Antibacterial constituents of the leaves of Dacryodes edulis

Dacryodes edulis is a dioecious, small to medium-sized tree, reaching 20 to 25 m high. Different parts of the plant are used to treat many diseases including skin infections, digestive tract disorder and dysentery. The leaves were macerated in 50% ethanol and the liquid extract concentrated to dryness. The dry extract was evaluated for antibacterial activity by using agar diffusion method. The extract was partitioned between water, ethyl acetate and butanol successively and further subjected to antibacterial testing. The most active fraction, ethyl acetate fraction, was purified through various chromatographic methods to obtain pure compounds identified by spectroscopic methods as ethylgallate and quercitrin. These compounds gave good antibacterial effects, while the minimum inhibitory concentrations of the fractions and the pure compounds ranged between 12.5 and 250 μg/ml. These phenolic compounds are reported for the first time in this plant.


INTRODUCTION
Dacryodes edulis is a dioecious, small to medium-sized tree reaching 20 to 25 m high, and it is low branching (Hutchinson and Dalziel, 1958).The decoction of the leaves of the plant is employed in traditional medicine in the treatment of certain disorders of the digestive tract, toothache and earache.The leaf and stem or stem bark are used to cure dysentery and anaemia (Ayuk et al., 1999).The root bark is used for leprosy in Congo Brazaville (Bouquet, 1969), while resin from the bark heals scars and other skin problems in Nigeria (Ekong and Okogun, 1969;Burkill, 1985).In Nigeria, the stem and root are also used as chewing sticks for oral hygiene, while the leaves are employed to cure skin diseases, such as rashes, scabies, ringworm and wound (Igoli et al., 2005;Ajibesin et al., 2008).The fruit and seed of the plant are rich in oil which contains lipid and fatty acid reported to exhibit considerable nutritional value (Obasi and Okoli, 1993;Kinkela et al., 2006).Different parts of the plant, such as the leaf, stem, root and fruit have been reported to produce essential oil of the monoterpene, sesquiterpene, diterpene and triterpene types (Ekong and Okogun, 1969;Jirovetz et al., 2003;Onocha et al., 1999).Antibacterial effect of the essential oil has been reported (Obame et al., 2008), but no report on the organic extract of the plant and its chemical constituents is available.
Thus, applying activity guided purification, this study aimed at validating the antibacterial effect of the plant and identifying the chemical constituents responsible for such effect.

Plant
The leaves (6 kg) of D. edulis were collected in June, 2005, at Ikot Ekpene in Akwa Ibom State, Nigeria.The plant was identified and authenticated by Dr. U. Essiett of the Department of Botany, University of Uyo, Uyo, Nigeria.Voucher specimen (KKA 21) was deposited in the Department of Pharmacognosy and Natural Medicine herbarium, Faculty of Pharmacy, University of Uyo, Uyo, Nigeria.

Extraction of the plant
The dried leaf powder (4 kg) of D. edulis was extracted by maceration using 50% EtOH (10 L).It was filtered and the marc was re-extracted with the fresh solvent mixture for 12 h (x2) and filtered.The filtrates were pooled together and concentrated to dryness in vacuo at 40°C to yield dry ethanol extract (80 g).

Antibacterial test
The bacteria used in this study were Bacillus cereus (NCIB 6349), Staphylococcus aureus (NCIB 8588), aeruginosa (NCIB 950) and Escherichia coli (NCIB 86).All the organisms were obtained from the Department of Microbiology, Obafemi Awolowo University, Ile-Ife, Nigeria.They were maintained on blood agar slants at 4°C prior to use.
The extract and the fractions were reconstituted in MeOH-H2O (1:1) to obtain a stock solution of 20 mg/ml.50 µl of this solution was introduced into each of the equidistant wells (8 mm) bored on the agar plate surface previously inoculated with each of the test organisms.A control well containing Gentamicin (5 µg/ml) was placed in each of the plates seeded with bacteria.The Petri plates were then incubated at 37°C for 24 h (Alade and Irobi, 1993;Igbinosa et al., 2009).Antibacterial activity was expressed as average diameter of the zones of inhibition calculated as a difference in diameter of the observed zones and those of the wells.

fractionation of D. edulis
The antibacterial principles were partitioned mostly into ethyl acetate fraction followed by butanol and aqueous fractions, respectively, and these were subjected to further fractionation using different chromatographic techniques.

Minimum inhibitory concentration (MIC)
The MIC was determined by incorporating various amounts (250 to 6.25 µg/ml) of the solution of the extracts and fractions into sets of test tubes containing the culture media.50 µl of the standard test bacterial broth cultures were added into each of the test tubes.The set of tubes containing a mixture of bacteria and the sample (extracts and fractions) were incubated at 37°C for 24 h (Cos et al., 2006).
A positive control tube containing only the growth medium of each of the organisms was also set up.The MIC was regarded as The lowest concentration of the extract or fraction that did not Ajibesin et al. 1783 permit any visible growth when compared with that of the control tubes.

Isolation and characterization
EtOAc, butanol and aqueous fractions of the plant species were subjected to TLC analysis, using solvent systems A, B, C and E, respectively and visualized under the UV light (λ 254 nm) before using 100% H2SO4 and FeCl3 solution as detecting spray reagents.The most active EtOAc fraction (18 g) showed phenolic components and was chromatographed on silica (Merck, 0.040 to 0.063 mm particle size) by accelerated gradient chromatography (AGC) column and eluted with C6H14 containing increasing amount EtOAc followed by increasing amount of CH3OH (9:1, 9:1).Six fractions coded A, B, C, D, E and F were obtained, two (A and C) of which showed significant antibacterial effects.The more active (A) of the two fractions was further fractionated on silica by repeated AGC, using C6H14 in gradient with CH2Cl2 and CH3OH (9.5:5, 9.5:5; 5:5, 9.8:0.2) to yield 1 (55 mg).The less active fraction was purified vacuum liquid chromatography (VLC) using CH2Cl2 in gradient with CH3OH and H2O (7:2:1), AGC (silica) and on Sephadex LH 20, eluted isocratically with EtOH.Final purification was carried out on silica by preparative thin layer chromatography (prep.TLC), using CH2Cl2-CH3OH (4:1) as mobile phase to yield 2 (10 mg).The two compounds were also subjected to antibacterial test.

RESULTS AND DISCUSSION
Two phenolic compounds were isolated from the leaves of D. edulis.Ethylgallate 1, a silky, fluffy, white crystal was isolated with the aid of accelerated gradient chromatography (AGC), while quercitrin 2 was separated with vacuum liquid chromatography (VLC), accelerated gradient chromatography (AGC), size exclusion chromatography (Sephadex LH-20) and preparative thin layer chromatography (prep.TLC).
The 1 HNMR spectrum of compound 1 gave a triplet at δ 1.33 representing three methyl protons, and a quartet at δ 4.28 indicating two methylene protons of O-CH 2 -CH 3 group.Presence of a sharp singlet at δ 7.08 corresponds to two aromatic protons, indicating the symmetrical nature of the molecule.It also suggests the attachment of hydroxyl group at 3-, 4-and 5-positions.These data agree with the literature values (Metha and Sharma, 1988;Adesina et al., 2000).On this basis, compound 1 was characterized as ethylgallate (Ethyl 3,4,5trihydroxybenzoate).
Quercitrin 2 exhibited band I and band II with the UV Gen, gentamicin, values are mean ± SD (n = 4); * P < 0.01 with respect to control.
The extract, fractions and the pure compounds isolated showed varying degrees of antibacterial activities against all the tested microorganisms (Tables 1 and 2).These test bacteria have been implicated in the pathogenesis of human infections (Duguid et al., 1978).The ethanol extract gave the highest activity against B. cereus with inhibition zone of 13 mm, followed by S. aureus with the inhibition zone of 10 mm.The good activity elicited by ethanol extract was partitioned into ethyl acetate fraction, which also showed significant activity against B. cereus with zone of inhibition of 12 mm and S. aureus with zone of inhibition of 11 mm.The butanol and aqueous fractions were less active.Previous studies on the antimicrobial activities of plants, such as different species of Acalypha and Gen: gentamicin different varieties of Lasianthera africana gave similar activity guided purification pattern (Adesina et al., 2000;Ajibesin and Bassey, 2011).Out of the six bulked fractions obtained from further purification of ethyl acetate fraction by column chromatography, bulked fraction A gave the best activity against all the test bacteria, followed by bulked fraction C (Table 2).These active bulked fractions A and C showed low minimum inhibitory concentration (MIC) values ranging from 100 to 250 µg/ml (Table 3).The most active bulked fraction A yielded ethylgallate which gave the highest antibacterial activity against the test microorganisms with the lowest MIC (12.5 µg/ml).This activity was higher than that of Gentamicin, the standard drug.Thus, its presence accounted for the antibacterial activity of the leaves of the plant.The antimicrobial properties of ethylgallate have been established in literature (Adesina et al., 2000;Lamikanra et al., 1990;Burapadaja and Bunchoo, 1995).Bulked fraction C gave quercitrin which showed less antibacterial activity with the MIC value of 50 µg/ml.Quercitrin has been isolated from plants, such as Piliostigma thonningii (Ibewuike, 1997;Bombardelli et al., 1973), Koelreuteria paniculata (Lin et al., 2002) and Hypericum caprifoliatum (Dall et al., 2003), and its antimicrobial activity widely reported in such plants.
The antibacterial effects of D. edulis leaf have been determined to be mainly due to ethylgallate, while quercitrin was also isolated as antibacterial principle showing less inhibitory activity.

Conclusion
The two compounds, ethylgallate and quercitrin have been identified from the leaves of D. edulis, and were found to be responsible for the antibacterial effects of the plant.This validates its use in traditional medicine for treating infections.

Table 1 .
Antibacterial activity of the extracts of D. edulis leaf.

Table 2 .
Antimicrobial activity of the fractions of D. edulis leaf.

Table 3 .
MIC of the fractions and phenolics isolated from D. edulis (µg/ml).