A 1000-year-old mystery solved: Unlocking the molecular structure for the medieval blue from Chrozophora tinctoria, also known as folium

Nabais e al., Sci. Ad . 2020; 6 : eaaz7772 17 Ap il 2020 SCIENCE ADVANCES | RESEARCH ARTICLE 1 o 8 CHEMISTRY A 1000-yea -old mys e y sol ed: Unlocking he molecula s uc u e o he medie al blue om Ch ozopho a inc o ia, also known as olium P. Nabais1, J. Oli ei a2*, F. Pina1, N. Teixei a2, V. de F ei as2*, N. F. B ás3, A. Clemen e4, M. Rangel5, A. M. S. Sil a6, M. J. Melo1* The molecula s uc u e o he medie al wa e colo known as olium has inally been sol ed in he 21s cen u y. The in e disciplina y app oach aken was he key o p oducing ex ac s ha had been p epa ed ollowing medie al ins uc ions, and shows he blue/pu ple ch omopho e as he majo dye in Ch ozopho a inc o ia ui s (shell). A mul i-analy ical cha ac e iza ion o i s s uc u e was made using HPLC-DAD-MS, GC-MS, NMR (1H, 13C, COSY, HSQC, HMBC, INADEQUATE), and compu a ional s udies. The esul s demons a e ha he blue compound co esponds o 6′-hyd oxy-4,4′-dime hoxy-1,1′-dime hyl-5′-{[3,4,5- ihyd oxy-6-(hyd oxyme hyl) e ahyd o-2H-py an-2-yl] oxy}-[3,3′-bipy idine]-2,2′,5,6(1H,1′H)- e aone, a he midin de i a i e, which we named ch ozopho idin. Expe imen al da a and compu a ional modeling s udies show ha his mono-glycosyla ed dime is ep esen ed by wo s able isome s (a opisome s). This is an indispensable piece o knowledge o he cha ac e iza ion o his medie al dye in wo ks o a such as medie al manusc ip illumina ions and o es ing i s s abili y and con ibu es o he p ese a ion o ou cul u al he i age. INTRODUCTION Ch ozopho a inc o ia in medie al and 19 h cen u y w i en sou ces The use o he plan Ch ozopho a inc o ia (L.) A.Juss. o p oduce colo s o illumina ed manusc ip s is ex ensi ely desc ibed in medie al w i en sou ces (1–4). Wha dis inguishes C. inc o ia om o he medie al na u al sou ces o dye o p oduce pain s is ha , un il now, he blue colo s uc u e emained elusi e (5–7), despi e he e o s by many g oups in he las decades o he 20 h cen u y and in o he 21s cen u y (8–11). To ackle his mys e y, ou in e disciplina y g oup assembled a eam o chemis s who ha e expe ise in na u al p oduc s iden i ica ion; conse a ion scien is s, wo king in he e- p oduc ion o medie al colo s; and a biologis wi h a g ea deal o bo anical and ield knowledge o he Po uguese lo a, who o e sees plan sou cing. This in e disciplina y app oach p o ed essen ial o sol ing he complex s uc u e o he blue dye. On he basis o he de ailed desc ip ions ha we e selec ed om h ee medie al ea ises, we planned he ield expedi ions and he sampling me hods o collec ing plan ma e ials. F ui s we e collec ed du ing July, Augus , and Sep embe 2017 and 2018 (un ipe and ipe) in sou he n Po ugal (G anja/Mou ão). C. inc o ia ex ac s we e p epa ed ollowing he ea ises’ ins uc ions, and he main colo an was isola ed, pu i ied, and cha ac e ized h ough a mul i-analy ical app oach: high-pe o mance liquid ch oma og aphy–high- esolu ion mass spec ome y–diode a ay de ec o (HPLC-HRMS-DAD), gas ch oma og aphy–mass spec ome y (GC-MS), nuclea magne ic es- onance (NMR) [1H, 13C, co ela ion spec oscopy (COSY), he e o- nuclea single-quan um cohe ence (HSQC), he e onuclea mul iple- bond co ela ion (HMBC), INADEQUATE], and elec on pa amagne ic esonance (EPR) (12,13). On he basis o he expe imen al esul s, he molecula s uc u e o he blue colo compound has inally been sol ed and will be discussed in his pape . Theo e ical calcula ions suppo ed his assignmen , and he p edic ed ul a iole - isible (UV-VIS) abso p ion spec a o e lapped well he expe imen al spec a. O he amilies o ch omopho es, p esen in mino amoun s, we e also de ec ed and cha ac e ized by HPLC-HRMS. C. inc o ia and i s uses we e well known in an iqui y and medie al imes. Howe e , he p ac ice o c ea ing he blue colo ell ou o use and i was los in he 19 h cen u y. I s medicinal p ope ies we e i s desc ibed by Diosco ides (De Ma e ia Medica, 1s cen u y) and we e also men ioned in medie al pha macopoeia ex s, and s udies ocusing on i s an i-in lamma o y p ope ies ha e been published ecen ly (14–16). The dyeing p ope ies o his species and hei applica ions a e a ascina ing subjec ha will be e isi ed, as i is ele an o his esea ch. In medie al imes, he blue and pu ple solu ions ex ac ed om C. inc o ia we e s o ed, a e adso p ion on o clo h and d ying, as wa e colo s (clo hle s), and we e applied as pain by cu ing a piece o clo h and ex ac ing i s colo wi h he app op ia e binding medium. Comple e desc ip ions o he plan , when o collec i , and how i was p ocessed a e ound in impo an medie al ea ises such as The book on how o make all he colou pain s o illumina ing books (15 h cen u y), Mon pellie libe di e sa um a cium (14 h cen u y), and Theophilus on di e s a s (12 h cen u y), he ea e e e ed o as Book o all colo pain s (1), Mon pellie (2), and Theophilus (3). In hese ea ises, only he ui s we e collec ed (Fig.1). The pain hus ob ained was named olium o o nasol ( u nsole); his la e desig- na ion is common o he blue/pu ple wa e colo s ob ained om 1REQUIMTE–Labo a ó io Associado pa a a Química Ve de, Faculdade de Ciências e Tecnologia, Uni e sidade NOVA de Lisboa, Campus Capa ica, 2829-516 Mon e de Capa ica, Po ugal. 2REQUIMTE–Labo a ó io Associado pa a a Química Ve de, Faculdade de Ciências, Uni e sidade do Po o, Rua do Campo Aleg e, s/n, 4169-007 Po o, Po ugal. 3REQUIMTE–UCIBIO, Faculdade de Ciências, Uni e sidade do Po o, Rua do Campo Aleg e, s/n, 4169-007 Po o, Po ugal. 4cE3c–Cen e o Ecology, E olu ion and En i onmen al Changes, Faculdade de Ciências, Uni e sidade de Lisboa, Campo G ande, 1749-016 Lisboa, Po ugal. 5REQUIMTE–Labo a ó io Associado pa a a Química Ve de, Ins i u o de Ciências Biomédicas de Abel Salaza , Uni e sidade do Po o. 6REQUIMTE–Labo a ó io Associado pa a a Química Ve de, Depa amen o de Química, and QOPNA, Uni e si y o A ei o, 3810-193 A ei o, Po ugal. *Co esponding au ho . Email: [email p o ec ed] (M.J.M.); [email p o ec ed] (J.O.); [email p o ec ed] (V.d.F.) Copy igh © 2020 The Au ho s, some igh s ese ed; exclusi e licensee Ame ican Associa ion o he Ad ancemen o Science. No claim o o iginal U.S. Go e nmen Wo ks. Dis ibu ed unde a C ea i e Commons A ibu ion NonComme cial License 4.0 (CC BY-NC). on Ma ch 4, 2021h p://ad ances.sciencemag.o g/Downloaded om Nabais e al., Sci. Ad . 2020; 6 : eaaz7772 17 Ap il 2020 SCIENCE ADVANCES | RESEARCH ARTICLE 2 o 8 lichens (e.g., Roccella inc o ia and Lasallia pus ula a), and conse- quen ly, C. inc o ia dyes emained o e e in e linked o hese lichens. Cla ke, in his c i ical edi ion o ex s w i en in he 14 h and 15 h cen u ies, goes u he and sugges s ha he e m o nasol was used gene ically o designa e any wa e -based colo s o ed in clo h (17). This had been al eady posi ed by Walle (9) in his p oposal o iden i y olium in manusc ip illumina ions based on molecula lu- o escence. Fo una ely, we we e able o selec some medie al eci- pes o p oduce C. inc o ia blues, because hey a e so de ailed and o e accu a e desc ip ions o he ui and ins uc ions o no b eak he ui s and ee he seeds, hus poin ing o he use o C. inc o ia (and no o lichens). We also ha e 19 h cen u y documen al e idence ha his medie al knowledge was p ese ed, un il nea ly he u n o ha cen u y, in he egion o G and-Galla gues (now, Galla gues- le- Mon ueux), in F ance. In 1842, Joly (18) published a clea , con- cise, and comple e ex on his subjec , including e e ences o ea lie wo ks such as Nissolle’s (19) publica ion ha o e s a p ecise desc ip ion o he plan , accompanied wi h an equally accu a e illus a ion. Joly’s ex is ema kable in se e al aspec s; i s , he isi s G and-Galla gues o in e iew a p ies by he name o Hugues and o ga he in o ma ion di ec ly om he make s; p ecisely du ing he pe iod when he plan should be collec ed (Augus /Sep embe ), “ e s la in de sep emb e 1838, e dans les de nie s jou s du mois d’aoû 1839.” Acco ding o Joly, in his egion, his ac i i y has been docu- men ed since 1600 (when eco ds a e a ailable), bu e en so, ac- co ding o Fa he Hugues, i is one o he mos mys e ious o c a s (18,20): “peu d’indus ies son aussi mys é ieuses: ceux qui l’exploi en n’en connaissen poin la des ina ion; ceux qui en p o i en n’en connaissen poin la p épa a ion, e ceux qui l’on déc i e n’on débi é que des mensonges, pa ce qu’ils ne ansc i aien que de ausses indica ions” [ ew indus ies a e so mys e ious: hose who exploi i do no know i s inal applica ions; hose who p o i by i do no know how o p epa e i , and hose who ha e desc ibed i ha e only old lies, because hey ha e only ansc ibed alse di ec ions] (18). Joly desc ibes he p ocess using he en i e plan o p oduce he da k blue colo and he inno a ions ha we e in oduced in he me- die al p ocess, and concludes ha , a ha ime, he clo hle s we e mainly sold o dye cheese inds ed in he Ne he lands (21): “mais il pa aî que l’usage en es bo né à donne aux c oû es du omage de Hollande ce e ein e ouge qui les dis ingue, (...) Il su i de empe les o mages dans un baque d’eau bleuie pa les chi ons, e de les en e i e p esque aussi ô pou les ai e séche ” [bu i seems ha he use is limi ed o gi ing he ind o Du ch cheese ha ed hue ha dis inguishes i , (...) All you ha e o do is soak he cheese in a bucke o wa e dyed blue by he clo hle s ( ags), and o emo e hem almos immedia ely o le hem d y] (18). Joly p oposes ha no only o he species could be es ed o dyeing mo e e icien ly, such as o he Ch ozopho a species, bu also Me cu ialis pe ennis (dog’s me cu y) and Me cu ialis omen osa. This is ex ao dina y, as now om his cu en p ojec , we know ha hese dyes sha e a common molecula s uc u e (Fig.1). Joly also es ed and concluded ha he p ecu so s o he blue may be ound in all he pa s o he plan and a e mo e abundan in ui s. He concluded, “Sous l’in luence de la ie, il exis e dans ces o ganes à l’é a incolo e; ap ès la mo du égé al, e sous l’in luence de l’oxygène a mosphé ique e d’une p omp e dessicca ion, il peu de eni bleu” [Unde he in luence o li e, i exis s in hese o ganisms in a colo less s a e; a e he dea h o he plan , and unde he in luence o a mosphe ic oxygen and a apid desicca ion, i becomes blue] (18), which is p ecisely he way he appea ance o he blue colo in M. pe ennis is desc ibed in mo e ecen s udies pe o med by Swan and Lo en z e al. (22–25). These au ho s ha e cha ac e ized he molecula s uc u e o he blue ch omopho e as will be desc ibed below. Medicinal p ope ies in Me cu ialis species and molecula s uc u es o i s blue colo s In 1985, Swan p oposed he i s molecula s uc u e o he “blue ch omogen” isola ed om M. pe ennis (dog’s me cu y), a pe ennial he b used in emedies o medicinal pu poses (22,23). Swan syn hesized 2,3,6- ihyd oxy-4-me hoxy-1-me hyl-py idinium ( ig. S1), a colo less compound in solu ion ha he could oxidize o a blue ansien colo . In his own wo ds: “To summa ize, i is sugges ed ha he ch omogen p esen in he colo less aqueous ex ac o Me cu ialis pe ennis (...) is compound he midin (2,3,6- ihyd oxy-4-me hoxy-1-me hyl-py idinium) and ha on oxida ion by ai o K3[Fe(CN)6] he blue ansien colo is due o a semiquinone- like (17), and ha a he momen when he solu ion u ns yellow i con ains compounds (16), (22), and (23).” ( ig. S1). On d ying, M. pe ennis plan s ake on a da k blue colo . As Swan could only ob ain a empo a y blue in solu ion, his made him doub ha his ch omogen could be he only sou ce o he blue colo . La e , in 2010 and 2014 (23,24), he ins abili y o he blue s uc u e in solu ion was s udied in de ail by Lo enz e al. (Fig.2). Tha esea ch allowed his g oup o con i m he molecula s uc u es p oposed by Swan as he o igin o he blue colo in M. pe ennis and o sugges in e con e sion mechanisms o he colo less he midin in o he blue he midin quinone and he la e in o yellow dime ic educed o ms, as desc ibed in de ail in he Supplemen a y Ma e ials and depic ed in Fig.2 (23,24). In 1984, Fo es e had al eady epo ed ha aqueous solu ions o he midin eadily gi e ise o a ansien blue adical-anion on Fig. 1. The molecule o his s udy, ch ozopho idin. Le : Close-up o C. inc o ia ui s (collec ed in Alen ejo, Po ugal) and clo hle s p epa ed wi h he juice o he ui s ollowing he ins uc ions in he Book o all colo pain s. Ligh g een ui s we e used in his s udy sho ly a e collec ion. Righ : Molecula s uc u es o he blue colo an s, he midin ( om M. pe ennis), and ch ozopho idin ( om C. inc o ia). Pho o c edi : Paula Nabais, Uni e sidade NOVA de Lisboa. on Ma ch 4, 2021h p://ad ances.sciencemag.o g/Downloaded om Nabais e al., Sci. Ad . 2020; 6 : eaaz7772 17 Ap il 2020 SCIENCE ADVANCES | RESEARCH ARTICLE 3 o 8 exposu e o ai , he iden i y o which has been es ablished by elec- on spin esonance spec oscopy (25). Plan desc ip ion C. inc o ia (L.) A.Juss. (Eupho biaceae) is an annual he b na i e o he Medi e anean egion, no h A ica, and cen al and sou hwes e n Asia. This species can be ound on d y and dis u bed lands, ude al habi a s, allows, and along he edges o cul i a ed ields, mos ly in limes one. The plan s a e 10 o 40cm all, g ay-g een, and omen ous (densely co e ed wi h s ella e hai s). S ems a e e ec and b anched and lea es a e al e na e, hombic o o a e, cunea e a he base, and wi h sinua e lea ma gin (26). C. inc o ia is monoecious, bea ing male and emale ep oduc i e o gans in di e en lowe s bu on he same plan . Flowe s a e g ouped in o spike-like acemes, wi h male lowe s a he op and emale lowe s a he base, usually soli a y. Male lowe s a e yellow and inconspicuous. Female lowe s exhibi a sphe ical o a y and pe als a e no p esen . Flowe ing occu s om May o Sep embe and ui ma u a ion is be ween July and Oc obe . Ma u e ui s a e da k g een, subsphe ic, and sligh ly lobed capsules, co e ed wi h whi e scales and 5 o 8mm in diame e (Fig.1 and ig. S2). Du ing dehiscence, each o he h ee single-seeded loculus opens in o wo al es, eleasing h ee g ay o ligh b own, ough- ex u ed obo a e seeds app oxima ely 4mm in size (26). Molecula s uc u es o colo an s in C. inc o ia In con as o he success ul disco e y o he molecula s uc u e o he blue in M. pe ennis (dog‘s me cu y), he wo ks published o C. inc o ia ha e been inconclusi e o ha e sugges ed inco ec s uc- u es. In 1997, Guineau (8) p oposed o cinol o he blue colo based on ime-o - ligh seconda y ion MS da a, which suppo ed he p e ious sugges ions o he p esence o lichen colo an s in C. inc o ia ha we e e iewed by Walle (9). As we will show in his pape , o cinol- based s uc u es a e no p esen in he ex ac s o he ui s o C. inc o ia (27). Mo e ecen ly, he dye was in es iga ed by Ace o’s g oup and, al hough hey did no p oposed any s uc u e o he blue dye, hey epo ed ha “ he mass spec um is domina ed by a peak a 266 m/z” (10). An hocyanins ha e also been p oposed, bu ne e iden i ied (9). In he pas 2 yea s, we ha e endea o ed o unlock he chemical s uc u e behind he blue/pu ple colo in C. inc o ia. This is an in- dispensable piece o knowledge o he p ese a ion o ou Eu opean cul u al he i age, in wo ks o a such as medie al manusc ip illu- mina ions whe e his dye could ha e been used. RESULTS Cha ac e iza ion o he blue dye ex ac ed om he ui s (shell) o C. inc o ia The blue ex ac o C. inc o ia, ob ained om he ui s as epo ed in Ma e ials and Me hods, was analyzed using HPLC-DAD and LC-MS (Table1 and ig. S3). The ex ac essen ially showed he p esence o one ch oma og aphic peak cha ac e ized by a maximum abso p ion wa eleng h, max, a ci ca 540 nm, indica i e o a pu ple/ bluish colo , and an ion mass mass/cha ge a io (m/z) a 501, in posi i e ion mode. The ollowing agmen s we e also de ec ed, a m/z 339, compa ible wi h he loss o a hexose moie y (shown o be an O-glucose linkage), and a m/z 307, 279, and 254, in he MS3 spec um, consis en wi h he loss o me hoxyl and me hyl g oups. In addi ion, an ion mass a m/z 334 wi h a MS2 spec um a m/z 172 (loss o 162 uni ied a omic mass uni ) and a MS3 spec um wi h wo agmen s a m/z 157 and 115 we e also de ec ed in he ex ac . The ion mass m/z 172 and he espec i e agmen s a m/z 157 and 115 we e desc ibed in he li e a u e by Lo enz e al. (23) and co espond o he midin p e sen in M. pe ennis. In his case, he ion mass a m/z 334 should co espond o he glycosyla ed o m o he midin (Fig.1). We did no obse e a peak a 266m/z, con a y o wha was obse ed by Ace o e al. (10) ha epo ed ha in hei ex ac s o C. inc o ia “ he mass spec um is domina ed by a peak a 266 m/z.” Fig. 2. Causes o colo in he midin ex ac ed om M. pe ennis. Con e sion o colo less he midin in o he blue he midin quinone and o ma ion o dime ic s uc u es as p oposed by Lo enz e al. (23, 24). on Ma ch 4, 2021h p://ad ances.sciencemag.o g/Downloaded om Nabais e al., Sci. Ad . 2020; 6 : eaaz7772 17 Ap il 2020 SCIENCE ADVANCES | RESEARCH ARTICLE 4 o 8 Adding o his, se e al an hocyanins we e also iden i ied as mino compounds in he ui s’ ex ac , peonidin-3-O-glucoside, and he couma oyla ed de i a i es o delphinidin, cyanidin, pela gonidin, and peonidin (Table 1 and igs. S3 and S4). These an hocyanins we e en a i ely iden i ied by mass spec a and he ch oma og aphic e- en ion ime compa ed o s anda ds. On he basis o i s exac mass (501.1371), he elemen al composi ion o he blue dye should co espond o C20H24O13N2 (500.41 gmol−1). On a i s a emp , and due o he p esence o a he midin-glucoside in he ex ac , a he midin-like s uc u e was conside ed o i . Con- a y o wha was epo ed o he ansien blue adical-anion o cyanohe midin in M. pe ennis ( ig. S1), he blue ex ac om C. inc o ia was s able o se e al days, poin ing o he absence o a adical species. Despi e his indica ion, EPR expe imen s we e pe o med and con- i med ha no adical species we e p esen in he aqueous solu ion o he pu i ied compound. To elucida e he s uc u e o he blue dye p esen in C. inc o ia ui s, he pu i ied compound was analyzed using one-dimensional (1D; 1H and 13C) and 2D (COSY, HSQC, HMBC and INADEQUATE) NMR in DMSO-d6/D2O (9:1). The assignmen o he p o on and ca bon chemical shi s (Table2) o he pu i ied compound showed he p esence o wo a opisome s (Fig.3). The p oposed s uc u es co espond o wo o ame s (a opisome 1 and a opisome 2), which we e pe cei ed in he 13C expe imen , wi h all he ca bon signals appea ing in duplica e and wi h he same in ensi y (Fig.3). The anome ic p o ons H-1′ om he wo isome s we e assigned o he wo double s (J=7.7 and 7.5 Hz, espec i ely) p esen a 4.32 and 4.20pa s pe million (ppm), espec i ely. P o ons H-2′, H-3′, H-4′, H-5′, H-6′a, and H-6′b o glucose o bo h isome s we e a - ibu ed o he signals p esen a 3.13, 3.10, 2.90, 3.16, 3.45, and 3.63 ppm, espec i ely, by hei co ela ions wi h each o he in he COSY spec um. Because o he supe imposi ion o signals in ol ing wo simila compounds, i was no possible o accu a ely a ibu e he chemical shi s and coupling cons an s o all he p o ons o bo h isome s. The O-CH3 p o ons p esen in ca bon C-4 and C-10 we e assigned o he single s p esen a 3.75/3.77 and 3.71/3.78 ppm, e- spec i ely, and he p o ons N-CH3 p esen in ings A and B o bo h isome s appea ed a 3.12 ppm as a single . The assignmen o he ca bon esonances was made possible using 2D NMR echniques (gHSQC, gHMBC, and INADEQUATE). Ca - bons C-1′, C-2′, C-3′, C-4′, C-5′, and C-6′ om he glucose moie y o a opisome s 1 and 2 we e assigned o 108.3/108.7, 74.1/74.1, 77.4/77.5, 75.0/75.0, 76.9/76.9, and 61.3/61.5 ppm h ough hei di ec co ela ions wi h he espec i e p o ons. Ca bons O-CH3 ( ings A and B) and N-CH3 ( ings A and B) om isome s 1/2 we e as- signed o 59.2/59.4, 60.8/60.1, 27.4/27.5, and 27.3/27.5 ppm (Table2). Ca bons C-5 om bo h a opisome s we e assigned o 122.0 and 122.3 ppm, by he long-dis ance co ela ion (HMBC) wi h he ano- me ic p o ons H-1′. Ca bons C-4 and C-10 om bo h a opisome s we e a ibu ed o 161.1/161.0 and 156.0/156.1 ppm, espec i ely, by hei long- ange co ela ions wi h me hyl g oups. Ca bons C-2, C-6, C-8, and C-12 om a opisome s 1 and 2 we e a ibu ed o 164.0/163.7, 159.0/158.8, 161.7/161.6, and 157.0/156.9 ppm, espec- i ely, and by hei long-dis ance co ela ion (HMBC spec um) wi h he p o ons p esen a he N-me hyl g oup. We could no make an unequi ocal assignmen o ca bons C-3 and C-9 h ough NMR, because hese wo ca bons do no p esen any p o on in hei icini y (Fig.3). Mo eo e , he chemical shi o ca bon C-11 could only be de e mined using a 2D 13C-13C INADEQUATE expe imen , whe e he co ela ion be ween his ca bon wi h ca bon C-12 was obse ed. The appea ance o ca bon C-11 a high chemical shi s concu s wi h a ca bonyl g oup in ha posi ion. The esul s a e consis en wi h he s uc u e o he blue compound co esponding o 6′-hyd oxy-4,4′-dime hoxy-1,1′-dime hyl-5′- {[3,4,5- ihyd oxy-6-(hyd oxyme hyl) e ahyd o-2H-py an-2-yl] oxy}-[3,3′-bipy idine]-2,2′,5,6(1H,1′H)- e aone, a he midin-mono- glycosyla ed dime ha we would he ea e designa e as ch ozopho idin (Fig.3). This is a no able esul , conside ing ha p e iously, in M. pe ennis, he dime ic o ms o he midin we e associa ed wi h yellow and yellow-b own solu ions, whe eas he blue colo was based on a monome ic adical ansien species. To ge u he insigh in o he p oposed s uc u e and as a means o p edic i s blue colo , compu a ional calcula ions we e pe o med. Compu a ional calcula ions By explo ing he con o ma ional space o ch ozopho idin, 10 en- e ge ically a o ed isome s (local minima) we e ound wi h E< 1.0 kcal mol−1 ( able S1 epo s hei ela i e in e nal ene gy and bo h 1 and 2 alues a e op imiza ion in an aqueous en i onmen ). Table 1. API-LC-ESI-MS/MS da a o he majo and mino compounds ex ac ed om C. inc o ia ui s. Molecula ion and espec i e agmen s MS2 and MS3 ob ained by a mosphe ic p essu e ioniza ion (API)–LC–elec osp ay ioniza ion (ESI)–MS/MS (posi i e ion mode) ound in he blue ex ac o C. inc o ia ui s. R (min) Iden i y [M+] [MS2] [MS3] 8.29 He midin-glycoside 334 172 157 15.63 Peonidin-3-glucoside 463 301 20.33 He midin-de i a i e (blue dye) 501 339 307; 279; 254 52.36 Delphinidin-3- couma oylglucoside 611 303 52.67 Cyanidin-3- couma oylglucoside 595 449 287 60.07 Pela gonidin-3- couma oylglucoside 579 433 271 64.23 Peonidin-3- couma oylglucoside 609 463 301 on Ma ch 4, 2021h p://ad ances.sciencemag.o g/Downloaded om Nabais e al., Sci. Ad . 2020; 6 : eaaz7772 17 Ap il 2020 SCIENCE ADVANCES | RESEARCH ARTICLE 5 o 8 The a ious isome s o ch ozopho idin can be assembled in wo g oups based on hei dihed al alues (g oup I composed o isome s 1, 3, 4, 5, 8, 9, and 10 ha ha e simila dihed al alues: 1=−61.1±1.5° and 2=83.0±0.9°, and g oup II composed by isome s 2, 6, and 7 ha ha e simila dihed al alues: 1=60.6±0.0° and 2=−80.9±0.3°). Each g oup can be ep esen ed by he mos he modynamically a- o ed a opisome s 1 and 2. A mix u e o bo h a opisome s is ex- pec ed because o hei e y small ene gy di e ence (0.004 kcal mol−1, which co esponds o 50.2% and 49.8% quan i ies o a opisome s 1 and 2, espec i ely, acco ding o he Maxwell-Bol zmann dis ibu ion, and using a empe a u e o 298 K). The analysis o he molecula s uc u es o bo h a opisome s in solu ion e ealed ha he elec- onic delocaliza ion p o ided by he pe pendicula o ien a ion o bo h a oma ic ings may be essen ial o hei o ma ion. Howe e , a opisome 2 has he B ing and he glucose g oup close o each o he o make one hyd ogen bond (as seen in Fig.4). This s e eo- chemical e ec may cause he sligh ene gy di e ence be ween he wo isome s. Time-dependen densi y unc ional heo y (TD-DFT) calcula ions we e pe o med o ob ain he UV-VIS abso p ion spec a o he wo a opisome s. max was e alua ed using a ious densi y unc- ionals (see able S4). I would appea ha in compa ison wi h ex- pe imen s, he Boese-Ma in o kine ics (BMK) densi y unc ional was he bes -pe o ming unc ional o bo h a opisome s (Fig.4 compa es hei p edic ed UV-VIS spec a wi h an expe imen al spec um a pH 7). Theo e ical and expe imen al spec a o e lap qui e well. The small disc epancy migh be he esul o e o s associa ed wi h he TD-DFT de e mina ion o max alues (28) and om he use o an implici sol en model app oach ins ead o an explici desc ip ion o he sol en molecules. The la e may a ec he in a- molecula in e ac ions and s abili y o he a opisome s (29). The spec al shi (max) o expe imen al da a ob ained in wa e (pH 7) is −23.8 and 5.0nm o a opisome s 1 and 2, espec i ely (Fig.4). Again, he hypsoch omic shi obse ed in he maximum abso p ion wa eleng h o a opisome 1in ela ion o a opisome 2 could be associa ed o he lowe p oximi y be ween he B ing and he glucose uni . The max o abso p ion, in he isible, is essen ially a ibu ed o he elec onic ansi ion om he highes occupied molecula o bi al (HOMO) o he lowes unoccupied molecula o bi al (LUMO). In bo h con o me s, he HOMO is sp ead h ough bo h A and B ings, while he LUMO is mainly dis ibu ed on B ing ( ig. S5). The ansi ion om HOMO o LUMO should ha e an in amolecula cha ge ans e cha ac e . Sligh MO dis ibu ion di e ences a e e- la ed wi h he ma ginally highe ene gy gap be ween HOMO and LUMO in isome 1in ela ion o isome 2 ( ig. S5). Wi h espec o he s abili y o he blue colo , i is in e es ing o no e ha he bond be ween he wo a oma ic ings may be iewed almos as a single bond (leng h o 1.464 Å in bo h con o me s). O e all, he UV-VIS spec a p edic ed o ch ozopho idin co - obo a es ha his dime is he cause o he blue/pu ple colo s (Fig.4). Po en ially, he blue colo is he esul o he abo emen ioned cha ge ans e . A opisome 1 A opisome 2 Fig. 3. Resul s o NMR (13C). S uc u e o he blue dye p esen in C. inc o ia ui s (shell), ch ozopho idin, and 13C zoomed spec a (bo om). Table 2. Essen ial NMR da a o he iden i ica ion o ch ozopho idin. 1H and 13C chemical shi s o he blue dye p esen in C. inc o ia ui s, de e mined in DMSO-d6:D2O (9:1). Posi ion  1Η (ppm); J (Hz)  13C (ppm)* A ing 1-N-CH33.12; s 27.4/27.5 2 C═O – 164.0/163.7 3 – 88.5/88.4 4 – 156.0/156.1 4-OCH33.71; 3.78; s 59.2/59.4 5 – 122.0/122.3 6 – 159.0/158.8 B ing 7-N-CH33.12; s 27.3/27.5 8 C═O – 161.7/161.6 9 – 128.4/127.7 10 – 161.1/161.0 10-OCH33.75; 3.77; s 60.8/61.0 11 C═O – 172.6/172.3 12 C=O – 157.0/156.9 Glucose moie y 1′ 4.32; d, 7.7/4.20; d, 7.5 108.3/108.7 2′ 3.13;†74.1/74.1 3′ 3.10;†77.4/77.5 4′ 2.90;†75.0/75.0 5′ 3.16;†76.9/76.9 6a′ 3.45; 61.3/61.5 6b′ 3.63; 61.3/61.5 *The ca bon signals a e duplica ed, which indica es ha a leas wo isome s a e p esen . †Un esol ed (supe posi ion). on Ma ch 4, 2021h p://ad ances.sciencemag.o g/Downloaded om Nabais e al., Sci. Ad . 2020; 6 : eaaz7772 17 Ap il 2020 SCIENCE ADVANCES | RESEARCH ARTICLE 6 o 8 DISCUSSION Knowledge was los on how o ex ac he blue colo om C. inc o ia, bu i was eco e ed h ough esea ch on medie al w i en sou ces on he a o pain ing, in pa icula , he a o illumina ing books (30,31). Following medie al ins uc ions, only he ui s we e col- lec ed be ween July and Sep embe , and ca e was aken o no g ind he seeds ha a e ound inside. The ex ac s hus ob ained p esen ed a blue dye as a majo ch omopho e ( ig. S3). Las , in he 21s cen u y, an in e disciplina y app oach led o he disco e y o a molecula s uc u e o he blue colo an ex ac ed om he ui s o C. inc o ia, namely, a he midin-based mono-glycosyla ed dime (Fig.3), named ch ozopho idin. Expe imen al da a and compu a ional quan um me- chanics s udies show ha his dime is ep esen ed by wo isome s, a opisome s 1 and 2 (Fig.4). Toge he wi h he blue he midin-based dime , se e al an hocyanins we e iden i ied o he i s ime (Table1 and igs. S3 and S4) and a e p esen as mino compounds. Ch ozo- pho idin was compa ed o he blue ound in a species om he same amily, M. pe ennis (dog’s me cu y), an old medicinal plan known mos ly h ough e hnomedicine (22). The blue ch omopho e in bo h species includes he he midin ing (Fig.1), bu in con as o he ansien adical species in M. pe ennis, he blue in C. inc o ia has a s able glycosyla ed dime ic s uc u e and is he e o e wa e soluble. To summa ize, his molecula s uc u e is key o iden i ying olium in wo ks o a and o s udying he s uc u al, elec onic, and eac i e p ope ies o his complex dye (32,33). This will pa e he way o e alua ing conse a ion condi ions and he de e mina ion and plan- ning o he bes p ese a ion s a egies. In conclusion, ch ozopho idin was used in ancien imes o make a beau i ul blue dye o pain ing, and i is nei he an an hocyanin— ound in many blue lowe s and ui s—no indigo, he mos s able na u al blue dye. I u ns ou o be in a class o i s own. Thus, we belie e ha his will be no ou inal wo d on his amazing plan and i s s o y and ha u he disco e ies will ollow soon. MATERIALS AND METHODS The ui s o C. inc o ia we e collec ed be ween July and Sep embe o 2016, 2017, and 2018, in Alen ejo (G anja/Mou ão), Po ugal (by A.C., F.P., M.J.M., and P.N.). The coo dina es we e p o ided by (34). F esh ui s (160 g) we e ex ac ed wi h 4li e s o me hanol:wa e (70:30, / ) du ing 2 hou s unde s i ing, gi ing ise o a deep blue–colo ed solu ion. Me hanol was e apo a ed unde acuum, and he ob ained c ude blue ex ac was analyzed by HPLC-DAD and LC-MS. Fo he cha ac e iza ion o he blue dye, he ex ac was u he pu i ied. In a i s s ep, by applying he ull ex ac o a e e sed- phase C18 silica gel in a Büchne unnel unde acuum (G3 po osi y) washed wi h deionized wa e , i was possible o obse e he elu ion o yellow compounds and hen he blue ac ion was eco e ed slowly wi h wa e . This blue ac ion was concen a ed in a o oe apo a o and hen was pu i ied, wice, by low-p essu e column ch oma og aphy using he same C18-RP gel as s a iona y phase (250mm × 16mm inside diame e ) and deionized wa e as a mobile phase. The pu i ied blue ac ion was lyophilized, and he blue powde ob ained was analyzed by NMR, HPLC-DAD, HPLC-DAD-MS, GC-MS, and EPR. Fig. 4. Analysis o he he modynamically a o ed a opoisome s o ch ozopho idin, in solu ion. Molecules a e depic ed as s icks and colo ed by a om ype. (A) A ing, B ing, and glucose ing a e colo ed in ed, o ange, and g een, espec i ely. Bo h 1 and 2 dihed als and hyd ogen bonds a e also indica ed. (B) Supe posi ion o he A ing o he wo molecules. Colo ed by elemen (a opisome 1) and g een (a opisome 2). (C) UV-VIS spec a (wa e , pH 7, blue) o ch ozopho idin compa ed wi h he p edic ed spec a [Boese-Ma in o kine ics (BMK) unc ional] o a opisome s 1 (black) and 2 (g een). on Ma ch 4, 2021h p://ad ances.sciencemag.o g/Downloaded om Nabais e al., Sci. Ad . 2020; 6 : eaaz7772 17 Ap il 2020 SCIENCE ADVANCES | RESEARCH ARTICLE 7 o 8 The ini ial geome y o ch ozopho idin was buil wi h he GaussView so wa e (35–37). Geome y op imiza ions we e ca ied ou , and he mos ene ge ically a o ed isome s (local minima wi h E<1.0 kcal mol−1) we e u he op imized in sol en . TD-DFT single-poin calcula ions we e applied o de e mine he UV-VIS ab- so p ion spec a o he wo mos ene ge ically s able a opisome s o ch ozopho idin. All calcula ions we e pe o med by he Gaussian 09 package (38). Fo comp ehensi e de ails, please see he Supple- men a y Ma e ials. 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Funding: This wo k was suppo ed by he Associa e Labo a o y o G een Chemis y-LAQV, which is inanced by na ional unds om FCT/MCTES (UID/QUI/50006/2019), and by p ojec STEMMA—F om singing o w i ing—su ey on ma e ial p oduc ion and ou es o Galician-Po uguese Ly ic, PTDC/LLT-EGL/30984/2017. J.O. and N.F.B. hank he FCT o hei IF g an (IF/00225/2015 and IF/01355/2014, espec i ely). N.T. hanks con ac REQUIMTE/EEC2018/PTDC/QUI-OUT/29925/2017, wi hin he p ojec “Polyphenols in on Ma ch 4, 2021h p://ad ances.sciencemag.o g/Downloaded om Nabais e al., Sci. Ad . 2020; 6 : eaaz7772 17 Ap il 2020 SCIENCE ADVANCES | RESEARCH ARTICLE 8 o 8 A : chemis y and biology hand in hand wi h conse a ion o cul u al he i age,” PTDC/ QUI-OUT/29925/2017, and P.N. hanks Ph.D. g an wi hin he CORES Ph.D. p og amme (PD/ BD/105895/2014). Au ho con ibu ions: Expe imen al wo k: P.N., J.O., F.P., N.T., A.C., M.R., A.M.S.S., and M.J.M.; compu a ional wo k: N.F.B.; p ojec de elopmen and unding: V.d.F., F.P., and M.J.M. Compe ing in e es s: The au ho s decla e ha hey ha e no compe ing in e es s. Da a and ma e ials a ailabili y: All da a needed o e alua e he conclusions in he pape a e p esen in he pape and/o he Supplemen a y Ma e ials. Addi ional da a ela ed o his pape may be eques ed om he au ho s. Submi ed 8 Oc obe 2019 Accep ed 22 Janua y 2020 Published 17 Ap il 2020 10.1126/sciad .aaz7772 Ci a ion: P. Nabais, J. Oli ei a, F. Pina, N. Teixei a, V. de F ei as, N. F. B ás, A. Clemen e, M. Rangel, A. M. S. Sil a, M. J. Melo, A 1000-yea -old mys e y sol ed: Unlocking he molecula s uc u e o he medie al blue om Ch ozopho a inc o ia, also known as olium. Sci. Ad . 6, eaaz7772 (2020). on Ma ch 4, 2021h p://ad ances.sciencemag.o g/Downloaded om , also known as oliumCh ozopho a inc o ia A 1000-yea -old mys e y sol ed: Unlocking he molecula s uc u e o he medie al blue om P. Nabais, J. Oli ei a, F. Pina, N. Teixei a, V. de F ei as, N. F. B ás, A. Clemen e, M. Rangel, A. M. S. Sil a and M. J. Melo DOI: 10.1126/sciad .aaz7772 (16), eaaz7772.6Sci Ad ARTICLE TOOLS h p://ad ances.sciencemag.o g/con en /a chi e/6/16/eaaz7772/1 MATERIALS SUPPLEMENTARY h p://ad ances.sciencemag.o g/con en /suppl/2020/04/13/6.16.eaaz7772.DC1 REFERENCES h p://ad ances.sciencemag.o g/con en /a chi e/6/16/eaaz7772/1#BIBL This a icle ci es 22 a icles, 0 o which you can access o ee PERMISSIONS h p://www.sciencemag.o g/help/ ep in s-and-pe missions Te ms o Se iceUse o his a icle is subjec o he is a egis e ed adema k o AAAS.Science Ad ancesYo k A enue NW, Washing on, DC 20005. The i le (ISSN 2375-2548) is published by he Ame ican Associa ion o he Ad ancemen o Science, 1200 NewScience Ad ances License 4.0 (CC BY-NC). Science. No claim o o iginal U.S. Go e nmen Wo ks. Dis ibu ed unde a C ea i e Commons A ibu ion NonComme cial Copy igh © 2020 The Au ho s, some igh s ese ed; exclusi e licensee Ame ican Associa ion o he Ad ancemen o on Ma ch 4, 2021h p://ad ances.sciencemag.o g/Downloaded om