Vinblastine production by the endophytic fungus Curvularia verruculosa from the leaves of Catharanthus roseus and its in vitro cytotoxicity against HeLa cell line.

The current study was to isolate endophytic fungi producing high yields of indole alkaloids such as vinblastine analogous to their host Cathranthus roseus. Endophytic fungi were isolated from the leaves of C. roseus, identified as Curvularia verruculosa by molecular techniques, and the sequence was deposited in NCBI (MK995628). Vinblastine producing endophytic fungus was grown in 1L Vinca medium for 21 days. The extract was examined for vinblastine by chromatographic techniques. TLC plates showed purple colour spot co-migrated with authentic vinblastine and Rf was calculated by HPTLC (Vin 1 vinblastine -0.75; authentic vinblastine-0.78), these results confirmed vinblastine presence in the Vin1 extract. Further, the TLC purified fungal extract was examined by LC-MS, which revealed the exact mass of vinblastine ([M + H]+m/z 811.51). The most important of the study is high yield production of vinblastine; hence, the extract analysed by HPLC revealed 182 µg/L vinblastine. The TLC purified fungal vinblastine was analysed for the cytotoxicity effect on HeLa cell line and it depicted a higher activity with IC50-8.5 µg/mL and apoptotic morphological changes were analysed. All the results revealed that the endophytic fungus Curvularia verruculosa produced vinblastine and for the first time in a surplus amount compared to other fungi.

Biotechnological advancements in Catharanthus roseus (L.) G. Don.

Catharanthus roseus (L.) G. Don, also known as Madagascar periwinkle or Sadabahar, is a herbaceous plant belonging to the family Apocynaceae. Being a reservoir for more than 200 alkaloids, it reserves a place for itself in the list of important medicinal plants. Secondary metabolites are present in its leaves (e.g., vindoline, vinblastine, catharanthine, and vincristine) as well as basal stem and roots (e.g., ajmalicine, reserpine, serpentine, horhammericine, tabersonine, leurosine, catharanthine, lochnerine, and vindoline). Two of its alkaloids, vincristine and vinblastine (possessing anticancerous properties), are being used copiously in pharmaceutical industries. Till date, arrays of reports are available on in vitro biotechnological improvements of C. roseus. The present review article concentrates chiefly on various biotechnological advancements based on plant tissue culture techniques of the last three decades, for instance, regeneration via direct and indirect organogenesis, somatic embryogenesis, secondary metabolite production, synthetic seed production, clonal fidelity assessment, polyploidization, genetic transformation, and nanotechnology. It also portrays the importance of various factors influencing the success of in vitro biotechnological interventions in Catharanthus and further addresses several shortcomings that can be further explored to create a platform for upcoming innovative approaches. KEY POINTS: • C. roseus yields anticancerous vincristine and vinblastine used in pharma industry. •In vitro biotechnological interventions prompted major genetic advancements. • This review provides an insight on in vitro-based research achievements till date. • Key bottlenecks and prospective research methodologies have been identified herein.

Synthesis of Bisindole Alkaloids from the Apocynaceae Which Contain a Macroline or Sarpagine Unit: A Review.

Bisindole natural products consist of two monomeric indole alkaloid units as their obligate constituents. Bisindoles are more potent with respect to their biological activity than their corresponding monomeric units. In addition, the synthesis of bisindoles are far more challenging than the synthesis of monomeric indole alkaloids. Herein is reviewed the enantiospecific total and partial synthesis of bisindole alkaloids isolated primarily from the Alstonia genus of the Apocynaceae family. The monomeric units belong to the sarpagine, ajmaline, macroline, vobasine, and pleiocarpamine series. An up-to-date discussion of their isolation, characterization, biological activity as well as approaches to their partial and total synthesis by means of both synthetic and biosynthetic strategies are presented.

[Purification and identification of impurities in vinorelbine bitartrate].

OBJECTIVE: To investigate the chemical constituents of impurities in Vinorelbine Bitartrate. METHODS: The impurities were isolated and purified by silica gel column chromatographies, and their structures were identified on the basis of their physicochemical properties and spectroscopic data. RESULTS: Three compounds were isolated from Vinorelbine Bitartrate, and their structures were identified as Vinorelbine Bitartrate 3',4'-epoxy vinorelbine (1), 3',4'-oxidevinoerlbine (2) and 6'-N-mthyl-17-bormovinoerlbine (3). CONCLUSION: Compounds 2 and 3 are obtained as the impurities in Vinorelbine Bitartrate for the first time.

[Alkaloids of Vinca rosea L. introduced to Western Georgia].

Vinca roseae L. (Саtharanthus rosea (L.) G. Don) was introduced at Kobuleti experimental station of medical plants. The object of investigation was the plant material of Vinca roseae L. collected in May, 2005., September, 2006 and October, 2009. Total alkaloids were obtained in accordance with Atta- ur-Rachman method. The variability of the quantitative and qualitative composition of total alkaloids and vincaleikoblastin (VLB) fraction during vegetation was studied. It was established that the maximal content of total alkaloids and VLB fraction of Vinca roseae L. is accumulated in the phase of secondary flowering, hence the collecting of a plant material is recommended to be made during the aforesaid vegetation phase as for this period it is a rather high output of a raw material, alkaloid complex and VLB faction. Alkaloids vinkaleikoblastin, ajmalicine and new epimer tetrahydroalstonine with С3Н-α- orientation were yielded, separated and identified using modern physical-chemical and spectral methods (13С NMR).

Negative-pressure cavitation extraction of four main vinca alkaloids from Catharanthus roseus leaves.

In the present study, an improved method termed negative-pressure cavitation extraction (NPCE) followed by reverse phase high-performance liquid chromatography (RP-HPLC) was developed for the extraction and quantification of vindoline (VDL), catharanthine (CTR), vincristine (VCR) and vinblastine (VLB) from Catharanthus roseus leaves. The optimized method employed 60-mesh particles, 80% ethanol, a negative pressure of -0.075 MPa, a solid to liquid ratio of 1:20, 30 min of extraction and three extraction cycles. Under these optimized conditions, the extraction yields of VDL, CTR, VCR and VLB are 0.5783, 0.2843, 0.018 and 0.126 mg/g DW, respectively. These extraction yields are equivalent to those from the well-known ultrasonic extraction method and higher than the yields from maceration extraction and heating reflux extraction. Our results suggest that NPCE-RP-HPLC represents an excellent alternative for the extraction and quantification of vinca alkaloids for pilot- and industrial-scale applications.

Synthesis and study of a molecularly imprinted polymer for specific solid-phase extraction of vinflunine and its metabolite from biological fluids.

A molecularly imprinted polymer (MIP) was synthesized in order to specifically extract vinflunine, an anticancer agent, and its metabolite (4-O-deacetylvinflunine) from bovine plasma and artificial urine by solid-phase extraction (SPE). Vinorelbine, a non-fluorinated analogue of vinflunine, was selected as a template for MIP synthesis. The selectivity of MIP versus the template (vinorelbine) and other alkaloids (catharanthine, vinblastine, vincristine, vinflunine and 4-O-deacetylvinflunine) was shown by a SPE protocol carried out with non-aqueous samples. A second protocol was developed for aqueous samples with two consecutive washing steps (AcOH-NH2 OH buffer (pH 7, I=10 mM)-MeOH mixture 95:5 v/v and ACN-AcOH mixture 99:1 v/v) and an elution step (MeOH-AcOH mixture 90:10 v/v). Thus, MIP-SPE of bovine plasma brought high recoveries, 81 and 89% for vinflunine and its metabolite, respectively. This protocol was slightly modified for artificial urine samples in order to obtain a good MIP/NIP selectivity; furthermore, elution recoveries were 73 and 81% for vinflunine and its metabolite, respectively. Repeatability was assessed in both biological matrices and RSD (%) were inferior to 4%. The MIP also showed a suitable linearity (r(2) superior to 0.99), between 0.25 and 10 µg/mL for plasma, and between 1 and 5 µg/mL for artificial urine.

Pathways Related to the Anti-Cancer Effects of Metabolites Derived from Cerrado Biome Native Plants: An Update and Bioinformatics Analysis on Oral Squamous Cell Carcinoma.

BACKGROUND: Oral cancer is a significant health problem worldwide. Oral squamous cell carcinoma (OSCC) is a malignant neoplasm of epithelial cells that mostly affects different anatomical sites in the head and neck and derives from the squamous epithelium or displays similar morphological characteristics. Generally, OSCC is often the end stage of several changes in the stratified squamous epithelium, which begin as epithelial dysplasia and progress by breaking the basement membrane and invading adjacent tissues. Several plant-based drugs with potent anti-cancer effects are considered inexpensive treatments with limited side effects for cancer and other diseases. OBJECTIVE: The aim of this review is to explore whether some Brazilian plant extracts or constituents exhibit anti-tumorigenic activity or have a cytotoxic effect on human oral carcinoma cells. METHODS: Briefly, OSCC and several metabolites derived from Brazilian plants (i.e., flavonoids, vinblastine, irinotecan, etoposide and paclitaxel) were used as keywords to search the literature on PubMed, GenBank and GeneCards. RESULTS: The results showed that these five chemical compounds found in Cerrado Biome plants exhibit anti-neoplastic effects. Evaluating the compounds revealed that they play a main role in the regulation of cell proliferation. CONCLUSION: Preserving and utilising the biodiversity of our planet, especially in unique ecosystems, such as the Cerrado Biome, may prove essential to preserving and promoting human health in modern contexts.

[Monomeric indole alkaloids from the aerial parts of Catharanthus roseus].

Catharanthus roseus (L.) G. Don is a plant of the Catharanthus genus of Apocynaceae which has been reported to have therapeutic effects of detoxication and anticancer. In order to further study the alkaloid constituents of C. roseus, the aerial parts of the plant were extracted with 95% EtOH, and then treated with 2% H2SO4 and NH3H2O to obtain total alkaloids. The total alkaloids were separated and purified by column chromatography over silica gel and prepared by high performance liquid chromatography (HPLC). Their structures were elucidated on the basis of physicochemical properties and spectral data. A new alkaloid together with five known compounds were isolated and identified as vindolinine B (1), lochnericine (2), horhammericine (3), vindorosine (4), vindoline (5), and coronaridine (6). Compound 1 is a new compound and named as vindolinine B.

In vitro cytotoxic activity of Salsola oppositifolia Desf. (Amaranthaceae) in a panel of tumour cell lines.

The aim of the present study was to evaluate for the first time the in vitro cytotoxic activity of fractions and isolated flavonols from Salsola oppositifolia Desf. (Amaranthaceae). The n-hexane fraction demonstrated an effective cytotoxic activity on the large lung carcinoma and amelanotic melanoma cell lines with IC50 values of 19.1 microg/ml and 24.4 microg/ml, respectively. Also the dichloromethane fraction exhibited cytotoxic activity against COR-L23 (IC50 30.4 microg/ml) and C32 (IC50 33.2 microg/ml) cells, while the EtOAc fraction demonstrated a selective cytotoxic activity against MCF-7 cells (IC50 67.9 microg/ml). The major active constituents of this fraction were isorhamnetin-3-O-glucoside (1) and isorhamnetin-3-O-rutinoside (2), which showed an interesting activity against the cell line MCF-7 with IC50 values of 18.2 and 25.2 microg/ml, respectively. Compound 2 exhibited a strong activity against the hormone-dependent prostate carcinoma LNCaP cell line with an IC50 of 20.5 microg/ml. Constituents of S. oppositifolia were identified by GC-MS and NMR analyses.

[Optimization of the polymerization conditions of vinblastine imprinted polymer by uniform design].

OBJECTIVE: To optimize the polymerization conditions of vinblastine (VLB) imprinted polymer. METHODS: The conditions were optimized by the method of uniform design. The major factors investigated included the amount of functional monomer (MAA) and the cross-linker (EDMA) and the progenic solvent (toluene or acetonitril). The adsorption rate of VLB on the solid-phase extraction (SPE) column packed with MIP was adopted as the response value. RESULTS: The optimal conditions were MAA 0.4 mmol, EDMA 1.6 mmol and using acetonitrile as the solvent. Under the conditions,the VLB imprinted polymer was synthesized and the absorption rate of VLB was 88.20%. The characterizations of the optimal MIP were determined by IR spectrometry and scanning electron microscope (SEM) analysis. CONCLUSION: It is possible to furtherly improve the nature of the polymer by optimizing the polymerization parameters with uniform design. The polymer synthesized under the optimal conditions exhibited high affinity to the target molecule VLB.

First report on a novel Nigrospora sphaerica isolated from Catharanthus roseus plant with anticarcinogenic properties.

This paper reports on the vinca alkaloid produced by a novel Nigrospora sphaerica isolated from Catharanthus roseus. Through liquid chromatography-mass spectrometry (LCMS), only the crude mycelia extract of this fungus was positive for determination of vinblastine. This vinca alkaloid was then purified by using high-performance liquid chromatography (HPLC) and tested for cytotoxicity activity using MTT assays. The breast cell line cancer (MDA-MB 231) was treated with a purified vinblastine which was intracellulary produced by N. sphaerica. The purified vinblastine from extracted leaf of C. roseus was used as a standard comparison. A positive result with a value of half maximal inhibitory concentration (IC50 ) of > 32 µg ml-1 was observed compared with standard (IC50 ) of 350 µg ml-1 only. It showed that a vinblastine produced by N. sphaerica has a high cytotoxicity activity even though the concentration of vinblastine produced by this endophytic fungus was only 0.868 µg ml-1 .

Industrial case study on alkaloids purification by pH-zone refining centrifugal partition chromatography.

The industrial potential of pH-zone refining centrifugal partition chromatography has been evaluated by studying the purification of pharmaceutical ingredients at the pilot scale. For the first time, a scale up methodology based on both column capacity and mass transfer efficiency as invariants was developed. The purification of catharanthine and vindoline from an industrial crude extract of aerial parts of Catharanthus roseus, was used as a case of study. Toluene/CH3CN/water (4/1/5, v/v/v) was selected as biphasic solvent system, triethylamine as retainer in the organic stationary phase and sulphuric acid as displacer in the aqueous mobile phase. The separation intensification was performed on a 36mL CPC column equipped with 832 partition twin-cells. The combined effects of four parameters (displacer and retainer concentrations for intensive parameters, flow rate and rotational speed for extensive parameters) were studied by design of experiment in order to maximize both recoveries and productivities. Then, scale change was done on two larger columns (305mL and 1950mL of capacity) equipped with only 231 and 238 partition cells. For this step, it has been shown that the global mass transfer coefficient k0a (the efficiency of a column design) and the stationary phase retention (the capacity of the column) were relevant and useful scale up invariants. A CPC model based on acid-base equilibriums and interfacial mass transfer in continuously stirred tank reactors in series was used to predict fully separations on larger CPC column at the optimized operating conditions and to guide the CPC user in its scale-up strategy. The experimental validation on pilot CPC column, by injecting up to 150g of Catharanthus roseus crude extract on the 1950mL column highlighted the preservation of the separation quality, the non-linear character of the scale up in centrifugal partition chromatography and that a productivity of about 4kg of processed crude extract per day can be reached by implementing developed methodology.

Original Vinca Derivatives: From P-Glycoprotein Substrates to P-Glycoprotein Inhibitors.

The first example of vinca derivatives 16-18 able to modulate P-glycoprotein (Pgp) efflux activity is reported. They were elaborated in two steps from vinorelbine 3 (VLN) by a modification of the velbenamine moiety. These compounds were able to decrease efficiently Pgp mediated influx and efflux of rhodamine-123 (Rho) and to restore the cytotoxicity of vinorelbine 3 (VLN) and doxorubicin (Dox) on K562R (dox-resistant) cell lines.

Modeling pH-zone refining countercurrent chromatography: a dynamic approach.

A model based on mass transfer resistances and acid-base equilibriums at the liquid-liquid interface was developed for the pH-zone refining mode when it is used in countercurrent chromatography (CCC). The binary separation of catharanthine and vindoline, two alkaloids used as starting material for the semi-synthesis of chemotherapy drugs, was chosen for the model validation. Toluene/CH3CN/water (4/1/5, v/v/v) was selected as biphasic solvent system. First, hydrodynamics and mass transfer were studied by using chemical tracers. Trypan blue only present in the aqueous phase allowed the determination of the parameters τextra and Pe for hydrodynamic characterization whereas acetone, which partitioned between the two phases, allowed the determination of the transfer parameter k0a. It was shown that mass transfer was improved by increasing both flow rate and rotational speed, which is consistent with the observed mobile phase dispersion. Then, the different transfer parameters of the model (i.e. the local transfer coefficient for the different species involved in the process) were determined by fitting experimental concentration profiles. The model accurately predicted both equilibrium and dynamics factors (i.e. local mass transfer coefficients and acid-base equilibrium constant) variation with the CCC operating conditions (cell number, flow rate, rotational speed and thus stationary phase retention). The initial hypotheses (the acid-base reactions occurs instantaneously at the interface and the process is mainly governed by mass transfer) are thus validated. Finally, the model was used as a tool for catharanthine and vindoline separation prediction in the whole experimental domain that corresponded to a flow rate between 20 and 60 mL/min and rotational speeds from 900 and 2100 rotation per minutes.

An Endophytic Fungus, Talaromyces radicus, Isolated from Catharanthus roseus, Produces Vincristine and Vinblastine, Which Induce Apoptotic Cell Death.

Endophytic fungi isolated from Catharanthus roseus were screened for the production of vincristine and vinblastine. Twenty-two endophytic fungi isolated from various tissues of C. roseus were characterized taxonomically by sequence analysis of the internal transcribed spacer (ITS) region of rDNA and grouped into 10 genera: Alternaria, Aspergillus, Chaetomium, Colletotrichum, Dothideomycetes, Eutypella, Eutypa, Flavodon, Fusarium and Talaromyces. The antiproliferative activity of these fungi was assayed in HeLa cells using the MTT assay. The fungal isolates Eutypella sp--CrP14, obtained from stem tissues, and Talaromyces radicus--CrP20, obtained from leaf tissues, showed the strongest antiproliferative activity, with IC50 values of 13.5 µg/ml and 20 µg/ml, respectively. All 22 endophytic fungi were screened for the presence of the gene encoding tryptophan decarboxylase (TDC), the key enzyme in the terpenoid indole alkaloid biosynthetic pathway, though this gene could only be amplified from T. radicus--CrP20 (NCBI GenBank accession number KC920846). The production of vincristine and vinblastine by T. radicus--CrP20 was confirmed and optimized in nine different liquid media. Good yields of vincristine (670 µg/l) in modified M2 medium and of vinblastine (70 µg/l) in potato dextrose broth medium were obtained. The cytotoxic activity of partially purified fungal vincristine was evaluated in different human cancer cell lines, with HeLa cells showing maximum susceptibility. The apoptosis-inducing activity of vincristine derived from this fungus was established through cell cycle analysis, loss of mitochondrial membrane potential and DNA fragmentation patterns.

In vitro metabolic transformations of vinblastine: oxidations catalyzed by human ceruloplasmin.

The dimeric Vinca alkaloid vinblastine (VLB) undergoes metabolic transformation to three products in a reaction catalyzed by the human serum copper oxidase ceruloplasmin. The enzyme reaction requires chlorpromazine as a shuttle oxidant, and the course of the oxidation reaction appears to be subject to the nature of the shuttle oxidant used. Preparative-scale incubations have resulted in the isolation of three products, which were characterized by chemical and spectral analyses. The metabolites were identified as the ring fission product catharinine, obtained by oxidation of the Iboga ring system; an enamine/ether derivative obtained by oxidation of the Aspidosperma portion of VLB; and a metabolite embodying the same structural changes in both parts of the vinblastine dimeric structure. Catharinine is identical with the product of VLB oxidation obtained by peroxidase oxidation. The other two products are new metabolites and are derivatives of VLB. All of the metabolites are less active than VLB when tested in vitro vs the human T-cell leukemic cell line (CRFF-CEM).

Isolation of vindoline from Catharanthus roseus by supercritical fluid extraction.

Vindoline was extracted from the leaves of Catharanthus roseus over the ranges of 35-70 degrees C and 100-300 bar using supercritical carbon dioxide with and without the addition of 3 wt % ethanol as a cosolvent. The vindoline contents in the extracts were determined by HPLC and identified by LC/MS. The remarkable highest vindoline concentration, 58 wt %, was obtained at the lowest temperature, 35 degrees C, and the highest pressure, 300 bar, of this study. The use of a cosolvent only slightly improved the extraction yields or selectivities at some experimental conditions.