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Industrial Crops and Products 32 (2010) 439444

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Optimization of anthocyanin, avonol and phenolic acid extractions from Delonix

regia tree owers using ultrasound-assisted water extraction
Flix Adj a,b,c,e , Yves F. Lozano a, , Paul Lozano a , Augustin Adima b , Farid Chemat d , Emile M. Gaydou e

CIRAD, UMR GPEB Gnie des Procds, Eau et Bioproduits, TA 40/16, 73 Avenue J.F. Breton, 34398 Montpellier Cedex 5, France
INP-HB Institut National Polytechnique Flix Houphout-Boigny, Laboratoire de Procds Industriels, de Synthse et de lEnvironnement (LAPISEN),
Unit Chimie de lEau et des Substances Naturelles, BP 1313 Yamoussoukro, Cote dIvoire
Universit dAbidjan-Cocody, UFR Biosciences, Laboratoire de Biochimie et Sciences des Aliments, 22 BP 582 Abidjan 22, Cote dIvoire
Universit dAvignon et des pays du Vaucluse, INRA, UMR A 408, Scurit et Qualit des produits dorigine vgtale, F-84000 Avignon, France
Institut des Sciences Molculaires de Marseille (ISM2), Equipe AD2EM (Groupe Phytochimie), UMR 6263, Universit Paul Czanne,
Avenue Escadrille Normandie Nimen, 13397 Marseille Cedex 20, France

a r t i c l e

i n f o

Article history:
Received 9 February 2010
Received in revised form 8 June 2010
Accepted 9 June 2010

Delonix regia

a b s t r a c t
Flowers of Delonix regia trees from Ivory Coast are traditionally macerated in water to prepare beverages
with benecial health properties mainly due to their polyphenolic contents. Different maceration procedures, mimicking the traditional process, were compared on the basis of polyphenol content followed by
HPLC-DAD analysis. Under optimized maceration ratio (R = plant sample weight/maceration media volume), plant extractions were compared, including acidied-water-based media and assisted-ultrasound
or stirring extraction procedures. Diffusion kinetics of polyphenol families (anthocyanins, avonols and
phenolic acids) were followed by HPLC and lead to nd optimum ratio (R = 1/100). Flavonol contents were
28.5, 31, and 33.5 mol g1 (expressed as quercetin equivalent), using acidied-water (0.01N) media with
sulphuric or citric acids. Anthocyanin and phenolic acid contents were within the same range for these
water media (5.6 mol g1 as cyanidin equiv. and 27.5 mol g1 as gallic acid (GA) equiv., respectively).
Ultrasound-assisted procedure shortened maceration time (three times shorter) but did not show higher
level of total polyphenol than stirring-assisted procedure (39 mol g1 GA equiv.). This eco-friendly and
low-cost extraction process will provide to African people, with safer quality and longer availability,
polyphenol-rich bio-products.
2010 Elsevier B.V. All rights reserved.

1. Introduction
Polyphenol compounds, including anthocyanins, avonols, and
phenolic acids, are among the most bioactive natural molecules
found in the plant kingdom because of their antioxidant activities
(Bruneton, 2002; Teow et al., 2007; Duh and Yen, 1997; Einbond et
al., 2003; Shahidi, 1997). Plant anthocyanins are also well-known
compounds for their functional property as natural colorants (Espin
et al., 2000; Mazza et al., 2004). In tropical countries, particularly in
Ivory Coast, Delonix regia (Flame tree), also called locally Flamboyant, is often used for traditional medicine (Ali et al., 1999)
prepared from various parts of the tree. Red owers contain anthocyanins (Nabieh and Moheb, 1976), which are generally extracted
at lab scale using hydro-alcoholic solvents (Campos et al., 2008;
Chirinos et al., 2007; Neungnapa et al., 2008), and at pilot plant
scale using acidied-water media (Adj et al., 2008). During the

Corresponding author. Tel.: +33 467614447; fax: +33 467616537.

E-mail address: (Y.F. Lozano).
0926-6690/$ see front matter 2010 Elsevier B.V. All rights reserved.

short blooming season of D. regia, African people use generally hot

water to prepare their plant extracts, to avoid health-problems due
to the natural bacteria load brought, either by the plant itself or by
water. Heating may also destroy, to a certain extend, the extracted
active molecules. Water maceration at room temperature requires
long contact times with the dried vegetal. Ultrasounds or mechanical stirring are applied as extraction technological helps, to reduce
time of polyphenol diffusion into water during plant maceration.
During sonication, the cavitation process causes the swelling of
cells or the breakdown of cell walls, which allow high diffusion
rates across the cell wall in the rst case or a simple washing-out
of the cell contents in the second. Besides the solvent, temperature and pressure, better recoveries of cell contents can be obtained
by optimizing ultrasounds application factors including frequency,
sonication power and time, as well as ultrasonic wave distribution.
It was claimed that ultrasounds reduce plant extraction time and
increase extraction yield (Chemat et al., 2008).
This work was conducted to estimate the value added by ultrasounds compared to water maceration of D. regia dried owers,
for producing red polyphenol extracts. The best extraction proce-


F. Adj et al. / Industrial Crops and Products 32 (2010) 439444

dure will be further coupled with other technology in a full process

line that will operate in local low technological environments, contributing to sustainable development of micro-economic activities
for African village communities.
2. Materials and methods
2.1. Plant material
Red owers of D. regia tree were harvested during the bloom
season (MarchJuly) in the centre part of Ivory Coast: plant material
A was harvested in 2006 and plant material B in 2008. The fresh
harvested owers were dried in the oven (40 C), packed in plastic
bags and sent to CIRAD laboratory (Montpellier, France) where they
were stored at 4 C until use.
2.2. Chemicals
All reagents were of analytical grade. Methanol (MeOH),
hydrochloric acid (HCl) (37%), sulphuric acid (H2 SO4 ) (96%), monohydrated citric acid, Folin-Ciocalteus phenol reagent, sodium
carbonate salt, formic acid were purchased from Carlo Erba (Spain).
Cyanidin, rutin, quercetin-3--d-glucoside and quercetin standards were purchased from Extrasynthese and Fluka (France). Gallic and protocatechuic acids were purchased from SigmaAldrich
(Germany). Acetonitrile (Carlo Erba, Spain) was of HPLC grade.
2.3. Apparatus
Ultrasound-assisted extraction (UAE) was performed with a PEX
3 Sonier (R.E.U.S., Contes, France) composed of an stainless steel
jug having 23 cm 13.7 cm internal dimensions with a maximal
capacity of 3 L, and a transducer, in the base of jug, operating at
a frequency of 25 kHz with maximum input power of 150 W. The
double layered mantle allowed us to control the temperature of
the medium by cooling/heating systems. The output power of the
generator is 150 W while the power dissipated in the medium is
about 60 W per kilogram, as measured by calorimetry.
pH was measured using a Testo 230 type 4 (Testo, France)
portable pH meter. Calibration was made with pH = 4 and pH = 7
standard buffer solutions.
An UVvis spectrophotometer with variable wavelength
(190800 nm), Jenway 6705 (Barloworld, France) equipped with
a recorder was used for total polyphenol analysis.
An Agilent Technologies HPLC (Agilent 1100 series) equipped
with a quaternary pump, an autosampler, a diode array detector
and a RP 18 column (250 mm 4.6 mm, 5 m, Cil Cluzeau, France),
equipped with a C18 guard cartridge, was used for polyphenol
identication and quantication. All samples were ltered using
a 0.45 m pore size membrane lter (Sartorius, Germany), before
HPLC analysis.
2.4. Maceration media
M1: watersulphuric acid medium. The extraction medium was
prepared according to Gorinstein (Gorinstein et al., 1999): 0.69 mL
of concentrated sulphuric acid (96%) was added to 2.5 L of deionised
water. Acid concentration of the extraction medium was 0.01N.
M2: watercitric acid medium. Crystals of monohydrated citric
acid (1.75 g) was dissolved into 2.5 L of deionised water. Acid concentration of the extraction medium was 0.01N.
M3: methanolwater medium acidied with hydrochloric acid.
1 mL of concentrated HCl (37%) was added into 1 L of methanol,
as previously described (Longo and Vasapollo, 2005).

2.5. Extraction procedures

P1: powdered-ower maceration. Dried D. regia owers were
turned into powder using a kitchen electric grinder. Weighted
aliquots of powdered plant material were macerated into various
volumes of extraction media in glass beakers to obtain different plant/media ratio R (g/ml), such as: R1 = 1/250, R2 = 1/200,
R3 = 1/100, R4 = 2/100 and R5 = 4/100. The maceration media were
slightly stirred to wet completely the powdered material and let
unstirred for 3 h maceration time, at room temperature.
P2: entire ower maceration. An aliquot of 25 g of material were
poured into 2.5 L of extraction medium, wet by gently stirring, and
then let unstirred to macerate for 3 h in a 3 L stainless steel beaker
P3: entire ower extraction with mechanical stirring. An aliquot
of 25 g of entire dried owers were poured into 2.5 L of extraction
medium, wet by gently stirring, and then let unstirred to macerate
for 3H in a 3 L stainless steel beaker capacity.
P4: entire ower extraction assisted by ultrasounds. P4 was
an ultrasound-assisted extraction (UAE) procedure based on P3,
including also mechanical stirring. An aliquot of 25 g of entire dried
owers were kept gently stirred with 2.5 L of extraction medium
for 1 h into a 3 L stainless beaker equipped with ultrasound (US)
capabilities, as described above.
Total extracted polyphenol content. Total polyphenol content was
determined according to the Folin-Ciocalteu method, reported by
Singleton and Rossi (1965) and modied by Wood et al. (2002).
To 30 L sample extract, 2.5 mL of diluted Folin-Ciocalteus phenol reagent (1/10) were added. The mixture was kept for 2 min
in the dark at room temperature and 2 mL of calcium carbonate
solution (75 g L1 ) were added. The mixture was heated at 50 C for
15 min then cooled down. The absorbance was measured at 760 nm
against water as blank. Analyses were performed in triplicate. Total
polyphenol content was quantied as gallic acid equivalents per
gram of dry weight (mol g1 GA equiv.).
HPLC analytical conditions. HPLC analytical conditions were
as follows: injection volume was 20 L; binary elution solvents
were A (deionised water/formic acid, 99.5/0.5; v/v) and B (acetonitrile/formic acid, 99.5/0.5; v/v), gradient was A (95%)/B (5%),
040 min; A (82%)/B (18%), 4045 min; A (70%)/B (30%), 4550 min;
A (60%)/B (40%), 5060 min; A (95%)/B (5%), 6065 min; ow rate
was 0.8 mL min1 ; column was a RP18. The run started with column
equilibration for 5 min with A (95%)/B (5%), and ended with column
washing cycle with C (deionised water/acetonitrile, 1:1, v:v) at ow
rate of 0.5 mL min1 for 10 min. UVvis spectra were recorded with
a DAD in the range  = 190600 nm during analysis. Specic chromatograms for anthocyanins, avonols and phenolic acids were
respectively recorded at  = 530, 325 and 280 nm. Sample analyses
were performed in duplicate.
HPLC polyphenol analysis. Polyphenol compounds were detected
at  = 530, 325 and 280 nm and quantied using external standard
method with standard calibration curves for cyanidin, quercetin
and gallic acid, respectively. Anthocyanins were quantied as
cyanidin equivalents (mol g1 C equiv. dry weight), avonols as
quercetin equivalents (mol g1 Q equiv. dry weight), and phenolic acids as gallic acid equivalents (mol g1 GA equiv. dry weight)
(Liu et al., 2000).

3. Results and discussion

D. regia tree owers were traditionally used for local preparation
of medicinal beverages. Few works have been done on phenolic composition of this vegetal besides anthocyanins which were
recently identied (Adj et al., 2008). Other polyphenols such as
avonol glycosides (rutin and quercetin-3--d-glucoside) and phe-

F. Adj et al. / Industrial Crops and Products 32 (2010) 439444


Table 1
Polyphenol contents of dried D. regia ower extracts obtained under M1/R3 and
M2/R3 extraction conditions.
Family compounds

Phenolic acidse

Fig. 1. Effect of dried powdered D. regia ower weight versus volume of acidiedwater media on total polyphenol extraction (mol L1 as gallic acid (GA) equiv.)
using maceration without stirring at room temperature (R1 = 1/250, R2 = 1/200,
R3 = 1/100, R4 = 2/100, R5 = 4/100 g mL1 , extraction conditions: M1: watersulphuric
acid 0.005 mol L1 media and M2: watercitric acid 0.0033 mol L1 media, P1:
powdered-ower maceration 3 h maceration time).

nolic acids (Gupta and Chandra, 1971) (gallic and protocatechuic

acids) were characterised by HLPC-DAD using standards, to evaluate the best maceration media used with the best extraction
procedures to prepare high polyphenol water extracts.
3.1. Optimization of the extraction conditions
Plant material samples harvested in 2006 (material A) were
used to determine the optimum ratio R (R = plant weight/extraction
media volume in g mL1 ) leading to a higher total polyphenol content with, either sulphuric acid (M1), or citric acid (M2), using
powdered-ower maceration (P1 extraction procedure). After 3 h
maceration time, total polyphenol contents were determined for
every extracted plant sample, using the total extracted polyphenol content analytical method. Fig. 1 shows the effect of dried
powdered-ower weight versus volume of acidied-water media
on total polyphenol extraction using maceration without stirring
at room temperature. It can be seen that, in both extraction conditions, i.e. M1/P1 (watersulphuric acid medium/powdered-ower
maceration) and M2/P1 (watercitric acid medium/powderedower maceration) with R3 = 1/100 g mL1 , the highest total
polyphenol content (5557 mol L1 as gallic acid (GA) equiv.)
is obtained. Although R4 = 2/100 g mL1 showed similar results
(5456 mol L1 GA equiv.) than R3, this ratio was not so different
than ratio R5 = 4/100 g mL1 , which showed very low polyphenol
content (16 and 47 equiv. mol for M1 and M2, respectively). As a
precautionary measure, we have selected R3 ratio to study polyphenol extraction efciency of the other procedures.



5.5 0.2
28.5 0.8
26.5 0.7

5.6 0.2
31.0 0.9
28.5 0.8

M1: watersulphuric acid 0.005 mol L1 medium/R3 = 1/100 g mL1 .

M2: watercitric acid 0.0033 mol L1 medium.
mol g1 as cyanidin equiv., max std error 7%.
mol g1 as quercetin equiv., max std error 5%.
mol g1 as gallic acid equiv., max std error 5%.

HPLC analyses of polyphenol of D. regia ower extracts showed

6 major compounds, as shown in Fig. 2, belonging to the 3 polyphenol families, anthocyanins: A1 (cyanidin 3-O-glucoside) and A2
(cyanidin 3-O-rutinoside) at  = 530 nm, avonols: F1 (rutin) and
F2 (quercetin-3--d-glucoside) at  = 325 nm and phenolic acids:
Ac1 (gallic acid) and Ac2 (protocatechuic acid) at  = 280 nm. The
chemical structures of anthocyanins were previously determined
(Adj et al., 2008) and the other compounds were identied using
standards and co-chromatography by matching the UVvis spectra given by HPLC-DAD. Polyphenols were therefore quantied in
extracts obtained with every extraction conditions. In the case of
powdered-ower maceration (P1) extraction conditions, polyphenol content slightly increased from using R1 = 1/250 g mL1 up
to R3 1/100 g mL1 ratio, then drastically decreased when using
R4 = 2/100 and R5 = 4/100 g mL1 . For both acidied media used, M1
(sulphuric) and M2 (citric), optimum extractions were found with
R3 for anthocyanins and phenolic acids and were in-between R3
and R4 for avonols as shown in Fig. 3. Whatever were the polyphenol chemical structures, polyphenols that diffused from the vegetal
cell walls into the maceration media seemed to reach maximum
content values at the end of the maceration time (3 h). Under P1
(powdered-ower maceration) procedure, acidied-water media
M1 (sulphuric acid) and M2 (citric acid), lead to the maximum
extraction level of each polyphenol families: maceration conditions
M1/R3 = 1/100 g mL1 gave generally slightly higher contents for
every polyphenol family than M2/R3 (Table 1).
3.2. Kinetic extraction of polyphenol with M3/R3 maceration
For analytical HPLC quantication of plant polyphenols, labscale extractions were generally made with acidied-methanol
extraction medium such as M3 (Campos et al., 2008; Chirinos et
al., 2007; Gorinstein et al., 1999). Kinetic of polyphenol extraction
was followed during 4 h maceration time under acidiedmethanolwater with hydrochloric acid (M3) at 1/100 g mL1 (R3)
with powdered-ower maceration (P1) conditions. As shown
in Fig. 4, anthocyanin and avonol contents increased until

Fig. 2. HPLC proles of polyphenol water-extracts of D. regia dried owers (Extraction conditionsM2/R3/P1: watercitric acid medium/R3 = 1/100 g mL1 /powdered-ower
maceration, A1: cyanidin 3-O-glucoside, A2: cyanidin 3-O-rutinoside, F1: rutin, F2: quercetin-3--d-glucoside, AC1: gallic acid and AC2: protocatechuic acid).


F. Adj et al. / Industrial Crops and Products 32 (2010) 439444

Fig. 3. Polyphenol contents of D. regia extracts obtained with P1/M1 and P1/M2 using various R ratios (mean of duplicate) (P1: powdered-ower maceration, M1:
watersulphuric acid medium, M2: watercitric acid medium, () anthocyanins as cyanidin equiv.; other polyphenols: () phenolic acids as gallic acid equiv., () avonols
as quercetin equiv.).

180 min maceration time, then decreased, slowly for anthocyanins and drastically for avonols. The maximum concentration
obtained was 5.5 mol g1 as cyaniding equiv. for anthocyanins
and 33.5 mol g1 as quercetin equiv. for avonols.
Liu et al. (2000) have reported that solvents with high polarity
and low strength, such as water, are not efcient to correctly extract
plant polyphenols. Comparing polyphenol contents of extracts (P1)
obtained with M3/R3 (alcohol-based medium) with M1/R3 and
M2/R3 (water-based media), it was found that anthocyanin contents were about the same amount (5.55.6 mol g1 (as cyaniding
equiv.)) for all media, and avonols contents slightly increased
from 28.5 up to 31 mol g1 (as quercetin equiv.), for M1/R3M2/R3, to 33.5 mol g1 (as quercetin equiv.), for M3/R3. Our
results showed that the 2 water-based maceration media, M1 and
M2, lead to extracts with the same polyphenol contents than those
of the alcohol-base maceration medium, M3, using in all cases
the same R3 ratio. It clearly appeared that polyphenol extraction,
using plant maceration in water-based media will be preferred
to methanol-based media to process large quantities of plant
polyphenol extracts. Water-based extraction will be suggested
for up-scaling extraction of natural and health-benet polyphenol
compounds from D. regia owers at pilot plant level for small-scale
local enterprises.
3.3. Procedure efciency within M1/R3 and M2/R3 maceration
Extraction polyphenol from D. regia dried owers (plant material B) were compared using 3 extraction procedures, including
extraction by simple maceration P2, stirring-assisted P3 and

ultrasound-assisted P4 extractions. Extractions were made using

the vegetal/medium ratio R3 = 1/100 (25 g/2.5 L) with the 2 waterbased maceration media, M1 and M2.
The difference between procedures was: P3 is similar to P2
with mechanical stirring added, and P4 is similar to P3 with ultrasounds applied. When the glands were subjected to more severe
stresses and localized high pressures induced by cavitation, as
in the case of ultrasound-assisted extraction (UAE), the pressure
build-up within the glands could have exceeded their capacity for
expansion, and caused their rupture more rapidly than in control
experiment. Conventional extraction involves diffusion of the plant
extract components across the unbroken gland wall due to the
increasing temperature in the medium. In the case of UAE, exudation of polyphenols from damaged cell walls and even cells, due
to a strong ultrasonic mechanical effect, this generally triggers an
instantaneous release of the plant extract components into the surrounding medium.
Extractions were made in the same 3 L stainless steel beaker of
the ultrasound device. For both P2 (entire ower maceration) and
P3 (entire ower extraction with mechanical stirring) procedures,
extraction durations were maintained for 3 h time. For P4 (entire
ower extraction assisted by ultrasounds), 1 h maceration time was
enough to reach the maximum polyphenol content in the extract.
With M1/R3 extraction conditions (watersulphuric acid 0.01N
media/R = 1/100 g mL1 ), P3 was the most efcient procedure to
extract polyphenols from D. regia material (total polyphenol content = 40 1 mol g1 as gallic acid (GA) equiv.). Similar contents of
total polyphenol extracted by P3 and P4 (39 2 and 40 1 mol g1
GA equiv., respectively), indicated that they were equivalent
extraction procedures (Table 2).
With M2/R3 extraction conditions (watercitric acid 0.01N
medium/R = 1/100), P3 was also found to be the most efcient
procedure to extract total polyphenol (39 2 mol g1 GA equiv.).
Nevertheless, P4 showed similar performance (33 2 mol g1 GA

Table 2
Effect of extraction procedures on total polyphenol content in dried D. regia ower
Extraction proceduresa



Total polyphenolc
P4 (UAE)
P3 (mechanical stirring)
P2 (maceration)

Fig. 4. Kinetics of anthocyanin and avonol derivatives extracted from

dried D. regia owers. (Maceration conditions: M3 = MeOHHCl 0.1%
medium/R3 = 1/100 g mL1 /P1: powdered-ower maceration, () anthocyanins as
cyanidin equiv., () avonols as quercetin equiv.).

39 2
40 1
27 1

33 2
39 2
26 1

P2: entire ower maceration, 3 h extraction time; P3: entire ower extraction
with mechanical stirring, 3 h extraction time; P4: entire ower extraction assisted
by ultrasounds, 1 h extraction time.
Extraction conditionsM1: watersulphuric acid medium; M2: watercitric
acid medium, R3 = 1/100 g mL1 .
mol g1 as gallic acid equiv.

F. Adj et al. / Industrial Crops and Products 32 (2010) 439444


Fig. 5. Variation of polyphenol content (Ct ) in dried D. regia ower extracts (a) and rst order law plotting kinetic (b) (Extraction conditions: M1/R3 and ultrasound-assisted
procedure P4).

equiv.) than P3, but P2 did not perform well (26 1 mol g1 GA
With both extraction conditions, P3 was found to have the highest polyphenol extraction capability among all procedures tested.
However, extraction time to reach the maximum polyphenol content was shorter for P4 (1 h) than for the 2 others (3 h), whatever
the maceration media used. Maximum level of polyphenol contents
reached by the 3 procedures was about 40 mol g1 GA equiv. On a
process duration basis, P4 appeared to be the most suitable extraction procedure for both maceration conditions (M1/R3 and M2/R3)
to obtain optimal polyphenol content in the shortest time. P4 did
not enhance polyphenol extraction yields, but only shortened drastically extraction duration time.
Moreover, Table 2 shows that maceration conditions M1/R3 are
better than M2/R3 on a basis of total polyphenol content of plant
extracts. So, sulphuric acid will be preferred to citric acid for acidication of water maceration medium.
Polyphenol content was monitored during extraction process
with P4/M1/R3 conditions. The kinetic curve of total polyphenol
content with time is given in Fig. 5a.
It was shown that ultrasound-assisted extraction of plant
polyphenol was a linear representation of the polyphenol content
versus time during the course of extraction, by plotting Ln(A) versus
time for the rst 30 min, where A = (C Ct )/(C C0 ) and C0 , Ct ,
and C are respectively extract polyphenol contents at start time t0 ,
at t, and at t , which was in reality set at 1 h extraction
time. According to Fick rst law, A = (C Ct )/(C C0 ) =
a exp (kt),
this relation can be transformed into the linear equation ln(A) = kt.
Plotting experimental data according to this last equation lead to
determine k values for every extraction conditions tested, which
was represented by the slope of the line obtained with a good
regression coefcient (R2 = 0.99) (Fig. 5b). This coefcient is also the
diffusivity Deff = 48 103 min1 of polyphenols in this maceration
conditions (P4/M1/R3).
For every other extraction conditions studied, a similar linear
regression was found, as shown in Table 3. Polyphenol content in
the extracts still followed a rst order kinetic law for the rst 30 min
maceration with P4/M1/R3 conditions and for the rst 60 min maceration using P2 and P3 procedures. One could notice that Deff
was found at least more than twice higher with P4 procedure
(Deff = 3545 103 min1 ) than with P3 and P2 procedures tested
(Deff = 821 103 min1 ).
These results raised the fact that ultrasound technology added
in P4 procedure increased the specic polyphenol Deff . The total

amount of polyphenol extracted with P4 was not much higher

than those extracted with P3 (similar as P4 without ultrasounds
use), according to values previously shown in Table 2, whatever
the extraction medium used, M1 or M2.
The content of every polyphenol families, i.e. anthocyanins,
avonols and phenolic acids, extracted using combination of 3 procedures and 2 extraction mediums M1 and M2, were quantied
by HPLC. Table 4 shows that P3 was the most efcient procedure
for anthocyanins extraction in M2 media (3.7 mol g1 C equiv.).
P4 gave similar but slightly lower results (3.2 mol g1 C equiv.).
For avonols extraction, P3/M1 conditions gave the highest concentration (28.9 mol g1 Q equiv.). For phenolic acids, P4/M1 was
Table 3
Regression equations for rst order kinetic law of polyphenol content in the extracts
obtained with various extraction conditions (procedure/medium).



Regression coefcient



Y = 0.048X + 0.060
Y = 0.035X + 0.021

r2 = 0.991
r2 = 0.988



Y = 0.021X 0.039
Y = 0.020X + 0.01

r2 = 0.993
r2 = 1



Y = 0.011X 0.118
Y = 0.008X 0.08

r2 = 0.91
r2 = 0.975

P2: entire ower maceration; P3: entire ower extraction with mechanical stirring; P4: entire ower extraction assisted by ultrasounds.
M1: watersulphuric acid 0.005 mol L1 media; M2: watercitric acid
0.0033 mol L1 medium.

Table 4
Comparative extraction efciency of polyphenols using maceration, stirring and
ultrasound procedures in either sulphuric or citric acids (HPLC polyphenol

Phenolic acidse














Extraction conditionsM1: watersulphuric acid medium; M2: watercitric
acid medium, R3 = 1/100 gm L1 .
ProceduresP2: entire ower maceration; P3: entire ower extraction with
mechanical stirring; P4: entire ower extraction assisted by ultrasounds.
mol g1 as cyanidin equiv., max std error 7%.
mol g1 as quercetin equiv., max std error 5%.
mol g1 as gallic acid equiv., max std error 5%.


F. Adj et al. / Industrial Crops and Products 32 (2010) 439444

the more efcient extraction conditions (42.6 mol g1 GA equiv.),

and P2 and P3 showed similar data.
Globally, M1/R3 maceration conditions (watersulphuric acid
media) lead to the highest polyphenol content in D. regia extracts,
except for anthocyanin which showed higher concentration with
citric acidied-water media using P3 or P4 extraction procedures.
Citric acid seemed to be more protective for anthocyanins than
did sulphuric acid. P3 and P4 appeared to be the best procedures
for optimising polyphenol extraction from dried D. regia owers,
and P4 showed the shortest extraction duration procedure due to
ultrasound extraction assistance. Water-based extraction will be
suggested for up-scaling extraction of natural and health-benet
polyphenol compounds from D. regia owers at pilot plant level for
small-scale local enterprises in developing countries.
Financial supports for this work were provided by the French
Embassy in Abidjan (Ivory Coast), CIRAD (DRS), and by the French
national network CNRS-INRA Chimie pour le Dveloppement
Adj, F., Lozano, Y.F., Meudec, E., Lozano, P., Adima, A., Agbo Nzi, G., Gaydou, E.M.,
2008. Anthocyanin characterization of pilot plant water extracts from Delonix
regia owers. Molecules 13, 12381245.
Ali, M.S., Azhar, I., Amtul, Z., Ahmad, V.U., Usmanghani, K., 1999. Antimicrobial
screening of some caesalpiniaceae. Fitoterapia 70, 299304.
Bruneton, J., 2002. Phytothrapie. Les donnes de lvaluation, 1st ed. Tec et Doc,
Editions mdicales internationales (EMI), Paris, pp. 1242.
Campos, D., Chirinos, R., Pedreschi, R., 2008. Extraction, recovery and purication of
natural antioxidants from Andean crops. Electron. J. Environ. Agric. Food Chem.
7, 32213225.

Chemat, F., Tomao, V., Virot, M., 2008. Ultrasound-assisted extraction in food analysis. In: Otles, S. (Ed.), Handbook of Food Analysis Instruments. CRC Press, New
York, pp. 8599.
Chirinos, R, Rogez, H., Campos, D., Pedreschi, R., Larondelle, Y., 2007. Optimization of extraction conditions of antioxidant phenolic compounds from mashua
(Tropaeolum tuberosum Ruiz et Pavon) tubers. Sep. Purif. Technol. 55, 217225.
Duh, P.-D., Yen, G.-C., 1997. Antioxidative activity of three herbal water extracts.
Food Chem. 60, 639645.
Einbond, L.S., Reynertson, K.A., Luo, X.D., Basile, M.J., Kennelly, E.J., 2003. Anthocyanin antioxidants from edible fruits. J. Food Chem. 84, 2328.
Espin, J.C., Soler-Rivas, C., Wichers, H.J., Garcia-Viguera, C., 2000. Anthocyanin-based
natural colorants: a new source of antiradical activity of foodstuff. J. Agric. Food
Chem. 48, 15881952.
Gorinstein, S., Zemser, M., Haruenkit, R., Chuthakorn, R., Grauer, F., Martin-Belloso,
O., 1999. Comparative content of total polyphenols and dietary ber in tropical
fruits and persimmon. J. Nutr. Biochem. 10, 367371.
Gupta, R.K., Chandra, S., 1971. Chemical investigation of Delonix regia owers. Ind. J.
Pharm. 33, 7475.
Liu, F.F., Ang, C.Y.W., Springer, D., 2000. Optimization of extractions for active components in Hypericum perforatum using response surface methodology. J. Agric.
Food Chem. 48, 33643371.
Longo, L., Vasapollo, G., 2005. Determination of anthocyanins in Ruscus aculeatus L.
berries. J. Agric. Food Chem. 53, 475479.
Mazza, G., Cacace, J.E., Colin, D.K., 2004. Methods of analysis for anthocyanins in
plants and biological uids. J. AOAC Int. 87, 129145.
Nabieh, A.M.S., Moheb, S.I., 1976. Anthocyanins of some leguminosae owers and
their effect on colour variation. Phytochemistry 15, 835836.
Neungnapa, R., Jia, Z., Xuewu, D., Bao, Y., Jianrong, L., Jiang, Y., 2008. Effects of various temperature and pH values on the extraction yield of phenolics from Litchi
Pericarp tissue and the antioxidant activity of the extracted anthocyanins. Int. J.
Mol. Sci. 9, 13331341.
Shahidi, F., 1997. Natural Antioxidants: Chemistry, Health Effects, and Applications.
AOCS Press, Urbana, IL, USA, pp. 1414.
Singleton, V.L., Rossi Jr., J.R., 1965. Colorimetry of total phenolics with
phosphomolybdic-phosphotungstic acid reagents. Am. J. Enol. Viticult. 16,
Teow, C.C., Truong, V.D., McFeeters, R.F., Thompson, R.L., Pecota, K.V., Yencho, G.C.,
2007. Antioxidant activities, phenolic and beta-carotene contents of sweet
potato genotypes with varying esh colours. Food Chem. 103, 829838.
Wood, J.E., Senthilmohan, S.T., Peskin, A.V., 2002. Antioxidant activity of
procyanidin-containing plant extracts at different pHs. Food Chem. 77, 155161.