Antimicrobial Activity And Interaction Of Toddalia Asiaticacoumarins With Two Known Drugs

ABSTRACT

Plant-derived compounds could exhibit a direct and/or an indirect antimicrobial

activity as antimicrobial resistance modifying compounds. Their effectiveness could

further be increased when combined with other bioactive compounds or antibiotics

(synergy). A systematic search for plant-derived bioactive compounds, including

those which can synergistically act with other bioactive compounds or antibiotics,

as resistance modifying agents represents a potential approach to overcoming

microbial resistance. Synergistic interaction of isolated coumarins from T. asiatica

had not been reported prior to the current study, thus the basis for this work. It was

also of interest to investigate possible synergistic effects of the isolated coumarins

with known antimicrobials. This study evaluated the antimicrobial activity of the

crude, isolated and combined coumarins from Toddalia asiatica. Further to this,

combination of the pure compounds and known antimicrobials were investigated for

possible synergy. The stem and root bark from the plant were collected from

Kakamega forest in Western Kenya and after drying separately, subjected to

sequential solvent extraction with organic solvents (n-Hexane, dichloromethane and

methanol). Chromatographic separation of the crude extracts followed by

characterization using spectroscopic techniques lead to the identification of six

compounds namely: toddalolactone (1), coumurrenol (2), 5, 7-dimethoxy- 6 - (3'-

methyl-,3'-hydroxyl–but–2-none) coumarine (3), toddaculin (5), and lupeol (6),

[from the stem bark] and 5, 7- dimethoxy – 8 - (3'-methyl-but-1',3'-diene)

coumarine (4) [from the root bark]. This is the first time compounds 3 and 6 are

being reported from T. asiatica stem bark. Compound 3 (coumarin) had never been

reported prior to this work. The crudes and isolated pure compounds 1, 2, 3 and 4

were evaluated for their antimicrobial activity against Staphylococcus aureus,

Escherichia coli, Penicillium digitatum and Rhizopus stolonifer using both dilution

and disc diffusion susceptibility methods. Antimicrobial activity of combined

compounds 2 and 3 was also investigated together with their combination with

standard drugs gentamycin and fluconazole against the same microbes. Analysis of

variance (ANOVA) was used to analyze the disc diffusion results. The individual

isolated compounds and all the crude extracts (apart from that of stem bark hexane)

showed considerable activity against all the organisms tested. Combinations of

compound 3 and fluconazole displayed additive effect in inhibiting the growth of

Penicillium digitatum with lower MIC of 125μg/mL compared to that of fluconazole

alone at 250 μg /mL. Combination of compounds 2 and 3 also showed additive

effect in inhibiting Rhizopus stolonifer lowering the MIC from 500μg/mL (for both

molecules) to 250 μg /mL. Interaction in antibacterial activity between compounds 2

and 3 lowered the MIC in action against Staphylococcus aureus to 250μg/mL

compared to individual compounds with MIC of 500μg/mL while showing additive

effect. Statistical data analysis of the bioassays using the Statistics Package for

Social Scientists (SPSS) illustrated significant variation of bioactivity between pure

and combined compounds 2 and 3 (p