Antioxidant flavonoids from Alhagi maurorum

Article abstract of DOI:10.1080/10286020903451724

A new flavonoid, isorhamnetin-3-O-[-α-L-rhamnopyranosyl-(1→3)]- β-D-glucopyranoside (1), along with two known flavonoids 3′-O-methylorobol (2) and quercetin 3-O-β-D-glucopyranoside (3), was isolated from Alhagi maurorum. Their structures were established with the help of mass spectrometry, 1D and 2D NMR spectroscopy, and in comparison with the literature data. Compounds 1 and 2 exhibited moderate antioxidant activity in the 2,2-diphenyl-1-picrylhydrazyl free radical scavenging assay.

Full text of DOI:10.1080/10286020903451724

Journal of Asian Natural Products Research
Vol. 12, No. 2, February 2010, 138–143
NOTE
Antioxidant flavonoids from Alhagi maurorum
Saeed Ahmada*, Naheed Riazb, Muhammad Saleemb, Abdul Jabbarb, Nisar-Ur-Rehmana
and Mohammad Ashrafa
aDepartment of Pharmacy, Faculty of Pharmacy and Alternative Medicine, Railway Campus, The
Islamia University of Bahawalpur, Bahawalpur 63100, Pakistan; Department of Chemistry, The
Islamia University of Bahawalpur, Baghdad-ul-Jadid Campus, Bahawalpur 63000, Pakistan
b
(Received 9 July 2009; final version received 29 October 2009)
A new flavonoid, isorhamnetin-3-O-[-a-L-rhamnopyranosyl-(1 ! 3)]-b-D-gluco-
pyranoside (1), along with two known flavonoids 30-O-methylorobol (2) and quercetin
3-O-b-D-glucopyranoside (3), was isolated from Alhagi maurorum. Their structures
were established with the help of mass spectrometry, 1D and 2D NMR spectroscopy,
and in comparison with the literature data. Compounds 1 and 2 exhibited moderate
antioxidant activity in the 2,2-diphenyl-1-picrylhydrazyl free radical scavenging assay.
Keywords: Alhagi maurorum; flavonoid glycosides; antioxidant; 2,2-diphenyl-1-
picrylhydrazyl radical scavenging
1. Introduction
alkaloids, flavonoids, and fatty acids are the
major active constituents of this genus [5,6].
The pharmacological importance of this
plant motivated us to explore this species for
its chemical constituents, which resulted in
the isolation of a new flavonoid glycoside
(1), along with 30-O-methylorobol (2) and
quercetin 3-O-b-D-glucopyranoside (3)
(Figure 1).
Alhagi maurorum is an undershrub, which is
60–100 cm tall, glabrous, or pubescent. It is
distributed in Pakistan, Kashmir, Iran,
Afghanistan, Russia, Turkey, Iraq, Syria,
Palestine, Cyprus, andAfrica[1]. Itiswidely
used in folk medicine as an anti-asthmatic,
aphrodisiac, antipyretic, appetizer, anti-
rheumatic, digestible, tonic, diuretic, demul-
cent, expectorant, laxative, cholagogue, and
refrigerant. It isalsousedforthetreatmentof
bronchitis, skin diseases, and piles [2]. The
flowers are used for the treatment of piles,
migraine, and warts. Essential oil from the
leaves of this plant is used for the treatment
of rheumatism [3]. Water extract of the roots
is used to enlarge the ureter and to remove
the kidney stones, whereas the methanolic
extract is known as an anti-diarrheal
agent [4]. A literature survey revealed that
2. Results and discussion
The methanolic extract of air-dried whole
plant material (8 kg) of A. maurorum was
evaporated in vacuo, suspended in H2O,
and successively partitioned with n-
hexane, chloroform, ethyl acetate, and n-
butanol. As a result of a series of column
chromatographic techniques of a chloro-
form-soluble fraction, compounds 1–3
were isolated as described in Section 3.
*Corresponding author. Email: rsahmed_iub@yahoo.com
ISSN 1028-6020 print/ISSN 1477-2213 online
q 2010 Taylor & Francis
DOI: 10.1080/10286020903451724
http://www.informaworld.com

Journal of Asian Natural Products Research
139
Figure 1. Structures of compounds 1–3.
Compound 1 was isolated as a yellow-
ish amorphous powder. The molecular
formula C28H32O16 was established
through HR-FAB-MS [positive mode]
showing a pseudo-molecular ion at m/z
625.1770 and common fragment ions in
FAB-MS at m/z 479 and 317 correspond-
ing to the loss of deoxyhexose [(MþH)2
146]þ and hexose [(MþH)2(146þ
162)]þ, respectively, from the molecular
ion. The UV spectrum of 1 showed
distinctive maxima at 254, 354, and 266
nm, similar to those of isorhamnetin-3-
O-glycoside [7]. The IR spectrum of 1
revealed the presence of hydroxyl groups
(3410 cm21), a methoxyl group (1590,
1055 cm21), and an aromatic system
(1610–1535 cm21). The 1H NMR spec-
trum of 1 (Table 1) showed distinctive
resonances for one methoxy group at d
3.93 and two anomeric protons at d 5.20
(1H, d, J ¼ 7.5 Hz) and 4.52 (1H, s). These
data combined with 13C NMR spectral
data allowed the glycosidic units to be
identified as b-glucopyranose and a-
rhamnopyranose, respectively. Acid
hydrolysis of 1 provided three products,
which were separated by a solvent

140
S. Ahmad et al.
Table 1. 1H and 13C NMR spectral data, HMBC, and COSY correlations of 1 (CD3OD).
Position
1Ha multiplicity (J, Hz)
13Cb (DEPT)
HMBCc (H ! C)
COSY-45
2
3
4
4a
5
6
7
8
–
–
–
–
–
158.8
135.4
179.2
105.5
162.8
100.1
166.4
95.0
158.4
122.9
114.5
148.3
150.8
116.1
124.0
104.4
72.0
84.8
72.5, 74.5
60.7
102.4
71.5
71.9
74.0
69.8
17.8
56.8
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
4a, 5, 7, 8
–
4a, 6, 7, 8a
6.19 d (1.5)
–
6.39 d (1.5)
–
8a
10
–
–
–
20
7.92 d (1.8)
–
2, 10, 30, 40, 60
30
–
–
40
–
–
500
6.90 d (8.5)
7.61 dd (8.5, 1.8)
5.20 d (7.5)
3.46 m
3.25 m
3.35–3.50 m
3.85–3.65 m
4.52 s
10, 30, 40, 600
H-50/H-600
600
100
200
300
400, 500
6
10, 20, 40, 5
H-50/H-600
3, 020 00, 300, 500
H-100/H-200
1 , 300, 400
H-200/H-3
1000, 200, 400, 500
H-300/H-400
–
–
300
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
1000
2000
3000
4000
5000
6000
OCH3
3.60 m
3.25 m
3.42 m
3.41 m
1.08 d (6.1)
3.93 s
Notes: a1H NMR spectrum was recorded at 500 MHz.
b13C NMR spectrum was recorded at 125 MHz.
cHMBC spectrum was recorded at 400 MHz.
extraction. The ethyl acetate layer contains
30-methylquercetin. The glycone could be
separated from the aqueous layer,
purified using preparative thin layer
chromatography by a solvent system
(EtOAc–MeOH–H2O–AcOH; 4:2:2:2),
and identified as D-glucose and L-rham-
nose through their optical rotation signs
and comparison of the retention time of
their trimethylsilyl (TMS) with the stan-
dards in gas chromatography. The inter-
glycosidic linkages and the site of attach-
ment between the glycoside and the
aglycone were determined from long-
range HMBC correlations, in which the
anomeric proton of b-Glu (d 5.20) showed
3J correlation with C-3 (d 135.4) of the
aglycone, confirming the site of attach-
ment of glucose at C-3 and Rha H-1000 (d
4.52) with Glu C-300 (d 84.8) and
characterizing the inter-glycosidic linkage
between these sugars as (1000 ! 300). The
‘D’ and ‘L’ configurations of b-Glu and
Rha were assumed by comparing the
coupling constant and 13C NMR spectral
data of several reported flavonoid glyco-
1
sides [8,9]. The aromatic region of the H
NMR spectrum showed two doublets at d
7.92 (1H, d, J ¼ 1.8 Hz), 6.90 (1H, d,
J ¼ 8.5 Hz) and a double doublet at d 7.61
(1H, dd, J ¼ 8.5, 1.8 Hz), supporting the
presence of the 1,3,4-trisubstituted ben-
zene ring. The downfield shift of H-20 (d
7.92) confirmed the position of the
methoxy group at C-30 [8], which was
further supported by HMBC 3J correlation

Journal of Asian Natural Products Research
141
of ZOMe (d 3.93) with C-3 (d 148.3). The
aromatic region also showed the signals of
two meta-coupled protons at d 6.39 (1H, d,
J ¼ 1.5 Hz) and 6.19 (1H, d, J ¼ 1.5 Hz).
The above-mentioned aromatic data
supported the presence of the isorhamnetin
nucleus in the molecule [8]. The 13C NMR
spectrum of 1 (Table 1) disclosed a total of
28 carbon signals; 2 methyl, 1 methylene,
15 methine, and 10 quaternary carbon
atoms. The downfield signals at d 179.2,
166.4, 158.4, 150.8, 148.3 indicated the
presence of a flavonoid nucleus with a
quercetin-like arrangement [8]. The sig-
nals for the anomeric carbons were
resonated at d 104.4 and 102.4, respect-
ively. The downfield signals of C-300 at d
84.8 indicated the attachment of both
sugars at C-300 position, confirming a
rungiose moiety in the molecule. On the
basis of these evidences, the structure of 1
was determined to be isorhamnetin 3-O-
rungioside. Although the glycone unit
rungiose is not common in natural
products, many natural flavonoid glyco-
sides have been reported in the literature to
have rungiose as a glycone part [10,11].
Compound 1 is a new natural product.
The structures of the known com-
pounds 2 [12] and 3 [13] were determined
by the comparison of spectroscopic data
and similar TLC profiles with the authentic
samples.
Table 2. Determination of DPPH scavenging
activity of 1 and 2.
S. no.
Sample code
IC50 (mg/ml)a
1
2
3
1
2
357.7 ^ 0.79
121.3 ^ 0.63
0.89 ^ 0.07
Quercetinb
Notes: aAll experiments were done in triplicate
(mean ^ SEM).
bStandard.
spectrophotometer (n in cm21). FAB-MS
and HR-FAB-MS spectra were recorded
on a Jeol JMS-HX 110 spectrometer with a
1
data system. The H NMR spectra were
recorded on Bruker AMX-400 and
500 MHz instruments using TMS as an
internal reference. The chemical shift
values are reported in ppm (d) units and
the scalar coupling constants (J) are in Hz.
The 13C NMR spectra were recorded at
125 MHz on the same instrument. Column
chromatography was carried out using
silica gel of 70–230 and 230–400 mesh.
Aluminum sheets precoated with silica gel
60 F254 (20 £ 20 cm, 0.2 mm thick;
E-Merck; purchased from a local author-
ized dealer of E-Merck) were used for
TLC to check the purity of the compounds
and were visualized under UV light (254
and 366 nm), followed by ceric sulfate as
the spraying reagent.
Compounds 1 and 2 were evaluated for
their antioxidant potential using 2,2-
diphenyl-1-picrylhydrazyl (DPPH) free
radical scavenging assay. Both compounds
exhibited low activity, with IC50 values of
357.7 and 121.3 mg/ml, respectively
(Table 2).
3.2 Plant material
The whole plant of A. maurorum (8 kg)
was collected from Cholistan Desert
(Bahawalpur) in May 2007 and identified
by Dr Muhammad Arshad, Plant Taxono-
mist, Cholistan Institute for Desert Studies
(CIDS), The Islamia University of Baha-
walpur, where a voucher specimen
(66/CIDS/07) is deposited.
3. Experimental
3.1 General experimental procedures
Optical rotations were measured on a
Jasco DIP-360 digital polarimeter. The UV
spectra were recorded on a Hitachi UV-
3200 spectrometer (lmax in nm). IR spectra
were recorded on a Shimadzu IR-460
3.3 Extraction and isolation
The shade-dried, ground whole plant
of A. maurorum (8 kg) was extracted

142
S. Ahmad et al.
with MeOH (3£ 33 liters). The combined
MeOH extract was concentrated, and
the residue (400g) was divided into n-
hexane-, chloroform-, ethyl acetate-, n-
butanol-, and water-soluble subfractions.
The chloroform-soluble subfraction (95 g)
was subjected to column chromatography
over silica gel, eluting with n-hexane, n-
hexane–chloroform, chloroform, chloro-
form–methanol, and methanol in increasing
order of polarity. The fractions obtained
from n-hexane–chloroform (3:7) showed a
strong UV-absorbing spot with some
impurities and were re-chromatographed
over silica gel using n-hexane–chloroform
(2.8:7.2) to obtain 30-O-methylorobol (2).
The fraction obtained from chloroform–
methanol (9.1:0.9) was chromatographed
over silica gel using chloroform–methanol
(9:1) as the eluent to provide quercetin 3-O-
b-D-glucopyranoside (3). The fraction
obtained from chloroform–methanol
(8.5:1.5) was chromatographed over silica
gel and eluted with chloroform–methanol
(8.4:1.6) to get compound 1.
system (EtOAc–MeOH–H2O–AcOH;
4:2:2:2) and was identified as D-glucose
and L-rhamnose by the sign of its optical
20
20
rotation (½aꢀD 252) and (½aꢀD þ8.1),
respectively. These sugars were also con-
firmed by comparison of the retention time
of their TMS ether (D-glucose a-anomer
4.1 min, b-anomer 7.8 min, and L-rhamnose
8.6 min) with the standards.
3.4 DPPH scavenging activity
The DPPH, stable free radical, was used
for the estimation of antioxidant activity
[14]. Compounds were dissolved in
DMSO, and 5 ml of each compound (final
concentration of 1 mg/ml) was mixed with
200 mM DPPH to make the total volume
up to 100 ml in a 96-well plate. The
contents were mixed and incubated at
378C for 30 min. The absorbance was
measured at 517 nm. Quercetin was used
as the control. The experiments were done
in triplicate. The percentage of DPPH
scavenging activity was calculated by the
formula given below. The IC50 value was
calculated by Ez-FitTM v. 5.03 (Perrella
Scientific, Amherst, NH, USA):
3.3.1 Compound 1
A yellowish amorphous powder (32 mg),
[a]D 225.75 (c ¼ 0.25, MeOH); IR nmax
(KBr): 3410, 1610–1535, 1055 cm21; UV
DPPH scavenging activity
¼ 100 2 ½ðOD of the test sample=
OD of the controlÞ £ 100ꢀ:
lmax (MeOH): 254, 354, 266 nm; FAB-MS
(positive-ion mode) m/z: 625 [MþH]þ,
479, 317; HR-FAB-MS (positive-ion
mode) m/z: 625.1770 (calcd for
C28H33O16, 625.1768); For the 1H and
13C NMR data, see Table 1.
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Journal of Asian Natural Products Research
143
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