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Catharanthine, a component of the anticancer drug vinblastine along with vindoline, disrupts the cell cycle by interfering with mitotic spindle formation. Apart from their antioxidant properties, vinca alkaloids like catharanthine inhibit phosphodiesterase activity and elevate intracellular cAMP levels. The aim of this study was to investigate how catharantine affects apoptosis and autophagy. This study conducted experiments on HepG2 liver carcinoma cells with varying doses of catharanthine to evaluate cell death rates and viability and determine the IC50 concentration via MTT assays. The apoptotic and autophagic effects of catharanthine were assessed using flow cytometry with annexin V and PI staining, while the expression of autophagy-related genes was analyzed through quantitative PCR. Additionally, molecular docking and molecular dynamics simulations were employed to further investigate catharanthine's impact on autophagy mechanisms. The study showed that catharanthine reduced oxidative stress and triggered apoptosis in HepG2 cells in a dose-dependent manner. Catharanthine also upregulated the expression of autophagy-related genes like LC3, Beclin1, and ULK1. Notably, catharanthine increased sirtuin-1 levels, a known autophagy inducer, while decreasing Akt expression compared to untreated cells. Molecular docking results indicated rapamycin had a stronger binding affinity with FRB (−10.7 KJ/mol−1) than catharanthine (−7.3 KJ/mol−1). Additionally, molecular dynamics simulations revealed that catharanthine interacted effectively with the FRB domain of mTOR, displaying stability and a strong binding affinity, although not as potent as rapamycin. In summary, besides its cytotoxic and pro-apoptotic effects, catharanthine activates autophagy signaling pathways and induces autophagic necrosis by inhibiting mTOR. [ABSTRACT FROM AUTHOR]

Since the role of Nrf2 in cancer cell survival has been highlighted, the pharmacological modulation of the Nrf2-Keap1 pathway may provide new opportunities for cancer treatment. This study purposed to use ubiquinone (Q10) as an antioxidant and catharanthine alkaloid as a cAMP inducer suppressing HepG2 cells by reducing Nrf2 level. The effects of Q10 and catharanthine on HepG2 cells in terms of viability were analyzed by MTT test. MTT results were used to determine the effective concentration of both drugs for the subsequent treatment and analysis. Subsequently, the effects of Q10 and catharanthine in a single and combined manner on oxidant/antioxidant status, apoptosis, metastasis, and drug resistance of HepG2 cells were investigated by related methods. Both Q10 and catharanthine decreased the level of oxidative stress products and increased antioxidant capacity in HepG2 cells. Nrf2 gene expression decreased by Q10, but catharanthine unexpectedly increased it. Following Nrf2 alterations, the expression levels of MMP-9 and MRP1 involved in metastasis and drug resistance were significantly and dose-dependently decreased by Q10, while catharanthine slightly increased both. However, both drugs increased caspase 3/7 activity and apoptosis rate, and the effect of Q10 on apoptosis was stronger than that of catharanthine. Most of the effects of the combination treatments were similar to those of the Q10 single treatment and indicated the dominant effect over the catharanthine component. Despite the antioxidant and apoptotic properties of both agents, Q10 was better than catharanthine in inducing apoptosis, counteracting drug resistance, and metastasis in HepG2 cells. [ABSTRACT FROM AUTHOR]

NAC transcription factors (TFs) are crucial to growth and defense responses in plants. Though NACs have been characterized for their role in several plants, comprehensive information regarding their role in Catharanthus roseus, a perennial ornamental plant, is lacking. Homology modelling was employed to identify and characterize NACs in C. roseus. In-vitro propagation of C. roseus plants was carried out using cell suspension and nodal culture and were elicitedwith two auxin-antagonists, 5-fluoro Indole Acetic Acid (5-F-IAA) and a-(phenyl ethyl-2-oxo)-Indole-Acetic-Acid (PEO-IAA) for the enhanced production ofmonoterpenoid indole alkaloids (MIAs) namely catharanthine, vindoline, and vinblastine. Analyses revealed the presence of 47 putative CrNAC genes in the C. roseus genome, primarily localized in the nucleus. Phylogenetic analysis categorized these CrNACs into eight clusters, demonstrating the highest synteny with corresponding genes in Camptotheca acuminata. Additionally, at least one defense or hormone-responsive cis-acting element was identified in the promoter region of all the putative CrNACs. Of the two elicitors, 5-F-IAA was effective at 200 µM to elicit a 3.07-fold increase in catharanthine, 2.76-fold in vindoline, and 2.4-fold in vinblastine production in nodal culture. While a relatively lower increase inMIAswas recorded in suspension culture. Validation of RNA-Seq by qRT-PCR showed upregulated expression of stressrelated genes (CrNAC-07 and CrNAC-24), and downregulated expression of growth-related gene (CrNAC-25) in elicited nodal culture of C. roseus. Additionally, the expression of genes involved in the biosynthesis of MIAs was significantly upregulated upon elicitation. The current study provides the first report on the role of CrNACs in regulating the biosynthesis of MIAs. [ABSTRACT FROM AUTHOR]

NAC transcription factors (TFs) are crucial to growth and defense responses in plants. Though NACs have been characterized for their role in several plants, comprehensive information regarding their role in Catharanthus roseus, a perennial ornamental plant, is lacking. Homology modelling was employed to identify and characterize NACs in C. roseus. In-vitro propagation of C. roseus plants was carried out using cell suspension and nodal culture and were elicited with two auxin-antagonists, 5-fluoro Indole Acetic Acid (5-F-IAA) and α-(phenyl ethyl-2-oxo)-Indole-Acetic-Acid (PEO-IAA) for the enhanced production of monoterpenoid indole alkaloids (MIAs) namely catharanthine, vindoline, and vinblastine. Analyses revealed the presence of 47 putative CrNAC genes in the C. roseus genome, primarily localized in the nucleus. Phylogenetic analysis categorized these CrNACs into eight clusters, demonstrating the highest synteny with corresponding genes in Camptotheca acuminata. Additionally, at least one defense or hormone-responsive cis-acting element was identified in the promoter region of all the putative CrNACs. Of the two elicitors, 5-F-IAA was effective at 200 µM to elicit a 3.07-fold increase in catharanthine, 2.76-fold in vindoline, and 2.4-fold in vinblastine production in nodal culture. While a relatively lower increase in MIAs was recorded in suspension culture. Validation of RNA-Seq by qRT-PCR showed upregulated expression of stress-related genes (CrNAC-07 and CrNAC-24), and downregulated expression of growth-related gene (CrNAC-25) in elicited nodal culture of C. roseus. Additionally, the expression of genes involved in the biosynthesis of MIAs was significantly upregulated upon elicitation. The current study provides the first report on the role of CrNACs in regulating the biosynthesis of MIAs.

Catharanthus roseus is a medicinal plant that produces indole alkaloids, which are utilized in anticancer therapy. Vinblastine and vincristine, two commercially important antineoplastic alkaloids, are mostly found in the leaves of Catharanthus roseus. ĸ-carrageenan has been proven as plant growth promoting substance for a number of medicinal and agricultural plants. Considering the importance of ĸ-carrageenan as a promoter of plant growth and phytochemical constituents, especially alkaloids production in Catharanthus roseus, an experiment was carried out to explore the effect of ĸ-carrageenan on the plant growth, phytochemicals content, pigments content, and production of antitumor alkaloids in Catharanthus roseus after planting. Foliar application of ĸ-carrageenan (at 0, 400, 600 and 800 ppm) significantly improved the performance of Catharanthus roseus. Phytochemical analysis involved determining the amount of total phenolics (TP), flavonoids (F), free amino acids (FAA), alkaloids (TAC) and pigments contents by spectrophotometer, minerals by ICP, amino acids, phenolic compounds and alkaloids (Vincamine, Catharanthine, Vincracine (Vincristine), and vinblastine) analysis uses HPLC. The results indicated that all examined ĸ-carrageenan treatments led to a significant (p ≤ 0.05) increase in growth parameters compared to the untreated plants. Phytochemical examination indicates that the spray of ĸ-carrageenan at 800 mg L−1 increased the yield of alkaloids (Vincamine, Catharanthine and Vincracine (Vincristine)) by 41.85 μg/g DW, total phenolic compounds by 3948.6 μg gallic/g FW, the content of flavonoids 951.3 μg quercetin /g FW and carotenoids content 32.97 mg/g FW as compared to the control. An amount of 400 ppm ĸ-carrageenan treatment gave the best contents of FAA, Chl a, Chl b and anthocyanin. The element content of K, Ca, Cu, Zn and Se increased by treatments. Amino acids constituents and phenolics compounds contents were altered by ĸ-carrageenan. [ABSTRACT FROM AUTHOR]

Catharanthus roseus (L.) G. Don is a plant belonging to the genus Catharanthus of the Apocynaceae family. It contains more than one hundred alkaloids, of which some exhibit significant pharmacological activities. Chitooligosaccharides are the only basic aminooligosaccharides with positively charged cations in nature, which can regulate plant growth and antioxidant properties. In this study, the leaves of Catharanthus roseus were sprayed with chitooligosaccharides of different molecular weights (1 kDa, 2 kDa, 3 kDa) and different concentrations (0.01 μg/mL, 0.1 μg/mL, 1 μg/mL and 10 μg/mL). The fresh weights of its root, stem and leaf were all improved after chitooligosaccharides treatments. More importantly, the chitooligosaccharides elicitor strongly stimulated the accumulation of vindoline and catharanthine in the leaves, especially with the treatment of 0.1 μg/mL 3 kDa chitooligosaccharides, the contents of them were increased by 60.68% and 141.54%, respectively. Furthermore, as the defensive responses, antioxidant enzymes activities (catalase, glutathione reductase, ascorbate peroxidase, peroxidase and superoxide dismutase) were enhanced under chitooligosaccharides treatments. To further elucidate the underlying mechanism, qRT-PCR was used to investigate the genes expression levels of secologanin synthase (SLS), strictosidine synthase (STR), strictosidine glucosidase (SGD), tabersonine 16-hydroxylase (T16H), desacetoxyvindoline-4-hydroxylase (D4H), deacetylvindoline-4-O-acetyltransferase (DAT), peroxidase 1 (PRX1) and octadecanoid-responsive Catharanthus AP2-domain protein 3 (ORCA3). All the genes were significantly up-regulated after chitooligosaccharides treatments, and the transcription abundance of ORCA3, SLS, STR, DAT and PRX1 reached a maximal level with 0.1 μg/mL 3 kDa chitooligosaccharides treatment. All these results suggest that spraying Catharanthus roseus leaves with chitooligosaccharides, especially 0.1 μg/mL of 3 kDa chitooligosaccharides, may effectively improve the pharmaceutical value of Catharanthus roseus. [ABSTRACT FROM AUTHOR]

The present study was carried out to silence the transcription factor genes ZCT1, ZCT2 and ZCT3 via lipofectamine based antisense LNA GapmeRs transfection into the protoplasts of established photomixotrophic cell suspensions. The photomixotrophic cell suspensions with a threshold of 0.5% sucrose were raised and established using two-tiered CO2 providing flasks kept under high light intensity. The photomixotrophic cell suspensions showed morphologically different thick-walled cells under scanning electron microscopic analysis in comparison to the simple thin-walled parenchymatous control cell suspensions. The LC–MS analysis registered the vindoline production (0.0004 ± 0.0001 mg/g dry wt.) in photomixotrophic cell suspensions which was found to be absent in control cell suspensions. The protoplasts were isolated from the photomixotrophic cell suspensions and subjected to antisense LNA GapmeRs silencing. Three lines, viz. Z1A, Z2C and Z3G were obtained where complete silencing of ZCT1, ZCT2 and ZCT3 genes, respectively, was observed. The Z3G line was found to show maximum production of vindoline (0.038 ± 0.001 mg/g dry wt.), catharanthine (0.165 ± 0.008 mg/g dry wt.) and vinblastine (0.0036 ± 0.0003 mg/g dry wt.). This was supported by the multifold increment in the gene expression of TDC, SLS, STR, SGD, d4h, dat, CrT16H and Crprx. The present work indicates the master regulation of ZCT3 knockdown among all three ZCTs transcription factors in C. roseus to enhance the terpenoid indole alkaloids production. The successful silencing of transcription repressor genes has been achieved in C. roseus plant system by using photomixotrophic cell cultures through GapmeR based silencing. The present study is a step towards metabolic engineering of the TIAs pathway using protoplast transformation in C. roseus. [ABSTRACT FROM AUTHOR]

Catharanthus roseus (L.) G. Don is a plant belonging to the genus Catharanthus of the Apocynaceae family. It contains more than one hundred alkaloids, of which some exhibit significant pharmacological activities. Chitooligosaccharides are the only basic aminooligosaccharides with positively charged cations in nature, which can regulate plant growth and antioxidant properties. In this study, the leaves of Catharanthus roseus were sprayed with chitooligosaccharides of different molecular weights (1 kDa, 2 kDa, 3 kDa) and different concentrations (0.01 μg/mL, 0.1 μg/mL, 1 μg/mL and 10 μg/mL). The fresh weights of its root, stem and leaf were all improved after chitooligosaccharides treatments. More importantly, the chitooligosaccharides elicitor strongly stimulated the accumulation of vindoline and catharanthine in the leaves, especially with the treatment of 0.1 μg/mL 3 kDa chitooligosaccharides, the contents of them were increased by 60.68% and 141.54%, respectively. Furthermore, as the defensive responses, antioxidant enzymes activities (catalase, glutathione reductase, ascorbate peroxidase, peroxidase and superoxide dismutase) were enhanced under chitooligosaccharides treatments. To further elucidate the underlying mechanism, qRT-PCR was used to investigate the genes expression levels of secologanin synthase (SLS), strictosidine synthase (STR), strictosidine glucosidase (SGD), tabersonine 16-hydroxylase (T16H), desacetoxyvindoline-4-hydroxylase (D4H), deacetylvindoline-4-O-acetyltransferase (DAT), peroxidase 1 (PRX1) and octadecanoid-responsive Catharanthus AP2-domain protein 3 (ORCA3). All the genes were significantly up-regulated after chitooligosaccharides treatments, and the transcription abundance of ORCA3, SLS, STR, DAT and PRX1 reached a maximal level with 0.1 μg/mL 3 kDa chitooligosaccharides treatment. All these results suggest that spraying Catharanthus roseus leaves with chitooligosaccharides, especially 0.1 μg/mL of 3 kDa chitooligosaccharides, may effectively improve the pharmaceutical value of Catharanthus roseus.

[Display omitted] • Isocatharanthine is a NaBH 4 reduction product of catharanthine. • Isocatharanthine was claimed to be an isomer of catharanthine. • Isocatharanthine is the N-borane derivative of catharanthine. • Structure of catharanthine borane was determined by NMR including 11B NMR. The reaction of catharanthine with sodium borohydride in methylene chloride was claimed to produce an isomer of the title compound. A spectroscopic re-examination of the product has revealed that it is catharanthine borane. [ABSTRACT FROM AUTHOR]

Iboga alkaloids, also known as coronaridine congeners, have shown promise in the treatment of alcohol and opioid use disorders. The objective of this study was to evaluate the effects of catharanthine and 18-methoxycoronaridine (18-MC) on dopamine (DA) transmission and cholinergic interneurons in the mesolimbic DA system, nicotine-induced locomotor activity, and nicotine-taking behavior. Utilizing ex vivo fast-scan cyclic voltammetry (FSCV) in the nucleus accumbens core of male mice, we found that catharanthine or 18-MC differentially inhibited evoked DA release. Catharanthine inhibition of evoked DA release was significantly reduced by both α4 and α6 nicotinic acetylcholine receptors (nAChRs) antagonists. Additionally, catharanthine substantially increased DA release more than vehicle during high-frequency stimulation, although less potently than an α4 nAChR antagonist, which confirms previous work with nAChR antagonists. Interestingly, while catharanthine slowed DA reuptake measured via FSCV ex vivo , it also increased extracellular DA in striatal dialysate from anesthetized mice in vivo in a dose-dependent manner. Superfusion of catharanthine or 18-MC inhibited the firing rate of striatal cholinergic interneurons in a concentration dependent manner, which are known to potently modulate presynaptic DA release. Catharanthine or 18-MC suppressed acetylcholine currents in oocytes expressing recombinant rat α6/α3β2β3 or α6/α3β4 nAChRs. In behavioral experiments using male Sprague-Dawley rats, systemic administration of catharanthine or 18-MC blocked nicotine enhancement of locomotor activity. Importantly, catharanthine attenuated nicotine self-administration in a dose-dependent manner while having no effect on food reinforcement. Lastly, administration of catharanthine and nicotine together greatly increased head twitch responses, indicating a potential synergistic hallucinogenic effect. These findings demonstrate that catharanthine and 18-MC have similar, but not identical effects on striatal DA dynamics, striatal cholinergic interneuron activity and nicotine psychomotor effects.

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Catharanthus roseus (C. roseus) plants were used to investigate the terpenoid indole alkaloids (TIAs) accumulation under the condition of PEG-induced drought stress. Multivariate analysis showed that 35% PEG6000 could induce more obvious and stable accumulation on proline (PRO) content and the relative water content (RWC). The results indicated that there were gradual increase and then decrease (p

NAC transcription factors (TFs) are crucial to growth and defense responses in plants. Though NACs have been characterized for their role in several plants, comprehensive information regarding their role in Catharanthus roseus , a perennial ornamental plant, is lacking. Homology modelling was employed to identify and characterize NACs in C. roseus . In-vitro propagation of C. roseus plants was carried out using cell suspension and nodal culture and were elicited with two auxin-antagonists, 5-fluoro Indole Acetic Acid (5-F-IAA) and α-(phenyl ethyl-2-oxo)-Indole-Acetic-Acid (PEO-IAA) for the enhanced production of monoterpenoid indole alkaloids (MIAs) namely catharanthine, vindoline, and vinblastine. Analyses revealed the presence of 47 putative CrNAC genes in the C. roseus genome, primarily localized in the nucleus. Phylogenetic analysis categorized these CrNACs into eight clusters, demonstrating the highest synteny with corresponding genes in Camptotheca acuminata . Additionally, at least one defense or hormone-responsive cis-acting element was identified in the promoter region of all the putative CrNAC s. Of the two elicitors, 5-F-IAA was effective at 200 µM to elicit a 3.07-fold increase in catharanthine, 2.76-fold in vindoline, and 2.4-fold in vinblastine production in nodal culture. While a relatively lower increase in MIAs was recorded in suspension culture. Validation of RNA-Seq by qRT-PCR showed upregulated expression of stress-related genes ( CrNAC-07 and CrNAC-24 ), and downregulated expression of growth-related gene ( CrNAC-25 ) in elicited nodal culture of C. roseus . Additionally, the expression of genes involved in the biosynthesis of MIAs was significantly upregulated upon elicitation. The current study provides the first report on the role of CrNACs in regulating the biosynthesis of MIAs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Ahmed, Sajjad, Gatasheh, Ibrahim, Huzafa, Khan, Situ, Abbasi and Hassan.)

Catharanthus roseus is known for its anticancer bisindole alkaloids, vinblastine and vincristine that are derived from the condensation of monomeric terpenoid indole alkaloids (TIAs), catharanthine and vindoline. The in planta content of TIAs varies with seasons and ontogeny making it difficult for growers to define the optimal parameters for harvesting for specific TIAs. Here, specific leaf TIAs were profiled in the plant at frequent intervals throughout the year covering various seasons under North-Indian conditions. Higher bisindole alkaloid content was observed during summer (June; average day temperature ≥ 40 °C), whereas vindoline content was higher in winter and spring (mid-December to mid-April). Further, the plant was analyzed for variation in ontogenic localization of TIAs in the leaves. The monomeric TIAs were present maximally in the top-level leaf-pairs, whereas the bisindole alkaloids were present maximally in the middle-level leaf-pairs. Analysis of relative abundance of individual TIAs in the total alkaloid pool indicated a clear partitioning between vindoline on one hand and bisindole alkaloids on the other. qRT-PCR-based expression analysis of 10 pathway genes (tdc , str , sgd , t16h , omt , t3o , t3r , nmt , d4h , dat) involved in the biosynthesis of key TIAs, like vindoline, indicated that the transcript abundance of all the genes, except sgd , was significantly higher in winter as compared to summer. Also, upon moving down from the top to the lower level leaf-pairs, the transcript abundance of all the genes decreased significantly. This study defines the seasonal and ontogenic parameters for harvesting of C. roseus leaves for optimal recovery of specific TIAs. • Catharanthus roseus produces many terpenoid indole alkaloids (TIAs). • Coupling of vindoline and catharanthine yields anticancer bisindole alkaloids. • Seasonal and ontogenic parameters defined for optimal recovery of specific TIAs. • Bisindoles – higher in summer; vindoline – higher in winter/spring. • Bisindoles – higher in middle-level leaves; monomers – higher in top-level leaves. [ABSTRACT FROM AUTHOR]

The aim of the study was to replace mineral fertilizers with organic and biological fertilizers to improve nutrient uptake, plant growth and the concentrations of some important secondary metabolites in periwinkle (Catharanthus roseus L.). Periwinkle plants were grown under different rates of N supply (0, 20 and 40 mg kg-1 soil) and biological treatments (Azospirillum, Azotobacter, Azospirillum plus Azotobacter, Azospirillum plus fungal compost, Azotobacter plus fungal compost, and fungal compost). The concentrations of pigments and nutrients were measured by spectrophotometry and flame photometry. Secondary metabolites were analyzed by high-performance liquid chromatography (HPLC). Data were recorded for plant growth and development parameters, nutrient uptake and some secondary metabolites of periwinkle plants. The results showed that the N-fertilizer and biological treatments significantly improved most growth attributes and nutrient uptake and increased the concentrations of secondary metabolites as compared to the control. Maximum concentrations of root ajmalicine (0.54 mg g-1 DW), leaf vinblastine (0.96 mg g-1 DW) and root catharanthine (2.38 mg g-1 DW) were obtained from the treatment with Azospirillum under N-fertilizer at 20 and 40 mg kg-1 soil. Azotobacter along with fungal compost under N-fertilizer at 40 mg kg-1 soil induced the maximum concentration of leaf vindoline (1.94 mg g-1 DW). The highest concentration of root alkaloids (1.11 mg g-1 DW) was obtained from the treatment with compost under 40 mg N kg-1 soil. Azospirillum, Azotobacter and fungal compost combined with the N-fertilizer improved many morphological and nutrient characteristics. In conclusion, the growth and metabolism of C. roseus were significantly positively affected by the organic and biological fertilizers. [ABSTRACT FROM AUTHOR]

Flowers of Tabernaemontana divaricata (L.) R. Br., (Apocynaceae) are used in traditional medicine for analgesic property. The present study was performed to isolate the active principles and investigate the mechanisms involved in the anti-nociception caused by T. divaricata flower methanolic extract (TDFME). The extract in the doses of 125, 250 and 500 mg/kg, p.o was subjected to various assays in acetic acid induced abdominal writhing and formalin induced paw licking test models. Naloxone, L-Arginine, Glibenclamide and Glutamate were used as inducers while Morphine, L-NAME, Methylene blue and Aspirin served as standard drugs. The phytochemical analysis led to the isolation of three indole alkaloids namely Voacangine, Catharanthine and O-acetyl Vallesamine. The anti-nociception produced by TDFME was attenuated significantly (p< 0.001) by the intra-peritoneal pretreatment of naloxone, L-Arginine and glibenclamide. The nociception produced by glutamate was inhibited by TDFME. TDFME also enhanced the antinociceptive activity of L-NAME when given in combination. However TDFME co-administration did not produce significant results with methylene blue indicating lack of cGMP involvement. These results indicate that TDFME produces anti-nociception action mediated by opioid, nitric oxide, K+-ATP and glutamate mechanisms and the effect is largely related to the indole alkaloids. [ABSTRACT FROM AUTHOR]

Monoterpenoid indole alkaloids (MIAs) possess a diversity of alkaloid skeletons whose biosynthesis is poorly understood. A bioinformatic search of candidate genes, combined with their virus-induced gene silencing, targeted MIA profiling and in vitro/in vivo pathway reconstitution identified and functionally characterized six genes as well as a seventh enzyme reaction required for the conversion of 19E-geissoschizine to tabersonine and catharanthine. The involvement of pathway intermediates in the formation of four MIA skeletons is described, and the role of stemmadenine-O-acetylation in providing necessary reactive substrates for the formation of iboga and aspidosperma MIAs is described. The results enable the assembly of complex dimeric MIAs used in cancer chemotherapy and open the way to production of many other biologically active MIAs that are not easily available from nature. [ABSTRACT FROM AUTHOR]

In contrast to plasma membrane-expressed nicotinic acetylcholine receptors (nAChRs), mitochondrial nAChRs function in an ion-independent manner by triggering intra-mitochondrial kinases that regulate the release of cytochrome c (Cyt c), an important step in cellular apoptosis. The aim of this study is to determine the structural requirements for mitochondrial α3β4* nAChR activation by measuring the modulatory effects of two noncompetitive antagonists of these receptors, (+)-catharanthine and (±)-18-methoxycoronaridine [(±)-18-MC], on Cyt c release from wild-type and α7−/− mice mitochondria. The sandwich ELISA results indicated that α3β4* nAChRs are present in liver mitochondria in higher amounts compared to that in brain mitochondria and that these receptors are up-regulated in α7−/− mice. Correspondingly, (±)-18-MC decreased Cyt c release from liver mitochondria of wild-type mice and from brain and liver mitochondria of α7−/− mice. The effect in wild-type mice mitochondria was mediated mainly by the Src-dependent pathway, regulating the apoptogenic activity of reactive oxygen species, while in α7−/− mice mitochondria, (±)-18-MC strongly affected the calcium-calmodulin kinase II-dependent pathway. In contrast, (+)-catharanthine was much less potent than (±)-18-MC and triggered several signaling pathways, suggesting the involvement of multiple nAChR subtypes. These results show for the first time that noncompetitive antagonists can induce mitochondrial α3β4* nAChR signaling, giving a more comprehensive understanding on the function of intracellular nAChR subtypes. [ABSTRACT FROM AUTHOR]

Main conclusion A Catharanthus roseus mutant accumulates high levels of ajmalicine at the expense of catharanthine and vindoline. The altered chemistry depends on increased expression and biochemical activities of strictosidine ß-glucosidase and ajmalicine synthase activities and reduced expression and biochemical activity of geissoschizine synthase. The Madagascar periwinkle [Catharanthus roseus (L.) G. Don] is a commercially important horticultural flower species and is a valuable source for several monoterpenoid indole alkaloids (MIAs), such as the powerful antihypertensive drug ajmalicine and the antineoplastic agents, vinblastine and vincristine. While biosynthesis of the common MIA precursor strictosidine and its reactive aglycones has been elucidated, the branch point steps leading to the formation of different classes of MIAs remain poorly characterized. Screening of 3600 ethyl methyl sulfonate mutagenized C. roseus plants using a simple thin-layer chromatography screen yielded a mutant (M2-0754) accumulating high levels of ajmalicine together with significantly lower levels of catharanthine and vindoline. Comparative bioinformatic analyses, virus-induced gene silencing, and biochemical characterization identified geissoschizine synthase, the gateway enzyme that controls flux for the formation of iboga and aspidosperma MIAs. The reduction of geissoschizine synthase transcripts in this high ajmalicine mutant, together with increased transcripts and enzyme activities of strictosidine β-glucosidase and of heteroyohimbine synthase, explains the preferential formation of ajmalicine in the mutant instead of catharanthine and vindoline that accumulates in the wild-type parent. Reciprocal crosses established that that the high ajmalicine phenotype is inherited as a Mendelian recessive trait. [ABSTRACT FROM AUTHOR]

We describe here a regeneration of plantlets of Catharanthus roseus (L.) G. Don from shoot tips and axillary buds. Shoot tips were excised from 7-day-old seedlings and were incubated in solid Nitsch and Nitsch (NN) medium supplemented with kinetin, benzyladenine (BA), indole-3-butyric acid (IBA) and β-indolylacetic acid (IAA) in various combinations. After two months in culture, regenerated rooted plantlets were cut and transferred to a new medium; the explants contained shoot tips or axillary buds. Four passages were done. We obtained about 200 rooting plantlets from one seedling. Then the plantlets were transferred to the soil and they grew under a foil tent. After five months of vegetation they were collected, dried and weighed. Chemical investigations of leaves of these plants were done. The vindoline and catharanthine were dominant alkaloids in the juvenile stage of plants (before blooming). Total amount of alkaloids, equal 2.95%, was gravimetrically determined in leaves of plants, after 4th passage, regenerated in vitro on NN medium supplemented with kinetin and IBA.

Isoquinuclidines constitute the central structural nucleus of numerous biologically active natural products, for example, iboga alkaloids such as ibogamine and catharanthine as well as non-indole-containing alkaloids such as the dioscorine and the cannivonines. Furthermore, in medicinal and pharmaceutical chemistry, the isoquinuclidine core is commonly employed as a rigid azabicyclic scaffold, thus providing significant precursors in the synthesis of numerous valuable alkaloids. Summarizing well-organized approaches to access the chiral isoquinuclidine structural centerpiece signifies a significant endeavor not only for developing biologically active natural products but also enhancing biological researches that can lead to possible drug discovery. Over time, the values and methodologies for the asymmetric synthesis of chiral isoquinuclidines are increasing; hence to advance asymmetric synthesis, this review combines and discusses the pros and cons of each synthesis techniques from 2008. This review should be helpful for promoting further developments of asymmetric synthetic methodologies and for medicinal chemistry. [ABSTRACT FROM AUTHOR]

The inhibitory activity of coronaridine congeners on human (h) α4β2 and α7 nicotinic acetylcholine receptors (AChRs) is determined by Ca 2+ influx assays, whereas their effects on neurons in the ventral inferior (VI) aspect of the mouse medial habenula (MHb) are determined by patch-clamp recordings. The Ca 2+ influx results clearly establish that coronaridine congeners inhibit hα3β4 AChRs with higher selectivity compared to hα4β2 and hα7 subtypes, and with the following potency sequence, for hα4β2: (±)-18-methoxycoronaridine [(±)-18-MC] > (+)-catharanthine > (±)-18-methylaminocoronaridine [(±)-18-MAC] ∼ (±)-18-hydroxycoronaridine [(±)-18-HC]; and for hα7: (+)-catharanthine > (±)-18-MC > (±)-18-HC > (±)-18-MAC. Interestingly, the inhibitory potency of (+)-catharanthine (27 ± 4 μM) and (±)-18-MC (28 ± 6 μM) on MHb (VI) neurons was lower than that observed on hα3β4 AChRs, suggesting that these compounds inhibit a variety of endogenous α3β4* AChRs. In addition, the interaction of bupropion with (−)-ibogaine sites on hα3β4 AChRs is tested by [ 3 H]ibogaine competition binding experiments. The results indicate that bupropion binds to ibogaine sites at desensitized hα3β4 AChRs with 2-fold higher affinity than at resting receptors, suggesting that these compounds share the same binding sites. In conclusion, coronaridine congeners inhibit hα3β4 AChRs with higher selectivity compared to other AChRs, by interacting with the bupropion (luminal) site. Coronaridine congeners also inhibit α3β4*AChRs expressed in MHb (VI) neurons, supporting the notion that these receptors are important endogenous targets for their anti-addictive activities. [ABSTRACT FROM AUTHOR]

Catharanthus roseus(C. roseus) plants were used to investigate the terpenoid indole alkaloids (TIAs) accumulation under the condition of PEG-induced drought stress. Multivariate analysis showed that 35% PEG6000 could induce more obvious and stable accumulation on proline (PRO) content and the relative water content (RWC). The results indicated that there were gradual increase and then decrease (p < .05) in the contents of vindoline (VIN) and catharanthine (CAT) under 35% PEG6000 stress, but the content of vinblastine (VBL) increased gradually. In addition, the expression levels of tryptophan decarboxylase (TDC), strictosidine synthase (STR) and deacetylvindoline-4-O-acetyltransferase (DAT) were upregulated in plants under 35% PEG6000 stress. Further correlation analysis indicated that CAT accumulation was significantly correlated with TDC gene expression, and VBL accumulation was significantly correlated with peroxidase (p < .05). Our results suggest that the cultivation ofC. roseusin drought stress would serve as effective treatment for accumulating TIAs. [ABSTRACT FROM PUBLISHER]

Our successful work for the synthesis of cyclopropanated vinblastine and its derivatives by the Simmons–Smith reaction was followed to build up further three-membered rings into the 14,15-position of the vindoline part of the dimer alkaloid. Halogenated 14,15-cyclopropanovindoline was prepared by reactions with iodoform and bromoform, respectively, in the presence of diethylzinc. Reactions of dichlorocarbene with vindoline resulted in the 10-formyl derivative. Unexpectedly, in the case of the dimer alkaloids vinblastine and vincristine, the rearranged products containing an oxirane ring in the catharanthine part were isolated from the reactions. The attempted epoxidation of vindoline and catharanthine also led to anomalous rearranged products. In the epoxidation reaction of vindoline, an o-quinonoid derivative was obtained, in the course of the epoxidation of catharanthine, a hydroxyindolenine type product and a spiro derivative formed by ring contraction reaction, were isolated. The coupling reaction of vindoline and the spiro derivative obtained in the epoxidation of catharanthine did not result in a bisindole alkaloid. Instead, two surprising vindoline trimers were discovered and characterized by NMR spectroscopy and mass spectrometry.

The research aim was to examine the enhancement of catharanthine content in Catharanthus roseus callus culture added with different concentration of Naphtalene Acetic Acid (NAA). NAA treatment produced callus that formed hairy roots. Fresh and dry weight of callus increased as the increasing of NAA concentration. The catharanthine content of C. roseus callus culture was increased by adding NAA as well. The highest catharanthine content was found in 2.5 ppm NAA added callus.

The research aim was to examine the enhancement of catharanthine content in Catharanthus roseus callus culture added with different concentration of Naphtalene Acetic Acid (NAA). NAA treatment produced callus that formed hairy roots. Fresh and dry weight of callus increased as the increasing of NAA concentration. The catharanthine content of C. roseus callus culture was increased by adding NAA as well. The highest catharanthine content was found in 2.5 ppm NAA added callus.

Many natural products, including vinblastine, have not been easily subjected to simplifications in their structures by synthetic means or modifications by late-stage semisynthetic derivatization in ways that enhance their biological potency. Herein, we detail a synthetic vinblastine that incorporates added benign complexity (ABC), which improves activity 10-fold, and is now accessible as a result of advances in the total synthesis of the natural product. The compound incorporates designed added molecular complexity but no new functional groups and maintains all existing structural and conformational features of the natural product. It constitutes a member of an analogue class presently inaccessible by semisynthetic derivatization of the natural product, by its late-stage functionalization, or by biosynthetic means. Rather, it was accessed by synthetic means, using an appropriately modified powerful penultimate single-step vindoline–catharanthine coupling strategy that proceeds with a higher diastereoselectivity than found for the natural product itself. [ABSTRACT FROM AUTHOR]

A quaternization reaction was observed at the tertiary nitrogen atom of the catharanthine part of vinblastine instead of the expected cyclopropanation in the Simmons–Smith reaction utilizing diiodomethane and diethylzinc. Investigating the energetic relations, thermodynamic stabilities and the interactions between the molecular orbitals by quantum chemical calculations elucidated the unexpected methylation and confirmed the reaction mechanism. Although the history of the Simmons–Smith reaction goes back more than half a century this represents the first time that the reaction and its possible side-reaction has been studied by DFT level calculations in a complex system. [ABSTRACT FROM AUTHOR]

A rapid and simple reversed phase liquid chromatographic system has been developed for simultaneous analysis of terpenoid indole alkaloids (TIAs) and their precursors. This method allowed separation of 11 compounds consisting of eight TIAs (ajmalicine, serpentine, catharanthine, vindoline, vindolinine, vincristine, vinblastine, and anhydrovinblastine) and three related precursors i.e., tryptophan, tryptamine and loganin. The system has been applied for screening the TIAs and precursors in Catharanthus roseus plant extracts. In this study, different organs i.e., flowers, leaves, stems, and roots of C. roseus were investigated. The results indicate that TIAs and precursor accumulation varies qualitatively and quantitatively in different organs of C. roseus . The precursors showed much lower levels than TIAs in all organs. Leaves and flowers accumulate higher level of vindoline, catharanthine and anhydrovinblastine while roots have higher level of ajmalicine, vindolinine and serpentine. Moreover, the alkaloid profiles of leaves harvested at different ages and different growth stages were studied. The results show that the levels of monoindole alkaloids decreased while bisindole alkaloids increased with leaf aging and upon plant growth. The HPLC method has been successfully applied to detect TIAs and precursors in different types of C. roseus samples to facilitate further study of the TIA pathway and its regulation in C. roseus plants. [ABSTRACT FROM AUTHOR]

Objective: Although catharanthine ( 1) is well known as a biosynthetic precursor of the anticancer alkaloid, vinblastine, its alternative metabolic pathways are unclear. Results: Biotransformation of 1 by suspension-cultured cells of Catharanthus roseus gave a new oxidative-cleavage product ( 2). The structure of 2 was determined as 3-hydroxy-4-imino-catharanthine by spectroscopic methods. Maximum conversion (9.75 %) of 2 was observed after 120 h adding 6 mg of 1/100 ml to 12-day-old suspension-cultured cells of C. roseus. Furthermore, qRT-PCR experiment was performed to reveal the effect of 1 on the expression of the genes in the biosynthetic pathway of TIA 1 up-regulated the transcript level of D4H whilst down-regulating the transcript levels of G10H, LAMT, GES, and IRS. Conclusion: A new metabolite of catharanthine, 3-hydroxy-4-imino-catharanthine, is reported. [ABSTRACT FROM AUTHOR]