A new isoflavone from Blepharis ciliaris of an Egyptian origin

Article abstract of DOI:10.1007/s00044-012-0228-2

A phytochemical study of the aerial parts of Blepharis ciliaris (L.) B.L. Burtt. led to the isolation of one new isoflavone glycoside caffeic acid ester: genistein-7-O-(6″-O-E-caffeoyl-β-d-glucopyranoside) (4), along with seven known compounds: methyl veratrate (1), methyl vanillate (2), protocatechuic acid (3), naringenin-7-O-(3″-acetyl-6″-E-p-coumaroyl- β-d-glucopyranoside) (5), naringenin-7-O-(6″-E-p-coumaroyl-β-d- glucopyranoside) (6), apigenin-7-O-(6″-E-p-coumaroyl-β-d- glucopyranoside) (7), and acteoside (8). Their structures were established on the basis of detailed analyses of physical, chemical, and spectral data. Compounds 1, 2, 3, 6, and 8 were isolated for the first time from this plant. The antioxidant activity of the different extracts as well as for some of the isolated compounds was evaluated.

Full text of DOI:10.1007/s00044-012-0228-2

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:2346–2350
DOI 10.1007/s00044-012-0228-2
ORIGINAL RESEARCH
A new isoflavone from Blepharis ciliaris of an Egyptian origin
•
Mohamed A. El-Shanawany Hanaa M. Sayed
•
•
Sabrin R. M. Ibrahim Marwa A. A. Fayed
•
•
Mohamed M. Radwan Samir A. Ross
Received: 31 May 2012 / Accepted: 5 September 2012 / Published online: 18 September 2012
Ó Springer Science+Business Media, LLC 2012
Abstract A phytochemical study of the aerial parts of
Blepharis ciliaris (L.) B.L. Burtt. led to the isolation of one
new isoflavone glycoside caffeic acid ester: genistein-7-O-(600-
O-E-caffeoyl-b-D-glucopyranoside) (4), along with seven
known compounds: methyl veratrate (1), methyl vanillate (2),
protocatechuic acid (3), naringenin-7-O-(300-acetyl-600-E-p-
coumaroyl-b-D-glucopyranoside) (5), naringenin-7-O-(600-E-p-
coumaroyl-b-D-glucopyranoside) (6), apigenin-7-O-(600-E-
p-coumaroyl-b-D-glucopyranoside) (7), and acteoside (8). Their
structures were established on the basis of detailed analyses
of physical, chemical, and spectral data. Compounds 1, 2, 3,
6, and 8 were isolated for the first time from this plant. The
antioxidant activity of the different extracts as well as for
some of the isolated compounds was evaluated.
Several plants of the family Acanthaceae are medicinally
useful and others are economically important as ornamentals
since they have large flowers with colorful petals and could
be considered as a sourceof natural dyes (Chopra, 1973). The
genus Blepharis is represented in Egypt (Sinai peninsula)
and Saudi Arabia only by Blepharis ciliaris (L.) B.L. Burtt.
which is known in arabic as Shawk-ul-Dab, Zughaf, Shuqaf
¨
(Boulos, 1981; Tackholm, 1974; Muschler, 1970; Rizk,
1986). The plant seeds (roasted or crushed) are applied on
sores, wounds, and boils as an antibacterial. The seeds are
also considered as diuretic, aphrodisiac, and expectorant
(Deshpande, 2006). The charcoal from the roots is
applied to the eyes to improve vision, hence the Arabic name
¨
‘‘Kohl-el-agouz’’ (Boulos, 1981; Tackholm, 1974). Previous
phytochemical studies of B. ciliaris (L.) resulted in the
isolation and identification of apigenin 7-O-(300-acetyl-600-
E-p-coumaroylglucoside), naringenin 7-O-(300-acetyl-600-
E-p-coumaroylglucoside), apigenin-7-O-glucoside, and 90-de-
carboxy-rosmarinic acid-40-O-(1 ? 4)-galactosylrhamnoside
(Afifi, 2003; Harraz et al., 1996).
Keywords Blepharis ciliaris ꢀ Isoflavone ꢀ Naringenin ꢀ
Apigenin ꢀ Acteoside ꢀ Antioxidant
Introduction
The family Acanthaceae includes about 346 genera and
around 4,300 species, distributed in tropics, the Mediterra-
nean, Australia, Central America, Brazil, Africa, and Indo-
Malaysia (Ghazanfar, 1994; Lawrence, 1968; Meikle, 1985).
Materials and methods
General
Melting points were uncorrected and carried out on an
Electrothermal 9100 Digital Melting Point apparatus
(Electrothermal Engineering Ltd, Essex, England). EI- and
FAB–MS were recorded on a Jeol the mass route JMS.600
H mass spectrometer. UV spectra were recorded in MeOH
on a Shimadzu 1601 UV/VIS spectrophotometer. The IR
spectra were measured on a Shimadzu Infrared-400 spec-
trophotometer (Kyoto, Japan). NMR spectra (chemical
shifts in ppm, coupling constants in Hz) were recorded on
M. A. El-Shanawany ꢀ H. M. Sayed ꢀ S. R. M. Ibrahim ꢀ
M. A. A. Fayed
Department of Pharmacognosy, Faculty of Pharmacy,
Assiut University, Assiut 71526, Egypt
M. M. Radwan ꢀ S. A. Ross (&)
National Center for Natural Products Research,
School of Pharmacy, University of Mississippi,
University, MS 38677, USA
e-mail: sross@olemiss.edu
123

Med Chem Res (2013) 22:2346–2350
2347
Varian Oxford NMRYH-400 using DMSO-d6 as solvent.
NMR spectra were referenced to the solvent signal
(2.49 ppm for 1H and 39.9 ppm for 13C). Column chro-
matographic separation was performed on silica gel 60
(0.04–0.063 mm), RP-18 (0.04–0.063 mm, Merck), and
Sephadex LH-20 (0.25–0.1 mm, Merck). TLC was per-
formed on precoated TLC plates with silica gel 60 F254
(0.2 mm, Merck). The solvent systems were used for
TLC analyses; CHCl3:MeOH (9.5:0.5, I), (9:1, II), (8:2,
III), (7:3, IV), and n-BuOH:acetone:formic acid:H2O
60:17:8:15 (system V). Authentic flavonoids and sugars
were obtained from the Department of Pharmacognosy,
Faculty of Pharmacy, Assiut University. 1,1-Diphenyl-2-
picrylhydrazyl radical (DPPH) and quercetin were pur-
chased from Sigma Chemical Co. (Germany).
10 % v/v H2SO4, and then heated to 110 °C. Flavonoidal
subfractions (EB-II to EB-V) were subjected to silica gel CC
(80 g, 100 9 4 cm) using CHCl3: MeOH gradients, com-
pounds 4 (200 mg, yellow needles, CHCl3:MeOH 9:1), 5
(25 mg, yellow powder, CHCl3:MeOH 8:2), 6 (100 mg, yel-
low powder, CHCl3:MeOH 8:2), and 7 (40 mg, pale yellow
powder, CHCl3:MeOH 8:2) were isolated. Reversed phase
CC of subfraction (EB-VI, 80 mg) (30 g, 60 9 3 cm) using
H2O:MeOH gradient elution gave compound 8 (30 mg, dark
red residue, H2O:MeOH 6:4). The other fractions were kept
for further investigation.
Genstein-7-O-(600-O-E-caffeoyl)-b-D-glucopyranoside
(4): Yellow needles (MeOH). Rf = 0.75 (solvent system
II). m.p 234 °C. UV kmax (MeOH) nm: 270, 301. IR (KBr):
c
max 3270, 1690, 1605, 1509 cm-1. NMR data (DMSO-d6,
400 and 100 MHz): see Table 1 FABMS m/z: 595
[M?H]?, 433 [M-(caffeoyl unit)?H]?, 271 [M-(caffeoyl-
unit?hexose unit)?H]?.
Plant materials
Blepharis ciliaris (L.) B.L. Burtt. aerial parts were col-
lected on May 2005 from Southern Sinai (Wadi Al-Ratam).
The plant was kindly identified by Dr. A. A. Fayed, Prof. of
Plant Taxonomy, Faculty of Science, Assiut University,
Assiut, Egypt. A voucher sample (BC-20051) was kept in
the herbarium of the Faculty of Pharmacy, Assiut Univer-
sity, Assiut, Egypt.
Antioxidant activity
The antioxidant activity was determined as previously out-
lined (Steffan et al., 2005) by the decrease in the absorption
of each of the isolated compounds or soluble fractions in
118 9 10-5 % DPPH solution (final concentration of the
sample in the cuvette was 20 lM for pure compounds and
0.25, 0.5, and 1.0 mg/mL for soluble fractions) monitored at
517 nm using a spectrophotometer. The absorbance of
DPPH in EtOH (with or without compounds) was measured
after 2 min. The antioxidant activity of each compound was
measured in relation to quercetin (as a reference antioxidant)
set as 100 % antioxidant activity. Determinations were
performed in triplicate. The antioxidant activity was calcu-
Chemistry
Extraction and isolation
The air-dried powdered aerial parts (3 kg) were exhaustively
extracted by cold percolation with MeOH. The alcoholic
extract was concentrated under reduced pressure to afford a
viscous brown residue (250 g). The residue was mixed with
500 mL distilled H2O and subjected to solvent fractionation
using n-hexane, CHCl3, EtOAc, and n-BuOH which were
separately concentrated yielding 70, 40, 40, and 50 g,
respectively. The CHCl3 fraction (40 g) was subjected to
silica gel column chromatography using CHCl3:MeOH gra-
dient elution. Ten subfractions were obtained (CB-I to CB-X).
All fractions were monitored by TLC and sprayed with FeCl3
and 10 % v/v H2SO4, and then heated to 110 °C. The sub-
fraction (CB-II) (2 g) was subjected to silica gel CC (60 g,
100 9 4 cm) using CHCl3:MeOH gradients, to afford 1
(25 mg, colorless needles, CHCl3:MeOH 9.5:0.5) and 2
(20 mg, colorless needles, CHCl3:MeOH 9.5:0.5). While the
subfraction CB-III (1 g) was subjected to silica gel CC (30 g,
100 9 1 cm) using CHCl3:MeOH gradient elution where
compound 3 (20 mg, pale yellow needles, CHCl3:MeOH 9:1)
was isolated; the EtOAc fraction(40 g) was subjected to silica
gel CC using CHCl3:MeOH gradient elution. Twelve sub-
fractions were obtained (EB-I to EB-XII). All subfractions
were monitored by TLC and sprayed by 5 % AlCl3 and
lated using the following equation:% Antioxidant activity ¼
Aꢂ with compound
Aꢂ of the blank
1 ꢁ
ꢃ 100 where, A° = Absorbance
Acid hydrolysis
A solution of the isolated glycoside (5 mg in 10 mL MeOH)
was treated with 3 % H2SO4 (1.5 mL) and heated at 100 °C
for 1 h. The aglycone was extracted with EtOAc, concen-
trated under reduced pressure, purified on Sephadex LH-20
column using MeOH, and identified by co-TLC with an
authentic sample using solvent system II. The sugars in the
aqueous layer were identified by co-PC (paper chromatog-
raphy) with authentic materials using solvent system V.
Results and discussion
In this paper, we report the isolation and structural eluci-
dation of one new isoflavone glucoside [genistein-7-O-(600-
O-E-caffeoyl-b-D-glucopyranoside)] (4), along with seven
123

2348
Med Chem Res (2013) 22:2346–2350
Table 1 1H and 13C-NMR data of 4 (DMSO-d6, 400 MHz)
absorption band at kmax 270 and 301 nm which are char-
acteristic for isoflavonoid (Mabry et al., 1970). The bath-
ochromic shift of the UV absorption bands I and II with
NaOCH3 and AlCl3 suggested the presence of 40- and
5- OH groups, respectively, and the absence of NaOAc
band II shift indicated that the 7-OH group was substituted
(Mabry et al., 1970). The IR spectrum indicated the pres-
ence of an a,b-unsaturated CO group at 1690 cm-1, OH
groups at 3270 cm-1, and aromatic ring at 1605 and
No.
dC (mult.)
dH [mult., J (Hz)]
2
156.9 (CH)
121.7 (C)
181.9 (C)
162.2 (C)
99.4 (CH)
164.4 (C)
94.6 (CH)
161.1 (C)
105.3 (C)
120.3 (C)
130.1 (CH)
115.8 (CH)
160.2 (C)
102.8 (CH)
73.0 (CH)
73.7 (CH)
69.9 (CH)
76.3 (CH)
63.4 (CH2)
124.6 (C)
115.98 (CH)
145.1 (C)
147.3 (C)
116.1 (CH)
120.3 d
8.43 s
3
–
4
–
5
–
6
6.45 brs
7
–
1
8
6.55 brs
1509 cm-1 (Silverstein and Webster, 1998). The HNMR
spectrum of 4 showed two singlet signals at dH 12.92 and
8.43 which were assigned to 5-chelated OH group and H-2
of isoflavone (Table 1) (Agrawal, 1989; Harborne, 1988).
The protons resonances at dH 7.33 (2H, d, J = 8.8 Hz) and
dH 6.67 (2H, d, J = 8.8 Hz) indicated the presence of a
para-disubstituted benzene moiety (ring B) as well as other
resonated protons at dH 6.55 and 6.45 (each 1H, brs) which
were assigned to H-8 and H-6, respectively (Agrawal,
1989; Harborne, 1988; Williams and Harborne, 1994). The
1HNMR spectrum of 4 also displayed an ABX system for a
1,3,4-tri-substituted aromatic ring at dH [6.81 (1H, d,
J = 3.2 Hz, H-2000), 7.90 (1H, d, J = 8.0 Hz, H-5000), 6.93
(1H, dd, J = 8.0, 3.2 Hz, H-6000)], and two trans-olefinic
protons at dH [7.45 (1H, d, J = 15.6 Hz, H-7000), 6.30 (1H,
d, J = 15.6 Hz, H-8000)], suggesting the presence of caf-
feoyl moiety (Chen and Yang, 2007; Du¨ru¨st et al., 2001).
Furthermore, anomeric proton for sugar moiety at dH 5.17
(1H, d, J = 8.0 Hz) indicated b-glucopyranose moiety
(Agrawal, 1992). The presence of genistein, b-D-glucose,
and caffeoyl moiety were confirmed by 13CNMR
(Table 1) and the multiplicity of each carbon was deter-
mined by DEPT experiments. Upon the hydrolysis of 4,
genistein, caffeic acid, and b-D-glucose were identified by
co-TLC alongside with authentic samples. On the basis of
the above results, it was assumed that 4 contained caf-
feoyl glucose linked at the 7-OH group of genistein.
Furthermore, the significant downfield shift of C-600 of
glucose (dC 63.4) indicated the attachment of caffeoyl
moiety at C-600 of glucose. On the basis of the previous
mentioned data, 4 was identified as genistein-7-O-(600-O-
E-caffeoyl)-b-D-glucopyranoside and considered as a new
natural product.
9
–
10
10
20, 60
30, 50
40
100
200
300
400
500
600
1000
2000
3000
4000
5000
6000
7000
8000
9000
5-OH
–
–
7.33 d (8.8)
6.67 d (7.2)
–
5.17 d (8.0)
3.14–5.03
–
6.81 d (3.2)
–
–
7.90 d (8.0)
6.93 dd (8.0, 3.2)
7.45 d (15.6)
6.30 d (15.6)
–
145.0 d
113.4 d
166.6 (C)
–
12.92
known compounds: methyl veratrate (1), methyl vanillate
(2), protocatechuic acid (3), naringenin-7-O-(300-acetyl-600-
E-p-coumaroyl-b-D-glucopyranoside) (5), naringenin-7-O-
(600-E-p-coumaroyl-b-D-glucopyranoside) (6), apigenin-7-
O-(600-E-p-coumaroyl-b-D-glucopyranoside) (7), and acteoside
(8) from the aerial parts of B. ciliaris. Compounds 1–3, 6,
and 8 are reported for the first time from this plant (Fig. 1).
The antioxidant activity of the crude extracts, and two of
the isolated compounds were evaluated.
The other isolated compounds were identified as methyl
veratrate (1) (Erickson and Miksche, 1974), methyl vanil-
late (2) (Wu et al., 1994), protocatechuic acid (3) (Gero-
thanassis et al., 1998; Sang et al., 2002), naringenin-
7-O-(300-acetyl-600-E-p-coumaroyl-b-D-glucopyranoside) (5)
(Harraz et al., 1996), naringenin-7-O-(600-E-p-coumaroyl-
b-D-glucopyranoside) (6) (Singh et al., 1999), apigenin-
7-O-(600-E-p-coumaroyl-b-D-glucopyranoside) (7) (Harraz
et al., 1996), and acteoside (8) (Pereira et al., 2008) by
comparison of their physical and spectral data with those in
literature.
Compound 4 was isolated as yellow needles and gave
positive tests for flavonoids (Harborne and Mabry, 1975;
Mabry et al., 1970; Markham, 1982). The FABMS spec-
trum showed [M?H]? at m/z 595 consistent with molec-
ular formula C30H27O13. The two significant fragments at
m/z 433 [M-caffeoyl?H]? and m/z 271 [M-caffeoyl,-
hexose,?H]? indicated that 4 is a favonoid with caffeoyl
and hexose units. The characteristic fragments in the mass
spectrum suggested it to be a genistein derivative (Mabry
et al., 1970). The UV spectrum in MeOH displayed
123

Med Chem Res (2013) 22:2346–2350
2349
8
O
O
OR1
2```
5```
7```
9```
7
1
OH
OH
3```
1```
8```
O
6
5
2
3
4```
6```
6``
4``
O
HO
HO
OR2
8
4
O
O
2``
9
1``
2
3
OH
O
7
OR
2`
5`
1 R = CH3, R1 = CH3, R2 = CH3
2 R = H, R1 = R2 = CH3
3 R = R1 = R2 = H
6
1`
4
10
3`
4
5
O
O
4`
OH
6`
OH
O
O
OH
OH
O
OH
HO
RO
OH
O
O
O
O
HO
OH
O
O
O
HO
OH
7
5
5
6
R = COCH3
R = H
HO
HO
6
4
OH
1
OH
3
O
8
2
7
9
OH
O
6``
O
4``
5``
6```
2``
H3C
4```
1``
O
3``
5```
OH
HO
HO
2` ``
O
1```
O
3```
OH
8`
8
7`
1`
6`
2`
3`
5`
HO
4`
OH
Fig. 1 Chemical structures of the isolated compounds
Agrawal PK (1992) NMR spectroscopy in the structural elucidation of
oligosaccharides and glycosides. Phytochemistry 31:3307–3330
Boulos L (1981) Medicinal plants of North Africa. Reference
Publications, Inc., Algonac, p 23
Chen P, Yang J (2007) Flavonol galactoside caffeiate ester and
homoisoflavones from Caesalpinia millettii Hook et ARN. Chem
Pharm Bull (Tokyo) 55:655–657
The results of antioxidant activity proved that the total
MeOH, EtOAc, and aqueous extracts of B. ciliaris exhib-
ited high antioxidant activities of 89, 86, and 85 %,
respectively, while compounds 4 and 6 showed no activity.
The activity of the different extracts may be due to the
presence of phenolic compounds (flavonoids, phenylprop-
anoids, and phenolic acids).
Chopra GL (1973) Angiosperms, 11th edn. S. L. Jain for S. Nagin &
Co., Jalandhar, pp 344–348
Deshpande DJ (2006) A handbook of medicinal herbs. Agrobios,
Jodhpur, pp 342–343
¨
˘
Du¨ru¨st N, Ozden S, Umur E, Du¨ru¨st Y, Ku¨c¸u¨kislamoglu M (2001)
The isolation of carboxylic acids from the flowers of Delphinium
formosum. Turkish J Chem 25:93–97
References
Erickson M, Miksche GE (1974) Two benzofurans obtained on
oxidative degradation of the moss Polytrichum Commune Hedw.
Acta Chemica Scandinavica 28:109–113
Gerothanassis IP, Exarchou V, Lagouri V, Troganis A, Tsimidou M,
Boskou D (1998) Methodology for identification of phenolic
Afifi MSA (2003) A novel 40-O-diglycoside of decarboxyrosmarinic
acid from Blepharis ciliaris. Pharm Biol 41:487–490
Agrawal PK (1989) Carbon-13 NMR of flavonoids. Elsevier, New
York
123

2350
Med Chem Res (2013) 22:2346–2350
acids in complex phenolic mixtures by high-resolution two-
dimensional nuclear magnetic resonance. Application to meth-
anolic extracts of two Oregano species. J Agric Food Chem
46:4185–4192
antibacterial compound from Lantana lilacina. Brazilian J
Pharmacogn 18:204–208
Rizk AM (1986) The phytochemistry of the Flora of Qatar. King
Print, Richmond, p 3
¨
Ghazanfar SA (1994) Handbook of Arabian medicinal plants. CRC
Press, Ann Arbor, p 8
Harborne JB (1988) The flavonoids. Advances in Research since
1980. Chapman & Hall, New York
Harborne JB, Mabry H (1975) The flavonoids. Chapman& Hall, New
York
Harraz FM, Pederson AT, Andersen M, Verotta L, Tato M (1996)
Acylated flavonoids from Blepharis ciliaris. Phytochemistry
43:521–525
Lawrence GHM (1968) Taxonomy of vascular plants, 12th printing.
MacMillan Co., New York, pp 707–709
Mabry TJ, Markham KR, Thomas MB (1970) The systematic
identification of flavonoids. Springer, New York
Markham K (1982) Techniques of flavonoid identification. Academic
Press, New York
Meikle RD (1985) Flora of cyprus, vol II. Bentham–Moxom Trust,
Kew
Muschler R (1970) A manual flora of Egypt. S-H Service Agency,
Inc., New York, pp 901–902
Sang S, Lapsley K, Rosen RT, HO C (2002) New prenylated benzoic
acid and other constituents from Almond hulls (Prunus amygd-
alus batsch). J Agric Food Chem 50:607–609
Silverstein RM, Webster FX (1998) Spectrometric identification of
organic compounds, 6th edn. John Wiley, New York
Singh VP, Bineeta Y, Pandey VB (1999) Flavanone glycosides from
Alhagi pseudalhagi. Phytochemistry 51:587–590
Steffan B, Watjen W, Michels G, Niering P, Wray V, Ebel R, Edrada
¨
RA, Kahl R, Proksch P (2005) Polyphenols from plants used in
traditional Indonesian medicine (Jamu): uptake and antioxidative
effects in rat H4IIE hepatoma cells. J Pharm Pharmacol 57:233–
240
¨
Tackholm V (1974) Students’ flora of Egypt, 2nd edn. Cairo
University, Cairo, pp 501–502
Williams CA, Harborne JB (1994) The flavonoids. Advances in
research since 1986. Chapman & Hall, New York
Wu TS, Ou LF, Teng CM (1994) Aristolochic acids, aristolactam
alkaloids and amides from Aristolochia kankauensis. Phyto-
chemistry 36:1063–1068
Pereira AC, Carvalho HWP, Silva GH, Oliveira DF, Figueiredo HCP,
Cavalheiro AJ, Carvalho DA (2008) Purification of an
123