Chinese workers have reported that 30 other species of Artemisia have been examined but none yield extracts with antimalarial activity (Anon., 1981). American workers have extracted 10 species of Artemisia but again, none (except A. annua) were found to contain artemisinin (Klayman et al., 1984). In India, screening of 13 Artemisia species was carried out but only A. annua contained artemisinin (Balachandran et al., 1987).
The Chinese literature does not provide details of isolation methods used to prepare artemisinin from A. annua but it does indicate that ethyl ether, petroleum ether and even gasoline have been used as solvents although extraction with hexane for several days at room temperature was also effective (Lou and Shen, 1987).
In the USA, A. annua extraction has been done using several low-boiling solvents such as dichloromethane, chloroform, ether and acetone; however, petroleum ether (30-60° C) was found to be the most selective and therefore considered to be the solvent of choice (Klayman et al, 1984). The extract prepared at room temperature using dried, powdered plant material is filtered and submitted to evaporative crystallisation. The crude product is purified by chromatography on silica gel with chloroform-ethyl acetate as the eluate. Recrystallisation of artemisinin may be effected using cyclohexane (Klayman et al, 1984) or 50% ethanol (Anon., 1979). In another report the plant material is extracted with ethanol; the extracts are filtered
and evaporated and the residue is extracted with hexane or cyclohexane; the extracts are then submitted to evaporative re-crystallisation. The amount of artemisinin recovered depends upon the efficiency of the extraction process and the initial concentration in the plant (Haynes and Vonwiller, 1994).
In an improved method, petroleum ether extracts were treated with a protic solvent to remove much of the accompanying waxes and then further purification of the extract over silica gel gave artemisinin (Klayman et al., 1984).
In India, air dried leaves and flowers of A. annua were extracted with petroleum ether or n-hexane. The concentrated residue was extracted with methanol/ethanol to decrease the bulk of the material which was then loaded onto a column of silica gel. Elution of the column with ethyl acetate/hexane afforded a fraction which on crystallisation from ethyl acetate/hexane gave pure needles of artemisinin (Thakur and Vishwakarma, 1990).
Very little information was found in the literature concerning the large scale isolation of artemisinin from the plant. One procedure reported involves the use of an Ito-multilayer separator extractor. In another large scale extraction procedure the hexane extract of dried leaves of A. annua was partitioned with aqueous acetoni-trile; after concentration, the acetonitrile phase was chromatographed on a silica gel column. Artemisinic acid, 13, arteannuin B, 3, and two other sesquiterpenes were isolated (Elsohly et al., 1990).
The limited availability of artemisinin as well as the demand for more potent antimalarial drugs has necessitated the development of synthetic methods for the production of artemisinin. Several total syntheses of artemisinin have been reported Avery et al., 1992; Liu et al., 1993; Ravindranathan, 1994; Ravindranathan et al., 1990; Schmidt and Hofheinz, 1983; Xu et al., 1986; Zhou and Xu, 1994). However, all of these give low yields of the final product and are not an economically viable alternative to plant extraction for commercial scale production. In order to overcome the disadvantage of multistep organic synthetic reactions, the preparation of artemisinin from close biosynthetic precursors has been tested as an alternative. Artemisinin could easily be obtained from artemisinic acid in an overall yield of about 30-35% (Acton and Roth, 1992; Haynes and Vonwiller, 1991). Assuming that the abundance of artemisinic acid in the source is about ten times that of artemisinin as has been reported in several studies on Chinese A. annua material, the relative amount of artemisinin would be increased by four fold. At present, selection and cropping of plant material will be the method of choice for increasing the production of artemisinin. The use of plant cell and tissue cultures has not yielded fruitful results so far.
ARTEMISININ DERIVATIVES AND ANALOGUES Dihydroartemisinin Derivatives
Since the endoperoxide group is an essential requirement for the antimalarial activity of artemisinin the main emphasis has been to prepare artemisinin derivatives and related compounds without disturbing the peroxy linkage. Reduction of 1 with sodium borohydride yields dihydroartemisinin, 35 in which the lactone function is converted into a lactol (hemiacetal) group (Jeremic et al., 1973; Cao et al., 1982), dihydroartemsinin is more potent than 1 against P. falciparum in vitro (Gu et al., 1980). The lactol, 35, has been used to prepare several hundred derivatives such as ethers 35a (Li et al., 1979; 1981), esters 35b, carbonates 35c, etc. The ethers 35a (Li et al., 1979; 1981), have the advantage of being more oil soluble than 1 (Dutta et al., 1987). Artemether, the /3-methylether, 36, is the most active compound of all the ether derivatives. Arteethers, the «-isomer 37 and /3-isomer 38 were prepared by Brossi and colleagues (Brossi et al., 1988; Buchs and Brossi, 1989), and these are equipotent and 2-3 times more potent than 1. Later, arteethers 37 and 38 were prepared independently by El-Feraly et al. (1992) and Vishwakarma et al. (1992) respectively. (Note that the names artemether and arteether refer to the ^-isomers unless otherwise stated). Not unexpectedly, the deoxy compound, 42, was found to be 100-300 times less potent in vitro than its peroxy precursor showing that, as with artemisinin the peroxy linkage is necessary for biological activity.
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