miR828a-CsMYB114 Module Negatively Regulates the Biosynthesis of Theobromine in Camellia sinensis.

Theobromine is an important quality component in tea plants (Camellia sinensis), which is produced from 7-methylxanthine by theobromine synthase (CsTbS), the key rate-limiting enzyme in theobromine biosynthetic pathway. Our transcriptomics and widely targeted metabolomics analyses suggested that CsMYB114 acted as a potential hub gene involved in the regulation of theobromine biosynthesis. The inhibition of CsMYB114 expression using antisense oligonucleotides (ASO) led to a 70.21% reduction of theobromine level in leaves of the tea plant, which verified the involvement of CsMYB114 in theobromine biosynthesis. Furthermore, we found that CsMYB114 was located in the nucleus of the cells and showed the characteristic of a transcription factor. The dual luciferase analysis, a yeast one-hybrid assay, and an electrophoretic mobility shift assay (EMSA) showed that CsMYB114 activated the transcription of CsTbS, through binding to CsTbS promoter. In addition, a microRNA, miR828a, was identified that directly cleaved the mRNA of CsMYB114. Therefore, we conclude that CsMYB114, as a transcription factor of CsTbS, promotes the production of theobromine, which is inhibited by miR828a through cleaving the mRNA of CsMYB114.

Comparative transcriptome analysis reveals key pathways and genes involved in trichome development in tea plant (Camellia sinensis).

Trichomes, which develop from epidermal cells, are considered one of the important characteristics of the tea plant [Camellia sinensis (L.) O. Kuntze]. Many nutritional and metabolomic studies have indicated the important contributions of trichomes to tea products quality. However, understanding the regulation of trichome formation at the molecular level remains elusive in tea plants. Herein, we present a genome-wide comparative transcriptome analysis between the hairless Chuyeqi (CYQ) with fewer trichomes and the hairy Budiaomao (BDM) with more trichomes tea plant genotypes, toward the identification of biological processes and functional gene activities that occur during trichome development. In the present study, trichomes in both cultivars CYQ and BDM were unicellular, unbranched, straight, and soft-structured. The density of trichomes was the highest in the bud and tender leaf periods. Further, using the high-throughput sequencing method, we identified 48,856 unigenes, of which 31,574 were differentially expressed. In an analysis of 208 differentially expressed genes (DEGs) encoding transcription factors (TFs), five may involve in trichome development. In addition, on the basis of the Gene Ontology (GO) annotation and the weighted gene co-expression network analysis (WGCNA) results, we screened several DEGs that may contribute to trichome growth, including 66 DEGs related to plant resistance genes (PRGs), 172 DEGs related to cell wall biosynthesis pathway, 29 DEGs related to cell cycle pathway, and 45 DEGs related to cytoskeleton biosynthesis. Collectively, this study provided high-quality RNA-seq information to improve our understanding of the molecular regulatory mechanism of trichome development and lay a foundation for additional trichome studies in tea plants.

Comparative Transcriptome Analysis of Agrobacterium tumefaciens Reveals the Molecular Basis for the Recalcitrant Genetic Transformation of Camellia sinensis L.

Tea (Camellia sinensis L.), an important economic crop, is recalcitrant to Agrobacterium-mediated transformation (AMT), which has seriously hindered the progress of molecular research on this species. The mechanisms leading to low efficiency of AMT in tea plants, related to the morphology, growth, and gene expression of Agrobacterium tumefaciens during tea-leaf explant infection, were compared to AMT of Nicotiana benthamiana leaves in the present work. Scanning electron microscopy (SEM) images showed that tea leaves induced significant morphological aberrations on bacterial cells and affected pathogen-plant attachment, the initial step of a successful AMT. RNA sequencing and transcriptomic analysis on Agrobacterium at 0, 3 and 4 days after leaf post-inoculation resulted in 762, 1923 and 1656 differentially expressed genes (DEGs) between the tea group and the tobacco group, respectively. The expressions of genes involved in bacterial fundamental metabolic processes, ATP-binding cassette (ABC) transporters, two-component systems (TCSs), secretion systems, and quorum sensing (QS) systems were severely affected in response to the tea-leaf phylloplane. Collectively, these results suggest that compounds in tea leaves, especially gamma-aminobutyrate (GABA) and catechins, interfered with plant-pathogen attachment, essential minerals (iron and potassium) acquisition, and quorum quenching (QQ) induction, which may have been major contributing factors to hinder AMT efficiency of the tea plant.

Transcriptomics analysis reveals the signal transduction mechanism of brassinolides in tea leaves and its regulation on the growth and development of Camellia sinensis.

BACKGROUND: Brassinosteroids (BRs) are a type of sterol plant hormone that play an important role in various biochemical and physiological reactions such as promoting cell growth, increasing biomass, and improving stress resistance. RESULTS: To investigate the regulatory and molecular mechanism of BRs on the growth and development of tea plants (Camellia sinensis L.), changes in cell structure and gene expression levels of tea leaves treated with exogenous BRs were analyzed by electron microscopy and high-throughput Illumina RNA-Seq technology. The results showed that the number of starch granules in the chloroplasts and lipid globules increased and thylakoids expanded after BR treatment compared with the control. Transcriptome analysis showed that in the four BR treatments (CAA: BR treatment for 3 h, CAB: BR treatment for 9 h, CAC: BR treatment for 24 h, and CAD: BR treatment for 48 h), 3861 (1867 upregulated and 1994 downregulated), 5030 (2461 upregulated and 2569 downregulated), 1626 (815 upregulated and 811 downregulated), and 2050 (1004 upregulated and 1046 downregulated) differentially expressed genes were detected, respectively, compared with CAK (BR treatment for 0 h). Using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, metabolic pathway enrichment analysis showed that the differentially expressed genes of CAA vs. CAK, CAB vs. CAK, CAC vs. CAK, and CAD vs. CAK significantly enriched the functional categories of signal transduction, cell cycle regulation, and starch, sucrose, and flavonoid biosynthesis and metabolism pathways. We also found that after spraying BR, the key genes for caffeine synthesis were downregulated. The results of qRT-PCR coincided with the findings of transcriptomic analysis. CONCLUSIONS: The present study improved our understanding of the effects of BRs on the growth and development of tea leaves and laid the foundation for the in-depth analysis of signal transduction pathways of BRs in tea leaves.

Development and Application of a Fast Gas Chromatographic Method Offer New Insights into l-theanine Production Regulation in Camellia sinensis L.

Tea is the most consumed beverage worldwide, and l-theanine in tea leaves significantly affects their flavor and market quality. We have developed and validated a fast and reliable gas chromatographic method with flame ionization detection (GC-FID) to quantify l-theanine after its extraction from Camellia sinensis (tea plant) and derivatization. The procedure was completed in 40 min, from extraction to chromatographic analysis, with a recovery rate of more than 93% and allowing a high sample throughput. The GC-FID intraday precision was within 0.57-2.28%, while the interday precision ranged from 1.57 to 13.48%. The intraday accuracy ranged from -6.84 to 5.26%, while the interday accuracy ranged from -1.08 to 3.12%. The limit of detection was 2.28 µg/mL, and the limit of quantification was 6.47 µg/mL. The GC-FID method was validated by high-performance liquid chromatography with UV detection (HPLC-UV) and was used to investigate the biosynthesis and regulation of l-theanine in tea plants. We found that plants fed with ethylamine significantly increased l-theanine concentrations in roots, while exogenous supplementation of glutamic acid, carbamide, and glutamine did not significantly affect the l-theanine level in roots. Our results also indicated that roots were not indispensable for the biosynthesis of l-theanine, which was detected in undifferentiated embryonic calluses in concentrations (g/100 g dry weight) as high as in leaves of whole plants (1.67 and 1.57%, respectively) and without any exogenous theanine precursor supplementation.

Seasonal and Differential Sesquiterpene Accumulation in Artemisia annua Suggest Selection Based on Both Artemisinin and Dihydroartemisinic Acid may Increase Artemisinin in planta.

Commercial Artemisia annua crops are the sole source of artemisinin (ART) worldwide. Data on seasonal accumulation and peak of sesquiterpenes, especially ART in commercial A. annua, is lacking while current breeding programs focus only on ART and plant biomass, but ignores dihydroartemisinic acid (DHAA) and artemisinic acid (AA). Despite past breeding successes, plants richer in ART are needed to decrease prices of artemisinin-combination therapy (ACT). Our results show that sesquiterpene concentrations vary greatly along the growing season and that sesquiterpene profiles differ widely among chemotypes. Field studies with elite Brazilian, Chinese, and Swiss germplasms established that ART peaked in vegetative plants from late August to early September, suggesting that ART is related to the photoperiod, not flowering. DHAA peaks with ART in Chinese and Swiss plants, but decreases, as ART increases, in Brazilian plants, while AA remained stable through the season in these genotypes. Chinese plants peaked at 0.9% ART, 1.6% DHAA; Brazilian plants at 0.9% ART, with less than 0.4% DHAA; Swiss plants at 0.8% ART and 1% DHAA. At single-date harvests, seeded Swiss plants produced 0.55-1.2% ART, with plants being higher in DHAA than ART; Brazilian plants produced 0.33-1.5% ART, with most having higher ART than DHAA. Elite germplasms produced from 0.02-0.43% AA, except Sandeman-UK (0.4-1.1% AA). Our data suggest that different chemotypes, high in ART and DHAA, have complementary pathways, while competing with AA. Crossing plants high in ART and DHAA may generate hybrids with higher ART than currently available in commercial germplasms. Selecting for high ART and DHAA (and low AA) can be a valuable approach for future selection and breeding to produce plants more efficient in transforming DHAA into ART in planta and during post-harvest. This novel approach could change the breeding focus of A. annua and other pharmaceutical species that produce more than one desired metabolite in the same pathway. Obtaining natural variants with high ART content will empower countries and farmers who select, improve, and cultivate A. annua as a commercial pharmaceutical crop. This selection approach could enable ART to be produced locally where it is most needed to fight malaria and other parasitic neglected diseases.

Preparative Separation of High-Purity Dihydroartemisinic Acid from Artemisinin Production Waste by Combined Chromatography.

In order to make full use of artemisinin production waste and thus to reduce the production cost of artemisinin, we developed an efficient and scalable method to isolate high-purity dihydroartemisinic acid from artemisinin production waste by combining anion-exchange resin with silica-gel column chromatography. The adsorption and desorption characteristics of dihydroartemisinic acid on 10 types of anion-exchange resin were investigated, and the results showed that the 717 anion-exchange resin exhibited the highest capacity of adsorption and desorption to dihydroartemisinic acid. Adsorption isotherms were established for the 717 anion-exchange resin and they fitted well with both Langmuir and Freundlich model. Dynamic adsorption and desorption properties of 717 anion-exchange resin were characterized to optimize the chromatographic conditions. Subsequently, the silica-gel column chromatography was performed and dihydroartemisinic acid with a purity of up to 98% (w/w) was obtained. Finally, the scale-up experiments validated the preparative separation of high-purity dihydroartemisinic acid from industrial waste developed in the present work. This work presented for the first time an isolation of dihydroartemisinic acid with a purity of 98% from Artemisia annua (A. annua) by-product, which adds more value to this crop and has the potential to lower the prices of anti-malarial drugs.

Isolation of Dihydroartemisinic Acid from Artemisia annua L. By-Product by Combining Ultrasound-Assisted Extraction with Response Surface Methodology.

Malaria is the most devastating parasitic disease worldwide. Artemisinin is the only drug that can cure malaria that is resistant to quinine-derived drugs. After the commercial extraction of artemisinin from Artemisia annua, the recovery of dihydroartemisinic acid (DHAA) from artemisinin extraction by-product has the potential to increase artemisinin commercial yield. Here we describe the development and optimization of an ultrasound-assisted alkaline procedure for the extraction of DHAA from artemisinin production waste using response surface methodology. Our results using this methodology established that NaOH at 0.36%, extraction time of 67.96 min, liquid-solid ratio of 5.89, and ultrasonic power of 83.9 W were the optimal conditions to extract DHAA from artemisinin production waste. Under these optimal conditions, we achieved a DHAA yield of 2.7%. Finally, we conducted a validation experiment, and the results confirmed the prediction generated by the regression model developed in this study. This work provides a novel way to increase the production of artemisinin per cultivated area and to reduce artemisinin production costs by recycling its commercial waste to obtain DHAA, an immediate precursor of artemisinin. The use of this technology may reduce the costs of artemisinin-based antimalarial medicines.

Determination of dihydroartemisinic acid in Artemisia annua L. by gas chromatography with flame ionization detection.

Dihydroartemisinic acid (DHAA) is the direct precursor to artemisinin, an effective anti-malaria compound from Artemisia annua L. (A. annua), and it can be transformed to artemisinin without the catalysis of enzyme. A rapid and sensitive analysis of DHAA in A. annua is needed to screen excellent plant resources aimed to improve artemisinin production. In order to develop a rapid and sensitive determination method for DHAA in plant, the extraction and analysis conditions were extensively investigated in the present work. As a result, extraction of powdered A. annua leaves at 55°C for 50 min with chloroform resulted in the highest yield of DHAA, with a recovery of >98%. The precision of this gas chromatographic procedure ranged from 1.22 to 2.94% for intra-day and from 1.69 to 4.31% for inter-day, respectively. The accuracy was 99.55-103.02% for intra-day and 98.86-99.98% for inter-day, respectively. The measured LOQ and LOD values of the proposed method reached 5.00 and 2.00 µg/mL, respectively. Validation indicated the method was robust, quick, sensitive and adequate for DHAA analysis.

Molecular cloning and characterization of a flavanone 3-Hydroxylase gene from Artemisia annua L.

Flavonoids were found to synergize anti-malaria and anti-cancer compounds in Artemisia annua, a very important economic crop in China. In order to discover the regulation mechanism of flavonoids in Artemisia annua, the full length cDNA of flavanone 3-hydroxylase (F3H) were isolated from Artemisia annua for the first time by using RACE (rapid amplification of cDNA ends). The completed open read frame of AaF3H was 1095 bp and it encoded a 364-amino acid protein with a predicted molecular mass of 41.18 kDa and a pI of 5.67. The recombinant protein of AaF3H was expressed in E. coli BL21(DE3) as His-tagged protein, purified by Ni-NTA agrose affinity chromatography, and functionally characterized in vitro. The results showed that the His-tagged protein (AaF3H) catalyzed naringenin to dihydrokaempferol in the present of Fe(2+). The Km for naringenin was 218.03 µM. The optimum pH for AaF3H reaction was determined to be pH 8.5, and the optimum temperature was determined to be 35 °C. The AaF3H transcripts were found to be accumulated in the cultivar with higher level of flavonoids than that with lower level of flavonoids, which implied that AaF3H was a potential target for regulation of flavonoids biosynthesis in Artemisia annua through metabolic engineering.

WITHDRAWN: Identification of an antimycin gene cluster and characterization of the tryptophan 2,3-dioxygenase from the deep sea-derived Streptomyces somaliensis HND1201.

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A Comparative Proteomic Analysis of the Buds and the Young Expanding Leaves of the Tea Plant (Camellia sinensis L.).

Tea (Camellia sinensis L.) is a perennial woody plant that is widely cultivated to produce a popular non-alcoholic beverage; this beverage has received much attention due to its pleasant flavor and bioactive ingredients, particularly several important secondary metabolites. Due to the significant changes in the metabolite contents of the buds and the young expanding leaves of tea plants, high-performance liquid chromatography (HPLC) analysis and isobaric tags for relative and absolute quantitation (iTRAQ) analysis were performed. A total of 233 differentially expressed proteins were identified. Among these, 116 proteins were up-regulated and 117 proteins were down-regulated in the young expanding leaves compared with the buds. A large array of diverse functions was revealed, including roles in energy and carbohydrate metabolism, secondary metabolite metabolism, nucleic acid and protein metabolism, and photosynthesis- and defense-related processes. These results suggest that polyphenol biosynthesis- and photosynthesis-related proteins regulate the secondary metabolite content of tea plants. The energy and antioxidant metabolism-related proteins may promote tea leaf development. However, reverse transcription quantitative real-time PCR (RT-qPCR) showed that the protein expression levels were not well correlated with the gene expression levels. These findings improve our understanding of the molecular mechanism of the changes in the metabolite content of the buds and the young expanding leaves of tea plants.

Comparative profiling of gene expression in Camellia sinensis L. cultivar AnJiBaiCha leaves during periodic albinism.

The AnJiBaiCha albino mutant tea cultivar has a reversible albino phenotype at low temperatures. Albino AnJiBaiCha leaves contain high levels of amino acids, which are important components affecting the quality of tea as a beverage. To examine the molecular mechanism of albinism and amino acid enrichment in AnJiBaiCha, we used the amplified fragment length polymorphism (cDNA-AFLP) technique to isolate genes that are differentially expressed during periodic albinism in AnJiBaiCha. A total of 127 transcript-derived fragments (TDFs) were successfully sequenced; among those, 60 TDFs showed high similarity to sequences with known functions, but 67 TDFs were not similar to any known genes. The identified transcripts include transcription factors, ubiquitination-related genes, chloroplast biogenesis genes, signal transduction genes, stress-related genes, cell cycle genes, and carbohydrate and energy metabolism genes. To validate the cDNA-AFLP results, quantitative real-time PCR was used to confirm the differential expression of six of the identified genes. The cDNA-AFLP and quantitative real-time PCR results correlated well, indicating that the cDNA-AFLP results are reliable. This study provides insights into the molecular mechanisms by which periodic albinism and amino acid accumulation take place in AnJiBaiCha.

Metabolic analysis of the increased adventitious rooting mutant of Artemisia annua reveals a role for the plant monoterpene borneol in adventitious root formation.

Adventitious root (AR) formation is a critical process for plant clonal propagation. The role of plant secondary metabolites in AR formation is still poorly understood. Chemical and physical mutagenesis in combination with somatic variation were performed on Artemisia annua in order to obtain a mutant with changes in adventitious rooting and composition of plant secondary metabolites. Metabolic and morphological analyses of the iar (increased adventitious rooting) mutant coupled with in vitro assays were used to elucidate the relationship between plant secondary metabolites and AR formation. The only detected differences between the iar mutant and wild-type were rooting capacity and borneol/camphor content. Consistent with this, treatment with borneol in vitro promoted adventitious rooting in wild-type. The enhanced rooting did not continue upon removal of borneol. The iar mutant displayed no significant differences in AR formation upon treatment with camphor. Together, our results suggest that borneol promotes adventitious rooting whereas camphor has no effect on AR formation.

Dynamic changes in catechin levels and catechin biosynthesis-related gene expression in albino tea plants (Camellia sinensis L.).

Tea (Camellia sinensis (L.) O. Kuntze) leaves are a major source of flavonoids that mainly belong to the flavan-3-ols or catechins and are implicated in a wide range of health benefits. Although the catechins in tea leaves were identified long ago, the regulatory mechanisms governing catechin biosynthesis remain unclear. In the present work, the dynamic changes of catechin levels and the expression profiles of catechin-related genes in albino tea plants were intensively examined. The amounts of most catechins decreased to their lowest levels in the albino phase, when epigallocatechingallate was the highest of the catechins compared to all catechins, and catechin the lowest. Enzyme assays indicated that phenylalanine ammonia-lyase (PAL) activity was positively correlated with the concentration of catechins (r = 0.673). Gene expression profiling by quantitative real-time reverse transcription-polymerase chain reaction showed that the transcript abundance of flavonoid biosynthetic genes followed a tightly regulated biphasic pattern, and was affected by albinism. These genes (PAL, C4H, 4CL, CHS, CHI, F3H, FLS, F3'H, F3'5'H, DFR, LAR, ANS and ANR) encode enzymes in flavonoid biosynthesis. The expression levels of PAL, F3H and FLS were correlated with the concentration of catechins and the correlation coefficients were -0.683, 0.687 and -0.602, respectively. Therefore, these results indicate that PAL might be a core regulator in the control of catechin biosynthesis in albino tea plants.

Simultaneous isolation of artemisinin and its precursors from Artemisia annua L. by preparative RP-HPLC.

It is still a major challenge to simultaneously isolate artemisinin and its precursors, especially dihydroartemisinic acid and artemisinic acid, from herbal Artemisia annua. A rapid, economical and automatical chromatographic separation process to isolate and purify artemisinin, dihydroartemisinic acid and artemisinic acid at the same time on a preparative scale was developed. The procedure included solvent extraction of ground Artemisia annua leaves by refluxing and purification of crude extract by preparative reverse-phase high-performance liquid chromatography (RP-HPLC). Fractions containing artemisinin and its precursors were collected and identified by gas chromatography and mass spectrometry. High purity of artemisinin, dihydroartemisinic acid and artemisinic acid was obtained by preparative HPLC with a C(18) column and 60% acetonitrile in water as the mobile phase. The techniques described here are useful tools for the preparative-scale isolation of artemisinin and its precursors in a fast, cost-effective and environmental friendly manner.

Proteomic analysis of young leaves at three developmental stages in an albino tea cultivar.

BACKGROUND: White leaf No.1 is a typical albino tea cultivar grown in China and it has received increased attention in recent years due to the fact that white leaves containing a high level of amino acids, which are very important components affecting the quality of tea drink. According to the color of its leaves, the development of this tea cultivar is divided into three stages: the pre-albinistic stage, the albinistic stage and the regreening stage. To understand the intricate mechanism of periodic albinism, a comparative proteomic approach based on two-dimensional electrophoresis (2-DE) and mass spectrometry was adopted first time to identify proteins that changed in abundance during the three developmental periods. RESULTS: The 2-DE results showed that the expression level of 61 protein spots varied markedly during the three developmental stages. To analyze the functions of the significantly differentially expressed protein spots, 30 spots were excised from gels and analyzed by matrix-assisted laser desorption ionization-time of flight-tandem mass spectrometry. Of these, 26 spots were successfully identified. All identified protein spots were involved in metabolism of carbon, nitrogen and sulfur, photosynthesis, protein processing, stress defense and RNA processing, indicating these physiological processes may play crucial roles in the periodic albinism. Quantitative real-time RT-PCR analysis was used to assess the transcriptional level of differentially expressed proteins. In addition, the ultrastructural studies revealed that the etioplast-chloroplast transition in the leaf cell of White leaf No. 1 was inhibited and the grana in the chloroplast was destroyed at the albinistic stage. CONCLUSIONS: In this work, the proteomic analysis revealed that some proteins may have important roles in the molecular events involved in periodic albinism of White leaf No. 1 and identificated many attractive candidates for further investigation. In addition, the ultrastructural studies revealed that the change in leaf color of White leaf No. 1 might be a consequence of suppression of the etioplast-chloroplast transition and damage to grana in the chloroplast induced by temperature. These results provide much useful information to improve our understanding of the mechanism of albinism in the albino tea cultivar.

Isolation and identification of novel genes involved in artemisinin production from flowers of Artemisia annua using suppression subtractive hybridization and metabolite analysis.

Malaria is a global health problem that threatens 300-500 million people and kills more than one million people annually. Artemisinin is highly effective against multidrug-resistant Plasmodium falciparum and it has been widely used as part of the artemisinin-based combination therapies against malaria. To elucidate the biosynthetic pathway of artemisinin and to clone related genes in Artemisia annua, differentially expressed genes between blooming flowers and flower buds were isolated and characterized by a combined approach of suppression subtractive hybridization (SSH) and metabolite analysis. A total of 350 cDNA clones from a subtractive cDNA library were randomly picked, sequenced and analyzed and 253 high-quality sequences were obtained. BLASTX comparisons indicated that about 9.9 % of the clones encoded enzymes involved in isoprenoid (including artemisinin) biosynthesis. The expression of 4 gene transcripts involved in artemisinin biosynthesis was examined by RT-PCR and the results confirmed the higher expression of these transcripts in blooming flowers than in flower buds. In addition, 2 putative transcript factors transparenta testa glabra 1 (TTG1) and ENHANCER OF GLABRA3 (GL3), which promote trichome initiation, were presented in the library. Finally, this study demonstrated that the increase of expression level of the putative TTG1 gene correlated with the improvement of glandular trichome density and artemisinin production in A. annua leaves. The subtractive cDNA library described in the present study provides important candidate genes for future research in order to increase the artemisinin content in A. annua.

Simple and rapid micro-scale quantification of artemisinin in living Artemisia annua L. by improved gas chromatography with electron-capture detection.

Malaria threatens 300-500 million people and kills more than one million people annually. Artemisinin has been widely used as part of the artemisinin-based combination therapies against malaria. However, its supply is seriously short due to very small amounts of production of artemisinin in Artemisia annua. Molecular biologic researches aimed at increasing the artemisinin yield in plant have received more and more attention and therefore corresponding quantification methods for artemisinin analysis are urgently needed. A variety of methods for determination of artemisinin have been developed but they cannot be applied when only very little plant material is available or the material should be kept live, which often occurs in molecular biologic researches. The present work developed a simple, fast and low toxic micro-scale analysis procedure for determination of artemisinin in a single leaf or flower of living Artemisia annua using improved gas chromatography with electron-capture detection. The recovery of >95% was achieved by vortex of a piece of fresh leaf in 1 mL ethyl acetate for 2 min at room temperature. This method provides a powerful tool for biosynthesis study of artemisnin, high-throughput screening high-yield clone in an early stage, or real-time quality control of Artemisia annua crop.

Affordable and sensitive determination of artemisinin in Artemisia annua L. by gas chromatography with electron-capture detection.

Artemisinin demand has increased sharply since the World Health Organization recommended its use as part of the artemisinin combination therapies in 2001. The area for the crop cultivation has expanded in Africa and Asia and simpler and affordable methods for artemisinin analysis are needed for crop quality control. This work presented a novel chromatographic method of artemisinin analysis using gas chromatography with electron-capture detection. The sample extraction and preparation involved a single-solvent one-step extraction, with samples being analyzed in the extraction solvent directly after extraction. This method was accurate and reproducible with over 97% recoveries. The limit of detection was less than 3 microg/mL and the limit of quantification was less than 9 microg/mL, allowing samples as low as 100mg dry weight to be analyzed for artemisinin. The method can be applied to quality control of commercial plant extracts and to artemisinin-derived pharmaceuticals.