Induction of alkaloid diversity in hybrid plant cell cultures

 The treatment of Rauwolfia serpentina x Rhazya stricta somatic hybrid cell suspension culture with 100 μM of methyl jasmonate led to a general increase in indole alkaloid content and to qualitative changes in the alkaloid pattern. The content of Six alkaloids were investigated with respect to their content in both the cell biomass and nutrition medium. Intracellular 17-O-acetyl-norajmaline content on the 5th day after treatment had increased about 40-fold compared with the control culture. The respective concentrations of the other alkaloids increased by a factor of two to five. In total 26 indole alkaloids were identified in extracts of the methyl jasmonate-treated culture by TLC, UV, MS and NMR data and comparison with reference alkaloids. The identification of macrophylline, yohimbine oxindole and yohimbine pseudoindoxyl has not been reported before in Rauwolfia serpentina or Rhazya stricta plants nor in cell cultures derived from these plants. Received: 17 June 1998 / Accepted: 7 December 1998     

Purification,cloning, functional expression and characterization of perakine reductase: the first example from the AKR enzyme family,extending the alkaloidal network of the plant <Emphasis Type="Italic">Rauvolfia</Emphasis>

Perakine reductase (PR) catalyzes an NADPH-dependent step in a side-branch of the 10-step biosynthetic pathway of the alkaloid ajmaline. The enzyme was cloned by a “reverse-genetic” approach from cell suspension cultures of the plant Rauvolfia serpentina (Apocynaceae) and functionally expressed in Escherichia coli as the N-terminal His₆-tagged protein. PR displays a broad substrate acceptance, converting 16 out of 28 tested compounds with reducible carbonyl function which belong to three substrate groups: benzaldehyde, cinnamic aldehyde derivatives and monoterpenoid indole alkaloids. The enzyme has an extraordinary selectivity in the group of alkaloids. Sequence alignments define PR as a new member of the aldo-keto reductase (AKR) super family, exhibiting the conserved catalytic tetrad Asp52, Tyr57, Lys84, His126. Site-directed mutagenesis of each of these functional residues to an alanine residue results in >97.8% loss of enzyme activity, in compounds of each substrate group. PR represents the first example of the large AKR-family which is involved in the biosynthesis of plant monoterpenoid indole alkaloids. In addition to a new esterase, PR significantly extends the Rauvolfia alkaloid network to the novel group of peraksine alkaloids. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. The nucleotide sequences reported in this article have been submitted to the Gene Bank under Accession No: AY766462.     

RLCC-isolation of Raucaffricine from its most efficient source — cell suspension cultures of Rauwolfia serpentina Benth

Raucaffricine (vomilenine-galactoside) was shown to be the major indole alkaloid of Rauwolfia serpentina Benth. cell suspension cultures grown in AP-medium (alkaloid production medium). Several grams of this glycoalkaloid can conveniently be isolated by RLCC (Rotation Locular Countercurrent Chromatography). A newly discovered enzyme efficiently converts the glycoalkaloid to its aglycon, vomilenine, which occupies a key function in the biosynthesis of ajmaline. This is the first demonstration of the occurrence of raucaffricine in Rauwolfia serpentina.     

Isoquinoline alkaloids from cell suspension cultures of Fumaria capreolata

Fumaria capreolata was taken into cell suspension culture. The culture yielded a biomass of about 12 g dry weight per liter of medium; the dried cells contained ca. 0.1% of alkaloids. Besides choline, the following ten known isoquinoline alkaloids were isolated from the cell extract in crystalline form: coptisine, dehydrocheilanthifoline; (+)-isoboldine; magnoflorine; N-methylcoclaurine; (+)-reticuline; (–)-pallidine; protopine; sanguinarine; (–)-scoulerine. This is the most diverse isoquinoline alkaloid spectrum thus far published for a cell suspension culture.     

Functional expression of an ajmaline pathway-specific esterase from <Emphasis Type="Italic">Rauvolfia</Emphasis> in a novel plant-virus expression system

Acetylajmalan esterase (AAE) plays an essential role in the late stage of ajmaline biosynthesis. Based on the partial peptide sequences of AAE isolated and purified from Rauvolfia cell suspensions, a full-length AAE cDNA clone was isolated. The amino acid sequence of AAE has the highest level of identity of 40% to putative lipases known from the Arabidopsis thaliana genome project. Based on the primary structure AAE is a new member of the GDSL lipase superfamily. The expression in Escherichia coli failed although a wide range of conditions were tested. With a novel virus-based plant expression system, it was possible to express AAE functionally in leaves of Nicotiana benthamiana Domin. An extraordinarily high enzyme activity was detected in the Nicotiana tissue, which exceeded that in Rauvolfia serpentina (L.) Benth. ex Kurz cell suspension cultures about 20-fold. This expression allowed molecular analysis of AAE for the first time and increased the number of functionally expressed alkaloid genes from Rauvolfia now to eight, and the number of ajmaline pathway-specific cDNAs to a total of six. The nucleotide sequence reported in this article has been submitted to the GenBank/EBI under GenBank Accession no. AY762990     

In silico investigation on alkaloids of Rauwolfia serpentina as potential inhibitors of 3-hydroxy-3-methyl-glutaryl-CoA reductase

Present work aimed to investigate the in silico activity of the alkaloids of roots of Rauwolfia serpentina as inhibitors of 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR). For this purpose, the three-dimensional (3D) structure of the protein HMGCR (PDB ID: 1HW9) was downloaded from Protein Data Bank (PDB) database, as a target enzyme. The structures of twelve alkaloids from the roots of R. serpentina were selected as ligands and docked with the selected HMGCR enzyme using Molegro Virtual Docker (MVD) software. The software ‘MVD’ computes the binding (atom) energies of selected protein (enzyme) and each ligand at minimum energetic conformation state by using the PLP (Piecewise Linear Potential) scoring mechanism. Docking results of twelve tested alkaloids showed that five alkaloids including compound 1 (ajmalicine), 2 (reserpine), 3 (indobinine), 4 (yohimbine), and 5 (indobine) have displayed the highest MolDock scores and best fit within the prominent active site residues (positioned between 684 and 692 of cis-loop) of HMGCR. According to the lowest MolDock energies obtained through non-covalent interactions of alkaloids with HMGCR, these are characterized to be the potential inhibitors of HMGCR. Therefore, the alkaloids from R. serpentina can effectively suppress the cholesterol biosynthesis pathway through inhibition of HMGCR and can serve as potential lead compounds for the development of new drugs for the treatment of hyperlipidaemia.     

Prospecting for Novel Plant-Derived Molecules of Rauvolfia serpentina as Inhibitors of Aldose Reductase,a Potent Drug Target for Diabetes and Its Complications

Aldose Reductase (AR) is implicated in the development of secondary complications of diabetes, providing an interesting target for therapeutic intervention. Extracts of Rauvolfia serpentina, a medicinal plant endemic to the Himalayan mountain range, have been known to be effective in alleviating diabetes and its complications. In this study, we aim to prospect for novel plant-derived inhibitors from R. serpentina and to understand structural basis of their interactions. An extensive library of R. serpentina molecules was compiled and computationally screened for inhibitory action against AR. The stability of complexes, with docked leads, was verified using molecular dynamics simulations. Two structurally distinct plant-derived leads were identified as inhibitors: indobine and indobinine. Further, using these two leads as templates, 16 more leads were identified through ligand-based screening of their structural analogs, from a small molecules database. Thus, we obtained plant-derived indole alkaloids, and their structural analogs, as potential AR inhibitors from a manually curated dataset of R. serpentina molecules. Indole alkaloids reported herein, as a novel structural class unreported hitherto, may provide better insights for designing potential AR inhibitors with improved efficacy and fewer side effects.     

Comparison of terpenoid indole alkaloid production and degradation in two cell lines of Tabernaemontana divaricata

Two cell lines of Tabernaemontana divaricata derived from the same suspension culture accumulate different amounts of the terpenoid indole alkaloids O-acetylvallesamine and voaphylline. [¹⁵N]O-acetylvallesamine and [¹⁵N]voaphylline were added to the suspension cultures to investigate whether the lack of accumulating capacity of one of the cell lines was due to a low biosynthetic ability or to high turnover rates. The difference was shown to be due to the inability of the cell culture to biosynthesize both alkaloids. Both cell lines were able to metabolize O-acetylvallesamine. This metabolisation occurred mainly during the stationary phase. The alkaloids added were chemically unstable under culture conditions. Under normal batch cell culture conditions chemical breakdown is thought to play a minor role in the total amount of compound transformed.     

Uncharacteristic alkaloid synthesis by suspension cultures of Cinchona pubescens fed with L-tryptophan

The growth and alkaloid production of a liquid suspension culture of Cinchona pubescens has been studied, particularly with attention to the effect on the alkaloid spectrum of feeding cultures with L-tryptophan. This treatment did not enhance the production of any of the known alkaloids of Cinchona. Above 2mmM, however, the presence of the amino acid was toxic, causing extreme acidification of the medium and cell death. Under these conditions a number of indole and quinoline derivatives accumulated. The principal component of the alkaloid fraction proved to be norharman; indole-3-aldehyde was also isolated. Both these products probably occur by uncharacteristic metabolism of L-tryptophan. Furthermore, evidence for the degradation of endogenous alkaloids was obtained, as 4-hydroxymethylquinoline was also isolated. None of the known quinoline alkaloids of Cinchona, which were present in untreated cells, could be detected after L-tryptophan treatment, even when large amounts of culture were analysed. It is concluded that, in this instance, Cinchona alkaloid production cannot be improved by feeding with a precursor.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indoleacetic acid - BA benzyladenine     

Fungal foliar diseases of Rauwolfia serpentina in wild,its seasonal occurrence,seed transmission and disease management

Rauwolfia serpentina is an important medicinal herb. Studies were conducted to determine causal organisms of foliar diseases in R. serpentina in Bhadra Wildlife Sanctuary during 2006–2009. The foliar disease incidence and its distribution and disease severity in nine state forest regions of the sanctuary were determined. The seedborne nature and transmission of the causal organism was also determined. The management of seedborne inoculum was done by seed dressing with fungicides. The effect of foliage infection on secondary metabolite content was also determined. Results of the present study indicated that Cercospora rauwolfiae is major leaf spot disease causing pathogen. The disease is homogeneously distributed through the study area. The foliar disease severity was high in Kagemanegiri forest during October–November. The minor leaf spot disease caused by Alternaria alternata occurred occasionally. Alternaria alternata is seedborne and seed transmitted and could be managed by seed treatment with Captra or Hyzeb. The secondary metabolites like alkaloids and steroids decreased with increase in foliar infection by C. rauwolfiae, while phenol and flavonoid contents increased. The study suggested that R. serpentina is affected in wild by C. rauwolfiae and A. alternata. The latter pathogen is seedborne and seed transmitted and controlled by seed treatment.     

Natural products and enzymes from plant cell cultures

Plants represent an unlimited source of natural products. Many of the recently detected phytochemicals exhibit remarkable bioactivities, ranging from anticancer activity, phosphodiesterase inhibition to cytotoxicity against HIV-infected cells. Cultivated plant cells produce at their unorganized, dedifferentiated stage secondary metabolites, but in very different amounts in so far as new compounds are concerned. In fact, more than 140 novel natural products are presently known from plant cell cultures, which also include new metabolites formed by biotransformation. The biotransformation capacity of suspended cells is described and recent high yielding transformations, like the formation of arbutin by hydroquinone-transformation withRauwolfia cells are discussed. As an example of alkaloid production by cell suspensions, the pattern of monoterpenoid indole alkaloids of the Indian medicinal plantRauwolfia serpentina Benth. is described and the so far 30 identified compounds are divided into eight groups which are biosynthetically closely related. Some of the key biosynthetic reactions leading to theRauwolfia alkaloids are discussed and an overview of the enzymes involved in the formation of the alkaloid ajmaline and proteins catalyzing side reactions of the ajmaline pathway are given.     

Catharanthus roseus: micropropagation and in vitro techniques

Different methods of in vitro culture of Catharanthus roseus provide new sources of plant material for the production of secondary metabolites such as indole alkaloids. Callus, cell suspension, plantlets, and transgenic roots cultured in the bioreactor are used in those experiments. The most promising outcomes include the production of the following indole alkaloids: ajmalicine in unorganised tissue, catharanthine in the leaf and cell culture in the shake flask and airlift bioreactor, and vinblastine in shoots and transformed roots. What is very important, enzymatic coupling of monomeric indole alkaloids, vindoline and catharanthine, is possible to form vinblastine in cell cultures. The method of catharanthine and ajmalicine production in the suspension culture in bioreactors has been successful. In this method, elicitation may be used acting on different metabolic pathways. Also of interest is the method of obtaining arbutin from the callus culture of C. roseus conducted with hydroquinone. The transformed root culture seems to be the most promising for alkaloid production. The genetically transformed roots, obtained by the infection with Agrobacterium rhizogenes, produce higher levels of secondary metabolites than intact plants. Also, whole plants can be regenerated from hairy roots. The content of indole alkaloids in the transformed roots was similar or even higher when compared to the amounts measured in studies of natural roots. The predominant alkaloids in transformed roots are ajmalicine, serpentine, vindoline and catharanthine, found in higher amounts than in untransformed roots. Transformed hairy roots have been also used for encapsulation in calcium alginate to form artificial seeds.     

A virus-induced gene silencing approach to understanding alkaloid metabolism in Catharanthus roseus

The anticancer agents vinblastine and vincristine are bisindole alkaloids derived from coupling vindoline and catharanthine, monoterpenoid indole alkaloids produced exclusively by the Madagascar periwinkle (Catharanthus roseus). Industrial production of vinblastine and vincristine currently relies on isolation from C. roseus leaves, a process that affords these compounds in 0.0003–0.01% yields. Metabolic engineering efforts to either improve alkaloid content or provide alternative sources of the bisindole alkaloids ultimately rely on the isolation and characterization of the genes involved. Several vindoline biosynthetic genes have been isolated, and the cellular and subcellular organization of the corresponding enzymes has been well studied. However, due to the leaf-specific localization of vindoline biosynthesis, and the lack of production of this precursor in cell suspension and hairy root cultures of C. roseus, further elucidation of this pathway demands the development of reverse genetics approaches to assay gene function in planta. The bipartite pTRV vector system is a Tobacco Rattle Virus-based virus-induced gene silencing (VIGS) platform that has provided efficient and effective means to assay gene function in diverse plant systems. A VIGS method was developed herein to investigate gene function in C. roseus plants using the pTRV vector system. The utility of this approach in understanding gene function in C. roseus leaves is demonstrated by silencing known vindoline biosynthetic genes previously characterized in vitro.     

Bisbenzylisoquinoline alkaloids in Berberis cell cultures

Callus cultures of 34 Berberis cell lines representing 33 species were screened for alkaloids using TLC and HPLC. Jatrorrhizine was always found to be a major constituent; two B. stolonifera lines were the richest in bisbenzylisoquinolines. The new alkaloid 2-norberbamunine was isolated from cultures of B. stolonifera V29 together with berbamunine, aromoline, berbamine, isotetrandrine and jatrorrhizine. The time course of bisbenzylisoquinoline accumulation was studied in B. stolonifera V29 suspension cultures and it was found that the concentrations of these alkaloids rose throughout the cell growth phase.     

Alkaloid formation in cell suspension cultures of Tabernaemontana divaricata after feeding of tryptamine and loganin

A cell suspension culture of Tabernaemontana divaricata, that had lost alkaloid production, was still capable of producing a similar pattern of alkaloids as directly after its initiation. When fed with early precursors, such as tryptamine and loganin, 57% of the precursors was converted into indole alkaloids such as strictosidine, vallesamine, O-acetylvallesamine and voaphylline. Apparently most of the cell factory has remained stable during the many years of subculturing. Only an early step of the biosynthesis the flux seems to be diverted to other pathways.     

Alkaloid production in elicited cell suspension cultures of Erythrina americana Miller

A number of species of the genus Erythrina are rich in secondary metabolites, particularly phenolics and alkaloids that exhibit interesting anti-inflammatory, anti-plasmodial, bactericidal, curariform and fungicidal activities. Unfortunately, the isolation of these compounds through the extraction of organs and seeds of whole plants is becoming more difficult since the natural growing areas of many of the species, and particularly of Erythrina americana, are being urbanised. Plant tissue culture not only constitutes a viable method through which to preserve the species, but may also represent a constant and stable source of target alkaloids. Currently, however, in vitro systems, and especially cell suspension cultures, only accumulate low levels of the desired products. A number of strategies for increasing production have been proposed, the most successful of which involves elicitation of suspension cells with plant growth regulators. In this context, excellent results have been reported following elicitation of cell cultures derived from different species and genera with jasmonic acid and methyl jasmonate. The present paper provides a brief review of the novel approaches to the use of Erythrina alkaloids that have recently been described, and of the advances that have been made in the formation of tissue cultures of E. americana and their subsequent elicitation in attempts to augment alkaloid accumulation.     

Effect of phytohormones on growth and alkaloid accumulation by a Catharanthus roseus cell suspension cultures fed with alkaloid precursors tryptamine and loganin

This work presents a study of the effect of different phytohormones on growth and accumulation of terpenoid indole alkaloids in a Catharanthus roseus cell suspension culture upon feeding with the precursors loganin and tryptamine. The phytohormones tested were 2,4-dichlorophenoxyacetic acid, salicylic acid, methyl jasmonate and abscisic acid. Among these only methyl jasmonate enhanced the accumulation of alkaloids. Abscisic acid did not enhance the accumulation of alkaloids but delayed the catabolism of strictosidine.     

Ligand structures of synthetic deoxa-pyranosylamines with raucaffricine and strictosidine glucosidases provide structural insights into their binding and inhibitory behaviours

Abstract

Insight into the structure and inhibition mechanism of O-β-d-glucosidases by deoxa-pyranosylamine type inhibitors is provided by X-ray analysis of complexes between raucaffricine and strictosidine glucosidases and N-(cyclohexylmethyl)-, N-(cyclohexyl)- and N-(bromobenzyl)-β-d-gluco-1,5-deoxa-pyranosylamine. All inhibitors anchored exclusively in the catalytic active site by competition with appropriate enzyme substrates. Thus facilitated prospective elucidation of the binding networks with residues located at <3.9?Å distance will enable the development of potent inhibitors suitable for the production of valuable alkaloid glucosides, raucaffricine and strictosidine, by means of synthesis in Rauvolfia serpentina cell suspension cultures.     

Chemical Constituents of Crinum asiaticum L. var. sinicum Baker and Their Cytotoxic Activities

A phytochemical investigation of the bulbs of Crinum asiaticum L. var. sinicum Baker resulted in the isolation of two new alkaloids, asiaticumines A and B ( 1 and 2 , resp.), together with 21 known compounds, including nine alkaloids, four amides, five phenolic compounds, and three flavonoids. All 23 compounds were isolated for the first time from Crinum asiaticum L. var. sinicum Baker . Their structures were elucidated by spectroscopic methods. In addition, ten alkaloids, 1 – 10 , were evaluated for their cytotoxic activities against human tumor cell lines A549, LOVO, HL‐60, and 6T‐CEM. Compounds 3, 4 , and 7 – 10 selectively showed remarkable inhibition against one or more of the tested cell lines.     

Alkaloid formation in cell suspension cultures of Tabernaemontana elegans after feeding of tryptamine and loganin or secologanin

A cell suspension culture of Tabernaemontana elegans lost its ability to produce alkaloids after a prolonged period of subculture. To determine whether it was still capable of performing the later steps of the alkaloid biosynthetic pathway, the culture was fed with tryptamine and loganin. The precursors and alkaloids were determined in the biomass and in the medium during a growth cycle. In this culture, an increase in the amount of serotonin was found in the biomass after feeding of tryptamine and loganin. Secologanin was detected in small amounts but strictosidine was not. Therefore, a limitation in alkaloid formation in this T. elegans cell line occured in the formation of secologanin from loganin. After feeding of secologanin alone, strictosidine, 10-hydroxy strictosidine, strictosidinic acid and two other indole alkaloids, as yet unidentified, were formed. However, the alkaloids originally produced by this cell line were not found. As the biosynthesis is impaired at several steps, it seems that the loss of productivity is more likely to be to a change on the level of the regulation of the pathway, than due to the loss of the capacity to express an individual biosynthetic gene of the pathway.