Artemisinin Content Research Articles

AaWIN1, an AP2/ERF protein, positively regulates glandular secretory trichome initiation in Artemisia annua

Exploring the genetic network of glandular trichomes and manipulating genes relevant to secondary metabolite biosynthesis are of great importance and value. Artemisinin, a key antimalarial drug ingredient, is synthesized and stored in glandular secretory trichomes (GSTs) in Artemisia annua. WIN/SHN proteins, a clade of AP2/ERF family, are known as regulators for cuticle biosynthesis. However, their function in glandular trichome development is less unknown. In this study, we identified a WIN/SHN gene from A. annua and named it as AaWIN1. AaWIN1 was predominantly expressed in buds, flowers and trichomes, and encoded a nuclear-localized protein. Overexpressing AaWIN1 in A. annua significantly increased the density of GST as well as the artemisinin content. Furthermore, AaGSW2 was reported to play an important role in promoting GST initiation, and the expression of AaGSW2 was induced in AaWIN1-overexpression lines. AaMIXTA1, a MYB protein positively regulating trichome initiation and cuticle biosynthesis, was confirmed to interact with AaWIN1. In addition, the ectopic expression of AaWIN1 resulted in slender and curled leaves, fewer trichomes, and rising expressions of cuticle biosynthesis genes in Arabidopsis thaliana. Taken together, based on phenotype observations, content measurements and gene expression detections, AaWIN1 was considered as a positive regulator for GST initiation in A. annua.

EVALUATION OF ARTEMISININ CONTENT AS AFFECTED BY SOIL AND WATER MANAGEMENT STRATEGIES UNDER GRAVITY-DRIP IRRIGATION SYSTEM I SAMARU, NIGERIA

ABSTRACT Malaria has been among the dangerous diseases not only in Nigeria but Africa as whole. Artemisia crop has an artemisinin, which is a strong anti- malaria drug. However, despite its importance in curing this disease, it is only produced under rain-fed but hardly produced under irrigation in Nigeria. The experiment consisted of three levels of irrigation water application at 100, 80, and 60% ETo (reference evapotranspiration); two mulch levels (rice straw mulch and no mulch) giving a total number of six treatments, replicated three times, totaling eighteen treatments laid in Randomized Complete Block Design (RCBD). The soil moisture was monitored throughout the crop growing season with access tubes installed at different incremental depth of 0-20 cm, 20-40 cm and 40-60 cm below the soil surface. The artemisinin content was determined in the Faculty of Pharmaceutical Sciences, Ahmadu Bello University, Zaria. The artemisinin content obtained ranges from 0.27 % w/w to 1.84 % w/w. The least artemisinin content was obtained from the treatment with irrigation at 60% ETo with no mulch. While the highest value was obtained with irrigation at 100% ETo with rice straw mulch. The artemisinin content of artemisia annua is affected by water stress. Irrigating the crop fully results in higher artemisinin content of artemisia annua followed by 20 % deficit. The least artemisinin content of artemisia annua was obtained at much water stress of 40 %. The study, recommends a full irrigation could be practiced for effective production of artemisia crop under drip irrigation. However, up to 40 % irrigation water deficit could be applied in areas of high water scarcity. Moreover, since rice straw is cost effective, it is recommended for use as mulching material for the cultivation of artemisia crop under irrigation.

Metabolic regulation and engineering of artemisinin biosynthesis in <i>A. annua</i>

Artemisinin is a potent anti-malarial sesquiterpene lactone that is naturally biosynthesized in glandular trichomes of a Chinese herbal plant, <italic>Artemisia annua</italic>. Although semi-synthesis of artemisinin in yeast has been feasible at present, <italic>A. annua</italic> plant is still the main commercial source of artemisinin. However, the content of artemisinin is low in <italic>A</italic>. <italic>annua</italic>, limiting its supply to the malarial victims. So it's crucial to elevate artemisinin production in <italic>A. annua</italic>. Up to date artemisinin biosynthetic pathway has been completely elucidated. And many regulatory factors, mediating diverse plant hormone or environmental signaling routes, have been identified to get involved in the regulation of artemisinin biosynthesis and glandular trichome formation. Understanding the regulatory mechanisms of artemisinin biosynthesis and glandular trichome formation will conduce to our practice for improving artemisinin production by metabolic engineering. In this review, the metabolic regulatory network with regard to artemisinin biosynthesis and glandular trichome formation is summarized, and the advance on metabolic engineering to increase artemisinin content in <italic>A. annua</italic> is also discussed.

Synergistic interaction of two bHLH transcription factors positively regulates artemisinin biosynthetic pathway in Artemisia annua L.

The wonder drug artemisinin, a sesquiterpene lactone endoperoxide from Artemisia annua is the million-dollar molecule required to curb the deadliest disease, Malaria. One of the major challenges even today is to increase the concentration of artemisinin within plants. The transcription factors are important regulators of plant secondary metabolites and have the potential to regulate key steps or the whole biosynthetic pathway. In this study, we have identified and characterized two bHLH transcription factors (Aa6119 and Aa7162) from A. annua. Both the transcription factors turned out to be transcriptionally active and nuclear-localised typical bHLH proteins. In our study, we found that Aa6119 specifically binds to the E-box element present on the promoter of artemisinin biosynthetic gene, AMORPHA-4,11-DIENE SYNTHASE (ADS). The protein-DNA interaction confirmed by Yeast One-hybrid assay was specific as Aa6119 was unable to bind to the mutated E-boxes of ADS. Further, Aa6119 interacted physically with Aa7162, which was confirmed in vitro by Yeast Two-hybrid assay and in vivo by Bimolecular Fluorescent complementation assay. Our quantitative expression studies have confirmed that Aa6119 and Aa7162 act synergistically in the regulation of artemisinin biosynthetic and trichome developmental genes. The higher accumulation of artemisinin content in the transient co-transformed transgenic plants than in the individual over-expression transgenic plants has further validated that Aa6119 and Aa7162 act positively and synergistically to regulate artemisinin accumulation.

Dual production of solar energy and important plant-derived active pharmaceutical ingredients: Artemisinin, a pilot study

• An simulated agrivoltaic system for the dual production of renewable energy and medicinal plants was investigated. • Artemisia annua plants that were shaded produced 20% more artemisinin than plants that were not shaded. • It seems possible to grow important high-value crops while also producing renewable photovoltaic energy. Agrophotovoltaics is a relatively new field of research where land is used for the production of both solar energy and various suitable (food) crops. Due to persistent rolling blackouts in South Africa, and a dire need to invest in renewable energy, a pilot study was conducted to test the feasibility to produce high value active pharmaceutical ingredient (API) yielding plants in combination with solar energy. In this study, solar panels were simulated by removing direct sunlight from Artemisia annua plants, and the concentration of the API, artemisinin, was determined and compared to control plants exposed to full sunlight. The impact of a low light regime on the biosynthesis of artemisinin was therefore investigated. Samples were collected from the shaded plants as well as from the control plants and artemisinin content quantified with liquid chromatography mass spectrometry. In addition, scanning electron microscopy analysis was conducted to determine the influence on the number and morphology of glandular trichomes which are believed to be the site of artemisinin production and/or storage. The shaded plants were found to contain roughly 20% more artemisinin as compared to the control plants. Scanning electron microscopy analysis could not establish a clear indication of the impact on the glandular trichomes due to a dense layer of filamentous trichomes obscuring the glandular trichomes in the sun exposed leave material. This pilot study indicates in principle that low maintenance solar panels may be used for energy generation and in practical enhancement of artemisinin content.

From Plant to Yeast-Advances in Biosynthesis of Artemisinin.

Malaria is a life-threatening disease. Artemisinin-based combination therapy (ACT) is the preferred choice for malaria treatment recommended by the World Health Organization. At present, the main source of artemisinin is extracted from Artemisia annua; however, the artemisinin content in A. annua is only 0.1–1%, which cannot meet global demand. Meanwhile, the chemical synthesis of artemisinin has disadvantages such as complicated steps, high cost and low yield. Therefore, the application of the synthetic biology approach to produce artemisinin in vivo has magnificent prospects. In this review, the biosynthesis pathway of artemisinin was summarized. Then we discussed the advances in the heterologous biosynthesis of artemisinin using microorganisms (Escherichia coli and Saccharomyces cerevisiae) as chassis cells. With yeast as the cell factory, the production of artemisinin was transferred from plant to yeast. Through the optimization of the fermentation process, the yield of artemisinic acid reached 25 g/L, thereby producing the semi-synthesis of artemisinin. Moreover, we reviewed the genetic engineering in A. annua to improve the artemisinin content, which included overexpressing artemisinin biosynthesis pathway genes, blocking key genes in competitive pathways, and regulating the expression of transcription factors related to artemisinin biosynthesis. Finally, the research progress of artemisinin production in other plants (Nicotiana, Physcomitrella, etc.) was discussed. The current advances in artemisinin biosynthesis may help lay the foundation for the remarkable up-regulation of artemisinin production in A. annua through gene editing or molecular design breeding in the future.

Improving artemisinin and essential oil production from Artemisia plant through in vivo elicitation with gamma irradiation nano-selenium and chitosan coupled with bio-organic fertilizers

Artemisia plants process potential bioactive secondary metabolites such as artemisinin and essential oil. They are useful in controlling potential pests and microbes and have a therapeutic effect. The commercial production of artemisinin and essential oil is limited to regarding the worldwide demand. Urgent attempts must be undertaken to improve the production of bioactive secondary metabolites. The objectives of this experiment were to increase the production and improve the quality of bioactive secondary metabolites in order to limit the use of agrochemicals. Therefore, a field experiment was conducted during the 2018 and 2019 seasons. Treatments included three elicitations (gamma irradiation, nano-selenium, and chitosan) and three kinds of fertilizers (NPK, Moringa leaf extract, and humic acid). The experiment was conducted as a factorial with a completely randomized block. The designs and treatments were arranged in a split–split plot with three replicates. A single application of elicitors showed that chitosan &amp;gt; nano-selenium, chitosan &amp;gt; gamma irradiation, and Moringa &amp;gt; humic acid &amp;gt; NPK in plants’ artemisinin and essential oil content, while the interaction showed a significant synergistic relationship between elicitors and the fertilizers in enhancing the quantity and quality of artemisinin and essential oil of Artemisia plants. Without using any pesticides, there was no infection that appeared in Artemisia plants, this could be due to the enhancement of bioactive secondary metabolite production.

MADS-box gene AaSEP4 promotes artemisinin biosynthesis in Artemisia annua.

The plant Artemisia annua is well known for its production of artemisinin, a sesquiterpene lactone that is an effective antimalarial compound. Although remarkable progress has been made toward understanding artemisinin biosynthesis, the effect of MADS-box family transcription factors on artemisinin biosynthesis is still poorly understood. In this study, we identified a MADS transcription factor, AaSEP4, that was predominantly expressed in trichome. AaSEP4 acts as a nuclear-localized transcriptional activator activating the expression of AaGSW1 (GLANDULAR TRICHOME-SPECIFIC WRKY1). Dual-luciferase and Yeast one-hybrid assays revealed that AaSEP4 directly bound to the CArG motif in the promoter region of AaGSW1. Overexpression of AaSEP4 in A. annua significantly induced the expression of AaGSW1 and four artemisinin biosynthesis genes, including amorpha-4,11-diene synthase (ADS), cytochrome P450 monooxygenase (CYP71AV1), double-bond reductase 2 (DBR2) and aldehyde dehydrogenase 1 (ALDH1). Furthermore, the results of high-performance liquid chromatography (HPLC) showed that the artemisinin content was significantly increased in the AaSEP4-overexpressed plants. In addition, RT-qPCR results showed that AaSEP4 was induced by methyl jasmonic acid (MeJA) treatment. Taken together, these results explicitly demonstrate that AaSEP4 is a positive regulator of artemisinin biosynthesis, which can be used in the development of high-artemisinin yielding A. annua varieties.

Influence of biotic and abiotic elicitors on artemisinin, quercetin, caffeic acid and essential oil production in Artemisia dracunculus L.

AbstractArtemisia dracunculus L. (tarragon) is valuable for its medicinal uses such as antimalarial, antibacterial and anticancer. Biotechnological methods need to be developed for the production of phytochemical compounds in plants with high and stable quality. Elicitation is one of the best tools for increasing secondary metabolites in various in vitro cultures. The current study determines the application of different concentrations of chitosan (25, 50, 100 and 150 mg/L) and UV‐C (5/10, 5/15, 10/10 and 10/15 min/cm) as biotic and abiotic elicitors in tarragon leaf callus cultures. The main aim was to increase the accumulation of artemisinin, quercetin, caffeic acid and essential oil content. For this purpose, callus formation rates and morphological features were also investigated. The results show that highest callus formation, weight and diameter were observed in UV‐C treated cultures. HPLC analyses revealed significant high accumulation of artemisinin (1.9 μg/g), quercetin (4.2 μg/g) and caffeic acid (2.9 μg/g) contents in 5/10 min/cm UV‐C treated samples. GC–MS analyses of callus cultures indicate high production of total amount of monoterpenes in all chitosan applications, best in 25 mg/L (90.75%). The major compound was β‐phellandrene in chemical composition of tarragon (19.61%) and increased the most in 10/15 min/cm UV‐C application (43.39%). The results show that the physical elicitor UV‐C can be effectively used in tarragon callus culture for enhancing pharmacologically active compounds in industrial production.

Basic Helix-Loop-Helix Transcription Factors AabHLH2 and AabHLH3 Function Antagonistically With AaMYC2 and Are Negative Regulators in Artemisinin Biosynthesis

Plants have evolved sophisticated systems for regulating the biosynthesis of specialized phytochemicals. Artemisinin, which is a sesquiterpene lactone widely used in anti-malaria treatment, is produced by the Artemisia annua L. plant. However, the artemisinin content in A. annua is low and difficult to meet market demands. Studies have shown that artemisinin biosynthesis in A. annua has complex temporal and spatial specificity and is under tightly transcriptional regulation. However, the mechanism of transcriptional regulation of artemisinin biosynthesis remains unclear. In this study, we identified two MYC-type bHLH transcription factors (AabHLH2 and AabHLH3) as novel regulators of artemisinin biosynthesis. These bHLH TFs act as transcription repressors and function redundantly to negatively regulate artemisinin biosynthesis. Furthermore, AabHLH2 and AabHLH3 are nuclear proteins that bind to DNA elements with similar specificity to that of AaMYC2, but lack the conserved activation domain, suggesting that repression is achieved by competition for the same cis-regulatory elements. Together, our findings reveal a novel artemisinin biosynthesis regulatory network, provide new insight into how specialized metabolites are modulated in plants, and propose a model in which different bHLH TFs coordinated in regulating artemisinin production in the plant. Finally, this study provides some useful target genes for metabolic engineering of artemisinin production via CRISPR/Cas9 gene editing.

Allele-aware chromosome-level genome assembly of Artemisia annua reveals the correlation between ADS expansion and artemisinin yield

Artemisia annua is the major natural source of artemisinin, an anti-malarial medicine commonly used worldwide. Here, we present chromosome-level haploid maps for two A. annua strains with different artemisinin contents to explore the relationships between genomic organization and artemisinin production. High-fidelity sequencing, optical mapping, and chromatin conformation capture sequencing were used to assemble the heterogeneous and repetitive genome and resolve the haplotypes of A. annua. Approximately 50,000 genes were annotated for each haplotype genome, and a triplication event that occurred approximately 58.12 million years ago was examined for the first time in this species. A total of 3,903,467–5,193,414 variants (SNPs, indels, and structural variants) were identified in the 1.5-Gb genome during pairwise comparison between haplotypes, consistent with the high heterozygosity of this species. Genomic analyses revealed a correlation between artemisinin concents and the copy number of amorpha-4,11-diene synthase genes. This correlation was further confirmed by resequencing of 36 A. annua samples with varied artemisinin contents. Circular consensus sequencing of transcripts facilitated the detection of paralog expression. Collectively, our study provides chromosome-level allele-aware genome assemblies for two A. annua strains and new insights into the biosynthesis of artemisinin and its regulation, which will contribute to conquering malaria worldwide.

The truncated AaActin1 promoter is a candidate tool for metabolic engineering of artemisinin biosynthesis in Artemisia annua L.

Malaria is a devastating parasitic disease with high levels of morbidity and mortality worldwide. Artemisinin, the active substance against malaria, is a sesquiterpenoid produced by Artemisia annua. To improve artemisinin content in the native A. annua plants, considerable efforts have been attempted, with genetic transformation serving as an effective strategy. Although, the most frequently-used cauliflower mosaic virus (CaMV) 35S (CaMV35S) promoter has proved to be efficient in A. annua transgenic studies, it appears to show weak activity in peltate glandular secretory trichomes (GSTs) of A. annua plants. Here, we characterized the 1727 bp fragment upstream from the translation start codon (ATG) of AaActin1, however, found it was inactive in tobacco. After removal of the 5’ intron, the truncated AaActin1 promoter (tpACT) showed 69% and 50% activity of CaMV35S promoter in transiently transformed tobacco and stably transformed A. annua, respectively. β-glucuronidase (GUS) staining analysis showed that the tpACT promoter was capable of directing the constant expression of a foreign gene in peltate GSTs of transgenic A. annua, representing higher activity than CaMV35S promoter. Collectively, our study provided a novel promoter available for metabolic engineering of artemisinin biosynthesis in A. annua.

The transcription factors TLR1 and TLR2 negatively regulate trichome density and artemisinin levels in Artemisia annua.

The important antimalarial drug artemisinin is biosynthesized and stored in Artemisia annua glandular trichomes and the artemisinin content correlates with trichome density; however, the factors affecting trichome development are largely unknown. Here, we demonstrate that the A. annua R2R3 MYB transcription factor TrichomeLess Regulator 1 (TLR1) negatively regulates trichome development. In A. annua, TLR1 overexpression lines had 44.7%-64.0% lower trichome density and 11.5%-49.4% lower artemisinin contents and TLR1-RNAi lines had 33%-93.3% higher trichome density and 32.2%-84.0% higher artemisinin contents compared with non-transgenic controls. TLR1 also negatively regulates the expression of anthocyanin biosynthetic pathway genes in A. annua. When heterologously expressed in Arabidopsis thaliana, TLR1 interacts with GLABROUS3a, positive regulator of trichome development, and represses trichome development. Yeast two-hybrid and pull-down assays indicated that TLR1 interacts with the WUSCHEL homeobox (WOX) protein AaWOX1, which interacts with the LEAFY-like transcription factor TLR2. TLR2 overexpression in Arabidopsis and A. annua showed that TLR2 reduces trichome development by reducing gibberellin levels. Furthermore, artemisinin contents were 19%-43% lower in TLR2-overexpressing A. annua plants compared to controls. These data indicate that TLR1 and TLR2 negatively regulate trichome density by lowering gibberellin levels and may enable approaches to enhance artemisinin yields.

Isolation and characterization of AaZFP1, a C2H2 zinc finger protein that regulates the AaIPPI1 gene involved in artemisinin biosynthesis in Artemisia annua.

AaZFP1, a C2H2-type transcription factor, was found to bind the AGT-N1-10-AGT box of AaIPPI1proand activate the expression of AaIPPI1 involved in artemisinin biosynthesis. Artemisinin, an endoperoxide sesquiterpene lactone, is a widely used antimalarial drug isolated from Artemisia annuaL. Isopentenyl pyrophosphate isomerase (AaIPPI1) catalyzes the interconversion of isopentenyl diphosphate and dimethylallyl diphosphate and is the key gene involved in the biosynthesis of artemisinin. However, the AaIPPI1 gene regulation network remains largely unknown. Here, we isolated the AaIPPI1 promoter (AaIPPI1pro) and predicted that it contains cis-elements involved in stress responses, including the TGACG motif (a methyl jasmonate-responsive element), GARE motif (a gibberellin-responsive element), ABRE (an abscisic acid-responsive element), TC-rich repeats (a stress-responsive element), and the AGT-N1-10-AGT box, which is the binding site of Cys/His2 zinc finger protein (C2H2 ZFP). The C2H2 ZFP gene AaZFP1 was discovered by screening a cDNA library using AaIPPI1pro as bait in yeast. AaZFP1 contains two conserved C2H2 regions, a nuclear localization domain (B box), a Leu-rich domain (L box), and a conserved DLN sequence (DLN box) close to its C terminus. A subcellular localization assay indicated that AaZFP1 protein is localized in the nucleus and cytoplasm. An electrophoretic mobility shift assay demonstrated that AaZFP1 binds to the AGT-N1-10-AGT box of AaIPPI1pro. A dual-luciferase assay indicated that AaZFP1 enhanced the promoter activity of AaIPPI1 in vivo. Transient overexpression of AaZFP1 in A. annua increased the expression of AaIPPI1 and the content of artemisinin. Our data demonstrated that AaZFP1 functions as a transcriptional activator that regulates the expression of AaIPPI1 by directly binding to its promoter. The present study provides insights into the transcriptional regulation of genes involved in artemisinin biosynthesis in A. annua.

Exogenous Strigolactone (GR24) Positively Regulates Growth, Photosynthesis, and Improves Glandular Trichome Attributes for Enhanced Artemisinin Production in Artemisia annua.

Artemisia annua is a medicinal plant particularly known for the production of a sesquiterpene lactone artemisinin; a specialty metabolite known for its efficacy in the treatment of malaria by killing different strains of Plasmodium falciparum due to radicals released upon the cleavage of its endoperoxide motif. Considering these facts and the immense medicinal value of artemisinin, the enhancement of in planta production of artemisinin is highly desirable. As strigolactones are known to regulate various aspects of plant growth and development, the effects of foliar spray of different concentrations of synthetic strigolactone analog GR24 (0, 0.5, 1, 2, 4, and 8 µM) on A. annua were studied. As compared to the control group, the foliar application of GR24 had a positive impact on general growth, photosynthesis, and other physiological indices with 4 µM GR24 showing the best results. The results indicate that GR24 application increased the plant biomass and various attributes related to photosynthesis, like total chlorophyll content, chlorophyll fluorescence, stomatal conductance, internal CO2, and net photosynthetic rate. Moreover, the activity of various enzymes related to photosynthesis like carbonic anhydrase, nitrate reductase, and RuBisCO was escalated. The GR24 also improved certain attributes related to glandular trichomes, with a significant enhancement in content and yield of artemisinin as compared to untreated plants.

INFLUENCE OF SOIL TYPE ON GROWTH AND ARTEMISININ CONTENT OF WORMWOOD (ARTEMISIA ANNUA L.) CHEN YOUNG VARIETY IN SOKOTO

Artemisia annua L. produce an array of complex secondary plant metabolite including artemisinin (ART), which kills the principal malarial parasite, Plasmodium falciparum, a compound of current interest in the treatments of drug resistant malaria. However, this compound remain expensive and hardy available on global scale. Synthesis of ART has been proved to be economically impossible. Therefore, increase in yield of natural occurring ART is necessary. The study evaluated the influence of soil types on germination, growth and artemisinin content of A. annua of Chen Young variety in Sokoto agro ecological region of Nigeria and. A greenhouse experiment was conducted in 2017 at Botanical garden of Usmanu Danfodiyo University Sokoto. The seeds were sown in plastic pots containing clay, sandy and loamy soils in completely randomized design with 4 replications. Germination percentage (G), Mean germination time (MT), Coefficient of variation of the germination time (CVt), Mean germination rate (MR), Uncertainty of germination (U) and Synchrony of germination (Z) were evaluated. Growth parameter and Artemisinin content were also determined. ART was determined and quantified with high performance liquid chromatography (HPLC) using calibration curve constructed by plotting the peak area against the concentration (5, 10, 15, 20, 25 µg/ml) of ART standard solutions. The results revealed that soil types had no significant effect on germination and growth parameters evaluated (P&lt;0.05). However, clay had the highest G, MT and CVt than sandy and loamy soils. The highest speed of germination per day was observed on sandy soil. Clay soil had the least values of U (2.277±0.2 bit) while the degree of overlapping germination was frequent in the clay (Z). The result obtained shows that treatment had no significant effect on the growth parameters evaluated (P&lt;0.05). Artemisinin content was significantly affected by soil type with the higher content in sandy soil (37.73 µg/ml) follow by clay and loamy soils with 17.90 and 15.70 µg/ml respectively. This study concludes that A. annua seeds can germinate and survived on different soils type in Savanna region of Nigeria. The study suggested the use of sandy soil in cultivation of the plant for its influence on high artemisinin content.

AaSPL9 affects glandular trichomes initiation by positively regulating expression of AaHD1 in Artemisia annua L.

Glandular trichomes can secrete and store a large number of secondary metabolites in plants, some of which are of high medicinal and commercial value. For example, artemisinin, isolated from Artemisia annua L. plants, and its derivatives have great high medicinal value. Previous research indicated that artemisinin was synthesized in the glandular trichomes on the leaves of A. annua. It is an important study direction to improve artemisinin yield by promoting the initiation and development of glandular trichome. In this study, SQUAMOSA promoter-binding protein-like 9 (AaSPL9) was identified. In AaSPL9 overexpression transgenic plants, the glandular trichomes density was increased by 45–60 %, and the content of artemisinin was increased by 33–60 %, indicating that AaSPL9 positively regulate the glandular trichomes initiation. Yeast one-hybrid(Y1H), Dual-luciferase (Dual-Luc), Electrophoretic Mobility Shift Assay (EMSA) demonstrated that AaSPL9 activated the expression of AaHD1 by combining directly the GTAC-box of the AaHD1 promoter. Taken together, we identified AaSPL9, a positive transcription factor, regulating the glandular trichome initiation in A. annua, and revealed a novel molecular mechanism by which a SPL protein to promote glandular trichome initiation.

AaCycTL Regulates Cuticle and Trichome Development in Arabidopsis and Artemisia annua L.

Artemisinin is an important drug for resistance against malaria. Artemisinin is derived from the glandular trichome of leaves, stems, or buds of the Chinese traditional herb Artemisia annua. Increasing the trichome density may enhance the artemisinin content of A. annua. It has been proven that cyclins are involved in the development of trichomes in tomato, Arabidopsis, and tobacco, but it is unclear whether the cyclins in A. annua influence trichome development. In this study, we showed that AaCycTL may regulate trichome development and affect the content of artemisinin. We cloned AaCycTL and found that it has the same expression files as the artemisinin biosynthesis pathway gene. We overexpressed AaCycTL in Arabidopsis, and the results indicated that AaCycTL changed the wax coverage on the surface of Arabidopsis leaves. The trichome density decreased as well. Using yeast two-hybrid and BiFC assays, we show that AaCycTL can interact with AaTAR1. Moreover, we overexpressed AaCycTL in A. annua and found that the expression of AaCycTL was increased to 82–195%. Changes in wax coverage on the surface of transgenic A. annua leaves or stems were found as well. We identified the expression of the artemisinin biosynthesis pathway genes ADS, CYP71AV1, and ALDH1 has decreased to 88–98%, 76–97%, and 82–97% in the AaCycTL-overexpressing A. annua lines, respectively. Furthermore, we found reduced the content of artemisinin. In agreement, overexpression of AaCycTL in A. annua or Arabidopsis may alter waxy loading, change the initiation of trichomes and downregulate trichome density. Altogether, AaCycTL mediates trichome development in A. annua and thus may serve to regulate trichome density and be used for artemisinin biosynthesis.

Determination, solvent extraction, and purification of artemisinin from Artemisia annua L.

Artemisinin, the main active compound in Artemisia annua L., has been used for antimalarial properties for centuries and currently attracting increasing interest for its antiviral activities. In addition, several recent publications indicated that this valuable compound can be effective on Sars-CoV-2 virus. In the study, a high-performance liquid chromatography (HPLC) method was optimized in terms of mobile phase compositions, column temperature and flow rate using response surface methodology for the determination of artemisinin from A. annua samples. The method was also validated for some parameters according to the Eurachem guideline. Validated method was applied on A. annua plant samples, cultivated in a controlled condition, and content of artemisinin was found in the range from 5825 to 7972 mg/kg (n = 20). Extraction conditions of artemisinin from the plant samples were also optimized. In the first step of the extraction, solvents with different polarities were applied to the samples for the evaluation of artemisinin solubility. Then, ethanol was chosen for extraction solvent due the high extraction yield and classification in safer chemical ingredients list by EPA. After the extraction, a purification step using various adsorbents was studied to remove remnant impurities such as chlorophyl. The results showed that powdered charcoal was found to be the most effective adsorbent. Amount of the adsorbent was also studied to evaluate for the reduction of chlorophyl without reducing the artemisinin concentration. Finally, purified solvent was dewaxed, evaporated, and dried under nitrogen to concentrate the artemisinin content. In conclusion, the optimized conditions could be regarded as a new alternative technique in pharmaceutical industry for the extraction of artemisinin from A. annua samples.

Artemisia annua L. hot-water extracts show potent activity in vitro against Covid-19 variants including delta

Ethnopharmacological relevanceFor millennia, Artemisia annua L. was used in Southeast Asia to treat “fever”. This medicinal plant is effective against multiple pathogens and is used by many global communities as a source of artemisinin derivatives that are first-line drugs to treat malaria caused by Plasmodium parasites.Aim of the studyThe SARS-CoV-2 (Covid-19) global pandemic has killed millions and evolved numerous variants, with delta being the most transmissible to date and causing break-through infections of vaccinated individuals. We further queried the efficacy of A. annua cultivars against new variants.Materials and methodsUsing Vero E6 cells, we measured anti-SARS-CoV-2 activity of dried-leaf hot-water A. annua L. extracts of four cultivars, A3, BUR, MED, and SAM, to determine their efficacy against five infectious variants of the virus: alpha (B.1.1.7), beta (B.1.351), gamma (P.1), delta (B.1.617.2), and kappa (B.1.617.1).ResultsIn addition to being effective against the original wild type (WT) WA1, A. annua cultivars A3, BUR, MED, and SAM were also potent against all five variants. IC50 and IC90 values based on measured artemisinin content ranged from 0.3 to 8.4 μM and 1.4–25.0 μM, respectively. The IC50 and IC90 values based on dried leaf weight (DW) used to make the tea infusions ranged from 11.0 to 67.7 μg DW and 59.5–160.6 μg DW, respectively. Cell toxicity was insignificant at a leaf dry weight of ≤50 μg in the extract of any cultivar.ConclusionsResults suggest that oral consumption of A. annua hot-water extracts (tea infusions) could potentially provide a cost-effective therapy to help stave off the rapid global spread of these variants, buying time for broader implementation of vaccines.