High Artemisinin Research Articles

Correlation and path analysis on artemisinin content and yield with different agronomic traits of Artemisia annua

To provide the basis for improving utilization of Artemisia annua germplasm resources and breeding variety, the interrelations between artemisinin content, artemisinin yield and agronomic traits of A. annua were studied. The artemisinin content and each agronomic trait of 63 A. annua germplasm resources were measured by the visual observation and measurement methods. And the correlation analysis, regression analysis and path analysis were adopted. The result showed that there were significant differences in the artemisinin content and yield of 63 germplasm resources from the main production region of A. annua. Correlation analysis showed that there were significantly positive correlation between leaf weight and artemisinin yield with stem and branch characters, but there were negative correlation between artemisinin content with leaf characters of A. annua plant. The artemisinin content of A. annua increased with the increasing of primary branch number, bottom secondary branch number, and bottom stem diameter, etc. On the other hand, it decreased with the increasing of top secondary branch number, secondary leaf axis length, and bottom branch diameter, etc. The artemisinin yield of A. annua increased with the increasing of artemisinin content, leaf weight, and bottom secondary branch number, etc., and decreased with the increasing of bottom branch diameter, middle secondary branch number, and stem weight, etc. Path analysis showed that the primary branch number and bottom secondary branch number had a direct positive effect on the artemisinin content of A. annua. But the top secondary branch number had a direct negative effect on the artemisinin content of A. annua. The leaf weight and artemisinin content had a direct positive effect on the artemisinin yield and the ratio of leaf/stem, branch weight and stem weight had a direct negative effect. On the breeding A. annua variety, it can take into account both high leaf yield and high artemisinin content. And it was strongly recommend that the plant with moderate plant height and crown, shortness pinnae and secondary leaf axis, less middle and top secondary branch, strong stem, higher primary branch number and bottom secondary branch number, and higher ratio leaf/stem could be selected for breeding new varieties with high leaf yield and high artemisinin content.

Effect of Different Soil Conditions on The Growth and Artemisinin Content of Artemisia annua L. Seedlings

Field plot trials involving 4 soil conditions (sand,upland soil,paddy soil and brown calcareous soil) were conducted under fertilized and un-fertilized conditions. Then the effect of soil conditions on the growth,biomass allocation and artemisinin content of Artemisia annua were investigated. The results showed that,A.annua could adequately acclimate to different soil conditions. It could grow and develop in sand,upland soil,paddy soil and brown calcareous soil,and respond to heterogeneity soil nutrient availability by adusting biomass partitioning to various organs. Under the low soil nutrient level,A.annua allocated more biomass to root,and its root mass fraction and root mass/crown mass were significantly increased. Under the high soil nutrient level,it allocated more biomass to leaf,and its leaf mass fraction was significantly increased. The growth and artemisinin content of A.annua were affected significantly by soil nutrient. Under un-fertilized condition,the height,basal diameter,total biomass,leaf biomass and artemisinin content of A.annua planted in paddy soil and calcareous soil were significantly higher than those planted in upland soil and sand,and the parameters presented above were significantly higher in upland soil than in sand. Whereas those parameters were not significantly different among soil conditions under fertilized condition,and those parameters were significantly higher than those under un-fertilized condition. Consequently,it can gain high artemisinin yield through suitable fertilizing.

Isolation, characterization and structural studies of amorpha - 4, 11-diene synthase (ADS(3963)) from Artemisia annua L.

With the escalating prevalence of malaria in recent years, artemisinin demand has placed considerable stress on its production worldwide. At present, the relative low-yield of artemisinin (0.01-1.1 %) in the source plant (Artemisia annua L. plant) has imposed a serious limitation in commercializing the drug. Amorpha-4, 11-diene synthase (ADS) has been reported a key enzyme in enhancing the artemisinin level in Artemisia annua L. An understanding of the structural and functional correlations of Amorpha-4, 11-diene synthase (ADS) may therefore, help in the molecular up-regulation of the enzyme. In this context, an in silico approach was used to study the ADS₃₉₆₃ (3963 bp) gene cloned by us, from high artemisinin (0.7-0.9% dry wt basis) yielding strain of A. annua L. The full-length putative gene of ADS₃₉₆₃ was found to encode a protein consisting of 533 amino acid residues with conserved aspartate rich domain. The isoelectric point (pI) and molecular weight of the protein were 5.25 and 62.2 kDa, respectively. The phylogenetic analysis of ADS genes from various species revealed evolutionary conservation. Homology modeling method was used for prediction of the 3D structure of ADS₃₉₆₃ protein and Autodock 4.0 version was used to study the ligand binding. The predicted 3D model and docking studies may further be used in characterizing the protein in wet laboratory.

Enhancement of Artemisinin Content through Four Cycles of Recurrent Selection with Relation to Heritability, Correlation and Molecular Marker inArtemisia annuaL.

Due to the high demand and low yield of the anti-malarial drug artemisinin in natural populations of Artemisia annua (Quinghao), an attempt has been made to enhance the artemisinin content through 4 cycles of recurrent selection (C(0)-C(3)) using selected genotypic and phenotypic traits. Based on their phenotypic and genotypic characteristics, the top 5% plants of each cycle were selected, and their seedlings were planted in poly-cross block to produce seeds for the subsequent generation. A significant increase in the artemisinin content (0.15% in C (0) to 1.16% in C (3), i.e., about 40% genetic gain over the generation) was observed. This enhancement was directly correlated with the plant height and branching intensity in all four cycles. Similarly, the PCV (phenotypic coefficient of variation) and GCV (genotypic coefficient of variation) have been observed to have a higher value for artemisinin content. The DNA marker (MAP 12) with relation to artemisinin was also identified for high yielding genotypes in all four cycles of selection. Over the four cycles of recurrent selection, the plant developed an oval appearance (Variety: CIM-Arogya) and a high artemisinin content (1.16%).

Synergistic re-channeling of mevalonate pathway for enhanced artemisinin production in transgenic Artemisia annua

To interfere with the endogenous gene encoding squalene synthase (SS) responsible for sterol biosynthesis, an antisense SS cDNA ( asSS) was introduced into the genome of A. annua. Following the decline of SS mRNA and synchronous reduction of total sterols, mRNA coding for amorpha-4,11-diene synthase (ADS) tremendously elevated, whereas mRNAs for sesquiterpene cyclases encompassing 8-epicedrol synthase (EPS), β-caryophyllene synthase (CS) and β-farnesene synthase (FS) declined or kept homeostasis, suggesting a bias in redirection of the partially blocked metabolic flow to amorpha-4,11-diene. By phenotyping the chimeric ADS promoter-fused β-glucuronidase gene ( pADS-GUS), overexpression of the pADS-GUS reporter was detected upon chilling exposure and ultraviolet (UV) irradiation. In transgenic plants that expose to chilling, ADS and EPS were dramatically up-regulated, but CS was down-regulated and FS and SS were unaffected, implying further chilling-induced diversion of carbon flux among sesquiterpenes. Accordingly, ADS and cytochrome P450 reductase (CPR) were also chilling-inducible, while cytochrome P450 monooxygenase (CYP71AV1) reflected a complicated chilling profile. A maximal artemisinin content was accounted for 16.6 mg/g or 3.7-fold increase in a chilled transgenic line. Fascinatingly, an extraordinarily high artemisinin content of 23.5 mg/g or 5.2-fold increase was measured after 15-month storage of the ground powder from the identical chilled transgenic tissue, demonstrating non-enzymatic conversion from a putative artemisinin precursor to artemisinin. Moreover, a relevance of chilling exposure with singlet oxygen ( 1O 2) emission and artemisinin overproduction was established. These results indicated that the mevalonate pathway has been first skewed to amorpha-4, 11-diene and shunted ultimately to artemisinin through a synergy of genetic modification with environmental superinduction.

The Molecular Cloning of Artemisinic Aldehyde Δ11(13) Reductase and Its Role in Glandular Trichome-dependent Biosynthesis of Artemisinin in Artemisia annua

At some point during biosynthesis of the antimalarial artemisinin in glandular trichomes of Artemisia annua, the Delta11(13) double bond originating in amorpha-4,11-diene is reduced. This is thought to occur in artemisinic aldehyde, but other intermediates have been suggested. In an effort to understand double bond reduction in artemisinin biosynthesis, extracts of A. annua flower buds were investigated and found to contain artemisinic aldehyde Delta11(13) double bond reductase activity. Through a combination of partial protein purification, mass spectrometry, and expressed sequence tag analysis, a cDNA clone corresponding to the enzyme was isolated. The corresponding gene Dbr2, encoding a member of the enoate reductase family with similarity to plant 12-oxophytodienoate reductases, was found to be highly expressed in glandular trichomes. Recombinant Dbr2 was subsequently characterized and shown to be relatively specific for artemisinic aldehyde and to have some activity on small alpha,beta-unsaturated carbonyl compounds. Expression in yeast of Dbr2 and genes encoding four other enzymes in the artemisinin pathway resulted in the accumulation of dihydroartemsinic acid. The relevance of Dbr2 to trichome-specific artemisinin biosynthesis is discussed.

NEW ARTEMISIA ANNUA HYBRIDS WITH HIGH ARTEMISININ CONTENT

NEW ARTEMISIA ANNUA HYBRIDS WITH HIGH ARTEMISININ CONTENT

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)

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

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).

Seasonal variation of artemisinin and its biosynthetic precursors in plants of Artemisia annua of different geographical origin: proof for the existence of chemotypes.

The time course of the levels of artemisinin, its biosynthetic precursors and the biosynthetically related sesquiterpenes was monitored during a vegetation period of Artemisia annua plants of different geographical origin. Considerable differences in contents of artemisinin and its direct precursors artemisinic acid and dihydroartemisinic acid were found between these A. annua's. For the first time the A. annua plants of different geographical origin were found to belong to different chemotypes. A chemotype with a high artemisinin level was found to have also a high dihydroartemisinic acid level but a relatively low artemisinic acid level. Reversibly, a chemotype with low levels of artemisinin and dihydroartemisinic acid contained a high artemisinic acid level. Artemisinic acid is considered to be the direct precursor of dihydroartemisinic acid in the biosynthetic pathway of artemisinin. The observed accumulation of artemisinic acid in one of the A. annua chemotypes may indicate the presence of a rate-limiting step in the biosynthetic pathway of artemisinin. The enzymatic reduction of artemisinic acid into dihydroartemisinic acid is probably a "bottle neck" in the biosynthetic pathway of artemisinin in varieties with high artemisinic acid and consequentially low artemisinin levels. After a night-frost period, the level of artemisinin was increased, in the Vietnamese A. annua plants, while the dihydroartemisinic acid level was decreased. This phenomenon is in accordance with our hypothesis that stress triggers the conversion of dihydroartemisinic acid to artemisinin. It is suggested that the presence of high levels of dihydroartemisinic acid may be an adaptation to stress conditions (e.g., night-frost), during which relatively high levels of 1O2 are formed. Dihydroartemisinic acid gives the plant protection by reacting with these reactive oxygen species yielding artemisinin as stable end-product.

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.

Effect of Plant Density on the Yields of Artemisinin and Essential Oil inArtemisia annuaCropped under Low Input Cost Management in North-Central India

A field experiment was conducted on Artemisia annua L. during the winter-summer season of 1995-96 at Lucknow, located at 26.5 degrees N, 80.5 degrees E and 120 m above mean sea level, representing a semiarid-subtropical climate with hot summer and fairly cool winters to study the effect of varying plant densities on the yields of essential oil and artemisinin under the conditions of no interculture and fertilizer applications. Results showed that A. annua at the population density of 2.22 x 10(5) plants ha(-1) yielded 7.4 kg of artemisinin and 91 kg of essential oil. It was also observed that at the same oil yield level, the yield of artemisinin increased by 1.5-, 2-, and 2.5-fold when the plant densities increased by about 2-, 4-, and 8-fold. Correspondingly, the suppression of weeds occurred by about 33, 133, and 333%, respectively. The plant architecture and canopy structure produced in high density A. annua plots presumably favoured the synthesis and accumulation of artemisinin resulting in artemisinin-rich, mature plants. It is recommended that, under subtropical agroclimatic conditions of north Indian plains, the A. annua should be cultivated at very high plant densities such as 2.22 x 10(5) plants ha(-1) forgetting high artemisinin yields.

Effect of Planting Time on the Yield of Essential Oil and Artemisinin in Artemisia annua under Subtropical Conditions

A field experiment was conducted during 1994–95 at the Research Farm of the Central Institute of Medicinal and Aromatic Plants (CIMAP), Lucknow, India to study the yield potential of Artemisia annua planted at different times on sandy loam soil in a subtropical environment. Results revealed that the crops planted between September to December produced significantly higher herbage yield measured directly, or after drying compared with that of February planted crop. September planted crop yielded the highest amount of artemisinin. Therefore, the plants established during pre-winter (August-September) weather and allowed to grow through the entire winter synthesized and accumulated more artemisinin than plants established during early (October-November) and late (February) winter periods. The crop planted in November (or the beginning of winter) was found to be significantly superior to a crop planted in August, January or February, both in herbage production and oil yield. The September planted crop yielded relatively less oil than the November planted crop but the artemisia ketone content of the oil of the former was better. It is concluded that the artemisinin content is dependent on the weather conditions, and high yields of artemisinin do not necessarily depend on high dry matter production by the crop. Under northern Indian plains conditions, the Artemisia annua crop should be planted in September for getting higher artemisinin yield with superior quality of oil as measured by the artemisia ketone content, and in November for obtaining maximum oil yield.

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.

Micropropagation ofArtemisia annuavia the Inflorescence

ABSTRACT Using nodal explants from inflorescence segments, an efficient protocol has been developed for a cyclic supply of in vitro micropropagated plants of high artemisinin-containing, superior, adult plant selections of Artemisia annua. Murashige and Skoog's medium (MS) supplemented with RT vitamins, 1.0 g/l casein hydrolysate and 1.0 mg/l BAP supported the conversion of terminal inflorescence heads into leafy structures with subsequent elongation of axillary shoots under continuous illumination of 3000 to 4000 lux. The regenerated shoots formed roots on MS medium supplemented with 5.0 mg/l IBA. Using the protocol 24 selected genotypes of Artemisia annua were propagated. Cloned plants showed excellent survival and establishment rates in soil under glass house conditions and could be successfully advanced to the field trials. The tissue culture procedure should enable raising nuclear base populations of high artemisinin yielding genotypes of Artemisia annua, selected at an advanced reproductive phase, a...

Relationship of Artemisinin Content of Tissue-Cultured, Greenhouse-Grown, and Field-Grown Plants ofArtemisia annua1

Artemisinin content of Artemisia annua L. plants grown under long days in a greenhouse was highly correlated (r = 0.931** and 0.954**, P = 0.01) with the same clones grown under long days in the field. The correlation of artemisinin content of clones grown in long days in tissue culture and in the greenhouse was r = 0.502*. Correlation of artemisinin content of tissue-cultured clones obtained two years apart was r = 0.61**. Broad-sense heritability estimates for artemisinin production based on vegetatively propagated clones derived from a random-mating population and grown in the greenhouse and field varied from 0.91 (greenhouse, individual basis) to 0.98 (combined greenhouse and field, family basis). These results indicate that genetic progress can be expected from intercrossing high artemisinin clones selected in the greenhouse under long days.

Selection of High Artemisinin YieldingArtemisia annua

Artemisia annua L. seeds from North Viet Nam were germinated and continuously propagated by tissue culture technique. Three-week-old plantlets were transplanted from the laboratory into the field under tropical climatic conditions and assayed for artemisinin at different stages of growth. The plants had short stature (100 cm), early flowering (14 weeks), short growth span (18-20 weeks), variable artemisinin content and produced no seeds. The highest artemisinin yield was found in the leaves of twelve (0.13-0.31%) to thirteen (0.12-0.39%) weeks old plants during pre-flowering periods. Plantlets in the test tubes were randomly selected and cloned. Statistical comparison of the artemisinin content from the various clones indicated that they may be grouped as high (0.41-0.42%), moderate (0.25-0.26%), and low (0.13%) artemisinin yielders.

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.

Selection and micropropagation of high artemisinin producing clones of Artemisia annua L.

AbstractAn in vitro micropropagation method has been used to clone Artemisia annua L. plants from axenic seedlings or field grown plants selected on the basis of morphological characteristics during the growing season. Clones were maintained in culture by continuous transfer every 4–6 weeks. The performance of the various clones in the field as well as their contents of artemisinin and related sesquiterpenes were compared with selected field grown plants. An internally standardized reverse‐phase HPLC method was developed for this evaluation. Artemisinic acid (III) was by far the major sesquiterpene for all clones tested, followed by arteannuin B (II) and then artemisinin (I). However, there was a high degree of variability between various clones for all sesquiterpenes tested. The high artemisinin producing clones are characterized as tall robust plants with long internodes, open branching, dense leaves and thick stems. Results from unorganized callus cultures initiated from the leaves of the corresponding clones did not indicate the formation of artemisinin or related sesquiterpenes in these cultures.