32

A
g
r
o
F
O
O
D

i
n
d
u
s
t
r
y

h
i
-
t
e
c
h

-

N
o
v
e
m
b
e
r
/
D
e
c
e
m
b
e
r

2
0
1
1

-

v
o
l

2
2

n

6
Polyphenols from
Adansonia Digitata
Extraction, antioxidant analysis and
total phenols content
SILVIA VERTUANI
1,2
*, EMANUELA SCALAMBRA
1
, SONIA MOLESINI
2
, LISA BUZZONI
2
,
ELISA DURINI
1
, GIANNI SACCHETTI
3
, STEFANO MANFREDINI
1,2
*
*Corresponding authors
1. University of Ferrara, Dept. of Pharmaceutical Sciences, via Fossato di Mortara 17-19, Ferrara, 44100, Italy
2. University of Ferrara, Ambrosia lab, via Mortara 171, Ferrara, 44121, Italy
3. University of Ferrara, Dept. of Biology and Evolution, Sect. Agro-technological and Pharmaceutical Resources (Agri Unife),
C.so Ercole I dEste 32, Ferrara, 44100, Italy
Peer-reviewed scientific article
Stefano Manfredini Silvia Vertuani
KEYWORDS: Adansoni Digitata, antioxidant activity, polyphenols, red fibre.
ABSTRACT: With the aim to confirm important nutritional properties, we here report the results obtained from baobab plant
tissues analysis (ORAC, DPPH, FRAP). The micronized red fibre display the highest antioxidant activity, among the tested
samples in all performed assays; methanolic extracts of this fibre (ME and HME) were prepared and tested in comparison to
selected plant extracts by ORAC, DPPH, FRAP and PCL assays, furthermore the total phenols content of ME and HME was
measured by Folin-Ciocalteu analysis and characterized by RP-HPLC. HME extract revealed higher antioxidant activity than
Blueberry extract (1 percent) in all performed texts and comparable antioxidant activity to that of Pomegranate extract (40
percent) in ORAC and DPPH assay; these results are probably due to the high total phenols content (708 14.2mgGAE/g)
of the extract.
INTRODUCTION
/nIicxiccnI:, Leccu:e cf Iheir Lenefcic| effecI cn hec|Ih
issues (1-8).and their involvement in prevention of oxidation
reactions of foods, pharmaceuticals and cosmetics, represent
an interesting study target. Currently to counteract oxidation
reaction in foods and cosmetics synthetic compounds, like
butylatedhydroxyanisole (BHA) and butylatedhydroxytoluene
(BHT), are commonly used. However, with regard to the
safety of these synthetic antioxidants the literature, even
cccumenIing ccnficIing cpinicn:, cfIen repcrI: :ice effecI:
(9-10). These evidences along with the attention of the
consumers, increasingly focused on safe and possibly natural
antioxidants, have fostered the research on plant sources.
In this context baobab (Adansonia Digitata L.) a deciduous
tree, of Bombacaceae family, native of central Africa or other
tropical climates, has attracted our interest since the year
2000. Baobab produces a nut-type fruit internally splitted into
:mc|| fcury, cehycrcIec cnc pcwcery :|ice: inc|ucing mu|Iip|e
:eec: cnc f|cmenI:, Ihe rec fLre. ln /fricc, LccLcL fruiI i:
used as food or natural refreshing drink; leaves, bark and fruits
of baobab are differently used for medicinal purposes (11, 12).
ln 2008, c|:c Lc:ec cn cur effcrI: cn Ihi: fe|c, Ihe Eurcpecn
Union has approved baobab fruit as a food ingredient under
Ihe EU: fccc |egi:|cIicn, reccgnizing LenefI cf LccLcL
consumption (13). Studies prompted from the ethno-
pharmacological usage of baobab, suggest that antioxidant
compounds in baobab have a pivotal role in determining
Lenefcic| effecI: cn humcn hec|Ih {14, 15). ln Ihi: regcrc, we
have recently reported the preliminary results of a study in
which several baobab constituent have been evaluated by
photochemiluminescence (PCL) for their radical scavenging
properties against superoxide anion, in comparison with
common fruit species with recognized antioxidant power (16).
Almost all the investigated baobab products were featured of
high integral antioxidant capacity (IAC), and in case of pulp
fruit also of high vitamin C concentration (16). The observation
that, notwithstanding the high ascorbic acid content, when
the fruit was left opened to the air the pulp remained whitish
fcr |cng Iime, wherec: Ihe pc|e Lrcwn fLre neIwcrk in Ihe nuI
became rapidly reddish, suggested us the presence of other
:ccrifcic| ccmpcnenI: IhcI mcy ccI c: ncIurc| pre:ervcIive:
toward ascorbic acid oxidation. Taking these suggestions,
we started the present investigation with the aim to provide
further evidences to the antioxidant properties of the plant by
the application of PCL complementary analysis methods such
are, DPPH, ORAC and FRAP assays. After that, we directed
cur cIIenIicn Ic Ihe :Iucy cf rec fLre, Leccu:e cf iI: high
cnIicxiccnI ccIiviIy. MeIhcnc|ic exIrccI: cf Ihi: fLre were
prepared and next assayed by Folin-Ciocalteu method and
cnIicxiccnI cnc|y:i:: fnc||y Ihe cifferenI pc|yphenc|: pre:enI in
Ihe exIrccI: were icenIifec Ly FF-HFLC ccup|ec wiIh cicce
array detector and quadrupole mass spectrometer.
MATERIALS AND METHODS
Scmp|e: cf 8ccLcL fruiI rec fLre were :upp|iec frcm 8ccLcL
Fruit Company Senegal, Thies, Senegal. Other study samples
were supplied by A.C.E.F., Fiorenzuola dArda, Italy.
Reactants, solvents and standards samples were purchased
from Sigma-Aldrich, Milan, Italy and Carlo Erba Reagenti, Milan,
Italy. PCL Kits (ACL) were purchased from Analitik Jena AG,
Jenc, Germcny. Irc|cx |{S)-{2)--hycrcxy-2,5,7,8-IeIrcmeIhy|-
chroman-2-carboxylic acid] (no. 39,192-1) was purchased
from Sigma-Aldrich, Taufkirchen, Germany.
Samples absorbance measurement (DPPH, FRAP and
Folin-Ciocalteu assays) were carried out on a UV-VIS
spectrophotometer ThermoSpectronic Helios g, Cambridge,
United Kingdom.
Extraction procedures
Methanolic Extract (ME). 50 g cf micrcnizec rec fLre wc:
mixec wiIh 500 mL cf meIhcnc| uncer crgcn cImc:phere fcr
1 h cI rccm IempercIure. Ihe :cmp|e wc: Ihen f|Ierec cnc
the pellet subjected to two further extractions. The obtained
supernatants fractions were combined dried under vacuum
cI |cw IempercIure {< 35C). Ihe cchievec meIhcnc|ic
exIrccI {ME) cmcunI wc:, cn cvercge, Ihe 35-40 percenI cf
the starting material.
A
n
t
i
o
x
i
d
a
n
t
s
34
A
g
r
o
F
O
O
D

i
n
d
u
s
t
r
y

h
i
-
t
e
c
h

-

N
o
v
e
m
b
e
r
/
D
e
c
e
m
b
e
r

2
0
1
1

-

v
o
l

2
2

n

6
A
n
t
i
o
x
i
d
a
n
t
s
concentration and the absorbance was measured by a
:pecIrcphcIcmeIer UV-VlS cI 517 nm, lC
50
values expressed
c: g/mL were ceIerminec Ly |inecr regre::icn cnc|y:i: cf
the results obtained at different sample concentrations.
3) Oxygen Radical Absorbance Capacity (ORAC ) assay.
This test was carried out on a Fluoroskan FL

ascent
{Ihermc Fi:her ScienIifc, lnc. Wc|Ihcm, M/) fc||cwing c
prccecure mccifec Ly u: {21) cnc Lc:ec cn c wcrk cf
Hcng eI c|. {22). 8riefy, in Ihe fnc| c::cy mixIure {0.2 mL
IcIc| vc|ume), fucre:cein :ccium :c|I {85 nM) wc: u:ec c:
a target of free radical attack with 2,2-azobis(2-amidino-
propane) dihydrochloride (AAPH) as a peroxyl radical
generator. Trolox was used to achieve a calibration curve.
The tested compounds were dissolved in a phosphate
buffer solution (pH=7,4) and prepared immediately before
Ihe experimenI:. Ihe fucre:cence mec:uremenI:, ccrriec
cuI cI 37C were reccrcec cI 5 min inIervc|: up 30 min
after the addition of AAPH. The ORAC values, calculated
as difference of the areas under the quenching curves
cf fucre:cein LeIween Ihe L|cnk cnc Ihe :cmp|e, cre
expressed as mol of Trolox per gram of product.
4) Ferric reducing ability of plasma (FRAP) assay. The ferric
recucing cLi|iIy wc: mec:urec ccccrcing Ic c mccifec
protocol described by Guihua and co-workers (23).
Samples were dissolved in the selected solvent (water
and/or methanol). The reagent for analysis was freshly
prepared by mixing the following solutions in the reported
ratio 10/1/1 (v:v:v) i) 0.1 M acetate buffer pH 3.6, ii) TPTZ 10
mmol/L in 40 mmol/ HCl, iii) ferric chloride 20 mmol/L. To a
1.9 mL of reagent were added 0.1 mL of sample extract
or solvent when blank was performed. Readings at the
cL:crpIicn mcximum {53 nm) were Icken cfIer 30 min.
using a UV-VIS spectrophotometer. Trolox solution was
used to perform the calibration curves. The FRAP values
cre expre::ec c: mc| euivc|enI: cf Irc|cx per grcm cf
product.
Statistical evaluations
The analysis were carried out in three replicates, average and
:IcIi:Iicc| :ignifccnce {SIucenI: I Ie:I: FS0.05) were given fcr
all data collected. One-way ANOVA and LSD post hoc Tukeys
hcne:I :ignifccnI cifference Ie:I were u:ec fcr ccmpcring Ihe
bioactive effects of different samples. All computations were
made using the statistical software STATISTICA 6.0 (StatSoft Italia srl).
HPLC Analysis
A Shimadzu (SCL-10Avp) liquid chromatography system
equipped with diode array detector (DAD) SPD-M10Avp and
quadrupole mass spectrometer (Shimadzu LCMS QP8000a) with
an APCI interface was used. Separation was performed on a
250 x 4. mm, 80 /, Fhencmenex Hycrc-FF cc|umn cI 25C. /
grccienI ccn:i:Iing cf :c|venI / {cceIic ccic 0.05 percenI in
wcIer) cnc :c|venI 8 {meIhcnc|) wc: cpp|iec cI c fcw rcIe cf
HydrolizedMethanolic Extract (HME).5 g cf ME were IrecIec
with 100 mL of sodium hydroxide solution (4 N) and keep on
stirring for 60 minutes at room temperature. The mixture was
Ihen ccicifec wiIh hycrcch|cric ccic 37 percenI unIi| pH 2 cnc
then extracted for three times with ethyl acetate. The organic
fractions were combined, dried on magnesium sulphate
cnhycrcu: c fnc||y Icken Ic cryne:: uncer vacuum. Hydrolized
meIhcnc|ic exIrccI {HME) cmcunI wc:, cn cvercge, Ihe 20-25
percent of the starting material.
Fur|0cot|on o| extrocts
2 g of the extract (ME or HME) were solubilized in methanol,
absorbed on silica gel (Macherey Nagel 0,63-0,20 mm (70-230
me:h) cnc purifec cn c g|c:: cc|umn {5x 50 cm) f||ec wiIh 120 g
of silica gel. Elution was performed using a linear gradient of
dichloromethane/methanol from 10/0 to 8/2, v/v. The most
:ignifccnI frccIicn: {/, 8, C, D, E), c: emergec Ly HFLC cnc|y:i:,
were obtained as follows. Fraction A collecting fractions (400 mL)
during the elution of 100 percent dichloromethane (10/0
dichloromethane/methanol); fraction B collecting fractions
(400 mL) during the elution of the 9.8/0.2 dichloromethane/
methanol mixture; fraction C collecting fractions (400 mL)
during the elution of the 9.6/0.4 dichloromethane/methanol
mixture; fraction D collecting fractions (400 mL) during the
elution of the 9/1 dichloromethane/methanol mixture; fraction
E collecting fraction (400 mL) during the elution of the 8/2
dichloromethane/methanol mixture.
Folin-Ciocalteu assay
All the extracts were evaluated for polyphenols content by the
Folin-Ciocalteu method (17).Gallic acid was used as standard
Ic cLIcin c cc|iLrcIicn curve. / 1.5 mL c|iucI cf wcIer-
ci|uIec Fc|in-Ciccc|Ieu recgenI {1/15) wc: cccec Ic Ihe ci|uIe
exIrccI: {20 mL). Ihe mixIure wc: incuLcIec fcr 5 min cI rccm
temperature and 300 mL of sodium carbonate solution (200 g/L)
was added. The mixture was then incubated for further 90 min
at room temperature and the absorbance was measured
cI 75 nm Ly c UV-VlS :pecIrcphcIcmeIer, cgcin:I c L|cnk
similarly prepared, containing distilled water instead of extract.
The results are expressed as milligrams of gallic acid equivalent
per grcm cf rec fLre {mg G/E/g cf fLre).
Antioxidant capacity assays
Vegetable materials and extracts were submitted to the
following methods in order to determine their antioxidant
activity.
1) Photochemiluminescence (PCL) assay. This analysis
involves the photochemical generation of superoxide
free radical (O
2
) combined with chemiluminescence
detection (18). The PCL method was carried out as
described by Popov and Lewin (19); it can be conducted
by two different protocols ACW and ACL, which permit the
measurement, respectively, of the antioxidant capacity
of the water- and lipid-soluble components; in this study,
measurements were conducted using the ACL protocol.
An exact quantity of Adansonia Digitata extract or fruit
extract samples was accurately solubilized in methanol or
methanol/water 1/1 and properly diluted with Reagent 1
of ACL. A 2.30mL portion of reagent 1 (solvent and dilution
recgenI), 0.2 mL cf recgenI 2 {Luffer :c|uIicn), 25 mL cf
recgenI 3{phcIc:en:iIizer), cnc frcm 5 mL Ic 25 mL cf
standard trolox solution 0.1mM (to obtain the calibration
curve) or sample solution were mixed and measured by
means of Photochem

. Fe:u|I: cre expre::ec c: mc|

equivalents of Troloxper gram of product.
2) 1,1-diphenyl-2-picryl-hydrazyl radical (DPPH) assay. It
was performed according to the method described by
Wang and co-workers (20). To a DPPH methanolic solution
{1.5 mL) wc: cccec 0.750 mL cf exIrccI: cI cifferenI
250 x 4. mm, 80 /, Fhencmenex Hycrc-FF cc|umn cI 25C. /
grccienI ccn:i:Iing cf :c|venI / {cceIic ccic 0.05 percenI in
wcIer) cnc :c|venI 8 {meIhcnc|) wc: cpp|iec cI c fcw rcIe cf
Baobab.
A
g
r
o
F
O
O
D

i
n
d
u
s
t
r
y

h
i
-
t
e
c
h

-

N
o
v
e
m
b
e
r
/
D
e
c
e
m
b
e
r

2
0
1
1

-

v
o
l

2
2

n

6
35
A
n
t
i
o
x
i
d
a
n
t
s
temperature, the best performance was achieved at room
temperature. In fact, at higher temperature, even shorting the
times (20 min), a decrease in the amount of total phenolos
was observed, probably due to partial degradation of these
molecules. Unexpectedly, the use of ultrasounds, in order to
:hcrIen exIrccIicn Iime cnc Ic enhcnce yie|c, cic ncI refecI c
:ignifccnI effciency imprcvemenI.
A second extract was then prepared
submitting ME to a basic hydrolysis, this
extract was also analysed by the Folin-
Ciocalteu assay; the result evidenced, as
expected, that the total polyphenols in
hydrolysed extracts were in higher amount
than in non-hydrolysed ones (Table 3
HME: 708 mgGAE/g; MRE: 383 mgGAE/g).
The ME and the HME extracts were also
analysed for the radical scavenging
activity by the ORAC, DPPH, FRAP and
PCL assays together with several plant or
fruit extracts known for their antioxidant
properties. In all performed tests ME and
HME extracts showed an antioxidant
capacity higher than red vine, blueberry
(1 percent anthocyanosides) and black
currant extracts (Table 3). Moreover
HME and ME extracts showed also
appreciable antioxidant capacity in
ORAC and DPPH assay if compared to
pomegranate extract (standardized in
40 percent ellagic acid) and green tea
extract (70 percent polyphenols), both
characterized by a well-known radical
scavenger activity (Table 3). With the
exception of the PCL analysis, HME and
ME provided also best results than BHT in
all performed assays.
1.0 mL/min c: fc||cw:: 0-5 percenI 8 |inecr frcm 0 Ic 2 min, 5-25
percenI 8 |inecr frcm 2 Ic 10 min, 25-40 percenI 8 |inecr frcm
10 Ic 20 min, 40-50 percenI 8 |inecr frcm 20 Ic 30 min, 50-100
percent B from 30 to 40 min, followed by washing with methanol
cnc re-eui|iLrcIicn. Scmp|e: were ci::c|vec in 0.05 percenI
acetic acid in water/ methanol 1:1, and 40 l of this solution
was injected in the column,
cfIer f|Iering Ly c 0.45 m f|Ier:
HFLC f|Ier: were purchc:ec frcm
Chemtek Analitica, Bologna, Italy.
The detection condition was
270 nm. In addition to their
UV :pecIrc, Ihe icenIifccIicn
of phenolic compounds was
also carried out by mass
spectrometry coupled to HPLC.
The APCI parameters were as
follows: negative mode APCI,
full scan, APCI temperature
400C, CDL IempercIure 250C,
CDL vc|Icge -25V .
RESULTS AND DISCUSSION
We recently investigated
baobab derivatives by
phot ochemi l umi nes cence
(PCL) and all investigated
tissues, in particular the fruit
fLre:, pre:enIec cn inIegrc|
antioxidant capacity (IAC)
higher than compared
antioxidants rich fresh fruit
(16). In order to give more
consistences to the obtained
data, additional investigations
have been here performed by
the use of further antioxidant
assays. With the exception
of the baobab seed powder
and baobab seeds peel, all
examined baobab derivatives
showed good antioxidant
potency in the performed
tests (Table 1), in particular
rec micrcnizec fruiI rec fLre
displayed best results in all
performed assays.
Based on their widespread
presence in vegetables, we
hypothesized that polyphenols
could play an important role
in determining the antioxidant
ccIiviIy cf LccLcL rec fLre. Cur
efforts were directed to the development of an extraction
meIhcc IhcI wcu|c en:ure high effciency in Ierm: cf
exIrccIec pc|yphenc|:. During Ihi: phc:e, Ihe infuence cf Ihe
solvent, temperature and the use of sonication equipment
were investigated to maximize the yield with the best time-
saving procedure (Table 2). The marker employed, to measure
exIrccIicn effciency in ecch experimenI, wc: Ihe IcIc| phenc|:
amount, determined with the Folin-Ciocalteu method.
DcIc cnc|y:i: :hcwec IhcI Ihe mc:I :ignifccnI vcricL|e wc: Ihe
kinc cf :c|venI emp|cyec. MeIhcnc|, re:u|Iec mcre effcienI
than acetone and methylene chloride at the same condition.
Considering the time of extraction, the most reasonable option
wc: Ic :e|ecI 0 minuIe: Leccu:e Ihe increc:e cf effciency
cI |cnger Iime wc: ncI :ignifccnI. Fegcrcing Ihe exIrccIicn
Tabl e 1. Anti oxi dant capaci ty assays of baobab
derivatives.
a
< LOQ (Limit Of Quantification)
Table 2. Influence of solvent and operative conditions on
the extraction yield of phenolic compounds from baobab
red fiber samples. Total amount of polyphenols was
determined with the Folin-Ciocalteu method on the
extracts obtained from a single 1 hour extraction.
*GAE= gallic acid equivalent
Table 3. Antioxidant activity and total polyphenols evaluation of ME, HME and selected extracts.
1
ExIrccI :Icnccrcizec in 17 cnc 257 cnIhccycnc:ice: re:pecIive|y:
2
Extract standardized in 70% polyphenols;
3
Extract
standardized in 40% of ellagic acid;
4
< LOQ (Limit Of Quantification)
Figure 1. ME chromatogram by HPLC-DAD at 270 nm.
36
A
g
r
o
F
O
O
D

i
n
d
u
s
t
r
y

h
i
-
t
e
c
h

-

N
o
v
e
m
b
e
r
/
D
e
c
e
m
b
e
r

2
0
1
1

-

v
o
l

2
2

n

6
A
n
t
i
o
x
i
d
a
n
t
s
and hydroxytyrosol (27). The chromatogram in Figure 3, shows
a satisfactory resolution for all standard compounds.
Although ME and HME extracts were both analysed
Ly HFLC, Ihe icenIifccIicn cf pc|yphenc|: wc: fnc||y
performed only on the HME fractions B and D because of
the higher polyphenols content detected and of the better
chromatograms resolution. Peak 1 for example (Figure
4) wc: icenIifec c: gc||ic ccic Ly ccmpcri:cn cf FI {.
min), UV spectra (maximum at 270 nm) and MS spectra (a
quasi-molecular ion [M-H]+ at m/z 169.2) with those of an
authentic sample (Table 4). In the same way the molecular
icn cI m/z 181.1, ceIecIec cn Ihe peck 7, wc: icenIifec c:
homovanillic acid.
The Rt, UV/VIS spectra and molecular ions of the peaks 10
and 11 matched those of the polyphenols p-coumaric acid
(m/z 163.1) and ferulic acid (m/z 193.1). Peak detected at
37.2 min, resulted corresponding to that of the cinnamic
ccic :Icnccrc {Figure 5, IcL|e 4).
In the analysed samples, it was not possible assert or exclude
the presence of further polyphenolic molecules such as
vanillic acid, o-coumaric acid, tyrosol and pyrocathecol.
In fact, for these compounds only two of the three selected
parameters (Rt and UV spectra or Rt and MS spectra)
matched those of the polyphenolic standards (data not
The antioxidant activity of HME, higher in all tests than ME, as
well as increased total content of phenols, suggested to us
that the basic hydrolysis promotes the release of polyphenols
present into the starting material in complexed form. This
re:u|I wc: ccnfrmec Ly HFLC cnc|y:i:: Ihe chrcmcIcgrcm:
comparison displayed an increase of polyphenols in HME
compared with ME extract, together with the improvement of
the chemicals detection (Figure 1 and Figure 2).
Ihe c|ecvcge cf Ihe e:Ier Lcnc: grecI|y :imp|ife: Ihe cnc|y:i:
by reducing the number of derivatives and increases stability
and reproducibility of the sample.
Since Ihe icenIifccIicn cf peck: i: c ciffcu|I Ic:k ccn:icering
the number of components and complexity of plant samples,
different methods were explored to fractionate the crude
extracts (ME and HME) as well as to remove undesired
inIerfering ccmpcnenI:. Severc| purifccIicn meIhcc: :uch
as the liquid-liquid extraction, the solid/liquid extraction, the
purifccIicn cn SFE ccrIricge: cnc Ihe e|uIicn Ihrcugh cc|umn
chromatography have been thoroughly investigated. The
resulting mixtures were all analysed by HPLC for qualitative
determination. Best results were obtained from column
chromatography fractionation and particularly by using
a linear gradient of organic solvents (dichloromethane/
methanol, 10/0 to 8/2, v/v).
The characterization step was carried out comparing the
chromatographic data of the obtained mixtures with those
of a set of standard polyphenols selected because of their
Lic|cgicc| :ignifccnce, recurrence in vegeIcL|e exIrccI: cnc
commercial availability (24-26). The polyphenolic structures
selected were: trans-cinnamic acid, p-coumaric acid,
caffeic acid, ferulic acid, o-coumaric acid, pyrocathecol,
p-hydroxybenzoic acid, vanillic acid, gallic acid,
protocatechuic acid, syringic acid, tyrosol, homovanillic acid
Figure 2. HME chromatogram by HPLC-DAD at 270 nm.
Figure 3. Fractionament of the standard phenolic compounds by HPLC-DAD
at 270 nm. Peak numbers: (1) gal l i c aci d; (2) hydroxytyrosol ; (3)
prcIcccIechuic ccic: {4) pyrcccIhecc|: {5) Iyrc:c|: {) p-hycrcxyLenzcic
acid; (7) homovanillic acid; (8) vanillic acid and caffeic acid; (9) syringic
ccic: {10) p-ccumcrin ccic: {11) feru|ic ccic: {13) c-ccumcric ccic: {15)
cinnamic acid. Peak numbers 12, 14 and 16 are degradation products of
hydroxytyrosol.
Table 4. Phenolic acids identified by HPLC-DAD/APCI-MS in
LccLcL rec fiLre {i
max
= 270 nm).
Figure 4. HME chromatogram after purification by chromatography on silica
gel (Fraction D). Peak numbers are referred to Figure 3.
Figure 5. HME chrcmcIcgrcm cfIer purificcIicn Ly chrcmcIcgrcphy cn :i|icc
gel (Fraction B). Peak numbers are referred to Figure 3.
A
g
r
o
F
O
O
D

i
n
d
u
s
t
r
y

h
i
-
t
e
c
h

-

N
o
v
e
m
b
e
r
/
D
e
c
e
m
b
e
r

2
0
1
1

-

v
o
l

2
2

n

6
S
e
z
i
o
n
e
37
shown). We have speculated that the reasons of this result could
be correlated to interference which makes impossible to perform
an accurate UV spectra or does not consent compound ionization.
Further studies are required to better investigate the presence of
Ihe:e cnc cIher mc|ecu|e: in Ihe LccLcL rec fLre.
CONCLUSION
We have comparatively examined several tissues from the baobab
plant and, to the best of our knowledge, this is the first report of analysis
on the constituents of baobab red fibre using a simple, direct and
rapid RP-HPLC method with concomitant evaluation of antioxidant
capacity of crude fractions by four different complementary
techniques. The results of this study correlate baobab red fibre
antioxidant activity to the presences of polyphenols and, in our
opinion, also confirms the hypothesis that they may work as sacrificial
molecules in preserving pulp integrity upon fruit storage and even
after exposure to light and air. It was also possible to obtain red
fibre extracts, rich in polyphenols, and with increased antioxidant
activity compared to the parent fibre. HME was the most interesting
extract exhibiting high antioxidant capacity by the presence of not
linked polyphenols, deriving from the hydrolysis process. In addition,
this extract showed an antioxidant potency comparable to that of
BHT in DPPH and FRAP tests and clearly higher in ORAC test (Table
3). The high antioxidant activity of HME deserves further interest
and supports the baobab fruit as a valuable source of functional
molecules. Furthermore this data confirm the potential of the fruit
and other plant tissues (leafs) as nutrient in food indust ry.
AKNOWLEDGMENTS
Authors wish to thank Alberto Casolari for careful technical assistance.
This work was supported by Ambrosialab srl with 2008 and 2009 grants
and lab facilities.
REFERENCES AND NOTES
1. H. Xiuzhen, L. Hongxiang et al., Int. J. Mol. Sci., 8, pp. 50-88 {2007).
2. L. Badimon, T. Padro et al., Cardiovascular Therapeutics, 28(4), pp. 202-215
(2010).
3. C.S. Yang, X. Wang, Nutrition and cancer; 62(7), pp. 931-937 (2010).
4. S. Pinto Mda, K. Shetty et al., J. Med. Food., 13(5), pp. 1036-1044 (2010).
5. R. Casado, A. Landa et al., Pharm Biol., 49(6), pp. 620-626 (2011).
6. Q. Meng, R. Ruan et al., Free Radic. Biol. Med., 44(6), pp. 1032-1041 (2008).
7. H. Masaki, Journal of Dermatological Science, 58, pp. 85-0 {2010).
8. A.T. Diplock, G. Crozier-Willi et al., Br J Nutr., 80(Suppl 1), S77S112 (1998).
9. Y.F. Sasaki, A. Kamaya et al., Mutat Res Genet Toxicol Environ Mutagen.,
519(1-2), pp. 103-119 (2002).
10. R. Kahl, H.Z. Kappus, Lebensm Unters Forsch, 196(4), pp. 329-338 (1993).
11. I.C. Obizoba, J.U. Anyika, Plants Foods Hum Nutr., 46, pp. 157-15 {14).
12. J. Kerharo, J.G. Adam, Plantes Mdicales et Toxiques, Editions Vigot Frres,
Paris (1974).
13. C||c|c| Jcu|nc| c| tne Fu|cpecn Un|cn, Ccmmi::icn Deci:icn 2008/575/EC.
14. F.M. Ramadan, S.A. Harraz, Fitoterapia, 65, pp. 418-422 (1994).
15. A. Tal-Dia, O. Sarr et al., Dakar Med., 42, pp. 68-73 (1997).
16. E. Besco, S. Manfredini et al., Food Chem., 102, pp. 1352-135 {2007).
17. V.L. Singleton, J.A. Rossi, Am J Enol Vitic., 16, pp. 144-158 {15).
18. G. Sacchetti, M.V. Muzzoli et al., Food Chem., 91, pp. 21-32 {2005).
19. a) I. Popov, G. Lewis, Method Enzymol., 300, pp. 437-45 {1): L) l. Fcpcv,
G. Lewis et al., Biomedica Biochimica Acta., 46(11), pp. 775-77 {187).
20. M. Wang, M. Rangarajan et al., Agric Food Chem., 46, pp. 4869-4873 (1998).
21. F. Pessina, G. Sgaragli et al., Naunyn Schmiedebergs Arch Pharmacol.,
370, pp. 521-528 {2004).
22. W. Hong, RL. Prior et al., J Agric Food Chem., 44, 701-705 {1).
23. X. Guihua, L. Donghong et al., Agric Food Chem., 55(2), pp. 330-335 {2007).
24. H.K. Obied, M.S. Allen et al., J Agric Food Chem., 53, pp. 823-837 {2005).
25. N. Allouche, I. Fki et al., J Agric Food Chem., 52, pp. 267-273 (2004).
26. L. Lesage-Messen, D. Navarro et al., Food Chem., 75, pp. 501-507 {2001).
27. P.G. Baraldi, D. Simoni et al., Liebigs Ann Chem., 4, pp. 684-686 (19 83).