Comparative analysis of Papaver somniferum genotypes having contrasting latex and alkaloid profiles.

Papaver somniferum produces therapeutically useful benzylisoquinoline alkaloids (BIAs) like papaverine, thebaine, codeine, and morphine that accumulate in its capsular latex. Morphine is a potent analgesic but is also abused as a narcotic, which has increased the demand for non-narcotic thebaine that can be converted into various analgesics. To curtail the narcotic menace, many distinct genotypes of the plant have been developed that are deficient in morphine and/or latex. Sujata is one such latex-less low alkaloid-producing variety developed from the alkaloid-rich gum harvest variety Sampada. Its utility for gene prospecting and studying differential gene regulation responsible for its low alkaloid, nutritive seed oil, and latex-less phenotype has been exploited in this study. BIA profiling of Sujata and Sampada capsules at the early and late stages indicated that except for thebaine, Sujata had a depressed alkaloid phenotype as compared to Sampada. Comparative transcript-based analysis of the two genotypes was carried out in the early stage capsule (higher thebaine) using subtractive hybridization and microarray. Interrogation of a P. somniferum array yielded many differentially expressing transcripts. Their homology-based annotation classified them into categories--latex related, oil/lipid related, alkaloid related, cell wall related, and others. These leads will be useful to characterize the highly sought after Sujata phenotype.

Differentially expressed genes during contrasting growth stages of Artemisia annua for artemisinin content.

Artemisia annua is the source of antimalarial phytomolecule, artemisinin. It is mainly produced and stored in the glandular secretory trichomes present in the leaves of the plant. Since, the artemisinin biosynthesis steps are yet to be worked out, in this investigation a microarray chip was strategized for the first time to shortlist the differentially expressing genes at a stage of plant producing highest artemisinin compared to the stage with no artemisinin. As the target of this study was to analyze differential gene expression associated with contrasting artemisinin content in planta and a genotype having zero/negligible artemisinin content was unavailable, it was decided to compare different stages of the same genotype with contrasting artemisinin content (seedling--negligible artemisinin, mature leaf--high artemisinin). The SCAR-marked artemisinin-rich (~1.2%) Indian variety 'CIM-Arogya' was used in the present study to determine optimal plant stage and leaf ontogenic level for artemisinin content. A representative EST dataset from leaf trichome at the stage of maximal artemisinin biosynthesis was established. The high utility small scale custom microarray chip of A. annua containing all the significant artemisinin biosynthesis-related genes, the established EST dataset, gene sequences isolated in-house and strategically selected candidates from the A. annua Unigene database (NCBI) was employed to compare the gene expression profiles of two stages. The expression data was validated through semiquantitative and quantitative RT-PCR followed by putative annotations through bioinformatics-based approaches. Many candidates having probable role in artemisinin metabolism were identified and described with scope for further functional characterization.

A transcriptomic approach for exploring the molecular basis for dosha-balancing property-based classification of plants in Ayurveda.

In Ayurveda, a healthy body is defined by a balance among the three doshas (Vata, Pitta, Kapha) and ailments result due to imbalances among them. It prescribes specific plant parts/tissues collected in a season-specific manner for curing dosha-related imbalances but the plants prescribed for treating a particular dosha imbalance belong to taxonomically diverse families and often contain similar classes of phytomolecules, making it difficult to provide a phytochemical validation for any similarity that might exist among them. This exploratory study hypothesised that plants of the same dosha-curing group may have similarity at the transcript level. For proving/disproving the hypothesis, cDNA-AFLP and specific expression subset analysis (SESA) were carried out on the Ayurveda-defined active tissues of four representative plants each of the three dosha-balancing groups. cDNA-AFLP analyses indicated that even though the plants belonging to a particular dosha-group may widely differ at the transcript level, there is a small fraction of transcripts that is monomorphic among their active tissues. SESA (Tester-active tissue cDNA; Driver-cDNA from other major tissue[s]) generated 803 subtractive ESTs from the twelve plants that yielded 150 unigenes upon assembly (of ESTs from each plant separately). Cross-plant EST assembly for plants in the same dosha group also corroborated the results. Although a distinct pattern of transcripts was not observed across all the plants in a particular dosha group, some commonalities were obtained that need further characterization towards searching for the hitherto elusive similarity among plants of the same group.

cDNA-AFLP-based numerical comparison of leaf and root organ cDNAs in Catharanthus roseus.

Comparative transcriptome study of the leaf and root tissues of Catharanthus roseus is a prerequisite for causing any favorable tissue-specific change in the secondary metabolism of this species. This study was aimed at comparative analysis of the leaf and root cDNAs in C. roseus, using a cDNA-AFLP approach. Using 64 primer combinations (EcoRI and MseI), a total of 784 distinct transcriptionally-defined fragments (TDFs) could be detected in the root and leaf tissue transcript populations. The leaf tissue yielded a larger number of TDFs than the root tissue (556 versus 464), indicating a greater variety of expressing genes in the leaf. The leaf-specific TDFs (320) outnumbered the root-specific TDFs (228), indicating a higher number of leaf-specific functions and the relative complexity of the leaf tissue vis-à-vis the root tissue. Among the 236 TDFs that were detected in both types of tissues, 42 had nearly equal expression levels in both the tissues (L=R). Common TDFs having higher expression levels in the leaf (L>R; 124) outnumbered those having higher expression levels in the root (L

Antiplasmodial activity of steroidal chalcones: evaluation of their effect on hemozoin synthesis and the new permeation pathway of Plasmodium falciparum-infected erythrocyte membrane.

Chalcone derivatives on an estradiol framework were evaluated for their ability to inhibit the growth and development of the malaria parasite Plasmodium falciparum. Out of twelve steroidal chalcones and one indanone derivative studied, three were found to have 50% growth inhibitory concentration less than 5µm and minimum inhibitory concentration for parasite development from ring to schizont stage as ≤20µm with best activity for gallic acid-based chalcone derivative 1 as 2.07 and 10µm, respectively. Two of the active derivatives 1 and 10 did not exhibit cytotoxicity against vero cells as evident by the good selectivity ratio. Study of structure-activity relationship indicated that increasing substitution in the benzoyl ring-enhanced antiplasmodial activity. Hemozoin synthesis of the parasite remained unaffected by these derivatives. These derivatives were also investigated for their effect on parasite-induced new permeation pathway in the erythrocyte membrane by sorbitol-induced hemolysis, and four derivatives 1, 2, 9, and 10 exhibited significant inhibition (>70%) at 20µm concentration. A positive correlation was also observed among the antiplasmodial activity and inhibition of new permeation pathway. These observations suggest that steroidal chalcones with selective activity for the parasite may be considered as antimalarial leads for further optimization and preclinical study.

Molecular profiling for genetic variability in Capsicum species based on ISSR and RAPD markers.

The taxonomic identity of Capsicum species is found to be difficult as it displays variations at morpho-chemical characters. Twenty-two accessions of six Capsicum species, namely, C. annuum, C. baccatum, C. chinense, C. eximium, C. frutescens, and C. luteum were investigated for phenotypic diversity based on flower color and for genetic differences by molecular makers. The genetic cluster analyses of 27 RAPD and eight ISSR primers, respectively, revealed genetic similarities in the ranges of 23-88% and 11-96%. Principal component analysis of the pooled RAPD and ISSR data further supports the genetic similarity and groupings. Different species showed variations in relation to corolla shade of flower. C. annuum accessions formed a single cluster in the molecular analysis as maintaining their flower characteristic. C. chinense accession shared flower features with the accessions of C. frutescens and were found to be closer at genotypic level. C. luteum was found to be rather closer to C. baccatum complex, both phenotypically and genetically. The only accession of C. eximium presenting purple flowers falls apart from the groupings. The floral characteristics and the molecular markers are found to be useful toward the delineation of the species specificity in Capsicum collection and identification of genetic stock.

AFLP marking and polymorphism among progenies of Gymnema sylvestre: an important medicinal plant of India.

The level of polymorphism among twelve selected progenies of Gymnema sylvestre was investigated through AFLP markers by multiplexing PCR reactions using 64 (8x8) primer combinations. Fourteen primer combinations were selected as the most suitable combination for G. sylvestre. Analysis of the 12 progenies with these 14 primer pairs produced 1689 fragments of which 972 (57.5%) were polymorphic and 485 (28.7%) were unique to a particular genotype. The number of fragments produced by individual primer pairs was in the range of 55 to 225. Out of these, polymorphic fragments were in the range of 34 (E-ACC/M-CAC) to 157 (E-AGG/M-CAG) and unique bands observed were 8 (E-ACC / M-CAC) to 69 (E-AGG/M-CAC). Different primer combinations detected different levels of polymorphism, ranging from 33% (E-AGG/ M-CAC) to 69.8% (E-AGG/ M-CAC). From the observations, it appears that the primer combinations E-AGG/M-CAC, E-AGG/CTG, E-AGG/CAG and E-ACA/CAT were the most informative for the detection of polymorphism among the progenies compared with others, since they produced a high number of unique fragments. The similarity coefficient ranged from 0.212 to 0.731. High similarity was observed between progeny S8 and S9 (73%) and high divergence between progenies S3 and S11. Among the selected progeny, S9 was found to be the most similar to the parent (63%), while genotype S11 was the most distant (36.9%).

AFLP studies on downy-mildew-resistant and downy-mildew-susceptible genotypes of opium poppy.

Downy mildew (DM) caused by Peronospora arborescens, is a serious disease in opium poppy (Papaver somniferum), which has a world-wide spread. The establishment of DM-resistant cultivars appears to be a sustainable way to control the In this paper, we present the results of a study aimed at the identification of amplified fragment length polymorphism (AFLP) markers for DM-resistance in opium poppy. Three opium poppy genotypes (inbred over about 10 years): Pps-1 (DM-resistant), Jawahar-16 (DM-susceptible) and H-9 (DM-susceptible) were crossed in a diallel manner and the F(1) progeny along with the parents were subjected to AFLP analysis of chloroplast (cp) and nuclear DNA with seven and nine EcoRI / MseI primer combinations, respectively. cpDNA AFLP analysis identified 24 Pps-1 (DM-resistant)-specific unique fragments that were found to be maternally inherited in both the crosses, Pps-1 x Jawahar-16 and Pps-1 x H-9. In the case of nuclear DNA AFLP analysis, it was found that 17 fragments inherited from Pps-1 were common to the reciprocal crosses of both (i) Pps-1 and Jawahar-16 as well as (ii) Pps-1 and H-9. This is the first molecular investigation on the identification of polymorphism between DM-resistant and DM-susceptible opium poppy genotypes and development of DM-resistant opium poppy genotypespecific AFLP markers. These AFLP markers could be used in future genetic studies for analysis of linkage to the downy mildew resistance trait.

Enhancement of artemisinin content through four cycles of recurrent selection with relation to heritability, correlation and molecular marker in Artemisia annua L.

Due to the high demand and low yield of the anti-malarial drug artemisinin in natural populations of Artemisia annua (Quinghao), an attempt has been made to enhance the artemisinin content through 4 cycles of recurrent selection (C(0)-C(3)) using selected genotypic and phenotypic traits. Based on their phenotypic and genotypic characteristics, the top 5% plants of each cycle were selected, and their seedlings were planted in poly-cross block to produce seeds for the subsequent generation. A significant increase in the artemisinin content (0.15% in C (0) to 1.16% in C (3), i.e., about 40% genetic gain over the generation) was observed. This enhancement was directly correlated with the plant height and branching intensity in all four cycles. Similarly, the PCV (phenotypic coefficient of variation) and GCV (genotypic coefficient of variation) have been observed to have a higher value for artemisinin content. The DNA marker (MAP 12) with relation to artemisinin was also identified for high yielding genotypes in all four cycles of selection. Over the four cycles of recurrent selection, the plant developed an oval appearance (Variety: CIM-Arogya) and a high artemisinin content (1.16%).

Influence of cellular differentiation and elicitation on intermediate and late steps of terpenoid indole alkaloid biosynthesis in Catharanthus roseus.

The invaluable antineoplastic bisindole alkaloids of Catharanthus roseus and their precursor, vindoline, are not produced in cell cultures. The intricacies involved in endogenous (cellular differentiation) and exogenous (elicitation) regulation of their biosynthesis need to be dissected out for favorable exploitation. This study aimed at elucidating the effect of Pythium aphanidermatum homogenate and methyl jasmonate (MeJa) on in vitro cultures (of cv. 'Dhawal') representing increasing level of differentiation (suspension < callus < shoots) in terms of alkaloid accumulation and transcript abundance of strictosidine beta-D: -glucosidase (SGD) and acetyl-CoA: 4-O-deacetylvindoline 4-O-acetyl-transferase (DAT) genes, representing intermediate and late steps, respectively, of terpenoid indole alkaloid biosynthesis. Elicitation of suspension cultures caused transcriptional upregulation of SGD and enhanced the accumulation of total alkaloids but did not produce vindoline as DAT transcripts were always found to be absent in suspension-cultured cells. Vindoline was also not detected in unelicited and MeJa-treated callus but appeared upon elicitation with fungal homogenate for 24 h that coincided with maximal DAT transcription. Transcript levels of both genes increased upon elicitation of callus but remained below levels present in the mature plant leaf. Elicitation caused appearance of vindoline in shoots and increased the transcript abundance of both genes beyond levels observed in the mature plant leaf. Differentiation was essential for expression of DAT but not SGD, and vindoline biosynthetic potential increased with it.

Antitubercular potential of plants: a brief account of some important molecules.

Mycobacterium tuberculosis is the most lethal pathogen causing tuberculosis in human. After the discovery of antitubercular drugs pyrazinamide, rifampicin, isoniazid, streptomycin, and ethambutol (PRISE), the disease was controlled for a limited period. However, over the course of their usage, the pathogen acquired resistance and evolved into multi-drug resistant, single-drug resistant, and extensive drug resistant forms. A good number of plant secondary metabolites are reported to have antitubercular activity comparable to the existing antitubercular drugs or sometimes even better in potency. A well-defined strategy is required to exploit these phytomolecules as antitubercular drugs. This review gives concise up-to-date information regarding the chemistry and pharmacology of plant-based leads and some insight into their structure-activity relationship.

Antioxidant potential of the root of Vetiveria zizanioides (L.) Nash.

Vetiveria zizanioides, an aromatic plant commonly known as vetiver has been used for various ailments. The essential oil of vetiver root has been shown to possess antioxidant activity. However, antioxidant potential of spent root extract has not been reported. Hence, in the present study, ferric reducing, free radical scavenging and antioxidant activity of two genotypes namely KS1 and gulabi of V. zizanioides L. Nash root were investigated using in vitro assays - the ferric reducing antioxidant power (FRAP), 1,1-diphenyl-2-picrylhydrazyl (DPPH), total phenolic content (TPC), total antioxidant capacity (TAC) and reducing power (RP). KS1 genotype showed higher FRAP values, DPPH inhibition, TPC and RP potential compared to gulabi and the antioxidant activity increased with the concentration of the extract (10-1000 microg/mL). A significant protective effect of cv KS1 (100 microg/mL) extract was also observed in reduced glutathione (GSH) and malondialdehyde (MDA) concentrations of erythrocytes subjected to oxidative stress by tert-butyl hydroperoxide (t-BHP) and hydrogen peroxide (H202). The cv KS1 showed better antioxidant activity, compared to cv gulabi indicating the possibility of exploring the presence of different phytoconstituents in the two varieties.

Antifungal activity of Glycyrrhiza glabra extracts and its active constituent glabridin.

Glabridin, an active constituent of Glycyrrhiza glabra roots, was found to be active against both yeast and filamentous fungi. Glabridin also showed resistance modifying activity against drug resistant mutants of Candida albicans at a minimum inhibitory concentration of 31.25-250 microg/mL. Although the compound was reported earlier to be active against Candida albicans, but this is the first report of its activity against drug resistant mutants.

Genetic variation revealed in the chloroplast-encoded RNA polymerase beta' subunit of downy mildew-resistant genotype of opium poppy.

Two accessions of opium poppy, Pps-1 (dark green leaves, highly resistant to downy mildew [DM]) and H-9 (yellowish green leaves, susceptible to DM), which originated from common progenitor SPS49 were selected, and their F(1) and F(2) progenies showed that leaf color trait was governed by single recessive nuclear gene, whereas DM resistance appeared to be the interaction between cytoplasmic and nuclear genes. Chloroplast DNA (cpDNA) analysis of these 2 accessions through arbitrarily-primed polymerase chain reaction generated a unique fragment in Pps-1. Subsequent sequence analysis upon cloning of this cpDNA fragment revealed its similarity with the plastid-encoded RNA polymerase beta' subunit (rpoCI). Full-length rpoCI DNA was therefore isolated from both the genotypes that was 2707 bp long with a 658-bp intron (436-1093) and a 2049-bp open reading frame encoding 682 amino acid long polypeptide. Comparative sequence analysis of the rpoC1 gene from both the genotypes, revealed 4 single-nucleotide substitutions at 4 positions that caused 3 amino acid changes in the protein sequence--1) A to C transversion at position 825 (Glu275Asp), 2) A to G transition at position 1203 (Ile401Met), and 3) T to C transition at position 1422 and G to A transition at position 1423 both in same codon of the reading frame (Ala475Thr). This investigation is the first report indicating base substitution changes in the plastid-encoded rpoCI gene in DM-resistant genotypes of opium poppy. This finding may lead to implication of possible role of RNA polymerase beta' subunit in resistance to DM caused by Peronospora arborescens.

Simultaneous quantification of withanolides in Withania somnifera by a validated high-performance thin-layer chromatographic method.

This paper describes a sensitive, selective, specific, robust, and validated densitometric high-performance thin-layer chromatographic (HPTLC) method for the simultaneous determination of 3 key withanolides, namely, withaferin-A, 12-deoxywithastramonolide, and withanolide-A, in Ashwagandha (Withania somnifera) plant samples. The separation was performed on aluminum-backed silica gel 60F254 HPTLC plates using dichloromethane-methanol-acetone-diethyl ether (15 + 1 + 1 + 1, v/v/v/v) as the mobile phase. The withanolides were quantified by densitometry in the reflection/absorption mode at 230 nm. Precise and accurate quantification could be performed in the linear working concentration range of 66-330 ng/band with good correlation (r2 = 0.997, 0.999, and 0.996, respectively). The method was validated for recovery, precision, accuracy, robustness, limit of detection, limit of quantitation, and specificity according to International Conference on Harmonization guidelines. Specificity of quantification was confirmed using retention factor (Rf) values, UV-Vis spectral correlation, and electrospray ionization mass spectra of marker compounds in sample tracks.

Expression of tropane alkaloids in the hairy root culture of Atropa acuminata substantiated by DART mass spectrometric technique.

Agrobacterium rhizogenes-mediated 'hairy root' cultures were established in Atropa acuminata. The chemical profiling of the hairy roots was carried out by a new mass spectrometric technique, direct analysis in real time (DART). The intact hairy roots were directly analyzed by holding them in the gap between the DART ion source and mass spectrometer. Two alkaloids, atropine and scopolamine, were characterized. The structural confirmation of the two alkaloids was made through their accurate molecular formula determinations. This is the first report of establishing hairy roots in A. acuminata as well as application of the DART technique for the chemical profiling of its hairy roots.

Analysis of hairy root culture of Rauvolfia serpentina using direct analysis in real time mass spectrometric technique.

The applicability of a new mass spectrometric technique, DART (direct analysis in real time) has been studied in the analysis of the hairy root culture of Rauvolfia serpentina. The intact hairy roots were analyzed by holding them in the gap between the DART source and the mass spectrometer for measurements. Two nitrogen-containing compounds, vomilenine and reserpine, were characterized from the analysis of the hairy roots almost instantaneously. The confirmation of the structures of the identified compounds was made through their accurate molecular formula determinations. This is the first report of the application of DART technique for the characterization of compounds that are expressed in the hairy root cultures of Rauvolfia serpentina. Moreover, this also constitutes the first report of expression of reserpine in the hairy root culture of Rauvolfia serpentina.

Antimicrobial potential of Glycyrrhiza glabra roots.

The present study was aimed to investigate antimicrobial potential of Glycyrrhiza glabra roots. Antimycobacterial activity of Glycyrrhiza glabra was found at 500 microg/mL concentration. Bioactivity guided phytochemical analysis identified glabridin as potentially active against both Mycobacterium tuberculosis H(37)Ra and H(37)Rv strains at 29.16 microg/mL concentration. It exhibited antimicrobial activity against both Gram-positive and Gram-negative bacteria. Our results indicate potential use of licorice as antitubercular agent through systemic experiments and sophisticated anti-TB assay.

Analysis of cell cultures of Taxus wallichiana using direct analysis in real-time mass spectrometric technique.

The applicability of a new mass spectrometric technique, DART (direct analysis in real time) has been studied in the analysis of the calli of Taxus wallichiana. The intact callus samples were directly analyzed by holding them in the gap between the DART source and mass spectrometer for measurements. Five C-14 oxygenated taxoids were characterized from the analysis of the calli of the Taxus wallichiana almost instantaneously. The confirmation of the structures of the identified taxoids was made through their accurate molecular formula determinations.

Separation and quantification of lignans in Phyllanthus species by a simple chiral densitometric method.

A sensitive, selective, and robust high-performance TLC (HPTLC) method using chiral TLC plates for qualitative and quantitative analysis of phyllanthin (A), hypophyllanthin (B), niranthin (C), and nirtetralin (D), the active lignans of Phyllanthus species, was developed and validated. The effectiveness and role of various stationary phases viz TLC silica gel 60F(254), HPTLC silica gel 60F(254), and chiral TLC plates in the quantitation were evaluated. A precoated chiral TLC plate was found suitable for the simultaneous analysis of four pharmacologically active lignans. For achieving good separation, the optimized mobile phase of n-hexane/acetone/1,4-dioxane (9:1:0.5 by volume) was used (R(f) = 0.30, 0.36, 0.41, and 0.48 for compounds A, B, C, and D, respectively). A densitometric determination of the above compounds was carried out in reflection/absorption mode at 620 nm. Optimized chromatographic conditions provide well-separated compact bands for the tested lignans. The calibration curves were found linear in the concentration range of 100-500 ng/band. Recoveries of A-D were 99.98, 100.51, 99.22, and 98.74%, respectively. The method was validated according to ICH guidelines. The method reported here is reproducible and applied for the quantitative analysis of the above lignans in the leaves of four Phyllanthus species, i. e., P. amarus, P. maderaspatensis, P. urinaria, and P. virgatus.