J. D. Phillipson

Antimalarial activity of Artemisia annua flavonoids from whole plants and cell cultures

SummaryCell suspension cultures developed from Artemisia annua exhibited antimalarial activity against Plasmodium faldparum in vitro both in the n-hexane extract of the plant cell culture medium and in the chloroform extract of the cells. Trace amounts of the antimalarial sesquiterpene lactone artemisinin may account for the activity of the n-hexane fraction but only the methoxylated flavonoids artemetin, chrysoplenetin, chrysosplenol-D and cirsilineol can account for the activity of the chloroform extract. These purified flavonoids were found to have IC50 values at 2.4 – 6.5 × 10−5M against P. falciparum in vitro compared with an IC50 value of about 3 × 10−8M for purified artimisinin. At concentrations of 5 × 10−6M these flavonoids were not active against P. falciparum but did have a marked and selective potentiating effect on the antiplasmodial activity of artemisinin.

Antiplasmodial activity of Artemisia annua plant cell cultures.

Extracts of Artemisia annua cultures have been assessed for in vitro activity against the malarial parasite Plasmodium falciparum. Callus and suspension cells and medium were analysed and examined for their activity at different stages of growth and development. Time-course experiments were carried out to investigate the influence of various basal media, plant growth regulators and light on both growth and possible artemisinin production. Two active fractions were obtained but artemisinin was not detected.

In vitro cultures of Cinchona species : Precursor feeding of C. ledgeriana root organ suspension cultures with L-Tryptophan.

The uptake of l-[methylene-14C]-tryptophan from culture medium into root organs of Cinchona ledgeriana and the subsequent incorporation of the radiolabel into quinine and quinidine is reported. In addition, feeding unlabelled l-tryptophan at levels of 500mg/l to the cultures results in a 5-fold increase in the yields of both quinoline alkaloids.

Studies on Ailanthus altissima cell suspension cultures: precursor feeding of L-[methylene-14C]tryptophan and L-tryptophan

The uptake of L-[methylene 14C]-tryptophan from culture medium and the subsequent incorporation of the radiolabel into canthin-6-one, 1-hydroxycanthin-6-one and 1-methoxycanthin-6-one has been demonstrated in cell suspension cultures of Ailanthus altissima. Efficient incorporation has been shown to depend significantly on the time of feeding. Furthermore, feeding of L-tryptophan, at levels of 500 mg/l resulted in improved alkaloid yields, particularly when fed during the lag phase of the growth cycle.

Studies on Ailanthus altissima cell suspension cultures. The effect of basal media on growth and alkaloid production.

Time-course experiments have been carried out to investigate the relationship between growth and alkaloid accumulation in A. altissima cell suspension cultures. Results indicate that the type of basal medium, viz. Murashige and Skoog, Linsmaier and Skoog, Schenk and Hildebrandt or Gamborgs B5, has a significant influence on both growth and alkaloid production.

Alkaloids from the seeds of Strychnos wallichiana Steud. ex DC. (Strychnos cinnamomifolia Thwaites var. wightii A. W. Hill)

The seeds of a south Indian sample of Strychnos wallichiana Steud. ex DC., previously analysed by Short (1924) under the name S. cinnamomifolia Thwaites and thought to contain mostly brucine with a little strychnine, have now been shown to contain a new base 4‐hydroxy‐3‐methoxystrychnine (I) as the major alkaloidal constituent. Smaller amounts of strychnine, 4‐hydroxystrychnine, brucine, vomicine, 4‐hydroxy‐3‐methoxy‐N‐methyl‐sec.‐pseudostrychnine (II), and nova‐cine and possibly also α‐colubrine are present.

The tertiary alkaloids of some Asian species of Strychnos

In our screening program for alkaloids, the extracts from more than 200 samples mostly from herbarium collections, belonging to 34 Asian Strychnos species, have been examined by t.1.c. and g.1.c. methods. The results obtained with S. nux-vomica L. and S. wallichiana Steud. ex DC. (S. colubrina L.) are particularly interesting in that:1. The alkaloid composition of the leaf and seed, irrespective of age (up to 300 years old) appeared to be unchanged. 2. Both species contained alkaloids of the following types:-Normal series: strychnine, brucine, strychnine N-oxide, brucine N-oxide; pseudo series: pseudostrychnine, pseudobrucine; N-methyl-pseudo series : icajine, vomicine, novacine. 3. Examination of different plant parts of the two species showed that in the root bark and root wood alkaloids of the normal series tend to predominate; in the stem bark pseudo and N-methyl-pseudo alkaloids are the most important; in the leaves the main alkaloids belong to the N-methyl-pseudo series (cf. Maier & Groger, 1968; Sefcovic, Dubravkova & Torto, 1968); and in the seeds again normal seriesbases Predominate. There is evidence that in S. nux-vomica the normal bases are formed in the roots (Schlatter, Waldner & others, 1969). Our data from S. nux-vomica and S. wallichiana suggest that as the alkaloids are transported up the plant through the wood they are gradually converted from bases of the normal series to bases of the pseudo and N-methyl-pseudo series, so that when they reach the leaves the Nmethyl-pseudo alkaloids predominate. It is possible that the reverse process may be taking place if the alkaloids descend from the leaves through the bark. Among the other species screened were:1. S. ignatii Berg., seed samples of which gave results very similar to those of S. nuxvomica. 2. S. nux-blanda A. W. Hill, leaf and seed samples of which contained small amounts of alkaloids similar in composition to those of S. nux-vomica except for the frequent occurrence of diaboline. 3. S. potatorum L.f., which contained diaboline as the major alkaloid in the leaves, seeds, and bark.

Metabolic TV-oxidation of atropine, hyoscine and the corresponding nor-alkaloids by guinea-pig liver microsomal preparations

Incubation of guinea‐pig liver microsomal preparations with atropine or hyoscine resulted in the formation of the corresponding nor‐alkaloids and both isomers of atropine N‐oxide from atropine and one isomer of hyoscine N‐oxide from hyoscine. Separate incubations of guinea‐pig liver microsomal preparations with nor‐atropine and nor‐hyoscine yielded the corresponding hydroxylamines. The N‐oxide and hydroxylamine metabolites were identified by comparison of their t.l.c. behaviour and m.s. with prepared compounds and also by their reduction to the corresponding tertiary or secondary amines.

Studies on Ailanthus altissima cell suspension cultures. Uptake of L-[methyl-14C]methionine and incorporation of label into 1-methoxycanthin-6-one

Time-course studies of the uptake of L-[methyl 14C]-methionine have shown rapid uptake by Ailanthus altissima cells when fed at weekly stages throughout the growth cycle. The radio-label from [14C]-methionine was shown to be incorporated into 1-methoxycanthin-6-one with the highest level of incorporation being achieved with cells fed late in the growth phase.

The characterization of alkaloid D, a new alkaloid from Euonymus europaea L., as armepavine

component of the glucoside and the lipid fractions, but tended to accumulate in mature tissues. Stigmasterol, the C-22 unsaturated isomer, accumulated in young leaves and immature flower buds. The proportions of campesterol were generally similar in most organs with the exception of the roots where larger quantities were detected. It was interesting to note that concentrations of cholesterol glucoside were highest in the younger organs, and it is possible that these tissues are the sites of sterol metabolism to the cardenolides and sapogenins. It is evident that biosynthesis of sterols occurs in the free form, and that either glucoside or ester formation occurs selectively at a stage when biosynthesis is complete, suggesting that a segregation of biological roles could lie behind this enzymatic selection. Free phytosterols have been implicated in the structure of cell and organelle membranes in association with phospholipids (Ansell & Hawthorne 1964; Evans 1971), and it has b&n suggested by Kemp, Goad & Mercer (1967) that ester sterols represent an intercellular transportation form. The areas of sterol requirement in the mature plant are the actively growing areas, and the export of sterols from mature leaves at the base of the plant could satisfy a heavy sterol requirement. This would involve the phloem transportation of sterols, possibly as the more hydrophilic glucosides, via the stem to the young leaves and developing inflorescence. The high phytosterol glucoside concentrations of these organs add weight to this suggestion.