Abstract

Ximenia americana root bark (Olacaceae) and Pavetta crassipes (Rubiaceae) leaf used in Nigerian traditional medicine were tested individually against clinical isolate of Mycobacterium tuberculosis by Lowenstein - Jensen method. Crude aqueous extracts of X. americana and P. crassipes exhibited minimum inhibitory activity (MIC) of 100 μg/mL and 200 μg/mL respectively.
Sequential screening with solvents of different polarities was used in evaluation tests to readily locate the source of the activity against tuberculosis and for conditions related to skin diseases since it was readily available. In general, antimicrobial screening of crude extracts gave MICs ranging from 31.25 μg/mL to > 5 mg/mL, with X. americana methanol extract being most active at 31.25 μg/mL against Staphylococcus aureus. In an effort to determine possible mechanisms of action, synergistic interaction studies between standard antibiotics and plant extracts were carried out with some synergy being observed between X. americana extract and streptomycin.
Hexane (MIC 60.6 μg/mL) and dichloromethane (MIC 30.5 μg/mL) fractions of X. americana exhibited 94.3 % and 96.4 % inhibition against M. tuberculosis H37HRv (virulent strain) while P. crassipes hexane fraction had 86.7% inhibition at > 64 μg/mL. Using HPLC, TLC, GC, 1D and 2D-NMR as well as mass spectral analyses it was possible to identify rutin and 5-O- caffeoyl quinic acid methyl ester from P. crassipes. It proved extremely difficult to identify compounds from LC and TLC fractions from the non-polar extracts of X. americana responsible for anti-TB activity. There was some spectroscopic evidence from these fractions for closely related phytosterol esters and individual compounds such as stigmast- 3, 5 - diene, stigmastane oleate and β-sitosterol.
Subsequent LC work with refractive index detection and SFC with evaporative light scattering data confirmed that the difficulties in assignment arose from the presence of non-UV absorbing non-volatile co-eluting compounds. Preparative
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SFC or SFC-MS with the aid of the NIST database would have been needed for identification.
Overall, these results lend some credence to the claims of the Nigerian remedy and potentially could be a source of assay biomarkers for monitoring its safety, efficacy and quality as required by IRCH (International Regulatory Co-operation for Herbal Medicines).