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. 2019 Mar 8;18(1):65.
doi: 10.1186/s12936-019-2694-1.

Bioassay-guided isolation and identification of gametocytocidal compounds from Artemisia afra (Asteraceae)

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Bioassay-guided isolation and identification of gametocytocidal compounds from Artemisia afra (Asteraceae)

Phanankosi Moyo et al. Malar J. .

Abstract

Background: Optimal adoption of the malaria transmission-blocking strategy is currently limited by lack of safe and efficacious drugs. This has sparked the exploration of different sources of drugs in search of transmission-blocking agents. While plant species have been extensively investigated in search of malaria chemotherapeutic agents, comparatively less effort has been channelled towards exploring them in search of transmission-blocking drugs. Artemisia afra (Asteraceae), a prominent feature of South African folk medicine, is used for the treatment of a number of diseases, including malaria. In search of transmission-blocking compounds aimed against Plasmodium parasites, the current study endeavoured to isolate and identify gametocytocidal compounds from A. afra.

Methods: A bioassay-guided isolation approach was adopted wherein a combination of solvent-solvent partitioning and gravity column chromatography was used. Collected fractions were continuously screened in vitro for their ability to inhibit the viability of primarily late-stage gametocytes of Plasmodium falciparum (NF54 strain), using a parasite lactate dehydrogenase assay. Chemical structures of isolated compounds were elucidated using UPLC-MS/MS and NMR data analysis.

Results: Two guaianolide sesquiterpene lactones, 1α,4α-dihydroxybishopsolicepolide and yomogiartemin, were isolated and shown to be active (IC50 < 10 μg/ml; ~ 10 μM) against both gametocytes and intra-erythrocytic asexual P. falciparum parasites. Interestingly, 1α,4α-dihydroxybishopsolicepolide was significantly more potent against late-stage gametocytes than to early-stage gametocytes and intra-erythrocytic asexual P. falciparum parasites. Additionally, both isolated compounds were not overly cytotoxic against HepG2 cells in vitro.

Conclusion: This study provides the first instance of isolated compounds from A. afra against P. falciparum gametocytes as a starting point for further investigations on more plant species in search of transmission-blocking compounds.

Keywords: Artemisia afra; Gametocytes; Malaria; Natural products; Plasmodium falciparum; Sesquiterpene lactone; Transmission-blocking.

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Figures

Fig. 1
Fig. 1
Bioassay-guided fractionation and isolation of gametocytocidal compounds 1 and 2 from Artemisia afra. Inhibition of in vitro viability of late-stage gametocytes (1.2–1.6% gametocytaemia, n = 1) of P. falciparum (NF54 strain) by crude extract and fractions of Artemisia afra, realised from a solvent–solvent partitioning step and b separation of chloroform fraction (column 1). c Typical microscopic pictures of macrogametocytes (condensed nuclei) and microgametocytes (scattered nuclei) used in pLDH assays. Full dose–response investigation of compound d 1 and e 2 against late stage gametocytes (1–2% gametocytaemia) of P. falciparum. IC50 values (μM), determined using the 72 + 72 h pLDH assay. Data are the mean ± SEM of three independent biological repeats in technical triplicates. Positive drug control for inhibition of viability of late-stage gametocytes were methylene blue and artemisinin. *Unpaired assays (n = 1, each carried out in technical triplicate, see Additional file 1: Fig. S2). MB methylene blue, Hex hexane, CE crude extract, Chl chloroform, EtOAc ethyl acetate, MeOH methanol, ART artemisinin
Fig. 2
Fig. 2
Pan-reactivity and cytotoxic investigations of Artemisia afra chloroform fraction, compounds 1 and 2. a IC50 values (μM), determined using the pLDH assay for asexuals (1% parasitaemia) and gametocytes (1–2% gametocytaemia), respectively. Data are the mean ± SEM of three independent biological repeats each done in technical triplicate. n = 2 for all asexual assays. Statistically significant differences between the IC50 values are indicated (*P < 0.05, unpaired Student’s t-test). b Cytotoxicity was investigated by the standard mammalian LDH leakage assay, with emetine used as positive reference control. *Unpaired assays (n = 3, each carried out in technical duplicate). Data are the mean ± SEM of three independent biological repeat experiments each performed in technical triplicate
Fig. 3
Fig. 3
Chemical structure of a compound 1, 1α,4α-dihydroxybishopsolicepolide and b selected HMBC and COSY correlations
Fig. 4
Fig. 4
Chemical structure of a compound 2, yomogiartemin and b selected HMBC and COSY correlations

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