Asian Pac J Trop Biomed 2014; 4(Suppl 1): S417-S423 S417

Asian Pacific Journal of Tropical Biomedicine

journal homepage: www.apjtb.com

Document heading doi:10.12980/APJTB.4.2014C1038 襃 2014 by the Asian Pacific Journal of Tropical Biomedicine. All rights reserved.

A ntioxidantand anti-acetylcholinesterase activities of extracts and

secondary metabolites from Acacia cyanophylla
Lotfi Ghribia , Hatem Ghouilaa , Amel Omrib , Malek Besbesb , Hichem Ben Janneta *
1 1 2 2 1

Laboratoire de Chimie hétérocyclique, Produits Naturels et Réactivité. Equipe: Chimie Médicinale et Produits Naturels. Faculté des Sciences de
1

Monastir, Université de Monastir 5019, Monastir, Tunisie

Laboratoire des Maladies Transmissibles et des Substances Biologiquement Actives, Faculté de Pharmacie, 5000 Monastir, Tunisie
2

PEER REVIEW ABSTRACT

Peer reviewer Objective: To investigate the antioxidant potential and anti-acetycholinesterase activity of
Prof. Vanessa Steenkamp, Department compounds and extracts from Acacia cyanophylla (A. cyanophylla).
of P harmacology, U niversity of Methods: Three polyphenolic compounds were isolated from ethyl acetate extract of A.
Pretoria, South Africa. cyanophylla flowers. They have been identified as isosalipurposide 1, quercetin 2 and naringenin
E-mail: Vanessa.steenkamp@up.ac.za 3. Their structures were elucidated by extensive spectroscopic methods including 1D and 2D NMR
experiments as well as ES-MS. The prepared extracts and the isolated compounds 1-3 were tested
for their antioxidant activity using 1’-1’-diphenylpicrylhydrazyl (DPPH) and 2,2'-azinobis(3-
Comments ethylbenzothiazoline-6-sulfonic acid) scavenging assays and reducing power. They have been
This article provides information on also investigated for inhibitory effect against acetylcholinesterase using the microplate assay.
the biological activity of the specific Results: In the DPPH test, the EtOAc extract of flowers exhibited the highest antioxidant effect
plant which can be taken further. (67.26 µg/mL). Isosalipurposide 1 showed a significant antiradical power against DPPH (81.9 µg/mL).
There is a need for the discovery and All extracts showed a dose-dependent acetylcholinesterase inhibition. In terms of the IC50 value,
development of novel/alternative the butanolic extract (16.03 µg/mL) was the most potent sample. Isosalipurposide 1 was found to
natural antioxidants and AChEIs that be active against AChE with an IC50 value of 52.04 µg/mL.
are safe, affordable and effective at a Conclusions: The results demonstrated the important antioxidant and anti-acetylcholinesterase
global level. activity of pure compounds and extracts from A. cyanophylla.
Details on Page S422

KEYWORDS
Acacia cyanophylla, Isosalipurposide, Naringenin, Quercetin, Antioxidant activity, Anti-
acetylcholinesterase activity

1. Introduction as alkaloids, flavonoid, tannins, saponins generally

produced by plants for their defense mechanisms have
The use of plants in traditional medicine for treating been implicated in the therapeutic properties of most
various ailments remains an integral part of the culture medicinal plants[2]. Plants therefore provide an invaluable
and traditions of a majority of the world’s population. In resourse useful as leads in the development of therapeutic
addition, factors such as the availability, affordability compounds[3]. Indeed, many plant-derived drugs are either
and accessibility of medicinal plants have led to their in the clinical trial phase or currently used in treatment
high demand and usage [1]. Secondary metabolites such of ailments such as Alzheimer’s disease, and cancer[4].

*Corresponding author: Ben Jannet Hichem, Professor, Laboratoire de Chimie Article history:
hétérocyclique, Produits Naturels et Réactivité. Equipe: Chimie Médicinale et Produits Received 10 Jan 2014
Naturels. Faculté des Sciences de Monastir, Université de Monastir 5019, Monastir, Tunisie. Received in revised form 25 Jan, 2nd revised form 10 Feb, 3rd revised form 20 Feb 2014
Tel: +21673500279/280 Accepted 15 Mar 2014
Fax: +21673500278 Available online 5 Apr 2014
E-mail: hichem.benjannet@yahoo.fr
Foundation project: Supported by the Ministry of High Study and Scientific Research
(MHSSR) of Tunisia (Grant No. 11/TM06).
S418 Lotfi Ghribia, Hatem Ghouila et al./Asian Pac J Trop Biomed 2014; 4(Suppl 1): S417-S423

Oxidative stress arises mainly from the over production acid, ferric chloride (FeCl3), acetylcholinesterase (AChE) type
of free radicals due to an imbalance in production of VI-S, from electric eel 349 U/mg solid, 411 U/mg protein,
antioxidants by the cells[5]. Natural products especially from 5,5’-dithiobis(2-nitrobenzoic acid), acetylthiocholine iodide,
plants sources have the ability to reduce oxidative stress by tris(hydroxymethyl) aminomethane, were obtained from
acting as antioxidants[6]. Presently, a number of treatments Sigma-Aldrich. All other chemicals were of analytical grade
are used against Alzheimer’s disease as well as to counter purity.
the effect of oxidative stress. These include the use of
acetylcholinesterase inhibitors (AChEIs) and high levels of 2.2. Plant material
antioxidants. Some adverse effects such as hepatotoxicity,
gastrointestinal disturbances, nausea, vomiting, diarrhea Flowers of A. cyanophylla were collected in the region of
and dizziness have been reported with the use of most Monastir (Tunisia) in March 2010. The plant was identified by
AChEIs[7]. There is also an increasing concern about the use Prof. Fethia Harzallah-Skhiri in the Laboratory of Vegetal
of some common antioxidants. Butylated hydroxyanisole Biology and Botanic, High Institute of Biotechnology of
(BHA) and butylated hydroxytoluene (BHT) for example, have Monastir, Tunisia and a voucher specimen (AC-10) was
been implicated in incidence of toxicity especially against deposited in the same laboratory.
animal DNA as well as liver damage and carcinogenesis[8].
C onsequently, there is need for the discovery and 2.3. Isolation and identification of compounds
development of novel/alternative natural antioxidants and
AChEIs that are safe, affordable and effective at a global The dried and powdered flowers (2.5 kg) of A. cyanophylla
level[9]. were extracted with the mixture methanol/water (7:3) at
The genus Acacia is quite large and is widespread in the room temperature for 48 h. The corresponding aqueous
warm subarid and arid parts of the world, relatively little is extract was obtained after filtration and evaporation of
known about the chemistry of most species. The most evident the organic solvent (MeOH) under reduced pressure. The
substances in many Acacia species are complex phenolic obtained aqueous extract was extracted successively with
compounds (condensed tannins), polysaccharides (gums) and dichloromethane, ethyl acetate and n-butanol to yield the
flavonoid[10]. The difficulty of structure elucidation and the corresponding extracts (Figure 1). From all crude extracts,
overall lack of toxicity of these substances undoubtedly have only ethyl acetate (25 g) was further subjected to silica gel
contributed to a dearth of chemical study of these plants. column chromatography eluting with CH2Cl2/MeOH gradients.
The situation is further complicated because identification of Six main fractions (250 mL 伊 6) were collected. Fraction 5 (19
Acacia species is difficult and their taxonomic relationships g) was purified by column chromatography over silica gel
are not clear[10]. Acacia cyanophylla (A. cyanophylla) is a using CH2Cl2/MeOH (9:1) as eluent to afford isosalipurposide 1
legume shrub species, it was introduced in Tunisia for range (18 g, 0.72%) and quercetin 2 (50 mg).
land rehabilitation, particularly in the semi arid zones. This Dried flowers (2.5 kg)
shrub represents a potential forage resource particularly
E xtraction with methanol/
during periods of drought. water (7:3) 48 h at room
T he most evident substances in A. cyanophylla are temperature filtration and
evaporation of the solvent
flavonoids such as quercetin and kaempferol, while presence
of other flavanols has been indicated[11]. Aqueous extract I

S everal studies have reported on the plant A. Extraction with ethylacetate

cyanophylla allowed the isolation of some phenolic at room tempertature
compounds[10,12]. Therefore, this present paper describes
the isolation, structure elucidation, antioxidant and Aqueous extract II Dichloromethane extract
anti-acetylcholinesterase activities of extracts and Extraction with ethylacetate (0.47%)
isosalipurposide 1, quercetin 2 and naringenin 3 isolated at room tempertature
from flowers of A. cyanophylla.
Aqueous extract III Ethylacetate extract Subjected
Extraction with butanol at to colum
(1%)
room tempreature chromatography
2. Materials and methods

Butanol extract
2.1. Chemicals Aqueous extract IV
(0.16%)

Figure 1. Extraction of various extracts of A. cyanophylla in solvents of

1,1-Diphenyl-2-picrylhydrazyl (DPPH), quercetin, 2,2’- increasing polarity.
azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS),
6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid Using the system MeOH/HCl concentrated at reflux for
(Trolox), potassium ferricyanide [K3Fe(CN)6], trichloroacetic 2 h, we managed the hydrolysis of glucopyranosyl system
 Lotfi Ghribia, Hatem Ghouila et al./Asian Pac J Trop Biomed 2014; 4(Suppl 1): S417-S423
S419

in C-2. We found from the spectral data of the obtained 2.4.2. Scavenging activity of ABTS radical cation
product explained below that the reaction evolved into a T he ABTS radical cation scavenging activity was
cyclization product 3 (naringenin) involving the phenol measured according to the method described by
group at C - 6 and the α, β-unsaturated ketone system Chakraborty (2010)[14]. ABTS was dissolved in water to a
(Figure 2). 7 mmol/ L concentration. T he ABTS radical cation was

2’
3’ OH
2’
3’ OH produced by adding to the ABTS stock solution 2 . 45
mmol/L potassium persulphate (final concentration). The
4’ 1 4’
HO 4 5 OH 1’ 8
6 5’ HO 7 9 O 5’
α
6’
MeOH, HCI
2 6’ completion of radical generation was obtained in the
dark at room temperature for 12-16 h. This solution was
3 β 6 3
2
1 2 h, reflux 10 4
5
OGlt O 1 OH O 3 then diluted with ethanol to adjust its absorbance at 734
Figure 2. Synthesis of naringenin 3 from isosalipurposide 1. nm to 0.70±0.02. To determine the scavenging activity, 50
T he FT - IR spectra were established using a B io- µL of diluted ABTS solution was added to 950 µL of plant
R ad spectrophotometer with a resolution of 4 cm and extracts and compound 1 (or water for the control), and the
-1

scanning a wavelength range from 400 to 4 000 cm -1 . absorbance at 734 nm was measured 6 min after the initial
H - NMR ( 300 MH z ) and C - NMR ( 75 MH z ) spectra were mixing, using ethanol as the blank. The percentage of
1 13

recorded in deuterated CD3OD on a Bruker AC-300. All inhibition was calculated by the equation:
chemical shifts were reported as δ values (mg/L). Double %IP= [(A0-A1)/A0] 伊 100
resonance, COSY and HMBC experiments were carried out W here A 0 is the absorbance of control and A 1 is the
for complete assignment of proton and carbon signals in absorbance of reaction mixture, respectively from a plot
the NMR spectra, whenever possible mass spectra were of concentration against %IP, a linear regression analysis
obtained with an automass multi thermo finingam ES-MS was performed to determine the IC50 value for each plant
spectrometer. Column chromatography was performed on extract. Tests were carried out in triplicate.
SDS silica gel 60 F254 (70-200 µm) using dichloromethane
and methanol mixture as eluents. 2.4.3. Reducing power assay
This assay has been performed following the method of
2.4. Antioxidant testing assays Oyaizu (1986) with a slight modification[15,16]. It was used
to assess the reducing power of different extracts and
The antioxidant activity of extracts and pure compounds compound 1. A volume of 1 mL of different concentrations
was addressed by employing following in vitro assays. of extracts and compound 1 (0.062-1.0 mg/mL) and 0.75
mL of distilled water were mixed with 1 mL of 0.2 mol/
2.4.1. DPPH scavenging assay L sodium phosphate buffer ( p H 6 . 6 ) and 1 m L ( 1 % ) of
T he extracts and compounds 1 - 3 were measured in potassium ferricyanide [ K 3 F e ( CN ) 6 ]. T he mixture was
terms of hydrogen donating or radical scavenging ability incubated at 50 °C. After 20 min of incubation, the reaction
using stable radical DPPH following the method given by mixture was acidified with 1 mL of trichloroacetic acid
Sarker et al. (2006) with little modifications[13]. To 1 mL of (10%). Finally, 0.25 mL of FeCl3 (0.1%) was added to this
DPPH, (80 µg/mL in methanolic solution) were added 1 mL solution. Distilled water was used as blank and for control.
of extracts or pure compounds (0.062-1.0 mg/mL). After Absorbance of this mixture was measured at 700 nm using
incubation of 30 min in darkness and at a temperature of a UV-spectrophotometer. Decreased absorbance indicates
25 °C, absorbance was read at 517 nm wavelength. ferric reducing power capability of sample. Tests were
A mixture of 0.5 mL of DPPH• solution and 0.5 mL of carried out in triplicate.
ethanol was taken as a blank. D ecrease in absorption
induced by the tested samples was compared to that of the 2.5. Acetylcholinesterase enzyme inhibitory activity
positive control quercetin. IC50 values calculated denote
the concentration required to scavenge 50 % of DPPH • Inhibition of acetylcholinesterase by the extracts and
radicals. Results were expressed in inhibition percentage compound 1 was investigated using the microplate assay.
at different sample concentrations (mg/mL) after 30 min. The assay is based on Falé method[17].
I nhibition of free radical DPPH in percent ( IP % ) was Briefly, 90 µL of 50 mmol/L Tris-HCl buffer, pH=8.30
calculated as follow: of mixture and 7 . 5 µL of acetylcholinesterase solution
%IP= [(A0-A1)/A0] 伊 100 containing 0 . 26 U /m L were mixed in a microwell plate
W here A 0 is the absorbance of the control reaction and left to incubate for 15 min. Subsequently, 22.5 µL of a
( containing all reagent except the test compound) , and solution of AHCI (0.023 mg/mL) and 142 µL of 3 mmol/L 5,5’-
A 1 is the absorbance of the test compound. E xtract dithiobis(2-nitrobenzoic acid) were added. The absorbance
concentration providing 50% inhibition (IC50) was calculated was read at 405 nm when the reaction reached equilibrium.
from the graph plotted from inhibition percentage against A control reaction was carried out using water instead of
extract concentration. Tests were carried out in triplicate. extract or compound 1 and it was considered 100% activity.
S420 Lotfi Ghribia, Hatem Ghouila et al./Asian Pac J Trop Biomed 2014; 4(Suppl 1): S417-S423

%IP=100-(A1/A0) 伊 100 5.16 mg/kg as a starting point for the interpretation of the
Where A1 is the absorbance of the extract containing H- H COSY spectrum. The above spectral data (Table 1)
1 1

reaction mixture and A0 is the absorbance of the reaction. helped in assigning the structure to the compound 1 as
IP is the percentage of inhibition. Tests were carried out isosalipurposide.
in triplicate and a blank with Tris-HCl buffer instead of Table 1
enzyme solution was used. In the case of the standards, a 1
H- (300 MHz, solvent CD3OD) and C- (75 MHz, solvent CD3OD) NMR data for
13

blank with methanol was carried out as tested samples were compound 1.
dissolved in this organic solvent. Atoms
Compound 1
H (m, J Hz) C
1 13

1 - 107.8
2.6. Statistical analysis 2 - 168.2
3 6.01 (d, 2.1 Hz) 98.8
All experiments were repeated at least three times. Results 4 - 162.2

are reported as mean±SE. 5 6.23 (d, 2.1 Hz) 96

6 - 166.3
1’ - 128.3
2’ 7.65 (d, 8.7 Hz) 132.2
3. Results 3’ 6.86 (d, 8.7 Hz) 117.3
4’ - 161.5

3.1. Characterization of compounds 5’ 6.86 (d, 8.7 Hz) 117.3

6’ 7.65 (d, 8.7 Hz) 132.2
1’’ 5.16 (d, 7.2 Hz) 102.2
The repeated column chromatograohy of ethyl acetate 2’’ 3.46-3.58 (m) 75.4
extract of flowers of A. cyanophylla resulted in the isolation 3’’ 3.46-3.58 (m) 78.9
of compound 1 and compound 2 (Figure 3). They are yellow 4’’ 3.46-3.58 (m) 71.5

solid, the first, weight 18 g (0.72%) and its molecular formula, 5’’ 3.46-3.58 (m) 78.8
b 3.76 (m) 62.8
C21H22O10, [M+Na] at m/z 457 and [M-H] at m/z 433 was
+ - 6’’a,
3.94 (m)
established by ES-MS. α 7.69 (d, 15.6 Hz) 128.9
3’ OH
OH 8.03 (d, 15.6 Hz) 144.6
2’
4’
β
5 OH
HO 4 6 OH
1’
5’ 2’ C=O - 194.8
H OH α 6’ 8 1
6’’ HO O 5’
4’’ 5’’ H O 3 2 1 β
HO 2 6’ S pectral data ( T able 2 ) of compounds 2 and 3 have
HO 1’’ O O
contributed to the proposal of the two structures of quercetin
6
3’’ H 2’’OH 4
3 OH
H H
Compound 1(isosalipurposide) 3’ OH O 2 and naringenin 3 (Figure 3).
2’ Compound 2 (quercetin)
4’ Table 2
8 1
1’
HO O H- (300 MHz, solvent CD3OD) and C-(75 MHz, solvent CD3OD) NMR data for
1 13
7 9 5’
2 6’
compounds 2 and 3.
6 3
10
5 4 Compound 2 Compound 3
Compound 3 (naringnin) Atoms
OH O H (m, J Hz) C H (m, J Hz) C
1 13 1 13

Figure 3. Structure of compounds 1, 2 and 3. 2 - 157.1 5.28 (dd, 13 Hz; 3 Hz) 80.8
3a, b - 136.1 2.64 (dd, 17.1 Hz; 3 Hz) 44.4
T he C and H - NMR spectra of 1 showed the typical
13 1
3.06 (dd, 17.1 Hz; 13 Hz)
signals for a β-D-glucopyranose with the doublet for the 4 - 176.2 - 198.1

anomeric proton H1’’ at δ 5.16 mg/kg and the resonance for 5 - 161.3 - 165.8
6.34 (d, 1.8 Hz) 98.1 5.87 (s) 97.5
the corresponding carbon C1’’ at δ 102.2 mg/kg (Table 1). The 6
7 - 164.4 - 168.7
IR spectrum displayed intense absorption bands for hydroxy 8 6.14 (d, 1.8 Hz) 93.3 5.87 (s) 96.6
(3 482 cm ), α,β-unsaturated carbonyl (1 624 cm ) and olefin
-1 -1
9 - 147.6 - 165.2
(1 591 cm ) functionalities. The H-NMR spectrum showed the
-1 1
10 - 103.4 - 103.7

signals of an olefin at δH 7.69 (H-α) and 8.03 (H-β), a para- 1’ - 123 - 131.5
7.69 (d, 2.1 Hz) 114.9 7.28 (d, 9.3 Hz) 129.4
substituted aromatic ring at δH 6.86 (H-3’ and H-5’) and 7.65 2’
3’ - 146.9 6.80 (d, 9.3 Hz) 116.7
(H-2’ and H-6’), a 1,2,4,6-tetrasubstitued aromatic ring at 4’ - 145.1 - 159.3
δH 6.01 (H-3), 6.23 (H-5). The 13C-NMR and DEPT 135 spectra 5’ 6.83 (d, 8.4 Hz) 115.1 6.80 (d, 9.3 Hz) 116.7
showed the existence of these structural moieties and one 6’ 7.59 (dd, 8.7 Hz; 2.1 Hz) 120.5 7.28 (d, 9.3 Hz) 129.4

conjugated ketone at δC 194.8 mg/kg. The HMBC spectrum,

which displayed correlation between the anomeric proton 3.2. Antioxidant acivity
of the β-D-glucopyranose at δH 5.16 mg/kg (1H, d, J=7.2 Hz,
H1’’) and C2 at δC 168.2 mg/kg, reinforced the deduction of the 3.2.1. DPPH radical scavenging activity
glycosilation site as C2. Assignment of the sugar resonances Compounds 1-3, dichloromethane, EtOAc, n-BuOH and
was achieved using the anomeric proton resonance at δH aqueous extracts were screened for DPPH radical scavenging
 Lotfi Ghribia, Hatem Ghouila et al./Asian Pac J Trop Biomed 2014; 4(Suppl 1): S417-S423
S421

activity. The concentrations of pure compounds 1-3 required 3.3. Anti-acetylcholinesterase activity
to neutralize 50% of DPPH range between 4.58 and 255.5 µg/mL
(Table 3). T he acetylcholinesterase inhibitory activity of
Table 3 isosalipurposide 1 and the organic extracts were measured
Total antioxidant activity of 1-3 and A. cyanophylla extracts against DPPH, using the microplate assay expressed as IC50 and values are
expressed in µg/mL. given in Table 5.
Samples IC50 (µg/mL) Table 5
1 81.90±0.81
Anti-acetylcholinesterase activity of isosalipurposide 1 and extracts from A.
Quercetin 2 4.58±0.19
cyanophylla.
Naringenin 3 255.5±1.31
Samples IC50 (µg/mL)
E1 164.64±5.76
1 52.04±1.91
E2 67.26±2.00
E1 20.01±0.78
E3 122.54±2.86
E2 27.12±1.35
E4 314.66±6.72
E3 16.03±0.64
Quercetin 4.77±0.18
a

Trolox
a
14.56±0.62 E1: Dichloromethane extract, E2: Ethyl acetate extract, E3: Butanol extract.

E1: Dichloromethane extract, E2: Ethyl acetate extract, E3: Butanol extract,
E4: Aqueous extract, : reference compounds.
a

4. Discussion

The ethyl acetate extract of A. cyanophylla on thin layer

3.2.2. ABTS radical cation scavenging activity
chromatography showed the presence of important number
C ompound 1 , dichloromethane, E t OA c, n- B u OH and
of components. The repeated column chromatography of
aqueous extracts were screened for ABTS radical scavenging
this mixture caused isolation of an important amount of
activity (Table 4).
Table 4 compound 1 (19 g, 0.072% yield) and compound 2 (50 mg).
Total antioxidant activity of A. cyanophylla extracts against ABTS, expressed T he samples were found to be analytically pure by
in µg/mL. thin layer chromatography. The samples was identified
Samples IC50 (µg/mL) as isosalipurposide and quercetin using spectroscopic
1 310.34±2.97 techniques viz. 1D, 2D-NMR and mass. Spectral data of
E1 78.37±0.18 compounds 2 and 3 (Figure 3) compared to the literature
E2 305.68±1.36 allowed us to establish their structures as quercetin and
315.91±1.85
E3
naringenin[18,19], respectively.
E4 399.40±3.57
T he geometry of the olefin of a chalcone moiety in
Trolox 200.02±0.74
a

compound 1 was determined to be E on the basis of the 1H-

E1: Dichloromethane extract, E2: Ethyl acetate extract, E3: Butanol extract,
H coupling constant (JH-α-H-β=15.6 Hz)[20]. Identification of
1

E4: Aqueous extract, : reference compound.

a

the sugar as β-D-glucopyranose was supported by the 3JH1’’-

H2’’ coupling constant of 7.2 Hz as well as by the trans di-
axial couplings between H2’’-H3’’, H3’’-H4’’ and H4’’-H5’’[21].
3.2.3. Reducing power assay
The assignment of the structure of compound 1 is in
This method gives a clear idea about the antioxidant
consonance with the previous report of Filippo (1978) and
power of pure products or extracts. Figure 4 portrays that
Hatem et al. (2012)[11,12]. For bio-evaluation studies, to the
compound 1, dichloromethane, EtOAc, n-BuOH and aqueous
best of our knowledge, no biological studies were performed
extracts reduced Fe3+ to ferrous ions (Fe2+) at 0.062-1.0 mg/mL
on this compound.
concentrations.
1.4 A review of the literature indicated that chalcones
1.2
showed anti-infective, anti-inflammatory and cytotoxic
properties[22,23]. Rupashree and Mitali (2011) reported that
Absorbance 700 nm

1
chalcones also had a leishmanicidal activity and anti-
0.8 tuberculosis effect as indicated by Hemshekhar et al. (2011)
0.6 [24,25].

0.4 Proton-radical scavenging action is an important attribute

of antioxidants, which is measured by DPPH radical
0.2
scavenging assay. DPPH is a free radical, a protonated
0
0 200 400 600 800 1000 1200
radical, has characteristic absorbance maximum at 517 nm,
Sample (µg/mL)
stable at room temperature, which produces a violet solution
E11 E2 E3 E4 BHT in ethanol. In presence of antioxidant compounds, the DPPH
Figure 4. Reducing power potential of 1 and extracts in reducing power assay. is reduced producing a yellow ethanol solution. It is known
E1: Dichloromethane extract, E2: Ethyl acetate extract, E3: Butanol extract, E4: Aqueous that blocking one or more phenol groups in the flavonoids
extract, BHT (Butylated hydroxytoluene): reference compound. and their derivatives such as chalcones inhibited in a
S422 Lotfi Ghribia, Hatem Ghouila et al./Asian Pac J Trop Biomed 2014; 4(Suppl 1): S417-S423

manner sometimes significant antioxidant powers[19]. For reducing power.

this reason and in order to improve the possible antioxidant Inhibition of AChE, the key enzyme in the breakdown of
activity of isosalipurposide 1, we thought to regenerate an acetylcholine, is considered one of the treatment strategies
additional phenol group fixed in C-2 via an acid hydrolysis against several neurological disorders such as Alzheimer’s
reaction (Figure 2). disease, senile dementia, ataxia and myasthenia gravis[34,35].
I n a preliminary analysis of plant extracts for their Plants have been traditionally used to enhance cognitive
antioxidant activity, we found that CH2Cl2, EtOAc, n-BuOH function and to alleviate other symptoms associated
and aqueous extracts of dried flowers of A. cyanophylla nowadays with Alzheimer’s disease[36]. The AChE inhibition
exhibited a significant activity. Antiradical properties of was determined using an adaptation of the method described
flowers of A. cyanophylla are given in Table 1. Quercetin 2 in the literature[17].
(4.58 µg/mL) has given the most important activity, followed I sosalipurposide 1 found to be active against
by isosalipurposide 1 (81.9 µg/mL) then naringenin 3 (255.5 acetylcholinesterase with an IC50 value of 52.04 µg/mL by
µg/mL) (Table 1). We note that the values of IC50 of quercetin comparison to some results cited in the literature. IC50 of
used as a reference and that isolated from flowers of A. dichloromethane (20.01 µg/mL), ethyl acetate (27.12 µg/
cyanophylla are almost similar[26]. Isosalipurposide 1 was mL) and butanol (16.03 µg/mL) extracts of A. cyanophylla
found to be three times more active than naringenin 3 in expressed in (Table 3) register encouraging results.
which the 2-OH may not contribute to this activity by its
implication in a hydrogen bond with the carbonyl function
forming a six center pseudo-ring[27]. The transformation Conflict of interest statement
of the chalcone 1 ( isosalipurposide) to the flavanone 3
(naringenin), led to the disappearance of the 6-OH group, and The authors report no conflicts of interest. The authors are
therefore the creation of the cycle C without the double bond alone responsible for the content and writing of the paper.
Cα-Cβ. These results are consistent with the relationships
between the structure of flavonoids and their ability to trap
free radicals. Indeed, numerous studies converge to suggest Acknowledgements
that the activity depends primarily on the following three
criteria[27,28], the presence of a catechol group in ring B (3‘, We are grateful to Dr Fethia Harzallah-S khiri, High
4’-OH), the C2-C3 double bond conjugated with the 4-oxo Institute of Biotechnology of Monastir, Tunisia for botanical
function and the presence of the 3-OH group. identification and to Mrs Amna Benzarti, Department of
The ethyl acetate extract had the highest antioxidant Chemistry, Faculty of Science of Monastir, for NMR analysis.
activity (67.26 µg/mL). This relatively high activity may be Supported by the Ministry of High Study and Scientific
due to its richness in phenolic compounds. The CH2Cl2, Research (MHSSR) of Tunisia (Grant No. 11/TM06).
n-BuOH and aqueous extracts present moderate activities
(164.64, 122.54, 314.66 µg/mL, respectively), probably due to
the weak amounts of phenolic compounds[29]. Comments
The ABTS method gives a measure of the antioxidant
activity of extracts or compound 1 by measuring the Background
reduction of the radical cation at 734 nm. Chakraborty The authors assessed the antioxidant potential and anti-
(2010) reported that the decolorization of the ABTS cation acetylcholinesterase activity of compounds and the crude
+.

reflects the capacity of an antioxidant to donate electrons or extract of A. cyanophylla.

hydrogen atoms to inactive this radical species[14].
C ompound 1 ( isosalipurposide ) showed an important Research frontiers
activity through its free OH groups (310.34 µg/mL). With ABTS, Testing of isolated compounds for activity for identification
the CH2Cl2 extract was found to be the more active one (78.37 of compounds will be used in future drug development.
µg/mL) compared to that of EtOAc, n-BuOH and aqueous
extracts (305.68, 315.91, 399.40 µg/mL, respectively). Related reports
Reducing power was measured by direct electron donation Articles on antioxidant and anti-acetylcholienesterase
of Fe3+ (CN−)6- Fe2+ (CN−)6[30]. The product was visualized by activity of some of the isolated compounds are available.
forming the intense blue color complex and then measured
at 700 nm. By increasing the concentration of the extracts Innovations and breakthroughs
and compound 1, we favor the greater the reduction of Fe3+ Determining biological activity of the isolated compounds
to Fe2+. This finding justifies the reducing power of these can be taken further.
samples[31-33]. The ethyl acetate extract from A. cyanophylla
might contain a higher amount of reductone, which could Applications
react with free radicals to stabilise and terminate radical Further tests to assess activity can be carried out in the
chain reactions. These results indicate that containing future study based on this paper.
flavonoids and polyphenols may play an important role in
 Lotfi Ghribia, Hatem Ghouila et al./Asian Pac J Trop Biomed 2014; 4(Suppl 1): S417-S423
S423

Peer review activity in herbal tea of Plectranthus barbatus (“falso boldo”).

This article provides information on the biological activity Food Chem 2009; 114: 798-805.
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