Red and Blue Light Promote the Accumulation of Artemisinin in Artemisia Annua L.

Artemisinin, which has been isolated from Artemisiaannua L., is the most effective antimalarial drug and has saved millions of lives. In addition, artemisinin and its derivatives have anti-tumor, anti-parasitic, anti-fibrosis, and anti-arrhythmic properties, which enhances the demand for these compounds. Improving the content of artemisinin in A.annua is therefore becoming an increasing research interest, as the chemical synthesis of this metabolite is not viable. Ultraviolet B and C irradiation have been reported to improve the artemisinin content in A.annua, but they are harmful to plant growth and development. Therefore, we screened other light sources to examine if they could promote artemisinin content without affecting plant growth and development. We found that red and blue light could enhance artemisinin accumulation by promoting the expression of the genes that were involved in artemisinin biosynthesis, such as amorpha-4,11-diene synthase (ADS) and cytochrome P450 monooxygenase (CYP71AV1) genes. Thus, in addition to being the main light sources for photosynthesis, red and blue light play a key role in plant secondary metabolism, and optimizing the combination of these light might allow for the productionof artemisinin-rich A.annua.

Cloning and characterization of DELLA genes in Artemisia annua.

Gibberellins (GA) are some of the most important phytohormones involved in plant development. DELLA proteins are negative regulators of GA signaling in many plants. In this study, the full-length cDNA sequences of three DELLA genes were cloned from Artemisia annua. Phylogenetic analysis revealed that AaDELLA1 and AaDELLA2 were located in the same cluster, but AaDELLA3 was not. Subcellular localization analysis suggested that AaDELLAs can be targeted to the nucleus and/or cytoplasm. Real-time PCR indicated that all three AaDELLA genes exhibited the highest expression in seeds. Expression of all AaDELLA genes was enhanced by exogenous MeJA treatment but inhibited by GA3 treatment. Yeast two-hybrid assay showed that AaDELLAs could interact with basic helix-loop-helix transcription factor AaMYC2, suggesting that GA and JA signaling may be involved in cross-talk via DELLA and MYC2 interaction in A. annua.

The activity of the artemisinic aldehyde Δ11(13) reductase promoter is important for artemisinin yield in different chemotypes of Artemisia annua L.

The artemisinic aldehyde double bond reductase (DBR2) plays an important role in the biosynthesis of the antimalarial artemisinin in Artemisia annua. Artemisinic aldehyde is reduced into dihydroartemisinic aldehyde by DBR2. Artemisinic aldehyde can also be oxidized by amorpha-4,11-diene 12-hydroxylase and/or aldehyde dehydrogenase 1 to artemisinic acid, a precursor of arteannuin B. In order to better understand the effects of DBR2 expression on the flow of artemisinic aldehyde into either artemisinin or arteannuin B, we determined the content of dihydroartemisinic aldehyde, artemisinin, artemisinic acid and arteannuin B content of A. annua varieties sorted into two chemotypes. The high artemisinin producers (HAPs), which includes the '2/39', 'Chongqing' and 'Anamed' varieties, produce more artemisinin than arteannuin B; the low artemisinin producers (LAPs), which include the 'Meise', 'Iran#8', 'Iran#14', 'Iran#24' and 'Iran#47' varieties, produce more arteannuin B than artemisinin. Quantitative PCR showed that the relative expression of DBR2 was significantly higher in the HAP varieties. We cloned and sequenced the promoter of the DBR2 gene from varieties of both the LAP and the HAP groups. There were deletions/insertions in the region just upstream of the ATG start codon in the LAP varities, which might be the reason for the different promoter activities of the HAP and LAP varieties. The relevance of promoter variation, DBR2 expression levels and artemisinin biosynthesis capabilities are discussed and a selection method for HAP varieties with a DNA marker is suggested. Furthermore, putative cis-acting regulatory elements differ between the HAP and LAP varieties.

Variations in antimalarial components of Artemisia annua Linn from three regions of Uganda.

INTRODUCTION: Artemisia annua plant from the family Asteracea is a powerful antimalarial plant introduced to Uganda around 2003. In addition to the artemisinin component, the plant also contains flavonoids which work in synergy to artemisinin against malaria parasites. The plant also contains aromatic oils which repel mosquitoes. In this paper we report the variations in antimalarial components of A. annua samples from the regions cultivating it in Uganda. METHODS: Artemisia annua samples were obtained from three regions that cultivated the plant at the time of this study. The samples were brought to laboratory, authenticated and processed. The levels of artemisinin, total flavonoids and aromatic components were quantified using high performance thin layer chromatography, ultra violet spectrophotometry and gas chromatography respectively. RESULTS: Artemisinin and total flavonoids levels were higher in samples obtained from high land areas (western and south western region) compared to that obtained from lowland regions (central) i.e 0.8% Vs 0.4% and 2.6% Vs 1.5% respectively. The aromatic oils (mosquito repellent components) were similar with camphor component being highest and levels ranging from 75.4% to 79.0%. CONCLUSION: Our findings show that the active components in Artemisia annua cultivated and used in the Uganda vary with geographical regions and this calls for standardisation by source.

[Genetic structure and genetic diversity of Artemisia annua varieties (strains) populations based on SCoT markers].

To reveal the genetic diversity and genetic structure in Artemisia annua varieties (strains) populations, we detected the genetic polymorphism within and among eight varieties (strains) populations (192 individuals) by the approach of Start Codon Targeted Polymorphism (SCoT). The associated genetic parameters were calculated by POPGENE1.31 and the relationship was constructed based on UPGMA method. The results showed that, using 20 screened primers, a total of 145 bands were produced, of which 122 were polymorphic loci. At species level, there was a high level of genetic diversity among eight varieties (strains) populations (PPB = 84.1% ,H = 0.217 3 and H(sp) = 0.341 9). However, at the variety (strains) population level, genetic diversity was lower, the average of genetic parameters was PPB = 41.9%, H = 0.121 5, H(pop) = 0.186 8. The Nei's genetic differentiation coefficient was 0.441 0, indicate that most of the genetic variation in this species existed within the variety populations. The gene flow (N(m) = 0.633 9) was less among populations, indicating that the degree of genetic differentiation was higher. Genetic similarity coefficient were changed from 0.755 1 to 0.985 7. By clustering analysis, eight varieties (strains) were clustered into two major categories and it was also showed the same or similar genetic background varieties (strains) have a tendency to gather in the same group. Results suggest that, in variety breeding, breeders should strengthen the exchange of bred germplasm and increase mutual penetration of excellent genes, which would broaden the genetic base of A. annua.

Fast and reliable artemisinin determination from different Artemisia annua leaves based alimentary products by high performance liquid chromatography-tandem mass spectrometry.

In many tropical countries malaria is endemic, causing acute illness and killing people, especially children. The availability of recommended malaria medicines is scant, even though these medicines are based on artemisinin, a compound extracted from the Artemisia annua plant that grows in many of these countries. New sources of treatment drawn from traditional medicine are therefore used, such as the tea infusion. An analytical method based on high-performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) was developed to quantify the artemisinin content of foods prepared with Artemisia annua leaves. A fast and reliable analytical method is described. The technique does not require any derivatisation prior to injection and offers excellent analytical intermediate precision. Robust qualitative and quantitative results were obtained using tea, biscuit or porridge specimens. Although further research is needed to define the potential therapeutic benefits of these alimentary formulations, the analytical method described can be employed in developing more convenient and appropriate foods for administering artemisinin to those infected with malaria.

Utilization of intron-flanking EST-specific markers in the genetic characterization of Artemisia annua genotypes from the trans-Himalayan region of Ladakh, India.

Genetic variation was assessed utilizing intron-flanking EST-specific markers among genotypes of Artemesia annua collected from two sampling sites viz. Nubra (9,600 ft) and Leh (11,500 ft) valleys of the trans-Himalayan region, Ladakh, India. The available ESTs (3,60,906) sequences of A. annua were aligned with the genomic sequences of Arabidopsis to developed 'intron-flanking' EST-PCR based primers. These primers anneal with the conserved region of exon (flanking to the intron) and amplified the introns. Out of the 39 primers selected and tested on 20 genotypes of A. annua, we successfully exploited 81 codominant intron length polymorphism (ILP) markers, with an average of 2.08 markers per primer and 92.04% polymorphism detection. Clustering of genotypes revealed distribution of genotypes into 2 distinct clusters with respect to their site of collection. Significantly, this study demonstrates that Arabidopsis genome sequence can be useful in developing gene-specific PCR-based markers for other non-model plant species like A. annua in the absence of genome sequences.

Volatile fingerprints of artemisinin-rich Artemisia annua cultivars by headspace solid-phase microextraction gas chromatography/ mass spectrometry.

The cultivar Anamed (A3) is a hybrid of Artemisia annua with a high content of the secondary metabolite artemisinin, a well-known antimalarial drug. Here we report for the first time the volatile profile of fresh leaves of this hybrid in comparison with that of Artemisia annua L. wild-type species. Evaluation and comparison of the volatile profiles of A. annua genotypes with different content in artemisinin were carried out by headspace solid-phase microextraction (HS-SPME) coupled with gas chromatography/mass spectrometry (GC/MS) that was performed on fresh leaves of the plants under investigation using a polydimethylsiloxane (PDMS) fiber. The chromatograms obtained from hybrids with a high content of artemisinin (A. annua cv. Anamed A3 and A. annua cv. Artemis F2) reveal the total absence of artemisia ketone, one of the major and characteristic compounds of the wild-type A. annua L., along with a significantly lower variety of volatile compounds. In conclusion, HS-SPME coupled with GC/MS is a very useful, non-destructive and efficient method to describe the volatile pattern of Artemisia annua cultivars. It represents a rapid screening method for the evaluation of volatile biomarkers like artemisia ketone, whose absence is typical of artemisinin-rich A. annua cultivars.

[Diversity and cluster analysis on agronomic traits of Artemisia annua germplasm resources in Yun-Gui plateau].

OBJECTIVE: To evaluate the diversity of germplasm resources of Artemisia annua and provide the basis for improving utilization of germplasm resources, the agronomic traits of germplasm resources of A. annua were studied in Yun-Gui plateau. METHOD: The agronomic traits of 67 A. annua germplasm resources were measured by the visual observation and measurement methods. And the germplasm resources were clustered using flexible-beta method to analysis their genetic background. RESULT: The result showed that 67 germplasm resources had a relatively wide variation on the 22 agronomic traits. Among 22 agronomic traits, the dry weight of branch had the greatest coefficient of variation, which was 53. 63, and the next were the dry weight of leaf, total plant weight, the length of pinnules and the length of leaflet, which were 42.74, 41.61, 39.54 and 39.22 respectively. The smallest coefficient of variation was the leaf corlor. Based the result of cluster analysis, these 67 germplasm resources were classed into 5 groups, and each group had its respective character. The first group showed early-maturing resources, dwarf stalk, slender rod, long bipinnata, high leaf-stem ratio and moderate leaf weight The third group showed late-maturing resources, tall and thick stalk, much-branch, bushy accessory pinna, high leaf weight and yield. The fifth group showed very late-maturing resources, strong lateral shoot, high leaf yield. CONCLUSION: There were significant genetic difference and diversity in the germplasm resources of A. annua. The result of cluster analysis showed that the resources of group 1, group 3 and group 5 were suitable as breeding material of A. annua.

[Studies on the chemical constituents of essential oil of Hunan Artemisia annua].

The chemical constituents of the essential oil of Hunan wild Artemisia annua L. in Xuefeng mountain region have been studied by means of the GC-MS. The essential oil was extracted from Artemisia annua L. by steam distillation. The chemical constituents were separated and identified by GC-MS, and the relative contents of them in the oil were determined with area normalization method. Forty-five components were separated and identified which accounted for 90% of total essential oil. These components identified from the essential oil indicate that Hunan wild Artemisia annua L. in Xuefeng mountain region is a good medical plant, and worth to exploiting.

[Determination of artemisinin in Artemisia annuna by HPLC-ELSD].

OBJECTIVE: A HPLC method was first established to determine artemisinin in Artemisia annuna by HPLC-ELSD. Artemisinin in Herba of Artemisia annuna from different places was determined by this new method. METHODS: The method utilized MeOH-H2O (75: 25) as mobile phase with flow rate of 1 ml/min, C18 (250 x 4.6 mm, 5 microm) column. The temperature of detector was 40 degrees C, the pressure of N2 was 3.5 bar, and gain value was 9. RESULTS: The calibration curves were linear within the range of 1 - 5 microg, and the average recovery was 99.33% (RSD = 1.97%). CONCLUSION: The method was rapid, convenient, and accurate. It can be used for the quality control of this herbal medicine.