Production and detection of artemisinin in Artemisia annua L.
Abstract
Artemisia annua L., native to China but naturalized in the United States, is the source of artemisinin (qinghaosu), an endoperoxide sesquiterpene lactone efficient against multidrug resistant strains of Plasmodium, the malarial parasite. A. annua is a short-day plant with a critical photoperiod of 13:31h and which flowers two weeks after induction. Artemisinin was detected in main, side stems, and leaves, but was 4-11 times higher in inflorescences at the full bloom stage; no artemisinin was detected in roots or pollen. Glandular trichomes, previously described in the leaves and reported to sequester artemisinin and artemisitene, were abundant in the corolla of florets and receptacles of capitula. In vitro shoot artemisinin was highest in semi-solid medium lacking benzyladenine. The presence of roots was associated with artemisinin content of shoots in vitro. Artemisinin was undetected in callus and liquid medium, but trace amounts were detected in cells of one of three clones. Artemisinin content of clones grown under long days in a greenhouse was highly correlated (r = 0.93$\sp{**}$ and 0.95$\sp{**}$) with the same clones grown in the field. The correlation of artemisinin content of clones grown under long days in tissue culture or in the greenhouse was r = 0.502$\sp{*}$. Correlation of artemisinin content of tissue-cultured clones obtained two years apart was r = 0.61$\sp{**}$. 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). An improved high performance liquid chromatography method with electrochemical detection (HPLC-EC) was established for analysis of dihydroartemisinin, artemisitene, and artemisinin from crude plant extracts, callus, and cell cultures of A. annua. This method was about 10-fold more sensitive and more reliable for the analysis of artemisinin than was GC. An enzyme-linked immunosorbant assay (ELISA) was developed for artemisinin analysis. Based on analysis of artemisinin standards, ELISA was ca. 400-fold more sensitive and six-fold faster than HPLC-EC. ELISA correlated highly with HPLC-EC analysis of crude plant extracts of different genotypes after 100- or 500-fold dilution without antibody purification. The ELISA, however, was less specific than HPLC-EC, because polyclonal antibodies cross-reacted with artemisinin-related compounds.
Degree
Ph.D.
Advisors
Janick, Purdue University.
Subject Area
Biochemistry|Botany|Plant propagation
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