Advances in dermatology and venereology ActaDV Acta Dermato-Venereologica ActaDV and 5-lipoxygenase(5-LOX) (6,7). The mainandactive inhibitedtheactivityofcyclooxygenase 2(COX-2) woad Moreover, in a cell-based assay, a lipophilic extract of and subchronic hapten-induced models of oedema (5). acute in and (4) inflammation airway allergen-induced extracts have demonstrated anti-inflammatory effects on inflammation and infections (1–3). In recent studies woad and hasalonghistoryofuseasmedicinalherbinskin times. Woad isusedforthemanufacture ofindigodye(1) This isan open access article under the CC BY-NC license.www.medicaljournals.se/acta Konstantin AGELOPOULOS T Von-Esmarch-Straße58, DE-48149, Münster, Münster, Germany. Hospital E-mail: [email protected] University Pruritus, Chronic for Corr: Acta DermVenereol 2020;100:adv00131. Accepted Apr2,2020;Epubaheadofprint6,2020 flammatory agent;contactdermatitis. anti-in- L; tinctoria Isatis eczema; atopic pruritus; Key words: in the treatment of inflamedandirritated skin. determination of their potential dermatological effects characterization of their specific properties will enable further and extracts such of production The nocytes. tact hypersensitivity animalmodel andhumankerati con a in properties anti-inflammatory have to shown and developed were extracts woad novel conclusion, tion was significantly inhibited in LAD2 cell cultures. In degranula cell mast human Furthermore, expression. cytokine pro-inflammatory inhibited extract ether um skin andhumankeratinocytepetrolemouse cultures, ear swelling, oedema and inflammatory cell density. In reduced significantly in resulted extract ether roleum lergic contact dermatitis. In the mouse model the pet andwastested inamouseeffects, model al of acute best the have to found was extract ether petroleum A matory effects using cyclooxygenase-2 activity assays. anti-inflam for screened and ingredients, active their Various extracts of properties. anti-inflammatory and ingredients active of extracts developed. Theaimof this studywasto develop novel have been withanalysis of activesubstances precise extracts well-defined no date, to However, effects. ry 1 Tobias and MastCellDegranulation inflammatory Effects via Inhibition of Interleukin-6, Interleukin-33 Isatis tinctoria L.-derived Petroleum Ether Extract Mediates Anti- for centuries and has demonstrated anti-inflammato demonstrated has and centuries for inmedicine Isatis tinctoria used (woad) hasbeen L. of Bioanalytical Sciences (IBAS), Anhalt University of Applied Sciences, Bernburg, Germany Journal Compilation ©2020ActaDermato-Venereologica. Department of Dermatology and Center for Chronic Pruritus, University Hospital Münster, Münster, and he dye plant sicaceae) has been cultivated in Europe since ancient osatn glpuo, eatet f emtlg ad Center and Dermatology of Department Agelopoulos, Konstantin LOTTS Isatis tinctoria L.,andto their characterize 1 , Kathrin Isatis tinctoriaL. (woad, family Bras L. were analysed for Isatis tinctoriawere analysed L. KABRODT 1 2 , Johanna INVESTIGATIVE REPORT HUMMEL 2 , ------BINDER Dorit lymphopoietin (TSLP) in lesional skininanatopic ec can also significantly reduce the level of thymic stromal inflammatory and anti-asthmatic properties. Tryptanthrin (11) and5-LOX(12)activity demonstratesanti- seems tobethemajorcompoundthatinhibitsCOX-2 tryptanthrin alkaloid these, Among 8–10). (6, recently substances inthewoadextractshavebeencharacterized was therefore to develop standardized woad extracts ally inchildrenandadults. The aimofthecurrentstudy especi skin, inflamed and/or irritated in use for effects anti-irritative and anti-pruritic anti-inflammatory, with known. There is ahighunmet need for topical emollients and thecompositionofactiveingredientsarelittle- wever, the mode of action of well-defined woad extracts ingredient ofemollientsforanti-irritativepurposes.Ho inflammation inamousemodel(17). ester wasrecentlyshowninultraviolet(UV)-induced cells (16). The benefit of topical p-coumaric acid methyl of hydrophobicity and alkyl chain bulkiness in RBL-2H3 tion, couldbedetected,whichisduetothemodulation anti-allergic effect, viainhibitionofmastcelldegranula For thewoadcompoundp-coumaricacidmethylester, an properties anti-inflammatory show and factor (TNF)-α, and interleukin (IL)-6 from macrophages, and prostaglandin E2 (PGE2), release of tumour necrosis woad, whichstronglyinhibitproductionofnitricoxide of indoleglucosinolates,areothermajorcompoundsin trile derivatives,productsfromtheenzymaticdegradation symptoms within this animal model (13). Indole acetoni zema animalmodel,andthuscanimprovetheclinical irritated, pruritic skin. of treatment the in use dermatological potential their of cise characterization of such extracts will enable evaluation in animal and human skin tissues. The production and pre propertiesanti-inflammatory their determine to and woad, of extracts well-characterized novel develop to aimed dy stu This developed. been have content substance active oftheir characterization precise with extracts defined no date, However, to studies. recent in effects inflammatory used in medicine for centuries, has demonstrated anti- The medicinal herb SIGNIFICANCE Extracts ofwoadhavebeenusedinresearchandas 2 , Ingo SCHELLENBERG Isatis tinctoria (woad), which has been Acta DermVenereol 2020;100: adv00131 2 Center of Life Sciences, Institute doi: 10.2340/00015555-3476 2 , Sonja (14, 15). 15). (14, in vivo STÄNDER 1

and 1/9 ------Advances in dermatology and venereology ActaDV Acta Dermato-Venereologica ActaDV www.medicaljournals.se/acta 1 extraction methodsandsolvents weretested.First, various extract, woad effective an for search the In Screening forapotentextract the firstcropwasusedforfurtherstudies. 1C). Therefore, a mixed genotype (GT 20.08.2013) from showed overallmorestabledrugtoextractratios(Fig. July 2013 and September 2013 were also analysed, and Mixed genotypesamplesat6harvestdatesbetween their yield of plant material and ratio of drug to extract. and 9werethemostpromisingsampleswithregardto second crop in 9 out of 10 cases (Fig. 1C). Genotypes 3 and extraction yields in the first crop compared with the TRYhigher revealed investigation However, the trend. distinct no shows concentration, TRY and yield tion 645, are listed in 32 DIN to according software SQS2000 by calculated substance. The limitofdetectionactivesubstances, improved from 222 ng/ml to ~30 ng/ml for each standard and sample preparation, the limit of quantitation could be hy (HPTLC). Due to optimization of the HPLC gradient (HPLC) andhigh-performancethin-layerchromatograp measured byhigh-performanceliquidchromatography the genotypes,andactivesubstanceconcentrations with regard to the parts ofthe plants, the harvest times and during the2-yeargrowthphase. These wereexamined from thewoadplantwereharvestedatdifferent times 2012 and 2014. Different materials (roots, stems, leaves) tivated byaspecializedagriculturalcompanybetween MATERIALS ANDMETHODS (see potential useindermatology. and to determine their anti-inflammatory properties, for with exactcharacterizationandhighreproducibility, 2/9 Woad wasplantedundercontrolledconditionsandcul extracts Profiling andanalyticalanalysisofIsatistinctoria RESULTS different cropsandharvestdates,regardingtheirextrac various puregenotypesofIsatistinctoriaL.leavesfrom The screeningofdifferent ethanolextractsobtainedfrom the weakpeakshowedaslightdeviation on thetimeaxis. pCM couldnotbeclearlyassignedintheextract.Here, extract in which TRY and IAN could be identified. Only from 190 nm to 600 nm. Fig. 1B shows a petroleum ether sample wasperformedbymatchingtheUV spectrum shown inFig.1A. is solution standard in compounds 3 the of separation acetonitrile (IAN) and tryptanthrin (TRY). The baseline identified: p-coumaric acid methyl ester (pCM), 3-indole https://www.medicaljournals.se/acta/content/abstract/10.2340/00015555-3476 Confirmation of TRY, IAN and pCM identity in a in identity pCM and TRY, IAN of Confirmation T. Lottsetal. Table SI 1 . Three mainsubstanceswere Appendix S1 1 - - - ) (95% inhibition at 20 µg/ml) was higher than inhibi than higher was µg/ml) 20 at inhibition (95% fraction 4.1 extract the of potential inhibitory The B). performance againsthuman COX-2enzyme(Fig.2A, Generated extractswere tested towardstheir inhibitory of thewoadextractsforpossible dermatologicaluse. during theextraction,aswellcolourandsmell was paid to high reproducibility and standardization nual solid-liquid and liquid-liquid extraction. Attention ma through 2013, in harvested leaves, from obtained were 4.1)) (extract fraction ether petroleum lipophilic and 3) (extract fraction acetate ethyl polar middle 2), shown). The other extracts (polar water fraction (extract vents, butdidnothavethedesiredresults(data different datesofharvest. 6 on genotypes mixed the to addition crop, in (S2) second and (S1) first from (GT) genotypes woad various of slash) (black concentration (TRY) ultraviolet (UV) spectra. (C) Yield of extraction (grey bars) and since tryptanthrin the 2 substances did not allow a clear assignment based solely on extract. the In (B) the ingredients pCM and IAN in were the woad detected plant, and by (B) retention the identified time, substances in the petroleum ether acid methyl ester (pCM), 3-indoleacetonitrile (IAN) and tryptanthrin (TRY) standard solution (3 µg/ml) and with peaks (HPLC) for the of Isatistinctoria screening chromatography 3 main liquid components p-cumaric High-performance 1. Fig. and carbondioxidewerealsotestedasadditionalsol dichloromethane isopropanol, step, first this In (ASE). extraction solvent accelerated by generated was 2012, an ethanolicextract(extract1) L.genotypes. of leaves,harvestedin HPLC chromatogram of a (A) - - - Advances in dermatology and venereology ActaDV Acta Dermato-Venereologica ActaDV the sametime(datanotshown). say, with a very low active ingredient concentration at as enzyme COX-2 the in µg/ml) 25 at inhibition (80% from freshwoadleavesalso showedahighinhibition inhibition of the COX-2 enzyme (Fig. 2B). A fat extract whereby a concentration of 7 µg/ml could achieve a 45% determined, was 4.2 extract of effectiveness dependent 89% inhibition at 28 µg/ml). In addition, a concentration- 4.2 extract vs. µg/ml 20 at inhibition 95% 4.1 (extract 4.1 extract as effects inhibitory similar showed 4.1) extract 4.2 (at a 1.5-fold concentration to that of extract assay,the In ng/mg. 100 below was 4.2 extract in pCM and IAN of content The µg/mg. TRY2.3 of to content higher yield of extract (3,066 mg/kg drug), but decreased 23-times a had which method, extraction optimized the by obtained was 4.2 Extract optimized. was process extraction the leaves, woad mg/kg 132 approximately tion ofthese(datanotshown). the inhibitionofwoadextractswithasimilarconcentra effects ofthe single standard substances werelower than on COX-2 similar to extract 3 (Fig. 2A). The inhibitory µg/ml) 596 at inhibition effect(~40% inhibitory range mid- a showed and µg/mg) TRY0.1 µg/mg; (22 IAN was tested in parallel, contained the highest amount of amount of IAN (< minor a TRYµg/mg and 5.3 approximately contained 4.1 extract tested The µg/ml). 20 at inhibition (47% 3 ml), extract2(10%inhibitionat20µg/ml)and µg/ 314 at inhibition (75% fraction 1 extract by tion Due to the small amount of extract 4.1, produced from 1 µg/mg). The fermented sap, which - - a cell proliferation assay (XTT) was performed. was A(XTT) gra assay proliferation cell a TRY, IAN and pCM on keratinocyte (Ha-CaT) viability, extracts 1, 3 and 4.2, as well as of the 3 main compounds range, uptoamaximumconcentration of100µg/ml. non-inhibitory the within 4.2 extract using performed high COX-2 inhibition. All subsequent experiments were candidate with a potential anti-inflammatory effect due to gamma-linolenic acidwerealsoincreasedhere(Fig.S1 the distributioninaleaffatextract.Palmiticacidand for found were Similarities fat. total the of n-6) (C18:3 was approximately 25% (C16:0) or approximately 40% acid (C18:3 n-6) in lipophilic extract 4.2. The proportion percentage in palmitic acid (C16:0) and gamma-linolenic promoting orproliferation-reducingeffect. proliferation- significant a showed tested ingredients TRY+pCM+IANwoad other the of None µM). 1 (each ficantly promote cell viability, just like a combination of shown in Fig. 2D, TRY (10 nM and 10 µM) could signi slightly, to the level of 70% surviving cells (p highest concentrationof1mg/mlreducedcellviability the only and 2C) Fig. µg/ml, 10 to µg/ml (100 trations concen lower for shown was effect inhibitory no 4.2, concentration of1mg/ml(datanotshown).Forextract (< 3 extract and control) vs viability the keratinocytes was observed for the extracts 1 (< to incubate HaCaTs for 24 h. A high cytotoxic effect on used was pCM TRY,or and IAN extracts the of dient experiments relative tothevehicle control(* are the means valuespresented The control. vehicle the of percent as Resultsshown the viability of keratinocytes (XTT assay) after 24 h 3-indoleacetonitrile incubation (IAN) (C and and p-cumaric (TRY), D). tryptanthrin acid compounds methyl the ester and (C) (pCM) 4.2 (D) extract of on effect The as means (B) Concentration-dependent effect of extract 4.2. Values are presented classification of the anti-inflammatory effect of extractsgeneral 1,the 2,for tests 3 and 4.1. Preliminary (A) shown. are material plant woad acetate 3 and petroleum ether 4.1/4.2), as well as fermented sap from ethyl 2, water 1, (ethanol extracts different of assay inhibitor COX-2 cell viability and assay. Fig. 2. Screening for anti-inflammatory potential of woad extracts In order to precisely determine the effects of the woad In summary,promising In a be to found was 4.2 extract Analysis of the fatty acid profile showed an increased Woad extractmediatesanti-inflammatoryeffects ±

standard deviation relating to uninhibited enzyme activity.enzyme uninhibited to relating deviation standard ±

standard error of the mean (SEM) of 4–6 independent The anti-inflammatory potential in a cell-free Acta DermVenereol 2020 p 10% viability) at a at viability) 10% < 0.05, ** < p 0.01). As < 0.01). 40% 3/9 1 ). - - - Advances in dermatology and venereology ActaDV Acta Dermato-Venereologica ActaDV of mouse ears treated with TRY+pCM+IAN or 4.2 using an antibody against CD4 and nuclear staining (DAPI). are presented as mean values compared with vehicle (DNFB only) control are shown, as well as the statistical analysis of (B) ear swelling and (C) oedema formation in the ears. Results www.medicaljournals.se/acta (DNFB). Representative haematoxylin and eosin staining in ears of wild-type mice (n model. mouse (CHS) hypersensitivity contact in cells CD4+ inflammatory of recruitment and formation oedema swelling, Ear 3. Fig. CD4-positive cells per 0.1 mm for TRY+pCM+IAN (p TRY+pCM+IAN for 50% of swelling ear reduced significantly a CHS, the of challenge) the of h 24 (within phase elicitation late the During ear. mouse the in response inflammatory and oedema acute inhibited significantly skin mouse on challenge after h 24 and challenge before min 30 ml per ear extract 4.2, or 0.25 mg/ear TRY+pCM+IAN, irritated and inflamed skin. Topical application of 100 µg/ model ofcontacthypersensitivity(CHS),asa the The vivo Anti-inflammatory effectofpetroleum etherextractin 4/9 4.2 (p 4.2 Isatis tinctoriaextractwasevaluatedinananimal potency and anti-inflammatory activity of activity anti-inflammatory and potency in vivo < Scale bars T. Lottsetal. 0.05) could be detected compared with the ve the with compared detected be could 0.05) : 100 µm. Woad extract 4.2 and tryptanthrin (TRY), 3-indoleacetonitrile (IAN) and 2 ± from 2–5 mice per group (n

standard error of the mean (SEM), * p < 0.05) and 51% for extract for 51% and 0.05) = 3 images per section). Plots are shown as means < 0.05, *** - = 8–12 mice per group) 48 h after challenge with 1-fluoro-2,4-dinitrobenzene (p (p 4.2 extract for skin treated the in cells T CD4-positive of number reduced the vehiclegroupcouldbedetectedforbothcom Here, a significantly lower ear thickness compared with 3A). (Fig. areas treated the in swelling in reduction a confirmed staining histological Subsequent (Fig. 3B). Fig. 3A, Fig. with extract 4.2 (p revealed alowernumberofmastcellsaftertreatment (p bination of TRY+pCM+IAN (p p diminish was h) 48 to (up phase inflammatory The group. hicle < p < < < 0.001) and 67% (extract 4.2; 0.05) (Fig.3D,E). 0.001) (Fig. 3C). Furthermore, histological analysis 0.001 vs. vehicle. (D) Representative immunofluorescence staining ed significantly, by 68% (TRY+pCM+IAN; 68% by significantly, ­ed Fig. S2 1 p-cumaric acid methyl ester (pCM) treated groups ). Immunohistochemistry revealed a revealed Immunohistochemistry ). < 0.01) or TRY+pCM+IAN (p < Scale bars 0.05) and for TRY+pCM+IANfor and 0.05) ±

SEM; *p : 50 µm and (E) total number of < < p 0.05 vs. vehicle (DNFB only). 0.001) and extract 4.2 < 0.001), respectively < 0.05; (A) - Advances in dermatology and venereology ActaDV Acta Dermato-Venereologica ActaDV *** 48 h after 1-fluoro-2,4-dinitrobenzene (DNFB) challenge (n challenge (DNFB) 1-fluoro-2,4-dinitrobenzene after h 48 (E) IL-33 and (F) ST2 in 4.2 or tryptanthrin (TRY), 3-indoleacetonitrile (IAN) and p-cumaric acid methyl ester (pCM)-treated mouse skin was measured IL-6, IL-4, (D) (C) (IL)-1beta, interleukin (B) (IFN)-γ, interferon (A) cytokines pro-inflammatory the of levels and mRNA Relative (A) pCM IAN inmouseear. TRY, compounds the or (4.2) extract ether petroleum with treatment after cytokines pro-inflammatory of Expression 4. Fig. human keratinocytesderivedfromhealthydonorsafter in IL-33 and IL-6 of expression investigated also we measured 48hafterchallenge(Fig.4A). expression IFN-γ increased the on effect an showed TRY+pCM+IAN nor 4.2 extract Neither skin. mouse could beobservedatthesiteofchallengeintreated (IFN)-γ interferon of expression Increased 4F). (Fig. (p 4.2 extract (IL1RL1=ST2) was down-regulated after treatment with 1 receptor-like 1 interleukin receptor IL-33 the of sion p 1bp (IL- 4.2 with treatment after skin mouse in found was IL-6 and (IL)-1b interleukin cytokines inflammatory pro- the of expression the in reduction significant A by petroleum etherextractandwoadingredients Cytokine expression invivoandvitrowere reduced by TRY+pCM+IAN (p affected significantly only was IL-33 cytokine alarmin the of mRNAexpression 4C). (Fig. 4.2 extract not but (p IL-4 of expression the inhibited control (Fig.4B, D). TRY+pCM+IAN also significantly < In order to validate the effects observed in mouse skin, p 0.05, IL-6 0.05, < < 0.001 vs.vehicle. 0.01, IL-6 0.01, < p 0.01) or the main compounds (p compounds main the or 0.01) p < < 0.001) compared with the vehicle the with compared 0.001) 0.001) or TRY+pCM+IAN (IL-1b TRY+pCM+IAN or 0.001) < 0.05, Fig. 4E), whereas expres < 0.01) in the mice, the in 0.01) = 8–11 per each group) and normalized against beta-actin (β-Actin). *p (β-Actin). beta-actin against normalized and group) each per 8–11 < 0.05) - β-hexosaminidase in LAD2 cells, which were stimula were which cells, LAD2 in β-hexosaminidase enzyme the of release by measured was Degranulation LAD2 mastcells Petroleum ether extract inhibits degranulation of human with thevehiclegroup. expression (p mRNA IL-33 induced in reduction significant a to led nation with pCM and IAN, or treatment with extract 4.2, S3 (Fig. expression IL-33 induce and concentration of IFN-γ as suitable to stimulate the cells ng/ml 20 a determined kinetics Dose 19). (18, nocytes centration comparedwiththevehicle(Fig.5B). ted either with the ion-carrier calcium ionophore (CI, ionophore calcium ion-carrier the with either ted decrease of 50% of decrease a to leading pronounced, more much was 4.2 extract of effect The (Fig. 5A). vehicle the with compared 50% expression of IL-6 in the keratinocytes to approximately TRY+pCM+IAN (p TRYwith Pretreatment (p IL-33). (for IFN-γ stimulation with the TLR3 agonist Poly I:C (for IL-6) or not inhibit induction of IL-33 expression, but, in combi IFN-γ has an effect on expression of IL-33 in kerati in IL-33 of expression on effect an has IFN-γ Woad extractmediatesanti-inflammatoryeffects < 0.01 and (p < < 0.05) reduced the Poly I:C-induced 0.01) for the highest extract con extract highest the for 0.01) p < 0.05; Fig. 5C, D) compared Acta DermVenereol 2020 1 ). TRY). did alone < 0.05, ** 0.05, = 0.07) or 0.07) p < 0.01 5/9 - - - - Advances in dermatology and venereology ActaDV Acta Dermato-Venereologica ActaDV www.medicaljournals.se/acta enzymatic assay. Data are shown as mean ester (pCM; each 1 µM). Shown is the spontaneous release (spon rel) vs. the release under stimulation (CI or SP) of β-hexosaminidase, measured by an pretreatment with: (A) petroleum ether extract 4.2 after cells LAD2 stimulated (SP) P- substance or (10–75 (CI) ionophore- Calcium treatment. extract woad by reduced is degranulation cell Mast 6. Fig. µg/ml) from woad or (B) tryptanthrin (TRY), 3-indole acetonitrile (IAN) and experiments. Expression level of IL-33 in primary human keratinocytes stimulated with interferon (IFN)-γ for 16 h and treated with (C) TRY (1 µM), (1 TRYTRY+pCM+IAN (each1µM)or(D)4.2(1,5,10µg/ml). (C) with treated and h 16 for (IFN)-γ interferon with stimulated keratinocytes human primary IL-33in of level Expression experiments. many conditions,suchasdiseasesassociatedwithchro in finding frequent a is skin irritated or pruritic Dry, DISCUSSION degranulation inoursetting(Fig.6B). in reduction significant a to however,led µM), 1 (each ml shown that emollients can modify the inflammation and appropriate or may induce side-effects (22). It has been steroids andtopicalcalcineurininhibitorsmightnotbe conditions, emollientuseisadvocated,whiletopical these In vulgaris. psoriasis or (AD) dermatitis atopic genodermatoses, andinintervalsbetweenrelapsesof nic pruritus (e.g. dry skin in elderly patients), paediatric S4 showed no effect on β-hexosaminidase release (see alone µM), 10 to nM TRY,pounds, (10 IAN and pCM com woad 3 Interestingly,the observed. was samples, (CI), respectively 30% (SP), compared with the untreated centration of extract 4.2 (75 µg/ml) an inhibition of 40% stimulation controlalone(Fig.6A). For the highest con 75 µg/ml, and µg/ml (50 CI- way,the concentration-dependent a in and significantly inhibited 4.2 Extract 21). 20, µM; 30 (SP,P substance neuropeptide with or 20) µM; 2 (PPIA). isomeraseA peptidylprolyl against Resultsnormalized µg/ml). were 10 5, (1, 4.2 (B) or µM) 1 (each TRY+pCM+IAN µM), (1 TRY (A) with treated and h 16 for I:C poly with stimulated keratinocytes human primary in (IL)-6 interleukin of expression and mRNA Relative pCM IAN in keratinocytes. TRY, compounds the or (4.2) extract ether petroleum with treatment after cytokines pro-inflammatory of Expression 5. Fig. 6/9 1 p ). Treatment with a combination of the 3 ingredients < 0.001) induced degranulation compared with the with compared degranulation induced 0.001) T. Lottsetal. p < 0.001) and SP- (50 µg/ml ±

standard error of the mean (SEM) from 4 independent experiments (** p < 0.01, 75 µg/ n ≥ 3 experiments;* Fig. - - - p optimized with regard to the content of the ingredients. solvent, numberanddurationofextractioncycles) Accelerated Solvent Extraction (temperature, content of were examinedwithdifferent extraction operationsusing extract intoanemollient.Smallscalesfromdriedleaves all importantfactorsforpossibleincorporationofthe also tookintoaccountthecolourandsmell. These are yield ofactiveingredientandstabilitytheextracts, was thethoroughanalysis,which,inadditiontoahigh study this of attribute important process. An extraction the for material plant raw optimal the find to order different harvestperiodsanddifferent genotypesin vitro anti-inflammatoryproperties. main ingredients,andanalysisoftheirinvivo and harvestingofwoad,isolationanalysisthe such preparations. This includedsystematicplanting for beneficial be might extracts (Isatis tinctoria L.) initiated thisstudyinordertoanalysewhetherwoad we Therefore, 4). (3, infections and diseases matory inflam of modification a such showed preparation root and leaf woad medicine, Chinese In potential. anti-inflammatory and anti-irritative mild with these are emollients promising most The (24). conditions lients shouldbepartofthetherapyregimeninthese chronic pruritus entities, such as AD (23). Thus, emol decrease the use of anti-inflammatory treatments in treatments anti-inflammatory of use the decrease < 0.05, ** This studyexaminedarangeofwoadmaterialfrom p < 0.01 vs.vehicle. p < 0.01, *** p < p-cumaric acid methyl 0.001 vs. untreated). n ≥ 3 - -

Advances in dermatology and venereology ActaDV Acta Dermato-Venereologica ActaDV be detectedinthetreatedmouse ears. This wasshown gation. In addition, a reduced number of mast cells could reduction inoedemaformation usinghistologicalinvesti dramatic a by confirmed was This response. effector CHS h 24–48 classical the in swelling ear in reduction TRY+pCM+IAN- mixture (1 µM each) showed a similar and (4.2) extract model (27,28).BoththeI.tinctoria the the bestcandidateforsubsequentstudies. as chosen was 4.2 extract process, extraction the from the lowcytotoxicpropertiesandhighyieldofextract effect ontheactivityofCOX-2enzyme(SR5,SR6). 4.2, increases expression of the COX-2 gene, but has no saturated fatty acid palmitic acid, also found in extract The SR4). (SR3, disease the of symptoms clinical the improves GLA adding but GLA, of level low a have and c-Jun N-terminal kinase-1 (SR2). Patients with AD phosphorylated extracellularsignal-regulatedkinase1/2 oxidative stressandthesignaltransductionpathwayof factor-kappaB andactivatorprotein-1bysuppressing nuclear inactivates GLA SR2). (SR1, activity enzyme COX-2 inhibit can and properties anti-inflammatory has GLA because interesting, particularly is finding new This present. percentage high a show (GLA) acid the polyunsaturatedfattyacid(n-6)gamma-linolenic extract, fat leaf the and 4.2 extracts, both In ingredient. our assay, withonlyaverylowconcentrationof active in activity COX-2 inhibit significantly can extract fat may notbeduetotheactiveingredientsalone. The leaf extracts the of effect anti-inflammatory observed the tion ofIANandpCMwasverylow. be detected at a significant amount, while the concentra most effective PE extract (4.2), however, only TRY could the activity of COX-2 and 5-LOX (6, 7). In the final and a lipophilicwoadextractisdescribedasaninhibitorof fat extract. theliterature,inwhich with consistent is This tested extracts (1, 2 and 3), the fermented other sap or the the leaf substances, single 3 the than effective more activity ofCOX-2inacell-freesystemandwasthereby say. Lipophilic extract 4.2 was able to strongly inhibit the addition, theextractswereexaminedinacellvitalityas effect. In anti-inflammatory potential their test to assay fat extractwerepreparedandusedinaCOX-2inhibitor ether, extracts 4.1 and 4.2), petroleum the fermented sap and a 3; leaf extract acetate ethyl 2; extract water 1; optimization steps, 4 different extracts (ethanolic extract and confirmedpreviousstudies(8). acid methyl ester (pCM) and 3-indole acetonitrile (IAN) (TRY),tryptanthrin compounds 4-coumaric main 3 the the literature (8, 9, 25, 26). In dried woad leaves we found compounds intheleaves,havealreadybeendescribed 70 including plant, woad the from Variousingredients The effect, anti-inflammatory potential strong the to Due that indicate profile acid fatty the from results The As a result of the extraction process and the subsequent I. tinctoria extract wasevaluated ina CHS animal in vivo potency and anti-inflammatory activity of - - - gene expression in the DNFB-treated mouse. Also, in nor TRY+pCM+IAN significantly reduce IL-1beta and IL-6 corresponding cytokines. with TRY mainly affect the Th2 cell population and the ingredients woad the date, to findings the to According (29). phenotype Th2 a to cells CD4+ T purified of tion differentia the TRYinhibits Furthermore, (29). fected epidermal keratinocytes from AD patients by IFN-γ (18, in only not induced be can IL-33 components. woad 3 gene expressionbythepetroleum etherextractandthe detected a significant inhibition in IFN-γ-induced IL-33 in lesional skin of AD (37). In human keratinocytes, we increased are ST2 receptor its and IL-33 that known is It 19). to (18, contribute ACD in and inflammation AD play a crucial role in the acute and chronic phases of may and family cytokine IL-1 the of member a is 33 mRNA(both) ST2 IL- CHS. during skin lesional the in (TRY+pCM+IAN) and IL-33 the reduced significantly show that treatment with extract 4.2 and TRY+pCM+IAN correlates withtheserumlevelsofIL-6(35,36). in somepruriticdiseaseswhichtheseverityofpruritus relationship between IL-6 and pruritus is also described of EASI) and diminish pruritus within 6 weeks (34). The antibodies against IL-6 reduce the signs of AD (reduction Monoclonal 34). (33, example for (AE), eczema atopic can produce IL-6, which is relevant to the acute phase of CD4+ T cells are prevalent in inflammatory lesions and 32). (31, dermatitis contact induced experimentally an levels oftheirtranscriptscorrelatewiththeintensity the and (ACD), dermatitis contact acute of formation the in role key a play cytokines Both (30). release IL-6 differentiation, apoptosisandinductionofCOX-2 1beta isinvolvedincellprocessessuchasproliferation, and IL-6 are important pro-inflammatory mediators; IL- IL-1beta I:C-induction. Poly after found was treatment ced IL-6 expression by extract 4.2 and TRY+pCM+IAN mal human epidermal keratinocytes a significantly redu cells, whereas IFN-γ production by Th1 cells is not af not is cells Th1 by production IFN-γ whereas cells, Th2 by IL-4 of production the reduces TRYalso alone IL-4 and IL-5 in allergen-induced airway inflammation. tinctoria extractinhibitsproduction of the Th2 cytokines Brattström et al. (4) demonstrated that a carbon dioxide while the 3 main components inhibited IL-4 expression. TRY+pCM+IAN, or 4.2 extract by influenced not was which demonstrated a similar reduction in inflammation. (5), inflammation skin of models mice subchronic and are inlinewithotherlipophilicwoadextractsacute results Our extracts. woad for time first the for shown of the untreated control As far as we know, this could be 4.2 and TRY+pCM+IAN) was reduced almost to the level number of CD4+ T cells at the site of treatment (extract the challenge, the after h 48 Furthermore, 4.2. extract both after treatment with the 3 active ingredients and with Furthermore, we were able to show that extract 4.2 and As new findings for woad extracts we were able to able were we extracts woad for findings new As The gene expression of IFN-γ in lesional mouse skin mouse lesional in IFN-γ of expression gene The Woad extractmediatesanti-inflammatoryeffects Acta DermVenereol 2020 7/9 I. I. - - - - Advances in dermatology and venereology ActaDV Acta Dermato-Venereologica ActaDV www.medicaljournals.se/acta extracts with a high content of TRY may be promising TRYof be content may high a with extracts sults ofthecurrentstudysuggest thatstandardizedwoad however,required; are skin pruritic and irritated re the preclinical and clinical studies in patients with inflamed, standing ofthemodeaction ofwoadextracts.Further studies. This couldbeausefulextensionofourunder to otheringredientswouldprovidethebasisforfurther the extract. A precise analysis of extract 4.2 with regard are due toadditional low-concentrations ofsubstances in rule outthatpartsoftheobservedeffects oftheextract with a consistently anti-inflammatory effect. We cannot for thevariabilityinrawmaterialtoproduceanextract extract toapetroleumethercouldcompensate displays highreproducibility. Fractionationofthecrude also extract defined and natural the TRY+pCM+IAN, the in effective more be to found was and IAN, and pCM TRY, ingredients woad 3 the to efficacy comparable model. mouse ACD an in efficacy its proven further has and activity, COX-2 responses, cell mast inhibiting IL-33, and IL-6 as such cytokines, inflammatory of regulation down- by properties anti-inflammatory displays that inflammatory reactions. our extractcaninhibitthemastcellsandreduceactual Thus, (40). and in ACD AD lesions skin and processes inflammatory of development the in involved are cells previously. effectorallergicAs for mast cells reactions, woad extractsonhumanmastcellshasnotbeenshown dependent manner. To ourknowledge,suchaneffect of concentration- a in cells LAD2 in β-hexosaminidase of degranulation, as it decreases CI- and SP-induced release demonstrated tobeaveryeffective inhibitorofmastcell 4.2 Extract secretion. β-hexosaminidase in reduction significant a to led however, pCM, and IAN gredients, in 2 other the with Combination cells. LAD2 line cell also hadnoeffect ondegranulationofthehumanmast alone TRYstudy, our In investigation. further require effect, whicharestillunclearforwoadextractsand may indicatedifferent mechanismsoftheinhibitory granulation inratbasophilicleukaemiacells(29). This de IgE-mediated in decrease a causes TRY contrast, In cells. mast marrow-derived bone murine in 2-one indolin- of results the confirmed (39) al. et Kiefer (38). whereas TRY does not cause (38), this induced degranulation cells mast peritoneal rat in demonstrated was release histamine C48/80-induced of inhibition 2-one, known toaffect mastcelldegranulation.Forindolin- of theregulatoryloop. expression and release of IL-33 may lead to interruption ents do not seem to directly affect IFN-γ, but decreased contribute tochronicityofdisease(18). Woad ingredi with activated T cells. This potentialregulatoryloopmay 19), but may increase IFN-γ release itself by interaction 8/9 In summary, we have developed a novel woad extract To date,onlyafewingredientsfromI.tinctoriaare in vitro experiments.Comparedwithacompositeof T. Lottsetal. , petroleum ether extract 4.2 showed 4.2 extract ether petroleum In vivo, - - - - - 13. 12. 15. 11. 16. 14. 10. REFERENCES Fund oftheUniversityMuenster. Gerlinde Schmitzfortheirexcellenttechnicalassistance. providing the cell for line LAD2. Kirshenbaum We A. acknowledge Dr Verenato grateful Blome and are We MD2). KF2981504 and MD2 KF2611103 (ZIM Germany Energy, and Affairs mic This work was supported by the Federal Ministry for Econo ACKNOWLEDGEMENTS irritated anddryskin. inflamed, in dermato-cosmetics in use for substances 4. 7. 3. 6. 9. 2. 5. 8. 1. We acknowledge support from the Open Access Publication Access Open the from support acknowledge We Han N-R, Moon P-D, Kim H-M, Jeong H-J. Tryptanthrin ame Brattström A, Schapowal A, Kamal MA, Maillet I, Ryffel B, Ryffel I, Maillet MA, Kamal A, Schapowal A, Brattström Oberthür C, Jäggi R, Hamburger M. HPLC based activity pro Danz H, Stoyanova S, Thomet OAR, Simon H-U, Dannhardt sts icoi. oorp. len e Rv 02 7: 2002; Rev Med Altern Monograph. tinctoria. Isatis Hamburger M. Isatis tinctoria – from the rediscovery of an of rediscovery the from – tinctoria Isatis M. Hamburger Shin J-S, Choi H-E, Kim S-D, Lee YS, Cho Y-W, Lee KT. Anti- Danz H, Stoyanova S, Wippich P, Brattström A, Hamburger M. T,Mohn comprehensivePlitzkoA M. metabo Hamburger I, Yeung H-C. Handbook of Chinese herbal formulas. 2nd edn. Recio M-C, Cerdá-Nicolás M, Potterat O, Hamburger M, Ríos Zhu F, Xu Z, Yonekura L, Yang R, Tamura H. Antiallergic acti K-T,Lee BJ, Kwon YH, et Lee J-S, Shin YoonIH, TH, Kwon Miceli N, Filocamo A, Ragusa S, Cacciola F, Dugo P, Mondello compounds fungicidal and W.Insecticidal Unger K, Seifert ur J. h wa pat n is y. s en London: edn. 1st dye. its and plant woad The JB. Hurry inhibits allergen-induced airway inflammation and hyper and inflammation airway allergen-induced inhibits (ITE) extract L. tinctoria Isatis extract plant The R. Moser 68: 875–880. on 2002; tryptanthrin Med Planta synthesis. leukotriene and prostaglandin of activity Inhibitory al. et H, Ulbrich G, 523–524. 333–344. 1: 2002; Reviews Phytochemistry phytopharmaceutical. anti-inflammatory novel a towards plant medicinal ancient inflammatory effects of 7-hydroxyl-1-methylindole-3-aceto principle in Isatis tinctoria. Planta Med 2001; 67: 411–416. Identification and isolation of the cyclooxygenase-2 inhibitory Rosemead, CA: Institute of ChineseMedicine; 1995. Med 2006;72: 539–546. Planta tryptanthrin. and extracts tinctoria Isatis lipophilic of vivo in activity antiallergic and Anti-inflammatory J-L. 23: 2571–2574. LPS-induced RAW 264.7 cells. Bioorg Med Chem in Lett 2013; productions PGE2 and oxide nitric on effects inhibitory their and derivatives indolyl-3-acetonitrile of Synthesis al. 2017; 14. Biodivers Chem Italy. Sicily, in growing (Brassicaceae) L. tinctoria Isatis leavesof cauline fractionfromphenolic-rich L, et al. Chemical characterization and biological activities of 44–48. 1994; Naturforschung für Zeitschrift tinctoria. Isatis from Oxford University Press; H.Milford; 1930. reactivity inmice. Phytomedicine 2010; 17:551–556. 2015; 79: 1178–1182. and bulkiness of alkyl side chain. Biosci Biotechnol Biochem vity of rosmarinic acid esters is modulated by hydrophobicity, kappaB activation. ChemBiol Interact 2014; 224: 68–77. through destabilizing mPGES-1 mRNA and suppressing NF- nitrile, a synthetic arvelexin derivative, on the macrophages Arch Biochem Biophys 2014;542:14–20. TSLP.of down-regulation through lioratesdermatitis atopic 2009; 70: 924–934. lite profiling of Isatis tinctoria leaf extracts. Phytochemistry leaf extracts. Fitoterapia 2005; 76: 324–332. filing for 5-lipoxygenase inhibitory activity in Isatis tinctoria ------Advances in dermatology and venereology ActaDV Acta Dermato-Venereologica ActaDV 27. 23. 26. 22. 19. 25. 21. 18. 28. 24. 20. 17. Honda T, Egawa G, Grabbe S, Kabashima K. Update of im of Update K. KabashimaT, S, GrabbeHonda G, Egawa Eichenfield LF, Tom WL, Berger TG, Krol A, Paller AS, Schwar brhr , abre M Tytnhi cnet n Isatis in content Tryptanthrin M. Hamburger C, Oberthür Nygaard U, Deleuran M, Vestergaard C. Emerging treatment aiuh K Ymmt S Htm E Iaa , uaa Y, Suyama Y, Inada E, Hitomi S, Yamamoto K, Taniguchi Fréchard A, Fabre N, Péan C, Montaut S, FauvelS, M-T,Montaut C, Péan FabreN, A, Fréchard Rollin Kulka M, Sheen CH, Tancowny BP, Grammer LC, Schleimer RP. Seltmann J, Werfel T, Wittmann M. Evidence for a regulatory eek-ick , aesaSceai M Przk W, Pyrczak M, Majewska-Szczepanik M, Zemelka-Wiącek Ständer S, Zeidler C, Augustin M, Bayer G, Kremer AE, Legat Gibbs BF, Wierecky J, Welker P, Henz BM, Wolff HH, Grabbe Song K, An SM, Kim M, Koh J-S, Boo YC. Comparison of the post-harvest treatments. Planta Med 2004; 70:642–645. tinctoria leaves—a comparative study of selected strains and (Basel) 2017;233: 333–343. options in atopic dermatitis: topical therapies. Dermatology Sugioka T, et al. Interleukin 33 is induced by tumor necro tumor T,by Sugioka induced is 33 Interleukin al. et tinctoria L.). Tetrahedron Letters2001;42:9015–9017. P,(Isatis woad from glucosinolates Novelindole-type al. et chemokine production. Immunology 2008;123:398–410. degranulationactivatecell Neuropeptides mast andhuman loop between IFN-γ and IL-33 in skin inflammation. Exp Dermatol 2013;22: 102–107. inflammation. skin in IL-33 and IFN-γ between loop Szczepanik M. Complementary methods for contact hyper contact for methods Complementary M. Szczepanik Ges 2017; 15: 860–872. chronic pruritus – update – short version. J Dtsch Dermatol FJ, et al. S2k Guidelines for the diagnosis and treatment of 10: 312–320. with anti-IgE and other secretagogues. Exp Dermatol 2001; stimulation following IL-8 and TNF-alpha generated and newly preformed release rapidly cells mast skin Human J. 63: 17–22. 2011; Sci Dermatol J permeabilities. skin their and ester methyl its and acid p-coumaric of effects antimelanogenic 2013; 387:270–275. Methods Immunol J mice. in evaluation (CHS) sensitivity Invest Dermatol 2013;133:303–315. J dermatitis. contact allergic of understanding the toward mune events in the murine contact hypersensitivity model: 2014; 71: 116–132. atopic dermatitis with topical therapies. J Am Acad Dermatol atopic dermatitis: Section 2. Management and treatment of zenberger K, et al. Guidelines of care for the management of Clin Immunol2013; 23: 428–434. contributes to allergic contact dermatitis. J Investig Allergol sis factor alpha and interferon gamma in keratinocytes and - - - - 38. 31. 37. 34. 30. 36. 35. 33. 32. 40. 39. 29. Rüster GU, Hoffmann B, Hamburger M. Inhibitory activity of Bonneville M, Chavagnac C, Vocanson M, Rozieres A, Bene Savinko T, Matikainen S, Saarialho-Kere U, Lehto M, WangM, Lehto U, T,Saarialho-KereSavinko S, Matikainen Navarini AA, French LE, Hofbauer GFL. Interrupting IL-6-re Hong H, Park Y-K, Choi M-S, Ryu N-H, Song D-K, Suh S-I Suh D-K, Song N-H, Ryu M-S, Choi Y-K,Park H, Hong Kimmel Kimmel M, Alscher DM, Dunst R, Braun N, Machleidt C, Kiefer Konda D, Chandrashekar L, Rajappa M, Kattimani S, Thappa Toshitani A, Ansel JC, Chan SC, Li SH, Hanifin JM. Increased Skov DA, Witherden AS, MacLeod P, Lovato MM, Nielsen aaai , no , iua , isn S Kwkm Y. Kawakami BS, Wilson M, Kimura T, Ando T,Kawakami P.Küenzi M, Hamburger A, Koryakina AC, Mertz S, Kiefer Iwaki K, Ohashi E, Arai N, Kohno K, Ushio S, Taniguchi M, et indolin-2-one indolin-2-one derivatives on compound 48/80-induced hista Invest Dermatol 2012;132:1392–1400. J factors. triggering by modulation and profiles expression dermatitis: atopic in ST2 and IL-33 al. et S, Lehtimäki G, Dermatol Sci2009; 56: 43–50. J glucosamine-hydrochloride. by fibroblasts skin human in MMP-13 and COX-2 of down-regulation Differential al. et Transplant 2006;21:749–755. Dial Nephrol patients. haemodialysis in pruritus uraemic of T, et al. The role of micro-inflammation in the pathogenesis association with pruritus severity, sleep quality and depres interleukin-6: and Serotonin PH. Ananthanarayanan DM, atopic dermatitis. J Invest Dermatol 1993; 100:299–304. interleukin 6 production by T cells derived from patients with dendritic epidermal T cells in contact hypersensitivity. J Im of IL-1β-dependentactivation al. et B, Dyring-Andersen L, 21: 666–678. 2009; Immunol Opin Curr dermatitis. atopic in cells Mast (E,Z)-3-(3’,5’-Dimethoxy-4’-hydroxy-benzylidene)-2-indoli RBL-2H3 cells. J Ethnopharmacol 2011; 134:450–459. degranulation and IL-4 production by rat basophilic leukemia al. Tryptanthrin inhibits Th2 development, and IgE-mediated 17: 24–28. 2015; Psychiatr J Asian nodularis. prurigo in severity sion 128: 1128–1130. 2011;Immunol Clin Allergy J superinfection. bacterial with associates but dermatitis atopic improves signaling ceptor munol 2014; 192:2975–2983. Invest Dermatol 2007;127:1430–1435. J dermatitis. contact allergic of severity and development tiere J, Pernet I, et al. Skin contact irritation conditions the 40: 143–147. none blocks mast cell degranulation. Eur J Pharm Sci 2010; mine release from mast cells. Pharmazie 2004; 59: 236–237. Woad extractmediatesanti-inflammatoryeffects Acta DermVenereol 2020 9/9 ------