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;Pectin administered to Uncaria tomentosa cell suspension cultures, was found to increase the production of triterpene acids (ursolic and oleanolic acid), however, neither growth nor sterol accumulation were affected. Cell cultures showed that pectin treatment caused a rapid threefold increase in the activities of enzymes involved in the biosynthesis of C 5 and C 30 isoprenoid, such as isopentenyl diphosphate isomerase and squalene synthase. The activity of a farnesyl diphosphatase, which could divert the flux of farnesyl diphosphate to farnesol, was two times lower in elicited than in control cells. Elicited cells also transformed more rapidly a higher percentage of [5-3 H]mevalonic acid into triterpene acids. Interestingly, addition of terbinafine, an inhibitor of squalene epoxidase, to elicited cell cultures inhibited sterol accumulation while triterpene production was not inhibited. These results suggest that in U. tomentosa cells, both the previously mentioned enzymes and those involved in squalene 2,3-oxide formation play an important regulatory role in the biosynthesis of sterols and triterpenes.

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The effect of oxidative stress on indole alkaloids accumulation by cell suspensions and root cultures of Uncaria tomentosa in bioreactors was investigated. Hydrogen peroxide (H2O2, 200 μM) added to U. tomentosa cell suspension cultures in shaken flasks induced the production of monoterpenoid oxindole alkaloids (MOA) up to 40.0 μg/L. In a stirred tank bioreactor, MOA were enhanced by exogenous H2O2 (200 μM) from no detection up to 59.3 μg/L. Root cultures grew linearly in shaken flasks with a μ=0.045 days−1 and maximum biomass of 12.08±1.24 g DW/L (at day 30). Roots accumulated 3α‐dihydrocadambine (DHC) 2354.3±244.8 μg/g DW (at day 40) and MOA 348.2±32.1 μg/g DW (at day 18). Exogenous addition of H2O2 had a differential effect on DHC and MOA production in shaken flasks. At 200 μM H2O2, MOA were enhanced by 56% and DHC by 30%; while addition of 800 and 1000 μM H2O2, reduced by 30–40% DHC accumulation without change in MOA. Root cultures in the airlift reactor produced extracellular H2O2 with a characteristic biphasic profile after changing aeration. Maximum MOA was 9.06 mg/L at day 60 while at this time roots reached ca. 1 mg/L of DHC. Intracellular H2O2 in root cultures growing in the bioreactor was 0.87 μmol/g DW compared to 0.26 μmol/g DW of shaken flasks cultures. These results were in agreement with a higher activity of the antioxidant enzymes superoxide dismutase and peroxidase by 6‐ and 2‐times, respectively. U. tomentosa roots growing in the airlift bioreactor were exposed to an oxidative stress and their antioxidant system was active allowing them to produce oxindole alkaloids.

Treatment of a Cinchona robusta How. cell suspension culture with a homogenate of Phytophthora cinnamomi resulted in cessation of growth and a rapid induction of the biosynthesis of anthraquinone-type phytoalexins. The strongest induction of anthraquinone biosynthesis was obtained when the elicitor was added in the early growth phase of the growth cycle. The accumulation of anthraquinones was accompanied by a tri-phasic response in the activity of isopentenyl diphosphate (IPP) isomerase (EC 5.3.3.2): phase I was characterised by a rapid induction of activity, reaching a maximum at 12 h after elicitation. During phase II, IPP isomerase rapidly decreased to levels below those found in untreated cells. At phase III, IPP isomerase activity increased again, reaching a second maximum at about 72 h after elicitation. During phase I, the activity of farnesyl diphosphate synthase (EC 2.5.1.10) was found to be suppressed. Extraction and assay conditions were optimised for IPP isomerase. The presence of Mn 2+ in the incubation buer resulted in a marked increase in the activity of the enzymes obtained from cells in phase I. The induction of IPP isomerase in combination with a concomitant inhibition of farnesyl diphosphate synthase might result in an ecient channeling of C 5 -precursors into phytoalexin biosynthesis.

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The alkaloids of Uncaria tomentosa micropropagated plantlets and root cultures were isolated and identified by NMR and mass spectrometry. Plantlets yielded pteropodine (1), isopteropodine (2), mitraphylline (3), isomitraphylline (4), uncarine F (5), speciophylline (6), rhynchophylline (7) and isorhynchophylline (8). In plantlets growing under continuous light, tetracyclic alkaloids 7 and 8 decreased from 20 ± 1.8 at 2 months to 2.2 ± 0.33 mg/g dry wt at 6 months, while the pentacyclic alkaloids 1-4 increased from 7.7 ± 1.4 to 15 ± 0.05 mg/g dry wt, supporting their biogenetic conversion. Micropropagated plantlets produced four times more alkaloids (27.6 ± 3.1 mg/g dry wt) than greenhouse plants. Plantlet roots yielded 3, 4, 8 and the glucoindole alkaloids 3α-dihydrocadambine (9) and dolichantoside (10), the last one not previously found in Uncaria.

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