High Artemisinin Content Research Articles

Cloning, E. coli Expression and Molecular Analysis of Amorpha‐4,11‐Diene Synthase from a High‐Yield Strain of Artemisia annua L.

AbstractIncreasing demand of artemisinin in the treatment of malaria has placed substantial stress on the total artemisinin supplies world‐wide, so more attention has been paid to increasing the content of artemisinin in the Artemisia annua L. plant. In this study, amorpha‐4,11‐diene synthase (ADS) cDNA (ads1) and genomics gene (gads1) were cloned from a high‐yield A. annua strain 001. The activity of ADS1 was confirmed by heterogeneous overexpression of ads1 and in vitro enzymatic incubation. Reverse transcript‐polymerase chain reaction results demonstrated that ads1 expressed in leaves, flowers and young stems, but not in roots. This organ‐specific expression pattern of ads1 is consistent with that of artemisinin accumulation in the plant. The gads1 has a complex organization including seven exons and six introns, and belongs to class III terpene synthase. DNA gel blotting revealed that the ADS gene has at least four copies in the genome of strain 001. The higher copy numbers might be one of the reasons for its high artemisinin content.(Managing editor: Wei Wang)

Effects of Overexpression of the Endogenous Farnesyl Diphosphate Synthase on the Artemisinin Content in Artemisia annua L.

AbstractArtemisinin is a novel effective antimalarial drug extracted from the medicinal plant Artemisia annua L. Owing to the tight market and low yield of artemisinin, there is great interest in enhancing the production of artemisinin. In the present study, farnesyl diphosphate synthase (FPS) was overexpressed in high‐yield A. annua to increase the artemisinin content. The FPS activity in transgenic A. annua was two‐ to threefold greater than that in non‐transgenic A. annua. The highest artemisinin content in transgenic A. annua was approximately 0.9% (dry weight), which was 34.4% higher than that in non‐transgenic A. annua. The results demonstrate the regulatory role of FPS in artemisinin biosynthesis.(Managing editor: Wei Wang)

ARTEMISININ PRODUCTION BY SHOOT CULTURE OF ARTEMISIA CINA BERG. EX POLJAKOV

Artemisinin is a secondary metabolite has a potential effect as an antimalaria. Research in artemisinin content in A. cina from shoot culture have not been reported. The aim of this research were:(i) to know the ability of A. cina shoot culture to produce artemisinin, (ii) to asses the effect of concentration of yeast extract, sucrose and their combination in promoting the production of artemisinin, (iii) to determine the highest artemisinin concentration produced by shoot culture in the medium containing different concentration of yeast extract, sucrose and their combination. The factorial of randomised complex block design (RCBD) was used in this experimental design. The first factor was concentration of sucrose ( 1%, 3%, 5%, 7%) and the second factor was concentration of yeast extract (0 mg/l, 100 mg/l, 200 mg/l, 300 mg/l). HPLC was used. To analyse quantitatively the artemisinin production, analysis of variance (ANOVA) and DMRT were used to analyse the data. The results showed that the addition of yeast extract and sucrose increased the concentration of artemisinin from the A. cina shoot culture. The highest artemisinin content produced was 14.035 mg/g dry weights which was produced by combination of 3 % sucrose concentration and 200 mg/l yeast extract. This result suggest that A. cina shoot culture could be used as a method to producing artemisinin. Key words : Artemisinin, shoot culture, Artemia cina Berg. Ex Poljakov

Exogenous GA3 and flowering induce the conversion of artemisinic acid to artemisinin in Artemisia annua plants

The contents of artemisinin and artemisinic acid were monitored in the Artemisia annua plants treated with GA3 at vegetative and flowering initiation stages. The highest artemisinin content was observed at full bloom. The decrease in artemisinic acid content occurred during the transition from the vegetative stage to the beginning of flowering. Endogenous GA3 content in the leaves peaked at full bloom. At the vegetative stage, in plants treated with various concentrations of GA3 , the content of artemisinin increased while that of artemisinic acid decreased. Apparently, the rate-limiting step in artemisinin biosynthesis was from artemisinic acid to artemisinin. The “bottleneck” of artemisinin biosynthesis was probably unlocked during the flowering or in the vegetative plants treated with GA3 , which triggered off the conversion of artemisinic acid to artemisinin.

The Effects of Different Plant Densities on Yield, Yield Components and Quality of Artemisia annua L. Ecotypes

ABSTRACT This study was conducted between 1997 and 1998 at Çukurova University, Faculty of Agriculture to determine the effects of different plant densities (5, 10, 15, 20, 25 and 30 plants/m2) on yield and yield components and quality of Artemisia annua L. ecotypes (Adana, Samankaya and Serinyol from Turkey). In the study, the plant height (cm), fresh weight (kg/da), dry weight (kg/da), dry leaf weight (kg/da), essential oil content (%), essential oil yield (l/da), artemisinin content (%), and artemisinin yield (kg/da) of ecotypes were investigated. The results of this study showed that the highest essential oil content and the highest essential oil yield were found in the Samankaya ecotype when planted at 15 plants/m2. However, the highest artemisinin content and highest artemisinin yield were found in the Serinyol ecotype when planted at 15 plants/m2.

The genetics of artemisinin content in Artemisia annua L. and the breeding of high yielding cultivars.

Artemisinin, the endoperoxide sesquiterpene lactone produced by the Chinese medicinal herb Artemisia annua, is very difficult to synthesise. Moreover, its production by mean of cell, tissue or organ cultures is very low. Presently, only its extraction from cultivated plants is viable. A large variation in artemisinin content has been observed in the leaves of plants from different origins. The genetic basis of this variation has been assessed and evidence for a quantitative inheritance of the artemisinin concentration presented. Additive genetic components were predominant, resulting in a high narrow-sense heritability estimate. Thus, goods results can be expected from mass selection for the breeding of lines of Artemisia annua rich in artemisinin. Yet, dominance variance is also present in the total genetic variability, indicating that crosses between selected genotypes should generate progenies with particularly high artemisinin content. As a matter of fact, selection and crossing, in wild populations, of genotypes with high artemisinin concentration resulted in hybrid lines containing up to 1.4 % artemisinin (on dry leaves basis).

TEST OF THE VIABILITY AND CONSERVATION OF THE POLLEN OF ARTEMISIA ANNUA L.

Artemisia annua L. synthesizes a molecule with very potent antimalarial properties: artemisinin. In order to improve the production of this valuable compound, breeding of strains with high artemisinin content are in progress. Such programs need the realisation of numerous hybridisations, sometime between genotypes whose flowering is difficult to synchronise. In this context, the possibility to conserve the pollen of the species would facilitate genetics studies and breeding programs. First, a medium has been developed to test the viability of the pollen. Efficient and rapid germination of the pollen grains can be achieved on a medium composed of 7 g/l agar, 300 g/l saccharose and 50 g/l boric acid in distilled water. Secondly, for conservation, good results have been obtained by storing the pollen grains at -25°C in 35 % relative humidity.

Isolation of high artemisinin-yielding clones of Artemisia annua

Abstract A random collection of 700 plants of a seed-raised population of Artemisia annua cv. Asha were screened for morphology and artemisinin and essential oil contents. Four morphologically distinct plant types were detected: short and early flowering, tall and early flowering, tall and late flowering, and dwarf and very late flowering. The artemisinin and essential oil were largely present in the inflorescence of all the types of plants. The artemisinin and the essential oil content in the dried herb (inflorescence + leaves) of these plant types ranged from 0.001 to 0.11% and 0.14 to 0.60%, respectively; the late flowering plants were generally richer in both artemisinin and the essential oil. Eleven individual adult plants were selected on the basis of their high artemisinin yield and in vitro regeneration response to a micropropagation procedure using young inflorescence segments as explants. The micro-cloned progenies of the selected plants were tested for their growth, morphology and artemisinin yield and homology in respect to these characters with the respective parent selections. The parent-micropropagated progeny characteristics were observed to be largely congruent. The artemisinin profiles of the micropropagated progenies at vegetative, preflowering and full bloom stages indicated that while highest artemisinin accumulation occurred at full bloom stage, the artemisinin content at preflowering stage was positively correlated with that at full bloom stage. Among the material cloned in vitro and tested in the field, clone numbers 166 and 187 yield 3.2- and 2.5-fold more artemisinin per plant as compared to the parent cultivar Asha, the former due to high herb yield and latter due to high artemisinin content per se.

Artemisinin, Related Sesquiterpenes, and Essential Oil inArtemisia annuaDuring a Vegetation Period in Vietnam

The active principle of ARTEMISIA ANNUA L., artemisinin, is currently being developed to a registered antimalarial drug. For production purposes, plants with a high artemisinin content are required. We followed the development of the artemisinin content and of the biosynthetically related sesquiterpenes artemisinic acid, arteannuin B, and artemisitene in A. ANNUA plants, during a vegetation period in Vietnam, where this species is indigenous. In addition, the essential oil content and composition were studied. Samples of leaves, buds, flowers, or post-bloom flowers and fruits were taken at different stages: vegetative (5, 6, and 8 months old), at mass formation of buds (9 months), at full bloom (10 months), and post-bloom (10S months). The highest artemisinin content (0.86% dry wt) was present in the leaves of 5 months-old plants. At this stage also the highest leaf yield was found. Subsequently, the artemisinin content gradually dropped. At the age of 5 months the highest artemisinic acid and arteannuin B contents, 0.16 and 0.08% dry wt, respectively, were found as well. Artemisitene was present at all stages of development, ranging from 0.002 to 0.09% dry wt. With 1.9% v/w, the essential oil content was maximal just before flowering and was composed of 55% monoterpenes and 45% sesquiterpenes. At all other stages (0.4 - 1.0% v/w oil) this ratio was ca. 30%/70%. The main components of the oil were camphor and germacrene-D.

Germplasm Variation in Artemisinin Content of Artemism annua Using an Alternative Method of Artemisinin Analysis from Crude Plant Extracts

Artemisinin content was evaluated in a germplasm collection of Artemisia annua using an HPLC-EC detector with a glassy carbon electrode. The highest artemisinin content (41.7%) was found in the leaves from the top 50-cm portion of the plant