BIOACTIVITY-GUIDED ISOLATION AND STRUCTURE ELUCIDATION OF ANTIMALARIAL TRITERPENES FROM Combretum zenkeriENGL & DIELS AND Combretum racemosumP. BEAUV. LEAVES

Abstract

Medicinal plants are rich sources of antimalarial compounds. Combretaceae family is known from ethnobotanical survey to possess broad spectrum of activities against different diseases including malaria. The increasing trend of resistance to many antimalarial agents including artemisinin and its derivatives has necessitated the need for new drug candidates. This study was, therefore, designed to validate the antimalarial potential of selected Combretaceae species, investigate the most active plant extracts by bioactivity-guided isolation and structure elucidation of the active principles. Methanol and acetone extracts of the leaves of ten Combretaceae species, collected from the University of Ibadan Botanical Garden, were obtained by Soxhlet method. These extracts were screened for inhibition of β-hematin synthesis monitored with UV-Visible spectrophotometer at 405 nm. The most active methanol extracts, Combretum racemosum (CRM) [FHI/108887] and Combretum zenkeri (CZM) [FHI/110277] were screened against chloroquine-sensitive D10 and chloroquine-resistant W2 Plasmodium falciparum strains using lactate dehydrogenase assay with chloroquine as the standard. Both extracts were successively partitioned into chloroform and n-butanol by solvent-solvent partitioning, fractions obtained were also investigated for anti-plasmodial activity. The chloroform fractions were subjected to flash and column chromatographic techniques for bioactive compound isolation. Isolated compounds were characterised by NMR and MS techniques (1D and 2D NMR, ESI-MS and HR-ESIMS) and subjected to anti-plasmodial screening. Structure-activity relationship (SAR) study of the isolated compounds was conducted. The IC50 was calculated by curve-fitting analysis. Statistical analyses were conducted using a two-tailed Student’s t test at α0.05. The CZM (IC50: 2.92±0.846 mg/mL) and CRM (IC50: 3.96±0.132 mg/mL) crude extracts had significant activities. The CRM [D10: IC50= 64.18±2.69 µg/mL (R2 = 0.99); W2: IC50= 65.80±14.85 µg/mL (R2 = 0.96)] and CZM [D10: IC50= 68.98±1.00 µg/mL (R2 = 0.95); W2: IC50= 69.68±3.09 µg/mL (R2 = 0.99)] crude extracts showed antiplasmodial activity. The CRM chloroform fraction (D10: IC50= 33.80±1.52 µg/mL; W2: IC50= 27.82±2.85 µg/mL) showed higher activity relative to the n-butanol fraction (D10: IC50= 78.08±7.29 µg/mL; W2: IC50= 78.12±14.98 µg/mL). The chloroform fraction of CZM (D10: IC50= 12.57±1.57 µg/mL; W2: IC50= 12.14±0.95 µg/mL); also had higher activity than n-butanol fraction (D10: IC50= 61.98±3.25 µg/mL; W2: IC50= 61.26±8.64 µg/mL). Phytochemical isolation from the C. racemosum chloroform fraction led to the identification of four ursane-type triterpenes: 19α-hydroxyasiatic acid, 6β, 23-dihydroxytormentic acid, madecassic acid, nigaichigoside F1; four oleanane-type triterpenes: arjungenin, combregenin, terminolic acid, arjunglucoside I, and abscisic acid. All isolated compounds exhibited antiplasmodial activity (17.19±4.34 ≤ IC50 ≤ 134.70±13.21 µg/mL) with madecassic acid showing significant activity [D10: IC50= 27.62±11.56 µg/mL (R2 = 0.96); W2: IC50= 17.19±4.34 µg/mL (R2 = 0.98)]; however, chloroquine standard showed higher activity (D10: IC50= 0.01±0.002 µg/mL; W2: IC50= 0.22±0.03 µg/mL) than madecasic acid. The chloroform fraction of C. zenkeri led to the isolation of two triterpenes, ursolic and oleanolic acids, with known antimalarial activities. The SAR showed that dehydroxylation at 6β- and/or 19α-positions in these triterpenes increased the antiplasmodial activity, while the geminal-dimethyl substitutions at position C-20 did not significantly impact the bioactivity. The antiplasmodial potential of Combretum racemosum and Combretum zenkeri was validated. Madecassic acid showed potential for antimalarial drug development.