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)

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.

PHYTOCHEMICAL AND ANTIBACTERIAL INVESTIGATIONS OF THE FRUITS OF Nauclea latifolia Smith (FAMILY: RUBIACEAE)