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J OURNAL OF FOREST PRODUCTS & INDUSTRIES, 2014, 3(5), 211-215 ISSN:2325–4513( PRINT) ISSN 2325 - 453X (ONLINE )

Research Article

Isolation and Identification of Two Flavonoids from Acacia

Nilotica (Leguminosae) Leaves
Bashir, H. S.*1; Mohammed, A. M.2, Magsoud, A. S. 3, Shaoub, A. M.4
1*
Bahri University College of Applied and Industrial Sciences
2
Sudan University of Sciences and Technology, Faculty of Sciences
3
Cairo University, College of Sciences
4
Khartoum University College of Pharmacy
(Received: July 22, 2014; Accepted: September 19, 2014)

 anti-inflammatort, antibacterial, antiallergic, antimutagenic,

Abstract— From ethyl acetate extract of acacia nioltica
(Leguminosae) leaves two flavonoids: querstin 3-galactosyl and
flavone: were isolated and their biological activities were
evaluated. The structures were established on the basis of their
spectral data (IR, UV, 1H NMR, 13C NMR and MS)
Index Terms — Acacia nilotica, Isolation, Flavonol glycoside,
Flavone aglycone and Biological activities.
I. INTRODUCTION
Acacia nilotica L. is a common, medium sized tree, locally
known as ‘Babul’ or ‘Kikar’ belonging to the family
Mimosaceae. Acacia nilotica is the most significant genus of
family Leguminosae firstly described by Linnaeus in 1773. It is
estimated that there are roughly 1380 species of Acacia
nilotica worldwide [1,2]. It grows to 15-18 m in height. Acacia
nilotica is also known as Gum Arabic tree, Babul, Egyptian
thorn. It is widely spread in subtropical and tropical Africa
from Egypt to Mauritania southwards to South Africa, and in
Asia eastwards to Pakistan and India [3]. The plant is
therapeutically used as Anti-cancer, antiscorbutic, astringent,
anti-oxidant, natriuretic, antispasmodial, diuretic, intestinal
pains and diarrhea, nerve stimulant, cold, congestion, coughs,
dysenter and fever [4]. Seeds have antimalarial, antidiabetic,
antihypertensive and antispasmodic activities. The leaves and
pods are an excellent fodder with anti-inflammatory properties
and are rich in protein. The pods have molluscicidal and
algicidal properties. The bark is used in the treatment of
hemorrhages, cold, diarrhea, tuberculosis and leprosy. The root
is used as an (aphrodisiac) and the flowers for treating syphilis
lesions [5].
Flavonoids are one of the largest groups of natural products
found in the plant kingdom [6, 7]. They are present in nearly
every part of the plant leaves, stems, roots, seeds and woods
[8]. Flavonoids contain fifteen carbon atoms in their basic
nucleus-flavan, arranged in a C6-C3-C6 configuration
consisting of two aromatic rings (A and B) linked by a three
carbon unit which may or may not form a third heterocyclic
ring (C) [9]. Flavonoids are large group of polyphenolic
substances with marked physiological potential including:
*E- mail of corresponding author: seham200996@hotmail.com
antiviral, antioxidant and anticancer [10, 11].
II. MATERIALS AND METHODS
Plant material
The leaves of Acacia nilotica (Leguminosae) were collected in
July, 2009 from Khartoum State and authenticated by Prof.
Hassan El sobky, Institute of Medicinal and Aromatic Herb,
National Research council, and a voucher sample was
deposited.
Methods
Extraction of active constituents
Powdered air-dried leaves (500 g) of Acacia nilotica were
macerated with ethyl alcohol (95%) till exhaustion. The
aqueous alcoholic extract was evaporated in vacuum at about
50 °C to afford 52.0 g of crude. The crude extract was
dissolved in water and partitioned with a series of organic
solvents: n-hexane (3 x 300 ml), dichloromethane (3 x 300 ml),
ethyl acetate and n-butanol (3 x 300).
Isolation of flavonoids
The ethyl acetate of acacia nilotica leaves was fractionated by
mini column using MeOH: CHCl3 (2:8 v: v) to elute three
fractions. These fractions were combined and purified by PC,
using Whatman 3mm sheets and acetic acid 15% as solvent
system. The products where further purified via Sephadex LH-
20 column eluting by methanol to afford two compounds.
Compound A1 (75 mg) as a yellow crystals, m.p. 180-185 °C, and
compound A2 (45 mg) is a white crystals, m.p. 155-157 °C.
Antimicrobial activity
Preparation of standard test organisms
One ml aliquots of 24 hours broth cultures of test organisms
(Bacillus subtilis, Staphylococcus aureus, Escherichia coli,
Pseudomonas aeruginosa and Neisseria gonorrhoeae) were
aseptically distributed onto nutrient agar slopes and incubated at
37 C for 24 hours. The bacterial growth was harvested and washed
off with sterile normal saline, to produce a suspension containing
about 108- 109 colony forming units per ml. The suspension was
stored in the refrigerator at 4 C till used. Serial dilution of stock
suspension, were made in sterile normal saline, and 0.02 ml
volumes of the appropriate dilution were transferred onto the
surface of dried nutrient agar and the drop was left to dry, and then
incubated at 37 C for 24 hours.
Testing for antibacterial activity
To determine the activity of methanolic extract and pure
component of Acacia nilotica against the five standard
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organisms. the cup-plate agar was adopted with some minor
modification. (2 ml) Of the standard bacteria stock suspension
were mixed with 200 ml of sterile molten nutrient agar which
was maintained at 45 C.
(20 ml) Aliquots of incubated agar were distributed into sterile
Petri dishes. The agar was left to settle and each plate was cut
and agar discs were removed. Alternated cups were filled with
0.1 ml sample of each of the extract (pure component) and
allowed to diffuse at room temperature for two hours. The Petri
dishes were then incubated in the upright position at 37 C for
18 hours. After incubation, the diameter of the resultant growth
inhibition zones were measured, and averaged [12].
III. RESULTS AND DISCUSSION
Spectral data of compound A1
The UV λmax (MeOH nm) 260, 295sh, 360 nm
(MeOH+NaOMe) 270, 326 and 410 nm (MeOH+NaOAc) 271,
323sh, 386 nm (MeOH+NaOAc+H3BO3) 263, 295sh, 379 nm
(MeOH+AlCl3) 275, 325sh, 425 nm (MeOH+AlCl3+HCl) 269,
371, 400 nm.
IR νmax (cm-1, KBr disc): 632- 759 (C-H, Ar bending), 1253
(C-O, ether), 1620 (C= C, Ar), 1705 (C=O), 2927 (C-H,
alkanes) and 3301- 3452 cm-1 (O-H).
1
H-NMR (500 MHz, DMSO-d6): δ (ppm): showed (figure: 1)
3.0- 3.5 (m, 6H) assigned for sugar protons; 6.15 (s, 1H) and
6.35 (s, 1H) for C6- and C8- H's respectively. The resonances at
6.79 and 7.53 were assigned for the aromatic H's of B-ring.

Figure (1) 1H-NMR spectrum data of compound A1

Figure (2) 13C-NMR spectrum data of compound A1


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The 13C NMR spectra of compound A1 in (figure: 2) showed
δc: 133.8 – 163.9 (C7, C5, C3, C2, C9, C4` and C3`), 177.9 (C4),
156.89 (C2), 156.66 (C9). The non-oxygenated carbon atoms
(C1`, C2`, C5`, C6`, C6, C8 and C10) appear in the range of
121.6- 93.6. Other carbon atoms bearing one oxygen
function in sugar moiety appear at 76.8 – 67.1 ppm.
Spectral data of compound A2
UV λmax (MeOH) 279, 355 nm (MeOH+NaOMe) 291, 373
nm (MeOH+NaOAc) 279, 377 nm
(MeOH+NaOAc+H3BO3) 284, 390 nm (MeOH+AlCl3) 288,
361, 390 nm (MeOH+AlCl3+HCl) 279, 343 sh, 374 nm.
213
IR (KBr) νmax : 667-767 (C-H, Ar bending), 1261 (C-O,
ether), 1539 (C= C, Ar), 1689 (C=O), 2923 (C-H, alkanes)
and 3398 cm-1 (O-H).
1
H-NMR (500 MHz, DMSO-d6): δ(ppm): showed (figure: 3)
δ 6.68 (d, 2H) assigned for C2`- and C6`-H's; δ 6.5 (d, 1H)
assigned for C8- H. The signals at 5.84 and 5.65 ppm are
characteristic of C3` and C5`-H's, while the resonances at δ
3.3, 3.54 and 3.78 ppm (m, 15H) accounts for a methoxyl
function.

Figure (3) 1H-NMR spectrum data of compound A2

Table 1. The antibacterial activity of crude and pure compounds extracted from Acacia nilotica.
Inhibition zone diameter (mm / mg sample)
Sample
Bacillus Escherichia Neisseria Pseudomonas Staphylococcus
subtilis coli gonorrhoeae (G-) aeruginosa aureus
(G+) (G-) (G-) (G+)

Tetracycline
Standard

27 31 33 29 26
Antibacterial agent

Plant crude 14 15 14 15 15

AL3 10 10 10 10 12

AL4 12 12 12 10 12

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The UV spectrum of compound A1 gave λ max (MeOH) 260,
360 nm due to the benzoyl and cinnamoyl chromophores of
the flavonol moiety [13]. The sodium methoxide spectrum
exhibited a bathochromic shift of 50 nm without decrease in
intensity indicating a 4`-hydroxyl group. The addition of
sodium acetate gave a bathochromic shift of about +11 nm
indicating the presence of 7-hydroxyl group. 19
bathochromic shift was observed in the boric acid spectrum
of A1 indicating a catechols moiety.
The 1H-NMR spectrum of compound A1 showed: δ 3.0- 3.5
(m, 6H) assigned for sugar protons; δ 6.15 (s, 1H) and δ 6.35
(s, 1H) accounting for C6- and C8- H's respectively. The
resonances at δ 6.79 and 7.53 ppm were assigned for the
214
aromatic H's of B-ring. 13C NMR 400 MHz, DMSO-d6) δ C:
133.8 – 163.9 (C7, C5, C3, C2, C9, C4` and C3`), 177.9 (C4),
156.89 (C2), 156.66 (C9). The non-oxygenated carbon atoms
(C1`, C2`, C5`, C6`, C6, C8 and C10) appear in the range of
121.6- 93.6. Other carbon atoms bearing one oxygen
function in sugar moiety appear at 76.8 – 67.1 ppm. The mass
spectrum of compound (A1) exhibited a molecular ion peak
at m/z 465 corresponding to the molecular formula C15H5 O7.
The fragments at m/z 152, 126 and m/z 150 indicated that the
A ring contained two hydroxyl groups and that the B-ring
also has two hydroxyl groups according to following retro
Diels- Alder process:

OH
OH
HO O

OH
OH O

The complete acid hydrolysis of compound A1 and
subsequent UV studies of aglycone using the shift reagent
AlCl3 indicated a 3 -OH group in the resultant aglycone and
this is precisely the site of glycosylation.
The following cumulative data suggest that compound A1
(may be because the positions of hydroxyls are not
confirmed) is a 5, 7, 3`, 4`-tetrahydroxyflavone
-3-O-galactosyl.
OH
HO O retro Diels- Alder fission supported the substitution pattern
OH
H3BO3. Thus catechol systems are absent. The AlCl3 spectrum
did not affored any detectable bathochromic shift suggesting
absence of C5-, C3-hydroxylation. The 1H-NMR spectrum of
compound A2 (figure 3) showed: δ 6.68 (d, 2H) assigned for
C2`- and C6`-H's; δ 6.5 (d, 1H) assigned for C8- H. The signals at
5.84 and 5.65 ppm are characteristic of C3` and C5`-H's, while
the resonances at δ 3.3, 3.54 and 3.78 ppm (m, 15H) accounts
for a methoxyl function.
The mass spectrum of compound A2 exhibited a molecular ion
peak at m/z 372 corresponding to a penta-methoxyflavone. The
proposed for assigned for A2, where the ions m/z 162 and m/z
210 were recorded in the electron beam

O
galactosyl
OH O
The UV spectrum of compound A2 gave λ max (MeOH) 279,
355 nm due to the benzoyl and cinnamoyl chromophores of the
flavonol moiety [13]. Band I in A2 absorbs at 355 nm
indicating a flavone or chalcone. But, chalcones are
distinguished (in their1H NMR spectrum) by a double doublet
(J = 17 Hz) in the range delta 6.7 – 7.4 and 7.3 – 7.7 ppm for α-
and β- H's respectively [14]. The 1H NMR spectrum of A2
(figure 3) did not reveal any signals for α- and β- protons. Thus
A2 is probably a flavone.
Only a slight bathochromic shift was observed in the sodium
methoxide spectrum indicating absence of a 3- and 4`-OH. The
sodium acetate spectrum was devoid of bathochromic shifts
indicating the absence of a 7-hydroxyl group. Also no
bathochromic shift was observed on addition of NaOAc/

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nilotica in table (1) showed moderate inhibition against all
five organisms, also compounds A1 and A2 showed moderate
inhibition against all five organisms.
IV. CONCLUSION
In the present study, two flavonoids: querstin 3-galactosyl
and flavone: were isolated from ethyl acetate extract of
acacia nioltica (Leguminosae) leaves, and purified using
different chromatographic techniques. These compounds
(A1 and A2) were identified via spectroscopic tools: IR, UV,
1
H NMR, 13C NMR and MS spectroscopy. These
compounds showed potent antibacterial activity. This would
be helpful to create awareness among people for taking
control measures based on, herbal plants against infectious
diseases.
ACKNOWLEDGMENTS
We are grateful to Professor Dr. Salwa Abd El Mgsoud, for
her interest in this study and for her valuable help and
support; our thanks also extend to the Cairo University for
technical assistance during this work.
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