Cow dung putrefaction via vermicomposting using Eisenia fetida and its influence on seed sprouting and vegetative growth of Viola wittrockiana (pansy).

The current research was conducted at Vermi-tech Unit, Muzaffarabad in 2018 to evaluate the efficacy of cow dung and vermicompost on seed sprouting, seedlings, and vegetative developmental parameters of Viola x wittrokiana (pansy). In the current study, vermicompost was produced using Eisenia fetida. Physicochemical parameters of vermicompost and organic manure were recorded before each experimentation. The potting experiment was designed and comprised of eight germination mediums containing different combinations of soil, sand, cow dung, and various concentrations of vermicompost such as 10% VC, 15% VC, 20% VC, 25% VC, 30% VC, and 35% VC. Seed sprouting and seedling developmental parameters were observed for 28 days while vegetative plant growth parameters were recorded after 10 weeks of transplantation in various vermicompost amended germination media. Pre and post-physicochemical analysis of germination media were also recorded to check their quality and permanency. The current findings showed that 30% VC germination media was an effective dose for early seed germination initiation and all seed germination parameters. However, the significant vegetative plant growth and flowering parameters of pansy occurred at 35% VC. Findings revealed that vermicompost not only enhanced the seed germination and growth of pansy but also improved soil health. These results indicate that vermicompost can be exploited as a potent bio-fertilizer for ornamental plant production.

Commercially Available Viola odorata Oil, Chemical Variability and Antimicrobial Activity.

Viola odorata L. oil is frequently recommended in the aromatherapeutic literature for treating respiratory, urinary, and skin infections; however, antimicrobial evidence is lacking. In addition, in aromatherapy, combinations of essential oils are predominantly utilized with the goal of achieving therapeutic synergy, yet no studies investigating the interaction of essential oil combinations with V. odorata oil exists. This study thus aimed to address these gaps by investigating the antimicrobial activity of three Viola odorata oil samples, sourced from different suppliers, independently and in combination with 20 different commercial essential oils, against micro-organisms involved in respiratory, skin, and urinary tract infections associated with global resistance trends. These pathogens include several of the ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp.) The chemical profile of the oils was determined using gas chromatography coupled with mass spectrometry. The minimum inhibitory concentrations (MIC) were determined using the broth micro-dilution method. The interactive profiles for the combinations were assessed by calculating the fractional inhibitory concentration index (ΣFIC). The main compounds varied across the three samples, and included phenethyl alcohol, isopropyl myristate, 2-nonynoic acid, methyl ester, α-terpineol, α-cetone, and benzyl acetate. The V. odorata oil samples displayed overall poor antimicrobial activity when tested alone; however, the antimicrobial activity of the combinations resulted in 55 synergistic interactions where the combination with Santalum austrocaledonicum resulted in the lowest MIC values as low as 0.13 mg/mL. The frequency of the synergistic interactions predominantly occurred against Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, and Enterococcus faecium with noteworthy MIC values ranging from 0.25-1.00 mg/mL. This study also reports on the variability of V. odorata oils sold commercially. While this warrants caution, the antimicrobial benefit in combination provides an impetus for further studies to investigate the therapeutic potential.

First detection of endopolyploidy in tapetal cells and chromosomal anomalies in meiocytes of Viola pilosa cytotypes (2n=20) from Pir Panjal (Himalayas).

The nine Viola pilosa Blume populations studied from Pir Panjal contained 20 chromosomes. This count is not reported so far in Indian populations. Currently, comparison of tapetal and meiotic cells revealed the existence of synchrony in different developmental phases. Young tapetal cells at prometaphase co-occurred with the pollen mother cells (PMCs) at diakinesis to metaphase, mature tapetal cells with disintegrated chromatin material co-occurred with tetrads and no tapetal cells were found at mature pollen stage. Cytological studies in young tapetal cells revealed most of these to be endopolyploid, with each having 40 chromosomes. While outnumbering somatic cells contained clear 40 chromosomes which seemed to be the outcome of endomitosis, a sizeable number of cells possessed 40 sticky chromosomes at metaphase. Later chromosomes are likely to form restitution nucleus. Mature tapetal cells, occurring singly/cytomictically connected (3.2-26.31%) or showing coalescence (10.5-22.8%), did not contain recognizable chromosomes. Instead, they were characterized by disintegrated nuclear content. Further, meiotic studies revealed that the present population contained all/outnumbering euploid cells (2n=20); many of which exhibited nearly regular behaviour. However, 6.5-26.9% meiocytes of eight populations and 47% cells of P-Khe population depicted aneuploidy/contained quadri-octavalents, with per cent pollen viabilities of these ranging from 38.6 to 49.9. Going by the normal tapetal development in V. pilosa, existence of various chromosomal anomalies seems to have accounted for the reduction in gametic fertility of this taxon.

Volumetric imaging of optically cleared and fluorescently labeled animal tissue (VIOLA) for quantifying the 3D biodistribution of nanoparticles at cellular resolution in tumor tissue.

Nanoparticle (NP) technology holds significant promise to mediate targeted drug delivery to specific organs in the body. Understanding the 3D biodistribution of NPs in heterogeneous environments such as the tumor tissue can provide crucial information on efficacy, safety and potential clinical outcomes. Here we present a novel end-to-end workflow, VIOLA, which makes use of tissue clearing methodology in conjunction with high resolution imaging and advanced 3D image processing to quantify the spatiotemporal 3D biodistribution of fluorescently labeled ACCURIN® NPs. Specifically, we investigate the spatiotemporal biodistribution of NPs in three different murine tumor models (CT26, EMT6, and KPC-GEM) of increasing complexity and translational relevance. We have developed new endpoints to characterize NP biodistribution at multiple length scales. Our observations reveal that the macroscale NP biodistribution is spatially heterogeneous and exhibits a gradient with relatively high accumulation at the tumor periphery that progressively decreases towards the tumor core in all the tumor models. Microscale analysis revealed that NP extravasation from blood vessels increases in a time dependent manner and plateaus at 72 h post injection. Volumetric analysis and pharmacokinetic modeling of NP biodistribution in the vicinity of the blood vessels revealed that the local NP density exhibits a distance dependent spatiotemporal biodistribution which provide insights into the dynamics of NP extravasation in the tumor tissue. Our data represents a comprehensive analysis of NP biodistribution at multiple length scales in different tumor models providing unique insights into their spatiotemporal dynamics. Specifically, our results show that NPs exhibit a dynamic equilibrium with macroscale heterogeneity combined with microscale homogeneity.

Efficacy and safety of add-on Viola odorata L. in the treatment of COVID-19: A randomized double-blind controlled trial.

ETHNOPHARMACOLOGICAL RELEVANCE: Severe Acute Respiratory Syndrome (SARS) due to the novel coronavirus has become the highest priority that threatens human health. This situation demands widespread vaccination and the innovation of new therapeutic methods. Despite drug discoveries, the need for approving new medicaments is felt because of adverse effects and lack of efficacy. Several medicinal plants including Viola odorata L. are recommended in traditional Persian medicine for alleviating respiratory infection symptoms. Recent studies showed anti-inflammatory, antioxidant, anti-asthmatic, antitussive, analgesic, and antibacterial activities of sweet violet. These enhance respiratory functions, reduce pulmonary inflammation, and decline mucous membrane edema. This study aimed to evaluate the efficacy of sweet violet syrup in alleviating the manifestations of COVID-19 infection. MATERIAL AND METHODS: A randomized parallel-group double-blind controlled trial was conducted at Al-Zahra general hospital, Isfahan, Iran. A total of 108 outpatients were enrolled in the study. The patients were randomly allocated to intervention and placebo groups, with 54 patients in each group. The allocation was concealed using sealed opaque envelopes. The intervention group received violet syrup and the control group received placebo syrup, an add-on to the conventional treatment. The outcomes were COVID-19 manifestations, such as dyspnea, cough, myalgia, headache, and diarrhea, considered as outcomes of the study and were evaluated twice using a visual analog scale before the intervention and after 7 days, at the end of the study. Patients were followed daily by phone calls to monitor proper drug consumption and possible side effects. RESULTS: No significant difference was between groups regarding demographic characteristics and vital signs before and after the treatment. Although all symptoms have improved significantly in both groups, patients who received violet syrup recovered faster and the mean severity scores of cough (P = 0.025), myalgia (P = 0.036), headache (P = 0.037), and diarrhea (P = 0.044) decreased greater in comparison to control group. CONCLUSION: This study, the first clinical trial on the effectiveness of Viola odorata on SARS-CoV-2 patients, showed that Viola odorata L. effectively controls prevalent manifestations of COVID-19 including cough, myalgia, headache, and diarrhea. Regarding this survey, the violet syrup can be mentioned as a complementary treatment for viral influenza-like infections in which cough, myalgia, headache, and diarrhea are prominent.

LC-MS/MS identification and structural characterization of isolated cyclotides from precursor sequences of Viola odorata L. petiole tissue using computational approach.

Viola odorata L., known for its pharmacological properties, produces a plethora of structurally stable peptides called cyclotides. Cyclotides are macrocyclic peptides with a unique topology containing a cyclic cystine knot motif. The objective of the present study was to identify the precursor sequences and respective cyclotide domains from the petiole tissue of V. odorata. The study is based on the isolation, identification, and characterization of the cyclic peptides using LC-MS/MS followed by database searching and processing. Our study detected 47 precursor sequences encoded for 15 reported cyclotides, 4 putative novel cyclotides, and 3 acyclotides from the petiole tissue. The novel sequences identified were based on the hydrophobic nature, disulfide bonds, conserved cysteine residues, and presence of cyclic peptide backbone. Four putative novel and three acyclotides were also characterized for their sequence and subfamilies. A protein diversity wheel was used to reveal the variation in the amino acid sequence and cysteine residue conservation in the isolated cyclotides. The results provide information about the number of cyclotides and acyclotides from the petiole tissue and their sequence diversity, which may constitute novel tools for future research on this plant species.

Megastigmane sesquiterpenoids from whole plants of Viola kunawurensis.

Investigation on the chemical constituents of Viola kunawurensis resulted in the isolation of seven undescribed megastigmane sesquiterpenoids including four bicyclic megastigmane glucosides, kunawuronoside A-D, two megastigmane glucosides, kunawuronoside E-F, and a megastigmane, kunawurone A, together with ten known megastigmane sesquiterpenoids. Their structures were established by comprehensive 1D, 2D-NMR and HRESIMS analyses, and their absolute configurations were determined by comparing their calculated ECD data with the experimental ones. Evaluations of the anti-inflammatory activity revealed that kunawuronoside A-D and compounds 14-15 inhibited COX-2 expression with inhibition rates ranging from 36.7% to 58.5%, while the NO production induced by lipopolysaccharide (LPS) was suppressed by the kunawuronoside A-D in a dose-dependent manner in RAW264.7 macrophage cell line.

Innate, High Tolerance to Zinc and Lead in Violets Confirmed at the Suspended Cell Level.

Many species of the Viola L. genus (violets) colonize areas with high concentrations of trace elements in the soil, e.g., nickel, cadmium, zinc, and lead. Although tolerance to heavy metals is a common phenomenon in violets, it is not clear whether this is the result of gradual microevolutionary processes as a part of the adaptation to the specific conditions, or whether the tolerance was inherited from the ancestor(s). We developed cell suspension cultures of five plant species: two non-metallophytes-Arabidopsis thaliana (Col-0) and Viola · wittrockiana, and three metallophytes-V. philippica, V. tricolor, and Silene vulgaris subsp. humilis for tolerance tests. The aim of the study was to measure the level of tolerance of violets in comparison with species from the other genera to verify the hypothesis of the high, innate tolerance of the former. We measured cell viability, non-enzymatic antioxidant content, and the accumulation of heavy metals after cell treatment with Zn or Pb. The results indicate they are innate and independent on the ecological status (metallophyte vs. non-metallophyte) and high in comparison with other species tolerance to Zn and Pb in violets. Viability of the cells after Zn and Pb (1000 µM) exposure for 72 h was the highest in violets. Antioxidant content, after heavy metal treatment, increased significantly, particularly in metallophyte violets, indicating their high responsivity to metals. In all species, lead was detected in the protoplasm of the cells, not in the vacuole or cell wall. All violets were characterized by the accumulation capacity of lead. Here, we clearly show that the physiological and biochemical studies conducted with the use of heavy metals on plant cells translate into the heavy metal tolerance of the species.

Rapid evolution of selfing syndrome traits in Viola arvensis revealed by resurrection ecology.

PREMISE: As part of global change, climate warming and pollinator decline are expected to affect plant phenology and plant-pollinator interactions. This paper aims at characterizing rapid evolution of life history traits and floral traits over two decades in the wild pansy (Viola arvensis), a common weed in agrosystems. METHODS: We used a resurrection ecology approach with genotypes sampled in 1991 and 2012 from a population in Burgundy (France). The species has a mixed mating system (hereafter: mixed selfer) and presents a floral polymorphism. To correct for maternal effects, we measured plant traits in the second generation in a common garden (after a refreshing generation) to characterize plant evolution during the two decades. In addition, historical population selfing rates in 1991 and 2012 were inferred from microsatellites markers through heterozygote deficiency and identity disequilibrium. RESULTS: Phenotypic data revealed a significant advance in flowering date, reduced flower sizes and a higher propensity of plants to set seed by autonomous selfing. Moreover, we detected a change in color morph frequency with an increase of the pale morph frequency. In accordance with phenotypic data, the neutral genetic data revealed an increase in historical selfing rates from 0.68 in 1991 to 0.86 in 2012. CONCLUSIONS: Taken together, such data suggest that the wild pansy, a mixed selfer, is evolving a selfing syndrome that may be the consequence of reduced pollinator activity in agrosystems.

The hematological and histological studies for the hepatoprotective-like effect of the hydromethanolic extract and the fractions of Viola serpens Wall.

Traditionally, Viola serpens has been used in the treatment of several human disorders including liver diseases without any scientific evidence. As the current therapies are not very effective and face challenges of unwanted effects and patient compliance, therefore more effective and safe agents are highly needed. The current study aimed to evaluate the hepatoprotective potential of the crude extract and subsequent fractions of the whole plant in the in-vivo model using various hematological and histopathological parameters followed by an HPLC study for the identification of phenolic compounds. Rabbits (1000-1200 g) were used in the study. Paracetamol (2g) was used to induce hepatotoxicity in experimental rabbits. The plant extract was used in two doses (150 and 300 mg/kg body weights) for eight days. The hematological parameters AST, ALT and ALP values were determined along with the histopathology of the liver. Phenolic compounds were identified by high-performance liquid chromatography (HPLC) Agilent-1260 infinity from their retention time, UV spectra and available standards while quantification was done taking the percent peak area. The doses 150 and 300 mg/kg body weight seemed to be more effective. The hematological values and the histopathological slides show the hepatoprotective effect of the plant. Regeneration indicated the presence of nuclei, nuclear cleaning, prominent nucleoli, RBC's, central veins and plates of hepatocytes. The HPLC studies revealed the presence of a number of phenicol compounds. The crude extract and the subsequent fractions of the plant possess strong hepatoprotective activity, providing a scientific rationale for its uses in the treatment of liver toxicities.

Application of chloroplast genome in the identification of Traditional Chinese Medicine Viola philippica.

BACKGROUND: Viola philippica Cav. is the only source plant of "Zi Hua Di Ding", which is a Traditional Chinese Medicine (TCM) that is utilized as an antifebrile and detoxicant agent for the treatment of acute pyogenic infections. Historically, many Viola species with violet flowers have been misused in "Zi Hua Di Ding". Viola have been recognized as a taxonomically difficult genera due to their highly similar morphological characteristics. Here, all common V. philippica adulterants were sampled. A total of 24 complete chloroplast (cp) genomes were analyzed, among these 5 cp genome sequences were downloaded from GenBank and 19 cp genomes, including 2 "Zi Hua Di Ding" purchased from a local TCM pharmacy, were newly sequenced. RESULTS: The Viola cp genomes ranged from 156,483 bp to 158,940 bp in length. A total of 110 unique genes were annotated, including 76 protein-coding genes, 30 tRNAs, and four rRNAs. Sequence divergence analysis screening identified 16 highly diverged sequences; these could be used as markers for the identification of Viola species. The morphological, maximum likelihood and Bayesian inference trees of whole cp genome sequences and highly diverged sequences were divided into five monophyletic clades. The species in each of the five clades were identical in their positions within the morphological and cp genome tree. The shared morphological characters belonging to each clade was summarized. Interestingly, unique variable sites were found in ndhF, rpl22, and ycf1 of V. philippica, and these sites can be selected to distinguish V. philippica from samples all other Viola species, including its most closely related species. In addition, important morphological characteristics were proposed to assist the identification of V. philippica. We applied these methods to examine 2 "Zi Hua Di Ding" randomly purchased from the local TCM pharmacy, and this analysis revealed that the morphological and molecular characteristics were valid for the identification of V. philippica. CONCLUSIONS: This study provides invaluable data for the improvement of species identification and germplasm of V. philippica that may facilitate the application of a super-barcode in TCM identification and enable future studies on phylogenetic evolution and safe medical applications.

Morphological studies of self-assembled cyclotides extracted from Viola odorata as novel versatile platforms in biomedical applications.

Self-assembling peptides have attracted researchers' attention recently. They are classified as biomedical materials with unique properties formed in response to environmental conditions. Cyclotides are macrocyclic plant-derived peptides containing 28-37 amino acids that have the ability to self-assemble. Herein, we investigated the effect of pH, time, and temperature on the self-assembling properties of the cyclotides extracted from Viola odorata. For this purpose, the cyclotides were dispersed in aqueous trifluoroacetic acid at pH 2, 4, or 6 and incubated at 25 or 37 °C for 1, 2, 3, 5, 7 or 10 days, and the morphology of the self-assembled structures was identified by optical microscopy, polarized optical microscopy, scanning electron microscopy, transmission electron microscopy, and fluorescence microscopy. At pH 2 and 4, the self-assembly process of cyclotides comprises a number of steps, starting with the formation of spherical peptide nanostructures followed by hierarchically assembled nanotubes, and then shifting to nanofibers after 10 days. At pH 6, amorphous structures were produced even after 10 days. The temperature also could affect the self-assembly mechanism of the cyclotides. At 25 °C, the spherical peptide micelles formed firstly and then merged to form nanotubes, while at 37 °C the cyclotides showed crystallization followed by an increase in length with time. The fluorescence microscopy images showed that the nanotubes could efficiently entrap the hydrophobic molecules of coumarin. This comparative study on the self-assembly of the cyclotides extracted from Viola odorata is the first example exploring the capacity of these cyclotides to adopt precise nanostructures. The nanotubes and nanofibers obtained with these cyclotides might find interesting applications in drug delivery and tissue engineering.

Transcriptomic comparison sheds new light on regulatory networks for dimorphic flower development in response to photoperiod in Viola prionantha.

BACKGROUND: Chasmogamous (CH)-cleistogamous (CL) dimorphic flowers are developed in Viola prionantha. However, the environmental and genetic factors necessary for the CH-CL transition are unknown. RESULTS: In the present work, short-day (SD) conditions induced CH flowers, whereas long days (LDs) triggered CL flowers in V. prionantha. Compared to fully developed CH flowers, CL flowers had less mature stamens, no nectar glands, and immature petals. Comparative transcriptomics revealed differentially expressed genes (DEGs) during CL and CH development. Core genes in the photoperiod pathway, such as V. prionantha orthologs of GIGANTEA (GI), CONSTANS (CO), and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1), which promote floral induction, were highly expressed in CL flowers, whereas UNUSUAL FLORAL ORGANS (UFO) and B-class MADS-box genes for floral organ identity and development showed an opposite alteration. Moreover, genes in the glycolytic process, sucrose metabolic process, and fatty acid biosynthetic process were all highly expressed in CH flowers. Interestingly, V. prionantha orthologs of the B-class MADS-box genes APETALA3 (AP3) and PISTILLATA (PI) might relate to these sugar-fatty acid processes and were co-expressed with GAIP-B-like and YABBY5 (YAB5), which regulate the development of the petal, stamen, and nectary. Compared to CH flowers, DEGs and hub genes in the most significantly correlated modules of the gene co-expression network, which are involved in abiotic and biotic responses, were upregulated in CL flowers. CONCLUSIONS: We proposed an integrative model for transcription regulation of genes in the photoperiod pathway, floral organ development, stress response, and sugar-fatty acid processes to determine CH-CL flower development in V. prionantha. Particularly, under LDs, activated GI may induce genes involved in the stress-response pathways, and then downregulated AP3 and PI or UFO to inhibit the sugar-fatty acid metabolic processes, together forming CL flowers. In contrast, CH flowers were produced under SDs. This work provides novel insights into the developmental evolution of dimorphic flowers in Viola.

Development of an alarm symptom-based risk prediction score for localized oesophagogastric adenocarcinoma (VIOLA score).

BACKGROUND: Gastroesophageal adenocarcinoma is a major contributor to global disease burden with poor prognosis even in resectable, regionally limited stages. Feasible prognostic tools are crucial to improve patient management, yet scarce. PATIENTS AND METHODS: Disease-related symptoms, patient, tumour, treatment as well as laboratory parameters at initial diagnosis and overall survival (OS) of patients with stage II and III gastroesophageal adenocarcinoma, who were treated between 1990 and 2020 at the Medical University of Vienna, were evaluated in a cross-validation model to develop a feasible risk prediction score. RESULTS: In total, 628 patients were included in this single-centre analysis. The final score ranked from 0 to 10 and included the factors sex (female +1), age, years (30-59 +1, >60 +2), underweight classified by body mass index (+2), location of the tumour (stomach +1), stage (III +2), stenosis in endoscopy (+1) and weight loss (+1). The score was grouped into low- (0-3), medium- (4-6) and high-risk (7+) subgroups. The median OS were 70.3 [95% confidence interval (CI) 51.2-111.8], 23.4 (95% CI 21.2-26.7) and 12.6 (7.0-16.1) months, respectively. The 1-year survival probabilities were 0.88 (95% CI 0.83-0.93), 0.75 (95% CI 0.70-0.79) and 0.54 (95% CI 0.39-0.74), whereas the 5-year survival probabilities were 0.57 (95% CI 0.49-0.66), 0.24 (95% CI 0.20-0.28) and 0.09 (95% CI 0.03-0.28), respectively. CONCLUSIONS: The VIennese risk prediction score for Oesophagogastric Localized Adenocarcinoma (VIOLA) risk prediction score poses a feasible tool for the estimation of OS in patients with regionally limited gastroesophageal adenocarcinoma and, thus, may improve patient management in clinical routine. Prospective analyses should be carried out to confirm our findings.

Immunomodulatory effects of cyclotides isolated from Viola odorata in an experimental autoimmune encephalomyelitis animal model of multiple sclerosis.

BACKGROUND: Multiple sclerosis (MS) is a demyelinating disease of the central nervous system that causes chronic inflammation. Cyclotides are small plant proteins with a wide range of biological activity, making them a target for researchers to investigate. This study was conducted to investigate the possible effects of cyclotide-rich fractions from Viola odorata as an immunomodulatory agent in an experimental autoimmune encephalomyelitis (EAE) model of MS. METHODS: At room temperature, the plant materials were subjected to maceration in methanol: dichloromethane (1:1; v/v) for 3 days. The extraction was repeated 3 times, and the final concentrated extract was partitioned 3 times by 1/2 volume of double-distilled water. The aqueous phases were separated and freeze-dried. Finally, the crude extract was fractionated by C18 silicagel using vacuum liquid chromatography, with mobile phases of 30%, 50% and 80% of ethanol: water, respectively. The 50%, and 80% fractions were analyzed by HPLC and MALDI-TOF analysis and administrated intraperitoneally to forty-five female C57BL/6 EAE-induced mice, at 5, 25, and 50 mg/kg doses. After 28 days, the animals were evaluated using EAE clinical scoring which was done every 3 days, cytokine levels, and myelination level. RESULTS: The results confirmed the presence of cyclotides in V. odorata based on their retention time and the composition of mobile phase in HPLC and the molecular weight of the peaks in MALDI-TOF analysis. It was observed that cyclotides, especially in the 80% fraction group at the dose of 50 mg/kg significantly reduced the clinical scores, inflammation, and demyelination in EAE mice compared with the normal saline group (P<0.05), and the results of this group were comparable with fingolimod (P>0.05). CONCLUSION: It could be concluded that V. odorata is a rich source of cyclotides which they could be extracted by an easily available process and also, they could be used as immunomodulatory agents in MS, with similar effects to fingolimod.

Viola yedoensis Makino formula alleviates DNCB-induced atopic dermatitis by activating JAK2/STAT3 signaling pathway and promoting M2 macrophages polarization.

BACKGROUND: Atopic dermatitis (AD), a common inflammatory skin disorder, severely affects the life quality of patients and renders heavy financial burden on patient's family. The Chinese medicine Viola yedoensis Makino formula (VYAC) has been widely used for treating various skin disorders. Previous studies have reported that VYAC is effective in relieving DNCB-induced AD and inflammation. However, the anti-inflammatory mechanism of VYAC is still ill-defined and poorly understood. This study aims to investigate the therapeutic effects of VYAC on DNCB-induced AD and to elucidate the underlying anti-inflammatory mechanisms. METHODOLOGY: VYAC were extracted with 70% ethanol and lyophilized for use. AD mice were established by DNCB. The therapeutic effects of VYAC were evaluated by oral administration VYAC (150, 300 and 600 mg/kg) daily in vivo. The histopathological and immunohistochemistry were used to analyze skin lesion and macrophages infiltration, RT-qPCR and Elisa were used to analyze the inflammatory factors in skin tissues and serum. To explore the underlying mechanism of VYAC against AD in vitro. RAW264.7 cells and bone-marrow-derived macrophages (BMDMs) were employed for macrophage polarization analysis. Flow cytometer, immunofluorescence and western blot were used to analyze M2 macrophages markers. STAT3 siRNA were transfected into both cells to validate the effects of VYAC-induced macrophages M2 polarization via JAK2/STAT3 signaling pathway. RESULTS: VYAC ameliorated skin lesion of DNCB-induced AD mice by decreased clinical scores and epidermal thickness, decreased the level of pro-inflammatory factors (IL-1ß, TNF-α and IL-18) and enhanced IL-10 anti-inflammatory factor level, inhibited macrophages infiltration and promoted M2 macrophages polarization in vivo. VYAC significantly promoted M2 macrophages polarization in vitro. It is observed that VYAC not only inhibited the phosphorylation of JAK2 and STAT3 in RAW264.7 cells and BMDMs, but also accelerated the translocation to the nucleus. What's more, VYAC reduced the polarization of M2 macrophage by activating JAK2/STAT3 signaling pathway was observed in both cells. CONCLUSIONS: Our findings demonstrate that VYAC significantly ameliorates skin lesion of DNCB-induced AD mice and reduces the levels of inflammatory factors by activating JAK2/STAT3 signaling pathway and promoting M2 macrophages polarization.

Polyphenolic Profiling, Antioxidant, and Antimicrobial Activities Revealed the Quality and Adaptive Behavior of Viola Species, a Dietary Spice in the Himalayas.

BACKGROUND: Himalayan Viola species (Banksha) are traditionally important herbs with versatile therapeutic benefits such as antitussive, analgesic, antipyretic, antimalarial, anti-inflammatory, and anticancerous ones. The current investigation was focused on exploring polyphenolic profiles, antioxidant, and antimicrobial potentials of wild viola species at 15 gradient locations (375-1829 m). METHODS: Morphological, physiochemical, and proximate analyses were carried out as per WHO guidelines for plant drug standardization. Total polyphenolic and flavonoid content were carried out using gallic acid and rutin equivalent. UPLC-DAD was used to profile the targeted polyphenols (gallic acid, vanillic acid, syringic acid, p-coumaric acid, ferulic acid, rutin, quercetin, luteolin, caffeic acid, and epicatechin). Similarly, all samples were screened for antioxidant and antimicrobial activity. Statistical analysis was used to correlate polyphenolic and targeted activities to assess Viola species adaptation behavior patterns. RESULTS: Viola canescens (V. canescens) and Viola pilosa (V. pilosa) were found abundantly at their respective sites. Among flowers and leaves, flowers of V. canescens and V. pilosa showed higher total polyphenolic and flavonoid content (51.4 ± 1.13 mg GAE/g and 65.05 ± 0.85 mg RE/g, and 33.26 ± 0.62 mg GAE/g and 36.10 ± 1.41 mg RE/g, respectively). Furthermore, UPLC-DAD showed the uppermost content of p-coumaric acid in flowers and ferulic acid in leaves, while rutin was significant in both the tissues. CONCLUSIONS: The adaptive behavior of Viola species showed variability in morphological characters with the altitudes, while targeted polyphenols and activities were significant at mid-altitudes. This research helps in the selection of right chemotype for agrotechnological interventions and the development of nutraceutical products.

Exploration of molecular mechanisms responsible for anti-inflammatory and anti-angiogenic attributes of methanolic extract of Viola betonicifolia.

Uncontrolled inflammation plays a central role in the pathogenesis of various diseases. Currently available anti-inflammatory agents on prolonged use may lead to ulcers or thrombus formation. The present study was designed to evaluate the anti-inflammatory, anti-arthritic and anti-angiogenic potentials of methanol extract of Viola betonicifolia using battery of in vivo models. Methanol extract of Viola betonicifolia (Vb.Me) was prepared through maceration. High performance liquid chromatography (HPLC) and gas chromatography mass spectrometery (GC-MS) were performed to identify bioactive compounds present in Vb.Me. In vivo safety profile of Vb.Me was evaluated following OECD 425 acute toxicity guidelines. Anti-inflammatory potential of Vb.Me at three different dose levels was evaluated in in vivo acute (carrageenan and, histamine-induced paw oedema), sub-chronic (cotton pellet-induced granuloma) and chronic (Complete Freund's adjuvant-induced arthritis) models. Blood and paws samples were collected to study effects of Vb.Me treatment on the expression of various pro- and anti-inflammatory genes (RT-PCR) and to study the histopathological changes at tissue levels. Effects of Vb.Me on neovasculature development were studied in ex-ovo chicken chorioallantoic membrane (CAM) assay. Quercetin and n-hexadecanoic were identified as one of the major bioactive molecules in HPLC and GC-MS analysis of Vb.Me. Toxicity data revealed that Vb.Me was safe for administration up to the dose of 2000 mg/kg. Findings of inflammatory models showed that Vb.Me produced time and dose-dependent effects. 500 mg/kg Vb.Me showed significantly (p < 0.05) better effects as compared with 125 and 250 mg/kg. 500 mg/kg Vb.Me also showed comparable anti-inflammatory effects with indomethacin in both acute and chronic models respectively. RT-PCR data exhibited significant (p < 0.05) down-regulation of IL-6, IL-1ß, NF-kß, TNF-α and COX-2 genes with simultaneous up-regulation of IL-4 and IL-10 genes in the blood samples of animals treated with 500 mg/kg of Vb.Me and 10 mg/kg of indomethacin respectively. CAM assay data revealed arrest of microvessel outgrowth in Vb.Me-treated eggs. Altogether, findings of the current study indicate that Vb.Me exerts in vivo anti-inflammatory and anti-angiogenic effects through regulation of expression of various pro- and anti-inflammatory genes. Synergist actions of various bioactive molecules in Vb.Me are proposed to be responsible for these attributes. However, further studies to standardize the extract and evaluation of its potential in various inflammation-induced diseases are warranted.

Procyanidin C1 from Viola odorata L. inhibits Na+,K+-ATPase.

Members of the Viola genus play important roles in traditional Asian herbal medicine. This study investigates the ability of Viola odorata L. extracts to inhibit Na+,K+-ATPase, an essential animal enzyme responsible for membrane potential maintenance. The root extract of V. odorata strongly inhibited Na+,K+-ATPase, while leaf and seeds extracts were basically inactive. A UHPLC-QTOF-MS/MS metabolomic approach was used to identify the chemical principle of the root extract's activity, resulting in the detection of 35,292 features. Candidate active compounds were selected by correlating feature area with inhibitory activity in 14 isolated fractions. This yielded a set of 15 candidate compounds, of which 14 were preliminarily identified as procyanidins. Commercially available procyanidins (B1, B2, B3 and C1) were therefore purchased and their ability to inhibit Na+,K+-ATPase was investigated. Dimeric procyanidins B1, B2 and B3 were found to be inactive, but the trimeric procyanidin C1 strongly inhibited Na+,K+-ATPase with an IC50 of 4.5 µM. This newly discovered inhibitor was docked into crystal structures mimicking the Na3E1∼P·ADP and K2E2·Pi states to identify potential interaction sites within Na+,K+-ATPase. Possible binding mechanisms and the principle responsible for the observed root extract activity are discussed.

The involvement of cyclotides in mutual interactions of violets and the two-spotted spider mite.

Plants employ different chemicals to protect themselves from herbivory. These defenses may be constitutive or triggered by stress. The chemicals can be toxic, act as repellents, phagosuppressants and/or phago-deterrents. The two-spotted spider mite (Tetranychus urticae) is a generalist arthropod herbivorous pest and its feeding causes extensive damage both to crops and wild plants. Cyclotides are cyclic peptides involved in host-plant defenses. A single Viola sp. can produce more than a hundred cyclotides with different biological activities and roles. The organ and tissue specific cyclotide patterns change over the seasons and/or with environment, but the role of biotic/abiotic stress in shaping them remains unclear. Here, we demonstrate the involvement of cyclotides in mutual interactions between violets and mites. We used immunohistochemistry and mass spectrometry imaging to show the ingested cyclotides in T. urticae and assess the Viola odorata response to mite feeding. Moreover, to assess how mites are affected by feeding on violets, acceptance and reproductive performance was compared between Viola uliginosa, V. odorata and Phaseolus vulgaris. We demonstrate that cyclotides had been taken in by mites feeding on the violets. The ingested peptides were found in contact with epithelial cells of the mite digestive system, in the fecal matter, feces, ovary and eggs. Mites preferred common bean plants (P. vulgaris) to any of the violet species; the latter affected their reproductive performance. The production of particular cyclotides in V. odorata (denoted by molecular weights: 2979, 3001, 3017, 3068, 3084, 3123) was activated by mite feeding and their levels were significantly elevated compared to the control after 5 and 21 days of infestation. Specific cyclotides may affect mites by being indigestible or through direct interaction with cells in the mite digestive tract and reproductive organs. A group of particular peptides in V. odorata appears to be involved in defense response against herbivores.