Melissa J. Towler

Production of Mouse Interleukin-12 Is Greater in Tobacco Hairy Roots Grown in a Mist Reactor Than in an Airlift Reactor

We compared the growth and productivity of a tobacco line of hairy roots that produces murine interleukin 12 (mIL‐12) grown in three different culture systems: shake flasks, an airlift reactor, and a scalable mist reactor. Of the total mIL‐12 produced by cultures grown in shake flasks (∼434.8 µg L−1), almost 21% was recovered from the medium. In contrast to roots harvested from shake flasks and the mist reactor, roots were not uniformly distributed in the airlift reactor. Roots formed a dense ring around the wall of the reactor and surrounding the central rising column of fine aeration bubbles. Root quality was also better in both the shake flasks and mist reactor than in the airlift reactor. There were more pockets of dark roots in the airlift reactor suggesting some of the roots were nutrient starved. Although the best root growth (7 g DW L−1) was in the shake flasks, both reactors produced about the same, but less dry mass, nearly 5 g DW L−1. Total mIL‐12 concentration was highest in the mist reactor at 5.3 µg g−1 FW, but productivity, 31 µg g−1 FW day−1 was highest in shake flasks. Roots grown in the mist reactor produced about 49.5% more mIL‐12 than roots grown in the airlift reactor. Protease activity in the media increased steadily during culture of the roots in all three systems. The comparisons of protease activity, protein and mIL‐12 levels done in the shake flask system suggest that the increase in proteases associated with progression into stationary phase is most detrimental to mIL‐12 concentration. This is the first description of the design and operation of a scalable version of a mist bioreactor that uses a plastic bag. This also the first report of reasonable production levels of functional mIL‐12, or any protein, produced by hairy roots grown in a mist reactor. Results will prove useful for further optimization and scale‐up studies of plant‐produced therapeutic proteins. Biotechnol. Bioeng. 2009;102: 1074–1086.

Alteration of biomass and artemisinin production in Artemisia annua hairy roots by media sterilization method and sugars

Transformed root cultures of Artemisia annua grown in autoclaved medium show large variations in biomass and artemisinin production regardless of the culture conditions or clonal type. However, using filter-sterilized sugars singly or in combination while holding the carbon level in the medium constant resulted in an unexpected variability in biomass production and artemisinin yield. Autoclaving results in variable hydrolysis of sucrose in the culture medium. Subsequent experiments using combinations of filter-sterilized sugars at a constant total carbon level in the medium showed a stimulation of artemisinin production by glucose. Growth in sucrose was equivalent to growth in fructose and significantly better than in glucose. These results suggest that sugars may be affecting terpenoid metabolism not only as carbon sources, but also as signal molecules.

Dried Whole Plant Artemisia annua as an Antimalarial Therapy

Drugs are primary weapons for reducing malaria in human populations. However emergence of resistant parasites has repeatedly curtailed the lifespan of each drug that is developed and deployed. Currently the most effective anti-malarial is artemisinin, which is extracted from the leaves of Artemisia annua. Due to poor pharmacokinetic properties and prudent efforts to curtail resistance to monotherapies, artemisinin is prescribed only in combination with other anti-malarials composing an Artemisinin Combination Therapy (ACT). Low yield in the plant, and the added cost of secondary anti-malarials in the ACT, make artemisinin costly for the developing world. As an alternative, we compared the efficacy of oral delivery of the dried leaves of whole plant (WP) A. annua to a comparable dose of pure artemisinin in a rodent malaria model (Plasmodium chabaudi). We found that a single dose of WP (containing 24 mg/kg artemisinin) reduces parasitemia more effectively than a comparable dose of purified drug. This increased efficacy may result from a documented 40-fold increase in the bioavailability of artemisinin in the blood of mice fed the whole plant, in comparison to those administered synthetic drug. Synergistic benefits may derive from the presence of other anti-malarial compounds in A. annua. If shown to be clinically efficacious, well-tolerated, and compatible with the public health imperative of forestalling evolution of drug resistance, inexpensive, locally grown and processed A. annua might prove to be an effective addition to the global effort to reduce malaria morbidity and mortality.

Dried whole-plant Artemisia annua slows evolution of malaria drug resistance and overcomes resistance to artemisinin

Significance Evolution of malaria parasite drug resistance has thwarted efforts to control this deadly disease. Use of drug combinations has been proposed to slow that evolution. Artemisinin is a favorite drug in the global war on malaria and is frequently used in combination therapies. Here we show that using the whole plant (Artemisia annua) from which artemisinin is derived can overcome parasite resistance and is actually more resilient to evolution of parasite resistance; i.e., parasites take longer to evolve resistance, thus increasing the effective life span of the therapy. Pharmaceutical monotherapies against human malaria have proven effective, although ephemeral, owing to the inevitable evolution of resistant parasites. Resistance to two or more drugs delivered in combination will evolve more slowly; hence combination therapies have become the preferred norm in the fight against malaria. At the forefront of these efforts has been the promotion of Artemisinin Combination Therapy, but despite these efforts, resistance to artemisinin has begun to emerge. In 2012, we demonstrated the efficacy of the whole plant (WP)—not a tea, not an infusion—as a malaria therapy and found it to be more effective than a comparable dose of pure artemisinin in a rodent malaria model. Here we show that WP overcomes existing resistance to pure artemisinin in the rodent malaria Plasmodium yoelii. Moreover, in a long-term artificial selection for resistance in Plasmodium chabaudi, we tested resilience of WP against drug resistance in comparison with pure artemisinin (AN). Stable resistance to WP was achieved three times more slowly than stable resistance to AN. WP treatment proved even more resilient than the double dose of AN. The resilience of WP may be attributable to the evolutionary refinement of the plant’s secondary metabolic products into a redundant, multicomponent defense system. Efficacy and resilience of WP treatment against rodent malaria provides compelling reasons to further explore the role of nonpharmaceutical forms of AN to treat human malaria.

The Flavonoids Casticin and Artemetin Are Poorly Extracted and Are Unstable in an Artemisia annua Tea Infusion

A number of flavonoids including casticin and artemetin from Artemisia annua have shown synergism with artemisinin against Plasmodium falciparum, but it is unclear if the flavonoids are also extracted into a tea infusion of the plant. Using a tea infusion preparation protocol that was reported to be highly effective for artemisinin extraction, we measured casticin and artemetin extraction. There was only a 1.8 % recovery of casticin in the infusion while artemetin was undetectable. After 24 hr storage at room temperature, casticin yield declined by 40 %. These results show that although a tea infusion of the plant may extract artemisinin, the polymethoxylated flavonoids casticin and artemetin are poorly extracted and lost with storage at room temperature and thus, the tea infusion appears to lose synergistic value.

Biomass Production of Hairy Roots of Artemisia annua and Arachis hypogaea in a Scaled-Up Mist Bioreactor

Hairy roots have the potential to produce a variety of valuable small and large molecules. The mist reactor is a gas phase bioreactor that has shown promise for low‐cost culture of hairy roots. Using a newer, disposable culture bag, mist reactor performance was studied with two species, Artemisia annua L. and Arachis hypogaea (peanut), at scales from 1 to 20 L. Both species of hairy roots when grown at 1 L in the mist reactor showed growth rates that surpassed that in shake flasks. From the information gleaned at 1 L, Arachis was scaled further to 4 and then 20 L. Misting duty cycle, culture medium flow rate, and timing of when flow rate was increased were varied. In a mist reactor increasing the misting cycle or increasing the medium flow rate are the two alternatives for increased delivery of liquid nutrients to the root bed. Longer misting cycles beyond 2–3 min were generally deemed detrimental to growth. On the other hand, increasing the medium flow rate to the sonic nozzle especially during the exponential phase of root growth (weeks 2–3) was the most important factor for increasing growth rates and biomass yields in the 20 L reactors. A. hypogaea growth in 1 L reactors was µ = 0.173 day−1 with biomass yield of 12.75 g DW L−1. This exceeded that in shake flasks at µ = 0.166 day−1 and 11.10 g DW L−1. Best growth rate and biomass yield at 20 L was µ = 0.147 and 7.77 g DW L−1, which was mainly achieved when medium flow rate delivery was increased. The mist deposition model was further evaluated using this newer reactor design and when the apparent thickness of roots (+hairs) was taken into account, the empirical data correlated with model predictions. Together these results establish the most important conditions to explore for future optimization of the mist bioreactor for culture of hairy roots. Biotechnol. Bioeng. 2010;107: 802–813.

Pharmacokinetics of artemisinin delivered by oral consumption of Artemisia annua dried leaves in healthy vs. Plasmodium chabaudi-infected mice.

ETHNOPHARMACOLOGICAL RELEVANCE The Chinese have used Artemisia annua as a tea infusion to treat fever for >2000 years. The active component is artemisinin. Previously we showed that when compared to mice fed an equal amount of pure artemisinin, a single oral dose of dried leaves of Artemisia annua (pACT) delivered to Plasmodium chabaudi-infected mice reduced parasitemia at least fivefold. Dried leaves also delivered >40 times more artemisinin in the blood with no toxicity. The pharmacokinetics (PK) of artemisinin delivered from dried plant material has not been adequately studied. MATERIALS AND METHODS Healthy and Plasmodium chabaudi-infected mice were oral gavaged with pACT to deliver a 100 mg kg(-1) body weight dose of artemisinin. Concentrations of serum artemisinin and one of its liver metabolites, deoxyartemisinin, were measured over two hours by GCMS. RESULTS The first order elimination rate constant for artemisinin in pACT-treated healthy mice was estimated to be 0.80 h(-1) with an elimination half-life (T½) of 51.6 min. The first order absorption rate constant was estimated at 1.39 h(-1). Cmax and Tmax were 4.33 mg L(-1) and 60 min, respectively. The area under the curve (AUC) was 299.5 mg min L(-1). In contrast, the AUC for pACT-treated infected mice was significantly greater at 435.6 mg min L(-1). Metabolism of artemisinin to deoxyartemisinin was suppressed in infected mice over the period of observation. Serum levels of artemisinin in the infected mice continued to rise over the 120 min of the study period, and as a result, the T½ was not determined; the Cmax and Tmax were estimated at ≥6.64 mgL(-1) and ≥120 min, respectively. Groups of healthy mice were also fed either artemisinin or artemisinin mixed in mouse chow. When compared at 60 min, artemisinin was undetectable in the serum of mice fed 100 mg AN kg(-1) body weight. When plant material was present either as mouse chow or Artemisia annua pACT, artemisinin levels in the serum rose to 2.44 and 4.32 mg L(-1), respectively, indicating that the presence of the plant matrix, even that of mouse chow, had a positive impact on the appearance of artemisinin in the blood. CONCLUSIONS These results showed that artemisinin and one of its drug metabolites were processed differently in healthy and infected mice. The results have implications for possible therapeutic use of pACT in treating malaria and other artemisinin-susceptible diseases.

The effect of roots and media constituents on trichomes and artemisinin production in Artemisia annua L.

Key messageRooting ofArtemisia annuaincreases trichome size on leaves and helps drive the final steps of the biosynthesis of the sesquiterpene antimalarial drug, artemisinin.AbstractArtemisia annua produces the antimalarial drug, artemisinin (AN), which is synthesized and stored in glandular trichomes (GLTs). In vitro-grown A. annua shoots produce more AN when they form roots. This may be a function not of the roots, but rather media components such as the phytohormones, α-naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BAP), or salts and sucrose used to maintain either rooted or unrooted shoot cultures. We investigated how three main media components altered artemisinic metabolite production, pathway gene transcripts, and GLT formation in both mature and developing leaves in rooted and unrooted cultures. Although transcript levels of AN biosynthetic genes were not altered, AN levels were significantly different, and there were major differences in both artemisinic metabolite levels and trichomes in mature versus developing leaves. For example, NAA induced higher AN production in rooted shoots, but only in mature leaves. In developing leaves, BAP increased GLT density on the leaf surface. When both phytohormones were present, GLTs were larger on young developing leaves, but smaller on mature leaves. Furthermore, although other media components increased GLT density, their size decreased on young leaves, but there was no effect on mature leaves. Roots also appeared to drive conversion of artemisinic precursors towards end products. These results suggest that, while the presence of roots affects AN and trichome production, phytohormones and other media constituents used for in vitro culture of A. annua also exert an influence.

Simulated digestion of dried leaves of Artemisia annua consumed as a treatment (pACT) for malaria.

ETHNOPHARMACOLOGICAL RELEVANCE Artemisinin (AN) is produced by Artemisia annua, a medicinal herb long used as a tea infusion in traditional Chinese medicine to treat fever; it is also the key ingredient in current artemisinin-based combination therapies (ACTs) effective in treating malaria. Recently we showed that dried leaves from the whole plant Artemisia annua that produces artemisinin and contains artemisinin-synergistic flavonoids seem to be more effective and less costly than ACT oral malaria therapy; however little is known about how digestion affects release of artemisinin and flavonoids from dried leaves. MATERIAL AND METHODS In the current study we used a simulated digestion system to determine how artemisinin and flavonoids are released prior to absorption into the bloodstream. Various delivery methods and staple foods were combined with dried leaves for digestion in order to investigate their impact on the bioavailability of artemisinin and flavonoids. Digestate was recovered at the end of the oral, gastric, and intestinal stages, separated into solid and liquid fractions, and extracted for measurement of artemisinin and total flavonoids. RESULTS Compared to unencapsulated digested dried leaves, addition of sucrose, various cooking oils, and rice did not reduce the amount of artemisinin released in the intestinal liquid fraction, but the amount of released flavonoids nearly doubled. When dried leaves were encapsulated into either hydroxymethylcellulose or gelatin capsules, there was >50% decrease in released artemisinin but no change in released flavonoids. In the presence of millet or corn meal, the amount of released artemisinin declined, but there was no change in released flavonoids. Use of a mutant Artemisia annua lacking artemisinin showed that the plant matrix is critical in determining how artemisinin is affected during the digestion process. CONCLUSIONS This study provides evidence showing how both artemisinin and flavonoids are affected by digestion and dietary components for an orally consumed plant delivered therapeutic and that artemisinin delivered via dried leaves would likely be more bioavailable if provided as a tablet instead of a capsule.

Artemisia annua dried leaf tablets treated malaria resistant to ACT and i.v. artesunate: Case reports

BACKGROUND Dried leaf Artemisia annua (DLA) has shown efficacy against Plasmodium sp. in rodent studies and in small clinical trials. Rodent malaria also showed resiliency against the evolution of artemisinin drug resistance. PURPOSE This is a case report of a last resort treatment of patients with severe malaria who were responding neither to artemisinin combination therapy (ACT) nor i.v. artesunate. STUDY DESIGN Of many patients treated with ACTs and i.v. artesunate during the 6 mon study period, 18 did not respond and were subsequently treated with DLA Artemisia annua. METHODS Patients were given a dose of 0.5g DLA per os, twice daily for 5d. Total adult delivered dose of artemisinin was 55mg. Dose was reduced for body weight under 30kg. Clinical symptoms, e.g. fever, coma etc., and parasite levels in thick blood smears were tracked. Patients were declared cured and released from hospital when parasites were microscopically undetectable and clinical symptoms fully subsided. RESULTS All patients were previously treated with Coartem® provided through Santé Rurale (SANRU) and following the regimen prescribed by WHO. Of 18 ACT-resistant severe malaria cases compassionately treated with DLA, all fully recovered. Of the 18, this report details two pediatric cases. CONCLUSIONS Successful treatment of all 18 ACT-resistant cases suggests that DLA should be rapidly incorporated into the antimalarial regimen for Africa and possibly wherever else ACT resistance has emerged.