Unveiling Dissolution Kinetics of CuO Nanofertilizer Using Bio-Based Ionic Liquids Envisaging Controlled Use Efficiency for Sustainable Agriculture.

The need for sustainable agriculture amid a growing population and challenging climatic conditions is hindered by the environmental repercussions of widespread fertilizer use, resulting in the accumulation of metal ions and the loss of micronutrients. The present study provides an approach to improve the efficiency of nanofertilizers by controlling the release of copper (Cu) ions from copper oxide (CuO) nanofertilizers through bioionic liquids based on plant growth regulators (PGR-ILs). A 7-day study was conducted to understand the kinetics of Cu ion release in aqueous solution of five different PGR-ILs, with choline ascorbate ([Cho][Asc]) or choline salicylate ([Cho][Sal]) leading to 200- to 700-fold higher dissolution of Cu ions in comparison to choline indole-3-acetate ([Cho][IAA]), choline indole-3-butyrate ([Cho][IBA]), and choline gibberellate ([Cho][GA3]). The tunable diffusion of Cu ions from CuO nanofertilizers using PGR-ILs is then applied in a foliar spray study, evaluating its impact on the growth phenotype, photosynthetic parameters, and carbon dioxide (CO2) sequestration in Nicotiana tabacum in a greenhouse. The results indicate that nanoformulations with lower concentrations of Cu ions in PGR-IL solutions exhibit superior outcomes in terms of plant length, net photosynthetic rate, dry biomass yield, and CO2 sequestration, emphasizing the critical role of dissolution kinetics in determining the effectiveness of PGR-IL-based nanoformulations for sustainable agriculture.

Do nanoparticles delivered to roots affect plant secondary metabolism? A comprehensive analysis in float seedling cultures of Hypericum perforatum L.

Since nanoparticles (NPs) released into the environment from household or industrial wastes and applied directly on plants as agrochemicals can accumulate in the rhizosphere, it is imperative to understand how these NPs affect plant secondary metabolism upon their contact with the roots of intact plants. Here, the effects of Pd, Au, ZnO and Fe2O3 NPs on secondary metabolism were comprehensively investigated in Hypericum perforatum L float seedlings by analyzing 41 major secondary metabolites using ultra-performance liquid chromatography coupled with photodiode array, fluorescence detector and high-resolution mass spectrometry (UPLC-PDA-FLR-HRMS). The results showed that exposure of H. perforatum roots to Pd, Au, ZnO and Fe2O3 NPs rapidly led to fluctuations in the levels of secondary metabolites. Although these fluctuations did not correlate with NP type, concentration and duration of treatment, a total of 22 compounds were significantly altered by the NPs tested. In particular, 1 ppm Au increased the content of quercetin 3-(2″-acetylgalactoside), cadensin G and leutoskyrin by 5.02-, 2.12- and 2.58-fold, respectively after 24 h; 25 ppm Pd NPs led to a 2.1-fold increase in miquelianin content after 6 h; 50 ppm Fe2O3 NPs increased the level of furohyperforin by 3.09-fold and decreased the content of miquelianin 5.22-fold after 24 h and 50 ppm ZnO led to a 2.13-fold increase in hypericin after 48 h. These results emphasise the need to understand the intricate interplay between NPs and plant secondary metabolism in order to enable safer and efficient applications of NPs in agriculture.

Single molecule real-time sequencing data sets of Hypericum perforatum L. plantlets and cell suspension cultures.

Hypericum is a large genus that includes more than 500 species of pharmacological, ecological and conservation value. Although latest advances in sequencing technologies were extremely exploited for generating and assembling genomes of many living organisms, annotated whole genome sequence data is not publicly available for any of the Hypericum species so far. Bioavailability of secondary metabolites varies for different tissues and the data derived from different cultures will be a valuable tool for comparative studies. Here, we report the single molecule real-time sequencing (SMRT) data sets of Hypericum perforatum L. plantlets and cell suspension cultures for the first time. Sequencing data from cell suspension cultures yielded more than 33,000 high-quality transcripts from 20 Gb of raw data, while more than 55,000 high-quality transcripts were obtained from 35 Gb of raw data from plantlets. This dataset is a valuable tool for comparative transcriptomic analysis and will help to understand the unknown biosynthetic pathways of high medicinal value in the Hypericum genus.

Suitability of Adenosine Derivatives in Improving the Activity and Stability of Cytochrome c under Stress: Insights into the Effect of Phosphate Groups.

It is well known that adenosine and its phosphate derivatives play a crucial role in biological phenomena such as apoptosis and cell signaling and act as the energy currency of the cell. Although their interactions with various proteins and enzymes have been described, the focus of this work is to demonstrate the effect of the phosphate group on the activity and stability of the native heme metalloprotein cytochrome c (Cyt c), which is important from both biological and industrial aspects. In situ and in silico characterizations are used to correlate the relationship between the binding affinity of adenosine and its phosphate groups with unfolding behavior, corresponding peroxidase activities, and stability factors. Interaction of adenosine (ADN), adenosine monophosphate (AMP), adenosine 5'-diphosphate (ADP), and adenosine 5'-triphosphate (ATP) with Cyt c increases peroxidase-like activity by up to 1.8-6.5-fold compared to native Cyt c. This activity is significantly maintained even after multiple stress conditions such as oxidative stress and the presence of a chaotropic agent such as guanidine hydrochloride (GuHCl). With binding affinities on the order of ADN < AMP < ADP < ATP, adenosine derivatives were found to stabilize Cyt c by varying the secondary structural features of the protein. Thus, in addition to being a fundamental study, the current work also proposes a way of stabilizing protein systems to be used for real-time biocatalytic applications.

Designing protein nano-construct in ionic liquid: a boost in efficacy of cytochrome C under stresses.

Herein, we present a simple approach to fabricate protein nanoconstructs by complexing cytochrome C (Cyt C) with silk nanofibrils (SNF) and choline dihydrogen phosphate ionic liquid (IL). The peroxidase activity of the IL modified Cyt C nanoconstruct (Cyt C + SNF + IL) increased significantly (2.5 to 10-fold) over unmodified Cyt C and showed enhanced catalytic activity and stability under harsh conditions, proving its potential as a suitable protein packaging strategy.

Presenting B-DNA as macromolecular crowding agent to improve efficacy of cytochrome c under various stresses.

Existence of numerous biomolecules results in biological fluids to be extremely crowded. Thus, Macromolecular crowding is an essential phenomenon to sustain active conformation of proteins in biological systems. Herein, double helical deoxyribonucleic acid (B-DNA) is presented for the first time as a biomacromolecular crowding system for sustainable packaging of cytochrome c (Cyt C). The peroxidase activity of Cyt C was investigated in the presence of various concentrations of B-DNA (from salmon milt). At an optimized concentration of 0.125 mg/mL B-DNA, an 11-fold higher catalytic activity was found than in native Cyt C with improved stability. Molecular docking and spectroscopic analyses revealed that electrostatic and H-bonding are the main interactions between DNA and Cyt C that affect the structural stability and activity of the protein. Moreover, the catalytic activity and stability of the protein were further investigated in the presence of severe process conditions by UV-visible, circular dichroism, and Fourier-transform infrared spectroscopies. Molecularly crowded Cyt C showed significantly higher activity and stability under severe environments such as high temperature (110 °C), oxidative stress, high pH (pH 10) and biological (trypsin) and chemical denaturants (urea) compared to bare Cyt C. The observed results support the suitability of DNA-based macromolecular crowding media as a viable and effective stabilizer of proteins against multiple stresses.

Prospective Application of Nanoparticles Green Synthesized Using Medicinal Plant Extracts as Novel Nanomedicines.

The use of medicinal plants in green synthesis of metal nanoparticles is increasing day by day. A simple search for the keywords "green synthesis" and "nanoparticles" yields more than 33,000 articles in Scopus. As of August 10, 2021, more than 4000 articles have been published in 2021 alone. Besides demonstrating the ease and environmental-friendly route of synthesizing nanomaterials, many studies report the superior pharmacological properties of green synthesized nanoparticles compared to those synthesized by other methods. This is probably due to the fact that bioactive molecules are entrapped on the surface of these nanoparticles. On the other hand, recent studies have confirmed the nano-dimension and biocompatibility of metal ash (Bhasma) preparations, which are commonly macerated with biological products and administered for the treatment of various diseases in Indian medicine since ancient times. This perspective article argues for the prospective medical application of green nanoparticles in the light of Bhasma.

Transcriptional responses of Hypericum perforatum cells to Agrobacterium tumefaciens and differential gene expression in dark glands.

Hypericum perforatum L. (St. John's wort) is a well-known medicinal plant that possesses secondary metabolites with beneficial pharmacological properties. However, improvement in the production of secondary metabolites via genetic manipulation is a challenging task as H. perforatum remains recalcitrant to Agrobacterium tumefaciens-mediated transformation. Here, the transcripts of key genes involved in several plant defence responses (secondary metabolites, RNA silencing, reactive oxygen species (ROS) and specific defence genes) were investigated in H. perforatum suspension cells inoculated with A. tumefaciens by quantitative real-time PCR. Results indicated that key genes from the xanthone, hypericin and melatonin biosynthesis pathways, the ROS-detoxification enzyme HpAOX, as well as the defence genes Hyp-1 and HpPGIP, were all upregulated to rapidly respond to A. tumefaciens elicitation in H. perforatum. By contrast, expression levels of genes involved in hyperforin and flavonoid biosynthesis pathways were markedly downregulated upon A. tumefaciens elicitation. In addition, we compared the expression patterns of key genes in H. perforatum leaf tissues with and without dark glands, a major site of secondary metabolite production. Overall, we provide evidence for the upregulation of several phenylpropanoid pathway genes in response to elicitation by Agrobacterium, suggesting that production of secondary metabolites could modulate H. perforatum recalcitrance to A. tumefaciens-mediated transformation.

Overexpression of polygalacturonase-inhibiting protein (PGIP) gene from Hypericum perforatum alters expression of multiple defense-related genes and modulates recalcitrance to Agrobacterium tumefaciens in tobacco.

Hypericum perforatum L is a remarkable source of high-value secondary metabolites with increasing applications in pharmaceutical industry. However, improvement in the production of secondary metabolites through genetic engineering is a demanding task, as H. perforatum is not amenable to Agrobacterium tumefaciens-mediated transformation. In this study, we identified a Polygalacturonase-inhibiting protein (PGIP) gene from a subtractive cDNA library of A. tumefaciens-treated H. perforatum suspension cells. The role of HpPGIP in defense against A. tumefaciens was analyzed in transgenic Nicotiana tabacum overexpressing HpPGIP alone or fused at the N-terminus to Phenolic oxidative coupling protein (Hyp-1), a gene that positively modulates resistance to A. tumefaciens. Furthermore, virus-induced gene silencing was employed to knock down the expression of the PGIP homologous in N. benthamiana. Results showed that Agrobacterium-mediated expression efficiency greatly decreased in both HpPGIP and Hyp-1-PGIP transgenic plants, as assessed by GUS staining assays. However, silencing of PGIP in N. benthamiana increased the resistance to A. tumefaciens rather than susceptibility, which correlated with induction of pathogenesis-related proteins (PRs). The expression of core genes involved in several defense pathways was also analyzed in transgenic tobacco plants. Overexpression of HpPGIP led to up-regulation of key genes involved in hormone signaling, microRNA-based gene silencing, homeostasis of reactive oxygen species, and the phenylpropanoid pathway. Overexpression of Hyp-1-PGIP seemed to enhance the effect of PGIP on the expression of most genes analyzed. Moreover, HpPGIP was detected in the cytoplasm, nucleus and the plasma membrane or cell wall by confocal microscopy. Overall, our findings suggest HpPGIP modulates recalcitrance to A. tumefaciens-mediated transformation in H. perforatum.

Nanoparticles Affect the Expression Stability of Housekeeping Genes in Plant Cells.

PURPOSE: We report on the expression stability of several housekeeping/reference genes that can be used in the normalization of target gene expression in quantitative real-time PCR (qRT-PCR) analysis of plant cells challenged with metal nanoparticles (NPs). MATERIALS AND METHODS: Uniform cell suspension cultures of Hypericum perforatum were treated with 25 mg/l silver and gold NPs (14-15 nm in diameter). Cells were collected after 0.5, 4.0, and 12 h. The total RNA isolated from the cells was analyzed for the stability of ACT2, ACT3, ACT7, EF1-α, GAPDH, H2A, TUB-α, TUB-ß, and 18S rRNA genes using qRT-PCR. The cycle threshold (Ct) values of the genes were analyzed using the geNorm, NormFinder, BestKeeper, and RefFinder statistical algorithms to rank gene stability. The stability of the top-ranked genes was validated by normalizing the expression of HYP1. RESULTS: The expression of the tested housekeeping genes varied with treatment duration and NP types. EF1-α in gold NP treatment and TUB-α and EF1-α in silver NP treatment ranked among the top three positions. However, none of the genes retained their top ranking with time and across NP types. CONCLUSION: EF1-α can be used as a reference for treatment involving both silver and gold NPs in H. perforatum cells. TUB-α can be used only for silver NP-treated cells. The expression instability of most of the housekeeping genes highlights the importance of systematic standardization of reference genes for NP treatment conditions to draw proper conclusions on the target gene expression.

Cordycepin Nanoencapsulated in Poly(Lactic-Co-Glycolic Acid) Exhibits Better Cytotoxicity and Lower Hemotoxicity Than Free Drug.

PURPOSE: Cordycepin, a natural product isolated from the fungus Cordyceps militaris, is a potential candidate for breast cancer therapy. However, due to its structural similarity with adenosine, cordycepin is rapidly metabolized into an inactive form in the body, hindering its development as a therapeutic agent. In the present study, we have prepared cordycepin as nanoparticles in poly(lactic-co-glycolic acid) (PLGA) and compared their cellular uptake, cytotoxicity and hemolytic potential with free cordycepin. MATERIALS AND METHODS: Cordycepin-loaded PLGA nanoparticles (CPNPs) were prepared by the double-emulsion solvent evaporation method. Physico-chemical characterization of the nanoparticles was done by zetasizer, transmission electron microscopy (TEM) and reverse-phase high-pressure liquid chromatography (RP-HPLC) analyses. Cellular uptake and cytotoxicity of CPNPs and free drug were tested in human breast cancer cells (MCF7). Hemolytic potential of both of these forms was evaluated in rat red blood cells (RBCs). RESULTS: Physico-chemical characterization revealed that CPNPs were spherical in shape, possessed a size range of 179-246 nm, and released the encapsulated drug sustainably over a period of 10 days. CPNPs exhibited a high level of cellular uptake and cytotoxicity than the free drug in MCF-7 cells. While CPNPs were not toxic to rat RBCs even at high concentrations, free cordycepin induced hemolysis of these cells at relatively low concentration. CONCLUSION: Our results reveal that delivery as CPNPs could enhance the clinical efficacy of cordycepin substantially.

Silver nanoparticles affect phenolic and phytoalexin composition of Arabidopsis thaliana.

Silver nanoparticles are widely used in industry, medicine, biotechnology and agriculture. As a consequence, these nanoparticles are reaching the environment as waste products, which might have a negative impact on the environment, especially on plants. This includes the elicitation of various biochemical processes in plants. In this article, we report on the changes in secondary metabolic profile of Arabidopsis thaliana seedlings subjected to silver nanoparticle treatment in vitro. Briefly, various sizes (10 nm, 40 nm and 100 nm in diameter) and concentrations (0.5-5.0 ppm) of silver nanoparticles were tested. Ultraperformance liquid chromatography coupled with ultraviolet and fluorescence detectors as well as hyphenated to a high-resolution mass spectrometer (UPLC-PDA-FLR, UPLC-HESI-HRMS) and HPLC - ion trap mass spectrometer (HPLC-ESI-MS/MS), were applied to identify and quantify secondary metabolites. To understand whether silver ions could induce changes in the secondary metabolite profile, seedlings treated with silver nitrate in concentrations equivalent to these of nanoparticles were also analysed. The results showed significant differences in the accumulation of phenolic and indole compounds between treatments. Silver nanoparticles and silver ions induced the biosynthesis of camalexin, hydroxycamalexin O-hexoside and hydroxycamalexin malonyl-hexoside. These compounds are important phytoalexins for Brassicaceae family (especially for Camelinae clad) and are also synthetized in response to biotic and abiotic stresses. Statistically significant changes have been also observed for five phenolic compounds and 5'Glucosyl-dihydroneoascorbigen in different treatment conditions.

Correction: Marslin, G., et al. Secondary Metabolites in the Green Synthesis of Metallic Nanoparticles. Materials 2018, 11, 940.

The authors have overlooked a few mistakes when rearranging the Table 1 and Table 2 and references at the final stages, which were carried-over to the published version of the review [...].

Elicitation as a tool to improve the profiles of high-value secondary metabolites and pharmacological properties of Hypericum perforatum.

OBJECTIVES: In this review, we aim at updating the available information on the improvement of the Hypericum perforatum L. (Hypericaceae) phytochemical profile and pharmacological properties via elicitation. KEY FINDINGS: Hypericum perforatum seedlings, shoots, roots, calli and cell suspension cultures were treated with diverse elicitors to induce the formation of secondary metabolites. The extracts of the elicitor-treated plant material containing naphthodianthrones, phloroglucinols, xanthones, flavonoids and other new compounds were quantitatively analysed and tested for their bioactivities. While hypericins were mainly produced in H. perforatum cultures containing dark nodules, namely shoots and seedlings, other classes of compounds such as xanthones, phloroglucinols and flavonoids were formed in all types of cultures. The extracts obtained from elicitor-treated samples generally possessed better bioactivities compared to the extract of control biomass. SUMMARY: Although elicitation is an excellent tool for the production of valuable secondary metabolites in H. perforatum cell and tissue cultures, its exploitation is still in its infancy mainly due to the lack of reproducibility and difficulties in scaling up biomass production.

Prediction and elucidation of factors affecting solubilisation of imatinib mesylate in lipids.

The physico-chemical properties of lipids influencing the solubilisation of imatinib mesylate (IM) in lipid matrix were evaluated and a statistical model to predict the same has been derived in the present study. After experimental quantification of IM solubility in various lipids, Hansen Hildebrand's total solubility parameters were calculated in order to study the role of various forces connected to lipid-drug interaction. To develop a relationship between the various descriptors of the lipids and experimental solubility of IM in lipids (% w/w), quantitative structure-solubility relationship (QSSR) was used. To generate equations that can predict the solubility of IM in lipids (%w/w), multiple linear regression was used. Amongst the various lipids tested, glyceryl monostearate and behenic acid solubilised the highest (6.19 ± 0.22%) and lowest (0.01 ± 0.01%) amounts of IM respectively. Our results suggested that alkyl chain length, polarity of the lipids, index of cohesive interaction in solids, estimated number of hydrogen bonds that would be accepted by the solute from water molecules in an aqueous solution, estimated number of hydrogen bonds that would be donated by the solute to water molecules in an aqueous solution and solvent accessible surface area collectively play a significant role in solubilising IM in the lipids. The equation developed could predict the solubility of IM in lipids with good accuracy (R2pred = 0.912).

Retraction: Organometallic Ag nanostructures prepared using Hypericum perforatum extract are highly effective against multidrug-resistant bacteria.

[This retracts the article DOI: 10.1039/C8RA05655B.].

CuO and CeO2 Nanostructures Green Synthesized Using Olive Leaf Extract Inhibits the Growth of Highly Virulent Multidrug Resistant Bacteria.

One of the major challenges of nano-biotechnology is to engineer potent antimicrobial nanostructures (NS) with high biocompatibility. Keeping this in view, we have performed aqueous olive leaf extract mediated one pot facile synthesis of CuO-NS and CeO2-NS. Prepared NS were homogenous, less than 26 nm in size, and small crystallite units as revealed by scanning electron microscopy (SEM) and X-ray diffraction (XRD) analyses. Fourier transform infrared spectroscopy (FTIR) of CuO-NS and CeO2-NS showed typical Cu-O prints around 592-660 cm-1 and Ce-O bond vibrations at 453 cm-1. The successful capping of CuO-NS and CeO2-NS by compounds present in the plant extract was further validated by high performance liquid chromatography (HPLC) and thermal gravimetric analysis (TGA). Active phyto-chemicals from the leaf extract simultaneously acted as strong reducing as well as capping agent in the NS synthesis. NS engineered in the present study showed antibacterial potential at extremely low concentration against highly virulent multidrug-resistant (MDR) gram-negative strains (Escherichia coli, Enterobacter cloacae, Acinetobacter baumannii and Pseudomonas aeruginosa), alarmed by World Health Organization (WHO). Furthermore, CuO-NS and CeO2-NS did not show any cytotoxicity on HEK-293 cell lines and Brine shrimp larvae indicating that the NS green synthesized in the present study are biocompatible.

Secondary Metabolites in the Green Synthesis of Metallic Nanoparticles.

The ability of organisms and organic compounds to reduce metal ions and stabilize them into nanoparticles (NPs) forms the basis of green synthesis. To date, synthesis of NPs from various metal ions using a diverse array of plant extracts has been reported. However, a clear understanding of the mechanism of green synthesis of NPs is lacking. Although most studies have neglected to analyze the green-synthesized NPs (GNPs) for the presence of compounds derived from the extract, several studies have demonstrated the conjugation of sugars, secondary metabolites, and proteins in these biogenic NPs. Despite several reports on the bioactivities (antimicrobial, antioxidant, cytotoxic, catalytic, etc.) of GNPs, only a handful of studies have compared these activities with their chemically synthesized counterparts. These comparisons have demonstrated that GNPs possess better bioactivities than NPs synthesized by other methods, which might be attributed to the presence of plant-derived compounds in these NPs. The ability of NPs to bind with organic compounds to form a stable complex has huge potential in the harvesting of precious molecules and for drug discovery, if harnessed meticulously. A thorough understanding of the mechanisms of green synthesis and high-throughput screening of stabilizing/capping agents on the physico-chemical properties of GNPs is warranted to realize the full potential of green nanotechnology.

Oral Delivery of Curcumin Polymeric Nanoparticles Ameliorates CCl4-Induced Subacute Hepatotoxicity in Wistar Rats.

Curcumin is the major bioactive compound of Curcuma longa, an important medicinal plant used in traditional herbal formulations since ancient times. In the present study, we report that curcumin nanoparticles (ηCur) protects Wistar rats against carbon tetrachloride (CCl4)-induced subacute hepatotoxicity. Nanoparticles of sizes less than 220 nm with spherical shape were prepared using PLGA and PVA respectively as polymer and stabilizer. Test animals were injected via intraperitoneal route with 1 mL/kg CCl4 (8% in olive oil) twice a week over a period of 8 weeks to induce hepatotoxicity. On the days following the CCl4 injection, test animals were orally administered with either curcumin or its equivalent dose of ηCur. Behavioural observation, biochemical analysis of serum and histopathological examination of liver of the experimental animals indicated that ηCur offer significantly higher hepatoprotection compared to curcumin.