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Charles, D.J., J.E. Simon, C.C. Shock, E.B.G. Feibert, and R.M. Smith. 1993. Effect of water stress and post-harvest handling on artemisinin content in the leaves of Artemisia annua L. p. 628-631. In: J. Janick and J.E. Simon (eds.), New crops. Wiley, New York.

Effect of Water Stress and Post-Harvest Handling on Artemisinin Content in the Leaves of Artemisia annua L.*

Denys J. Charles, James E. Simon, Clinton C. Shock, Erik B.G. Feibert, and Robin M. Smith

  1. METHODOLOGY
  2. RESULTS AND DISCUSSION
  3. REFERENCES
  4. Table 1
  5. Table 2
  6. Table 3
  7. Fig. 1
  8. Fig. 2
  9. Fig. 3
Artemisia annua L., (Asteraceae) is a highly aromatic annual herb that has potential value as a source of artemisinin and essential oils (Simon et al. 1990). Artemisinin is a secondary plant product that has been found to have strong anti-malarial properties with little or no side effects (Klayman 1985). New anti-malarial drugs are important due to the increasing resistance of the malaria causing protozoans to current drugs (WHO 1981). Extensive research has been carried out over the last decade towards the characterization, isolation, synthesis, and pharmacology of artemisinin and its derivatives because of their effectiveness against both chloroquine and mafloquine resistant Plasmodium falciparum associated with cerebral malaria (Klayman 1985; Schmid and Hofheinz 1983; Xu et al. 1986). While artemisinin can be synthesized, the synthetic compound is unlikely to be economically competitive with the naturally produced compound (Schmid and Hofheinz 1983; Xu et al. 1986). Of the total artemisinin in the plant, 89% is found in the leaves (Charles et al. 1989).

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.

METHODOLOGY

This report consists of three studies conducted in Ontario, Oregon. The first, a preliminary field trial, conducted in 1989, examined whether the time of harvest could influence artemisinin content. Seeds were sown in the greenhouse on March 30 and transplanted to the field Owyhee silt loam on May 3 into 1.12 m rows with 0.91 m between plants. Mechanical cultivation and manual hoeing kept the field weed free. The trial was fertilized with a total of 167 kg N/ha as broadcast urea and water-run UAN (urea ammonium nitrate). Leaf samples were collected from 20 plants in each of 27 lines on 3 harvest dates. All leaf samples were air dried in the shade and the stems removed by hand.

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.

RESULTS AND DISCUSSION

Harvest date had a significant effect on artemisinin content, with July 26 harvest giving the highest artemisinin content (Table 3). Higher artemisinin content well before flower bud formation was not expected and a further examination of seasonal changes in artemisinin accumulation is needed. However, this data and our current studies do show that artemisinin content does change within the plant over the growing season. We expected that the increase in artemisinin content would have been related to the contribution of flowers.

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.

REFERENCES

*The authors gratefully acknowledge financial support for 1989 and 1990 research provided by the Malheur County Regional Economic Development Strategy Board. Oregon Agricultural Experiment Station, Technical Paper No. 9812; and from the Purdue Agricultural Experiment Station.
Table 1. Water stress treatments for Artemisia annua at Ontario, Oregon, 1990.

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
Plants harvested on Aug. 20, Sept. 5, and Sept 17.

Table 2. Effects of six drying treatments and four durations on the artemisinin content in Artemisia annua leaves, 1990.

Air temp. (°C) Sample temp. (°C) Artemisinin content (% dry weight)
Drying method Avg. air RH (%) avg. max. avg. max. 12h 24h 36h 48h
Sun 30.9 23.5 30.0 25.0 42.2 0.08±0.06 0.09±0.03 0.10±0.03 0.12±0.06
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
30°C 52.8 32.1 35.0 31.9 34.4 0.07±0.05 0.06±0.01 0.06±0.01 0.05±0.03
50°C 36.3 49.6 53.9 49.5 52.8 0.05±0.02 0.05±0.02 0.06±0.02 0.12±0.08
80°C ND 79.9 80.0 70.9 80.0 0.13±0.11 0.08±0.04 0.06±0.02 0.06±0.03
Plants harvested on Aug. 20, Sept. 5, and Sept 17.

Table 3. Artemisinin content in air dried leaves at different harvest datesz, 1989.

Harvest date Days after
planting		 Growth stage Artemisininy
content
(% dry wt)
June 30 90 Active growth 0.06
July 26 117 Active growth 0.15
September 5 158 Visible flower bud 0.06
LSD (0.05) 0.04
zMeans are from 20 plants in each of 27 lines sampled on three dates.
yOnly leaves were analyzed, flowers were not present.


Fig. 1. Decline in artemisinin content from Artemisia annua with average soil water potential (x) two weeks before plant sampling (P < 0.01). Data are from all plant samples from the water stress trial at Ontario, Oregon, taken Aug. 20 and Sept. 5, 1990.

Fig. 2. Effects of different ambient air drying treatments on leaf moisture of Artemisia annua, LSD (0.05) = 4.1 for treatment x duration. For the air outside and air inside treatments, leaf material was placed in paper bags, Ontario, Oregon, 1990.

Fig. 3. Effect of different forced air drying treatments on leaf moisture of Artemisia annua, LSD (0.05) = 4.1 for treatment x duration, Ontario, Oregon, 1990.
Last update September 18, 1997 aw