Industrial Crops and Products 89 (2016) 316–322

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Industrial Crops and Products

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In-vivo edema inhibition of Hyoscyamus albus antioxidant extracts

rich in calystegines
Lynda Bourebaba a,∗ , Giuseppe Sullini b,c , Jose A. Mendiola c , Yasmina Bourebaba d ,
Amirouche Deghima a , Naima Oukil a , Fatiha Bedjou a
a
Laboratoire de Biotechnologies végétales et d’Ethnobotanique, Faculté des Sciences de la Nature et de la Vie, Université de Bejaia, 06000 Bejaia, Algérie
b
Dipart. di Scienze del Farmaco e dei Prodotti per la Salute (S.C.I.F.A.R.), University of Messina, Viale Annunziata, 98168 Messina, Italy
c
Foodomics Laboratory, Bioactivity and Food Analysis Department, Institute of Food Science Research (CIAL-CSIC), Campus de Cantoblanco, Calle Nicolás
Cabrera 9, 28049 Madrid, Spain
d
Laboratoire d’Ecologie Microbienne, Faculté des Sciences de la Nature et de la Vie, Université de Bejaia, 06000 Bejaia, Algérie

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

Article history: Calystegines are polyhydroxylated alkaloids that mimic carbohydrate structures. In this study, nortropane
Received 31 December 2015 alkaloids were extracted from Hyoscyamus albus in order to evaluate their antioxidant and In-vivo anti-
Received in revised form 22 April 2016 inflammatory activities. Gas chromatography-mass spectrometry analysis indicated the presence of
Accepted 25 April 2016
several calystegines in the plant (leaves, flowers and seeds), and highlighted the existence of calyste-
gines A5 and B4 in seeds and flowers; in fact the occurrence of these two compounds in the plant has
Keywords:
been showed for the first time in this study. Antioxidant tests (DPPH, ABTS, reducing power assay and
Hyoscyamus albus
inhibition of human erythrocyte hemolysis mediated by peroxyl free radicals) showed potent activity
Calystegines
GC–MS
of seeds calystegines with an EC50 of 60.83 ␮g/ml in DPPH scavenging test, TEAC value of 111.4 ␮mol
Antioxidant Trolox/g in ABTS assay and gave 54.03% human membrane RBCs hemolysis protection at 2.5 mg/ml. Sim-
Anti-inflammatory ilar trend was found in anti-inflammatory test, where H. albus extracts showed strong activity, being
Mice seeds extract the strongest. This extract enriched in calystegines inhibited mice paw edema induced
by carrageenan at 100 and 200 mg/kg 2 h after injection comparably to the standard Indomethacin. In
conclusion, seeds extract had the strongest antioxidant and antiinflammatory activity, which can be
related with the highest content of calystegines compared to leaves and flowers and this can support the
traditional uses of this plant in many countries.
© 2016 Elsevier B.V. All rights reserved.

1. Introduction
Calystegines are a family of polyhydroxylated nortropane alka-
loids that were first isolated from Calystegia sepium, and due to
their hydrophilic nature they have been overlooked for a long
time in well-known drugs like belladonna leaf (Csuk et al., 2008;
Dräger, 2002). These compounds are classified into three groups
based on the numbers of hydroxyl groups present on the bicyclic
Abbreviations: AAPH, 2,2 -Azobis(2-amidinopropane) dihydrochloride; ABTS,
2,2 -azino-bis(3-ethylbenzothiazoline-6-sulphonic acid); BHA, butylhydroxyanisol;
BHT, butylhydroxytoluene; CNS, central nervous system; DPPH, 1,1-diphenyl-2-
picrylhydrazyl; DW, Dry Weight; GC–MS, gas chromatography–mass spectrometry;
HPLC, high-performance liquid chromatography; IL-1, interleukin 1; IL-6, inter-
leukin 6; PBS, Phosphate Buffered Saline; TEAC, Trolox Equivalent Antioxidant
Capacity; TNF-␣, Tumor Necrosis Factor alpha; W/V, Weight/Volume.
∗ Corresponding author.
E-mail address: lynda06bourebaba@gmail.com (L. Bourebaba).
ring system: those carrying three hydroxyl groups are designated
A, while calystegines containing four or five hydroxyl groups are
designated B and C, respectively (Rasmussen and Jensen, 2011).
Since calystegines mimic monosaccharides structures, many of
them show significant inhibition of glycoside hydrolases, in partic-
ular ␤-glucosidase and both ␣ and ␤-galactosidases (Asano et al.,
2000). Most of the calystegines were isolated from Solanaceae
familly, specifically Solanum melongena and Solanum tuberosum
were reported to contain calystegine A3 and calystegine B2 which
were also found in the following genera: Atropa, Datura, Lycium,
Physalis, Hyoscyamus and Mandragora (Bekkouche et al., 2001;
Kvasnička et al., 2008). Species of Hyoscyamus genus are important
medicinal plants represented by three main members: Hyoscya-
mus muticus, Hyoscyamus niger and Hyoscyamus albus. These plants
contain several tropan and nortropan alkaloids such as atropine,
scopolamine, hyoscyamine and calystegines (Mahfouze and Ottai,
2011; Nejadhabibvash et al., 2012).

http://dx.doi.org/10.1016/j.indcrop.2016.04.067
0926-6690/© 2016 Elsevier B.V. All rights reserved.
 L. Bourebaba et al. / Industrial Crops and Products 89 (2016) 316–322
Hyoscyamus albus L. or white henbane is a Mediteranean plant
belonging to Solanaceae family which ranks as one of the most
important to human beings (Goullé et al., 2004). It is an herbaceous
plant of 40–90 cm high characterized by a poisonous smell, it flow-
ers generally appears in July to give a fruit with small dark seeds
(Hammiche et al., 2013). Hyoscyamus albus has been used in folk
medicine in many countries essentially as a sedative of CNS, anti-
neuralgic, in Parkinson’s disease for its antispasmodic and analgesic
properties and as anti-inflammatory, antihelmintic, antipyretic and
anti-tumor remedy (Begum et al., 2010).
Several studies have shown the presence of several calystegines
essentially A3 and B2 in Hyoscyamus species (Asano et al., 2000); due
to the occurrence of these alkaloids in these plants and the great
therapeutic interest of these molecules, the interest of this study
was to evaluate the biological activities of calystegines extracted
from Hyoscyamus albus growing in Algeria.
2. Materials and methods
2.1. Plant material
Hyoscyamus albus leaves, flowers and seeds were collected from
wild plants growing in Bejaia (Algeria) in March and August 2014.
The plant material was washed and dried in a ventilated room
(30 ± 3 ◦ C) and then ground to a fine powder.
2.2. Chemicals
Solvents used for extraction and GC–MS analysis were from
HPLC grade (≥ 99%) purchased from Merk-Prolabo; 2,2-Diphenyl-
1-picrylhydrazyl hydrate (DPPH, ≥ 99%), Ascorbic acid, Butylated
hydroxyanisole (BHA ≥ 98.5%), 3,5-Di-tert-4-butylhydroxytoluene
(BHT ≥ 99%), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic
acid (Trolox, 98%), 2,2 -azino-bis(3-ethylbenzothiazoline-
6-sulfonic acid (ABTS, ≥ 99%), ␣,˛ -Azodiisobutyramidine
dihydrochloride (AAPH ≥ 97%), ␫-Carrageenan, Potassium fer-
ricyanide, Trichloroacetic acid (TCA), Ferric chloride (FeCl3 ) were
purchased from Sigma-Aldrich (Germanny).
2.3. Extraction
Calystegines are a group of nonesterified hydroxylated
nortropane alkaloids, and due to their hydrophilic nature, they are
not extracted by conventional methods and are generally isolated
from vegetal aqueous extracts by fractionation on ion exchange
column (Dräger, 2002). The extraction protocol was based in the
one described by Bekkouche et al. (2001). Briefly, air-dried and
powdered plant material was extracted three times by macerat-
ing in water/methanol (50/50, v/v) for 24 h. After centrifugation,
the supernatant was evaporated to dryness and applied to a cation
exchange column (Amberlite IR 120B, H+ form). The column was
washed with water to remove non-binding contaminants. Calyste-
gines and bound compounds were eluted with 2 N NH4 OH. The
concentrated residue was then applied to an anion exchange col-
umn (Dowex 1 × 2, Cl− form) and eluted with water to obtain a
purified plant extract that contains total calystegines.
2.4. Gas chromatography-mass spectrometry analysis
2.4.1. Derivatization
In order to analyze calystegines by GC, a derivatization proce-
dure was applied to form trimethylsilyl (TMS) ether derivatives. The
different purified plant extracts were dissolved in pyridine (50 ␮l),
mixed with 50 ␮l of trimethylsilyltrifluoroacetamide (MSTFA) and
317
kept at 60 ◦ C for 30 min; the reaction mixtures were then cooled
and subjected to GC–MS analysis.
2.4.2. GC–MS procedure
To characterize the calystegines of the different Hyoscyamus
albus extracts, GC–MS was employed using the modified method of
Bekkouche et al. (2001). A GCMS-QP2010 plus system (Shimadzu,
Kyoto, Japan) equipped with a DB–5 ms column (30 m × 0.25 mm
I.D. × 0.25 ␮m df, Quadrex Corporation, Woodbridge, CT) was used.
The separation was performed according to the following oven
temperature program: initial temperature was 100 ◦ C and held for
5 min, then it was raised to 300 ◦ C at 10 ◦ C/min, and this value was
maintained for 5 min. The injection volume was 0.5 ␮l in split mode
(split ratio 1:10) with the injector temperature at 250 ◦ C.
The carrier gas was He at 36.5 cm/s. The MS detection parame-
ters were: interface temperature, 280 ◦ C; Ion source temperature,
250 ◦ C; mass range, m/z 50–600; scan speed, 2500 amu/seg; and
event time, 0.20 seg. The data collection and handling were
performed using the GCMS solution (ver. 2.50SU3, Shimadzu) soft-
ware.
Several ions were monitorized in SIM: 217 m/z was used as a
common ion to monitor calystegines B2 , B3 , B4 and C1 ; 229m/z
for monitoring calystegine B2 ; 244 m/z for calystegine B4 ; 189 m/z
for calystegine B1 , 156 m/z for calystegines A3 and A5 ; 375 m/z for
calystegine C1 ; and 390 m/z to monitor calystegine N1 .
2.5. Antioxidant activity determination
2.5.1. DPPH radical scavenging assay
The DPPH radical scavenging activity was determined accord-
ing to the method described by Lai et al. (2009). Briefly, 1 ml of
0.1 mmol/l DPPH radical solution was mixed with 3 ml of various
concentrations of extracts dissolved in methanol. The mixture was
then vortexed vigorously and was incubated for 30 min in the dark
at room temperature. For the control, 3 ml of methanol was used.
The absorbance was measured at 517 nm. BHA and BHT, two well-
known antioxidants, were used as references. All measurements
were done in triplicate. The DPPH radical-scavenging activity was
calculated as follows:
Radical-scavenging activity(%) = (AC − AS /AC ) × 100 (1)
Where AC and AS are respectively the control and the sample
absorbance.
2.5.2. TEAC assay
The antioxidant activity of Hyoscyamus albus extracts was deter-
mined using the Trolox Equivalents Antioxidant Capacity (TEAC)
assay (Re et al., 1999). The ABTS•+ radical cation was produced by
reacting 7 mM ABTS and 2.45 mM potassium persulfate in the dark
at room temperature for 16 h before use. After addition of 100 ␮L
of samples to 1900 ␮L of diluted ABTS•+ solution the absorbance
reading was taken 7 min after at 734 nm. Trolox was used as the
reference standard and the results were expressed as TEAC values
(mmol trolox/g of extract).
2.5.3. Reducing power assay
The reducing power was determined according to the method of
Oyaizu (1986). Various concentrations of H. albus extracts (2.5 ml)
were mixed with 2.5 ml of 200 mmol/l sodium phosphate buffer
(pH 6.6) and 2.5 ml of 1% potassium ferricyanide. The mixture was
incubated at 50 ◦ C for 20 min, then 2.5 ml of 10% trichloroacetic
acid (w/v) were added, and the mixture was centrifuged at 650 rpm
for 10 min. The upper layer (5 ml) was mixed with 5 ml deionized
water and 1 ml of 0.1% of ferric chloride, and the absorbance was
measured at 700 nm (higher absorbance indicates higher reducing
power). The assays were carried out in triplicate and the results
318 L. Bourebaba et al. / Industrial Crops and Products 89 (2016) 316–322

Table 1
Identified calystegines and their amount in Hyoscyamus albus extracts.

Compound number Calystegines Retention time (min) Retention Index Leaves extract Flowers extract Seeds extract (␮g/ g DW) Referencesa
(␮g/ g DW) (␮g/ g DW)

1 Calystegine A5 13.2 1535 117.36 ± 5.18 95.42 ± 7.5 45.91 ± 9.24 12, 13
2 Calystegine A3 15.8 1729 NPb NDc 54.90 ± 3.95 12, 13, 14, 15
3 Calystegine A5 glycoside 16.1 1735 NP 5.84 ± 3.95 197.76 ± 21.96 13
4 Calystegine B4 18.12 1934 NP ND 212.54 ± 8.12 12
5 Calystegine B1 18.34 1949 NP 2.60 ± 3.16 91.25 ± 5.71 12, 13, 14
6 Calystegine N1 19.05 2016 54.99 ± 6.41 53.20 ± 3.34 180.22 ± 10.9 5, 15
7 Calystegine B2 19.70 2074 NP ND 87.14 ± 6.35 12, 13, 14
a
References used for identification.
b
NP: Not present.
c
ND: Not determined.

were expressed as mean values ± standard deviations. Trolox was
tested in the same conditions at different concentrations in order to
calculate TEAC values for tested extracts and results were expressed
as mmol trolox/g extract.
2.5.4. In-vitro assay for inhibition of human erythrocyte
hemolysis mediated by peroxyl free radicals
The in-vitro inhibition of human erythrocyte hemolysis by H.
albus extracts was evaluated according to the procedures described
by Yuan et al. (2005). The human erythrocyte hemolysis was per-
formed by the thermolabile azo-compound AAPH as the free radical
initiator. A volume of 100 ␮l of 5% (v/v) suspension of erythrocytes
in PBS was added to 200 ␮l of 100 mmol/l AAPH solution (in PBS pH
7.4) and 100 ␮l of the three extracts with different concentrations.
The reaction mixture was shaken gently while being incubated at
37 ◦ C for 3 h. After that, the reaction mixture was diluted with 8 ml
of PBS and centrifuged at 2000g for 10 min. The absorbance (A)
of the resulting supernatant was measured at 540 nm by a spec-
trophotometer. Likewise, the reaction mixture was treated with
8 ml of distilled water to obtain a complete hemolysis (B). The
absorbance of it supernatant (B) was measured at the same con-
dition. The inhibitive ratio (%) was calculated using the following
formula:
Inhibitiveratio(%) = [1-(A/B)] × 100
2.6. Anti-inflammatory activity
2.6.1. Animals
Eight groups of six adult male SWISS Albino mice (20–25 g)
were purchased from Pasteur Institute in Algeria and used
for the experiment. They were housed in acrylfiber cages
(48 cm × 35 cm × 22 cm) at a controlled room (temperature
25 ± 3 ◦ C and humidity 50 ± 10%) and were kept on a 12 h light: 12 h
dark cycle. They were fed with standard diet and water ad libitum
and acclimated 10 days before they were used and fasted for 12 h
before testing. All experiments were approved by the Animal Ethics
Committee of the University of Bejaia (Algeria) and conducted
in strict compliance with internationally accepted principles for
laboratory animals (directive N◦ 2010/63/EU of 22 September
2010 which updates and replaces directive N◦ 86/609/EEC of 24
November 1986).
2.6.2. Experimental procedure
Paw edema was induced by injecting 0.05 ml of 1% carrageenan
in physiological saline into the subplantar tissues of the right hind
paw of each mouse. The extracts (100 and 200 mg/kg) were admin-
istered orally 60 min prior to carrageenan injection. The paw edema
was measured (mm) before and then at 1, 2, 3, 4 and 5 h, after car-
rageenan injection. Mean values of treated groups were compared
with mean values of a control group. Indomethacin (20 mg/kg) was
used as the reference drug (Winter et al., 1962). The percentage
inhibition for each mouse and each group was obtained as follows:
%Inhibition
Increase in paw edema (Control) − Increase in paw edema (Test)
= × 100 (3)
Increase in paw edema (Control)
3. Results
3.1. Characterization results
Leaves, flowers and seeds extracts of Hyoscyamus albus were
analyzed to identify the present calystegines by GC–MS (Fig. 1).
Their retention indexes and MS fragmentation pattern were used
for identification, Table 1. GC–MS analysis of H. albus extracts
showed that seeds extract contains most kinds of calystegines in
highest amounts in comparison with leaves and flowers extracts.
Calystegines A5 , and N1 are the two common alkaloids in the
three extracts with a maximum concentration of 180.22 ± 10.9 ␮g
Calystegine N1 /g DW in seeds and 117.36 ± 5.18 ␮g calystegine A5 /g
DW found in leaves. The characterization allowed the identification
of calystegine B4 for the first time in this plant, and, as shown in
(2) Table 1, this alkaloid is present in highest proportion in seeds. It is
also present in flowers but at less concentration.
3.2. Antioxidant results
3.2.1. DPPH and ABTS scavenging activity
Antioxidant activity against free radicals DPPH and ABTS of H.
albus extracts was evaluated at different concentrations and the
results are shown in Fig. 2. Seeds extract was most active in the two
tests with a maximum inhibition of 93.61% at 200 ␮g/ml and 23.52%
at 1 mg/ml for DPPH and ABTS radicals respectively, while leaves
and flowers extracts gave only 65.89% and 70.37% DPPH inhibition
and 9.71% and 17.75% ABTS inhibition at the same concentrations.
This high activity could be related with the high amount of calyste-
gines present in extracts.
The EC50 corresponds to the concentration able to inhibit
50% of DPPH free radical, and TEAC indicates the TROLOX quan-
tity that exercises the same activity of 1 mM of extract. In this
way, seeds extract showed the highest antioxidant activity, that
means the lowest EC50 and the highest TEAC values (60.83 ␮g/ml
and 111.4 ␮mol Trolox/g extract respectively). In contrast, leaves
and flowers extracts had lower activities with an EC50 of 196.55
and 190.22 ␮g/ml and TEAC of 38.33 and 82.03 ␮mol TROLOX/g
extract respectively). Common used antioxidants, BHA and BHT,
were employed as reference compounds showing the following
antioxidant activities: EC50 = 0.97 and 3.89 ␮g/ml; TEAC = 6130 and
5950 ␮mol TROLOX/g compound for BHA and BHT respectively.
 L. Bourebaba et al. / Industrial Crops and Products 89 (2016) 316–322 319

Fig. 1. GC–MS chromatograms of Hyoscyamus albus extracts (A: Leaves extract, B: Flowers extract and C: Seeds extract). 217 m/z: calystegines B; 229 m/z: calystegine B2;
156 m/z: calystegines A; 390 m/z: calystegine N1; 375 m/z: calystegine C1; 189 m/z: calystegine B1; 71 m/z: octadecane; 1: calystegine A5; 2: calystégine A3; 3: calystegine
A5 Gly.; 4: calystegine B4; 5: calystegine B1; 6: calystegine N1; 7: calystegine B2.

3.2.2. Reducing power Fig. 3 represents the reducing power of H. albus extracts as
The reducing power was the second antioxidant activity mea- absorbance versus concentrations, and Table 2 shows TEAC val-
surement assay tested with H. albus seeds, flowers and leaves. ues of the different extracts and Ascorbic acid used as reference
320 L. Bourebaba et al. / Industrial Crops and Products 89 (2016) 316–322
A B
Fig. 2. Radical scavenging activity of Hyoscyamus albus extracts (A: DPPH assay; B: ABTS assay).
Fig. 3. Reducing power of Hyoscyamus albus calystegines.
compound. Fe3+/ ferricyanide complex reduction ability of Hyoscya-
mus albus extracts was concentration dependent with a maximum
absorbance of 0.495, 1.186 and 1.475 AU for leaves, flowers and
seeds extracts respectively at 5 mg/ml. TEAC values indicated that
seeds extract has better activity (178.6 ␮mol Trolox/g extract) with
only small difference compared to flowers extract (145.70 ␮mol
Trolox/g extract). Results showed also that leaves exerted the low-
est ferric reduction capacity (49 ␮mol Trolox/g extract).
3.2.3. In-vitro inhibition of human erythrocyte hemolysis
Hyoscyamus albus extracts were investigated for their capacity
to protect normal Human erythrocytes against In-vitro hemolysis
caused by aqueous peroxyl radicals, Fig. 4 shows the inhibitory
effect of different concentrations of extracts on an AAPH-induced
hemolysis of 5% (v/v) Human erythrocytes for 3 h. Human erythro-
cytes hemolysis inhibition by calystegines was dose dependant,
especially for seeds extract, that showed the highest activity against
thermostable peroxyl radicals with a maximum of 54.03% hemol-
ysis inhibition at 2.5 mg/ml. Leaves and flowers extracts presented
Table 2
Reducing power of H. albus extracts. Results showed in TEAC values. Ascorbic acid
was used as control.
Samples TEAC (␮mol Trolox/g extract)
Leaves. 49, 9
Flowers. 145, 70
Seeds. 178, 6
Ascorbic acid 11564, 18
more or less a similar effect much lower than that of seeds (22.6
and 28.03% hemolysis inhibition at the same concentration).
3.3. In-vivo anti-inflammatory activity
Mice hind paw edema was induced by a sub-plantard injection
of carrageenan salin solution; this caused an increase of foot pad
circumference of group control up to 1.78 ± 0.26 mm at 4 h post-
phlogistic agent injection (Table. 3). In general, H. albus extracts
showed potent and fast in-vivo anti-inflammatory effect with inhi-
bition rates ranging from 21.3% to 58.33%. All samples acted at the
2nd hour of carrageenan injection unlike the standard that acted
at the 3rd hour with an inhibition of 63.98%. Seeds and flowers
were most active at 200 mg/kg with predominance of seeds extract
which showed paw edema inhibition at 58.33%, 54.81% for flowers
and only 30.75% for leaves extract rich in calystegines.
4. Discussion
In this study, Leaves, Flowers and Seeds of Hyoscyamus albus
(Solanaceae) were investigated for their calystegines content,
antioxidant and in-vivo anti-inflammatory activities. More than 14
different calystegine structures have been published and most of
them have been isolated from Solanaceae (Keiner and Dräger, 2000).
Calystegines of A, B and N group were found in Hyoscyamus albus
extracts; according to literature, we have identified calystegines
A3 , A5 , A5 glycoside, B1 , B2 , B4 and N1 in seeds extract, the same
calystegines with less amounts in flowers and only calystegines A3
and N1 in leaves extract. Dräger et al. (1994) indicated that calyste-
gines occur not only in the roots of Solanaceae plants but also in the
leaves and found that in Hyoscyamus albus the content of calyste-
gines of the B-group is even higher. In the present study it was
demonstrated that H. albus seeds and flowers are even richer in
calystegines than leaves. The presence of A3 , N1 and other calyste-
gines of the B-group in leaves of Hyoscyamus albus L. has already
been previously reported (Bekkouche et al., 2001). In this study,
occurrence of calystegine B4 in this species has been demonstrated
for the first time.
Hyoscyamus albus calystegines showed potent antioxidant
activity, and due to their high degree of hydroxylated substitu-
tions, the high antioxidant potential could be related to their several
OH-moieties. In fact, Asano et al. (1997) demonstrated that an
increase in degree of hydroxylation of the nortropane ring results
in enhanced of biological activities of calystegines, which could
explain the differences in efficiency observed between the different
extracts. In all tests, seeds extract was most active. GC–MS analysis
allowed us to determine the presence of several calystegines in the
 L. Bourebaba et al. / Industrial Crops and Products 89 (2016) 316–322 321

60
Leaves_Caly.

% of hemol ysi s inhi bi ti on

Flowers_Caly.
Seeds_Caly.
40

20

0
5

5
1
75
0.

1.

2.
0.
Con centration s (mg/ml)

Fig. 4. Inhibitory effect on AAPH-induced hemolysis of 5% (v/v) Human erythrocytes.

three extracts, and indicated a highest content of calystegine B4 in
seeds, a lower concentration in flowers and absence of this com-
pound in leaves. The highest degree of hydroxylated pattern of this
compound (4-OH) and the specific position of these substituents
could explain the highest antioxidant efficiency of seeds extract.
Two main mechanisms by which antioxidants can play their
protective role have been proposed. In the first one, the free radical
removes a hydrogen atom from the antioxidant that itself becomes
a radical, this mechanism is referred to as H-atom transfer. A higher
stability of the radical ArO• corresponds to a better efficiency of the
antioxidant ArOH, so that it is unlikely to react with the substrate
(Leopoldini et al., 2011). In this mechanism, the bond dissociation
enthalpy (BDE) of the O H bonds is an important parameter in the
evaluation of the antioxidant action, because the weaker the OH
bond the easier will be the reaction of free radical inactivation.
In the second mechanism (the one-electron transfer), the
antioxidant can give an electron to the free radical becoming itself
a radical cation. Also in this case, the radical cation arising from
the electron transfer must be stable, so it does not react with sub-
strate molecules (Leopoldini et al., 2011). Some studies provide
important information on the structure–activity relationship of the
antioxidants and stressed the importance of the ortho-hydroxyl
and ortho-electron donating groups (possibly para– as well) that
exhibit pronounced antioxidant activity (Lee et al., 2015; Murias
et al., 2005)
In this study and based on the common uses of Hyoscyamus
albus in folk medicine, an In-vivo model of acute inflammation
(mice paw edema induced by the injuring agent carrageenan) was
used to investigate the supposed anti-inflammatory effect of H.
albus calystegines. This experiment is a widely used model to test
new anti-inflammatory drugs and is generally used to evaluate the
anti-edematous effect of natural products (Buapool et al., 2013).
As shown in results section, H. albus extracts demonstrated potent
anti-inflammatory effect for the three different parts tested, and
seems that they play their action at the 2nd hour of edema induc-
tion.
Carrageenan-induced edema causes the release of several
inflammatory mediators such as histamine, serotonin, bradykinin
and prostaglandins. The development of an edema induced by car-
rageenan is a three phase event; the first occurring between 0 and
1.5 h after injection, is caused by the release of histamine and sero-
tonin, the second is mediated by bradykinin after 1.5–2.5 h and
finally, the third phase, after 2.5–6 h, has been correlated with
an increased production of prostaglandins (Buapool et al., 2013).
Local and/or systemic inflammation is associated with enhanced
levels of the pro-inflammatory cytokines TNF-␣, IL-1, and IL-6
(Cuzzocrea et al., 1999). The initial phase of edema, which is
not inhibited by nonsteroidal anti-inflammatory drugs (NSAIDs)
such as indomethacin or aspirin but in this case inhibited by H.
albus calystegines, has been attributed to the release of histamine,
5-hydroxytryptamine (5-HT) and bradykinin. The second accel-
erating phase of swelling has not only been correlated with the
elevated production of prostaglandins, but more recently has been
attributed to the induction of inducible cyclooxygenase (COX-2) in
the hind paw (Nantel et al., 1999).
The anti-inflammatory effect of calystegines can occur via
several mechanisms prominent of which is inhibition of pro-
inflammatory cytokine-dependent up-regulation of adhesion
molecule expression (Kelly and Whe, 1994). Since histamine
mediates the early stages of vasodilation and increased vascular
permeability which characterize the acute inflammatory response,
it has been established that leukocytes and platellets are largely
implicated in histamine release during this process (Chacko et al.,
2011). Leukocytes recruitment in acute and chronic inflamma-
tion is characterized by sequential cell adhesion and activation
events. Migration processes of these cells recruit granulocytes,

Table 3
Effect of H. albus calystegines on carrageenan-induced mice paw edema. Percentage of inhibition rate shown in parenthesis.

Group Dose (mg/kg, p.o.) Edema value (mm) and inhibition rate (%)

1h 2h 3h 4h 5h

Control – 1.22 ± 0.16 1.42 ± 0.17 1.44 ± 0.47 1.78 ± 0.33 1.44 ± 0.26
Leaves 100 1.01 ± 0.3(17.35) 0.94 ± 0.23 (34.6) 0.97 ± 0.26 (31.92) 1.3 ± 0.28(26.96) 1.28 ± 0.22(10.89)
Flowers 100 0.96 ± 0.20(21.45) 0.98 ± 0.29(30.76) 1.025 ± 0.24(28.81) 1.38 ± 0.16(22.28) 1.3 ± 0.16(9.72)
Seeds. 100 0.8 ± 0.13(34.43) 0.825 ± 0.35(41.90) 0.91 ± 0.30(36.92) 1.09 ± 0.21(38.67) 1.19 ± 0.16(17.25)
Leaves 200 0.81 ± 0.21(33.74) 0.98 ± 0.29(30.75) 1.13 ± 0.07(21.3) 1.34 ± 0.17(24.63) 1.175 ± 0.25(18.40)
Flowers. 200 0.64 ± 0.15(47.40) 0.64 ± 0.16(54.81) 0.875 ± 0.27(39.34) 0.94 ± 0.36(47.10) 0.81 ± 0.34(43.67)
Seeds 200 0.57 ± 0.24(53.55) 0.6 ± 0.19 (58.33) 0.63 ± 0.22 (55.4) 0.81 ± 0.32(54.59) 0.78 ± 0.45(45.60)
Indomethacin 20 0.825 ± 0.25(43.75) 0.775 ± 0.19(57.92) 0.63 ± 0.42(63.98) 0.68 ± 0.17(58.16) 0.875 ± 0.20(38.95)
322 L. Bourebaba et al. / Industrial Crops and Products 89 (2016) 316–322
including polymorphonuclear leukocytes in particular but also
monocytes and dendritic cells, to acute inflammatory sites. The
selectins that are implicated in leukocytes migration and recruit-
ment to vascular endothelium are type I membrane-spanning
glycoproteins. Expression of some of them (as E-selectin) is largely
confirmed; where it is induced via transcription-dependent mech-
anisms directed by interleukin (IL)-1b, tumor necrosis factor alpha
(TNF-␣) and other inflammatory mediators. Cell adhesion inter-
actions involved the presence of specific counter-receptors with
specific glycan-based post-translational modification, which is the
consequence of a series of ordered glycosyltransferase dependent
events characterized by glycan polymer elongation or branching
(Lowe, 2003). Alkaloids, such as calystegines, mimicking sugars
in size and shape are now believed to be widespread and of cru-
cial importance in plants and microorganisms. The presence of
the nitrogen atom mimicking the positive charge of the glycosyl
cation intermediate in the enzyme-catalyzed glycoside hydrolysis,
possible anti-inflammatory effect of Hyoscyamus albus calystegines
could be related with glycosidase inhibition pathway as on glyco-
syltransferases (Borges de Melo et al., 2006).
5. Conclusions
The presence of several calystegines in leaves, flowers and seeds
of Hyoscyamus albus has been demonstrated by means of GC–MS;
furthermore, the occurrence of calystegines A5 and B4 has been
shown for the first time in this plant. Moreover, antioxidant activity
of seeds, flowers and leaves extracts has been tested by differ-
ent methods, being seeds extract which had the strongest activity,
which can be related with the highest content of calystegines
compared to leaves and flowers. Similar trend was found in anti-
inflammatory test, where H. albus extracts showed strong activity,
being seeds extract the strongest.
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