Phytochemical Investigation Of Zanthoxylum Holstzianum For Antiplsmodial, Larvicidal And Antinociceptive Principles

Abstract

In this study, secondary metabolites from the root and stem bark of Zanthoxylum holstzianum were evaluated for antiplasmodial and mosquito larvicidal activities. In addition, the anti-nociceptive activities of the crude extract and some pure compounds were determined using tail flick method on animal model (mice). The air-dried and pulverized stem and root bark of Z. holstzianum was exhaustively extracted with CH2Cl2/MeOH (1:1) at room temperature. The stem bark extract was subjected to a combination of chromatographic techniques and resulted into isolation of eight compounds. These were five benzophenanthridine alkaloids; [8-acetonyldihydrochelerythrine (3), nitidine (6), dihydrochelerythine (2), norchelerythrine (2), arnottianamide (7)]; a 2-quinolone alkaloid [N-methylflindersine (8)]; a symmetrical lignan [4,4’-dihydroxy-3,3’-dimethoxylignan-9,9’-diyl diacetate (10)] and a C-C linked dimer of a benzophenanthridine and 2-quinoline [holstzianoquinoline (9)]. Similarly, the root bark afforded eight compounds. These included five benzophenanthridine alkaloids [1, 2, 3, 7, 9-demethyloxychelerythrine (5)], a benzophenathridine and 2-quinoline dimer (9), a symmetrical acetone bridged dimer of two benzophenanthridine alkaloids [chelerythridimerine (4)] and a 2-quinoline (8). Overall, ten compounds were isolated from the stem and root bark of Z. holstzianum. Of these, compound 10 and 6 were only found in the stem bark while compounds 4 and 5 were only found in the root bark. Compounds 1, 2, 3, 6, 7, 8 and 9 were found in both parts. Holstzianoquinoline (9) is a new compound and this is the second report of a C-C linked dimer of a benzophenanthridine and a 2-quinoline from nature. Compounds 4 and 10 are reported here for the first time from this genus. The identification of the isolated compounds was based on spectroscopic evidence, which included 1H NMR, 13C NMR, HMBC, HSQC, COSY, NOESY and MS. The CH2Cl2/MeOH (1:1) stem bark extract (crude) and five isolates were tested for in vitro antiplasmodial activity against chloroquine resistant (W2) and chloroquine sensitive (D6), Plasmodium falciparum strains. The crude extract showed potent activity with IC50 values of 2.5 ± 0.3 and 2.6 ± 0.3 μgml-1 against W2 and D6 strains, respectively. Nitidine (6) showed potent activity with IC50 values of 0.11 ± 0.01 μgml-1 for both W2 and D6 while norchelerythrine (2) showed potent activity with IC50 value of 0.15 ± 0.01 μgml-1 against D6 strain but with moderate activity of 7.3 ± xvi 0.3 μgml-1 against W2 strain. Dihydrochelerythrine (1) showed moderate activity of 3.8 ± 0.7 μgml-1 and 3.2 ± 0.8 μgml-1 and similarly 8-Acetonyldihydrochelerythrine with activity of 4.0 ± 0.2 and 3.8 ± 0.7 μgml-1 against W2 and D6 strains, respectively. N-methylflindersine (8) showed a weak activity against both W2 and D6. Further, the crude extracts and some of the isolated compounds were tested for larvicidal activity against the early fourth instar mosquito larvae of Aedes aegypti. The root bark extract showed moderate activity (LC50 = 43.9 ± 5.89, LC90 = 61.0 ± 9.77 μgml-1 at 24 h and LC50 = 32.9 ± 3.66 and LC90 = 50.0 ± 9.12 μgml-1 at 48 h) while the stem bark extract showed weak activity. Two pure compounds showed high activity; N-methylflindersine (8) having a potent larvicidal activity (LC50 = 4.8 ± 0.3, LC90 = 6.9 ± 0.3 μgml-1 at 24 h and LC50 = 2.9 ± 0.3, LC90 = 5.13 ± 0.22 μgml-1 at 48 h) while 9-demethyloxychelerythrine (5) showed activity (LC50 = 12.6 ± 0.8, LC90 = 19.6 ± 1.5 μgml-1 at 24 h and LC50 = 6.5 ± 1.1, LC90 = 13.4 ± 1.87 μgml-1 at 48 h). Holstzianoquinoline (9) showed moderate activity while dihydrochelerythrine and 8-Acetonyldihydrochelerythrine had no activity at 20 μgml-1. In addition, the crude extract (stem and root bark) showed statistically significant antinociceptive effect (P<0.05) at a dose of 100 and 200 mg kg-1 while N-methylflindersine, and dihydrochelerythrine showed extremely statistically significant antinociceptive effect (p<0.0001) at a dose of 100 and 50 mgkg-1 . Holstzianoquinoline showed extremely significant effect (p<0.0001) and highly significant effect (p<0.001) for 100 and 50 mgkg-1 d