Ephedra Species
Botanically, Ephedra () is a member of the smallest and most problematic division of flowering plants, the Gnetopsida, and major questions remain unanswered about the taxonomy of the Gnetopsida and the evolutionary relationships of the different genera within the division. Ephedra is the largest and most widely distributed genus in the Gnetopsida, a subgroup of the gymnosperms. Many anatomical and reproductive characters of Ephedra are angiosperm-like. Recent molecular and chemical studies support the view that the Gnetopsida are the closest living relatives of the angiosperms but that the angiosperms are not derived from them.
Pharmacologically, Ephedra has been the main botanical source of the active alkaloids l-ephedrine (E) and d-pseudoephedrine (PE) for thousands of years, with records of its medicinal use dating to 5000 years b.p.. The alkaloids E and PE remain important drugs today – the current world consumption of d-pseudoephedrine salts (PE-sulphate and PE-hydrochloride) stands at 1000-2000 tonnes per annum with a value of approximately $100-200 million.
Powdered Ephedra stems are used in traditional herbal medicines as a hypertensive aid to treat asthma, nose and lung congestion, hay fever, and several other ailments. E. sinica and E. equisitina are the two main species exploited. The role of l-ephedrine and d-pseudoephedrine in modern medicine has been changing slowly as new applications for these alkaloids are realised. Acting as potent vasoconstrictors, they can be used to elevate blood pressure and increase heart and respiratory rate. They are very close to adrenaline in structure and pharmaceutical activity, but have the advantage that they can be administered orally. Uses for the Ephedra alkaloids continue to expand. L-ephedrine (E) is currently under investigation as a therapeutic agent for the treatment of obesity. L-ephedrine has been linked with toxic psychosis with the result that the demand is increasing for the stereoisomer, d-pseudoephedrine (PE), which has fewer side effects than 1-ephedrine and a weaker, longer-lasting effect upon the central nervous and cardiac systems. A recent development is the introduction by Schering-Plough of a new combination product containing pseudoephedrine sulphate and Loratadine, a non-sedating antihistamine.
China and Pakistan are the main producers. Attempts have been made to cultivate Ephedra in Australia, England, Kenya and the United States, but these ventures failed, largely for economic reasons.
Although it is currently possible to manufacture these compounds from benzaldehyde via a yeast fermentation and catalytic reduction, several species of Ephedra continue to be exploited for these high-demand pharmaceutically active compounds. Therefore, the continued importance of these well-known Ephedra alkaloids appears assured for some time to come, and there is the prospect that other pharmacologically active agents may be found in Ephedra and other members of the Gnetopsida.
Distribution of the Ephedrines
From the definitions outlined by Southon and Buckingham (1989), the Ephedra alkaloids, 1-ephedrine (E), nor-ephedrine (NE), d-pseudoephedrine (PE) and nor-pseudoephedrine (NPE), can be classed as protoalkaloids. This definition is supported by Hegnauer (1988), who classes alkaloids according to their biogenetic pathways. These compounds are also referred to as phenylalkylamines or phenylpropanoids, terms which give more information on their structure. Nevertheless, with these words of caution, we will refer loosely to the ephedrines as alkaloids.
The literature shows that there appears to be wide variation in alkaloid content between species. However, several problems arise in assessing alkaloid yields of Ephedra. Firstly, the compounds 1-ephedrine (E), d-pseudoephedrine (PE), nor-ephedrine (NE), nor-pseudoephedrine (NPE), methyl-ephedrine (ME), and methyl-pseudoephedrine (MPE) are difficult to separate and identify.
Many published reports of alkaloid production by Ephedra () have relied upon TLC; some do not refer to the analytical method used. In fact, TLC is not sufficiently discriminating on its own to differentiate between the Ephedra alkaloid stereoisomers. Moreover, ethanol extracts of parent plant and in vitro-grown tissues often contain compounds which cochromatograph with E and PE standards and stain positively with ninhydrin, but which HPLC analysis reveals are not phenylalkylamines. The reported occurrence and levels of alkaloids in the Ephedra genus, especially in the older references depending only on TLC, must therefore be questioned.
Analytical techniques such as HPLC, which accurately determine and quantify these compounds, are quite recent developments. It is now feasible to survey the distribution of phenylalkylamines throughout the Ephedra genus and to construct chemical profiles within and between species. Such analysis would enable the critical testing of the suggestion that there is evidence of chemical races within the species Ephedra distachya which differ in their alkaloid composition. It may be possible to distinguish between genetic and environmental factors influencing alkaloid synthesis.
It is also recognised that there is a marked seasonal variation in the alkaloid yields of Ephedra (). Furthermore, by our own observation, uneven sampling of plant tissues and the age of the tissues sampled affects the recorded alkaloid levels. Kasahara et al. (1985) found that the total alkaloid content in nodes of several Ephedra species was only 40% of the concentration found in the internodes (approximately 0.29 and 0.69% dry wt., respectively). Variation in alkaloid content also occurs along the length of the stem with low quantities in the very young tip growth and the basal, woodier tissues.
It is still not known where the site/s of synthesis of the compounds are located in the plant and it is important to note that high levels in certain tissues may indicate sites of accumulation rather than of production. As much of this chapter concerns the secondary metabolism of Ephedra, we suggest Gottleib and Kubitzki (1984) for a review of flavonoid, terpenoid and other constituents of this genus.
In summary, there is strong evidence that the age and type of tissue sampled, environmental growth conditions and the method of analysis used all strongly influence the alkaloid content measured for that species.
Ephedra Species: Summary and Conclusion
Our interest was to establish whether an enzyme(s) exists which interconverts ephedrine and pseudoephedrine, hence the choice of Ephedra for our studies. The infection of Ephedra with Agrobacterium rhizogenes led to the production of tumorous tissues capable of low-level, but stable, alkaloid production. Further work in this direction could prove beneficial in establishing a model experimental system.
Kutchan (1995) states that many of the problems with elucidating plant secondary biosynthetic pathways have often stemmed from the complexity of the molecules being studied. With Ephedra the problems are almost the reverse. Due to the relative simplicity of the ephedrine alkaloid molecules and their apparent similarity to many compounds produced by both parent plant and in vitro cultures, it was difficult to monitor their production. In our experience, HPLC/mass spectroscopy could not distinguish several compounds isolated from collected HPLC fractions or TLC spots. A more specific method for the identification of these compounds is required. We raised antibodies to E and PE for identifying the presence of alkaloids in plant, culture and cell-free extracts and for locating them in plant sections and cells. An ELISA assay for the determination of ephedrine was developed, but further work would be required to clean up the antiserum to ensure its monospecificity towards ephedrine. Using radiolabelled Ephedra alkaloids may reveal whether interconversion occurs between the alkaloids and could help trace the fate of any intermediates. It may prove unnecessary to develop an in vitro system by applying such radiolabelled compounds through the base of cut stems or by painting them onto the stem surface. This approach, using intermediates from the early steps of the pathway, has yielded most of the current information on E/PE biosynthesis.
The problems with elucidating the ephedrine/pseudoephedrine bio-synthetic pathway are twofold. Firstly, there is the need for a system in which the alkaloids are being actively biosynthesised and to which precursors and cofactors can be added and in which the flux of compounds can be traced. Secondly, there is a need to develop systems for separating, isolating and identifying the compounds involved. An in vitro system capable of producing ephedrine and pseudoephedrine at commercial levels from Ephedra cultures is not possible at present. What is sought is a system capable of detectable alkaloid production, even at a low level, as long as it is reliable. This would offer a model suitable for enzyme studies to elucidate secondary biosynthetic pathways. Theoretically, this is easier in vitro, as cultured tissues are more homogeneous, contain fewer phenolics, and precursor or elicitor feeding and environmental conditions are more easily controlled. If it proves possible to identify enzymes catalysing the reactions in the ephedrine and pseudoephedrine biosynthetic pathway, it may become possible to isolate DNA encoding these proteins. The problem has been to develop cultures which reliably produce ephedrine and pseudophedrine. Reevaluation of culture conditions and media is one possibility.
The study of alkaloid biosynthesis has recently started to fulfil some of the early promises. Biosynthetic pathways are being characterised, e.g. tropane alkaloids from several Solonaceae species, benzophenanthridine alkaloid synthesis in Eschscholtzia californica (). The cDNA for enzymes from plant secondary biosynthetic pathways are being expressed in other organisms, e.g. strictosidine synthase from Rauwolfia serpentina, has been expressed in Escherichia coli (); the berberine bridge enzyme from Eschscholtzia californica has been expressed in insect cell culture. If it proves possible to identify enzymes catalysing the reactions in the 1-ephedrine and d-pseudoephedrine biosynthetic pathway, it may subsequently lead to the isolation of cDNA encoding these proteins. It has been envisaged that such DNA could be inserted into the bacterial genome and be expressed. With Ephedra, it may be sufficient to express only the final step or few steps of the alkaloid biosynthetic pathway in a fermentation system. Cheap, bulk precursors could be fed to the system or cheap ephedrine to a system that converts it to the more valuable pseudoephedrine (if such a step is enzymatically catalysed).
Such approaches are commercially valid where the product is of high value and the market demand hard to satisfy from natural sources, especially where chemical synthesis is not a viable option. Novel compounds from in vitro systems could be similarly exploited. Each case requires evaluation upon its own merits. In the case of Ephedra we are dealing with pharmaceutical compounds which command a low price but are of value on account of the size of the market. With a growing world population and a growing per capita demand for medicines, the ephedrine/pseudoephedrine market will probably continue to expand, especially in its role as an antiasthmatic. Producing Ephedra alkaloids by a fermentation system may prove necessary if demand outstrips the rate of production by current means.
Studies to improve natural methods of production should parallel biotechnological research. In this and other studies, protocols have been established for improved identification of alkaloid profiles in plants. High alkaloid-yielding or high pseudoephedrine-producing individuals can be identified or, perhaps, engineered by molecular techniques, e.g. by preventing catabolism or blocking competitive pathways. Micropropagation methods could then be used to produce large numbers of clones to supplement the alkaloid yield of the natural population. This approach could improve the economics of the natural production of the ephedrine alkaloids.
Selections from the book: “Medicinal and Aromatic Plants X”, (1998).
