Indole alkaloids in clonal propagules of Rauwolfia serpentina benthe ex kurz

Plantlets were succesfully regenerated from shoot cultures of Rauwolfia serpentina initiated from auxillary meristems on medium containing BA (4.44 M) + NAA (0.54 M). Rooting was initiated in White's basal medium supplemented with NAA (0.54 M). Tissue culture derived piants of R. serpentina (RSTC) were similar to normal plants (RS) in their morphological characteristics and chemical consitution. The biomass of the RSTC plants was higher (47.11 gms) than the normal plant (18.23 gms) on a dry weight basis. Five RSTC plants were cloned and the cloned plants were similar in biomass and alkaloid content to the normal plant.Abbreviations BA benzyl adenine - NAA naphthalene acetic acid - MS Murashige and Skoog - TLC thin layer chromatography - HPLC high performance liquid chromatography - F.W. fresh weight - D.W. dry weight - RS normal field grown plant established from stem cuttings - RSTC tissue culture plants established from shoot cultures - R reserpine - Aj ajmalicine - A ajmaline - S serpentine     

Indole alkaloids from cell suspension cultures of Tabernaemontana divaricata and Tabernanthe iboga

From cell suspension cultures of Tabernaemontana divaricata and Tabernanthe iboga grown under standard conditions, six monoterpenoid indole alkaloids have been isolated and identified. T. divaricata synthesized apparicine, catharanthine, coronaridine, conoflorine, tubotaiwine and vinervine, whereas T. iboga produced tubotaiwine and conoflorine. Both cultures are a reasonable source for conoflorine, which is expected to be a good candidate for studying the mechanism of Aspidosperma type alkaloid formation at the cell-free level.     

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.     

7-Dehydrositosterol from Rauwolfia serpentina

7-Dehydrositosterol has been isolated from the roots of Rauwolfia serpentina.     

Ajmalicidine an alkaloid from Rauwolfia serpentina

A new heteroyohimban alkaloid, ajmalicidine, has been isolated from the roots of Rauwolfia serpentina of Thai origin. Its structure has been elucidated as 1-carbomethoxy-17α-hydroxy-16-decarbomethoxy 16,17-dihydro ajmalicine through chemical and spectral studies.     

Triggered efflux of protoberberine alkaloids from cell suspension cultures ofThalictrum rugosum

Cell cultures ofThalictrum rugosum released their protoberberine alkaloids into the medium, when cells were transferred to fresh medium lacking phosphate. The nutritional factors required and the impact of the cells' physiological state for the alkaloid excretion were analyzed. Cell cultures, having released their alkaloids into the medium, continued to grow when the alkaloid containing medium was replaced by fresh growth medium.     

催吐萝芙木中生物碱的研究

从催吐萝芙木根的乙醇提取物中分离得到15个吲哚生物碱,利用波谱(ESI-MS,1H NMR,13C NMR)等技术分别鉴定为利血平(1),四氢鸭脚木碱(2),异山德维辛碱(3),利血平酸甲酯(4),萝芙木碱(5),山德维辛碱(6),异育亨宾(7),霹雳萝芙木碱(8),α-育亨宾(9),育亨宾(10),催吐萝芙木定(11),四叶萝芙新碱(12),harman(13),mauiensine(14),12-hydroxymauiensine(15)。其中化合物13 ~15是首次从该植物中分离到。     

Selection of cell lines for the production of rosmarinic acid from cell suspension cultures of Orthosihon stamineus benth

Summary Cell suspension cultures of Orthosiphon stamineus were established from friable calluses produced from leaf pieces of in vitro plantlets that were derived from nodal segments of the mother plants collected from three different geographical locations. Eight lines were eventually selected after seven subculture cycles based on the growth characteristic (plant height) of the plantlets from the three locations: two fast-growing lines (>5.1 cm tall), three intermediate-growing lines (3.1–5.0 cm tall), and three slow-growing lines (<3.0 cm tall). All eight lines grew well in liquid Murashige and Skoog medium supplemented with 4.5 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 5.4 μM 1-naphthaleneacetic acid (NAA). All cell lines exhibited the same growth pattern but produced different maximum cell biomass when cultured in this medium. The time of harvesting the plant cells from the culture medium and the geographical source of the original plant material were both found to affect the production of rosmarinic acid (RA) in cell cultures. Two cell lines were successfully selected and identified to produce high amounts of RA. These cell lines were a fast-growing cell line from Air Itam, Penang and an intermediate-growing cell line from Relau Agriculture Research Centre, Penang which could produce 5% [(w/w) dry weight] and 4.5% [(w/w) dry weight] of RA, respectively.     

Purification, partial amino acid sequence and structure of the product of raucaffricine-O-beta-D-glucosidase from plant cell cultures of Rauwolfia serpentina

Plant cell suspension cultures of Rauwolfia produce within 1 week approximately 250 nkat/l of raucaffricine-O-beta-D-glucosidase. A five step procedure using anion exchange chromatography, chromatography on hydroxylapatite, gel filtration and FPLC-chromatography on Mono Q and Mono P delivered in a yield of 0.9% approximately 1200-fold enriched glucosidase. A short protocol employing DEAE sepharose, TSK 55 S gel chromatography and purification on Mono Q gave a 5% recovery of glucosidase which was 340-fold enriched. SDS-PAGE showed a Mr for the enzyme of 61 kDa. The enzyme is not glycosylated. Structural investigation of the enzyme product, vomilenine, demonstrated that the alkaloid exists in aqueous solutions in an equilibrium of 21(R)- and 21(S)-vomilenine in a ratio of 3.4:1. Proteolysis of the pure enzyme with endoproteinase Lys C revealed six peptide fragments with 6-24 amino acids which were sequenced. The two largest fragments showed sequences, of which the motif Val-Thr-Glu-Asn-Gly is typical for beta-glucosidases. Sequence alignment of these fragments demonstrated high homologies to linamarase from Manihot esculenta (81% identity) or to beta-glucosidase from Prunus avium (79% identity). Raucaffricine-O-beta-D-glucosidase seems to be a new member of the family 1 of glycosyl hydrolases.     

Molecular and chemical characterization of plants regenerated from Ri-mediated hairy root cultures of Rauwolfia serpentina

Hairy root lines were induced from leaf explants of Rauwolfia serpentina known to contain high levels of reserpine (0.0882 % DW) content. Out of five high yielding hairy root lines, three (R1, R14 and R15) exhibited spontaneous regeneration of shoots after 6–8 weeks in liquid B5 medium. Excised regenerated shoots underwent robust shoot proliferation when cultured on Murashige and Skoog (MS) medium supplemented with 0.1 mg/l naphthanleneacetic acid (NAA) and 1.0 mg/l 6-benzyladenine. When shoots were transferred to a root induction medium, consisting of MS basal medium and 1.0 mg/l NAA, all rooted within 2–3 weeks. Of a total of 45 plants developed from three different hairy root lines, 30 were successfully acclimatized and transferred to the green house. Almost 90 % of these plants grown in the green house showed no observed phenotypic differences, while 10 % were stunted and grew poorly, in comparison to non-transformed plants. Phenotypic assessment of regenerated plants for plant length, number of nodes and intermodal lengths, number of leaves per node, leaf color, leaf size, number of flowering shoots, flower size, fruit size, lateral root branching and root biomass was conducted. Polymerase chain reaction and Southern blot hybridization revealed that all plants derived from hairy roots carried the Ri T_L-DNA fragment. Moreover for plants derived from transgenic hairy root line R14, presence of more than a single transgene copy number was observed, and this might have contributed to observed abnormal phenotypes. Analysis of reserpine content revealed that roots of regenerated plants had similar levels (0.0889 % DW) to those of their corresponding hairy roots.     

Indole alkaloids from Alstonia scholaris

A new indole alkaloid, alstonamine and a sitsirikine type indole alkaloid, rhazimanine, have been isolated from the leaves of Alstonia scholaris.     

Indole alkaloids from Rauvolfia media

Four monomeric indole alkaloids have been isolated from the bark of Rauvolfia media. Three of them are the known cabucine, reserpiline and mauiensine; the fourth is a new alkaloid, 12-hydroxymauiensine.     

Indole alkaloids from Ervatamia chinensis

Four vobasinyl–ibogan type bisindole alkaloids, ervachinines A–D (1–4), along with 12 known terpenoid indole alkaloids, were isolated from the whole plant of Ervatamia chinensis. Their structures were elucidated by analysis of spectroscopic data, including 1D and 2D NMR, and the absolute configurations of 1–4 were determined by CD exciton chirality method. All of the compounds were evaluated for in vitro cytotoxicity against five human cancer cell lines: HL-60, SMMC-7721, A-549, MCF-7 and SW480. Bisindole alkaloids 1–6 exhibited inhibitory effects, with IC₅₀ values comparable to those of cisplatin.     

Indole alkaloids from Hunteria zeylanica

Fourteen indole alkaloids were isolated and identified from the leaves and bark of Hunteria zeylanica: isocorymine, vobasine, (+)-eburnamenine, eburnamine, isoeburnamine, O-ethyleburnamine, O-methyleburnamine, O-methylisoeburnamine, pleiocarpamine, dihydrocorynantheol, yohimbol, epiyohimbol, tuboxenine and hydroxy-17-decarbomethoxy-16-dihydroepiajmalicine. O-Ethyleburnamine, O-methylisoeburnamine, epiyobimbol and hydroxy-17-decarbomethoxy-16-dihydroepiajmalicine, although known products, were isolated for the first time from a natural source.     

Formation of edulinine and furoquinoline alkaloids from quinoline derivatives by cell suspension cultures of Ruta graveolens

The formation of furoquinoline alkaloids and of edulinine, elaborated by cell suspension cultures of Ruta graveolens, was found to occur by way of 4-hydroxy-2-quinolone. Other substrates transformed to furoquinolines included 4-hydroxy- and 4-methoxy-3-(3-methyl-2-butenyl)-2-quinolone, known earlier as natural precursors in studies with whole plants. Involvement of dictamnine as a natural precursor of 8-methoxydictamnine (γ-fagarine) and skimmianine was proved using ¹⁴C-labelled compounds. Edulinine in the cell suspensions was formed from such substrates as 4-hydroxy-N-methyl-2-quinolone, 4-hydroxy-3-(3-methyl-2-butenyl)-N-methyl-2-quinolone and its 4- methyl ether; this is probably the natural biosynthetic sequence. Changes in alkaloid yields were noted upon prolonged subculturing.     

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.     

Isochorismate hydroxymutase from Rubiaceae cell suspension cultures

The enzyme catalysing the isomerisation of chorismic to isochorismic acid (isochorismate hydroxymutase E.C. 5.4.99.6) has been detected in protein preparations of various cell suspension cultures derived from plants of Rubiaceae species.Abbreviations OSB o-Succinylbenzoic acid - Tris Tris(hydroxymethyl)aminomethane - DEAE Diethylaminoethyl cellulose     

Isolation of organelles from carrot cell suspension cultures

Summary Organelles isolated from carrot cell suspension cultures by density gradient centrifugation and identified by their specific marker enzymes were found at the following mean densities on the sucrose gradient: microbodies 1.25 g/cm³ (catalase), mitochondria 1.18 (fumarase), endoplasmic reticulum 1.09 g/cm³ (NADH-cytochrome c reductase). Further enzyme assays were done for characterization of microbodies from carrot cultures.This work was supported by the Deutsche Forschungsgemeinschaft.