SUPPLEMENTARY MATERIAL
Phytochemical analysis and bioactivity of the aerial parts of Abutilon
theophrasti (Malvaceae), a medicinal weed
N.Z. Mamadalievaa*, F. Sharopovb, J-P. Giraultc, M. Winkb and R. Lafontd
a
Institute of the Chemistry of Plant Substances, Mirzo Ulugbek Str 77, Tashkent 100170,
Uzbekistan; bHeidelberg University, Institute of Pharmacy and Molecular Biotechnology, Im
Neuenheimer Feld 364, 69120 Heidelberg, Germany;
c
Université Paris Descartes,
Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, CNRS UMR 8601,
45 rue des Saints-Pères, F-75270 Paris Cedex 06, France; dSorbonne Université, Université
Pierre et Marie Curie, IBPS-BIOSIPE, 7 Quai Saint Bernard, Case Courrier 29, 75252 Paris
Cedex 05, France
*
Corresponding author. E-mail: nmamadalieva@yahoo.com
Abstract
Phytochemical investigations of aerial parts of Abutilon theophrasti yielded (6S,9R)-roseoside
(1) and (6S,9S)-roseoside (2) which are new for the genus. The elucidation of the chemical
structures was established by mass spectrometry, 1D and 2D NMR experiments. Although
methanol extracts contained 48.5±7.2 mg of caffeic acid equivalents (CAE) and 15.87±4.6 mg
of quercetin equivalents (QE), the antioxidant activity, as revealed by DPPH and ABTS
assays, was of medium strength (EC50 of 306.2±16.3 and 394.3±14.8 µg/mL, respectively). A.
theophrasti extract inhibits soybean 5-LOX with IC50 value 2.89±0.2 mg/mL. The
cytotoxicity of the methanol extract against MCF-7, CCRF-CEM and CEM/ADR5000 cancer
cells resulted in IC50 values of 505.8±34.7 µg/mL for MCF-7, 75.6±7.1 µg/mL for CCRFCEM, and 89.5±13.4 µg/mL for CEM/ADR 5000 cells.
Keywords: Abutilon theophrasti, roseoside, NMR, mass-spectroscopy, antioxidant, antiinflammatory, cytotoxicity
Experimental part
Plant extraction. Aerial parts of Abutilon theophrasti Medic were collected from Tashkent
region (Uzbekistan) in August 2008 and authenticated by Dr A.M. Nigmatullaev, Laboratory
of Herbal Plants, Institute of the Chemistry of Plant Substances, Tashkent, where a voucher
specimen (N 20084123) was deposited.
The aerial parts were washed thoroughly and dried in the shade. The dried plant material was
then powdered. The powder (1.5 kg) was extracted with MeOH thrice (6 L each time for 4
hours) at room temperature and the extract was concentrated in a rotary vacuum evaporator at
40 °C. After evaporation of the solvent, 87 g residue was obtained (5.8 % of the weight of the
aerial parts), which was then dissolved in water and filtered, extracted first with chloroform,
and next successively with n-butanol. The butanol extract was concentrated using a vacuum
evaporator and dried. The yield of the butanol extract (21.5 g) was 1.43 %.
Isolation of roseosides. The dried butanol extract of A. theophrasti (21.5 g) was subjected to
column chromatography on silica gel KSK and L (63-100 µm), Czech Republic. The column
(4.0 × 100 cm) was eluted with СHCl3–CH3OH (25:1 v/v and 9:1 v/v) and fraction A was
obtained containing a mixture of compounds 1 and 2. The purity was monitored using thinlayer chromatography (TLC). TLC was conducted on plates precoated with Silufol F254. Spots
on TLC plates were visualized under UV light (λ=254 and 360 nm). The eluting solvent
system for column chromatography used was СHCl3 - CH3OH (25:1; v/v), then (9:1; v/v).
Solvents were evaporated in vacuo with a rotary evaporator IR - 1М2. For the separation of
(6S,9R)-roseoside (1) and (6S,9S)-roseoside (2) a NP-HPLC Column Zorbax-SIL (5 µm,
250×4.6 mm) was used, with cyclohexane-isopropanol-water (100:30:1.5 v/v/v), at a flowrate of 1 mL/min. The chemical structure of compounds 1 and 2 was determined by UV, mass
spectrometry and 1D- and 2D-NMR spectroscopy (Table S1, Figure S1).
Spectroscopic methods. High resolution p-ESI mass spectra were acquired with a ultra-highresolution mass-spectrometer, the hybrid linear ion trap LTQ-Orbitrap (Thermo Fisher
Scientific, Les Ulis, France) as previously described (Blais et al., 2010). NMR Spectroscopy
was performed on a Bruker AVANCE II 500 Spectrometer in D2O for initial mixture of major
and minor compounds and in CD3OD for isolated major and minor compounds (500 MHz for
1
H and 125 Mhz for
13
C) at 300K. Presaturation of the solvent was used for all 1D and
1
1
homonuclear 2D H experiments. 2D-Experiments ( H, TOCSY [spin-lock time 15 and
1
13
1 1
13
35ms], NOESY [mixing time 500ms], H, C, HSQC [correlation one bond J H- C] and
1
13
2 1
13
3 1
13
HMBC [correlation H- C via two or three bonds via J H- C or J H- C]) were performed
by Pulse Field Gradient (PFG) methods using standard Bruker software. The sample was
lyophilized once and dissolved in CD3OD. The chemical shifts are ± 0.01 ppm for 1H and ±
0.2 ppm for 13C. Spectra were referenced with respect to residual HCD2OD at 3.31ppm for 1H
and 49.0 ppm for 13C, used as internal reference for the proton and carbon shifts.
Compounds 1 and 2 were tentatively identified on the basis of the work of Sohn et al. (2008),
who used Circular Dichroism and NMR analyses, as (6S,9R)-dihydroxy-megastigma-4,7dien-3-one-9-O-β-D-glucopyranoside (1) and (6S,9S)-dihydroxy-megastigma-4,7-dien-3-one9-O-β-D-glucopyranoside (2) (Figure 1), and from comparison of their spectral properties
with literature values (Murai et al., 2001; Sohn et al., 2008; Wang et al., 2012).
(6S,9R)-Roseoside (1) − C19H30O8, Rt=18 min; UV λmax (MeOH): 236-237 nm; ESI-MS
(positive mode) m/z: 409 [M+Na]+; 301, 185; ESI-HR-MS m/z 409.18399 [M+Na]+
(expected 409.18329 for C19H30O8Na); 1H and 13C NMR (Table S1).
(6S,9S)-Roseoside (2) − C19H30O8, Rt=19.5 min; UV λmax (MeOH): 236-237 nm; ESI-MS
(positive mode) m/z: 409 [M+Na]+; 301; ESI-HR-MS m/z 409.18366 [M+Na]+ (expected
409.18329 for C19H30O8Na); 1H and 13C NMR (Table S1).
Determination of total phenolic content. The total phenolic content was determined
according to the Folin-Ciocalteu method as described by Folin and Ciocalteu (1997) and
Vermerris and Nicholson (2007). Extracts were dissolved in methanol at the concentration of
10 mg/mL. 20 µL of serial dilutions with a concentration range of 0 to 200 µg/mL were
pipetted into wells of a 96 well microtitre plate to which 100 µL Folin-Ciocalteu reagent were
then added. After 5 min 80 µL of 7.5 % (w/v) sodium carbonate solution were added and the
plate was mixed well. The plate was kept in the dark at room temperature for 2 h (Zhang et al.
2006; Fujita et al. 2012). The absorbance was measured at 750 nm with a Biochrom Asys
UVM 340 Microplate Reader (Biochrom company). Based on absorbance measurements
obtained with a standard solution (caffeic acid), a calibration curve was constructed. Results
were expressed as mg caffeic acid equivalents (CAE)/g of extract.
Determination of total flavonoid content. Total flavonoid content was determined by the
aluminum colorimetric method (Harborne 1973) using quercetin as a reference standard.
Briefly, the methanol extract (150 μL, 0.4 mg/mL) was mixed with 2% (w/w) AlCl3 (100 μL)
in 96-well plates. Absorbance was measured at 430 nm with a Biochrom Asys UVM 340
Microplate Reader after 30 min of incubation at room temperature. All determinations were
performed in triplicate with reference to a sample blank value (without aluminum chloride).
The calibration curve was obtained using concentrations of quercetin (4 to 80 μg/mL). Results
were expressed as mg quercetin equivalents (QE)/g of extract.
DPPH radical-scavenging assay. In the DPPH radical-scavenging assay (Blois 1958), 100 μL
of extract (diluted in methanol to a concentration range of 7.8 to 1000 µg/ml) was mixed with
100 μL 0.2 mM DPPH in methanol in wells of a 96-well plate. The plate was kept in the dark
for 15 min, after which the absorbance of the solution was measured at 515 nm in a Biochrom
Asys UVM 340 Microplate Reader. Appropriate blanks (methanol) and standards (ascorbic
acid in methanol) were assessed simultaneously. This method follows closely that used by
previous workers (Brand-Williams et al. 1995; Clarke et al. 2013). The inhibitory activity (in
%) was calculated by using the following expression:
DPPH scavenging (%) = 100 × [(Abs sample + DPPH) - (Abs sample blank)]/[(Abs DPPH)
- (Abs methanol)]
The EC50 value is defined as the amount of extract needed to scavenge 50% of DPPH
radicals.
ABTS radical-scavenging assay. The ABTS radical-scavenging activity was determined
using the method as described by Brangoulo and Molan (2010). 38 mg of ABTS reagent and
6.5 mg of potassium persulphate were dissolved in 10 mL of deionised purified water. The
solution was keep in the dark at room temperature for 16 h to form the stable ABTS •+ radical
cation. The ABTS•+ solution was further diluted with deionised purified water to an
absorbance value between 2.0 and 2.4 at 645 nm.
Trolox standard (5 mg/mL) was prepared in absolute ethanol a few minutes prior to analysis.
A 96-well microplate was loaded with 100 μL methanolic extract of A. theophrasti (with
concentration range 5.0-0.005 mg/mL) or trolox or water blank and mixed with 100 μL
ABTS•+ solution. Absorbance was read at 645 nm using a spectrophotometer (Biochrom Asys
UVM 340 Microplate Reader). The activity was calculated as for the DPPH assay.
Determination of the 5-lipoxygenase activity. Soybean 5-lipoxygenase (5-LOX) was used for
the conversion of linoleic acid to hydroperoxy linoleic acid. The kinetics of reaction were
monitored spectrophotometrically at 234 nm at 10-second intervals using a spectrophotometer
(Biowave II UV/VIS). Briefly, 0.1 M phosphate buffer (1 mL) pH 9.0, containing 6.25 µL of
5-LOX (7.9U/mL) and 20 µL of 10 different concentrations (0.04-10 mg/mL) of extracts in
ethanol were incubated at room temperature for 10 minutes. The reaction was initiated by
addition of 20 µL 62.5 µM sodium linoleate (Baylac and Racine, 2003). 5-LOX inhibition
was determined by comparison of rates of reaction of samples relative to blank sample using
the formula
(E − S)/E × 100,
where E is the activity of enzyme without test sample and S is the activity of enzyme with
test sample. The IC50 of the positive control nordihydroguaiaretic acid (NDGA) was 27
μg/mL.
Cell culture. Cytotoxic activities of the plant extracts were investigated on MCF-7 (human
breast adenocarcinoma), CCRF-CEM (human T lymphoblast leukaemia) and CEM/ADR5000
(adriamycin-resistant leukaemia) cell lines. MCF-7 cells were maintained in DMEM complete
media (L-glutamine supplemented with 10 % heat-inactivated foetal bovine serum, 100 U/mL
penicillin, and 100 µg/mL streptomycin). CCRF-CEM and CEM/ADR5000 cells were grown
in RPMI 1640 media which were supplemented with 1% L-glutamine, 100 U/ml penicillin,
100 µg/mL streptomycin and 10% heat-inactivated foetal bovine serum. Cells were grown at
37°C in a humidified atmosphere of 5% CO2.
MTT assay. Cytotoxicity of the samples was determined in triplicate using the 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability assay (Mosmann
1983). The A. theophrasti extract was serially doubly diluted with medium from 1.0 mg/mL
to 0.002 mg/mL, and 100 μL liquid of each concentration was applied to the wells of a 96well plates. Cells (2 × 104 cells/well of exponentially growing MCF-7 cells and 3 × 104
cells/well for CCRF-CEM and CEM/ADR5000 cells) were seeded in a 96-well microplate
(Greiner Labortechnik). Cells were incubated with the samples for 24 h (MCF-7) and 48 h
(CCRF-CEM and CEM/ADR5000) before the medium was removed and replaced with fresh
medium containing 0.5 mg/mL MTT. The formazan crystals were dissolved in 100 µL DMSO
4 h later; the absorbance was measured at 570 nm with a Biochrom Asys UVM 340
Microplate Reader. The cell viability rate (%) of three independent experiments was
calculated by the following formula:
Cell viability rate (%) = (OD of treated cells) / (OD of control cells) × 100 %
The IC50 values were calculated. Doxorubicin was used as positive control.
Statistical analysis. All experiments were carried out three times. The IC50 was determined as
the drug concentration which resulted in a 50 % reduction in viability or inhibition of the
biological activity. IC50 values were calculated using a four parameter logistic curve (Sigma
Plot 11.0).
Acknowledgements
The authors would like to thank the DAAD for a scholarship to N.Z. Mamadalieva, and Dr
Laurence Dinan for critical reading of the manuscript.
References
Baylac S, Racine P. 2003. Inhibition of 5-lipoxygenase by essential oils and other natural
fragrant extracts. International Journal Aromatherapy. 13: 138–142.
Blais C, Blasco T, Maria A, Dauphin-Villemant C, Lafont R. 2010. Characterization of
ecdysteroids in Drosophila melanogaster by enzyme immunoassay and nano-liquid
chromatography-tandem mass spectrometry. Journal of Chromatography B. 878: 925932.
Blois MS. 1958. Antioxidant determinations by the use of a stable free radical. Nature. 181:
1199-1200.
Brand-Williams W, Cuvelier ME, Berset C. 1995. Use of free radical method to evaluate
antioxidant activity. Lebensmittel - Wissenschaft und Technologie. 28: 25-30.
Brangoulo HL, Molan PC. 2010. Assay of the antioxidant capacity of foods using an iron(II)catalysed lipid peroxidation model for greater nutritional relevance. Food Chemistry.
125: 1126-1130.
Clarke G, Ting KN, Wiart C, Fry J. 2013. High correlation of 2,2-diphenyl-1-picrylhydrazyl
(DPPH) radical scavenging, ferric reducing activity potential and total phenolics content
indicates redundancy in use of all three assays to screen for antioxidant activity of
extracts of plants from the Malaysian rainforest. Antioxidants. 2: 1-10.
Folin O, Ciocalteu V. 1927. On tyrosine and tryptophan determinations in proteins. The
Journal of Biological Chemistry. 73: 627-650.
Fujita N, Saito Y, Ito T, Mizuguchi H, Endo M, Ogata T. 2012. Folin-Chiocalteu colorimetric
analysis using a scanner for rapid determination of total polyphenol content in many test
samples. Studies in Science and Technology. 1: 139-142.
Harborne JB. 1973. Phytochemical methods, A guide to modern techniques of plant analysis.
London, Chapman and Hall Ltd.
Mosmann T. 1983. Rapid colorimetric assay for cellular growth and survival: application to
proliferation and cytotoxicity assays. Journal of Immunological Methods. 65: 55-63.
Murai Y, Kashimura S, Tamezawa S, Hashimoto T, Takaoka S, Asakawa Y, Kiguchi K,
Murai F, Tagawa M. 2001. Absolute configuration of (6S,9S)-roseoside from Polygonum
hydropiper. Planta Medica. 67: 480-481.
Sohn YM, Chin Y-W, Yang MH, Kim J. 2008. Terpenoid constituents from the aerial parts of
Asplenium scolopendrium. Natural Product Sciences. 14: 265-268.
Vermerris W, Nicholson R. 2007. Phenolic compound biochemistry. Springer, 276 pp.
Wang Y-S, Liao Z, Zhu H-K, Feng X-F, Jiang K-M, Huang R, Zhu N, Yang J-H. 2012.
Megastigmane O-glucopyranosides from Litsea glutinosa. Chemistry of Natural
Compounds. 48: 346-349.
Zhang Q, Zhang J, Shen J, Silva A, Dennis DA, Barrow CJ. 2006. A simple 96-well
microplate method for estimation of total polyphenolic content in seaweeds. Journal of
Applied Phycology. 18: 445-450.
Table S1. 13C NMR (solvent CD3OD, T= 300K, referenced to  (13CD3OD) = 49.0 ppm) and
1
H NMR (solvent CD3OD, unless otherwise stated, T = 300K; referenced to  (HCD2OD) =
3.31 ppm, data of roseosides 1 and 2 recorded at 500.13 MHz (1H) and 125 MHz (13C)
Multiplicity
13C
C
CH2
C-1
C-2
C
C-3
13C ppm
1
2
42.2
42.0
50.4
50.2
1H
1
—
—
2-Hax and 2.15 (d, 17)
2-He
2.52 (d, 17.1)
2
—
2.17 (d, 16.5)
2.60 (d, 16.5)
—
—
CH
C-4
4-H
5.86 (s)
5.87 (s br, w1/2 =4)
C
C-5
—
—
—
C
C-6
—
—
—
CH
C-7
7-H
5.87
5.97 (d, 15.6)
CH
C-8
8-H
5.86
5.73 (d,d, 15.6, 7.3)
CH
C-9
9-H
4.42 (qui, 5.5) 4.53 (qui, 6.4)
CH3
C-10
10-Me
1.29 (d, 6.2) 1.29 (d, 6)
CH3
C-11
11-Me
1.039 (s)
1.039 (s)
CH3
C-12
12-Me
1.033 (s)
1.018 (s)
CH3
C-13
13-Me
1.92 (d, 1.1)
1.943 (d, 1.1)
CH
C-1’
H-1’
4.34 (d, 7.9) 4.27 (d, 7.7)
CH
C-2’
H-2’
3.17 (d,d, 9, 3.19
7.7)
CH
C-3’
77.9
77.8
H-3’
3.34
3.27
CH
C-4’
71.0
71.2
H-4’
3.24
3.20
CH
C-5’
77.8
77.7
H-5’
3.23
3.15
CH2
C-6’
62.3
62.1
H-6’ and 3.62 (d,d, 12, 3.63 (d,d, 11.7, 6.1)
H-6’’
5) and 3.85 and 3.85 (d,d, 11.9,
(d,d, 12, 1.8) 1.8)
Multiplicity of signals: s - singlet; d - doublet; t - triplet; qui- quintuplet; m – multiplet; br broad signal; w1/2: width at half-height in Hz; δ in ppm; ax: axial; e: equatorial; * - signal not
detected (too low concentration of the sample)
200.8
126.8
166.8
79.7
131.2
134.9
77.0
21.0
23.2
24.3
19.3
102.4
74.8
*
126.6
166.9
79.4
133.4
133.3
74.3
21.5
23.7
24.2
18.1
100.7
74.7
—
1H ppm
Figure S1. Analytical NP-HPLC separation of (6S,9R)-roseoside (1) as major metabolite
(86% of the total) and (6S,9S)-roseoside (2) as minor one (14% of the total)
Figure S2. Cytotoxic activity of a methanol extract of A. theophrasti extract as compared to
doxorubicin (IC50 values in µg/mL) in MCF-7, CCRF-CEM and CEM/ADR 5000 cells
(results from an MTT assay).