PHYTOCHEMICAL AND ANTIBACTERIAL INVESTIGATIONS OF THE FRUITS OF Nauclea latifolia Smith (FAMILY: RUBIACEAE)
PHYTOCHEMICAL AND ANTIBACTERIAL INVESTIGATIONS OF THE FRUITS OF Nauclea latifolia Smith (FAMILY: RUBIACEAE)
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Date
2016-02
Authors
FADIPE, LABAKE AJOKE
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Abstract
Ripe and unripe fruits of Nauclea latifolia Smith (Family Rubiaceae) used traditionally
in the treatment of dysentery, diarrhoea and other bacterial infections were investigated
for their phytochemical content and antibacterial efficacy. Extraction, partitioning and
further re-extraction of the air-dried ripe (r) and unripe (u) fruits gave rise to extracts
(coded rP, uP, rM, uM), partitioned-soluble fractions (coded C, E, B, A) and soluble
fractions (coded rD, uD, uC, rC, uE, rE, rA, uA, rR, uR). Phytochemical screening of
extracts and fractions using standard methods revealed the presence of alkaloids,
flavonoids, steroidal nucleus, saponins, coumarins and tannins. Purification of the ethyl
acetate partitioned-soluble fraction of the methanol extract of the ripe fruits (E), its
column fraction (E-2) and column sub-fraction (E-2f) led to the isolation of a
benzaldehyde derivative, identified as 2- (2‟-ethyl-3‟-tertbutoxypropyl) benzaldehyde
(coded E-2f1a). Similar purification of the acetone soluble-fraction of the ripe fruits
(rA), its column fraction (rA-5) and sub-fraction (rA-5a) afforded a phthalate derivative,
identified as di- (ethylhexyl) phthalate, (DEHP), coded rA-5a1. Chromatographic
separations of the ethyl acetate-soluble fraction of the methanol extract of the unripe
fruits (uE), its column fractions (uE-1 to uE-5) and column sub-fractions (uE-2 and uE3)
led to the isolation of (i) same phthalate as was isolated from the ripe fruits, also
identified as DEHP, coded uE-2a1 (but of lesser quantity than in the ripe fruits), (ii) an
unsaturated fatty acid ester derivative, identified as ethenyl pentadecanoate, coded uE2a2,
and (iii) a phytosterol, identified as β-sitosterol, coded uE-3a2a. Characterization of
all compounds was based on the use of physical, colour reactions, spectroscopic
parameters (IR, UV, 1H-NMR,13C-NMR, DEPT-135 and GC-MS) and literature search. Antibacterial assay of the extracts (100 mg/ml) and soluble fractions (50 mg/ml) in
comparison with chloramphenicol, erythromycin and tetracycline (1 mg/ml each)
against two Gram-positive (Bacillus subtilis and Staphylococcus aureus) and four
Gram-negative bacteria (Escherichia coli, Klebsiella pneumoniae, Pseudomonas
aeruginosa and Salmonella typhi) was carried out using the agar-well diffusion method.
The diameter of zones of inhibition (active, if ≥ 14 mm), calculated percent activities,
calculated bacterial susceptibility index, minimum inhibitory concentrations, and
minimum bactericidal concentrations for the soluble fractions of the unripe fruits ranged
from 12.3 – 21.2 mm, 50 - 100 %, 75 – 100 %, 6.25 – 25 mg/ml and 12.5 – 50 mg/ml
respectively, while, it ranged from 7.33 – 21.2 mm, 0 – 83.3 %, 0 – 50 %, 6.25 – 25
mg/ml, 12.5 – 50 mg/ml; 6.33 – 17.6 mm, 0 – 66.7 %, 0 – 50 %, 12.5 – 50 mg/ml and
12.5 – 50 mg/ml for the soluble fractions and partitioned-soluble fractions of the ripe
fruits respectively. The values for the crude extracts of both fruits also ranged from 6.67
- 15.3 mm, 0 - 50 %, 0 - 25 %, 12.5 - 25 mg/ml and 100 mg/ml. All values obtained in
comparison with those of the standard whose values ranged from 9.60 – 26.2 mm, 83.3
– 100 %, 33.3 – 100 %, 0.20 – 0.50 mg/ml, 0.20 – 0.50 mg/ml respectively, indicates
that the soluble fractions of the unripe fruits displayed higher activity than the soluble-
and partitioned-soluble fractions of the ripe fruits and crude extracts of both ripe and
unripe fruits. The diethyl ether- (uD) and ethyl acetate- (uE) soluble fractions of the
unripe fruits displayed higher broad-spectrum activity better than that exhibited by some
of the standard drugs against some of the test organisms.
The column fractions (rA-1 to rA-8; 6.21 – 19.0 mm) and column sub-fractions (rA-5a
to rA-5c; 5.22 – 18.5 mm) of the acetone-soluble fraction of the ripe fruits (rA) at 20
mg/ml each, showed zones of inhibition (mm) that was higher than rA (7.0 - 10.1 mm) at 50 mg/ml. Also, the column fractions (E-1 to E-6; 6.80 – 18.8 mm) and sub-fractions
(E-2a to E-2h; 5.70 – 18.3 mm) of the ethyl acetate partitioned-soluble fraction of the
ripe fruits (E) also at 20 mg/ml showed higher zones of inhibition (mm) than E (8.25 –
17.6 mm) at 50 mg/ml, while the column fractions (uE-1 to uE-5; 8.50 – 14.8 mm) and
column sub-fractions (uE-2a; 6.6 -13.8 mm) of the ethyl acetate soluble fraction of the
unripe fruits (uE), also at 20 mg/ml showed lower antibacterial activity than uE (17.3 –
21.5 mm) at 50 mg/ml. An activity that was of broad-spectrum when compared with
that displayed by erythromycin (9.60 - 26.2 mm) at 1 mg/ml
DEHP (100 µg/ml) exhibited similar antibacterial activity against B. subtilis (17.0 mm)
in comparison with erythromycin (15.1 mm) at 1 mg/ml, while it was less active against
S. aureus (15.3 mm; erythromycin, 22.2 mm). β-sitosterol at 100 µg/ml was not too
active (10.4 - 13.5 mm) against all tested strains (erythromycin, 9.60 - 26.2 mm) but
was only moderately active against E. coli (15.1 mm; erythromycin, 22.6 mm).
The antibacterial studies of the ripe and unripe fruits of N. latifolia validate the
ethnomedicinal uses of the fruits of the plant.
Description
A DISSERTATION SUBMITTED TO THE SCHOOL OF POSTGRADUATE
STUDIES,
AHMADU BELLO UNIVERSITY, ZARIA
IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE AWARD
OF A
DOCTOR OF PHILOSOPHY IN PHARMACEUTICAL AND MEDICINAL
CHEMISTRY
DEPARTMENT OF PHARMACEUTICAL AND MEDICINAL CHEMISTRY,
FACULTY OF PHARMACEUTICAL SCIENCES
AHMADU BELLO UNIVERSITY, ZARIA
NIGERIA
Keywords
PHYTOCHEMICAL,, ANTIBACTERIAL INVESTIGATIONS,, FRUITS,, Nauclea latifolia Smith,, (FAMILY RUBIACEAE),