Degradation of lucidin: New insights into the fate of this natural pigment present in Dyer's madder (Rubia tinctorum L.) during the extraction of textile artefacts

Article abstract of DOI:10.1016/j.dyepig.2018.03.023

The complex mixtures of colorants present in different madder species can provide significant information about which plant species or technique was used to dye the fibres of historical textile artefacts, hence, when extracting and analysing colorants from textile artefacts as much of this information as possible should be preserved. Historical textiles are most commonly extracted with 37% hydrochloric acid: methanol: water (2:1:1, v/v/v), but this solvent system hydrolyses dye glycosides and may also induce chemical reactions. One of the primary components in Dyers’ madder (Rubia tinctorum L.) is lucidin primeveroside, but it is rarely seen in artefacts, nor is the corresponding aglycon lucidin. It has been demonstrated that the hydrochloric acid method causes hydrolysis of anthraquinone glycosides to their aglycon counterpart. Herein it is demonstrated that lucidin is not stable in such acidic conditions and degrades rapidly to xanthopurpurin. This is confirmed by HPLC, LC-MS and ¹H NMR, which also provide evidence of the mechanism of degradation being a retro-aldol process.

Full text of DOI:10.1016/j.dyepig.2018.03.023

Dyes and Pigments 154 (2018) 290–295
Contents lists available at ScienceDirect
Dyes and Pigments
journal homepage: www.elsevier.com/locate/dyepig
Degradation of lucidin: New insights into the fate of this natural pigment
present in Dyer's madder (Rubia tinctorum L.) during the extraction of textile
artefacts
Lauren Forda,b, Christopher M. Rayner , Richard S. Blackburn
b
a,∗
a
School of Design, University of Leeds, Leeds LS2 9JT, UK
b
School of Chemistry, University of Leeds, Leeds LS2 9JT, UK
A R T I C L E I N F O
A B S T R A C T
Keywords:
Madder
Rubia tinctorum
Lucidin
Dyes
Textiles
Extraction
Reactivity
HPLC analysis
The complex mixtures of colorants present in different madder species can provide significant information about
which plant species or technique was used to dye the fibres of historical textile artefacts, hence, when extracting
and analysing colorants from textile artefacts as much of this information as possible should be preserved.
Historical textiles are most commonly extracted with 37% hydrochloric acid: methanol: water (2:1:1, v/v/v), but
this solvent system hydrolyses dye glycosides and may also induce chemical reactions. One of the primary
components in Dyers' madder (Rubia tinctorum L.) is lucidin primeveroside, but it is rarely seen in artefacts, nor is
the corresponding aglycon lucidin. It has been demonstrated that the hydrochloric acid method causes hydro-
lysis of anthraquinone glycosides to their aglycon counterpart. Herein it is demonstrated that lucidin is not stable
in such acidic conditions and degrades rapidly to xanthopurpurin. This is confirmed by HPLC, LC-MS and 1
H
NMR, which also provide evidence of the mechanism of degradation being a retro-aldol process.
1
. Introduction
insight on how they were dyed and the plant species from which the
dye originated, hence, it is important to limit the damage to the col-
orant molecule in the extraction process. However, extraction of arte-
facts is not straightforward as the dyes are strongly bound to the sub-
strate via a mordant metal (typically Al3+); the most common literature
extraction procedure uses a 37% hydrochloric acid: methanol: water
(2:1:1, v/v/v) mixture [3–8], as the strong acid enables displacement of
the dye molecules from their mordant metal complex [9]. However,
such conditions may also induce a chemical reaction, hence, it is vital
that fundamental understanding of the reactivity of such natural dyes is
developed alongside the analysis of the components within the mixture.
If the conditions of extraction and analysis of these dyed textiles
changes the ratios of the compounds present, or modifies their struc-
ture, then valuable information on that artefact will be lost or poten-
tially misinterpreted.
Only relatively recently has there been significant evidence con-
firming the primary anthraquinone components in Rubia tinctorum roots
as the glycosides ruberythric acid (1) and lucidin primeveroside (3)
[10–14]; the majority of literature has pointed to alizarin (2) as the
major anthraquinone present, and whilst it does occur in the plant, it is
in much lower concentrations than its glycoside [11,12,14]. We have
previously suggested [15] that acidic conditions used in extraction and
Natural colorants are complex mixtures of many different molecules
and plant dyes are often a mixture of aglycons of the parent colorant
moiety and their glycosidic counterparts. The ratio of the abundance of
these molecules can provide significant information about which plant
species was used to dye the fibres or the technique used for the dyeing
process. In the context of historical textiles, this information is of
paramount importance for conservation and restoration purposes, as
well as the generation of information on the ethnographic origins of the
artefacts.
Colorants obtained from the roots of Dyers' madder (Rubia tinctorum
L.), are grouped collectively in the Colour Index as C. I. Natural Red 8,
and have been used as a red dyestuff for centuries. Over 35 anthra-
quinonoid compounds have been reported to be extractable from
madder roots [1], however, many of these compounds are artefacts of
inherent reactivity during analytical extraction methods and are sus-
pected as not being not present in planta; for example, anthraquinones
that contain a 2-methoxymethyl- or a 2-ethoxyethyl group are formed
during extraction with hot methanol or ethanol, respectively [1,2].
When extracting and analysing colorants from textile artefacts as much
information should be preserved as possible in order to gain better
∗
Corresponding author.
E-mail address: r.s.blackburn@leeds.ac.uk (R.S. Blackburn).
https://doi.org/10.1016/j.dyepig.2018.03.023
Received 10 January 2018; Received in revised form 14 February 2018; Accepted 13 March 2018
Available online 14 March 2018
0143-7208/ © 2018 Elsevier Ltd. All rights reserved.

L. Ford et al.
Dyes and Pigments 154 (2018) 290–295
Fig. 1. Possible inter-relationships between anthraquinone compounds found in Rubia tinctorum based on chemical or biochemical interconversion.
analysis of dyes in previous studies may have led to observations that
alizarin was the primary component, it being the product of ruberythric
acid hydrolysis (1 → 2). However, despite high concentrations of lu-
cidin primeveroside (3) in Rubia tinctorum roots [11,12,14], the aglycon
lucidin (4) is rarely detected (and then only in low and trace con-
centrations in planta and in textile artefacts [10–12,16]) even when
acidic conditions are used that would promote hydrolysis (3 → 4); it is
suspected that the reactive nature of lucidin means that it is readily
converted to other compounds. As Fig. 1 shows, lucidin (4) can be
oxidised to nordamnacanthal (5), and studies have suggested this is
catalysed by endogenous oxidase enzymes in the plant [17,18]. It is
possible that nordamnacanthal (5) can form munjistin by the action of
endogenous oxidase; subsequently xanthopurpurin (7) may be formed
through decarboxylation of munjistin (6). It has also been proposed
degradation unique. As described in our previous research [15], use of
the 37% hydrochloric acid: methanol: water (2:1:1, v/v/v) solvent
system causes hydrolysis of anthraquinone glycosides present in
madder root, with the result that only aglycons are detected in back
extraction experiments.
Herein it is suggested that when such acidic methods of extraction
are used to solvate the dye compounds, degradation of the aglycon
lucidin may also occur. The purpose of the research described is to
study the degradation of lucidin under the conditions of extraction in-
volved with the common 37% hydrochloric acid: methanol: water
(2:1:1, v/v/v) solvent method.
2. Materials and methods
[
19] that another enzymatic reaction can occur that converts lucidin
2.1. Materials and solvents
into the quinone methide (Fig. 1). It is thought this intermediate may be
able to be formed by acidic conditions, but the actual intermediate is
too difficult to isolate due to it being a very strong electrophile and
addition at the double bond by any nucleophile is highly likely [12].
However, these enzymes are probably denatured in the dyeing
process and hence this mode of degradation probably is not responsible
for these compounds in historic artefacts. Mouri & Laursen [12] re-
cently confirmed that, unless R. tinctorum roots were “warmed in water”
for prolonged periods (hence, providing enzymatic incubation condi-
tions), significant concentrations of anthraquinone glycosides were
present in the dyebath and on dyed wool fibre. They demonstrated that
steaming madder roots or boiling them in water for 30 s was sufficient
to deactivate the hydrolytic enzymes. An initial extraction process by
boiling the madder root was typically performed in the Japanese Ku-
saki-zome dyeing method and typical European madder dyeing pro-
cesses historically involved heating the dyebath to 75–80 °C, which
would most probably also denature the endogenous enzymes present
All chemicals were purchased from Sigma-Aldrich. All solvents used
were of HPLC grade and also purchased from Sigma-Aldrich. HPLC
grade water was obtained by distillation on site.
2
.2. General procedures and instrumentation
1
Nuclear magnetic resonance (NMR) spectra recorded for H NMR at
00.13 MHz and 500.21 MHz and 13C at 75.45 MHz on a Bruker
3
DPX300 and DRX500 spectrometer. Chemical shifts are given in parts
per million (ppm) downfield of tetramethylsilane (singlet at 0 ppm) for
proton resonances. The proton coupling constants are corrected and
given in Hz and expressed, e.g. as multiplicities, singlet (s), broad
singlet (bs), doublet (d), double doublet (dd), triplet (t) and quartet (q).
High resolution electrospray (ESI+) mass spectrometry was performed
on a Bruker MaXis Impact spectrometer, m/z values are reported in
Daltons to four decimal places. Liquid Chromatography with Mass
Spectrometry (LC-MS) was carried out for analysis synthetic references.
LC analyses were carried out at room temperature on a Phenomenex
Hyperclone C18 column, 5 μm particle size, 250 × 4.6 mm I.D. column
equipped with a pre-column. Chromatography was carried out using
two solvents: (A) water and 0.1% formic acid solution and (B) acet-
onitrile and 0.1% formic acid solution. A linear gradient programme
was applied: of 0–3 min 0–100% increase of solvent B. The flow rate
[
20].
We have previously suggested [15] that xanthopurpurin may also be
formed directly from lucidin (4) through an acid (or base)-catalysed
loss of formaldehyde through a retro-aldol type process (Fig. 1), but
there is no literature to support this proposal. The absence of lucidin in
the analysis of artefacts dyed with madder is rarely acknowledged, or it
is stated that lucidin is degraded into unknown products [9]. Lucidin is
the only commonly reported anthraquinone detected in the roots of
Rubia tinctorum to contain a primary alcohol, which could make its
−
1
during the experiment was 1.0 ml min . Injections were made by a
Basic Marathon autosampler equipped with a 20 μl loop. The method
291

L. Ford et al.
Dyes and Pigments 154 (2018) 290–295
was carried out on an Agilent 1200 LC using a Bruker HCT Ultra Ion
Trap for the MS detection and a Diode Array Detector. The ESI (elec-
trospray ionisation) parameters in the negative ion mode were as fol-
lows: spray voltage 4000 V (applied to the spray tip needle), dry gas
Lucidin (4) was collected as a yellow amorphous solid, 21 mg,
−1
87.5% yield, m.p. 301–305 °C. IR (ATR), ν (cm ): 3400, 1634, 1558,
1365, 1338. λmax (log ε) in MeOH: 410 nm (3.66). 1H NMR (500 MHz,
DMSO): δ 11.33 (s, 1H, OH), 8.22 (dd, J = 7.5, 1.5 Hz, 1H), 8.15 (dd,
J = 7.0, 1.5 Hz, 1H), 7.77 (app td, J = 1.6, 7.2 Hz, 1H), 7.74 (app td,
J = 1.6, 7.2 Hz, 1H), 7.26 (s, 1H), 4.83 (broad s, 1H, OH), 4.55 (s, 2H,
3
−1
10 dm min , dry temperature 365 °C, capillary 60 nA, nebulizer 65
psi, nebulising gas N . UV/visible spectrophotometry was carried out
2
13
using a Jasco V-530 UV/visible/NIR spectrophotometer at 2 nm inter-
vals. Spectral properties and wavelength of maximum absorbance
CH
2
). C NMR (126 MHz, MeOD) δ 159.9, 159.6, 159.4, 159.1, 158.7,
158.4, 119.4, 117.1, 114.8, 112.6, 54.7, 54.5, 54.3, 54.2, 54.0. HRMS:
-
(
λ
max) were evaluated. Infrared spectra were recorded on a Bruker
15 10 5
m/z (ESI-) calculated for C H O [M-H] :269.0528; found [M-
-
Alpha Platinum ATR. Samples were analysed in the solid phase and
absorption maxima (νmax) are given in wave numbers (cm ) to the
H] :269.0464. HPLC retention time and mass data of negative ion can
be found in Table 1.
−
1
nearest whole wavenumber.
2
2
.4. Chemical degradation of lucidin
2
2
.3. Synthesis of references for chemical components of madder root
.4.1. With methanol
Pure lucidin (2 mg) synthesised and purified as described above was
.3.1. Xanthopurpurin
This method was based on that of Murti et al. [21]. Anhydrous
aluminium chloride (4.8 g, 40 mmol) and sodium chloride (1.2 g,
0 mmol) were heated to 150 °C until molten. To this, a mixture of
phthalic anhydride (1.84 g, 8 mmol) and resorcinol (0.80 g, 8 mmol)
was added slowly. The temperature was then slowly increased to 165 °C
and maintained for 4 h. The reaction mixture was then cooled to 0 °C
and 2 M aqueous hydrochloric acid solution was added and stirred for
dissolved in 37% hydrochloric acid: methanol: water (2:1:1, v/v/v)
(0.5 ml) and heated to 100 °C for 15 min. After this time an aliquot of
the reaction mixture was taken for LC-MS and HPLC analysis. The re-
maining reaction mixture was evaporated to dryness and re-dissolved in
deuterated acetone for NMR analysis. Deuterated DMSO was also
evaluated as a solvent for NMR analysis, but deuterated acetone pro-
vided better solubility. Deuterated methanol could not be used as it
would interfere with the results forming the methyl ether adduct.
2
1
5 min. The reaction mixture was then heated to reflux for 30 min, after
which it was cooled to room temperature and extracted with ethyl
acetate (3 × 30 ml). The ethyl acetate extracts were then washed suc-
cessively with saturated sodium bicarbonate solution (30 ml), dried
with magnesium sulphate and evaporated to dryness.
2.4.2. Without methanol
Pure lucidin (2 mg) synthesised and purified as described above was
dissolved in 37% hydrochloric acid: water (1:1, v/v) (0.5 ml) and he-
ated to 100 °C for 15 min. After this time an aliquot was taken for LC-
MS and HPLC analysis. The remaining reaction mixture was evaporated
to dryness and re-dissolved in deuterated acetone for NMR analysis.
Xanthopurpurin (7) was collected as a yellow/orange amorphous
−
1
solid, 28 mg, 1.2% yield, m.p. 261–264 °C. IR (ATR), ν (cm ): 3360,
1
1
(
633, 1598, 1451, 1258. λmax (log ε) in MeOH: 412 nm (4.15). H NMR
500 MHz, DMSO): δ 12.76 (s, 1H, OH), 11.32 (s, 1H, OH), 8.23 (dd,
J = 7.5, 1.7 Hz, 1H), 8.18 (dd, J = 7.5, 1.7 Hz, 1H), 7.95 (app td,
J = 1.7, 7.6 Hz, 1H), 7.92 (app td, J = 1.7, 7.6 Hz, 1H), 7.15 (d,
J = 2.3 Hz, 1H), 6.62 (d, J = 2.3 Hz, 1H). C NMR (101 MHz, MeOD) δ
58.2, 157.8, 157.4, 157.0, 134.0, 133.8, 126.6, 126.2, 118.9, 116.0,
8 4
13.2, 110.4, 108.1, 107.5. HRMS: m/z (ESI-) calculated for C14H O
3. Results and discussion
1
3
The HPLC chromatogram of lucidin after heating in 37% hydro-
chloric acid: water (1:1, v/v) (Fig. 2) showed a decrease in the lucidin
peak (4) and a peak appearance at retention time 11.57 min (7); this
new peak has the same UV/vis data and retention time as that of the
synthesised xanthopurpurin standard (Table 1). The LC-MS also shows
two peaks with the same molecular weights as lucidin (m/z = 269) and
xanthopurpurin (m/z = 239). These results suggest that under aqueous
acidic conditions that mimic those used in textile back extractions [20],
lucidin is partially degraded to xanthopurpurin.
It is hypothesised that in this case the reaction probably proceeds
through a retro-aldol type mechanism (Scheme 1). The ability of this
reaction to occur is unique to aromatic systems containing multiple
hydroxyl groups as hydroxyl groups on an aromatic ring are electron
donating, and usually in equilibrium with a low concentration of the
keto tautomer. The keto tendency of the hydroxyl groups in positions
one and three on the lucidin aromatic ring in acidic conditions drive the
reverse aldol condition. The electron donating ability of the other hy-
droxyl group in the ring also provides stabilisation to the ketone tau-
tomer. The loss of formaldehyde also provides an entropic driving force
for this potentially reversible reaction.
1
1
[
-
-
M-H] :239.0423; found [M-H] :239.0354. HPLC retention time and
mass data of negative ion can be found in Table 1.
2.3.2. Lucidin
In order to gain a better understanding of the effect that 37% hy-
drochloric acid has on lucidin during extraction, a pure sample of lu-
cidin was synthesised to observe any changes in its structure under the
back extraction conditions. This method was based on that of Murti
et al. [21]. Xanthopurpurin (20 mg, 0.08 mmol) was dissolved in 5%
aqueous sodium hydroxide solution (0.5 ml). Aqueous formaldehyde
37% (30 μl, 0.4 mmol, 5 equivalents) was then added and stirred at
room temperature for 3 h and the reaction was monitored by LC-MS.
Once completion was observed the solution was precipitated with 10%
aqueous hydrochloric acid solution (∼1 ml) until a yellow precipitate
was observed. The yellow precipitate was then extracted with ethyl
acetate (3 × 1 ml), dried with magnesium sulphate and then evapo-
rated to dryness. This was then separated on a short flash silica column
with 70% ethyl acetate, 30% hexane.
The HPLC chromatogram of the madder extract prepared using 37%
Table 1
Compounds identified by HPLC-DAD and LC-MS in hydrochloric acid degradation of lucidin.
Solvent
Anthraquinone derivative assigned to
HPLC peak
Retention time UV λmax values for compound
Molecular ion, m/z, from LC-
(min)
identification (nm)
MS [M-H]-
3
7% hydrochloric acid: water (1:1, v/v)
lucidin (4)
xanthopurpurin (7)
lucidin (4)
xanthopurpurin (7)
lucidin methyl ether (8)
9.9
11.6
9.9
11.6
12.3
244.6, 280.9
243.5, 280.7
244.5, 280.2
243.5, 280.7
244.5, 281.3
269.0
239.0
269.4
239.4
283.4
3
7% hydrochloric acid: methanol: water
(2:1:1, v/v/v)
292

L. Ford et al.
Dyes and Pigments 154 (2018) 290–295
Fig. 2. HPLC chromatogram of lucidin after heating in 37% hydrochloric acid: water (1:1,
v/v).
Fig. 3. HPLC chromatogram of lucidin after heating in 37% hydrochloric acid: methanol:
water (2:1:1, v/v/v).
hydrochloric acid: methanol: water (2:1:1, v/v/v) (Fig. 3) showed a
decrease in the lucidin peak (4) and formation of the xanthopurpurin
peak (7). However, in this reaction there was also the formation of a
third peak (Table 1) which has the largest peak height and peak area in
the chromatogram. LC-MS also showed three peaks in the chromato-
gram; lucidin (m/z = 269), xanthopurpurin (m/z = 239) and the third
peak which gives a mass of m/z = 283, which corresponds to the me-
thyl ether of lucidin (8) [13]. It is well documented that ether products
can be formed when using alcohol in the solvent system when ex-
tracting dye compounds and in extraction from textile artefacts
[
1,2,13]. The methyl ether product is expected to be much more stable
than the methyl hydroxyl present in lucidin; the primary alcohol in
lucidin can potentially be displaced by nucleophilic attack of alcoholic
solvents such as ethanol or methanol. Under dry or acidic conditions
this primary alcohol is most likely removed as water and formation of
the quinone methide can take place. The quinone methide is very sus-
ceptible to nucleophilic addition [12] and this process is reversible –
addition of water will reform lucidin whereas attack by methanol will
form the lucidin methyl ether. It is important to note that whilst lucidin
can undergo the retro-aldol process, this is not feasible for the methyl
ether, and reformation of the quinone methide would be the main re-
action pathway available. LC-MS also showed evidence of the formation
of a dimeric species (9) with the mass of m/z = 491.
Fig. 4. Stacked 1H NMR of lucidin breakdown experiments. From top to bottom: H
2
O:
HCl; MeOH: H O: HCl; xanthopurpurin standard; lucidin standard.
2
hydroxyl groups being labile for exchange with deuterium providing
further evidence for the presence of the keto-equilibrium form.
1
The H NMR spectra shown in Fig. 4 shows an increase in the proton
signals highlighted in the coloured box; this proton corresponds to the
aromatic signal between the two hydroxyl groups in xanthopurpurin
[
22]. The appearance of this signal shows that lucidin has degraded to
xanthopurpurin most likely through the retro-aldol type reaction pro-
posed. There is also evidence for the formation of formaldehyde (as
indicated in Scheme 1). Under the reaction conditions, where water (or
methanol) is present, formaldehyde would not be expected to be ob-
It is noted herein that the UV/vis spectra of each peak was very
similar for these compounds. For this reason, mass spectra data and H
1
NMR were used to fully assign the peaks and observe the reaction as it
proceeds. The NMR experiments are not trivial to analyse as lucidin has
limited solubility in most solvents or is only soluble at low concentra-
tions. In addition to this the NMR signal corresponding to xantho-
purpurin can appear diminished due to the proton between the two
served as it would exist primarily as the hydrate (HOCH
equilibrium, and resulting 1H NMR signals are quite sensitive to con-
centration, temperature and pH, but it is known that HOCH OH has a
chemical shift of ca. 4.6 ppm and the oligomer HOCH OCH OH slightly
2
OH). This
2
2
2
Scheme 1. Mechanism of the proposed retro-aldol type
mechanism of lucidin in acidic aqueous conditions.
293

L. Ford et al.
Dyes and Pigments 154 (2018) 290–295
deuterated product and, hence, not visible in the 1H NMR spectrum
[
24]. However, the mass spectrum shows the lucidin methyl ether ion
(
m/z = 283 + 3) for the deuterated methanol adduct, which confirms
formation of the lucidin methyl ether product.
Degradation of lucidin into xanthopurpurin is important for the field
of conservation scientists and conservators. Previously the presence of
xanthopurpurin has been used as an indicator that the sample probably
contained munjistin, which is a carboxylic acid derivative of xantho-
purpurin [12]. Munjistin can be easily decarboxylated to form xan-
thopurpurin [18], this reactivity is again due to the keto tendency of the
hydroxyl groups on the ring. Munjistin is a marker pigment which can
be used to prove the presence of R. cordifolia in dyed textiles [20],
therefore, by employing an acid extraction process information relevant
to the analysis of the dyed materials is potentially lost as munjistin is
decarboxylated to xanthopurpurin and lucidin is degraded to xantho-
purpurin, causing problems in determining whether the original arte-
fact was dyed with R. tinctorum or R. cordifolia. This is further com-
plicated by the fact that xanthopurpurin and alizarin elute very closely
on a C18 column and hence could co-elute as one peak in many systems.
The two compounds also have the same mass, hence, if their UV/vis
spectra are not analysed in detail they could be mistaken for the same
compound and hence alizarin could be wrongly assigned in the sample.
Fig. 5. Stacked 1H NMR spectra showing meta-coupling of the aromatic signals: xan-
thopurpurin standard (blue; top); H O: HCl (green; middle); MeOH: H O: HCl (red;
2 2
bottom). (For interpretation of the references to color in this figure legend, the reader is
referred to the Web version of this article.)
higher around 4.7 ppm [23]. There are definitely appropriate (if small)
signals in this region of the spectrum consistent with this explanation
however unambiguous assignment has not been possible so far. It is
feasible that liberated formaldehyde may re-add to xanthopurpurin at a
different position (e.g. C4 rather than C2) to form an isomeric product,
although the presence of the adjacent carbonyl makes this centre much
less nucleophilic, and so is unlikely. Other reactions, such as O-alky-
lation on phenolic groups, are feasible, but there is no evidence to
suggest this could occur at anything more than a small equilibrium
concentration.
4. Conclusions
It has been demonstrated herein that lucidin is not stable in acidic
conditions and degrades rapidly to xanthopurpurin when heated in
aqueous acid conditions that are typically used for extraction of his-
torical textiles dyed with madder. This is confirmed by HPLC, LC-MS
and 1H NMR, which have also provided support for the proposed me-
chanism of degradation being a retro-aldol process. Different madder
varieties and species and different origins have different chromato-
graphic profiles in planta, hence, the most effective artefact extraction
technique would be the one that preserves the colorants in the dyeings
in the form as applied. As most existing methods cause some form of
acid-catalysed degradation to colorant moieties, it is vital to future
development of analytical techniques to examine historical textiles, that
milder and effective extraction techniques are developed to enable
better-informed identification of the original dyestuff and to provide
more information about the botanic, geographic and ethnographic
origins of the dyes.
Fig. 5 shows the 1H NMR spectra expanded in the aromatic region
which displays the appearance of meta coupling (2–3 Hz), which in-
dicates that there is a hydrogen in the meta position of the ring, further
confirming the degradation to xanthopurpurin (which displays this
1
meta coupling). Fig. A1 shows the H NMR spectra of lucidin methyl
ether and displays integration between the singlet (H4, 7.30 ppm) and
the methyl group corresponding to the methyl ether (OCH , 3.27 ppm).
3
Upon closer inspection, the meta-coupling constant between the two
protons on the xanthopurpurin can be observed in the degradation
studies, which further confirms the degradation of lucidin into xan-
thopurpurin. A singlet corresponding to lucidin present in the break-
down experiments is also observed, indicating that the breakdown of
lucidin is not complete and some remains in the reaction medium. This
result is confirmed in the HPLC chromatograms (Figs. 2 and 3) where
lucidin is still present in both of these experiments, but in lower con-
centrations.
Acknowledgments
Funding: The authors would like to thank The Clothworkers'
Foundation for the provision of financial support to LF to allow her to
conduct her PhD studies.
This reaction was also examined in deuterium oxide and deuterated
methanol, however, this resulted in the deuterated reaction product of
the lucidin methyl ether (m/z = 286), seen in Fig. 6, which caused
problems in analysis by NMR due to the methanol adduct being the
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://dx.
doi.org/10.1016/j.dyepig.2018.03.023.
Fig. 6. Mass spectrum of deuterated methyl ether product of lucidin.
294

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Dyes and Pigments 154 (2018) 290–295
References
chromatography-photodiode array detection. Ultrason Sonochem 2009;16:75–82.
12] Mouri C, Laursen R. Identification of anthraquinone markers for distinguishing
Rubia species in madder-dyed textiles by HPLC. Microchim Acta 2012;179:105–13.
13] Henderson RL, Rayner CM, Blackburn RS. Isolation and extraction of lucidin pri-
meveroside from Rubia tinctorum L. and crystal structure elucidation.
Phytochemistry 2013;95:105–8.
[
[
[
1] Derksen GCH, Niederlander HAG, van Beek T. Analysis of anthraquinones in Rubia
tinctorum L. by liquid chromatography coupled with diode-array UV and mass
spectrometric detection. J Chromatogr A 2002;978:119–27.
[
2] Wijnsma R, Verpoorte R. Anthraquinones in the rubiaceae. Hill RA, Krebs HC,
editors. Progress in the chemistry of organic natural products, vol. 49. Wein:
Springer-Verlag; 1986. p. 78–149.
[
14] Ford L, Rayner CM, Blackburn RS. Isolation and extraction of ruberythric acid from
Rubia tinctorum L. and crystal structure elucidation. Phytochemistry
2
015;117:168–73.
[3] Wouters J. High performance liquid chromatography of anthraquinones: analysis of
plant and insect extracts and dyed textiles. Stud Conserv 1985;30:119–28.
[4] Wouters J, Maes L, Germer R. The identification of haematite as a red colorant on an
Egyptian textile from the second millennium BC. Stud Conserv 1990;35:89–92.
[5] Karapanagiotis I, Daniilia S, Tsakalof A, Chryssoulakis Y. Identification of red nat-
ural dyes in post-byzantine icons by HPLC. J Liquid Chromatogr Rel Technol
[
15] Ford L, Henderson RL, Rayner CM, Blackburn RS. Mild extraction methods using
aqueous glucose solution for the analysis of natural dyes in textile artefacts dyed
with Dyer's madder (Rubia tinctorum L.). J Chromatogr A 2017;1487:36–46.
16] Yasui Y, Takeda N. Identification of a mutagenic substance, in Rubia tinctorum L.
[
[
[
(
madder) root, as lucidin. Mutat Res 1983;121:185–90.
17] Aburjai T, Natsheh FM. Plants used in cosmetics. Phytotherapy Res
003;17:987–1000.
2
005;28:739–49.
2
[
[
[
[
6] Balakina G, Vasiliev V, Karpova E, Mamatyuk V. HPLC and molecular spectroscopic
18] Derksen GCH, Naayer M, van Beek TA, Capelle A, Haaksman IK, van Doren HA, de
Groot AE. Chemical and enzymatic hydrolysis of anthraquinone glycosides from
madder roots. Phytochem Anal 2003;14:137–44.
investigations of the red dye obtained from an ancient Pazyryk textile. Dyes Pigm
2
006;71:54–60.
7] Lech K, Jarosz M. Novel methodology for the extraction and identification of natural
dyestuffs in historical textiles by HPLC-UV-Vis-ESI MS. Case study: chasubles from
the Wawel Cathedral collection. Anal Bioanal Chem 2011;399:3241–51.
8] Liu J, Guo D, Zhou Y, Wu Z, Li W, Zhao F, Zheng X. Identification of ancient textiles
from Yingpan, Xinjiang, by multiple analytical techniques. J Archaeol Sci
[
[
[
[
[
[
19] Zhang X, Laursen R. Application of LC-MS to the analysis of dyes in objects of
historical interest. Int J Mass Spectrom 2009;284:108–14.
20] Blackburn RS. Natural dyes in madder (Rubia spp.) and their extraction and analysis
in historical textiles. Color Technol 2017;133:449–62.
21] Murti VVS, Seshadri TR, Sivakumaran S. A study of madder, the roots of Rubia
tinctorum linn. Indian J Chem 1970;8:779–82.
22] Akrawi OA, Hussain M, Langer P. Site-selective Suzuki-Miyaura reactions of the bis
2
011;38:1763–70.
9] Sanyova J, Reisse J. Development of a mild method for the extraction of anthra-
quinones from their aluminum complexes in madder lakes prior to HPLC analysis. J
Cult Heritage 2006;7:229–35.
(triflate) of 1,3-dihydroxyanthraquinone. Tetrahedron Lett 2011;52:1093–5.
23] Rivlin M, Eliav U, Navon G. NMR studies of proton exchange kinetics in aqueous
formaldehyde solutions. J Magn Reson 2014;242:107–12.
24] Abraham RJ, Fisher J, Loftus P. Introduction to NMR spectroscopy. Oxford: John
Wiley & Sons; 1988.
[
10] Boldizsár I, Szűcs Z, Füzfai Zs Molnár-Perl I. Identification and quantification of the
constituents of madder root by gas chromatography and high-performance liquid
chromatography. J Chromatogr A 2006;1133:259–74.
[11] Cuoco G, Mathe C, Archier P, Chemat F, Vieillescazes C. A multivariate study of
ultrasound-assisted extraction of madder dyes and characterisation by liquid
295