Resistance mitigating effect of Artemisia annua phytochemical extracts in cultures of Plasmodium falciparum and in -Plasmodium berghei and Plasmodium yoelii

Abstract

The emergence and spread of multidrug resistant Plasmodium falciparum has severely limited therapeutic options for the treatment of malaria. With ever-increasing failure rates associated with chloroquine or sulphadoxine-pyrimethamine treatment, attention has turned to the few alternatives, which include quinine, mefloquine and recently the use of artemisinin. Artemisinin derivatives, particularly in combination with other drugs, are thus increasingly used to treat malaria, reducing the probability that parasites resistant to their components will emerge. Although stable resistance to artemisinin is yet to be reported from laboratory or field isolates, its emergence would be disastrous because of lack of alternative treatments. The project was designed to demonstrate resistance-mitigating effects of phytochemicals in the extract of Artemisia annua relative to pure artemisinin against the malaria parasite Plasmodium falciparum and on rodent malaria parasite Plasmodium berghei ANKA and P. yoelii. For the in vitro experiments selection was undertaken on two cultures of P. falciparum, a CQ-sensitive strain D6 (originally from Sierra Leone) and a CQ-resistant W2 (strain from Indochina), by exposing them to A. annua phytochemical extract and the pure artemisinin over 50 cycles at doses initially required to give 50% mortality of the parasites. Dose-response effects of the extract and the pure artemisinin were determined after 10, 20, 30, and 40 exposure cycles and compared to determine if significant difference in percentage mortality of the parasites with increasing exposure cycles. The in-vivo experiments were carried out by inoculating mice with the murine Plasmodium parasites and thereafter given the test drugs using the 4 day test. After 4 days parasitaemia in test mice and control was determined to calculate the effective doses ED50 and the ED9O. The ED90 was utilized to study resistance under drug pressure and this gave gave rise to “resistant” parasites that were used for molecular characterization. Emergence of resistance of the phenotypes was determined by a quantitative Peter’s 4-day test. DNA was extracted from resistant parasites and sensitive parasites using DNA extraction kit by Roche and then amplified using PCR method. The amplicons were then purified before sequencing. The nucleotide sequences of the possible genetic modulators of Artemisinin and Artemisia annua extract resistance (mdr1, cg10, tctp, and atpase 6) of sensitive and “resistant” parasites were compared. On analyzing the sequences, one mutation was detected on PfMDR 86, in parasites exposed to artemisinin at IC50. No mutations were detected with TCTP, cg10 and MDR 1034. However the in-vitro results showed an increase in (IC50) values where W2 parasites exposed to artemisinin at (IC50) showed a twenty six fold and parasites exposed to artemisinin at (IC90) a ten fold increase was realized. W2 parasites exposed to the blend at (IC50) and at (IC90) showed a twenty nine fold and thirty six fold increases respectively when tested with artemisinin but W2 parasites exposed to Artemisia annua remained sensitive to Artemisia annua extract. A rather interesting finding was that parasites exposed to the Artemisia annua and tested with the extract remained sensitive but parasites exposed to the Artemisia annua and tested with artemisinin showed some level of resistance. . A similar trend was observed with D6 parasites in that those exposed the Artemisia annua extract and tested with the extract remained sensitive to the blend The findings indicated that artemisinin resistance will finally occur and the use of Artemisia annua whole extract would be one of the ways of mitigating against resistance development.