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.
SUPPLEMENTARY MATERIALS
Supplemen a y Ma e ials o his a icle is a ailable a h p://ad ances.sciencemag.o g/cgi/
con en / ull/6/16/eaaz7772/DC1
REFERENCES AND NOTES
1. M. J. Melo, R. Cas o, P. Nabais, T. Vi o ino, The book on how o make all he colou pain s
o illumina ing books: Un a elling a Po uguese Heb ew illumina o s’ manual. He i . Sci.
6, 44 (2018).
2. M. Cla ke, Mediae al Pain e s’ Ma e ials and Techniques: The Mon pellie Libe Di e sa um
A cium (A che ype Publica ions, 2011), pp. 110–111.
3. J. G. Haw ho ne, C. S. Smi h, Theophilus on Di e s A s: The Fo emos Medie al T ea ise on
Pain ing, Glassmaking and Me alwo k (Do e Publica ions, 1979), pp. 38–40.
4. F. B unello, De A e Illuminandi e Al i T a a i Sulla Tecnica Della Minia u a Medie ale (Ne i
Pozza Edi o e, 1992), pp. 63–67.
5. M. J. Melo, P. Nabais, R. A aújo, T. Vi o ino, The conse a ion o medie al manusc ip
illumina ions: A chemical pe spec i e. Phys. Sci. Re . 4, 20180017 (2019).
6. I. Degano, Liquid ch oma og aphy: Cu en applica ions in he i age science and ecen
de elopmen s. Phys. Sci. Re . 4, 20180009 (2018).
7. F. Pozzi, M. Leona, Su ace-enhanced Raman spec oscopy in a and a chaeology.
J. Raman Spec osc. 47, 67–77 (2015).
8. B. Guineau, Le olium des enlumineu s, une couleu aujou d'hui dispa ue: Ce que nous
appo en les ex es su l'o igine e la ab ica ion de ce e couleu , son p océdé
d'emmagasinage su un mo ceau d'é o e e son emploi dans l'enluminu e médié ale.
Iden i ica ion de olium dans des pein u es du IXe s., du Xe s. e du débu du XIe s.
A chéologie Médié ale 26, 23–44 (1996).
9. A. Walle , Ch ozopho a inc o ia Juss. P oblems in iden i ying an illumina ion colo an .
Res au a o 11, 141–155 (1990).
10. M. Ace o, A. A ais, F. Ma sano, A. Agos ino, G. Fenoglio, A. Idone, M. Gulmini,
A diagnos ic s udy on olium and o chil dyes wi h non-in asi e and mic o-des uc i e
me hods. Spec ochim. Ac a A Mol. Biomol. Spec osc. 142, 159–168 (2015).
11. C. K ekel, hesis, Ludwig-Maximilians-Uni e si ä München, Munich, Ge many
(1996).
12. J. Oli ei a, J. Aze edo, A. M. S. Sil a, N. Teixei a, L. C uz, N. Ma eus, V. de F ei as,
Py anoan hocyanin dime s: A new amily o u quoise blue an hocyanin-de i ed
pigmen s ound in Po wine. J. Ag ic. Food Chem. 58, 5154–5159 (2010).
13. J. Oli ei a, C. San os-Buelga, A. M. S. Sil a, V. de F ei as, N. Ma eus, Ch oma ic
and s uc u al ea u es o blue an hocyanin-de i ed pigmen s p esen in Po wine.
Anal. Chim. Ac a 563, 2–9 (2006).
14. A. Menghini, Ed., De Ma e ia Medica, Il Diosco ide di Napoli (Aboca Edizioni, 2016), ol. 1,
pp. 413.
15. O. Meung, La Pha macie des Moines: T ai é de la Ve u des Plan es (Mace lo idus) (Édi ions
Paleo, 2011), pp. 127.
16. H. M. Abdallah, F. M. Almowallad, A. Esma , I. A. Sheha a, E. A. Abdel-Sa a ,
An i-in lamma o y ac i i y o la onoids om Ch ozopho a inc o ia. Phy ochem. Le . 13,
74–80 (2015).
17. M. Cla ke, The c a e o lymmyng and The mane o s eynyng: Middle English ecipes o
pain e s, s aine s, sc ibes, and illumina o s (Ea ly English Tex Socie y, Ox o d, 2016),
pp. 44: ‘ u nesole, o nesole , ou nesole, u sole; n. any colo o di ec dye ex ac ed om
a lowe o be y ha is s o ed by being abso bed in o a clo hle , o be subsequen ly
eleased by imme sion in a medium o e.g. an illumina o 's colo , ypically pu ple o blue
(no a bo anical name, i.e. no e e ing o he u nsole plan s Ch ozopho a inc o ia
(Juss.) o Helo opium spp., al hough he o me plan i sel may be used as he colo
sou ce)’.
18. M. N. Joly, Reche ches su la Fab ica ion du Tou nesol en D apeaux, e su le P incipe
Colo an du Ch ozopho a inc o ia (Ad. de jussieu; C o on inc o ium, Linné) Employé à
ce e Fab ica ion. Ann. Chim. Phys. 111–126 (1842).
19. M. Nissolle, Desc ip ion du Ricinoïdes, ex quâ pa a u Tou nesol Gallo um, Ins . Rei He b.
App. 565, in His oi e de l’Academie Royale des Sciences (Imp ime ie Royale Pa is, 1712),
pp. 332–338.
20. We we e no ye able o ha e access o he publica ion o Fa he Hugues.
21. On he o he hand, a “ ou nesol en pains” was impo ed om he Ne he lands, which
was p epa ed wi h di e se species o lichens (Roccella inc o ia, used in he pas , had
been subs i u ed by Lecano a a a ea - cudbea ).
22. G. A. Swan, Isola ion, s uc u e, and syn hesis o he midin, a ch omogen om Me cu ialis
pe ennis L. J. Chem. Soc. Pe kin T ans. 1, 1757–1766 (1985).
23. P. Lo enz, M. H adecky, M. Be ge , J. Be ams, U. Meye , F. C. S in zing, Lipophilic
cons i uen s om ae ial and oo pa s o Me cu ialis pe ennis L. Phy ochem. Anal. 3,
234–245 (2010).
24. P. Lo enz, J. Con ad, S. Ducks ein, D. R. Kamme e , F. C. S in zing, Chemis y o he midin:
Insigh s om ex ac ion expe imen s wi h he main alkaloid o Me cu ialis pe ennis L.
acked by GC/MS and LC/MSn. Hel . Chim. Ac a 97, 1606–1623 (2014).
25. A. R. Fo es e , Au oxida ion o he midin: An ESR s udy. Expe ien ia 40, 688–689
(1984).
26. C. Benedí, in Flo a Ibe ica 8, S. Cas o iejo, C. Aedo, M. Laínz, F. Muñoz Ga mendia,
G. Nie o Feline , C. Pai a, J. Benedí, Eds. (Real Ja dín Bo ánico, CSIC, 1997), pp. 196–199.
27. M. J. Melo, P. Nabais, M. Guima ães, R. A aújo, R. Cas o, M. C. Oli ei a, I. Whi wo h,
O ganic dyes in illumina ed manusc ip s: A unique cul u al and his o ic eco d.
Philos. T ans. A Ma h. Phys. Eng. Sci. 374, 20160050 (2016).
28. P. T ouillas, F. Di Meo, J. Gie schne , M. Lina es, J. C. Sancho-Ga cía, M. O yepka, Op ical
p ope ies o wine pigmen s: Theo e ical guidelines wi h new me hodological
pe spec i es. Te ahed on 71, 3079–3088 (2015).
29. P. T ouillas, J. C. Sancho-Ga cía, V. De F ei as, J. Gie schne , M. O yepka, O. Dangles,
S abilizing and modula ing colo by copigmen a ion: Insigh s om heo y
and expe imen . Chem. Re . 116, 4937–4982 (2016).
30. N. Eas augh, V. Walsh, T. Chaplin, R. Siddall, Pigmen Compendium: A Dic iona y o
His o ical Pigmen s (Else ie Bu e wo h-Heinemann, 2004), p. 372.
31. D. Ca don, Na u al Dyes: Sou ces, T adi ion, Technology and Science (A che ype
Publica ions, 2007).
32. C. Miliani, L. Monico, M. J. Melo, S. Fan acci, E. M. Angelin, A. Romani, K. Janssens,
Pho ochemis y o a is s’ dyes and pigmen s: Towa ds be e unde s anding and
p e en ion o colo change in wo ks o a . Angew. Chem. In . Ed. 57, 7324–7334 (2018).
33. P. Lo enz, M. Bunse, S. Saue , J. Con ad, F. C. S in zing, D. R. Kamme e , Con e sion
o plan seconda y me aboli es upon e men a ion o Me cu ialis pe ennis L. Ex ac s
wi h wo lac obac e ia s ains. Fe men a ion 5, 42 (2019).
34. M. Po o, A. J. Pe ei a, C. Tauleigne Gomes, J. L. Vi o ino, Dis ibu ion o
Ch ozopho a inc o ia (L.) Ra . Flo a-On: Flo a de Po ugal In e ac i a, Sociedade
Po uguesa de Bo ânica (2016); h p://www. lo a-on.p /#wCh ozopho a+ inc o ia
[accessed 7 Janua y 2016].
35. F. Di Meo, J. C. Sancho-Ga cía, O. Dangles, P. T ouillas, Highligh s on an hocyanin
pigmen a ion and copigmen a ion: A ma e o la onoid -s acking complexa ion
o be desc ibed by DFT-D. J. Chem. Theo y Compu . 8, 2034–2043 (2012).
36. E. H. Anoua , J. Gie schne , J.-L. Du oux, P. T ouillas, UV/Visible spec a o na u al
polyphenols: A ime-dependen densi y unc ional heo y s udy. Food Chem. 131, 79–89
(2012).
37. J. Oli ei a, P. A aújo, A. Fe nandes, N. F. B ás, N. Ma eus, F. Pina, V. F ei as, In luence
o he s uc u al ea u es o amino-based py anoan hocyanins on hei acid-base
equilib ia in aqueous solu ions. Dyes, Pigm. 141, 479–486 (2017).
38. M. J. F isch, G. W. T ucks, H. B. Schlegel, G. E. Scuse ia, M. A. Robb, J. R. Cheeseman,
G. Scalmani, V. Ba one, G. A. Pe e sson, H. Naka suji, X. Li, M. Ca ica o, A. Ma enich,
J. Bloino, B. G. Janesko, R. Gompe s, B. Mennucci, H. P. H a chian, J. V. O iz, A. F. Izmaylo ,
J. L. Sonnenbe g, D. Williams-Young, F. Ding, F. Lippa ini, F. Egidi, J. Goings, B. Peng,
A. Pe one, T. Hende son, D. Ranasinghe, V. G. Zak zewski, J. Gao, N. Rega, G. Zheng,
W. Liang, M. Hada, M. Eha a, K. Toyo a, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima,
Y. Honda, O. Ki ao, H. Nakai, T. V e en, K. Th ossell, J. A. Mon gome y J ., J. E. Pe al a,
F. Oglia o, M. Bea pa k, J. J. Heyd, E. B o he s, K. N. Kudin, V. N. S a o e o , T. Kei h,
R. Kobayashi, J. No mand, K. Ragha acha i, A. Rendell, J. C. Bu an , S. S. Iyenga , J. Tomasi,
M. Cossi, J. M. Millam, M. Klene, C. Adamo, R. Cammi, J. W. Och e ski, R. L. Ma in,
K. Mo okuma, Ö. Fa kas, J. B. Fo esman, D. J. Fox, Gaussian 09 (Gaussian Inc., 2009).
Acknowledgmen s: We hank Z. Aze edo o he LC-ESI-MS analysis, M. And ade o he NMR
analysis, M. C. Oli ei a o he i s HRMS spec a and an hocyanins’ iden i ica ion, and E. Eis o
p o iding he plan M. pe ennis. 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