Antimicrobial compounds from root, stem bark and seeds of Melia volkensii

Abstract Three compounds, toosendanin (1), kulactone (2) and scopoletin (3), were isolated from either the root bark and/or the stem bark of Melia volkensii. Their structures were determined on the basis of spectroscopic data generated and by comparison with data from the literature. 1 and 2, isolated for the first time from M. volkensii, exhibited significant (p < 0.05) activity against Escherichia coli with minimum inhibitory concentration of 12.5 μg/mL, close to that of neomycin (6.25 μg/mL). The compounds also exhibited high activity against Aspergillus niger (MIC 6.25 μg/mL compared to 2.5 μg/mL for clotrimazole). Dichloromethane and methanol seed, hexane stem bark and methanol root bark extracts exhibited activities towards Escherichia coli, Staphylococcus aureus, Aspergillus niger and Plasmodium falciparum, respectively. Antimicrobial activity of the plant towards A. niger, P. falciparum and S. aureus is reported for the first time in the current work. Graphical abstract


Introduction
Continuous search for new molecules with therapeutic importance is necessitated by development of resistance by pathogens to current medicines (Achan et al. 2011;Nanyunja et al. 2011;Shey et al. 2011;White 2012). Several plants are reliable sources of new efficacious molecules (Alshawsh et al. 2009;Kunle et al. 2012;Phuwapraisirisan et al. 2013). Melia volkensii, a woody tree from semi-arid areas of East Africa, belonging to the genus Melia (Meliaceae), has been used traditionally for the treatment of diarrhoea, pain, skin rashes and eczema (Cantrell et al. 1999;Akanga 2008). Previous investigation on M. volkensii has concentrated ABSTRACT Three compounds, toosendanin (1), kulactone (2) and scopoletin (3), were isolated from either the root bark and/or the stem bark of Melia volkensii. Their structures were determined on the basis of spectroscopic data generated and by comparison with data from the literature. 1 and 2, isolated for the first time from M. volkensii, exhibited significant (p < 0.05) activity against Escherichia coli with minimum inhibitory concentration of 12.5 μg/mL, close to that of neomycin (6.25 μg/mL). The compounds also exhibited high activity against Aspergillus niger (MIC 6.25 μg/mL compared to 2.5 μg/mL for clotrimazole). Dichloromethane and methanol seed, hexane stem bark and methanol root bark extracts exhibited activities towards Escherichia coli, Staphylococcus aureus, Aspergillus niger and Plasmodium falciparum, respectively. Antimicrobial activity of the plant towards A. niger, P. falciparum and S. aureus is reported for the first time in the current work. on anticancer activity (Lingling et al. 1998;Zhao et al. 2010) which contradicts traditional use, mainly pointing to antimicrobial activity (Cantrell et al. 1999;Akanga 2008). However, recent investigation on extracts of Melia Azedarach has shown antimicrobial activity (Zahoor et al. 2015). It was therefore in our interest to carry out both phytochemical and antimicrobial investigations on M. volkensii focusing on antimicrobial activity. This study revealed for the first time the existence of toosendanin (1) and scopoletin (3) in this plant together with kulactone (2) (re-isolated). The antifungal, antibacterial and antiplasmodial activities of the crude extracts, and the isolated compounds are also reported.

Results and discussion
Compound 1 was obtained as white rod-shaped crystals from the root bark using MeOH/CH 2 Cl 2 . Its MS (m/z 574 for molecular ion), 1 H-and 13 C-NMR data (Table S1, Figures S2 and S3) compared well with those in the literature (Tada et al. 1999;Su et al. 2011) enabling its identification as toosendanin. This is the first report of this compound in M. volkensii.
Compound 2 was obtained as colourless needle-shaped crystals from the root bark using Hex/CH 2 Cl 2 mixture. Its HRMS (m/z 452.3281 molecular mass), 1 H-and 13 C-NMR data (Table S1, Figures S4 and S5) and comparison of the spectra data with those in the literature (Kelecom et al. 1996;Tan et al. 2010) led to its identification as kulactone.
The third compound was identified as scopoletin (3) based on spectroscopic data (Table S2, Figures S6 and S7) obtained and comparison of the spectra data with those in the literature (Carpinella et al. 2005) and is being reported for the first time in M. volkensii.
The dichloromethane root and stem bark crude extracts showed significant activity against Aspergillus niger with average inhibition zones of 10.75 ± 2.04 and 10.30 ± 0.53 mm, respectively. The pure compounds 1 and 2 also showed significant (p < 0.05) inhibition towards growth of A. niger (Table 1). The bioactivities for 1, 2 and clotrimazole were comparable (MICs 6.25, 6.25 and 2.5 μg/mL, respectively). Test of the dichloromethane stem bark extract against Staphylococcus aureus showed medium activity with average inhibition zone of 7.00 ± 2.30 mm. The same extract exhibited low activity against Escherichia coli with  Table 2. the minimum inhibitory concentration (MIc) of pure compounds against Escherichia coli.

Sample
Dilutions of sample (μg/mL) 100 50 25 12.5 6.25 2.5 toosendanin average inhibition zone of 4.11 ± 1.24 mm. 1 and 2 showed no significant activity against S. aureus but exhibited considerable potency against E. coli (MICs of 12.5 μg/mL; p > 0.05) two times less active than neomycin (6.25 μg/mL; Table 2). It is interesting to note that since the activities of the two compounds were not significantly different against the gram negative bacteria, the furan ring, hydroxyl, epoxide and acetyl groups on 1 seemed not to contribute to its bioactivity. All extracts from seeds showed inhibition towards the growth of Plasmodium falciparum with dichloromethane extract showing the highest percentage inhibition (66.6%; p < 0.05; Figure S1). The antiplasmodial activity could be attributed to a blend of phytochemicals working together in the extracts against the parasite. On the other hand, there could be specific compounds in crude extracts which upon isolation could yield higher activity.

Conclusion
This study has revealed the presence of three secondary metabolites namely toosendanin (1) and scopoletin (3) being reported for the first time in this plant together with kulactone (2) (re-isolated). Methanol extract showed the highest activity against both S. aureus and A. niger, while dichloromethane extracts were most active against E. coli. In addition, dichloromethane and methanol extracts from seeds showed highest activity against P. falciparum. 1 and 2 exhibited significant (p < 0.05) activity against E. coli and A. niger with MICs values of 12.5 and 6.25 μg/mL, respectively. These activities were comparable to those of neomycin and clotrimazole. The presence of toosendanin (1), known for its insecticidal activity, as the major compound in M. volkensii explains the reported larvicidal activity exhibited by the seed extracts.