Combining metabolomic analysis and microarray gene expression analysis in the characterization of the medicinal plant Chelidonium majus L.

BACKGROUND AND OBJECTIVE: Even though herbal medicines have played an important role in disease management and health for many centuries, their present frequent use is challenged by the necessity to determine their complex composition and their multitarget mode of action. In the present study, modern methods were investigated towards their potential in the characterization of herbal substances. As a model the herbal substance Chelidonii herba was used, for which several reports on liver toxicities exist. Extracts of Chelidonii herba with different solvents were characterized phytochemically and functionally by experiments with HepG2 liver cells. METHODS: Chelidonii herba was extracted with four solvents of different polarity (dichloromethane, water, ethanol, and ethanol 50% (V/V); four replicates each). The different extracts were characterized metabolomically by (1)H-NMR fingerprinting analysis and principal component analysis (PCA). The content of alkaloids was additionally determined by RP-HPLC. Functional characterization was achieved by the determination of cell proliferation and by transcriptomics techniques (Whole Genome Gene Expression Microarrays v2, Agilent Technologies) in HepG2 cells after exposure to the different extracts (four experimental replicates each). RESULTS: Based on data from (1)H-NMR fingerprints and RP-HPLC analyses the different extracts showed a divergent composition of constituents depending on the solvent used. HepG2 liver cells responded differentially to the four extracts. Microarray analysis revealed a significant regulation of genes and signal cascades related to biotransformation. Also liver-toxic signal cascades were activated. Neither the activated genes nor the proliferation response could be clearly related to the differing alkaloid content of the extracts. CONCLUSION: Different manufacturing processes lead to different herbal preparations. A systems biology approach combining a metabolomic plant analysis with a functional characterization by gene expression profiling in HepG2 cells is an appropriate strategy to characterize variations in plant extracts. Safety assessments of herbal substances may benefit from such complementary analyses.

Generation and characterisation of monoclonal antibodies to oleanolic acid.

Monoclonal antibodies to oleanolic acid, a pentacyclic triterpene, were generated using the hybridoma fusion method described by Kohler and Milstein . Protein conjugates of the target molecule for immunisation were prepared either by directly linking the isoprenoid to different protein carriers via the carboxylic function or after introduction of a succinic spacer between the protein carrier and position 3 of the target molecule. Antibodies of three different cell lines were further analysed by competitive ELISA and were shown to be directed either against both rings A and B or to ring E of the pentacyclic system. Thus these antibodies can be used in the specific detection of oleanolic acid itself or of a broad range of oleanolic acid derivatives sharing a specific structural moiety. Therefore these antibodies may be interesting tools for the screening of putative terpenoid-containing plants.

Accumulation of furanic labdane diterpenes in Marrubium vulgare and Leonurus cardiaca.

Accumulation of furanic labdane diterpenes has been investigated in different parts of field-grown plants of MARRUBIUM VULGARE (Lamiaceae) and LEONURUS CARDIACA (Lamiaceae). Furanic labdane diterpenes were produced and accumulated only in the aerial parts. Greatest amounts were measured in leaves and flowers. Up to 4 mg furanic labdane diterpenes per g fresh weight were found. Accumulation of furanic labdane diterpenes in plantlets seemingly depends on a developmental programme. No furanic labdane diterpenes were detected in plantlets during the first four to five weeks following germination. At this time the leaves became more differentiated and the number of trichomes on leaves was obviously increasing. Young leaves and buds contained most furanic labdane diterpenes. It was proven that at least a part of the non-volatile furanic labdane diterpenes is stored in peltate glandular trichomes. NMR signals of marrubiin were investigated with correlated spectra. Some (1)H- and (13)C-NMR assignments reported in literature were revised.

Biosynthesis of the labdane diterpene marrubiin in Marrubium vulgare via a non-mevalonate pathway.

The biosynthesis of the furanic labdane diterpene marrubiin has been studied in plantlets and shoot cultures of Marrubium vulgare (Lamiaceae). The use of [2-14C]acetate, [2-14C]pyruvate, [2-14C]mevalonic acid and [U-14C]glucose incorporation experiments showed that the labelling of sterols in etiolated shoot cultures of M. vulgare was in accordance with their biosynthesis via the acetate-mevalonate pathway. In contrast, the incorporation rates of these precursors into the diterpene marrubiin could not be explained by biosynthesis of this compound via the acetate-mevalonate pathway. Cultivation of etiolated shoot cultures of M. vulgare on medium containing [1-13C]glucose and subsequent 13C-NMR spectroscopy of marrubiin led to the conclusion that the biosynthesis of marrubiin follows a non-mevalonate pathway. All isoprenic units of 13C-labelled marrubiin were enriched in those carbons that correspond to positions 1 and 5 of a putative precursor isopentenyl diphosphate. This labelling pattern from [1-13C]glucose is consistent with an alternative pathway via trioses, which has already been shown to occur in Eubacteria and Gymnospermae. The labdane skeleton is a precursor of many other skeletal types of diterpenes. Therefore it becomes obvious that in connection with the few known examples of a non-mevalonate pathway to isoprenoids the formation of some isoprenoids in plants via a non-mevalonate pathway might be quite common.

Biosynthesis of [14C]geranylgeranyldiphosphate by a prenyl transferase system from a mutant strain of Gibberella fujikuroi.

[14C]Geranylgeranyldiphosphate was produced by a prenyl transferase system extracted from the mutant strain SG4 of Gibberella fujikuroi (wild-type strain IMI 58289), which is blocked in carotenoid biosynthesis. The fungus was grown in liquid medium containing only potato-dextrose broth. Crude extracts of 12- to 14-day-old mycelia in Tris buffer, pH 8.5, were centrifuged at 100,000g. The supernatant was desalted and used to produce [4,8,12,16-14C]geranylgeranyldiphosphate from R,S-[2-14C]mevalonic acid. Maximum yield of [14C]geranylgeranyldiphosphate was obtained using mycelia from the stationary growth phase with incubation of the enzyme preparation at pH 8.5 for 5 h. Routinely, 65% of the biogenetic available R-mevalonic acid was converted to [14C]geranylgeranyldiphosphate, which was identified by cochromatography with authentic [1-3H]geranylgeranyldiphosphate by means of radio-TLC, radio-HPLC, and hydrolysis to geranylgeraniol.

Establishment of callus, cell suspension and shoot cultures of Leonurus cardiaca L. and diterpene analysis.

Callus cultures, cell suspension cultures and shoot cultures of Leonurus cardiaca L. (Motherwort) were established and growth conditions optimized. Shoot cultures showed constant growth whether in the dark or under continuous light, accumulating varying amounts of the furanic labdane diterpenes leosibiricin, preleosibirin, leosibirin and isoballotenol acetate, which are also present in the soil-grown plants. Only traces of leosibiricin were detected in callus cultures, while cell suspension cultures did not produce any furanic diterpenes. A small amount of furanic labdane diterpenes was found in the medium of shoot cultures. Callus and shoot culture induction of several other Lamiaceae species is also described.