Sourcing from Plants
Several approaches could be envisaged to source 20E or other ecdysteroid analogues, but, currently, isolation from an ecdysteroid-accumulating plant species is the only scientifically and financially viable option. Currently, only 20E is commercially available in large amounts, so this will be used as the example in the discussion below:
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It is perhaps worth reminding ourselves first of the criteria that an ideal plant source should fulfil:
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The plant should accumulate a high amount of 20E;
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The plant should have a simple ecdysteroid profile (ideally just 20E);
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The plant should be easy and rapid to grow in accessible areas of the world;
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The ecdysteroids should be present in the aerial portions (allowing the roots to regenerate the plant);
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The plant matrix should be amenable to the ready purification of ecdysteroids;
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Purification and isolation of 20E should not involve expensive chromatographic methods;
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The plant should not be susceptible to pests and diseases;
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The species should not be rare or protected;
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Culture, harvesting and processing costs should be low; initial processing should take place at the culture site.
Clearly, no plant species will fulfil all these criteria fully, but the closer it comes, the more culturally and commercially viable the challenge of isolating adequate amounts of 20E will become.
Table 1 provides data for the concentrations of 20E in specified parts of selected ecdysteroid-accumulating species. Most ecdysteroid-accumulating species contain between 0.01% and 0.1% of the dry weight as ecdysteroids, whereas the few identified high-accumulators contain 1% and above. The roots of
Cyanotis spp. can contain up to 5% of its dry weight as ecdysteroids, largely as 20E
[165]. Mature stems of
Diploclisia glaucescens were found to contain 3.2% of its dry weight as 20E
[166], but the collection of the stems of this South Asian climber precludes this as a feasible source of large amounts of 20E.
The relationship between the presence of phytoecdysteroids and plant taxonomy is complex, but high accumulation is associated with certain species in the genera Achyranthes (Amaranthacea), Cyanotis (Commelinaceae), Pfaffia (Amaranthaceae), Rhaponticum (syn. Leuzea/Stemmacantha; Asteraceae), Serratula (Asteraceae) and Silene (Caryophyllaceae).
The vast majority of currently available commercial sources of 20E are derived from Cyanotis spp., Pfaffia spp., Rhaponticum (Leuzea) spp. or Serratula spp. In addition, turkesterone (11α-hydroxy20E) from Ajuga turkestanica is commercially available.
It has been shown in several ecdysteroid-accumulating species that ecdysteroid levels are additionally influenced by environmental factors (e.g., temperature, nutritional factors, invertebrate predation
[35]). Thus, to maximise 20E content in a chosen species, it is not only necessary to select a high producing genetic line (cultivar) but also to optimize the growth conditions and to consider treating the plants with appropriate elicitors (e.g., methyl jasmonate to mimic insect or fungal attack) at suitable times in development.
Various researchers have started to explore a range of culture methods in vitro (e.g., micropropagation, callus cultures, plant cell culture, hairy-root cultures, transformed yeast fermentation) in the hope of obtaining more amenable ecdysteroid-producing systems. To date, only the hairy roots of
Ajuga reptans var.
atropurpurea [167] consistently and reliably produce ecdysteroids, but the culture is too expensive to provide a commercial source of 20E. Additionally, the hairy roots of some other ecdysteroid-producing species do not contain ecdysteroids, so this cannot be viewed as a general approach. Transforming yeast to biosynthesise ecdysteroids, as has been previously done for certain vertebrate steroids
[168][169], is an attractive prospect but is currently confounded by our incomplete knowledge of the ecdysteroid biosynthetic pathways in either invertebrates or plants and the probable number of genes involved in the complete pathway(s)
[3].
The major requirements in the processing of plant material to obtain a natural product in adequate amounts for commercial use are simplicity, efficiency of extraction and purification, reproducibility and cost-effectiveness. As explained above, the choice of plant material is key since not only should it contain a large amount of the target molecule, but the plant matrix should be readily extractable and not contain components (e.g., large amounts of polysaccharides) that make processing difficult or significant amounts of close analogues of the target molecule, which would be difficult to separate out without moderate-to-high-resolution chromatographic methods. Given that a suitable plant source will contain typically 1–2% of its dry weight as 20E and that a pharmaceutical/medicinal dose of 20E is likely to be in the 100 mg–2 g/day range, it is necessary to be able to process tonnes of plant material. Clearly, it would be highly advantageous if the harvested plant material could be cleaned, dried, broken up (to reduce volume and increase the surface area) and subjected to initial extraction as close to the site of harvesting as possible, as the mass of the initial extract is probably only 1–2% of the fresh weight of the plant material and, therefore, much more readily and cost-effectively transportable. The extraction and purification have to be optimised with regard to the physico-chemical properties of the target molecule (generally already known or readily determinable) and the nature of the plant matrix (generally only vaguely understood). Owing to the large number of hydroxyl groups in 20E, it is highly soluble in alcohols, so the dried, powdered plant material will usually be extracted with methanol (preferred as it is cheaper) or ethanol (if the product is to be BIO), with or without prior extraction with a non-polar solvent, such as petroleum ether, to de-fat the plant material. Extraction may occur with heating, stirring or maceration for a defined time, all of which need to be optimised. Other methods, such as super critical fluid extraction or bi-phasic extraction, could be used, but these are generally more expensive and would only be cost-effective if the target molecule is potent (low daily dose required) and high-value, which is not the case for 20E.
Figure 12 provides a flow diagram of a representative processing method for Cyanotis sp. roots, where the dried plant material is refluxed thrice with ethanol and the pooled extracts are filtered before being passed through a macroporous resin to absorb plant compounds and vacuum-dried to yield a powder containing 90% 20E.
Figure 12. A representative flow diagram for the large-scale extraction and purification of 20E from roots of
Cyanotis sp. (taken from
[170]).
This preparation can then be recrystallised twice to bring the purity of the 20E to >97%. A comparison of the RP-HPLC profiles of the 90% and 98% 20E preparations is shown in the inset to Figure 10. Most of the minor peaks in the chromatograms correspond to other ecdysteroids, which are very difficult to separate fully from 20E by crystallisation. This underlines the need to start with plant material that contains essentially only 20E.
Owing to the need for optimisation at each stage of the extraction and purification, the process should be developed in stages, going from small-scale (100 g—a few kg of dry plant material) in the laboratory through increasing medium-scale stages (e.g., 10–500 kg), before being applied at the industrial scale (>1 tonne), so that difficulties can be identified and resolved early on and any problems of scale-up can be dealt with. A thorough cost analysis needs to be performed throughout the scale-up procedure to ensure that the target molecule can be brought to market at a viable cost.
For pharmaceutical-grade preparations, the API has to be prepared by a standardised procedure to a defined level of purity (e.g., >97%), and all impurities at levels of >0.5% must be identified, quantified and assessed for toxicity and effects.