International Journal of Organic Chemistry, 2011, 1, 132-141
doi:10.4236/ijoc.2011.13020 Published Online September 2011 (http://www.SciRP.org/journal/ijoc)
Two Flavones from Acanthospermum hispidum DC and
Their Antibacterial Activity
Theresa I. Edewor, Abass A. Olajire*
Department of Pure and Applied Chemistry, Ladoke Akintola University of Technology, Ogbomoso, Nigeria
Received May 28, 2011; revised June 28, 2011; accepted July 6, 2011
Abstract
Two new flavones, namely 5,7,2’,5’-tetrahydroxy-3,4’-dimethoxyflavone and 5’-acetoxy-5,7,2’-trihydroxy-3,
4’-dimethoxyflavone were successfuly isolated from the leaves of Acanthospermum hispidum DC and iden-
tified by UV-Vis, IR, 1H-NMR and EI-MS techniques. Both compounds exhibited antibacterial activity
against Salmonella typii, Staphylococcus aureus, Klebsiella pneumoniae, Proteus mirabilis, Bacillus subtilis,
Pseudomonas aeruginosa and Shigella dysenteriae with minimum inhibition concentration (MIC) ranging
from 0.001 - 0.20 but was inactive against Escherichia coli, Corybacterium pyogenes and Proteus vulgaris.
Keywords: Acanthospermum hispidum DC, Flavonoids, Antibacterial, Spectroscopy
1. Introduction
Although the main use of plants is directed to nutritional
purposes; however they are also used at various religious
functions, for economic purpose and more importantly
for medicinal purposes [1]. Nowadays, pathogenic mi-
croorganisms are acquiring multiple resistances to exist-
ing antibiotics at an alarming rate globally [2]. All these
factors necessitated the search for alternative medicine of
natural origin for the treatment of microbial infections
and traditional folk medicine is being used as a guide.
Many herbs used in folklore for the treatment of diseases
in different parts of the world are being screened for an-
timicrobial activities and the results obtained from these
scientific studies so far have rationalized the tradomedi-
cal use of many plants and plant parts [3-5].
Acanthospermum hispidum DC is a member of the
family of plants called Asteraceae. It is found in East,
West and Central Africa [6]. In USA, it is found in
Georgia, Virginia, Florida, New Jersey and Alabama [7].
Other countries where it is found include Paraguay, Ar-
gentina, Bolivia, India, Brazil, Nicaragua, Honduras,
Hawaii, Australia and Venezuela [8].
In the specific case of A. hispidum, there is a huge
volume of literature related to research on numerous
chemical components extracted from this plant, as well
as chemotaxonomic and phytotherapeutic investigations.
The oldest phytochemical studies dated to 1975 when
investigations concerning sesquiterpene lactones with
potential biological activities were reported [9] and in
1976 further studies on A. hispidum were published
[10,11]. The sesquiterpene lactones found in A. hispidum
were chemically distinct from other sesquiterpenoids due
to the presence of a α-methylene-γ-lactone system, many
of them containing α,β-unsaturated carbonyl and epo-
xides, which are part of a larger family of compounds
with a wide spectrum of biological activities. Investiga-
tions of species of Acanthospermum have led to the iso-
lation of cis-cis-germacranolides and melampolides [8].
Additional compounds isolated from A. hispidum are
alkaloids [12], polyphenolic constituents (flavones, caf-
feic acid and phenylpropans, acanthospermol galactoside
[13], sesquiterpene hydrocarbons, β-caryophyllene, α-hu-
mulene, bicyclogermacrene, germancrene D, α-isabolol,
nonanal, carvacrol and methyl carvacrol [8], saponins
and lipids [14-20].
Many studies have shown that flavonoids play impor-
tant pharmacological roles against various human dis-
eases, such as cardiovascular disease, cancer, inflame-
mation and allergies [21,22]. The aim of the present
study consists of the investigation of the flavonoidal con-
stituents of the ethyl acetate fraction of the methanolic
leaf extract of Acanthospermum hispidum DC that grows
in Nigeria. The antibacterial activity of the isolated
compounds is also reported.

133 T. I. EDEWOR ET AL.
2. Experimental
2.1. General
Melting points were determined using a Stuart Model
SMPI (MG155) instrument and are corrected against the
temperatures of certified reference standards. The ele-
mental analyses were carried out using a Carlo Erba 1106
Elemental analysis. The IR spectra were recorded on a
Perkins Elmer 257 spectrophotometer. UV-Vis spectra
were determined using a Pye Unicam, Hetr spectropho-
tometer. 1HNMR spectra were recorded in Methanol-d4
on a Varian Oxford NMR YH 200 instrument operating
at 500 MHz. δ values are given in ppm, using TMS as
internal standard. EI mass spectra were measured on a
Bruker FTMS 4.7 T mass spectrometer. Merck silica gel
was used for TLC. Spots were detected on TLC plates by
using iodine tank. Column chromatography (CC) was
carried out on silica gel 60 (70 - 230 mesh).
2.2. Plant Material
The plant material used in this study was the leaf of A.
hispidum. This material was taken to Department of Pure
and Applied Biology of Ladoke Akintola University of
Technology, Ogbomoso, Nigeria for identification.
2.3. Extraction and Isolation
The dried plant leaves (1 Kg) was first extracted with
n-hexane for 24 h, then with MeOH for 24 h using cold
extraction method. The crude extract was subjected to
accelerated gradient chromatography (AGC) to afford
100 fractions of 10 ml each. Fractions 74 - 77 were
bulked together and coded as EA1 then further separated
on open column using EtOAc: MeOH 9:1 v/v as eluent.
This afforded 20 fractions coded as EA1(A-M). Frac-
tions EA1(F-H) were bulked together based on similar Rf
values and further chromatographed on open column
using EtOAc: DCM: MeOH 5:4:1 v/v/v as eluent to yield
10 fractions coded EA1(F-H) (1 - 10). Fractions EA1(F-
H)(10) were recrystalized from methanol to yield com-
pound 1 (467mg) as a white amorphous powder (found:
C, 58.99; H, 4.06; O, 36.95; Calc. for C17H14O8: C, 58.96;
H, 4.05; O, 36.99), m.p: 169˚C - 171˚C; MW: 346; UV
λmax MeOH (nm) logε: 259 (3.02), 325 (2.12); IR (KBr)
cm–1: 3422, 1645, 1560, 1412, 1162, 1086; 1HNMR
(500MHz): 6.20(s), 6.33(s), 6.56(s), 6.91(s), 3.71(s)(3H),
3.88(s)(3H); EI-MS, (70Ev): m/z: 346 [M]+ (38), 329
(42), 315 (100), 303 (30), 194 (20), 174(30), 167 (18),
153 (32), 69 (77).
Fractions EA1(I, J) were combined and further puri-
fied on open column using EtOAc: MeOH: CHCl3 6:1:1
v/v/v as eluent to afford 10 fractions coded EA1 (I,
J)(1-10). Fractions EA1(I, J)(5) were recrystalized from
methanol to afford compound 2 (449mg) as a white
amorphous powder ((found: C, 58.80; H, 4.12; O, 37.08.
Calc. for C19H16O9: C, 58.77; H, 4.15; O, 37.08), m.p:
182oC - 184oC; MW: 388; UV λmax MeOH (nm) logε:
255 (3.31), 338 (2.34); IR (KBr) cm–1 : 341, 1772, 1646,
1436, 1162, 1086; 1H-NMR (500MHz): 6.20(s), 6.28(s),
6.85(s), 7.12(s), 3.61(s)(2H), 3.90(s)(3H), 2.15(s)(3H);
EI-MS, 70Ev, m/z: 388[M]+, (33), 346 (75), 329 (35),
315 (100), 194 (20), 179 (30), 167 (18), 153 (32), 69
(77).
2.4. Organisms
Clinical isolates of Salmonella typii, Staphylococcus
aureus, Klebsiella pneumoniae, Proteus mirabilis, Ba-
cillus subtilis, Corynebacterium pyogenes, Proteus vul-
garis, Escherichia coli and Pseudomonas aeruginosa
were obtained from Baptist Medical Center, Ogbomoso,
Nigeria.
2.5. Preparation of the Medium
Nutrient agar medium was prepared by dissolving 2.8 g
of nutrient agar in 100 ml of distilled water. The medium
was sterilized in an autoclave at 121˚C for 15 minutes. It
was cooled to 45˚C and poured into sterile Petri dishes to
solidify.
2.6. Preparation of Test Samples
10 mg each of the extracts was dissolved in 10 ml each
of redistilled solvents (chloroform, ethyl acetate metha-
nol and distilled water). The activity of Streptomycin, a
standard antibiotic was also determined and used as the
positive control; 10.0 mg of it was dissolved in 10 ml of
distilled water. Distilled water and redistilled solvents
were used as negative control.
2.7. Disc Diffusion Test
Disc diffusion method was employed [23]. This involved
the use of filter paper disc as carrier for the antibacterial
agents. Sterilized discs cut from Whatman No. 1 filter
paper were impregnated with solutions of the antibacte-
rial agents. The solvent was evaporated and the disc
dried properly. The nutrient agar medium was inoculated
with the test organism and the impregnated disc placed
on the surface of the nutrient agar. The antibacterial
agent upon contact with the agar diffused into all direc-
tions. The ability of the test organism to grow or not in
the presence of the test sample was then determined

T. I. EDEWOR ET AL.
134
within 24 hours by measuring the zones of inhibition.
The plates were incubated upside down at 37oC. All tests
were done in quadruplicate and the antibacterial activity
was expressed as a mean of inhibition diameters (mm)
produced by the leaf extracts with streptomycin as the
standard antibiotic.
2.8. Minimum Inhibitory Concentration (MIC)
The minimum inhibitory concentration (MIC) of the ex-
tracts against the sensitive microorganisms was deter-
mined using the disc diffusion method. Serial dilutions of
the isolated compounds were prepared (10.0, 5.0, 2.5,
1.25, 0.625, 0.313 and 0.156 mg/ml). Each of the in-
nocula was poured into each Petri-dish and the agar was
later poured and allowed to set. Wells were made using
the sterile 3 mm cork borer. Serial dilutions of the iso-
lated compounds were added into the marked wells. The
plates were incubated at 37˚C for 24 h. The growth was
observed to determine the sensitivity of the microorgan-
ism using clear zones of no microbial growth. The least
concentration of the extracts that had inhibitory effect
was taken as the minimum inhibitory concentration (MIC)
of that extract against such microorganism.
3. Results and Discussion
3.1 Structural Elucidation
Our flavones were isolated from the ethyl acetate frac-
tion obtained from fractionation of the crude methanolic
leaf extract of A. hispidum. Compounds 1 and 2 were
obtained as white amorphous powder after repeated
column chromatography. Their assigned structure is
shown in Scheme 1.
Thus, the structure of compound 1 was established
based on elemental analysis, UV-Vis., IR, 1HNMR and
MS data. The EI-MS of compound 1 exhibited a mo-
lecular ion peak at m/z 346 [M]+ (calc. 346.291) as well
as elemental analysis (found: C, 58.99; H, 4.06, O, 36.95;
required: C, 58.96; H, 4.05, O, 36.99). Data provided by
1H-NMR (Table 1) indicates the presence of four aro-
O
H
OH
H
OH
OH
H
H
O
OMe
OMe
12
3
4
5
6
789
10
2' 3'
4'
5'
6'
1'
R
1: R = OH
2: R = OCOCH3
Scheme 1. Structure of Compounds 1 and 2.
matic protons (δH 6.20 (s), 6.33(s), 6.56(s), 6.91(s))
which are typical of protons at C-6, C-8, C-3’ and C-6’
of a flavone skeleton [24] and two methoxy signals at δH
3.71(s)(3H) and 3.88(s)(3H). Based on the structural
pieces of information from 1HNMR, coupled with the
use of the ‘rule of 13’ [25], the molecular formula was
established to be C17H14O8.
The IR spectrum (KBr) of 1 revealed the presence of
hydroxyl (3422 cm–1) and carbonyl (1645 cm–1) groups.
Bands in the range 1645 - 1065 cm–1 are typical of a fla-
vone skeleton [24].
Next, the UV spectrum of 1 exhibited absorption
maxima at 259 nm (band II) and 350 nm (band I) that are
characteristic absorption bands of a flavone skeleton [17].
No shift in band I was observed after the addition of
AlCl3 and AlCl3/HCl, suggesting a hydroxyl-keto com-
plex formation at the C-5 hydroxyl [17, 26]. The UV
spectral data recorded with various shift reagents clearly
indicated a free hydroxyl group at C(5) and C(7) with no
ortho- dihydroxyl groups on rings A and B. These find-
ings were also supported by observation of an EI-MS
fragment at m/z 153 [A1 + 1]+, which accounts for a ring
A fragment with free hydroxyl groups at C(5) and C(7)
[27]. Characteristic losses of [M – 17]+ at m/z 329 and
[M-31]+ at m/z 315 were signs of 2’-OH and a 3-OMe
system [17]. The second methoxy group was placed in
position 4’ based on an EI-MS fragment at m/z 167 [B2]+
which accounts for a ring B fragment. Thus, compound 1
was finally identified as
5,7,2’,5’-tetrahydroxy-3,4’-dimethoxyflavone. The
fragmentation pattern is as shown in Scheme 2.
Similarly, the structure of compound 2 was established
based on elemental analysis, UV-Vis., IR, 1HNMR and
MS data. The EI-MS of compound 2 gave a molecular
ion peak at m/z 388 [M]+ (calc: 38.328) as well as the
elemental analysis (found: C, 58.80; H, 4.12; O, 37.08;
required: C, 58.77, H 4.15; O, 37.08). The 1H-NMR data
(Table 1) exhibited a flavonoid pattern and showed sig-
nals at δH (ppm) 6.20(1H, s), 6.28 (1H, s), 6.85(1H, s)
and 7.12(1H, s) typical of protons at C-6, C-8, C-3’ and
C-6’ of a flavone skeleton [24]. Chemical shifts at 3.90
ppm and 2.15 ppm suggested that there are substitutions
Table 1. Relevant proton NMR data of compounds 1 and 2
(500 MHz. [D4] MeOH. r.t).
Compound 1 Compound 2
Carbon position 1H (δ) 1H (δ)
6 6.20(s) 6.20(s)
8 6.33(s) 6.28(s)
3’ 6.56(s) 6.85(s)
6’ 6.91(s) 7.12(s)
MeO-3 3.71(s)(3H) 3.61(s)(3H)
MeO-4’ 3.88(s)3H) 3.90(s)(3H)
AcO-5’ - 2.15(s)(3H)

T. I. EDEWOR ET AL.
135
O
H
H
OH
H
O
H
OMe
OMe
O
HO
HO
O
H
H
OH
OMe
H
H
OMe
OH
O
HO
O
H
H
OH
H
H
OMe
OH
O
HO
H
H
OH
OMe
H
H
OH O
HO
H
H
OH
HO
H
H
OMe
OH
HO
H
OH
H
OH
H
H
OH
OMe
OMe
HO
OH
O
HO
O
H
OH
H
OMe
MeO
H
OH
H
OMe
OH
H
H
OH
O
+
m/z = 329
m/z = 315
m/z = 303
++
H
Molecular ion = 346
m/z = 153
.
-OCH3
. +
+
.
-C=OCH3
+
.
O+
m/z = 167
OH+
m/z = 194
A1 + 1+
B2 +
+
+
m/z = 159m/z = 174
OH+
+
.
.
O
+
O
+
m/z = 69
Scheme 2. Fragmentation pattern for compound 1.

T. I. EDEWOR ET AL.
136
O
H
H
OH
H
H
OMe
OMe
O
O
HO
HO
CH3
O
O
H
H
OH
H
O
H
OMe
OMe
O
HO
HO
O
H
H
OH OMe
H
H
OMe
OH
O
HO
H
O
H
OH
OMe
H
H
OH O
HO
HO
H
H
OH
HO
H
H
OH
OMe
HO
H
H
OH
H
H
OH
OMe
HO HO
MeO
H
OH
H
OH
OMe
O
H
CH2CO
Molec u lar ion =3 88m/z = 346
OH
m/z = 329
m/z = 315
++
-OCH3
+
.
+
+
+m/z = 194
..
+
+
+OH
m/z = 153
O
m/z = 167
..
O
.
+
m/z = 179
+
O
OH+
.
.
m/z = 69
Scheme 3. Fragmentation pattern for compound 2.

T. I. EDEWOR ET AL.
137
in the B ring of the flavonoid ((MeO-4’) and (AcO-5’)).
Based on the structural pieces of information from
1HNMR and mass spectrum coupled with the ‘rule of
13’[25], the molecular formula was established to be
C19H16O9. The IR spectrum (KBr) indicated the presence
of hydroxyl (3418 cm-1) and chromone (1646 cm–1)
groups. Bands in the range 1650 - 1065 cm–1 are typical
for a flavone skeleton [24]. The band at 1772 cm-1 indi-
cated that compound 2 had an acetyl group. The UV
spectrum exhibited absorption maxima at 255 nm (band II)
and 338 nm (band I) that are characteristic absorption
bands of a flavone skeleton [17]. No shift in band I was
observed after the addition of AlCl3 and AlCl3/HCl, sug-
gesting a hydroxyl-keto complex formation at the C-5
hydroxyl [17,26]. Fragments at m/z 329 [M-COCH2-17]+
and at m/z 315 [M-COCH2-31]+ revealed a OH substitu-
tion in C(2’) and a OCH3 group in C(3). UV spectral data
supported these findings by confirming free OH groups
at C(5) and C(7) and no free OH group at C(4’) as well
as no ortho-dihydroxy substitution in rings A and B.
From these results, compound 2 was identified as
5’-acetoxy-5,7,2’-trihydroxy-3,4’-dimeth- oxyflavone.
The fragmentation pattern for compound 2 is as shown in
Scheme 3.
3.2 Antibacterial Screening
The antibacterial screening of the isolated compounds
inhibited the activity of the following bacteria: Salmo-
nella typii, Staphylococcus aureus, Klebsiella pneumo-
niae, Proteus mirabilis, Bacillus subtilis, Pseudomonas
aeruginosa and Shigella dysenteriae with MIC ranging
from 0.001 - 0.2 mg/ml but was inactive against Ech-
erichia coli, Corybacterium pyogenes and Proteus vul-
garis (Table 2).
Thus, compound 2 showed a higher activity against all
the screened bacteria except for S. dysentery. This activ-
ity was more pronounced against Gram-positive than
Gram-negative bacteria. This could be as a result of the
morphological differences between this micro-organ-
isms. The Gram-positive bacteria have an outer pepti-
doglycan layer which is not an effective permeability
barrier [28] which makes it to be more susceptible to the
compounds under investigation while Gram-negative
bacteria have an outer phospholipidic membrane that
carries the lipopolysaccharide components which makes
the cell wall impermeable to lipophilic solutes [29].
Therefore the inactivity against P. vulgaris could be re-
lated to the high lipid content of the cell wall of P. vul-
garis, which may have trapped the compounds while its
inhibition against S. aureus is as a result of the cell wall
lacking a lipid layer and therefore could be penetrated by
the isolated compounds. The results indicated that the
presence of hydroxyl groups at positions C-5 and C-7
was very important for bioactivity.
Now, comparing the inhibitory activity of compounds
1 and 2 against S. aureus and P. vulgaris showed that
most of the activity was related to the presence of hy-
droxyl groups at positions 3’, 4’, 5’ in ring B and at C-3.
However, it was observed that the presence of C-2, C-3
double bond was not crucial for antibacterial activity [23]
and the most active antibacterial were reported to be
compounds such as Epigallocatechin and dihydro-
robinetin with hydroxyl groups at C-5 and C-7 and three
substitutions in ring B which is also observed with com-
pounds 1 and 2.
Table 2. Antibacterial activities of compounds 1 and 2.
TEST ORGANISM Zones of MIC inhibition (mm) (mg/ml) Zones of MIC inhibition (mm) (mg/ml)
BACTERIA Compound 1 Compound 2
Bacillus subtillis 30.00 0.02 40.50 0.20
Staphylococcus aureus 20.00 0.01 45.00 0.001
Kliebsiella pneumoniae 25.00 0.01 60.00 0.001
Corynebacterium pygenes 0.00 0.0 0.00 0.00
Proteus mirabilis 30.00 0.01 70.00 0.001
Shigella dysenteriae 45.00 0.20 35.00 0.20
Escherichia coli 0.00 0.00 00.00 0.00
Proteus vulgaris 0.00 0.00 0.00 0.00
Pseudomonas aeruginosa 40.00 0.01 50.00 0.01
Salmonella typhii 40.00 0.10 30.00 0.10
Zones of inhibition are mean of quadruplicate determinations.

T. I. EDEWOR ET AL.
138
4. Conclusions
The isolation and spectroscopic characterization of anti-
bacterial flavonoids from the methanolic leaf extract of A.
hispidum DC was described. The antibacterial flavonoids
are flavones and were identified as 5,7,2’,5’-tetrahydro-
xy-3,4’-dimethoxyflavone and 5’-acetoxy-5,7,2’-trihy-
droxy-3,4’-dimethoxyflavone.
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140
Appendices
Appendix 1. UV spectra for compound 1 with classical shift
regents.
Appendix 2. UV-visible spectra for compound 1 with clas-
sical shift regents.
Appendix 3. IR spectrum of compound 1.
Appendix 4. 1H-NMR spectrum of compound 1 (500 MHz,
methanol-d4)
Appendix 5. EI-MS spec t r um of compound 1

141 T. I. EDEWOR ET AL.
Appendix 6. UV-visible spectrum for compound 2 with
classical shift regents
Appendix 7. UV-visible spectrum for compound 2 with
classical shift regents.
Appendix 8. IR spectrum of compound 2.
Appendix 9. 1H-NMR spectrum of compound 2 (500 MHz,
methanol-d4).
Appendix 10. EI-MS spectr um of compound 2.