A. Wilhelm Alfermann

Sustainable bioproduction of phytochemicals by plant in vitro cultures: anticancer agents

Due to their complex structure with several chiral centres important anticancer agents are still extracted from plants and not synthesized chemically on a commercial scale. Sustainable bioproduction of the compounds of interest may be achieved by plant in vitro cultures. Undifferentiated callus and suspension cultures, which can be cultivated in large bioreactors easily, very often fail to accumulate the compounds of interest, whereas shoot and root cultures as well hairy roots normally produce the same compounds as in the appropriate organs. The production of anticancer compounds, such as the alkaloids vinblastine, vincristine, paclitaxel (Taxol ® ), camptothecin, or the lignan podophyllotoxin, by plant in vitro cultures is reviewed. Taxanes can be produced in bioreactors using cell suspensions of various Taxus species with good yields; presently paclitaxel is produced on a commercial scale by Phyton Biotech (Germany). Camptothecin has low yields in suspension cultures of Camptotheca acuminata or Nothapodytes foetida (0.0003–0.01%), but a good production (0.1–0.3% dry wt) in root and hairy root cultures of Ophiorrhiza pumila , O. mungos and C. acuminata . Podophyllotoxin can be produced in cell suspension and root as well as hairy root cultures of Podophyllum and various Linum species up to 130 mg/l ( Linum album cell suspensions); its derivative 6-methoxypodophyllotoxin is accumulated in hairy roots of L. persicum up to about 500 mg/l. The in vitro production of dimeric indole alkaloids in Catharanthus roseus has failed so far both in undifferentiated and differentiated in vitro cultures. In cases where in vitro cultures show good yields, they can be employed in biotechnology for the sustainable production of valuable products.

Biosynthesis of podophyllotoxin in Linum album cell cultures

Abstract. Cell cultures of Linum album Kotschy ex Boiss. (Linaceae) showing high accumulation of the lignan podophyllotoxin (PTOX) were established. Enzymological studies revealed highest activities of phenylalanine ammonia-lyase, cinnamyl alcohol dehydrogenase, 4-hydroxycinnamate:CoA ligase and cinnamoyl-CoA:NADP oxidoreductase immediately prior to PTOX accumulation. To investigate PTOX biosynthesis, feeding experiments were performed with [2-13C]3′,4′-dimethoxycinnamic acid, [2-13C]3′,4′-methylenedioxycinnamic acid (MDCA), [2-13C]3′,4′,5′-trimethoxycinnamic acid, [2-13C]sinapic acid, [2-13C]- and [2,3-13C2]ferulic acid. Analysis of the metabolites by HPLC coupled to tandem mass spectrometry revealed incorporation of label from ferulic acid into PTOX and deoxypodophyllotoxin (DOP). In addition, MDCA was also unambiguously incorporated intact into PTOX. These observations suggest that in L. album both ferulic acid and methylenedioxy-substituted cinnamic acid can be incorporated into lignans. Furthermore, it appears that, in this species, the hydroxylation of DOP is a rate-limiting point in the pathway leading to PTOX.

Lignans in flowering aerial parts of Linum species - chemodiversity in the light of systematics and phylogeny.

The aerial parts of 54 accessions representing 41 Linum species and four species of related genera were analysed for lignans by means of HPLC-ESI/MS-MS-UV/DAD. In total, 64 different lignans of the aryltetralin-, arylnaphthalene-, aryldihydronaphthalene-, dibenzylbutyrolactone-, and furofuran type were identified. According to their lignan profile, the Linum species can be divided in two groups accumulating as major lignan types either cyclolignans of the aryltetralin-series on one hand, or aryldihydronaphthalenes/arylnaphthalenes, on the other. Five of the investigated Linum species did not contain any detectable amounts of these lignans under the chosen analytical conditions. Furthermore, none of the lignans identified in Linum species was detectable in representatives of three related genera, namely, Reinwardtia (Linaceae, Linoideae), Hugonia and Indorouchera (Linaceae, Hugonioideae). The two species groups differing in the types of the dominating cyclolignans comprise representatives of the major taxonomic sections. Representatives of sections Syllinum, Cathartolinum and Linopsis accumulate mainly aryltetralins while those of sections Linum and Dasylinum were found to contain mainly aryldihydronaphthalenes/-naphthalenes. These phytochemical data correlate very well with a recent study on the molecular phylogeny of Linum/Linaceae, where a subdivision of Linum into two major clades comprising representatives of the two mentioned groups was found. Thus, the distribution of lignans apparently reflecting phylogenetic interrelations at the infrageneric level, a plausible scenario for the evolution of lignan biosynthesis in the genus Linum can now be presented.

Tropane alkaloid patterns in plants and hairy roots of Hyoscyamus albus

Abstract Hyoscyamus albus is a solanaceous plant forming tropane alkaloids. The roots as the site of biosynthesis show a rich alkaloid spectrum of at least 26 compounds, whereas the alkaloid composition of the aerial parts is less complex. Some of the alkaloids, i.e. 3-propionyloxytropane, 3-isobutyryloxytropane, 3β-tigloyloxynortropane and 6-hydroxy-3-phenyl-acetoxytropane are described as components of H. albus for the first time. Three hitherto unknown compounds were tentatively identified as N-methylpyrrolidinyl-cuscohygrine, occurring in two isomeric forms, and as 3α-(p-hydroxyphenyl)lactoyloxytropane (4′-hydroxylittorine). Compared with plant roots, hairy roots show a rather similar but not identical alkaloid pattern. The low proportion of scopolamine in hairy roots is especially remarkable.

Taxol® and baccatin III production in suspension cultures of Taxus baccata and Taxus wallichiana in an airlift bioreactor

Summary Suspension cultures of Taxus baccata var. fastigiata and Taxus wallichiana were grown in a 20-L airlift bioreactor running for 28 days in a batch mode, and their growth rate and capacity to accumulate taxol® and baccatin III were measured. T. wallichiana culture showed a greater accumulation of taxol® and baccatin III than T. baccata culture (factor of 2.0 and 1.3, respectively) when both Taxus suspension cultures were in the highest productive state (from day 24 to day 28). This difference was mainly due to the higher capacity of T. wallichiana cells to synthesise the taxanes considered; another contributory factor was an increase in biomass in T. wallichiana culture relative to T. baccata culture. The highest yields of taxol® (21.04 mg L −1 ) and baccatin III (25.67 mg L −1 ) achieved for T. wallichiana culture exceeded those reported for other Taxus sp. suspension cultures. Approximately 40 % of the taxol® and 67 % of the baccatin III were released into the extracellular medium. Paclitaxel is the generic name for taxol®, which is a registered trademark of Bristol-Myers Squibb.

Metabolic Profiling of Lignan Variability in Linum species of Section Syllinum native to Bulgaria

Lignans in eighteen samples of Linum species ( L. tauricum ssp. tauricum, serbicum, bulgaricum and linearifolium; L. elegans; L. flavum ssp. sparsiflorum, L. capitatum var. laxiflorum), all members of the section Syllinum occurring in Bulgaria, were analysed by HPLC-ESI/MS and HPLC-UV/DAD. The ESI/MS fragmentation pathways recently established for aryltetralin lignans are now extended to ester and glycoside derivatives. In total, 22 different lignans, mainly of the aryltetralin type, were identified. 6-Methoxypodophyllotoxin and its glucoside were present as major constituents in all samples. Differences between the investigated taxa were observed especially with respect to the accumulation of 6-deoxy-7-hydroxy-aryltetralins such as podophyllotoxin and of 6-hydroxy-7-deoxy-aryltetralin lignans of the peltatin type. The distribution of aryltetralin lignans with different oxygenation patterns in the various samples, and correlations between the chemical data and the molecular phylogeny based on an analysis of ITS sequences of the investigated species are discussed.

Semicontinuous cultivation of photoautotrophic cell suspension cultures in a 20 l airlift-reactor

An airlift-bioreactor system was established for semicontinuous growth of photosynthetically active plant cell suspension cultures in a controlled environment. The bioreactor unit was constructed as a conventional, internal draught tube airlift-reactor, which is characterized by a H D-1 ratio of 2.9, a ratio of the cross-sectional area of the riser to the cross-sectional area of the downcomer of 0.25 and a surface area of 0.435 m2 for illumination. Cultivation experiments could be scaled up to working volumes of maximal 20 1. Sixteen fluorescent tubes were fixed around the outer glass cylinder to provide cells continuously with light. An external cooling device was used to keep the temperature constantly at 27°C. Agitation as well as supply with CO2 was performed by injecting air enriched with CO2 through a ring-shaped sparger at the bottom of the vessel. A first set of experiments was carried out with a photoautotrophic culture of Chenopodium rubrum L. Cell material adapted to large scale culture conditions was used to inoculate a modified MS medium (Murashige & Skoog 1962) without any organic constituents. Under these conditions a biomass increase of 1870% was achieved in 18 days. Several physiological parameters (e.g. pigmentation, photosynthetic O2 evolution, carbohydrate content) were measured routinely to elucidate the growth characteristics of large-scale grown Chenopodium cells. Electron microscopic photographs from different phases of culture growth clearly demonstrate the pattern of cellular development. Special emphasis was placed upon the differentiation of chloroplast ultrastructure. The presented data confirm the feasibility of large-scale culture techniques with photosynthetic active plant cell cultures.

Accumulation of lignans in suspension cultures of Linum mucronatum ssp. armenum (Bordz.) davis

Abstract For the first time callus and suspension cultures of Linum mucronatum ssp. armenum were initiated, grown in darkness at 25 °C and analyzed for lignans. 6-Methoxy-podophyllotoxin was the main lignan besides smaller amounts of podophyllotoxin isolated and identified by chromatographic methods as well as by 1H NMR.

Linum mucronatum: Organ to Organ Lignan Variations

The percentage of podophyllotoxin (PTOX) and its congener lignans were measured by HPLC in Linum mucronatum ssp. mucronatum (Linaceae) fresh plant organs. The highest amounts of PTOX (0.595 ± 0.060% g/g dry wt) and 6-methoxypodophyllotoxin (MPTOX) (1.491 ± 0.125% g/g dry wt) were found in the plant sexual organs. Whereas, the highest levels of β-peltatin, 5′-demethoxy-MPTOX and yatein were found in not developed buds, petals and sepals, respectively.

Identification of Tropane Alkaloids in Hairy Root Cultures of Hyoscyamus albus

Abstract Tropane alkaloids are typical natural products of solanaceous plants. The patterns of these alkaloids from hairy roots of Hyoscyamus albus were determined by GC-MS analysis. 18 alka-loidal compounds were detected, six of them only in trace amounts. Some of these alkaloids, namely hygrine, 3a-acetoxytropane, 3 β-acetoxytropane and 6-hydroxylittorine as well as the trace compounds N-methylpyrrolidinylhygrine, phenylacetoxytropane and two isomers of feruloyloxytropane were not previously reported to be components of this species. The major compounds formed were found to be hyoscyamine and littorine.