Artemisia: Plant Cultural Techniques

2016

Plant Establishment

Natural stands

In China Artemisia annua traditionally has been harvested from wild natural self seeded stands. Although no specific crop production statistics are available, because of a confidentiality policy of Chinese authorities, it is believed that the bulk of Chinese production still comes from wild stands. These stands are the source of much of the artemisinin derived drugs used in China and probably the bulk of those drugs exported elsewhere (WHO, 1994) although some selected lines of Artemisia annua are cultivated as a row crop in Szechwan Province (). Ideally the harvesting of raw material for medicinal drug production from wild stands is not a good policy (). The plant material in wild stands is typically very variable in its content of the required medicinal constituents and this has an impact on the economics of drug extraction. Added to this the continual encroachment and elimination of wild stands will ultimately limit the source of genetic variability which is vital to the development of improved seed lines (). Another negative factor against utilisation of wild stands is that transport distances often become uneconomic with a crop such as Artemisia annua with a relatively low artemisinin content and a large bulk of material required.

Where plants traditionally have been harvested from the wild for centuries there is most likely a general conservative reluctance in many quarters to believe that plantation grown crops could have equal quality characeristics. Because Artemisia annua has been grown in China for its medicinal qualities for many centuries the belief in the superiority of the wild grown plant may be very deeply entrenched. A. comparison of the quality of wild grown and plantation grown Artemisia annua plants in key production areas in China would be a useful manoeuvre to help overcome this barrier. No such comparisons of wild and plantation grown Artemisia annua appear to have been published elsewhere. However the analogy of wild and plantation grown Gentiana lutea L. plants which have both medicinal and liquor industry uses may supply a useful model for this type of comparison ().

Transplanting

Because supplies of high quality Artemisia annua seed have been generally limited up to the present time most experimental programs have utilised transplanting as the preferred method of establishment (). These transplants have taken the form of cuttings () but in most other studies the investigators have used some form of cellular tray system such as Speedlings® (). This system involves germinating Artemisia annua seed in shallow (5-6 cm deep) seed trays using a sterilised potting mix such as 2 parts of sand, 2 parts of peat and 1 part vermiculite which has had the pH adjusted to pH 6.0-7.0 and a low to moderate quantity of complete nutrient fertiliser added after sterilisation. The seeds are sprinkled uniformly onto the surface of this mixture and covered very lightly with vermiculite (1-2 mm) and germinated in a greenhouse. The surface must be kept moist and not let dry out. When the plantlets are about 2 cm high (4-5 true leaves) – which takes about 3 weeks – they are pricked out, taking care not to damage the roots, and transplanted into the cellular trays using the same mixture as above or whatever alternative container system may be used. The plantlets are then grown on in the greenhouse in these containers for about an additional 4 weeks or until the young plants have about 10 true leaves and are about 10-14 cm high. After this the plants are hardened off outside or in a shade house for 3-5 days and then transplanted to the field. There is no published information on the optimum size for transplanting Artemisia annua plantlets but the key point is that they should be robust enough to cope with mechanical transplanting systems but not so tall that they become spindly and susceptible to wind damage. Until the transplanted seedlings start to actively grow away the soil should be kept moist. It would be a useful exercise to compare the optimum age and size of transplants in each environment in which Artemisia annua is introduced.

Seed drilling

If supplies of seed were freely available, the direct drilling of seed would be the most economical method of plant establishment provided the length of the growing season and other environmental factors were suitable and weeds could be controlled (see Herbicides and Plant population density). In Australia (Tasmania) direct drilling of seed gave leaf dry matter and artemisinin yields very similar to the yields from transplanting in 2 out of 3 field experiment (). In these experiments seed drilling and transplanting took place in mid October (spring). Both treatments matured at the same time and were harvested at the early bud stage in late February (summer) four and a half months later (). There is little information on the possibility of autumn sowing of seed and the over-wintering survival of Artemisia annua seedlings in geographical zones where this technique may be appropriate (). However the performance of self sown seeds of a Yugoslavian strain of Artemisia annua which germinated in the field in autumn (April) in Tasmania (Forthside 41° 12′ S) may add some information. This seed germinated in late April and the young seedlings survived the winter very well. They grew away strongly in spring but did not flower until late February in the following year (J.C. Laughlin, unpublished data). In Germany Artemisia annua has been direct drilled successfully in field experiments with drilling operations taking place in the third week of June and full bloom in late August (). Also in Vietnam, Artemisia annua was direct drilled in January with floral induction in October.

Direct drilling of Artemisia annua seed can be carried out by various methods ranging from the basic hand application or simple hand pushed single row seeders to sophisticated combined multi row seed and fertiliser drills. In all of these operations the soil needs to be cultivated down to a fine tilth and consolidated by rolling where appropriate. Because Artemisia annua seed is so small (10-15,000 per gram) it needs to be diluted either by some inert material or by mixing with an appropriate neutral fertiliser. In Tasmanian experiments the technique of mixing a 50:50 blend of fine ground limestone and superphosphate with Artemisia annua seed gave successful establishment (). This technique is useful in many situations ranging from basic hand sowing to machine drilling and has the advantage of not requiring a sophisticated drill with a specialised small seed facility. Although the 50:50 fertiliser limestone blend is neutral, the seed should only be mixed through it immediately before drilling. Ideally the drilled area should be irrigated soon after sowing if soil moisture is less than optimal. In this technique of mixing seed and fertiliser it is important to have some detailed knowledge of (i) laboratory germination and (ii) field emergence and survival in order to use a seed sowing rate that avoids the problems of either very sparse stands or very thick stands which may necessitate hand or machine thinning.

Depth of sowing is also critical with a small seeded crop such as Artemisia annua. In Tasmanian studies, a depth of drilling of 5 mm below the surface gave good emergence and establishment (J.C. Laughlin, unpublished data). However it is important, with a shallow drilling depth such as this, that the soil be cultivated down to a fine tilth to avoid seeds being sown on the surface and thus be in danger of losses in dry weather. For this reason also it is important to irrigate soon after drilling so that young emerging seedlings do not suffer moisture stress as well as obviating any possibility of fertiliser “burn” (see Irrigation). An alternative technique to mixing seed and fertiliser has been the drilling of a finely broken up mixture of seed and other inert floral part of Artemisia annua (). Ultimately the success of any techniques of direct seed drilling depend on a detailed knowledge of local soil and environmental conditions such as rainfall and temperature.

Time of Establishment

The choice of time of establishment is an important decision which must allow a sufficient period for rapid early growth and the production of a vigorous vegetative framework before the commencement of flowering. In continental climates good growth and yields were obtained from establishment in late spring in Switzerland () and early summer in Germany () and in the USA (). In a cool temperate maritime climate in Australia (Tasmania) a field experiment compared transplanting in the spring months of October and November with early summer in December. Although all transplants flowered at the same time, the leaf dry matter yield from October transplants were double and fourfold those from November and December transplants respectively (). Although the concentration of artemisinin was the same for these three times of establishment the concentration of artemisinic acid decreased by 25% and 50% respectively from November and December transplanting compared to the concentration in October transplants (). Later field experiments in Tasmania compared winter transplanting in July and August with spring transplanting in September and October. There were no differences in either leaf dry matter, artemisinin or artemisinic acid yield between any of these times of transplanting (). This experiment showed that there was no point in transplanting earlier than October in Tasmania with the associated problem of weed control. It also showed that although July and August transplants had 2 and 3 months of growth respectively by October, when day length had increased to 13:30 h, flowering did not occur until February of the following year when day length had decreased to 13:30 h again. In addition to absolute day length the factor of decreasing day length per se also may be of some significance in triggering flowering.

In the USA (Indiana) a greenhouse experiment at a constant temperature of 27° C with cuttings of a Chinese selection showed that Artemisia annua was a short day plant and that floral induction was initiated when the photoperiod was 13:31 h. In a related field experiment bud formation occurred two weeks later at a day length of 12:57 h (). Temperature x photoperiod interactions were not studied in this greenhouse investigation. However floral induction and flowering seem to show a very marked shift from this model in warmer tropical and subtropical climates. In India at Lucknow, (26° 52′ N) seedlings of the same European selection which were used in the Tasmanian field experiment described above () were transplanted in the relatively cool winter period of mid December (). In this experiment, bud formation (preflowering) occurred at a day length of 11:16 h with full flowering and maximum dry matter yield of leaves + flowers and artemisinin about 6 weeks later on 26 March at a day length of 12:15 h (). Later work on establishment times concluded that the optimum transplanting time on the north Indian plains was mid October (). Another field experiment in Vietnam with an Artemisia annua seed source native to Langson in Northern Vietnam was carried out near Hanoi and it studied the pattern of dry matter yield and artemisinin content over a growing season (). In this experiment the seed was direct drilled into field plots at a density of 25 plants/m2 (200 mm x 200 mm) on 10 January. Maximum dry matter yield of leaves (5.3t/ha), maximum artemisinin concentration (0.86%) and maximum artemisinin yield (45.4 kg/ha) occurred at the vegetative stage on 15 June at a day length of 13:24 h (). The plants remained vegetative until the preflowering stage on 15 October when there was a dry matter leaf yield of 3.8t/ha and artemisinin concentration of 0.42% at a day length of 11:41 h (). The conclusion from this study is that with a strain of Artemisia annua adapted to the tropical Vietnamese environment an adequate biomass at a high concentration of artemisinin was achieved before the onset of flowering.

The adaptability of Vietnamese seed selections from the northern latitudes near Hanoi is illustrated by their performance in the climatic environment of Tasmania (). Tissue cultured plants from Vietnamese selections were grown-on in the greenhouse in mid December 1994. These plants were transferred to the field to a number of locations near Devonport in mid February 1995 and grew vegetatively throughout the autumn. They survived the winter without any problem and continued good vegetative growth throughout the following spring with some achieving a height of two metres. Flowering did not commence until late February 1996, but only on some plants. None of the flowers from these plants set viable seed ().

In Madagascar at Antananarivo (Latitude 18° 52’S) at an elevation of ca. 1500 metres above sea level A. Annua plantlets resulting from a cross between plants of Chinese and Vietnamese origin were transplanted into field plots on 12 March at a spacing of 50 x 70 cm. Mature plants were harvested on 4 August and leaf dry matter yield of 4.7 t/ha and an artemisinin yield of 41.3 kg/ha were obtained (). In Penang, Malaysia (5° 30′ N) a seed line of Artemisia annua obtained from the latitude of Hanoi in Vietnam was set out in field plots to study the performance at low latitudes and about sea level. Three week old plantlets were transplanted into field plots and flowering occurred at 14 weeks with maximum artemisinin (0.39%) one week before flowering (). Although detailed yields of leaf dry matter were not studied in this experiment plants grew to one metre tall. Even at these low latitudes (0-10°) it would be a useful exercise to explore the yield potential Artemisia annua may have in lower temperature areas at higher altitudes. It may also be possible, in these tropical climates with very rapid growth, to consider the possibility of two following crops as has been suggested for Vietnam () and Brazil ().

Plant Nutrition

Major elements

There are only a small number of recorded studies of the vegetative growth responses of Artemisia annua to the specific major elements nitrogen, phosphorus and potassium or of their effects on the concentration of artemisinin and related compounds. Good yields of total plant and leaf dry matter relative to a wide range of published yield figures, have been obtained in Mississippi USA where a complete fertiliser mixture containing 100 kg N, 100 kg P and 100 kg K/ha was broadcast and worked uniformly through the soil (WHO, 1988). Similarly in Australia (Tasmania) good to high plant and dry leaf yields have been obtained with a mixed fertiliser containing 60 kg N, 60 kg P and 50 kg K/ha pre-drilled in bands 150 mm apart and about 50 below seed and 75 mm below transplants (). The technique of banding fertiliser is to be generally recommended in soils where phosphorous fixation is a problem. The manoeuvre of pre-drilling fertiliser in 150 mm rows prior to drilling seed or transplanting plantlets allows very simple and inexpensive drilling equipment to be used and obviates the need for sophisticated and expensive drills which place fertiliser and seed in the one operation. In this technique the fertiliser bands can never be more than 75 mm (half the row width) laterally displaced from the plant row when seed is drilled at random in a second operation parallel to the fertiliser and closer to the surface (Laughlin, 1978).

Although there are no specific experimental data on field responses of Artemisia annua to phosphorous or potassium there is some specific evidence of the response of Artemisia annua to nitrogen. Trials in the USA (Indiana) compared different rates of nitrogen fertiliser supplying zero, 67 and 135 kg N/ha and obtained the highest total plant yield with 67 kg N/ha (). In India, sand culture studies with an American strain of Artemisia annua (Washington) also showed that nitrogen deficiency was associated with a large decrease in artemisinin (). Similar hydroponic studies in Brazil concluded that the omission of nitrogen or phosphorus drastically reduced plant growth and dry matter production (). Later field trials with nitrogen fertiliser compared 0, 32, 64 and 91 kg N/ha applied as urea (). In this trial the dry matter leaf yield increased up to the highest rate of N but the concentration of artemisinin was reduced by 22% so that maximum economic yield of artemisinin was given by 64 kg N/ha. This represented a 50% increase in artemisinin yield above zero N.

Because nitrogen is a very mobile element it can easily be leached out of the root zone specially in areas of high or concentrated rainfall periods. This leaching effect may well be very significant in tropical and sub-tropical regions and in these situations the method and timing of nitrogen fertiliser application may be very important. Banding of nitrogen near the seed or plant row may give less leaching than broadcasting and uniform mixing. Split applications of nitrogen or slow release nitrogen may be other means whereby leaching is minimised, especially if the growing season is a long one. For example in the growth pattern of Artemisia annua in Vietnam near Hanoi the plants remained vegetative from seed drilling in January until September (). The optimum time of harvest in this experiment was mid June and at this stage the mean total rainfall between January and June was 533 mm with more than twice this quantity possible in very wet years (). In situations such as this the splitting of the total quantity of nitrogen into two, three or more doses may be an appropriate method to adopt. Ideally some form of leaf or tissue analysis to determine the critical concentration of N at which a vegetative growth or artemisinin (or artemisinic acid) concentration response would be obtained could be the ultimate to aim for because soil analyses for N (unlike P and K) are often unreliable.

It has been shown that some strains of Artemisia annua are sensitive to soil pH below 5.0-5.5 (). A. comparison of different forms of nitrogen such as urea and the neutral calcium ammonium nitrate with the more acidic ammonium sulphate and ammonium nitrate may be useful trials to carry out with Artemisia annua. Ammonium sulphate has been compared with ammonium nitrate in a field experiment on sandy soil in Switzerland (). When 90 kg N/ha was applied both forms of nitrogen increased leaf dry matter yield and artemisinin yield by about 50%. However a hydroponic nutrient culture experiment suggested that, under some circumstances nitrogenous fertiliser in the nitrate form may give higher yields of artemisinin (mg/plant) than the ammonium form (). More work needs to be done on this aspect on a range of soil types in the field.

Micro elements

In China a range of growing media and nutrient treatments were tested for their effect on the synthesis of artemisinin. There were no effects on artemisinin from any of these treatments (). However Indian sand culture experiments with the American strain Washington showed that both copper and boron deficiencies were related to large decreases in artemisinin (). Similarly in Brazilian studies the omission of any of the elements N, P, K, Ca, Mg, or S from nutrient solutions limited artemisinin and artemisinic acid production (). Where experience has shown that boron or copper deficiencies can occur with other crops, particularly on lighter soils in specific regions, standard soil tests for these elements would be a wise precaution if the cultivation of Artemisia annua was to be attempted.

Soil Acidity

Cultivated crops show well defined patterns for those that are tolerant or intolerant of acid or alkaline soils. However there are only a few studies of the effect of soil pH on the vegetative growth of Artemisia annua and on artemisinin concentration. In Australia (Tasmania) a field experiment studied the effect of zero and lOt/ha of fine ground limestone (calcium carbonate) on the growth of Chinese and Yugoslavian strains of Artemisia annua on a red krasnozem soil of pH 5.0 in the top 500 mm (). The 10t/ha limestone treatment increased soil pH from 5.0 to 5.5. The leaf dry matter yield of the Yugoslavian strain was increased from 1.0t/ha to 6.5t/ha while the Chinese strain increased from 4.5t/ha to 8.0t/ha. The concentrations of neither artemisinin nor artemisinic acid were affected by the change in soil pH. These results suggested that there were large differences in strain (genotype) susceptibility to soil pH. The responses of the Chinese and Yugoslavian strains of Artemisia annua to a wide range of soil pH were later studied in a greenhouse pot experiment using the same krasnozem soil as the above field experiment. Fine ground calcium hydroxide at the equivalent of zero, 1, 2.5, 5, 10, 20 and 40 t/ha was uniformly mixed through the soil to give mean soil pH values of 5.0, 5.2, 5.3, 5.4, 6.0, 7.4 and 8.2 respectively. Both strains of Artemisia annua grew well in the soil pH range 5.4 to 7.4 but the Chinese strain was much more tolerant of both very high (8.2) and very low (5.0) pH conditions than the Yugoslavian strain (). In Indian pot culture experiments, with Artemisia annua grown on soils of widely varying pH, the oil yields at pH 4.9 and 9.9 were respectively about 75% and 25% of those grown on soils of pH 7.9-8.9 ().

The response of plants to soil pH is not generally to the applied limestone per se but to the increased or decreased availability of other nutrients. In the above study on krasnozem soil in Tasmania the results may imply that the Yugoslavian strain was intolerant of high levels of manganese or aluminium at low soil pH as is the case with a number of other crops on this soil type. The work of Srivastava and Sharma () which drew an association between boron and copper and artemisinin concentration may also have implications for soil amelioration practices by lime application. On some light soils the application of lime can lower the availability of boron. It may be useful for further studies to more closely examine the response of Artemisia annua to the combined effects of copper, boron and lime application. An alternative to the sometimes costly strategy of ameliorating soil pH by lime application may be the selection of strains of Artemisia annua which are not only adapted to the local environment but also tolerant of extremes of soil pH below 5.5 and above 7.5.

Plant Population Density

Plant population density and its components of inter and intra-row spacings () are of considerable importance in determining yield and the practicability of both weed control () and harvesting (). If inter-row cultivation were to be used to control weeds before the rows close then inter-row spacings of 0.5 to 1.0 metres may be appropriate. Similarly wide intra-row spacings may also be appropriate. However, if effective herbicides were available, then yield per unit area could be increased by using much higher plant population densities. In some earlier studies, low densities of 1 plant/m2 () and 2.5 plants/m2 were used and gave yields of 1-4 t/ha of dried leaf. In other studies higher densities have been used. Simon et al. () in the USA compared 3, 7 and 11 plants/m2 and obtained the highest biomass at the highest density. In Australia (Tasmania) a field experiment with a Yugoslavian strain compared 1, 5, 10, 15 and 20 plants/m2 at a November transplanting and found that leaf dry matter yield increased up to a density of 20 plants/m2 (). However yield at 10 plants/m2 was about 90% of the maximum of 6.8t/ha (). High densities of 25 plants/m2 were also used in a field experiment in Vietnam to give a maximum leaf dry matter yield of 5.3t/ha (). In the above Australian study () plant population density had no effect on the concentration of either artemisinin or artemisinic acid in the Yugoslavian strain of Artemisia annua which was used and therefore yields reflected leaf dry matter yield (). However plant density may have different effects on the concentration of anti-malarial constituents in other climatic regions and with other strains of Artemisia annua this area of study may be worth investigating. On the north Indian plains it has been recommended that Artemisia annua should be cultivated at a high plant density of about 22 plants/m2 (). The effect of variation in rectangularity (the ratio of inter- to intra-plant spacings) at constant plant population density () has not been studied with Artemisia annua and may also be worth investigating.

Herbicides

Weeds are a constant problem for crop production throughout the world and any system of Artemisia annua cultivation must give careful thought to weed control. In small areas of cultivation in developing countries hand control of weeds may be appropriate and if this system is used, row spacings wide enough to allow easy access, should be used (). Similarly, if inter-row cultivation by hand pushed or tractor drawn implements is to be used, careful thought must be given to row spacing to allow easy access while the crop is small and before the rows close. This system may also be the only practical one available even in developed economies where stringent regulations governing the registration of herbicides for new crops demand lengthy lead times and investigations ().

If Artemisia annua is to be established from seed, weed control in the early stages of growth is even more critical than with transplants. The young seedlings which develop from the tiny Artemisia annua seed () are very small and can easily be choked out by weeds in the first days and weeks of growth. In this situation weed control with herbicides is certainly the most convenient and efficient method. Some detailed experiments on the use of herbicides with Artemisia annua have been carried out. Application of 2.2 kg active ingredient (a.i.)/ha napropamide before transplanting gave good weed control without phytoxicity in the USA (). More detailed field studies have been carried out in the USA where a range of herbicides was tested (). Chloramben was very effective when applied at 2.2 kg a.i./ha before emergence also trifluralin at 0.6 kg a.i./ha incorporated before transplanting followed by fluazifol at 0.2 + 0.2 kg a.i./ha broadcast after emergence and acifluorfen at 0.6 kg a.i./ha after emergence. All of these treatments gave good weed control without any significant reduction in leaf yield or concentration of artemisinin (). Arteether, a derivative of artemisinin, has been shown to be a very effective growth inhibitor of dicotyledenous weeds ().

Diseases and Pests

Very few significant plant diseases or pests of Artemisia annua have been reported in the literature to date. In a wide selection of experiments in the USA ranging from harvest at the green vegetative stage to seed production and utilisation as a dried flower arrangement no obvious plant pathological symptoms were observed (). The only pests observed in these trials were caterpillars but with no visible feeding injury. In Australia (Tasmania) across a similar wide range of experiments, the only disease observed in some trials was a very low incidence (<1% of plants) of Sclerotinia stem infection on the lower third of the plant (J.C. Laughlin, unpublished data). The symptoms took the form of conspicuous white fungal patches on the surface of the main stem. The possibility of Sclerotinia stem infection and appropriate control measures should be considered when Artemisia annua is grown in plantations at high plant densities. Under these conditions the build up in localised humidity could induce the infection (). In Saudi Arabia Orobanche cernua was identified as a root parasite of Artemisia annua with the potential to cause yield losses ().

Irrigation

Whether Artemisia annua is transplanted as seedlings or directly drilled into the field as seed it is important that soil moisture be adequate. In many situations frequent light irrigations are necessary to ensure good safe establishment and the frequency of these water applications will depend very much on soil type and climate. Another compelling reason to ensure that soil moisture does not become low at establishment or at later stages is the possibility of fertiliser “burn”. This problem is caused by a detrimental osmotic effect from high concentrations of soluble mobile elements such as nitrogen and sometimes potassium (). Water stress at critical periods of growth can cause yield reductions in many plants. With Artemisia annua, water stress during the two weeks before harvest gave a reduced leaf artemisinin concentration (). More work on the irrigation requirements of Artemisia annua needs to be done to define critical growth stages in a range of environments, especially with relation to nitrogen application and its effect on leaf dry matter yield and the concentration of both artemisinin and artemisinic acid.

Growth Regulators

The effects of growth regulators on the yield and artemisinin concentration of Artemisia annua have had very little study. In India triacontanol at 1.0 and 1.5 mg/litre significantly increased artemisinin levels, plant height and fresh weight. Chlormquat at 100 and 1500 mg/litre also increased artemisinin levels. When applied at 1500 and 2000 mg/litre plant height was reduced (). The application of GA3 (25 or 50 mg/litre) has also significantly increased plant fresh weight, artemisinin concentration and oil yield (). The possibilities of increasing artemisinin levels and reducing plant height have important practical implications. If ultimately Artemisia annua may be grown in plantations at high densities to maximise leaf yield () the plant height is generally increased and is typically between two and three metres tall (). Plants of this height may be a problem for machine harvesting in situ by leaf stripping () and a significant reduction in height may be a practical advantage. Growth retardants such as cycocel (CCC), dimethylsulphoxide (DMSO and maleic hydrazide (MH) could show useful effects in this regard. Related to this technique of leaf stripping, plant desiccants such as diquat and paraquat may also be worth investigating for their impact on ease of leaf removal and artemisinin content.

A study of the endogenous plant growth substances in Artemisia annua may give useful leads as to which of the large range of plant growth substances available may give the best effect when applied exogenously. Another potentially important area in which growth regulators may play a significant role is in seed set and seed germination. Although some strains of Artemisia annua may grow vegetatively very well when tested in a new environment the seed set and/or germination may be poor (see Time of establishment). This aspect may be very important in establishing cultivation of Artemisia annua from direct seeding. A. sustainable source of seed would be an important pre-requisite for economical production rather than having to rely on the importation of seed from another country or region (see Seed drilling). The growth substances uracil and 5-bromouracil have been effective in the seed set of Papaver somniferum () and could well be investigated with Artemisia annua.

Distribution of Artemisinin in Artemisia annua

The distribution of artemisinin in the Artemisia annua plant varies between its constituents of leaf, main stem, lateral branches, roots, flowers and seeds. The concentration of artemisinin within these constituents also changes over time as the plant develops during the vegetative phase and again as the plant moves into the flowering stage of growth. These changes in concentration over time are sometimes reflected in changes in the plant profile from the top to the bottom of the plant. The relative differences in concentration of artemisinin between main stem, lateral branches, roots, leaves, flowers and seed appear to be fairly similar between ecotypes. There is evidence that artemisinin is localised in special cell structures of Artemisia annua. Duke and Paul () showed that biseriate glandular trichomes were present along both sides of the leaf midrib as well as on abaxial surfaces of the leaf and on the stem. Later work () concluded that these glandular trichomes were the site of sequestration of artemisinin. Similarly, Ferreira and Janick () showed that biseriate glandular trichomes were common in the bracts, receptacles and florets of the capitulum of Artemisia annua and that sequestration of artemisinin also occurred in these structures. These workers also hypothesised that artemisinin may also be synthesised within the glandular trichomes.

Distribution between plant components

In experiments in the USA it was found just prior to flowering that about 89% of the total plant artemisinin was in the leaves with only about 10% in the lateral branches (). In terms of specific concentration of artemisinin these workers found 0.15% in the upper leaves, 0.04% in side shoots, trace amounts in the main stem and none in the roots. After flowering 0.04% of artemisinin was found in seeds. A. similar pattern of distribution was found by Acton et al. (). Later greenhouse and field experiments in which plants were sequentially harvested from the vegetative stage to flowering and seed set showed a similar relative distribution of artemisinin in the vegetative stage (). These workers also showed that the concentration of artemisinin in the inflorescences was 4-11 fold that in leaves and that the content of artemisinin in the seed was mainly associated with floral remnants and debris.

Changes in artemisinin over time

Most researchers have found, in sequential harvesting studies, that the concentration of artemisinin in the leaves increases as the plant develops in the vegetative phase. In some experiments artemisinin concentration peaked just before flowering (). Other workers agree that artemisinin concentration increases during the vegetative phase but have found that peak artemisinin was achieved later during full flowering (). The differences between the two times of peak artemisinin may be attributable to climatic conditions, ecotype, cultural practices or a combination of these factors. However, as the time of peak artemisinin concentration is of considerable importance to optimum yield of artemisinin it would be a wise precaution for this point to be determined experimentally for any new area of production rather than to rely only on published figures. The difference between the time of peak artemisinin concentration in the data of Singh et al. () and Laughlin () illustrates this point. The same European strain was used in both studies but with peaks at different stages: namely full bloom () and late vegetative () respectively. Although a knowledge of the time of peak artemisinin concentration is one of the necessary conditions to maximise yield of artemisinin it is not sufficient in itself. It must be coupled with a knowledge of the way that the dry matter yields of leaves and flowers change over time (see Time of harvest). In the data of Laughlin () although the concentration of artemisinin peaked at the late vegetative stage and decreased by about 25% at flowering the dry matter yield of leaves and flowers about doubled over the same period. Because of this, the yield per unit area of artemisinin was at a maximum at full bloom ().

Changes down the plant profile

The variation of artemisinin concentration down the plant profile varies considerably between strain origin. In the USA a field experiment (plant density unspecified) with an accession of Artemisia annua of Chinese origin sampled leaves from the top, middle and bottom thirds of the plant. The concentration and yield of artemisinin in the top third of the plant was about double that from the middle and lower thirds when sampled in the vegetative phase just before flowering (). In Australia (Tasmania) a similar field experiment – grown at a density of 10 plants/m2 - showed different patterns with a Yugoslavian and Chinese strain. The leaves of the Yugoslavian strain were harvested at a range of times from early vegetative to full bloom for each quarter of the stem from top to bottom of the plant. At the vegetative stage just prior to flowering the concentration of artemisinin was very similar in each quarter. The earliest time of harvest with the Chinese strain was early bud and at this stage the concentration from the top to bottom quarters increased from 0.12%, 0.17%, 0.19% to 0.22% (). In another study in the USA the leaves of six Chinese clones of Artemisia annua were taken from the top, middle and bottom thirds of the stems at both vegetative and flowering stages (). Artemisinin was evenly distributed. It is apparent that there are large differences in the profile of artemisinin distribution in the various Artemisia annua strains. Plant population density may be an important factor in the difference between the results of Laughlin () and those of Charles et al. () and Ferreira and Janick (); however these profile distribution patterns should be explored in developing harvesting strategies in new areas of Artemisia annua production ().

Artemisinic Acid: Precursor of Artemisinin

In addition to artemisinin some studies also have shown that its precursor artemisinic acid (= arteannuic acid = qinghao acid) may be a viable supplementary or alternative method of obtaining artemisinin (). Methods of conversion of artemisinic acid to artemisinin with efficiencies of about 18% () and 40% () have been developed. The concentration of artemisinic acid in Artemisia annua is strongly influenced by strain selection. In European and Chinese selections the concentration of artemisinic acid can be up to tenfold that of artemisinin (). However in a Vietnamese strain Woerdenbag et al. () found that, in the vegetative stage, the opposite was the case with the concentration of artemisinin being up to tenfold that of artemisinic acid.

Changes in artemisinic acid over time

There are only a limited number of studies which have related the concentration of artemisinic acid and dry matter yield of leaves to plant developmental stages by sequential harvesting from the vegetative stage to flowering. In an Australian (Tasmanian) study a Yugoslavian strain of Artemisia annua was transplanted in the field at the optimum time in October. The mean concentration of artemisinic acid in the leaves from the whole plant peaked in mid February – just before early bud and about the same time as artemisinin – and then decreased about threefold at full bloom (). In another Australian field experiment a Chinese selection of Artemisia annua which was transplanted in October gave a similar pattern of artemisinic acid accumulation which peaked just before the early bud stage. However in this case, the concentration of artemisinic acid only decreased by about 30% at full bloom (). In a study of the changes in artemisinic acid over a vegetation period in Vietnam, plants were sampled from the time of maximum leaf yield (mid June) to just before flowering (mid October). The mean leaf concentration of artemisinic acid decreased almost threefold (0.16% to 0.06%) over this period and to 0.02% at full bloom (10 November) ().

The data from all three experiments suggest that the concentration of artemisinic acid peaks at about the late vegetative or early bud stages and then decreases. The extent of the decreases vary greatly between strain selections and as with artemisinin the final yield of artemisinic acid hinges on the relative changes in the dry matter yield of leaves and flowers as well. The maximum yield of artemisinic acid in the Vietnamese study () and in the Australian study with the Yugoslavian strain () would have been in the vegetative stage. In the Australian study this was because artemisinic acid concentration decreased threefold while leaf + flower dry matter only doubled between the late vegetative and full bloom stages of growth. In contrast to artemisinin, the time of maximum artemisinic acid yield was influenced more by the decrease in acid concentration than by the increase in leaf + flower dry matter, (see Time of harvest, Method of harvest and Oil production).

Oil Production

Artemisia annua is the source of an essential oil which is located mainly in the leaves and flowers. In eastern Europe where Artemisia annua has been established as a weed for a lengthy period the oil has been extracted commercially in Hungary, Rumania and Bulgaria and has found a limited market for perfumery and as an anti-bacterial (). In Hungary a field experiment studying the potential of Artemisia annua as a source of oil found that the concentration of oil was at a maximum at full bloom and obtained oil yields of 20-40 kg/ha (). In Bulgaria other experiments showed that all parts of Artemisia annua contained the oil but it was mainly concentrated in the flowers with a maximum concentration of 3.2% in dried flowers at full bloom (). In the Ukrainian SSR a late maturing selection gave an oil yield of 111 kg/ha with a high perfume quality (). In later more detailed experiments it was confirmed that oil was mainly concentrated in the leaves and flowers with only trace amounts in the main stem, side branches and roots () with the highest concentration at flowering (). In excess of sixty individual chemical constituents have been reported in the oil distilled from Artemisia annua with a marked variation in composition between the selection used (). Artemisia ketone made the major contribution (68.5%) in a Chinese selection () but it formed a maximum of only 4.4% in a Vietnamese strain ().

Up to the present time the volume of Artemisia annua oil traded has been relatively small. However with the increased interest in Artemisia annua for the production of antimalarial drugs the potential volume of oil available is much greater and may induce greater interest from the perfumery or general chemical industries. The extraction of oil from Artemisia annua by steam distillation can denature many of the non-volatile sesquiterpenes (). However there may be strategies whereby a dual purpose use of Artemisia annua for antimalarial drugs and oil could be carried out. In a field experiment in Australia (Tasmania), European, USA and Chinese selections of Artemisia annua were harvested at the late vegetative and full bloom stages of growth. Whole plants were chipped into small segments and oil extracted by steam distillation for one hour. A comparison of distilled and non-distilled plants showed that while artemisinin was either completely denatured or showed only minute traces in the distilled plants, artemisinic acid was unaffected by distillation (). These results were similar for all selections of Artemisia annua and at both times of harvest. Depending on the concentration of artemisinic acid relative to artemisinin and on the concentration of oil it may be economic to consider a dual purpose production with artemisinin obtained by conversion from artemisinic acid (see Artemisinic acid, Time of Harvest and Method of harvest).

Time of Harvest

The optimum time of harvest of Artemisia annua will depend on the key target compound which is required. On the assumption that artemisinin is the main objective then maximum yield of artemisinin was at full bloom in most cases (). However this was not the case with a Vietnamese selection in a trial in that country where maximum yield was at the vegetative stage (). If artemisinic acid were the main objective then the limited amount of data available at present suggest that the late vegetative stage would give maximum yield (). In the situation where oil was the main objective or a dual purpose objective of oil and artemisinic acid then full bloom would be the preferred time. In the latter case of dual purpose use it would be important to use a selection of Artemisia annua which did not show a large decrease in artemisinic acid between the late vegetative stage and full bloom ().

Methods of Harvest

Hand harvest

There is little published detailed information on the method of harvesting Artemisia annua. In China and Vietnam, the two countries where the plant has been traditionally grown, it is reported that the crop is cut at the base, left to dry and then the leaves are removed by some form of shaking ().

Mowing

One of the earliest attempts at mechanical harvesting was that of Dr E. Croom of the University of Mississippi, USA who grew a crop of two hectares of Artemisia annua for the production of one kilogram of artemisinin for World Health Organisation experiments (). In this project the crop, which was grown in rows three feet apart (ca. 90cm), was successfully cut at the base with a tractor drawn mower (see Post harvest treatment).

Leaf stripping

Because artemisinin and artemisinic acid and oil are all concentrated mainly in the leaves and flowers the ideal harvesting technique would be some form of mechanical stripping of these components. Some preliminary trials of this method were carried out in Australia (Tasmania) using a standard “Mather and Platt” green bean harvester. Although obvious blockages occurred in these rudimentary trials with a completely unmodified machine the system showed some promise and should be explored further with suitable modifications (). Other techniques which should be explored and which could facilitate the possibility of leaf stripping as a viable method of harvest may be the use of leaf desiccants and growth (height) retardants (see Growth regulators). The importance of testing this method of harvest is because of the high percentage of main stem and lateral branch components with low artemisinin which must be handled in some way in alternative methods of harvest. At the late vegetative stage the fresh leaf contribution to total above ground plant yield was only 26.9% in a Vietnamese study () and 29% in Tasmania (). In the Tasmanian study where maximum artemisinin yield occurred at full bloom the composite fresh yield of leaves and flowers contributed 46% to total plant yield ().

Forage harvesting

In this technique the fresh plant is cut close to the base, or at any required height, mechanically chopped into small segments and blown into a trailing or following bin (). The second stage in this method involves kiln or some alternative form of drying before the separation of leaf (plus flowers) from stem segments by sieving. This system has been given a very preliminary screening in Tasmania () and although feasible it involves the obvious problem of cartage, handling and drying (see Post-harvest treatment). In any system of harvesting such as this it would be important that the drying and post-harvest handling installation be close to the area of production. The harvested plant mass in this system can heat up quickly and could result in losses of artemisinin. There are no published studies on this aspect but it is an area which merits investigation.

Cutting and field wilting

In this method plants are cut at or near the base and then left to lose moisture for a period of time appropriate to the climatic region of production. The next stage is to pick up the wilted plants with a forage harvester () which also has the facility for breaking the stem into pieces and blowing them into a trailing or following bin. This system has been successfully tested in Tasmania () and the advantage is the large loss of moisture (up to 50% or more) and hence the reduction in cartage and drying costs and time. The adaptability of this system will depend on the climatic conditions in the region of production, mainly temperature, rainfall, humidity and possibly ultra violet effects.

Post Harvest Treatment

Shade drying

In the USA, where two hectares were initially harvested with a tractor drawn mower, whole plants were transported to a large shaded enclosure and hung up for drying (). In India () where Artemisia annua has been introduced for experimental purposes the plants are harvested by cutting at the base, mechanically chipped into small segments and dried in the shade prior to leaf removal by sieving (). In more recent field studies in Madagascar (Antananarivo) whole plants were also dried under cover before leaf removal ().

Field (sun) drying

The wilting of Artemisia annua in the field after harvest with exposure to direct sunlight is believed to reduce artemisinin. Large decreases occurred when plants were dried in the open in India at Lucknow (). In comparisons of the effect of sun and shade drying in Oregon, USA, leaf (or branch) samples were dried (i) in open sunshine, (ii) in open sunshine protected by paper bags, and (iii) air dried under cover at ambient temperature. Air drying under cover gave the highest artemisinin concentration with sun drying the lowest and dried in bags intermediate between the two (). In this study the maximum air temperature was 30° C and the maximum sample temperatures were: sun 42.2° C, shaded 22.8° C and bagged 35.6° C. In another similar comparison of drying methods in Australia (Tasmania) whole plants were cut off at the base and either (i) sun dried in the open, (ii) shade dried or (iii) leaves were removed and oven dried at 35° C immediately after harvest. In this study the sun dried treatments were left for 7, 14 and 21 days and the shade dried for 21 days. The maximum air temperature in this trial was 22° C. There were no differences in artemisinin or artemisinic acid concentration between any of these treatments (). Maximum air temperatures may be the simple explanation for the differences between these two studies. However, given that sun drying would be most economical, if feasible, more detailed comparisons need to be carried out in a range climatic zones, especially sub-tropical, to confirm or otherwise the suspected effects of sun drying. To date no data are available for the effect of sun drying on artemisinic acid in hot continental or tropical environments and the effect of drying whole intact plants may give different results from drying stripped leaves or detached branches. Wilting and drying Artemisia annua plants in the sun appears to have no detrimental effects on oil content (). This is another incentive to obtain more data on the effect of sun drying on artemisinic acid because of the possibilities of dual purpose harvesting of these two products (see Oil production).

Oven (kiln) drying

Oven or kiln drying may be an expedient necessity in some areas where Artemisia annua could be cultivated. Although air drying of Artemisia annua has been commonly practised in experimental programs there are not many examples in the literature of detailed results from oven drying. In the study of Charles et al. (), drying in a forced fan oven at 30° C, 50° C and 80° C was included in the comparison with sun and air drying. Plants were dried at these temperatures for 12, 24, 36 and 48 hours respectively. Although generally the results of this study suggested that oven drying gave lower artemisinin than shade drying the variability was such that further studies are well warranted. In Australia (Tasmania) leaves of Artemisia annua were dried in a forced fan oven at 35° C, 45° C, 55° C and 65° C for 48 hours. On a relative scale the artemisinin concentration of leaves after these drying temperatures were 100, 97, 90 and 75 respectively. No comparison with air drying was made in this study ().

In a study in the USA, shoots of Artemisia annua were oven dried at 40° C and compared with indoor air drying and freeze drying. Indoor air drying gave the highest artemisinin concentration (0.13%), oven drying next (0.10%) and freeze drying lowest (0.02%) (). In this study some supplementary investigation was given to the effect of microwave drying. Drying fresh leaves for 2 minutes at 100% power virtually eliminated artemisinin while 5 minutes at 50% power reduced artemisinin concentration by about 50%. In this study () air drying gave 30% more artemisinin than oven drying at 40° C. There may be situations where the cost of drying and the reduction in artemisinin may be acceptable penalties for the speed and efficiency of production flow. The answer to this scenario would depend on the particular cost structure in the region of production. Here again it would be important to know what effect oven drying may have on the concentration of artemisinic acid in that this may be an alternative route to artemisinin.

Pelleting and crude extract production

In a number of situations the extraction of artemisinin or artemisinic acid may take place at a considerable distance from the site of Artemisia annua production: possibly in another country. In this event not only land but sea or air transport may be necessary and the volume and weight of the Artemisia annua plant material could be a problem. A. crude extract with an organic solvent such as hexane is a possible solution to this problem in that the volume of the extract would only be 5-10% of the original Artemisia annua plant material. If this method were to be used, pelleting of the Artemisia annua plant material () may be necessary to allow efficient movement of the plant material during the counter-current extraction process. It may be possible that the physical texture of the Artemisia annua leaf material could be suitable for extraction without pelleting but this could only be determined by experiment. In preliminary pelleting trials in Australia (Tasmania), in which the only heat involved was in the grinding and compression phases of the operation, there was no reduction in either artemisinin or artemisinic acid concentration of the final pellets compared with the original Artemisia annua leaf material (). However the feasibility of extraction from original unpelleted Artemisia annua plant material was not attempted in this study and experiments to establish this possibility would be well worthwhile.

Cost of Production

The cost of production of Artemisia annua in both the cultivation and post-harvest phases will vary widely with region and method of production used. However an obvious necessity is the availability of strains of Artemisia annua with a high artemisinin and/or artemisinic acid content which are adapted to the region of production. This is a vital necessity in order to make the production of antimalarial drugs profitable for the producer and affordable to the end user.

 

Selections from the book: “Artemisia”. Edited by Colin W. Wright. Series: “Medicinal and Aromatic Plants — Industrial Profiles”. 2002.