Cytohistological and phytochemical study of madder root extracts obtained by ultrasonic and classical extractions.

Abstract

Madder (Rubia tinctorum) has been used since ancient times as a source of pigments for dyeing and painting. Madder dyes are localised in roots and the native chemical population is composed of glycosiled and aglycone compounds. The aim of this study is to elaborate an efficient extraction process without any chemical denaturation of dyes.
To compare an optimised ultrasonic process, using for madder dye extraction, with two conventional procedures and to determine the efficiency of ultrasound on these vegetable matrix.
Madder roots were extract in a methanol-water mixture in 37 : 63 (v/v) for ultrasound and 80 : 20 (v/v) for reflux and agitation. HPLC-PAD analyses showed the anthraquinone proportion for each extraction process and their denaturing effects. Finally, cytohistological observations were made to show the consequence of each process on the cell organisation in madder roots.
The results showed that the amount of extracted dyes was higher with UAE than with agitation and reflux. HPLC-PAD analysis revealed that the anthraquinone composition differed according to the extraction procedure. The UAE extracts presented an important richness in terms of anthraquinonic compounds that suggests a preserving effect. Cytohistological observations showed that the main alterations concerned the cell walls of phloem. After UAE the walls exhibited numerous pitted areas reflecting an ultrasound-induced cavitation that enhances the extraction effectiveness of this method.
The study has shown the improvement of madder roots extraction both quantitatively and qualitatively using the efficiency of ultrasound-assisted extraction in comparison with magnetic agitation and reflux techniques.

Full text

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484
Copyright © 2009 John Wiley & Sons, Ltd.
Phytochem. Anal. 2009, 20, 484–490
Research Article
Received: 15 September 2008;Revised: 5 June 2009; Accepted: 18 June 2009Published online in Wiley Interscience: 22 September 2009
(www.interscience.wiley.com) DOI 10.1002/pca.1150
Cytohistological and phytochemical study of
madder root extracts obtained by ultrasonic
and classical extractions
Guillaume Cuoco,a Carole Mathe,a* Paul Archier,a Mohamed El Maâtaouib
and Cathy Vieillescazesa
ABSTRACT:
Introduction – Madder (Rubia tinctorum) has been used since ancient times as a source of pigments for dyeing and painting.
Madder dyes are localised in roots and the native chemical population is composed of glycosiled and aglycone compounds.
The aim of this study is to elaborate an effi cient extraction process without any chemical denaturation of dyes.
Objective – To compare an optimised ultrasonic process, using for madder dye extraction, with two conventional procedures
and to determine the effi ciency of ultrasound on these vegetable matrix.
Methodology – Madder roots were extract in a methanol–water mixture in 37 : 63 (v/v) for ultrasound and 80 : 20 (v/v) for refl ux
and agitation. HPLC-PAD analyses showed the anthraquinone proportion for each extraction process and their denaturing
eff ects. Finally, cytohistological observations were made to show the consequence of each process on the cell organisation in
madder roots.
Results – The results showed that the amount of extracted dyes was higher with UAE than with agitation and refl ux. HPLC-PAD
analysis revealed that the anthraquinone composition diff ered according to the extraction procedure. The UAE extracts pre-
sented an important richness in terms of anthraquinonic compounds that suggests a preserving eff ect. Cytohistological
observations showed that the main alterations concerned the cell walls of phloem. After UAE the walls exhibited numerous
pitted areas refl ecting an ultrasound-induced cavitation that enhances the extraction eff ectiveness of this method.
Conclusion – The study has shown the improvement of madder roots extraction both quantitatively and qualitatively using
the effi ciency of ultrasound-assisted extraction in comparison with magnetic agitation and refl ux techniques. Copyright ©
2009 John Wiley & Sons, Ltd.
Keywords: Rubia tinctorum; ultrasound extraction; anthraquinones; HPLC-PAD; cytohistology
* Correspondence to: Laboratoire de Chimie Bio-Organique et des
Systèmes Moléculaires Vectoriels, Université d’Avignon et des Pays de
Vaucluse, Faculté des Sciences, 33 rue Louis Pasteur, F-84000 Avignon,
France. E-mail: carole.mathe@univ-avignon.fr
a Laboratoire de Chimie Bio-Organique et des Systèmes Moléculaires
Vectoriels, Université d’Avignon et des Pays de Vaucluse, Faculté des
Sciences, 33 rue Louis Pasteur, F-84000 Avignon, France
b INRA, UMR 406, F-84914 Avignon Cedex 9, France
Introduction
Natural tinctures may represent an elegant solution to control
the ecological and health risks engendered by the massive use
of synthetic dyes, particularly in the textile industry (Angelini
et al., 1997). Accordingly, there is great interest in tinctorial plants
today. Madder (Rubia tinctorum L.) is a member of the Rubiaceae
family that has historically been used since ancient times (∼2000
BC) as a source of pigments for textile dyeing (cotton, wool and
silk) (Novotná et al., 1999; Surowiec et al., 2006; Clementi et al.,
2007) and for painting (Miliani et al., 1998). It is still employed
today, for example, madder lake has been found in a collection
of Russian avant-garde painting dating from the twenieth century
(Cuoco et al., 2008). Madder compounds have also been shown
to protect textiles against pests due to an antifeedant action
(Morimoto et al., 2002). While several species of the Rubia genus
are reported in the literature, R. tinctorum, growing from
Mediterranean Europe to Asia, is the most used tinctorial madder
(Angelini et al., 1997; Mischenko et al., 1999; Cardon, 2003).
Underground parts of this species are known to accumulate
various tinctorial secondary metabolites basically composed of
an anthraquinonic (anthracen-9,10-dione) skeleton, correspond-
ing to heterosidic and aglycone molecules (Schweppe, 1992; De
Santis and Moersi, 2007). The latter are alizarin (1,2-dihydroxyan-
thraquinone), purpurin (1,2,4-trihydroxyanthraquinone), pseu-
dopurpurin (1,2,4-trihydroxy-3-carboxyanthraquinone), lucidin
(1,3-dihydroxy-2-hydroxymethylanthraquinone), xanthopurpu-
rin (1,3-dihydroxyanthraquinone) and rubiadin (1,3-dihydroxy-2-
methylanthraquinone). The heterosidic dyes are composed of
compounds with an anthraquinonic part and a glycosidic one,
corresponding to a primeverose (6-O-b-D-xylopyranosyl-b-D-
glucose). The major heterosidic dyes are lucidin-3-O-b-D-pri-
meveroside, ruberythric acid (alizarin-2-O-b-D-primeveroside)
and galiosin (pseudopurpurin-1-O-b-D-primeveroside; Table 1).
During the biosynthetic pathway madder cells initially form het-

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Cytohistological and phytochemical study of madder root extracts
Phytochem. Anal. 2009, 20, 484–490 Copyright © 2009 John Wiley & Sons, Ltd. www.interscience.wiley.com/journal/pca
485
Table 1. Chemical dyes composition of madder roots
O
HO
HO
OH
O
O
HO
HO
OH
R5
CH2
O
O
R2
R3
R1
R4
PrimeveroseAnthraquinone aglycone moiety
No:
1
2
3
4
5
6
7
8
9
Common name
Lucidin primeveroside
Ruberythric acid
Galiosin
Rubiadin primeveroside
Pseudopurpurin
Lucidin
Alizarin
Xanthopurpurin
Purpurin
R1
OH
OH
Primeverose
OH
OH
OH
OH
OH
OH
R2
CH2OH
Primeverose
OH
CH3
OH
CH2OH
OH
H
OH
R3
Primeverose
H
COOH
Primeverose
COOH
OH
H
OH
H
R4
H
H
OH
H
OH
H
H
H
OH
tR (min)
5.45
5.98
10.17
10.43
13.98
18.63
19.53
21.60
22.63
Absorption maxima (nm)
203, 265, 406
199, 261, 334, 418
201, 255, 288, 434
203, 269, 412
203, 253, 439
203, 245, 280, 414
199, 248, 428
200, 245, 281, 416
203, 256, 294, 481
erosidic compounds that are also the aglycone dye precursors.
Then, an enzymatic hydrolysis of these precursors takes place to
produce the diff erent aglycones (Derksen et al., 2003; Nakanishi
et al., 2005). This richness of madder makes it interesting for
dyeing textiles, in comparison with the use of synthetic dyes such
as alizarin. Angelini et al. (1997) reported that the best coloration
is obtained from three-year-old plants.
The classical extraction method of madder dyes is a refl ux of
tissues, with a water–alcohol mixture for more than 1 h (Derksen
et al., 2002; Boldizsár et al., 2006). This process is long and often
gives analyte decomposition, for example the glycosiled
anthraquinonic compounds of madder. To overcome these dis-
advantages, an alternative procedure using ultrasound has been
developed for the extraction of R. tinctorum dyes (Cuoco et al.,
2009). Ultrasound assisted extraction (UAE) proved to be an effi -
cient, non-denaturing technique for plant primary and second-
ary metabolites purifi cation (reviewed in Vinatrou, 2001; Vilkhu
et al., 2008).
In the present paper a chromatographic study using an HPLC-
PAD system has been carried out to assess the anthraquinonic
composition extracted from madder tissues via UAE in compari-
son with two classical methods: magnetic agitation and refl ux. A
cytohistological study was conducted by light microscopy to
reveal the eff ects of each extraction process at cell and tissue
levels.
Experimental
Plant material
Dye-containing tissues of R. tinctorum were purchased from Okhra
(Roussillon, France). They essentially consisted of dried, root-bearing rhi-
zomes and fi ne roots. Just before use for extraction experiments they
were ground in a mixer and calibrated to 0.5 mm fragments using a sieve
shaker (Idar-Oberstein, Germany). This granulometric size proved to be
optimal for extraction via UAE (Cuoco et al., 2009).
Chemicals (reagents)
Solvents and reagents were all of analytical grade and were purchased
from Merck (Darmstadt, Germany). Alizarin and purpurin were purchased
from Acros Organics (Geel, Belgium). Lucidin primeveroside, ruberythric
acid, rubiadin primeveroside and rubiadin were kindly provided by
Professor V. Golicov (Russian Research Institute for Cultural and Natural
Heritage, Moscow, Russia). Lucidin primeveroside was hydrolysed (hydro-
chloric acid) to obtain lucidin, the structure of which was characterised
on the basis of chemical and spectral evidence including two-dimen-
sional NMR experiments (COSY and NOESY 1H-1H; HMQC and HMBC) and
mass spectrometric techniques (EI, HR-MS).
Extraction procedures
Ultrasound was applied by means of a PEX 3 (REUS, Contes, France) soni-
fi er (25 kHz, 150 W), composed of an inox jug with a maximum capacity
of 3 L. The actual ultrasonic power dissipated to the system, experimen-
tally determined, was 42 W (Cuoco et al., 2009). For UAE extraction 20 g
of crushed madder tissue was used with 500 mL methanol–water mixture
(37 : 63, v/v).
The classical extractions used 6 g of crushed madder tissues which
were extracted with 150 mL methanol–water (80 : 20 and 37 : 63, v/v)
during 1 h. The fi rst method applied a refl ux condenser and the second
a magnetic agitation.
For the three extraction processes, the fi ltered extract (1 mL) was taken
for the HPLC-PAD analysis, and the remaining phase was evaporated to
dryness to determine the corresponding yield.
Liquid chromatography–photodiode array detection
HPLC-PAD analysis was carried out in a Waters liquid chromatograph
consisting of a high-pressure ternary pump (Waters 600), a vacuum
degasser, a high pressure manual injector valve (20 µL injection loop)
and a photodiode array detection (PAD) system Waters 2996. The
liquid chromatography was equipped with a Symmetry Shield
RP18 (Symmetry Columns, 5 µm, 4.6 × 250 mm) and controlled by
Empower 2 software.

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Phytochem. Anal. 2009, 20, 484–490
486
The HPLC separation was performed at 35°C with a binary elution
mixture composed of acetonitrile (A) and double-distilled water (B) con-
taining 0.01% trifl uoroacetic acid (TFA). Chro matography was carried out
for 30 min at a continuous fl ow-rate of 0.7 mL/min. The gradient program
was as follows: 0–5 min, 30% A and 70% B; 5–10 min, 30–70% A and
70–30% B; 10–20 min, 70% A and 30% B; 20–25 min, 70–100% A and
30–0% B; 25–30 min, 100% A. The chromatograms were acquired at
450 nm. Each sample was injected in triplicate.
Cytohistology
After each extraction procedure, samples of madder tissues were col-
lected and processed for cytohistological observations in light micros-
copy. They were fi xed in formalin–acetic acid–alcohol (1 : 1 : 8; v/v/v),
rinsed overnight in tap water and stored in 70% alcohol until required.
They were then dehydrated in a graded alcohol series (80–100%) and
embedded in methacrylate resin (Kultzer, Wehrheim, Germany). Sections
(3 µm thickness) were serially cut using an RM 2165 rotation microtome
(Leica Microsystems, Wetzlar, Germany) collected on microscope slides,
and stored at 40°C for 48 h. They were then stained to specifi cally visual-
ise polysaccharides and proteins using the cytochemical periodic acid-
Schiff ’s reagent (PAS) test and naphthohol blue black (NBB), respectively
(for details, see El Maâtaoui and Pichot, 1999). Polysaccharides (cellulose,
starch) turn red due to PAS reaction whereas insoluble proteins (nucleo-
proteins) turn blue due to NBB. After staining, the slides were dried and
coverslipped in Sugipath Micromount medium (Sugipath, Richemond,
Canada). Observations and photographs were made using a light micro-
scope (Leica DM 2000) equipped with a digital camera (Leica DFC 30 FX).
Approximately 15 fragments were analysed for each treatment.
Results and Discussion
HPLC-PAD analysis
In a previous study, a UAE process was carried out for the extrac-
tion of madder red dyes. Four parameters were optimised: root
size (0.5 mm), extraction time (18 min), temperature (36°C) and
solvent composition (37 : 63 methanol : water, v/v) (Cuoco et al.,
2009).
This method was compared with a classical method using
refl ux extraction (Boldizsár et al., 2006) and with an extraction
procedure using a magnetic agitation. According the specialised
literature (Derksen et al., 2002; Boldizsár et al., 2006), in both proc-
esses, the red dyes were extracted in a mixture composed of
80 : 20 methanol : water (v/v) for 1 h. Table 2 shows the extraction
conditions used for the three techniques tested. The yields
(weight of relative extracted dyes from madder tissues reported
to the weight of dry sample) obtained from each extraction
process were compared (Table 2). Refl ux and agitation processes
produced similar yields (respectively 60.25 and 58.71%), while
ultrasound method produced 63.31%.
However, in order to ensure correct interpretation of the
obtained results, refl ux and agitation experiments were also
carried out in the solvent mixture 37 : 63 methanol : water (v/v).
The corresponding yields were respectively 61.85% for refl ux
and 58.16% for agitation. These data were equivalent to the
results from 80 : 20 methanol : water (v/v) conditions (Table 2),
but they were lower than those obtained by using ultrasound
procedure (63.31%). Therefore, the solvent mixture infl uences
the extraction yield but ultrasound allows an increase of the
quantity of extracted madder dye. The eff ect of ultrasound on
the cells, as described below, may explain the higher yield
obtained with this method, at a lower temperature and a shorter
extraction time.
These dyes, as are many natural compounds, are sensitive
to diff erent factors. Humidity (Derksen et al., 2004) and especially
a high temperature (Cardon, 2003; Cuoco et al., 2009) during
the extraction process may denature these anthraquinonic
compounds. These degradations can occur by hydrolysis of
heterosidic precursors to give aglycones and/or cause decar-
boxylation of some molecules. For example, the presence of
purpurin can be explained by a double degradation process
of galiosin via the hydrolysis of the heterosidic precursor
synthesizing pseudopurpurin, which undergoes decarboxylation
to form purpurin. Moreover, native madder enzymes, after
drying madder tissues, are still able to convert anthraquinones
(Derksen et al., 2004).
The chemical composition of the extracts obtained via the
diff erent methods was compared by HPLC-PAD at 450 nm
(Fig. 1). In these extracts, nine compounds were detected and
identifi ed (using standard compounds and UV/Visible spectrum).
They corresponded to anthraquinonic compounds in agreement
with the literature (Schweppe, 1992; Derksen et al., 2002; Boldizsár
et al., 2006).
Refl ux and ultrasound extractions showed the same chroma-
tographic profi le and all of these experiments were carried out
at the same concentration; it is thus possible to compare com-
pound areas from the obtained chromatograms. Figure 2 repre-
sents the average peak area of each studied anthraquinone as a
function of the extraction process used. The same proportions of
madder sample and solvent mixture were employed for all of
these experiments (20 g of madder in 500 mL of solvents for UAE
and 6 g of madder in 150 mL of solvents for the others extraction
processes). Therefore, this fi gure permits direct comparison of
the total amounts of extracted dyes by sample.
Heterosidic compounds (1, 2, 3 and 4) and aglycones (5, 7 and
9) were detected. Xanthopurpurin (8) was only present in the
extract issued from refl ux and galiosin (3) was not detected in
the chromatogram of magnetic agitation extraction. To draw
the correct conclusion, it is necessary to compare several
experiments in diff erent experimental conditions, but all of the
Table 2. Extraction conditions and yield for each process
Solvent (MeOH : H2O)
80 : 20
37 : 63
80 : 20
37 : 63
37 : 63
Time (min)
60
60
60
60
18
Temperature (°C)
75
84
25
25
36
Root size (mm)
0.5
0.5
0.5
0.5
0.5
Mean yield (%)
60.25
61.85
58.71
58.16
63.31
s (%)
1.6673
0.1979
0.2868
1.99
1.6407
RSD (%)
2.77
0.32
0.49
3.42
2.59
Refl ux
Agitation
Ultrasound

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Cytohistological and phytochemical study of madder root extracts
Phytochem. Anal. 2009, 20, 484–490 Copyright © 2009 John Wiley & Sons, Ltd. www.interscience.wiley.com/journal/pca
487
AU
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
Minutes
2.004.006.008.00 10.0012.00 14.0016.0018.00 20.00 22.0024.00
a
1
2
3
4 5
6
7
9
AU
0.000
0.010
0.020
0.030
0.040
0.050
0.060
0.070
0.080
0.090
Minutes
2.004.00 6.008.0010.00 12.0014.0016.0018.00 20.00 22.0024.00
b
1
2
3
4 5 6
7
8
9
AU
0.000
0.010
0.020
0.030
0.040
0.050
0.060
0.070
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0.090
0.100
0.110
0.120
Minutes
2.00 4.00 6.00 8.0010.0012.00 14.0016.00 18.0020.0022.0024.00
c
1
2
4
6
7
Figure 1. HPLC-PAD chromatogram at 450 nm of madder extract obtained by ultrasound (a), refl ux (b) and agitation (c).
parameters do not simultaneously change. Moreover, it is impor-
tant to consider the extract amount from dry weighed matter, i.e.
the extraction yield, but also the proportion of each extracted
anthraquinone. In general, the refl ux procedure permitted
extraction of all of the dyes but it extracted fewer heterosidic
compounds, suggesting its denaturing eff ects. The magnetic agi-
tation process did not extract all of the studied compounds.
Comparatively to the other extraction method, magnetic agita-
tion was not optimal because only fi ve compounds were
extracted (1, 2, 4, 6 and 7); they were in appreciable amounts
but these extracts are not representative of the complete native
composition of madder. Moreover, it can be considered a less
aggressive physical method, so magnetic agitation could be a
satisfactory alternative method to extract madder dyes, contrary
to the refl ux process. Ultrasound extracted all of the dyes and in
good proportions. In general, the quantity of anthraquinonic
dyes was increased with UAE process over refl ux (Fig. 2). The UAE
process proved to be the best method to extract madder dyes,
giving a higher yield and preserving the native chemical compo-
sition. Similar results have been reported in other plant systems
(Hemwimol et al., 2006; Jacques et al., 2007).
Cytohistology
Preliminary microscopic observations of freehand sections
from fresh tissues of R. tinctorum showed that, although the
dyes were present within parenchyma cells of cortical tissues,

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Phytochem. Anal. 2009, 20, 484–490
488
a
X
P
P
B
b
P
d
*
*
X
P
B
c
Figure 2. Average peak area comparison of anthraquinones total amount for the three extraction processes. LuP = lucidin primeveroside,
RuAc = ruberythric acid, Gal = Galiosin, RuP = rubiadin primeveroside, Pseu = pseudopurpurin, Luc = lucidin, Aliz = alizarin and Purp = purpurin.
Figure 3. Longitudinal sections showing cytohistological behaviours of madder tissues after extraction procedures. Polysaccharides (cellulose in cell
walls) were stained (red) by PAS reaction and proteins were stained (blue) by naphtohol blue black. Prior to extraction (a) phloem elements (P) exhibit
a normal architecture with healthy cell walls surrounding the cell bodies in which nuclei (arrowheads) and cytoplasmic traces (arrows) are recognisable.
X = xylem. After extraction via magnetic agitation (b) phloem cells (P) conserved their normal appearance but the cell walls gained in thickness. B =
bark. Tissue fragments that have been subjected to refl ux and ultrasound extractions (c and d) show destroyed phloem (P) areas displaying fl attened
elements leading to drastic diminution of cell volume. With ultrasound treatment bleb-like structures (d, asterisks) have been seen to form in cell wall
material. Magnifi cation: ×450, a–d. P = phloem elements; X = xylem; B = bark; *bleb-like structures.