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.

Hepatotoxic effect of subacute vincristine administration activates necrosis and intrinsic apoptosis in rats: protective roles of broccoli and Indian mustard.

This study investigated the hepatotoxic effect of long-term vincristine (VCR) administration in rats and to assess if an individual or combined therapy with Indian mustard and broccoli afforded protection. Signs of hepatotoxicity, including altered liver architecture and higher serum levels of ALT and AST, were seen in VCR-treated rats. Concomitantly, the impaired antioxidant potential and higher mRNA levels of IL-12 and IL-4, which are markers of apoptosis, were seen in rat livers. VCR treatment induced hepatocyte apoptosis, shown by the up-regulation of mRNA and protein levels of 53, increased protein levels of cleaved caspase-3 and Bax, and reduced levels of intracellular ATP and BCl-2mRNA and protein. Although individual administration of mustard or broccoli partially ameliorated all these responses, the combined therapy of both extracts resulted in the maximum improvement. Thus, the long-term administration of VCR is hepatotoxic and induces apoptosis; however, the combined therapy of both extracts mitigated these effects.

Multivariate optimization of solvent bar microextraction combined with HPLC-UV for determination of trace amounts of vincristine in biological fluids.

In the current work, an efficient method named solvent bar microextraction-high performance liquid chromatography-UV detection (HPLC-UV) was developed for preconcentration and determining the trace amount of vincristine (VCR) in biological samples such as plasma and urine. Briefly, VCR was extracted from an aqueous sample with pH 10.7 (donor phase) into 1-octanol as the supported liquid membrane (SLM) which is inserted into the pores of the hollow fiber and followed by back extraction into an aqueous receiving phase (pH=3.1). Studying the factors affecting the extraction performance in order to achieve a high extraction efficiency, requires the design of experiments (DOE) approach. In this regards, diverse factors' effects including the pH value of donor and acceptor phases, extraction time, extraction temperature, stirring rate and salt content of the donor phase were considered. The optimum experimental condition was as following: pH of the source phase, 10.7; pH of the receiving phase, 3.1; stirring rate, 1000rpm; extraction temperature, 51°C; extraction time, 60min and 11.3% w/v NaCl in the sample solution. Under the optimal; extraction condition, a favorable preconcentration factor equal to 98.5 was achieved. The linearity range was obtained in the domain of 0.05-5mgL-1. The limits of detection and quantification were 0.015 and 0.05mgL-1. Within-day and between-day RSDs of the proposed SBME method were 4.1% and 12.5%, respectively. Finally, the applicability of the implemented SBME method was evaluated by the extraction and quantification of VCR from biological samples such as urine and plasma and satisfactory results were obtained.

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.

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.

Isolation, purification and characterization of vinblastine and vincristine from endophytic fungus Fusarium oxysporum isolated from Catharanthus roseus.

Endophytic fungi reside in a symbiotic fashion inside their host plants, mimic their chemistry and interestingly, produce the same natural products as their hosts and are thus being screened for the production of valuable compounds like taxol, camptothecin, podophyllotoxin, etc. Vinblastine and vincristine are excellent anti-cancer drugs but their current production using plants is non-abundant and expensive. In order to make these drugs readily available to the patients at affordable prices, we isolated the endophytic fungi from Catharanthus roseus plant and found a fungus AA-CRL-6 which produces vinblastine and vincristine in appreciable amounts. These drugs were purified by TLC and HPLC and characterized using UV-Vis spectroscopy, ESI-MS, MS/MS and (1)H NMR. One liter of culture filtrate yielded 76 µg and 67 µg of vinblastine and vincristine respectively. This endophytic fungal strain was identified as Fusarium oxysporum based upon its cultural and morphological characteristics and internal transcribed spacer (ITS) sequence analysis.

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.

Cytotoxicity of vincristine on the 5637 cell line is enhanced by combination with conferone.

Bladder cancer is one of the most common cancers worldwide, with the highest incidence in industrialized countries. There are three major histological subtypes of bladder cancer: transitional cell carcinoma (TCC) (> 90%), squamous cell carcinoma (< 10%) and adenocarcinoma (1-2%). The present study was carried out to assess the effects of conferone, a sesquiterpene coumarin isolated from Ferula badrakema, on a TCC subline, 5637 cells. In order to test the effects of conferone, 5637 cells were treated with different concentrations (16, 32, 64, 128 microg/ml) of conferone. The results indicated that conferone did not have any significant cytotoxic effect on these neoplastic cells. To determine the combining effects, the cells were cultured in the presence of different concentrations of conferone (16, 32, 64, 128 microg/ ml) and vincristine (30, 40, 50 microg/ml) in combination. The morphological changes were then observed and cytotoxicity effects were studied using the MTT assay 24, 48 and 72 h following drug administration. The cells were more rounded and granulated after treatments with both drugs in comparison to vincristine only. The results of the MTT assay confirmed the morphological observations. After 48 h of combined treatment with 40 microg/ml vincristine and 16 microg/ml conferone, the cytotoxicity of vincristine was increased by 23.6%.

Isolation and characterization of antineoplastic alkaloids from Catharanthus roseus L. Don. cultivated in Egypt.

Vinblastine and vincristine (the antileukemic agents) were isolated, in a pure form, from Catharanthus roseus L. Don., cultivated in Egypt, by several chromatographic techniques. Five modified methods for the preparation of total alkaloids were carried out. All the isolated mixtures were evaluated by HPLC and HPTLC analyses. The antineoplastic alkaloids; vinblastine and vincristine, were isolated by the use of vacuum liquid chromatographic column on silica gel : aluminium oxide (1:1) mixed bed vacuum liquid chromatography (VLC), Charcoal column, and finally purified by centrifugally accelerated radial chromatography (Chromatotrone).

[Advances in the study of vincristine: an anticancer ingredient from Catharanthus roseus].

Vincristine is a dimer-indo-alkaloid which is extracted from the leaves of Catharanthus roseus. It is effective to treat acute lymphocytic cell leukemia, Hodgkin disease and non-Hodgkin disease clinically. But the severe side effects, such as neurotoxic and tissue damage, limit its application. In this paper, we summarize physical, chemical, pharmacological and pharmacokinetical properties of VCR and advances in decreasing its side effects. In clinic, association with other medication is adopted. In pharmaceutics, people adopt some new methods and technology such as conjugation with the antibody, encapsulation in liposomes or controlled release films.

Novel bisindole derivatives of Catharanthus alkaloids with potential cytotoxic properties.

Pantropical plant Catharanthus roseus (L) G. Don is known as a source of valuable bisindole alkaloids: vinblastine (VBL) and vincristine (VCR), oncolytics widely used as sulfates in therapy of malignant diseases. They are biosynthesized in the plant from monoindolic vindoline and catharanthine, both derived from L-tryptophan and loganine units. In the course of phytochemical screening of this plant cultivated in Poland and considered as a home source of VBL and VCR we developed a new isolation method based on the solid phase extraction. Mild conditions used during the isolation procedure enabled the isolation of some labile compounds and so the monomeric alkaloids with high yields. Vindoline and so its congeners and subjected to various oxidative conditions gave 15,15'-bisindolic derivatives in quite good yield. Biological activities of the above mentioned bisindolic compounds are under study.

Effects of various principles from Chinese herbal medicine on rhodamine123 accumulation in brain capillary endothelial cells.

AIM: To search for novel effective P-glycoprotein (P-gp) reversal agents in the blood-brain barrier (BBB). METHODS: Using rhodamine123 (Rh123) to examine the functional activity of P-gp in cultured bovine brain capillary endothelial cells (BCEC) and screen various principles on P-gp modulation in BBB. RESULTS: All of tested compounds (1-10 micromol/L) increased the intracellular accumulation of Rh123 in a concentration-dependent manner. The rank order of these agents in increasing Rh123 accumulation in BCEC was: cyclosporin A (CsA) > tetrandrine (Tet) > vincrinstine (VCR) approximate, equals flunarizine (Flu) > dl-tetrahydropalmatine (dl-THP) > dauricine (DRC) > azithromycin (Azi) > verapamil (Ver) approximate, equals berbamine (BBM) > daurisoline (DRS) > berberine (BBR) approximate, equals doxorubicin (Dox) > l-tetrahydropalmatine (l-THP) > tetramethylpyrazine (TMP). These agents at concentration of 10 micromol/L increased Rh123 accumulation by 346 %, 203 %, 136 %, 129 %, 115 %, 103 %, 92 %, 87 %, 81 %, 75 %, 67 %, 67 %, 63 %, and 54 %, respectively. The effects of CsA, Tet, Ver, Flu, Azi, and dl-THP on cellular accumulation of Rh123 in BCEC were reversible. When CsA, Tet, Ver, Flu, Azi, or dl-THP-pretreated BCEC were examined at 48, 36, 24, 36, 36, or 12 h, respectively, after removing the agent, the amount of cellular Rh123 accumulation in BCEC returned to control levels (no drug treatment). CONCLUSION: The functional activity of P-gp on the blood-brain barrier could be modulated by various MDR-reversing agents and some principles with low toxicity extracted from medicinal herbs, such as some isoquinoline alkaloids without permanent modifying effects on the intrinsic level of P-gp function.

Determination of free and liposome-associated doxorubicin and vincristine levels in plasma under equilibrium conditions employing ultrafiltration techniques.

A thorough understanding of the pharmacodynamic relationships associated with toxicity and efficacy behavior of liposome-encapsulated anticancer agents such as doxorubicin and vincristine will rely on the ability to accurately separate and quantify the free and liposome-associated drug fractions in plasma after administration. We have investigated the use of ultrafiltration as a method of isolating free doxorubicin and vincristine from liposomal drug under equilibrium conditions and compared it to previously developed nonequilibrium procedures based on solid-phase extraction. Adsorption of drugs dissolved in saline to the ultrafiltration devices resulted in concentration-dependent ultrafiltrate drug recoveries ranging from 41 to 96%. However, concentration-independent quantitative recovery of vincristine in saline solutions could be obtained by passivating the ultrafiltration devices with PEG-8000 and device drug adsorption was ameliorated for both agents by plasma. The ultrafiltration method provided a more reliable separation of free and protein-bound drug, whereas solid-phase extraction yielded artificially high free drug concentrations due to process-induced protein-bound drug complex dissocation. Also, coelution of liposomes with the free drug fraction using solid-phase extraction was 64- to 418-fold higher than observed with ultrafiltration. Taken together, these properties indicated a significantly increased degree of accuracy in measuring the amount of free doxorubicin and vincristine in samples containing liposomal formulations employing ultrafiltration compared to solid-phase extraction. The importance of this improvement was highlighted by observations that determinations of free drug concentrations in the plasma of mice injected with liposomal doxorubicin and vincristine were 3- to 12-fold higher using solid-phase extraction compared to ultrafiltration. Finally, the ultrafiltration procedure is rapid, versatile, and can be used for a wide range of drug and liposome concentrations and free drug/liposomal drug ratios.

Liquid chromatographic isolation of vincristine and vinblastine.

A fundamentally new method is described for the separation of the dimeric indole alkaloids vincristine and vinblastine from monomeric indole alkaloid impurities. This method uses an RP-18 high-performance liquid chromatography column with a methanol-water mobile phase containing an inorganic acid and an unusually low concentration of inorganic buffer. By keeping the buffer concentration low, the elution of all indole alkaloids is retarded, but the dimeric ones are retarded more than the monomeric ones. A theoretical model developed to explain this behavior postulates that the anions of the buffer solubilize the protonated indole alkaloids by pairing with them. Lowering the buffer concentration reduces the availability of pairing ions and thus decreases the mobile phase affinity of protonated alkaloids, particularly those having a 2+ charge. A similar approach may be applicable in other situations where ionogenic organic compounds having a particular valence must be separated from related compounds having different valences, or from non-ionogenic compounds.