Influence of spacing and drying methods on concentration of artemisinin in artemisia annua

Abstract

Artemisia annua L. from the family Asteraceae is an annual medicinal plant and has been used to make herbal remedies in Asia for thousands of years. Artemisinin is a sesquiterpene lactone, isolated from aerial parts of Artemisia annua, with the highest concentrations being in flowers and leaves. In addition to potent anti-malarial activity, artemisinin possesses anti-cancer, anti-schistosomiatic, anti-hepatitis B, anti-HIV, anti-leishmanial and herbicidal activities. Low artemisinin production (0.01-2%) from A. annua is a major constraint in commercialisation of the drug for control of malaria. Worldwide, efforts have been underway to improve the concentration of artemisinin using conventional breeding, biochemical, physiological, molecular and hairy-root culture techniques, however all these methods are not economical. Cultural practices like spacing and pruning have limitation in improving artemisinin concentration and these may help in increasing the concentrations of artemisinin. Study was conducted at the experimental farm of the Agricultural Research Council – Vegetable and Ornamental Plants, Roodeplaat Pretoria. The objective of this study was to determine whether spacing, pruning and their interactions would have any effect on the concentrations of artemisinin, growth and yield of A. annua and whether drying methods would have an effect on the concentrations of artemisinin in A. annua. Since there was only one field trial, all sub-objectives were addressed at once (Chapter 3). Fresh seeds of A. annua were obtained from the ARC-VOP gene bank and sown in seedling trays in September 2014. Uniform eight-week-old seedlings were hardened-off, transplanted in November 2014 in 10 cm deep holes and then pruned ten weeks after transplanting. Treatments for Experiment 1, viz., 3 × 4 factorial experiment were laid out in a randomised complete block design, with four replications (n = 48). The two factors of the experiment were (a) spacing [0.5 × 1 m2 (standard: 0.50 m2), 0.5 × 0.7 m2 (small: 0.35 m2) 0.5 × 0.5 m2 (smaller: 0.25 m2) and 0.3 × 0.7 m2 (smallest: 0.21 m2)] and (b) pruning [no pruning (control), removing the apical bud and removing shoots three nodes from the bottom]. The plants were irrigated using overhead sprinklers system for two hours three times per week. Four readings for growth variables (plant height, stem diameter and chlorophyll content) were collected with one week interval. Plants were harvested after 180 days from planting, and leaves, stems and roots were separated weighed and oven dried at 40 ºC for 72 h. In Experiment 2 (drying methods), treatments, namely, 100% sun, 100% shade, 50% shade, freeze and oven drying were arranged in completely randomised design with four replicates (n = 20). The treatments were exposed for a week, to full sunlight, 50% shade-drying under a shade net that allows 50% light penetration, 100% shade under enclosed room at ambient (24-25 ºC) temperature, oven drying for 24 h at 40 ºC, and freeze-drying for three days. Freeze-drying had significant effect on artemisinin concentration of 1.941%. It was followed by oven (1.738%) and 100% shade drying (1.657%) and the lowest artemisinin concentration (1.412%) was obtained from 50% shade drying. The smaller spacing of 0.25 m2 in combination with apical bud removal had a significant effect on artemisinin concentration, producing artemisinin concentration of 0.193%. Spacing had a significant effect on stem diameter, fresh leaf mass and dry leaf mass but had no effect on plant height and chlorophyll content. Pruning had a significant effect on plant height and chlorophyll content and had no effect on stem diameter. The small spacing of 0.35 m2 had the highest fresh and dry leaf mass of 17.99 and 9.62 t/ha. The interaction of spacing and pruning had no significant effect on the growth and yield of A. annua. The results from this study suggested that cultural and processing practices may have direct effects in the concentration of artemisinin, growth and yield of A. annua. The results xiv provided some understanding on how agronomic and processing practices can be used to increase artemisinin content in A. annua and understand the interaction between different agronomic practices and thereby allowing the development of economic methods for A. annua post-harvest handling. Future work should focus on implementing various pruning techniques to trigger stress and indirectly secondary metabolites