Bignanoside A “A new neolignan glucoside” and bignanoside B “A new iridoid glucoside” from Bignonia binata leaves

Article abstract of DOI:10.1016/j.phytol.2019.12.009

One new neolignan glucoside; bignanoside A (1) and one new iridoid glucoside; bignanoside B (2), and one new lignan rhamnoside; (-)-1yoniresinol 3α-O-α-L-rhamnopyranoside (3) reported for the first time, alongside with seven known compounds were isolated from Bignonia binata leaves. The structures of the isolated compounds were elucidated by various spectroscopic methods, including 1D and 2D NMR experiments, as well as HR-ESI-MS. Compound 2 had a potent DPPH radical scavenging activity with IC₅₀ value of 18.34 ± 0.81 μM. Likewise, compounds 4 and 9 exhibited antitrypanosomal activity with IC₅₀ value of 29.12 ± 1.21 and 17.80 ± 0.32 μM, respectively, against Trypanosoma brucei brucei S427.

Full text of DOI:10.1016/j.phytol.2019.12.009

PhytochemistryLetters35(2020)200–205
Contents lists available at ScienceDirect
Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
Bignanoside A "A new neolignan glucoside" and bignanoside B "A new
iridoid glucoside" from Bignonia binata leaves
Basma Khalaf Mahmouda, Mamdouh Nabil Samya,*, Ashraf Nageeb Elsayed Hameda,
Usama Ramadan Abdelmohsena,b, Dina Hajjarc, Yoshi Yamanod, Sachiko Sugimotod,
Katsuyoshi Matsunamid, Mohamed Salah Kamela,e
a Department of Pharmacognosy, Faculty of Pharmacy, Minia University, 61519 Minia, Egypt
b Department of Botany II, Julius-von-Sachs Institute for Biological Sciences, University of Würzburg, Würzburg, Germany
c Division of Chemical & Life Sciences and Engineering and Division of Applied Mathematics and Computer Science, King Abdullah University of Science and Technology,
Thuwal 23955-6900, Saudi Arabia
d Department of Pharmacognosy, Graduate School of Biomedical & Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan
e Department of Pharmacognosy, Faculty of Pharmacy, Deraya University, Universities Zone, 61111 New Minia City, Egypt
A R T I C L E I N F O
A B S T R A C T
Keywords:
One new neolignan glucoside; bignanoside A (1) and one new iridoid glucoside; bignanoside B (2), and one new
lignan rhamnoside; (-)-1yoniresinol 3α-O-α-L-rhamnopyranoside (3) reported for the first time, alongside with
seven known compounds were isolated from Bignonia binata leaves. The structures of the isolated compounds
were elucidated by various spectroscopic methods, including 1D and 2D NMR experiments, as well as HR-ESI-
Bignoniaceae
Bignonia binata
Bignanoside A
Bignanoside B
MS. Compound 2 had a potent DPPH radical scavenging activity with IC50 value of 18.34
0.81 μM. Likewise,
(-)-Lyoniresinol 3α-O-α-L-rhamnopyranoside
compounds 4 and 9 exhibited antitrypanosomal activity with IC50 value of 29.12
respectively, against Trypanosoma brucei brucei S427.
1.21 and 17.80 0.32 μM,
1. Introduction
2. Results and discussion
Bignonia L. is the fifth largest genus in the tribe Bignonieae
(Bignoniaceae), with 31 lianas species distributed from Argentina to
USA (Zuntini et al., 2015a, b). Many species were reported to have
various secondary metabolites including; phenylethanoids, phenolics,
lignans, flavonoids, coumarins and xanthones (Mahmoud et al., 2019;
da Rocha et al., 2017; Martin et al., 2008, 2007). Most species are used
in folk medicine for treating a wide range of ailments, such as skin
ailments like fungal infections, boils, psoriasis and eczema, dysentery,
ringworm, tapeworm, malaria, diabetes, pneumonia, venereal diseases
and ulcers (Atawodi and Olowoniyi, 2015). B. binata Thunb. (syn:
Clytostoma binatum Thunb.) is a polymorphic and polyphyletic liana and
distributed from Mexico to Argentina (Zuntini et al., 2015a), The lack of
reports about any phytochemical investigation of B. binata encouraged
us to investigate the isolation and characterization of active con-
stituents from leaves of B. binata. As a result, ten compounds (1-10)
were isolated and characterized. All of the isolated compounds were
reported for the first time from this plant. In addition, the DPPH radical
scavenging, cytotoxic and antitrypanosomal activities of the isolated
compounds were described.
Extraction, fractionation and isolation of B. binata leaves, using
various chromatographic procedures led to the identification of ten
compounds, including two new compounds
2.1. Structure elucidation of the new compounds
24
Compound 1 [α]D +19.37 (c = 0.16, MeOH), was obtained as
yellow powder. Its molecular formula was established as C28H36O12
from the positive ion mode HR-ESI-MS. The IR spectrum of 1 displayed
absorption bands at 3123 cm−1 for hydroxyl groups. The 1H-NMR
spectrum of 1 (Table 1), showed the presence of three aromatic signals,
including one singlet signal was equivalent to two protons at δH 6.72
(2H, H-2,6) and two broad singlet aromatic protons at δH 6.96 (1H, H-
2') and 7.07 (1H, H-6'), which were attributable to two sets of tetra-
substituted benzene rings, besides other three singlet signals corre-
sponding to four methoxyls at δH 3.36 (3H, OCH3-9'), 3.82 (6H, OCH3-
3,5) and 3.89 (3H, OCH3-3'). In addition to, two trans olefinic proton
signals at δH 6.59 (d, J = 16.0 Hz, H-7') and 6.21 (m, H-8'). Ad-
ditionally, the 1H-NMR data revealed the presence of three doublet
⁎ Corresponding author.
E-mail address: mamdouhnabil.2006@yahoo.com (M.N. Samy).
https://doi.org/10.1016/j.phytol.2019.12.009
Received 11 September 2019; Received in revised form 8 December 2019; Accepted 16 December 2019
1874-3900/©2019PhytochemicalSocietyofEurope.PublishedbyElsevierLtd.Allrightsreserved.

B.K. Mahmoud, et al.
PhytochemistryLetters35(2020)200–205
Table 1
1H and APT spectral data of compound 1 (400 MHz and 100 MHz, respectively;
CD3OD).
No.
δH (m, J in Hz)
δC
1
–
132.5
104.5
149.5
134.4
149.5
104.5
89.9
2
6.72 (s)
–
3
4
–
5
–
6
6.72 (s)
5.64 (d, 6.7)
3.73 (m)
3.69 (d, 6.7)
3.82 (s)
–
7
8
52.9
9
63.0
OCH3-3,5
57.0
1′
132.5
112.5
145.7
150.4
130.3
117.0
134.5
124.5
74.5
2′
6.96 (br s)
–
3′
4′
–
5′
–
6′
7.07 (br s)
6.59 (d, 16.0)
6.21 (m)
4.07 (d, 6.2)
3.89 (s)
3.36 (s)
4.39 (d, 7.8)
3.2–4.3 (m)
3.2–4.3 (m)
3.2–4.3 (m)
3.2–4.3 (m)
3.2–4.3 (m)
7′
8′
9′
OCH3-3'
56.9
OCH3-9'
58.1
1″
2"
3″
4″
5″
6″
104.4
75.3
78.4
71.8
78.2
62.9
proton signals at δH 5.64 (d, J = 6.7 Hz, H-7), 4.07 (2H, d, J = 6.2 Hz,
H-9') and 3.69 (2H, d, J = 6.7 Hz, H-9), besides one multiplet proton
signal at δH 3.73 (H-8). Also, the 1H-NMR spectrum showed the pre-
sence of a doublet anomeric proton signal at δH 4.39 (H-1") was as-
signable to glucopyranosyl moiety with β-configuration, which was
deduced from the coupling constant (7.8 Hz) (Samy et al., 2017). The D-
configuration of the β-glucopyranosyl unit was deduced by HPLC ana-
lysis, using chiral detector. The resonance of C-8 at δC 52.9, besides the
coupling constant (6.7 Hz) of H-7 at δH 5.64, indicating the trans iso-
mers of the dihydrobezofuran neolignan (Wang et al., 2010; Li et al.,
Fig. 1. Structures of the isolated compounds 1–3 from B. binata leaves.
1997), additionally comparing the NMR spectroscopic data of
1
(Table 1) with those reported in the literature was in agreement with
(+)-dehydrodiconiferyl alcohol-4-O-β-D-glucopyranoside (Changzeng
and Zhongjian, 1997). The presence of methoxy groups at δH 3.82
(OCH3-3,5) and 3.36 (OCH3-9') attached to C-3,5 and C-9', respectively,
was confirmed by HMBC correlations. The anomeric proton signal at δH
4.39 (H-1") of β-D-glucopyranosyl moiety was correlated to C-4 at δC
134.4, indicating the glycosylation at C-4 (Fig. 2). All the previous as-
signments were completely substantiated with the help of APT, 1H-1H
COSY, HSQC, and HMBC experiments, therefore compound 1 was
characterized as (+)-5, 9'- dimethoxy dehydrodiconiferyl alcohol-4-O-
β-D-glucopyranoside and named as bignanoside A (Fig. 1).
Fig. 2. Significant COSY and HMBC correlations of 1.
signal at δC 151.9 (C-8) and one triplet carbon signal at δC 111.9 (C-10),
revealing the presence of an exocyclic methylene carbon in 13C-NMR,
which was confirmed by the resonance of the two methylene proton
signals of H-10 at δH 5.10 (1H, t, J = 2.5 Hz, H-10a) and 5.22 (1H, t,
J = 2.7 Hz, H-10b) in 1H-NMR. The 1H-NMR showed an anomeric
proton signal 4.26 (d, J = 7.9 Hz, H-1'), assignable to β-D-glucopyr-
anosyl moiety, in which the β- configuration was concluded from the
coupling constant (7.9 Hz) and the D-configuration of the β-glucopyr-
anosyl unit was deduced by HPLC analysis, using chiral detector. All
previously mentioned NMR data was in agreement with those reported
in the literature for strictoloside (Ye et al., 2010; Boros and Stermitz,
1990), in addition to the presence of an additional 9 carbon signals. Six
of them were attributed for trisubstituted benzene ring, concluded from
three aromatic proton signals at δH 6.76 (1H, d, J = 8.2 Hz, H-5"), 6.93
(1H, dd, J = 8.2, 2.0 Hz, H-6") and 7.02 (1H, d, J = 2.0 Hz, H-2”) in 1H-
NMR spectrum. Additionally, two olefinic carbons at δC 147.8 (C-7")
and 115.3 (C-8"), resonated at δH 7.58 (1H, d, J = 15.9 Hz, H-7") and
6.30 (1H, d, J = 15.9 Hz, H-8") in 1H-NMR and one carbonyl carbon
signal at δC 169.5 that were characteristic for a caffeoyl moiety (Xiong
23
Compound 2 [α]D − 6.0 (c = 0.1, MeOH), was obtained as an
amorphous yellow powder and having the molecular formula
C26H30O14, which had been determined by HR-ESI-MS positive mode.
The IR spectrum of 2 showed absorption bands at 3360 cm−1 for hy-
droxyl, (1696 and 1627 cm−1) for carbonyl groups and (1285, 1164
and 1079 cm−1) for an aromatic system. The 1H-NMR of compound 2
(Table 2) revealed the presence of a singlet signal at δH 7.48 of an
olefinic proton (H-3) and one hemiacetal proton signal at δH 5.27 (H-1,
d, J = 6.6). This was confirmed from 13C-NMR spectrum which showed
an olefinic carbon signal at δC 155.2 (C3), one quaternary carbon at δC
113.1 (C-4) and one hemiacetal carbon at δC 97.2 (C-1), in addition to
the presence of one carbonyl carbon signal at δC 168.2 (C-11), which
were characteristic signals of the iridoid skeleton (Ye et al., 2010).
Additionally, the downfield shift of C-7 at δC 72.4, indicating the pre-
sence of hydroxyl group, besides the resonance of quaternary carbon
201

B.K. Mahmoud, et al.
PhytochemistryLetters35(2020)200–205
Table 2
Table 3
1H and 13C -NMR spectral data of compound 2 (500 MHz and 125 MHz, re-
1H and 13C -NMR spectral spectral data of compound 3 (400 and 100 MHz,
spectively; CD3OD).
respectively CD3OD).
No.
δH (J in Hz)
δC
No.
δH (J in Hz)
δC
1
3
4
5
6
5.27 (d, 6.6)
97.2
1
2.63 (dd, 14.9, 11.7)
33.7
7.48 (s)
155.2
113.1
70.3
2
1.69 (m)
40.8
–
3
2.12 (m)
46.7
–
4
4.34 (d, 5.5)
43.1
1.88 (dd,12.2, 10.4)
2.88 (dd, 12.2, 6.8)
4.09 (m)
46.6
5
–
147.7
139.0
148.8
107.9
130.3
126.3
66.5
6
–
7
72.4
7
–
8
–
151.9
54.0
8
6.61 (s)
9
3.10 (m)
9
–
10
5.10 (t, 2.5)
5.22 (t, 2.7)
–
111.9
10
2α
–
3.64 (dd, 10.6, 4.4)
11
168.2
52.2
3.48 (dd, 10.6, 6.9)
OCH3-11
3.71 (s)
3α
3.72 (dd, 11.2, 3.5)
70.2
1′
4.26 (d,7.9)
overlapped
3.79 (m)
100.6
74.8
3.36 (m)
3.38 (s)
3.88 (s)
–
2′
OCH3-5
60.3
3′
77.9
OCH3-7
56.7
4′
3.77 (m)
72.2
1′
134.7
107.0
149.2
139.4
149.2
107.0
57.0
5′
3.21 (m)
76.4
2′
6.38 (s)
–
6′
4.42 (dd, 5.9, 12.1)
–
64.8
3′
1″
2"
3″
4″
5″
6″
7″
8″
9″
128.1
115.7
147.3
150.2
123.6
117.0
147.8
115.3
169.5
4′
–
7.02 (d, 2.0)
–
5′
–
6′
6.38 (s)
3.76 (s)
4.68 (br s)
–
OCH3-3', 5'
6.76 (d, 8.2)
6.93 (dd, 8.2, 2.0)
7.58 (d, 15.9)
6.30· (d, 15.9)
–
1″
2"
3″
4″
5″
6″
102.5
72.7
72.4
74.1
70.4
1.33 (d, 6.2)
18.3
m; multiplet or overlapped.
2.2. Identification of known compounds
Compounds 4 and 5 were identified by comparison of their physical
and chromatographic properties with an authentic samples and were
identified as ursolic acid (4) and β-sitosterol 3-O-β-D-glucopyranoside
(5), while the structures of the other isolated compounds were eluci-
dated by various spectroscopic methods, including 1D and 2D NMR
experiments (1H-NMR, 13C-NMR, DEPT, APT, COSY, HSQC, and
HMBC), as well as HR-ESI-MS analysis as acteoside (6) (Pereira et al.,
2008), isoacteoside (7) (Chen et al., 2013), adenosine (8) (Ciuffreda
et al., 2007), theviridoside (9) (Martin et al., 2007) and (+)-lyonir-
esinol 3α-O-β-D-glucopyranoside (10) (Ohashi et al., 1994) (Fig. 1).
Fig. 3. Significant HMBC correlations of 2.
et al., 1996). The downfield resonance of the of C-6' of the β-D-gluco-
pyranosyl moiety at δC 64.8 and the upfield shift of C-5' at δC 76.4,
indicated the attachment of the caffeoyl moiety at C-6', which was
confirmed from the long range correlation between H-6' of the gluco-
pyranosyl moiety at δH 4.42 and the carbonyl carbon (C-9") at δC 169.5
in HMBC (Fig. 3). Thus, the structure of 2 was confirmed as 6'-caffeoyl
strictoloside, and named as bignanoside B.
2.3. Biological assay of the isolated compounds
Compounds 1–10 were tested for their 1,1-diphenyl-2-picrylhy-
drazyl (DPPH) radical-scavenging activity and tumor cell growth in-
hibitory activities toward A549 by means of the 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. While, compounds
(3-10) were evaluated for their antitrypanosomal activity against
Trypanosoma brucei brucei S427 (Table 4).
26
Compound 3 [α]D −33.3 (c = 0.27, MeOH), was obtained as
yellow residue, its absolute configuration was found to be levorotatory
based on its negative optical rotation. The 1H-NMR and DEPT-Q spec-
tral data of 3 (Table 3), were similar to those of (-)-1yoniresinol 3α-O-β-
D-glucopyranoside (Ohashi et al., 1994; Tian et al., 2012), except for
the presence of one anomeric proton signal at δH 4.68 (1H, br s, H-1")
and one doublet methyl signal at δH 1.33 (3H, d, J = 6.0 Hz, H3-6”) in
1H-NMR, suggesting the presence of rhamnose instead of glucose. Re-
viewing the available literature, only (+)-1yoniresinol 3α-O-α-L-
rhamnopyranoside was previously isolated from Eucalyptus maideni
(Tian et al., 2012) and Ulmus thomasii (Hostettler and Seikel, 1969). It is
the first time for isolation of this compound from natural source.
Consequently, it was identified as (-)-1yoniresinol 3α-O-α-L-rhamno-
pyranoside.
Compounds 2, 3, 6 and 10 displayed DPPH radical scavenging ac-
tivity with IC50 of 18.34
0.81, 89.40
4.80, 37.00
1.20 and
72.10
20.30 μM, respectively, comparable with that of the standard
2.20 μM). Only compound 4 showed weak cytotoxic
trolox (16.60
activity toward A549 with IC50 of 45.00
5.80 μM. On the other
hand, the antitrypanosomal activity evaluation revealed that com-
pounds 4 and 9 exhibited the highest activity against T. brucei brucei
S427, with IC50 values of 29.12
1.21 and 17.80
0.32 μM, re-
spectively, as shown in Table 4. Whereas, the antitrypanosomal activity
of theviridoside (9) is reported for the first time.
202

B.K. Mahmoud, et al.
PhytochemistryLetters35(2020)200–205
Table 4
polarimeter. IR and UV spectra were measured on Horiba FT-710
Fourier transform infrared and JASCO V-520 UV/Vis spectro-
photometers, respectively. 1H and 13C NMR spectra were recorded on
Bruker Avance 400 MHz, 500 MHz and 600 MHz instruments in DMSO-
DPPH radical scavenging, cytotoxic and antitrypanosomal activities of the
isolated compounds.
No.
IC50 (μM)
d
6 and CD3OD. HR-ESI mass spectrum was taken on a LTQ Orbitrap XL
DPPH
A549
Trypanosoma brucei
mass spectrometer. Solvents used in this work, e.g., petroleum ether
(pet. ether; B.p. 60–80 °C), dichloromethane (DCM), ethyl acetate
(EtOAc), methanol (MeOH), and ethanol (EtOH), were purchased from
El-Nasr Company for Pharmaceuticals and Chemicals, Egypt and were
distilled before use. Acetonitrile (CH3CN) and MeOH of high perfor-
mance liquid chromatography (HPLC) grade were used for HPLC se-
parations and purifications and were obtained from SDFCL sd Fine-
Chem Limited, India. Deuterated solvents (Sigma-Aldrich, Germany),
including methanol (CD3OD) and dimethyl sulfoxide (DMSO-d6), were
used for nuclear magnetic resonance (NMR) spectroscopic analyses.
Column chromatography (CC) was performed using silica gel 60 (El-
Nasr Company for Pharmaceuticals and Chemicals, Egypt; 60–120
mesh) or sephadex LH-20 (0.25–0.1 mm, GE Healthcare, Sweden),
while silica gel GF254 for thin layer chromatography (TLC) (El-Nasr
Company for Pharmaceuticals and Chemicals, Egypt) was employed for
vacuum liquid chromatography (VLC)
brucei S427
1
> 100
18.34
89.40
> 100
> 100
37.00
> 100
> 100
> 100
72.10
16.60
–
> 100
> 100
> 100
45.00
> 100
> 100
> 100
> 100
> 100
> 100
> 100
0.90
NT
2
0.81
4.80
NT
3
> 100
4
5.80
29.12
> 100
> 100
> 100
> 100
17.80
> 100
–
1.21
5
6
1.20
7
8
9
0.32
0.08
10
20.30
2.20
Trolox
Doxorubicin
α-Difluoromethyl
ornithine
0.02
–
–
–
34.59
NT: Not tested. Data are presented as mean
SD (n = 3).
HPLC purifications were performed on KNAUER HPLC (smart line
pump 1000, degasser, diode array detector) with UV Detector, using
semi-prep RP-18 column (5 μm, 10 × 250 mm; Waters XBridge,
Germany), while an analytical Gemini-NX RP-18 column (5 μm,
4.60 × 100 mm; Phenomenex, Germany) was used for analytical pur-
poses. Ultraviolet lamp (UVP, LLC, USA) was used for visualization of
spots on thin layer chromatograms at 254 and/or 365 nm. Thin layer
chromatography (TLC) analyses were carried out using pre-coated silica
gel 60 GF254 plates (E. Merck, Darmstadt, Germany; 20 × 20 cm,
0.25 mm in thickness). Spots were visualized by spraying with 10 %
sulfuric acid in methanol followed by heating at 110 °C on a hot plate.
The vacuum liquid chromatography (VLC) technique was performed on
dry silica gel for TLC packed column (6 × 30 cm, 90 g) in the room
temperature and then the sample was loaded as solute and the elution
produced by the aid of water vacuum pump.
2.4. Chemotaxonomic significance
Ten secondary metabolites were isolated from leaves of B. binata,
including one neolignan (1), two iridoids (2 and 9), two lignans (3 and
10), one triterpene (4), one sterol (5), two phenylethanoids (6 and 7)
and one nucleoside (8). Among them, only compound (3) was reported
in the family Bignoniaceae for the first time and compound (8) was new
from the genus Bignonia. The obtained results reflected the chemical
diversity of B. binata and provide evidence for further chemotaxonomic
studies. This is the first comprehensive chemical investigation of leaves
of B. binate, where all the isolated compounds were reported for the first
time from this plant.
In this study two phenylethanoids (6 and 7) were previously ob-
tained from genus Bignonia. Among them, acteoside (6) was previously
reported in Tecoma stans (Syn. B. stans) (Ramirez et al., 2016) and Pi-
thecoctenium crucigerum (Syn. B. crucigera) (Martin et al., 2007), while
isoacteoside (7) was previously isolated from P. crucigera (Martin et al.,
2007).
3.2. Plant material
The leaves of B. binata were collected in December 2015, from El-
Zohria botanical garden, Giza, Egypt. Authentication of the plant was
identified by Prof. Dr. Nasser Barakat, Professor of Botany, Faculty of
Science, Minia University. A voucher specimen (Mn-ph-Cog-033) has
been deposited in the Herbarium of Pharmacognosy Department,
Faculty of Pharmacy, Minia University, Minia, Egypt.
Theviridoside (9) was previously gained from B. crucigera (Martin
et al., 2007). Additionally, (+)-lyoniresinol 3α-O-β-D-glucopyranoside
(10), and β-sitosterol 3-O-β-D-glucopyranoside (5) were isolated from
Macfadyena unguis-cati (Syn. B. unguis-cati) (Chen et al., 2017; Duarte
et al., 2000).
Likewise, ursolic acid (4) was previously isolated from B. unguis-cati
(Chen et al., 2017), in addition to Arrabidaea triplinervia (Syn. B. tri-
plinervia) (Leite et al., 2006) and Arrabidaea samydoides (Syn. B. samy-
doides) (Pauletti et al., 2003), which were various species of the same
genus, revealing the close phylogenetic relationship between these
Bignonia species.
3.3. Extraction and isolation
The air dried, powdered leaves (2.7 kg) of B. binata were extracted
by maceration with 95 % ethanol and concentrated under reduced
pressure to give solvent-free residue (420 g), which was then suspended
in 400 ml of distilled water, and defatted with pet. ether, followed by
partitioning with EtOAc (300 ml each×6) and then the solvents were
separately evaporated under vacuum, affording pet. ether (75 g) and
EtOAc fractions (125 g). Finally, the remaining mother liquor was
concentrated under reduced pressure to afford aqueous fraction (200 g).
An aliquot of the EtOAc fraction of B. binata leaves was subjected to
fractionation using VLC technique. In which, it is eluted initially with
EtOAc and then the polarity was increased gradually in 10 % by MeOH
till EtOAc-MeOH 70:30. Each polarity was collected and concentrated
under reduced pressure affording four subfractions. The first subfrac-
tion E-I (2.0 g) was rechromatographed on a silica gel column chro-
matography (CC) (Ф = 20 mm, L = 40 cm) using gradient mixtures of
pet. ether-EtOAc gradient mixtures affording compounds 4 (50.0 mg)
and 5 (40.0 mg).
On the other hand, adenosine (8) was reported in the genus Bignonia
for the first time and previously isolated from other genus in the family
Bignoniaceae, including Oroxylum indicum (Wei et al., 2013), indicating
chemotaxonomic homogeneity between the genera Bignonia and Orox-
ylum.
It is noteworthy that compounds 1–3 have not been isolated from
any other plant, which enhances the chemical diversity of natural
compounds and gives evidence for the value of chemotaxonomic studies
of B. binata.
3. Materials and methods
3.1. General experimental procedures
Optical rotation of the compounds was obtained on a JASCO P-1030
The second subfraction E-II (30.0 g) was further fractionated on
203

B.K. Mahmoud, et al.
PhytochemistryLetters35(2020)200–205
silica gel CC (Ф = 60 mm, L = 77 cm), using EtOAc-MeOH gradient
mixtures to afford five subfractions. Subfraction E-II-2 was chromato-
graphed on silica gel VLC (Ф = 40 mm, L = 40 cm), which was gra-
diently eluted with DCM-MeOH to give eight subractions, of which
subraction E-II-2-3 was subjected to silica gel CC using DCM-MeOH
(92:8) isocratic elution to give three subfractions, of which the second
subfraction E-II-2-3-2 (0.53 g) was subjected to sephadex LH-20 CC
using MeOH, followed by HPLC purification using H2O-MeOH (95:5)
for 5 min, followed by a linear gradient to 100% MeOH within 55 min
and finally with a further isocratic condition of MeOH for 5 min at a
flow rate of 2 ml/min to yield compounds 1 (5.0 mg; Rt = 21.69), 3
(15.0 mg; Rt = 21.69).
3.5. Evaluation of antioxidant activity
The antioxidant potential of the isolated compounds (3-10) was
evaluated by using DPPH radical scavenging method. Whereas, the
absorbance with various concentrations of the test compounds were
dissolved in MeOH (100 μl) in a 96-well microtiter plate and was
measured at 515 nm as Ablank. Then, 200 μM DPPH solution (100 μl)
was added to each well, followed by incubation in the dark at room
temperature for 30 min. The absorbance was measured again as Asample
.
Trolox was used as positive control with IC50 of 16.6
2.2 μM.
The % inhibition was calculated using the following equation:
% inhibition=[1-(Asample−Ablank)/(Acontrol−Ablank)]×100
Additionally, subraction E-II-2-6 (2.0 g) was rechromatographed on
sephadex LH-20 CC using MeOH, followed by HPLC using H2O-MeOH-
CH3CN (95:4:1) for 5 min, followed by a linear gradient to H2O-MeOH-
CH3CN (10:65:25) within 60 min at a flow rate of 2 ml/min, resulted in
the isolation of compounds 6 (60.0 mg; Rt = 23.07) and 7 (10.0 mg;
Rt = 25.05). While, subraction E-II-2-7 (2.9 g) was subjected to se-
phadex LH-20 CC using MeOH, afforded two subractions. Subfraction E-
II-2-7-a (0.43 g) was recrystallized by MeOH to afford compound 8
(19.5 mg), while E-II-2-7-b (1.9 g) was rechromatographed on reversed
phase (RP18) CC, followed by HPLC purification using H2O–MeOH
(90:10) for 5 min, followed by a linear gradient to 100 % MeOH within
60 min at a flow rate of 2 ml/min., yielded compounds 9 (100.0 mg;
Rt = 27.09), 2 (5.0 mg; Rt = 25.02) and 10 (25.0 mg; Rt = 20.27).
Where Acontrol is the absorbance of the control reaction mixture (con-
taining DMSO and all reagents except for the test compounds and
compounds.
IC50 was determined as the concentration of sample required to
inhibit the formation of the DPPH radical by 50 % (Samy et al., 2015).
3.6. Human cancer cell growth inhibition assay
This assay was performed using a human lung cancer cell line
(A549) and the viability was estimated by means of the colorimetric
MTT assay. Dulbecco’s modified Eagle medium supplemented with 10
% heat-inactivated FBS and 100 μg/mL of kanamycin was used as the
cell culture medium. The test compounds were dissolved in DMSO at
concentration of 10 mM and then added to the wells of 96-well mi-
crotiter plates to the final concentration of 1 %. A549 cells (5 × 103
cells/well) were cultured in a 5 % CO2 incubator at 37 °C for 72 h and
then a MTT solution was added to each well and the plates were in-
cubated for a further 1.5 h. Then the formazan precipitates were dis-
solved in DMSO and the optical density value for each well was mea-
sured at 540 nm with a microplate reader. Doxorubicin was used as a
3.3.1. Bignanoside A
24
Yellow powder. [α]D +19.37 (c = 0.16, MeOH); IR νmax (film)
cm–1: 3123, 2885, 1557, 1540, 1508, 1455, 1314, 1200, 1081, 1043;
UV λmax (MeOH) nm (log ε): 213 (4.16), 255 (3.94), 350 (3.94), 365
(4.06); 1H-NMR (400 MHz, CD3OD) and 13C-NMR (100 MHz, CD3OD):
Table 1; HR-ESI-MS (positive-ion mode) m/z: 587.2123 [M + Na]+
(Calcd for C28H36O12Na: 587.2104).
positive control (IC50: 0.90
The cell growth inhibition was calculated using the following
equation:
0.02 μM).
3.3.2. Bignanoside B
23
Pale yellow amorphous powder. [α]D − 6.0 (c = 0.1, MeOH); IR
% Inhibition = [1 ‒ (Asample ‒ Ablank) / (Acontrol ‒ Ablank)] × 100
νmax (film) cm–1: 3360, 1696 and 1627, 1285, 1164, 1079; UV λmax
(MeOH) nm (log ε): 197 (0.73), 220 (0.11), 242 (0.07) and 332 (0.06);
1H-NMR (500 MHz, CD3OD) and 13C-NMR (125 MHz, CD3OD): Table 2;
HR-ESI-MS (positive-ion mode) m/z: 589.1525 [M + Na]+ (Calcd for
Where Acontrol is the absorbance of the control reaction mixture con-
taining DMSO and all reagents except for the test compound. IC50 was
determined as the concentration of sample required to inhibit the for-
mation of MTT formazan by 50 % (Samy et al., 2014).
C
26H30O14Na: 589.1533).
3.7. Evaluation of antitrypanosomal activity
3.3.3. (-)-Lyoniresinol 3α-O-α-L-rhamnopyranoside
26
Pale yellow amorphous powder. [α]D −33.3 (c = 0.27, MeOH);
The isolated compounds were tested against Trypanosoma brucei
brucei S427 in the blood stream form. Whereas, samples were initially
screened at a single concentration of 20 μM (n = 3). The pure com-
pounds were prepared as 10 mM stock solution in 100 % DMSO and
diluted with HMI-9 (10 % FBS) medium. Controls included a sterility
control, 0.2 % DMSO and a concentration range of suramin as a positive
control. Suramin gave an antitrypanosomal activity against T. brucei
brucei at an MIC of 0.11 μM. Trypanosomes were counted using a
haemocytometer and diluted to a concentration of 3 × 104 trypano-
somes/mL (HMI-9 (10 % FBS)), 100 μL of this suspension was added to
each well. The assay plate was incubated at 37 °C with a humidified
atmosphere containing 5 % CO2 for 48 h. Then 20 μL of Alamar blue
IR νmax (film) cm–1: 3340, 2800, 1612, 1517, 1500, 1460, 1320, 1208,
1105, 1048, 980, 752; UV λmax (MeOH) nm (log ε): 225 (1.83), 272
(4.54), 283 (4.65); 1H-NMR (400 MHz, CD3OD) and 13C-NMR
(100 MHz, CD3OD): Table 3; HR-ESI-MS (positive-ion mode) m/z:
589.2261 [M + Na]+ (Calcd for C28H38O12Na: 589.2255).
3.4. Analysis of the sugar moiety
About 1 mg of compounds 1–3 were hydrolyzed with 1 M HCl
(1.0 ml) at 80 °C for 2 h. The reaction mixtures were neutralized with
Amberlite IRA96SB (OH−), then partitioned with an equal amount of
EtOAc (1.0 ml), and then water layers were analyzed for their sugar
components. The sugars were evaluated by HPLC on an amino column
[Shodex Asahipak NH2P-50 4E (4.6 mm × 250 mm), CH3CN-H2O (3:1),
1 ml/min], using chiral detector (JASCO OR-2090plus), in comparison
with an authentic sugars (D-glucose and L-rhamnose). Compounds 1
and 2 presented the peak for D-(+)-glucose at retention time of
8.32 min, while compound 3 provided the peak for L-(−)-rhamnose at
retention time of 5.38 min.
was added and the incubation continued for
a further 24 h.
Fluorescence was then determined using a Wallac Victor 2 microplate
reader (Excitation 530 nm Emission 590 nm). The results were calcu-
lated as % of the DMSO control values (Raheem et al., 2019).
4. Conclusion
The present study revealed the phytochemical analysis of B. binata
204

B.K. Mahmoud, et al.
PhytochemistryLetters35(2020)200–205
leaves, led to the isolation and identification of ten compounds, in-
cluding two new compounds with eight known ones, revealing the di-
verse classes of secondary metabolites in the plant. Besides, evaluation
of DPPH radical scavenging, cytotoxic and antitrypanosomal activities
of the isolated compounds.
stereochemistry at C-7 and C-8 in arylcoumaran neolignans. Phytochemistry 46,
929–934.
Mahmoud, B.K., Hamed, A.N.E., Samy, M.N., Kamel, M.S., 2019. Phytochemical and
biological overview of genus "Bignonia" (1969-2018). J. Adv. Biomed. Pharm. Sci. 2,
83–97.
Martin, F., Hay, A.-E., Corno, L., Gupta, M.P., Hostettmann, K., 2007. Iridoid glycosides
from the stems of Pithecoctenium crucigerum (Bignoniaceae). Phytochemistry 68,
1307–1311.
Declaration of Competing Interest
Martin, F., Hay, A.-E., Cressend, D., Reist, M., Vivas, L., Gupta, M.P., Carrupt, P.-A.,
Hostettmann, K., 2008. Antioxidant C-glucosylxanthones from the leaves of
Arrabidaea patellifera. J. Nat. Prod. 71, 1887–1890.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Ohashi, K., Watanabe, H., Okumura, Y., Uji, T., Kitagawa, I., 1994. Indonesian medicinal
plants. XII. Four isomaric lignan-glucosides from the bark of Aegle marmelos
(Rutaceae). Chem. Pharm. Bull. 42, 1924–1926.
Pauletti, P.M., Bolzani, V.D.S., Young, M.C.M., 2003. Chemical constituents of Arrabidaea
samydoides (Bignoniaceae). Química Nova 26 (5), 641–643.
Appendix A. Supplementary data
Pereira, A.C., Carvalho, H.W., Silva, G.H., Oliveira, D.F., Figueiredo, H.C., Cavalheiro,
A.J., Carvalho, D.A., 2008. Purification of an antibacterial compound from Lantana
lilacina. Rev. Bras. Farmacogn. 18, 204–208.
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.phytol.2019.12.009.
Raheem, D.J., Tawfike, A.F., Abdelmohsen, U.R., Edrada-Ebel, R., Fitzsimmons-Thoss, V.,
2019. Application of metabolomics and molecular networking in investigating the
chemical profile and antitrypanosomal activity of British bluebells (Hyacinthoides
non-scripta). Sci. Rep. 9, 2547.
References
Ramirez, G., Zamilpa, A., Zavala, M., Perez, J., Morales, D., Tortoriello, J., 2016.
Chrysoeriol and other polyphenols from Tecoma stans with lipase inhibitory activity.
J. Ethnopharmacol. 185, 1–8.
Atawodi, S.E.-O., Olowoniyi, O.D., 2015. Pharmacological and therapeutic activities of
Kigelia africana (Lam.) Benth. Annu. Res. Rev. Biol. 5, 1–17.
Samy, M.N., Sugimoto, S., Matsunami, K., Otsuka, H., Kamel, M., 2014. One new flavo-
noid xyloside and one new natural triterpene rhamnoside from the leaves of
Syzygium grande. Phytochem. Lett. 10, 86–90.
Boros, C.A., Stermitz, F.R., 1990. Iridoids. An updated review. Part I. J. Nat. Prod. 53,
1055–1147.
Changzeng, W., Zhongjian, J., 1997. Lignan, phenylpropanoid and iridoid glycosides from
Pedicularis torta. Phytochemistry 45, 159–166.
Samy, M.N., Khalil, H.E., Sugimoto, S., Matsunami, K., Otsuka, H., Kamel, M., 2015.
Amphipaniculosides A-D, triterpenoid glycosides, and amphipaniculoside E, an ali-
phatic alcohol glycoside from the leaves of Amphilophium paniculatum.
Phytochemistry 115, 261–268.
Chen, L., Chen, D., Zheng, Z., Liu, S., Tong, Q., Xiao, J., Lin, H., Ming, Y., 2017. Cytotoxic
and antioxidant activities of Macfadyena unguis-cati L. aerial parts and bioguided
isolation of the antitumor active components. Ind. Crops Prod. 107, 531–538.
Chen, C., Zhao, X.-H., Yue, H.-L., Li, Y.-L., Chen, T., 2013. Separation of phenylpropanoid
glycosides from a Chinese herb by HSCCC. J. Chromatogr. Sci. 52, 395–399.
Ciuffreda, P., Casati, S., Manzocchi, A., 2007. Complete 1H and 13C NMR spectral as-
signment of α‐and β‐adenosine, 2′‐deoxyadenosine and their acetate derivatives.
Magn. Reson. Chem. 45, 781–784.
Samy, M.N., Fahim, J.R., Sugimoto, S., Otsuka, H., Matsunami, K., Kamel, M.S., 2017.
Chodatiionosides A and B: two new megastigmane glycosides from Chorisia chodatii
leaves. J. Nat. Med. 71, 321–328.
Tian, L.W., Xu, M., Li, Y., Li, X.Y., Wang, D., Zhu, H.T., Yang, C.R., Zhang, Y.J., 2012.
Phenolic compounds from the branches of Eucalyptus maideni. Chem. Biodivers. 9,
123–130.
da Rocha, C.Q., de-Faria, F.M., Marcourt, L., Ebrahimi, S.N., Kitano, B.T., Ghilardi, A.F.,
Ferreira, A.L., de Almeida, A.C.A., Dunder, R.J., Souza-Brito, A.R.M., 2017.
Gastroprotective effects of hydroethanolic root extract of Arrabidaea brachypoda:
evidences of cytoprotection and isolation of unusual glycosylated polyphenols.
Phytochemistry 135, 93–105.
Wang, Y.H., Sun, Q.Y., Yang, F.M., Long, C.L., Zhao, F.W., Tang, G.H., Niu, H.M., Wang,
H., Huang, Q.Q., Xu, J.J., 2010. Neolignans and caffeoyl derivatives from Selaginella
moellendorffii. Helv. Chim. Acta 93, 2467–2477.
Wei, X.N., Lin, B.B., Xie, G.Y., Li, J.W., Qin, M.J., 2013. Chemical constitunents of seeds of
Oroxylum indicum. Zhongguo Zhong Yao Za Zhi 38, 204–207.
Xiong, Q., Kadota, S., TANI, T., NAMBA, T., 1996. Antioxidative effects of phenyletha-
noids from Cistanche deserticola. Biol. Pharm. Bull. 19, 1580–1585.
Ye, M., Zhao, Y., Norman, V.L., Starks, C.M., Rice, S.M., Goering, M.G., O’Neil‐Johnson,
M., Eldridge, G.R., Hu, J.F., 2010. Antibiofilm phenylethanoid glycosides from
Penstemon centranthifolius. Phytother. Res. 24, 778–781.
Duarte, D., Dolabela, M., Salm, C., Raslan, D., Oliveiras, A., Nenninger, A., Wiedemann,
B., Wagner, H., Lombardi, J., Lopes, M., 2000. Chemical characterization and bio-
logical activity of Macfadyena unguis-cati (Bignoniaceae). J. Pharm. Pharmacol. 52,
347–352.
Hostettler, F.D., Seikel, M.K., 1969. Lignans of Ulmus thomasii heartwood—II: lignans
related to thomasic acid. Teterahedron 25, 2325–2337.
Zuntini, A.R., Taylor, C.M., Lohmann, L.G., 2015a. Deciphering the Neotropical Bignonia
binata species complex (Bignoniaceae). Phytotaxa 219, 69–77.
Leite, J.P.V., Oliveira, A.B., Lombardi, J.A., Filho, J.D., Chiari, E., 2006. Trypanocidal
activity of triterpenes from Arrabidaea triplinervia and derivatives. Biol. Pharm. Bull.
29, 2307–2309.
Zuntini, A.R., Taylor, C.M., Lohmann, L.G., 2015b. Problematic specimens turn out to be
two undescribed species of Bignonia (Bignoniaceae). PhytoKeys 7.
Li, S., Iliefski, T., Lundquist, K., Wallis, A.F., 1997. Reassignment of relative
205