsof plants or substances are having potential to promote

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 319795 9 pageshttpdxdoiorg1011552014319795

2 BioMed Research International

Day 0 Day 7 Day 12 Day 16

Con

trol

(a)

Day 0 Day 7 Day 12 Day 16

Min

oxid

il

(b)

Day 0 Day 7 Day 12 Day 16

A ra

dix

(c)

Day 0 Day 7 Day 12 Day 16

E a

lba

(d)

Day 0 Day 7 Day 12 Day 16

P gi

nsen

g

(e)

Figure 1 Stimulation of hair growth by different natural extract in nude mice Specific concentrations of different extract in vehicleformulation were daily applied topically (119899 = 5) on the back of nude mice skin (a) control (b) minoxidil (c) A radix (d) E alba and(e) P ginseng during one hair growth phase

hair growth as well In particular only little candidate plantplays great role to enhance hair growth whose efficaciesare now under investigation Therefore recent research wasunder taken to investigate of some known plants those weretraditionally acclaimed and purported in oriental medicinesuch as A radix [5] P ginseng [6 7] and E alba [8 9] exertto promote hair growth

Moreover E alba has also been reported with multiplemedicinal uses and is known for numerous biological activi-ties It is an enormous source of several secondary metabo-lites such as polypeptides polyacetylenes and triterpenes[10] flavonoids phytosterols and coumestans [11] Phyto-chemical coumestans represent an important class of naturaloxygenated aromatic compound including wedelolactoneand demethylwedelolactone and saponins responsible forthe main medicinal effects of E alba such as its antihepatitisC virus [12] antitumor activity [13] and neuropharmaco-logical activity [14] and its anticancer effect shows antipro-liferative and cytotoxic effects determined using MTT assay[15]

Taken together this bioresource information we under-take nude mice as a model to get more precise result for hair

biology from different well-known medicinal plant extractsas these mice express the role of Foxn1 in mammalian skinbiology responsible for structural abnormality of hair shaftdue to abnormal keratinization [16]Moreover nudemice arealso frequently used in skin pharmacology to serve for skinpenetration assays [17] as the follicular penetration route actsas an entry pathway for compounds into the skin [18]

E alba is widely reported traditional medicine and well-being functional food thus it prompted us to evaluate hairgrowth promoting activity alongwith other plantsThereforethis approach may incorporate some novel findings that maypromote us to explore extensive bioactive compounds fromcrude extract for prevention of hair loss as well as enhancinghair growth

2 Materials and Methods

21 Plant Materials and Preparation of Extraction Aerialparts (leaves stems and blooms) of E alba and dried rootsof A radix and P ginseng were purchased from the JecheonMedicinal Herb Association South Korea All the samples

BioMed Research International 3

Table 1 Botanical origins plant parts and percentage yields of investigated specimens

Botanical name (family) Chinese name Common name Plant part Extraction yield ( ww)Eclipta alba (Asteraceae) Han lian cao False daisy Leaves stems and blooms 1240Asiasarum sieboldii (Aristolochiaceae) Shashin Chinese wild ginger root Roots 752Panax ginseng (Araliaceae) Hong-Shen Red ginseng Roots 3833

0

1

2

3

4

1st cycle 2nd clycle Mean of cycles

ControlMinoxidilA radix

E albaP ginseng

a

ab

ab

a

b

a

ab

b

a

b

a

b

ab ab

a

Hai

r cov

erag

e are

a (sc

ale0

ndash4)

Figure 2 Effect of different plant extract on hair coverage areain athymic nude mice Mice were evaluated on a scale of ldquo0ndash4rdquoparameters as follows ldquo0rdquo = complete alopecia ldquo1rdquo = lt25 haircoverage ldquo2rdquo = 25ndash50 hair coverage ldquo3rdquo = 50ndash75 hair coverageldquo4rdquo = full hair coverage The different letters (a and b) within acolumn indicate significant differences (119875 lt 005) determined byDuncanrsquos multiple range test

were authenticated by Associate Professor Ki Hwan Bae (Col-lege of Pharmacy ChungnamNational University) where thevoucher specimens were deposited The dried plant sam-pleswere ground into powder (500 g) and extracted with 95methanol at 60∘C The methanol extract was concentratedand dried in a vacuum evaporator under reduced pressurebelow 60∘CThe resulting residue was weighed and dissolvedin a vehicle mixture (propylene glycol ethanol dimethylsulfoxide 67 30 3 vv)The extract yieldswere determinedas ldquordquo of dry crude extracts (Table 1)

22 Chromatographic Analysis HPLC-grade acetonitrile waspurchased from Merck Co (Merck Darmstadt Germany)Deionized water was purified by Milli-Q system (MilliporeBedford MA USA) Other chemicals were of reagent gradeStandard wedelolactone powder was purchased from theSigma-AldrichCoThepurity of the standardwas over 98 asindicated by the manufacturer Calibration standard sampleswere prepared from the stock solutions by adding 05ndash15mgmL of wedelolactone inMeOH to obtain appropriateddilutions which were filtered through Millipore membranebefore use Chromatographic analysis was performed onthe Shimadzu (Japan) HPLC apparatus with a column of

Discovery C18 (ODS 25 cm times 46mm 5 120583m Supelco USA)and a Guard Column Security Guard Cartridge system (Phe-nomenex USA) The mobile phase consisted of acetonitrilewater (ACN vv) at a flow rate of 1mLmin as follows20ndash45 (up to 15min) 45ndash100 (up to 30min) and 100(up to 40min)Themethod was performed with an injectionvolume of 20 120583L and detection wavelength at 365 nm

23 Toxicity Studies Toxicity studies were carried out byapplying the extract to the nude mice skin in concentrationsof up to 5 for 7 days and did not show any toxic side effectslike erythema on skin surface Thus the prepared extractswere considered safe for topical application on nude mice

24 Balbc Nude Mice Athymic Balbc male nude miceat 7 weeks of age were purchased from Dae-Han Biolink(Eumsung Korea) and maintained under 12 h light darkperiods at 24 plusmn 2∘C and were housed and handled underaseptic conditions Experiment was performed in accordancewith approved institutional protocols by the InstitutionalAnimal Care and Use Committee (IACUC) ChungnamNationalUniversity Daejeon SouthKoreaThe authorizationcode number is CNU-00244

25 Treatment Protocol of Different Natural Extracts on NudeMice Five animals were allocated to five randomized groupsfor evaluation of hair growth and hair changing pattern ofnude mice Topical application was performed daily with asolution of A radix E alba and P ginseng extracts (25) ina vehicle formulation andorminoxidil (2) until completionof two full hair growth cycles

26 Measurement of Hair Density and Length After dailytreatment in each hair growth cycle the images from specificregions of neck back and hip were captured two times perweek using KONG Bom-Viewer Plus (Seoul Korea) Aboutsix images of 80times and 200times were exploited to measure thehair length and hair density respectively using Bom-ViewerImage Analyzer

27 Evaluation of Hair Existing Area Thehair existing area ofnude mice was systematically evaluated for each mouse threetimes per week At this time point mice were scored on ascale of ldquo0rdquo to ldquo4rdquo for percent body coverage of hair ldquo0rdquo =complete alopecia ldquo1rdquo = lt25 hair coverage ldquo2rdquo = 25ndash50hair coverage ldquo3rdquo = 50ndash75 hair coverage ldquo4rdquo = normal hairdensity and full hair coverage

28 Histologic Assessment of Hair Growth Mice were sac-rificed in anagen phase and skin samples were fixed in

4 BioMed Research International

Control Minoxidil A radix E alba P ginseng1

st ha

ir cy

cle2

nd h

air c

ycle

(A) (B) (C) (D) (E)

(A998400) (B998400) (C998400) (D998400) (E998400)

0

10

20

30

40

50

60

70

80

90

1st hair cycle 2nd hair cycle

a

c

a

b

c c

a

b

a

cH

air d

ensit

y (36

mm

2ar

ea)

ControlMinoxidilA radix

E albaP ginseng

000

020

040

060

080

100

120

140

160

180

200

1st cycle 2nd cycle

d

b

c

a b

a

b

c

b

c

Hai

r len

gth

(mm

)

ControlMinoxidilA radix

E albaP ginseng

(G)(F)

Figure 3 Evaluation of hair length and hair density in cycles Image of nude mice hair on skin surface treated with control ((A) (A1015840))minoxidil ((B) (B1015840)) A radix ((C) (C1015840)) E alba ((D) (D1015840)) and P ginseng ((E) (E1015840)) in first and second hair cycles Hair length (F) and hairdensity (G) shown in column The different letters (a b c and d) within a column indicate significant differences (119875 lt 00001) determinedby Duncanrsquos multiple range test Digital images were takenby KONG Bom-Viewer Plus with 80times magnification lens Hair length and hairdensity were measured using Bom-Viewer Image analyzer (119899 = 10)

10 buffered formalin for histological analysis Paraffin-embedded 4 120583m sections were stained with hematoxylinand eosin (HampE) The nude mice HF morphology andthe number of HFs were evaluated microscopically in 5fields per section of the dorsal skin at a magnification oftimes100

29 BrdU Immunohistochemistry BrdU labeling cell prolif-eration was detected by intraperitoneal injection of bromod-eoxyuridine (BrdU Sigma USA) with 50mgkg body weightat a concentration of 5mgmL in PBS BrdU incorporationwas detected by IHC staining of paraffin-embedded sec-tions with mouse anti-BrdU primary antibody (Santa CruzBiotechnology Santa Cruz CA USA) Skin sections thatwere not incubated with primary antibodies were used as

a negative control Detection of follicular keratinocyte BrdUlabeling was done at magnification times100 and times400

210 Statistical Analysis Statistical analysis was performedusing SAS Systems ver 93 and analysis of variance (ANOVA)was used for comparisons among all groups All data areexpressed as means plusmn SD unless otherwise is noted Aprobability level of 5was considered significant Differencesbetween two groups were analyzed by t-test ANOVA wasfollowed after Duncanrsquos multiple comparison tests

3 Results

31 Differences in Activity of Plant Extracts on ChangingHair Growth Pattern of Nude Mice After daily treatment

BioMed Research International 5

Control Minoxidil A radix E alba P ginseng

(A) (B) (C) (D) (E)

(A998400) (B998400) (C998400) (D998400) (E998400)

Figure 4Histological features of the skin specimen of different extract andor vehicle control treatedmiceMacroscopic observation of hair inthe skin surface of (A) control (B)minoxidil (C)A radix (D) E alba and (E) P ginseng in early hair existing phase Microscopic observationof longitudinal sections of (A1015840) control (B1015840) minoxidil (C1015840) A radix (D1015840) E alba and (E1015840) P ginseng treated mice skin was harvested andfixed in buffered formalin for routine hematoxylin and eosin (HampE) stained histology (119899 = 7) The broken line indicates defective HFs at thelevel of sebaceous gland ((A1015840) (B1015840) (C1015840) and (E1015840)) Note that the straight hair shafts that emerge from the skin surface of minoxidil and Ealba treated mouse ((B) (D) and Arrows (B1015840) (D1015840)) Scale bar 100 120583m

hair growth was documented on experimental day 7 Sys-tematic monitoring of hair growth showed that A radix(Figure 1(c)) and vehicle treated control mice (Figure 1(a))exhibit extremely sparse and transient hair coat On the otherhand minoxidil and E alba extract treated mice hair growthfirst documented from the frontal part of the head as earlyas 7 days and became dense thicker and longer that evenlyextended to the shoulder and posterior part of the backduring experimental day 12 The maximum hair coat wasevident in E alba extract treated group on experimental day16 (Figures 1(d) and 1(b)) In contrast A radix (Figure 1(c))and P ginseng (Figure 1(e)) treated mice found rapid hair losswith increasing time point and eventually remained only fewsparse abortive short regional hair thatmoved caudally to thetail region

32 Effect onHair CoverageArea Theeffects on hair coveragearea of all the treated groups were precisely estimated foreach mouse in both first and second hair growth cycle bygiving them a score from ldquo0rdquo to ldquo4rdquo The maximum hairgrowth score was significantly (119875 lt 0001) increased in micetreated with minoxidil and E alba than control group duringfirst and second hair growth cycles (Figure 2) On the otherhand A radix treated group also consistently effective forinducing hair coverage area (225plusmn082) in first hair cycle buttransient hair coat and progressive hair loss turn to similarscore (125 plusmn 05) with that of control group (125 plusmn 05) insecond hair cycleTherewas no significant difference in termsof the hair coverage area among P ginseng and control group

33 Effect on Hair Length and Hair Density In terms ofthe measurement of hair length the remarkable effect wasobserved in E alba extract treated group A significant

increase (119875 lt 00001) of hair lengthwas observed in both firstand second hair growth cycles while being compared withthat of control group (Figure 3(F)) Similar effect was alsofound while evaluating hair density (Figure 3(G)) Althoughminoxidil treated group showed similar hair density with Ealba treated group (Figure 3(G)) the irregular and deformedhair was found in minoxidil treated mice (Figure 3(B)) Onthe other hand E alba treated mice hair (Figures 3(D) and3(D1015840)) was straight and thicker and smother than othertreatment groups Although the hair length of A radixand P ginseng extract treated mice increased significantlythan control group in earlier hair cycle it became similarwith that of control group afterward (Figure 3(F)) On theother hand in terms of hair density A radix treated groupfound significant effect compared to control but there wasno difference of hair density of P ginseng and controlgroup (Figure 3(G)) Moreover irregularly distributed singledeformed hair shaft was also observed on the skin surface inbothA radix (Figures 3(C) and 3(C1015840)) and P ginseng (Figures3(E) and 3(E1015840)) treatedmice similar to control group (Figures3(A) and 3(A1015840))

34 Effect of Plant Extracts on the Development and Mor-phogenesis of Nude Mice HF Histological examination ofthe skin specimen revealed differences between control andtreated mice in the morphology of the developing HFsOn experimental day 10 minoxidil (Figure 4(B1015840)) A radix(Figure 4(C1015840)) and E alba treated mice (Figure 4(D1015840))showed an early onset of anagen hair morphogenesis com-pared with that of control (Figure 4(A1015840)) confirming themacroscopic observation (Figures 4(B) 4(C) and 4(D)) Onthe other hand P ginseng extract treated mice skin specimen(Figure 4(E1015840)) showed similar stage of hair morphogenesis

6 BioMed Research International

Control

(a)

Minoxidil

(b)

A radix

(c)

E alba

(d)

P ginseng

(e)

0

10

20

30

40

50

60

70

Subcutis Total hair follicle

a

bc

cc

b

a

d

bc

b

cd

Num

ber o

f hai

r fol

licle

s (pe

r fiel

d)

ControlMinoxidilA radix

E albaP ginseng

(f)

Figure 5 Increase in number of HFs in the dorsal skin of nude mice treated with different plant extract Representative photomicrographs ofthe transverse sections of nudemice skin (a) control (b)minoxidil (c)A radix (d) E alba and (e) P ginsengwere stained with hematoxylin-eosin (HampE) (f)The number of hair follicles in subcutis with respect to the total number of HFs per field (119899 = 10) Values aremeanplusmn standarddeviation (SD) The different letters (a b c and d) within a column indicate significant differences (119875 lt 00001) determined by Duncanrsquosmultiple range test Scale bar 100 120583m

with that of control mice Following the guidelines proposedby [19] we determined that the control and P ginsengtreated mice HFs were on stage 6 (Figures 4(A1015840) and 4(E1015840))whereas E alba extract treated mice HFs were clearly at stage8 (Figure 4(D1015840)) Negligible numbers of hair shafts whichemerged from the HFs and moderately atrophic were alsoobserved in the skin ofA radix P ginseng and vehicle treatedmice (Figures 4(C) 4(E) and 4(A)) Moreover the hairshafts of those were defective (often twisted) with dilationof the hair canal and apparently had insufficient structuralrigidity to emerge through the epidermis (Figures 4(A1015840) and4(E1015840)) On the other hand E alba treatedmice seemed tohave a propensity for follicular accumulation of keratin andstructural rigidity to penetrate the epidermis (Figure 4(D))

35 Number of Hair Follicles Histologic assessment wasperformed to investigate the progression of hair follicles innude mice since there was an increase in the number ofhair follicles observed in the deep subcutis during the anagenphase In the representative transverse sections the numbers

of hair follicles were increased significantly in E alba (119875 lt00001) and minoxidil (119875 lt 0005) extract treated groupscompared to the control (Figure 5(f))

36 BrdU Immunohistochemistry To further investigate themolecularmechanismunderlying the ability ofE alba extractto form nude mice hair follicles we performed immunohis-tochemistry with BrdU antibodies of the back skin of nudemice We found that E alba treated mice skin exhibits BrdUpositive keratinocyte in the HFs particularly a single celllayer that surrounds the innermost layer of the outer rootsheath as shown in Figures 6(B) and 6(B1015840) Moreover Ealba treated nude mice exhibited a significant (119875 lt 00001)increase in the number of BrdU-labeled keratinocyte peranagen follicle versus control mice (Figure 6(C)) On theother hand there was no significant difference in the numberof BrdU-labeled epithelial cells per sebaceous gland

37 Identification and Quantification of Wedelolactone in Ealba Extract Additionally HPLC analysis was performed to

BioMed Research International 7

0

5

10

15

20

25

30

HF SG

Num

ber o

f Brd

U p

ositi

ve ce

lls

lowastlowastlowast

(C)

ControlE alba

Control E alba

SBSB

HF

(A) (B)

(A998400) (B998400)

Figure 6 BrdU-labeled proliferating cells in anagen hair folliclesControl (A) and E alba extract treated (B) follicles are nuclear BrdUstaining (Arrows red-brown) Scale bar 100 120583m (B) and 50 120583m ((A)(A1015840) and (B1015840)) Number of BrdU positive cells (C) Data shown aremeans of five replicate samples (lowastlowastlowast119875 lt 0001)

illustrate the methanol extract of E alba (Figure 7) and wasfound to contain principle constituents like wedelolactoneThe existence of the wedelolactone was quantified by 18in methanol extract It has been reported that various bioac-tive phytochemicals found in the E alba extract includingwedelolactone demethylwedelolactone and saponins areresponsible for the main medicinal effects of E alba suchas antitumor activity [13] antihepatitis C virus [12] andneuropharmacological activity [14] and its anticancer effectshows antiproliferative and cytotoxic effects determinedusing MTT assay [15]

00 50 100 150 200 250 300 3500

250

500

750

1000

1250 Standard wedelolactone

(min)

(mV

)

(a)

00 50 100 150 200 250 300 3500

250500750

1000125015001750

Wedelolactone

(min)(m

V)

(b)

Figure 7 HPLC chromatogram (a) Standard wedelolactone (b)HPLC chromatogram of methanol extract of E alba

4 Discussion

Medicinal plants may have potential by identifying activebiological components and their successful use might openreverse pharmacology a new approach to drug discoveryGiven the known therapeutic properties of E alba extractwe have performed enormous screening of different plantextracts on BalbC nude mice model and this has beenused as a unique model to study the hair growth mechanism[20 21]

Among all the plant extracts E alba showed outstandinghair growth promoting potential Topical applicationwas per-formed on the back of nude mice skin daily up to completionof two hair growth generationsThemacroscopic appearanceand the local distribution of hair were precisely observedin each individual group and showed clearly distinguishablefeature The regional distribution of the ldquoabortiverdquo anagenhair pattern in nude strains corresponded to the wave-likepattern of hair generations [22] The hair growth area ofE alba treated mice is distinctly visible and simultaneouslycovered the maximum part of the back In contrast Aradix P ginseng treated mice with increasing age rapidlydeveloped hair loss recognizable from the boundaries ofhair covering However a few uneven hairs are visibleparticularly in nude mice skin surface which referred to asldquoabortive hair growthrdquo [23] During anagen growth phasewe were able to observe a comparable growth pattern inE alba treated mice with other treated and control mice(Figure 1) These phenomena may suggest that E albaimproved the disordered keratinization of nude mouse hairfollicles because original control hair shafts are short and

8 BioMed Research International

irregular in shape suggesting some disordered keratinizationof the HF [24 25] The precise genetic defect of nude miceFoxn1mutation expression involved abnormal keratinizationas the cause for defective cell type in the hair development[26ndash28] Our observation suggests that treatment with E albaacts transiently to normalize keratinocyte of nude mice HFleading to fully formed visible HF with increased hair cover-age area An increase in the number and size of HFs has beenconsidered as an indicator for the transition of hair growthfrom telogen to anagen [9 19]These results suggest that top-ical application of E alba extract could stimulate the forma-tion of hair follicles and induce an earlier anagen phase com-pared to either the control or other treated groups (Figure 4)To elucidate the molecular mechanism underlying the abilityof E alba extract the BrdU immunohistochemistry resultsuggests an enhanced keratinocyte proliferation in anagenhair follicles [29] In our present studiesmacroscopic analysisof hair growth parameters included changing pattern ofnude mice hair growth hair coverage area and hair lengthHistological study of different plant extracts along withcontrol and minoxidil groups was investigated and foundto have fully formed HFs in E alba treated group as wellas increasing the number of HFs The active phase of HFcycling is also accompanied by increase in size and numberof follicles resulting in an enlargement of the subcutis layer[19] Given the vital role of hair restorer efficacy of E albaextract further HPLC analysis was carried out and showedthat wedelolactone and other components exist in methanolextract In this experiment none of the plant extracts causedadverse dermatological effects such as erythema rednessdrying or scaling and dermatitis as it was case withminoxidilgroup at the skin site of nude mice

It has been argued that combination of several con-stituents of a plant extract may act synergistically rather thansingle compound [30] The interaction is possibly due to thebinding of more than one compound at different sites on thesame target or receptor Further detailed clinical trials andstudies will be necessary to investigate what components inE alba extract contribute to its efficacy since whole E albaextract rather than individual components was used here toestablish its biological activity against alopecia

5 Conclusion

E alba extract containing active ingredients provided moresafety and compatibility which has more accessibility inpharmaceutical and cosmetic science and may promote theherbal medicine with minimal or no side effects or toxicitiesfor hair growth therapy Taking together all the investigationindicates that plant extract with suitable vehiclemay be usefulas an alternative topical medicine to minoxidil therapy

Conflict of Interests

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

Acknowledgments

The authors would like to express special thanks to Dr KangJuChoi a great generous person for sharing pearls of wisdomwith them which is gratefully acknowledged This study wassupported by a grant from the DBIO Science and TechnologyCorporation

References

[1] S Tiede J E Kloepper D A Whiting and R Paus ldquoTheldquofollicular trochanterrdquo an epithelial compartment of the humanhair follicle bulge region in need of further characterizationrdquoBritish Journal of Dermatology vol 157 no 5 pp 1013ndash10162007

[2] V A Randall ldquoAndrogens and human hair growthrdquo ClinicalEndocrinology vol 40 no 4 pp 439ndash457 1994

[3] H-J Park N Zhang and D K Park ldquoTopical applicationof Polygonum multiflorum extract induces hair growth ofresting hair follicles through upregulating Shh and 120573-cateninexpression in C57BL6 micerdquo Journal of Ethnopharmacologyvol 135 no 2 pp 369ndash375 2011

[4] C J Girman T Rhodes F R W Lilly et al ldquoEffects of self-perceived hair loss in a community sample of menrdquo Dermatol-ogy vol 197 no 3 pp 223ndash229 1998

[5] S S Rho S J Park S L Hwang et al ldquoThe hair growthpromoting effect of Asiasari radix extract and its molecularregulationrdquo Journal of Dermatological Science vol 38 no 2 pp89ndash97 2005

[6] H Matsuda M Yamazaki Y Asanuma and M Kubo ldquoPromo-tion of hair growth by ginseng radix on culturedmouse vibrissalhair folliclesrdquo Phytotherapy Research vol 17 no 7 pp 797ndash8002003

[7] S Park W-S Shin and J Ho ldquoFructus panax ginseng extractpromotes hair regeneration in C57BL6 micerdquo Journal ofEthnopharmacology vol 138 no 2 pp 340ndash344 2011

[8] R K Roy M Thakur and V K Dixit ldquoHair growth promotingactivity of Eclipta alba in male albino ratsrdquo Archives of Derma-tological Research vol 300 no 7 pp 357ndash364 2008

[9] K Datta A T Singh A Mukherjee B Bhat B Ramesh and AC Burman ldquoEclipta alba extract with potential for hair growthpromoting activityrdquo Journal of Ethnopharmacology vol 124 no3 pp 450ndash456 2009

[10] H Wagner B Geyer Y Kiso H Hikino and G S RaoldquoCoumestans as the main active principles of the liver drugsEclipta alba and Wedelia calendulaceardquo Planta Medica vol 5pp 370ndash374 1986

[11] A M S Pereira B W Bertoni A Menezes Jr P S Pereiraand S C Franca ldquoSoil pH and production of biomass andwedelolactone in field grown Eclipta albardquo Journal of HerbsSpices and Medicinal Plants vol 6 no 1 pp 43ndash48 1998

[12] D Manvar M Mishra S Kumar and V N Pandey ldquoIdentifi-cation and evaluation of anti Hepatitis C virus phytochemicalsfrom Eclipta albardquo Journal of Ethnopharmacology vol 144 no3 pp 545ndash554 2012

[13] Q-M Liu H-Y Zhao X-K Zhong and J-G Jiang ldquoEcliptaprostrata L phytochemicals isolation structure elucidationand their antitumor activityrdquo Food andChemical Toxicology vol50 no 11 pp 4016ndash4022 2012

[14] V DThakur and S AMengi ldquoNeuropharmacological profile ofEclipta alba (Linn) Hasskrdquo Journal of Ethnopharmacology vol102 no 1 pp 23ndash31 2005

BioMed Research International 9

[15] H Chaudhary V Dhuna J Singh S S Kamboj and S SeshadrildquoEvaluation of hydro-alcoholic extract of Eclipta alba for itsanticancer potential an in vitro studyrdquo Journal of Ethnophar-macology vol 136 no 2 pp 363ndash367 2011

[16] L Mecklenburg B Tychsen and R Paus ldquoLearning fromnudity lessons from the nude phenotyperdquo Experimental Der-matology vol 14 no 11 pp 797ndash810 2005

[17] G A Simon and H I Maibach ldquoRelevance of hairless mouse asan experimental model of percutaneous penetration in manrdquoSkin Pharmacology and Applied Skin Physiology vol 11 no 2pp 80ndash86 1998

[18] R Toll U Jacobi H Richter J Lademann H Schaefer and UBlume-Peytavi ldquoPenetration profile of microspheres in follicu-lar targeting of terminal hair folliclesrdquo Journal of InvestigativeDermatology vol 123 no 1 pp 168ndash176 2004

[19] R Paus S Muller-Rover C van der Veen et al ldquoA compre-hensive guide for the recognition and classification of distinctstages of hair follicle morphogenesisrdquo Journal of InvestigativeDermatology vol 113 no 4 pp 523ndash532 1999

[20] M Sawada N Terada H Taniguchi R Tateishi and YMori ldquoCyclosporin A stimulates hair growth in nude micerdquoLaboratory Investigation vol 56 no 6 pp 684ndash686 1987

[21] S Watanabe A Mochizuki K Wagatsuma M Kobayashi YKawa and H Takahashi ldquoHair growth on nude mice due tocyclosporin Ardquo The Journal of Dermatology vol 18 no 12 pp714ndash719 1991

[22] K Militzer ldquoHair growth pattern in nude micerdquo Cells TissuesOrgans vol 168 no 4 pp 285ndash294 2001

[23] U Iversen and O H Iversen ldquoCycles of hair growth in hairlessmicerdquoActa PathologicaMicrobiologica Scandinavica vol 69 no1 pp 50ndash62 1967

[24] R H Rigdon and A A Packchanian ldquoHistologic study of theskin of congenitally athymic ldquonuderdquo micerdquo Texas Reports onBiology amp Medicine vol 32 no 3-4 pp 711ndash723 1974

[25] Y Hozumi T Imaizumi and S Kondo ldquoEffect of cyclosporinon hair-existing area of nude micerdquo Journal of DermatologicalScience vol 7 pp S33ndashS38 1994

[26] S P Flanagan ldquoldquoNuderdquo a new hairless gene with pleiotropiceffects in the mouserdquo Genetical Research vol 8 no 3 pp 295ndash309 1966

[27] J L Brissette J Li J Kamimura D Lee and G P Dotto ldquoTheproduct of the mouse nude locus whn regulates the balancebetween epithelial cell growth and differentiationrdquo Genes andDevelopment vol 10 no 17 pp 2212ndash2221 1996

[28] L Mecklenburg R Paus Z Halata L S Bechtold P Fleckmanand J P Sundberg ldquoFOXN1 is critical for onycholemmalterminal differentiation in nude (Foxn1119899119906) micerdquo Journal ofInvestigative Dermatology vol 123 no 6 pp 1001ndash1011 2004

[29] H Pinkus T Iwasaki and Y Mishima ldquoOuter root sheathkeratinization in anagen and catagen of the mammalian hairfollicle A seventh distinct type of keratinization in the hairfollicle trichilemmal keratinizationrdquo Journal of Anatomy vol133 no 1 pp 19ndash35 1981

[30] H Wagner and G Ulrich-Merzenich ldquoSynergy researchapproaching a new generation of phytopharmaceuticalsrdquo Phy-tomedicine vol 16 no 2-3 pp 97ndash110 2009

Submit your manuscripts athttpwwwhindawicom

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BioMed Research International

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Pharmaceutics

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MEDIATORSINFLAMMATION

of

2 BioMed Research International

Day 0 Day 7 Day 12 Day 16

Con

trol

(a)

Day 0 Day 7 Day 12 Day 16

Min

oxid

il

(b)

Day 0 Day 7 Day 12 Day 16

A ra

dix

(c)

Day 0 Day 7 Day 12 Day 16

E a

lba

(d)

Day 0 Day 7 Day 12 Day 16

P gi

nsen

g

(e)

Figure 1 Stimulation of hair growth by different natural extract in nude mice Specific concentrations of different extract in vehicleformulation were daily applied topically (119899 = 5) on the back of nude mice skin (a) control (b) minoxidil (c) A radix (d) E alba and(e) P ginseng during one hair growth phase

hair growth as well In particular only little candidate plantplays great role to enhance hair growth whose efficaciesare now under investigation Therefore recent research wasunder taken to investigate of some known plants those weretraditionally acclaimed and purported in oriental medicinesuch as A radix [5] P ginseng [6 7] and E alba [8 9] exertto promote hair growth

Moreover E alba has also been reported with multiplemedicinal uses and is known for numerous biological activi-ties It is an enormous source of several secondary metabo-lites such as polypeptides polyacetylenes and triterpenes[10] flavonoids phytosterols and coumestans [11] Phyto-chemical coumestans represent an important class of naturaloxygenated aromatic compound including wedelolactoneand demethylwedelolactone and saponins responsible forthe main medicinal effects of E alba such as its antihepatitisC virus [12] antitumor activity [13] and neuropharmaco-logical activity [14] and its anticancer effect shows antipro-liferative and cytotoxic effects determined using MTT assay[15]

Taken together this bioresource information we under-take nude mice as a model to get more precise result for hair

biology from different well-known medicinal plant extractsas these mice express the role of Foxn1 in mammalian skinbiology responsible for structural abnormality of hair shaftdue to abnormal keratinization [16]Moreover nudemice arealso frequently used in skin pharmacology to serve for skinpenetration assays [17] as the follicular penetration route actsas an entry pathway for compounds into the skin [18]

E alba is widely reported traditional medicine and well-being functional food thus it prompted us to evaluate hairgrowth promoting activity alongwith other plantsThereforethis approach may incorporate some novel findings that maypromote us to explore extensive bioactive compounds fromcrude extract for prevention of hair loss as well as enhancinghair growth

2 Materials and Methods

21 Plant Materials and Preparation of Extraction Aerialparts (leaves stems and blooms) of E alba and dried rootsof A radix and P ginseng were purchased from the JecheonMedicinal Herb Association South Korea All the samples

BioMed Research International 3

Table 1 Botanical origins plant parts and percentage yields of investigated specimens

Botanical name (family) Chinese name Common name Plant part Extraction yield ( ww)Eclipta alba (Asteraceae) Han lian cao False daisy Leaves stems and blooms 1240Asiasarum sieboldii (Aristolochiaceae) Shashin Chinese wild ginger root Roots 752Panax ginseng (Araliaceae) Hong-Shen Red ginseng Roots 3833

0

1

2

3

4

1st cycle 2nd clycle Mean of cycles

ControlMinoxidilA radix

E albaP ginseng

a

ab

ab

a

b

a

ab

b

a

b

a

b

ab ab

a

Hai

r cov

erag

e are

a (sc

ale0

ndash4)

Figure 2 Effect of different plant extract on hair coverage areain athymic nude mice Mice were evaluated on a scale of ldquo0ndash4rdquoparameters as follows ldquo0rdquo = complete alopecia ldquo1rdquo = lt25 haircoverage ldquo2rdquo = 25ndash50 hair coverage ldquo3rdquo = 50ndash75 hair coverageldquo4rdquo = full hair coverage The different letters (a and b) within acolumn indicate significant differences (119875 lt 005) determined byDuncanrsquos multiple range test

were authenticated by Associate Professor Ki Hwan Bae (Col-lege of Pharmacy ChungnamNational University) where thevoucher specimens were deposited The dried plant sam-pleswere ground into powder (500 g) and extracted with 95methanol at 60∘C The methanol extract was concentratedand dried in a vacuum evaporator under reduced pressurebelow 60∘CThe resulting residue was weighed and dissolvedin a vehicle mixture (propylene glycol ethanol dimethylsulfoxide 67 30 3 vv)The extract yieldswere determinedas ldquordquo of dry crude extracts (Table 1)

22 Chromatographic Analysis HPLC-grade acetonitrile waspurchased from Merck Co (Merck Darmstadt Germany)Deionized water was purified by Milli-Q system (MilliporeBedford MA USA) Other chemicals were of reagent gradeStandard wedelolactone powder was purchased from theSigma-AldrichCoThepurity of the standardwas over 98 asindicated by the manufacturer Calibration standard sampleswere prepared from the stock solutions by adding 05ndash15mgmL of wedelolactone inMeOH to obtain appropriateddilutions which were filtered through Millipore membranebefore use Chromatographic analysis was performed onthe Shimadzu (Japan) HPLC apparatus with a column of

Discovery C18 (ODS 25 cm times 46mm 5 120583m Supelco USA)and a Guard Column Security Guard Cartridge system (Phe-nomenex USA) The mobile phase consisted of acetonitrilewater (ACN vv) at a flow rate of 1mLmin as follows20ndash45 (up to 15min) 45ndash100 (up to 30min) and 100(up to 40min)Themethod was performed with an injectionvolume of 20 120583L and detection wavelength at 365 nm

23 Toxicity Studies Toxicity studies were carried out byapplying the extract to the nude mice skin in concentrationsof up to 5 for 7 days and did not show any toxic side effectslike erythema on skin surface Thus the prepared extractswere considered safe for topical application on nude mice

24 Balbc Nude Mice Athymic Balbc male nude miceat 7 weeks of age were purchased from Dae-Han Biolink(Eumsung Korea) and maintained under 12 h light darkperiods at 24 plusmn 2∘C and were housed and handled underaseptic conditions Experiment was performed in accordancewith approved institutional protocols by the InstitutionalAnimal Care and Use Committee (IACUC) ChungnamNationalUniversity Daejeon SouthKoreaThe authorizationcode number is CNU-00244

25 Treatment Protocol of Different Natural Extracts on NudeMice Five animals were allocated to five randomized groupsfor evaluation of hair growth and hair changing pattern ofnude mice Topical application was performed daily with asolution of A radix E alba and P ginseng extracts (25) ina vehicle formulation andorminoxidil (2) until completionof two full hair growth cycles

26 Measurement of Hair Density and Length After dailytreatment in each hair growth cycle the images from specificregions of neck back and hip were captured two times perweek using KONG Bom-Viewer Plus (Seoul Korea) Aboutsix images of 80times and 200times were exploited to measure thehair length and hair density respectively using Bom-ViewerImage Analyzer

27 Evaluation of Hair Existing Area Thehair existing area ofnude mice was systematically evaluated for each mouse threetimes per week At this time point mice were scored on ascale of ldquo0rdquo to ldquo4rdquo for percent body coverage of hair ldquo0rdquo =complete alopecia ldquo1rdquo = lt25 hair coverage ldquo2rdquo = 25ndash50hair coverage ldquo3rdquo = 50ndash75 hair coverage ldquo4rdquo = normal hairdensity and full hair coverage

28 Histologic Assessment of Hair Growth Mice were sac-rificed in anagen phase and skin samples were fixed in

4 BioMed Research International

Control Minoxidil A radix E alba P ginseng1

st ha

ir cy

cle2

nd h

air c

ycle

(A) (B) (C) (D) (E)

(A998400) (B998400) (C998400) (D998400) (E998400)

0

10

20

30

40

50

60

70

80

90

1st hair cycle 2nd hair cycle

a

c

a

b

c c

a

b

a

cH

air d

ensit

y (36

mm

2ar

ea)

ControlMinoxidilA radix

E albaP ginseng

000

020

040

060

080

100

120

140

160

180

200

1st cycle 2nd cycle

d

b

c

a b

a

b

c

b

c

Hai

r len

gth

(mm

)

ControlMinoxidilA radix

E albaP ginseng

(G)(F)

Figure 3 Evaluation of hair length and hair density in cycles Image of nude mice hair on skin surface treated with control ((A) (A1015840))minoxidil ((B) (B1015840)) A radix ((C) (C1015840)) E alba ((D) (D1015840)) and P ginseng ((E) (E1015840)) in first and second hair cycles Hair length (F) and hairdensity (G) shown in column The different letters (a b c and d) within a column indicate significant differences (119875 lt 00001) determinedby Duncanrsquos multiple range test Digital images were takenby KONG Bom-Viewer Plus with 80times magnification lens Hair length and hairdensity were measured using Bom-Viewer Image analyzer (119899 = 10)

10 buffered formalin for histological analysis Paraffin-embedded 4 120583m sections were stained with hematoxylinand eosin (HampE) The nude mice HF morphology andthe number of HFs were evaluated microscopically in 5fields per section of the dorsal skin at a magnification oftimes100

29 BrdU Immunohistochemistry BrdU labeling cell prolif-eration was detected by intraperitoneal injection of bromod-eoxyuridine (BrdU Sigma USA) with 50mgkg body weightat a concentration of 5mgmL in PBS BrdU incorporationwas detected by IHC staining of paraffin-embedded sec-tions with mouse anti-BrdU primary antibody (Santa CruzBiotechnology Santa Cruz CA USA) Skin sections thatwere not incubated with primary antibodies were used as

a negative control Detection of follicular keratinocyte BrdUlabeling was done at magnification times100 and times400

210 Statistical Analysis Statistical analysis was performedusing SAS Systems ver 93 and analysis of variance (ANOVA)was used for comparisons among all groups All data areexpressed as means plusmn SD unless otherwise is noted Aprobability level of 5was considered significant Differencesbetween two groups were analyzed by t-test ANOVA wasfollowed after Duncanrsquos multiple comparison tests

3 Results

31 Differences in Activity of Plant Extracts on ChangingHair Growth Pattern of Nude Mice After daily treatment

BioMed Research International 5

Control Minoxidil A radix E alba P ginseng

(A) (B) (C) (D) (E)

(A998400) (B998400) (C998400) (D998400) (E998400)

Figure 4Histological features of the skin specimen of different extract andor vehicle control treatedmiceMacroscopic observation of hair inthe skin surface of (A) control (B)minoxidil (C)A radix (D) E alba and (E) P ginseng in early hair existing phase Microscopic observationof longitudinal sections of (A1015840) control (B1015840) minoxidil (C1015840) A radix (D1015840) E alba and (E1015840) P ginseng treated mice skin was harvested andfixed in buffered formalin for routine hematoxylin and eosin (HampE) stained histology (119899 = 7) The broken line indicates defective HFs at thelevel of sebaceous gland ((A1015840) (B1015840) (C1015840) and (E1015840)) Note that the straight hair shafts that emerge from the skin surface of minoxidil and Ealba treated mouse ((B) (D) and Arrows (B1015840) (D1015840)) Scale bar 100 120583m

hair growth was documented on experimental day 7 Sys-tematic monitoring of hair growth showed that A radix(Figure 1(c)) and vehicle treated control mice (Figure 1(a))exhibit extremely sparse and transient hair coat On the otherhand minoxidil and E alba extract treated mice hair growthfirst documented from the frontal part of the head as earlyas 7 days and became dense thicker and longer that evenlyextended to the shoulder and posterior part of the backduring experimental day 12 The maximum hair coat wasevident in E alba extract treated group on experimental day16 (Figures 1(d) and 1(b)) In contrast A radix (Figure 1(c))and P ginseng (Figure 1(e)) treated mice found rapid hair losswith increasing time point and eventually remained only fewsparse abortive short regional hair thatmoved caudally to thetail region

32 Effect onHair CoverageArea Theeffects on hair coveragearea of all the treated groups were precisely estimated foreach mouse in both first and second hair growth cycle bygiving them a score from ldquo0rdquo to ldquo4rdquo The maximum hairgrowth score was significantly (119875 lt 0001) increased in micetreated with minoxidil and E alba than control group duringfirst and second hair growth cycles (Figure 2) On the otherhand A radix treated group also consistently effective forinducing hair coverage area (225plusmn082) in first hair cycle buttransient hair coat and progressive hair loss turn to similarscore (125 plusmn 05) with that of control group (125 plusmn 05) insecond hair cycleTherewas no significant difference in termsof the hair coverage area among P ginseng and control group

33 Effect on Hair Length and Hair Density In terms ofthe measurement of hair length the remarkable effect wasobserved in E alba extract treated group A significant

increase (119875 lt 00001) of hair lengthwas observed in both firstand second hair growth cycles while being compared withthat of control group (Figure 3(F)) Similar effect was alsofound while evaluating hair density (Figure 3(G)) Althoughminoxidil treated group showed similar hair density with Ealba treated group (Figure 3(G)) the irregular and deformedhair was found in minoxidil treated mice (Figure 3(B)) Onthe other hand E alba treated mice hair (Figures 3(D) and3(D1015840)) was straight and thicker and smother than othertreatment groups Although the hair length of A radixand P ginseng extract treated mice increased significantlythan control group in earlier hair cycle it became similarwith that of control group afterward (Figure 3(F)) On theother hand in terms of hair density A radix treated groupfound significant effect compared to control but there wasno difference of hair density of P ginseng and controlgroup (Figure 3(G)) Moreover irregularly distributed singledeformed hair shaft was also observed on the skin surface inbothA radix (Figures 3(C) and 3(C1015840)) and P ginseng (Figures3(E) and 3(E1015840)) treatedmice similar to control group (Figures3(A) and 3(A1015840))

34 Effect of Plant Extracts on the Development and Mor-phogenesis of Nude Mice HF Histological examination ofthe skin specimen revealed differences between control andtreated mice in the morphology of the developing HFsOn experimental day 10 minoxidil (Figure 4(B1015840)) A radix(Figure 4(C1015840)) and E alba treated mice (Figure 4(D1015840))showed an early onset of anagen hair morphogenesis com-pared with that of control (Figure 4(A1015840)) confirming themacroscopic observation (Figures 4(B) 4(C) and 4(D)) Onthe other hand P ginseng extract treated mice skin specimen(Figure 4(E1015840)) showed similar stage of hair morphogenesis

6 BioMed Research International

Control

(a)

Minoxidil

(b)

A radix

(c)

E alba

(d)

P ginseng

(e)

0

10

20

30

40

50

60

70

Subcutis Total hair follicle

a

bc

cc

b

a

d

bc

b

cd

Num

ber o

f hai

r fol

licle

s (pe

r fiel

d)

ControlMinoxidilA radix

E albaP ginseng

(f)

Figure 5 Increase in number of HFs in the dorsal skin of nude mice treated with different plant extract Representative photomicrographs ofthe transverse sections of nudemice skin (a) control (b)minoxidil (c)A radix (d) E alba and (e) P ginsengwere stained with hematoxylin-eosin (HampE) (f)The number of hair follicles in subcutis with respect to the total number of HFs per field (119899 = 10) Values aremeanplusmn standarddeviation (SD) The different letters (a b c and d) within a column indicate significant differences (119875 lt 00001) determined by Duncanrsquosmultiple range test Scale bar 100 120583m

with that of control mice Following the guidelines proposedby [19] we determined that the control and P ginsengtreated mice HFs were on stage 6 (Figures 4(A1015840) and 4(E1015840))whereas E alba extract treated mice HFs were clearly at stage8 (Figure 4(D1015840)) Negligible numbers of hair shafts whichemerged from the HFs and moderately atrophic were alsoobserved in the skin ofA radix P ginseng and vehicle treatedmice (Figures 4(C) 4(E) and 4(A)) Moreover the hairshafts of those were defective (often twisted) with dilationof the hair canal and apparently had insufficient structuralrigidity to emerge through the epidermis (Figures 4(A1015840) and4(E1015840)) On the other hand E alba treatedmice seemed tohave a propensity for follicular accumulation of keratin andstructural rigidity to penetrate the epidermis (Figure 4(D))

35 Number of Hair Follicles Histologic assessment wasperformed to investigate the progression of hair follicles innude mice since there was an increase in the number ofhair follicles observed in the deep subcutis during the anagenphase In the representative transverse sections the numbers

of hair follicles were increased significantly in E alba (119875 lt00001) and minoxidil (119875 lt 0005) extract treated groupscompared to the control (Figure 5(f))

36 BrdU Immunohistochemistry To further investigate themolecularmechanismunderlying the ability ofE alba extractto form nude mice hair follicles we performed immunohis-tochemistry with BrdU antibodies of the back skin of nudemice We found that E alba treated mice skin exhibits BrdUpositive keratinocyte in the HFs particularly a single celllayer that surrounds the innermost layer of the outer rootsheath as shown in Figures 6(B) and 6(B1015840) Moreover Ealba treated nude mice exhibited a significant (119875 lt 00001)increase in the number of BrdU-labeled keratinocyte peranagen follicle versus control mice (Figure 6(C)) On theother hand there was no significant difference in the numberof BrdU-labeled epithelial cells per sebaceous gland

37 Identification and Quantification of Wedelolactone in Ealba Extract Additionally HPLC analysis was performed to

BioMed Research International 7

0

5

10

15

20

25

30

HF SG

Num

ber o

f Brd

U p

ositi

ve ce

lls

lowastlowastlowast

(C)

ControlE alba

Control E alba

SBSB

HF

(A) (B)

(A998400) (B998400)

Figure 6 BrdU-labeled proliferating cells in anagen hair folliclesControl (A) and E alba extract treated (B) follicles are nuclear BrdUstaining (Arrows red-brown) Scale bar 100 120583m (B) and 50 120583m ((A)(A1015840) and (B1015840)) Number of BrdU positive cells (C) Data shown aremeans of five replicate samples (lowastlowastlowast119875 lt 0001)

illustrate the methanol extract of E alba (Figure 7) and wasfound to contain principle constituents like wedelolactoneThe existence of the wedelolactone was quantified by 18in methanol extract It has been reported that various bioac-tive phytochemicals found in the E alba extract includingwedelolactone demethylwedelolactone and saponins areresponsible for the main medicinal effects of E alba suchas antitumor activity [13] antihepatitis C virus [12] andneuropharmacological activity [14] and its anticancer effectshows antiproliferative and cytotoxic effects determinedusing MTT assay [15]

00 50 100 150 200 250 300 3500

250

500

750

1000

1250 Standard wedelolactone

(min)

(mV

)

(a)

00 50 100 150 200 250 300 3500

250500750

1000125015001750

Wedelolactone

(min)(m

V)

(b)

Figure 7 HPLC chromatogram (a) Standard wedelolactone (b)HPLC chromatogram of methanol extract of E alba

4 Discussion

Medicinal plants may have potential by identifying activebiological components and their successful use might openreverse pharmacology a new approach to drug discoveryGiven the known therapeutic properties of E alba extractwe have performed enormous screening of different plantextracts on BalbC nude mice model and this has beenused as a unique model to study the hair growth mechanism[20 21]

Among all the plant extracts E alba showed outstandinghair growth promoting potential Topical applicationwas per-formed on the back of nude mice skin daily up to completionof two hair growth generationsThemacroscopic appearanceand the local distribution of hair were precisely observedin each individual group and showed clearly distinguishablefeature The regional distribution of the ldquoabortiverdquo anagenhair pattern in nude strains corresponded to the wave-likepattern of hair generations [22] The hair growth area ofE alba treated mice is distinctly visible and simultaneouslycovered the maximum part of the back In contrast Aradix P ginseng treated mice with increasing age rapidlydeveloped hair loss recognizable from the boundaries ofhair covering However a few uneven hairs are visibleparticularly in nude mice skin surface which referred to asldquoabortive hair growthrdquo [23] During anagen growth phasewe were able to observe a comparable growth pattern inE alba treated mice with other treated and control mice(Figure 1) These phenomena may suggest that E albaimproved the disordered keratinization of nude mouse hairfollicles because original control hair shafts are short and

8 BioMed Research International

irregular in shape suggesting some disordered keratinizationof the HF [24 25] The precise genetic defect of nude miceFoxn1mutation expression involved abnormal keratinizationas the cause for defective cell type in the hair development[26ndash28] Our observation suggests that treatment with E albaacts transiently to normalize keratinocyte of nude mice HFleading to fully formed visible HF with increased hair cover-age area An increase in the number and size of HFs has beenconsidered as an indicator for the transition of hair growthfrom telogen to anagen [9 19]These results suggest that top-ical application of E alba extract could stimulate the forma-tion of hair follicles and induce an earlier anagen phase com-pared to either the control or other treated groups (Figure 4)To elucidate the molecular mechanism underlying the abilityof E alba extract the BrdU immunohistochemistry resultsuggests an enhanced keratinocyte proliferation in anagenhair follicles [29] In our present studiesmacroscopic analysisof hair growth parameters included changing pattern ofnude mice hair growth hair coverage area and hair lengthHistological study of different plant extracts along withcontrol and minoxidil groups was investigated and foundto have fully formed HFs in E alba treated group as wellas increasing the number of HFs The active phase of HFcycling is also accompanied by increase in size and numberof follicles resulting in an enlargement of the subcutis layer[19] Given the vital role of hair restorer efficacy of E albaextract further HPLC analysis was carried out and showedthat wedelolactone and other components exist in methanolextract In this experiment none of the plant extracts causedadverse dermatological effects such as erythema rednessdrying or scaling and dermatitis as it was case withminoxidilgroup at the skin site of nude mice

It has been argued that combination of several con-stituents of a plant extract may act synergistically rather thansingle compound [30] The interaction is possibly due to thebinding of more than one compound at different sites on thesame target or receptor Further detailed clinical trials andstudies will be necessary to investigate what components inE alba extract contribute to its efficacy since whole E albaextract rather than individual components was used here toestablish its biological activity against alopecia

5 Conclusion

E alba extract containing active ingredients provided moresafety and compatibility which has more accessibility inpharmaceutical and cosmetic science and may promote theherbal medicine with minimal or no side effects or toxicitiesfor hair growth therapy Taking together all the investigationindicates that plant extract with suitable vehiclemay be usefulas an alternative topical medicine to minoxidil therapy

Conflict of Interests

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

Acknowledgments

The authors would like to express special thanks to Dr KangJuChoi a great generous person for sharing pearls of wisdomwith them which is gratefully acknowledged This study wassupported by a grant from the DBIO Science and TechnologyCorporation

References

[1] S Tiede J E Kloepper D A Whiting and R Paus ldquoTheldquofollicular trochanterrdquo an epithelial compartment of the humanhair follicle bulge region in need of further characterizationrdquoBritish Journal of Dermatology vol 157 no 5 pp 1013ndash10162007

[2] V A Randall ldquoAndrogens and human hair growthrdquo ClinicalEndocrinology vol 40 no 4 pp 439ndash457 1994

[3] H-J Park N Zhang and D K Park ldquoTopical applicationof Polygonum multiflorum extract induces hair growth ofresting hair follicles through upregulating Shh and 120573-cateninexpression in C57BL6 micerdquo Journal of Ethnopharmacologyvol 135 no 2 pp 369ndash375 2011

[4] C J Girman T Rhodes F R W Lilly et al ldquoEffects of self-perceived hair loss in a community sample of menrdquo Dermatol-ogy vol 197 no 3 pp 223ndash229 1998

[5] S S Rho S J Park S L Hwang et al ldquoThe hair growthpromoting effect of Asiasari radix extract and its molecularregulationrdquo Journal of Dermatological Science vol 38 no 2 pp89ndash97 2005

[6] H Matsuda M Yamazaki Y Asanuma and M Kubo ldquoPromo-tion of hair growth by ginseng radix on culturedmouse vibrissalhair folliclesrdquo Phytotherapy Research vol 17 no 7 pp 797ndash8002003

[7] S Park W-S Shin and J Ho ldquoFructus panax ginseng extractpromotes hair regeneration in C57BL6 micerdquo Journal ofEthnopharmacology vol 138 no 2 pp 340ndash344 2011

[8] R K Roy M Thakur and V K Dixit ldquoHair growth promotingactivity of Eclipta alba in male albino ratsrdquo Archives of Derma-tological Research vol 300 no 7 pp 357ndash364 2008

[9] K Datta A T Singh A Mukherjee B Bhat B Ramesh and AC Burman ldquoEclipta alba extract with potential for hair growthpromoting activityrdquo Journal of Ethnopharmacology vol 124 no3 pp 450ndash456 2009

[10] H Wagner B Geyer Y Kiso H Hikino and G S RaoldquoCoumestans as the main active principles of the liver drugsEclipta alba and Wedelia calendulaceardquo Planta Medica vol 5pp 370ndash374 1986

[11] A M S Pereira B W Bertoni A Menezes Jr P S Pereiraand S C Franca ldquoSoil pH and production of biomass andwedelolactone in field grown Eclipta albardquo Journal of HerbsSpices and Medicinal Plants vol 6 no 1 pp 43ndash48 1998

[12] D Manvar M Mishra S Kumar and V N Pandey ldquoIdentifi-cation and evaluation of anti Hepatitis C virus phytochemicalsfrom Eclipta albardquo Journal of Ethnopharmacology vol 144 no3 pp 545ndash554 2012

[13] Q-M Liu H-Y Zhao X-K Zhong and J-G Jiang ldquoEcliptaprostrata L phytochemicals isolation structure elucidationand their antitumor activityrdquo Food andChemical Toxicology vol50 no 11 pp 4016ndash4022 2012

[14] V DThakur and S AMengi ldquoNeuropharmacological profile ofEclipta alba (Linn) Hasskrdquo Journal of Ethnopharmacology vol102 no 1 pp 23ndash31 2005

BioMed Research International 9

[15] H Chaudhary V Dhuna J Singh S S Kamboj and S SeshadrildquoEvaluation of hydro-alcoholic extract of Eclipta alba for itsanticancer potential an in vitro studyrdquo Journal of Ethnophar-macology vol 136 no 2 pp 363ndash367 2011

[16] L Mecklenburg B Tychsen and R Paus ldquoLearning fromnudity lessons from the nude phenotyperdquo Experimental Der-matology vol 14 no 11 pp 797ndash810 2005

[17] G A Simon and H I Maibach ldquoRelevance of hairless mouse asan experimental model of percutaneous penetration in manrdquoSkin Pharmacology and Applied Skin Physiology vol 11 no 2pp 80ndash86 1998

[18] R Toll U Jacobi H Richter J Lademann H Schaefer and UBlume-Peytavi ldquoPenetration profile of microspheres in follicu-lar targeting of terminal hair folliclesrdquo Journal of InvestigativeDermatology vol 123 no 1 pp 168ndash176 2004

[19] R Paus S Muller-Rover C van der Veen et al ldquoA compre-hensive guide for the recognition and classification of distinctstages of hair follicle morphogenesisrdquo Journal of InvestigativeDermatology vol 113 no 4 pp 523ndash532 1999

[20] M Sawada N Terada H Taniguchi R Tateishi and YMori ldquoCyclosporin A stimulates hair growth in nude micerdquoLaboratory Investigation vol 56 no 6 pp 684ndash686 1987

[21] S Watanabe A Mochizuki K Wagatsuma M Kobayashi YKawa and H Takahashi ldquoHair growth on nude mice due tocyclosporin Ardquo The Journal of Dermatology vol 18 no 12 pp714ndash719 1991

[22] K Militzer ldquoHair growth pattern in nude micerdquo Cells TissuesOrgans vol 168 no 4 pp 285ndash294 2001

[23] U Iversen and O H Iversen ldquoCycles of hair growth in hairlessmicerdquoActa PathologicaMicrobiologica Scandinavica vol 69 no1 pp 50ndash62 1967

[24] R H Rigdon and A A Packchanian ldquoHistologic study of theskin of congenitally athymic ldquonuderdquo micerdquo Texas Reports onBiology amp Medicine vol 32 no 3-4 pp 711ndash723 1974

[25] Y Hozumi T Imaizumi and S Kondo ldquoEffect of cyclosporinon hair-existing area of nude micerdquo Journal of DermatologicalScience vol 7 pp S33ndashS38 1994

[26] S P Flanagan ldquoldquoNuderdquo a new hairless gene with pleiotropiceffects in the mouserdquo Genetical Research vol 8 no 3 pp 295ndash309 1966

[27] J L Brissette J Li J Kamimura D Lee and G P Dotto ldquoTheproduct of the mouse nude locus whn regulates the balancebetween epithelial cell growth and differentiationrdquo Genes andDevelopment vol 10 no 17 pp 2212ndash2221 1996

[28] L Mecklenburg R Paus Z Halata L S Bechtold P Fleckmanand J P Sundberg ldquoFOXN1 is critical for onycholemmalterminal differentiation in nude (Foxn1119899119906) micerdquo Journal ofInvestigative Dermatology vol 123 no 6 pp 1001ndash1011 2004

[29] H Pinkus T Iwasaki and Y Mishima ldquoOuter root sheathkeratinization in anagen and catagen of the mammalian hairfollicle A seventh distinct type of keratinization in the hairfollicle trichilemmal keratinizationrdquo Journal of Anatomy vol133 no 1 pp 19ndash35 1981

[30] H Wagner and G Ulrich-Merzenich ldquoSynergy researchapproaching a new generation of phytopharmaceuticalsrdquo Phy-tomedicine vol 16 no 2-3 pp 97ndash110 2009

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

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Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

BioMed Research International 3

Table 1 Botanical origins plant parts and percentage yields of investigated specimens

Botanical name (family) Chinese name Common name Plant part Extraction yield ( ww)Eclipta alba (Asteraceae) Han lian cao False daisy Leaves stems and blooms 1240Asiasarum sieboldii (Aristolochiaceae) Shashin Chinese wild ginger root Roots 752Panax ginseng (Araliaceae) Hong-Shen Red ginseng Roots 3833

0

1

2

3

4

1st cycle 2nd clycle Mean of cycles

ControlMinoxidilA radix

E albaP ginseng

a

ab

ab

a

b

a

ab

b

a

b

a

b

ab ab

a

Hai

r cov

erag

e are

a (sc

ale0

ndash4)

Figure 2 Effect of different plant extract on hair coverage areain athymic nude mice Mice were evaluated on a scale of ldquo0ndash4rdquoparameters as follows ldquo0rdquo = complete alopecia ldquo1rdquo = lt25 haircoverage ldquo2rdquo = 25ndash50 hair coverage ldquo3rdquo = 50ndash75 hair coverageldquo4rdquo = full hair coverage The different letters (a and b) within acolumn indicate significant differences (119875 lt 005) determined byDuncanrsquos multiple range test

were authenticated by Associate Professor Ki Hwan Bae (Col-lege of Pharmacy ChungnamNational University) where thevoucher specimens were deposited The dried plant sam-pleswere ground into powder (500 g) and extracted with 95methanol at 60∘C The methanol extract was concentratedand dried in a vacuum evaporator under reduced pressurebelow 60∘CThe resulting residue was weighed and dissolvedin a vehicle mixture (propylene glycol ethanol dimethylsulfoxide 67 30 3 vv)The extract yieldswere determinedas ldquordquo of dry crude extracts (Table 1)

22 Chromatographic Analysis HPLC-grade acetonitrile waspurchased from Merck Co (Merck Darmstadt Germany)Deionized water was purified by Milli-Q system (MilliporeBedford MA USA) Other chemicals were of reagent gradeStandard wedelolactone powder was purchased from theSigma-AldrichCoThepurity of the standardwas over 98 asindicated by the manufacturer Calibration standard sampleswere prepared from the stock solutions by adding 05ndash15mgmL of wedelolactone inMeOH to obtain appropriateddilutions which were filtered through Millipore membranebefore use Chromatographic analysis was performed onthe Shimadzu (Japan) HPLC apparatus with a column of

Discovery C18 (ODS 25 cm times 46mm 5 120583m Supelco USA)and a Guard Column Security Guard Cartridge system (Phe-nomenex USA) The mobile phase consisted of acetonitrilewater (ACN vv) at a flow rate of 1mLmin as follows20ndash45 (up to 15min) 45ndash100 (up to 30min) and 100(up to 40min)Themethod was performed with an injectionvolume of 20 120583L and detection wavelength at 365 nm

23 Toxicity Studies Toxicity studies were carried out byapplying the extract to the nude mice skin in concentrationsof up to 5 for 7 days and did not show any toxic side effectslike erythema on skin surface Thus the prepared extractswere considered safe for topical application on nude mice

24 Balbc Nude Mice Athymic Balbc male nude miceat 7 weeks of age were purchased from Dae-Han Biolink(Eumsung Korea) and maintained under 12 h light darkperiods at 24 plusmn 2∘C and were housed and handled underaseptic conditions Experiment was performed in accordancewith approved institutional protocols by the InstitutionalAnimal Care and Use Committee (IACUC) ChungnamNationalUniversity Daejeon SouthKoreaThe authorizationcode number is CNU-00244

25 Treatment Protocol of Different Natural Extracts on NudeMice Five animals were allocated to five randomized groupsfor evaluation of hair growth and hair changing pattern ofnude mice Topical application was performed daily with asolution of A radix E alba and P ginseng extracts (25) ina vehicle formulation andorminoxidil (2) until completionof two full hair growth cycles

26 Measurement of Hair Density and Length After dailytreatment in each hair growth cycle the images from specificregions of neck back and hip were captured two times perweek using KONG Bom-Viewer Plus (Seoul Korea) Aboutsix images of 80times and 200times were exploited to measure thehair length and hair density respectively using Bom-ViewerImage Analyzer

27 Evaluation of Hair Existing Area Thehair existing area ofnude mice was systematically evaluated for each mouse threetimes per week At this time point mice were scored on ascale of ldquo0rdquo to ldquo4rdquo for percent body coverage of hair ldquo0rdquo =complete alopecia ldquo1rdquo = lt25 hair coverage ldquo2rdquo = 25ndash50hair coverage ldquo3rdquo = 50ndash75 hair coverage ldquo4rdquo = normal hairdensity and full hair coverage

28 Histologic Assessment of Hair Growth Mice were sac-rificed in anagen phase and skin samples were fixed in

4 BioMed Research International

Control Minoxidil A radix E alba P ginseng1

st ha

ir cy

cle2

nd h

air c

ycle

(A) (B) (C) (D) (E)

(A998400) (B998400) (C998400) (D998400) (E998400)

0

10

20

30

40

50

60

70

80

90

1st hair cycle 2nd hair cycle

a

c

a

b

c c

a

b

a

cH

air d

ensit

y (36

mm

2ar

ea)

ControlMinoxidilA radix

E albaP ginseng

000

020

040

060

080

100

120

140

160

180

200

1st cycle 2nd cycle

d

b

c

a b

a

b

c

b

c

Hai

r len

gth

(mm

)

ControlMinoxidilA radix

E albaP ginseng

(G)(F)

Figure 3 Evaluation of hair length and hair density in cycles Image of nude mice hair on skin surface treated with control ((A) (A1015840))minoxidil ((B) (B1015840)) A radix ((C) (C1015840)) E alba ((D) (D1015840)) and P ginseng ((E) (E1015840)) in first and second hair cycles Hair length (F) and hairdensity (G) shown in column The different letters (a b c and d) within a column indicate significant differences (119875 lt 00001) determinedby Duncanrsquos multiple range test Digital images were takenby KONG Bom-Viewer Plus with 80times magnification lens Hair length and hairdensity were measured using Bom-Viewer Image analyzer (119899 = 10)

10 buffered formalin for histological analysis Paraffin-embedded 4 120583m sections were stained with hematoxylinand eosin (HampE) The nude mice HF morphology andthe number of HFs were evaluated microscopically in 5fields per section of the dorsal skin at a magnification oftimes100

29 BrdU Immunohistochemistry BrdU labeling cell prolif-eration was detected by intraperitoneal injection of bromod-eoxyuridine (BrdU Sigma USA) with 50mgkg body weightat a concentration of 5mgmL in PBS BrdU incorporationwas detected by IHC staining of paraffin-embedded sec-tions with mouse anti-BrdU primary antibody (Santa CruzBiotechnology Santa Cruz CA USA) Skin sections thatwere not incubated with primary antibodies were used as

a negative control Detection of follicular keratinocyte BrdUlabeling was done at magnification times100 and times400

210 Statistical Analysis Statistical analysis was performedusing SAS Systems ver 93 and analysis of variance (ANOVA)was used for comparisons among all groups All data areexpressed as means plusmn SD unless otherwise is noted Aprobability level of 5was considered significant Differencesbetween two groups were analyzed by t-test ANOVA wasfollowed after Duncanrsquos multiple comparison tests

3 Results

31 Differences in Activity of Plant Extracts on ChangingHair Growth Pattern of Nude Mice After daily treatment

BioMed Research International 5

Control Minoxidil A radix E alba P ginseng

(A) (B) (C) (D) (E)

(A998400) (B998400) (C998400) (D998400) (E998400)

Figure 4Histological features of the skin specimen of different extract andor vehicle control treatedmiceMacroscopic observation of hair inthe skin surface of (A) control (B)minoxidil (C)A radix (D) E alba and (E) P ginseng in early hair existing phase Microscopic observationof longitudinal sections of (A1015840) control (B1015840) minoxidil (C1015840) A radix (D1015840) E alba and (E1015840) P ginseng treated mice skin was harvested andfixed in buffered formalin for routine hematoxylin and eosin (HampE) stained histology (119899 = 7) The broken line indicates defective HFs at thelevel of sebaceous gland ((A1015840) (B1015840) (C1015840) and (E1015840)) Note that the straight hair shafts that emerge from the skin surface of minoxidil and Ealba treated mouse ((B) (D) and Arrows (B1015840) (D1015840)) Scale bar 100 120583m

hair growth was documented on experimental day 7 Sys-tematic monitoring of hair growth showed that A radix(Figure 1(c)) and vehicle treated control mice (Figure 1(a))exhibit extremely sparse and transient hair coat On the otherhand minoxidil and E alba extract treated mice hair growthfirst documented from the frontal part of the head as earlyas 7 days and became dense thicker and longer that evenlyextended to the shoulder and posterior part of the backduring experimental day 12 The maximum hair coat wasevident in E alba extract treated group on experimental day16 (Figures 1(d) and 1(b)) In contrast A radix (Figure 1(c))and P ginseng (Figure 1(e)) treated mice found rapid hair losswith increasing time point and eventually remained only fewsparse abortive short regional hair thatmoved caudally to thetail region

32 Effect onHair CoverageArea Theeffects on hair coveragearea of all the treated groups were precisely estimated foreach mouse in both first and second hair growth cycle bygiving them a score from ldquo0rdquo to ldquo4rdquo The maximum hairgrowth score was significantly (119875 lt 0001) increased in micetreated with minoxidil and E alba than control group duringfirst and second hair growth cycles (Figure 2) On the otherhand A radix treated group also consistently effective forinducing hair coverage area (225plusmn082) in first hair cycle buttransient hair coat and progressive hair loss turn to similarscore (125 plusmn 05) with that of control group (125 plusmn 05) insecond hair cycleTherewas no significant difference in termsof the hair coverage area among P ginseng and control group

33 Effect on Hair Length and Hair Density In terms ofthe measurement of hair length the remarkable effect wasobserved in E alba extract treated group A significant

increase (119875 lt 00001) of hair lengthwas observed in both firstand second hair growth cycles while being compared withthat of control group (Figure 3(F)) Similar effect was alsofound while evaluating hair density (Figure 3(G)) Althoughminoxidil treated group showed similar hair density with Ealba treated group (Figure 3(G)) the irregular and deformedhair was found in minoxidil treated mice (Figure 3(B)) Onthe other hand E alba treated mice hair (Figures 3(D) and3(D1015840)) was straight and thicker and smother than othertreatment groups Although the hair length of A radixand P ginseng extract treated mice increased significantlythan control group in earlier hair cycle it became similarwith that of control group afterward (Figure 3(F)) On theother hand in terms of hair density A radix treated groupfound significant effect compared to control but there wasno difference of hair density of P ginseng and controlgroup (Figure 3(G)) Moreover irregularly distributed singledeformed hair shaft was also observed on the skin surface inbothA radix (Figures 3(C) and 3(C1015840)) and P ginseng (Figures3(E) and 3(E1015840)) treatedmice similar to control group (Figures3(A) and 3(A1015840))

34 Effect of Plant Extracts on the Development and Mor-phogenesis of Nude Mice HF Histological examination ofthe skin specimen revealed differences between control andtreated mice in the morphology of the developing HFsOn experimental day 10 minoxidil (Figure 4(B1015840)) A radix(Figure 4(C1015840)) and E alba treated mice (Figure 4(D1015840))showed an early onset of anagen hair morphogenesis com-pared with that of control (Figure 4(A1015840)) confirming themacroscopic observation (Figures 4(B) 4(C) and 4(D)) Onthe other hand P ginseng extract treated mice skin specimen(Figure 4(E1015840)) showed similar stage of hair morphogenesis

6 BioMed Research International

Control

(a)

Minoxidil

(b)

A radix

(c)

E alba

(d)

P ginseng

(e)

0

10

20

30

40

50

60

70

Subcutis Total hair follicle

a

bc

cc

b

a

d

bc

b

cd

Num

ber o

f hai

r fol

licle

s (pe

r fiel

d)

ControlMinoxidilA radix

E albaP ginseng

(f)

Figure 5 Increase in number of HFs in the dorsal skin of nude mice treated with different plant extract Representative photomicrographs ofthe transverse sections of nudemice skin (a) control (b)minoxidil (c)A radix (d) E alba and (e) P ginsengwere stained with hematoxylin-eosin (HampE) (f)The number of hair follicles in subcutis with respect to the total number of HFs per field (119899 = 10) Values aremeanplusmn standarddeviation (SD) The different letters (a b c and d) within a column indicate significant differences (119875 lt 00001) determined by Duncanrsquosmultiple range test Scale bar 100 120583m

with that of control mice Following the guidelines proposedby [19] we determined that the control and P ginsengtreated mice HFs were on stage 6 (Figures 4(A1015840) and 4(E1015840))whereas E alba extract treated mice HFs were clearly at stage8 (Figure 4(D1015840)) Negligible numbers of hair shafts whichemerged from the HFs and moderately atrophic were alsoobserved in the skin ofA radix P ginseng and vehicle treatedmice (Figures 4(C) 4(E) and 4(A)) Moreover the hairshafts of those were defective (often twisted) with dilationof the hair canal and apparently had insufficient structuralrigidity to emerge through the epidermis (Figures 4(A1015840) and4(E1015840)) On the other hand E alba treatedmice seemed tohave a propensity for follicular accumulation of keratin andstructural rigidity to penetrate the epidermis (Figure 4(D))

35 Number of Hair Follicles Histologic assessment wasperformed to investigate the progression of hair follicles innude mice since there was an increase in the number ofhair follicles observed in the deep subcutis during the anagenphase In the representative transverse sections the numbers

of hair follicles were increased significantly in E alba (119875 lt00001) and minoxidil (119875 lt 0005) extract treated groupscompared to the control (Figure 5(f))

36 BrdU Immunohistochemistry To further investigate themolecularmechanismunderlying the ability ofE alba extractto form nude mice hair follicles we performed immunohis-tochemistry with BrdU antibodies of the back skin of nudemice We found that E alba treated mice skin exhibits BrdUpositive keratinocyte in the HFs particularly a single celllayer that surrounds the innermost layer of the outer rootsheath as shown in Figures 6(B) and 6(B1015840) Moreover Ealba treated nude mice exhibited a significant (119875 lt 00001)increase in the number of BrdU-labeled keratinocyte peranagen follicle versus control mice (Figure 6(C)) On theother hand there was no significant difference in the numberof BrdU-labeled epithelial cells per sebaceous gland

37 Identification and Quantification of Wedelolactone in Ealba Extract Additionally HPLC analysis was performed to

BioMed Research International 7

0

5

10

15

20

25

30

HF SG

Num

ber o

f Brd

U p

ositi

ve ce

lls

lowastlowastlowast

(C)

ControlE alba

Control E alba

SBSB

HF

(A) (B)

(A998400) (B998400)

Figure 6 BrdU-labeled proliferating cells in anagen hair folliclesControl (A) and E alba extract treated (B) follicles are nuclear BrdUstaining (Arrows red-brown) Scale bar 100 120583m (B) and 50 120583m ((A)(A1015840) and (B1015840)) Number of BrdU positive cells (C) Data shown aremeans of five replicate samples (lowastlowastlowast119875 lt 0001)

illustrate the methanol extract of E alba (Figure 7) and wasfound to contain principle constituents like wedelolactoneThe existence of the wedelolactone was quantified by 18in methanol extract It has been reported that various bioac-tive phytochemicals found in the E alba extract includingwedelolactone demethylwedelolactone and saponins areresponsible for the main medicinal effects of E alba suchas antitumor activity [13] antihepatitis C virus [12] andneuropharmacological activity [14] and its anticancer effectshows antiproliferative and cytotoxic effects determinedusing MTT assay [15]

00 50 100 150 200 250 300 3500

250

500

750

1000

1250 Standard wedelolactone

(min)

(mV

)

(a)

00 50 100 150 200 250 300 3500

250500750

1000125015001750

Wedelolactone

(min)(m

V)

(b)

Figure 7 HPLC chromatogram (a) Standard wedelolactone (b)HPLC chromatogram of methanol extract of E alba

4 Discussion

Medicinal plants may have potential by identifying activebiological components and their successful use might openreverse pharmacology a new approach to drug discoveryGiven the known therapeutic properties of E alba extractwe have performed enormous screening of different plantextracts on BalbC nude mice model and this has beenused as a unique model to study the hair growth mechanism[20 21]

Among all the plant extracts E alba showed outstandinghair growth promoting potential Topical applicationwas per-formed on the back of nude mice skin daily up to completionof two hair growth generationsThemacroscopic appearanceand the local distribution of hair were precisely observedin each individual group and showed clearly distinguishablefeature The regional distribution of the ldquoabortiverdquo anagenhair pattern in nude strains corresponded to the wave-likepattern of hair generations [22] The hair growth area ofE alba treated mice is distinctly visible and simultaneouslycovered the maximum part of the back In contrast Aradix P ginseng treated mice with increasing age rapidlydeveloped hair loss recognizable from the boundaries ofhair covering However a few uneven hairs are visibleparticularly in nude mice skin surface which referred to asldquoabortive hair growthrdquo [23] During anagen growth phasewe were able to observe a comparable growth pattern inE alba treated mice with other treated and control mice(Figure 1) These phenomena may suggest that E albaimproved the disordered keratinization of nude mouse hairfollicles because original control hair shafts are short and

8 BioMed Research International

irregular in shape suggesting some disordered keratinizationof the HF [24 25] The precise genetic defect of nude miceFoxn1mutation expression involved abnormal keratinizationas the cause for defective cell type in the hair development[26ndash28] Our observation suggests that treatment with E albaacts transiently to normalize keratinocyte of nude mice HFleading to fully formed visible HF with increased hair cover-age area An increase in the number and size of HFs has beenconsidered as an indicator for the transition of hair growthfrom telogen to anagen [9 19]These results suggest that top-ical application of E alba extract could stimulate the forma-tion of hair follicles and induce an earlier anagen phase com-pared to either the control or other treated groups (Figure 4)To elucidate the molecular mechanism underlying the abilityof E alba extract the BrdU immunohistochemistry resultsuggests an enhanced keratinocyte proliferation in anagenhair follicles [29] In our present studiesmacroscopic analysisof hair growth parameters included changing pattern ofnude mice hair growth hair coverage area and hair lengthHistological study of different plant extracts along withcontrol and minoxidil groups was investigated and foundto have fully formed HFs in E alba treated group as wellas increasing the number of HFs The active phase of HFcycling is also accompanied by increase in size and numberof follicles resulting in an enlargement of the subcutis layer[19] Given the vital role of hair restorer efficacy of E albaextract further HPLC analysis was carried out and showedthat wedelolactone and other components exist in methanolextract In this experiment none of the plant extracts causedadverse dermatological effects such as erythema rednessdrying or scaling and dermatitis as it was case withminoxidilgroup at the skin site of nude mice

It has been argued that combination of several con-stituents of a plant extract may act synergistically rather thansingle compound [30] The interaction is possibly due to thebinding of more than one compound at different sites on thesame target or receptor Further detailed clinical trials andstudies will be necessary to investigate what components inE alba extract contribute to its efficacy since whole E albaextract rather than individual components was used here toestablish its biological activity against alopecia

5 Conclusion

E alba extract containing active ingredients provided moresafety and compatibility which has more accessibility inpharmaceutical and cosmetic science and may promote theherbal medicine with minimal or no side effects or toxicitiesfor hair growth therapy Taking together all the investigationindicates that plant extract with suitable vehiclemay be usefulas an alternative topical medicine to minoxidil therapy

Conflict of Interests

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

Acknowledgments

The authors would like to express special thanks to Dr KangJuChoi a great generous person for sharing pearls of wisdomwith them which is gratefully acknowledged This study wassupported by a grant from the DBIO Science and TechnologyCorporation

References

[1] S Tiede J E Kloepper D A Whiting and R Paus ldquoTheldquofollicular trochanterrdquo an epithelial compartment of the humanhair follicle bulge region in need of further characterizationrdquoBritish Journal of Dermatology vol 157 no 5 pp 1013ndash10162007

[2] V A Randall ldquoAndrogens and human hair growthrdquo ClinicalEndocrinology vol 40 no 4 pp 439ndash457 1994

[3] H-J Park N Zhang and D K Park ldquoTopical applicationof Polygonum multiflorum extract induces hair growth ofresting hair follicles through upregulating Shh and 120573-cateninexpression in C57BL6 micerdquo Journal of Ethnopharmacologyvol 135 no 2 pp 369ndash375 2011

[4] C J Girman T Rhodes F R W Lilly et al ldquoEffects of self-perceived hair loss in a community sample of menrdquo Dermatol-ogy vol 197 no 3 pp 223ndash229 1998

[5] S S Rho S J Park S L Hwang et al ldquoThe hair growthpromoting effect of Asiasari radix extract and its molecularregulationrdquo Journal of Dermatological Science vol 38 no 2 pp89ndash97 2005

[6] H Matsuda M Yamazaki Y Asanuma and M Kubo ldquoPromo-tion of hair growth by ginseng radix on culturedmouse vibrissalhair folliclesrdquo Phytotherapy Research vol 17 no 7 pp 797ndash8002003

[7] S Park W-S Shin and J Ho ldquoFructus panax ginseng extractpromotes hair regeneration in C57BL6 micerdquo Journal ofEthnopharmacology vol 138 no 2 pp 340ndash344 2011

[8] R K Roy M Thakur and V K Dixit ldquoHair growth promotingactivity of Eclipta alba in male albino ratsrdquo Archives of Derma-tological Research vol 300 no 7 pp 357ndash364 2008

[9] K Datta A T Singh A Mukherjee B Bhat B Ramesh and AC Burman ldquoEclipta alba extract with potential for hair growthpromoting activityrdquo Journal of Ethnopharmacology vol 124 no3 pp 450ndash456 2009

[10] H Wagner B Geyer Y Kiso H Hikino and G S RaoldquoCoumestans as the main active principles of the liver drugsEclipta alba and Wedelia calendulaceardquo Planta Medica vol 5pp 370ndash374 1986

[11] A M S Pereira B W Bertoni A Menezes Jr P S Pereiraand S C Franca ldquoSoil pH and production of biomass andwedelolactone in field grown Eclipta albardquo Journal of HerbsSpices and Medicinal Plants vol 6 no 1 pp 43ndash48 1998

[12] D Manvar M Mishra S Kumar and V N Pandey ldquoIdentifi-cation and evaluation of anti Hepatitis C virus phytochemicalsfrom Eclipta albardquo Journal of Ethnopharmacology vol 144 no3 pp 545ndash554 2012

[13] Q-M Liu H-Y Zhao X-K Zhong and J-G Jiang ldquoEcliptaprostrata L phytochemicals isolation structure elucidationand their antitumor activityrdquo Food andChemical Toxicology vol50 no 11 pp 4016ndash4022 2012

[14] V DThakur and S AMengi ldquoNeuropharmacological profile ofEclipta alba (Linn) Hasskrdquo Journal of Ethnopharmacology vol102 no 1 pp 23ndash31 2005

BioMed Research International 9

[15] H Chaudhary V Dhuna J Singh S S Kamboj and S SeshadrildquoEvaluation of hydro-alcoholic extract of Eclipta alba for itsanticancer potential an in vitro studyrdquo Journal of Ethnophar-macology vol 136 no 2 pp 363ndash367 2011

[16] L Mecklenburg B Tychsen and R Paus ldquoLearning fromnudity lessons from the nude phenotyperdquo Experimental Der-matology vol 14 no 11 pp 797ndash810 2005

[17] G A Simon and H I Maibach ldquoRelevance of hairless mouse asan experimental model of percutaneous penetration in manrdquoSkin Pharmacology and Applied Skin Physiology vol 11 no 2pp 80ndash86 1998

[18] R Toll U Jacobi H Richter J Lademann H Schaefer and UBlume-Peytavi ldquoPenetration profile of microspheres in follicu-lar targeting of terminal hair folliclesrdquo Journal of InvestigativeDermatology vol 123 no 1 pp 168ndash176 2004

[19] R Paus S Muller-Rover C van der Veen et al ldquoA compre-hensive guide for the recognition and classification of distinctstages of hair follicle morphogenesisrdquo Journal of InvestigativeDermatology vol 113 no 4 pp 523ndash532 1999

[20] M Sawada N Terada H Taniguchi R Tateishi and YMori ldquoCyclosporin A stimulates hair growth in nude micerdquoLaboratory Investigation vol 56 no 6 pp 684ndash686 1987

[21] S Watanabe A Mochizuki K Wagatsuma M Kobayashi YKawa and H Takahashi ldquoHair growth on nude mice due tocyclosporin Ardquo The Journal of Dermatology vol 18 no 12 pp714ndash719 1991

[22] K Militzer ldquoHair growth pattern in nude micerdquo Cells TissuesOrgans vol 168 no 4 pp 285ndash294 2001

[23] U Iversen and O H Iversen ldquoCycles of hair growth in hairlessmicerdquoActa PathologicaMicrobiologica Scandinavica vol 69 no1 pp 50ndash62 1967

[24] R H Rigdon and A A Packchanian ldquoHistologic study of theskin of congenitally athymic ldquonuderdquo micerdquo Texas Reports onBiology amp Medicine vol 32 no 3-4 pp 711ndash723 1974

[25] Y Hozumi T Imaizumi and S Kondo ldquoEffect of cyclosporinon hair-existing area of nude micerdquo Journal of DermatologicalScience vol 7 pp S33ndashS38 1994

[26] S P Flanagan ldquoldquoNuderdquo a new hairless gene with pleiotropiceffects in the mouserdquo Genetical Research vol 8 no 3 pp 295ndash309 1966

[27] J L Brissette J Li J Kamimura D Lee and G P Dotto ldquoTheproduct of the mouse nude locus whn regulates the balancebetween epithelial cell growth and differentiationrdquo Genes andDevelopment vol 10 no 17 pp 2212ndash2221 1996

[28] L Mecklenburg R Paus Z Halata L S Bechtold P Fleckmanand J P Sundberg ldquoFOXN1 is critical for onycholemmalterminal differentiation in nude (Foxn1119899119906) micerdquo Journal ofInvestigative Dermatology vol 123 no 6 pp 1001ndash1011 2004

[29] H Pinkus T Iwasaki and Y Mishima ldquoOuter root sheathkeratinization in anagen and catagen of the mammalian hairfollicle A seventh distinct type of keratinization in the hairfollicle trichilemmal keratinizationrdquo Journal of Anatomy vol133 no 1 pp 19ndash35 1981

[30] H Wagner and G Ulrich-Merzenich ldquoSynergy researchapproaching a new generation of phytopharmaceuticalsrdquo Phy-tomedicine vol 16 no 2-3 pp 97ndash110 2009

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

4 BioMed Research International

Control Minoxidil A radix E alba P ginseng1

st ha

ir cy

cle2

nd h

air c

ycle

(A) (B) (C) (D) (E)

(A998400) (B998400) (C998400) (D998400) (E998400)

0

10

20

30

40

50

60

70

80

90

1st hair cycle 2nd hair cycle

a

c

a

b

c c

a

b

a

cH

air d

ensit

y (36

mm

2ar

ea)

ControlMinoxidilA radix

E albaP ginseng

000

020

040

060

080

100

120

140

160

180

200

1st cycle 2nd cycle

d

b

c

a b

a

b

c

b

c

Hai

r len

gth

(mm

)

ControlMinoxidilA radix

E albaP ginseng

(G)(F)

Figure 3 Evaluation of hair length and hair density in cycles Image of nude mice hair on skin surface treated with control ((A) (A1015840))minoxidil ((B) (B1015840)) A radix ((C) (C1015840)) E alba ((D) (D1015840)) and P ginseng ((E) (E1015840)) in first and second hair cycles Hair length (F) and hairdensity (G) shown in column The different letters (a b c and d) within a column indicate significant differences (119875 lt 00001) determinedby Duncanrsquos multiple range test Digital images were takenby KONG Bom-Viewer Plus with 80times magnification lens Hair length and hairdensity were measured using Bom-Viewer Image analyzer (119899 = 10)

10 buffered formalin for histological analysis Paraffin-embedded 4 120583m sections were stained with hematoxylinand eosin (HampE) The nude mice HF morphology andthe number of HFs were evaluated microscopically in 5fields per section of the dorsal skin at a magnification oftimes100

29 BrdU Immunohistochemistry BrdU labeling cell prolif-eration was detected by intraperitoneal injection of bromod-eoxyuridine (BrdU Sigma USA) with 50mgkg body weightat a concentration of 5mgmL in PBS BrdU incorporationwas detected by IHC staining of paraffin-embedded sec-tions with mouse anti-BrdU primary antibody (Santa CruzBiotechnology Santa Cruz CA USA) Skin sections thatwere not incubated with primary antibodies were used as

a negative control Detection of follicular keratinocyte BrdUlabeling was done at magnification times100 and times400

210 Statistical Analysis Statistical analysis was performedusing SAS Systems ver 93 and analysis of variance (ANOVA)was used for comparisons among all groups All data areexpressed as means plusmn SD unless otherwise is noted Aprobability level of 5was considered significant Differencesbetween two groups were analyzed by t-test ANOVA wasfollowed after Duncanrsquos multiple comparison tests

3 Results

31 Differences in Activity of Plant Extracts on ChangingHair Growth Pattern of Nude Mice After daily treatment

BioMed Research International 5

Control Minoxidil A radix E alba P ginseng

(A) (B) (C) (D) (E)

(A998400) (B998400) (C998400) (D998400) (E998400)

Figure 4Histological features of the skin specimen of different extract andor vehicle control treatedmiceMacroscopic observation of hair inthe skin surface of (A) control (B)minoxidil (C)A radix (D) E alba and (E) P ginseng in early hair existing phase Microscopic observationof longitudinal sections of (A1015840) control (B1015840) minoxidil (C1015840) A radix (D1015840) E alba and (E1015840) P ginseng treated mice skin was harvested andfixed in buffered formalin for routine hematoxylin and eosin (HampE) stained histology (119899 = 7) The broken line indicates defective HFs at thelevel of sebaceous gland ((A1015840) (B1015840) (C1015840) and (E1015840)) Note that the straight hair shafts that emerge from the skin surface of minoxidil and Ealba treated mouse ((B) (D) and Arrows (B1015840) (D1015840)) Scale bar 100 120583m

hair growth was documented on experimental day 7 Sys-tematic monitoring of hair growth showed that A radix(Figure 1(c)) and vehicle treated control mice (Figure 1(a))exhibit extremely sparse and transient hair coat On the otherhand minoxidil and E alba extract treated mice hair growthfirst documented from the frontal part of the head as earlyas 7 days and became dense thicker and longer that evenlyextended to the shoulder and posterior part of the backduring experimental day 12 The maximum hair coat wasevident in E alba extract treated group on experimental day16 (Figures 1(d) and 1(b)) In contrast A radix (Figure 1(c))and P ginseng (Figure 1(e)) treated mice found rapid hair losswith increasing time point and eventually remained only fewsparse abortive short regional hair thatmoved caudally to thetail region

32 Effect onHair CoverageArea Theeffects on hair coveragearea of all the treated groups were precisely estimated foreach mouse in both first and second hair growth cycle bygiving them a score from ldquo0rdquo to ldquo4rdquo The maximum hairgrowth score was significantly (119875 lt 0001) increased in micetreated with minoxidil and E alba than control group duringfirst and second hair growth cycles (Figure 2) On the otherhand A radix treated group also consistently effective forinducing hair coverage area (225plusmn082) in first hair cycle buttransient hair coat and progressive hair loss turn to similarscore (125 plusmn 05) with that of control group (125 plusmn 05) insecond hair cycleTherewas no significant difference in termsof the hair coverage area among P ginseng and control group

33 Effect on Hair Length and Hair Density In terms ofthe measurement of hair length the remarkable effect wasobserved in E alba extract treated group A significant

increase (119875 lt 00001) of hair lengthwas observed in both firstand second hair growth cycles while being compared withthat of control group (Figure 3(F)) Similar effect was alsofound while evaluating hair density (Figure 3(G)) Althoughminoxidil treated group showed similar hair density with Ealba treated group (Figure 3(G)) the irregular and deformedhair was found in minoxidil treated mice (Figure 3(B)) Onthe other hand E alba treated mice hair (Figures 3(D) and3(D1015840)) was straight and thicker and smother than othertreatment groups Although the hair length of A radixand P ginseng extract treated mice increased significantlythan control group in earlier hair cycle it became similarwith that of control group afterward (Figure 3(F)) On theother hand in terms of hair density A radix treated groupfound significant effect compared to control but there wasno difference of hair density of P ginseng and controlgroup (Figure 3(G)) Moreover irregularly distributed singledeformed hair shaft was also observed on the skin surface inbothA radix (Figures 3(C) and 3(C1015840)) and P ginseng (Figures3(E) and 3(E1015840)) treatedmice similar to control group (Figures3(A) and 3(A1015840))

34 Effect of Plant Extracts on the Development and Mor-phogenesis of Nude Mice HF Histological examination ofthe skin specimen revealed differences between control andtreated mice in the morphology of the developing HFsOn experimental day 10 minoxidil (Figure 4(B1015840)) A radix(Figure 4(C1015840)) and E alba treated mice (Figure 4(D1015840))showed an early onset of anagen hair morphogenesis com-pared with that of control (Figure 4(A1015840)) confirming themacroscopic observation (Figures 4(B) 4(C) and 4(D)) Onthe other hand P ginseng extract treated mice skin specimen(Figure 4(E1015840)) showed similar stage of hair morphogenesis

6 BioMed Research International

Control

(a)

Minoxidil

(b)

A radix

(c)

E alba

(d)

P ginseng

(e)

0

10

20

30

40

50

60

70

Subcutis Total hair follicle

a

bc

cc

b

a

d

bc

b

cd

Num

ber o

f hai

r fol

licle

s (pe

r fiel

d)

ControlMinoxidilA radix

E albaP ginseng

(f)

Figure 5 Increase in number of HFs in the dorsal skin of nude mice treated with different plant extract Representative photomicrographs ofthe transverse sections of nudemice skin (a) control (b)minoxidil (c)A radix (d) E alba and (e) P ginsengwere stained with hematoxylin-eosin (HampE) (f)The number of hair follicles in subcutis with respect to the total number of HFs per field (119899 = 10) Values aremeanplusmn standarddeviation (SD) The different letters (a b c and d) within a column indicate significant differences (119875 lt 00001) determined by Duncanrsquosmultiple range test Scale bar 100 120583m

with that of control mice Following the guidelines proposedby [19] we determined that the control and P ginsengtreated mice HFs were on stage 6 (Figures 4(A1015840) and 4(E1015840))whereas E alba extract treated mice HFs were clearly at stage8 (Figure 4(D1015840)) Negligible numbers of hair shafts whichemerged from the HFs and moderately atrophic were alsoobserved in the skin ofA radix P ginseng and vehicle treatedmice (Figures 4(C) 4(E) and 4(A)) Moreover the hairshafts of those were defective (often twisted) with dilationof the hair canal and apparently had insufficient structuralrigidity to emerge through the epidermis (Figures 4(A1015840) and4(E1015840)) On the other hand E alba treatedmice seemed tohave a propensity for follicular accumulation of keratin andstructural rigidity to penetrate the epidermis (Figure 4(D))

35 Number of Hair Follicles Histologic assessment wasperformed to investigate the progression of hair follicles innude mice since there was an increase in the number ofhair follicles observed in the deep subcutis during the anagenphase In the representative transverse sections the numbers

of hair follicles were increased significantly in E alba (119875 lt00001) and minoxidil (119875 lt 0005) extract treated groupscompared to the control (Figure 5(f))

36 BrdU Immunohistochemistry To further investigate themolecularmechanismunderlying the ability ofE alba extractto form nude mice hair follicles we performed immunohis-tochemistry with BrdU antibodies of the back skin of nudemice We found that E alba treated mice skin exhibits BrdUpositive keratinocyte in the HFs particularly a single celllayer that surrounds the innermost layer of the outer rootsheath as shown in Figures 6(B) and 6(B1015840) Moreover Ealba treated nude mice exhibited a significant (119875 lt 00001)increase in the number of BrdU-labeled keratinocyte peranagen follicle versus control mice (Figure 6(C)) On theother hand there was no significant difference in the numberof BrdU-labeled epithelial cells per sebaceous gland

37 Identification and Quantification of Wedelolactone in Ealba Extract Additionally HPLC analysis was performed to

BioMed Research International 7

0

5

10

15

20

25

30

HF SG

Num

ber o

f Brd

U p

ositi

ve ce

lls

lowastlowastlowast

(C)

ControlE alba

Control E alba

SBSB

HF

(A) (B)

(A998400) (B998400)

Figure 6 BrdU-labeled proliferating cells in anagen hair folliclesControl (A) and E alba extract treated (B) follicles are nuclear BrdUstaining (Arrows red-brown) Scale bar 100 120583m (B) and 50 120583m ((A)(A1015840) and (B1015840)) Number of BrdU positive cells (C) Data shown aremeans of five replicate samples (lowastlowastlowast119875 lt 0001)

illustrate the methanol extract of E alba (Figure 7) and wasfound to contain principle constituents like wedelolactoneThe existence of the wedelolactone was quantified by 18in methanol extract It has been reported that various bioac-tive phytochemicals found in the E alba extract includingwedelolactone demethylwedelolactone and saponins areresponsible for the main medicinal effects of E alba suchas antitumor activity [13] antihepatitis C virus [12] andneuropharmacological activity [14] and its anticancer effectshows antiproliferative and cytotoxic effects determinedusing MTT assay [15]

00 50 100 150 200 250 300 3500

250

500

750

1000

1250 Standard wedelolactone

(min)

(mV

)

(a)

00 50 100 150 200 250 300 3500

250500750

1000125015001750

Wedelolactone

(min)(m

V)

(b)

Figure 7 HPLC chromatogram (a) Standard wedelolactone (b)HPLC chromatogram of methanol extract of E alba

4 Discussion

Medicinal plants may have potential by identifying activebiological components and their successful use might openreverse pharmacology a new approach to drug discoveryGiven the known therapeutic properties of E alba extractwe have performed enormous screening of different plantextracts on BalbC nude mice model and this has beenused as a unique model to study the hair growth mechanism[20 21]

Among all the plant extracts E alba showed outstandinghair growth promoting potential Topical applicationwas per-formed on the back of nude mice skin daily up to completionof two hair growth generationsThemacroscopic appearanceand the local distribution of hair were precisely observedin each individual group and showed clearly distinguishablefeature The regional distribution of the ldquoabortiverdquo anagenhair pattern in nude strains corresponded to the wave-likepattern of hair generations [22] The hair growth area ofE alba treated mice is distinctly visible and simultaneouslycovered the maximum part of the back In contrast Aradix P ginseng treated mice with increasing age rapidlydeveloped hair loss recognizable from the boundaries ofhair covering However a few uneven hairs are visibleparticularly in nude mice skin surface which referred to asldquoabortive hair growthrdquo [23] During anagen growth phasewe were able to observe a comparable growth pattern inE alba treated mice with other treated and control mice(Figure 1) These phenomena may suggest that E albaimproved the disordered keratinization of nude mouse hairfollicles because original control hair shafts are short and

8 BioMed Research International

irregular in shape suggesting some disordered keratinizationof the HF [24 25] The precise genetic defect of nude miceFoxn1mutation expression involved abnormal keratinizationas the cause for defective cell type in the hair development[26ndash28] Our observation suggests that treatment with E albaacts transiently to normalize keratinocyte of nude mice HFleading to fully formed visible HF with increased hair cover-age area An increase in the number and size of HFs has beenconsidered as an indicator for the transition of hair growthfrom telogen to anagen [9 19]These results suggest that top-ical application of E alba extract could stimulate the forma-tion of hair follicles and induce an earlier anagen phase com-pared to either the control or other treated groups (Figure 4)To elucidate the molecular mechanism underlying the abilityof E alba extract the BrdU immunohistochemistry resultsuggests an enhanced keratinocyte proliferation in anagenhair follicles [29] In our present studiesmacroscopic analysisof hair growth parameters included changing pattern ofnude mice hair growth hair coverage area and hair lengthHistological study of different plant extracts along withcontrol and minoxidil groups was investigated and foundto have fully formed HFs in E alba treated group as wellas increasing the number of HFs The active phase of HFcycling is also accompanied by increase in size and numberof follicles resulting in an enlargement of the subcutis layer[19] Given the vital role of hair restorer efficacy of E albaextract further HPLC analysis was carried out and showedthat wedelolactone and other components exist in methanolextract In this experiment none of the plant extracts causedadverse dermatological effects such as erythema rednessdrying or scaling and dermatitis as it was case withminoxidilgroup at the skin site of nude mice

It has been argued that combination of several con-stituents of a plant extract may act synergistically rather thansingle compound [30] The interaction is possibly due to thebinding of more than one compound at different sites on thesame target or receptor Further detailed clinical trials andstudies will be necessary to investigate what components inE alba extract contribute to its efficacy since whole E albaextract rather than individual components was used here toestablish its biological activity against alopecia

5 Conclusion

E alba extract containing active ingredients provided moresafety and compatibility which has more accessibility inpharmaceutical and cosmetic science and may promote theherbal medicine with minimal or no side effects or toxicitiesfor hair growth therapy Taking together all the investigationindicates that plant extract with suitable vehiclemay be usefulas an alternative topical medicine to minoxidil therapy

Conflict of Interests

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

Acknowledgments

The authors would like to express special thanks to Dr KangJuChoi a great generous person for sharing pearls of wisdomwith them which is gratefully acknowledged This study wassupported by a grant from the DBIO Science and TechnologyCorporation

References

[1] S Tiede J E Kloepper D A Whiting and R Paus ldquoTheldquofollicular trochanterrdquo an epithelial compartment of the humanhair follicle bulge region in need of further characterizationrdquoBritish Journal of Dermatology vol 157 no 5 pp 1013ndash10162007

[2] V A Randall ldquoAndrogens and human hair growthrdquo ClinicalEndocrinology vol 40 no 4 pp 439ndash457 1994

[3] H-J Park N Zhang and D K Park ldquoTopical applicationof Polygonum multiflorum extract induces hair growth ofresting hair follicles through upregulating Shh and 120573-cateninexpression in C57BL6 micerdquo Journal of Ethnopharmacologyvol 135 no 2 pp 369ndash375 2011

[4] C J Girman T Rhodes F R W Lilly et al ldquoEffects of self-perceived hair loss in a community sample of menrdquo Dermatol-ogy vol 197 no 3 pp 223ndash229 1998

[5] S S Rho S J Park S L Hwang et al ldquoThe hair growthpromoting effect of Asiasari radix extract and its molecularregulationrdquo Journal of Dermatological Science vol 38 no 2 pp89ndash97 2005

[6] H Matsuda M Yamazaki Y Asanuma and M Kubo ldquoPromo-tion of hair growth by ginseng radix on culturedmouse vibrissalhair folliclesrdquo Phytotherapy Research vol 17 no 7 pp 797ndash8002003

[7] S Park W-S Shin and J Ho ldquoFructus panax ginseng extractpromotes hair regeneration in C57BL6 micerdquo Journal ofEthnopharmacology vol 138 no 2 pp 340ndash344 2011

[8] R K Roy M Thakur and V K Dixit ldquoHair growth promotingactivity of Eclipta alba in male albino ratsrdquo Archives of Derma-tological Research vol 300 no 7 pp 357ndash364 2008

[9] K Datta A T Singh A Mukherjee B Bhat B Ramesh and AC Burman ldquoEclipta alba extract with potential for hair growthpromoting activityrdquo Journal of Ethnopharmacology vol 124 no3 pp 450ndash456 2009

[10] H Wagner B Geyer Y Kiso H Hikino and G S RaoldquoCoumestans as the main active principles of the liver drugsEclipta alba and Wedelia calendulaceardquo Planta Medica vol 5pp 370ndash374 1986

[11] A M S Pereira B W Bertoni A Menezes Jr P S Pereiraand S C Franca ldquoSoil pH and production of biomass andwedelolactone in field grown Eclipta albardquo Journal of HerbsSpices and Medicinal Plants vol 6 no 1 pp 43ndash48 1998

[12] D Manvar M Mishra S Kumar and V N Pandey ldquoIdentifi-cation and evaluation of anti Hepatitis C virus phytochemicalsfrom Eclipta albardquo Journal of Ethnopharmacology vol 144 no3 pp 545ndash554 2012

[13] Q-M Liu H-Y Zhao X-K Zhong and J-G Jiang ldquoEcliptaprostrata L phytochemicals isolation structure elucidationand their antitumor activityrdquo Food andChemical Toxicology vol50 no 11 pp 4016ndash4022 2012

[14] V DThakur and S AMengi ldquoNeuropharmacological profile ofEclipta alba (Linn) Hasskrdquo Journal of Ethnopharmacology vol102 no 1 pp 23ndash31 2005

BioMed Research International 9

[15] H Chaudhary V Dhuna J Singh S S Kamboj and S SeshadrildquoEvaluation of hydro-alcoholic extract of Eclipta alba for itsanticancer potential an in vitro studyrdquo Journal of Ethnophar-macology vol 136 no 2 pp 363ndash367 2011

[16] L Mecklenburg B Tychsen and R Paus ldquoLearning fromnudity lessons from the nude phenotyperdquo Experimental Der-matology vol 14 no 11 pp 797ndash810 2005

[17] G A Simon and H I Maibach ldquoRelevance of hairless mouse asan experimental model of percutaneous penetration in manrdquoSkin Pharmacology and Applied Skin Physiology vol 11 no 2pp 80ndash86 1998

[18] R Toll U Jacobi H Richter J Lademann H Schaefer and UBlume-Peytavi ldquoPenetration profile of microspheres in follicu-lar targeting of terminal hair folliclesrdquo Journal of InvestigativeDermatology vol 123 no 1 pp 168ndash176 2004

[19] R Paus S Muller-Rover C van der Veen et al ldquoA compre-hensive guide for the recognition and classification of distinctstages of hair follicle morphogenesisrdquo Journal of InvestigativeDermatology vol 113 no 4 pp 523ndash532 1999

[20] M Sawada N Terada H Taniguchi R Tateishi and YMori ldquoCyclosporin A stimulates hair growth in nude micerdquoLaboratory Investigation vol 56 no 6 pp 684ndash686 1987

[21] S Watanabe A Mochizuki K Wagatsuma M Kobayashi YKawa and H Takahashi ldquoHair growth on nude mice due tocyclosporin Ardquo The Journal of Dermatology vol 18 no 12 pp714ndash719 1991

[22] K Militzer ldquoHair growth pattern in nude micerdquo Cells TissuesOrgans vol 168 no 4 pp 285ndash294 2001

[23] U Iversen and O H Iversen ldquoCycles of hair growth in hairlessmicerdquoActa PathologicaMicrobiologica Scandinavica vol 69 no1 pp 50ndash62 1967

[24] R H Rigdon and A A Packchanian ldquoHistologic study of theskin of congenitally athymic ldquonuderdquo micerdquo Texas Reports onBiology amp Medicine vol 32 no 3-4 pp 711ndash723 1974

[25] Y Hozumi T Imaizumi and S Kondo ldquoEffect of cyclosporinon hair-existing area of nude micerdquo Journal of DermatologicalScience vol 7 pp S33ndashS38 1994

[26] S P Flanagan ldquoldquoNuderdquo a new hairless gene with pleiotropiceffects in the mouserdquo Genetical Research vol 8 no 3 pp 295ndash309 1966

[27] J L Brissette J Li J Kamimura D Lee and G P Dotto ldquoTheproduct of the mouse nude locus whn regulates the balancebetween epithelial cell growth and differentiationrdquo Genes andDevelopment vol 10 no 17 pp 2212ndash2221 1996

[28] L Mecklenburg R Paus Z Halata L S Bechtold P Fleckmanand J P Sundberg ldquoFOXN1 is critical for onycholemmalterminal differentiation in nude (Foxn1119899119906) micerdquo Journal ofInvestigative Dermatology vol 123 no 6 pp 1001ndash1011 2004

[29] H Pinkus T Iwasaki and Y Mishima ldquoOuter root sheathkeratinization in anagen and catagen of the mammalian hairfollicle A seventh distinct type of keratinization in the hairfollicle trichilemmal keratinizationrdquo Journal of Anatomy vol133 no 1 pp 19ndash35 1981

[30] H Wagner and G Ulrich-Merzenich ldquoSynergy researchapproaching a new generation of phytopharmaceuticalsrdquo Phy-tomedicine vol 16 no 2-3 pp 97ndash110 2009

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

BioMed Research International 5

Control Minoxidil A radix E alba P ginseng

(A) (B) (C) (D) (E)

(A998400) (B998400) (C998400) (D998400) (E998400)

Figure 4Histological features of the skin specimen of different extract andor vehicle control treatedmiceMacroscopic observation of hair inthe skin surface of (A) control (B)minoxidil (C)A radix (D) E alba and (E) P ginseng in early hair existing phase Microscopic observationof longitudinal sections of (A1015840) control (B1015840) minoxidil (C1015840) A radix (D1015840) E alba and (E1015840) P ginseng treated mice skin was harvested andfixed in buffered formalin for routine hematoxylin and eosin (HampE) stained histology (119899 = 7) The broken line indicates defective HFs at thelevel of sebaceous gland ((A1015840) (B1015840) (C1015840) and (E1015840)) Note that the straight hair shafts that emerge from the skin surface of minoxidil and Ealba treated mouse ((B) (D) and Arrows (B1015840) (D1015840)) Scale bar 100 120583m

hair growth was documented on experimental day 7 Sys-tematic monitoring of hair growth showed that A radix(Figure 1(c)) and vehicle treated control mice (Figure 1(a))exhibit extremely sparse and transient hair coat On the otherhand minoxidil and E alba extract treated mice hair growthfirst documented from the frontal part of the head as earlyas 7 days and became dense thicker and longer that evenlyextended to the shoulder and posterior part of the backduring experimental day 12 The maximum hair coat wasevident in E alba extract treated group on experimental day16 (Figures 1(d) and 1(b)) In contrast A radix (Figure 1(c))and P ginseng (Figure 1(e)) treated mice found rapid hair losswith increasing time point and eventually remained only fewsparse abortive short regional hair thatmoved caudally to thetail region

32 Effect onHair CoverageArea Theeffects on hair coveragearea of all the treated groups were precisely estimated foreach mouse in both first and second hair growth cycle bygiving them a score from ldquo0rdquo to ldquo4rdquo The maximum hairgrowth score was significantly (119875 lt 0001) increased in micetreated with minoxidil and E alba than control group duringfirst and second hair growth cycles (Figure 2) On the otherhand A radix treated group also consistently effective forinducing hair coverage area (225plusmn082) in first hair cycle buttransient hair coat and progressive hair loss turn to similarscore (125 plusmn 05) with that of control group (125 plusmn 05) insecond hair cycleTherewas no significant difference in termsof the hair coverage area among P ginseng and control group

33 Effect on Hair Length and Hair Density In terms ofthe measurement of hair length the remarkable effect wasobserved in E alba extract treated group A significant

increase (119875 lt 00001) of hair lengthwas observed in both firstand second hair growth cycles while being compared withthat of control group (Figure 3(F)) Similar effect was alsofound while evaluating hair density (Figure 3(G)) Althoughminoxidil treated group showed similar hair density with Ealba treated group (Figure 3(G)) the irregular and deformedhair was found in minoxidil treated mice (Figure 3(B)) Onthe other hand E alba treated mice hair (Figures 3(D) and3(D1015840)) was straight and thicker and smother than othertreatment groups Although the hair length of A radixand P ginseng extract treated mice increased significantlythan control group in earlier hair cycle it became similarwith that of control group afterward (Figure 3(F)) On theother hand in terms of hair density A radix treated groupfound significant effect compared to control but there wasno difference of hair density of P ginseng and controlgroup (Figure 3(G)) Moreover irregularly distributed singledeformed hair shaft was also observed on the skin surface inbothA radix (Figures 3(C) and 3(C1015840)) and P ginseng (Figures3(E) and 3(E1015840)) treatedmice similar to control group (Figures3(A) and 3(A1015840))

34 Effect of Plant Extracts on the Development and Mor-phogenesis of Nude Mice HF Histological examination ofthe skin specimen revealed differences between control andtreated mice in the morphology of the developing HFsOn experimental day 10 minoxidil (Figure 4(B1015840)) A radix(Figure 4(C1015840)) and E alba treated mice (Figure 4(D1015840))showed an early onset of anagen hair morphogenesis com-pared with that of control (Figure 4(A1015840)) confirming themacroscopic observation (Figures 4(B) 4(C) and 4(D)) Onthe other hand P ginseng extract treated mice skin specimen(Figure 4(E1015840)) showed similar stage of hair morphogenesis

6 BioMed Research International

Control

(a)

Minoxidil

(b)

A radix

(c)

E alba

(d)

P ginseng

(e)

0

10

20

30

40

50

60

70

Subcutis Total hair follicle

a

bc

cc

b

a

d

bc

b

cd

Num

ber o

f hai

r fol

licle

s (pe

r fiel

d)

ControlMinoxidilA radix

E albaP ginseng

(f)

Figure 5 Increase in number of HFs in the dorsal skin of nude mice treated with different plant extract Representative photomicrographs ofthe transverse sections of nudemice skin (a) control (b)minoxidil (c)A radix (d) E alba and (e) P ginsengwere stained with hematoxylin-eosin (HampE) (f)The number of hair follicles in subcutis with respect to the total number of HFs per field (119899 = 10) Values aremeanplusmn standarddeviation (SD) The different letters (a b c and d) within a column indicate significant differences (119875 lt 00001) determined by Duncanrsquosmultiple range test Scale bar 100 120583m

with that of control mice Following the guidelines proposedby [19] we determined that the control and P ginsengtreated mice HFs were on stage 6 (Figures 4(A1015840) and 4(E1015840))whereas E alba extract treated mice HFs were clearly at stage8 (Figure 4(D1015840)) Negligible numbers of hair shafts whichemerged from the HFs and moderately atrophic were alsoobserved in the skin ofA radix P ginseng and vehicle treatedmice (Figures 4(C) 4(E) and 4(A)) Moreover the hairshafts of those were defective (often twisted) with dilationof the hair canal and apparently had insufficient structuralrigidity to emerge through the epidermis (Figures 4(A1015840) and4(E1015840)) On the other hand E alba treatedmice seemed tohave a propensity for follicular accumulation of keratin andstructural rigidity to penetrate the epidermis (Figure 4(D))

35 Number of Hair Follicles Histologic assessment wasperformed to investigate the progression of hair follicles innude mice since there was an increase in the number ofhair follicles observed in the deep subcutis during the anagenphase In the representative transverse sections the numbers

of hair follicles were increased significantly in E alba (119875 lt00001) and minoxidil (119875 lt 0005) extract treated groupscompared to the control (Figure 5(f))

36 BrdU Immunohistochemistry To further investigate themolecularmechanismunderlying the ability ofE alba extractto form nude mice hair follicles we performed immunohis-tochemistry with BrdU antibodies of the back skin of nudemice We found that E alba treated mice skin exhibits BrdUpositive keratinocyte in the HFs particularly a single celllayer that surrounds the innermost layer of the outer rootsheath as shown in Figures 6(B) and 6(B1015840) Moreover Ealba treated nude mice exhibited a significant (119875 lt 00001)increase in the number of BrdU-labeled keratinocyte peranagen follicle versus control mice (Figure 6(C)) On theother hand there was no significant difference in the numberof BrdU-labeled epithelial cells per sebaceous gland

37 Identification and Quantification of Wedelolactone in Ealba Extract Additionally HPLC analysis was performed to

BioMed Research International 7

0

5

10

15

20

25

30

HF SG

Num

ber o

f Brd

U p

ositi

ve ce

lls

lowastlowastlowast

(C)

ControlE alba

Control E alba

SBSB

HF

(A) (B)

(A998400) (B998400)

Figure 6 BrdU-labeled proliferating cells in anagen hair folliclesControl (A) and E alba extract treated (B) follicles are nuclear BrdUstaining (Arrows red-brown) Scale bar 100 120583m (B) and 50 120583m ((A)(A1015840) and (B1015840)) Number of BrdU positive cells (C) Data shown aremeans of five replicate samples (lowastlowastlowast119875 lt 0001)

illustrate the methanol extract of E alba (Figure 7) and wasfound to contain principle constituents like wedelolactoneThe existence of the wedelolactone was quantified by 18in methanol extract It has been reported that various bioac-tive phytochemicals found in the E alba extract includingwedelolactone demethylwedelolactone and saponins areresponsible for the main medicinal effects of E alba suchas antitumor activity [13] antihepatitis C virus [12] andneuropharmacological activity [14] and its anticancer effectshows antiproliferative and cytotoxic effects determinedusing MTT assay [15]

00 50 100 150 200 250 300 3500

250

500

750

1000

1250 Standard wedelolactone

(min)

(mV

)

(a)

00 50 100 150 200 250 300 3500

250500750

1000125015001750

Wedelolactone

(min)(m

V)

(b)

Figure 7 HPLC chromatogram (a) Standard wedelolactone (b)HPLC chromatogram of methanol extract of E alba

4 Discussion

Medicinal plants may have potential by identifying activebiological components and their successful use might openreverse pharmacology a new approach to drug discoveryGiven the known therapeutic properties of E alba extractwe have performed enormous screening of different plantextracts on BalbC nude mice model and this has beenused as a unique model to study the hair growth mechanism[20 21]

Among all the plant extracts E alba showed outstandinghair growth promoting potential Topical applicationwas per-formed on the back of nude mice skin daily up to completionof two hair growth generationsThemacroscopic appearanceand the local distribution of hair were precisely observedin each individual group and showed clearly distinguishablefeature The regional distribution of the ldquoabortiverdquo anagenhair pattern in nude strains corresponded to the wave-likepattern of hair generations [22] The hair growth area ofE alba treated mice is distinctly visible and simultaneouslycovered the maximum part of the back In contrast Aradix P ginseng treated mice with increasing age rapidlydeveloped hair loss recognizable from the boundaries ofhair covering However a few uneven hairs are visibleparticularly in nude mice skin surface which referred to asldquoabortive hair growthrdquo [23] During anagen growth phasewe were able to observe a comparable growth pattern inE alba treated mice with other treated and control mice(Figure 1) These phenomena may suggest that E albaimproved the disordered keratinization of nude mouse hairfollicles because original control hair shafts are short and

8 BioMed Research International

irregular in shape suggesting some disordered keratinizationof the HF [24 25] The precise genetic defect of nude miceFoxn1mutation expression involved abnormal keratinizationas the cause for defective cell type in the hair development[26ndash28] Our observation suggests that treatment with E albaacts transiently to normalize keratinocyte of nude mice HFleading to fully formed visible HF with increased hair cover-age area An increase in the number and size of HFs has beenconsidered as an indicator for the transition of hair growthfrom telogen to anagen [9 19]These results suggest that top-ical application of E alba extract could stimulate the forma-tion of hair follicles and induce an earlier anagen phase com-pared to either the control or other treated groups (Figure 4)To elucidate the molecular mechanism underlying the abilityof E alba extract the BrdU immunohistochemistry resultsuggests an enhanced keratinocyte proliferation in anagenhair follicles [29] In our present studiesmacroscopic analysisof hair growth parameters included changing pattern ofnude mice hair growth hair coverage area and hair lengthHistological study of different plant extracts along withcontrol and minoxidil groups was investigated and foundto have fully formed HFs in E alba treated group as wellas increasing the number of HFs The active phase of HFcycling is also accompanied by increase in size and numberof follicles resulting in an enlargement of the subcutis layer[19] Given the vital role of hair restorer efficacy of E albaextract further HPLC analysis was carried out and showedthat wedelolactone and other components exist in methanolextract In this experiment none of the plant extracts causedadverse dermatological effects such as erythema rednessdrying or scaling and dermatitis as it was case withminoxidilgroup at the skin site of nude mice

It has been argued that combination of several con-stituents of a plant extract may act synergistically rather thansingle compound [30] The interaction is possibly due to thebinding of more than one compound at different sites on thesame target or receptor Further detailed clinical trials andstudies will be necessary to investigate what components inE alba extract contribute to its efficacy since whole E albaextract rather than individual components was used here toestablish its biological activity against alopecia

5 Conclusion

E alba extract containing active ingredients provided moresafety and compatibility which has more accessibility inpharmaceutical and cosmetic science and may promote theherbal medicine with minimal or no side effects or toxicitiesfor hair growth therapy Taking together all the investigationindicates that plant extract with suitable vehiclemay be usefulas an alternative topical medicine to minoxidil therapy

Conflict of Interests

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

Acknowledgments

The authors would like to express special thanks to Dr KangJuChoi a great generous person for sharing pearls of wisdomwith them which is gratefully acknowledged This study wassupported by a grant from the DBIO Science and TechnologyCorporation

References

[1] S Tiede J E Kloepper D A Whiting and R Paus ldquoTheldquofollicular trochanterrdquo an epithelial compartment of the humanhair follicle bulge region in need of further characterizationrdquoBritish Journal of Dermatology vol 157 no 5 pp 1013ndash10162007

[2] V A Randall ldquoAndrogens and human hair growthrdquo ClinicalEndocrinology vol 40 no 4 pp 439ndash457 1994

[3] H-J Park N Zhang and D K Park ldquoTopical applicationof Polygonum multiflorum extract induces hair growth ofresting hair follicles through upregulating Shh and 120573-cateninexpression in C57BL6 micerdquo Journal of Ethnopharmacologyvol 135 no 2 pp 369ndash375 2011

[4] C J Girman T Rhodes F R W Lilly et al ldquoEffects of self-perceived hair loss in a community sample of menrdquo Dermatol-ogy vol 197 no 3 pp 223ndash229 1998

[5] S S Rho S J Park S L Hwang et al ldquoThe hair growthpromoting effect of Asiasari radix extract and its molecularregulationrdquo Journal of Dermatological Science vol 38 no 2 pp89ndash97 2005

[6] H Matsuda M Yamazaki Y Asanuma and M Kubo ldquoPromo-tion of hair growth by ginseng radix on culturedmouse vibrissalhair folliclesrdquo Phytotherapy Research vol 17 no 7 pp 797ndash8002003

[7] S Park W-S Shin and J Ho ldquoFructus panax ginseng extractpromotes hair regeneration in C57BL6 micerdquo Journal ofEthnopharmacology vol 138 no 2 pp 340ndash344 2011

[8] R K Roy M Thakur and V K Dixit ldquoHair growth promotingactivity of Eclipta alba in male albino ratsrdquo Archives of Derma-tological Research vol 300 no 7 pp 357ndash364 2008

[9] K Datta A T Singh A Mukherjee B Bhat B Ramesh and AC Burman ldquoEclipta alba extract with potential for hair growthpromoting activityrdquo Journal of Ethnopharmacology vol 124 no3 pp 450ndash456 2009

[10] H Wagner B Geyer Y Kiso H Hikino and G S RaoldquoCoumestans as the main active principles of the liver drugsEclipta alba and Wedelia calendulaceardquo Planta Medica vol 5pp 370ndash374 1986

[11] A M S Pereira B W Bertoni A Menezes Jr P S Pereiraand S C Franca ldquoSoil pH and production of biomass andwedelolactone in field grown Eclipta albardquo Journal of HerbsSpices and Medicinal Plants vol 6 no 1 pp 43ndash48 1998

[12] D Manvar M Mishra S Kumar and V N Pandey ldquoIdentifi-cation and evaluation of anti Hepatitis C virus phytochemicalsfrom Eclipta albardquo Journal of Ethnopharmacology vol 144 no3 pp 545ndash554 2012

[13] Q-M Liu H-Y Zhao X-K Zhong and J-G Jiang ldquoEcliptaprostrata L phytochemicals isolation structure elucidationand their antitumor activityrdquo Food andChemical Toxicology vol50 no 11 pp 4016ndash4022 2012

[14] V DThakur and S AMengi ldquoNeuropharmacological profile ofEclipta alba (Linn) Hasskrdquo Journal of Ethnopharmacology vol102 no 1 pp 23ndash31 2005

BioMed Research International 9

[15] H Chaudhary V Dhuna J Singh S S Kamboj and S SeshadrildquoEvaluation of hydro-alcoholic extract of Eclipta alba for itsanticancer potential an in vitro studyrdquo Journal of Ethnophar-macology vol 136 no 2 pp 363ndash367 2011

[16] L Mecklenburg B Tychsen and R Paus ldquoLearning fromnudity lessons from the nude phenotyperdquo Experimental Der-matology vol 14 no 11 pp 797ndash810 2005

[17] G A Simon and H I Maibach ldquoRelevance of hairless mouse asan experimental model of percutaneous penetration in manrdquoSkin Pharmacology and Applied Skin Physiology vol 11 no 2pp 80ndash86 1998

[18] R Toll U Jacobi H Richter J Lademann H Schaefer and UBlume-Peytavi ldquoPenetration profile of microspheres in follicu-lar targeting of terminal hair folliclesrdquo Journal of InvestigativeDermatology vol 123 no 1 pp 168ndash176 2004

[19] R Paus S Muller-Rover C van der Veen et al ldquoA compre-hensive guide for the recognition and classification of distinctstages of hair follicle morphogenesisrdquo Journal of InvestigativeDermatology vol 113 no 4 pp 523ndash532 1999

[20] M Sawada N Terada H Taniguchi R Tateishi and YMori ldquoCyclosporin A stimulates hair growth in nude micerdquoLaboratory Investigation vol 56 no 6 pp 684ndash686 1987

[21] S Watanabe A Mochizuki K Wagatsuma M Kobayashi YKawa and H Takahashi ldquoHair growth on nude mice due tocyclosporin Ardquo The Journal of Dermatology vol 18 no 12 pp714ndash719 1991

[22] K Militzer ldquoHair growth pattern in nude micerdquo Cells TissuesOrgans vol 168 no 4 pp 285ndash294 2001

[23] U Iversen and O H Iversen ldquoCycles of hair growth in hairlessmicerdquoActa PathologicaMicrobiologica Scandinavica vol 69 no1 pp 50ndash62 1967

[24] R H Rigdon and A A Packchanian ldquoHistologic study of theskin of congenitally athymic ldquonuderdquo micerdquo Texas Reports onBiology amp Medicine vol 32 no 3-4 pp 711ndash723 1974

[25] Y Hozumi T Imaizumi and S Kondo ldquoEffect of cyclosporinon hair-existing area of nude micerdquo Journal of DermatologicalScience vol 7 pp S33ndashS38 1994

[26] S P Flanagan ldquoldquoNuderdquo a new hairless gene with pleiotropiceffects in the mouserdquo Genetical Research vol 8 no 3 pp 295ndash309 1966

[27] J L Brissette J Li J Kamimura D Lee and G P Dotto ldquoTheproduct of the mouse nude locus whn regulates the balancebetween epithelial cell growth and differentiationrdquo Genes andDevelopment vol 10 no 17 pp 2212ndash2221 1996

[28] L Mecklenburg R Paus Z Halata L S Bechtold P Fleckmanand J P Sundberg ldquoFOXN1 is critical for onycholemmalterminal differentiation in nude (Foxn1119899119906) micerdquo Journal ofInvestigative Dermatology vol 123 no 6 pp 1001ndash1011 2004

[29] H Pinkus T Iwasaki and Y Mishima ldquoOuter root sheathkeratinization in anagen and catagen of the mammalian hairfollicle A seventh distinct type of keratinization in the hairfollicle trichilemmal keratinizationrdquo Journal of Anatomy vol133 no 1 pp 19ndash35 1981

[30] H Wagner and G Ulrich-Merzenich ldquoSynergy researchapproaching a new generation of phytopharmaceuticalsrdquo Phy-tomedicine vol 16 no 2-3 pp 97ndash110 2009

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

6 BioMed Research International

Control

(a)

Minoxidil

(b)

A radix

(c)

E alba

(d)

P ginseng

(e)

0

10

20

30

40

50

60

70

Subcutis Total hair follicle

a

bc

cc

b

a

d

bc

b

cd

Num

ber o

f hai

r fol

licle

s (pe

r fiel

d)

ControlMinoxidilA radix

E albaP ginseng

(f)

Figure 5 Increase in number of HFs in the dorsal skin of nude mice treated with different plant extract Representative photomicrographs ofthe transverse sections of nudemice skin (a) control (b)minoxidil (c)A radix (d) E alba and (e) P ginsengwere stained with hematoxylin-eosin (HampE) (f)The number of hair follicles in subcutis with respect to the total number of HFs per field (119899 = 10) Values aremeanplusmn standarddeviation (SD) The different letters (a b c and d) within a column indicate significant differences (119875 lt 00001) determined by Duncanrsquosmultiple range test Scale bar 100 120583m

with that of control mice Following the guidelines proposedby [19] we determined that the control and P ginsengtreated mice HFs were on stage 6 (Figures 4(A1015840) and 4(E1015840))whereas E alba extract treated mice HFs were clearly at stage8 (Figure 4(D1015840)) Negligible numbers of hair shafts whichemerged from the HFs and moderately atrophic were alsoobserved in the skin ofA radix P ginseng and vehicle treatedmice (Figures 4(C) 4(E) and 4(A)) Moreover the hairshafts of those were defective (often twisted) with dilationof the hair canal and apparently had insufficient structuralrigidity to emerge through the epidermis (Figures 4(A1015840) and4(E1015840)) On the other hand E alba treatedmice seemed tohave a propensity for follicular accumulation of keratin andstructural rigidity to penetrate the epidermis (Figure 4(D))

35 Number of Hair Follicles Histologic assessment wasperformed to investigate the progression of hair follicles innude mice since there was an increase in the number ofhair follicles observed in the deep subcutis during the anagenphase In the representative transverse sections the numbers

of hair follicles were increased significantly in E alba (119875 lt00001) and minoxidil (119875 lt 0005) extract treated groupscompared to the control (Figure 5(f))

36 BrdU Immunohistochemistry To further investigate themolecularmechanismunderlying the ability ofE alba extractto form nude mice hair follicles we performed immunohis-tochemistry with BrdU antibodies of the back skin of nudemice We found that E alba treated mice skin exhibits BrdUpositive keratinocyte in the HFs particularly a single celllayer that surrounds the innermost layer of the outer rootsheath as shown in Figures 6(B) and 6(B1015840) Moreover Ealba treated nude mice exhibited a significant (119875 lt 00001)increase in the number of BrdU-labeled keratinocyte peranagen follicle versus control mice (Figure 6(C)) On theother hand there was no significant difference in the numberof BrdU-labeled epithelial cells per sebaceous gland

37 Identification and Quantification of Wedelolactone in Ealba Extract Additionally HPLC analysis was performed to

BioMed Research International 7

0

5

10

15

20

25

30

HF SG

Num

ber o

f Brd

U p

ositi

ve ce

lls

lowastlowastlowast

(C)

ControlE alba

Control E alba

SBSB

HF

(A) (B)

(A998400) (B998400)

Figure 6 BrdU-labeled proliferating cells in anagen hair folliclesControl (A) and E alba extract treated (B) follicles are nuclear BrdUstaining (Arrows red-brown) Scale bar 100 120583m (B) and 50 120583m ((A)(A1015840) and (B1015840)) Number of BrdU positive cells (C) Data shown aremeans of five replicate samples (lowastlowastlowast119875 lt 0001)

illustrate the methanol extract of E alba (Figure 7) and wasfound to contain principle constituents like wedelolactoneThe existence of the wedelolactone was quantified by 18in methanol extract It has been reported that various bioac-tive phytochemicals found in the E alba extract includingwedelolactone demethylwedelolactone and saponins areresponsible for the main medicinal effects of E alba suchas antitumor activity [13] antihepatitis C virus [12] andneuropharmacological activity [14] and its anticancer effectshows antiproliferative and cytotoxic effects determinedusing MTT assay [15]

00 50 100 150 200 250 300 3500

250

500

750

1000

1250 Standard wedelolactone

(min)

(mV

)

(a)

00 50 100 150 200 250 300 3500

250500750

1000125015001750

Wedelolactone

(min)(m

V)

(b)

Figure 7 HPLC chromatogram (a) Standard wedelolactone (b)HPLC chromatogram of methanol extract of E alba

4 Discussion

Medicinal plants may have potential by identifying activebiological components and their successful use might openreverse pharmacology a new approach to drug discoveryGiven the known therapeutic properties of E alba extractwe have performed enormous screening of different plantextracts on BalbC nude mice model and this has beenused as a unique model to study the hair growth mechanism[20 21]

Among all the plant extracts E alba showed outstandinghair growth promoting potential Topical applicationwas per-formed on the back of nude mice skin daily up to completionof two hair growth generationsThemacroscopic appearanceand the local distribution of hair were precisely observedin each individual group and showed clearly distinguishablefeature The regional distribution of the ldquoabortiverdquo anagenhair pattern in nude strains corresponded to the wave-likepattern of hair generations [22] The hair growth area ofE alba treated mice is distinctly visible and simultaneouslycovered the maximum part of the back In contrast Aradix P ginseng treated mice with increasing age rapidlydeveloped hair loss recognizable from the boundaries ofhair covering However a few uneven hairs are visibleparticularly in nude mice skin surface which referred to asldquoabortive hair growthrdquo [23] During anagen growth phasewe were able to observe a comparable growth pattern inE alba treated mice with other treated and control mice(Figure 1) These phenomena may suggest that E albaimproved the disordered keratinization of nude mouse hairfollicles because original control hair shafts are short and

8 BioMed Research International

irregular in shape suggesting some disordered keratinizationof the HF [24 25] The precise genetic defect of nude miceFoxn1mutation expression involved abnormal keratinizationas the cause for defective cell type in the hair development[26ndash28] Our observation suggests that treatment with E albaacts transiently to normalize keratinocyte of nude mice HFleading to fully formed visible HF with increased hair cover-age area An increase in the number and size of HFs has beenconsidered as an indicator for the transition of hair growthfrom telogen to anagen [9 19]These results suggest that top-ical application of E alba extract could stimulate the forma-tion of hair follicles and induce an earlier anagen phase com-pared to either the control or other treated groups (Figure 4)To elucidate the molecular mechanism underlying the abilityof E alba extract the BrdU immunohistochemistry resultsuggests an enhanced keratinocyte proliferation in anagenhair follicles [29] In our present studiesmacroscopic analysisof hair growth parameters included changing pattern ofnude mice hair growth hair coverage area and hair lengthHistological study of different plant extracts along withcontrol and minoxidil groups was investigated and foundto have fully formed HFs in E alba treated group as wellas increasing the number of HFs The active phase of HFcycling is also accompanied by increase in size and numberof follicles resulting in an enlargement of the subcutis layer[19] Given the vital role of hair restorer efficacy of E albaextract further HPLC analysis was carried out and showedthat wedelolactone and other components exist in methanolextract In this experiment none of the plant extracts causedadverse dermatological effects such as erythema rednessdrying or scaling and dermatitis as it was case withminoxidilgroup at the skin site of nude mice

It has been argued that combination of several con-stituents of a plant extract may act synergistically rather thansingle compound [30] The interaction is possibly due to thebinding of more than one compound at different sites on thesame target or receptor Further detailed clinical trials andstudies will be necessary to investigate what components inE alba extract contribute to its efficacy since whole E albaextract rather than individual components was used here toestablish its biological activity against alopecia

5 Conclusion

E alba extract containing active ingredients provided moresafety and compatibility which has more accessibility inpharmaceutical and cosmetic science and may promote theherbal medicine with minimal or no side effects or toxicitiesfor hair growth therapy Taking together all the investigationindicates that plant extract with suitable vehiclemay be usefulas an alternative topical medicine to minoxidil therapy

Conflict of Interests

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

Acknowledgments

The authors would like to express special thanks to Dr KangJuChoi a great generous person for sharing pearls of wisdomwith them which is gratefully acknowledged This study wassupported by a grant from the DBIO Science and TechnologyCorporation

References

[1] S Tiede J E Kloepper D A Whiting and R Paus ldquoTheldquofollicular trochanterrdquo an epithelial compartment of the humanhair follicle bulge region in need of further characterizationrdquoBritish Journal of Dermatology vol 157 no 5 pp 1013ndash10162007

[2] V A Randall ldquoAndrogens and human hair growthrdquo ClinicalEndocrinology vol 40 no 4 pp 439ndash457 1994

[3] H-J Park N Zhang and D K Park ldquoTopical applicationof Polygonum multiflorum extract induces hair growth ofresting hair follicles through upregulating Shh and 120573-cateninexpression in C57BL6 micerdquo Journal of Ethnopharmacologyvol 135 no 2 pp 369ndash375 2011

[4] C J Girman T Rhodes F R W Lilly et al ldquoEffects of self-perceived hair loss in a community sample of menrdquo Dermatol-ogy vol 197 no 3 pp 223ndash229 1998

[5] S S Rho S J Park S L Hwang et al ldquoThe hair growthpromoting effect of Asiasari radix extract and its molecularregulationrdquo Journal of Dermatological Science vol 38 no 2 pp89ndash97 2005

[6] H Matsuda M Yamazaki Y Asanuma and M Kubo ldquoPromo-tion of hair growth by ginseng radix on culturedmouse vibrissalhair folliclesrdquo Phytotherapy Research vol 17 no 7 pp 797ndash8002003

[7] S Park W-S Shin and J Ho ldquoFructus panax ginseng extractpromotes hair regeneration in C57BL6 micerdquo Journal ofEthnopharmacology vol 138 no 2 pp 340ndash344 2011

[8] R K Roy M Thakur and V K Dixit ldquoHair growth promotingactivity of Eclipta alba in male albino ratsrdquo Archives of Derma-tological Research vol 300 no 7 pp 357ndash364 2008

[9] K Datta A T Singh A Mukherjee B Bhat B Ramesh and AC Burman ldquoEclipta alba extract with potential for hair growthpromoting activityrdquo Journal of Ethnopharmacology vol 124 no3 pp 450ndash456 2009

[10] H Wagner B Geyer Y Kiso H Hikino and G S RaoldquoCoumestans as the main active principles of the liver drugsEclipta alba and Wedelia calendulaceardquo Planta Medica vol 5pp 370ndash374 1986

[11] A M S Pereira B W Bertoni A Menezes Jr P S Pereiraand S C Franca ldquoSoil pH and production of biomass andwedelolactone in field grown Eclipta albardquo Journal of HerbsSpices and Medicinal Plants vol 6 no 1 pp 43ndash48 1998

[12] D Manvar M Mishra S Kumar and V N Pandey ldquoIdentifi-cation and evaluation of anti Hepatitis C virus phytochemicalsfrom Eclipta albardquo Journal of Ethnopharmacology vol 144 no3 pp 545ndash554 2012

[13] Q-M Liu H-Y Zhao X-K Zhong and J-G Jiang ldquoEcliptaprostrata L phytochemicals isolation structure elucidationand their antitumor activityrdquo Food andChemical Toxicology vol50 no 11 pp 4016ndash4022 2012

[14] V DThakur and S AMengi ldquoNeuropharmacological profile ofEclipta alba (Linn) Hasskrdquo Journal of Ethnopharmacology vol102 no 1 pp 23ndash31 2005

BioMed Research International 9

[15] H Chaudhary V Dhuna J Singh S S Kamboj and S SeshadrildquoEvaluation of hydro-alcoholic extract of Eclipta alba for itsanticancer potential an in vitro studyrdquo Journal of Ethnophar-macology vol 136 no 2 pp 363ndash367 2011

[16] L Mecklenburg B Tychsen and R Paus ldquoLearning fromnudity lessons from the nude phenotyperdquo Experimental Der-matology vol 14 no 11 pp 797ndash810 2005

[17] G A Simon and H I Maibach ldquoRelevance of hairless mouse asan experimental model of percutaneous penetration in manrdquoSkin Pharmacology and Applied Skin Physiology vol 11 no 2pp 80ndash86 1998

[18] R Toll U Jacobi H Richter J Lademann H Schaefer and UBlume-Peytavi ldquoPenetration profile of microspheres in follicu-lar targeting of terminal hair folliclesrdquo Journal of InvestigativeDermatology vol 123 no 1 pp 168ndash176 2004

[19] R Paus S Muller-Rover C van der Veen et al ldquoA compre-hensive guide for the recognition and classification of distinctstages of hair follicle morphogenesisrdquo Journal of InvestigativeDermatology vol 113 no 4 pp 523ndash532 1999

[20] M Sawada N Terada H Taniguchi R Tateishi and YMori ldquoCyclosporin A stimulates hair growth in nude micerdquoLaboratory Investigation vol 56 no 6 pp 684ndash686 1987

[21] S Watanabe A Mochizuki K Wagatsuma M Kobayashi YKawa and H Takahashi ldquoHair growth on nude mice due tocyclosporin Ardquo The Journal of Dermatology vol 18 no 12 pp714ndash719 1991

[22] K Militzer ldquoHair growth pattern in nude micerdquo Cells TissuesOrgans vol 168 no 4 pp 285ndash294 2001

[23] U Iversen and O H Iversen ldquoCycles of hair growth in hairlessmicerdquoActa PathologicaMicrobiologica Scandinavica vol 69 no1 pp 50ndash62 1967

[24] R H Rigdon and A A Packchanian ldquoHistologic study of theskin of congenitally athymic ldquonuderdquo micerdquo Texas Reports onBiology amp Medicine vol 32 no 3-4 pp 711ndash723 1974

[25] Y Hozumi T Imaizumi and S Kondo ldquoEffect of cyclosporinon hair-existing area of nude micerdquo Journal of DermatologicalScience vol 7 pp S33ndashS38 1994

[26] S P Flanagan ldquoldquoNuderdquo a new hairless gene with pleiotropiceffects in the mouserdquo Genetical Research vol 8 no 3 pp 295ndash309 1966

[27] J L Brissette J Li J Kamimura D Lee and G P Dotto ldquoTheproduct of the mouse nude locus whn regulates the balancebetween epithelial cell growth and differentiationrdquo Genes andDevelopment vol 10 no 17 pp 2212ndash2221 1996

[28] L Mecklenburg R Paus Z Halata L S Bechtold P Fleckmanand J P Sundberg ldquoFOXN1 is critical for onycholemmalterminal differentiation in nude (Foxn1119899119906) micerdquo Journal ofInvestigative Dermatology vol 123 no 6 pp 1001ndash1011 2004

[29] H Pinkus T Iwasaki and Y Mishima ldquoOuter root sheathkeratinization in anagen and catagen of the mammalian hairfollicle A seventh distinct type of keratinization in the hairfollicle trichilemmal keratinizationrdquo Journal of Anatomy vol133 no 1 pp 19ndash35 1981

[30] H Wagner and G Ulrich-Merzenich ldquoSynergy researchapproaching a new generation of phytopharmaceuticalsrdquo Phy-tomedicine vol 16 no 2-3 pp 97ndash110 2009

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

BioMed Research International 7

0

5

10

15

20

25

30

HF SG

Num

ber o

f Brd

U p

ositi

ve ce

lls

lowastlowastlowast

(C)

ControlE alba

Control E alba

SBSB

HF

(A) (B)

(A998400) (B998400)

Figure 6 BrdU-labeled proliferating cells in anagen hair folliclesControl (A) and E alba extract treated (B) follicles are nuclear BrdUstaining (Arrows red-brown) Scale bar 100 120583m (B) and 50 120583m ((A)(A1015840) and (B1015840)) Number of BrdU positive cells (C) Data shown aremeans of five replicate samples (lowastlowastlowast119875 lt 0001)

illustrate the methanol extract of E alba (Figure 7) and wasfound to contain principle constituents like wedelolactoneThe existence of the wedelolactone was quantified by 18in methanol extract It has been reported that various bioac-tive phytochemicals found in the E alba extract includingwedelolactone demethylwedelolactone and saponins areresponsible for the main medicinal effects of E alba suchas antitumor activity [13] antihepatitis C virus [12] andneuropharmacological activity [14] and its anticancer effectshows antiproliferative and cytotoxic effects determinedusing MTT assay [15]

00 50 100 150 200 250 300 3500

250

500

750

1000

1250 Standard wedelolactone

(min)

(mV

)

(a)

00 50 100 150 200 250 300 3500

250500750

1000125015001750

Wedelolactone

(min)(m

V)

(b)

Figure 7 HPLC chromatogram (a) Standard wedelolactone (b)HPLC chromatogram of methanol extract of E alba

4 Discussion

Medicinal plants may have potential by identifying activebiological components and their successful use might openreverse pharmacology a new approach to drug discoveryGiven the known therapeutic properties of E alba extractwe have performed enormous screening of different plantextracts on BalbC nude mice model and this has beenused as a unique model to study the hair growth mechanism[20 21]

Among all the plant extracts E alba showed outstandinghair growth promoting potential Topical applicationwas per-formed on the back of nude mice skin daily up to completionof two hair growth generationsThemacroscopic appearanceand the local distribution of hair were precisely observedin each individual group and showed clearly distinguishablefeature The regional distribution of the ldquoabortiverdquo anagenhair pattern in nude strains corresponded to the wave-likepattern of hair generations [22] The hair growth area ofE alba treated mice is distinctly visible and simultaneouslycovered the maximum part of the back In contrast Aradix P ginseng treated mice with increasing age rapidlydeveloped hair loss recognizable from the boundaries ofhair covering However a few uneven hairs are visibleparticularly in nude mice skin surface which referred to asldquoabortive hair growthrdquo [23] During anagen growth phasewe were able to observe a comparable growth pattern inE alba treated mice with other treated and control mice(Figure 1) These phenomena may suggest that E albaimproved the disordered keratinization of nude mouse hairfollicles because original control hair shafts are short and

8 BioMed Research International

irregular in shape suggesting some disordered keratinizationof the HF [24 25] The precise genetic defect of nude miceFoxn1mutation expression involved abnormal keratinizationas the cause for defective cell type in the hair development[26ndash28] Our observation suggests that treatment with E albaacts transiently to normalize keratinocyte of nude mice HFleading to fully formed visible HF with increased hair cover-age area An increase in the number and size of HFs has beenconsidered as an indicator for the transition of hair growthfrom telogen to anagen [9 19]These results suggest that top-ical application of E alba extract could stimulate the forma-tion of hair follicles and induce an earlier anagen phase com-pared to either the control or other treated groups (Figure 4)To elucidate the molecular mechanism underlying the abilityof E alba extract the BrdU immunohistochemistry resultsuggests an enhanced keratinocyte proliferation in anagenhair follicles [29] In our present studiesmacroscopic analysisof hair growth parameters included changing pattern ofnude mice hair growth hair coverage area and hair lengthHistological study of different plant extracts along withcontrol and minoxidil groups was investigated and foundto have fully formed HFs in E alba treated group as wellas increasing the number of HFs The active phase of HFcycling is also accompanied by increase in size and numberof follicles resulting in an enlargement of the subcutis layer[19] Given the vital role of hair restorer efficacy of E albaextract further HPLC analysis was carried out and showedthat wedelolactone and other components exist in methanolextract In this experiment none of the plant extracts causedadverse dermatological effects such as erythema rednessdrying or scaling and dermatitis as it was case withminoxidilgroup at the skin site of nude mice

It has been argued that combination of several con-stituents of a plant extract may act synergistically rather thansingle compound [30] The interaction is possibly due to thebinding of more than one compound at different sites on thesame target or receptor Further detailed clinical trials andstudies will be necessary to investigate what components inE alba extract contribute to its efficacy since whole E albaextract rather than individual components was used here toestablish its biological activity against alopecia

5 Conclusion

E alba extract containing active ingredients provided moresafety and compatibility which has more accessibility inpharmaceutical and cosmetic science and may promote theherbal medicine with minimal or no side effects or toxicitiesfor hair growth therapy Taking together all the investigationindicates that plant extract with suitable vehiclemay be usefulas an alternative topical medicine to minoxidil therapy

Conflict of Interests

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

Acknowledgments

The authors would like to express special thanks to Dr KangJuChoi a great generous person for sharing pearls of wisdomwith them which is gratefully acknowledged This study wassupported by a grant from the DBIO Science and TechnologyCorporation

References

[1] S Tiede J E Kloepper D A Whiting and R Paus ldquoTheldquofollicular trochanterrdquo an epithelial compartment of the humanhair follicle bulge region in need of further characterizationrdquoBritish Journal of Dermatology vol 157 no 5 pp 1013ndash10162007

[2] V A Randall ldquoAndrogens and human hair growthrdquo ClinicalEndocrinology vol 40 no 4 pp 439ndash457 1994

[3] H-J Park N Zhang and D K Park ldquoTopical applicationof Polygonum multiflorum extract induces hair growth ofresting hair follicles through upregulating Shh and 120573-cateninexpression in C57BL6 micerdquo Journal of Ethnopharmacologyvol 135 no 2 pp 369ndash375 2011

[4] C J Girman T Rhodes F R W Lilly et al ldquoEffects of self-perceived hair loss in a community sample of menrdquo Dermatol-ogy vol 197 no 3 pp 223ndash229 1998

[5] S S Rho S J Park S L Hwang et al ldquoThe hair growthpromoting effect of Asiasari radix extract and its molecularregulationrdquo Journal of Dermatological Science vol 38 no 2 pp89ndash97 2005

[6] H Matsuda M Yamazaki Y Asanuma and M Kubo ldquoPromo-tion of hair growth by ginseng radix on culturedmouse vibrissalhair folliclesrdquo Phytotherapy Research vol 17 no 7 pp 797ndash8002003

[7] S Park W-S Shin and J Ho ldquoFructus panax ginseng extractpromotes hair regeneration in C57BL6 micerdquo Journal ofEthnopharmacology vol 138 no 2 pp 340ndash344 2011

[8] R K Roy M Thakur and V K Dixit ldquoHair growth promotingactivity of Eclipta alba in male albino ratsrdquo Archives of Derma-tological Research vol 300 no 7 pp 357ndash364 2008

[9] K Datta A T Singh A Mukherjee B Bhat B Ramesh and AC Burman ldquoEclipta alba extract with potential for hair growthpromoting activityrdquo Journal of Ethnopharmacology vol 124 no3 pp 450ndash456 2009

[10] H Wagner B Geyer Y Kiso H Hikino and G S RaoldquoCoumestans as the main active principles of the liver drugsEclipta alba and Wedelia calendulaceardquo Planta Medica vol 5pp 370ndash374 1986

[11] A M S Pereira B W Bertoni A Menezes Jr P S Pereiraand S C Franca ldquoSoil pH and production of biomass andwedelolactone in field grown Eclipta albardquo Journal of HerbsSpices and Medicinal Plants vol 6 no 1 pp 43ndash48 1998

[12] D Manvar M Mishra S Kumar and V N Pandey ldquoIdentifi-cation and evaluation of anti Hepatitis C virus phytochemicalsfrom Eclipta albardquo Journal of Ethnopharmacology vol 144 no3 pp 545ndash554 2012

[13] Q-M Liu H-Y Zhao X-K Zhong and J-G Jiang ldquoEcliptaprostrata L phytochemicals isolation structure elucidationand their antitumor activityrdquo Food andChemical Toxicology vol50 no 11 pp 4016ndash4022 2012

[14] V DThakur and S AMengi ldquoNeuropharmacological profile ofEclipta alba (Linn) Hasskrdquo Journal of Ethnopharmacology vol102 no 1 pp 23ndash31 2005

BioMed Research International 9

[15] H Chaudhary V Dhuna J Singh S S Kamboj and S SeshadrildquoEvaluation of hydro-alcoholic extract of Eclipta alba for itsanticancer potential an in vitro studyrdquo Journal of Ethnophar-macology vol 136 no 2 pp 363ndash367 2011

[16] L Mecklenburg B Tychsen and R Paus ldquoLearning fromnudity lessons from the nude phenotyperdquo Experimental Der-matology vol 14 no 11 pp 797ndash810 2005

[17] G A Simon and H I Maibach ldquoRelevance of hairless mouse asan experimental model of percutaneous penetration in manrdquoSkin Pharmacology and Applied Skin Physiology vol 11 no 2pp 80ndash86 1998

[18] R Toll U Jacobi H Richter J Lademann H Schaefer and UBlume-Peytavi ldquoPenetration profile of microspheres in follicu-lar targeting of terminal hair folliclesrdquo Journal of InvestigativeDermatology vol 123 no 1 pp 168ndash176 2004

[19] R Paus S Muller-Rover C van der Veen et al ldquoA compre-hensive guide for the recognition and classification of distinctstages of hair follicle morphogenesisrdquo Journal of InvestigativeDermatology vol 113 no 4 pp 523ndash532 1999

[20] M Sawada N Terada H Taniguchi R Tateishi and YMori ldquoCyclosporin A stimulates hair growth in nude micerdquoLaboratory Investigation vol 56 no 6 pp 684ndash686 1987

[21] S Watanabe A Mochizuki K Wagatsuma M Kobayashi YKawa and H Takahashi ldquoHair growth on nude mice due tocyclosporin Ardquo The Journal of Dermatology vol 18 no 12 pp714ndash719 1991

[22] K Militzer ldquoHair growth pattern in nude micerdquo Cells TissuesOrgans vol 168 no 4 pp 285ndash294 2001

[23] U Iversen and O H Iversen ldquoCycles of hair growth in hairlessmicerdquoActa PathologicaMicrobiologica Scandinavica vol 69 no1 pp 50ndash62 1967

[24] R H Rigdon and A A Packchanian ldquoHistologic study of theskin of congenitally athymic ldquonuderdquo micerdquo Texas Reports onBiology amp Medicine vol 32 no 3-4 pp 711ndash723 1974

[25] Y Hozumi T Imaizumi and S Kondo ldquoEffect of cyclosporinon hair-existing area of nude micerdquo Journal of DermatologicalScience vol 7 pp S33ndashS38 1994

[26] S P Flanagan ldquoldquoNuderdquo a new hairless gene with pleiotropiceffects in the mouserdquo Genetical Research vol 8 no 3 pp 295ndash309 1966

[27] J L Brissette J Li J Kamimura D Lee and G P Dotto ldquoTheproduct of the mouse nude locus whn regulates the balancebetween epithelial cell growth and differentiationrdquo Genes andDevelopment vol 10 no 17 pp 2212ndash2221 1996

[28] L Mecklenburg R Paus Z Halata L S Bechtold P Fleckmanand J P Sundberg ldquoFOXN1 is critical for onycholemmalterminal differentiation in nude (Foxn1119899119906) micerdquo Journal ofInvestigative Dermatology vol 123 no 6 pp 1001ndash1011 2004

[29] H Pinkus T Iwasaki and Y Mishima ldquoOuter root sheathkeratinization in anagen and catagen of the mammalian hairfollicle A seventh distinct type of keratinization in the hairfollicle trichilemmal keratinizationrdquo Journal of Anatomy vol133 no 1 pp 19ndash35 1981

[30] H Wagner and G Ulrich-Merzenich ldquoSynergy researchapproaching a new generation of phytopharmaceuticalsrdquo Phy-tomedicine vol 16 no 2-3 pp 97ndash110 2009

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

8 BioMed Research International

irregular in shape suggesting some disordered keratinizationof the HF [24 25] The precise genetic defect of nude miceFoxn1mutation expression involved abnormal keratinizationas the cause for defective cell type in the hair development[26ndash28] Our observation suggests that treatment with E albaacts transiently to normalize keratinocyte of nude mice HFleading to fully formed visible HF with increased hair cover-age area An increase in the number and size of HFs has beenconsidered as an indicator for the transition of hair growthfrom telogen to anagen [9 19]These results suggest that top-ical application of E alba extract could stimulate the forma-tion of hair follicles and induce an earlier anagen phase com-pared to either the control or other treated groups (Figure 4)To elucidate the molecular mechanism underlying the abilityof E alba extract the BrdU immunohistochemistry resultsuggests an enhanced keratinocyte proliferation in anagenhair follicles [29] In our present studiesmacroscopic analysisof hair growth parameters included changing pattern ofnude mice hair growth hair coverage area and hair lengthHistological study of different plant extracts along withcontrol and minoxidil groups was investigated and foundto have fully formed HFs in E alba treated group as wellas increasing the number of HFs The active phase of HFcycling is also accompanied by increase in size and numberof follicles resulting in an enlargement of the subcutis layer[19] Given the vital role of hair restorer efficacy of E albaextract further HPLC analysis was carried out and showedthat wedelolactone and other components exist in methanolextract In this experiment none of the plant extracts causedadverse dermatological effects such as erythema rednessdrying or scaling and dermatitis as it was case withminoxidilgroup at the skin site of nude mice

It has been argued that combination of several con-stituents of a plant extract may act synergistically rather thansingle compound [30] The interaction is possibly due to thebinding of more than one compound at different sites on thesame target or receptor Further detailed clinical trials andstudies will be necessary to investigate what components inE alba extract contribute to its efficacy since whole E albaextract rather than individual components was used here toestablish its biological activity against alopecia

5 Conclusion

E alba extract containing active ingredients provided moresafety and compatibility which has more accessibility inpharmaceutical and cosmetic science and may promote theherbal medicine with minimal or no side effects or toxicitiesfor hair growth therapy Taking together all the investigationindicates that plant extract with suitable vehiclemay be usefulas an alternative topical medicine to minoxidil therapy

Conflict of Interests

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

Acknowledgments

The authors would like to express special thanks to Dr KangJuChoi a great generous person for sharing pearls of wisdomwith them which is gratefully acknowledged This study wassupported by a grant from the DBIO Science and TechnologyCorporation

References

[1] S Tiede J E Kloepper D A Whiting and R Paus ldquoTheldquofollicular trochanterrdquo an epithelial compartment of the humanhair follicle bulge region in need of further characterizationrdquoBritish Journal of Dermatology vol 157 no 5 pp 1013ndash10162007

[2] V A Randall ldquoAndrogens and human hair growthrdquo ClinicalEndocrinology vol 40 no 4 pp 439ndash457 1994

[3] H-J Park N Zhang and D K Park ldquoTopical applicationof Polygonum multiflorum extract induces hair growth ofresting hair follicles through upregulating Shh and 120573-cateninexpression in C57BL6 micerdquo Journal of Ethnopharmacologyvol 135 no 2 pp 369ndash375 2011

[4] C J Girman T Rhodes F R W Lilly et al ldquoEffects of self-perceived hair loss in a community sample of menrdquo Dermatol-ogy vol 197 no 3 pp 223ndash229 1998

[5] S S Rho S J Park S L Hwang et al ldquoThe hair growthpromoting effect of Asiasari radix extract and its molecularregulationrdquo Journal of Dermatological Science vol 38 no 2 pp89ndash97 2005

[6] H Matsuda M Yamazaki Y Asanuma and M Kubo ldquoPromo-tion of hair growth by ginseng radix on culturedmouse vibrissalhair folliclesrdquo Phytotherapy Research vol 17 no 7 pp 797ndash8002003

[7] S Park W-S Shin and J Ho ldquoFructus panax ginseng extractpromotes hair regeneration in C57BL6 micerdquo Journal ofEthnopharmacology vol 138 no 2 pp 340ndash344 2011

[8] R K Roy M Thakur and V K Dixit ldquoHair growth promotingactivity of Eclipta alba in male albino ratsrdquo Archives of Derma-tological Research vol 300 no 7 pp 357ndash364 2008

[9] K Datta A T Singh A Mukherjee B Bhat B Ramesh and AC Burman ldquoEclipta alba extract with potential for hair growthpromoting activityrdquo Journal of Ethnopharmacology vol 124 no3 pp 450ndash456 2009

[10] H Wagner B Geyer Y Kiso H Hikino and G S RaoldquoCoumestans as the main active principles of the liver drugsEclipta alba and Wedelia calendulaceardquo Planta Medica vol 5pp 370ndash374 1986

[11] A M S Pereira B W Bertoni A Menezes Jr P S Pereiraand S C Franca ldquoSoil pH and production of biomass andwedelolactone in field grown Eclipta albardquo Journal of HerbsSpices and Medicinal Plants vol 6 no 1 pp 43ndash48 1998

[12] D Manvar M Mishra S Kumar and V N Pandey ldquoIdentifi-cation and evaluation of anti Hepatitis C virus phytochemicalsfrom Eclipta albardquo Journal of Ethnopharmacology vol 144 no3 pp 545ndash554 2012

[13] Q-M Liu H-Y Zhao X-K Zhong and J-G Jiang ldquoEcliptaprostrata L phytochemicals isolation structure elucidationand their antitumor activityrdquo Food andChemical Toxicology vol50 no 11 pp 4016ndash4022 2012

[14] V DThakur and S AMengi ldquoNeuropharmacological profile ofEclipta alba (Linn) Hasskrdquo Journal of Ethnopharmacology vol102 no 1 pp 23ndash31 2005

BioMed Research International 9

[15] H Chaudhary V Dhuna J Singh S S Kamboj and S SeshadrildquoEvaluation of hydro-alcoholic extract of Eclipta alba for itsanticancer potential an in vitro studyrdquo Journal of Ethnophar-macology vol 136 no 2 pp 363ndash367 2011

[16] L Mecklenburg B Tychsen and R Paus ldquoLearning fromnudity lessons from the nude phenotyperdquo Experimental Der-matology vol 14 no 11 pp 797ndash810 2005

[17] G A Simon and H I Maibach ldquoRelevance of hairless mouse asan experimental model of percutaneous penetration in manrdquoSkin Pharmacology and Applied Skin Physiology vol 11 no 2pp 80ndash86 1998

[18] R Toll U Jacobi H Richter J Lademann H Schaefer and UBlume-Peytavi ldquoPenetration profile of microspheres in follicu-lar targeting of terminal hair folliclesrdquo Journal of InvestigativeDermatology vol 123 no 1 pp 168ndash176 2004

[19] R Paus S Muller-Rover C van der Veen et al ldquoA compre-hensive guide for the recognition and classification of distinctstages of hair follicle morphogenesisrdquo Journal of InvestigativeDermatology vol 113 no 4 pp 523ndash532 1999

[20] M Sawada N Terada H Taniguchi R Tateishi and YMori ldquoCyclosporin A stimulates hair growth in nude micerdquoLaboratory Investigation vol 56 no 6 pp 684ndash686 1987

[21] S Watanabe A Mochizuki K Wagatsuma M Kobayashi YKawa and H Takahashi ldquoHair growth on nude mice due tocyclosporin Ardquo The Journal of Dermatology vol 18 no 12 pp714ndash719 1991

[22] K Militzer ldquoHair growth pattern in nude micerdquo Cells TissuesOrgans vol 168 no 4 pp 285ndash294 2001

[23] U Iversen and O H Iversen ldquoCycles of hair growth in hairlessmicerdquoActa PathologicaMicrobiologica Scandinavica vol 69 no1 pp 50ndash62 1967

[24] R H Rigdon and A A Packchanian ldquoHistologic study of theskin of congenitally athymic ldquonuderdquo micerdquo Texas Reports onBiology amp Medicine vol 32 no 3-4 pp 711ndash723 1974

[25] Y Hozumi T Imaizumi and S Kondo ldquoEffect of cyclosporinon hair-existing area of nude micerdquo Journal of DermatologicalScience vol 7 pp S33ndashS38 1994

[26] S P Flanagan ldquoldquoNuderdquo a new hairless gene with pleiotropiceffects in the mouserdquo Genetical Research vol 8 no 3 pp 295ndash309 1966

[27] J L Brissette J Li J Kamimura D Lee and G P Dotto ldquoTheproduct of the mouse nude locus whn regulates the balancebetween epithelial cell growth and differentiationrdquo Genes andDevelopment vol 10 no 17 pp 2212ndash2221 1996

[28] L Mecklenburg R Paus Z Halata L S Bechtold P Fleckmanand J P Sundberg ldquoFOXN1 is critical for onycholemmalterminal differentiation in nude (Foxn1119899119906) micerdquo Journal ofInvestigative Dermatology vol 123 no 6 pp 1001ndash1011 2004

[29] H Pinkus T Iwasaki and Y Mishima ldquoOuter root sheathkeratinization in anagen and catagen of the mammalian hairfollicle A seventh distinct type of keratinization in the hairfollicle trichilemmal keratinizationrdquo Journal of Anatomy vol133 no 1 pp 19ndash35 1981

[30] H Wagner and G Ulrich-Merzenich ldquoSynergy researchapproaching a new generation of phytopharmaceuticalsrdquo Phy-tomedicine vol 16 no 2-3 pp 97ndash110 2009

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

BioMed Research International 9

[15] H Chaudhary V Dhuna J Singh S S Kamboj and S SeshadrildquoEvaluation of hydro-alcoholic extract of Eclipta alba for itsanticancer potential an in vitro studyrdquo Journal of Ethnophar-macology vol 136 no 2 pp 363ndash367 2011

[16] L Mecklenburg B Tychsen and R Paus ldquoLearning fromnudity lessons from the nude phenotyperdquo Experimental Der-matology vol 14 no 11 pp 797ndash810 2005

[17] G A Simon and H I Maibach ldquoRelevance of hairless mouse asan experimental model of percutaneous penetration in manrdquoSkin Pharmacology and Applied Skin Physiology vol 11 no 2pp 80ndash86 1998

[18] R Toll U Jacobi H Richter J Lademann H Schaefer and UBlume-Peytavi ldquoPenetration profile of microspheres in follicu-lar targeting of terminal hair folliclesrdquo Journal of InvestigativeDermatology vol 123 no 1 pp 168ndash176 2004

[19] R Paus S Muller-Rover C van der Veen et al ldquoA compre-hensive guide for the recognition and classification of distinctstages of hair follicle morphogenesisrdquo Journal of InvestigativeDermatology vol 113 no 4 pp 523ndash532 1999

[20] M Sawada N Terada H Taniguchi R Tateishi and YMori ldquoCyclosporin A stimulates hair growth in nude micerdquoLaboratory Investigation vol 56 no 6 pp 684ndash686 1987

[21] S Watanabe A Mochizuki K Wagatsuma M Kobayashi YKawa and H Takahashi ldquoHair growth on nude mice due tocyclosporin Ardquo The Journal of Dermatology vol 18 no 12 pp714ndash719 1991

[22] K Militzer ldquoHair growth pattern in nude micerdquo Cells TissuesOrgans vol 168 no 4 pp 285ndash294 2001

[23] U Iversen and O H Iversen ldquoCycles of hair growth in hairlessmicerdquoActa PathologicaMicrobiologica Scandinavica vol 69 no1 pp 50ndash62 1967

[24] R H Rigdon and A A Packchanian ldquoHistologic study of theskin of congenitally athymic ldquonuderdquo micerdquo Texas Reports onBiology amp Medicine vol 32 no 3-4 pp 711ndash723 1974

[25] Y Hozumi T Imaizumi and S Kondo ldquoEffect of cyclosporinon hair-existing area of nude micerdquo Journal of DermatologicalScience vol 7 pp S33ndashS38 1994

[26] S P Flanagan ldquoldquoNuderdquo a new hairless gene with pleiotropiceffects in the mouserdquo Genetical Research vol 8 no 3 pp 295ndash309 1966

[27] J L Brissette J Li J Kamimura D Lee and G P Dotto ldquoTheproduct of the mouse nude locus whn regulates the balancebetween epithelial cell growth and differentiationrdquo Genes andDevelopment vol 10 no 17 pp 2212ndash2221 1996

[28] L Mecklenburg R Paus Z Halata L S Bechtold P Fleckmanand J P Sundberg ldquoFOXN1 is critical for onycholemmalterminal differentiation in nude (Foxn1119899119906) micerdquo Journal ofInvestigative Dermatology vol 123 no 6 pp 1001ndash1011 2004

[29] H Pinkus T Iwasaki and Y Mishima ldquoOuter root sheathkeratinization in anagen and catagen of the mammalian hairfollicle A seventh distinct type of keratinization in the hairfollicle trichilemmal keratinizationrdquo Journal of Anatomy vol133 no 1 pp 19ndash35 1981

[30] H Wagner and G Ulrich-Merzenich ldquoSynergy researchapproaching a new generation of phytopharmaceuticalsrdquo Phy-tomedicine vol 16 no 2-3 pp 97ndash110 2009

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of