For several years, we have been evaluating the plant's production potential, determining its horticultural characteristics, and developing a rapid assay to determine artemisinin content from crude plant materials for use in selection and breeding (Shock and Stieber 1987; Charles et al. 1990; Simon et al. 1990). Essential oil composition was also characterized in order to evaluate Artemisia annua as a source of aroma chemicals for the fragrance industry (Charles et al. 1991). Selection of lines for high artemisinin content began in 1989 at Purdue University and Oregon State University at the Malheur Experiment Station.
Artemisinin is unstable due to its endoperoxy group and the chemical analysis is difficult. Most secondary products can be altered by environmental factors as well as post-harvest handling practices, yet little is known about the stability of artemisinin when subjected to either pre- or post-harvest environmental changes. The objective of this study was to examine the influence of soil water stress and drying techniques on the retention of artemisinin.
The next two studies were conducted in 1990 on a Greenleaf silt loam with a pH of 6.7, 1.6% organic matter, 28 meq/100 g CEC, 17 ppm potassium, 1996 ppm calcium, 362 ppm magnesium, 186 ppm sodium, 1.5 ppm zinc, 7.2 ppm iron, 26.7 ppm manganese, 1.1 ppm copper, and 0.5 ppm boron. The herbicides, Dual (2.2 kg ai/ha) and Treflan (0.56 kg ai/ha) were preplant incorporated on 2 May for weed control. Seeds were greenhouse sown on 29 Apr. and transplanted to the field on 29 May. Plants were 0.5 m apart, with 1.12 m between rows. For the water stress study, each plot consisted of three rows wide and 27 m long with 4 replications in a randomized complete block design. The crop was irrigated regularly with furrow irrigation. The crop was fertilized June 25 with 167 kg/ha of N in the form of urea. N-Serve at 0.84 kg ai/ha had been applied on the urea immediately before fertilization to conserve nitrogen.
Treatments for the water stress trial were imposed in July. After July 1, the timing of irrigations (frequency and duration) was based on the soil water potential according to treatment criteria. The four treatments consisted of plots irrigated so as to maintain low, mild, moderate water stress, and low stress followed by mild then moderate stress (Table 1). Soil water potential was monitored in each plot with the use of four granular matrix sensors (Watermark Soil Moisture Sensors, Model 200X, Irrometer Inc., Riverside, CA) with two sensors placed 15 cm deep and two sensors placed 45 cm deep in the planted row within the harvest area of each plot. Criterion for irrigation of a plot was based on the average water potential of the four sensors in each plot. Granular matrix sensor resistance was calibrated against tensiometer measurements of soil water potential in the field. Sensors were read several times a week and before all irrigations.
Leaf samples for artemisinin content were collected from 12 plants in the center of each plot on 20 Aug., 5 Sept. (first bud), and Sept. 5 (onset of flowering) from the soil water stress study. A total of 12 contiguous plants from the center rows of each plot were harvested on Sept. 17 from the soil water stress study to determine fresh weight yield. Plants were harvested at the soil surface. Plant height, fresh weight, plant dry weight, dry leaf weight, leaf to stem ratio, and artemisinin content were determined.
Six drying treatments were examined: sun dried, sun dried shaded in paper bag, air dried indoors, and artificial drying using forced air heat at 30°, 50°, and 80°C (Table 2). For each drying treatment, plant samples were dried for 0, 12, 24, 36, and 48 h. To reduce error from interplant variation, composite samples consisting of branches from thirty adjacent plants were used for every treatment in each of five replicates. Air temperature, relative humidity, and sample temperatures were determined, except at 80°C where relative humidity was not determined. Leaves from each treatment were evaluated for water and artemisinin content. All artemisinin concentrations reported are based on the harvest of all leaves from whole plants.
Stress has been shown to induce the synthesis of a number of secondary or natural plant products (Fluck 1955; Gershenzon 1984). To investigate the possible effect of water stress on the accumulation of artemisinin, four different water stress treatments were investigated. Artemisinin content was determined for plants harvested on Aug. 20, Sept. 5, and Sept. 17. Season long (July 1 to Sept. 17) water stress was not related to artemisinin content, plant or leaf yields. Water stress during the two weeks before harvest was associated with reduced plant height (p = 0.014). Regression analysis revealed that greater soil water stress (lower soil water potential) during the two weeks before harvest lead to reduced leaf artemisinin content (Fig. 1).
Since postharvest handling of artemisia plants could affect artemisinin content, six drying techniques over different time periods were examined to identify the best drying method. Drying method and duration had highly significant effects on leaf moisture (Fig. 2, 3). Artemisinin contents were retained to a greater extent when plants were dried under ambient conditions compared to forced air at 30° to 80°C, except when dried at 80°C for the shortest time period (12 h) (Table 2). Prolonged drying generally resulted in further losses in artemisinin. In conclusion, these results strongly suggest that both plant water status and post-harvest handling can influence the retention of artemisinin.
J. Ess. Oil Res. 3:33-39.
|Treatment||Irrigation criteria (kPa)||Timing|
|Low stress||-50||from July 1|
|Mild stress||-100||from July 1|
|Moderate stress||-150||from July 1|
|Stress before harvest||-50||from July to 8 Aug.|
|then||-150||from Aug. 8 to Sept. 5|
|Air temp. (°C)||Sample temp. (°C)||Artemisinin content (% dry weight)|
|Drying method||Avg. air RH (%)||avg.||max.||avg.||max.||12h||24h||36h||48h|
|Air dried outside||30.9||23.5||30.0||24.4||35.6||0.15±0.12||0.17±0.10||0.04±0.02||0.08±0.02|
|Air dried inside||35.6||23.2||28.9||19.5||22.8||0.15±0.07||0.19±0.08||0.12±0.09||0.09±0.06|
|Harvest date||Days after |
(% dry wt)
|June 30||90||Active growth||0.06|
|July 26||117||Active growth||0.15|
|September 5||158||Visible flower bud||0.06|