Food Chemistry 125 (2011) 193–200

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Food Chemistry
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Antioxidant constituents from Lawsonia inermis leaves: Isolation, structure

elucidation and antioxidative capacity
Anis Ben Hsouna a, Mohamed Trigui a, Gérald Culioli b, Yves Blache b, Samir Jaoua a,c,⇑
a
Laboratory of Biopesticides, Centre of Biotechnology of Sfax, P.O. Box: ‘‘1177”, 3038 Sfax, Tunisia
b
Laboratoire MAPIEM (EA 4323), Equipe «Biofouling and Substances Naturelles Marines», Université du Sud Toulon-Var, Avenue de l’Université, BP 20132,
F-83957 La Garde Cedex, France
c
University of Qatar, College of Arts and Sciences, P.O. Box 2713, Doha, Qatar

a r t i c l e i n f o a b s t r a c t

Article history: The antioxidant potential of different fractions of Lawsonia inermis (Lythraceae) was investigated. The n-
Received 18 March 2010 butanolic fraction showed the highest yield of extraction; it also exhibited a strong antioxidant activity in
Received in revised form 23 August 2010 the DPPH assay and a potent capacity in preventing linoleic acid oxidation. Five phenolic glycosides were
Accepted 25 August 2010
identified in this fraction. The structure of a new compound was established as 1,2,4-trihydroxynaphtha-
lene-1-J-b-D-glucopyranoside. In addition, the known 2,4,6-trihydroxyacetophenone-2-J-b-D-glucopy-
ranoside was described for the first time in this species. The three other compounds, lalioside (2,3,4,
Keywords:
6-tetrahydroxyacetophenone-2-J-b-D-glucopyranoside), lawsoniaside (1,2,4-trihydroxynaphthalene-
Lawsonia inermis
Naphthalene glycosides
1,4-di-J-b-D-glucopyranoside) and luteolin-7-J-b-D-glucopyranoside, have been previously reported in
Acetophenone glycosides L. inermis. The antioxidant activity of these glycosides was evaluated by DPPH and b-carotene assays,
1,2,4-Trihydroxynaphthalene-1-O-b-D- and compared to those of commercial standards. 1,2,4-Trihydroxynaphthalene-1-J-b-D-glucopyranoside
glucopyranoside was the most active in the DPPH free-radical scavenging test (EC50 = 6.5 lg/ml) and showed a moderate
Antioxidant activities inhibition in the b-carotene bleaching assay. Chemical components of L. inermis have good antioxidant
capacities and this species could be used as a potential source of new natural antioxidants.
Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction or broadly lanceolate, entire, acute or obtuse, 2–3 cm long and 1–

Antioxidants play an important role in preventing undesirable
changes in flavour and nutritional quality of foods and may reduce
the risk of a wide spectrum of chronic diseases, such as carcinogen-
esis, diabetes, or inflammation (Vasundhara, Vijay, & Jagan, 2008).
Synthetic antioxidants are widely used as food additives, but their
application has been reassessed because of the formation of possi-
ble toxic or carcinogenic components during their degradation
(Barlow, 1990; Linderschmidt, Trylka, Good, & Witschi, 1986).
Hence, identification and isolation of new antioxidants from natu-
ral sources has become an active area of research. A number of nat-
ural products, such as flavonoids, coumarins, curcuminoids or
terpenes, isolated from plants have shown potent antioxidant
activity and low toxicity (Yang et al., 2007).
Tunisian flora is known for its diversity of medicinal plants such
as Lawsonia inermis (syn. Lawsonia alba), commonly known as ‘hen-
na’. This plant is a glabrous, much branched shrub or small tree
with greyish-brown bark. Leaves are opposite, sub-sessile, elliptic
⇑ Corresponding author. Address: Centre of Biotechnology of Sfax, Laboratory of
Biopesticides, P. O. Box ‘‘1177”, Sfax 3038, Tunisia. Tel./fax: +216 74874446.
E-mail address: Samir.jaoua@cbs.rnrt.tn (S. Jaoua).
2 cm wide (Muhammad & Muhammad, 2005). This plant is fre-
quently cultivated in India, Middle East and along the African
coasts of the Mediterranean Sea. Besides its use in cosmetics for
staining hands and as a hair dye, the leaves are used as a prophy-
lactic agent against skin diseases (Ahmed et al., 2000).
Phytochemical investigations of L. inermis have shown predom-
inantly the presence of phenolic compounds (coumarins, flavo-
noids, naphthalene and gallic acid derivatives) which could be
glycosylated (Siddiqui, Kardar, Ali, & Khan, 2003). Other com-
pounds, such as triterpenoids, steroids and aliphatic hydrocarbons
have been also isolated from this plant (Siddiqui & Kardar, 2001;
Siddiqui et al., 2003). However, no correlation has been proved be-
tween the biochemical composition of this plant and its therapeu-
tic uses. The screening of 20 plant species used by Yemeni
traditional healers to treat infectious diseases showed that the
ethyl acetate extract of L. inermis was found to be the most active
against both Gram+ and Gram bacteria (Ali, Juelich, Kusnick, &
Lindequist, 2001). In addition, an ethanolic extract of the whole
plant possessed antifungal (Misra & Dixit, 1979) and antitubercular
activities (Bhatnagar et al., 1961). The decoction of bark and leaves
has been found to inhibit trypsin enzymes (Prasad & Gupta, 1967)
and showed anti-inflammatory activity (Singh, Shrivastava, Modi,

0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2010.08.060
194 A.B. Hsouna et al. / Food Chemistry 125 (2011) 193–200
& Saifi, 1982). The bark was also known to alleviate serum trans-
aminase levels and to restore normal bile flow.
The aim of the present study was to evaluate the antioxidant
and radical-scavenging activities of n-hexane, chloroform, ethyl
acetate, n-butanol and water fractions obtained from the hydroal-
coholic extract of L. inermis leaves. The amounts of total phenolics
in these fractions were also determined. As the n-butanolic fraction
showed the strongest antioxidant activity, it was subjected to fur-
ther fractionation by flash chromatography (FC). The chemical
composition of the most active fractions was analysed by LC/
DAD-ELSD-ESI/MSn and five phenolic glycosides were isolated by
semi-preparative reversed-phase HPLC. NMR and MS analyses of
these compounds allowed the unambiguous characterisation of
their chemical structure. The purified molecules were the subject
of antioxidant activity evaluation by measuring their capacity to
scavenge DPPH and to inhibit the bleaching of b-carotene.
2. Materials and methods
2.1. Chemicals
2,2-Diphenyl-1-picrylhydrazyl (DPPH), butylated hydroxytolu-
ene (BHT), b-carotene, linoleic acid, ascorbic acid, Folin–Ciocalteu
reagent, Tween 20 and sodium carbonate were purchased from
Sigma–Aldrich Co. (St. Louis, MO). All solutions were freshly pre-
pared in distiled water. The solvents used for chromatographic
studies were all of HPLC grade and were from Fisher Scientific
(Illkirch, France) while extraction and partition were realised with
solvents from Sigma–Aldrich.
2.2. Plant material
L. inermis leaves were collected from Sfax (Tunisia, 35.23°N and
11.11°E) in May 2008. After the botanical identification of the spe-
cies, a voucher specimen (LBPes 03) was deposited in the Herbar-
ium of the Laboratory of Biopesticides (Centre of Biotechnology
of Sfax, Tunisia) for future reference.
2.3. Phytochemical study of L. inermis
2.3.1. Preparation of plant extracts
The L. inermis fresh leaves were dried and fine powdered. The ob-
tained powder (100 g) was extracted by maceration into 600 ml of
aqueous ethanol (ethanol/water, 4:1, v/v) with occasional shaking,
at room temperature. After 3 days, the extract was filtered through
a 45-lm filter and was concentrated under vacuum. The resulted
residue (22 g) was suspended in 100 ml of distiled water and
sequentially partitioned into n-hexane (3  250 ml), chloroform
(3  250 ml), ethyl acetate (3  250 ml) and then n-butanol
(3  250 ml). The resulting five solutions were concentrated in vacuo
to dryness, to give n-hexane (1.92 g), chloroform (0.28 g), ethyl ace-
tate (1.97 g), n-butanol (6.41 g) and water (8.84 g) soluble fractions.
These fractions were kept at 4 °C in the dark until further analysis.
2.3.2. Fractionation of the n-butanolic fraction using flash
chromatography
Separation of phenolic constituents from the n-butanolic frac-
tion of L. inermis was carried out by FC on a Spot Flash (Armen
Instruments; Saint Ave, France) chromatographic system. For frac-
tionation, 1.5 g of this extract were dissolved in 5 ml of methanol
and mixed with 20 g of reversed-phase silica gel (Sepra C18e,
50 lm; Phenomenex, Torrance, CA). Methanol was removed by
evaporation under reduced pressure at 35 °C. The obtained dry ex-
tract of henna was then loaded on a guard column on the top of the
reversed-phase cartridge (SuperVarioFlash D26-RP18 model, 40–
63 lm, 37 g; Merck, Darmstadt, Germany) used for the fraction-
ation. The separation process was carried out using a ternary sol-
vent system: water (solvent A), methanol (solvent B) and
dichloromethane (solvent C). After an initial isocratic step with
90% of A and 10% of B from 0 to 2 min, a linear gradient up to
100% of B from 2 to 15 min, an isocratic step with 100% of B from
15 to 17 min, and a linear gradient up to 100% of C from 17 to
20 min, an isocratic step with 100% of C was finally used from 20
to 25 min. Concerning the flow rate, an initial linear ramp was used
from 2 to 20 ml/min in 2 min and then this final flow rate was
maintained during all the fractionation process. The separation
was monitored by UV detection at 280 nm. All sub-fractions were
automatically collected, evaporated and then weighed.
2.3.3. LC-DAD-ELSD-ESI/MSn analysis
In order to check the purity of the constituents and to deter-
mine their molecular mass, all sub-fractions obtained after the FC
fractionation of the n-butanolic extract were analysed by LC-
DAD-ELSD-ESI/MSn. Their chromatographic profiles were com-
pared with those of the crude n-butanolic fraction. For this exper-
iment, a LaChrom Elite HPLC (VWR-Hitachi, Fontenay-sous-Bois,
France) was used, which comprised an L-2130 quaternary pump,
an L-2200 autosampler and an L-2300 column oven. Detection
was performed with an L2455 PDA detector and an evaporative
light scattering detector (ELSD; Chromachem model; Eurosep, Cer-
gy-Saint Christophe, France) coupled to an ion trap mass spectrom-
eter fitted with an electrospray ionisation (ESI) interface (Esquire
6000; Bruker Daltonics, Bremen, Germany). Operating conditions
for MS analysis were: dry temperature, 350 °C; capillary voltage,
4000 V; nebuliser, 50 psi; dry gas, helium at 12 l/min. Ion trap
full-scan analysis was conducted from m/z 50 to 1200 with an
upper fill time of 200 ms. One milligram of each fraction was dis-
solved in 1 ml of a mixture of methanol/water (1:1, v/v) and a
10-ll sample volume was injected into the chromatographic
system.
Compounds separation was achieved using an analytical re-
versed-phase column (Gemini C6-Phenyl, 250  3 mm, 5 lm, Phe-
nomenex) maintained at 30 °C. The gradient elution was optimised
and consisted of a binary mobile phase in which eluent A was 1%
(v/v) formic acid in water and eluent B was 1% (v/v) formic acid
in acetonitrile. The elution was started with 30% of B and ascended
to 50% of B in 20 min (linear ramp) and 100% B in 5 min (linear
ramp). This composition was maintained for 5 min until re-equili-
bration of the system to the initial conditions (5 min). The flow rate
was fixed at 0.5 ml/min.
2.3.4. Purification of phenolic glycosides by semi-preparative HPLC
The main chemical components of selected sub-fractions ob-
tained from the n-butanolic fraction of henna were separated by
semi-preparative reversed-phase HPLC. This purification was rea-
lised on a Biotek-Kontron (Montigny-le-Bretonneux, France) HPLC
system equipped with a ternary pump (Model 525), an auto sam-
pler (Model 560) and a column oven (Model 582). The detection
was made with a UV detector (LDC Analytical, Model 3100) at
280 nm. The separation of the main constituents of these fractions
was achieved on a semi-preparative reversed-phase column (Gem-
ini C6-Phenyl, 250  10 mm, 5 lm, Phenomenex) with the same
gradient of elution previously used for the LC–MS analysis.
2.3.5. Identification of phenolic glycosides
The structures of the phenolic glycosides isolated in this study
were determined through extensive spectrometric analysis (ESI/
MS, 1D and 2D-NMR) and by comparison with data reported in
the literature. High resolution mass spectra (HR-ESI/MS) were ob-
tained on an LTQ Orbitrap mass spectrometer (Thermo Finnigan,
San José, CA). 1D (1H, 13C and DEPT) and 2D (COSY 1H–1H, HSQC,
 A.B. Hsouna et al. / Food Chemistry 125 (2011) 193–200
HMBC and NOESY 1H–1H) NMR spectra were obtained at 400 and
100 MHz for 1H and 13C, respectively, on an Avance 400 MHz
NMR spectrometer (Bruker BioSpin, Rheinstetten, Germany) in
DMSO-d6, D2O or CD3OD. All chemical shifts were referenced to
the solvent peaks (dH 2.50 and dC 39.5 for DMSO-d6, dH 3.31 and
dC 49.0 for CD3OD and dH 4.79 for D2O).
2.4. Determination of the total phenolic content
Total phenolic content (TPC) was determined using the Folin–
Ciocalteu method (Waterman & Mole, 1994) adapted to a micro-
scale. Briefly, 10 ll of diluted sample solution were shaken for
5 min with 50 ll of Folin–Ciocalteu reagent. Then 150 ll of 20%
Na2CO3 were added and the mixture was shaken once again for
1 min. Finally, the solution was brought up to 790 ll by the addi-
tion of distiled water. After 90 min, the absorbance at 760 nm
was evaluated using a spectrophotometer (SmartSpecTm3000;
Bio-Rad; Hercules, CA). Gallic acid was used as an internal standard
for the calibration curve. The phenolic content was expressed as
mg of gallic acid equivalent (GAE)/gram of dry sample using the
linear equation based on the calibration curve.
2.5. Antioxidant assays
2.5.1. Determination of DPPH radical-scavenging capacity
Radical-scavenging activity of the different fractions was deter-
mined using DPPH radical as a reagent, according to the method of
Kirby and Schmidt (1997), with some modifications. Briefly, 1 ml of
a 4% (w/v) solution of DPPH radical in ethanol was mixed with
500 ll of sample solution in ethanol (different concentrations).
The mixture was incubated for 20 min in the dark at room temper-
ature. Scavenging capacity was read spectrophotometrically by
monitoring the decrease of the absorbance at 517 nm. Lower absor-
bance of the reaction mixture indicates higher free radical-scaveng-
ing activity. Ascorbic acid was used as standard. The percent DPPH
scavenging effect was calculated using the following equation:
DPPH scavenging effect ð%Þ ¼ ðAcontrol  Asample =Acontrol Þ  100:
where Acontrol is the absorbance of the control reaction, where the
sample is replaced by 500 ll ethanol. Tests were carried out in
triplicate.
2.5.2. b-Carotene bleaching assay
The antioxidant activity was determined according to the b-car-
otene bleaching method described by Pratt (1980). A stock solution
of b-carotene/linoleic acid mixture was prepared by dissolving
0.5 mg of b-carotene in 1 ml of chloroform with 25 ll of linoleic
acid and 200 mg of Tween 20. Chloroform was completely evapo-
rated, using a vacuum evaporator. Then, 100 ml of distiled water,
saturated with oxygen (30 min), were added and the obtained
solution was vigorously shaken.
Four millilitres of this reaction mixture were dispensed into test
tubes and 200 ll of each sample, prepared at different concentra-
tions, were added. The emulsion system was incubated for 2 h at
50 °C. The same procedure was repeated with BHT as positive con-
trol, and a blank as a negative control. After this incubation period,
the absorbance of each mixture was measured at 490 nm. Antiox-
idant activity in b-carotene bleaching model as a percentage (A%)
was calculated with the following equation:
A% ¼ 1  ðA0  At =A00  A0t Þ  100;
where A0 and A00 are absorbances of the sample and the blank,
respectively, measured at zero time, and At and A0t are absorbances
195
of the sample and the blank, respectively, measured after 2 h. All
tests were carried out in triplicate.
2.6. Statistical analysis
Experimental results from this study were expressed as
means ± standard deviation of three parallel measurements. The
significance of difference was calculated by Student’s t-test, and
values p < 0.05 were considered to be significant. Correlation and
regression analysis was carried out using Microsoft Excel.
3. Results and discussion
3.1. Recovery percent and phenolic content of L. inermis fractions
The crude aqueous ethanolic extract of L. inermis leaves com-
pounds was fractionated by sequential extraction with solvents
of increasing polarity: n-hexane, chloroform, ethyl acetate, n-buta-
nol and water. It is well known that phenolic substances contribute
directly to the antioxidant activity of plant extracts (Rice-Evans,
Miller, & Paganga, 1996). Therefore, it was quite important to eval-
uate the total phenolic content (TPC) of these five fractions. Extrac-
tion yield, TPC, EC50 of DPPH assay and b-carotene bleaching
method of the different solvent fractions obtained from L. inermis
hydroalcoholic extract are given in Table 1. The amount of extract-
able compounds, expressed as percentage by weight of dried pow-
der, ranged from 0.29% for the chloroform extract to 6.74% and
8.84% for the n-butanol and water extracts, respectively. The TPC
of each fraction was determined according to the Folin–Ciocalteu
method and expressed as mg GAE/g of extract. As shown in Table
1, the highest levels of TPC were found in the water extract, fol-
lowed by ethyl acetate and n-butanol extracts. In contrast, TPC of
n-hexane fraction was the lowest. This result was in agreement
with recent findings of Tunalier, Kosar, Kupeli, Calis, and Baser
(2007) who demonstrated that the yield of aqueous extracts of Ly-
thrum salicaria (Lythraceae) was significantly higher than those ob-
tained with organic solvents. This variation might be due to the
better solubility of phenolic constituents in water. Additionally, a
linear correlation between the extraction yield and the total ex-
tracted phenolics was observed (r2 = 0.923).
3.2. Antioxidant activity of L. inermis fractions
3.2.1. DPPH free radical-scavenging activity of L. inermis fractions
The imbalance between reactive oxygen species (ROS) and anti-
oxidant defence mechanisms leads to oxidative modification in
cellular membrane or intracellular molecules. Phytochemicals like
phenolics, commonly found in plants, could constitute strong nat-
ural antioxidants and could play an important role in health. The
DPPH radical-scavenging assay was used to evaluate the ability
of a compound or an extract to scavenge free radicals and to pro-
mote the formation of the non-radical form DPPH-H by hydro-
gen-donating action (Chung, Chien, Teng, & Chou, 2006). Free
radical-scavenging capacities of the different fractions, measured
by DPPH assay, are shown in Fig. 1a.
The scavenging activity of the five fractions tested was com-
pared to those of ascorbic acid, used as positive control, and was
found to be concentration-dependent. This activity can be evalu-
ated by the determination of the EC50 values, corresponding to
the amount of extract required to scavenge 50% of DPPH radicals
present in the reaction mixture. High EC50 values indicate low anti-
oxidant activity. As shown in Table 1, the ethyl acetate fraction was
the most potent radical scavenger, followed by water and n-butan-
olic fractions. Chloroform and n-hexane fractions did not show any
antioxidant activities.
196
Table 1
The yield extraction, the total phenolic content, the evaluation of the EC50 values in the DPPH free radical-scavenging and b-carotene bleaching assay of L. inermis leaves crude
fractions. Ascorbic acid was used as standard.
Yield (%) Total phenolic contenta (mg GAE/g)b
n-Hexane 1.92 50 ± 5.0
Chloroform 0.28 73 ± 4.0
Ethyl acetate 1.97 367 ± 19
n-Butanol 6.41 286 ± 8.0
Water 8.84 444 ± 38
Ascorbic acid – –
a
Each value represents the mean ± SD of three experiments.
b
(mg GAE/g): mg of gallic acid equivalent per g of dry plant extract.
120
100
Scavenging activities (%)
80
60
40
20
0
H CHCL3
Fig. 1a. The DPPH free radical-scavenging activity of L. inermis leaves fractions at different concentrations (lg/ml). H: n-hexane, CHCl3: chloroform, EA: ethyl acetate, n-
BuOH: n-butanol, W: water, A. acid: Ascorbic acid. Each value represents the mean ± SD of three experiments.
It has been reported that the free radical-scavenging activity is
greatly influenced by the phenolic composition of the sample (Che-
ung, Cheung, & Ooi, 2003). Thus, the antioxidant activity of the n-
butanol, water and ethyl acetate fractions may be attributed to
their phenolic content. Although the TPC of the aqueous fraction
was significantly higher (p = 0.034) than that of the ethyl acetate
fraction, its antioxidant activity was lower. Tunalier et al. (2007)
reported that the EC50 values from L. salicaria extracts (a Lythracea)
were found between 0.1 and 2.7 mg/ml. Our results demonstrate
that L. inermis extracts, particularly ethyl acetate, n-butanol and
water fractions, have a better performance against DPPH radical
and therefore showed higher antioxidant activities.
3.2.2. Antioxidant activity of L. inermis fractions measured by b-
carotene bleaching method
The inhibitory effect on lipid peroxidation was determined by
the b-carotene/linoleic acid bleaching test. This test simulates the
oxidation of the membrane lipid components in the presence of
antioxidants inside the cells. Lipid peroxidation is one of the major
causes of quality deterioration in lipid-containing foods. It affects
colour, flavour, texture, and nutritive value of foods. b-Carotene,
in this model system, may be oxidised in the absence of an antiox-
idant and the test solution undergoes rapid discoloration. The pres-
ence of extract with antioxidant activity can hinder the extent of b-
carotene bleaching by neutralising the linoleate-free radical
formed in the system (Jayaprakasha, Singh, & Sakariah, 2001). As
shown in Fig. 1b, n-hexane, ethyl acetate, n-butanol and water
fractions exhibited antioxidant activity in a concentration-depen-
dent manner, with the exception of the chloroform fraction. This
fraction was inactive at concentrations less than 200 lg/ml. Based
on the comparison of the EC50 values, the order of the antioxidant
A.B. Hsouna et al. / Food Chemistry 125 (2011) 193–200
DPPHa EC50 (lg/ml) b-Carotenea bleaching assay EC50 (lg/ml)
>200 11.8 ± 0.3
>200 >200
4.8 ± 0.2 38.7 ± 0.7
9.0 ± 0.3 52.0 ± 3.0
7.6 ± 0.1 16.5 ± 1.5
3.5 ± 0.2 –
EA n-ButOH W A. acid
1 5 10 20 50 (µg/ml)
activities was found to be n-hexane > water > ethyl acetate > n-
butanol (Table 1).
3.3. Purification and identification of compounds from L. inermis
leaves
LC-DAD-ELSD ESI/MSn analysis of the water fraction showed the
presence of tannin compounds, widely known, from the literature,
to have antioxidant and other biological activities. Of the several
fractions obtained from the hydroalcoholic extract of L. inermis
leaves, the n-butanolic was found to have an excellent DPPH radi-
cal-scavenging activity and to inhibit lipid peroxidation. Active
compounds in the n-butanol extract were possibly different mole-
cules to those found in the water extract. Thus, the n-butanolic
fraction was chosen for the identification of potential compounds
with antioxidant activity.
After a preliminary survey of the chromatographic profile of this
fraction by LC-DAD-ELSD-ESI/MSn, its fractionation by FC was per-
formed, in order to isolate its main components. From the n-butan-
olic extract (1.5 g), 41 fractions were generated. Fractions 7 and 10,
eluted with 65% and 60% water in MeOH, respectively, were re-
chromatographed by semi-preparative reversed-phase HPLC, in or-
der to isolate their components. This procedure separated three
compounds: 1 (5 mg), 2 (9 mg) and 4 (7 mg) from fraction 7, while
fraction 10 afforded two compounds: 3 (17 mg) and 5 (15 mg). The
chemical structures of these constituents of henna leaves were
identified on the basis of NMR spectroscopic and mass spectromet-
ric analyses.
Compound 1 was obtained as a white powder and its molecular
formula C14H18O10 was deduced from ESIMS (m/z 347.3 [M+H]+)
and 13C NMR spectra. These data together with a complete NMR
 A.B. Hsouna et al. / Food Chemistry 125 (2011) 193–200 197

120

Inhibition percentage (%)

100

80

60

40

20

0
H CHCL3 EA n-ButOH Water BHT

1 2,5 5 10 20 50 (µg/ml)

Fig. 1b. Antioxidant activities of different fractions of L. inermis and BHT at different concentrations (lg/ml). The values represent the percent of inhibition of autoxidation of
the linoleic acid/b-carotene emulsion. Measurements were carried out in triplicate. H: n-hexane, CHCl3: chloroform, EA: ethyl acetate, n-BuOH: n-butanol, W: water, A. acid:
Ascorbic acid. Each value represents the mean ± SD of three experiments.

analysis showed that this compound was lalioside (2,3,4,6-tetra-
hydroxyacetophenone-2-J-b-D-glucopyranoside). The confirma-
tion of spectral data was made by comparison with the literature
(Takeda & Fatope, 1988).
Compound 2, a white amorphous material, possessed a molec-
ular formula of C22H28O13, which was established on the basis of
its HRESIMS spectrum (m/z 523.14227 [M+Na]+, D 0.12 ppm).
Based on NMR analysis and thanks to the comparison of the data
with the reported literature values (Takeda & Fatope, 1988), com-
pound 2 was identified as lawsoniaside (1,2,4-trihydroxynaphtha-
lene-1,4-di-J-b-D-glucopyranoside).
Compound 3 was isolated as a white powder with a molecular
formula of C21H20O11 (ESIMS; m/z 449.1 [M+H]+) and NMR data
consistent with those of luteolin-7-J-b-D-glucopyranoside (Lin,
Chen, Tseng, Lee, & Chen, 2009).
Compound 4, a white powder, had a molecular formula of
C14H18O9 determined by ESIMS (m/z 331.1 [M+H]+) as well as 13C
NMR data. The comparison of its MS and NMR data with those
described in the literature (Lee, Hong, Kwak, Pyo, & Jee, 1996) al-
lowed us to identify unambiguously 4 as 2,4,6-trihydroxyaceto-
phenone-2-J-b-D-glucopyranoside.
The new compound 5 was isolated as a white powder and its
molecular formula was defined as C16H18O8 by HRESIMS (m/z
361.08951 [M+Na]+, D 0.33 ppm). The 1H NMR spectrum of 5 in
CD3OD exhibited the presence of five aromatic protons ascribable
to: (i) an isolated proton at dH 6.50 (s, H-3) and, (ii) four protons
at dH 7.22 (ddd, J = 8.5, 7.5 and 1.0 Hz, H-6), 7.40 (ddd, J = 8.5, 7.5
and 1.0 Hz, H-7), 8.03 (dd, J = 8.5 and 1.0, H-5) and 8.33 (dd, J =
8.5 and 1.0, H-8) with an ABCD coupling pattern implying the
occurrence of a 1,2-disubstituted benzene moiety. The 1H NMR
spectrum showed also characteristic signals of a sugar moiety be-
tween dH 3.26 and 4.59 including one anomeric signal (dH 4.59, d,
J = 8.0 Hz, H-10 ). The sugar appeared to be b-D-glucopyranose
according to 13C NMR data (Table 2), where signals belonging to
the sugar moiety were four methines at dC 71.0 (C-40 ), 75.6 (C-
20 ), 78.0 (C-30 ) and 78.3 (C-50 ), one methylene at dC 62.2 (C-60 )
and one anomeric methine at dC 108.4 (C-10 ). In addition to the sig-
nals due to the glucosidic part, the 13C NMR and DEPT spectra of 5
exhibited 10 sp2 carbon signals between dC 101.3 and 152.6,
including five methine and five quaternary carbons. On the basis
of these data, 1H–1H COSY and HMBC experiments (Table 2) were
used to establish the structure of the aglycone part as 1,2,4-tri-
hydroxynaphthalene. The HMBC spectrum also showed the con-
nectivity between the sugar moiety and the aglycone part,
thanks to the correlation between C-1 and H-10 . A NOE cross-peak
between H-8 and H-10 confirmed that glucose moiety was involved

Table 2
The NMR spectral data of compound 5.
13 a 1
Position C DEPT Hb HMBC COSY 1H–1H NOESY 1H–1H
1 132.4 C – – – –
2 147.7 C – – – –
3 101.3 CH 6.50 s C-1, C-2, C-4, C-4a – –
4 152.6 C – – – –
4a 121.6 C – – – –
5 123.1 CH 8.03 dd (8.5, 1.0) C-4, C-7, C-8a H-6 –
6 123.1 CH 7.22 ddd (8.5, 7.5, 1.0) C-4a, C-6 H-5, H-7 –
7 127.5 CH 7.40 ddd (8.5, 7.5, 1.0) C-5, C-8a H-6, H-8 –
8 122.3 CH 8.33 dd (8.5, 1.0) C-1, C-4a, C-6 H-7 H-10 ,
8a 131.0 C – – – –
10 108.4 CH 4.59 d (8.0) C-1, C-20 , C-3, C-50 H-20 H-8, H-30 , H-50
20 75.6 CH 3.61 dd (9.0, 8.0) C-10 , C-30 H-10 , H-30 –
30 78.0 CH 3.44 t (9.0) C-20 , C-40 H-20 , H-40 H-10 , H-50
40 71.0 CH 3.52 t (9.0) C-20 , C-30 , C-50 , C-60 H-30 , H-50 H-60
50 78.3 CH 3.26 ddd (9.0, 4.5, 2.5) C-30 H-40 , H-60 H-10 , H-30
60 62.2 CH2 a: 3.85 dd (12.0, 2.5)b: 3.77 dd (12.0, 4.5) C-40 , C-50 H-50 H-40
a
Chemical shifts are shown in the d scale (in ppm); measured at 100 MHz in CD3OD.
b
Chemical shifts are shown in the d scale (in ppm) with J values in parentheses (in Hz); measured at 400 MHz in CD3OD.
198 A.B. Hsouna et al. / Food Chemistry 125 (2011) 193–200

H OH 3.4. Antioxidative properties of the compounds isolated from the L.

HO
inermis leaves
H OH OH
HO
OH
H 3.4.1. DPPH radical-scavenging activity
H H
O It is well known that the mechanism of reaction between an
H H antioxidant and DPPH radical depends on the structural conforma-
H
O OH
tion of the antioxidant. For phenolic compounds, the free radical-
1'
R OH O H scavenging activity is dependent on the presence of free OH groups
8 1 especially at C-3 in flavonoid structures (Sharififar, Dehghn-Nudeh,
HO O H OH
& Mirtajaldini, 2009). In this study, the DPPH radical-scavenging
2
capacities of the five compounds isolated from L. inermis leaves
were evaluated and compared to that of ascorbic acid, used as po-
4 sitive control. The second aim of this part of the work was to con-
OH O OR firm possible structure–antioxidant activity relationships and the
hydrogen-donating ability of the isolated compounds (Cao, Sofic,
1 R=OH 2 R=β-D-glucopyranoside & Prior, 1997).
4 R=H 5 R=H As shown in Fig. 3, the compounds isolated from L. inermis dem-
onstrated a concentration-dependent antiradical activity, by
OH reducing the stable radical DPPH. Among the tested compounds,
H
HO OH
5, 2 and 1 showed potent antioxidant activities and the relative or-
der of DPPH scavenging capacity was found to be: 5 > 2 > 1 > 3 > 4
as depicted in Table 3. Our results showed a significant difference
H
H H in the activity of the naphthalene derivatives 5 and 2. This finding
O suggests that antioxidant activity may be influenced by the num-
HO OH ber of hydroxyls in the aromatic rings of the compound as previ-
H ously reported by Sroka and Cisowski (2003). The comparison of
O O
HO
the results obtained with compounds 5 and 2 showed that the anti-
oxidant capacities of phenolic compounds increased with the num-
ber of hydroxyl groups attached to the aromatic core. Singh,
O’Malley, and Popelier (2005) reported that ortho and para substi-
O OH tution of phenols by electron-donating groups reduced the bond
dissociation energies and increased the transfer rate of hydrogen
3 atom to peroxyl radicals.
Subsequently, it could be noticed that the O-glycosidation of the
Fig. 2. The chemical structures of the phenolic glucosides identified from L. inermis
naphthalene core contributed to the reduction of the antioxidant
leaves.
activity. In the case of 2, the substitution of the 4-hydroxyl group
with a glycosyl group caused a decrease of 23% of the antioxidant
in glycosidation with the naphthalene aglycone at the C-1 hydroxyl capacity when compared to compound 5. In addition, this struc-
group. Therefore, compound 5 was identified as 1,2,4-trihydroxy- ture–activity relationship could also be observed with the aceto-
naphthalene-1-J-b-D-glucopyranoside. phenone-derived compounds 1 and 4. Compound 1 which
Among these compounds, 4 was isolated from this plant species contains three hydroxyl groups showed a higher scavenging power
for the first time, while it is the first report, to our knowledge, of 5 than compound 4, which bears only two hydroxyl groups. There-
as a natural product. The chemical structures of the isolated and fore, an additional hydroxyl group confers to the molecule a better
identified compounds from the n-butanolic part of henna leaves radical-scavenging activity, due to the facts that: (i) more hydro-
hydroalcoholic extracts are depicted in Fig. 2. gen atoms of the phenolic hydroxyl groups could be donated to

120

100
Scavenging activities (%)

80

Compound 1
60
Compound 2
Compound 3
40 Compound 4
Compound 5
Ascorbic acid
20

0
0 10 20 30 40 50 60 70 80 90 100 110
Concentration (µg/ml)

Fig. 3. The DPPH radical-scavenging activity of compounds isolated from L. inermis, expressed as percentages of inhibition (%), versus the positive control (ascorbic acid). Data
represent the means ± SD of three experiments.
 A.B. Hsouna et al. / Food Chemistry 125 (2011) 193–200
Table 3
The antioxidant activities of compounds 1–5 on DPPH and b-carotene assays.
Compounds EC50 DPPH % Inhibitiona of lipid
assaya (lg/ peroxidation at
ml) 100 lg/ml
Lalioside (1) 10.0 ± 0.5 0 of antioxidative natural compounds.
Lawsoniaside (2) 8.0 ± 0.6 18.5 ± 0.1
Luteolin-7-J-b-D-glucopyranoside 17.0 ± 0.7 61.5 ± 0.4
(3)
2,4,6-Trihydroxyacetophenone- 54.0 ± 1.2 14.0 ± 0.1
2-J-b-D-glucopyranoside (4)
1,2,4-Trihydroxynaphthalene-1- 6.5 ± 0.8 12.0 ± 0.5
J-b-D-glucopyranoside (5)
Ascorbic acid 3.5 ± 0.2 n.d.b
BHT n.d.b 79.0 ± 0.5
a
Each value represents the mean ± SD of three experiments.
b
Not determined.
stabilise the free radicals, and (ii) the bond dissociation of the
phenoxy group was easiest when the aromatic system was substi-
tuted by electron-donating groups.
3.4.2. Protection of b-carotene from degradation
The antioxidant activity of compounds 1–5 was also investi-
gated on the b-carotene/linoleic acid model system. This activity
was compared to BHT, a synthetic antioxidant reference widely
used in the protection of food. The effects of these five compounds
on b-carotene consumption are shown in Table 3. In this assay, the
highest antioxidative effect was obtained with BHT, which com-
pletely inhibited b-carotene consumption during incubation. The
lowest activity against b-carotene consumption was observed for
compounds 4 and 5 and the highest activity for compound 3.
Compound 1 did not show any antioxidant activity in this assay.
Therefore, the order of b-carotene assay was found to be:
BHT > 3 > 2 > 4 > 5 > 1, as shown in Table 3. We conclude that the
highest activity for compound 3 could be due to its flavonoid struc-
ture together with the number of hydroxyl groups on the flavonoid
nucleus (Foti, Piattelli, Baratta, & Ruberto, 1996). Moreover, the
solubility of a compound in the reaction environment may affect
its activity. In this case, a hydrophobic compound is more available
to the b-carotene bleaching assay in water emulsion and may be
more active. Thus, the solubility of aglycones in emulsion phase
is more important than those of glycosides, which naturally move
within the aqueous phase of an oil–water emulsion (Weng &
Wang, 2000). Hence, in comparison to BHT, lower antioxidant
activities of glycosides isolated in our study may be due to the sol-
ubility effect and the number of the free hydroxyl groups in pheno-
lic compounds.
The constituents of henna act as free radical scavengers that
may prevent phospholipid membrane peroxidation and protect
immuno-compromised cells from free radical damage. This may
explain the previously reported anti-sickling activity, through pro-
tecting the membrane of normal and sickle erythrocytes (Clarke,
Jones, & Martin, 1986).
4. Conclusion
In the present study, it was found that fractions of the hydroal-
coholic extract obtained from leaves of L. inermis possessed antiox-
idant and radical-scavenging activities. To the best of our
knowledge, this is the first report of such activities from this plant.
These fractions were tested by two distinct assays, in order to
determine which were the most active. Our results revealed that
the n-butanolic fraction showed significant antioxidant and free
radical-scavenging capacities. Furthermore, five compounds were
identified and isolated from this fraction. Among them, the new
199
compound 5 showed a potent antioxidant activity in DPPH assay
while compound 3 demonstrated the highest inhibition of oxida-
tion of the b-carotene/linoleic acid system.
Both the antioxidant activity and the phenolic contents found in
L. inermis leaves make this plant species a suitable potential source
Acknowledgements
This work was supported by grants from the Tunisian ‘‘Ministry
of Higher Education, Scientific Research and Technology” and from
ARCUS-CERES MAEE program. The authors wish to thank D. Bonho-
mme and Dr. A. Ortalo-Magné (Laboratoire MAPIEM, USTV, France)
for their technical assistance during the isolation and characterisa-
tion of pure compounds, and J.-M. Guigonis (IFR 50, Faculté de
Médecine, Nice, France) for recording the HRESIMS spectra. We
thank also, Dr. M. Siella Trigui and Dr. R. Jarraya-Mezghanni for
their suggestions and help to improve the project paper.
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