Identification of an indigo precursor from leaves of Isatis tinctoria (Woad)

Article abstract of DOI:10.1016/S0031-9422(01)00335-1

Indole is presumably a product of indole-3-glycerol phosphate catabolism in Isatis tinctoria. It is oxidized into indoxyl and stored in young leaves as indigo precursor. Further oxidation and dimerization of indoxyl produces indigoid pigments. In this work, we describe an HPLC method dedicated to the identification and quantification of indigoid pigments (indigo, indirubin, isoindigo and isoindirubin) and indigo precursors produced in I tinctoria (Woad). This work, carried out with two cultivars of I tinctoria, has confirmed that the quantity of indigo precursors is dependent on the species and the harvest period. In addition we have shown for the first time that young leaves of I. tinctoria, harvested in June contained a new indigo precursor in addition to isatan B (indoxyl-5-ketogluconate) and indican (indoxyl-β-D-glucoside). We suggest the name isatan C for this new indigo precursor in I. tinctoria. Its chemical characteristics point to an dioxindole ester with PM of 395. We have shown that isatan C reacts with isatan B increasing the red pigment production.

Full text of DOI:10.1016/S0031-9422(01)00335-1

Phytochemistry 58 (2001) 897–904
www.elsevier.com/locate/phytochem
Identification of an indigo precursor from leaves of
Isatis tinctoria (Woad)
a,
a
b
a
Thierry Maugard *, Estelle Enaud , Patrick Choisy , Marie Dominique Legoy
aLaboratoire de G e´ nie Prot e´ ique et Cellulaire, EA3169, Universit e´ de La Rochelle, Avenue Michel Cr e´ peau, 17042 La Rochelle cedex 1, France
bCRITT Horticole 18, rue de l‘Arsenal, 17300 Rochefort, France
Received 23 April 2001; received in revised form 24 July 2001
Abstract
Indole is presumably a product of indole-3-glycerol phosphate catabolism in Isatis tinctoria. It is oxidized into indoxyl and stored
in young leaves as indigo precursor. Further oxidation and dimerization of indoxyl produces indigoid pigments. In this work, we
describe an HPLCmethod dedicated to the identification and quantification of indigoid pigments (indigo, indirubin, isoindigo and
isoindirubin) and indigo precursors produced in I. tinctoria (Woad). This work, carried out with two cultivars of I. tinctoria, has
confirmed that the quantity of indigo precursors is dependent on the species and the harvest period. In addition we have shown for
the first time that young leaves of I. tinctoria, harvested in June contained a new indigo precursor in addition to isatan B (indoxyl-5-
ketogluconate) and indican (indoxyl-b-d-glucoside). We suggest the name ‘‘isatan C’’ for this new indigo precursor in I. tinctoria.
Its chemical characteristics point to an dioxindole ester with PM of 395. We have shown that isatan Creacts with isatan B
increasing the red pigment production. # 2001 Elsevier Science Ltd. All rights reserved.
Keywords: Woad; Isatis tinctoria; Brassicaceae; Indigo; Indirubin; Isoindirubin; Isatan; Indican
1
. Introduction
exposed to the air. The liberated indoxyl is sponta-
neously oxidized by oxygen yielding indigo. Isatin is
generated from indoxyl in an oxygen-rich environment
as a side reaction. The condensation of indoxyl with
isatin produces indirubin which is a by-product of bio-
synthetic indigo production. It is well known that plants
contain, in addition to trans-indigo (indigotin, blue) and
trans-indirubin (isoindigotin, red), certain impurities
such as cis-indigo (blue), cis-indirubin (isoindirubin,
red), indigo brown (isoindigo), indigo gluten, indigo
yellow and traces of flavonoids that makes natural
indigo a more pleasing tinge than synthetic indigo (Per-
kin and Bloxam, 1907; Perkin, 1907).
In the present work, a qualitative and quantitative
HPLCmethod is described for the identification and the
separation of indigoid pigments and precursors present
in I. tinctoria leaves. We show for the first time, that I.
tinctoria produces three indigo precursors, isatan B as a
major precursor, indican as a minor precursor and an
other precursor that we named ‘‘isatan C’’. Eventually,
this work describes the possible effect of isatan Con the
red pigments production.
The blue dye indigo (indigotin) is one of the oldest
natural dyestuffs known to human beings (Ensley et al.,
983). Indigo has, for centuries, been obtained from a
1
variety of plant sources such as Indigofera (tropical
species, Africa, Asia, East Indies, South America),
Polygonum tinctorium (Far East, China, Korea) and
Isatis tinctoria (Woad) (Europe). Indigo is an artefact of
secondary metabolism. Indigofera tinctoria and P. tinc-
toriuni contain indican (indoxyl-b-d-glucoside) which
serves as a starting material for indigo production,
whereas I. tinctoria (Woad) contains isatan B (indoxyl-
b-ketogluconate), as a major indigo precursor and indi-
can as minor a indigo precursor (Epstein et al., 1967;
Maier et al., 1990). In old leaves, precursors are broken
down by hydrolases when the leaves are damaged and
*
Corresponding author. Tel.: +33-5-46-45-82-77; fax: +33-5-46-
5-82-47.
E-mail address: tmaugard@univ-lr.fr (T. Maugard).
4
0
031-9422/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(01)00335-1

898
T. Maugard et al. / Phytochemistry 58 (2001) 897–904
2
. Results and discussion
Compounds 6, 7 and 8: HPLC t 19.3, 21.6 and 22.2
R
min respectively. UV spectra of these three compounds
correspond to flavonoid spectra.
2
.1. Extraction and identification of compounds from
Isatis tinctoria
Compound 4: HPLC t 14.9 min. The mass spectrum
R
+
+
3
indicated 396, assigned as MH , 398, assigned as MH
+
An extract of I. tinctoria (IT BM) harvested on 27th
June 2000 is displayed in Fig. 1.
Extraction and HPLCanalyses were performed as
described in Experimental. The major compounds pro-
duced in I. tinctoria were identified by their UV and
mass spectra:
and 378, assigned as MH –H O. High resolution–mass
2
spectrometry indicated 777 assigned as [2(M–
+
H O)+Na] .
2
A
possible molecular formula is
218,
C H O N (M, 395). The UV spectrum (UV l
2
0
13
8
1
max
280, Fig. 3) was similar to isatan B, indican and indoxyl
acetate spectra. The amount of material isolated has not
permitted the acquisition of a 1H NMR spectrum.
Alkaline media, in presence of oxygen, induce the
synthesis of indigoid pigments out of isatan B and
indoxylacetate, whereas indican remains unhydrolysed.
This instability in alkaline media is consistent with the
ester bound of isatan B, indoxyl acetate and compound
4. On the basis of these results, we conclude that com-
pound 4 is a new indigo precursor present in young
leaves of I. tinctoria. Indeed, compound 4 is probably
composed of dioxindole moiety (UV lmax 218, 280)
which binds an organic acid through ester bound. This
new indigo precursor was named isatan C.
Compound 1: Indican. HPLC tR 7.6 min, UV lmax
+
2
18, 280. MS: m/z 296 (MH ). HPLC t , mass spec-
R
trum and UV spectrum were identical to those of com-
mercially available indican (Sigma).
Compound 2: Isatan B. HPLC tR 9.2 min, UV lmax
18, 280. MS: m/z 310 (MH ). No standard was avail-
+
2
able for isatan B. Consequently, it was necessary to
produce pure isatan B and characterize it using NMR.
1
H NMR (D O) ꢀ (ppm) 3.42 (s, 2H, H-1), 3.71 (d, 1H,
2
H-4 or H-7), 3.73 (d, 1H, H-4 or H-7), 4.85 (d, 1H, H-4
or H-7), 6.98 (d, 1H, H-4 or H-7), 7.00 (d, 1H, H), 7.12
(
1
t, 1H, H-4 or H-7), 7.31 (d, 1H, H-5 or H-6), 7.58 (d,
H, H-5 or H-6).
Acetone extract of young leaves of I. tinctoria (har-
vested June 27 2000), plated on TLCwere developed by
fuming ammonia vapour (pH>13). One band devel-
Compound 3: Isatin. HPLC tR 11.9 min, UV lmax
+
2
10, 244, 305. MS: m/z 148 (MH ). HPLC t , mass
R
spectrum and UV spectrum were identical to those of
commercially available isatin (Sigma).
oped a red/brown colour at R 0.7, one band developed
f
a blue color at R 0.6 and one band developed a brown
f
Compound 5: Indoxyl. HPLC tR 18.3 min, UV lmax
colour at R 0.2. These three compounds are three
f
+
2
40 nm. MS: m/z 134 (MH ). HPLC t , mass spectrum
indigo precursors of woad, indican (R 0.2), isatan B (Rf
f
R
and UV spectrum were identical to those of product
generated by the hydrolysis of commercial 3-acetoxindole
0.6) and isatan C( R 0.7).
f
Compounds 9, 10, 11, 12 and 13: the indigoid pig-
ments produced in I. tinctoria were separated into five
(
indoxyl acetate) by the lipase of Rhizomucor miehei.
Fig. 1. HPLCanalysis of an extract of Isatis tinctoria leaves harvested on 27 June 2000. Chromatograms were recorded at 280 nm. HPLC analysis
was performed as described in Experimental.

T. Maugard et al. / Phytochemistry 58 (2001) 897–904
899
Fig. 2. UV/visible spectrocopy of pigments 9, 10, 11, 12 and 13 formed in Isatis tinctoria. The pigments were seperated by HPLC, and UV/visible
spectra were recorded. The spectrum of pigment 9 is typical of isoindirubin, pigment 10 corresponds to isoindigo, 11 and 12 are two isomers of
indigo and 13 corresponds to indirubin.
compounds, of which two were blue (11 and 12, HPLC
t : 34–36 min), two were red/purple (9, HPLC t : 23
min, 13, HPLC t : 37.5 mm) and one was brown (10,
young leaves contain higher concentrations of indigo
precursors than older ones. This study confirms that the
quantity of indigo precursors is dependent of the culti-
vars and of the period of harvest. It is important to
notice that isatan B was observed in I. tinctoria leaves,
from the harvest of April, whereas indican and com-
pound 4 (isatan C) were observed and consequently
quantified only from the harvest of the end of June.
R
R
R
HPLC t : 24.5 min). Pigments 11 and 13 were much
R
more predominant. The UV/visible spectra of compounds
1
1 and 13 (UV 603 and 552 nm lmax respectively) match
the literature for trans-indigo (indigotin) and trans-
indirubin (isoindigotin), respectively (Fearon and Bog-
gust, 1950; Friedmann et al., 1950). The UV/visible
spectra of compounds 9, 10 and 12 (Fig 2, UV lmax 552
nm, 500 nm and 603 nm respectively) correspond to
cis-indirubin (isoindirubin), isoindigo (indigo brown)
and cis-indigo, respectively. Mass spectrum yielded an
apparent MH+ ion at m/z 263 for compounds 9, 10, 11,
2.3. Transformation of indigo precursors in alkaline
media
The conversion of indigo precursors into indigo was
followed by HPLC. Products were detected using a UV
detector at 280, 550 (red pigments) and 603 nm (blue
pigments) (Fig. 5, Table 1, Scheme 1). HPLCanalysis
was performed as described in Experimental.
12 and 13, indicating a molecular mass of 262. Frag-
mentation of the m/z 263 ion in each case yielded m/z
2
35 (ÀCO) and 219 (ÀCOÀNH ). The same ions were
2
seen when standard indigo (Sigma) was fragmented in
the same way. We noted that cis-indirubin and cis-
indigo had exactly the same UV/visible spectrum and
Pure isatan B treated with an alkaline medium
(NaOH solution 0.05 M, in water/CH CN 90/10, v/v.
3
pH 13), produces indoxyl since the ester bond sponta-
neously brakes down. Indoxyl is then oxidized by air
yielding indigo (11) (HPLC t : 32 min, blue).
+
mass spectrum (MS: m/z 263, MH ) as trans-indirubin
and trans-indigo, respectively. Isoindirubin (cis) is an
isomer of indirubin (trans), as cis-indigo is an isomer of
trans-indigo (indigotin) (Fig. 2).
R
2.2. Quantification of indigo precursors from Isatis
tinctoria
The amount of indigo precursors present in old leaves
and young leaves of two cultivars of I. tinctoria (IT BM
and IT MC) was estimated at A280 (Fig. 4). The con-
centrations were evaluated with leaves harvested on 9,
1
5 and 30 May 2000 and 27 June 2000. The two culti-
vars of I. tinctoria contain similar but not identical
concentrations of indigo precursors. We may notice that
indigo precursors’ quantity present in the young leaves,
increased during time. Moreover, the amount of pre-
cursors present in IT MCyoung leaves is higher than
the amount present in IT BM young leaves. Finally,
Fig. 3. Spectral characterization of compound 4. UV spectrum online
from HPLC: (a) indican, (b) indoxyl acetate, (c) isatan B, (d) com-
pound 4.

900
T. Maugard et al. / Phytochemistry 58 (2001) 897–904
Fig. 4. Concentration of indigo precursors present in old leaves and young leaves of two cultivars of Isatis tinctoria. We consider young leaves as
those located on the apex and old leaves those located underneath. Young leaves may be easily differentiated from old ones since they are light green
and smaller than old leaves which are dark green. The concentrations were measured on leaves harvested on 9, 15 and 30 May 2000 and, finally, on
27 June 2000. Extraction and quantification were performed as described in Experimental.
Fig. 5. HPLCanalysis of the transformation of indigo precursors. Ch romatograms were recorded at 280 nm. HPLCanalysis was performed as
described in Experimental.
Table 1
Alkaline formation of indigoid pigments
Indigo (11)
603, area units
Indirubin (13)
Isoindirubin (9)
A
A552, area units
A552,area units
Isatan B
Isatan C300
8000
200
5
5000
–
Isatan B and Isatan C5000
Isatan B and Isatin
Isatan Cand Isatin
200
100
6800
870
3000
1000
–
50
3
Indigo precursors were incubated with NaOH solution 0.05 M, in water/CH CN 90/10, v/v, pH 13. After 30 min, mixtures were analysed by HPLC.
HPLCanalysis was performed as described in Experimental. Dye production was analysed by recording A 603 for blue pigments and A552 for red
pigments.

T. Maugard et al. / Phytochemistry 58 (2001) 897–904
901
Scheme 1. Chemical pigments formation.
Isatan B added to isatin solution in 0.05 M NaOH,
produces indoxyl which is oxidized yielding indigo. In
addition, indoxyl reacts with isatin yielding indirubin
These results confirm that isatan Chydrolysis pro-
duces dioxindole that may react with isatin yielding
isoindirubin (9) and indirubin (13). Isatan Cis an indigo
precursor and more specifically a precursor of red pig-
ments present in I. tinctoria, and its chemical nature was
established as dioxindole ester.
(13) (HPLC t : 34 min, red). We noticed that indigo
R
appeared to be the major pigment produced. When
higher concentrations of isatin were used, pigment pro-
duction decreased. Under these conditions, isatin
appeared to be an inhibitor of the pigments production.
Isatan Cin alkaline solution (0.05 M NaOH) pro-
duces dioxindole since the ester bond brakes down.
Dioxindole is then oxidized by air yielding three pig-
2.4. Discussion
The metabolism of indigo precursors is still under
study. It is likely that indole is a product of indole-3-
glycerol phosphate (Xia and Zenk, 1992) but not of
tryptophan catabolism in vegetables. The indole ring
could be oxidised by cytochrome P450 enzyme produ-
cing indoxyl and dioxindole. P450s are monooxygenases
that catalyze the oxidation of a remarkably broad range
of endogenous and exogenous chemicals. In plants they
play important roles in biosynthetic pathways, including
those of sterols, isoprenoids, alkaloids, oxygenated fatty
acids, and phenylpropanoids (Bolwell et al., 1994; Durst
and O’Keefe, 1995; Schuler, 1996). Indeed there is not
yet evidence for the existence in plants of naphtalene
dioxygenase or xylene mono-oxygenase (Ensley et al.,
1983; Mermod et al., 1986), enzymes which are known
to oxidize indole to indoxyl in some bacteria. In
young leaves plants protect indoxyl, a highly unstable
intermediate, from spontaneous oxidation to indigo by
glycosylation to yield indican or by addition of 5-keto-
gluconate to yield isatan B. Further oxydation transforms
ments, indigo (11) and indirubin (13) (HPLC t : 34 min)
R
as minor pigments, and another major red pigment,
isoindirubin (9) (HPLC t : 24.5 min). The same result is
R
obtained with a solution of isatin reduced by NaBH in
4
alkaline medium. Indeed, the product generated by
NaBH reduction of isatin is dioxindole (HPLC t : 7.5
4
R
min) as confirmed by HPLC t and UV spectra. Diox-
R
indole may react with isatin in alkaline medium yielding
isoindirubin and indirubin.
The reaction of isatan B and isatan Cin alkaline
medium produces indoxyl and dioxindole, which may
be oxidized by air yielding indigo (11), indirubin (13)
and isoindirubin (9).
A solution of oxindole and isatin in an alkaline med-
ium do not produce any pigment. The condensation of
oxindole with isatin may only be achieved in acidic media.
In these conditions, the solution produces isoindigo, a
brown pigment (10) (HPLC t : 25.6 min, l
500 nm).
max
R

902
T. Maugard et al. / Phytochemistry 58 (2001) 897–904
indoxyl to dioxindole. As indoxyl, plants may protect
dioxindole by addition of an organic acid to yield isatan
C. In the case of indican, this pathway has been located
in vacuoles in which indican is synthesized from UDP-
glucose and indoxyl (Minami et al., 2000; Marcinek et
al., 2000). These indigo precursors are broken down by
hydrolases when the leaves are old or damaged. Indoxyl
liberated from isatan B and indican in old leaves is oxi-
dized spontaneously by air yielding indigo. Isatin is
generated from indoxyl and/or dioxindole in an oxygen-
rich environment as a side reaction. Dioxindole is liber-
ated from isatan C. The condensation of indoxyl with
isatin produces indirubin, whereas the condensation of
dioxindole with isatine yields isoindirubin and indir-
ubin, which are by-products of the biosynthesis of
indigo (Scheme 2).
3. Conclusion
In this work, an HPLCmethod is described to iden-
tify and quantify the pigments (indigo, indirubin and
isoindirubin) and the indigo precursors produced in I.
tinctoria. Our work with two cultivars of I. tinctoria has
Scheme 2. Pigment formation in Isatis tinctoria.

T. Maugard et al. / Phytochemistry 58 (2001) 897–904
903
confirmed that the quantity of indigo precursors is
dependent of the cultivars and harvest period. We have
shown for the first time that young leaves of I. tinctoria,
harvested from June, produced a new indigo precursor
in addition to isatan B and indican. The name ‘‘isatan
C’’ is proposed to describe this new indigo precursor in
woad. Its chemical nature was tentatively established as
dioxindole ester with PM of 395 and possible molecular
formula C H O N. However its structure is not com-
pletely specified due to low amounts of the precursor
and to higher instability. Finally, we have shown that
isatan Cincreases the pigments production and espe-
cially isoindirubin. These results confirm that I. tinctoria
harvested in June produced isatan Cas indigo precursor
which favours the production of red pigments, indirubin
and isoindirubin.
Isoindigo was prepared by reaction of isatin with
1
oxindole in acid media and identified by H NMR, UV
spectrum and mass analysis (coupled to HPLC) (Hoes-
sel et al., 1999; Russel and Kaup, 1969). Oxindole (1
ꢀ
mmol) and isatin (1 mmol) were stirred at 95 Cin a
mixture of glacial acetic acid (3 ml) and concentrated
hydrochloric acid (50 ml). After 3 h, the precipitate
(dark brown crystals) was filtered off and washed with
methanol and diethyl ether. Yield: 90%, UV l
550, MS: m/z 263 (MH ).
2
0
13
8
max
+
4.3. Plant material
Two cultivars of I. tinctoria (IT BM and IT MC) were
grown in a field of CRITT Horticole (St Savin, France).
The date of seeding was 23 March 2000. In this paper,
we consider young leaves as those located on the apex
and old leaves those located underneath. Young leaves
may be easily differentiated from old ones since they are
light green and smaller than old leaves which are dark
green. Leaves were harvested from June to September.
The leaves were weekly for indigo quantification. Four
overall harvests were carried out monthly.
4
. Experimental
4
.1. Biological and chemical material
Indigo, indican, isatin, oxindole and 3-acetoxy-indole
indoxyl acetate), were obtained from Sigma Chemical
(
Co. (USA). Isatan B and isatan C were obtained from
an extract of woad. The extraction procedure is descri-
bed below.
4.4. Extraction
Reactions were conducted in screw-cap glass vials. In
standard procedure, fresh leaves were ground in a
mortar and suspended in acetone/acetic acid 1% v/v
4
.2. Chemical pigment synthesis
ꢀ
(10 ml/g of fresh leaves) and stirred at 24 Cfor 4 h. After
Indirubin was prepared by reaction of isatin with 3-
filtration, acetone was evaporated under reduced pres-
sure. The residue was dissolved in a small volume of meth-
anol and analyzed by TLCand HPLCor HPLC–MS.
Pigment concentrations and yields of indigo pre-
cursors produced from I. tinctoria were quantified by
HPLCanalysis with reference to a standard curve of
products dissolved in methanol.
acetoxy-indole in alkaline media. Under argon, isatin (1
mmol) and sodium carbonate (2.4 mmol) were added to
a solution of indoxyl acetate (1 mmol) in methanol (5
ml) (Hoessel et al., 1999; Russel and Kaup, 1969). The
mixture was stirred for 1 h at room temperature and
filtered off after 24 h hold. The residue was washed with
methanol and cold water until neutral reaction of the
filtrate. The product (dark violet crystals) was dried
over potassium hydroxide in the dessicator. Indirubin
4.5. TLC analysis
1
was identified by H NMR, UV spectrum and mass
TLCwas used for qualitative analysis on silica gel 60
F plates (Merck). Elution was carried out with the mix-
ture: ethyl acetate/acetone (1/1, v/v). Conversion of the
indoxyl glycosides (indigo precursors) into indigo and
indirubin on TLCplates were achieved by fuming with
analysis (coupled to HPLC). Indirubin: yield 85%, UV
lmax 552 nm, M: m/z 263 (MH+).
Isoindirubin was prepared by reaction of isatin with
NaBH in alkaline media. Isatin (1 mmol) and NaBH
4
0.5 mmol) were stirred at 25 Cin methanol (3 ml).
4
ꢀ
(
NH vapor from a concentrated solution (pH >13).
3
After 1 h, isatine (1 mmol) and 1 ml of NaOH 0.1 M
were added. The mixture was stirred at room tempera-
ture and was filtered after 12 h hold, The residue was
washed with methanol and cold water. Products (two
dark violet crystals) were dried over potassium hydrox-
ide in a dessicator. Pigments were identified according
to the UV spectrum and mass analysis (coupled to
4.6. Separation and identification of Indigo precursors
and pigments from Isatis tinctoria by HPLC
Separation and identification of indigo precursors and
pigments was conducted using an HPLCsystem from
Hewlett Packard (processor, pump, UV detector and
injector model 1100). Analyses were carried out with an
Ultrasep C18 (250Â4 mm, 6 m) reverse phase column
HPLC). Indirubin: yields 85%, UV l
552 nm, MS:
552
max
+
m/z 263 (MH ); Isoindirubin: yield 10%, UV l
nm, MS: m/z 263 (MH ).
max
+
ꢀ
from ICS (France), at 50 Cwith a flow rate of 0.85 ml/

904
T. Maugard et al. / Phytochemistry 58 (2001) 897–904
min using a gradient made of solvent A (CH OH/TFA
3
analyses were recorded using an HPLCcoupled to an
API III+ (Sciex, Thornhillm, L3T 1P2, ON. Canada).
We have used the same column and gradient system as
described above.
0
0
.2%) and solvent B (H O/TFA 0.2%). Programme:
2
–10 min: 85% B/ 15% A to 75% B/ 25% A; 10–30
min: 75 to 30% B; 30–40 min: hold at 30% B; 40–45
min: 30 to 85% B. Products were detected and quanti-
fied using a diode array detector at 280 nm. During the
chromatographic analysis, UV/visible spectra of each
individual peak were acquired online using a diode
array detector. Further characterisation of purified
peaks was achieved by HPLC–MS analyses, with com-
parisons to the A280 traces (online) obtained with rapid-
scanning UV analysis.
Acknowledgements
This project was financially supported by the CRITT
Horticole, France.
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4
.7. Hydrolysis of indigo precursors
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¨
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ꢀ
50 Cwith a flow rate of 2.3 ml/min and with a gradient
composed of solvent A (CH CN/TFA 0.2%) and sol-
3
Mermod, N., Harayama, S., Timmis, K.N., 1986. New route to bac-
terial production of indigo. BioTechnology 4, 321–326.
Minami, Y., Nishimura, O., Hara-Nishimura, I., Nishimura, M.,
Matsubara, H., 2000. Tissue and intracellular localization of indican
and the purification and characterization of indican synthase from
indigo plants. Plant Cell Physiol. 41, 218–225.
vent B (H O/TFA 0.2%). Programme: 0–10 min: 85%
2
B/ 15% A to 75% B/ 25% A; 10–30 min: 75 to 30% B;
0–40 min: hold at 30% B; 40–45 min: 30 to 85% B.
3
Products were detected using a UV detector at 280, 550
and 603 nm.
Perkin, A.G., 1907. Constituents of natural indigo. Part II. J. Chem.
Soc., 435–440.
4
.9. Structural analysis
Perkin, A.G., Bloxam, P., 1907. Some constituents of natural indigo.
Part I. J. Chem. Soc., 279–288.
1H NMR were recorded on a Jeol-JNM LA400 (400
Russel, G.A., Kaupp, G., 1969. Oxidation of carbanions. IV. Oxida-
tion of indoxyl to indigo in basic solution. J. Am. Chem. Soc. 91,
MHz) spectrometer (Laboratoire Commun d’Analyse,
Universite de La Rochelle), with an internal reference of
tetramethylsilane. HPLC–MS analysis was carried out
in the Centre Regional de Mesure Physiques de L’Ouest
CRMPO), Universite de Rennes, France. HPLC–MS
3
851–3859.
´
Schuler, M.A., 1996. Plant cytochrome P450 monooxygenases. Crit.
Rev. Plant Sci. 15, 235–284.
´
Xia, Z.K., Zenk, M.H., 1992. Biosynthesis of indigo precursors in
higher plants. Phytochemistry 31, 2695–2697.
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