Characterization of a class III peroxidase from Artemisia annua: relevance to artemisinin metabolism and beyond.

KEY MESSAGE: A class III peroxidase from Artemisia annua has been shown to indicate the possibility of cellular localization-based role diversity, which may have implications in artemisinin catabolism as well as lignification. Artemisia annua derives its importance from the antimalarial artemisinin. The -O-O- linkage in artemisinin makes peroxidases relevant to its metabolism. Earlier, we identified three peroxidase-coding genes from A. annua, whereby Aa547 showed higher expression in the low-artemisinin plant stage whereas Aa528 and Aa540 showed higher expression in the artemisinin-rich plant stage. Here we carried out tertiary structure homology modelling of the peroxidases for docking studies. Maximum binding affinity for artemisinin was shown by Aa547. Further, Aa547 showed greater binding affinity for post-artemisinin metabolite, deoxyartemisinin, as compared to pre-artemisinin metabolites (dihydroartemisinic hydroperoxide, artemisinic acid, dihydroartemisinic acid). It also showed significant binding affinity for the monolignol, coniferyl alcohol. Moreover, Aa547 expression was related inversely to artemisinin content and directly to total lignin content as indicated by its transient silencing and overexpression in A. annua. Artemisinin reduction assay also indicated inverse relationship between Aa547 expression and artemisinin content. Subcellular localization using GFP fusion suggested that Aa547 is peroxisomal. Nevertheless, dual localization (intracellular/extracellular) of Aa547 could not be ruled out due to its effect on both, artemisinin and lignin. Taken together, this indicates possibility of localization-based role diversity for Aa547, which may have implications in artemisinin catabolism as well as lignification in A. annua.

Diarylheptanoids Rich Fraction of Alnus nepalensis Attenuates Malaria Pathogenesis: In-vitro and In-vivo Study.

Diarylheptanoids from Alnus nepalensis leaves have been reported for promising activity against filariasis, a mosquito-borne disease, and this has prompted us to investigate its anti-malarial and safety profile using in-vitro and in-vivo bioassays. A. nepalensis leaf extracts were tested in-vitro against chloroquine-sensitive Plasmodium falciparum NF54 by measuring the parasite specific lactate dehydrogenase activity. Among all, the chloroform extract (ANC) has shown promising anti-plasmodial activity (IC50 8.06 ± 0.26 µg/mL). HPLC analysis of ANC showed the presence of diarylheptanoids. Efficacy and safety of ANC were further validated in in-vivo system using Plasmodium berghei-induced malaria model and acute oral toxicity in mice. Malaria was induced by intra-peritoneal injection of P. berghei infected red blood cells to the female Balb/c mice. ANC was administered orally at doses of 100 and 300 mg/kg/day following Peter's 4 day suppression test. Oral administration of ANC showed significant reduction of parasitaemia and increase in mean survival time. It also attributed to inhibition of the parasite induced pro-inflammatory cytokines as well as afford to significant increase in the blood glucose and haemoglobin level when compared with vehicle-treated infected mice. In-vivo safety evaluation study revealed that ANC is non-toxic at higher concentration. Copyright © 2016 John Wiley & Sons, Ltd.

Latex-less opium poppy: cause for less latex and reduced peduncle strength.

A genotype 'Sujata' developed earlier at CSIR-CIMAP from its parent 'Sampada' is considered to be the latex-less variety of Papaver somniferum. These two genotypes are contrasting in terms of latex and stem strength. Earlier we have carried out microarray analysis to identify differentially expressing genes from the capsules of the two genotypes. In this study, the peduncles of the two genotypes were compared for the anatomy revealing less number of laticifers in the cortex and vascular bundles. One of the important cell wall-related genes (for laccase) from the microarray analysis showing significantly higher expression in 'Sampada' capsule was taken up for further characterization in the peduncle here. It was functionally characterized through transient overexpression and RNAi suppression in 'Sujata' and 'Sampada'. The increase in acid insoluble lignin and total lignin in overexpressed tissue of 'Sujata', and comparable decrease in suppressed tissue of 'Sampada', along with corresponding increase and decrease in the transcript abundance of laccase confirm the involvement of laccase in lignin biosynthesis. Negligible transcript in phloem compared to the xylem tissue localized its expression in xylem tissue. This demonstrates the involvement of P. somniferum laccase in lignin biosynthesis of xylem, providing strength to the peduncle/stem and preventing lodging.

Lipid Nanocapsule: A Novel Approach to Drug Delivery System Formulation Development.

Nanocapsules are polymeric nanoparticles encased in a polymeric coating composed of a predominantly non-ionic surfactant, macromolecules, phospholipids, and an oil core. Lipophilic drugs have been entrapped using various nanocarriers, including lipid cores, likely lipid nanocapsules, solid lipid nanoparticles, and others. A phase inversion temperature approach is used to create lipid nanocapsules. The PEG (polyethyleneglycol) is primarily utilised to produce nanocapsules and is a critical parameter influencing capsule residence time. With their broad drug-loading features, lipid nanocapsules have a distinct advantage in drug delivery systems, such as the capacity to encapsulate hydrophilic or lipophilic pharmaceuticals. Lipid nanocapsules, as detailed in this review, are surface modified, contain target-specific patterns, and have stable physical and chemical properties. Furthermore, lipid nanocapsules have target-specific delivery and are commonly employed as a marker in the diagnosis of numerous illnesses. This review focuses on nanocapsule synthesis, characterisation, and application, which will help understand the unique features of nanocapsules and their application in drug delivery systems.

4-coumarate: CoA ligase partitions metabolites for eugenol biosynthesis.

Biosynthesis of eugenol shares its initial steps with that of lignin, involving conversion of hydroxycinnamic acids to their corresponding coenzyme A (CoA) esters by 4-coumarate:CoA ligases (4CLs). In this investigation, a 4CL (OS4CL) was identified from glandular trichome-rich tissue of Ocimum sanctum with high sequence similarity to an isoform (OB4CL_ctg4) from Ocimum basilicum. The levels of OS4CL and OB4CL_ctg4-like transcripts were highest in O. sanctum trichome, followed by leaf, stem and root. The eugenol content in leaf essential oil was positively correlated with the expression of OS4CL in the leaf at different developmental stages. Recombinant OS4CL showed the highest activity with p-coumaric acid, followed by ferulic, caffeic and trans-cinnamic acids. Transient RNA interference (RNAi) suppression of OS4CL in O. sanctum leaves caused a reduction in leaf eugenol content and trichome transcript level, with a considerable increase in endogenous p-coumaric, ferulic, trans-cinnamic and caffeic acids. A significant reduction in the expression levels was observed for OB4CL_ctg4-related transcripts in suppressed trichome compared with transcripts similar to the other four isoforms (OB4CL_ctg1, 2, 3 and 5). Sinapic acid and lignin content were also unaffected in RNAi suppressed leaf samples. Transient expression of OS4CL-green fluorescent protein fusion protein in Arabidopsis protoplasts was associated with the cytosol. These results indicate metabolite channeling of intermediates towards eugenol by a specific 4CL and is the first report demonstrating the involvement of 4CL in creation of virtual compartments through substrate utilization and committing metabolites for eugenol biosynthesis at an early stage of the pathway.

Simultaneous quantification of diterpenoids in Premna integrifolia using a validated HPTLC method.

A sensitive, selective and robust densitometric high-performance thin layer chromatographic method was developed and validated for the determination of diterpenoids in the root bark of Premna integrifolia. Diterpenoids 1ß,3α,8ß-trihydroxy-pimara-15-ene (A), 6α,11,12,16-tetrahydroxy-7-oxo-abieta-8,11,13-triene (B) and 2α,19-dihydroxy-pimara-7,15-diene (C) were used as chemical markers for the standardization of P. integrifolia plant extracts. The separation was performed on silica gel 60F(254) high-performance thin layer chromatography plates using hexane/acetone/ethylacetate (60:20:20 v/v) as mobile phase. The quantitation of diterpenoids was carried out using densitometric reflection/absorption mode at 475 nm after post-chromatographic derivatization using vanillin-sulfuric acid reagent. A precise and accurate quantification can be performed for compounds A, B and C in the linear working concentration range of 1-10 µg/spot with good correlations (r(2) =0.9985, 0.9996 and 0.9992, respectively). The method was validated for peak purity, precision, robustness, limit of detection (LOD) and quantitation (LOQ) etc., as per the International Conference on Harmonization (ICH) guidelines. Specificity of quantitation was confirmed using retention factor (R(f)) and spectra correlation of markers in standard and sample tracks. The method reported here is simple and reproducible which may be applied for quantitative analysis of above diterpenoids in the root bark of P. integrifolia.

Promises of Lipid-based Drug Delivery Systems in the Management of Breast Cancer.

Breast cancer is one of the leading types among the common non-cutaneous malignancies in women. All the curative methods available for its treatment are minimal due to their toxicity issues and dose-related side effects. Various evolving nanotechnology techniques displayed the opportunity to target breast cancer. One such delivery system is lipid-based drug delivery systems (LDDS). This concept is constrained only for the laboratory scale should be shifted to the industrial level targeting the nanomedicine with clinical benefits. This work tried to portray the advancements in the LDDS along with the lipid-based excipients, advantages, disadvantages and applications. It even helped in highlighting the recently developed lipid-based nanocarriers for breast cancer management.

Complex basilar artery fenestration aneurysm successfully treated with single flow diverter using novel "crossing flow diverter technique" - A rare case report and review of literature.

Basilar artery fenestration aneurysms are very rare and endovascular management of large and complex aneurysms is extremely challenging. Most of these type of cases are managed with stent assisted coiling, dual flow diverters (FD) and single FD with additional coiling of aneurysm and occlusion of one of the vertebral artery. Here, we report a case of large complex basilar artery fenestration aneurysm successfully treated with single FD using novel technique called "crossing flow diverter technique" without any additional coiling of aneurysm or occlusion of vertebral artery. Using this technique cost of procedure and procedural complexity inherent with other above mentioned techniques can be significantly reduced.

Pharmacophore modeling, docking and the integrated use of a ligand- and structure-based virtual screening approach for novel DNA gyrase inhibitors: synthetic and biological evaluation studies.

Fluoroquinolones, a class of compound, act via inhibiting DNA gyrase and topoisomerase IV enzymes. This is an important class of drugs with high success rates for the treatment of tuberculosis and other bacterial infections. An indirect drug design approach was used to develop a meaningful pharmacophore model using the HypoGen module of Discovery Studio 2.0 on a set of 27 structurally diverse compounds with a wide range of biological activity (5 log units). The best hypothesis had three hydrogen bond acceptors (HBA) and one hydrophobic (Hy) moiety, showing r = 0.95, and it predicts the test set of 44 compounds well, with r 2 = 0.823. The same features (acceptor and hydrophobic functionality) were validated at the binding site of the DNA gyrase active site using GOLD version 3.0.1 and Molegro Virtual Docker, which showed corresponding hydrogen bond interactions and also π-π stacking interactions that correlated well with the PIC50 values (r 2 = 0.6142). The thoroughly validated model was used to screen an extensive database of 0.25 million compounds to identify potential leads. The validated model was implemented for the identification, design, synthesis, and biological evaluation of leads. Ten new chemical entities were synthesized based on our scaffold hopping techniques from the identified virtual screening and tested against the tuberculosis bacterium to obtain preliminary MIC values. The results showed that 3 out of 10 synthesized compounds exhibited good MICs, from 1.25 to 50 µM. This proves the robustness and applicability of the developed model, which is a promising tool for identifying new topoisomerase II inhibitors for the treatment of tuberculosis.

Quantitation of dietary dihydrochalcones in Indian crabapple (Malus sikkimensis) using validated high-performance liquid chromatography.

In the present study, a systematic validated method was developed for the determination of two key dietary dihydrochalcones (DHC) viz. phloridzin (PZ) and phloretin (PT) in the leaves of Sikkim crabapple (Malus sikkimensis) using HPLC-Photo Diode Array (PDA). Chromatographic separation was optimized on a C18 column using a gradient elution of water/acetonitrile with the flow rate of 1.0 mL/min at 25°C at 280 nm. Sample preparation approach is rapid and energy efficient, and it requires no pre-concentration before analysis. Validation showed a good analytical performance in terms of specificity, linearity (r2 > 0.999), precision (% RSD < 1.08), recovery (97-100.4%) and sensitivities (limits of detection: 12.48 and 14.95 ng/mL; limit of quantification: 41.61 and 49.85 ng/mL) of PZ and PT, respectively. Developed approach was employed for targeted phytochemical analysis in the bark and fruits of M. sikkimensis. The PZ content in the bark and leaves was highest (12-13 mg/100 mg), about 90-fold higher than fruits. PT was only present in the leaves (0.57 mg/100 mg). The comparative data on PZ and PT content in various wild apple species/cultivar from different countries have also been discussed. The reliability of the validated method was established by analyzing global and expanded uncertainties in two DHC determinations in wild apple. The present method fulfills the technical requirement of ISO 17025:2017 for quality control of M. sikkimensis.

Iridoid glycosides from Gmelina arborea.

Three iridoid glycosides 6-O-(3''-O-benzoyl)-alpha-L-rhamnopyranosylcatalpol (1a), 6-O-(3''-O-trans-cinnamoyl)-alpha-L-rhamnopyranosylcatalpol (2a) and 6-O-(3''-O-cis-cinnamoyl)-alpha-L-rhamnopyranosylcatalpol (3a) were isolated from aerial parts of Gmelina arborea and structures were elucidated by spectral analysis. Additionally a known iridoid 6-O-(3'', 4''-O-dibenzoyl)-alpha-L-rhamnopyranosylcatalpol (4) was also isolated and identified.

Iridoid glycoside-based quantitative chromatographic fingerprint analysis: a rational approach for quality assessment of Indian medicinal plant Gambhari (Gmelina arborea).

A sensitive, selective and robust qualitative and quantitative densitometric high-performance thin layer chromatographic method was developed and validated for the determination of iridoid glycoside in the aerial part of Gambhari (Gmelina arborea). Iridoid gycoside 6-O-(2'',3''-dibenzoyl)-alpha-l-rhamnopyranosylcatalpol (IG) was used as a chemical marker for the standardization of G. arborea plant extracts. The separation was performed on aluminum Kieselgel 60F254 TLC plates using chloroform-methanol as mobile phase. The quantitation of IG was carried out using the densitometric reflection/absorption mode at 240 and 430 nm after post-chromatographic derivatization with vanillin-sulphuric acid reagent. A precise and accurate quantification can be performed in the linear working concentration range of 1000-5000 ng/spot with good correlation (r2=0.994). The method was validated for peak purities, precision, robustness, limit of detection (LOD) and quantitation (LOQ), etc., as per ICH guidelines. Specificity of quantitation was confirmed using retention factor (R(f)), UV-vis spectral correlation and ESI-MS spectra of marker compound (IG) in sample track.

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.

Genetic variability, associations, and path analysis of chemical and morphological traits in Indian ginseng [Withania somnifera (L.) Dunal] for selection of higher yielding genotypes.

BACKGROUND: The study was carried out to assess the genetic variability present in ashwagandha and to examine the nature of associations of various traits to the root yield of the plant. METHODS: Fifty-three diverse genetic stocks of ashwagandha (Withania somnifera) were evaluated for 14 quantitative characteristics. Analysis of variance, correlation, and path coefficient analysis were performed using the mean data of 2 years. RESULTS: Analysis of variance revealed that the genotypes differed significantly for all characteristics studied. High heritability in conjunction with high genetic advance was observed for fresh root weight, 12 deoxywithastramonolide in roots, and plant height, which indicated that selection could be effective for these traits. Dry root weight has a tight linkage with plant height and fresh root weight. Further, in path coefficient analysis, fresh root weight, total alkaloid (%) in leaves, and 12 deoxywithastramonolide (%) in roots had the highest positive direct effect on dry root weight. CONCLUSION: Therefore, these characteristics can be exploited to improve dry root weight in ashwagandha genotypes and there is also scope for the selection of promising and specific chemotypes (based on the alkaloid content) from the present germplasm.

AromaDb: A Database of Medicinal and Aromatic Plant's Aroma Molecules With Phytochemistry and Therapeutic Potentials.

In traditional, herbal medicine, and aromatherapy, use of essential oils and their aroma compounds have been known since long, for the management of various human diseases. The essential oil is a mixture of highly complex, naturally occurring volatile aroma compounds synthesized by medicinal and aromatic plants as secondary metabolites. Essential oils widely used in pharmaceutical, cosmetic, sanitary, food industry and agriculture for their antibacterial, antiviral, antifungal, antiparasitic, insecticidal, anticancer, neuroprotective, psychophysiological, and anti-aging activities. Moreover, volatile aroma compounds comprise a chemically diverse class of low molecular weight organic compounds with significant vapor pressure. However, aroma compounds produced by plants, mainly attract pollinators, seed dispersers and provide defense against pests or pathogens. However, in humans, about 300 active olfactory receptor genes are involved to detect thousands of different aroma compounds and modulates expression of different metabolic genes regulating human psychophysiological activity, brain function, pharmacological signaling, and therapeutic potential. Keeping in mind this importance, present database, namely, AromaDb (http://bioinfo.cimap.res.in/aromadb/) covers information of plant varieties/chemotypes, essential oils, chemical constituents, GC-MS profile, yield variations due to agro-morphological parameters, trade data, aroma compounds, fragrance type, and bioactivity details. The database includes 1,321 aroma chemical structures, bioactivities of essential oil/aroma compounds, 357 fragrance type, 166 commercially used plants, and their high yielding 148 varieties/chemotypes. Also includes calculated cheminformatics properties related to identification, physico-chemical properties, pharmacokinetics, toxicological, and ecological information. Also comprises interacted human genes affecting various diseases related cell signaling pathways correlating the use of aromatherapy. This database could be a useful resource to the plant's growers/producers, an aroma/fragrance industrialist, health professionals, and researchers exploring the potential of essential oils and aroma compounds in the development of novel formulations against human diseases.

A recurrent neural network for hierarchical control of interconnected dynamic systems.

A recurrent neural network for the optimal control of a group of interconnected dynamic systems is presented in this paper. On the basis of decomposition and coordination strategy for interconnected dynamic systems, the proposed neural network has a two-level hierarchical structure: several local optimization subnetworks at the lower level and one coordination subnetwork at the upper level. A goal-coordination method is used to coordinate the interactions between the subsystems. By nesting the dynamic equations of the subsystems into their corresponding local optimization subnetworks, the number of dimensions of the neural network can be reduced significantly. Furthermore, the subnetworks at both the lower and upper levels can work concurrently. Therefore, the computation efficiency, in comparison with the consecutive executions of numerical algorithms on digital computers, is increased dramatically. The proposed method is extended to the case where the control inputs of the subsystems are bounded. The stability analysis shows that the proposed neural network is asymptotically stable. Finally, an example is presented which demonstrates the satisfactory performance of the neural network.

Inducing mutations through γ-irradiation in seeds of Mucuna pruriens for developing high L-DOPA-yielding genotypes.

PURPOSE: To develop elite genotypes in Mucuna pruriens (L.) DC with high L-DOPA (L-3, 4 dihydroxyphenylalanine) yields, with non-itching characteristics and better adaptability by applying γ-irradiation. Molecular and chemical analysis was performed for screening based on specific characteristics desired for developing suitable genotypes. MATERIALS AND METHODS: Developed, mutant populations were analyzed for L-DOPA % in seeds through TLC (thin layer chromatography), and the results obtained were validated with the HPLC (High performance liquid chromatography). The DNA (Deoxyribonucleic acid) was isolated from the leaf at the initial stage and used for DNA polymorphism. RNA (Ribonucleic acid) was isolated from the leaf during maturity and used for expression analysis. RESULTS: The selected mutant T-I-7 showed 5.7% L-DOPA content compared to 3.18% of parent CIM-Ajar. The total polymorphism obtained was 57% with the molecular marker analysis. The gene expression analysis showed higher fold change expression of the dopadecarboxylase gene (DDC) in control compared to selected mutants (T-I-7, T-II-23, T-IV-9, T-VI-1). CONCLUSION: DNA polymorphism was used for the screening of mutants for efficient screening at an early stage. TLC was found suitable for the large-scale comparative chemical analysis of L-DOPA. The expression profile of DDC clearly demonstrated the higher yields of L-DOPA in selected mutants developed by γ-irradiation in the seeds of the control.

Heteroyohimbinoid type oxindole alkaloids from Mitragyna parvifolia.

The leaves of the plant Mitragyna parvifolia have afforded two alkaloids, 16,17-dihydro-17beta-hydroxy isomitraphylline (1) and 16, 17-dihydro-17beta-hydroxy mitraphylline (2), together with two known alkaloids, isomitraphylline (3) and mitraphylline (4). The structures of 1 and 2 were elucidated using 1D and 2D NMR spectral methods, including (1)H-(1)HCOSY, DEPT, HSQC, and HMBC. Mitraphylline was the main alkaloid constituent.

Liquid chromatographic determination of sennosides in Cassia angustifolia leaves.

A simple liquid chromatographic method was developed for the determination of sennosides B and A in leaves of Cassia angustifolia. These compounds were extracted from leaves with a mixture of methanol-water (70 + 30, v/v) after defatting with hexane. Analyte separation and quantitation were achieved by gradient reversed-phase liquid chromatography and UV absorbance at 270 nm using a photodiode array detector. The method involves the use of an RP-18 Lichrocart reversed-phase column (5 microm, 125 x 4.0 mm id) and a binary gradient mobile-phase profile. The various other aspects of analysis, namely, peak purity, similarity, recovery, repeatability, and robustness, were validated. Average recoveries of 98.5 and 98.6%, with a coefficient of variation of 0.8 and 0.3%, were obtained by spiking sample solution with 3 different concentration solutions of standards (60, 100, and 200 microg/mL). Detection limits were 10 microg/mL for sennoside B and 35 microg/mL for sennoside A, present in the sample solution. The quantitation limits were 28 and 100 microg/mL. The analytical method was applied to a large number of senna leaf samples. The new method provides a reliable tool for rapid screening of C. angustifolia samples in large numbers, which is needed in breeding/genetic engineering and genetic mapping experiments.

Kinematic analysis of a flexible six-DOF parallel mechanism.

In this paper, a new type of six-degrees of freedom (DOF) flexible parallel mechanism (FPM) is presented. This type of parallel mechanism possesses several favorable properties: (1) its number of DOFs is independent of the number of serial chains which make up the mechanism; (2) it has no kinematical singularities; (3) it is designed to move on rails, and therefore its workspace is much larger than that of a conventional parallel manipulator; and (4) without changing the number of DOFs and the kinematics of the mechanisms, the number of the serial chains can be reconfigured according to the needs of the tasks. These properties make the mechanism very preferable in practice, especially for such tasks as joining huge ship blocks, in which the manipulated objects vary dramatically both in weights and dimensions. Furthermore, the mechanism can be used as either a fully actuated system or an underactuated system. In the fully actuated case, the mechanism has six DOF motion capabilities and manipulation capabilities. However, in the underactuated case, the mechanism still has six DOF motion capabilities, but it has only five DOF manipulation capabilities. In this paper, both the inverse and forward kinematics are studied and expressed in a closed form. The workspace and singularity analysis of the mechanism are also presented. An example is presented to illustrate how to calculate the kinematics of the mechanism in both fully-actuated and underactuated cases. Finally, an application of such a mechanism to manufacturing industry is introduced.