Dynamic Optimization and Non-linear Model Predictive Control to Achieve Targeted Particle Morphologies.

An event-driven approach based on dynamic optimization and nonlinear model predictive control (NMPC) is investigated together with inline Raman spectroscopy for process monitoring and control. The benefits and challenges in polymerization and morphology monitoring are presented, and an overview of the used mechanistic models and the details of the dynamic optimization and NMPC approach to achieve the relevant process objectives are provided. Finally, the implementation of the approach is discussed, and results from experiments in lab and pilot-plant reactors are presented.

Cellular engineering of Artemisia annua and Artemisia dubia with the rol ABC genes for enhanced production of potent anti-malarial drug artemisinin.

BACKGROUND: Malaria is causing more than half of a million deaths and 214 million clinical cases annually. Despite tremendous efforts for the control of malaria, the global morbidity and mortality have not been significantly changed in the last 50 years. Artemisinin, extracted from the medicinal plant Artemisia sp. is an effective anti-malarial drug. In 2015, elucidation of the effectiveness of artemisinin as a potent anti-malarial drug was acknowledged with a Nobel prize. Owing to the tight market and low yield of artemisinin, an economical way to increase its production is to increase its content in Artemisia sp. through different biotechnological approaches including genetic transformation. METHODS: Artemisia annua and Artemisia dubia were transformed with rol ABC genes through Agrobacterium tumefacienes and Agrobacterium rhizogenes methods. The artemisinin content was analysed and compared between transformed and untransformed plants with the help of LC-MS/MS. Expression of key genes [Cytochrome P450 (CYP71AV1), aldehyde dehydrogenase 1 (ALDH1), amorpha-4, 11 diene synthase (ADS)] in the biosynthetic pathway of artemisinin and gene for trichome development and sesquiterpenoid biosynthetic (TFAR1) were measured using Quantitative real time PCR (qRT-PCR). Trichome density was analysed using confocal microscope. RESULTS: Artemisinin content was significantly increased in transformed material of both Artemisia species when compared to un-transformed plants. The artemisinin content within leaves of transformed lines was increased by a factor of nine, indicating that the plant is capable of synthesizing much higher amounts than has been achieved so far through traditional breeding. Expression of all artemisinin biosynthesis genes was significantly increased, although variation between the genes was observed. CYP71AV1 and ALDH1 expression levels were higher than that of ADS. Levels of the TFAR1 expression were also increased in all transgenic lines. Trichome density was also significantly increased in the leaves of transformed plants, but no trichomes were found in control roots or transformed roots. The detection of significantly raised levels of expression of the genes involved in artemisinin biosynthesis in transformed roots correlated with the production of significant amounts of artemisinin in these tissues. This suggests that synthesis is occurring in tissues other than the trichomes, which contradicts previous theories. CONCLUSION: Transformation of Artemisia sp. with rol ABC genes can lead to the increased production of artemisinin, which will help to meet the increasing demand of artemisinin because of its diverse pharmacological and anti-malarial importance.

Multivariate data analysis and metabolic profiling of artemisinin and related compounds in high yielding varieties of Artemisia annua field-grown in Madagascar.

An improved liquid chromatography-tandem mass spectrometry (LC-MS/MS) protocol for rapid analysis of co-metabolites of A. annua in raw extracts was developed and extensively characterized. The new method was used to analyse metabolic profiles of 13 varieties of A. annua from an in-field growth programme in Madagascar. Several multivariate data analysis techniques consistently show the association of artemisinin with dihydroartemisinic acid. These data support the hypothesis of dihydroartemisinic acid being the late stage precursor to artemisinin in its biosynthetic pathway.

Comparative cytotoxicity of artemisinin and cisplatin and their interactions with chlorogenic acids in MCF7 breast cancer cells.

In parts of Africa and Asia, self-medication with a hot water infusion of Artemisia annua (Artemisia tea) is a common practice for a number of ailments including malaria and cancer. In our earlier work, such an extract showed better potency than artemisinin alone against both chloroquine-sensitive and -resistant parasites. In this study, in vitro tests of the infusion in MCF7 cells showed high IC50 values (>200 µM). The combination of artemisinin and 3-caffeoylquinic acid (3CA), two major components in the extract, was strongly antagonistic and gave a near total loss of cytotoxicity for artemisinin. We observed that the interaction of 3CAs with another cytotoxic compound, cisplatin, showed potentiation of activity by 2.5-fold. The chelation of cellular iron by 3CA is hypothesized as a possible explanation for the loss of artemisinin activity.

The effect of O-methylated flavonoids and other co-metabolites on the crystallization and purification of artemisinin.

Methoxylated flavonoids casticin, artemetin and retusin were identified as putative causative factors for low crystallization yields of artemisinin from extracts. Comparative profiling of biomass grown in different countries found elevated levels (∼60% higher) of artemetin in the East African biomass, which also demonstrates poor crystallization yields. The single compound and the combined doping experiments at 0, 25 and 50 µg mL⁻¹ doping levels showed that artemetin (50 µg mL⁻¹) caused a reduction in the amount of artemisinin crystallized by ca. 60%. A combination of the three flavonoids at 50 µg mL⁻¹ almost completely inhibited crystallization, reducing the yield by 98%. Treatment of extracts by adsorbents efficiently resolves the problem of low crystallization yield.

Anti-plasmodial polyvalent interactions in Artemisia annua L. aqueous extract--possible synergistic and resistance mechanisms.

Artemisia annua hot water infusion (tea) has been used in in vitro experiments against P. falciparum malaria parasites to test potency relative to equivalent pure artemisinin. High performance liquid chromatography (HPLC) and mass spectrometric analyses were employed to determine the metabolite profile of tea including the concentrations of artemisinin (47.5±0.8 mg L(-1)), dihydroartemisinic acid (70.0±0.3 mg L(-1)), arteannuin B (1.3±0.0 mg L(-1)), isovitexin (105.0±7.2 mg L(-1)) and a range of polyphenolic acids. The tea extract, purified compounds from the extract, and the combination of artemisinin with the purified compounds were tested against chloroquine sensitive and chloroquine resistant strains of P. falciparum using the DNA-intercalative SYBR Green I assay. The results of these in vitro tests and of isobologram analyses of combination effects showed mild to strong antagonistic interactions between artemisinin and the compounds (9-epi-artemisinin and artemisitene) extracted from A. annua with significant (IC50 <1 µM) anti-plasmodial activities for the combination range evaluated. Mono-caffeoylquinic acids, tri-caffeoylquinic acid, artemisinic acid and arteannuin B showed additive interaction while rosmarinic acid showed synergistic interaction with artemisinin in the chloroquine sensitive strain at a combination ratio of 1:3 (artemisinin to purified compound). In the chloroquine resistant parasite, using the same ratio, these compounds strongly antagonised artemisinin anti-plasmodial activity with the exception of arteannuin B, which was synergistic. This result would suggest a mechanism targeting parasite resistance defenses for arteannuin B's potentiation of artemisinin.

A rapid method for the determination of artemisinin and its biosynthetic precursors in Artemisia annua L. crude extracts.

A rapid high-pressure liquid chromatography (HPLC) tandem mass spectrometry (TQD) method for the determination of artemisinin, 9-epi-artemisinin, artemisitene, dihydroartemisinic acid, artemisinic acid and arteannuin B in Artemisia annua extracts is described. Detection and quantification of 9-epi-artemisinin in crude extracts are reported for the first time. In this method all six metabolites are resolved and eluted within 6 min with minimal sample preparation. A recovery of between 96.25% and 103.59% was obtained for all metabolites analysed and the standard curves were linear (r(2)>0.99) over the concentration range of 0.15-10 µg mL(-1) for artemisinin, 9-epi-artemisinin, artemisitene and arteannuin B, and the range of 3.75-120 µg mL(-1) for dihydroartemisinic acid and artemisinic acid. All validation indices were satisfactory, showing the method to be robust, quick, sensitive and adequate for a range of applications including high throughput (HTP) analysis.