Involvement of RPL11 in the enhancement of P53 stability by a podophyllum derivative, a topoisomerase II inhibitor.

In previous work, we presented experimental and theoretical evidence that D-3F or 4-N-(2-Amino-3-fluoropyridine)-4-deoxidation-4'-demethylepipofophyllotoxin induced G2 /M phase arrest and apoptosis, purportedly by increasing the expression of P53. However, the precise mechanism of D-3F action is currently unknown. Here, we investigated the mechanism by which D-3F treatment induces increased expression of P53. This study showed that D-3F definitively inhibited the activity of topoisomerase II in a dose-dependent manner and resulted in DNA damage. The results were in overall agreement with modeling and docking studies performed on D-3F. In addition, D-3F increased the levels of P53 and P21 in HeLa cells in a dose-dependent manner, this in turn prolonged the half-life of P53. Taken together, these data suggested that D-3F-mediated transient enhancement of P53 stabilization may be critical for the P53/P21 signalling pathway leading to G2 /M phase arrest on HeLa cells. Furthermore, D-3F downregulated the phosphorylation of E3 ubiquitin-protein ligase murine double minute 2 (Mdm2) at Ser166, inhibited Mdm2-mediated ubiquitination of P53, and released 60S ribosomal protein L11 (RPL11) from the nucleolus into the nucleoplasm. To conclude, the topoisomerase II inhibitor D-3F causes P53 to accumulate in HeLa cell lines by enhancing its stability as a result of DNA-damage induced RPL11 relocalization and subsequent blocking of the P53-Mdm2 feedback loop.

Recent Advances in the Chemistry and Biology of Podophyllotoxins.

Podophyllotoxin and its related aryltetralin cyclolignans belong to a family of important products that exhibit various biological properties (e.g., cytotoxic, insecticidal, antifungal, antiviral, anti-inflammatory, neurotoxic, immunosuppressive, antirheumatic, antioxidative, antispasmogenic, and hypolipidemic activities). This Review provides a survey of podophyllotoxin and its analogues isolated from plants. In particular, recent developments in the elegant total chemical synthesis, structural modifications, biosynthesis, and biotransformation of podophyllotoxin and its analogues are summarized. Moreover, a deoxypodophyllotoxin-based chemosensor for selective detection of mercury ion is described. In addition to the most active podophyllotoxin derivatives in each series against human cancer cell lines and insect pests listed in the tables, the structure-activity relationships of podophyllotoxin derivatives as cytotoxic and insecticidal agents are also outlined. Future prospects and further developments in this area are covered at the end of the Review. We believe that this Review will provide necessary information for synthetic, medicinal, and pesticidal chemistry researchers who are interested in the chemistry and biology of podophyllotoxins.

Transcriptome-wide identification and characterization of CAD isoforms specific for podophyllotoxin biosynthesis from Podophyllum hexandrum.

Podophyllotoxin (ptox) is a therapeutically important lignan derived from Podophyllum hexandrum and is used as a precursor for the synthesis of anticancer drugs etoposide, teniposide and etopophose. In spite of its enormous economic significance, genomic information on this endangered medicinal herb is scarce. We have performed de novo transcriptome analysis of methyl jasmonate (MeJA)-treated P. hexandrum cell cultures exhibiting enhanced ptox accumulation. The results revealed the maximum up-regulation of several isoforms of cinnamyl alcohol dehydrogenase (CAD). CAD catalyzes the synthesis of coniferyl alcohol and sinapyl alcohol from coniferaldehyde (CAld) and sinapaldehyde respectively. Coniferyl alcohol can produce both lignin and lignan while sinapyl alcohol produces only lignin. To isolate the CAD isoforms favoring ptox, we deduced full length cDNA sequences of four CAD isoforms: PhCAD1, PhCAD2, PhCAD3 and PhCAD4 from the contigs of the transcriptome data. In vitro enzyme assays indicated a higher affinity for CAld over sinapaldehyde for each isoform. In silico molecular docking analyses also suggested that PhCAD3 has a higher binding preference with CAld over sinapaldehyde, followed by PhCAD4, PhCAD2, and PhCAD1, respectively. The transgenic cell cultures overexpressing these isoforms independently revealed that PhCAD3 favored the maximum accumulation of ptox as compared to lignin followed by PhCAD4 and PhCAD2, whereas, PhCAD1 favored both equally. Together, our study reveals transcriptome-wide identification and characterization of ptox specific CAD isoforms from P. hexandrum. It provides a useful resource for future research not only on the ptox biosynthetic pathway but on overall P. hexandrum, an endangered medicinal herb with immense therapeutic importance.

Expression analysis of biosynthetic pathway genes vis-à-vis podophyllotoxin content in Podophyllum hexandrum Royle.

Podophyllum hexandrum Royle is known for its vast medicinal properties, particularly anticancer. It contains higher amount of podophyllotoxin (4.3 %), compared to Podophyllum peltatum (0.025 %) and other plant species; as a result, it has been used worldwide in the preparation of various drugs including anticancer, antimalarial, antiviral, antioxidant, antifungal, and so on. Currently, Etoposide (VP-16-213), Vumon® (Teniposide; VM-26), Etopophos®, Pod-Ben- 25, Condofil, Verrusol, and Warticon are available in the market. Due to highly complex synthesis and low cell culture yields of podophyllotoxin (0.3 %), the supply of raw material cannot be met due to increasing industrial demands. The knowledge on podophyllotoxin biosynthetic pathway vis-à-vis expression status of genes is fragmentary. Quantitative expression analysis of 21 pathway genes has revealed 9 genes, namely SD, PD, PCH, CM, CMT, CAD, CCR, C4H, and ADH, that showed increase in transcript abundance up to 1.4 to 23.05 folds, respectively, vis-à-vis podophyllotoxin content in roots (1.37 %) and rhizomes (3.05 %) of P. hexandrum. In silico analysis of putative cis-regulatory elements in promoter regions of overexpressed genes showed the presence of common Skn-1 motif and MBS elements in CMT, CAD, CCR, C4H, and ADH genes, thereby, suggesting their common regulation. The outcome of the study has resulted in the identification of suitable candidate genes which might be contributing to podophyllotoxin biosynthesis that can act as potential targets for any genetic intervention strategies aimed at its enhanced production.

An efficient in vitro system for somatic embryogenesis and podophyllotoxin production in Podophyllum hexandrum Royle.

Podophyllum hexandrum Royle known as Indian mayapple is an important medicinal plant found only in higher altitudes (2,700 to 4,200 m) of the Himalayas. The highly valued anticancer drug Podophyllotoxin is obtained from the roots of this plant. Due to over exploitation, this endemic plant species is on the verge of extinction. In vitro culture for efficient regeneration and the production of podophyllotoxin is an important research priority for this plant. Hence, in the present study, an efficient plant regeneration system for mass multiplication through somatic embryogenesis was developed. We have screened P. hexandrum seeds collected from three different regions in the Himalayas to find their regenerative potentials. These variants showed variation in germination percentage as well as somatic embryogenic frequency. The seeds collected from the Milam area of Pithoragarh district showed better germination response (99.3%) on Murashige and Skoog (MS) medium fortified with Gibberellic acid (GA3 [5 mg/l]) and higher direct somatic embryogenic frequency (89.6%). Maximum production of embryogenic callus (1.2 g fresh weight [FW]) was obtained when cotyledons containing the direct somatic embryo clusters were cultured in MS medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D [1.5 mg/l]) after 4 week of culture in complete darkness. In the present investigation, somatic embryogenesis was accomplished either by direct organogenesis or callus mediated pathways. The latter method resulted in a higher frequency of somatic embryo induction in hormone-free MS medium yielding 47.7 embryos/50 mg of embryogenic callus and subsequent germination in MS medium supplemented with GA3 (5 mg/l). Seventy-nine percent of embryos attained complete maturity and germinated into normal plants with well-developed roots. Systematic histological analysis revealed the origin of somatic embryo and their ontogenesis. The higher level of podophyllotoxin (1.8 mg/g dry weight [DW]) was recorded in germinated somatic embryos when compared to field grown plants. The present system can be widely used for mass propagation, transgenic recovery, and podophyllotoxin production for commercial utilization.

Relationship between podophyllotoxin accumulation and soil nutrients and the influence of Fe2+ and Mn2+ on podophyllotoxin biosynthesis in Podophyllum hexandrum tissue culture.

Podophyllotoxin (PDT) and its derivatives, which are isolated from the Podophyllum species, are widely used in the clinical setting. The present study was designed to analyze the correlation between PDT levels in the rhizomes of Podophyllum hexandrum (P. hexandrum) and Dysosma versipellis (D. versipellis) and the nutrients in soil. We also aimed to investigate the influence of Fe(2+) and Mn(2+) on the enzyme activity of phenylalanine ammonia lyase (PAL), cinnamyl alcohol-dehydrogenase (CAD), and deoxypodophyllotoxin 6-hydroylase (DOP6H) and PDT accumulation via P. hexandrum tissue culture. The results showed that PDT accumulation was positively correlated with the NO3(-), PO4(3-), Na(+), Fe, and Mn levels and was negatively correlated with the SO4(2-) and K(+) levels, while the correlation with the Mg(2+), Ca(2+), Cu and Zn levels was not significant. The Fe(2+) and Mn(2+) levels were associated with the increased activity of PAL and CAD at 3-18 days; Fe(2+) enhanced the activity levels by 2.66- and 1.76-fold, respectively, and Mn(2+) was associated with a 1.68- and 1.10-fold increase in activity levels, respectively, compared with the control (CK) at 18 days. DOP6H activity was enhanced by Mn(2+), but it was not significantly affected by Fe(2+). Finally, PDT production was enhanced approximately 60% and 34% by Fe(2+) and Mn(2+), respectively, compared with CK at 16 days. These observations may be useful for the generation of PDT and related lignans via commercial cultivation as well as cell and tissue culture of P. hexandrum and other related plant resources.

Next generation sequencing in predicting gene function in podophyllotoxin biosynthesis.

Podophyllum species are sources of (-)-podophyllotoxin, an aryltetralin lignan used for semi-synthesis of various powerful and extensively employed cancer-treating drugs. Its biosynthetic pathway, however, remains largely unknown, with the last unequivocally demonstrated intermediate being (-)-matairesinol. Herein, massively parallel sequencing of Podophyllum hexandrum and Podophyllum peltatum transcriptomes and subsequent bioinformatics analyses of the corresponding assemblies were carried out. Validation of the assembly process was first achieved through confirmation of assembled sequences with those of various genes previously established as involved in podophyllotoxin biosynthesis as well as other candidate biosynthetic pathway genes. This contribution describes characterization of two of the latter, namely the cytochrome P450s, CYP719A23 from P. hexandrum and CYP719A24 from P. peltatum. Both enzymes were capable of converting (-)-matairesinol into (-)-pluviatolide by catalyzing methylenedioxy bridge formation and did not act on other possible substrates tested. Interestingly, the enzymes described herein were highly similar to methylenedioxy bridge-forming enzymes from alkaloid biosynthesis, whereas candidates more similar to lignan biosynthetic enzymes were catalytically inactive with the substrates employed. This overall strategy has thus enabled facile further identification of enzymes putatively involved in (-)-podophyllotoxin biosynthesis and underscores the deductive power of next generation sequencing and bioinformatics to probe and deduce medicinal plant biosynthetic pathways.

Factors affecting podophyllotoxin yield in the ex situ grown Podophyllum hexandrum, an endangered alpine native of the western Himalayas.

This study reports an appreciable yield of podophyllotoxin (PDT) in P. hexandrum plants grown ex situ under polyhouse conditions of a temperate locale. The PDT content of below-ground parts was affected by both plant age and growth period. However, only the effect of plant age on PDT content was significant. Thus, the highest amounts of PDT were recorded in the below-ground parts of 2-year-old plants harvested during the late-growth period (LGP). High total soluble sugars in the below-ground parts during the early growth period (EGP) and the highest nitrate and nitrate reductase in the leaves of 2-year-old plants during the peak-growth period (PGP) indicated higher mobilization and assimilation of starch and nitrate. Probably the surplus carbon and nitrogen gained during the PGP were diverted from aerial parts to below-ground parts during the LGP and in turn contributed to the synthesis of higher amounts of PDT. This study shows that commercial cultivation of P. hexandrum is possible under ex situ temperate conditions.

Stress-mediated adaptive response leading to genetic diversity and instability in metabolite contents of high medicinal value: an overview on Podophyllum hexandrum.

Podophyllum hexandrum, known for its diversified clinical importance particularly for antineoplastic activity and valuable source for biological protection against high doses of radiation, has its unique position in the plant kingdom. Detailed understanding of mechanism and opportunity of chemical manipulations has amplified the scope of its bioactivity. Podophyllotoxin, the major active principle of this plant, has passed through various structural deviations with the basic aim of making the end product clinically more effective with minimal toxicity. However, over exploitation and limited growth has categorized this plant under endangered species. Depending upon the geographical variations, different species and subspecies of this plant have been explored. Morphological variations and quantitative differences in active principles are the major concern of its unstable medicinal value in whole and semifractionated preparations. The current review has addressed the issues related to the genetic diversity of P. hexandrum, extrinsic and intrinsic stresses responsible for its diversified nature, chemical modifications to enhance its multitasking bioactivity, and efforts for its cultivation and production of important metabolites to avoid collection of wild species due to its critically endangered nature.

The role of biotechnology in the production of the anticancer compound podophyllotoxin.

Podophyllotoxin is a plant-derived compound found in Podophyllum sp. that is used to produce semi-synthetic anticancer pharmaceuticals such as etoposide, teniposide, and etoposide phosphate. This chapter describes the role of biotechnology to produce podophyllotoxin and our attempts to domesticate Podophyllum peltatum L., also known as the American mayapple. The domestication research on mayapple included surveys of the natural population, identification of high yielding genotypes, propagation, cultivation, sustainable harvest procedures and the development of protocols for in vitro germplasm bank.

Podophyllum hexandrum modulates gamma radiation-induced immunosuppression in Balb/c mice: implications in radioprotection.

Aqueous extract of Podophyllum hexandrum (RP-1), which has been reported to render more than 82% survival against whole body lethal (10 Gy) gamma-irradiation in mice, was further investigated for its immunomodulatory potential. In this study, no significant change could be scored in peritoneal macrophages survival up to 8th day after whole body irradiation. RP-1 treatment (200 mg/kg body weight, i.p.) alone or 2 h before whole body irradiation enhanced macrophage survival significantly (p<0.05) as compared to irradiated control mice. In irradiated animals, there was significant (p<0.01) reduction in splenocyte survival and proliferation as revealed by 3H-TdR method. RP-1 treatment (200 mg/kg) alone or 2 h before irradiation countered the decrease in survival of splenocytes and proliferation significantly (p<0.05) as compared to irradiated control group. Whole body irradiation also significantly (p<0.05) reduced the population of CD4+ and CD8+ T cells and bone marrow GM-CFU at 24 h and 72 h post-irradiation intervals, respectively, as compared to unirradiated control. RP-1 treatment 2 h before whole body irradiation countered the decrease in CD4+ and CD8+ T cells populations and CGM-CFU. Nitric oxide free radicals generation was enhanced significantly (p<0.05) in the supernatant of peritoneal macrophage cultures exposed to 2 Gy gamma radiation ex vivo in comparison to unirradiated control, which was reduced by pre-irradiation (-2 h) administration of RP-1. Whole body irradiation (10 Gy) also reduced the serum titres of IL-3, IL-1 and various IgG isotypes observed at different post-irradiation time interval. RP-1 treatment alone or before whole body irradiation countered radiation induced decrease in the titre of IL-1, IL-3 and IgG's in the serum of mice. These findings indicate immunostimulatory potential of RP-1.

The endophytic fungus Trametes hirsuta as a novel alternative source of podophyllotoxin and related aryl tetralin lignans.

The aryl tetralin lignans are synthesized by Podophyllum sps. and are in great demand worldwide due to their use in synthesis of topoisomerase inhibitors. However, the sustained production of these aryl tetralin lignans requires large-scale harvesting from the natural environments, which has resulted in the plant-endangered status. In view of the difficulties in their total chemical synthesis, cultivation and failure of metabolic engineering approaches, there is a need to search for alternative sources of production of aryl tetralin lignans. We unequivocally established the methodology for isolation, identification, and characterization of a novel fungal endophyte (Trametes hirsuta) that produces aryl tetralin lignans consistently as shown by HPLC, LC-MS, LC/MS-MS and (1)H NMR. The lignans produced by the microorganism are biologically active, and exhibit potent antioxidant, anticancer and radioprotective properties. This strategy promises to improve the production of these therapeutically important compounds at lower costs.

Crystal structures of apo-form and binary/ternary complexes of Podophyllum secoisolariciresinol dehydrogenase, an enzyme involved in formation of health-protecting and plant defense lignans.

(-)-Matairesinol is a central biosynthetic intermediate to numerous 8-8'-lignans, including the antiviral agent podophyllotoxin in Podophyllum species and its semi-synthetic anticancer derivatives teniposide, etoposide, and Etopophos. It is formed by action of an enantiospecific secoisolariciresinol dehydrogenase, an NAD(H)-dependent oxidoreductase that catalyzes the conversion of (-)-secoisolariciresinol. Matairesinol is also a plant-derived precursor of the cancer-preventative "mammalian" lignan or "phytoestrogen" enterolactone, formed in the gut following ingestion of high fiber dietary foodstuffs, for example. Additionally, secoisolariciresinol dehydrogenase is involved in pathways to important plant defense molecules, such as plicatic acid in the western red cedar (Thuja plicata) heartwood. To understand the molecular and enantiospecific basis of Podophyllum secoisolariciresinol dehydrogenase, crystal structures of the apo-form and binary/ternary complexes were determined at 1.6, 2.8, and 2.0 angstrom resolution, respectively. The enzyme is a homotetramer, consisting of an alpha/beta single domain monomer containing seven parallel beta-strands flanked by eight alpha-helices on both sides. Its overall monomeric structure is similar to that of NAD(H)-dependent short-chain dehydrogenases/reductases, with a conserved Asp47 forming a hydrogen bond with both hydroxyl groups of the adenine ribose of NAD(H), and thus specificity toward NAD(H) instead of NADP(H). The highly conserved catalytic triad (Ser153, Tyr167, and Lys171) is adjacent to both NAD(+) and substrate molecules, where Tyr167 functions as a general base. Following analysis of high resolution structures of the apo-form and two complex forms, the molecular basis for both the enantio-specificity and the reaction mechanism of secoisolariciresinol dehydrogenase is discussed and compared with that of pinoresinol-lariciresinol reductase.

Biotechnological aspects of the production of the anticancer drug podophyllotoxin.

The natural lignan podophyllotoxin, a dimerized product of two phenylpropanoid moieties which occurs in a few plant species, is a pharmacologically important compound for its anticancer activities. It is used as a precursor for the chemical synthesis of the anticancer drugs etoposide, teniposide and etopophose. The availability of this lignan is becoming increasingly limited because of the scarce occurrence of its natural sources and also because synthetic approaches for its production are still commercially unacceptable. Biotechnological production using cell culture may be considered as an alternative source. Selection of the best performing cell line, its maintenance and stabilization are necessary prerequisites for its production in bioreactors and subsequent scale-up of the cultivation process to the industrial level. Scale-up of growth and product yield depends on a multitude of factors, such as growth medium, physicochemical conditions, seed inoculum, type of reactor and processing conditions. The composition of the growth medium, elicitors and precursors, etc. can markedly influence the production. Optimum levels of parameters that facilitate high growth and product response in cell suspensions of Podophyllum hexandrum have already been determined by statistical design. P. hexandrum cells have successfully been cultivated in a 3-l stirred-tank bioreactor under low shear conditions in batch and fed-batch modes of operation. The batch kinetic data were used to identify the mathematical model which was then used to develop nutrient-feeding strategies for fed-batch cultivation to prolong the productive log phase of cultivation. An improvement in the production of podophyllotoxin to 48.8 mg l(-1) in a cell culture of P. hexandrum was achieved, with a corresponding volumetric productivity of 0.80 mg l(-1) day(-1), when the reactor was operated in continuous cell-retention mode. Efforts are being made to further enhance its production levels by the development of hairy root culture or by varying the channeling of precursors towards the desired biosynthetic pathway by molecular approaches.

Immunomodulatory and cytoprotective role of RP-1 in gamma-irradiated mice.

RP-1 has been reported to provide protection against lethal gamma-irradiation in mice. The present study was undertaken to understand its mechanism of action, especially with respect to modulation of radiation-induced changes in immune cell function, plasma antioxidant potential, cell cycle perturbations, apoptosis in mouse bone marrow cells, and micronuclei frequency in mice reticulocytes. 2 Gy reduced mitogenic response of splenic lymphocytes significantly at 48 h. Pre-irradiation RP-1 treatment significantly countered the radiation-induced loss of splenocyte proliferation. RP-1 treatment, with or without radiation, suppressed macrophage activation as compared to control. Irradiation decreased plasma antioxidant status significantly (p < 0.05) at 1 and 2 h (4.8 +/- 0.224 and 4.9 +/- 0.057 mM Fe2+) as compared to control (6.29 +/- 0.733 mM Fe2+) that was countered by RP-1 pre-treatment significantly (p < 0.05). RP-1 and irradiation individually caused G2 delay in bone marrow cells. RP-1 pre-treatment augmented radiation-induced G2 delay and elicited significant (p < 0.05) recovery in S-phase fraction at 48 h in comparison to irradiated group. Radiation-induced apoptosis (3%) was significantly higher than the control. RP-1 pre-treatment further enhanced apoptosis frequency (7.2%) in bone marrow cells. RP-1 pre-treatment significantly (p < 0.05) reduced (1.23%) the radiation-induced MN frequency (2.9%) observed at 48 h post-irradiation interval. Since the radioprotective manifestation of RP-1 is mediated through multiple mechanisms, needs further investigation.

Production of podophyllotoxin using cross-species coculture of Linum flavum hairy roots and Podophyllum hexandrum cell suspensions.

Novel cross-species coculture systems using Linum flavum hairy roots and Podophyllum hexandrum cell suspensions were applied for in vitro production of podophyllotoxin. The hairy roots and suspensions were cocultured in Linsmaier and Skoog medium in dual shake flasks and dual bioreactors. In separate experiments, coniferin feeding was shown to be an effective strategy for increasing the accumulation of podophyllotoxin in P. hexandrum suspensions. Because roots of L. flavum are a natural source of coniferin, hairy roots of this species were used in coculture with P. hexandrum to provide an in situ supply of coniferin. Compared with P. hexandrum suspensions cultured alone in shake flasks or bioreactors, podophyllotoxin concentrations in cocultured P. hexandrum cells were increased by 240% and 72% in dual shake flask and dual bioreactor systems, respectively. The availability and stability of coniferin in the medium are the most likely factors limiting podophyllotoxin synthesis in coculture. Intensification of the coculture process is required to further improve total podophyllotoxin accumulation on a volumetric basis.

Radiation protection of HepG2 cells by Podophyllum hexandrum Royale.

Radioprotection by an aqueous extract of Podophyllum hexandrum (RP-1) was investigated in HepG2 cells by evaluating colony forming efficacy (CFE), redox status of mitochondria, reactive oxygen species (ROS), generation of nitric oxide (NO), peroxidation of lipids and intracellular glutathione. Lower concentrations of RP-1 (0.1 and 1 microg/ml) rendered maximum radioprotection when administered 1 or 2 h before irradiation. Higher concentrations (5 and 10 microg/ml) however were less effective when administered 1 or 2 h before irradiation, but were more effective with increased time intervals (4 or 8 h) between RP-1 administration and irradiation. RP-1 pre-treatment also significantly inhibited radiation-induced MTT reduction in a concentration and time-dependent manner by decreasing gamma radiation-induced leakage of electrons from electron transport chain. Pre-irradiation administration of RP-1 significantly reduced both ROS and NO generation and enhanced glutathione levels, thereby inhibiting lipid peroxidation.

Enhanced production of podophyllotoxin by Podophyllum hexandrum using in situ cell retention bioreactor.

The rhizomes of the rare plant Podophyllum hexandrum contain podophyllotoxin, which is a precursor of the anticancer drugs etoposide and teniposide. Batch cultivation of Podophyllum hexandrum was conducted using optimized medium in a 3 L bioreactor, which resulted in biomass and podophyllotoxin concentrations of 21.4 g/L and 13.8 mg/L in 24 and 26 days, respectively. The batch kinetics was used to identify the mathematical model. The model was extrapolated to identify the nutrient feeding rate (150 mL/d) and substrate concentration (105 g/L) in the incoming feed for nonlimiting and noninhibitory glucose concentration in the cell retention bioreactor. An improvement in cell growth to 53 g/L and intracellular podophyllotoxin accumulation of 48.8 mg/L was achieved in 60 days, when the bioreactor was operated in continuous cell retention cultivation mode.

Effect of major nutrients on podophyllotoxin production in Podophyllum hexandrum suspension cultures.

The effect of major medium ingredients (sugar, nitrogen source and phosphate) in Podophyllum hexandrum suspension cultures was investigated in order to increase the production of podophyllotoxin, the raw material in the synthesis of anticancer drugs. Amongst B5, Eriksson, MS, Nitsch, Street and White's medium, MS medium resulted in high growth and podophyllotoxin accumulation. The optimum level of nitrogen was found to be 60 mM, with a combination of ammonium salts and nitrate in the ratio of 1:2. The highest level of podophyllotoxin was obtained at 60 g glucose/l and at 1.25 mM phosphate after 30 days. Statistical design was adopted to determine the optimum levels of the parameters for cell growth and podophyllotoxin production.

Optimization of culture parameters for production of podophyllotoxin in suspension culture of Podophyllum hexandrum.

The root explants of the germinated seedlings of Podophyllum hexandrum were grown in MS medium supplemented with indole acetic acid (IAA) (2 mg/L) and activated charcoal (0.5%), and healthy callus culture was obtained after incubation for 3 wk at 20 degrees C. The cultivation of plant cells in shake flask was associated with problems such as clumping of cells and browning of media, which were solved by the addition of pectinase and polyvinylpyrrolidone. The effect of major media components and carbon source was studied on the growth and podophyllotoxin production in suspension culture. It was found that glucose was a better carbon source than sucrose and that NH4+:NO3- ratio (total nitrogen concentration of 60 mM) and PO4(3-) did not have much effect on the growth and product formation. The relative effect of culture parameters (inoculum level, pH, IAA, glucose, NH4+:NO3- ratio, and PO4(3-)) on the overall growth and product response of the plant cell suspension culture was further investigated by Plackett-Burman design. This indicated that inoculum level, glucose, IAA, and pH had significant effects on growth and production of podophyllotoxin. To identify the exact optimum concentrations of these parameters on culture growth and podophyllotoxin production, central composite design experiments were formulated. The overall response equations with respect to growth and podophyllotoxin production as a function of these culture parameters were developed and used to determine the optimum concentrations of these parameters, which were pH 6.0, 1.25 mg/L of IAA, 72 g/L of glucose, and inoculum level of 8 g/L.