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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 antiviral, antioxidant and anticancer [10, 11].
(Leguminosae) leaves two flavonoids: querstin 3-galactosyl and II. MATERIALS AND METHODS
flavone: were isolated and their biological activities were Plant material
evaluated. The structures were established on the basis of their The leaves of Acacia nilotica (Leguminosae) were collected in
spectral data (IR, UV, 1H NMR, 13C NMR and MS)
July, 2009 from Khartoum State and authenticated by Prof.
Hassan El sobky, Institute of Medicinal and Aromatic Herb,
Index Terms — Acacia nilotica, Isolation, Flavonol glycoside,
National Research council, and a voucher sample was
Flavone aglycone and Biological activities. deposited.
Methods
I. INTRODUCTION Extraction of active constituents
Acacia nilotica L. is a common, medium sized tree, locally Powdered air-dried leaves (500 g) of Acacia nilotica were
known as ‘Babul’ or ‘Kikar’ belonging to the family macerated with ethyl alcohol (95%) till exhaustion. The
Mimosaceae. Acacia nilotica is the most significant genus of aqueous alcoholic extract was evaporated in vacuum at about
family Leguminosae firstly described by Linnaeus in 1773. It is 50 °C to afford 52.0 g of crude. The crude extract was
estimated that there are roughly 1380 species of Acacia dissolved in water and partitioned with a series of organic
nilotica worldwide [1,2]. It grows to 15-18 m in height. Acacia solvents: n-hexane (3 x 300 ml), dichloromethane (3 x 300 ml),
nilotica is also known as Gum Arabic tree, Babul, Egyptian ethyl acetate and n-butanol (3 x 300).
thorn. It is widely spread in subtropical and tropical Africa Isolation of flavonoids
from Egypt to Mauritania southwards to South Africa, and in The ethyl acetate of acacia nilotica leaves was fractionated by
Asia eastwards to Pakistan and India [3]. The plant is mini column using MeOH: CHCl3 (2:8 v: v) to elute three
therapeutically used as Anti-cancer, antiscorbutic, astringent, fractions. These fractions were combined and purified by PC,
anti-oxidant, natriuretic, antispasmodial, diuretic, intestinal using Whatman 3mm sheets and acetic acid 15% as solvent
pains and diarrhea, nerve stimulant, cold, congestion, coughs, system. The products where further purified via Sephadex LH-
dysenter and fever [4]. Seeds have antimalarial, antidiabetic, 20 column eluting by methanol to afford two compounds.
antihypertensive and antispasmodic activities. The leaves and Compound A1 (75 mg) as a yellow crystals, m.p. 180-185 °C, and
pods are an excellent fodder with anti-inflammatory properties compound A2 (45 mg) is a white crystals, m.p. 155-157 °C.
and are rich in protein. The pods have molluscicidal and Antimicrobial activity
algicidal properties. The bark is used in the treatment of Preparation of standard test organisms
hemorrhages, cold, diarrhea, tuberculosis and leprosy. The root One ml aliquots of 24 hours broth cultures of test organisms
is used as an (aphrodisiac) and the flowers for treating syphilis (Bacillus subtilis, Staphylococcus aureus, Escherichia coli,
lesions [5]. Pseudomonas aeruginosa and Neisseria gonorrhoeae) were
Flavonoids are one of the largest groups of natural products aseptically distributed onto nutrient agar slopes and incubated at
found in the plant kingdom [6, 7]. They are present in nearly 37 C for 24 hours. The bacterial growth was harvested and washed
every part of the plant leaves, stems, roots, seeds and woods off with sterile normal saline, to produce a suspension containing
[8]. Flavonoids contain fifteen carbon atoms in their basic about 108- 109 colony forming units per ml. The suspension was
nucleus-flavan, arranged in a C6-C3-C6 configuration stored in the refrigerator at 4 C till used. Serial dilution of stock
consisting of two aromatic rings (A and B) linked by a three suspension, were made in sterile normal saline, and 0.02 ml
carbon unit which may or may not form a third heterocyclic volumes of the appropriate dilution were transferred onto the
ring (C) [9]. Flavonoids are large group of polyphenolic surface of dried nutrient agar and the drop was left to dry, and then
substances with marked physiological potential including: incubated at 37 C for 24 hours.
Testing for antibacterial activity
*E- mail of corresponding author: seham200996@hotmail.com To determine the activity of methanolic extract and pure
component of Acacia nilotica against the five standard
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J OURNAL OF FOREST PRODUCTS & INDUSTRIES, 2014, 3(5), 211-215 ISSN:2325–4513( PRINT) ISSN 2325 - 453X (ONLINE )

organisms. the cup-plate agar was adopted with some minor III. RESULTS AND DISCUSSION
modification. (2 ml) Of the standard bacteria stock suspension Spectral data of compound A1
were mixed with 200 ml of sterile molten nutrient agar which The UV λmax (MeOH nm) 260, 295sh, 360 nm
was maintained at 45 C. (MeOH+NaOMe) 270, 326 and 410 nm (MeOH+NaOAc) 271,
(20 ml) Aliquots of incubated agar were distributed into sterile 323sh, 386 nm (MeOH+NaOAc+H3BO3) 263, 295sh, 379 nm
Petri dishes. The agar was left to settle and each plate was cut (MeOH+AlCl3) 275, 325sh, 425 nm (MeOH+AlCl3+HCl) 269,
and agar discs were removed. Alternated cups were filled with 371, 400 nm.
0.1 ml sample of each of the extract (pure component) and IR νmax (cm-1, KBr disc): 632- 759 (C-H, Ar bending), 1253
allowed to diffuse at room temperature for two hours. The Petri (C-O, ether), 1620 (C= C, Ar), 1705 (C=O), 2927 (C-H,
dishes were then incubated in the upright position at 37 C for alkanes) and 3301- 3452 cm-1 (O-H).
1
18 hours. After incubation, the diameter of the resultant growth H-NMR (500 MHz, DMSO-d6): δ (ppm): showed (figure: 1)
inhibition zones were measured, and averaged [12]. 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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J OURNAL OF FOREST PRODUCTS & INDUSTRIES, 2014, 3(5), 211-215 ISSN:2325–4513( PRINT) ISSN 2325 - 453X (ONLINE )
The 13C NMR spectra of compound A1 in (figure: 2) showed IR (KBr) νmax : 667-767 (C-H, Ar bending), 1261 (C-O,
δc: 133.8 – 163.9 (C7, C5, C3, C2, C9, C4` and C3`), 177.9 (C4), ether), 1539 (C= C, Ar), 1689 (C=O), 2923 (C-H, alkanes)
156.89 (C2), 156.66 (C9). The non-oxygenated carbon atoms and 3398 cm-1 (O-H).
1
(C1`, C2`, C5`, C6`, C6, C8 and C10) appear in the range of H-NMR (500 MHz, DMSO-d6): δ(ppm): showed (figure: 3)
121.6- 93.6. Other carbon atoms bearing one oxygen δ 6.68 (d, 2H) assigned for C2`- and C6`-H's; δ 6.5 (d, 1H)
function in sugar moiety appear at 76.8 – 67.1 ppm. assigned for C8- H. The signals at 5.84 and 5.65 ppm are
Spectral data of compound A2 characteristic of C3` and C5`-H's, while the resonances at δ
UV λmax (MeOH) 279, 355 nm (MeOH+NaOMe) 291, 373 3.3, 3.54 and 3.78 ppm (m, 15H) accounts for a methoxyl
nm (MeOH+NaOAc) 279, 377 nm function.
(MeOH+NaOAc+H3BO3) 284, 390 nm (MeOH+AlCl3) 288,
361, 390 nm (MeOH+AlCl3+HCl) 279, 343 sh, 374 nm.

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

OH
OH
HO O

OH
OH O

The complete acid hydrolysis of compound A1 and H3BO3. Thus catechol systems are absent. The AlCl3 spectrum
subsequent UV studies of aglycone using the shift reagent did not affored any detectable bathochromic shift suggesting
AlCl3 indicated a 3 -OH group in the resultant aglycone and absence of C5-, C3-hydroxylation. The 1H-NMR spectrum of
this is precisely the site of glycosylation. compound A2 (figure 3) showed: δ 6.68 (d, 2H) assigned for
The following cumulative data suggest that compound A1 C2`- and C6`-H's; δ 6.5 (d, 1H) assigned for C8- H. The signals at
(may be because the positions of hydroxyls are not 5.84 and 5.65 ppm are characteristic of C3` and C5`-H's, while
confirmed) is a 5, 7, 3`, 4`-tetrahydroxyflavone the resonances at δ 3.3, 3.54 and 3.78 ppm (m, 15H) accounts
-3-O-galactosyl. for a methoxyl function.
OH The mass spectrum of compound A2 exhibited a molecular ion
peak at m/z 372 corresponding to a penta-methoxyflavone. The
HO O retro Diels- Alder fission supported the substitution pattern
OH 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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J OURNAL OF FOREST PRODUCTS & INDUSTRIES, 2014, 3(5), 211-215 ISSN:2325–4513( PRINT) ISSN 2325 - 453X (ONLINE )
nilotica in table (1) showed moderate inhibition against all comprehensive review on ethnopharmacological claims.
five organisms, also compounds A1 and A2 showed moderate International Journal of Pharmacy and Life Sciences, 2(6),
inhibition against all five organisms. 0976-7126.
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IV. CONCLUSION and antimicrobial studies of Acacia species (Mimosaceae).
In the present study, two flavonoids: querstin 3-galactosyl Journal of Medicinal Plants Research, 2(12), 378-386.
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acacia nioltica (Leguminosae) leaves, and purified using Trado-Medic Books, Owerri.
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ACKNOWLEDGMENTS [10] Pal, R. S., Ariharasivakumar, G., Girhepunjhe, K. and
We are grateful to Professor Dr. Salwa Abd El Mgsoud, for Upadhay, A. (2009): International Journal of Pharmacy
her interest in this study and for her valuable help and and Pharmaceutical Sciences, 1, 136-140.
support; our thanks also extend to the Cairo University for [11] Marchand, L. L. (2002): Cancer preventive affects of
technical assistance during this work. flavonoids- a review. Biomedicine and Pharmacotherapy,
56, 296-301.
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