We found conclusive evidence that orally ingested, powdered dried leaves of whole plant A. annua kills malaria parasites more effectively than a comparable dose of pure drug. In our primary analysis we used dried A. annua leaves containing 14.8 mg artemisinin per gram of dried leaves and compared parasitemia over time in mice treated with either low-dose, whole plant A. annua (WPLO), low-dose, pure drug artemisinin (ANLO), or placebo (CON). Only 24 hr after treatment, dead parasites (with condensed dark pigment) were observed in mice treated with WPLO; 30 hours after treatment, parasitemia was <0.001% (). Mice treated with WPLO showed significantly lower parasitemia than those treated with ANLO from 12 to 72 hours post-treatment (). Mice treated with ANLO did not show significant difference in parasitemia from those administered a placebo at any time point.
Giemsa stained blood smears from mice showing erythrocytes infected with P. chabaudi, 30
P. chabaudi parasitemia of three treatment groups across all experimental replicates: low-dose whole plant A. annua
In a subsequent dose-response analysis, we added high-dose comparison groups, ANHI
. Each of the four treatment groups experienced significantly lower parasitemia than the control mice (). All mice treated with WPLO
responded strongly in the first 24 hr following gavage, showing the lowest level of parasitemia at 30 hours post-gavage; only 3 of 26 WPLO
-treated mice (12%) had parasitemia ≥3%. However, 36 hours after gavage, 38% of the WPLO
-treated mice were above this threshold. Notably, treatment with WPLO
was just as effective in reducing parasitemia as was treatment with ANHI
for the first 72 hours post treatment (). Thereafter, WPLO
-treated mice had significantly higher parasitemia. Although the suppression of parasitemia was significant in the three treatment groups after a single dose treatment, low dose WPLO
resulted in faster recrudescence than either WPHI
(), suggesting that multiple treatments at this dose would be necessary for a curative effect. Considering that a normal course of ACT treatment for human malaria currently requires several doses, spread over several days, a similar regime may also be effective in the case of lower dose WP administration. Pharmacokinetics will be needed to determine serum levels of the drug. No differences between the WPHI
groups were detected after treatment. This is consistent with the short half-life seen in the pure drug in human patients and the prescribed need for multiple doses per day for several days 
Dose comparisons of WP and AN treatments from the third replicate of data: WPLO
Although the precise mechanism of its anti-malarial activity remain unproven, artemisinin is suspected (like other drugs, including chloroquine) to interfere with heme detoxification, a crucial requirement for parasite survival in erythrocytes. Plasmodium
parasites digest hemoglobin, producing heme as a byproduct. Free heme molecules are toxic, so parasites sequester it in the form of hemozoin polymers in unique digestive vacuoles. Artemisinin is a sesquiterpene lactone with a crucial endoperoxide bridge 
, and in the presence of heme, this bridge is broken, thereby releasing free radicals harmful to the parasite 
The empirical evidence for increased anti-malarial activity of WP relative to AN might be explained by differential bioavailability of the artemisinic compounds. Weathers et al. showed that mice treated with a WP equivalent of 1.2 mg/kg AN reached their highest concentration of artemisinin in the blood (87 µg/L) 30 min after gavage, whereas mice treated with AN did not reach their the maximum concentration (74 µg/L) until much later (≥60 min) 
. Moreover, poor solubility and high metabolic breakdown of artemisinin by hepatic and intestinal cytochrome P enzymes (CYP P450 and CYP3A4) may reduce its bioavailability when administered in pure form 
. Infusions made from whole plant A. annua
showed marked inhibition of the intestinal and hepatic CYP enzymes by flavonoids and/or other compounds 
. Hence, inhibition of metabolic enzymes correlates with greater bioavailability of artemisinin, which is consistent with our findings that WP demonstrates greater parasite killing activity than a comparable pure drug treatment.
Whole plant (WP) A. annua
may also have enhanced antimalarial activity due to synergism among particular plant compounds and artemisinin 
. Among these compounds are many flavonoids, of which at least six (artemetin, casticin, chrysosplenetin, chrysosplenol-D, cirsilineol, and eupatorin) are of interest for their antimalarial roles. The synergism between artemisinin and these flavonoids may be due to their ability to potentiate the activity of artemisinin. When each of these six flavonoids was combined individually with artemisinin, the IC50
of AN against P. falciparum
decreased by 20–50%, demonstrating an apparent synergy between the sesquiterpene lactone, artemisinin, and those six methoxylated flavonoids 
. The precise mechanism of flavonoids in activating artemisinin is not fully understood, however it has been reported that A. annua
methoxylated flavonoids enhance the formation of the artemisinin-heme complex 
, which increases the release of free radicals.
Two other major A. annua
flavonoids, myricetin and quercetin, are known to inhibit mammalian thioredoxin reductase, which is critical for cellular redox control 
. Thioredoxin reductase is also essential for the P. falciparum
erythrocytic stage 
; therefore, inhibition of this parasite enzyme by myricetin and quercetin may work in synergy with artemisinin against P. falciparum
In addition to the bioavailability and potentiation attributes of WP, there are other compounds in A. annua
that may act to reduce parasitemia independent of artemisinin. Liu et al (1992) reported the antimalarial activity of several A. annua
flavonoids delivered in vitro
as isolated compounds, in the absence of artemisinin 
. Moreover, antimalarial activity has been documented for related plant species that do not produce artemisinin 
. Among the compounds in A. annua
not yet fully investigated are more than a dozen other sesquiterpenes, some of which have shown promise for killing parasites in rodent models 
Determining the mechanisms for increased efficacy of WP will require further investigation, but it seems certain that the constituent compounds contained within A. annua comprise a complex set of interactions and synergies yet to be described. Given the complex nature of the plant and its many components, WP may not necessarily be considered a simple monotherapy. While the temptation might be to consider WP as merely an alternative delivery mechanism for artemisinin, our results strongly indicate that WP is unique and may represent an innovative combination therapy. We refer to this as a plant Artemisinin Combination Therapy (pACT). A pACT can be distinguished from other combinational therapies where the drug components do not necessarily work synergistically because their combinations are artificially contrived. The pACT comprises a biologically complex entity, in which the combinations are result of evolutionary processes that would have attributes of redundancy and resiliency that make combination therapies selectively advantageous to simple monotherapies. Refinements of these combinations by evolutionary processes ensures they are robust.
The novelty of the WP pACT cannot be overemphasized as there is a common misconception that this therapy has been tested previously. It has not. The WP therapy tested in the present study should not be confused with tea or infusion therapy. Whole plant A. annua (WP) tested here against murine malaria, uses the plant leaves, dried under controlled conditions and ingested by the host. Such a preparation of A. annua has never been tested against malaria parasites (in humans, mice or otherwise).
Because A. annua
has long been used to make tea to treat fever in Asia 
, several investigators have proposed to re-establish the use of A. annua
tea for rapid treatment of malaria 
. These teas have major shortcomings. First, large volumes of tea must be consumed over short periods to ensure adequate ingestion of drug, a nontrivial matter considering the bitter taste of the tea, especially for pediatric patients. Moreover, while a 5 min boiling water extraction yields about 90% of the plant's artemisinin 
, this is not an effective process for extracting key flavonoids 
. Our analysis of hot water tea extracts following the optimized protocol described by van der Kooy and Verpoorte 
, showed loss of about 99% of some of the original flavonoids that reportedly synergize with artemisinin 
Not only does WP differ from teas and infusions in terms of its efficacy and pharmaceutical properties, but also because of its preparation, it can be carefully controlled and preserved. We previously proposed development of a new form of anti-malarial therapy based on dried, encapsulated A. annua
leaves as an inexpensive, dose-controlled, rapid delivery of the drug to treat uncomplicated cases of malaria and other neglected tropical diseases for which artemisinin has been shown to be effective 
. Dosage can be controlled because dried WP A. annua
can be homogenized and assayed for artemisinin content prior to encapsulation. Capsule size and number can be adjusted based on artemisinin content and patient weight.
Our purpose in the present study was to determine whether this “generally regarded as safe” (GRAS) herb 
is effective in killing malaria parasites in vivo
. Extrapolating appropriate human dosage from experimental evidence in mouse models will require additional investigation, however it is generally accepted that this extrapolation does not scale linearly with respect to body mass. Better indicators that allow for allometry include use of total body surface area 
and/or take full consideration of physiochemical properties of the drugs and species involved 
. As an example of this non-linear relationship, we can look to the results from ANLO
treatment administered to mice in our study. ANLO
mice received a dose of 24 mg/kg, which exceeds the current WHO single therapeutic dose (20 mg/kg) for treating human malaria, however, in our study this dose of pure drug had very little effect against mouse malaria parasites. This is most likely due to metabolic differences and rates of uptake of oral drug between mice and humans.
Single oral dosages of AN proven effective against human P. falciparum
malaria range from 100–500 mg 
, which corresponds to 6–33 grams of WP assuming a 1.5% artemisinin content. Assuming an average tablet size of 1 gram, delivery of a comparable dose of WP seems plausible. However, our data suggests that WP requires a smaller overall amount of artemisinin since even the WPLO
effectively reduced malaria after just a single dose. Moreover, it is important to bear in mind that while WPLO
was the lowest concentration in our study, it does not necessarily represent a minimum effective dose.
Notwithstanding challenges to be overcome and further research needed, our preliminary investigations hold great promise for easing the burden of high cost and limited availability currently confronting use of artemisinin-based semi-synthetic derivatives. While production costs for pharmaceuticals are not generally publically available, it is possible to estimate potential savings associated with using WP vs. AN by looking to the estimated efficiencies of processing AN from the plant. Kindermans et al 
estimate the yield for extraction and purification of drug from A. annua
to be 50–80% efficient. This is consistent with general understanding of downstream processing costs wherein product losses increase with the number of unit operations (unit ops) 
. All things being equal (e.g. artemisinin content for a given cultivar), this would suggest a 20–50% savings realized by forgoing the extraction and purification steps, as would be the case for production of WP. An example of the steps involved in production of AN from whole plant is provided by de Vries et al., and compared with steps for producing WP (). Current production of AN by extraction from A. annua
has seven more unit ops than the one associated with direct use of the whole plant, and this net loss in efficiency translates to higher costs per product unit (). Given the multiplicative principle of downstream processing, even if each processing step has relatively high efficiency, the overall efficiency will be less. For example, even if each of the seven additional steps for production of AN was 95% efficient, the overall efficiency would be merely 69.8%, which means that less than three quarters of the starting material is realized as drug. Such a loss translates to higher cost for the delivered drug. This simple analysis does not even consider the additional costs for reagents, labor, and energy that are required for processing the extract (), which de Vries et al. 
estimated may comprise up to 22.9% of the total manufacturing costs. This 22.9% represents additional cost savings for the use of WP because those inputs are no longer required.
Comparison of unit operations required for production of extracted AN and production of WP.
And while our data are far from representing a clinical trial, they do provide preliminary indications that the WP therapy requires a far smaller dose than the corresponding amount of pure artemisinin. Indeed, our experiments indicate that a dose of WP has a five-fold increase in anti-malarial activity over that of the corresponding amount of AN. This increased activity per unit of plant mass not only affects the dosing regimen but also has profound economic impacts if the WP approach should prove useful on a large scale to treat human malaria.
Much work remains to determine feasibility and efficiency of bringing whole plant A. annua into the arsenal in the fight against malaria. Among the challenges to be faced are some botanical obstacles, not the least of which is that this plant readily outcrosses, making it difficult to maintain high artemisinin content in the plant using seed saving methods which are standard agricultural practice in the developing world. Moreover, to date all efforts at improving the A. annua crop have focused on plant breeding and agricultural methods to maximize artemisinin content and in so doing to increase efficiencies and drive down costs. The use of the whole plant as therapy may represent a paradigm shift in this regard, since it may well be the case that effectiveness of WP is not wholly dependent on artemisinin content. New plant breeding strategies would have to be considered to optimize plant performance and maximize efficacy. The potential for an inexpensive malaria therapy that by its very nature possesses great resilience to parasite resistance, makes this investment of effort well worthwhile.