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Two new prenylated flavonoids named sinoflavonoids NJ and NK (1–2), along with ten known compounds were isolated from the fruits of Podophyllum hexandrum Royle. The chemical structures were determined through NMR spectroscopic data and MS analysis. Sinoflavonoid NJ (1) with an unusual 5,11-dioxabenzo[b]fluoren-10-one skeleton was firstly reported from Berberidaceae. The isolated flavonoids were tested with LPS-induced RAW 264.7 mouse macrophages model for their anti-inflammatory activity. Sinoflavonoid NJ (1) showed the most potent inhibition on nitric oxide production with IC50 value as 0.06 μM. [ABSTRACT FROM AUTHOR]

Podophyllum hexandrum—an endangered, high-altitude medicinal herb is a vital source of the therapeutically important lignan viz. podophyllotoxin (PTOX). PTOX is widely used as a precursor for semi-synthesis of anticancer drugs etoposide, teniposide and etopophos. Although next generation sequencing has been employed to characterize the genes involved in PTOX biosynthesis; the complete biosynthetic pathway is yet to be known. miRNAs play pivotal roles in secondary metabolites biosynthesis besides plant growth and development. Till date, several novel miRNAs have been identified in different plant species, but novel miRNAs and their regulatory functions still remains unknown in P. hexandrum. Here, two small RNA libraries were prepared from control and MeJA treated cell cultures of P. hexandrum. High-throughput sequencing of these small RNA libraries yielded 59 novel miRNAs in control and 66 novel miRNAs in treated P. hexandrum apart from known miRNAs. The identified miRNAs targeted different mRNAs. Kyoto Encyclopedia of Genes and Genomes pathway analysis unveiled the role of these miRNAs in various secondary metabolites biosynthetic pathways. qRT-PCR analysis showed lower expression of a set of miRNAs identified in MeJA treated cell culture. The current study expands our knowledge of novel miRNAs mediated secondary metabolites, especially PTOX biosynthesis, in P. hexandrum. [ABSTRACT FROM AUTHOR]

Podophyllum hexandrum Royle (Berberidaceae), a high valued medicinal plant with Podophyllotoxin as a pharmacologically active compound. The present study aimed to understand the variability in metabolomic and antioxidant potential of the rhizome and leaves extracts of P. hexandrum. The secondary metabolites from the rhizome and leaves were extracted, quantified and identified using various methods viz. soxhlet reflux extraction method, Folin–Ciocalteu method (λ max 765 nm), Aluminum chloride-colorimetric method (λ max 415 nm), GC-MS and LC-QTOF-HRMS techniques. The antioxidant activity was evaluated using the 2,2′-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging method, hydroxyl radical scavenging method, superoxide anion scavenging method, hydrogen peroxide scavenging method and reducing power method. Among the different solvent extracts, the methanolic extract of the rhizome exhibited the highest total phenolic content (164.39 ± 2.92 μg/mL), while the hexane extract showed the lowest content (69.4 ± 2.8 μg/mL). The total flavonoid content was highest in all rhizome extracts compared to the leaf extracts. The GC-MS analysis reported various therapeutic important compound and most of the compound were reported in the rhizome extract. The pharmacologically active compound Podophyllotoxin was only reported in the rhizome extract. Similar to GC-MS analysis, the LC-QTOF-HRMS also reported maximum number of compounds in the rhizome extract. The methanolic extract demonstrated the best antioxidant activity in all the assays conducted. Furthermore, the rhizome displayed the highest antioxidant activity, which can be attributed to its higher polyphenolic content. [Display omitted] • The methanolic extract of the rhizome of P. hexandrum exhibited the highest (164.39 ± 2.92 μg/mL) total phenolic content. • The GC-MS analysis reported most of the compounds including Podophyllotoxin in the rhizome extract. • The LC-QTOF-HRMS was used first time to analyze phytoconstituents of P. hexandrum's rhizome (14) and leaf extract (7). • The methanolic extract demonstrated the best antioxidant activity in all the samples. • The highest antioxidant activity of the rhizome extract can be attributed to its higher polyphenolic content. [ABSTRACT FROM AUTHOR]

Podophyllum hexandrum, a perennial alpine herb produces the anti-cancer metabolite podophyllotoxin (PPT) that is in large part responsible for the plant’s endangered species status. Since PPT commercial production via chemical synthesis, biotechnological intervention and/or cultivation by ex situ conditions have yet to meet the ever-increasing demand for this potent anticancer drug, identifying cultivation practices to improve PPT accumulation is essential. While P. hexandum is indigenous to the mountainous Himalayan region of Asia, effect of elevations on plant growth and PPT accumulation has not been systematically investigated. Here is reported plant growth and PPT production at two elevations: 2300 and 3300 m. To dissect genetic versus environmental conditions responsible for enhanced growth at the higher elevation, plants adapted to each elevation were transplanted to the alternative site. Aerial and rhizome dry-weight was 1.4- to 2.0-fold and 1.2- to 2.0-fold greater, respectively at the 3300 m versus the 2300-m site for 3- to 5-year-old plants. Other growth parameters including leaf area, rhizome length/diameter, number of petioles, root and fruit per plant and fruit dry weight per plant showed an increased value at the higher elevation. PPT content in the aerial portions and rhizomes for all years studied was greater at the 3300 m with 2.2- to 5.3-fold and 2.2- to 3.5-fold on a per plant basis compared to the 2300-m site. Based on the plant’s perennial and fruiting characteristics, a sustainable harvesting scheme that includes the plant’s aerial portions, rhizomes and seeds is proposed for improving PPT yield without over harvesting of this endangered species. [ABSTRACT FROM AUTHOR]

Podophyllotoxin is a high value metabolite having anticancerous activity and procured from a medicinal plant Podophyllum hexandrum. The escalating demand for podophyllotoxin necessitates development of alternate production platforms. Cell cultures have been tried in the past, however, podophyllotoxin yields were very low (0.3%). Multiple genes of biosynthetic pathway have been correlated with podophyllotoxin content. Understanding regulation of podophyllotoxin is, therefore, essential to design a suitable genetic intervention strategy. Six potential miRNAs were identified in NGS transcriptomes of P. hexandrum for which eight potential mRNA targets were discerned. The identified miRNAs and their mRNA targets (UDP glycosyltransferase, flavonol synthase, glyceraldehyde 3-phosphate dehydrogenase, peroxidase, malate dehydrogenase, phosphoenolpyruvate carboxylase, WRKY 37 and MYBF1 transcription factors) were further tested for any correlation through qRT-PCR. miR396b, miR2673a, miR828b and miR2910 established negative correlation with their mRNA targets through downregulation via complementary miRNA:mRNA base pairing. This is the first report on identification, characterisation, validation and expression analysis of miRNAs and their targets in P. hexandrum. [ABSTRACT FROM AUTHOR]

Podophyllotoxin is isolated as a sustainable natural bioresource from the roots and rhizomes of Podophyllum hexandrum and Juniperus Sabina . In continuation of our program aimed at the discovery of biorenewable natural product-based pesticides, a series of 4α-acyloxy derivatives of 2'-chloro-5-bromopicropodophyllotoxin or 2',5-dibromopicropodophyllotoxin were synthesized by structural modification of podophyllotoxin, and evaluated as insecticidal and acaricidal agents against two serious agricultural pests, Mythimna separata Walker and Tetranychus cinnabarinus Boisduval. Among all derivatives, compounds 6a and 7a exhibited more promising insecticidal and acaricidal activities than podophyllotoxin, and the final mortality rates of 6a , 7a and 1 against M. separata (at 1 mg/mL) and T. cinnabarinus (at 0.5 mg/mL) were 50%/53.3%/36.7%, and 33%/41.8%/1.6%, respectively. This suggested that introduction of the acetoxy moiety at the C-4 position of 2'-chloro-5-bromopicropodophyllotoxin/2',5-dibromopicropodophyllotoxin was necessary for the insecticidal and acaricidal activities. [ABSTRACT FROM AUTHOR]

Background and aim: The endangered Podophyllum hexandrum is an important industrial source of podophyllotoxin, which is a precursor for the anticancer drugs etoposide and teniposide. Attempts to obtain podophyllotoxin through cell cultures or chemical synthesis have still a long way to go before being economical feasible. The objective of this study was to increase the root formation and podophyllotoxin production of P. hexandrum cultivated in a glasshouse. Methods: Root formation and podophyllotoxin production of P. hexandrum in sand or peat-perlite soil at 15 °C or 25 °C was determined. Furthermore, the influence of methyl jasmonate on the podophyllotoxin production was determined. Results: More root formation was observed in peat-perlite soil than in sand soil. Furthermore, root formation was higher at 15 °C than at 25 °C. This resulted in the highest podophyllotoxin production per plant in peat-perlite at 15 °C (160 ± 22 mg/plant d.w.). Furthermore, methyl jasmonate treatment of the leaves increased the podophyllotoxin production in the roots by 21%. Conclusion: We were able to cultivate P. hexandrum in a glasshouse in the Netherlands and improve the root formation and podophyllotoxin production. This paves the way for large-scale cultivation of P. hexandrum in the temperate latitudes for the production of the pharmaceutical interesting podophyllotoxin. [ABSTRACT FROM AUTHOR]

Podophylloxin (ptox), primarily obtained from Podophyllum hexandrum, is the precursor for semi-synthetic anticancer drugs viz. etoposide, etopophos, and teniposide. Previous studies established that methyl jasmonate (MeJA) treated cell culture of P. hexandrum accumulate ptox significantly. However, the molecular mechanism of MeJA induced ptox accumulation is yet to be explored. Here, we demonstrate that MeJA induces reactive oxygen species (ROS) production, which stimulates ptox accumulation significantly and up regulates three ROS-responsive ptox biosynthetic genes, namely, PhCAD3, PhCAD4 (cinnamyl alcohol dehydrogenase), and NAC3 by increasing their mRNA stability. Classic uncoupler of oxidative phosphorylation, carbonylcyanide m-chlorophenylhydrazone, as well as H2O2 treatment induced the ROS generation and consequently, enhanced the ptox production. However, when the ROS was inhibited with NADPH oxidase inhibitor diphenylene iodonium and Superoxide dismutase inhibitor diethyldithio-carbamic acid, the ROS inhibiting agent, the ptox production was decreased significantly. We also noted that, MeJA up regulated other ptox biosynthetic pathway genes which are not affected by the MeJA induced ROS. Further, these ROS non-responsive genes were controlled by MeJA through the down regulation of five secondary metabolites biosynthesis specific miRNAs viz. miR172i, miR035, miR1438, miR2275, and miR8291. Finally, this study suggested two possible mechanisms through which MeJA modulates the ptox biosynthesis: primarily by increasing the mRNA stability of ROS-responsive genes and secondly, by the up regulation of ROS non-responsive genes through the down regulation of some ROS non-responsive miRNAs. [ABSTRACT FROM AUTHOR]

The article focuses on a study for identification and characterization of novel UniGene-derived microsatellite markers in Podophyllum hexandrum. Topics discussed include economic impact of Podophyllum hexandrum, information on population genetic, extraction of expressed sequence tag using SeqMan DNAStar Lasergene, and analysis of population genetic structure and gene flow.

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. [ABSTRACT FROM AUTHOR]

DNA damage can be assessed by the quantitation of γH2AX foci that form at DSB sites. This study examines the generation and persistence of γH2AX foci, variability in foci size after acute and fractionated radiation exposure, and the effect of pretreatment with a safe radioprotective formulation termed G-003M on foci generation and persistence. G-003M contains a combination of podophyllotoxin and rutin hydrate, and was administered intramuscularly to rabbits 1 hr prior to Co60 gamma irradiation. Rabbits were assigned to one of the following treatment groups: untreated, G-003M alone, irradiated (single dose 8 Gy, fractionated 2 Gy/day for 4 days or single dose 2 Gy) or G-003M preadministration followed by radiation exposure. Foci continuously persisted for a week in peripheral blood mononuclear cells of rabbits exposed to a single 8 Gy dose. However, the number of foci gradually decreased after reaching a maximum at 1 h. In rabbits exposed to fractionated radiation, foci detected 1 hr after the final exposure were significantly larger ( P < 0.001) than in rabbits exposed to a single 8 Gy dose, but disappeared completely after 24 h. In both groups, foci reappeared on days 11-15 in terminally ill animals. G-003M pretreatment significantly ( P < 0.05) attenuated the formation of γH2AX foci in all irradiated rabbits. This study reveals that γH2AX focus assessment could be used to confirm radiation exposure, that focus size reflects the type of radiation exposure (acute or fractionated), that the re-appearance of foci is a strong indicator of imminent death in animals, and that G-003M provides protection against radiation. Environ. Mol. Mutagen. 57:455-468, 2016. © 2016 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

The present study was conceptualized with the aim of developing a safe radioprotector for human application against radiation induced toxicity. For this study, a formulation (G-002M) prepared by combining three active principles isolated from rhizomes of Podophyllum hexandrum, was evaluated for its potential to protect genomic DNA of human blood cells exposed to different doses of radiation (5,7&10Gy). Blood samples were pretreated (-1hr to exposure) with G-002M. Parameters of Premature Chromosome Condensation (PCC) assay like PCC-index, PCC-rings and PCC-fragments were used to estimate radiation induced chromosomal aberrations. Radiation (7Gy) induce ROS generation and its modulation by G-002M was determined by flow-cytometry and fluorescent microscopy while apoptosis (0,2,24&48 hr) was analyzed using TUNEL assay. Effect on spindle organization in G2/M arrested cells by all the three compounds individually was studied using immunofluorescence microscopy. Irradiation caused dose dependent linear increase in PCC-rings and fragments, while decline in PCC index. G-002M pretreatment significantly decreased these chromosomal aberrations at all the radiation doses and assisted cell survival as indicated by increased PCC index compared with radiation only group. Significant decrease in radiation induced intracellular ROS (45 ± 3%) and apoptosis (49.9%) was also exhibited by the formulation. On podophyllotoxin treatment, most of the cells have shown blocked spindles however, depicted normal arrangement. G-002M also demonstrated a highly significant Dose Modifying Factor or DMF (PCC-rings: 2.27 and PCC-fragments: 1.60). Present study based on many parameters along with DMF study, strongly suggests that G-002M is an effective formulation with a potential to minimize chromosomal damage even at very high radiation doses. [ABSTRACT FROM AUTHOR]

Purpose: To investigate the protective role of a novel formulation, prepared by a combination of three active principles isolated fromPodophyllum hexandrum(G-002M), against radiation- mediated hematopoietic suppression and cytogenetic aberrations in lethally irradiated mice. Materials and methods: G-002M, a combination of podophyllotoxin, podophyllotoxin-β-D glucoside and rutin, was administered intramuscularly in mice (− 1 h) to radiation (9 Gy) exposure. The animals were autopsied at different time intervals for further studies. Results: Loss of bone marrow progenitor cells, altered myeloid/erythroid ratio, serum erythropoietin and pancytopenia in irradiated mice was found significantly (p< 0.001) ameliorated in G-002M pre-administered mice within 30 d. Bcl-2 (B-cell lymphoma 2) and BAX (Bcl-2-associated X) protein expression was also positively (p< 0.001) countered in these mice. Chromosomal aberrations in 30 d were found remarkably (p< 0.001) reduced in marrow of G-002M pretreated mice. Accelerated antioxidants, reduced DNA damage, stimulated lymphocyte proliferation and minimal cellular atrophy in spleen were some of the other key features observed in G-002M administered mice. Conclusions: Reduction in hematopoietic aplasia and chromosomal aberrations, besides, early recovery in bone marrow and spleen of G-002M pretreated mice, could be attributed to its free radical scavenging, DNA protecting and apoptotic proteins modulating ability against radiation. [ABSTRACT FROM PUBLISHER]

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. [ABSTRACT FROM AUTHOR]

In the present study, micropropagation of Podophyllum hexandrum, an endangered species of Pakistan was carried out on basal media containing different combinations of auxins and cytokinins as an attempt for its conservation. The results showed that the media containing BAP and NAA enhanced shoot induction from rhizome segments. Maximum number of shoots was observed in media having higher concentrations of BAP and NAA i.e. 11.0 mg L-1 and 5.3mg L-1 respectively. On an average 84% test tubes were observed showing initiation of shoots within 24 ± 0.83a days, media containing 3.0 mg L-1IBA was found to be the best for rooting. RAPD analysis using 10-mer primers was also carried out to check genetic stability of the in vitro conserved plants in comparison to the mother plants kept in the green house as the control. The results showed no genetic alterations when compared in term of number of bands and their respective sizes thus confirming that the plants conserved in vitro did not go through any genetic changes. [ABSTRACT FROM AUTHOR]

This study is aimed at the development of a safe radioprotective formulation to minimize human sufferings during accidental nuclear exposures. In the current study, a combination of three active principles, namely podophyllotoxin, podophyllotoxin beta-D-glucoside, and rutin (G-002M), isolated from Podophyllum hexandrum rhizomes, has been evaluated for its radioprotective potential and mode of action. Total body protection studies have demonstrated that a single prophylactic dose of G-002M delivered more than 85% survival in mice exposed to a lethal (9 Gy) dose of gamma radiation, and significantly protected the radiosensitive hematopoietic and gastrointestinal organs. Studies have also revealed a reduction in free radical generation, lipid peroxidation, protein carbonylation, and cell death in mouse intestine after G-002M treatment, while GSH was observed to be enhanced in the same tissue. Redox-sensitive transcription factor (Nrf2) activation and subsequent upregulation of heme oxygenase-1 (HO-1) and SOD-1 revealed the cytoprotective role of G-002M. A histological examination of the jejunum pretreated with the formulation also demonstrated less damage to the villi, crypts, and the mucosal layers. These observations reiterated that the reduction in the ROS levels, protection of cellular macromolecules, and activation of the antioxidant signaling pathway may have been the principle factors involved in G-002M- mediated protection against radiation-induced tissue impairment. The potentially safe and effective radioprotective characteristics of this new combination are encouraging for further studies for human application. [ABSTRACT FROM AUTHOR]

We investigated the root of Podophyllum hexandrum as a potential source of lead bioactive metabolites with anticancer activity. The present study led to the isolation of six known aryltetralin-type lignans designated as 4′-demethyl-deoxypodophyllotoxin ( 1 ), podophyllotoxin ( 2 ), 4′-demethyl-podophyllotoxin ( 3 ), podophyllotoxin-4- O -β- d -glucopyranoside ( 4 ), 4′-demethyl-deoxypodophyllotoxin-4- O -β- d -glucopyranoside ( 5 ), 4′-demethyl-podophyllotoxin-4- O -β- d -glucopyranoside ( 6 ), along with three known flavones Kaempferol ( 7 ), Quercetin ( 8 ), Astragalin ( 9 ) from the root of P. hexandrum . Compounds ( 1–9 ) exhibited the remarkable cytotoxic potential in diverse cancer cell lines. 5 therapeutic potential was extensively studied first time which exhibiting antiproliferative and ROS generating activity than its non-glycoside analogue 1 . Furthermore, 5 augmented the apoptotic cascades in MCF-7 breast cancer cells, viz. nuclear condensation, membrane blebbing, probably by destabilizing the micro-tubular protein tubulin. Strikingly, our docking study and in vitro assays demonstrate that 5 binds to and modulate checkpoint kinase-2, a key cell cycle regulatory protein in normal and cancer cells. [ABSTRACT FROM AUTHOR]

The objective of this study was to establish a simple and efficient method for high frequency of somatic embryogenesis and plant regeneration of Podophyllum hexandrum and podophyllotoxin (PTOX) production. Embryogenic callus (100 mg FW) cultured on 0.75 strength MS medium with 4% sucrose and 3 g/l PVP produced maximum number of somatic embryos (1089) after five weeks of culture under dark condition. When these somatic embryos were subcultured to the same MS medium with 1 mg/l ABA, the embryos matured after 2 weeks of culture. The MS medium supplemented with1.0 mg/l GA3 led to high frequency germination (91.1%). The accumulation of PTOX varied in somatic embryos, matured embryos and germinated embryos. Highest PTOX accumulation (2.8 mg/l) was recorded in somatic embryos cultured in 0.75 strength MS medium with 8% sucrose. Regenerated plantlets were successfully acclimatized in the growth chamber. The protocol established in this study will be helpful for large scale production of podophyllotoxin. [ABSTRACT FROM AUTHOR]

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 (GA [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 GA (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. [ABSTRACT FROM AUTHOR]

Radiation exposure is a serious threat to biomolecules, particularly DNA, proteins and lipids. Various exogenous substances have been reported to protect these biomolecules. In this study we explored the effect of pre-treatment with G-002M, a mixture of three active derivatives isolated from the rhizomes of Podophyllum hexandrum, on DNA damage response in irradiated human blood leukocytes. Blood was collected from healthy male volunteers, preincubated with G-002M and then irradiated with various doses of radiation. Samples were analyzed using flow cytometry to quantify DNA double strand break (DSB) biomarkers including γ-H2AX, P53BP1 and levels of ligase IV. Blood samples were irradiated in vitro and processed to determine time and dose-dependent kinetics. Semiquantitative RT-PCR was performed at various time points to measure gene expression of DNA -PKcs, Ku80, ATM, and 53BP1; each of these genes is involved in DNA repair signaling. Pre-treatment of blood with G-002M resulted in reduction of γ-H2AX and P53BP1 biomarkers levels and elevated ligase IV levels relative to non-G-002M-treated irradiated cells. These results confirm suppression in radiation-induced DNA DSBs. Samples pre-treated with G-002M and then irradiated also showed significant up-regulation of DNA-PKcs and Ku80 and downregulation of ATM and 53BP1 gene expressions, suggesting that G-002M plays a protective role against DNA damage. The protective effect of G-002M may be due to its ability to scavange radiation-induced free radicals or assist in DNA repair. Further studies are needed to decipher the role of G-002M on signaling molecules involved in radiation-induced DNA damage repair pathways. Environ. Mol. Mutagen. 55:436-448, 2014. © 2014 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

To determine the possibility of generating the Podophyllotoxin accumulation using Podophyllum hexandrum adventitious roots derived from root segments, several nutrient constituents (carbon sources, media strength, initial medium pH, ammonium and nitrate proportion and phosphate ratio) were evaluated in culture. The maximum biomass accumulation was recorded in 0.50 MS medium containing 3 mg/l indole-3-butyric acid and 2 % sucrose, and the maximum accumulation of Podophyllotoxin was documented in the same strength of MS medium with 6 % of sucrose. When the initial medium pH was on 6 in the optimized MS medium, the biomass and Podophyllotoxin accumulations were highest. The lower concentration of ammonium (10 mM) in combination with a moderate concentration of nitrate (20 mM) was found ideal for maximum accumulations of biomass and Podophyllotoxin. Maximum Podophyllotoxin accumulation (6.4 mg/g dry weight) was recorded at the higher concentration of phosphate (2.25 mM), and lower concentration of phosphate (1.25 mM) showed highest growth accumulation. The outcome of the present work will be helpful for the large-scale cultivation of adventitious root for the production of Podophyllotoxin. [ABSTRACT FROM AUTHOR]

The composition of lignans extracted fromPodophyllum hexandrumrhizomes was studied by sequential extraction with supercritical CO2,ethyl acetate modified CO2and methanol modified CO2. The results were compared with the extracts obtained by Accelerated Solvent Extraction (ASE) and soxhlet's methods. The lignan contents comprised of Podophyllotoxin, deoxypodophyllotoxin, 4′-demethylpodophyllotoxin, Picropodophyllotoxin, Isopicropodophyllotoxin, Podophllotoxin-β-D-glucopyranoside, and 4′-demethylpodophyllotoxin β-D-glucopyranoside in the extracts ofPodophyllum hexandrumrhizomes obtained by different methods was studied. There was a variation in the concentration of lignan in the extracts obtained by different methods of extraction. Podophyllotoxin formed the major component (36.55%) of the extract obtained by SFE and picropodophyllotoxin, isopicropodophyllotoxin, deoxypodophyllotoxin, and 4′-demethylpodophyllotoxin were present in 6.82, 1.51, 1.46, and 0.85%, respectively, whereas 4′-demethylpodophyllotoxin β-D-glucopyranoside was 1.89% in the extract obtained by ASE, which is higher than SFE extracts. Soxhlet's derived extract contains better concentration of isopicropodophyllotoxin (24.49%) than SFE. A simple, isocratic, and reliable analytical HPLC-UV (DAD) method was developed for simultaneous identification and quantification of different seven lignans in the extracts of different extraction techniques. [ABSTRACT FROM AUTHOR]

Podophyllotoxin, an aryltetralin lignan, is the source of important anticancer drugs etoposide, teniposide, and etopophos. Roots/rhizome of Podophyllum hexandrum form one of the most important sources of podophyllotoxin. In order to understand genes involved in podophyllotoxin biosynthesis, two suppression subtractive hybridization libraries were synthesized, one each from root/rhizome and leaves using high and low podophyllotoxin-producing plants of P. hexandrum. Sequencing of clones identified a total of 1,141 Expressed Sequence Tags (ESTs) resulting in 354 unique ESTs. Several unique ESTs showed sequence similarity to the genes involved in metabolism, stress/defense responses, and signalling pathways. A few ESTs also showed high sequence similarity with genes which were shown to be involved in podophyllotoxin biosynthesis in other plant species such as pinoresinol/lariciresinol reductase. A full length coding sequence of pinoresinol/lariciresinol reductase (PLR) has been cloned from P. hexandrum which was found to encode protein with 311 amino acids and show sequence similarity with PLR from Forsythia intermedia and Linum spp. Spatial and stress-inducible expression pattern of PhPLR and other known genes of podophyllotoxin biosynthesis, secoisolariciresinol dehydrogenase ( PhSDH), and dirigent protein oxidase ( PhDPO) have been studied. All the three genes showed wounding and methyl jasmonate-inducible expression pattern. The present work would form a basis for further studies to understand genomics of podophyllotoxin biosynthesis in P. hexandrum. [ABSTRACT FROM AUTHOR]

Podophyllotoxin, an aryltetralin lignan, is the source of important anticancer drugs etoposide, teniposide, and etopophos. Roots/rhizome of Podophyllum hexandrum form one of the most important sources of podophyllotoxin. In order to understand genes involved in podophyllotoxin biosynthesis, two suppression subtractive hybridization libraries were synthesized, one each from root/rhizome and leaves using high and low podophyllotoxin-producing plants of P. hexandrum. Sequencing of clones identified a total of 1,141 Expressed Sequence Tags (ESTs) resulting in 354 unique ESTs. Several unique ESTs showed sequence similarity to the genes involved in metabolism, stress/defense responses, and signalling pathways. A few ESTs also showed high sequence similarity with genes which were shown to be involved in podophyllotoxin biosynthesis in other plant species such as pinoresinol/lariciresinol reductase. A full length coding sequence of pinoresinol/lariciresinol reductase (PLR) has been cloned from P. hexandrum which was found to encode protein with 311 amino acids and show sequence similarity with PLR from Forsythia intermedia and Linum spp. Spatial and stress-inducible expression pattern of PhPLR and other known genes of podophyllotoxin biosynthesis, secoisolariciresinol dehydrogenase ( PhSDH), and dirigent protein oxidase ( PhDPO) have been studied. All the three genes showed wounding and methyl jasmonate-inducible expression pattern. The present work would form a basis for further studies to understand genomics of podophyllotoxin biosynthesis in P. hexandrum. [ABSTRACT FROM AUTHOR]

Background The Himalayan or Indian Mayapple (Podophyllum hexandrum Royle) produces podophyllotoxin, which is used in the production of semisynthetic anticancer drugs. High throughput transcriptome sequences or genomic sequence data from the Indian Mayapple are essential for further understanding of the podophyllotoxin biosynthetic pathway. Results 454 pyrosequencing of a P. hexandrum cell culture normalized cDNA library generated 2,667,207 raw reads and 1,503,232 high quality reads, with an average read length of 138 bp. The denovo assembly was performed by Newbler using default and optimized parameters. The optimized parameter generated 40, 380 assembled sequences, comprising 12,940 contigs and 27,440 singlets which resulted in better assembly as compared to default parameters. BLASTX analysis resulted in the annotation of 40,380 contigs/singlet using a cut-off value of ⩽1E-03. High similarity to Medicago truncatula using optimized parameters and to Populus trichocarpa using default parameters was noted. The Kyoto encyclopedia of genes and genomes (KEGG) analysis using KEGG Automatic Annotation Server (KAAS) combined with domain analysis of the assembled transcripts revealed putative members of secondary metabolism pathways that may be involved in podophyllotoxin biosynthesis. A proposed schematic pathway for phenylpropanoids and podophyllotoxin biosynthesis was generated. Expression profiling was carried out based on fragments per kilobase of exon per million fragments (FPKM). 1036 simple sequence repeats were predicted in the P. hexandrum sequences. Sixty-nine transcripts were mapped to 99 mature and precursor microRNAs from the plant microRNA database. Around 961 transcripts containing transcription factor domains were noted. High performance liquid chromatography analysis showed the peak accumulation of podophyllotoxin in 12-day cell suspension cultures. A comparative qRTPCR analysis of phenylpropanoid pathway genes identified in the present data was performed to analyze their expression patterns in 12-day cell culture, callus and rhizome. Conclusions The present data will help the identification of the potential genes and transcription factors involved in podophyllotoxin biosynthesis in P. hexandrum. The assembled transcripts could serve as potential candidates for marker discovery and conservation, which should form the foundations for future endeavors. [ABSTRACT FROM AUTHOR]

Human exposure to radiation has expanded considerably in recent years, due to a wide range of medical, agricultural, and industrial applications. Despite its beneficial utilities, radiation is also known to have a deleterious effect on cells and tissues, largely through the creation of free radicals, which cause severe damage to biological systems through processes such as DNA double/single-strand fragmentation, protein modification, and upregulation of lipid peroxidation pathways. In addition, radiation damages genetic material while inducing hereditary genotoxicity. Developing measures to counter radiation-induced damage is thus considered to be of significant importance. Considering the inherent capability of plants to survive radiative conditions, certain plants and natural compounds have been the subject of investigations to explore and harness their natural radioprotective abilities. Podophyllum hexandrum, an Indian medicinal plant with several known traditional phytotherapeutic uses, is considered in particular to be of immense therapeutic importance. Recent studies have been conducted to validate its radioprotective potential alongside discovering its protective mechanisms following γ-radiation-induced mortality and disorder in both mice and human cells. These findings show that Podophyllum and its constituents/natural compounds protect the lungs, gastrointestinal tissues, hemopoietic system, and testis by inducing DNA repair pathways, apoptosis inhibition, free radical scavenging, metal chelation, anti-oxidation and anti-inflammatory mechanisms. In this review, we have provided an updated, comprehensive summary of ionizing radiations and their impacts on biological systems, highlighting the mechanistic and radioprotective role of natural compounds from Podophyllum hexandrum. [Display omitted] • Radiation causes genotoxicity and hereditary consequences by damaging genetic material. • Novel radioprotectants from Podophyllum hexandrum and its active components are summarized. • Radioprotective agents provide significant protection against the harmful effects of radiation. • The mode of radioprotective action mechanisms has been reviewed. • Podophyllum hexandrum and its derived bioactive constituents have the potentials to become novel radioprotective agents. [ABSTRACT FROM AUTHOR]

Considerable interest has centred on the Podophyllum-based lignans as lead compounds for the development of new drugs. The successful introduction of the anticancer drugs etoposide® and teniposide® and the development of new derivatives such as etopophos® have created a demand for podophyllotoxin. Currently it is obtained from the rhizomes and roots of wild populations of Podophyllum hexandrum, and thus the availability of this natural product is limited. There is an urgent need for a maintainable supply of P. hexandrum plants, a rare and threatened species. In the present work, a protocol was developed for the sterilisation and germination of seeds of P. hexandrum. Ripe fruits were harvested from seed-derived plants cultivated at the University of Nottingham. Seeds were removed from fruits, left for 20 – 30 min in running water, and washed three times with sterile, reverseosmosis water. Seeds were surface sterilised in (5, 10, 15 and 20%; v:v) Domestos bleach solution with 0.2% (v:v) Tween 20 for 5, 10, 15 and 20 min, followed by three washes in sterile, reverse-osmosis water. The effect of storage as a pre-treatment for the germination of seeds was also investigated. Sterilised seeds were kept in Petri dishes and maintained in the dark at 22 ± 1°C for 30 d. Ten stored seeds were cultured per dish containing a moist sterile filter paper disk. Cultures were incubated in the dark (22 ± 1 °C). Seeds with emerged radicles were individually transferred onto full-strength MS medium containing IAA (0.00875 mg l-1) in combination with kinetin (0.03 mg l-1) and folic acid (0.01 mg l-1) designated BGS medium. Seeds were also transferred onto full-strength MS medium lacking growth regulators (MSO medium). Media were supplemented with 3.0% (v:v) sucrose and solidified with 0.8% (w:v) agar. Cultures were maintained under diffuse light (3.8 µmol m-2 s-1) at 22 ± 1°C for three weeks and then transferred to a 16 h photoperiod under fluorescent light (42 µmol m-2 s-1) at 22 ± 1°C. Seed sterilisation was best achieved with 20% (v:v) Domestos and 0.2% (v:v) Tween 20 for 20 min. The results from these studies confirm that a post-harvest ripening period of 30 d was required for in vitro seed germination. If seeds are stored in moist and dark conditions, spontaneous germination occurs within 35 to 40 d. Axenic cultures were successfully established either on full-strength BGS medium with growth regulators or fullstrength MS medium lacking growth regulators. However, the overall growth of plants showing a normal morphology was superior on the latter medium. The in vitro-grown seedlings can be used as an alternative source of plant material for tissue culture experiments.

Abstract: 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 Fe2+ and Mn2+ 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 , , Na+, Fe, and Mn levels and was negatively correlated with the and K+ levels, while the correlation with the Mg2+, Ca2+, Cu and Zn levels was not significant. The Fe2+ and Mn2+ levels were associated with the increased activity of PAL and CAD at 3–18 days; Fe2+ enhanced the activity levels by 2.66- and 1.76-fold, respectively, and Mn2+ 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 Mn2+, but it was not significantly affected by Fe2+. Finally, PDT production was enhanced approximately 60% and 34% by Fe2+ and Mn2+, 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. [Copyright &y& Elsevier]

An efficient Agrobacterium-mediated genetic transformation method has been developed for the medicinal plant Podophyllum hexandrum Royle, an important source of the anticancer agent podophyllotoxin. Highly proliferating embryogenic cells were infected with Agrobacterium tumefaciens harbouring pCAMBIA 2301, which contains npt II and gusA as selection marker and reporter genes, respectively. The transformed somatic embryos and plantlets were selected on Murashige and Skoog (MS) basal medium containing kanamycin and germination medium, respectively. GUS histochemical analysis, polymerase chain reaction and Southern blot hybridisation confirmed that gusA was successfully integrated and expressed in the P. hexandrum genome. Compared with cefotaxime, 200 mg l timentin completely arrested Agrobacterium growth and favoured somatic embryo development from embryogenic cells. Among the different Agrobacterium strains, acetosyringone concentrations and co-cultivation durations tested, embryogenic callus infected with A. tumefaciens EHA 105 and co-cultivated for 3 days on MS basal medium containing 100 μM acetosyringone proved to be optimal and produced a transformation efficiency of 29.64 % with respect to germinated GUS-positive plantlets. The Agrobacterium-mediated genetic transformation method developed in the present study facilitates the transference of desirable genes into P. hexandrum to improve the podophyllotoxin content and to enhance other useful traits. [ABSTRACT FROM AUTHOR]

Objective To test possible antioxidant activity of n-hexane extract of Podophyllum hexandrum under in vitro and in vivo conditions. Methods The in vitro antioxidant activity was evaluated by the ability of the extract to interact with the stable free radical DPPH, Superoxide (O2), Hydroxyl (OH- ), Hydrogen peroxide (H202) radicals, and reducing power ability of the extract was also evaluated. Under in vivo conditions the extract was evaluated for its hepatoprotective activity by measuring different biochemical parameters, such as serum alanine aminotransaminase, serum aspartate aminotransaminase and serum lactate dehydrogenase and antioxidant enzymes. Antioxidant status was estimated by determining the activities of antioxidative enzymes, glutathione reductase (GR), glutathione peroxidase (GPx), glutathione-S-transferase (GST) and superoxide dismutase (SOD), and by determining the levels of reduced glutathione (GSH) and thiobarbituric acid reactive substances (TBARS). Results Hexane extract of P. hexandrum exhibited good radical scavenging capacity in neutralization of DPPH, O2", OH", and H2O2 radicals in a dose dependent manner, n-hexane extract of Podophyllum hexandrum at the doses of 20, 30, and 50 mg/kg-day produced hepatoprotective effect by decreasing the activity of serum marker enzymes, while it significantly increased the levels of glutathione (GSH), glutathione peroxidase (GPx), glutathione reductase (GR), super oxide dismutase (SOD), and glutathione-S-transferase (GST) in a dose dependant manner. The effect of n-hexane extract was comparable to that of standard antioxidant vitamin E. Conclusion The extract of Podophyllum hexandrum possess free radical scavenging activity under in vitro conditions and could protect the liver tissue against CCI4 induced oxidative stress probably by increasing antioxidant defense activities. [ABSTRACT FROM AUTHOR]

Abstract: Now-a-days synthesis and characterization of silver nanoparticles (AgNPs) through biological entity is quite interesting to employ AgNPs for various biomedical applications in general and treatment of cancer in particular. This paper presents the green synthesis of AgNPs using leaf extract of Podophyllum hexandrum Royle and optimized with various parameters such as pH, temperature, reaction time, volume of extract and metal ion concentration for synthesis of AgNPs. TEM, XRD and FTIR were adopted for characterization. The synthesized nanoparticles were found to be spherical shaped with average size of 14nm. Effects of AgNPs were analyzed against human cervical carcinoma cells by MTT Assay, quantification of ROS, RT-PCR and western blotting techniques. The overall result indicates that AgNPs can selectively inhibit the cellular mechanism of HeLa by DNA damage and caspase mediated cell death. This biological procedure for synthesis of AgNPs and selective inhibition of cancerous cells gives an alternative avenue to treat human cancer effectively. [Copyright &y& Elsevier]

Tea, a beverage crop, is a rich source of polyphenols and polysaccharides which greatly attribute to its importance. However, oxidation and precipitation of these compounds during nucleic acids extraction is a limitation to molecular biology and genomic studies. On isolation of total RNA from root tissue using established protocols, difficulties were encountered in terms of purity and quantity of isolated RNA or some of the methods were time-consuming and also yields were low. The present communication combines a phenol-based RNA isolation protocol with a cetyltrimethylammonium bromide-based procedure with appropriate modifications. This protocol successfully isolated RNA from tap root tissue in 2-3 h as compared with 16 h reported by the previous method. Also, RNA yield was higher by more than fourfold. The RNA isolated by this protocol was successfully used for downstream applications such as RT-PCR and the construction of suppression subtractive hybridization library. The developed protocol worked well with other plant tissue with high polyphenols and polysaccharides contents. [ABSTRACT FROM AUTHOR]

A new quantitative method using thin-layer chromatography silica gel 60F254 plates as the stationary phase and the mobile phase consisting of toluene-ethyl acetate-acetic acid (15.0:7.5:0.5, v/v) was employed for simultaneous determination of lignans and flavonoid in Podophyllum hexandrum. Densitometric determination of the constituents was performed at 254 nm in reflectance/absorbance mode. The method was validated for precision, accuracy, specificity, robustness, and recovery. The linear regression analysis data for the calibration plots showed a good linear relationship (r2 = 0.9903-0.9976) in the calibration range of 1-8 µg per band for 4'-Odemethylpodophyllotoxin (1), podophyllotoxin (2), and podophyllotoxone (4), and 2-10 µg per band for kaempferol (3) and deoxypodophyllotoxin (5) with respect to peak area. Limits of detection and quantitation were in the range of 250-617 ng per band and 856-1974 ng per band. The average recovery for 4'-O-demethylpodophyllotoxin, podophyllotoxin, kaempferol, podophyllotoxone, and deoxypodophyllotoxin was 96.38 ± 1.92 to 101.84 ± 1.05%, indicating the excellent reproducibility. Podophyllotoxin was found in highest content (9.92 µg mg-1) and podophyllotoxone in the lowest content (0.94 µg mg-1). The proposed method is rapid, simple, precise, specific, sensitive, accurate, and robust. [ABSTRACT FROM AUTHOR]

Objective: Current study had aimed to assess the role of alcoholic extract of Podophyllum hexandrum (G-001M) in minimizing radiation mediated injuries in gastrointestinal (GI) tissue of lethally irradiated mice. Methods: G-001M (6 mg/kg b.w) was administered intramuscularly 1 h prior to 10Gy whole body irradiation in strain 'A' mice (total 279 mice were used in current study divided into various experimental groups). Biochemical indicators of oxidative stress such as lipid peroxidation, superoxide dismutase (SOD), reduced glutathione (GSH) and glutathione reductase (GR) were measured in jejunum at different time intervals in various experimental groups. Histological changes in jejunum of all the groups were evaluated from 24 h to 30 days using Hematoxylin & Eosin staining and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. Results: Histological observations showed increase in cellular apoptosis in crypts of jejunum along with regression of mitosis, depletion of viable crypts and crypt/villous cells in irradiated mice (10Gy). Pretreatment of G-001M improved crypt survival, lowered apoptotic activity and enhanced mitotic index in intestine of irradiated animals. Increase in lipid peroxidation and depression in the levels of endogenous antioxidants like SOD, GSH and GR were observed after lethal radiation exposure. Prophylactic administration of G-001M in whole body irradiated mice significantly inhibited lipid peroxidation and enhanced antioxidants level in jejunum. Conclusion: Current finding demonstrates significant protection delivered to gastrointestinal tract of mice exposed to lethal doses of gamma radiation by prophylactically administered Podophyllum hexandrum extract (G-001M) [ABSTRACT FROM AUTHOR]

The present study was undertaken to evaluate the effect of the aqueous extract of Podophyllum hexandrum against free radical-mediated damage and also explore its anticancer activity. The extract exhibited significant activity in scavenging 1, 1-diphenyl-2-picryl-hydrazyl radicals, •OH radical-mediated DNA damage, and lipid peroxide production in rat liver microsomes. The extract was also tested for its reducing abilities. The activity of liver marker enzymes and antioxidant defense enzymes in rat liver homogenate was assessed in control and carbon tetrachloride (CCl4)-treated animals. It was observed that CCl4-induced changes viz., increases in the activities of aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase, a decrease in reduced glutathione as well as decreases in the activities of superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, and glutathione-S-transferase. All these parameters showed reversal when pretreated with aqueous extract of P. hexandrum. Podophylotoxin and etoposide are the two known anticancer agents derived from P. hexandrum and interestingly the aqueous extract of P. hexandrum showed a typical DNA ladder formation in HL-60 cells confirming its role as an inducer of apoptosis. The results obtained suggest that the plant extract exhibits inhibition of and free radical production and lipid peroxidation, increase in antioxidant enzyme activities, revealing its antioxidant properties, and is also able to show potent anticancer activity as depicted by its ability to cause fragmentation of DNA. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: Podophyllum hexandrum Royle (P. hexandrum) is a perennial herb and widely used in clinic. The present study was designed to separate and identify the biochemical composition and antioxidant capacity of extracts from P. hexandrum rhizome. Methods: The ethyl acetate and ethanol extracts from P. hexandrum rhizome were analyzed by GC-MS (gas chromatography–mass spectrometry), and the antioxidant capacity of the extracts and the components was tested by using the DPPH (2, 2-diphenylpicrylhydrazyl) and FRAP (Ferric reducing/antioxidant power) assays. Results: The rhizome extracts had greater antioxidant capacity than the petiole extracts in DPPH and FRAP assays. About 16 kinds of main reactive oxygen components were identified in the extracts. Components of PADE (Phthalic acid, diisobutyl ester), BADE (1,2-Benzenedicarboxylic acid, diisooctyl ester), Polyneuridine, PODD (Podophyllotoxin, deoxy), β-Sitosterol and POD (Podophyllotoxin) showed the antioxidant capacity in some degree. PODD, POD, and Polyneuridine showed stronger antioxidant capacity with the IC50 and FRAP values of 9.61 ± 0.81 and 2923.98 ± 21.89 μM, 9.98 ± 0.24 and 2847.27 ± 14.82 μM, and 13.37 ± 0.35 and 2404.32 ± 36.88 μM, respectively, than the positive control ASA (Ascorbic acid) with the values of 60.78 ± 1.22 and 1267.5 ± 30.24 μM (P < 0.01). Conclusions: PODD, POD, and Polyneuridine are very critical for the antioxidant capacity in the extract of P. hexandrum rhizome. These results provide useful biochemical basis and information for the potential use of this plant. [ABSTRACT FROM AUTHOR]

Plants used in traditional medicine have stood up to the test of time and contributed many novel compounds for preventive and curative medicine to modern science. India is sitting on a gold mine of well recorded and traditionally well practiced knowledge of herbal medicine. Specially, plants growing at high altitude in Himalayan pastures are time-honored sources of health and general well being of local inhabitants. As of today, Himalayan plants are a major contributor to the herbal pharmaceutical industry both of India and other countries. Plants growing at higher altitudes are subjected to an assault of diverse testing situations including higher doses of mutagenic UV-radiation, physiological drought, desiccation and strong winds. Plants interact with stressful environments by physiological adaptation and altering the biochemical profile of plant tissues and producing a spectrum of secondary metabolites. Secondary metabolites are of special interest to scientists because of their unique pharmacophores and medicinal properties. Secondary metabolites like polyphenols, terpenes and alkaloids have been reported to possess antimutagenic and anticancer properties in many studies. The fundamental aspiration of the current review is to divulge the antimutagenic/anticancer potential of five alpine plants used as food or medicine by the populations living at high altitudes. [ABSTRACT FROM AUTHOR]

Objectives: The effect of Podophyllum hexandrum methanolic extract and α-tocopherol in reducing oxidative stress in male albino rats was evaluated. Materials and Methods: Lipid peroxidation was monitored by measuring malondialdehyde (MDA) level in different tissues of rats. Activities of free radical scavenging enzymes (superoxide dismutase, glutathione peroxidase and catalase) were determined using H2O2 decomposition. Results: Results showed that administration of H2O2 (0.1%) in drinking water of the rats, for 25 weeks, increased the malondialdehyde levels in liver, kidney and lung tissues of all the rats. However, rats receiving Podophyllum hexandrum extract and α-tocopherol had lower MDA levels in a dose dependent manner, which indicates decreased lipid peroxidation in these rats. Increase in the catalase activity appears to be a response to H2O2 accumulation. The decrease in the activity of catalase and increase in the activity of superoxide dismutase and glutathione peroxidase in different organs of the rats receiving Podophyllum hexandrum extract and α-tocopherol indicates the protective effect of the plant in combating oxidative stress undergone by the rats. Objectives: Rhizome of Podophyllum hexandrum have been ethnomedically claimed to possess a wide array of biological activities including anticancer activity. Materials and Methods: To verify the folklore claim, this study was performed in a six Human carcinoma cell lines, Lung (A-549), Prostate (PC-3), Colon (Colo-25), Breast (MCF- 7), Neuroblastoma (IMR-32) and CNS (SF-295) MCF-7 and MDA-MB-231. Results: Methanol and 70% ethanolic extracts of the rhizome of Podophyllum hexandrum showed highest cytotoxic effect on MCF-7 (Breast) and Colo-25 (Colon) cell line, as determined with sulforhodamine-B (SRB) assay. Conclusions: These findings 1- showed that Podophyllum hexandrum extract may ameliorate H2O2 induced oxidative stress by decreasing lipid peroxidation via alteration of the antioxidant defense system of the rats. 2- these data also showed the anticancer activity of the plant extracts on different human cancer cell lines. However, further investigation is needed to assess the molecular mechanisms mediated anticancer activities of this plant. [ABSTRACT FROM AUTHOR]

Podophyllum hexandrum is an important high altitude medicinal plant from Himalaya. Somatic chromosomes of this species were studied to delineate and physical mapping of repetitive rDNA sites to provide landmarks in chromosome identification. The karyotype formula of this species was found to be 6m + 2sm + 2st + 2t with secondary constriction in the chromosome 1 and 7. The FISH analysis of rDNA sites showed 4 sites for 18S rDNA and 2 sites for 5S rDNA. The chromosome number 1, 2, 5 and 6 can be identified based on 18S rDNA sites in their short arm and chromosome 1 and 2 can be identified by 5S rDNA site in the centromere region. The estimated genome size of this plant is 16.07 pg (1C). [ABSTRACT FROM AUTHOR]

Total synthesis of podophyllotoxin is an expensive process and availability of the compound from the natural resources is an important issue for pharmaceutical companies that manufacture anticancer drugs. In order to facilitate reasoned scientific decisions on its management and conservation for selective breeding programme, genetic analysis of 28 populations was done with 19 random primers, 11 ISSR primers and 13 AFLP primer pairs. A total of 92.37 %, 83.82 % and 84.40 % genetic polymorphism among the populations of Podophyllum were detected using RAPD, ISSR and AFLP makers, respectively. Similarly the mean coefficient of gene differentiation (Gst) were 0.69, 0.63 and 0.51, indicating that 33.77 %, 29.44 % and 26 % of the genetic diversity resided within the population. Analysis of molecular variance (AMOVA) indicated that 53 %, 62 % and 64 % of the genetic diversity among the studied populations was attributed to geographical location while 47 %, 38 % and 36 % was attributed to differences in their habitats using RAPD, ISSR and AFLP markers. An overall value of mean estimated number of gene flow (Nm) were 0.110, 0.147 and 0.24 from RAPD, ISSR and AFLP markers indicating that there was limited gene flow among the sampled populations. [ABSTRACT FROM AUTHOR]

Management of radiation-induced reactive oxygen/nitrogen species requires a holistic approach to mitigate the deleterious effects of free radicals. Flora of the Himalayas, which prevails under extreme climatic conditions, has been explored for its potential utility to develop radioprotective drugs. The Himalayan high altitude medicinal plant, Podophyllum hexandrum Royle, was selected on the basis of its unique properties, and a novel fractionated nonpolar extract (REC-2003) was prepared and evaluated for radioprotective efficacy, in vitro as well as in vivo. The free radical scavenging activity of REC-2003 was found to be > 75% (20 μg/ml) with maximum superoxide scavenging activity (57.56 ± 1.38%) recorded at 1 mg/ml concentration (tetrazolium-based estimation). More than 30% inhibition of nitric oxide radicals was observed at concentrations > 0.5 mg/ml, while hydroxyl radical scavenging activity (deoxy-D-ribose assay) exhibited a dose-dependent (100–600 μg/ml) increase. Significantly high (90%) protection to human erythrocytes was observed at 75 μg/ml, which was found to be the most optimized dose. Similarly, more than 90% inhibition was observed against lipid peroxidation (evaluated by estimating levels of malondialdehyde). The significant antihemolytic potential of REC-2003 could be attributed to its ability to scavenge free radicals, reduce peroxidative stress on lipid membranes, and render protection to DNA (evaluated using plasmid relaxation assay). All these activities holistically contributed toward the radioprotective ability. REC-2003 (8 mg/kg BW; intraperitoneal (i.p.), −30 min) rendered > 80% total-body protection in Swiss Albino Strain ‘A’ mice [against lethal radiation (10 Gy)] in a 30-day survival assay. Phytochemical characterization of the constituents of REC-2003 revealed the presence of polyphenolics (flavonoids). The characterized constituents also included the aryl-tetralin lignans like podophyllotoxin, its glycoside, 4′-demethyl derivative, and epi-podophyllotoxin. The optimized requisite single dose (8 mg/KgBW; i.p., −30 min) for obtaining significant radioprotection is reasonably low and establishes its future utility as a dietary supplement in the medical management of free radical-mediated diseases and specifically for rescue missions during nuclear and radiological emergencies (NREs). [ABSTRACT FROM AUTHOR]

Podophyllotoxin is the active ingredient in the rhizome of an endangered Indian medicinal herb, Podophyllum hexandrum. Podophyllotoxin content in the P. hexandrum differs greatly in different natural habitats. The podophyllotoxin content reached more than 6.62% when soil pH value was about 4.82, soil organic carbon (C) was more than 3.23%, and nitrogen (N) content was more than 2.7% of soil dry weight. Available phosphorous (P) content of more than 0.419% and potassium (K) content of more than 1.56% resulted in low podophyllotoxin content. The linear relationship detected between podophyllotoxin and soil nutrients, environmental factors, and altitude suggested that further optimization of these factors are important in the conservation and exploitation of P. hexandrum in the northwestern Himalayan region, Himachal Pradesh, India. In this regard, like artificial neural network (ANN) and multiple linear regression (MLR), the prediction model used in this study to map the effect of these factors on podophyllotoxin yield will be helpful. [ABSTRACT FROM AUTHOR]

The present study was carried out to evaluate the radioprotective efficacy of Podophyllum hexandrum fraction (REC-2006) in hepatoma cell lines having different p53 statuses. Higher radioresistance was observed in the HepG2 (p53++) cell line in comparison to the Hep3B (p53--) cell line, indicating a plausible role of p53 in radioresistance. REC-2006 exhibited nearly twice the survival in p53-expressing HepG2 cells compared with p53-negative Hep3B cells. REC-2006 treatment alone induced p53 expression as compared with untreated controls. However, REC-2006 reduced p53 expression when treated 2 hours before irradiation as compared with the irradiated HepG2 controls, indicating that REC-2006 modulates the expression of p53 to mitigate its apoptotic effect. Induction of p21 in the REC-2006 + radiation treatment group downregulated the expression of cyclin E and CDK2, leading to a delay in the G1 phase of HepG2 cells, which provided time for DNA repair or related processes. However, no significant difference in CDC2 expression in both cell lines suggested that G2 phase arrest might not be the only responsible factor for REC-2006-mediated radioprotection. Significant induction of PCNA and GADD45 expression in HepG2 cells suggested that REC-2006 increased the percentage survival of HepG2 cells by increasing the span of time as well as efficacy for repair processes. In conclusion, REC-2006 modulated the expression of p53 and thereby promoted cell cycle arrest in the G1 phase, encouraging cell proliferation and DNA repair and thus providing significantly higher protection against acute g-radiation in the HepG2 cell line. [ABSTRACT FROM AUTHOR]

Twenty eight populations of Podophyllum hexandrum Royle were selected to study genetic relationship using RAPD and ISSR markers from north-western Himalayas, Himachal Pradesh, India. Nineteen RAPD primers and 11 ISSR primers amplified a total of 131 and 68 scorable bands, of which 92.37% and 83.82%, respectively, were polymorphic. The mean coefficient of gene differentiation (Gst) was 0.6933 and 0.6296, indicating that Nei's gene diversity of 33.77% and 29.44% reside in all the populations. Estimated value of gene flow for RAPD (Nm = 0.11059), for ISSR (Nm = 0.1470) individually, and the combination of RAPD+ISSR (Nm = 0.1211) markers indicated that there was limited gene flow among the populations. The dendrogram obtained from UPGMA analysis revealed grouping of populations with respect to their forest division, except with Kullu forest division. The existence variation among 28 populations based on percentage of polymorphic bands (PPB) was proved to be coupled with geographical altitude (r = 0.474). The genetic similarity matrices generated by ISSR and RAPD markers were highly correlated (r = 0.721 at P = 0.001), showed similar estimation between the 2 systems. Both markers were equally useful in providing some understanding about the genetic relationship of different populations in Podophyllum L. [ABSTRACT FROM AUTHOR]