A newly identified ß-amyrin synthase gene hypothetically involved in oleanane-saponin biosynthesis from Talinum paniculatum (Jacq.) Gaertn.

Talinum paniculatum or Javanese ginseng in Indonesia is a plant widely used as a traditional medicine. The genus Talinum produces oleanane-type saponins, such as talinumoside I. The first aim of this study was to isolate the probable gene encoding ß-amyrin synthase (bAS), a key enzyme involved in the cyclization of 2,3-oxidosqualene producing the backbone of the oleanane-type saponin ß-amyrin and characterize the gene sequence and the predicted protein sequence using in silico approach. The second aim was to analyze the correlation between the TpbAS gene expression level and saponin production in various plant organs. Thus, TpbAS was isolated using degenerate primers and PCR 5'/3'-Rapid Amplification of cDNA Ends (RACE), then the gene sequence and the predicted protein were in silico analyzed using various programs. TpbAS expression level was analyzed using reverse transcriptase PCR (RT-PCR), and saponin content was measured using a spectrophotometer. The results showed that the full-length TpbAS gene consists of 2298 base pairs encoding for a 765-amino acid protein. From in silico study, the (GA)n sequence was identified in the 5'-untranslated regions and predicted to be a candidate of the gene expression modulator. In addition, functional RNA motifs and sites analysis predicted the presence of exon splicing enhancers and silencers within the coding sequence and miRNA target sites candidate. Amino acid sequence analysis showed DCTAE, QW, and WCYCR motifs that were conserved in all classes of oxidosqualene cyclase enzymes. Phylogenetic tree analysis showed that TpbAS is closely related to other plant oxidosqualene cyclase groups. Analysis of TpbAS expression and saponin content indicated that saponin is mainly synthesized and accumulated in the leaves. Taken together, these findings will assist in increasing the saponin content through a metabolic engineering approach.

Triterpenoid Saponins from Maesa argentea Leaves.

Within an ongoing research program on saponins with potential antileishmanial activity, four previously undescribed saponins were isolated from Maesa argentea leaves and identified by LC-MS/MS, GC-MS, and 1D and 2D NMR spectroscopy as 3ß-O-{([ß-D-glucopyranosyl-(1 → 2)-α-L-rhamnopyranosyl-(1 → 2)-ß-D-galactopyranosyl-(1 → 3)]-[ß-D-galactopyranosyl-(1 → 2)]-ß-D-glucuronopyranosyl)}-21ß-angeloyloxy-22α-butanoyloxy-13ß,28-oxidoolean-16α,28α-diol (1), 3ß-O-{([ß-D-glucopyranosyl-(1 → 2)-α-L-rhamnopyranosyl-(1 → 2)-ß-D-galactopyranosyl-(1 → 3)]-[ß-D-galactopyranosyl-(1 → 2)]-ß-D-glucuronopyranosyl)}-21ß,22α-angeloyloxy-13ß,28-oxidoolean-16α,28α-diol (2), 3ß-O-{([ß-D-glucopyranosyl-(1 → 2)-α-L-rhamnopyranosyl-(1 → 2)-ß-D-galactopyranosyl-(1 → 3)]-[ß-D-galactopyranosyl-(1 → 2)]-ß-D-glucuronopyranosyl)}-21ß-angeloyloxy-22α-(E)-cinnamoyloxy-13ß,28-oxidoolean-16α,28α-diol (3), and 3ß-O-{([α-L-rhamnopyranosyl-(1 → 2)-ß-D-galactopyranosyl-(1 → 3)]-[ß-D-galactopyranosyl-(1 → 2)]-ß-D-glucuronopyranosyl)}-21ß-angeloyloxy-22α-(E)-cinnamoyloxy-13ß,28-oxidoolean-16α,28α-diol (4). Leaf material was obtained from a germinated seed that was clonally propagated using in vitro tissue culturing. Compounds 1-4 showed structural similarity with maesasaponins and maesabalides reported before from other Maesa spp. All four compounds showed in vitro activity against Plasmodium falciparum K1 and Leishmania infantum at micromolar concentrations. However, the observed inhibitory action must be considered nonspecific since they were also cytotoxic in the same concentration range.

Evaluation of the anti-angiogenic activity of saponins from Maesa lanceolata by different assays.

Angiogenesis, in which a vascular network is established from pre-existing vessels, is a complex multistep process. Mechanisms underlying angiogenesis can be investigated using a variety of in vitro, ex vivo and in vivo approaches. Evaluation of several promising plants and plant metabolites, including terpenoids, revealed promising anti-angiogenic activity. Since the maesasaponins displayed anti-angiogenic activity in the chick chorioallantoic membrane (CAM) assay, their activity was further investigated in several test systems. The rat aorta ring assay was compared with the placental vein assay and then selected for the ex vivo investigation of the saponins. Besides their effect on the viability of HUVEC, the anti-angiogenic capacity of the compounds was also investigated in an in vivo zebrafish assay. The activity of the saponins in the viability assay was more pronounced than in the rat aorta ring assay and similar to the effect observed in the CAM assay. The use of different test systems, however, implies different results in the case of saponins.

Agroinfiltration of intact leaves as a method for the transient and stable transformation of saponin producing Maesa lanceolata.

UNLABELLED: A method has been developed to genetically transform the medicinal plant Maesa lanceolata. Initially, we tested conditions for transient expression of GFP-bearing constructs in agroinfiltrated leaves. Leaf tissues of M. lanceolata were infiltrated with Agrobacterium tumefaciens carrying a nuclear-targeted GFP construct to allow the quantification of the transformation efficiency. The number of transfected cells was depended on the bacterial density, bacterial strains, the co-cultivation time, and presence of acetosyringone. The transient transformation assay generated the highest ratio of transfected cells over non-transfected cells upon 5 days post-infiltration using A. tumefaciens strain LBA4404 at an OD600 = 1.0 in the presence of 100 µM acetosyringone and in the absence of a viral suppressor construct. In a second series of experiments we set up a stable transformation protocol that resulted in the regeneration of kanamycin-resistant plants expressing nuclear GFP. This transformation protocol will be used to introduce overexpression and RNAi constructs into M. lanceolata plants that may interfere with triterpenoid saponin biosynthesis. KEY MESSAGE: We have developed a transformation protocol for saponin producing Maesa lanceolata. Using the protocol reported here, now we are able to generate the tools for the modification of saponin production.

Modulation of triterpene saponin production: in vitro cultures, elicitation, and metabolic engineering.

Saponins are secondary metabolites that are widely distributed in the plant kingdom and are often the active components in medicinal herbs. Hence, saponins have a potential for the pharmaceutical industry as antibacterial, virucidal, anti-inflammatory, and anti-leishmanial drugs. However, their commercial application is often hindered because of practical problems, such as low and variable yields and limited availability of natural resources. In vitro cultures provide an alternative to avoid problems associated with field production; they offer a system in which plants are clonally propagated and yield is not affected by environmental changes. Additionally, treatment of in vitro cultures with elicitors such as methyl jasmonate may increase the production of saponins up to six times. In vitro cultures are amenable to metabolic engineering by targeting specific genes to enhance saponin production or drive production towards one specific class of saponins. Hitherto, this approach is not yet fully explored because only a limited number of saponin biosynthesis genes are identified. In this paper, we review recent studies on in vitro cultures of saponin-producing plants. The effect of elicitation on saponin production and saponin biosynthesis genes is discussed. Finally, recent research efforts on metabolic engineering of saponins will also be presented.